Structure-Activity Relationships of Quinoxaline-Based 5-HT3A and 5-HT3AB Receptor-Selective Ligands

Article abstract of DOI:10.1002/cmdc.201300032

Until recently, discriminating between homomeric 5-HT₃A and heteromeric 5-HT₃AB receptors was only possible with ligands that bind in the receptor pore. This study describes the first series of ligands that can discriminate between these receptor types at the level of the orthosteric binding site. During a recent fragment screen, 2-chloro-3-(4-methylpiperazin-1-yl)quinoxaline (VUF10166) was identified as a ligand that displays an 83-fold difference in [³H]granisetron binding affinity between 5-HT₃A and 5-HT₃AB receptors. Fragment hit exploration, initiated from VUF10166 and 3-(4-methylpiperazin-1-yl)quinoxalin-2-ol, resulted in a series of compounds with higher affinity at either 5-HT₃A or 5-HT₃AB receptors. These ligands reveal that a single atom is sufficient to change the selectivity profile of a compound. At the extremes of the new compounds were 2-amino-3-(4-methylpiperazin-1-yl)quinoxaline, which showed 11-fold selectivity for the 5-HT₃A receptor, and 2-(4-methylpiperazin-1-yl)quinoxaline, which showed an 8.3-fold selectivity for the 5-HT₃AB receptor. These compounds represent novel molecular tools for studying 5-HT₃ receptor subtypes and could help elucidate their physiological roles.

Full text of DOI:10.1002/cmdc.201300032

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DOI: 10.1002/cmdc.201300032
Structure–Activity Relationships of Quinoxaline-Based 5-
HT3A and 5-HT3AB Receptor-Selective Ligands
Andrew J. Thompson,[b] Mark H. P. Verheij,[a] Jacqueline E. van Muijlwijk-Koezen,[a]
Sarah C. R. Lummis,*[b] Rob Leurs,[a] and Iwan J. P. de Esch[a]
Until recently, discriminating between homomeric 5-HT3A and
heteromeric 5-HT3AB receptors was only possible with ligands
that bind in the receptor pore. This study describes the first
series of ligands that can discriminate between these receptor
types at the level of the orthosteric binding site. During
a recent fragment screen, 2-chloro-3-(4-methylpiperazin-1-yl)-
quinoxaline (VUF10166) was identified as a ligand that displays
an 83-fold difference in [3H]granisetron binding affinity be-
tween 5-HT3A and 5-HT3AB receptors. Fragment hit explora-
tion, initiated from VUF10166 and 3-(4-methylpiperazin-1-yl)-
quinoxalin-2-ol, resulted in a series of compounds with higher
affinity at either 5-HT3A or 5-HT3AB receptors. These ligands
reveal that a single atom is sufficient to change the selectivity
profile of a compound. At the extremes of the new com-
pounds were 2-amino-3-(4-methylpiperazin-1-yl)quinoxaline,
which showed 11-fold selectivity for the 5-HT3A receptor, and
2-(4-methylpiperazin-1-yl)quinoxaline, which showed an 8.3-
fold selectivity for the 5-HT3AB receptor. These compounds
represent novel molecular tools for studying 5-HT3 receptor
subtypes and could help elucidate their physiological roles.
Introduction
5-HT3 receptors are ligand-gated ion channels that are respon-
sible for fast synaptic neurotransmission in the central (CNS)
and peripheral nervous systems (PNS). They are involved in
physiological functions as diverse as the vomiting reflex, pain
processing, reward, cognition, and anxiety, and modulate the
release of neurotransmitters such as acetylcholine, cholecysto-
kinin, dopamine, GABA, glutamate, and serotonin itself.[1] To
date, five different subunits (5-HT3A–5-HT3E) have been identi-
fied, but the homomeric 5-HT3A- and heteromeric 5-HT3AB-
containing receptors are the most fully characterized.[1,2] 5-
HT3A receptors are located primarily in the CNS, while 5-HT3AB
receptors may be more abundant in the PNS.[1,3]
The two 5-HT3 receptor subtypes (5-HT3A and 5-HT3AB) can
be distinguished by differences in their 5-HT concentration–re-
sponse curves (increased EC50 values and shallower Hill slopes),
increased single channel conductance (5-HT3A=sub-pS; 5-
HT3AB=16-30 pS), increased rate of desensitization, decreased
relative Ca2+ permeability, and different current–voltage rela-
tionships (5-HT3A is inwardly rectifying, 5-HT3AB is linear).[1b,5]
Pharmacologically distinguishing 5-HT3A from 5-HT3AB recep-
tors has historically required the use of compounds that bind
in the pore, such as bilobalide, ginkgolide, and picrotoxinin.[6]
In contrast, competitive ligands usually have very similar affini-
ties at 5-HT3A and 5-HT3AB receptors. Recently, however, a qui-
noxaline compound (VUF10166) was identified that showed
differences in both its binding affinity and functional proper-
ties at 5-HT3A and 5-HT3AB receptors (Figure 1).[7] Detailed
studies of VUF10166 showed that these differences may stem
from a second, allosteric, site only found in the 5-HT3AB recep-
tor, the occupation of which may increase the rate of ligand
dissociation from the adjacent orthosteric site.
5-HT3 receptors are members of the Cys-loop family of neu-
rotransmitter-gated receptors that all share a pentameric struc-
ture of subunits surrounding a central ion-conducting pore.
Each subunit has an extracellular domain, four transmembrane
a helices (one of which contributes to the ion conducting
pore) and an intracellular domain.[4] The agonist/competitive
antagonist (orthosteric) binding site is located at the interface
of two adjacent subunits and is formed by the convergence of
three loops (loops A–C) from the principal (or +) subunit and
three b sheets (loops D–E) from the adjacent complementary
(or ꢀ) subunit.
The actions of a range of quinoxalines have also been previ-
ously studied at both 5-HT3A and native receptors and re-
vealed that these compounds can be relatively potent (sub-
micromolar affinities) as antagonists, agonists, and partial ago-
[a] Dr. M. H. P. Verheij,+ Dr. J. E. van Muijlwijk-Koezen, Prof. R. Leurs,
Dr. I. J. P. de Esch
Amsterdam Institute for Molecules Medicines and Systems (AIMMS)
Division of Medicinal Chemistry, Faculty of Sciences
VU University Amsterdam, Amsterdam (The Netherlands)
[b] Dr. A. J. Thompson,+ Prof. S. C. R. Lummis
Department of Biochemistry, University of Cambridge, Cambridge (UK)
E-mail: sl120@cam.ac.uk
Figure 1. a) Structure of compound 1; b) general structure for all analogues;
c) VUF10166.
[+] These authors contributed equally to this work.
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nists, with potential as novel therapeutics.[8] There is
particular interest, for example, in developing qui-
noxalines which are impermeable to the blood–brain
barrier that would target peripheral 5-HT3 receptor-
s.[8a] None of these studies, however, have evaluated
ligand affinities at specific 5-HT3 receptor subtypes. In
this manuscript, we report the synthesis and binding
affinities of a series of quinoxalines and demonstrate
subtle differences in structure–activity relationships
(SAR) for the 5-HT3A and 5-HT3AB receptor subtypes
using competition binding on recombinantly ex-
pressed receptors in HEK293 cells.
Scheme 1. Synthesis of quinoxalines. Reagents and conditions: a) R2COCO2H, CH3OH, RT,
30 min; b) POCl3, 1008C, 1 h; c) N-methylpiperazine, mw, 1208C, or N-methylpiperazine,
EtOAc, mw, 1608C, 15 min.
Results and Discussion
Chemistry
The pharmacophore features of lead compound
1 and
VUF10166, and the effects of these features on 5-HT3A and 5-
HT3AB receptor affinities, was explored by screening a series of
compounds that contain the quinoxaline scaffold (Figure 1b).
