Design, synthesis, and structure-activity relationships of highly potent 5-HT3 receptor ligands

Article abstract of DOI:10.1021/jm300801u

The 5-HT₃ receptor, a pentameric ligand-gated ion channel (pLGIC), is an important therapeutic target. During a recent fragment screen, 6-chloro-N-methyl-2-(4-methyl-1,4-diazepan-1-yl)quinazolin-4-amine (1) was identified as a 5-HT₃R hit fragment. Here we describe the synthesis and structure-activity relationships (SAR) of a series of (iso)quinoline and quinazoline compounds that were synthesized and screened for 5-HT₃R affinity using a [³H]granisetron displacement assay. These studies resulted in the discovery of several high affinity ligands of which compound 22 showed the highest affinity (pK_i > 10) for the 5-HT₃ receptor. The observed SAR is in agreement with established pharmacophore models for 5-HT₃ ligands and is used for ligand-receptor binding mode prediction using homology modeling and in silico docking approaches.

Full text of DOI:10.1021/jm300801u

Article
pubs.acs.org/jmc
Design, Synthesis, and Structure−Activity Relationships of Highly
Potent 5‑HT3 Receptor Ligands
Mark H. P. Verheij,†,‡ Andrew J. Thompson,‡,§ Jacqueline E. van Muijlwijk-Koezen,†
Sarah C. R. Lummis,§ Rob Leurs,† and Iwan J. P. de Esch*,†
†Leiden/Amsterdam Center of Drug Research (LACDR), Amsterdam Institute for Molecules Medicines and Systems (AIMMS),
Division of Medicinal Chemistry, Faculty of Sciences, VU University Amsterdam, Amsterdam, The Netherlands.
§Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom
S
* Supporting Information
ABSTRACT: The 5-HT3 receptor, a pentameric ligand-gated ion channel
(pLGIC), is an important therapeutic target. During a recent fragment screen, 6-
chloro-N-methyl-2-(4-methyl-1,4-diazepan-1-yl)quinazolin-4-amine (1) was identi-
fied as a 5-HT3R hit fragment. Here we describe the synthesis and structure−
activity relationships (SAR) of a series of (iso)quinoline and quinazoline
compounds that were synthesized and screened for 5-HT3R affinity using a
[3H]granisetron displacement assay. These studies resulted in the discovery of
several high affinity ligands of which compound 22 showed the highest affinity (pKi
> 10) for the 5-HT3 receptor. The observed SAR is in agreement with established
pharmacophore models for 5-HT3 ligands and is used for ligand−receptor binding
mode prediction using homology modeling and in silico docking approaches.
fined.19−25 The consensus is that 5-HT3 receptor ligands
1. INTRODUCTION
share three pharmacophore features: an aromatic part (A), a
basic moiety (B) and an intervening hydrogen bond acceptor
clinical use to prevent emesis during chemotherapy-induced,
5-HT3 receptor antagonists like alosetron and granisetron are in
(C) moiety (Figure 1b). Interestingly, Jørgensen and co-
workers recently published 5-HT3A receptor ligands that lack a
positively charged basic moiety, and it is suggested that these
compounds exert their effects via an allosteric site of the
receptor.26
radiotherapy-induced and postoperative nausea and vomiting
and to alleviate the effects of irritable bowel syndrome (IBS).1,2
Recent studies have also indicated that the 5-HT3 receptor is
involved in depression3 and may play a role in a range of other
indications such as schizophrenia, anxiety, substance abuse and
addiction, bulimia and pruritus. Moreover, the 5-HT3 receptor
is thought to modulate analgesia, inflammation and cognitive
processes.4 The 5-HT3 receptor belongs to the Cys-loop
receptor family of ion channels,5 which include nicotinic
acetylcholine (nACh), GABAA, and glycine receptors. All of
these receptors consist of five subunits that surround a central
ion conducting pore.6 At present, no high resolution structures
are available for Cys-loop receptors, but the availability of
crystal structures of the closely related acetylcholine binding
protein (AChBP)7 has significantly improved our under-
standing of the extracellular domain. Together with the results
from mutagenesis studies8−15 it is now acknowledged that the
ligand binding site is situated in the extracellular domain at the
interface of two subunits. The principal subunit contributes
three loops (A−C), while the complementary side contributes
three beta-sheets (“loops” D−F) from the adjacent subunit
(Figure 1a). 5-HT3 receptors can be homomeric (all A
subunits) or heteromeric (A + B to E), but the exact subunit
composition/stoichiometry of the latter type is not yet
clear.16,17 As early as 1989, a ligand-based pharmacophore for
5-HT3 receptor ligands was constructed.18 During the last two
decades, this pharmacophore has been continuously re-
Recently, we performed a cell-based fragment screen of 1010
fragments on the human 5-HT3A receptor resulting in a total of
70 hits.27 Within this set, the higher affinity ligands contained
bicyclic heteroaromatic structures, that is, quinoxalines,
quinazolines or quinolines. In the literature, fused six-
membered aromatic rings have been described as 5-HT3
receptor ligands, such as quipazine, which contains a quinoline
scaffold.28 Quipazine analogues have also been described as
multicyclic aryl piperazine 5-HT3R ligands by Anzini and co-
workers.29−32 In our screen, the quinazoline scaffold,
VUF10434 (Figure 1c) was identified as having the highest
affinity (18 nM) and a ligand efficiency (LE) of 0.5 (calculated
as the ratio of Gibbs free energy in kcal to the amount of non-
hydrogen atoms). Here, we examine the SAR of VUF10434 (1)
by designing, synthesizing and testing a range of new
derivatives and exploring their putative water-mediated binding
modes in a 5-HT3A receptor binding site homology model.
Received: May 21, 2012
© XXXX American Chemical Society
A
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Figure 1. (a) Extracellular domains of two adjacent subunits of the 5-HT3 receptor. (b) Pharmacophore features for 5-HT3 receptor antagonists. A,
B and C indicate respectively an aromatic part, a basic moiety and a hydrogen bond acceptor. (c) Structure of hit fragment VUF10434 (1).
Starting from 2-aminobenzoic acid (23), 2-thioxo-2,3-
dihydroquinazolin-4(1H)-one (24) was synthesized using a
procedure described in literature33 (Scheme 2). This
intermediate is allowed to react with methyl iodide to create
a more reactive intermediate (25) that is used in the
subsequent substitution reaction with N-methylpiperazine
which results in the formation compound 26 (Scheme 2).
Compounds 30 and 31 (Scheme 3) were synthesized by
reacting commercially available 2,4-dibromoquinoline (27)
2. CHEMISTRY
Quinazolines and (iso)quinolines were either obtained from
our in-house compound library or were synthesized as
described below. 2,4-Dichloroquinazoline (2) was reacted
with methylamine to yield the corresponding 4-substituted
intermediate (5) which was directly used without further
purification in the substitution reaction with N-methylpiper-
azine to form compound 21 (R2 = NMe, R3 = H) (Scheme 1).
a
Scheme 1. Preparation of Quinazoline Compounds 6−22
a
Scheme 3. Preparation of Quinoline Compounds 30 and 31
a
Reagents: (a) Aqueous NH4OH, 140 °C; (b) MeNH2, DiPEA,
EtOAc, 100 °C; (c) N-methylpiperazine, THF or neat, 160 °C.
with either ammoniumhydroxide or methylamine followed by
coupling of N-methylpiperazine. This synthesis route yielded in
the first step mixtures of both the 2- as well as the 4-substituted
regio isomers (for 28 and 29). Since these two regioisomers
were difficult to separate, it was decided to use them as
regioisomeric mixtures in the substitution reaction with N-
methylpiperazine. The obtained regioisomeric mixtures (for 30
and 31) could be separated by column chromatography. Both
compound 30 and 31 were obtained as pure regioisomers
whose identity was confirmed by 2D H NMR (NOESY, see
Supporting Information, Figure S2).
2-Methoxynaphtalene (32) was substituted with N-methyl-
piperazine using n-butyllithium as the base to create compound
33 in a good yield (Scheme 4).
a
Reagents: (a) amine (R2H) EtOH or EtOAc, rt; (b) Bu3SnH,
Pd(PPh3)4, toluene, 100 °C; (c) N-methylpiperazine, EtOAc, 140 °C;
(d) (i) MeNH2, EtOAc, DiPEA, rt, (ii) amine (R1H), EtOAc, DiPEA,
120 °C; (e) (i) MeNH2, N-methylpyrrolidone, DiPEA, rt, (ii) amine
(R1H), N-methylpyrrolidone, DiPEA, 150 °C.
1
2,4,6-Trichloroquinazoline (3) was reacted with tributyltin
hydride and tetrakis(triphenylphosphine)palladium(0) to yield
2,6-dichloroquinazoline (4) which in turn was substituted with
N-methylpiperazine under microwave irradiation to yield 11
(R2 = H, R3 = 6-Cl).
a
Scheme 2. Preparation of 2-(4-Methylpiperazin-1-yl)quinazolin-4(3H)-one (26)
a
Reagents: (a) SOCl2, reflux; (b) NH4SCN, acetone, rt; (c) CH3I, aqueous NaOH, rt; (d) N-methylpiperazine, 160 °C.
B
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Scheme 4. Preparation of 1-Methyl-4-(naphthalen-2-
yl)piperazine (33)
3. BIOCHEMICAL EVALUATION AND SAR STUDIES
a
Target compounds were evaluated using competition with the
5-HT3-specific radioligand [3H]granisetron34 and most com-
pounds displayed high affinity (expressed here as pKi) for the 5-
HT3A receptor (Tables 1−5). With the quinazoline scaffold of
compound 1 as a starting point, the role of the basic moiety as a
key feature in the 5-HT3 pharmacophore first was explored. A
series of N-methylhomopiperazine replacements were inves-
tigated (Table 1) by introducing several other cyclic amines.
