Design and synthesis of novel arylpiperazine derivatives containing the imidazole core targeting 5-HT2A receptor and 5-HT transporter

Article abstract of DOI:10.1021/jm200682b

Serotonin antagonist reuptake inhibitor (SARI) drugs that block both 5-HT₂ receptors and the serotonin transporters have been developed. The human 5-HT_2A/2C receptor has been implicated in several neurological conditions, and potent

Full text of DOI:10.1021/jm200682b

ARTICLE
pubs.acs.org/jmc
Design and Synthesis of Novel Arylpiperazine Derivatives Containing
the Imidazole Core Targeting 5-HT2A Receptor and 5-HT Transporter
Hee Jeong Seo,† Eun-Jung Park,† Min Ju Kim,† Suk Youn Kang,† Suk Ho Lee,† Hyun Jung Kim,† Ki Nam Lee,†
Myung Eun Jung,† MinWoo Lee,† Mi-Soon Kim,† Eun-Jung Son,† Woo-Kyu Park,‡ Jeongmin Kim,† and
Jinhwa Lee*,†
†Research Center, Green Cross Corporation, 303 Bojeong-dong, Giheung-gu, Yongin 446-770, Korea
‡Pharmacology Research Center, Korea Research Institute of Chemical Technology, 100 Jang-Dong, Yuseong-Gu,
Daejeon 305-343, Korea
S Supporting Information
b
ABSTRACT:
Serotonin antagonist reuptake inhibitor (SARI) drugs that block both 5-HT2 receptors and the serotonin transporters have been
developed. The human 5-HT2A/2C receptor has been implicated in several neurological conditions, and potent selective 5-HT2A/2C
ligands may have therapeutic potential for treatment of CNS diseases such as depression. An imidazole moiety usually provides good
pharmacokinetic properties as a drug substance, and thus considerable efforts have been devoted to develop imidazole derivatives
into drug candidates. The imidazole series of compounds was evaluated against 5-HT2A/2C and serotonin reuptake inhibition. A few
of the compounds in the series showed promising IC50 values and antidepressant-like effect in in vivo forced swimming test (FST).
On the basis of these results, further lead optimization studies resulted in identifying promising compounds potentially for
therapeutic use.
’ INTRODUCTION
Recently, there has been the achievement of an important
development of an antidepressant which interacts with dual or
multiple targets.7
Depression, especially major depression, is an extremely
serious disease affecting about 121 million people and is one of
the leading causes of disability worldwide. And unipolar depres-
sion is predicted to be the second main cause of disability in 2020
by the World Health Organization.1,2 Over the past several
decades, the synaptic actions of monoamine neurotransmitters
such as norepinephrine (NE) and serotonin (SER, 5-HT) were
considered as important indications to psychiatric disease,
including anxiety and depression.3,4 Various psychotropic drugs
which are associated with these neurotransmitters have been
individually developed. Selective serotonin reuptake inhibitors
(SSRIs) are a relatively newer class of antidepressants for treating
depression. SSRIs such as fluoxetine, sertraline, paroxetine, and
citalopram have been the most widely prescribed antidepressants
since 1980s.5 All SSRIs strongly and selectively inhibit 5-HT
reuptake by the presynaptic neuron, thus increasing 5-HT
concentration at the synapse. Although SSRIs offer a more
favorable profile, they also have some side adverse effects
including anxiety, sedation, headache, tremor, insomnia, and
sexual dysfunction. More importantly, there are troublesome
facts that SSRIs are generally effective only for less than two-third
patients and have undesirably long therapeutic onset time.6
5-HT1 autoreceptors are widely distributed in the brain, and
also serotonin transporters (SERT) are known to have a major
role in the control of synaptic 5-HT levels.8 Blockade of 5-HT1A/B/D
autoreceptors, with or without concomitant SERT inhibition,
rapidly increases brain 5-HT levels and consequently might
be able to provide a fast onset of antidepressant/anxiolytic action
relative to current therapies.9 Arylpiperazine is a core fragment of
a good number of active compounds displaying various pharma-
cological effects. The most frequently studied of arylpiperazine
derivatives, so-called long-chain arylpiperazines, have been found
as serotonin receptor ligands, especially 5-HT1A and 5-HT2A
ones. Their general chemical structure contains an alkyl chain
attached to the N4 atom of the piperazine moiety and a terminal
amide or an imide fragment.10
Because numerous side effects are associated with nonselec-
tive binding at postsynaptic 5-HT receptors, addition of 5-HT
receptor antagonistic component to 5-HT reuptake inhibitor has
Received: May 30, 2011
Published: August 08, 2011
r
2011 American Chemical Society
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Figure 1. Chemical structures of representative antidepressant compounds.
Figure 2. Preparation of target compounds using peptide bond formation.
been proposed.11ꢀ13 The level of synaptic 5-HT was expected to
increase with this approach and thus eventually achieve rapid onset
time. Along the line, various compounds have been proposed and
developed as potential antidepressant with dual activity (Figure 1).
For instance, Eli Lilly’s LY367265 (1-[2-[4-(6-fluoro-1H-indol-3-
yl)-3,6-dihydro-1(2H)-pyridinyl]ethyl]-5,6-dihydro-1H,4H-[1,2,5]-
thiadiazolo[4.3.2-ij]quinoline-2,2,-dioxide, 1) shows excellent
binding affinities (Ki = 2.3 nM for SERT; Ki = 0.81 nM for
5-HT2A).14 Yamanouchi has also discovered YM992 ((S)-2-((7-
fluoro-2,3-dihydro-1H-inden-4-yloxy)methyl)morpholine mono-
hydrochloride, 2) as an antidepressant with moderate affinities
for SERT/5-HT2A (Ki = 21 and 86 nM, respectively).15 Bristol-
Myers Squibb’s nefazodone (1-(3-(4-(3-chlorophenyl)piperazin-
1-yl)propyl)-3-ethyl-4-(2-phenoxyethyl)-1H-1,2,4-triazol-5(4H)-
one, 3) has been described as having a similar mode of action
with an improved side effect profile.16 3 had advantages over
other antidepressants, including reduced possibility to disturb
sleep or sexual dysfunction and ability to treat patients who did
not respond properly to other antidepressant drugs. However,
Bristol-Myers Squibb discontinued the sale in 2004 with adverse
hepatic events including liver failure.16c,d More recently,
aripiprazole (7-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butoxy)-
3,4-dihydroquinolin-2(1H)-one, 4),17,18 whichhad been approved
for the treatment of atypical antipsychotics, was also approved by
the FDA as an adjunct to treat major depressive disorder in
adults. Unlike other FDA-approved atypical antipsychotics
antagonizing the D2 receptor, 4 appears as a 5-HT1A partial
agonist, 5-HT2A antagonist, and 5-HT2C partial agonist. In
addition, it has moderate affinity for the serotonin transporter.