Intermediates 4–5 were synthesized via a two-step ring forma-
tion between 2-amino aniline 2 or 3 and the appropriate 2-oxo
carboxylic acids (Scheme 1). After conversion into the corre-
sponding 2-chloroquinoxalines with phosphorylchloride, subse-
quent nucleophilic aromatic substitution with N-methylpipera-
zine under microwave conditions gave compounds 6 and 7 in
moderate to good yields.
Scheme 2. Synthesis of compounds 10 and 11. Reagents and conditions:
a) 2m NH3 in EtOH, mw, 1008C, 120 min; b) N-methylpiperazine, THF, mw,
1508C, 40 min; c) N-methylpiperazine, Et3N, THF, 808C, 96 h.
Starting from commercially available chloro-quinoxalines 8
and 9, different synthetic routes were followed to synthesize
compounds 10 and 11 (Scheme 2). Compound 10 was synthe-
sized through two subsequent nucleophilic aromatic substitu-
tion reactions. First, the amine moiety was introduced by react-
ing compound 8 with ammonia in ethanol. Subsequently, the
N-methylpiperazine group was introduced. Both reac-
tions were performed under microwave conditions.
Compound 11 was created in a similar manner, al-
though conventional heating was used for this syn-
thesis.
Scheme 3. Synthesis of 2-bromo-3-(4-methylpiperazin-1-yl)quinoxaline (14).
Reagents and conditions: a) PBr5, toluene, 1608C, 3 h; b) N-methylpiperazine,
Et3N, toluene, reflux, 6 h.
Commercially available quinoxaline-2,3(1H,4H)-
dione (12) was treated with phosphorous pentabro-
mide to form 2,3-dibromoquinoxaline (13), which
was then allowed to react with N-methylpiperazine
in toluene at reflux to yield compound 14
(Scheme 3). For compounds 16, 18, 19, and 21, 2,3-
dichloroquinoxaline (8) or 2-chloroquinoxaline (15)
were reacted with the corresponding amines using
various solvents and temperatures to yield 16, 17,
19, and 20 in good yields. Boc-protected intermedi-
Scheme 4. Synthesis of compounds 16, 18, 19, and 21. Reagents and conditions: a) N-
methylhomopiperazine, Et3N, toluene, reflux, overnight; b) tert-butyl methyl(pyrrolidin-3-
yl)carbamate, K2CO3, DMF, 908C, 4 h; c) 4m HCl in dioxane, RT, overnight.
ates 17 and 20 were subsequently deprotected with a 4m so-
lution of hydrochloric acid in dioxane to give compounds 18
and 21 (Scheme 4). The regioselective synthesis of compound
22 was described earlier by our group.[9] Here, we used this
compound as a precursor in the synthesis of compound 23
(Scheme 5).
Scheme 5. Synthesis of compound 23. Reagents and conditions: a) POCl3,
DiPEA, toluene, reflux, 20 h.
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Biochemical evaluation and SAR studies
and R3 positions of the quinoxaline core scaffold. We found
that several quinoxaline compounds show clear differences in
their binding affinities at the two receptor subtypes, and the
subtype preference differs within the series.
SAR of quinoxaline compounds for the 5-HT3A receptors
Target compounds were evaluated using competition binding
with the 5-HT3-specific ligand [3H]granisetron; the results are
summarized in Table 1. SAR data in this table are presented
with a focus on different substitution patterns at the R1, R2,
First, the SAR of the series will be described for the 5-HT3A
receptor subtype. The alcohol moiety at the R2 position implies
that compound 1 can adopt two different tautomeric states. It
Table 1. Competition binding affinities for quinoxalines at 5-HT3A and 5-HT3AB receptors with respect to substitutions at R1, R2, and R3.
Compd
R1
R2
R3
H
pKi (A)
n
pKi (AB)
n
Fold diff.[a]
1
OH
8.93ꢁ0.21
11
9.36ꢁ0.06
11
+2.7
28
OMe
H
H
H
H
H
H
H
H
H
H
H
9.18ꢁ0.16
9.00ꢁ0.22
6.27ꢁ0.20
7.06ꢁ0.07
6.89ꢁ0.12
8.53ꢁ0.11
8.59ꢁ0.19
9.20ꢁ0.12
7.35ꢁ0.15
9.31ꢁ0.16
9.82ꢁ0.26
6
7
4
2
4
5
5
5
3
6
7
8.34ꢁ0.10
8.24ꢁ0.10
6.71ꢁ0.30
6.77ꢁ0.39
7.12ꢁ0.13
7.51ꢁ0.32
8.42ꢁ0.09
8.84ꢁ0.46
7.42ꢁ0.39
8.85ꢁ0.16
7.90ꢁ0.49
7
7
4
2
4
5
5
8
3
7
6
ꢀ7.1
ꢀ5.8
+2.8
ꢀ2.0
+1.7
ꢀ11
29
30
31
32
10
NH2
Me
Et
26
ꢀ1.5
ꢀ2.3
+1.2
ꢀ2.9
ꢀ83
6
27
CF3
Br
14
VUF10166
Cl
16
Cl
H
9.11ꢁ0.26
3
8.34ꢁ0.23
3
ꢀ5.9
18
23
11
Cl
Cl
Cl
H
6.69ꢁ0.16
8.95ꢁ0.18
8.09ꢁ0.34
3
7
6
6.67ꢁ0.02
7.85ꢁ0.08
7.48ꢁ0.16
3
14
6
ꢀ1.0
ꢀ13
6-Cl
6,7-Cl
ꢀ4.1
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Table 1. (Continued)
Compd
R1
R2
R3
pKi (A)
n
pKi (AB)
n
Fold diff.[a]
22
OH
7-Cl
7.50ꢁ0.13
3
8.14ꢁ0.21
3
+4.3
34
24
33
7
OH
H
6,7-Cl
H
7.30ꢁ0.39
8.21ꢁ0.26
7.79ꢁ0.20
8.41ꢁ0.29
6
5
3
6
7.14ꢁ0.26
9.13ꢁ0.30
6.99ꢁ0.31
8.07ꢁ0.27
9
5
3
5
ꢀ1.4
+8.3
ꢀ6.3
ꢀ2.2
H
6-Cl
6,7-Cl
H
19
21
H
H
H
H
8.09ꢁ0.20
7.49ꢁ0.08
5
6
7.48ꢁ0.13
7.11ꢁ0.15
5
5
ꢀ4.0
ꢀ2.4
[a]+/ꢀ refer to an increase or decrease at 5-HT3AB relative to 5-HT3A.
seems that the tautomeric form in which the aromatic nitro-
gen atom represents a hydrogen bond donor is not involved
in binding, as the conversion of the R2 alcohol functionality of
1 into a methoxy (compound 28) or ethoxy (compound 29)
group results in compounds with comparable affinities. How-
ever, larger ether analogues are not favorable for binding, as
observed for compounds 30–32 in which the cyclohexyl,
phenyl, and benzyl ether derivatives have ~100-fold lower
affinities. When the hydroxy group of 1 at R2 was changed to
a different polar moiety (e.g., an amine moiety, as in com-
pound 10), the high affinity was maintained. A decreased affin-
ity was observed for compound 26, which incorporates
a methyl group (which is electron-donating) at the R2 position,
relative to VUF10166. Addition of an electron-withdrawing CF3
group (compound 27) results in an even larger decrease in
5-HT3A receptor affinity. Compounds that have chlorine or bro-
mine atoms at this position have sub-nanomolar affinities
(VUF10166 and 14), indicating that the SAR in this position is
very subtle, and an interplay between inductive and resonance
effects cannot be ruled out.
tion R3 (6,7-Cl, 11) results in another ~10-fold decrease. Again,
VUF10166 (R3 =H) shows the highest affinity for the 5-HT3A re-
ceptor. For compounds with R2 =OH (1), a similar trend is ob-
served. Affinity at the 5-HT3A receptor is highest for R3 =H (1)
and decreases significantly for both compound 22 (R3 =6-Cl)
and 34 (R3 =6,7-Cl), which both have a pKi in the mid-nano-
molar range.