The N-methylpiperazine analogue 6 shows a 16-fold increase in
affinity. All other compounds, including rigid tertiary amine 7,
rigid secondary amines (8, 9) and flexible tertiary amine 10,
show reduced affinity for the 5-HT3A receptor. Thus, for this
series of compounds, the N-methyl piperazine moiety at the R1
position results in the highest affinity.
a
Reagents: (a) N-Methylpiperazine, n-BuLi, 0 °C, then 2-methox-
ynaphthalene (32), THF, rt.
To investigate the role of the position of the heteroatom in
the aromatic ring, 34 was reacted with deprotonated N-
methylpiperazine in order to obtain 35 (Scheme 5).
Next, keeping the N-methylpiperazine moiety at the R1-
position, we explored the SAR associated with the R2 position
(Table 2). Removal of the substituent in the R2 position (i.e.,
R2 = H), leading to compound 11, results in a ∼10-fold
reduction in affinity. The same reduction in affinity is observed
for the removal or addition of a methyl group on the aniline
nitrogen atom (compounds 12, 13). Interesting is the ∼500-
fold lower affinity of compound 48 (Table 2). This compound
can adopt distinct tautomeric states (Figure 2) and
consequently nitrogen atom N3 of compound 48b is no
longer capable of interacting as a HBA.
Scheme 5. Preparation of 3-(4-Methylpiperazin-1-
yl)quinoline (35)
a
Analogs that contain more bulky groups at position R2,
including saturated ring systems (15) and aromatic moieties
(17), all show lower affinities than the lead compound (Table
2). Introduction of a large polar group results in high affinity 5-
HT3 compounds 18 and 20. The SAR at both the R1 and R2
position follow similar trends, as observed for the histamine
H4R,35−37 and agrees with our earlier reports that fragment
library screening indicates a remarkable degree of binding site
similarities of the 5-HT3AR and H4R proteins.38
In Table 3, the effect of the 6-chloro atom on 5-HT3 receptor
affinity is shown. Replacement of the 6-chloro atom by a
hydrogen atom results in a ∼5-fold reduction in affinity
(compare 6 and 21 respectively) when R2 = NMe. Replacement
of R3 = 6-Cl by R3 = H results in analogs that have a higher
affinity for the 5-HT3A receptor when R2 = H, OH or NH2 (the
affinity increases ∼5, ∼10, and ∼100 fold, respectively).
Interestingly, for the H4R the latter modification leads to a
reduction in affinity of more than 10-fold, marking a clear
difference in SAR for 5-HT3 and H4R receptors.35 Ultimately,
the SAR for these receptors is different, as can be deduced by
comparing the different affinities of compounds 6, 8−10, 12−
20, 22 and 48−5235−37 which have been synthesized, evaluated
a
Reagents: (a) NaNH2, t-BuOH, THF, 0 °C, N-methylpiperazine, 40
°C, then 3-bromoquinoline (34), 0 °C to rt.
For the synthesis of compounds 39 and 40 a similar
approach was used as for the synthesis of compounds 30 and
31. First, the commercially available 1,3-dichloroisoquinoline
(36) was reacted with either ammoniumhydroxide or methyl-
amine to yield 37 or 38 respectively as the single 1-substituted
regioisomers. Both intermediates were then used without
purification in the reaction with N-methylpiperazine to yield 39
and 40 as the single regioisomers (Scheme 6).
Compound 41 was reacted with a mixture of potassium
nitrate and sulfuric acid that in situ yields the reactive nitric acid.
This yielded a mixture of regioisomers with the 5- and 8-nitro-
compounds (42, 43) as the main products. These regioisomers
were separated by column chromatography and each individual
regioisomers was subsequently reacted with N-methylpiper-
azine to yield nitro analogs 44 and 45. These intermediates in
turn were reduced to the corresponding anilines (46, 47) with
palladium on carbon and hydrogen gas (Scheme 7).
a
Scheme 6. Preparation of isoquinoline compounds 39 and 40
a
Reagents: (a) NH3 in MeOH, 120 °C; (b) MeNH2, DiPEA, EtOH, 100 °C; (c) N-methylpiperazine, 220 °C.
C
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a
Scheme 7. Preparation of Quinolines 46 and 47
a
Reagents: (a) H2SO4, KNO3, 0 °C to rt; (b) N-methylpiperazine, 140 °C; (c) H2, Pd/C, MeOH, rt.
Table 1. 5-HT3A Receptor Binding Affinities (pKi) of
Compounds 1, 6−10
Table 2. 5-HT3A Receptor Binding Affinities (pKi) of
Compounds with Different Substituents at R2
a
Determined by radioligand competition using [3H]granisetron.35
for H4R affinity and published by our group earlier.
Importantly, compound 2235 is now identified as a 5-HT3
ligand with subnanomolar affinity and ∼40 000 fold selectivity
for the 5-HT3A receptor over the H4R.
a
Determined by radioligand competition using [3H]granisetron
The role of the H-bond acceptor moiety was explored using
the ligands in Table 4. As already illustrated by the
pharmacophore model in Figure 1B, a distance of ∼5 Å
between the basic moiety and the H-bond acceptor is essential.
This is in line with our present findings, as the distance between
these pharmacophore features in the minimal global energy
conformation of our new compounds is calculated to be ∼5 Å.
The highest affinity compounds are those where the
heteroaromatic nitrogen atom is positioned next to the N-
methylpiperazine group. These are compounds 49 (N1 = 4.96
Å, N3 = 4.97 Å), 51 (4.71 Å) and 52 (4.75 Å), with the highest
affinity compound (51) having a pKi > 9. The affinity of
compound 50 is 3−10 fold lower than compounds 49, 51 and
52; here the distance of N1 to the basic nitrogen atom is 4.79 Å
while the distance for N4 is 6.42 Å. The distance between the
basic nitrogen atom and the H-bond acceptor in compound 35
is 6.53 Å, and affinity drops >250 fold when compared to
(iso)quinolines 51 and 52. The importance of this
pharmacophore feature is supported by the fact that compound
Figure 2. Tautomers of compound 48.
33, which has no H-bond acceptor in the ring, shows similar
affinity as compound 35.
For quinazolines (Table 3) R2 = NH2 in combination with R3
= H results in a significant increase in affinity (22). Therefore,
the same derivatization strategy was applied to the correspond-
ing (iso)quinolines (Table 5). Here, 4-NMe substitution of the
quinoline scaffold (31) gives a ∼30-fold drop in affinity. In
addition, the 4-NH2 analogue (30) also shows a loss of affinity
D
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Table 3. Effect of R3 = Cl or R3 = H on the 5-HT3AR Affinity
Table 5. 5-HT3A Receptor Binding Affinities (pKi) of
Different (Methyl)aniline Compounds
a
#
R2
R3
pKi
11
49
48
26
12
22
6
H
Cl
H
8.13 0.27
8.87 0.10
6.24 0.24
7.33 0.12
7.92 0.09
10.29 0.15
8.95 0.05
8.53 0.05
H
OH
OH
NH2
NH2
NMe
NMe
Cl
H
Cl
H
Cl
21
H
a
Determined by radioligand competition using [3H]granisetron.
Table 4. 5-HT3A Receptor Binding Affinities (pKi) of
Compounds 33, 35 and 49−52
a
Determined by radioligand competition using [3H]granisetron.
HT2B receptors for which 31% and 43% inhibition is observed
respectively (Table 6 and Supporting Information, Tables S1−
S3).
Table 6. Cross-target Pharmacology of Compound 22 at a
Concentration of 0.1 μM
a
Determined by radioligand competition using [3H]granisetron.
% inhibition of control specific
a
target
radioligand
[3H]muscimol
binding s.e.m.
GABAA1
<15
b
(α1,β2,γ2)
although to a lesser extent. In order to explore the effect of the
aniline moiety when positioned on the second aromatic ring of
the quinoline moiety, two additional compounds were
synthesized. The compound that has the aniline functionality
at the 5-position of the quinoline scaffold resulted in compound
46. The 5-HT3AR affinity dropped ∼1000-fold and ∼100-fold
when compared to compounds 51 and 30, respectively. The 8-
aniline (47) derivative shows a comparable affinity to
compound 46. Finally, a similar approach for the isoquinoline
scaffold results in compounds 40 and 39. Here, addition of a 2-
NMe moiety (compound 40) results in a ∼5-fold lower affinity.
The 2-NH2 analogue (compound 39), however, shows a 140-
fold decrease in affinity when compared to the parent
isoquinoline compound 52.
c
Glycine
[3H]strychnine
[3H]cytisine
[3H]epibatidine
[3H]8-OH-DPAT
<15
<15
b
nACh (α4β2)
b
nACh (α7)
31
1
0
b
5-HT1A
<15
<15
c
5-HT1B
[
125I]CYP (+30 μM
isoproterenol)
c
5-HT1D
[3H]serotonin
<15
<15
43
b
5-HT2A
[
[
[
125I]( )DOI
125I]( )DOI
125I]( )DOI
b
5-HT2B
b
5-HT2C
<15
<15
<15
<15
b
5-HT4E
[3H]GR113808
[3H]LSD
[3H]LSD
b
5-HT6
b
5-HT7
To reassure that the most active compound (22) has no
cross-target affinities, this compound was subjected to a broader
pharmacological screening panel at a concentration of 1000
times its Ki for 5-HT3AR. Compound 22 shows no affinity for
other closely related receptors, except for nACh (α7) and 5-
a
Results are expressed as percent inhibition of control specific binding
obtained in the presence of compound 22. Results showing an
inhibition <15% are considered non binding and are displayed as <15
(see Supporting Information for more details). Human receptor. Rat
receptor.
b
c
E
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Figure 3. AChBP-based homology model of the human 5-HT3A receptor binding site (protein carbon atoms and cartoon representation in white).
