Isoindol-1-one analogues of 4-(2'-methoxyphenyl)-1-[2'-[N-(2'-pyridyl)- p-iodobenzamido]ethyl]piperazine (p-MPPI) as 5-HT1(A) receptor ligands

Article abstract of DOI:10.1021/jm970296s

In developing radioiodinated antagonists for in vivo imaging of 5- HT(1A) receptors with SPECT, a series of new arylpiperazine benzamido derivatives, including 4-(2'-methoxyphenyl)-l-[2'-[N-(2_-pyridyl)-p- iodobenzamido]ethyl]piperazine (p-MPPI, 31) (K(d) = 0.36 nM), as potential ligands for 5-HT₁(A) receptors were reported previously. However, rapid in vivo metabolism may have caused the breakdown of the amide bond of [¹²³I]- 31 and rendered this agent obsolete as an in vivo imaging agent in humans. To improve the in vivo stability of 31, a series of cyclized amide analogues were designed and synthesized. In vitro binding, metabolic stability, and in vivo biodistribution of these new derivatives were investigated. Several five-membered-ring isoindol-1-ones displayed very high in vitro binding affinity, especially 2-{2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl}-6- nitro-3-phenyl-2,3-dihydroisoindol-1-one, 15, 3-hydroxy-6-iodo-2-{2-[4-(2- methoxyphenyl)piperazin-1-yl]ethyl}-3-phenyl-2,3-dihydroisoindol-1-one, 18, and 6-iodo-2-{2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl)}-3-phenyl-2,3- dihydroisoindol-1-one, 21, which showed K(i) values of 0.05, 0.65, and 0.07 nM, respectively. The affinities for 5-HT₁(A) receptors of other cyclized amide derivatives, 5-(4-bromophenyl)-1-{2-[4-(2-methoxyphenyl)piperazin-1- yl]ethyl}pyrrolidin-2-one, 25, 5-(4-iodophenyl)-1,{2-[4-(2- methoxyphenyl)piperazin-1-yl]ethyl}pyrrolidin-2-one, 27, and 2-{2-[4-(2- methoxyphenyl)piperazin-1-yl]ethyl}-2,3-dihydroisoindol-1-one, 29, were 1.09, 2.54, and 14.9 nM, respectively. Compared to [¹²⁵I]-31, iodinated cyclized amide derivatives [¹²⁵I]-21 and [¹²⁵I]-27 displayed a slower metabolism in human liver microsomal and cytosolic preparations. Biodistribution of [¹²⁵I]-21 and [¹²⁵I]-27 in rats (after an iv injection) displayed moderate to low brain uptakes with little or no specific localization in hippocampal region, where 5-HT₁(A) receptors are concentrated. These data indicate that the new iodinated ligands showed high binding affinities and better metabolic stability but displayed unexpectedly low selective binding to 5-HT₁(A) receptors in vivo. Additional structural modifications may be needed to correct the unfavorable properties displayed for these iodinated cyclized amide derivatives for in vivo biodistribution in rats.

Full text of DOI:10.1021/jm970296s

J . Med. Chem. 1998, 41, 157-166
157
Isoin d ol-1-on e An a logu es of
4-(2′-m eth oxyp h en yl)-1-[2′-[N-(2′′-p yr id yl)-p-iod oben za m id o]eth yl]p ip er a zin e
(p-MP P I) a s 5-HT1A Recep tor Liga n d s
Zhi-Ping Zhuang,† Mei-Ping Kung,† Mu Mu,† and Hank F. Kung*,†,‡
Departments of Radiology and Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania 19104
Received May 6, 1997
In developing radioiodinated antagonists for in vivo imaging of 5-HT1A receptors with SPECT,
a series of new arylpiperazine benzamido derivatives, including 4-(2′-methoxyphenyl)-1-[2′-
[N-(2′′-pyridyl)-p-iodobenzamido]ethyl]piperazine (p-MPPI, 31) (Kd ) 0.36 nM), as potential
ligands for 5-HT1A receptors were reported previously. However, rapid in vivo metabolism
may have caused the breakdown of the amide bond of [123I]-31 and rendered this agent obsolete
as an in vivo imaging agent in humans. To improve the in vivo stability of 31, a series of
cyclized amide analogues were designed and synthesized. In vitro binding, metabolic stability,
and in vivo biodistribution of these new derivatives were investigated. Several five-membered-
ring isoindol-1-ones displayed very high in vitro binding affinity, especially 2-{2-[4-(2-
methoxyphenyl)piperazin-1-yl]ethyl}-6-nitro-3-phenyl-2,3-dihydroisoindol-1-one, 15, 3-hydroxy-
6-iodo-2-{2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl}-3-phenyl-2,3-dihydroisoindol-1-one, 18,
and 6-iodo-2-{2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl}-3-phenyl-2,3-dihydroisoindol-1-one,
21, which showed Ki values of 0.05, 0.65, and 0.07 nM, respectively. The affinities for 5-HT1A
receptors of other cyclized amide derivatives, 5-(4-bromophenyl)-1-{2-[4-(2-methoxyphenyl)-
piperazin-1-yl]ethyl}pyrrolidin-2-one, 25, 5-(4-iodophenyl)-1-{2-[4-(2-methoxyphenyl)piperazin-
1-yl]ethyl}pyrrolidin-2-one, 27, and 2-{2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl}-2,3-dihydro-
isoindol-1-one, 29, were 1.09, 2.54, and 14.9 nM, respectively. Compared to [125I]-31, iodinated
cyclized amide derivatives [125I]-21 and [125I]-27 displayed a slower metabolism in human liver
microsomal and cytosolic preparations. Biodistribution of [125I]-21 and [125I]-27 in rats (after
an iv injection) displayed moderate to low brain uptakes with little or no specific localization
in hippocampal region, where 5-HT1A receptors are concentrated. These data indicate that
the new iodinated ligands showed high binding affinities and better metabolic stability but
displayed unexpectedly low selective binding to 5-HT1A receptors in vivo. Additional structural
modifications may be needed to correct the unfavorable properties displayed for these iodinated
cyclized amide derivatives for in vivo biodistribution in rats.
In tr od u ction
(negatively coupled to forskolin-stimulated adenylyl
cyclase activity) and in vivo (hypothermia and serotonin
behavior syndromes such as rapid jaw movements and
forepaw treading).6-9
Serotonin receptors play important roles in normal
brain function. One of the serotonin receptor subtypes,
5-HT1A, plays an important function as the somatoden-
dritic autoreceptor (presynaptic) in the dorsal raphe
nucleus and as a postsynaptic receptor for 5-HT in
terminal areas.1 Buspirone and ipsapirone, which are
agonists and display high affinity to 5-HT1A receptors
(IC50 ) 60 and 35 nM, respectively), are presently being
used as antianxiety agents.2,3 It is potentially useful
to develop ligands that are selective for the 5-HT1A
subtype to facilitate the study and characterization of
this receptor.4,5 The most commonly used ligand, 8-OH-
DPAT (8-hydroxy-2-(N,N-di-n-propylamino)tetralin), is
a potent 5-HT1A agonist, and the tritium-labeled com-
pound is the ligand of choice for 5-HT1A receptor binding
studies (Kd ) 0.5 nM, rat hippocampal homogenates).
It produces full 5-HT1A receptor agonist activity in vitro
Several early front-runners of pure antagonists are
arylpiperazine derivatives such as NAN-190, which
displays high affinity for 5-HT1A receptors (Ki ) 0.6 nM),
but also has a high potency for the R1 receptor.10-12 In
addition, NAN-190 displays partial agonist-like activity
in a radioligand binding assay.13-15 BMY7378 appears
to exhibit the same partial agonist property.10 Another
* Address for correspondence: Hank F. Kung, Ph.D., Departments
of Radiology and Pharmacology, University of Pennsylvania, 3700
Market Street, Room 305, Philadelphia, PA 19104. Tel: (215) 662-
3096. Fax: (215) 349-5035. E-mail: kunghf@sunmac.spect.upenn.edu.
† Department of Radiology.
