Structure-activity relationships for the binding of arylpiperazines and arylbiguanides at 5-HT3 serotonin receptors

Article abstract of DOI:10.1021/jm9603936

Arylpiperazines are nonselective agents that bind at 5-HT₃ serotonin receptors with moderate to high affinity, whereas 1-phenylbiguanide is a low- affinity but more selective 5-HT₃ agonist. In an attempt to enhance the affinity of th

Full text of DOI:10.1021/jm9603936

J . Med. Chem. 1996, 39, 4017-4026
4017
Str u ctu r e-Activity Rela tion sh ip s for th e Bin d in g of Ar ylp ip er a zin es a n d
Ar ylbigu a n id es a t 5-HT3 Ser oton in Recep tor s
Małgorzata Dukat,† Ashraf A. Abdel-Rahman,† Abd M. Ismaiel,† Stacy Ingher,‡ Milt Teitler,‡
Laszlo Gyermek,§ and Richard A. Glennon*,†
Department of Medicinal Chemistry, Medical College of Virginia, Virginia Commonwealth University,
Richmond, Virginia 23298-0540, Department of Pharmacology, Albany Medical College, Albany, New York 12208, and
Department of Anesthesiology, UCLA School of Medicine/ Harbor UCLA Medical Center, Torrance, California 90509-2910
Received J une 3, 1996X
Arylpiperazines are nonselective agents that bind at 5-HT3 serotonin receptors with moderate
to high affinity, whereas 1-phenylbiguanide is a low-affinity but more selective 5-HT3 agonist.
In an attempt to enhance the affinity of the latter agent, and working with the assumption
that similarities might exist between the binding of the two types of agents, we formulated
structure-activity relationships for the binding of the arylpiperazines and then incorporated
those substituents, leading to high affinity for the arylpiperazines, into 1-phenylbiguanide. A
subsequent investigation examined the structure-activity relationships of the arylbiguanides
and identified arylguanidines as a novel class of 5-HT3 ligands. Although curious similarities
exist between the structure-activity relationships of the arylpiperazines, arylbiguanides, and
arylguanidines, it cannot be concluded that all three series of compounds are binding in the
same manner. Furthermore, upon investigating pairs of compounds in the three series, the
arylpiperazines behaved as 5-HT3 antagonists (von Bezold-J arisch assay) whereas the
arylbiguanides and arylguanidines acted as 5-HT3 agonists.
Selective ligands are typically required for character-
izing the nature and function of neurotransmitter
receptors, and it is fortunate that high-affinity 5-HT3
serotonin receptor ligands have been identified/devel-
oped. This population of serotonin receptors has re-
cently attracted widespread attention because of its
possible involvement in chemotherapy-induced emesis,
migraine, pain management, and certain mental dis-
orders.1-6 Most of the agents currently available,
however, are 5-HT3 antagonists. On the basis that
metoclopramide (1) might produce some of its effects
through a 5-HT3 mechanism, Fozard7 developed the first
5-HT3-selective antagonist: MDL-72222. Since then,
numerous other 5-HT3 antagonists have been described;
most of these antagonists are aryl- or heteroarylamide
derivatives. The structure-activity relationships of
5-HT3 antagonists have been extensively reviewed.3-5
demonstrated that the N,N,N-trimethyl quaternary
amine derivative of 5-HT binds with 10 times the
affinity of 5-HT, possesses a much more restrictive
binding profile, and behaves as a 5-HT3 agonist.8,10
Two other groups of agents that bind at 5-HT3
receptors include the 1-arylpiperazines and the 1-aryl-
biguanides. However, whereas 5-HT3 structure-activ-
ity relationships have been formulated for 5-HT3 an-
tagonists, e.g., ref 5, and for 5-HT-related derivatives,
e.g., ref 10, relatively little is known about the arylpip-
erazines and arylbiguanides. In fact, 1-phenylbiguanide
(2) was the only arylbiguanide available as a seroton-
ergic agent for a number of years, and as a class, the
arylpiperazines have never been specifically categorized
as 5-HT3 agonists or antagonists. One reason for the
apparent lack of interest in the arylpiperazines as 5-HT3
ligands is their high affinity for other populations of
5-HT receptors.3,11 Quipazine (3), a nonselective sero-
tonergic agent, was the first arylpiperazine demon-
strated to bind at 5-HT3 receptors.12 Quipazine has
been demonstrated to be an agonist in certain prepara-
tions13 but an antagonist in others.14-16
Much less is known about 5-HT3 agonists. Agents
commonly employed as 5-HT3 agonists include serotonin
(5-HT) and 2-methyl 5-HT.3 Serotonin binds with
modest affinity at 5-HT3 receptors (Ki ) ca. 750 nM)
and is a nonselective agent;3,8 2-methyl 5-HT, which
binds with somewhat lower affinity (Ki ) 1350 nM) than
5-HT, displays greater selectivity for 5-HT3 receptors
and retains agonist activity.9 With the exception of
5-HT3 receptors, most populations of 5-HT receptors are
G-protein-coupled receptors;3 5-HT3 receptors are ion
channel receptors. Consequently, many of the agents
that bind at 5-HT3 receptors are fairly unique struc-
turewise, and most agents that bind at other popula-
tions of 5-HT receptors display low affinity for 5-HT3
receptors and vice versa.1-5 Quaternized amines, for
example, bind at 5-HT3 receptors but not at other
populations of 5-HT receptors.4 Indeed, it has been
In the late 1980s we undertook a study aimed at
identifying novel 5-HT3 receptor agonists. We endeav-
ored to formulate structure-activity relationships for
the binding of arylpiperazines at 5-HT3 receptors and
reported the results of the first such study in 1989.17
[3H]Quipazine was briefly used as a radioligand to label
5-HT3 receptors,18 and it was the radioligand employed
† Virginia Commonwealth University.
‡ Albany Medical College.
§ UCLA School of Medicine/Harbor UCLA Medical Center.
X Abstract published in Advance ACS Abstracts, September 1, 1996.
S0022-2623(96)00393-7 CCC: $12.00 © 1996 American Chemical Society

4018 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 20
Dukat et al.
by us in our structure-activity study.17 However,
subsequent anecdotal reports suggested that [3H]quip-
azine may not provide reliable results, and there now
is evidence that this radioligand may be unstable under
the conditions of the binding assay.19 [3H]Quipazine is
no longer used to label 5-HT3 receptors. Nevertheless,
our earlier investigation identified two arylpiperazines,
1-(3-chlorophenyl)piperazine (mCPP, 4) and 1-(2-naph-
thyl)piperazine (2-NP, 5), as interesting agents for
further study.17 Although 4 and 5 bind with high
affinity at 5-HT3 receptors (5-HT3 Ki ) 20 and 30 nM,
respectively), they are nonselective agents in that they
bind at other populations of 5-HT receptors.11 In 1987,
Ireland and Tyers15 described 1-phenylbiguanide (2) as
a novel 5-HT3 agonist. Although 1-phenylbiguanide
displays relatively low affinity for 5-HT3 receptors (Ki
> 1000 nM), it represented the first 5-HT3-selective
agonist. Thus, 1-phenylbiguanide is a selective but low-
affinity agonist, whereas certain arylpiperazines rep-
resent higher affinity but nonselective agents. Even
though there is little evidence or a priori reason to
suspect that arylpiperazines such as 1-phenylpipera-
zine, and arylbiguanides, such as 1-phenylbiguanide,
should bind at 5-HT3 receptors in a similar manner, this
concept served as a starting point for our initial inves-
tigations, that is, if arylpiperazines and arylbiguanides
utilize a common aromatic binding site on 5-HT3 recep-
tors, it might be possible to extrapolate the structure-
activity relationships formulated for the arylpiperazines
to the arylbiguanides so as to enhance their affinity for
5-HT3 receptors. Early work in our laboratory, based
on the results of our initial structure-activity study on
arylpiperazines, led to two novel arylbiguanides: 1-(3-
chlorophenyl)biguanide (6) and 1-(2-naphthyl)biguanide
(7). Some of our preliminary results have been re-
ported.20
commercially available, or were prepared according to
literature procedures (see the Experimental Section for
details). Pyridine derivative 11 was synthesized by
catalytic hydrogenation of the product obtained from the
Grignard reaction of 2-methoxybromobenzene and N-ben-
zyl-4-piperidone. The quaternary compounds 14 and 16
were synthesized by exhaustive N-methylation of the
corresponding phenylpiperazines with iodomethane.