However, it also has a number of side effects including headache,
nausea, constipation, anxiety, restlessness, insomnia, nervous-
ness, and so on. In this regard, there are still urgent unmet
medical needs on the development of novel drugs with better
developability characteristics, i.e. improved pharmacologic prop-
erties and reduced side effects.
We have previously reported a series of arylpiperazine-con-
taining heteroaryl derivatives targeting serotonin 5-HT2A,
5-HT2c, and the serotonin transporter as
a potential
antidepressant.19 The pyrrole series showed outstanding in vitro
activity profiles and in vivo antidepressant-like activity on a
forced swimming test, but it also suffered from a relatively strong
hERG channel inhibition and false positive effects measured via
spontaneous locomotor activity.19a,b On the other hand, the
pyrimidine series showed the favorable spontaneous locomotor
activity as well as improved hERG channel inhibition profile but
tended to exhibit diminished binding affinity against 5-HT2A,
5-HT2c, and the serotonin transporter.19c On the basis of these
earlier data, we embarked on exploring imidazole series, hoping
that we would be able to collect only positive aspects out of the
above pyrrole and pyrimidine series. With this in mind, the
general structure of target compounds A can be readily prepared
by typical amide coupling of imidazole 4-carboxylic acid B with
arylpiperazinyl alkyl amine C at the final stage as shown in
Figure 2. As a continuation of our investigation, we wish to
describe the design, synthesis, and biological evaluation of novel
arylpiperazine-containing imidazole 4-carboxamide derivatives
targeting serotonin 5-HT2A, 5-HT2C, and the serotonin trans-
porter as a potential antidepressant.
’ CHEMISTRY
The first approach toward a key imidazole intermediate is
described in Scheme 1. Thus, ethyl acetoacetate (5) was sub-
jected to a reaction with sodium nitrite in acetic acid to give ethyl
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Scheme 1a
Scheme 3a
a Reagents: (a) NaNO2, AcOH, ꢀ10 °C to rt, 2 h, 70%; (b) 7, Pd/C, H2,
EtOH, rt, 12 h, 86%; (c) 9, TFAorAcOH, butyronitrile, microwave, 140°C,
a Reagents: (a) 18, AlCl3, toluene, reflux, 5 h, 60%; (b) 20, Na2CO3,
40 min, 76%; (d) LiOH H2O, THF/H2O(1/1), heat, 12 h, 90%.
3
toluene, reflux, 4 h, 37%; (c) LiOH H2O, THF/H2O(1/1), heat, 12 h,
3
86%.
Scheme 2a
Scheme 4a
a Reagents: (a) POCl3, DMA, 45 °C, 50%; (b) NaH, EtOH, rt, then
reflux, 1 h; (c) 26, AcOH, rt, 12 h; (d) (NH4)2SO4, AcOH, HMDS,
reflux; (e) NaOH, aq MeOH, reflux.
a Reagents: (a) 9, NaHMDS, THF, rt; (b) 14, NaHCO3, i-PrOH, reflux,
55%; (c) LiOH H2O, THF/H2O(1/1), heat, 12 h.
3
form 22 was prepared from the ester 21 using lithium hydroxide,
followed by acidification, as shown in Scheme 3.
2-(hydroxyimino)-3-oxobutanoate (6) in 70% yield. The com-
pound 6 was reacted with an anhydride such as acetic anhydride
(7) in the presence of a reducing agent such as hydrogen and a
catalyst such as Pd on carbon (Pd/C) in EtOH to give an amide
8.20 The amide 8 was reacted with aniline 9 in acetonitrile under
microwave irradiation to produce an imidazole derivative 10 in
76% yields. Hydrolysis of ester 10 with lithium hydroxide
produced compound 11 as shown in Scheme 1.
The imidazole acid derivative 16 was synthesized by adopting
a method reported by Solvay.21 Thus, benzonitrile (12) was
reacted with aniline (9) in the presence of sodium bis-
(trimethylsilyl)amide (NaHMDS) to produce a corresponding
arylbenzamidine 13. Subsequent reaction of the resulting aryl-
benzamidine 13 with R-bromoketone 14 generated an inter-
mediate ethyl 5-methyl-1,2-diphenyl-1H-imidazole-4-carboxylate
(15) in 55% yield. Hydrolysis of ester 15 with lithium hydroxide
produced the corresponding lithium carboxylate 16 as shown in
Scheme 2.
Another approach toward imidazole intermediate is described
in Scheme 4. The original reaction sequence was published by F.
Hoffmann-La Roche.23 N-Acetylglycine (23) and phosphorus
oxychloride are mixed, to which dimethylacetamide was added
dropwise slowly at low temperature (exothermic). The reaction
was then stirred and warmed at 45 °C. In this way, (E)-
4-(1-(dimethylamino)ethylidene)-2-methyloxazol-5(4H)-one (24)
was generated in 50% yield. Oxazolone 24 was cleaved with
in situ generated ethoxide to produce (E)-ethyl 2-acetamido-
3-(dimethylamino)but-2-enoate (25). Next, an amine 26 was
reacted with 25 in acetic acid at room temperature overnight to
give a Michael adduct 27. The crude intermediate 27 was then
refluxed together with fine powdered ammonium sulfate in
hexamethyldisilazane at 145 °C to provide the corresponding
imidazoles of structure 28 in 55ꢀ73% yields for the three steps
from the oxazolone 24. The ester 28 was hydrolyzed with sodium
hydroxide to produce 29 uneventfully.