‡ Department of Pharmacology.
S0022-2623(97)00296-3 CCC: $15.00 © 1998 American Chemical Society
Published on Web 01/15/1998

158 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 2
Zhuang et al.
Sch em e 1
Sch em e 2
arylpiperazine, WAY100135, was recently reported.16,17
However, it was quickly surpassed by a similar com-
pound, WAY100635, which displayed a higher binding
affinity (Kd ) 0.10 nM) and better selectivity18,19 while
exhibiting no partial agonist property.20,21 Recently,
newer versions of arylpiperazines as 5-HT1A receptor
ligands have been reported.22 To develop radioiodinated
ligands for in vivo imaging of 5-HT1A receptors, we have
previously reported a series of new arylpiperazine
benzamido derivatives, including the antagonist ligand,
31,23 and the iodinated agonist ligand, 8-OH-PIPAT.24
Results of in vitro binding and adenylyl cyclase assays
for 31 demonstrated that it is an excellent “pure”
antagonist with high affinity (Kd ) 0.36 nM) and
selectivity for 5-HT1A receptors.25 Evaluation of the in
vivo properties of 31 and the related p-fluoro derivative,
p-MPPF, in rats clearly demonstrated that they act as
5-HT1A receptor antagonists.26,27 The novel iodinated
compound, 31, is the first selective 5-HT1A receptor
antagonist which can be labeled with iodine-125 and
iodine-123. The 125I-labeled ligand is a useful probe for
pharmacological characterization for 5-HT1A receptors,
whereas the 123I-labeled ligand is potentially useful for
in vivo imaging of 5-HT1A receptors by single photon
emission computed tomography (SPECT).
contrast, when using ketanserin, a selective 5-HT2
receptor antagonist, as the chasing agent, SPECT
images displayed no in vivo displacement of activity
localized in hippocampus of monkey brain. The data
strongly suggest that in vivo hippocampal binding of
[
123I]-31 is selective to 5-HT1A receptors. Surprisingly,
preliminary studies in humans with [123I]-31 displayed
low brain uptake after 20 min post iv injection.36 Initial
SPECT images (0-20 min) were close to those of brain
perfusion images. It is likely that the low brain uptake
observed in the human subject could be due to a rapid
metabolism (amide hydrolysis), whereas in other species
(i.e., rats and monkeys), the metabolic pathway (cleav-
age of amide bond by liver enzymes) appears to be much
slower.37 To improve the in vivo stability and bioavail-
ability, isoindol-1-one and other cyclic amides were
prepared. The rationale for choosing this ring system
is that the cyclized amide bond may extend in vivo
stability to allow delivery of the agent into specific brain
regions for receptor-directed binding without extensive
in vivo degradation. The synthesis, in vitro binding,
metabolic stability, and in vivo biodistribution studies
of these new ligands are reported herein.
Ch em istr y
Recent reports have indicated that [3H]- or [11C]-
WAY100635, a close analogue of 31, displayed good in
vivo biodistribution properties suitable for imaging with
positron emission tomography (PET).28-34 Regional
distribution of 5-HT1A receptors in normal human brain
has been reported.8,30,35 As expected, [11C]WAY100635
exhibited high density in the hippocampal region. A
SPECT study carried out in monkeys after an iv
injection of [123I]-31 clearly indicated selective brain
uptake in the frontal cortex, cingulate gyrus, hippocam-
pus, and entorhinal cortex, regions with high 5-HT1A
receptor density.36 In vivo displacement experiments
in monkey using SPECT demonstrated that hippocam-
pal localization is saturable with cold 31. The displace-
ment experiment showed the same competitive binding
using (()-8-OH-DPAT, a 5-HT1A receptor agonist. On
The synthesis of a group of isoindol-1-ones, cyclic
amide analogues of 4-(2′-methoxyphenyl)-1-[2′-[N-(2′′-
pyridyl)-p-iodobenzamido]ethyl]piperazine (p-MPPI, 31),
as 5-HT1A receptor ligands was achieved by reactions
shown in Schemes 1-5. Commercially available phenan-
thridinone 4 and bromide 3 were coupled in the presence
of NaH to produce compound 5 in moderate yield (45%).
Alternatively, a reductive condensation reaction was
carried out between γ-keto acid 1238 or 1439 or γ-keto
ester 9, 24, or 28 and the amine 740 using a HOAc-
MeOH, NaCNBH3 system or under a refluxing condition
in benzene with a catalytic amount of p-TsOH followed
by NaBH4 as the reducing agent. When the nitro
derivative of benzoylbenzoate methyl ester, 14, was
condensed with amine 7 under the same reductive
amination condition, in addition to the expected fused

Cyclic Amides as 5-HT1A Receptor Ligands
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 2 159
Sch em e 3
Sch em e 4
Ta ble 1. Comparison of Inhibition Constants, Ki (nM), of
ring derivative 16 (8% yield), a second product, unde-
hydrated compound 15, was obtained (29%). This side
product persisted even after the reaction was refluxed
for 3 days. Compound 16 was reduced by a hydrogena-
tion reaction in the presence of Pd/C as the catalyst,
under which the 6-nitro group was successfully con-
verted to a 6-amino group (compound 17). The diazo-
tization reaction converted the 6-amino to 6-iodo to give
compound 18 (yield 16%). This reaction gave an unex-
pected product, 19 (yield 39%), on which an extra nitro
group was added on the 2-methoxyphenyl ring. This
side product probably was produced due to the nitration
reaction derived by an oxidation of sodium nitrite under
the reaction condition. Using the similar reaction
sequence, the desired product compound 21 (yield 19%)
was prepared, while the yield for the side product with
an extra nitro group, compound 22, was 41%.
The bromo compound, 25, which was obtained by
reductive amination with γ-keto ester 24 and amine 7,
was converted to the iodo compound 27 via the tin
intermediate formed through normal Pd-catalyzed reac-
tion. Similarly, 2-(methoxycarbonyl)benzaldehyde re-
acted with amine 7 gave another fused ring compound
29. All of the reactions were not optimized, and in
general they gave poor to moderate yields.
5-HT1A Receptor Ligandsa
compd
Ki (nM)
compd
Ki (nM)
5
10
13
15
16
17
18
8.9 ( 1.20
19
21
22
25
27
29
31
9.3 ( 0.83
0.069 ( 0.006
51.7 ( 16
1.09 ( 0.13
2.54 ( 0.59
14.9 ( 0.40
0.2 ( 0.05
0.5 ( 0.80
6.5 ( 0.94
0.045 ( 0.021
0.34 ( 0.03
1.85 ( 1.06
0.65 ( 0.13
a
Specific inhibition of [125I]-31 binding to rat hippocampal
membrane preparations. Values are the mean ( SD of three
independent experiments, each performed in duplicate.
receptors, comparable to or exceeding that of the parent
compound, 31. 2-{2-[4-(2-Methoxyphenyl)piperazin-1-
yl]ethyl}-6-nitro-3-phenyl-2,3-dihydroisoindol-1-one, 15,
3-hydroxy-6-iodo-2-{2-[4-(2-methoxyphenyl)piperazin-1-
yl]ethyl}-3-phenyl-2,3-dihydroisoindol-1-one, 18, and
6-iodo-2-{2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl}-
3-phenyl-2,3-dihydroisoindol-1-one, 21, showed Ki val-
ues of 0.05, 0.65, and 0.07 nM, respectively (Table 1).