The piperazine derivatives 17,22 18, and 1922,23 were
prepared using the procedure described by Brewster et
al.24 where bis(2-chloroethyl)amine hydrochloride was
used to alkylate the appropriate aniline. 2-[1-(4-Meth-
ylpiperazinyl)]pyridines 24-26 were obtained by alky-
lation of N-methylpiperazine with the corresponding
halopyridine.25 The N,N-dimethyl analog of arylbiguan-
ide 34 was prepared by the general and well-known
reaction of an aromatic amine mineral acid salt and
appropriately substituted dicyandiamide. The reverse
amide 36 and amide 37 were prepared by reaction of
the appropriate amine with acid halides which were
then converted by Gabriel synthesis26 to the phthalim-
ides and hydrolyzed with hydrazine hydrate to the
corresponding amines. Although the free base and
nitrate of compound 44 have been reported,27 we
synthesized its oxalate salt in a manner similar to that
of 39, that is, the appropriate halide was converted to
its nitrile and then reduced with LiAlH4 to the corre-
sponding amine. The N-methyl (55) and N,N-dimethyl
(56) derivatives of compound 54 are mentioned in the
literature; however, we were unable to find any physical
characterization of these compounds. The secondary
amine 55 was prepared from 54 via a carbamate
intermediate which was subsequently reduced to the
desired target with LiAlH4. The tertiary amine 56 was
prepared from 54 by reductive methylation using an
Eschweiler-Clarke procedure. Guanidine 60 was pre-
pared by the general method described by Short and
Darby.28
Due to our use of [3H]quipazine in the original
arylpiperazine structure-activity study, we were no
longer certain that the formulated structure-activity
relationships were valid. We wished to repeat these
studies using a newer radioligand to label 5-HT3 recep-
tors and to continue our investigations of the arylbi-
guanides. While our work was in progress, Kilpatrick
et al.12 reported that 6 is a high-affinity 5-HT3 agonist.
Morain et al.21 have more recently described some of
their structure-activity studies with aryl-modified aryl-
biguanides. However, apart from their study, a sys-
tematic and comprehensive structure-activity investi-
gation of arylbiguanides has not yet been reported.
The purpose of the present report, then, is severalfold.
First, we describe a structure-affinity study on arylpip-
erazines using [3H]GR65630 as radioligand and, where
possible, compare the present findings with the origi-
nal17 structure-affinity findings. Second, we describe
the results of a structure-affinity study with the
arylbiguanides where we investigate modifications of
both the aryl and biguanide portions of the molecule.
Finally, we examine the functional activity of certain
selected compounds in order to determine if they are
5-HT3 receptor agonists or antagonists. During the
course of this investigation, arylguanidines were identi-
fied as a novel class of 5-HT3 agonists.
Resu lts
Ar ylp ip er a zin es. Radioligand binding data for the
arylpiperazine derivatives are presented in Table 1. Of
the compounds examined, 10 are common to this and
the original17 study. This allowed some comparisons to
be made between the two studies. Of the 10 compounds,
binding data for five (3-5, 20, 21) differed between the
two investigations by only 2- or 3-fold. However, the
affinity of the parent phenylpiperazine 8 is approxi-
mately 7-fold lower than reported earlier, and the
affinities of 9, 10, 12, and 13 are >23-, 8-, 5-, and 18-
fold lower. While there generally seems to be a good
qualitative agreement between the two studies, the
original study overestimated the affinity of certain
arylpiperazines for 5-HT3 receptors. The only case
where this may have been of consequence is with 8
because, being the parent member of the series, its
affinity was compared to that of other arylpiperazines
in making structure-activity comparisons. Thus, al-
though some of the quantitative statements made in the
original study may now need to be revised, many of the
original conclusions still seem to apply.
1-Phenylpiperazine (8) binds with low affinity (Ki )
3000 nM; Table 1). Replacement of the less basic
nitrogen atom of 8 with a carbon atom results in reduced
affinity (9, Ki > 10 000 nM). Methoxy substitution at
the phenyl 2- or 4-position (10, 12) has little effect on
Ch em istr y
Many of the compounds necessary for this investiga-
tion were on hand as a result of previous studies, were

SAR for Binding at 5-HT3 Serotonin Receptors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 20 4019
Ta ble 2. Binding of Arylbiguanides at 5-HT3 Receptors
Ta ble 1. Binding of Aryl-Modified Piperazines at 5-HT3
Receptors
compd
R
Ki, nM (SEM)
compd
R
Y
R′
Ki, nM (SEM)
2
27
6
28
29
30
31
32
33
7
phenyl
2-Cl-phenyl
3-Cl-phenyl
4-Cl-phenyl
3-NO2-phenyl
3-Me-phenyl
4-Me-phenyl
3-Cl-4-Me-phenyl
2-OMe-5-Cl-phenyl
2-naphthyl
1200 (40)
62 (4)
17 (4)
8
9
H
H
N
CH
N
C-OH
N
N
N
N
N
N
H
H
H
H
H
H
H
Me2
H
Me2
H
3000 (50)
>10000
10
11
12
13
4
14
15
16
17
18
19
2-OMe
2-OMe
4-OMe
4-Cl
3-Cl
3-Cl
3-CF3
3-CF3
2,4-(OMe)2
2-Me, 4-OMe
2-OMe, 5-Cl
2500 (530)
>10000
200 (20)
220 (40)
780 (50)
>10000
225 (25)
126 (33)
12 (2)
1640 (480)
2160 (170)
62 (5)
+
+
1450 (380)
1390 (425)
>10000
N
N
N
>10000
H
H
660 (10)
40 (4)
(20, Ki ) 175 nM) and found it to bind with 17-fold
higher affinity than the parent phenylpiperazine 8. 1-(2-
Naphthyl)piperazine (2-NP, 5, Ki ) 32 nM) binds with
even higher affinity and with an affinity approximately
100 times greater than that of 8. The lack of affinity of
22 suggests that the N4 nitrogen atom is required for
binding. Introduction of a nitrogen atom into the ring
as with quipazine (3) and its N-methyl derivative 21
(Ki ) 2 and 3 nM, respectively) produces the highest
affinity members of the series. Finally, the quinoline-
type nitrogen atom of 3 was incorporated into 4 (Ki )
62 nM) to afford the 6-chloro-2-pyridyl derivative 23;
compound 23 (Ki ) 15 nM) was found to bind with 4
times the affinity of 4 and 200 times the affinity of 8.
The related N-methyl derivative 24 (Ki ) 38 nM) binds
with similar affinity as does the 6-iodo counterpart 26
(Ki ) 48 nM). In contrast, the 6-methyl derivative 25
(Ki ) 560 nM) binds with reduced affinity.
20
5
3
21
22
H
H
H
Me
H
175 (15)
32 (8)
2 (1)
CH
N
N
3 (1)
>10000
N
The present findings essentially confirm (at least
qualitatively) and extend the results of our earlier
structure-activity study.17 Most importantly, there
appears to be something unique about the 3-position of
the phenylpiperazines because the presence of a chloro
group at this position enhances affinity by about 50-
fold. This effect cannot be mimicked by incorporation
of a trifluoromethyl group. However, fusion of the c-face
of 8 with a benzene ring, to give 2-NP (5), results in a
high-affinity compound. Affinity is further enhanced by
introduction of a ring nitrogen (i.e., 3, Ki ) 2 nM). A
ring nitrogen atom also enhances the affinity of 4 (i.e.,
23) but to a somewhat lesser extent. Perhaps the most
unexpected finding is that quaternization of the termi-
nal amine of the arylpiperazines (i.e., 14, 16) results in
reduced affinity; this is unlike what was seen with 5-HT
itself.8
23
24
25
26
Cl
Cl
Me
I
N
N
N
N
H
15 (5)
38 (1)
560 (90)
48 (3)
Me
Me
Me
affinity, whereas 2,4-dimethoxy substitution (i.e., 17) is
detrimental to binding. Replacement of the less basic
nitrogen atom of 10 with a C-OH group (i.e., 11) also
reduces affinity. Introduction of a 4-chloro group (i.e.,
13) seems inconsequential relative to 8, whereas intro-
duction of the 3-chloro (i.e., 4, Ki ) 62 nM) enhances
affinity by 50-fold. The 3-trifluoromethyl derivative 15
binds with 2 times the affinity of its parent 8 but with
a 22-fold lower affinity than the corresponding 3-chloro
derivative 4. The influence of the m-chloro substituent
is also seen when the 2-methoxy-5-chloro compound 19
(Ki ) 40 nM) is compared with the monosubstituted
2-methoxy compound 10 (Ki ) 2500 nM). As mentioned
in the introduction, the quaternary amine analog of
5-HT binds with 10-fold higher affinity than 5-HT itself.