(a) Overlay of the waters participating in the structural water network in several different AChBP crystal structures (red: 2WNC; blue: 2BYR; cyan:
2PGZ; green: 2BYS; yellow: 2XYT). (b−d) Binding poses for the 5-HT3 receptor antagonists. (b) Tropisetron (gold sticks). (c) Compound 22
(green sticks). (d) Compound 35 (green sticks).
Although protein structural information of cys-loop receptors
is very limited, the emerging crystallographic data on AChBP
structures, in combination with 5-HT3R site directed muta-
genesis studies allow some preliminary considerations with
respect to protein−ligand interactions. A homology model of
the 5-HT3A receptor binding site was constructed using the
tropisetron bound AChBP crystal structure (PDB ID: 2WNC)
as a template.39 The derived binding orientation of tropisetron
is in agreement with published site-directed mutagenesis
studies. The basic tropane moiety of tropisetron interacts
with the carbonyl backbone of W183 and is positioned in an
aromatic cavity consisting of W18311,40,41 and Y2348,42 from
the principle subunit, and W9013,43 of the complementary
subunit. The basic nitrogen atom of tropisetron is positioned at
4.5 Å from E129,9 a residue that is critical for both serotonin
and granisetron binding. The indole ring of tropisetron is in
close proximity to R92,13,43 which can interact with this moiety
through a cation-π interaction, and has been previously
identified to interact with the indazole ring of granisetron.15,43
Finally, five water molecules from the template cocrystal
structure (wat1−wat5) were included; these form a structural
water network that interacts with both the carbonyl moiety of
tropisetron and the receptor.39 We found that these water
molecules have a high level of conservation across the AChBP
structures that are cocrystallized with small antagonists (Figure
3a).44 Compounds were docked into the homology model with
GOLD45 using standard settings and the resulting poses were
scored with GOLDscore.46
From the observed docking poses of compounds 22 and 35
(Figure 3c−d) and other novel ligands (data not shown) we
speculate that the basic moiety of these ligands interacts with
the same residues as observed for the basic moiety of the
reference ligand tropisetron (Figure 3b). In the binding model,
heteroaromatic HBA (N3) of the quinazoline ring of
compound 22 can interact with a structural water molecule
(wat2) in the binding site (Figure 3c) similar to the carbonyl
oxygen atom of tropisetron. This interaction is also possible for
other ligands where the distance between the basic nitrogen
atom and the HBA is ∼5 Å. Therefore, this protein−ligand
model is able to accommodate the classic ligand-based 5-HT3R
pharmacophore (Figure 1b) by suggesting that the hydrogen
bond acceptor feature (C) of the ligands binds to the protein
via conserved water molecules, thereby explaining the subtle
differences in SAR with respect to this pharmacophore feature.
F
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mL) and extracted with EtOAc. The combined organic extracts were
dried over anhydrous sodium sulfate, filtered and concentrated under
reduced pressure to yield 444 mg of 5 (2.29 mmol, 91%) as a white
solid. H NMR (250 MHz, CDCl3) δ ppm 7.86−7.61 (m, 3H), 7.44
(m, 1H), 6.11 (br s, 1H), 3.22 (d, J = 4.9, 3H).
Future crystallization studies of 5-HT3R ligands with AChBP
might lead to additional insights with respect to binding mode,
although the ultimate proof via crystallization studies of the
actual 5-HT3R remains a scientific challenge. Nevertheless,
careful SAR studies and ligand-based design approaches in
combination the use of AChBP derived structural information
and site-directed mutagenesis might lead to insights that enable
structure-based drug design approaches that have proven to be
efficient for water-soluble drug targets.
1
6-Chloro-2-(3-(dimethylamino)azetidin-1-yl)-N-methylqui-
nazolin-4-amine (7). Methanamine in ethanol (33%, w/v, 0.11 mL,
0.86 mmol) and DiPEA (0.61 mL, 3.5 mmol) were added to a
suspension of 2,4,6-trichloroquinazoline (3) (200 mg, 0.86 mmol) in
EtOAc (2 mL) and stirred at rt until TLC indicated complete
conversion. Then N,N-Dimethyl-3-azetidinamine dihydrochloride
(150 mg, 0.87 mmol) was added and the resulting suspension was
heated by microwave irradiation at 120 °C for 30 min. The resulting
mixture was diluted with water and extracted with EtOAc. The
combined organic extracts were washed with brine and dried over
anhydrous sodium sulfate, filtered and concentrated under reduced
pressure. The crude oil was purified over SiO2 (EtOAc/MeOH/Et3N
= 90/5/5, v/v/v) to yield 98 mg of 7 (2.29 mmol, 91%) as a beige
CONCLUSION
■
In summary, optimization of fragment hit (1) has led to the
identification of several novel 5-HT3 receptor ligands with
(sub)nanomolar affinities that are comparable to some of the
most potent 5-HT3 ligands described to date. These ligands
match the known pharmacophore descriptors for 5-HT3
ligands. We found that the N-methylpiperazine group at the
R1 position is favorable, and that a hydrogen bond acceptor at
∼5 Å from the basic nitrogen is essential for high affinity
binding. Compound 22 was identified as the ligand with the
highest 5-HT3 receptor affinity in this study (pKi = 10.29) and
showed at least a-1000 fold selectivity over related targets. The
high affinity and previously reported receptor specificity of
closely related compounds47 suggests that some of these
compounds will make ideal candidates for future in vivo studies.
1
solid. H NMR (500 MHz, CDCl3) δ ppm 7.47−7.38 (m, 3H), 5.54
(br s, 1H), 4.23−4.16 (m, 2H), 4.06−3.99 (m, 2H), 3.24−3.14 (m,
1H), 3.11 (d, J = 4.8 Hz, 3H), 2.22 (s, 6H); 13C NMR (126 MHz,
CDCl3) δ ppm 160.56, 159.70, 150.40, 132.90, 127.22, 125.65, 120.25,
111.31, 55.99, 54.31, 41.91, 28.01; LCMS: ret. time 2.06 min, purity
>99%, [M + H]+ 292.05; HRMS m/z: [M + H]+ calcd for
C14H19ClN5: 292.1323, found: 292.1312.
6-Chloro-2-(4-methylpiperazin-1-yl)quinazoline (11). Tribu-
tyltin hydride (312 mg, 1.07 mmol) was added dropwise to a round-
bottom flask containing 2,4,6-trichloroquinazoline (3) (250 mg, 1.07
mmol) in dry toluene (5 mL). Then tetrakis (triphenylphosphine)-
palladium(0) (60 mg, 0.05 mmol) was added and the reaction mixture
was stirred at 100 °C for 1 h. Next, toluene was removed under
reduced pressure, the residue was dissolved in DCM (5 mL) and
hydrolyzed with a saturated solution of potassium fluoride. The
mixture was stirred vigorously for 30 min, filtered over a pad of Celite
and washed with DCM. The aqueous phase was extracted with DCM
and the combined organic extracts were dried over anhydrous
magnesium sulfate, filtered and concentrated under reduced pressure.
The residue was purified over SiO2 (Hept/DCM = 50/50 to 0/100, v/
v) yielding 30 mg of 4. This crude intermediate was then added to a
microwave tube containing N-methylpiperazine (1 mL, 9.0 mmol) and
EtOAc (3 mL). The resulting mixture was heated at 140 °C for 30 min
under microwave irradiation. The solvent and excess of N-
methylpiperazine were removed under reduced pressure and the
residue was purified over SiO2 (KPNH) (DCM/EtOAc = 90/10 to
60/40, v/v) yielding 20 mg of 11 (0.08 mmol, 7% over two steps) as a
EXPERIMENTAL SECTION
■
Chemistry. Chemicals and solvents were purchased from Aldrich
and used as received. Unless indicated otherwise, all reactions were
carried out under an inert atmosphere of dry N2. TLC analyses were
performed with Merck F254 alumina silica plates using UV
visualization or staining. Column purifications were carried out
automatically using the Biotage equipment. All HRMS spectra were
recorded on Bruker microTOF mass spectrometer using ESI in
1
positive ion mode. H NMR spectra were recorded on a Bruker 250
(250 MHz) or a Bruker 500 (500 MHz) spectrometer. Data are
reported as follows: chemical shift, integration, multiplicity (s = singlet,
d = doublet, t = triplet, br = broad, m = multiplet), and coupling
constants (Hz). Chemical shifts are reported in ppm with the natural
abundance of deuterium in the solvent as the internal reference
(CHCl3 in CDCl3: δ 7.26 and CH3OH in CH3OD: δ 3.31, (CH3)2SO
in (CD3)2SO: δ 2.50). 13C NMR spectra were recorded on a Bruker
500 (126 MHz) spectrometer with complete proton decoupling.