The affinities of other cyclized amide derivatives, 5-(4-
bromophenyl)-1-{2-[4-(2-methoxyphenyl)piperazin-1-yl]-
ethyl}pyrrolidin-2-one, 25, 5-(4-iodophenyl)-1-{2-[4-(2-
methoxyphenyl)piperazin-1-yl]ethyl}pyrrolidin-2-one, 27,
and 2-{2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl}-2,3-
dihydroisoindol-1-one, 29, were 1.09, 2.54, and 14.9 nM,
respectively. The in vitro binding data strongly sug-
gested that the formation of a cyclic amide ring system
Biologica l Resu lts
Several isoindol-1-ones containing a cyclic amide ring
displayed very high in vitro binding affinity for 5-HT1A

160 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 2
Zhuang et al.
Sch em e 5
(isoindol-1-one) was acceptable in maintaining the
ligand affinity to 5-HT1A receptors.
fractions (approximately 40% degradation in the pres-
ence of 1.3 mg of cytosol protein; Figure 1). Unlike [125I]-
31, [125I]-21 and [125I]-27 were not metabolized signifi-
cantly by the same human liver microsomal or cytosolic
preparations. The results, demonstrating an increased
metabolic stability for cyclized amide derivatives, are
consistent with the observation reported for the steri-
cally hindered derivatives of WAY100635.37,41 In a
control experiment using rat or monkey liver microso-
mal preparations, [125I]-31 displayed little decomposition
after in vitro incubation (unpublished results, data not
shown).
Despite high in vitro binding affinity and better
metabolic stability, the biodistribution of [125I]-21 in rats
(after an iv injection) displayed a lower brain uptake
than that observed for the parent compound [125I]-31
(Table 2). The brain uptake (percentage dose/organ)
was 0.26, 0.11, and 0.04 for [125I]-21 as compared to 1.22,
0.22, and 0.12 for [125I]-31 at 2, 30, and 60 min,
respectively. The data on regional brain uptake indi-
cated that the compound, [125I]-21, did not show any
specific uptake in the hippocampal region, which con-
tains a high density of 5-HT1A receptors (hippocampus/
cerebellum ratios were 0.87, 1.12, and 1.06 vs 1.35, 3.28,
and 3.17 for [125I]-21 and [125I]-31, respectively). The
ligand appears to be more lipophilic than [125I]-31 (PC
) 1800 ( 300 and 2400 ( 400 for [125I]-31 and [125I]-
21, respectively, and the HPLC retention times were
7.6 and 11.2 min for [125I]-31 and [125I]-21, respectively,
on the reversed-phase PRP-1 column). The high lipo-
philicity of [125I]-21 was further evidenced by the high
nonspecific binding (∼30% around the Kd value) in the
Two cyclized amide ligands, 21 and 27, which contain
the iodo group on the molecules, were subjected for
radiolabeling and further in vitro and in vivo evalua-
tions. Radioiodinated compounds [125I]-21 and [125I]-
27 were successfully prepared using the corresponding
tributyltin intermediates, and the products were suc-
cessfully purified by HPLC (yield 60-80%, radiochemi-
cal purity >95%). The identities of the ligands were
confirmed with the cold standards on HPLC with
similar retention times (tR ) 11.2 and 6.4 min for [125I]-
21 and [125I]-27, respectively) with an isocratic solvent
system of CH3CN:5 mM DMGA (pH 7.4), 90:10, eluting
at a flow rate of 1 mL/min.
The studies carried out for [11C]WAY100635, a po-
tential PET ligand for imaging 5-HT1A receptors, clearly
indicated rapid metabolism of this tracer (amide hy-
drolysis) by human liver (but not by rat liver).37,41 As
an analogue of WAY100635, [125I]-31 may encounter a
rapid degradation by human liver enzymes, which was
evidenced by the successful signal and imaging for
5-HT1A receptors in rats and monkeys but not in
humans.36,42 To improve in vivo metabolic stability of
the ligands, we synthesized a series of cyclized amide
derivatives as shown in the present study. These
cyclized amide derivatives together with 31 were tested
for their metabolic stability using a metabolic model
system derived from human liver. [125I]-31, as expected,
was metabolized rapidly by both human liver microso-
mal fraction (>60% degradation in the presence of 0.75
mg of microsomal protein) and by human liver cytosolic

Cyclic Amides as 5-HT1A Receptor Ligands
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 2 161
Ta ble 2. Biodistribution of [125I]-21 and [125I]-27 in Rats after iv Injectiona
[
125I]-21
[
125I]-27
organb or
regionc
2 min
30 min
60 min
2 min
30 min
60 min
blood
2.84 ( 0.07
1.42 ( 0.08
9.45 ( 0.73
2.05 ( 0.26
4.01 ( 0.24
0.26 ( 0.07
21.36 ( 2.4
3.66 ( 0.54
0.073 ( 0.003
0.26 ( 0.01
(1.22 ( 0.22)
1.92 ( 0.36
0.30 ( 0.06
14.35 ( 1.71
0.70 ( 0.05
2.32 ( 0.01
0.22 ( 0.03
18.65 ( 1.69
5.80 ( 0.21
0.05 ( 0.009
0.11 ( 0.001
(0.22 ( 0.01)
1.22 ( 0.04
0.15 ( 0.0001
8.83 ( 1.94
0.56 ( 0.01
1.42 ( 0.16
0.15 ( 0.02
10.76 ( 0.28
4.96 ( 0.99
0.06 ( 0.003
0.04 ( 0.007
(0.12 ( 0.005)
5.60 ( 0.35
0.77 ( 0.04
14.07 ( 4.77
1.54 ( 0.09
3.02 ( 0.12
0.27 ( 0.01
19.49 ( 3.79
6.45 ( 1.45
0.06 ( 0.01
0.71 ( 0.02
3.45 ( 0.28
0.33 ( 0.03
19.08 ( 9.13
0.75 ( 0.04
2.20 ( 0.32
0.22 ( 0.001
23.02 ( 2.23
7.41 ( 1.80
0.04 ( 0.01
0.21 ( 0.009
2.08 ( 0.42
0.18 ( 0.04
9.05 ( 1.00
0.42 ( 0.06
1.00 ( 0.24
0.15 ( 0.03
17.95 ( 2.54
6.93 ( 0.06
0.03 ( 0.005
0.08 ( 0.003
heart
muscle
lung
kidney
spleen
liver
skin
thyroid
brain
brain
cerebellum
striatum
hippocampus
cortex
0.202 ( 0.006
0.204 ( 0.009
0.177 ( 0.011
0.242 ( 0.012
0.87
0.080 ( 0.011
0.093 ( 0.007
0.090 ( 0.005
0.081 ( 0.003
1.12
0.035 ( 0.005
0.035 ( 0.002
0.037 ( 0.005
0.028 ( 0.004
1.06
0.46 ( 0.04
0.55 ( 0.006
0.47 ( 0.018
0.57 ( 0.009
1.02
0.12 ( 0.003
0.15 ( 0.009
0.13 ( 0.004
0.14 ( 0.002
1.08
0.051 ( 0.007
0.053 ( 0.008
0.045 ( 0.013
0.048 ( 0.007
0.88
HP /CB
HP /CB
(1.35)
(3.28)
(3.17)
a
b
Values for previously reported [125I]-31 were listed in parentheses for comparison. Percentage dose/organ, average of three rats (
SD. c Regional distribution (percentage dose/g).
philicity and the high nonspecific binding of this ligand
may lead to the low specific localization in brain regions
such as the hippocampus, where 5-HT1A receptors are
concentrated.
Selective in vitro autoradiographic localization of
[
125I]-21 in hippocampal and cortical regions where
5-HT1A receptors are concentrated was, however, clearly
illustrated (Figure 2). The background level (nonspecific
binding due to high lipophilicity) of section labeling of
[
125I]-21 was higher than that of [125I]-31 under the same
conditions. In addition, it required a longer time period
(18 h in ice) to wash out the nonspecific binding of the
ligand from the sections compared to the short interval
of 15 min that was required for [125I]-31. This is an
additional line of evidence suggesting that the high
lipophilicity may lead to high nonspecific binding, even
under conditions in which the blood-brain barrier is
not a factor.