Consequently, we prepared the N,N-dimethyl quater-
nary amine analogs of 4 and 15; however, affinity was
decreased in both cases (i.e., 14 and 16, Ki ) 1450 and
> 10 000 nM, respectively).
Ar ylbigu a n id es. 1. Ar yl Su bstitu tion . 1-Phenyl-
biguanide (PBG; 2) and several of its aryl-substituted
derivatives were examined (Table 2). PBG (2) binds
with low affinity (Ki ) 1200 nM). Incorporation of a
chloro group at the 2-, 3-, or 4-position results in
enhanced affinity (27, 6, 28, Ki ) 62, 17, and 200 nM,
respectively). The effect of the 3-chloro group could not
be mimicked by replacement with a (nearly equilipo-
philic) methyl group (i.e., 30, Ki ) 780 nM) or the
electron-withdrawing nitro group (i.e., 29, Ki ) 220 nM).
The 4-methyl derivative 31 lacks affinity; however,
introduction of a chloro group to give the 3-chloro-4-
methyl derivative 32 (Ki ) 225 nM) again improves
binding. The 2-methoxy-5-chloro derivative 33 (Ki )
126 nM) binds with 10 times the affinity of 2. The
Introduction of the 2-methyl-4-methoxy substituents
seems to enhance affinity (18, Ki ) 660 nM) relative to
the 4-methoxy derivative 12 (Ki ) 1640 nM) suggesting
that substitution may be tolerated at the 2-position.
Consequently we examined 1-(1-naphthyl)piperazine

4020 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 20
Ta ble 3. Binding of Biguanide Derivatives at 5-HT3 Receptors
Dukat et al.
compd
A
B
C
D
E
X
Ki, nM (SEM)
17 (4)
>10000
1250 (22)
>10000
6
34
35
36
37
38
39
40
41
42
43
44
45
NH
NH
NH
NH
C(dO)
NH
CH2
CH2
CH2
CH2
CH2
CH2
NH
C(dNH)
C(dNH)
C(dO)
C(dO)
NH
CH2
CH2
CH2
CH2
NH
NH
NH
C(dNH)
C(dNH)
C(dNH)
CH2
CH2
CH2
NH2
NMe2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
2-Cl
4-Cl
3-Cl
3-Cl
3-Cl
CH2
CH2
CH2
CH2
NH
>10000
>10000
>10000
CH2
C(dNH)
C(dNH)
C(dNH)
NH2
40 (2)
NH
NH
150 (30)
243 (27)
1200 (12)
CH2
NH
C(dNH)
CH2
NH2
CH2
C(dNH)
NH2
>10000
35 (5)
2-naphthyl derivative 7 (Ki ) 12 nM) binds with 100-
fold greater affinity than 2.
the biguanide chain as in 36-39 also abolishes affinity.
Interestingly, the N-(2-phenylethyl)guanidine derivative
40 binds with high affinity (Ki ) 40 nM) suggesting that
the intact biguanide moiety is not required for binding.
The affinity of the 3-chloro derivative 40 was compared
with those of its 2- and 4-chloro positional isomers 41
and 42, respectively. The 2-chloro derivative 41 and the
4-chloro derivative 42 bind with approximately 4- and
6-fold lower affinity, respectively, than 40. Shortening
of the phenylethyl portion of 40 to benzyl (43, Ki ) 1200
nM) reduces affinity by 30-fold, and further modifica-
tion, as with 44, abolishes affinity. In an investigation
of the binding requirements of 5-HT3 antagonists, Rizzi
et al.30 found that the deschloro derivative of 43 (i.e.,
N-benzylguanidine; Ki > 10 000 nM) does not bind at
5-HT3 receptors. The higher affinity of 43 suggests once
again that the m-chloro group contributes to binding.
However, removing the ethyl chain of 40 to give the (3-
chlorophenyl)guanidine derivative 45 (Ki ) 35 nM)
results in a high-affinity compound. Because 45 is
essentially a biguanide lacking the terminal amidine
portion of the molecule, its structure-activity relation-
ships were examined in further detail.
There are some similarities and differences between
the binding of the arylpiperazines and the arylbiguan-
ides. In both series, introduction of a 3-chloro group
results in enhanced affinity (50- and 70-fold) as com-
pared to the parent aryl-unsubstituted compound. In
both series, the 2-naphthyl derivatives bind with 100-
fold greater affinity than their respective parent. In
both series, the electron-withdrawing chloro group
cannot be replaced with another electron-withdrawing
group with retention of affinity. However, 2-methoxy-
5-chloro substitution results in a 75-fold increase in
affinity in the arylpiperazine series, whereas it produces
only a 10-fold increase in affinity in the arylbiguanide
series. At this time, it cannot be conclusively stated that
the aromatic portions of the two series of agents are
binding in a similar manner (or at the same site);
however, there are certain curious trends to suggest that
this may be possible.
Morain et al.21 recently published the results of their
structure-activity studies on arylbiguanides. Although
they examined a total of 24 aryl-modified biguanides,
there are only three substituted phenylbiguanide de-
rivatives common to the two investigations. For these
three compounds, Ki values are nearly identical between
the two studies: 27 (Ki ) 69 nM21 as compared to 62
nM in Table 2), 6 (13 nM21 as compared to 17 nM), and
28 (170 nM21 as compared to 200 nM). Morain et al.21
found that the 3-chloro group could not be replaced by
a trifluoromethyl group with retention of high affinity
(i.e., this replacement resulted in a 54-fold decrease in
affinity); they further identified the 2,3,5-trichloro
derivative of 2 (Ki ) 0.44 nM) as the highest affinity
member of the series. Krijzer and Tulp29 had earlier
reported that the 3,4-dichloro derivative of phenylbi-
guanide is a high-affinity (Ki ) 40 nM) 5-HT3 agonist;
Morain et al.21 reported a Ki of 20 nM for this compound.
All of these findings attest to the importance of a
m-chloro group.
Ar ylgu a n id in es. Compound 45 appears to be the
first member of a novel class of serotonergic ligands;
we investigated the effect of both aryl substitution and
modification of the guanidine moiety (Table 4). Removal
of the 2-chloro group to give the parent 1-phenylguani-
dine (46, Ki ) 2340 nM) reduces affinity by nearly 70-
fold. Of the three different chloro-substituted com-
pounds (45, 47, 48), 3-chloro substitution seems to be
optimal. As seen with the other series of compounds,
the 3-chloro substituent cannot be replaced by trifluo-
romethyl with retention of affinity; the trifluoromethyl
derivative 49 (Ki ) 5770 nM) binds with 165-fold lower
affinity than 45. Likewise, replacement of this chloro
substituent with methyl or methoxy groups (50, 51)
results in low-affinity compounds.
Conversion of the guanidine moiety of 45 to a urea
(i.e., 52) abolishes affinity; most other modifications of
the guanidine moiety also abolish affinity (i.e., 53-56).