The imidazole derivative 22 was prepared by reaction of a
conventional method,22 for example, by reacting a nitrile 18 with
an aniline derivative 17 using aluminum chloride to produce
N-(4-chlorophenyl)-butyrimidamide (19). Subsequent reaction
of the resulting compound 19 with ethyl 3-bromo-2-oxopro-
panoate (20) provided an intermediate ethyl 1-(4-chloro-
phenyl)-2-propyl-1H-imidazole-4-carboxylate (21). An acid
As shown in Scheme 5, the synthesis of 3-(4-(2,3-dichlorophe-
nyl)piperazin-1-yl)propan-1-amine (33) and the like, wherein the
alkyl chain between the piperazine and the terminal amine corre-
sponds to two carbons through four carbons, commenced with
N-(2-bromoethyl)phthalimide, N-(3-bromopropyl)phthalimide
(30) and N-(4-bromobutyl)phthalimide by adopting a reported
procedure.24 For example, N-(3-bromopropyl)phthalimide (30)
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Scheme 5a
a Reagents: (a) K2CO3, DMF, rt, 8 h, 80%; (b) (i) H2NNH2 H2O, EtOH, rt, 6 h, (ii) 2 M HCl solution in diethyl ether, 76%.
3
Scheme 6a
a Reagents: (a) TEA, THF, 80 °C, 92%; (b) (i) H2NNH2 H2O, EtOH, rt, 6 h, (ii) 2 M HCl solution in diethyl ether, 95%.
3
Scheme 7a
a Reagents: (a) (i) EDCI, HOBt, NMM, DMF, rt, 12 h, (ii) 2 M HCl solution in diethyl ether.
was reacted with 1-(2,3-dichlorophenyl)piperazine (31) in the
presence of potassium carbonate in a suitable solvent such as DMF
at rt afforded the corresponding alkylated product 32 in 80% yield.
Hydrazinolysis of 32, followed by treatment of HCl solution,
generated 3-(4-(2,3-dichlorophenyl)piperazin-1-yl)propan-1-amine
as a HCl salt form (33) in 76% yield.
dichlorophenyl)piperazin-1-yl)propan-2-ol (36) as a white solid
in 95% yield.
Generally, a target compound was prepared by amide bond
formation of acid B and amine C by use of EDCI, HOBt, and
NMM in a suitable solvent such as DMF to generate A as shown
in Scheme 7.
To increase hydrophilicity for compounds such as 3-(4-(2,3-
dichlorophenyl)piperazin-1-yl)propan-1-amine (33), a com-
pound such as 1-amino-3-(4-(2,3-dichlorophenyl)piperazin-1-
yl)propan-2-ol (36) was prepared as shown in Scheme 6. Thus,
commercially available N-(2,3-epoxypropyl)phthalimide (34)
was treated with 1-(2,3-dichlorophenyl)piperazine (31) in the
presence of base such as triethylamine in a suitable solvent such
as THF at 80 °C produced the alcohol 35 in about 91% yield.
Subsequently, hydrazinolysis of 35 generated 1-amino-3-(4-(2,3-
’ RESULTS AND DISCUSSION
The binding affinity of current compounds against 5-HT2A,
5-HT2C receptor, and serotonin transporter, stably expressed in
CHO-K1 cells, was evaluated by displacement binding using
[3H]ketanserin, [3H]mesulergine, and [3H]imipramine, respec-
tively, as radioligands.25 The initial work was focused on explora-
tion of substitution on the central imidazole moiety and the size
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of the linker connecting the central imidazole ring to a 2,3-
dichlorophenylpiperazine moiety. We decided to use 2,3-dichlor-
ophenylpiperazine as an initial arylpiperazine, adopting a struc-
tural motif shown in 4. The binding affinity against 5-HT2A/2C
and inhibition against SERT are shown in Table 1. Most
compounds tested displayed IC50 < 1 μM, implying that this
series of arylpiperazinyl imidazole 4-carboxamide might hold
promise as a potentially viable antidepressant. Especially, 2,5-
dimethyl-1-phenyl-1H-imidazole (40) or 1-(2,3-dihydrobenzo-
[b][1,4]dioxin-6-yl)-2,5-dimethyl-1H-imidazole (57) shows po-
tent and balanced activity profiles against 5-HT2A/2C and SERT
(IC50 = 18.6, 12.1, and 10.2 nM for 40 vs IC50 = 8.63, 14.6, and
9.17 nM for 57, respectively). As the linker size reduces by one
carbon, the activity profiles appear to become deteriorated as
shown in 37 (IC50 = 31, 66, and 129 nM, respectively). Most of
phenyl derivatives on the 1-position of the imidazole ring appear
to be tolerated. Thus, physicochemical properties can be modu-
lated by modifying this moiety later. Also, different groups such
as methyl, ethyl, n-propyl, or phenyl can be introduced at
2-position of the imidazole ring without hampering overall in
vitro activity profiles. A methyl (40) or n-propyl group (42)
usually appears to be more favorable than an ethyl group (41),
but in some cases (47, 55, and 58), a n-propyl group on C-2
imidazole ring was observed to diminish in vitro 5-HT2A/2C and
SERT, implying that increased lipophilicity at this position might
disrupt binding to 5-HT2A/2C receptors.
Some of the compounds were further evaluated with other
subtypes that constitute the serotonin receptor family and
dopamine receptors. The results are shown in Table 2. Interest-
ingly, both compounds 37 and 40 proved to be highly bound to
either 5-HT1A or 5-HT7 as well. In particular, compound 40
showed good activity against 5-HT receptors and SERT across
the board (IC50 = 4.9ꢀ19 nM) except 5-HT6 (IC50 = 136 nM).
Such compounds might have the synergistic antidepressant
properties because the blockade of 5-HT7 receptors has been
recently proposed as an alternative therapy for depression. On
the contrary, binding affinity against dopamine receptors was
only marginal, thereby providing good selectivity over dopamine
D2/3/4 receptors while maintaining the key target interactions.
Meanwhile, a radioligand binding assay for the hERG potassium
channel was adopted to see if our compounds may have
inhibitory activity and potential cardiotoxicity.26 Compound 37
turned out not to be a significant inhibitor of hERG channel
(IC50 > 10 μM). But as the linker size becomes elongated (38),
the hERG profile gets slightly worse (IC50 = 8.11 μM) although
these data are not too much problematic as shown in Table 2.