The same modifications to R1 and R3 were made for the
most simple 2-N-methylpiperazine quinoxaline compound of
the series (R2 =H, 24), which has a pKi of 8.21. Addition of
chlorine atoms at position R3 (33, 7) results in compounds with
similar affinity at the 5-HT3A receptor. Finally, replacement of
the N-methylpiperazine group of compound 24 with an
N-methylhomopiperazine group (19) has no effect on 5-HT3A
receptor affinity, but for R1 =N-methylpyrrolidin-3-amine (21),
a small decrease in affinity is observed. This is different to
what is observed for R2 =Cl, where basic moieties other than
the N-methylpiperazine group resulted in more pronounced
differences in affinity (e.g., compare 19 and 21 with 16 and
18, respectively).
For R2 =Cl (VUF10166), different basic moieties were intro-
duced. A small drop in affinity results from replacing R1 =N-
methylpiperazine (VUF10166) with R1 =N-methylhomopipera-
zine (16), but a ~1000-fold drop in affinity is observed for R1 =
N-methylpyrrolidin-3-amine (18). As the methylpiperazine
moiety leads to the most potent compounds at 5-HT3A recep-
tors, this basic group was used in the R1 position when explor-
ing the effects of different chlorine substitution patterns at the
R3 position. Addition of a 6-Cl at R2 (compound 23) causes
a ~10-fold drop in affinity, and a second chlorine atom at posi-
Affinity differences at 5-HT3A and 5-HT3AB receptors
The affinity of compound 1 is slightly higher (2.7-fold) for 5-
HT3AB receptors than for 5-HT3A receptors. Methoxy and
ethoxy analogues 28 and 29 both show a 10-fold decrease in
affinity at 5-HT3AB receptors relative to compound 1; in con-
trast, these modifications do not result in a change in affinity
at the 5-HT3A receptor. The larger ether analogues 30–32 have
pKi values of ~7 for the 5-HT3AB receptor, which are similar to
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their affinities for the homomeric receptor. Replacement of the
alcohol moiety with an amine moiety (compound 10) resulted
in a large decrease in affinity (~70-fold) for 5-HT3AB receptors
but had little effect on the affinity for 5-HT3A receptors. Similar
affinities are observed at both the 5-HT3A and 5-HT3AB recep-
tors for compounds that have methyl and ethyl substituents in
the R2 position (i.e., 26 and 6, respectively), as well as for tri-
fluoromethyl derivative 27. For the halogen-substituted com-
pounds, a different trend is observed. For R2 =Br, the pKi for 5-
HT3AB receptors is close to 9, which is similar to that for 5-
HT3A receptors, but the affinity of VUF10166 (R2 =Cl) is signifi-
cantly decreased at 5-HT3AB receptors, resulting in a ~100-fold
difference relative to 5-HT3A receptors. The effect of replacing
R1 =N-methylpiperazine (VUF10166) for R1 =N-methylhomopi-
perazine while R2 =Cl (16) is negligible at 5-HT3AB receptors,
which is again different to what is observed at 5-HT3A recep-
tors. For R1 =N-methylpyrrolidin-3-amine (18), a >10-fold de-
crease in affinity for 5-HT3AB receptors is observed. 5-HT3AB re-
ceptor affinities for compounds with a chlorine atom at posi-
tion R2 (VUF10166, 23, and 11) do not change substantially
when increasing numbers of chlorine atoms are added at the
R3 position, although compound 11 has the lowest 5-HT3AB re-
ceptor affinity of this subset. This is different to what is ob-
served for the 5-HT3A receptor affinities of these compounds,
where addition of chlorine at R3 resulted in a large decrease in
affinity. When R2 =OH, compound 1 (R3 =H) had the highest
5-HT3AB receptor affinity, compound 22 (R3 =6-Cl) showed
a ~10-fold decrease in affinity, and a further ~10-fold decrease
in affinity was observed for compound 34 (R3 =6,7-Cl). 5-HT3A
receptor affinities shown by 22 and 34 are similar. When R2 =
H, the highest 5-HT3AB receptor affinity was observed for R3 =
H (24), but a ~100-fold drop in affinity was observed for R3 =
6-Cl (33) and only a ~10-fold drop for R3 =6,7-Cl (7). The effect
on 5-HT3AB receptor affinity when replacing R1 =N-methyl-
piperazine (24) for R1 =N-methylhomopiperazine (19), while
R2 =H, is a ~45-fold decrease in affinity. For compound 21,
a similar lowering in 5-HT3AB receptor affinity is observed. This
is in contrast to what is observed for 5-HT3A receptors and can
primarily be attributed to the sub-nanomolar affinity of com-
pound 24 at 5-HT3AB receptors, which is almost 10-fold higher
than its 5-HT3A receptor affinity.
alkyl (26, 6) and ether analogues (28 and 29) also have high af-
finities, whereas incorporation of larger ether groups at R2 re-
sults in decreased affinity. However, for R2 =Cl (VUF10166) and
NH2 (10), only 5-HT3AB receptor affinity is decreased, resulting
in 100- and 10-fold selectivity for 5-HT3A over 5-HT3AB recep-
tors, respectively. Different substitution patterns at the R3 posi-
tion also caused marked changes. For example, when R2 =Cl,
replacement of R3 =H (VUF10166) with a chlorine atom results
in a ~10-fold (R3 =6-Cl, 23) or ~100-fold (R3 =6,7-Cl, 11) de-
crease in affinity for the 5-HT3A receptor, but this replacement
does not have a large effect on 5-HT3AB receptor affinity.
When R2 =H, the affinity for 5-HT3A receptors does not show
a large difference upon addition of chlorine atoms to the R3
position, but the 5-HT3AB receptor affinity changes significant-
ly. It can be concluded that, in either case, the greatest 5-HT3
receptor subtype selectivity is achieved for R1 =N-methylpiper-
azine.
5-HT3 receptor binding sites
Orthosteric binding sites in 5-HT3AB receptors could theoreti-
cally exist at A+Aꢀ, A+Bꢀ, B+Aꢀ, and B+Bꢀ interfaces,
but the majority of 5-HT3 receptor-competitive ligands only
bind to an A+Aꢀ interface.[4,10] There is evidence, however,
that at least one of the quinoxaline compounds studied here
(VUF10166) binds to both an A+Aꢀ and an A+Bꢀ interface;
binding to the A+Bꢀ interface may decrease the affinity of li-
gands binding to the A+Aꢀ site by allosterically increasing
the rate of ligand dissociation.[7] Other quinoxalines may simi-
larly bind to both sites; thus, to identify potential interactions,
we constructed models of the two interfaces.
Homology models were based on a tropisetron-bound
AChBP crystal structure (PDB code: 2WNC) as no quinoxaline-
bound Cys-loop receptor structure has been solved to date,
and tropisetron is the closest structurally related compound to
those described here (Figure 3). Tropisetron is an antagonist at
the 5-HT3 receptor but can act as an agonist at some nACh re-
ceptors;[11] thus, it is an ideal choice from the available struc-
tures as quinoxalines can act as both agonists and antagonists
at 5-HT3 receptors, though they were not evaluated in this
study. As with all homology models, caution must be applied
in data interpretation, especially now that recent electron mi-
croscope images of the nACh receptor have shown that the
difference between the structure of unbound and agonist-
bound binding site sites is less than that observed in AChBP.[12]
Nevertheless, it is likely that our compounds adopt a broadly
similar orientation to tropisetron; therefore, the models serve
as means of identifying residues that could potentially be re-
sponsible for the differences in affinities of the quinoxaline li-
gands at 5-HT3A and 5-HT3AB receptors. As discussed below,
several of the identified residues are known to interact with
a range of 5-HT3 receptor ligands (Figures 2 and 3).[4] Some of
these are the same in both A+Aꢀ and A+Bꢀ binding sites
and are unlikely to be responsible for differences in affinity,
while others are different and may provide possible explana-
tions for the varied ligand affinities at the two receptor sub-
types.