The amidine 57 (Ki ) 1200 nM), in which one of the
nitrogen atoms of 45 has been replaced by a methylene
group, binds with 35-fold reduced affinity, whereas
cyclization to the imidazoline 58 abolishes affinity. The
2-pyridyl analog 59 is inactive, but interestingly the
2. Bigu a n id e Mod ifica tion . With the 3-chloro
group present, the biguanide portion of 6 (Ki ) 17 nM)
was modified (Table 3). Introduction of a terminal N,N-
dimethyl group (i.e., 34) abolishes affinity. Replacement
of the N2 nitrogen atom with oxygen (35, Ki ) 1250 nM)
decreases affinity by 73-fold. Further modification of

SAR for Binding at 5-HT3 Serotonin Receptors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 20 4021
Ta ble 4. Binding of Arylguanidines at 5-HT3 Receptors
Ta ble 5. 5-HT3 Receptor Stimulant Potencies in Rat (von
Bezold-J arisch Reflex)
relative potencya
compd
n
potency
range
2
4
5
6
7
5
3
3
21
4
7
0.45
<0.02
<0.02
1.0
0.5
0.74
1.0
0.3-0.6
compd
A
B
C
X
Ki, nM (SEM)
46
47
45
48
49
50
51
52
53
54
55
56
57
58
NH
NH
NH
NH
NH
NH
NH
NH
Nd
C(dNH) NH2
C(dNH) NH2
C(dNH) NH2
C(dNH) NH2
C(dNH) NH2
C(dNH) NH2
C(dNH) NH2
H
2340 (150)
190 (90)
35 (5)
0.3-0.7
0.5-1.3
0.5-1.5
2-Cl
3-Cl
4-Cl
3-CF3
3-Me
3-OMe
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
45
61
5
320 (150)
5700 (2300)
6520 (1035)
1600 (300)
>10000
a
Stimulant potency relative to compound 6 ) 1. Compound 6
produced relfex bradycardia in all preparations at concentrations
of 0.9-20 µg/kg and was used as a reference standard in all
experiments. Tropisetron at doses of 2-10 µg/kg markedly
reduced or fully antagonized the stimulant effects of 2, 6, 7, 45,
and 61. Compounds 4 and 5 produced no detectable effect.
C(dO)
NH2
NH2
NH2
NH-Me
N(Me)2
CH
>10000
CH2 CH2
CH2 CH2
CH2 CH2
>10000
>10000
>10000
Ta ble 6. 5-HT3 Receptor Stimulant Potencies in the Rabbit
Bladder Preparation
CH2 C(dNH) NH2
1200 (96)
>10000
compd
n
minimal dosea
2
5
6
7
45
61
2
1
6
4
4
2
1-10
30
6.6
1-10
30-150
2.5-15
a
Minimal effective dose in µg/kg. The minimal effective dose
for 6 was determined to be 6.6 ( 1.8 µg/kg. Potencies of other
compounds are reported as dose ranges. Compound 4 was not
examined. The stimulant effect of all compounds was antagonized
by tropisetron at doses of 5-10 µg/kg; antagonism of 61 was not
examined.
59
60
61
NH
NH
NH
C(dNH) NH2
C(dNH) NH2
C(dNH) NH2
>10000
2910 (740)
25 (2)
that the two piperazines 4 and 5 lack agonist activity
and possess some antagonist character. In contrast,
both arylbiguanides and both arylguanidines act as
5-HT3 agonists.
3-pyridyl analog displays about the same affinity (60,
Ki ) 2910 nM) as the unsubstituted parent 46. The
2-naphthyl derivative 61 (Ki ) 25 nM) binds with nearly
100-fold higher affinity than 46.
In the rabbit bladder assay,32 compound 6 produced
a stimulant effect with a minimal effective dose of 6.6
((1.8) µg/kg (Table 6). Compounds 2 and 7 produced
similar effects at the 1-10 µg/kg dose range. Compound
45 was somewhat less potent (minimal effective dose
range ) 30-150 µg/kg), whereas 61 (2.5-15 µg/kg)
seems nearly equipotent with 6. The stimulant effects
of each of these compounds was antagonized by tro-
pisetron. At 30 µg/kg, the piperazine derivative 5
displayed agonist activity; although its potency is low,
its effect was antagonized by tropisetron. Compound 4
was not examined. These results show that the two
arylbiguanides and the two arylguanidines act as ago-
nists in this preparation. In conclusion, both assays
reveal that the two arylbiguanides and the two aryl-
guanidines are 5-HT3 agonists.
This class of compounds is rather interesting for
several reasons. As with the arylpiperazines and the
arylbiguanides, introduction of a 3-chloro group results
in enhanced affinity and replacement of phenyl with
2-naphthyl increases affinity by 100-fold. Also, as with
the other two series, the 3-chloro substituent cannot be
replaced with a trifluoromethyl group without a signifi-
cant decrease in affinity. Differences also exist. For
example, whereas introduction of a 4-chloro group
enhances affinity by about 7-fold in the latter two series,
it has essentially no effect in the arylpiperazine series;
also, replacement of the phenyl group by 2-pyridyl
enhances the affinity of the arylpiperazines but reduces
the affinity of the arylguanidines.
F u n ction a l Stu d ies. Compounds 2, 4-7, 45, and
61 were examined in the von Bezold-J arisch assay.31
After several preliminary trials, compound 6 was sub-
sequently used as control in all other experiments; 6
consistently produced the bradycardic response at doses
of 0.9-20 µg/kg. Results for the other compounds
examined are reported in Table 5 relative to 6. 1-Phen-
ylbiguanide (2) and the naphthyl biguanide 7 were
about one-half as potent as 6; guanidine 45 was some-
what more potent than, and 61 was essentially equipo-
tent with, 6. The agonist effects of these compounds
could be antagonized by the 5-HT3 antagonist tro-
pisetron (data not shown). The two piperazine deriva-
tives 4 and 5 were ineffective as agonists at doses of up
to 200 µg/kg. In fact, at doses of 20-120 µg/kg, both
agents were able to attenuate the effects of 6 when given
in combination (data not shown). It would seem then
Discu ssion
Previously, it was not known with certainty whether
or not arylpiperazines represented 5-HT3 agonists or
antagonists. Even in the absence of knowledge con-
cerning the functional activity of these agents, it was
not unreasonable to suspect that there may be some
structural similarity between the arylpiperazines and
arylbiguanides with respect to the manner in which they
interact with 5-HT3 receptors, that is, it is possible that
structural relationships might exist between the two
structure types even if one class represents agonists and
the other antagonists. We expected that structure-
activity information derived from the high-affinity but
nonselective arylpiperazines might be applied to en-

4022 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 20
Dukat et al.
hancing the affinity of the more 5-HT3-selective, yet low-
affinity, 1-phenylbiguanide (2). We posed the question:
Is there any structural similarity between arylpiper-
azines and arylbiguanides? One can envision that
arylbiguanides, particularly in a protonated form, might
exist as hydrogen-bonded structures (e.g., 62). The
delocalized cationic center of 62 might mimic the
4-position nitrogen atom of an arylpiperazine at the
receptor, and molecular modeling studies have shown
that the two structures can be superimposed.20 Inter-
estingly, 25 years ago, Shapiro and co-workers33 pro-
posed such a cyclic conformation to account for some of
the ultraviolet absorption spectra of various arylbiguan-
ides. Although this cyclic structure represents only one
of numerous solution conformations, and is not sup-
ported by the solid state structures of such compounds
as determined by X-ray crystallography,34-36 it does
offer a reasonable explanation for why structural simi-
larities might exist.
Structure-activity studies with arylbiguanides iden-
tified several arylguanidines as high-affinity 5-HT3
ligands. As with the arylpiperazines and arylbiguan-
ides, the 3-chloro derivative 45 and the naphthyl
derivative 61 are among the higher affinity members
of the series. From this perspective, these are two
features that are shared by all three series of com-
pounds. All three series may bind in a common manner,
but this is difficult to envision. Obviously, additional
work needs to be done. However, in comparing the
high-affinity arylbiguanides and arylguanidines, the
only nonaromatic feature shared by the two series is
that substituent “C” (see Tables 3 and 4) contains NH
(e.g., 6, 40, 45). But, this alone is insufficient for
binding (e.g., see 54, Ki > 10 000 nM). The guanidino
moiety (i.e., as found in 6, 40, and 45) is evidently
important for binding. However, if the guanidino por-
tion of all three compounds is overlayed, the aromatic
portions cannot be aligned. In contrast, if the aromatic
portions are aligned (and there is reason to believe this
is possible in some cases on the basis of parallel
substituent effects), the “C-substituent” NH is common
and may constitute a center of diffuse charge with which
to interact at a common feature of the receptor.