Another iodinated and cyclized amide analogue, [125I]-
27, was similarly evaluated for its in vitro and in vivo
binding properties. The Scatchard analysis of the
saturation experiment with [125I]-27 in rat hippocampal
homogenates resulted in a Kd value of 1.0 nM (data not
shown), which is comparable to the Ki value obtained
with the unlabeled 27 (Ki ) 2.54 nM; see Table 1).
However, [125I]-27 also displayed comparable in vitro
binding to dopamine D2 receptors (Kd ) 1.7 nM, data
not shown) which could confound the detection of 5-HT1A
receptors in vivo. Initial brain uptake of [125I]-27 (0.71%
dose/organ at 2 min postinjection) was comparable to
the value obtained with [125I]-31. However, because of
the lack of selective localization in the rat hippocampal
region in vivo, this cyclized amide analogue, [125I]-27,
is not useful as an in vivo imaging agent.
In conclusion, a new series of isoindol-1-ones ana-
logues of p-MPPI, 31 (4-(2′-methoxyphenyl)-1-[2′-[N-(2′′-
pyridyl)-p-iodobenzamido]ethyl]piperazine), as potential
ligands for 5-HT1A receptors was reported. Several new
novel ligands, 15, 18, 21, and 27, displayed high binding
affinity to 5-HT1A receptors as measured by in vitro
binding study. Among these cyclized amide derivatives,
two iodinated ligands, 21 and 27, showed an improved
metabolic stability over that of 31 using the human liver
enzyme model system. However, in vivo biodistribution
F igu r e 1. Metabolism of p-MPPI, 31, and analogues 21 and
27 by human liver microsomal and cytosolic preparations. The
incubations time was 10 min at 37 °C, and the concentrations
of the radioligands used were between 5 and 6 nM.
saturation experiment in vitro (Kd ) 0.2 nM, data not
shown). Consequently, it is likely that the high lipo-

162 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 2
Zhuang et al.
F igu r e 2. In vitr o a u tor a d iogr a p h y sh ow in g [125I]-21 loca lized in 5-HT1A con cen tr a ted a r ea s su ch a s cor tex (CX),
la ter a l sep ta l n u cleu s (LSD), d en ta te gyr u s (DG), a n d CA1, CA2 of h ip p oca m p a l r egion s.
studies of [125I]-21 and [125I]-27 in rats after an iv
injection suggested that these compounds did not local-
added. To the solution was added NaBH4 (100 mg) in solid
form at 0 °C. The mixture was stirred at room temperature
for 1 h. MeOH was removed, and water was added. The
ize in the target area of the brain (hippocampus) where
aqueous phase was extracted with CH2Cl2. The combined
the 5-HT1A receptor concentration is high. Further
organic phases were dried over Na2SO4, filtered, and concen-
structural modification is warranted to improve the in
vivo selectivity for this series of 5-HT1A receptor ligands.
trated to give a crude product which was purified by PTLC
(Hex/EtOAc, 1:1) to give 10 mg of desired product 10 (4.6%).
IR (film, νmax): 3048, 2935, 2815, 1695, 1611, 1592, 1469,
1237, 1023 cm-1
.
1H NMR (CDCl3, δ): 2.53-2.72 (7H, m,
Exp er im en ta l Section
NCH2, CONCHaHb), 3.02-3.09 (4H, m, NCH2), 3.84 (3H, s,
CH3O), 4.10 (1H, d, t, J ) 14.3, 5.7 Hz, CONCHaHb), 5.76 (1H,
s, NCHPh), 6.85 (1H, d, J ) 7.8 Hz, ArH), 6.90-7.00 (3H, m,
ArH), 7.13-7.46 (8H, m, ArH), 7.90 (1H, d, d, J ) 5.2, 2.0 Hz,
ArH). MS: m/ z 428 (M+ + 1), 279, 236, 205, 150, 124.
2-{2-[4-(2-Meth oxyp h en yl)p ip er a zin -1-yl]eth yl}-3-p yr i-
d in -2-yl-2,3-d ih yd r oisoin d ol-1-on e (13). To a solution of
2-[4-(2-methoxyphenyl)piperazinyl]ethylamine, 7 (118 mg, 0.5
mmol), and acetic acid (0.2 mL) in methanol (2 mL) was added
2-[2-(hydroxycarbonyl)benzoyl]pyridine, 12 (114 mg, 0.5 mmol),
followed by NaCNBH3 (32 mg, 0.5 mmol). The mixture was
stirred at room temperature for 3 days. Water was added.
The mixture was made basic with NaOH (1 M) and extracted
with CH2Cl2. The organic layers were dried and evaporated
to give a crude product that was purified by PTLC to give 48
mg of product 13 (22%).
Proton NMR spectra were obtained on Bruker AMX300 or
AMX500 spectrometers. The chemical shifts were reported in
ppm downfield from the tetramethylsilane standard. Infrared
spectra were recorded on Mattson Polaris FT-IR spectrometer.
Low- and high-resolution mass spectra were carried out on
the VG mass spectrometer (Model ZAB-E) and MS-50 with
NH3 and isobutane as gases at the Department of Chemistry,
University of Pennsylvania, and the Nebraska Center for Mass
Spectrometry. Elemental analyses were performed by Atlantic
Microlabs, Inc., Atlanta, GA, or by the Department of Chem-
istry, University of Pennsylvania.
5-{2-[4-(2-Met h oxyp h en yl)p ip er a zin -1-yl]et h yl}-5H -
p h en a n th r id in -6-on e (5). To a suspension of NaH (24 mg,
0.6 mmol, 60% dispersion in mineral oil) in a mixed solvent of
DMF (1 mL) and toluene (0.5 mL) was added phenanthridi-
none, 4 (100 mg, 0.5 mmol). The mixture was heated at 90
°C in an oil bath for 1 h. 2-[4-(2-Methoxyphenyl)piperazinyl]-
ethyl bromide, 3 (150 mg, 1 equiv), in DMF (1 mL) was added
dropwise, and the mixture was stirred at 90 °C for 4 h. Water
was added after the reaction mixture was cooled to room
temperature and extracted with methylene chloride. The
organic layer was dried over Na2SO4, filtered, and concentrated
to produce a crude product that was purified by PTLC (Hex/
EtOAc, 1:1) twice to give product 5 (94 mg, 45%).
IR (film, νmax): 3048, 2944, 2832, 1690, 1611, 1590, 1498,
1463, 1237, 1023 cm-1 1H NMR (CDCl3, δ): 2.53-2.94 (7H,
.
m, NCH2, CONCHaHb), 3.04-3.16 (4H, m, NCH2), 3.84 (3H,
s, CH3O), 4.23 (1H, d, t, J ) 14.4, 6.2 Hz, CONCHaHb), 5.95
(1H, s, NCHPh), 6.84 (1H, d, J ) 7.8 Hz, ArH), 6.88-7.04 (3H,
m, ArH), 7.21-7.35 (2H, m, ArH), 7.44-7.51 (2H, m, ArH),
7.63 (1H, d, t, J ) 7.68, 1.8 Hz, ArH), 7.71-7.93 (2H, m, ArH),
8.65 (1H, d, d, d, J ) 4.9, 1.7, 0.9 Hz, ArH). MS: m/ z 429
(M+ + 1), 205.
IR (film, cm-1): 3058, 2938, 2817, 1646, 1606, 1589, 1502,
1237, 1141 cm-1
.