Before we move on to the in vivo efficacy study, we screened
our compounds particularly against spontaneous locomotor
activity to identify false positives. Although several compounds
in Table 1 looked very good for in vitro activity, they appeared to
have hyperactivity. For example, compounds 37 or 49 showed a
high spontaneous locomotor activity (SLA) value (14802 (
3275 at 50 mg/kg for 37; 13040 ( 4261 at 50 mg/kg for 49,
respectively). Thus, to reduce SLA values, we decided to
introduce 2,3-dimethylphenylpiperazine or 2-methyl-3-chloro-
phenylpiperazine which is less hydrophobic while maintain the
structural motif as an approach and we also decided to keep the
three-carbon distance between imidazole-amide and phenylpi-
perazine because they appear to provide better activity than the
corresponding two-carbon linker. Also four-carbon linker was
conceivable at this point, but we hesitated to explore its
possibility because it looked so lengthy and too flexible that they
would not be desirable from the development perspective even if
they would be potent. All compounds thus designed and
prepared were evaluated. The binding affinity against 5-HT2A/2C
and inhibition against SERT are shown in Table 3. Notably, this
Table 1. Binding Affinity to Serotonin Receptor 5-HT2A,
5-HT2C and Serotonin Transporter (SERT) (IC50, nM)
Table 2. Profiles of Interesting Compounds via Competition Binding Assay at Serotonin Receptors, SERT, Dopamine Receptors
(IC50, nM), and Properties of hERG Channels (IC50, μM)
serotonin (5-HT) receptor
dopamine receptor
compd
5-HT1A
5-HT2A
5-HT2C
5-HT6
5-HT7
serotonin transpoter
D2
D3
D4
hERG IC50 (μM)
37
40
3.9
4.9
31
19
66
12
1682
136
6.8
11
129
10
908
2189
3942
>10
8.11
1369
493
>10000
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Table 3. Binding Affinity to Serotonin Receptor 5-HT2A,
5-HT2C and Serotonin Transporter (SERT) (IC50, nM)
to have hypoactivity (SLA value =5409 ( 3319 cm at 50 mg/kg).
Replacement of the 2,3-dimethylpiperazine in 92 with 2-methyl-3-
chloro-piperazineprovidedthe compound 91 with an encouraging
overallactivity profile (IC50 = 29 nMfor 5-HT2A, IC50 = 39 nM for
5-HT2C, IC50 = 18 nM for SERT). Disappointingly, further
investigation of the compound with SLA revealed its hyperactivity
(SLA value =10189 ( 628 cm at 50 mg/kg). Among 45
compounds in Table 3, 13 compounds show IC50 < 10 nM against
SERT, and 8 compounds show IC50 < 20 nM against 5-HT2A,
while none of the compounds show IC50 < 30 nM against 5-HT2C.
It is notable that replacement of 2,3-dichloropiperazine with 2,3-
dimethylpiperazine boosted in vitro inhibitory activity against the
serotonin transporter while maintaining binding affinity against
5-HT2A. Thus, this series appears to become more like SSRI
compounds thathappentoadditionallyblock5-HT2A atthispoint.
There are reported studies indicating that the antidepressants
which occupy 5-HT2 receptors in the brain at clinical doses and
block mainly 5-HT2A responses augment the clinical response to
SSRIs in treatment-resistant patients.27,28
To decrease clogP and increase polarity and thus possibly
improve solubility of the compounds in the series, we decided to
introduce a hydroxyl group on the linker chain. Because intro-
duction of a hydroxyl group might affect the overall efficacy and
false positives profiles, the corresponding 2,3-dichlorophenylpi-
perazine analogues were also synthesized and evaluated.
Introduction of hydroxyl group into the linker chain also revealed
a range of interesting profiles and subtle effects on intrinsic activity as
shown in Table 4. Hydroxyl compound 106 afforded about 2-fold
drop in 5-HT2A and 5-fold drop in 5-HT2C but with slightly
increased intrinsic activity at SERT compared to the corresponding
deoxy analogue 43. A 2-F-Phenyl analogue 122 afforded about a
6-fold drop in 5-HT2A and a 9-fold drop in 5-HT2C but maintained
SERT affinity. 2,3-Dihydrobenzo[b][1,4]dioxinylmoietyonC-1on
the imidazole ring (138) gave the most desirable intrinsic activity
profiles yet observed, maintaining high affinities at 5-HT2A and
SERT but again accompanied by decreased 5-HT2C intrinsic
activity. Overall, introduction of hydroxyl group into the linker led
to the discovery of compounds with good SERT inhibitory activity
with additional moderate 5-HT2A receptor antagonism.
Interesting compounds were further evaluated in vivo with
immobility in forced swimming test (FST) on mice.29 Fluoxetine
(N-methyl-3-phenyl-3-[4-(trifluoromethyl)phenoxy]propan-1-
amine), venlafaxine ((RS)-1-[2-dimethylamino-1-(4-methoxy-
phenyl)-ethyl]cyclohexanol), paroxetine ((3S,4R)-3-[(2H-1,3-
benzodioxol-5-yloxy)methyl]-4-(4-fluorophenyl)piperidine), and
sertraline ((1S,4S)-4-(3,4-dichlorophenyl)-N-methyl-1,2,3,4-tetra-
hydronaphthalen-1-amine) were used as reference compounds
for comparison. The results are shown in Figure 3. At 50 mg/kg,
des-methyl at C-5 of imidazole (81) showed virtually no
appreciable immobility effect. This phenomenon was also
observed inanother des-methylanalogue69 which is devoidof any
antidepressant-like effect at 25 mg/kg. Thus, a substituent on C-5
imidazole ring appears to be minimal requirement for in vivo
efficacy against FST model on mice. Compared to sertraline,
which demonstrated the most impressive immobility performance
among the reference compounds tested at 50 mg/kg, compounds
46, 57, 68, 70, and 131 showed superior in vivo immobility data.
Among them, compounds 68, 70, and 131 also displayed nice
dose-dependency. As oral dose reduced down from 25 to 10 mg/
kg, virtually all the compounds tested showed insufficient anti-
depressant activity. Either 70 or 131 demonstrated merely
moderate in vivo efficacy (immobility ∼80%) (see Figure 3).
modification significantly reduced intrinsic activity against
5-HT2A/2C in an order of magnitude. This phenomenon is
exemplified in dimethylphenylpiperazine analogue 59 (IC50
91 nM for 5-HT2A, IC50 = 170 nM for 5-HT2C) compared to the
dichlorophenylpiperazine 40 (IC50 = 18.6 nM for 5-HT2A, IC50
=
=
12.1 nM for 5-HT2C). However, its inhibition activity against
SERT is not decreased but rather maintained or even increased as
exemplified in 40 (IC50 = 6 nM). Among the compounds
demonstrating IC50 < 20 nM in Table 3, compound 75 appears
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Table 4. Binding Affinity to Serotonin Receptor 5-HT2A,
5-HT2C, and Serotonin Transporter (SERT) (IC50, nM)
In passing, an interesting observation during exploration of
this series is described in Table 5. When hydroxyl group was
introduced onto the linker chain in the molecule, it was
observed that SERT affinity becomes improved in conjunction
with a drop in 5-HT2A/2C. Notably, significant increases in
intrinsic activity at SERT were observed for compounds with
(R)-configuration as shown in Table 5. Thus, compounds 143
and 145 showed subnanomolar SERT affinities in this series for
the first time (IC50 = 0.64 nM, IC50 = 0.75 nM, respectively).