Chemical shifts are reported in ppm with the solvent resonance
resulting from incomplete deuteration as the internal reference
(CDCl3: δ 77.16, CH3OD: δ 49.00, (CD3)2SO: δ 39.52). Systematic
names for molecules according to IUPAC rules were generated using
the Chemdraw AutoNom program. Purity was determined using a
Shimadzu HPLC/MS workstation with a LC-20AD pump system,
SPD-M20A diode array detection, and a LCMS-2010 EV liquid
chromatography mass spectrometer. The column used is an Xbridge
C18 5 μm column (100 mm ×4.6 mm). Compound purities were
calculated as the percentage peak area of the analyzed compound by
UV detection at 230 nm. Solvents used were the following: solvent B =
MeCN 0.1% Formic Acid; solvent A = water 0.1%. The analysis was
conducted using a flow rate of 1.0 mL/min, start 5% B, linear gradient
to 90% B in 4.5 min, then 1.5 min at 90% B, linear gradient to 5% B in
0.5 min and then 1.5 min at 5% B, total run time of 8 min.
Compounds 6, 8−10, 12−20, 22 and 48−52 were synthesized by our
group as described by Smits et al.35−37
1
yellow solid. H NMR (500 MHz, CDCl3) δ ppm 8.92 (s, 1H), 7.62
(d, J = 2.2 Hz, 1H), 7.59−7.56 (m, 1H), 7.50 (d, J = 9.0 Hz, 1H),
4.02−3.93 (m, 4H), 2.56−2.47 (m, 4H), 2.36 (s, 3H); 13C NMR (126
MHz, CDCl3) δ 160.47, 159.23, 150.89, 134.80, 127.40, 127.37,
126.01, 119.87, 55.01, 46.22, 43.92; LCMS: ret. time 2.71 min, purity
97%, [M + H]+ 263.05, HRMS m/z: [M + H]+ calcd for C13H16ClN4:
263.1058, found: 263.1048.
N-Methyl-2-(4-methylpiperazin-1-yl)quinazolin-4-amine
(21). 5 (400 mg, 2.07 mmol) was added to a microwave tube
containing N-methylpiperazine (1.6 mL, 14.5 mmol) and EtOAc (5
mL). The resulting mixture was heated at 140 °C for 120 min under
microwave irradiation. The reaction mixture was diluted with H2O (10
mL) and extracted with EtOAc. The combined organic extracts were
dried over anhydrous sodium sulfate, filtered, concentrated under
reduced pressure and purified over SiO2 (EtOAc/Hept/Et3N = 80/
15/5, v/v) yielding 330 mg of compound 21 (1.28 mmol, 62%) as a
white solid. 1H NMR (250 MHz, CDCl3) δ ppm 7.60−7.33 (m, 3H),
7.03−6.96 (m, 1H), 5.74 (br s, 1H), 4.09−3.80 (m, 4H), 3.07 (d, J =
4.8 Hz, 3H), 2.60−2.36 (m, 4H), 2.31 (s, 3H); 13C NMR (126 MHz,
CDCl3) δ ppm 160.46, 159.09, 151.99, 132.38, 125.69, 120.85, 120.78,
110.66, 55.23, 46.29, 43.84, 27.97; LCMS: ret. time 1.49 min, purity
>99%, [M + H]+ 258.05; HRMS m/z: [M + H]+ calcd for C14H20N5:
258.1713, found: 258.1681.
2-Chloro-N-methylquinazolin-4-amine (5). Methanamine in
ethanol (40%, w/v, 0.24 mL, 2.76 mmol) and DiPEA (357 mg, 2.76
mmol) were added to a suspension of 2,4-dichloroquinazoline (2)
(500 mg, 2.51 mmol) in EtOH (20 mL) and stirred at rt. After 4.5 h
the reaction mixture was concentrated under reduced pressure to a
volume of 1 mL. The resulting mixture was diluted with water (25
2-Thioxo-2,3-dihydroquinazolin-4(1H)-one (24). A suspension
of anthranilic acid (23) (10.0 g, 73.0 mmol) in thionyl chloride (40.0
G
dx.doi.org/10.1021/jm300801u | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
mL) was heated at reflux for 2 h. The thionyl cloride was removed
under reduced pressure, DCM (20 mL) was added to the residue and
was also removed under reduced pressure (repeated three times).
Next, the crude acid chloride was dissolved in acetone (20 mL) and
added dropwise to a suspension of NH4SCN (5.75 g, 75.5 mmol) in
acetone (10 mL). The reaction mixture was stirred at rt for 45 min and
purity >99%, [M + H]+ 243.10; HRMS m/z: [M + H]+ calcd for
C14H19N4: 243.1604, found: 243.1596.
N-Methyl-2-(4-methylpiperazin-1-yl)quinolin-4-amine (31).
Methanamine in MeOH (40%, w/v, 0.6 mL, 7.00 mmol) was added
to a microwave tube containing 1,4-dibromoquinoline (27) (100 mg,
0.35 mmol), DiPEA (50 mg, 0.38 mmol) and EtOAc (2 mL). The
resulting mixture was heated at 100 °C under microwave irradiation
for 6 h. The reaction mixture was concentrated under reduced
pressure, dissolved in N-methylpiperazine (5 mL) and heated at 160
°C under microwave irradiation for 1 h. Then the mixture was diluted
with H2O (10 mL) and extracted with EtOAc (3 × 30 mL). The
combined organic extracts were washed with water, dried over
anhydrous sodium sulfate, filtered, concentrated under reduced
pressure and purified over SiO2 (EtOAc/MeOH/Et3N = 96/2/2, v/
v/v) yielding 46 mg of 31 (0.18 mmol, 51%) as a white solid. 1H NMR
(500 MHz, CDCl3) δ ppm 7.65 (d, J = 8.4 Hz, 1H), 7.52−7.43 (m,
2H), 7.20−6.93 (m, 1H), 5.92 (s, 1H), 4.89 (s, 1H), 3.79−3.69 (m,
4H), 3.00 (d, J = 5.0 Hz, 3H), 2.58−2.51 (m, 4H), 2.35 (s, 3H); 13C
NMR (126 MHz, CDCl3) δ ppm 158.99, 151.51, 148.11, 129.21,
127.30, 121.08, 118.92, 115.39, 85.14, 55.21, 46.27, 45.25, 30.06.
LCMS ret. time 1.83 min, purity 97%, [M + H]+ 257.05; HRMS m/z:
[M + H]+ calcd for C15H21N4: 257.1761, found: 257.1767.
1-Methyl-4-(naphthalen-2-yl)piperazine (33). A solution of n-
butyllithium in hexanes (2.5 N, 8.8 mL, 22.0 mmol) was added
dropwise to a stirred solution of N-methylpiperazine (2.0 mL, 18.1
mmol) in THF while maintaining a temperature around 0 °C. The
reaction mixture was kept at 0 °C for another 30 min and subsequently
stirred at rt for 1 h. Then 2-methoxynaphtalene (32) (3.16 g, 20.0
mmol) was added and the reaction mixture was stirred at rt for another
16 h. The reaction mixture was poured over an aqueous HCl solution
(10%, 100 mL), basidified with an aqueous solution of NaOH (2.5 M)
and extracted with DCM. The combined organic extracts were dried
over anhydrous sodium sulfate, filtered, concentrated under reduced
pressure and purified over SiO2 (EtOAc/Et3N = 95/5, v/v) yielding
3.56 g (15.7 mmol, 79%) of 33 as a white solid. 1H NMR (250 MHz,
CDCl3) δ ppm 7.79−7.59 (m, 3H), 7.43−7.31 (m, 1H), 7.30−7.18
(m, 2H), 7.10 (d, J = 2.4 Hz, 1H), 3.36−3.21 (m, 4H), 2.69−2.49 (m,
4H), 2.36 (s, 3H); 13C NMR (63 MHz, CDCl3) δ ppm 149.16, 134.66,
128.71, 128.55, 127.45, 126.77, 126.28, 123.38, 119.38, 110.27, 55.19,
49.51, 46.20. LCMS: ret. time 3.01 min, purity >99%, [M + H]+
226.95; HRMS m/z: [M + H]+ calcd for C15H19N2: 227.1543, found:
227.1536.
3-(4-Methylpiperazin-1-yl)quinoline (35). t-BuOH (1.48 g, 20.0
mmol) in THF (6 mL) was slowly added to a stirred suspension of
NaNH2 (1.56 g, 40.0 mmol) in THF (6 mL) at 0 °C. The resulting
suspension was stirred at 40 °C for 2 h. N-methylpiperazine (2.0 g,
20.0 mmol) was added and the resulting suspension was stirred at 40
°C for 2 h. Then, the reaction mixture was cooled to 0 °C and a
solution of 3-bromoquinoline (34) (2.08 g, 10.0 mmol) in THF (5
mL) was added. The resulting mixture was stirred at rt for 1 h. Next,
the reaction mixture was cooled to 0 °C and hydrolyzed with H2O (20
mL). The resulting mixture was extracted with DCM, the combined
organic extracts were dried over Na2SO4, filtered, concentrated under
reduced pressure and purified over SiO2 (EtOAc/MeOH = 99/1 to
90/10, v/v) yielding 890 mg (3.91 mmol, 19%) of 35 as an off white
solid. 1H NMR (250 MHz, CDCl3) δ ppm 8.85 (d, J = 2.8, 1H), 8.08−
7.97 (m, 1H), 7.72 (m, 1H), 7.60−7.45 (m, 2H), 7.40 (d, J = 2.8, 1H),
3.48−3.32 (m, 4H), 2.79 − 2.65 (m, 4H), 2.46 (s, 3H); 13C NMR (63
MHz, CDCl3) δ ppm 144.96, 144.79, 142.99, 128.96, 128.84, 126.92,
126.59, 126.39, 116.73, 54.87, 49.14, 46.13. LCMS: ret. time 2.18 min,
purity 99%, [M + H]+ 227.95; HRMS m/z: [M + H]+ calcd for
C14H18N3: 228.1495, found: 228.1483.