1H NMR (CDCl3, δ): 2.84 (2H, t, J ) 7.9
2-{2-[4-(2-Meth oxyph en yl)piper azin -1-yl]eth yl}-6-n itr o-
3-p h en yl-2,3-d ih yd r oisoin d ol-1-on e (15) a n d 3-Hyd r oxy-
2-{2-[4-(2-m eth oxyp h en yl)p ip er a zin -1-yl]eth yl}-6-n itr o-
3-p h en yl-2,3-d ih yd r oisoin d ol-1-on e (16). To a mixture of
2-benzoyl-5-nitrobenzoic acid, 14 (1.36 g, 5 mmol), and 2-[4-
(2-methoxyphenyl)piperazinyl]ethylamine, 7 (1.18 g, 5 mmol),
in benzene (20 mL) was added p-toluenesulfonic acid (200 mg).
The mixture was stirred under reflux for 3 days. The solvent
was removed on vacuum, and the residue was dissolved in
MeOH (30 mL). NaBH4 (500 mg) was added in portions at 0
°C in an ice bath. The mixture was stirred at room temper-
ature for 1 h. MeOH was removed, and water was added. The
separated aqueous phase was extracted with CH2Cl2. The
combined organic phases were dried over Na2SO4 and concen-
trated to give a crude product which was purified by MPLC
(Hex/EtOAc, 1:1) to give 3-hydroxy-2-{2-[4-(2-methoxyphenyl)-
piperazin-1-yl]ethyl}-6-nitro-3-phenyl-2,3-dihydroisoindol-1-
one, 16 (690 mg, 29%), and 2-{2-[4-(2-methoxyphenyl)piperazin-
1-yl]ethyl}-6-nitro-3-phenyl-2,3-dihydroisoindol-1-one, 15 (187
mg, 8%).
Hz, CONCH2CH2), 2.90 (4H, s, br, NCH2), 3.17 (4H, s, br,
NCH2), 3.87 (3H, s, OCH3), 4.62 (2H, t, J ) 7.9 Hz, CONCH2-
CH2), 6.86 (1H, d, d, J ) 7.8, 1.4 Hz, ArH), 6.90-7.04 (3H, m,
ArH), 7.33 (1H, d, d, d, J ) 8.1, 6.4, 1.8 Hz, ArH), 7.51-7.62
(3H, m, ArH), 7.77 (1H, d, d, d, J ) 8.4, 7.2, 1.5 Hz, ArH),
8.30 (2H, t, d, J ) 8.1, 1.1 Hz, ArH), 8.55 (1H, d, d, J ) 8.0,
1.3 Hz, ArH). Anal. (C26H27N3O2‚1/2H2O) Calcd: C, 73.91; H,
6.69; N, 9.94. Found: C, 73.62; H, 6.47; N, 9.82.
2-{2-[4-(2-Meth oxyph en yl)piper azin -1-yl]eth yl}- 3-ph en -
yl-2,3-d ih yd r oisoin d ol-1-on e (10). To a solution of 2-ben-
zoylbenzoic acid, 8 (2.8 g, 12.4 mmol), in methanol (25 mL)
was added H2SO4 (concentrated, 1 mL) dropwise at room
temperature. The mixture was refluxed for 3 h. Methanol
was removed after cooling. Water was added, and the mixture
was extracted with ethyl acetate. The organic layer was
washed with saturated NaHCO3 solution followed by saturated
NaCl solution, dried, and evaporated to give 2.9 g of methyl
2-benzoylbenzoate, 9, which was used for the next reaction
without further purification.
To a mixture of 2-[4-(2-methoxyphenyl)piperazinyl]ethyl-
amine, 7 (118 mg, 0.5 mmol), and methyl 2-benzoylbenzoate,
9 (120 mg, 0.5 mmol), in benzene (10 mL) was added p-TsOH
(50 mg) in solid form. The mixture was refluxed for 3 days.
The solvent was removed after cooling, and MeOH (5 mL) was
3-Hyd r oxy-2-{2-[4-(2-m et h oxyp h en yl)p ip er a zin -1-yl]-
eth yl}-6-n itr o-3-p h en yl-2,3-d ih yd r oisoin d ol-1-on e (16). IR
(film, νmax): 2950, 2828, 1706, 1592, 1530, 1498, 1345, 1243
cm-1 1H NMR (CDCl3, δ): 2.46 (1H, d, d, J ) 12.6, 1.7 Hz,
.
CONCHaHb), 2.72-3.17 (10H, m, NCH2), 3.86 (3H, s, CH3O),

Cyclic Amides as 5-HT1A Receptor Ligands
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 2 163
4.22 (1H, d, t, J ) 13.0, 2.4 Hz, CONCHaHb), 6.88 (1H, d, d, J
) 7.6, 1.2 Hz, ArH), 6.93-7.08 (3H, m, ArH), 7.33-7.46 (6H,
m, ArH), 8.33 (1H, d, d, J ) 8.3, 2.1 Hz, ArH), 8.67 (1H, d, d,
2,3-dihydroisoindol-1-one, 20, which was used to run the next
reaction without further purification.
To a mixture of 2-{2-[4-(2-methoxyphenyl)piperazin-1-yl]-
ethyl}-6-amino-3-phenyl-2,3-dihydroisoindol-1-one, 20 (125
mg, 0.28 mmol), HCl (concentrated, 0.4 mL), and ice (0.6 g)
was added a solution of NaNO2 (40 mg, 2 equiv) dropwise at
0 °C in an ice bath. The mixture was stirred at 0 °C for 30
min and then added to a solution of KI (700 mg) in water (3
mL) dropwise at room temperature. The mixture became a
foam, which was stirred at room temperature for 30 min and
extracted with CH2Cl2. The organic phase was dried and
evaporated to give a crude product that was purified by PTLC
(Hex/EtOAc, 1:1) and then HPLC (reverse-phase column,
MeCN/NH4OAc [0.1 M], 60:40) to give 6-iodo-2-{2-[4-(2-meth-
oxy-5-nitrophenyl)piperazin-1-yl]ethyl}-3-phenyl-2,3-dihydro-
isoindol-1-one, 22 (70 mg, 41%), 6-iodo-2-{2-[4-(2-methoxyphen-
yl)piperazin-1-yl]ethyl}-3-phenyl-2,3-dihydroisoindol-1-one, 21
(30 mg, 19%).
J
) 2.1, 0.4 Hz, ArH). HRMS: calcd (C27H28N4O5) m/ z
488.2060 (M+), found m/ z 488.2073 (M+).
2-{2-[4-(2-Meth oxyph en yl)piper azin -1-yl]eth yl}-6-n itr o-
3-p h en yl-2,3-d ih yd r oisoin d ol-1-on e (15). IR (film, νmax):
2934, 2821, 1702, 1598, 1533, 1502, 1341, 1237 cm-1 1H NMR
.
(CDCl3, δ): 2.50-2.84 (7H, m, NCH2, CONCHaHb), 3.00-3.10
(4H, m, NCH2), 3.84 (3H, s, CH3O), 4.13 (1H, d, t, J ) 14.5,
5.4 Hz, CONCHaHb), 5.94 (1H, s, NCHPh), 6.86 (1H, d, d, J )
7.4, 1.1 Hz, ArH), 6.88-7.07 (4H, m, ArH), 7.13-7.18 (2H, m,
ArH), 7.32-7.45 (4H, m, ArH), 8.33 (1H, d, d, J ) 8.3, 2.2 Hz,
ArH), 8.73 (1H, d, J ) 2.1 Hz, ArH); HRMS: calcd (C27H28N4O4)
m/ z 472.2110 (M+), found m/ z 472.2125 (M+).
3-Hyd r oxy-6-iod o-2-{2-[4-(2-m eth oxyp h en yl)p ip er a zin -
1-yl]eth yl}-3-p h en yl-2,3-d ih yd r oisoin d ol-1-on e (18) a n d
3-Hyd r oxy-6-iod o-2-{2-[4-(2-m eth oxy-5-n itr op h en yl)p ip -
er azin -1-yl]eth yl}-3-ph en yl-2,3-dih ydr oisoin dol-1-on e (19).