The affinity difference between (R)- and (S)-configuration is
approximately an order of magnitude. However, it is noted that
racemic compounds 104 or 124 rather gave the highest activity
in in vivo antidepressant activity in forced swimming test on
mice as shown in Figure 4.
To further evaluate this series of compounds, some of the
representative compounds have been tested for hERG inhibi-
tion, cytotoxicity at HT22, genotoxicity (Ames test), and
inhibition against a panel of cytochrome P450 isoforms,
respectively. For instance, compound 68 showed relatively
strong inhibition against hERG (IC50 = 1.3 μM). Also 68
demonstrated moderate inhibitory activity against CYP 2C9
(IC50 = 2.9 μM) and 2D6 (IC50 = 5.4 μM), while 68 lacks
inhibitory activity against 1A2 or 3A4. 68 appeared to show neither
cytotoxicity nor mutagenicity as shown in Table 6. The profile has
been improved in dihydrobenzodioxin 100. For example, 100
improved hERG inhibition in approximately 7-fold, showing IC50
=
8.7 μM. Inhibition against CYP 2D6 was reduced in greater than
3-fold. However, all of three compounds showed moderate liability
against the CYP 2C9 isoform, displaying IC50 = 2.9ꢀ4.4 μM.
Subsequently, the pharmacokinetic properties of 57 were
measured in male SD rats. After oral administration of 5 mg/kg
of 57 to rats, a Cmax of 0.208 μg/mL was obtained at 15 min.
The elimination half-life of 57 following oral administration was
0.37 h in rats. Compound 57 showed only moderate oral
bioavailability (F = 14.92%) in rats. This profile became even
worse in 2-methoxyphenylimidazole 71 as shown in Table 7,
suggesting its solubility-limited absorption.
’ CONCLUSION
In summary, serotonin antagonist reuptake inhibitor (SARI)
drugs that block both 5-HT2 receptors and the serotonin transpor-
ters have been developed. We investigated a series of arylpiperazine
containing imidazole 4-carboxamide derivatives for the treatment of
depressive disorders. The human 5-HT2A/2C receptor has been
implicated in several neurological conditions, and potent selective
5-HT2A/2C ligands may have therapeutic potential for treatment of
CNS diseases such as depression. The imidazole series of com-
pounds was evaluated against 5-HT2A/2C and serotonin reuptake
inhibition. A few of the compounds in the series showed promising
in vitro IC50 values and in vivo antidepressant-like effect in the
forced swimming test (FST). On the basis of these results, current
imidazole series of compounds is being used as tool compound to
identify a development candidate for promising antidepressant.
The second in vivo test involves spontaneous locomotor
activity test. The results are illustrated in Figure 3. Dichlorophe-
nylpiperazine compound 106 was observed to show obvious
hyperactivity. Additional dichlorophenylpiperazine analogues
43, 46, and 138 also appear to have hyperactivity. At this point,
it was observed that as arylpiperazine was altered from dichlor-
ophenylpiperazine into the corresponding dimethylphenylpiper-
azine, some degree of spontaneous locomotor activity was
decreased as exemplified in compounds 69, 100, and 103.
Encouragingly, 3-chloro-2-methylphenylpiperazine analogues
68, 70, and 131, which we had introduced as novel arylpiperazine
analogues to reduce hyperactivity with maintaining antidepres-
sant activity, did not show hyperactivity but normal activity.
’ EXPERIMENTAL SECTION
Chemistry. Unless otherwise noted, all starting materials were
obtained from commercial suppliers and used without further purifica-
tion. All references to ether are to diethyl ether; brine refers to a
saturated aqueous solution of NaCl. Unless otherwise indicated, all
temperatures are expressed in °C. All reactions are conducted under an
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Figure 3. Antidepressants activity immobility in forced swimming test on mice (FST, %) and spontaneous locomotor activity (SLA, cm). Effects of the
drugs on immobility in forced swimming test on mice. a) Drugs (50, 25, and 10 mg/kg) were injected orally (po) 60 min before the testing, and total
duration of immobility was recorded during the last 5 min of the 6-min testing period. (Values are means ( SEM). b) Locomotor activities of the mice
treated with compounds or vehicle were counted for 30 min by Activity Analyzer. Compound (50 mg/kg) or vehicle was administered orally (po) 60 min
before the test. Data were expressed as mean ( SEM of 6ꢀ7 mice.
inert atmosphere at room temperature unless otherwise noted, and all
solvents are of the highest available purity unless otherwise indicated.
Microwave reaction was conducted with a Biotage Initiator microwave
synthesizer. 1H NMR and 13C NMR spectra were recorded on Bruker
400 MHz AVANCE III. Chemical shifts were expressed in parts per
million (ppm, δ units). Coupling constants are in units of hertz (Hz).
Splitting patterns describe apparent multiplicities and are designated as s
(singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad).
Mass spectra were obtained with either a Micromass, Quattro LC triple
quadruple tandem mass spectometer, ESI or Agilent, 1200LC/MSD,
ESI. For preparative HPLC, ca. 100 mg of a product was injected in 1 mL
of DMSO onto a SunFire Prep C18 OBD 5 μm 19 mm ꢁ 100 mm
column with a 10 min gradient from 10% CH3CN to 90% CH3CN in
H2O (purification systems from Gilson, Inc. or Waters, Inc.). The tested
compounds were determined to be >95% pure by HPLC. Flash
chromatography was carried using Merck Silica Gel 60 (230ꢀ400
mesh). A Biotage SP1 FLASH purification system or Biotage Isolera
FLASH purification system was used for normal phase column chro-
matography with ethyl acetate/hexane or tetrahydrofuran/hexane. Most
of the reactions were monitored by thin-layer chromatography on 0.25
mm E. Merck silica gel plates (60F-254), visualized with UV light using a
5% ethanolic phosphomolybdic acid or p-anisaldehyde solution. Except
where otherwise noted, all reactions were run under an inert atmosphere
of nitrogen gas using anhydrous solvents.