Table 1 shows that compound 24 shows the highest selectiv-
ity for 5-HT3AB over 5-HT3A receptors (~10-fold), and VUF
10166 has the highest selectivity for 5-HT3A over 5-HT3AB re-
ceptors (~100-fold). The difference between these two com-
pounds is solely the atom at position R2, R2 =H for compound
24 and R2 =Cl for VUF10166. When the hydrogen atom is re-
placed with a chlorine atom, the 5-HT3A receptor affinity in-
creases ~40-fold, while the affinity for 5-HT3AB receptors de-
creases ~20-fold. Both of these compounds comprise the
N-methylpiperazine moiety, which is the preferred basic group
for selectivity. For 5-HT3A receptor affinity, R2 =Cl (VUF10166) is
superior, with Br (14), Et (6), OMe (28), and OEt (29) having
similar lower affinities. For the 5-HT3AB receptor, an alcohol
moiety at position R2 (as observed for compound 1) is pre-
ferred, but a hydrogen atom at R2 also results in high affinity
(compound 24). At 5-HT3AB receptors, R2 =Br and the smaller
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Figure 2. Protein sequence alignment for Ac-AChBP, 5-HT3A, and 5-HT3B. Residues illustrated in Figure 3 are highlighted. Identical residues are shown in
beige, dissimilar residues of the complementary side of the 5-HT3A subunit in orange, and residues for the 5-HT3B receptor are shown in green. Accession
numbers for the Ac-AChBP, 5-HT3A and 5-HT3B subunits are Q8WSF8, P46098, and O95264, respectively. Note that the numbering of the 5-HT3A and 5-HT3B
residues corresponds to the mouse numbering in order to allow comparison with other work.[11]
have shown that both Glu129 and Trp183 are essential for
5-HT function and granisetron binding.[13]
Because the principle faces of both A+Aꢀ and A+Bꢀ bind-
ing sites are identical, we must look toward the Aꢀ and Bꢀ in-
terfaces for differences between the two binding sites. Of the
differing residues, AIle71/BPhe71, AArg92/BGln92 and AGln151/BGlu151 are
closest to tropisetron, and might also be expected to interact
with the structurally related quinoxaline ligands described
here. AIle71/BPhe71 lies in the b1-strand, close to binding loop A.
However, AIle71 mutations to Ala and Leu had no effect on gra-
nisetron binding affinity, suggesting the residue at this location
does not affect ligand binding.[13] Conversely substitution of
Figure 3. a) Overlay of homology models for the A+Aꢀ (protein carbon
AArg92 changed the affinities of several 5-HT3 ligands, including
atoms in orange) and A+Bꢀ (protein carbon atoms in green) binding sites
5-HT and granisetron, and we have previously speculated that
containing tropisetron (purple ball and stick) and a network of structural
water molecules (oxygen atoms as red balls). Hydrogen bonds for ligand–
a cation–p interaction could exist between AArg92 and the aro-
matic parts of (iso)quinolines and quinazolines, as it does with
granisetron.[10,15] As this type of interaction would be absent in
an A+Bꢀ site (as Gln is a neutral residue), quinoxalines might
adopt quite a distinct orientation in this binding pocket. In
support of this speculation, we have previously shown that in-
troduction of a Cys substitution at this location has no effect
on 5-HT or granisetron, but eliminates the allosteric effects of
VUF10166 in heteromeric receptors.[7] Similarly, the change in
charge at location 151 (AGln151/BGlu151) could have a significant
effect on ligand binding properties. Although the effect of this
residue has not yet been studied, mutation of the closely locat-
ed BTyr153 residue also eliminates the allosteric effects of
VUF10166, showing that this region influences binding at the
B interface.[7] For VUF10166, the effects of these B-substitutions
are known to alter both the binding properties and the func-
tional response, but for the other quinoxalines studied here, it
has yet to be determined whether the differing binding affini-
ties also translate into functional changes.
receptor, ligand–solvent, and solvent–solvent interactions are shown as
green dotted lines. Residue annotation for identical residues is in black and,
for divergent residues, orange corresponds to 5-HT3A subunits and green to
5-HT3B subunits. b) Structures of tropisetron and VUF10166 to highlight
their similarities.
Studies of the 5-HT3A receptor have identified an aromatic
binding cavity formed by residues Trp90 (loop D), Trp183
(loop B) and Tyr234 (loop C), mutagenesis of which effects
both 5-HT activation and the binding of 5-HT3 receptor-com-
petitive antagonists.[13] Our homology models predict that
both A+Aꢀ and A+Bꢀ binding sites contain these residues,
providing an aromatic environment to accommodate the posi-
tively charged moiety that is a well-known pharmacophore
feature of 5-HT3 ligands.[14] Another pharmacophore feature,
a hydrogen bond acceptor (HBA), is observed in both models
as an interaction between the carbonyl oxygen atom of tropi-
setron and wat2 from a water network that has been observed
in AChBP crystal structures; water molecules in this network
are also stabilized by interactions with the backbone of the
protein and the side chain of Tyr234. In both binding sites, the
positively charged moieties of tropisetron are also stabilized by
ionic interactions with Glu129 (loop A), and by a hydrogen
bonding interaction between the protonated nitrogen atom
and the carbonyl backbone of Trp183. Mutagenesis studies
Conclusions
In summary, most quinoxaline compounds examined here
show no difference in their affinities at 5-HT3A and 5-HT3AB re-
ceptors, and we suggest that these compounds may only bind
to the A+A binding site that is found in both receptor types,
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dried over MgSO4 and concentrated under reduced pressure to
yield 133 mg of 5 (0.62 mmol, 13%) as a dark-brown solid: H NMR
(250 MHz, DMSO) d=8.19 (s, 1H), 8.04 (s, 1H), 7.45 ppm (s, 1H).
consistent with all other 5-HT3 receptor-competitive li-
gands.[10,16] Some, however, show significant differences and
thus may bind to the A+Bꢀ interface as has previously been
shown for VUF10166.[10] These novel ligands could be valuable
in both experimental and computer-aided drug design, with
potential for the development of novel therapeutic agents.
1
2-Ethyl-3-(4-methylpiperazin-1-yl)quinoxaline (6): A solution of 4
(1.64 g, 9.39 mmol) in phosphoryl trichloride (100 mL) was stirred
at 1008C for 1 h. The reaction mixture was then concentrated
under reduced pressure. H2O was added to the remaining solid,
then the mixture was extracted with CH2Cl2. The organic layers
were combined, dried over Na2SO4, and concentrated under re-
duced pressure to yield 1.59 g (8.24 mmol, 88%) of 2-chloro-3-eth-
ylquinoxaline as a dark-pink solid: 1H NMR (250 MHz, CDCl3) d=
8.10–8.03 (m, 1H), 8.02–7.95 (m, 1H), 7.79–7.67 (m, 2H), 3.17 (q,
J=7.5 Hz, 2H), 1.44 ppm (t, J=7.5 Hz, 3H). Next, 2-chloro-3-ethyl-
quinoxaline (555 mg, 2.88 mmol) was dissolved in N-methylpipera-
zine (2 mL), and the resulting solution was heated at 1208C for
15 min using microwave (mw) radiation. After cooling to room
temperature, excess N-methylpiperazine was removed under re-
duced pressure, and the product was purified over SiO2 (EtOAc/
Et3N, 96:4, v/v) to yield 566 mg of 6 (2.21 mmol, 77%) as a yellow
solid: 1H NMR (250 MHz, CDCl3) d=7.95–7.87 (m, 1H), 7.85–7.78
(m, 1H), 7.61–7.45 (m, 2H), 3.43–3.31 (m, 4H), 2.97 (q, J=7.4 Hz,
2H), 2.68–2.58 (m, 4H), 2.38 (s, 3H), 1.41 ppm (t, J=7.4 Hz, 3H);
13C NMR (126 MHz, CDCl3) d=155.74, 154.15, 139.92, 138.88,
128.68, 128.01, 127.27, 126.57, 55.04, 49.67, 46.24, 27.70,
12.64 ppm; LCMS: tR =2.82 min, purity 95%, [M+H]+ 257.00;
HRMS m/z: [M+H]+ calcd for C15H21N4: 257.1761, found: 257.1763.