62
When our work began, it was known that 1-phenyl-
biguanide represents a 5-HT3 agonist; however, the
functional activity of arylpiperazines as a class was
unclear. More recently, it has been shown that quip-
azine acts as a 5-HT3 antagonist in certain instances
and as an agonist in others (see the introduction).
Specifically, quipazine behaves as a 5-HT3 agonist as
reflected by its ability to increase [14C]guanidinium
uptake in NG 108-15 cells;13 NG 108-15 cells also served
as the source of 5-HT3 receptors for the present inves-
tigation. The possibility exists, then, that certain aryl-
piperazines might constitute 5-HT3 agonists. Never-
theless, for the purpose of the present investigation, we
wished to specifically determine if the m-chlorophenyl
and 2-naphthyl analogs in the piperazine, biguanide,
and guanidine series act as 5-HT3 agonists or antago-
nists, that is, we wished to compare the actions of
arylpiperazines 4 and 5 with those of arylbiguanides 6
and 7 and arylguanidines 45 and 61. The commonly
used 1-phenylbiguanide (2) was included for purpose of
comparison. The von Bezold-J arisch reflex is an in vivo
assay that consists of a reflex and transient bradycardia
evoked by 5-HT that activates vagal afferent nerve
terminals within the heart; as an assay, it represents
the first and still the most convenient in vivo assay for
5-HT3 receptor function.31 The effect can be induced by
5-HT and 5-HT3 agonists such as 2-methyl 5-HT and
1-phenylbiguanide (2), and the effect evoked by these
agents is antagonized by 5-HT3 antagonists.31 The
actions of 5-HT on isolated ganglia are complex and may
involve, at least in part, 5-HT3 receptors.31 In an older
study, it was shown that 1-phenylbiguanide stimulates
pelvic ganglia producing bladder stimulation in ani-
mals.32 This effect, which is also evoked by 5-HT, can
be antagonized by the 5-HT3 antagonist tropisetron
(unpublished data).
The results of our original structure-activity studies
on arylpiperazines were essentially confirmed and
extended in the present investigation. Taken together,
they suggest that 1-phenylpiperazine (8, Ki ) 3000 nM)
binds with modest affinity and that introduction of a
3-chloro group, or replacement of the phenyl ring with
a 2-naphthyl group, enhances affinity by 50-100-fold
(i.e., 4 Ki ) 62 nM, 5 Ki ) 32 nM, respectively).
Replacement of phenyl by a 2-quinolinyl group, to afford
quipazine, results in the highest affinity member of the
series (3, Ki ) 2 nM). The present results also support
the original suggestion that the presence of the 4-posi-
tion piperidine nitrogen atom is critical for 5-HT3
binding (i.e., 22, Ki > 10 000).
Armed with this information, we prepared and ex-
amined the 3-chloro derivative of 1-phenylbiguanide (2,
Ki ) 1200 nM) and found it to bind with markedly
enhanced affinity (6, Ki ) 17 nM). Kilpatrick et al.12
have independently reported that 6 binds with high
affinity at 5-HT3 receptors. 1-(2-Naphthyl)biguanide (7,
Ki ) 12 nM) also binds with high affinity. In both series,
the effect of the chloro group could not be mimicked by
another electron-withdrawing group. Morain et al.21
observed a similar phenomenon upon replacement of the
chloro group with a trifluoromethyl group in the biguan-
ide series and have suggested that the inductive and
mesomeric properties of the chloro group may play a
major role in the acidic character of the molecules and
that this might be important for 5-HT3 binding. They
also suggested that delocalization of electrons between
the biguanide moiety and the aromatic ring contributes
to binding due to a decrease in affinity upon insertion
of a methylene group between the two components.
However, as shown in Table 3, compound 40 (Ki ) 40
nM), which lacks an NH substituent attached directly
to the 3-chlorophenyl ring, still binds with high affinity.
Although aryl substitution may influence the acidity of
an attached nitrogen, the presence of such a nitrogen
attached directly to the aromatic ring does not appear
to be critical for binding.
Functional studies with 1-phenylbiguanide (2), the
3-chloro and naphthyl derivatives of phenylpiperazine
(i.e., 4 and 5), phenylbiguanide (i.e., 6 and 7), and
phenylguanidine (i.e., 45 and 61) indicate that both
arylpiperazines behave as antagonists whereas the

SAR for Binding at 5-HT3 Serotonin Receptors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 20 4023
arylbiguanides and arylguanidines act as 5-HT3 ago-
nists in the von Bezold-J arisch assay. The stimulant
effects of the agonists were effectively antagonized by
the 5-HT3 antagonist tropisetron. In the rabbit bladder
assay, again the arylbiguanides and the arylguanidines
served as agonists, and their effects could be antago-
nized by tropisetron.
Initially, it may seem surprising that the arylguan-
idines bind at 5-HT3 receptors and, further, that they
are 5-HT3 agonists, in light of a report by Morain et al.21
that guanidines lack affinity for 5-HT3 receptors (Ki )
36 000->100 000 nM); however, the compounds exam-
ined by Morain and co-workers were heterocyclic (i.e.,
benzimidazole, benzothiazole, benzoxazole; e.g., 63 where
X ) NH, S, and O) guanidines and not simple arylguan-
idines of the type examined here. Rizzi et al.30 have
found that even the structurally simpler N-benzylguani-
dine lacks affinity for 5-HT3 receptors. But the same
group developed guanidine 64 as a 5-HT3 antagonist and
demonstrated that it binds with high affinity (Ki ) 3.3
nM).38 Interestingly, they also found in the von Bezold-
J arisch assay that 64 is a mixed agonist/antagonist.
important for binding led to the synthesis of the corre-
sponding arylbiguanide derivatives 6 and 7 (ref 20 and
present study). Of the arylbiguanides investigated
(Tables 2 and 3), none bind with higher affinity than 6
and 7. A detailed structure-activity investigation
identified arylguanidines as retaining 5-HT3 affinity,
and subsequently, the (m-chlorophenyl)guanidine 45
and the 2-naphthylguanidine 61 were found to be the
highest affinity members of the series. Finally, all four
of these compounds (6, 7, 45, 61) were shown to act as
5-HT3 agonists (it might be noted, however, that the
selectivity of these compounds for 5-HT3 versus other
populations of 5-HT receptors has yet to be determined).
Thus, these studies provide structure-activity data on
several classes of 5-HT3 ligands, indicate how arylguan-
idines may be structurally related to the arylbiguanides,
and show that the arylguanidines 45 and 61 can act as
5-HT3 agonists. These results should also prove useful
in future molecular modeling studies aimed at under-
standing how 5-HT3 ligands interact with 5-HT3 recep-
tors.
Exp er im en ta l Section
Syn th esis. Melting points were determined on a Thomas-
Hoover melting point apparatus and are uncorrected. Proton
magnetic resonance spectra were recorded on either a J EOL
FX90Q or QE300 spectrometer with tetramethylsilane as an
internal standard. Assigned structures are consistent with
spectral data. Elemental analysis was performed by Atlantic
Microlab (Norcross, GA), and values are within 0.4% of theory.
Compounds 3 maleate,39 5 HCl,39 10 HCl,40 20 HCl,39 21
dimaleate,41 and 22 HCl39 were previously prepared in our
laboratories. The arylpiperazine 23 HCl was prepared ac-
cording to the method of Lumma and Saari.42 The arylbiguan-
ides 6 HCl,43,44 7 HCl,46 27 HCl,44 28 HCl,44 29 HCl,44 30 HCl,43
31 HCl,43 32 HCl,45 and 33 HCl45 were prepared according to
literature procedures. The modified analogs phenylbiguanide
35 HCl,45 38 2HCl,47 40 hemisulfate,48 41 hemisulfate,48 42
hemisulfate,28 43 HCl,28 and 45 nitrate49 are known, and
methods of preparation were employed from cited references.