Ethyl 2-(Hydroxyimino)-3-oxobutanoate (6). To a solution of ethyl
acetoacetate (5, 30 g, 0.23 mol) in acetic acid (35 mL) was added slowly
sodium nitrite (18.0 g, 0.25 mol) in cold water (40 mL) at ꢀ10 °C. The
reaction mixture was stirred for 1 h, then cold water (120 mL) was added and
then stirred for 3 h. The mixture was extracted with diethyl ether (300 mL).
The organic layer was washed with saturated NaHCO3 (400 mL ꢁ 2), then
dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The
obtained product (21.46 g, white solid) was used for the next step without
purification. 1H NMR (400 MHz, DMSO-d6) δ 13.20 (s, 1H), 4.21 (q, J =
7.2 Hz, 2H), 2.32 (s, 3H), 1.92 (t, J = 6.8 Hz, 3H).
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Table 5. Binding Affinity to Serotonin Receptor 5-HT2A, 5-HT2C and Serotonin Transporter (SERT) (IC50, nM)
in vitro
compd
R1
Ph
R2
R4
R5
5HT2A
5HT2C
SERT
104
143
144
124
145
146
131
147
148
Me
Me
Me
Me
Me
Me
Ph
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Cl
racemic
247.6
72.5
646
579
270
244
26.7
95
2741
227
5
Ph
(R)-configuration
(S)-configuration
racemic
0.64
5.08
7.59
0.75
10.3
478.4
257
Ph
1779
>10000
193
2-F-Ph
2-F-Ph
2-F-Ph
4-F-Ph
4-F-Ph
4-F-Ph
(R)-configuration
(S)-configuration
racemic
1031
>10000
479
Ph
Cl
(R)-configuration
(S)-configuration
Ph
Cl
20.1
534
232
with aqueous K2CO3. The organic layer was dried over anhydrous
MgSO4, filtered, and concentrated in vacuo. The crude product was
purified by flash column chromatography (Biotage SP1 FLASH
purification system) to provide the title compound (4.97 g, 76%) as
1
a yellow solid. H NMR (400 MHz, CDCl3) δ 7.57ꢀ7.51 (m, 3H),
7.20ꢀ7.18 (m, 2H), 4.41 (q, J = 6.8 Hz, 2H), 2.31 (s, 3H), 2.22 (s, 3H),
1.42 (t, J = 7.2 Hz, 3H); MH+ 245.
Lithium 2,5-Dimethyl-1-phenyl-1H-imidazole-4-carboxylate (11).
To a solution of ethyl 2,5-dimethyl-1-phenyl-1H-imidazole-4-carboxy-
late (10, 4.97 g, 20.3 mmol) in THF/water (15/15 mL) was added
lithium hydroxide monohydrate at room temperature. The reaction
mixture was stirred at 55 °C for 12 h, and then the mixture was
concentrated in vacuo. The crude solid was washed with diethyl ether
to provide the title compound (4.06 g, 90%) as a white solid. The
Figure 4. Antidepressants Activity immobility in forced swimming test
on mice (FST, %). Effects of drugs on immobility in forced swimming test
on mice. Drugs (50, 25, and 10 mg/kg) were injected orally (po) 60 min
before the testing, and total duration of immobility was recorded during
the last 5 min of the 6-min testing period. (Values are means ( SEM).
1
obtained product was used for the next step without purification. H
NMR (400 MHz, CD3OD) δ 7.59ꢀ7.54 (m, 3H), 7.31ꢀ7.28 (m, 2H),
2.26 (s, 3H), 2.14 (s, 3H). MS m/z: 217 (MH+ ꢀ Li+).
N-Phenylbenzimidamide (13). To NaHMDS (49 mL, 48.5 mmol,
1.0 M solution in THF) was added dropwise a solution of aniline (9,
4.5 mL, 48.5 mmol) in anhydrous THF (10 mL) under N2. After 20 min,
a solution of benzonitrile (12, 5.0 mL, 48.5 mmol) in anhydrous THF
(10 mL) was slowly added. The reaction mixture was stirred for 12 h,
poured into cold water, and extracted with EtOAc. The organic layer was
dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The
crude product was used for the next step without purification.
Ethyl 5-Methyl-1,2-diphenyl-1H-imidazole-4-carboxylate (15). A
mixture of N-phenylbenzimidamide (13, 1.0 g, 5.10 mmol), 3-bromo-
2-oxovalerate (14, 1.3 g, 6.12 mmol), and NaHCO3 in i-PrOH was
stirred at 90 °C for 12 h. The reaction mixture was concentrated under
reduced pressure, and then the residue was diluted with EtOAc and
washed with H2O. The organic layer was dried over anhydrous MgSO4,
filtered, and concentrated in vacuo. The crude product was purified by
flash column chromatography (Biotage Isolera FLASH purification
system) to provide the title compound (0.86 g, 55%) as a yellow solid.
Lithium 5-Methyl-1,2-diphenyl-1H-imidazole-4-carboxylate (16).
Following the procedure for compound 11, the desired compound
was obtained as a white solid. 1H NMR (400 MHz, CD3OD) δ
7.52ꢀ7.48 (m, 3H), 7.34ꢀ7.31 (m, 2H), 7.26ꢀ7.17 (m, 5H), 2.36 (s,
3H). MS m/z: 279 (MH+ ꢀ Li+).
Ethyl 2-Acetamido-3-oxobutanoate (8). To a mixture of ethyl
2-(hydroxyimino)-3-oxobutanoate (6, 9.32 g, 58.5 mmol) and Pd/C
(400 mg, palladium on carbon, 10 wt %, support activated carbon, wet,
Degussa type E101 NE/W) in EtOH was added acetic anhydride
(7, 11.0 mL, 117.0 mmol) at room temperature. The reaction mixture
was stirred at room temperature under H2 for 15 h. The palladium was
filtered off (Filter aid, Celite 521 AW), and then the filtrate was
concentrated in vacuo. The crude product was purified by flash column
chromatography (Biotage SP1 FLASH purification system) to provide
the title compound (9.48 g, 86%) as a white solid. 1H NMR (400 MHz,
CDCl3) δ 6.62 (br s, 1H), 5.25 (d, J = 6.4 Hz, 1H), 4.28 (q, J = 7.2 Hz,
2H), 2.39 (s, 3H), 2.07 (s, 3H), 1.32 (t, J = 7.2 Hz, 3H).