The mixture of 3-hydroxy-2-{2-[4-(2-methoxyphenyl)piperazin-
1-yl]ethyl}-6-nitro-3-phenyl-2,3-dihydroisoindol-1-one, 16 (550
mg, 1.2 mmol), and Pd/C (10%, 100 mg) in a mixed solvent
(60 mL, EtOAc/EtOH, 5:1) was hydrogenated at 40 psi for 1
h. The mixture was then filtered and evaporated to give a
white solid (3-hydroxy-2-{2-[4-(2-methoxyphenyl)piperazin-1-
yl]ethyl}-6-amino-3-phenyl-2,3-dihydroisoindol-1-one, 17; 500
mg) that was clean enough to run in the next reaction without
further purification.
To a mixture of 3-hydroxy-2-{2-[4-(2-methoxyphenyl)piper-
azin-1-yl]ethyl}-6-amino-3-phenyl-2,3-dihydroisoindol-1-one, 17
(200 mg, 0.45 mmol), HCl (concentrated, 0.4 mL), and ice (0.6
g) was added a solution of NaNO2 (62 mg, 2 equiv) dropwise
at 0 °C in an ice bath. The mixture was stirred at 0 °C for 30
min and then added to a solution of KI (700 mg) in water (3
mL) dropwise at room temperature. The mixture became a
foam which was stirred at room temperature for 30 min and
extracted with CH2Cl2. The organic phase was dried and
evaporated to give a crude product which was purified by
PTLC (Hex/EtOAc, 1:1) followed by HPLC (reverse-phase
column, MeCN/NH4OAc [0.1 M], 60:40) to give 3-hydroxy-6-
iodo-2-{2-[4-(2-methoxy-5-nitrophenyl)piperazin-1-yl]ethyl}-3-
phenyl-2,3-dihydroisoindol-1-one, 19 (106 mg, 39%), and 3-hy-
droxy-6-iodo-2-{2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl}-
3-phenyl-2,3-dihydroisoindol-1-one, 18 (40 mg, 16%).
6-Iod o-2-{2-[4-(2-m et h oxy-5-n it r op h en yl)p ip er a zin -1-
yl]eth yl}-3-ph en yl-2,3-dih ydr oisoin dol-1-on e (22). IR (film,
ν
max): 2937, 2821, 1688, 1582, 1513, 1247, 1095, 1023 cm-1
.
1H NMR (CDCl3, δ): 2.54-2.68 (6H, m, NCH2), 3.03 (1H, d, t,
J ) 14.4, 5.7 Hz, CONCHaHb), 3.21 (4H, br, NCH2), 3.94 (3H,
s, CH3O), 4.10 (1H, d, t, J ) 14.5, 5.9 Hz, CONCHaHb), 5.67
(1H, s, NCHPh), 6.88 (1H, d, J ) 8.9 Hz, ArH), 6.94 (1H, d, J
) 8.0 Hz, ArH), 7.13-7.16 (2H, m, ArH), 7.36-7.40 (3H, m,
ArH), 7 71 (1H, d, J ) 2.49 Hz, ArH), 7.78 (1H, d, d, J ) 8.0,
1.6 Hz, ArH), 7.86 (1H, d, d, J ) 8.8, 2.5 Hz, ArH), 8.24 (1H,
d, J ) 1.4 Hz, ArH). HRMS: calcd (C27H27N4O4I) m/ z
599.1105 (M+ + 1), found m/ z 599.1136 (M+ + 1).
6-Iod o-2-{2-[4-(2-m eth oxyp h en yl)p ip er a zin -1-yl]eth yl}-
3-p h en yl-2,3-d ih yd r oisoin d ol-1-on e (21). IR (film, νmax):
2938, 2817, 1688, 1584, 1502, 1237, 1029 cm-1
.
1H NMR
(CDCl3, δ): 2.38-2.66 (6H, m, NCH2), 2.90-2.99 (5H, m,
NCH2), 3.78 (3H, s, CH3O), 4.00 (1H, d, t, J ) 14.4, 5.6 Hz,
CONCHaHb), 5.66 (1H, s, NCHPh), 6.78 (1H, d, J ) 7.6 Hz,
ArH), 6.82-6.96 (4H, m, ArH), 7.05-7.08 (2H, m, ArH), 7.27-
7.34 (3H, m, ArH), 7.69 (1H, d, d, J ) 8.0, 1.5 Hz, ArH), 8.16
(1H, d, J ) 1.3 Hz, ArH). HRMS: calcd (C27H28N3O2I) m/ z
554.1305 (M+ + 1), found: m/ z 554.1302 (M+ + 1).
5-(4-Br om oph en yl)-1-{2-[4-(2-m eth oxyph en yl)piper azin -
1-yl]eth yl}-p yr r olid in -2-on e (25). To a solution of (4-
bromobenzoyl)propanoic acid, 23 (1 g, 3.9 mmol), in methanol
(25 mL) was added H2SO4 (concentrated, 1 mL) dropwise at
room temperature. The mixture was refluxed for 0.5 h.
Methanol was removed after cooling. Water was added, and
the mixture was extracted with methylene chloride. The
organic layer was washed with saturated NaHCO3 solution
followed by saturated NaCl solution, dried, and evaporated to
give 1.07 g of methyl (4-bromobenzoyl)propionate, 24, which
was used for the next reaction without further purification.
To a mixture of 2-[4-(2-methoxyphenyl)piperazinyl]ethyl-
amine, 7 (235 mg, 1 mmol), and methyl (4-bromobenzoyl)-
propionate, 24 (271 mg, 1 mmol), in benzene (10 mL) was
added p-TsOH (100 mg) in solid form. The mixture was
refluxed with a Dean-Stark overnight. Benzene was removed,
and MeOH (5 mL) was added. NaBH4 (50 mg) was added at
0 °C, and the mixture was stirred at room temperature for 1
h. MeOH was removed, and water was added. The mixture
was extracted with CH2Cl2-MeOH (9:1). The combined
organic phases were dried over NaSO4 and filtered. The
filtrate was concentrated to give a crude product which was
purified by PTLC (CH2Cl2/MeOH, 95:5) to give 143 mg of
desired product 25 (31%).
1H NMR (CDCl3, δ): 1.75-1.84 (1H, m), 2.40-2.59 (9H, m),
2.73 (1H, d, t, J ) 14.3, 6.3 Hz, COCHaHb), 3.04 (4H, br), 3.85
(3H, s, OCH3), 3.85 (1H, d, t, J ) 14.1, 6.5, COHCHaHb), 4.81
(1H, m, CONCHAr), 6.86 (1H, d, J ) 7.4 Hz, ArH), 6.93 (2H,
m, ArH), 7.01 (1H, d, d, d, J ) 7.8, 5.4, 3.8 Hz, ArH), 7.10
(2H, d, t, J ) 8.4, 2.2 Hz, BrArH), 7.50 (2H, d, t, J ) 8.4, 2.2
Hz, BrArH). MS: m/ z 458 (M+ + 1), 205.