Experimental Section
Chemistry: Chemicals and solvents were purchased from Sigma–
Aldrich and used as received. Unless indicated otherwise, all reac-
tions 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 car-
ried out automatically using the Biotage equipment. All HRMS
spectra were recorded on Bruker microTOF mass spectrometer
1
using ESI in 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, multiplici-
ty (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 in-
ternal reference (CHCl3 in CDCl3: d=7.26 ppm and CH3OH in
CH3OD: d=3.31 ppm, (CH3)2SO in (CD3)2SO: d=2.50 ppm). 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 deuter-
ation as the internal reference (CDCl3: d=77.16 ppm, CH3OD: d=
49.00 ppm, (CD3)2SO: d=39.52 ppm). Systematic names for mole-
cules according to IUPAC rules were generated using the Chem-
Draw AutoNom program. Purity was determined using a Shimadzu
HPLC/MS workstation with a LC-20AD pump system, SPD-M20A
diode array detection, and an LCMS-2010 EV mass spectrometer.
An Xbridge C18 5 mm column (100 mmꢁ4.6 mm) was used. Com-
pound purities were calculated as the percentage peak area of the
analyzed compound by UV detection at 230 nm. Solvents used
were as follows: solvent B=CH3CN 0.1% formic acid; solvent A=
H2O 0.1%. The analysis was conducted using a flow rate of
1.0 mLminꢀ1, starting at 5% B with a linear gradient to 90% B in
4.5 min, then 1.5 min at 90% B with a linear gradient to 5% B in
0.5 min, and then 1.5 min at 5% B, with a total run time of 8 min.
Compounds 24–34 were synthesized by our group as described by
Smits et al.[9]
6,7-Dichloro-2-(4-methylpiperazin-1-yl)quinoxaline (7): A solution
of 5 (133 mg, 0.62 mmol) in phosphoryl trichloride (50 mL) was
stirred at 1008C for 1 h. The reaction mixture was concentrated
under reduced pressure, H2O was added to the remaining solid
this, and the mixture was extracted with CH2Cl2. The organic layers
were combined, dried over Na2SO4, and concentrated under re-
duced pressure to yield 66 mg (0.28 mmol, 46%) of 2,6,7-trichloro-
1
quinoxaline: H NMR (250 MHz, CDCl3) d=8.77 (s, 1H), 8.25 (s, 1H),
8.15 ppm (s, 1H). Then, 2,6,7-trichloroquinoxaline (66 mg,
0.28 mmol) was dissolved in EtOAc (2 mL), N-methylpiperazine
(0.1 mL, 0.90 mmol) was added, and the resulting solution was
heated at 1608C for 1 h using microwave radiation. After cooling
to room temperature, EtOAc and excess N-methylpiperazine were
removed under reduced pressure, and the product was purified
over SiO2 (EtOAc/Et3N, 96:4, v/v) to yield 36 mg of 7 (0.12 mmol,
1
43%) as a light-brown solid: H NMR (250 MHz, CDCl3) d=8.55 (s,
1H), 7.96 (s, 1H), 7.77 (s, 1H), 3.85–3.79 (m, 4H), 2.59–2.52 (m, 4H),
2.37 ppm (s, 3H); 13C NMR (126 MHz, CDCl3) d=152.87, 142.50,
139.15, 136.67, 134.50, 131.10, 128.26, 127.62, 54.60, 48.80,
46.07 ppm; LCMS: tR =3.39 min, purity >99%, [M+H]+ 296.90;
HRMS m/z: [M+H]+ calcd for C13H15Cl2N4: 297.0668, found:
297.0662.
3-Ethyl-3,4-dihydroquinoxalin-2(1H)-one (4): Benzene-1,2-diamine
(2) (1.07 g, 28.4 mmol) and 2-oxobutanoic acid (2.90 g, 28.4 mmol)
were dissolved in 50 mL CH3OH, and the resulting solution was
stirred overnight at room temperature. The resulting precipitate
was collected via filtration over a Bꢂchner funnel. The precipitate
was washed with cold CH3OH and dried in a vacuum oven to yield
2.25 g (12.9 mmol, 46%) of 4 as an off-white solid: 1H NMR
(500 MHz, CDCl3) d=11.25 (s, 1H), 7.84 (d, J=8.1 Hz, 1H), 7.52–
7.45 (m, 1H), 7.36–7.31 (m, 1H), 7.28 (d, J=7.8 Hz, 1H), 3.01 (q, J=
7.4 Hz, 2H), 1.38 ppm (t, J=7.4 Hz, 3H); 13C NMR (126 MHz, CDCl3)
d=162.54, 156.24, 132.91, 130.97, 129.57, 128.85, 124.09, 115.31,
26.85, 10.85 ppm.
3-(4-Methylpiperazin-1-yl)quinoxalin-2-amine (10): 2,3-Dichloro-
quinoxaline (8) (1.99 g, 10.0 mmol) was dissolved in a 2m NH3 solu-
tion in EtOH (5.5 mL) and heated in the microwave at 1008C for
2 h. The solvent was then removed under reduced pressure, and
the residue was purified over SiO2 (CH2Cl2/EtOAc, 100:0 to 60:40,
v/v) to give 300 mg (1.67 mmol, 17%) of 3-chloroquinoxalin-2-
amine. Next, 3-chloroquinoxalin-2-amine (150 mg, 0.84 mmol) and
N-methylpiperazine (1.0 mL, 9.02 mmol) were dissolved in THF
(4 mL). The resulting mixture was heated under microwave condi-
tions at 1508C for 40 min, quenched with H2O, and extracted with
EtOAc. The organic layers were combined, dried (Na2SO4), and con-
centrated under reduced pressure. The product was crystallized
from EtOAc to give 100 mg (0.41 mmol, 49%) of 10 as a dark-
6,7-Dichloro-3,4-dihydroquinoxalin-2(1H)-one (5): 4,5-dichloro-
benzene-1,2-diamine (3) (842 mg, 4.76 mmol) and 2-oxoacetic acid
(715 mg, 4.83 mmol) were dissolved in CH3OH (50 mL) and stirred
at room temperature for 30 min. The mixture was concentrated
under reduced pressure, H2O was added, and the resulting mixture
was extracted with EtOAc. The combined organic phases were
1
yellow solid: H NMR (250 MHz, CDCl3) d=7.76 (d, J=7.8 Hz, 1H),
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7.60 (d, J=7.8 Hz, 1H), 7.50–7.33 (m, 2H), 4.98 (s, 2H), 3.56–3.27
(m, 4H), 2.76–2.51 (m, 4H), 2.46–2.29 ppm (m, 3H); 13C NMR
(126 MHz, CDCl3) d=148.39, 147.69, 138.94, 137.41, 127.34, 127.26,
125.25, 125.08, 55.07, 48.54, 46.24 ppm; LCMS: tR =2.25 min, purity
>99%, [M+H]+ 244.00; HRMS m/z: [M+H]+ calcd for C13H18N5:
244.1557, found: 224.1552.