Ethyl 2,5-Dimethyl-1-phenyl-1H-imidazole-4-carboxylate (10). To
a solution of ethyl 2-acetamido-3-oxobutanoate (8) (5.0 g, 26.7 mmol)
and aniline (9, 7.3 mL, 80.1 mmol) in butyronitrile (10 mL) was added
trifluoroacetic acid (6.2 mL, 80.1 mmol) at room temperature. The
reaction mixture was irradiated in a microwave reactor (Biotage
Initiator) for 40 min at 140 °C. The mixture was concentrated under
reduced pressure. The residue was diluted with CH2Cl2 and washed
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Table 6. In Vitro Toxicity of Compound 68, 70, and 100
Table 7. Pharmacokinetic Parameters and Brain/Plasma Ratio of Compounds 57 and 71 after Oral (5 mg/kg) and iv (1 mg/kg)
Administration to Rat
compd
57
71
PK parameters
iv (1 mg/kg, n = 3)
oral (5 mg/kg, n = 3)
iv (1 mg/kg, n = 3)
oral (5 mg/kg, n = 3)
C
max (μg/mL)
0.245 ( 0.032
0.001 ( 0.000
0.208 ( 0.117
0.000 ( 0.000
0.250 ( 0.000
0.372 ( 0.012
10.811 ( 2.986
1.631 ( 1.246
0.408 ( 0.056
0.004 ( 0.001
0.065 ( 0.046
0.000 ( 0.000
0.250 ( 0.000
0.121 ( 0.038
1.312 ( 0.839
0.262 ( 0.168
Clast (μg/mL)
Tmax(h)
t1/2 (h)
0.438 ( 0.064
10.931 ( 2.019
10.931 ( 2.019
0.226 ( 0.019
7.611 ( 0.488
7.611 ( 0.488
AUCinf (min μg/mL)
3
AUCinf/dose
BA (%)
14.92
3.45
N-(4-Chlorophenyl)butyrimidamide (19). To a solution of butyro-
nitrile (18, 7.5 mL, 86.2 mmol) and AlCl3 in toluene was added
4-chloroaniline (17, 10.0 g, 78.4 mmol). The reaction mixture was
stirred at 115 °C for 5 h. The mixture was diluted with water (200 mL)
and extracted with EtOAc (200 mL). The aqueous layer was neutralized
with saturated NaHCO3 (500 mL) and extracted with EtOAc (300 mL ꢁ 2).
The organic layer was dried over anhydrous MgSO4, filtered, and concen-
trated in vacuo. The obtained product (9.2 g, pale-brown solid) was used for
the next step without purification.
CD3OD) δ 7.58ꢀ7.56 (m, 2H), 7.49 (s, 1H), 7.43ꢀ7.40 (m, 2H),
2.63ꢀ2.59 (m, 2H), 1.70ꢀ1.61 (m, 2H), 0.86 (t, J = 7.6 Hz, 3H). MS m/z:
265 (MH+ ꢀ Li+).
4-[1-Dimethylamino-eth-(Z)-ylidene]-2-methyl-4H-oxazole-5-one
(24). N-Acetylglycine (23, 10 g, 85.5 mmol) was dissolved in N,N0-
dimethylacetamide (20 mL, 21.4 mmol), and POCl3 (19.6 mL, 21.4
mmol) was added dropwise slowly at 0 °C. The reaction mixture was
stirred at 50 °C for 3 h and then cooled to room temperature. CH2Cl2
(50 mL) was added and the mixture poured into iceꢀwater. The
resulting solution was basified with ammonium hydroxide to over than
pH 8. The organic extracts were washed with 50 mL of water, dried over
MgSO4, filtered, and evaporated under reduced pressure. The residue
was purified by normal phase preparative column, and the desired
compound was obtained as an orange solid (6.7 g, 47%). MH+ 169.
(E)-Ethyl 2-Acetamido-3-(dimethylamino)but-2-enoate (25). 4-[1-Di-
methylamino-eth-(Z)-ylidene]-2-methyl-4H-oxazole-5-one (24, 7.54 g, 44.8
mmol) was dissolved in ethanol (50 mL), and sodium hydride (179 mg, 4.5
mmol, 60% dispersion in mineral oil) wad added at room temperature. The
solution was refluxed for 1 h. The solvent was evaporated, and the crude
product was used without any further purification for the next step. MH+ 215.
(E)-Ethyl 2-Acetamido-3-(cyclopentylamino)but-2-enoate (27).
(E)-Ethyl 2-acetamido-3-(dimethylamino)but-2-enoate (25, 1 g,
4.67 mmol) and cyclopentylamine (26, 0.5 mL) were stirred at room
temperature in AcOH (10 mL) for overnight. The reaction mixture
was diluted slowly with water (10 mL) and evaporated under reduced
pressure to obtain the desired product as a dark-brown oil, which
Ethyl 1-(4-Chlorophenyl)-2-propyl-1H-imidazole-4-carboxylate (21).
To a solution of N-(4-chlorophenyl)butyrimidamide (19, 3.5 g, 17.80
mmol) and NaHCO3 (3.14 g, 37.38 mmol) in i-PrOH was added ethyl
bromopyruvate (20, 4.7 mL, 37.38 mmol) under N2. The reaction mixture
was stirred at 85 °C for 72 h, and then AcOH (15 mL)was added. After 4 h,
the mixture was concentrated under reduced pressure. The residue was
diluted with H2O and extracted with CH2Cl2. The organic layer was washed
with saturated NaHCO3 and 1N HCl solution and then dried over anhydrous
MgSO4, filtered, and concentrated in vacuo. The crude product was purified
by flash column chromatography (Biotage Isolera FLASH purification
1
system) to provide the title compound (1.28 g, 25%) as a brown oil. H
NMR (400 MHz, CDCl3) δ 7.62 (s, 1H), 7.51ꢀ7.47 (m, 2H), 7.26ꢀ7.23
(m, 2H), 4.39 (q, J = 7.2 Hz, 2H), 2.62 (t, J = 7.6 Hz, 2H), 1.71ꢀ1.61
(m, 2H), 1.39 (t, J = 6.8 Hz, 3H), 0.86 (t, J = 7.2 Hz, 3H). MH+ 293.