The arylguanidines 46 nitrate,50 47 nitrate,51 48 nitrate,52 49
nitrate,53 50 hemisulfate,53 51,54 52,55 53 HCl,56 54 HCl,57 57
HCl,58 58 HCl,59 59 hemisulfate,60 and 6161 were synthesized
using the literature procedures. Compounds 2, 4, 8, 9, 12, 13,
and 15 were commercially available (Aldrich Chemical Co.).
4-Hydr oxy-4-(2-m eth oxyp h en yl)pip er idin e Hyd r och lo-
r id e (11). A solution of 2-methoxybromobenzene (2.0 g, 10.7
mmol) in anhydrous Et2O (10 mL) was added to a mixture of
Mg (0.29 g, 11.8 mmol) and a catalytic amount of I2 in
anhydrous Et2O (20 mL) under N2 at room temperature. The
stirred mixture was heated at reflux for 5 h. To this cloudy
solution was added N-benzyl-4-piperidone (2.0 g, 10.7 mmol)
in anhydrous Et2O (15 mL) at 0 °C, and the mixture was
heated at reflux (water bath) for 1.5 h. The crude product was
isolated by acid-base extraction and purified by distillation
(bp 87-95 °C/0.55 mmHg). This oily product was used in the
next step without further characterization. A solution of this
intermediate (0.5 g) in absolute EtOH (60 mL) was debenzy-
lated by hydrogenation over 30% Pd/C (0.1 g) for 24 h, at 45
psi. The heterogeneous mixture was filtered through a Celite
pad to removed the catalyst. The filtrate was evaporated
under reduced pressure to afford a white solid. The crude free
base was converted to the HCl salt and recrystallized from
absolute EtOH to yield 0.25 g (70%) of 11: mp 231-233 °C;
1H-NMR (DMSO-d6) δ 2.5-3.3 (m, 9H, CH2, CH), 3.82 (s, 3H,
CH3), 6.85-7.6 (m, 4H, Ar-H), 9.05 (bs, 1H, NH). Anal.
(C12H17NO2‚HCl) C,H,N.
These results suggest that with the appropriate sub-
stituents guanidines can bind at 5-HT3 receptors and
that they may contribute to agonist action. Further-
more, a search of the literature reveals that guanidine
(and biguanide) derivatives were reported over 40 years
ago as possessing serotonergic agonist activity, but this
work seems to have laid dormant. For example, certain
biguanides produce a fall in blood pressure and heart
rate through what was speculated to be a serotonergic
mechanism (see Fastier37 for a review). Gyermek32
demonstrated that 61 (and 8) mimics 5-HT in stimulat-
ing the inferior mesenteric ganglion of the cat and the
pelvic ganglia of the dog and that these effects are
inhibited by 5-HT antagonists. Perhaps the significance
of these studies went unrecognized because they were
reported prior to the discovery of 5-HT3 receptors. With
the realization that arylguanidines bind at 5-HT3 recep-
tors, that they act as agonists in the von Bezold-J arisch
assay, and that their effects can be antagonized by the
5-HT3 antagonist tropisetron, it seems that we have
rediscovered a “new” class of 5-HT3 agonists. Indeed,
it may be the presence of the guanidine moiety that
imparts mixed agonist/antagonist activity to 64, a
compound originally designed as a 5-HT3 antagonist.
In summary, then, we have examined the structure-
activity relationships for the binding of arylpiperazines
at 5-HT3 receptors and, where possible, compared the
results with those from our original17 investigation. It
would seem that the original binding assay using [3H]-
quipazine as radioligand provided results that overes-
timated the affinities of certain compounds. Neverthe-
less, the qualitative, if not quantitative, conclusions are
consistent between the two studies. The identification
of m-chlorophenyl and 2-naphthyl substituents as being
1-(3-Ch lor op h en yl)-4,4-d im eth ylp ip er a zin iu m Iod id e
(14). Compound 14 was prepared as a white crystalline
material in 31% yield by alkylation of 1-(3-chlorophenyl)-
piperazine with MeI in a manner similar to that described for
16: mp 199-201 °C (MeOH); 1H-NMR (DMSO-d6) δ 3.2 (s,

4024 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 20
Dukat et al.
6H, CH3) 3.5 (s, 8H, CH2), 6.8-7.4 (m, 4H, Ar-H). Anal.
absolute EtOH afforded 2.34 g (70%) of the title compound 34
as white crystals: mp 258-259 °C. Anal. (C10H14N5 HCl)
C,H,N.
N-(3-Am in op r op ion yl)-3-ch lor oa n ilin e Oxa la te (36).
The title compound 36 was prepared in a similar manner to
that of 37 in 15% yield: mp 169-170 °C (EtOH:anhydrous
Et2O). Anal. (C9H11N2ClO‚C2H2O4‚0.5 H2O) C,H,N.
N-(2-Am in oeth yl)-3-ch lor oben za m id e Hyd r och lor id e
(37). 3-Chlorobenzoyl chloride (1.75 g, 10 mmol) in THF (10
.
(C12H18N2ClI) C,H,N.
1-[3-(Tr iflu or om eth yl)p h en yl]-4,4-d im eth ylp ip er a zin -
iu m Iod id e (16). A mixture of 1-(R,R,R-trifluoro-m-tolyl)-
piperazine (1.15 g, 5 mmol), MeI (1.43 g, 10 mmol), and K2CO3
(0.35 g, 2.5 mmol) was heated at reflux for 3 h. The reaction
mixture was poured onto 5 mL of H2O and washed with 3 ×
10-mL portions of Et2O. The product that precipitated from
the aqueous solution upon cooling was collected by filtration
and recrystallized from MeOH to give 0.51 g (27%) of 16 as
white crystals: mp 224-225 °C; 1H-NMR (DMSO-d6) δ 3.1 (s,
6H, CH3), 3.6 (s, 8H, CH2), 7.0-7.6 (m, 4H, Ar-H); 13C-NMR
(DMSO-d6) δ 150.13 (ArC-N), 130.49 (ArC-CF3), 119.59 (CF3),
116.17 (ArC), 116.12 (ArC), 111.86 (ArC), 111.81 (ArC), 60.44
mL) was added in
a dropwise manner to a solution of
2-bromoethylamine (2.04 g, 10 mmol) and NEt3 (2.02 g, 20
mmol) in THF (20 mL). The mixture was allowed to stir at
room temperature for 6 h; then the precipitate (NEt3‚HBr) was
removed by filtration. The solvent was removed in vacuo, and
the residue was dissolved in Et2O (15 mL), washed with H2O
(3 × 15 mL), and dried (MgSO4). After removal of the solvent,
the residue was recrystallized from benzene:hexane (5:1) to
afford 1.00 g (33%) of N-(2-bromoethyl)-3-chlorobenzamide (mp
82-84 °C) as an intermediate which was used in the next step
without further characterization.
Potassium phthalimide (0.70 g, 3.8 mmol) was added to a
solution of the above intermediate bromide (1.00 g, 3.8 mmol)
in dry DMF (3 mL). The reaction was slightly exothermic, with
the temperature rising to 80 °C. Stirring was allowed to
continue for 12 h, and the temperature was slowly decreased
to 25 °C. After the addition of CHCl3 (20 mL), the mixture
was poured into H2O (100 mL). The aqueous phase was
separated and extracted with CHCl3 (2 × 10 mL). The
combined extract was washed with 0.2 N NaOH (20 mL) and
H2O (20 mL) and dried (Na2SO4). The solvent was removed
in vacuo, and the white crystalline residue was triturated with
Et2O (30 mL) to give 0.50 g (40%) of N-(phthalimidoethyl)-3-
chlorobenzamide (mp 165-168 °C).
Hydrazine hydrate (0.08 g, 1.5 mmol) was added to a
solution of the above phthalimide (0.50 g, 1.5 mmol) in 95%
EtOH (10 mL). The reaction mixture was heated at reflux
for 3 h and then cooled to 0 °C (ice bath). The cold solution
was acidified to pH 1-2 with ice-cold concentrated HCl and
filtered. The solid was washed with 95% EtOH (3 × 5 mL).