5-[4-(Tr ibu tylsta n n yl)p h en yl]-1-{2-[4-(2-m eth oxyp h en -
yl)p ip er a zin -1-yl]eth yl}p yr r olid in -2-on e (26). A mixture
of 5-(4-bromophenyl)-1-{2-[4-(2-methoxyphenyl)piperazin-1-yl]-
3-H yd r oxy-6-iod o-2-{2-[4-(2-m et h oxy-5-n it r op h en yl)-
p ip e r a zin -1-yl]e t h yl}-3-p h e n yl-2,3-d ih yd r oisoin d ol-1-
on e (19). IR (film, νmax): 2959, 2923, 2846, 1700, 1589, 1513,
1445, 1336, 1261, 1023 cm-1 1H NMR (CDCl3, δ): 2.45 (1H,
.
d, d, J ) 14.6, 2.6 Hz, CONCHaHb), 2.60-3.32 (10H, m, NCH2),
3.95 (3H, s, CH3O), 4.16 (1H, d, d, J ) 13.2, 2.3 Hz, CON-
CHaHb), 6.93 (1H, d, J ) 8.9 Hz, ArH), 7.00 (1H, d, d, J ) 8.0,
0.5 Hz, ArH), 7.31-7.45 (5H, m, ArH), 7.74 (1H, d, J ) 2.5
Hz, ArH), 7.79 (1H, d, d, J ) 8.0, 1.6 Hz, ArH), 7.90 (1H, d, d,
J ) 8.8, 2.5 Hz, ArH), 8.19 (1H, d, d, J ) 1.6, 0.5 Hz, ArH).
MS: m/ z 615 (M+ + 1).
3-Hyd r oxy-6-iod o-2-{2-[4-(2-m eth oxyp h en yl)p ip er a zin -
1-yl]eth yl}-3-p h en yl-2,3-d ih yd r oisoin d ol-1-on e (18). IR
(film, νmax): 3060, 2948, 2834, 1708, 1594, 1498, 1449, 1371,
1237 cm-1 1H NMR (CDCl3, δ): 2.36 (1H, d, d, J ) 14.6, 2.6
.
Hz, CONCHaHb), 2.62-3.08 (10H, m, NCH2), 3.78 (3H, s,
CH3O), 4.10 (1H, d, d, d, J ) 13.9, 3.4, 1.6 Hz, CONCHaHb),
6.80 (1H, d, J ) 7.7 Hz, ArH), 6.85-7.00 (4H, m, ArH), 7.24-
7.37 (5H, m, ArH), 7.70 (1H, d, d, J ) 8.0, 1.6 Hz, ArH), 8.10
(1H, d, d, J ) 1.5, 4.2 Hz, ArH). MS: m/ z 570 (M+ + 1).
6-Iod o-2-{2-[4-(2-m eth oxyp h en yl)p ip er a zin -1-yl]eth yl}-
3-p h en yl-2,3-d ih yd r oisoin d ol-1-on e (21) a n d 6-Iod o-2-{2-
[4-(2-m eth oxy-5-n itr oph en yl)piper azin -1-yl]eth yl}-3-ph en -
yl-2,3-d ih yd r oisoin d ol-1-on e (22). A mixture of 2-{2-[4-(2-
methoxyphenyl)piperazin-1-yl]ethyl}-6-nitro-3-phenyl-2,3-di-
hydroisoindol-1-one, 15 (140 mg, 0.3 mmol), and Pd/C (10%,
50 mg) in a mixed solvent (20 mL, EtOAc/EtOH, 5:1) was
hydrogenated at 40 psi for 1 h. The mixture was filtered, and
the filtrate was evaporated to give a white solid (136 mg), 2-{2-
[4-(2-methoxyphenyl)piperazin-1-yl]ethyl}-6-amino-3-phenyl-

164 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 2
Zhuang et al.
ethyl}pyrrolidin-2-one, 25 (50 mg, 0.11 mmol), bis(tributyltin)
(0.2 mL), and (Ph3P)4Pd (10 mg) in 1,4-dioxane (5 mL) was
stirred under reflux overnight. The solvent was removed, and
the residue was purified by PTLC (EtOAc as developing
solvent) to give 53 mg (73%) of the desired tin compound.
1H NMR (CDCl3, δ): 0.88 (4.5H, t, J ) 7.1 Hz, CH3), 0.92
(4.5H, t, J ) 7.1 Hz, CH3), 1.05 (4H, t, J ) 8 Hz, CH2), 1.36
(8H, m, CH2), 1.56 (6H, m, CH2), 1.80 (1H, m), 2.43-2.57 (9H,
m), 2.78 (1H, d, t, J ) 13.9, 6.6 Hz, COCHaHb), 3.03 (4H, m),
3.84 (3H, s, OCH3), 3.85 (1H, d, t, J ) 13.9, 6.6, CONCHaHb),
4.76 (1H, m, CONCHAr), 6.90 (3H, ArH), 7.15 (1H, m, ArH),
7.49 (3H, m, ArH), 7.70 (1H, d, t, J ) 8.4, 2.2 Hz, ArH).
5-(4-Iod op h en yl)-1-{2-[4-(2-m eth oxyp h en yl)p ip er a zin -
1-yl]eth yl}p yr r olid in -2-on e (27). Thirty-four milligrams
(0.05 mmol) of the tin compound, 26, was dissolved in
chloroform (5 mL), to which was added a solution of iodine in
chloroform (0.1 M) dropwise at room temperature until the
color was persistent. The mixture was stirred at room
temperature for 5 min. KF (3 mL, 1 M in MeOH) and NaHSO3
(3 mL, 5% solution) were added successively. The mixture was
stirred at room temperature for 5 min and extracted with
methylene chloride. The organic phase was dried over Na2-
SO4 and filtered. The filtrate was concentrated to give a crude
product that was purified by PTLC (EtOAc as developing
solvent) to give 12 mg of compound (48%).
1H NMR (CDCl3, δ): 1.75-1.83 (1H, m), 2.37-2.58 (9H, m),
2.73 (1H, d, t, J ) 14.1, 6.3 Hz, COCHaHb), 3.03 (4H, br), 3.85
(3H, s, OCH3), 3.85 (1H, d, t, J ) 14.1, 6.4, CONCHaHb), 4.79
(1H, m, CONCHAr), 6.86 (1H, d, J ) 7.4 Hz, ArH), 6.93 (2H,
m, ArH), 6.97 (1H, m, ArH), 7.01 (2H, d, t, J ) 8.4, 2.3 Hz,
BrArH), 7.70 (2H, d, t, J ) 8.4, 2.2 Hz, BrArH). MS: m/ z
506 (M+ + 1), 380, 205.
2-{2-[4-(2-Meth oxyp h en yl)p ip er a zin -1-yl]eth yl}-2,3-d i-
h yd r oisoin d ol-1-on e (29). To a mixture of 2-[4-(2-methoxy-
phenyl)piperazinyl]ethylamine, 7 (95 mg, 0.4 mmol), and
2-(methoxycarbonyl)benzaldehyde, 28 (55 mg, 0.34 mmol), in
benzene (10 mL) was added p-TsOH (50 mg) in solid form. The
mixture was refluxed with a Dean-Stark overnight. Benzene
was removed, and MeOH (5 mL) was added. NaBH4 (50 mg)
was added at 0 °C, and the mixture was stirred at room
temperature for 1 h. MeOH was removed, and water was
added. The mixture was extracted with CH2Cl2/MeOH (9:1).
The combined organic phases were dried over NaSO4, filtered,
and concentrated to give a crude product that was purified by
PTLC (EtOAc as developing solvent) to give 44 mg of desired
product 29 (37%).
re-extracted with ethyl acetate (3 × 1 mL). The final no-
carrier-added products were evaporated to dryness and redis-
solved in 100 µL of 50% EtOH with 100 µg of ascorbic acid
added as an antioxidant. The radiochemical purity of the
product was evaluated on HPLC, and the identity was
confirmed with the coelution on HPLC with the unlabeled
standard.
P a r t it ion Coefficien t Det er m in a t ion .
[
125I]Ligand
(500000-800000 cpm) was mixed with 3 g each of 1-octanol
and buffer (pH 7.0 or pH 7.4, 0.1 M phosphate) in a test tube.
The test tube was vortexed for 3 min at room temperature and
then centrifuged for 5 min to separate the 1-octanol and buffer
layers. The radioactivity of approximately 1 g samples from
each layer was counted in a gamma counter (Packard 5000).