3-(4-Methylpiperazin-1-yl)isoquinolin-1-amine (39). A solu-
tion of ammonia in methanol (7N, 10 mL, 70.0 mmol) was added to a
microwave tube containing 1,3-dichloroisoquinoline (36) (400 mg,
2.02 mmol). The resulting mixture was heated at 120 °C for 6 h under
microwave irradiation. The reaction mixture was concentrated under
reduced pressure and the crude product (37) (180 mg) was added to a
microwave tube containing N-methylpiperazine (5.0 mL, 45.0 mmol).
The resulting mixture was heated at 220 °C for 2 h under microwave
the solid was collected by filtration over a Buchner filter. This solid was
̈
then suspended in an aqueous solution of NaOH (10% w/w) (70 mL)
and filtered over a Buchner filter. Water (70 mL) was added to the
̈
residue and the resulting mixture was acidified to pH 2 with aqueous
2N HCl, the resulting solid was collected via filtration over a Buchner
̈
funnel. After washing with a H2O/MeO (50:50) solution (100 mL)
and subsequent drying under vacuum at 35 °C. The product, 10.0 g of
24, was obtained as a beige solid and used in the next step without
further purification.
2-(Methylthio)quinazolin-4(3H)-one (25). A solution of meth-
yliodide (17.9 g, 126 mmol) in MeOH (80 mL) was added to a
suspension of 24 (10.0 g, 56.2 mmol) in an aqueous solution of NaOH
(1% w/w) (80 mL). After 1 h at rt the pH was adjusted to pH 7 with
an aqueous solution of HCl (1 M) and the solvents were removed
under reduced pressure. A total of 7.0 g 25 was obtained as an off
white solid and used in the synthesis of 26 without further purification.
2-(4-Methylpiperazin-1-yl)quinazolin-4(3H)-one (26). Com-
pound 25 (2.00 g, 10.4 mmol) was added to a microwave tube
containing N-methylpiperazine (10.0 mL, 111 mmol). The resulting
mixture was heated at 160 °C under microwave irradiation for 30 min.
The excess of N-methylpiperazine was removed under reduced
pressure and the residue was suspended in H2O (30 mL). The
insoluble material was dissolved by addition of an aqueous NaOH
solution (10% w/w, 10 mL). After filtration over a Buchner filter to
̈
remove insolubles, the filtrate was concentrated under reduced
pressure and purified over SiO2 (KPNH) (EtOAc) yielding 1.08 g
1
of 26 (4.43 mmol, 43%) as an off white solid. H NMR (250 MHz,
MeOD) δ ppm 8.04 (dd, J = 8.0, 1.2 Hz, 1H), 7.70−7.60 (m, 1H),
7.46−7.39 (m, 1H), 7.33−7.22 (m, 1H), 3.85−3.73 (m, 4H), 2.65−
2.55 (m, 4H), 2.39 (s, 3H). 13C NMR (63 MHz, CDCl3) δ ppm
165.68, 151.35, 150.44, 134.97, 126.25, 125.35, 122.68, 116.85, 54.79,
46.20, 45.14; LCMS: ret. time 2.04 min, purity >99%, [M + H]+
244.95; HRMS m/z: [M + H]+ calcd for C13H17N4O: 245.1397,
found: 245.1401.
2-Bromoquinolin-4-amine and 4-bromoquinolin-2-amine
(28). (mixture of both regio isomers). 2,4-Dibromoquinoline (27)
(860 mg, 3.00 mmol) was added to a microwave tube containing an
aqueous solution of ammonium hydroxide (28−30%, w/v, 10 mL).
The resulting mixture was heated at 140 °C under microwave
irradiation for 120 min. The reaction mixture was diluted with water
(15 mL) and subsequently extracted with DCM (3 × 15 mL). The
combined organic extracts were dried over anhydrous magnesium
sulfate, filtered and concentrated under reduced pressure. The
resulting crude product was purified over SiO2 (Hept/EtOAc = 50/
50, v/v) yielding 378 mg (1.70 mmol, 57%) of a regioisomeric mixture
containing 2-(4-methylpiperazin-1-yl)quinolin-4-amine and 4-(4-
methylpiperazin-1-yl)quinolin-2-amine.
2-(4-Methylpiperazin-1-yl)quinolin-4-amine (30). The regioi-
someric mixture (28) (378 mg, 1.70 mmol) was added to a microwave
tube containing THF (4 mL) and N-methylpiperazine (1.20 g, 12.0
mmol). The resulting mixture was heated at 160 °C for 90 min under
microwave irradiation. The reaction mixture was quenched with a
saturated aqueous solution of NaHCO3 (4 mL) and extracted with
EtOAC (100 mL). The combined organic extracts were then washed
with H2O. The combined organic extracts were dried over Na2SO4,
filtered and concentrated under reduced pressure. The residue was
purified over SiO2 (KPNH) (DCM/MeOH = 95/5, v/v) yielding 100
1
mg (0.41 mmol) of 30 as an off white solid. H NMR (250 MHz,
CDCl3) δ ppm 7.66 (d, J = 7.8 Hz, 1H), 7.55 (d, J = 8.2 Hz, 1H),
7.52−7.45 (m, 1H), 7.19−7.13 (m, 1H), 6.16 (s, 1H), 4.51 (br s, 2H),
3.72−3.63 (m, 4H), 2.57−2.45 (m, 4H), 2.34 (s, 3H); 13C NMR (63
MHz, CDCl3) δ ppm 158.65, 150.48, 148.63, 129.63, 127.23, 121.30,
119.91, 115.41, 90.34, 55.13, 46.24, 45.14; LCMS: ret. time 5.24 min,
H
dx.doi.org/10.1021/jm300801u | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
irradiation. Then the excess N-methylpiperazine was removed under
reduced pressure and the crude product was purified over SiO2
(EtOAc/Et3N = 98/2, v/v) to yield 102 mg of 39 (0.42 mmol, 21%
over 2 steps) as a dark green oil. 1H NMR (500 MHz, CDCl3) δ ppm
7.60 (d, J = 8.2 Hz, 1H), 7.48 (d, J = 8.2 Hz, 1H), 7.44−7.40 (m, 1H),
7.17−7.12 (m, 1H), 6.22 (s, 1H), 4.97 (s, 2H), 3.69−3.32 (m, 4H),
2.65−2.46 (m, 4H), 2.36 (s, 3H); 13C NMR (126 MHz, CDCl3) δ
ppm 155.50, 155.21, 140.46, 130.05, 125.86, 122.63, 122.10, 112.78,
91.00, 55.02, 46.25, 46.19. LCMS: ret. time 1.86 min, purity >99%, [M
+ H]+ 243.00; HRMS m/z: [M + H]+ calcd for C14H19N4: 243.1604,
found: 243.1604.
MHz, CDCl3) δ ppm 8.68 (d, J = 9.7 Hz, 1H), 8.00−7.90 (m, 2H),
7.55 (dd, J = 8.4, 7.8 Hz, 1H), 7.16 (d, J = 9.7 Hz, 1H), 3.86−3.78 (m,
4H), 2.59−2.50 (m, 4H), 2.37 (s, 3H); 13C NMR (126 MHz, CDCl3)
δ ppm 157.07, 148.77, 145.73, 133.34, 133.02, 127.56, 119.82, 115.52,
112.26, 54.91, 46.21, 44.66; LCMS: ret. time 3.62 min, purity >99%,
[M + H]+ 273.10; HRMS m/z: [M + H]+ calcd for C14H17N4O2:
273.1333, found: 273.1337.
2-(4-Methylpiperazin-1-yl)-8-nitroquinoline (45). 2-chloro-8-
nitroquinoline (43) (800 mg, 3.84 mmol) was dissolved in N-
methylpiperazine (10 mL). Potassium carbonate (530 mg, 3.84 mmol)
was added. The solution was stirred overnight at 140 °C. The reaction
mixture turned brown/dark red. The reaction mixture was quenched
with water and extracted with ethyl acetate. The red organic layer was
collected, dried over anhydrous sodium sulfate and concentrated under
vacuum to obtain a brown oil which was purified by column
chromatography (EtOAc/Et3N = 96/4, v/v) to obtain 45 (989 mg,
3.63 mmol, 95%) as a light brown solid. 1H NMR (250 MHz, CDCl3)
δ ppm 7.93−7.87 (m, 2H), 7.74 (dd, J = 8.0, 1.3 Hz, 1H), 7.23−7.15
(m, 1H), 7.04 (d, J = 9.3 Hz, 1H), 3.85−3.78 (m, 4H), 2.56−2.48 (m,
4H), 2.35 (s, 3H); 13C NMR (126 MHz, CDCl3) δ ppm 157.56,
145.42, 139.94, 137.29, 131.48, 124.53, 124.17, 120.03, 110.60, 54.85,
46.16, 44.57; LCMS: ret. time 2.70 min, purity >99%, [M + H]+
273.05; HRMS m/z: [M + H]+ calcd for C14H17N4O2: 273.1333,
found: 273.1341.