Evaporation of the combined filtrate and washings gave 0.05
g (16%) of compound 37: mp 205-207 °C, after recrystalliza-
tion from absolute EtOH. Anal. (C9H11N2ClO‚HCl) C,H,N.
4-(3-Ch lor op h en yl)bu tyla m in e F u m a r a te (39). The
method was adapted from 44. 3-(3-Chlorophenyl)propyl bro-
mide62 was converted to its nitrile and then reduced with
LiAlH4 and treated with an ethereal solution of fumaric acid
to afford 0.02 g (6%) of the title compound 39: mp 169-170
°C. Anal. (C10H14NCl‚C4H4O4) C,H,N.
3-(3-Ch lor op h en yl)p r op yla m in e Oxa la t e (44). 3-(3-
Chlorophenyl)ethanol (1.00 g, 6.4 mmol) in 48% HBr (10 mL)
and H2SO4 (3 drops) was heated at reflux for 3 h. The reaction
mixture was cooled to room temperature, poured into H2O (60
mL), extracted with CHCl3 (3 × 25 mL), and dried (MgSO4).
The solvent was removed in vacuo to afford 1.25 g (89%) of
3-(3-chlorophenyl)ethyl bromide as an oil. IR spectral data
indicated lack of an OH signal.
((CH2)2N+), 50.75 ((CH3)2N+), 42.00 ((CH2)2N). Anal. (C13H18
N2F3I) C,H,N.
-
1-(2-Meth yl-4-m eth oxyp h en yl)p ip er a zin e Hyd r och lo-
r id e (18). Anhydrous K2CO3 (4 g, 29 mmol) and bis(2-
chloroethyl)amine hydrochloride (5.2 g, 29 mmol) were added
to a stirred solution of freshly distilled 2-methyl-4-methoxy-
aniline (4 g, 29 mmol) in diglyme (25 mL) at room temperature.
The reaction mixture was heated at reflux for 18 h, cooled to
room temperature, and poured into H2O (100 mL). The
aqueous mixture was made basic (ca. pH 14) with a saturated
KOH solution and extracted with EtOAc (3 × 100 mL). The
combined organic extracts were washed with H2O (3 × 100
mL) and dried (MgSO4). The solvent was removed under
reduced pressure to give an oily residue. Vacuum distillation
afforded 3.6 g (60%) of 18 as a free base, bp 105 °C/0.05 mmHg.
Preparation of the hydrochloride salt gave 18 as white
crystals: mp 255 °C dec; 1H-NMR (CDCl3) δ 2.15 (s, 1H, NH),
2.30 (s, 3H, Ar-CH3), 2.55-3.30 (m, 8H, N-CH2), 3.75 (s, 3H,
OCH3), 6.8 (d, 2H, J ) 9.0 Hz, Ar-H), 7.10 (d, 1H, J ) 9.0 Hz,
Ar-H). Anal. (C12H18N2O.HCl) C,H,N.
6-Ch lor o-2-[1-(4-m eth ylp ip er a zin yl)]p yr id in e Ma lea te
(24). A solution of 2,6-dichloropyridine (1.5 g, 10.1 mmol),
N-methylpiperazine (1.0 g, 10 mmol), and NEt3 (1.0 g, 10
mmol) in n-BuOH (100 mL) was heated at reflux for 20 h and
allowed to cool to room temperature. The NEt3‚HCl was
removed by filtration, and the filtrate was evaporated to
dryness under reduced pressure. The oily residue was dis-
solved in Et2O (100 mL), and the solution was washed well
with H2O (3 × 50 mL) and dried (MgSO4). The filtrate was
treated with a methanolic solution of maleic acid; the precipi-
tate was collected by filtration and recrystallized from absolute
EtOH to give 2.0 g (62%) of the crystalline maleate salt: mp
144-146 °C. Anal. (C10H14ClN3‚C4H4O4) C,H,N.
6-Meth yl-2-[1-(4-m eth ylp ip er a zin yl)]p yr id in e Ma lea te
(25). Compound 25 was obtained by alkylation of N-meth-
ylpiperazine with 6-chloro-2-picoline in a manner similar to
that described for 24. The product was recrystallized from
absolute EtOH to yield 0.12 g (49%) of 25: mp 140-141 °C.
Anal. (C11H17N3‚C4H4O4) C,H,N.
6-Iodo-2-[1-(4-m eth ylpiper azin yl)]pyr idin e Maleate (26).
A mixture of 2,6-diiodopyridine (0.32 g, 1 mmol), N-methylpip-
erazine (0.1 g, 1 mmol), and NEt3 (0.1 g, 1 mmol) in n-BuOH
(20 mL) was heated under reflux for 16 h; the solution was
allowed to cool to room temperature and filtered, and the
filtrate was evaporated to dryness under reduced pressure. The
solid residue was treated with dilute HCl (10%), and the
solution was again filtered. The filtrate was made alkaline
to pH 8-9 with NaOH and extracted with Et2O (3 × 15 mL).
The combined Et2O extracts were dried (MgSO4), filtered, and
treated with a saturated solution of maleic acid in Et2O. The
precipitate was collected by filtration, dried, and recrystallized
from absolute EtOH/anhydrous Et2O to give 0.1 g (35%) of 26
as a crystalline solid: mp 138-139 °C. Anal. (C10H14IN3‚C4-
H4O4) C,H,N.
The above intermediate (1.25 g, 5.7 mmol) in MeOH (10 mL)
was added at once to a solution of NaCN (0.56 g, 11.3 mmol)
in H2O (1 mL). The reaction mixture was heated at gentle
reflux (water bath) for 30 min; then the solvent was removed
in vacuo, and the residue was dissolved in Et2O (5 mL). The
precipitate was removed by filtration, and the filtrate was
evaporated to dryness to give 0.55 g (58%) of 3-(3-chlorophen-
yl)ethyl nitrile as an oily intermediate. IR spectroscopy
indicated the presence of a CN signal at 2242 cm-1
.
The above nitrile (0.17 g, 10 mmol) in dry THF (5 mL) was
added in a dropwise manner to a stirred and cooled (ice bath)
suspension of LiAlH4 (0.05 g, 13 mmol) in dry THF (3 mL)
under an N2 atmosphere. After the addition was complete,
the reaction mixture was heated at reflux for 2 h. Excess
LiAlH4 was decomposed by the addition of H2O (0.2 mL), 15%
NaOH (0.2 mL), and H2O (0.6 mL), and the inorganic material
was removed by filtration. The solution was dried (MgSO4),
and THF was removed under reduced pressure to afford an
oily residue of the title compound. The crude product was
purified by Kugelrohr distillation to afford 0.07 g (41%) of 44
N,N-Dim eth yl-(3-ch lor op h en yl)bigu a n id e Hyd r och lo-
r id e (34). A mixture of 3-chloroaniline hydrochloride (1.97
g, 12 mmol) and dimethyl dicyandiamide (1.37 g, 12 mmol) in
H2O (10 mL) was heated at reflux for 2 h and filtered while
hot. After cooling in an ice bath, the aqueous solution was
allowed to stand at 5 °C overnight. The white precipitated
product was collected by filtration. Recrystallization from

SAR for Binding at 5-HT3 Serotonin Receptors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 20 4025
as a free base. The free base was converted to its oxalate salt
and recrystallized from absolute EtOH: mp 148-152 °C.
Anal. (C9H12ClN‚C2H2O4‚0.5 H2O) C,H,N.
scintillation counter in 5 mL of aqueous counting scintillant
(Ecoscint, National Diagnostic). Data from binding assays
were plotted as log concentration vs percent inhibition and
analyzed by nonlinear least squares techniques in which 100%
maximal inhibition was assumed at high test compound
concentrations. The IC50 values obtained from such data
treatment were used to calculate apparent inhibition constants
from the following equation: Ki ) IC50/1 + ([c]/KD), where [c]
is the concentration of radioligand employed in the binding
assay and KD is its receptor dissociation constant (KD ) 0.7
nM for [3H]GR65630).