28.06 ppm; LCMS: tR =2.89 min, purity >99%, [M+H]+ 277.10;
HRMS m/z: [M+H]+ calcd for C14H18ClN4: 277.1215, found:
277.1210.
tert-Butyl
(1-(3-chloroquinoxalin-2-yl)pyrrolidin-3-yl)(methyl)-
carbamate (17): 2,3-dichloroquinoxaline (0.54 g, 2.71 mmol) was
dissolved in DMF (30 mL). K2CO3 (0.37 g, 2.71 mmol) and tert-butyl
methyl(pyrrolidin-3-yl)carbamate (0.49 g, 2.47 mmol) were added,
and the mixture was stirred at 908C for 4 h. The mixture was
cooled to room temperature, diluted with H2O, and extracted with
Et2O. The combined organic layers were dried over MgSO4 and
concentrated under reduced pressure to give 0.68 g of 17, which
was directly used in the synthesis of 18.
2,6,7-Trichloro-3-(4-methylpiperazin-1-yl)quinoxaline
(11):
2,3,6,7-Tetrachloroquinoxaline (9) (1,23 g, 4.58 mmol) was dissolved
in THF (50 mL). N-methylpiperazine (0.58 mL, 4.58 mmol) and trie-
thylamine (0.65 mL, 4.66 mmol) were added, and the mixture was
stirred at 808C for 96 h, quenched with H2O, and extracted with
EtOAc. The organic layers were combined, dried over MgSO4, and
concentrated under reduced pressure to yield 1.02 g (3.08 mmol,
67%) of 11 as a light-brown solid: 1H NMR (250 MHz, CDCl3) d=
7.95 (s, 1H), 7.92 (s, 1H), 3.68–3.59 (m, 4H), 2.66–2.58 (m, 4H),
2.38 ppm (s, 3H); 13C NMR (126 MHz, CDCl3) d=152.87, 142.50,
139.15, 136.67, 134.50, 131.10, 128.26, 127.62, 54.60, 48.80,
46.07 ppm; LCMS: tR =3.64 min, purity >99%, [M+H]+ 330.85;
HRMS m/z: [M+H]+ calcd for C13H14Cl3N4: 331.0279, found:
331.0271.
1-(3-Chloroquinoxalin-2-yl)-N-methylpyrrolidin-3-amine
(18):
Compound 17 (0.40 g) was dissolved in dioxane (10 mL) and
stirred at room temperature. A 4m solution of HCl in dioxane
(20 mL) was added dropwise, and precipitation was observed. The
resulting suspension was stirred overnight and subsequently fil-
tered over a Bꢂchner funnel, and the residue was washed with 1,4-
dioxane. The residue was then dried under reduced pressure to
yield 202 mg of 18 as a light-yellow solid (0.68 mmol, 61%):
1H NMR (500 MHz, CDCl3) d=7.87–7.74 (m, 2H), 7.68 (ddd, J=8.4,
7.1, 1.4 Hz, 1H), 7.58–7.46 (m, 1H), 4.27–4.10 (m, 3H), 4.10–3.92 (m,
2H), 2.83 (s, 3H), 2.61–2.45 (m, 1H), 2.37–2.23 ppm (m, 1H);
13C NMR (126 MHz, CDCl3) d=147.42, 137.60, 136.72, 136.02,
131.08, 127.39, 126.50, 123.29, 57.92, 52.40, 48.69, 31.08,
27.60 ppm; LCMS: tR =2.90 min, purity 99%, [M+H]+ 263.05;
HRMS m/z: [M+H]+ calcd for C13H16ClN4: 263.1058, found:
263.1055.
2,3-Dibromoquinoxaline (13): Quinoxaline-2,3-diol (12) (2.96 g,
18.3 mmol) and pentabromophosphorane (17.06 g, 39.6 mmol)
were suspended in toluene (200 mL) and heated at 1608C for 3 h.
After cooling to room temperature, ice water (200 mL) was added
to the solution, and the mixture was stirred vigorously for 30 min.
The mixture was extracted with toluene, washed with 1n NaOH
(100 mL), dried over MgSO4, filtered, and concentrated under
vacuum. The crude product was purified over SiO2 (CH2Cl2/n-hep-
tanes, 1:2, v/v) to give 505 mg (12.7 mmol, 70%) of 13 as a beige
solid: 1H NMR (250 MHz, CDCl3) d=8.09–8.00 (m, 2H), 7.86–
7.77 ppm (m, 2H).
2-(4-Methyl-1,4-diazepan-1-yl)quinoxaline (19): 2-Chloroquinoxa-
line (15) (2.97 g, 18.0 mmol), N-methyl-1,4-diazepane (3.3 mL,
24.0 mmol), and Et3N (2.5 mL, 18.0 mmol) were dissolved in toluene
(50 mL). The solution was stirred overnight at reflux. After cooling
to room temperature, H2O was added, and the resulting mixture
was extracted with toluene, dried over MgSO4, and concentrated
under vacuum. The crude residue was purified over SiO2 (EtOAc/
Et3N, 98:2, v/v) to give 3.12 g (12.9 mmol, 71%) of 19 as an off-
2-Bromo-3-(4-methylpiperazin-1-yl)quinoxaline (14): 2,3-Dibro-
moquinoxaline (13) (505 mg, 1.75 mmol), N-methylpiperazine
(176 mg, 1.75 mmol), and Et3N (177 mg, 1.75 mmol) were dissolved
in toluene (50 mL). The solution was stirred at 1608C for 6 h. After
cooling to room temperature, H2O was added, and the emulsion
was extracted with toluene. The combined organic extracts were
dried over MgSO4 and concentrated under vacuum. The crude
product was purified over SiO2 (EtOAc/Et3N, 98:2, v/v) to give
1
white solid: H NMR (250 MHz, CDCl3) d=8.47 (s, 1H), 7.86 (d, J=
8.2 Hz, 1H), 7.65 (d, J=8.4 Hz, 1H), 7.59–7.49 (m, 1H), 7.39–7.29
(m, 1H), 4.02–3.93 (m, 2H), 3.86 (t, J=6.3 Hz, 2H), 2.77 (dd, J=5.7,
4.2 Hz, 2H), 2.65–2.53 (m, 2H), 2.39 (s, 3H), 2.15–2.00 ppm (m, 2H);
13C NMR (126 MHz, CDCl3) d=151.59, 142.03, 136.46, 134.92,
129.96, 128.65, 126.33, 123.98, 58.18, 57.25, 46.73, 46.55, 46.24,
27.48 ppm; LCMS: tR =2.40 min, purity >99%, [M+H]+ 243.00;
HRMS m/z: [M+H]+ calcd for C14H19N4: 243.1604, found: 243.1608.
377 mg (1.23 mmol, 70%) of 14 as
a
beige solid: 1H NMR
(250 MHz, CDCl3) d=7.90 (dd, J=8.3, 1.2 Hz, 1H), 7.83 (dd, J=8.3,
1.1 Hz, 1H), 7.70–7.61 (m, 1H), 7.58–7.48 (m, 1H), 3.63–3.55 (m,
4H), 2.69–2.61 (m, 4H), 2.39 ppm (s, 3H); 13C NMR (126 MHz, CDCl3)
d=153.60, 140.03, 139.18, 134.98, 130.25, 127.86, 127.40, 127.19,
54.70, 49.48, 46.18 ppm; LCMS: tR =2.69 min, purity >99%, [M+
H]+ 306.90; HRMS m/z: [M+H]+ calcd for C13H16BrN4: 307.0553,
found: 307.0552.
tert-Butyl
methyl(1-(quinoxalin-2-yl)pyrrolidin-3-yl)carbamate
(20): 2-Chloroquinoxaline (1.79 g, 10.9 mmol) was dissolved in DMF
(50 mL). K2CO3 (1.51 g, 10.9 mmol) and tert-butyl methyl(pyrrolidin-
3-yl)carbamate (2.00 g, 10.0 mmol) were added, and the mixture
was stirred at 908C for 6 h. The mixture was cooled to room tem-
perature, diluted with H2O, and extracted with Et2O. The combined
organic layers were dried over MgSO4 and concentrated under re-
duced pressure to give 3.28 g of 20, which was directly used in the
synthesis of 21.