N-Methyl-3-(4-methylpiperazin-1-yl)isoquinolin-1-amine
(40). Methanamine in ethanol (33%, w/v, 1.3 mL, 15.0 mmol) was
added to a microwave tube containing 1,3-dichloroisoquinoline (36)
(500 mg, 2.52 mmol), DiPEA (522 mg, 4.04 mmol) and EtOH (5
mL). The resulting mixture was heated at 100 °C under microwave
irradiation for 6 h and at rt for 16 h. The reaction mixture was
concentrated under reduced pressure, diluted with H2O (10 mL) and
extracted with EtOAc (3 × 100 mL). The combined organic extracts
were washed with water and brine, dried over anhydrous sodium
sulfate, filtered and concentrated under reduced pressure. The crude
product (38) (450 mg) was added to a microwave tube containing N-
methylpiperazine (3.5 g, 35.0 mmol). The resulting mixture was
heated at 220 °C for 20 min under microwave irradiation. Then the
mixture was diluted with H2O (10 mL) and extracted with EtOAc (3
× 100 mL). The combined organic extracts were washed with water,
dried over anhydrous sodium sulfate, filtered, concentrated under
reduced pressure and purified over SiO2 (DCM/EtOAc/Et3N = 50/
49/1, v/v/v) yielding 228 mg of 40 (0.89 mmol, 35% over 2 steps) as
2-(4-Methylpiperazin-1-yl)quinolin-5-amine (46). 2-(4-meth-
ylpiperazin-1-yl)-5-nitroquinoline (44) (206 mg, 0.76 mmol) was
dissolved in methanol (50 mL). Pd/C 5 wt % (30 mg) was added to
the solution and the resulting suspension was stirred overnight at rt
under a hydrogen gas atmosphere. Hereafter, the mixture was filtered
over Celite and concentrated under reduced pressure to obtain 46
1
a light brown solid. H NMR (500 MHz, CDCl3) δ ppm 7.54 (d, J =
1
(143 mg, 0.59 mmol, 81%) as a brown solid. H NMR (500 MHz,
8.2 Hz, 1H), 7.46 (d, J = 8.2 Hz, 1H), 7.42 − 7.35 (m, 1H), 7.15 −
7.06 (m, 1H), 6.10 (s, 1H), 5.17 (s, 1H), 3.63 − 3.54 (m, 4H), 3.13 (d,
J = 4.8 Hz, 3H), 2.63 − 2.53 (m, 4H), 2.37 (s, 3H); 13C NMR (126
MHz, CDCl3) δ ppm 155.53, 154.95, 140.16, 129.53, 125.88, 121.71,
121.34, 113.35, 88.60, 55.09, 46.33, 46.10, 28.68; LCMS: ret. time 1.94
min, purity 95%, [M+H]+ 257.10; HRMS m/z: [M+H]+ calcd for
C15H21N4: 257.1761, found: 257.1757.
2-Chloro-5-nitroquinoline (42) and 2-Chloro-8-nitroquino-
line (43). 2-chloroquinoline (41) (9.47 g, 57.9 mmol) was dissolved
in sulfuric acid (62 mL, 1.16 mol) while stirring at 0 °C. After 15 min,
potassium nitrate (6.23 g, 61.6 mmol) dissolved in sulfuric acid (20
mL) was slowly added to the solution. The mixture was stirred for 2 h
while maintaining the temperature below 10 °C. Subsequently, the
mixture was stirred overnight at rt and poured over ice (200 g). The
CDCl3) δ ppm 7.93 (d, J = 9.3 Hz, 1H), 7.35−7.30 (m, 1H), 7.18 (d, J
= 8.4 Hz, 1H), 6.92 (d, J = 9.3 Hz, 1H), 6.53 (d, J = 7.4 Hz, 1H), 4.01
(s, 2H), 3.81−3.70 (m, 4H), 2.59−2.50 (m, 4H), 2.36 (s, 3H); 13C
NMR (126 MHz, CDCl3) δ ppm 157.30, 148.82, 142.21, 130.97,
130.11, 117.84, 113.00, 107.81, 107.14, 55.05, 46.26, 45.02; LCMS:
ret. time 0.96 min, purity 99%, [M + H]+ 243.10; HRMS m/z: [M +
H]+ calcd for C14H19N4: 243.1604, found: 243.1599.
2-(4-Methylpiperazin-1-yl)quinolin-8-amine (47). 2-(4-meth-
ylpiperazin-1-yl)-8-nitroquinoline (43) (203 mg, 0.75 mmol) was
dissolved in methanol (50 mL). Pd/C 5 wt % (30 mg) was added to
the solution and the resulting suspension was stirred overnight at rt
under a hydrogen gas atmosphere. Hereafter, the mixture was filtered
over Celite and was concentrated over vacuum to obtain 47 (168 mg,
1
0.69 mmol, 93%) as a brown solid. H NMR (250 MHz, MeOD) δ
resulting suspension was filtered over a Buchner funnel and washed
̈
ppm 7.93 (d, J = 9.1 Hz, 1H), 7.14 (d, J = 9.1 Hz, 1H), 7.07−7.00 (m,
2H), 6.94−6.89 (m, 1H), 3.87−3.76 (m, 4H), 2.88−2.77 (m, 4H),
2.54 (s, 3H); 13C NMR (126 MHz, CH3OH+D2O) δ ppm 156.95,
143.24, 139.14, 138.53, 124.87, 124.47, 117.51, 112.90, 111.07, 55.36,
45.36, 45.35; LCMS: ret. time 2.07 min, purity 98%, [M + H]+ 243.10;
HRMS m/z: [M + H]+ calcd for C14H19N4: 243.1604, found:
243.1599.
Radioligand Binding. 5-HT3AR. HEK293 cells stably expressing 5-
HT3AR were scraped into 1 mL of ice-cold HEPES buffer (10 mM,
pH 7.4) and frozen. After thawing, they were washed with HEPES
buffer and homogenized using a fine-bore syringe. Fifty microliters of
cell membranes were incubated in 0.5 mL HEPES buffer containing
0.7 nM [3H]granisetron (∼Kd) and differing concentrations of the test
compound. Competition binding (8 point) was performed on at least
three separate plates of transfected cells. Nonspecific binding was
determined using 1 mM quipazine. Reactions were incubated for at
least 24 h at 4 °C, to allow compounds with slow kinetics to
equilibrate. Incubations were terminated by vacuum filtration using a
Brandel cell harvester (Alpha Biotech Ltd., London, UK) onto GF/B
filters presoaked in 0.3% polyethyleneimine. Radioactivity was
determined by scintillation counting. Data were fit according to the
equation:
twice with ice water (200 mL) and ethanol (40 mL). The residual solid
was dissolved in dichloromethane (50 mL) and dried with anhydrous
sodium sulfate, filtered and concentrated under reduced pressure. The
obtained 10.4 g crude product was purified by column chromatog-
raphy over SiO2 (EtOAc/Hept = 5/95, v/v) to obtain 42 (2.70 g, 13
mmol, 22%) as a white solid and 43 (4.06 g, 19.5 mmol, 34%) as an
off-white solid. 2-Chloro-5-nitroquinoline (42): 1H NMR (250 MHz,
CDCl3) δ ppm 8.99 (d, J = 9.2 Hz, 1H), 8.43−8.31 (m, 2H), 7.84 (t, J
= 8.1 Hz, 1H), 7.64 (d, J = 9.2 Hz, 1H); 13C NMR (126 MHz, CDCl3)
δ ppm 152.47, 148.02, 145.43, 135.54, 134.95, 128.82, 125.45, 124.89,
119.91; LCMS: ret. time 4.56 min, purity >99%, [M + H]+ 220.00. 2-
1
Chloro-8-nitroquinoline (43): H NMR (250 MHz, CDCl3) δ ppm
8.20 (d, J = 8.7 Hz, 1H), 8.12−8.01 (m, 2H), 7.64 (t, J = 7.9 Hz, 1H),
7.54 (d, J = 8.7 Hz, 1H); 13C NMR (126 MHz, CDCl3) δ ppm 153.56,
147.15, 138.98, 138.62, 131.75, 127.61, 125.78, 124.93, 124.55; LCMS:
ret. time 4.35 min, purity >99%, [M + H]+ 208.90.
2-(4-Methylpiperazin-1-yl)-5-nitroquinoline (44). 2-chloro-5-
nitroquinoline (42) (800 mg, 3.84 mmol) was dissolved in N-
methylpiperazine (10 mL). The solution was stirred at 140 °C
overnight. The reaction mixture was quenched with water and
extracted with ethyl acetate. The red organic layer was collected, dried
over anhydrous sodium sulfate, filtered and concentrated under
reduced pressure to obtain a brown oil which was purified by column
chromatography over SiO2 (EtOAc/Et3N = 96/4, v/v) to obtain 44
Bmax − Bmin
BL = Bmin
+
H(log L50−log L)
1 + 10n
1
(892 mg, 3.28 mmol, 85%) as a light brown solid. H NMR (250
I
dx.doi.org/10.1021/jm300801u | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
where L is the concentration of ligand present; BL is the binding in the
presence of ligand concentration L; Bmin is the binding when L = 0;
Bmax is the binding when L = ∞, L50 is the concentration of L which
gives a binding equal to (Bmax + Bmin)/2; and nH is the Hill coefficient.
Ki values were estimated from IC50 values using the Cheng-Prusoff
equation48 Ki = IC50/(1+[L]/Kd) where Ki is the equilibrium
dissociation constant for binding of the unlabeled antagonist, IC50 is
the concentration of antagonist that blocks half the specific binding,
[L] is the free concentration of radioligand and Kd is the equilibrium
dissociation constant of the radioligand.
Non 5-HT3AR. Off-target binding of compound 22 was assessed at
13 additional targets (Table 6). With the exception of nACh(α7), all
single point radioligand competition binding was performed by
Cerep.49 The results are expressed as a percent inhibition of control
specific binding (100-(measured specific binding/control specific
binding)*100) obtained in the presence of compound 22 (see
Supporting Information, Tables S1−S3 for more details).