The partition coefficient (PC) was determined by calculating
the ratio of counts per minute per gram of octanol to that of
buffer. Samples from the octanol layer were repartitioned
until consistent PC values were obtained. The measurements
were repeated three times.
Bin d in g Assa ys. The rat hippocampal membrane homo-
genates were prepared as described previously.25 Binding
assays were performed in glass tubes (12 × 75 mm) in a final
volume of 0.25 mL. In saturation experiments, aliquots (100
µL corresponding to 20-30 µg of protein) of membrane
suspensions were mixed with 50 mM Tris-HCl, pH 7.4,
containing 0.1% BSA and radioligands of 0.05-2.0 nM [125I]-
21 or [125I]-27. Competition experiments were performed using
0.2 nM [125I]-31 and 8-10 concentrations (10-10-10-5 M) of
competing drugs (serially diluted in 50 mM Tris buffer
containing 0.1% BSA). Nonspecific binding was defined with
1 µM 31. Incubation was carried out for 20 min at 37 °C and
then terminated by separation of bound from free radioligand
by filtration through glass fiber filters (Schleicher & Schuell
No. 25, Keene, NH) presoaked with 1% polyethylenimine. The
filters were then washed three times with 3 mL of ice-cold 20
mM Tris buffer and counted in a gamma counter (Packard
5000) with 70% efficiency. The results of saturation and
competition experiments were subjected to nonlinear regres-
sion analysis using EBDA43 to obtain Kd and IC50 values.
Meta bolic Sta bility. Human liver microsomal and cyto-
solic fractions purchased from Dr. Ted Inaba (University of
Toronto) were used as a model of human liver metabolism for
evaluating the stability of various tracer ligands. The radio-
iodinated ligands (2-3 µCi in 40 µL of 50 mM Na phosphate,
pH 7.4) were incubated at 37 °C for 10 min with 160 µL of
phosphate buffer containing various amount of cytosolic or
microsomal fractions. At the end of the incubation, the amount
of unmetabolized [125I]-31 or [125I]-27 was evaluated directly
by thin-layer chromatography (TLC) with Whatman silica gel
plates (PESIL G/UV) and a solvent system of EtOAc/MeOH/
triethylamine, 85:15:5. Due to the lipophilic property of [125I]-
21, ethyl acetate (3 × 1 mL) extraction was carried out in the
presence of 10 µg “cold” carrier followed by analysis on TLC.
Similar procedures were applied to [125I]-31 for the purpose of
comparison. Control samples were carried out by mixing small
amount of ligands (1-2 µCi) with microsomal or cytosolic
fractions without incubation, followed by the same procedures
used for the incubation samples to determine the extraction
efficiency and the total recovery yield.
Biod istr ibu tion in Ra ts. The animal studies were con-
ducted according to protocols approved by the University of
Pennsylvania Institutional Animal Care and Use Committee.
Male Sprague-Dawley rats (225-300 g) allowed free access
to food and water were used for in vivo biodistribution
studies.44,45 While under ether anesthesia, 0.2 mL of a saline
solution containing [125I]-21 or [125I]-27 (10-20 µCi) was
injected directly into the femoral vein of the rats. The rats
were then sacrificed by cardiac excision at various time points
postinjection. The organs of interest were removed and
weighed, and the radioactivity was counted with an automatic
gamma counter (Packard 5000). The percentage dose per
organ was calculated by a comparison of the tissue counts to
suitably diluted aliquots of the injected material. Total
activities of blood and muscle were calculated under the
1H NMR (CDCl3, δ): 2.73 (6H, m), 3.08 (4H, m), 3.79 (2H,
t, J ) 6.5 Hz, CONCH2), 3.85 (3H, s, OCH3), 4.53 (2H, s,
CONCH2Ar), 6.85 (1H, d, J ) 7.5 Hz, ArH), 6.91 (2H, m, ArH),
7.00 (1H, m, ArH), 7.42-7.55 (3H, m, ArH), 7.86 (1H, m, ArH).
HRMS: calcd (C21H25N3O2) m/ z 352.2025 (M+ + 1), found
352.2036 (M+ + 1).
6-(Tr ibu tylstan n yl)-2-{2-[4-(2-m eth oxyph en yl)piper azin -
1-yl]eth yl}-3-p h en yl-2,3-d ih yd r oisoin d ol-1-on e (30).
A
mixture 6-iodo-2-{2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl}-
3-phenyl-2,3-dihydroisoindol-1-one, 21 (22 mg, 0.04 mmol), bis-
(tributyltin) (0.2 mL), and (Ph3P)4Pd (10 mg) in 1,4-dioxane
(2 mL) was stirred under reflux overnight. The solvent was
removed, and the residue was purified by PTLC (EtOAc/Hex,
1:1, as developing solvent) to give 8 mg (28%) of the desired
tin compound 30.
1H NMR (200 Hz, CDCl3, δ): 0.87 (9H, t, J ) 7.1 Hz, CH3),
1.07 (4H, t, J ) 7.9 Hz, CH2), 1.32 (8H, m, CH2), 1.53 (6H, m,
CH2), 2.62 (6H, m, NCH2), 3.06 (4H, m, NCH2), 3.18 (1H, m,
CONCHab), 3.85 (3H, s, CH3O), 4.10 (1H, m, CONCH6a), 5.75
(1H, s, NCHPh), 6.93 (3H, m, ArH), 7.14 (3H, m, ArH), 7.33
(4H, m, ArH), 7.54 (1H, d, J ) 7.2 H, ArH), 8.00, (1H, s, ArH).
Ra d iola belin g. No-carrier-added [125I]-21 and [125I]-27
were prepared by an iododestannylation reaction similar to
the procedure reported previously for [125I]-31.25 Hydrogen
peroxide (50 µL, 3% w/v) was used as the oxidant, and the
products were purified by HPLC using a reverse-phase column
(PRP-1 column, Hamilton Co., Reno, Nevada). The fractions
containing the desired product were collected, condensed, and

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assumption that they were 7% and 40% of the total body
weight, respectively. Regional brain distribution in rats was
measured after an iv injection of the radioactive tracer.
Samples from different brain regions (cortex, striatum, hip-
pocampus and cerebellum) were dissected, weighed, and
counted. The percentage dose per gram of each sample was
calculated by comparing the sample counts with the counts of
the diluted initial dose. The ratio of uptake in each region
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region with that of the cerebellum, which was used as a
background region containing no 5-HT1A receptors.
In Vitr o Au tor a d iogr a p h y. Rats were decapitated and
brains were removed immediately. Brains were placed in OTC
embedding medium (Miles Laboratory, Elkhart, IN) and frozen
with powdered dry ice. Coronal sections of 20 µm thickness
were cut at -20 °C in a cryostat (Hacker Instruments,
Fairfield, NJ ) and thaw-mounted onto gelatin-coated glass
slides. The sections were desiccated at 4 °C for 3 h and then
kept at -70 °C until use.
The rat brain sections were thawed, brought to room
temperature, and preincubated in Tris-HCl buffer, pH 7.5, at
room temperature for 30 min. The sections were then incu-
bated for 90 min at room temperature in the same buffer
containing 0.1 nM [125I]-21 followed by washing with cold
buffer for 18 h (sections were kept in ice) with one change of
buffer. Adjacent sections were labeled similarly, but in the
presence of 1 µM 31 to define nonspecific binding. The sections
after washing were dipped in ice-cold distilled water to remove
buffer salts before drying with a stream of cold air. Dried
sections were apposed to DuPont X-ray films for 18 h, and the
analysis of the autoradiograms was carried out using a
computer-based image analysis system (NIH Image, version
1.61).
Ack n ow led gm en t. The authors would like to thank
Dr. Alan Wilson for his helpful discussion and Ms. Dana
Frederick and Ms. Susan West for their assistance in
preparing the manuscript. This work supported by
grants awarded by the National Institutes of Health
(NS35120 and MH49125).
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