2-Chloro-3-(4-methyl-1,4-diazepan-1-yl)quinoxaline (16): 2,3-Di-
chloroquinoxaline (8) (1.00 g, 5.0 mmol), N-methyl-1,4-diazepane
(0.86 mL, 7.50 mmol), and Et3N (0.70 mL, 5.00 mmol) were dissolved
in toluene (50 mL). The solution was stirred overnight at reflux.
After cooling to room temperature, H2O was added, and the result-
ing mixture was extracted with toluene, dried over MgSO4, and
concentrated under vacuum. The crude was purified over SiO2
(EtOAc/Et3N, 99:1, v/v) to give 1.01 g (3.65 mmol, 73%) of 16 as
N-Methyl-1-(quinoxalin-2-yl)pyrrolidin-3-amine (21): Compound
20 (2.95 g) was dissolved in dioxane (20 mL) and stirred at room
temperature. A solution of 4m HCl in dioxane (10 mL) was added
dropwise, and precipitation was observed. The suspension was
stirred overnight and filtered over a Bꢂchner funnel. The residue
was washed with 1,4-dioxane and dried under vacuum to yield
1
a yellow oil: H NMR (500 MHz, CDCl3) d=7.87–7.76 (m, 1H), 7.76–
7.69 (m, 1H), 7.58 (ddd, J=8.4, 7.0, 1.4 Hz, 1H), 7.44 (ddd, J=8.3
7.0, 1.4 Hz, 1H), 3.90–3.86 (m, 2H), 3.86–3.81 (m, 2H), 2.90–2.83 (m,
2H), 2.70–2.64 (m, 2H), 2.42 (s, 3H), 2.10 ppm (dt, J=7.0, 6.0 Hz,
2H); 13C NMR (126 MHz, CDCl3) d=151.91, 140.04, 139.18, 137.27,
130.04, 127.52, 126.40, 126.04, 58.57, 57.51, 50.61, 50.33, 46.93,
1
1.33 g of 21 as a beige solid (5.02 mmol, 56%): H NMR (500 MHz,
DMSO) d=9.69–9.52 (m, 2H), 8.58 (s, 1H), 7.89–7.82 (m, 1H), 7.72
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(d, J=8.2 Hz, 1H), 7.67–7.58 (m, 1H), 7.45–7.37 (m, 1H), 4.00–3.85
(m, 4H), 3.78–3.69 (m, 1H), 2.65–2.58 (m, 3H), 2.46–2.29 ppm (m,
2H); 13C NMR (126 MHz, DMSO) d=149.63, 140.28, 138.07, 136.38,
130.79, 129.12, 125.36, 124.68, 57.49, 49.25, 45.20, 31.42,
27.86 ppm; LCMS: tR =2.41 min, purity 97%, [M+H]+ 229.10;
HRMS m/z: [M+H]+ calcd for C13H17N4: 229.1448, found: 229.1454.
constructed by homology modeling using MOE (version 2010.10,
Chemical Computing Group, Montreal). The sequence of the
human 5-HT3AB gene (O95264) was aligned with the sequence of
the 5-HT3A gene (P46098) using the “Protein Align” option in MOE
(standard settings) and was adjusted manually. The final sequence
alignment is shown in Figure 2. The 5-HT3A receptor homology
model was selected to serve as the template. Structural waters lo-
cated in the binding pocket of the original crystal structure (PDB
code: 2WNC)[19] form a conserved protein–ligand hydrogen bond
interaction network in several other AChBP crystals (e.g., 2BYR,
2PGZ, 2BYS, 2XYT) and were included in the 5-HT3AB receptor
model. The template backbone, the ligand, and the water mole-
cules were fixed, and ten receptor models were constructed based
on the template backbone. During this construction, the ligand
and waters of the original co-crystal structure were considered as
an additional restraint using the “Environment” option within MOE.
The structural quality of the models was checked using the evalua-
tion modules in MOE; protein geometry of receptor atoms was
evaluated for bond lengths, bond angles, atom clashes, and con-
tact energies. Ramachandran plots were used to check the Phi and
Psi angles of all residues. The best model 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.
3,6-Dichloro-2-(4-methylpiperazin-1-yl)quinoxaline (23): DiPEA
(0.38 mL, 2.15 mmol) and POCl3 (2.00 mL, 21.5 mmol) were added
to a solution of 7-chloro-3-(4-methylpiperazin-1-yl)quinoxalin-2(1H)-
one (22) (300 mg, 1.08 mmol) in toluene (20 mL). The resulting
mixture was stirred at reflux for 20 h, after which the mixture was
concentrated under reduced pressure. H2O (50 mL) and 1m
NaOH(aq) (10 mL) were added to the crude product, and the result-
ing mixture was extracted with CH2Cl2. The combined organic
layers were washed with brine (50 mL), dried over NaSO4, and con-
centrated under reduced pressure. The crude product was purified
over SiO2 (EtOAc/Et3N, 50:1, v/v) to give 270 mg of 23 (0.91 mmol,
84%) as a yellow solid: 1H NMR (500 MHz, CDCl3) d=7.85 (d, J=
2.3 Hz, 1H), 7.75 (d, J=8.9 Hz, 1H), 7.57 (dd, J=8.9, 2.3 Hz, 1H),
3.69–3.54 (m, 4H), 2.72–2.56 (m, 4H), 2.38 ppm (s, 3H); 13C NMR
(126 MHz, CDCl3) d=152.58, 142.61, 138.75, 138.30, 132.59, 130.91,
128.14, 126.71, 54.70, 48.94, 46.15 ppm; LCMS: tR =3.40 min, purity
<99%, [M+H]+ 196.90; HRMS m/z: [M+H]+ calcd for C13H15Cl2N4:
297.0668, found: 297.0671.
Radioligand binding: This was carried out as previously de-
scribed.[6,7,10] Briefly, HEK293 cells expressing either 5-HT3A or 5-
HT3AB receptors 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. For com-
petition binding experiments, 50 mL of cell membranes were incu-
bated in 0.5 mL HEPES buffer containing a final concentration of
0.7 nm [3H]granisetron (~Kd), both with and without the test com-
pound. To ensure that there were no changes in the Kd of
[3H]granisetron, which could influence the Ki values of competing
ligands, saturation binding curves were run in parallel with compe-
tition studies. Competition binding experiments using ten concen-
trations of ligands were performed on at least three separate
plates of cells. Nonspecific binding was determined using 1 mm
quipazine. Reactions were incubated for at least 24 h at 48C to
allow compounds with slow kinetics to equilibrate. Experiments on
5-HT3A and 5-HT3AB receptors were run in parallel. Incubations
were terminated by vacuum filtration using a Brandel cell harvester
(Alpha Biotech Ltd., London, UK) onto GF/B filters pre-soaked in
0.3% polyethyleneimine. Radioactivity was determined by scintilla-
tion counting. Data were fit according to Equation (1):
Acknowledgements
This work was supported by a grant from the Wellcome Trust
[081925] to S.C.R.L. and an EU FP7 grant (NeuroCypres) to I.d.E.
and S.C.R.L.. S.C.R.L. is a Wellcome Trust Senior Research Fellow
in Basic Biomedical Science. We thank Danny van Willigen and
Linda Silvestri for excellent technical assistance.
Keywords: Cys loops
· heteromers · homomers · 5-HT3
receptors · quinoxalines · serotonin · subtypes
[1] a) J. Walstab, G. Rappold, B. Niesle, Pharmacol. Ther. 2010, 128, 146–
169; b) A. J. Thompson, S. C. Lummis, Curr. Pharm. Des. 2006, 12, 3615–
3630; c) A. J. Thompson, S. C. Lummis, Expert Opin. Ther. Targets 2007,
11, 527–540.