Homology Modeling. A model of the 5-HT3A receptor binding
site was constructed by homology modeling using MOE (version
2010.10, Chemical Computing Group Montreal),50 based on the
tropisetron bound AChBP X-ray crystal structure determined at 2.20 Å
resolution (PDB code: 2WNC).39 The sequence of the human 5-
HT3A gene (Q7KZM7) was aligned with the Aplysia californica gene
(Q8WSF8) using the “Protein Align” option in MOE (standard
settings) and adjusted manually. The final sequence alignment is given
in Supporting Information, Figure S2. Chains A and E of the original
PDB structure were selected to serve as the template. Structural waters
located in this binding pocket of the 2WNC crystal structure form a
conserved protein−ligand H-bond interaction network in several other
AChBP crystals containing small competitive antagonists (e.g., 2BYR,
2PGZ, 2BYS, 2XYT44), and were included in the 5-HT3A receptor
model. The template backbone, the ligand and the water molecules
were fixed and 10 preliminary receptor models were constructed based
on the template backbone. During this construction, the ligand and
waters were considered as an additional restraint using the
“Environment” option within MOE. The structural quality of the
models was checked using the evaluation modules in MOE. During
this evaluation the focus was on the binding site region of the model.
The protein geometry of receptor atoms was evaluated for their bond
lengths, bond angles, atom clashes and contact energies. Ramachan-
dran plots were used to check the Phi and Psi angles of all residues.
Model 1 was selected for further refinement, hydrogen atoms were
added, partial atomic charges were calculated and the protein was
minimized around the fixed ligand and static water molecules using the
Amber99 force field in MOE.
Molecular Docking. Ligands were protonated according to
physiological pH using the MMFF94 force field and MOE software.
Relevant tautomers of the ligands were created and subsequently the
three-dimensional structures were energy minimized and converted
into mol2 files using Molecular Network’s Corina.51 Docking studies
were performed with the docking program GOLD (Version 5.0,
CCDC, Cambridge, UK)45 using default settings. The protein binding
site was defined by a radius of 12 Å around W183 (atom NE1) of the
principle subunit. A total of 30 dockings were set up for each ligand
run with a root-mean-square deviation (rmsd) tolerance of 1.5 Å for
early termination. Docking scores were calculated with the GoldScore
scoring function.
AUTHOR INFORMATION
Corresponding Author
*Phone: +31205987841. Fax: +31205987610. E-mail: i.de.
esch@vu.nl. Division of Medicinal Chemistry, Faculty of
Sciences, VU University Amsterdam, Room: G-379a, De
Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
■
Author Contributions
‡These authors contributed equally to this work.
Author Contributions
M.H.V. and A.J.T. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
M.V. and I.d.E. would like to acknowledge EEC grant
(Neurocypres) for financial support. This work was further-
more supported by COST Action BM0806COST. S.C.R.L. and
A.J.T. are Wellcome Trust funded (081925). There are no
competing interests.
ABBREVIATIONS USED
■
AChBP, acetylcholine binding protein; pLGIC, pentameric
ligand-gated ion channel; IBS, irritable bowel syndrome; 5-HT,
5-hydroxy tryptamine; nACh, nicotinic acetylcholine; GABA, γ-
aminobutyric acid; ( ) DOI, ( ) 2,5-dimethoxy-4-iodoam-
phetamine; 8-OH-DPAT, 8-hydroxy-2-(di-N-propylamino)-
tetralin; MeOH, methanol; EtOH, ethanol; EtOAc, ethyl
acetate; DiPEA, diisopropyl ethyl amine; t-BuOH, tert-
butoxide; n-BuLi, n-butyllithium; DCM, dichloromethane;
Hept, heptanes; MeNH2, methylamine; HBA, hydrogen bond
acceptor; ESI, electron-spray ionization; UV, ultraviolet;
LCMS, liquid chromatography mass spectrometry
REFERENCES
■
(1) Hsu, E. S. A review of granisetron, 5-hydroxytryptamine3
receptor antagonists, and other antiemetics. Am. J. Therap. 2010, 17
(5), 476−486.
(2) Spiller, R. C. Targeting the 5-HT3 receptor in the treatment of
irritable bowel syndrome. Curr. Opin. Pharmacol. 2011, 11 (1), 68−74.
(3) Rajkumar, R.; Mahesh, R. The auspicious role of the 5-HT3
receptor in depression: a probable neuronal target? J. Psychopharmacol.
2010, 24 (4), 455−469.
(4) Thompson, A. J.; Lummis, S. C. The 5-HT3 receptor as a
therapeutic target. Expert Opin. Ther. Targets 2007, 11 (4), 527−540.
(5) Maricq, A. V.; Peterson, A. S.; Brake, A. J.; Myers, R. M.; Julius,
D. Primary structure and functional expression of the 5HT3 receptor,
a serotonin-gated ion channel. Science (New York, N.Y 1991, 254
(5030), 432−437.
(6) Unwin, N. Acetylcholine receptor channel imaged in the open
state. Nature 1995, 373 (6509), 37−43.
(7) Brejc, K.; van Dijk, W. J.; Klaassen, R. V.; Schuurmans, M.; van
Der Oost, J.; Smit, A. B.; Sixma, T. K. Crystal structure of an ACh-
binding protein reveals the ligand-binding domain of nicotinic
receptors. Nature 2001, 411 (6835), 269−276.
(8) Beene, D. L.; Price, K. L.; Lester, H. A.; Dougherty, D. A.;
Lummis, S. C. Tyrosine residues that control binding and gating in the
5-hydroxytryptamine3 receptor revealed by unnatural amino acid
mutagenesis. J. Neurosci. 2004, 24 (41), 9097−9104.
(9) Price, K. L.; Bower, K. S.; Thompson, A. J.; Lester, H. A.;
Dougherty, D. A.; Lummis, S. C. R. A hydrogen bond in loop a is
critical for the binding and function of the 5-HT3 receptor.
Biochemistry 2008, 47 (24), 6370−6377.
(10) Suryanarayanan, A.; Joshi, P. R.; Bikadi, Z.; Mani, M.; Kulkarni,
T. R.; Gaines, C.; Schulte, M. K. The loop C region of the murine 5-
ASSOCIATED CONTENT
■
S
* Supporting Information
Overview of the 2D NOESY H NMR interactions found for
1
compounds 30 and 31. The sequence alignment between the
human 5-HT3A gene (Q7KZM7) and the Aplysia californica
gene (Q8WSF8). Radioligand binding studies for the receptors
shown in Table 6. This material is available free of charge via
the Internet at http://pubs.acs.org.
J
dx.doi.org/10.1021/jm300801u | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
HT3A receptor contributes to the differential actions of 5-
hydroxytryptamine and m-chlorophenylbiguanide. Biochemistry 2005,
44 (25), 9140−9149.
(29) Anzini, M.; Cappelli, A.; Vomero, S.; Giorgi, G.; Langer, T.;
Hamon, M.; Merahi, N.; Emerit, B. M.; Cagnotto, A.; Skorupska, M.;
et al. Novel, potent, and selective 5-HT3 receptor antagonists based on
the arylpiperazine skeleton: synthesis, structure, biological activity, and
comparative molecular field analysis studies. J. Med. Chem. 1995, 38
(14), 2692−2704.
(30) Cappelli, A.; Anzini, M.; Vomero, S.; Canullo, L.; Mennuni, L.;
Makovec, F.; Doucet, E.; Hamon, M.; Menziani, M. C.; De Benedetti,
P. G.; Bruni, G.; Romeo, M. R.; Giorgi, G.; Donati, A. Novel potent
and selective central 5-HT3 receptor ligands provided with different
intrinsic efficacy. 2. Molecular basis of the intrinsic efficacy of
arylpiperazine derivatives at the central 5-HT3 receptors. J. Med. Chem.
1999, 42 (9), 1556−1575.
(31) Cappelli, A.; Anzini, M.; Vomero, S.; Mennuni, L.; Makovec, F.;
Doucet, E.; Hamon, M.; Bruni, G.; Romeo, M. R.; Menziani, M. C.; De
Benedetti, P. G.; Langer, T. Novel potent and selective central 5-HT3
receptor ligands provided with different intrinsic efficacy. 1. Mapping
the central 5-HT3 receptor binding site by arylpiperazine derivatives. J.
Med. Chem. 1998, 41 (5), 728−741.
(32) Cappelli, A.; Gallelli, A.; Manini, M.; Anzini, M.; Mennuni, L.;
Makovec, F.; Menziani, M. C.; Alcaro, S.; Ortuso, F.; Vomero, S.
Further studies on the interaction of the 5-hydroxytryptamine3 (5-
HT3) receptor with arylpiperazine ligands. development of a new 5-
HT3 receptor ligand showing potent acetylcholinesterase inhibitory
properties. J. Med. Chem. 2005, 48 (10), 3564−3575.
(33) Somers, F.; Ouedraogo, R.; Antoine, M.-H.; de Tullio, P.;
Becker, B.; Fontaine, J.; Damas, J.; Dupont, L.; Rigo, B.; Delarge, J.;
Lebrun, P.; Pirotte, B. Original 2-Alkylamino-6-halogenoquinazolin-
4(3H)-ones and KATP Channel Activity. J. Med. Chem. 2001, 44 (16),
2575−2585.
(34) Nelson, D. R.; Thomas, D. R. [3H]-BRL 43694 (granisetron), a
specific ligand for 5-HT3 binding sites in rat brain cortical membranes.
Biochem. Pharmacol. 1989, 38 (10), 1693−1695.
(35) Smits, R. A.; de Esch, I. J.; Zuiderveld, O. P.; Broeker, J.; Sansuk,
K.; Guaita, E.; Coruzzi, G.; Adami, M.; Haaksma, E.; Leurs, R.