2. von Bezold -J a r isch Br a d yca r d ia Reflex in Ra ts.
White albino Harlan rats (200-300 g) were anesthetized with
ethylurethane (1.5 g/kg, ip). The trachea was cannulated, and
when necessary, artificial ventilation was provided. A tail vein
was cannulated with a no. 25 “butterfly ST” needle (Abbott
Laboratories, N. Chicago, IL) for iv injection. Needle (no. 25)
electrodes were inserted subcutaneously to record ECG (Mod-
ule AEC 3101, Western Graphtec (WG) Co., Irvine, CA). Lead
II ECG signals were coupled with a Cardiotachograph (KF
3101-02 module), and responses were recorded on a WG
Linearcorder WR 3101 polygraph. A constant body temper-
ature was maintained. Agents were dissolved in normal
saline, and stock solutions were stored under refrigerated
conditions; appropriate dilutions were made prior to use.
Agents were injected in dose ranges of 1-200 µg/kg in a total
volume of 0.3 mL and “washed in” with an additional 0.4 mL
of normal saline. The reflex bradycardic responses were
elicited at 15-20-min intervals. Minimal effective doses of the
agents (producing 10-20% bradycardia) were determined by
interpolation between two to three doses for each agent. To
identify the bradycardic response with 5-HT3 receptor stimula-
tion, each stimulant agent was tested at least once at 2 and
20 min after the administration of the 5-HT3 antagonist
tropisetron (5-10 µg/kg, iv).
3. Ra bbit Bla d d er Exp er im en ts. White male adult New
Zealand rabbits (1.8-2.5 kg) were anesthetized with 1.2 g/kg
of ethylurethane (ip) and, when necessary, supplemented with
sodium pentobarbital (10-20 mg/kg) injected into a marginal
ear vein. The trachea was cannulated for artificial ventilation.
The bladder was exposed from a lower midline incision of the
abdomen, and a no. 12 gauge plastic catheter was inserted into
the apex of the bladder. The urethra was clamped off.
Pressure changes of the bladder were monitored via a Gould
P 2310 pressure transducer with a WG, AUT 3510 pressure
module on a WG polygraph. Agents were injected with close
intraarterial administration into the cannulated femoral artery
in the 1-200 µg/kg dose range in each animal at 15-20-min
intervals. Minimal effective doses producing twitchlike con-
tractions were determined as described earlier in dog and cat
preparations.64 To identify the bladder contractions as being
5-HT3 receptor mediated, tropisetron (5-10 µg/kg) was ad-
ministered intraarterially 1 min prior to the administration
of an effective dose of each stimulant agent to produce complete
blockade.
N-Met h yl-2-(3-ch lor op h en yl)et h yla m in e H yd r och lo-
r id e (55). Methyl chloroformate (0.31 g, 3.3 mmol) in dry THF
(5 mL) was added dropwise to a solution of 2-(3-chlorophenyl)-
ethylamine (0.50 g, 3.2 mmol) and NEt3 (0.66 g, 6.5 mmol) in
dry THF (5 mL) at 0 °C under an N2 atmosphere. The solution
was allowed to stir at 0 °C for 30 min and at room temperature
for 14 h. The NEt3‚HCl was removed by filtration, and the
solvent was removed in vacuo. The residue was dissolved in
H2O (15 mL) and then extracted with Et2O (3 × 10 mL). The
combined ethereal portions were washed with 10% HCl (15
mL), brine (15 mL), and H2O (20 mL), dried (K2CO3), and
evaporated to afford 0.65 g (96%) of N-carbomethoxy-2-(3-
chlorophenyl)ethylamine as an oil which was used in the next
step without further characterization.
A solution of the above N-carbomethoxy intermediate (0.45
g, 2.1 mmol) in dry THF (4 mL) was added in a dropwise
manner to a suspension of LiAlH4 (0.11 g, 2.7 mmol) in THF
(3 mL) at 0 °C under an N2 atmosphere. After the addition
was complete, the mixture was heated at reflux for 2 h. The
excess LiAlH4 was decomposed by the addition of H2O (0.5 mL),
15% NaOH (0.5 mL), and H2O (1.5 mL), and the inorganic
material was removed by filtration. The solution was dried
(MgSO4), and the THF was removed under reduced pressure
to afford 0.24 g (67%) of the title compound 55 as an oil. The
free base was converted to its HCl salt: mp 134-135 °C
(EtOH:Et2O). Anal. (C9H12ClN‚HCl) C,H,N.
N,N-Dim eth yl-2-(3-ch lor op h en yl)eth yla m in e Oxa la te
(56). Formic acid (88%, 0.69 g, 15 mmol), followed by
formaldehyde (36%, 0.39 g, 13 mmol), was slowly added to 2-(3-
chlorophenyl)ethylamine (0.78 g, 5 mmol) at 0 °C. The
resulting solution was stirred at 80 °C for 24 h, cooled to 0 °C,
and acidified with 6 N HCl (2 mL). The mixture was extracted
with Et2O (3 × 10 mL), basified to pH 10 by the addition of
50% aqueous NaOH, and extracted with Et2O (3 × 10 mL).
The pooled ether extracts were washed with H2O (5 mL) and
dried (MgSO4), and solvent was removed under reduced
pressure. The resulting residue was dissolved in EtOH:Et2O
(1:1) (2 mL) and treated with an ethereal solution of oxalic
acid to give 0.38 g (28%) of the title compound 56: mp 144-
146 °C. Anal. (C10H14ClN‚C2H2O4) C,H,N.
N-(3-P yr id yl)gu a n id in e Hyd r och lor id e (60). The title
compound was prepared by the general method of Short and
Darby.28 A mixture of 3-aminopyridine (0.47 g, 3.5 mmol) and
cyanamide (0.18 g, 4.3 mmol) in concentrated HCl (2 mL) was
heated in an oil bath to 180 °C over a 1-h period and held at
that temperature for 3 h. The residue was triturated with
MeOH:Et2O; then the solid was washed with acetone (to
remove unreacted amine) and recrystallized from EtOH:Et2O
to give 0.40 g (38%) of the title compound 60: mp 210-213
°C. Anal. (C6H8N4‚2HCl) C,H,N.
P h a r m a cology. 1. Ra d ioliga n d Bin d in g. NG 108-15
cells, which express the 5-HT3 receptor,63 were obtained from
Dr. Marshall Nirenberg (National Institutes of Health). The
cells were grown in Dulbecco’s modified Eagle’s medium with
fetal bovine serum (10%), penicillin/streptomycin (200 units/
mL), L-glutamine (2 nM/mL), hypoxanthine (25 µM), amino-
pterin (0.5 µM), and thymidine (4 µM), in cell culture flasks,
to confluence. The cells were harvested in 50 mM Tris HCl
buffer, 0.5 mM EDTA, and 10 nM MgSO4 and centrifuged at
9000g for 25 min. The pellet was resuspended in buffer,
incubated at 37 °C for 15 min, and centrifuged again at 9000g
for 20 min. The pellets were stored at -40 °C until used.
Radioligand binding assays were performed in triplicate in
a 2.0-mL volume containing 1 nM [3H]GR65630 (New England
Nuclear); 1 µM tropisetron (ICS 205-930) was used to define
nonspecific binding, with varying concentrations of competing
drug and membranes prepared from NG 108-15 cells (100 mg
of protein/mL). A 64% specific binding signal was produced
using 1 nM [3H]GR65630 (84.2 Ci/mmol). Assay tubes were
incubated for 30 min at room temperature; suspensions were
filtered on Schleicher & Schuell 32 glass fiber filters (presoaked
in 0.1% poly(ethylenimine)) and washed with 10 mL ice-cold
buffer. The filters were counted by a Beckman 3801 liquid
Ack n ow led gm en t. We would like to express our
appreciation to the Egyptian Channel Program that
supported A.A.A.-R., to R. M. Slusher and A. Damoda-
ran who assisted with some of the syntheses, and to N.
Nguyen and J . Chiang who assisted with the in vivo
pharmacological assays.
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