[2] a) X. Q. Hu, R. W. Peoples, J. Biol. Chem. 2008, 283, 6826–6831; b) B.
Niesler, J. Kapeller, C, Hammer, G. Rappold, Pharmacogenomics 2008,
9, 501–504.
[3] A. A. Jensen, P. A. Davies, H. Brauner-Osborne, K. Krzywkowski, Trends
Pharmacol. Sci. 2008, 29, 437–444.
[4] A. J. Thompson, H. A. Lester, S. C. Lummis, Q. Rev. Biophys. 2010, 43,
449–499.
Bmax ꢀ Bmin
ð1Þ
BL ¼ Bmin
þ
Hðlog L50ꢀlog LÞ
1 þ 10n
[5] a) S. P. Kelley, J. I. Dunlop, E. F. Kirkness, J. J. Lambert, J. A. Peters, Nature
2003, 424, 321–324; b) A. E. Dubin, R. Huvar, M. R. D’Andrea, J. Pyati,
J. Y. Zhu, K. C. Joy, S. J. Wilson, J. E. Galindo, C. A. Glass, L. Luo, M. R.
Jackson, T. W. Lovenberg, M. G. Erlander, J. Biol. Chem. 1999, 274,
30799–810.
[6] a) A. J. Thompson, R. K. Duke, S. C. Lummis, Mol. Pharmacol. 2011, 80,
183–190; b) P. Das, G. H. Dillon, Brain Res. Mol. Brain Res. 2003, 119,
207–12.
in which 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=1, L50 is the concentration of L
which gives a binding equal to (Bmax +Bmin)/2, and nH is the Hill co-
efficient. Ki values were estimated from IC50 values using the
Cheng–Prusoff equation[17] Ki =IC50/(1+[L]/Kd), for which Ki is the
equilibrium dissociation constant for binding of the unlabeled an-
tagonist, 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.
[7] A. J. Thompson, M. H. P. Verheij, I. J. P. de Esch, S. C. R. Lummis, J. Phar-
macol. Exp. Ther. 2012, 341, 350–359.
[8] a) S. Butini, R. Budriesi, M. Hamon, E. Morelli, S. Gemma, M. Brindisi, G.
Borrelli, E. Novellino, I. Fiorini, P. Ioan, A. Chiarini, A. Cagnotto, T. Menni-
ni, C. Fracasso, S. Caccia, G. Campiani, J. Med. Chem. 2009, 52, 6946–
6950; b) G. Campiani, A. Cappelli, V. Nacci, M. Anzini, S. Vomero, M.
Hamon, A. Cagnotto, C. Fracasso, C. Uboldi, S. Caccia, S. Consolo, T.
Mennini, J. Med. Chem. 1997, 40, 3670–3678; c) R. Mahesh, R. V. Peru-
Homology modeling: Construction of the homomeric 5-HT3A recep-
tor binding site model has been previously described.[18] Using the
same approach, a model of the 5-HT3AB receptor binding site was
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CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
mal, P. V. Pandi, Bioorg. Med. Chem. Lett. 2004, 14, 5179–5181; d) R.
Mahesh, T. Devadoss, D. K. Pandey, S. Bhatt, S. K. Yadav, Bioorg. Med.
Chem. Lett. 2010, 20, 6773–6776; e) A. Monge, J. A. Palop, J. C. Del Cas-
tillo, J. M. Caldero, J. Roca, G. Romero, J. Del Rio, B. Lasheras, J. Med.
Chem. 1993, 36, 2745–2750.
[14] a) A. Asagarasu, T. Matsui, H. Hayashi, S. Tamaoki, Y. Yamauchi, M. Sato,
Chem. Pharm. Bull. 2009, 57, 34–42; b) A. Cappelli, M. Anzini, S.
Vomero, L. Mennuni, F. Makovec, E. Doucet, M. Hamon, M. C. Menziani,
P. G. De Benedetti, G. Giorgi, C. Ghelardini, S. Collina, Bioorg. Med. Chem.
2002, 10, 779–801; c) R. D. Clark, A. B. Miller, J. Berger, D. B. Repke, K. K.
Weinhardt, B. A. Kowalczyk, R. M. Eglen, D. W. Bonhaus, C. H. Lee, J.
Med. Chem. 1993, 36, 2645–2657; d) S. Evans, A. Galdes, M. Gall, Phar-
macol. Biochem. Behav. 1991, 40, 1033–1040; e) M. Hibert, R. Hoffmann,
R. Miller, A. Carr, J. Med. Chem. 1990, 33, 1594–1600; f) R. Mahesh, R. V.
Perumal, P. V. Pandi, Biol. Pharm. Bull. 2004, 27, 1403–1405.
[9] R. A. Smits, H. D. Lim, A. Hanzer, O. P. Zuiderveld, E. Guaita, M. Adami, G.
Coruzzi, R. Leurs, I. J. P. de Esch, J. Med. Chem. 2008, 51, 2457–2467.
[10] A. J. Thompson, K. L. Price, S. C. R. Lummis, J. Physiol. 2011, 589, 4243–
4257.
[11] R. L. Papke, J. K. P. Papke, G. M. Rose, Bioorg. Med. Chem. Lett. 2004, 14,
1849–1853.
[15] D. Yan, M. M. White, Mol. Pharmacol. 2005, 68, 365–371.
[12] N. Unwin, Y. J. Fujiyoshi, J. Mol. Biol. 2012, 422, 617–34.
[13] a) D. L. Beene, G. S. Brandt, W. Zhong, N. M. Zacharias, H. A. Lester, D. A.
Dougherty, Biochemistry 2002, 41, 10262–10269; b) D. L. Beene, K. L.
Price, H. A. Lester, D. A. Dougherty, S. C. Lummis, J. Neurosci. 2004, 24,
9097–90104; c) K. L. Price, S. C. Lummis, J. Biol. Chem. 2004, 279,
23294–23301; d) K. L. Price, K. S. Bower, A. J. Thompson, H. A. Lester,
D. A. Dougherty, S. C. R. Lummis, Biochemistry 2008, 47, 6370–6377;
e) A. J. Thompson, M. Lochner, S. C. Lummis, Biophys. J. 2008, 95, 5728–
5736; f) A. J. Thompson, K. L. Price, D. C. Reeves, S. L. Chan, P. L. Chau,
S. C. Lummis, J. Biol. Chem. 2005, 280, 20476–20482; g) P. Venkatara-
man, P. Joshi, S. P. Venkatachalan, M. Muthalagi, H. S. Parihar, K. S.
Kirschbaum, M. K. Schulte, BMC Biochem. 2002, 3, 16; h) D. Yan, M. K.
Schulte, K. E. Bloom, M. M. White, J. Biol. Chem. 1999, 274, 5537–5541.
[16] J. Peters, M. Cooper, M. Livesey, J. Carland, J. Lambert in Ion Channels:
From Structure to Function (Eds.: J. N. C. Kew, C. H. Davies), Oxford Uni-
versity Press, 2010, pp. 231–251.
[17] Y. Cheng, W. H. Prusoff, Biochem. Pharmacol. 1973, 22, 3099–30108.
[18] M. H. P. Verheij, A. J. Thompson, J. E. van Muijlwijk-Koezen, S. C. R.
Lummis, R. Leurs, I. J. P. de Esch, J. Med. Chem. 2012, 55, 8603–14.
[19] R. E. Hibbs, G. Sulzenbacher, J. Shi, T. T. Talley, S. Conrod, W. R. Kem, P.
Taylor, P. Marchot, Y. Bourne, EMBO J. 2009, 28, 3040–3051.
Received: January 22, 2013
Revised: March 15, 2013
Published online on May 2, 2013
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ChemMedChem 2013, 8, 946 – 955 955