Discovery of quinazolines as histamine H4 receptor inverse agonists
using a scaffold hopping approach. Journal of medicinal chemistry 2008,
51 (24), 7855−7865.
(36) Smits, R. A.; Lim, H. D.; Hanzer, A.; Zuiderveld, O. P.; Guaita,
E.; Adami, M.; Coruzzi, G.; Leurs, R.; de Esch, I. J. Fragment based
design of new H4 receptor-ligands with anti-inflammatory properties
in vivo. J. Med. Chem. 2008, 51 (8), 2457−2467.
(37) Smits, R. A.; Lim, H. D.; Stegink, B.; Bakker, R. A.; de Esch, I. J.;
Leurs, R. Characterization of the histamine H4 receptor binding site.
Part 1. Synthesis and pharmacological evaluation of dibenzodiazepine
derivatives. J. Med. Chem. 2006, 49 (15), 4512−4516.
(38) Verheij, M. H. P.; de Graaf, C.; de Kloe, G. E.; Nijmeijer, S.;
Vischer, H. F.; Smits, R. A.; Zuiderveld, O. P.; Hulscher, S.; Silvestri,
L.; Thompson, A. J.; van Muijlwijk-Koezen, J. E.; Lummis, S. C. R.;
Leurs, R.; de Esch, I. J. P. Fragment library screening reveals
remarkable similarities between the G protein-coupled receptor
histamine H4 and the ion channel serotonin 5-HT3A. Bioorg. Med.
Chem. Lett. 2011, 21 (18), 5460−5464.
(39) Hibbs, R. E.; Sulzenbacher, G.; Shi, J.; Talley, T. T.; Conrod, S.;
Kem, W. R.; Taylor, P.; Marchot, P.; Bourne, Y. Structural
determinants for interaction of partial agonists with acetylcholine
binding protein and neuronal alpha7 nicotinic acetylcholine receptor.
EMBO J. 2009, 28, 3040−3051.
(40) Beene, D. L.; Brandt, G. S.; Zhong, W.; Zacharias, N. M.; Lester,
H. A.; Dougherty, D. A. Cation-pi interactions in ligand recognition by
serotonergic (5-HT3A) and nicotinic acetylcholine receptors: the
anomalous binding properties of nicotine. Biochemistry 2002, 41 (32),
10262−10269.
(11) Thompson, A. J.; Lochner, M.; Lummis, S. C. Loop B is a major
structural component of the 5-HT3 receptor. Biophys. J. 2008, 5728−
5736.
(12) Thompson, A. J.; Padgett, C. L.; Lummis, S. C. Mutagenesis and
molecular modeling reveal the importance of the 5-HT3 receptor F-
loop. J. Biol. Chem. 2006, 281 (24), 16576−16582.
(13) Thompson, A. J.; Price, K. L.; Reeves, D. C.; Chan, S. L.; Chau,
P. L.; Lummis, S. C. Locating an antagonist in the 5-HT3 receptor
binding site using modeling and radioligand binding. J. Biol. Chem.
2005, 280 (21), 20476−20482.
(14) Venkataraman, P.; Venkatachalan, S. P.; Joshi, P. R.; Muthalagi,
M.; Schulte, M. K. Identification of critical residues in loop E in the 5-
HT3ASR binding site. BMC Biochem. 2002, 3, 15.
(15) Yan, D.; White, M. M. Spatial orientation of the antagonist
granisetron in the ligand-binding site of the 5-HT3 receptor. Mol.
Pharmacol. 2005, 68 (2), 365−371.
(16) Barrera, N. P.; Herbert, P.; Henderson, R. M.; Martin, I. L.;
Edwardson, J. M. Atomic force microscopy reveals the stoichiometry
and subunit arrangement of 5-HT3 receptors. Proc. Natl. Acad. Sci.
U.S.A. 2005, 102 (35), 12595−12600.
(17) Thompson, A. J.; Price, K. L.; Lummis, S. C. R. Cysteine
modification reveals which subunits form the ligand binding site in
human heteromeric 5-HT3AB receptors. J. Physiol. 2011, 589 (17),
4243−4257.
(18) Schmidt, A. W.; Peroutka, S. J. Three-dimensional steric
molecular modeling of the 5-hydroxytryptamine3 receptor pharmaco-
phore. Mol. Pharmacol. 1989, 36 (4), 505−511.
(19) Asagarasu, A.; Matsui, T.; Hayashi, H.; Tamaoki, S.; Yamauchi,
Y.; Sato, M. Design and Synthesis of Piperazinylpyridine Derivatives as
Novel 5-HT(1A) Agonists/5-HT(3) Antagonists for the Treatment of
Irritable Bowel Syndrome (IBS). Chem. Pharm. Bull. (Tokyo) 2009, 57
(1), 34−42.
(20) Cappelli, A.; Anzini, M.; Vomero, S.; Mennuni, L.; Makovec, F.;
Doucet, E.; Hamon, M.; Menziani, M. C.; De Benedetti, P. G.; Giorgi,
G.; Ghelardini, C.; Collina, S. Novel potent 5-HT(3) receptor ligands
based on the pyrrolidone structure: synthesis, biological evaluation,
and computational rationalization of the ligand-receptor interaction
modalities. Bioorg. Med. Chem. 2002, 10 (3), 779−801.
(21) Clark, R. D.; Miller, A. B.; Berger, J.; Repke, D. B.; Weinhardt,
K. K.; Kowalczyk, B. A.; Eglen, R. M.; Bonhaus, D. W.; Lee, C. H. 2-
(Quinuclidin-3-yl)pyrido[4,3-b]indol-1-ones and isoquinolin-1-ones.
Potent conformationally restricted 5-HT3 receptor antagonists. J. Med.
Chem. 1993, 36 (18), 2645−2657.
(22) Evans, S.; Galdes, A.; Gall, M. Molecular modeling of 5-HT3
receptor ligands. Pharmacol., Biochem. Behav. 1991, 40 (4), 1033−
1040.
(23) Hibert, M.; Hoffmann, R.; Miller, R.; Carr, A. Conformation-
activity relationship study of 5-HT3 receptor antagonists and a
definition of a model for this receptor site. J. Med. Chem. 1990, 33 (6),
1594−1600.
(24) Mahesh, R.; Perumal, R. V.; Pandi, P. V. Pharmacophore based
synthesis of 3-chloroquinoxaline-2-carboxamides as serotonin3 (5-
HT3) receptor antagonist. Biol. Pharm. Bull. 2004, 27 (9), 1403−1405.
(25) Thompson, A. J.; Lummis, S. C. 5-HT3 receptors. Curr. Pharm.
Des 2006, 12 (28), 3615−3630.
(26) Jørgensen, C. G.; Frølund, B.; Kehler, J.; Jensen, A. A. Discovery
of benzamide analogues as a novel class of 5-HT3 receptor agonists.
ChemMedChem 2012, 6 (4), 725−736.
(27) Thompson, A. J.; Verheij, M. H.; Leurs, R.; De Esch, I. J.;
Lummis, S. C. An efficient and information-rich biochemical method
design for fragment library screening on ion channels. Biotechniques
2010, 49 (5), 822−829.
(28) Hong, E.; Sancilio, L. F.; Vargas, R.; Pardo, E. G. Similarities
between the pharmacological actions of quipazine and serotonin. Eur.
J. Pharmacol. 1969, 6 (3), 274−280.
(41) Duffy, N. H.; Lester, H. A.; Dougherty, D. A. Ondansetron and
Granisetron binding orientation in the 5-HT(3) receptor determined
by unnatural amino acid mutagenesis. ACS Chem. Biol. 2012,
DOI: 10.1021/cb300246j.
K
dx.doi.org/10.1021/jm300801u | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(42) Price, K. L.; Lummis, S. C. The role of tyrosine residues in the
extracellular domain of the 5-hydroxytryptamine3 receptor. J. Biol.
Chem. 2004, 279 (22), 23294−23301.
(43) Yan, D.; Schulte, M. K.; Bloom, K. E.; White, M. M. Structural
features of the ligand-binding domain of the serotonin 5HT3 receptor.
J. Biol. Chem. 1999, 274 (9), 5537−5541.
(44) PDB: 2BYR, 2PGZ, 2BYS, 2XYT.
(45) Verdonk, M. L.; Chessari, G.; Cole, J. C.; Hartshorn, M. J.;
Murray, C. W.; Nissink, J. W. M.; Taylor, R. D.; Taylor, R. Modeling
water molecules in protein−ligand docking using GOLD. J. Med.
Chem. 2005, 48 (20), 6504−6515.
(46) Verdonk, M. L.; Cole, J. C.; Hartshorn, M. J.; Murray, C. W.;
Taylor, R. D. Improved protein-ligand docking using GOLD. Proteins
2003, 52 (4), 609−623.
(47) Thompson, A. J.; Verheij, M.; De Esch, I.; Lummis, S. C. R.,
VUF10166, a novel compound with differing activities at 5-HT3A and
5-HT3AB receptors. J. Pharmacol. Exp. Ther. 2012.
(48) Cheng, Y.; Prusoff, W. H. Relationship between the inhibition
constant (K1) and the concentration of inhibitor which causes 50%
inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 1973,
22 (23), 3099−108.
(49) Cerep, Le bois l’Eveq
cerep.fr.
̂
ue 86600 Celle l’Evescault France. www.
(50) MOE, Version 2010.10; Chemical Computing Group, Inc.:
Montreal, Canada, 2010.
(51) Corina, version 3.46; Molecular Networks GmbH: Erlangen,
Germany, 2006.
L
dx.doi.org/10.1021/jm300801u | J. Med. Chem. XXXX, XXX, XXX−XXX