Lithium 1-(4-Chlorophenyl)-2-propyl-1H-imidazole-4-carboxylate
(22). Following the procedure for compound 11, the desired compound
was obtained as a pale-brown solid (3.00 g, 86%). 1H NMR (400 MHz,
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could be used without any further purification for the next step.
MH+ 256.
chloride (459 mg, 1.27 mmol), EDCI (305 mg, 1.59 mmol), and HOBt
(286 mg, 2.12 mmol) in anhydrous DMF (5 mL) was added NMM
(580 μL, 5.30 mmol). The reaction mixture was stirred at room
temperature for 15 h. The mixture was concentrated under reduced
pressure. The residue was diluted with EtOAc and washed with saturated
NaHCO3. The organic layer was dried over anhydrous MgSO4, filtered,
and concentrated in vacuo. The crude product was purified by pre-
parative HPLC (purification system, Gilson Inc.) and followed by
treatment of 2 M HCl solution in diethyl ether generated to provide
the title compound (267 mg, 46%) as a white solid. 1H NMR (400 MHz,
DMSO-d6) δ 10.52 (s, 1H), 8.45 (s, 1H), 7.59ꢀ7.20 (m, 13H), 6.97 (dd,
J = 7.2, 2.4 Hz, 1H), 3.81ꢀ3.68 (m, 2H), 3.65ꢀ3.51 (m, 2H), 3.50ꢀ3.31
(m, 4H), 2.77ꢀ2.60 (m, 4H), 2.45 (s, 3H), 2.09ꢀ1.95 (m, 2H). 13C
NMR (100 MHz, DMSO-d6) δ 161,4, 149.5, 144.3, 134.9, 134.8, 132.7,
130.1, 129.9, 129.8, 128.8, 128.6, 128.3, 128.0, 127.4, 127.0, 126.0, 125.2,
119.8, 53.6, 51.1, 47.6, 35.9, 23.7, 10.2. MS m/z: 548 (MH+ ꢀ 2HCl);
positive HR-FAB-MS m/z 548.1990 (MH+ ꢀ 2HCl) (calcd for C30H31-
Cl2N5O: 548.1984).
Ethyl 1-Cyclopentyl-2,5-dimethyl-1H-imidazole-4-carboxylate (28).
Ammonium sulfate (100 mg) was added to a solution of (E)-ethyl
2-acetamido-3-(cyclopentylamino)but-2-enoate (27, 1.5 g, 5.9 mmol)
and hexamethyldisilazane (15 mL) and refluxed overnight at 150 °C. The
reactionmixture was evaporatedand extractedwithEtOAcandwater. The
organic layer was evaporated, and the residue was purified with 20%
methanol in CH2Cl2 to produce as a light-brown solid (1.0 g, 71.4%). 1H
NMR (400 MHz, CDCl3) δ 4.31 (q, J = 6.5 Hz, 2H), 3.77 (m, 1H), 2.59
(s, 3H), 2.33 (s, 3H), 2.15ꢀ2.06 (m, 2H), 1.83ꢀ1.80 (m, 2H), 1.72ꢀ1.54
(m, 2H), 1.32 (t, J = 6.5 Hz, 3H). MH+ 237.
2-(3-(4-(2,3-Dichlorophenyl)piperazin-1-yl)propyl)isoindoline-1,3-dione
(32). The mixture of 1-(2,3-dichlorophenyl)piperazine hydrochloride
(31, 10.0 g, 37.4 mmol), N-(3-bromopropyl)phthalimide (30, 9.09 g,
33.9 mmol), and potassium carbonate (11.7 g, 84.8 mmol) in DMF was
stirred at room temperature. The mixture was concentrated under
reduced pressure. The residue was diluted with CH2Cl2 and washed
with water. The organic layer was dried over anhydrous MgSO4, filtered,
and concentrated in vacuo. The obtained product was washed with
EtOH to provide the title compound (11.2 g, 80%) as a white solid. 1H
NMR (400 MHz, CDCl3) δ 7.88ꢀ7.83 (m, 2H), 7.74ꢀ7.69 (m, 2H),
7.15ꢀ7.09 (m, 2H), 6.82 (dd, J = 7.6, 2.4 Hz, 1H), 3.80 (t, J = 7.2 Hz,
2H), 2.93ꢀ2.87 (m, 4H), 2.62ꢀ2.56 (m, 4H), 2.51 (t, J = 5.6 Hz, 2H),
1.94ꢀ1.87 (m, 2H). MH+ 418; mp 119.2 °C.
Biological Methods. Measurement of Binding Affinity for Ser-
otonin 5-HT2A and 5-HT2C Receptors. Receptor binding affinities of the
compounds for serotonin receptors were measured by the method
described in the literature.25
For serotonin 5-HT2A binding, an aliquot of human recombinant
serotonin 5-HT2A receptor (PerkinElmer Life and Analytical Sciences,
USA) expressed in CHO-K1 cells (5 μg/well) and 1 nM [3H]Ketanserin
(Perkin-Elmer) were used in the presence of mianserin (20 μM) as
nonspecific. The reaction mixture was incubated for 60 min at 27 °C
using 50 mM Tris-HCl (pH 7.4) buffer containing 4 mM CaCl2 and
0.1% ascorbic acid and harvested through filtermate A glass fiber filter
(Wallac, Finland) presoaked in 0.5% polyethyleneimine (PEI) by
MicroBeta Filtermate-96 harvester (PerkinElmer) to terminate the
reaction and then washed with ice-cold 50 mM Tris-HCl buffer solution
(pH 7.4). The filter was then covered with MeltiLex, sealed in a sample
bag, and dried in an oven. The radioactivity retained in the filter was
finally counted using MicroBeta Plus (Wallac).
3-(4-(2,3-Dichlorophenyl)piperazin-1-yl)propan-1-amine Dihydro-
chloride (33). To a suspension of 2-(3-(4-(2,3-dichlorophenyl)piperazin-
1-yl)propyl)isoindoline-1,3-dione (32, 11.1 g, 26.5 mmol) in EtOH was
added hydrazine monohydrate. The reaction mixture was stirred at room
temperature for 6 h and then the white solid filtered off. The filtrate was
concentrated under reduced pressure. The residue was diluted with
CH2Cl2 and washed with saturated NaHCO3. The organic layer was
dried over anhydrous MgSO4, filtered, and concentrated in vacuo. To the
crude product in diethyl ether was added 2 M HCl solution in diethyl
ether (10 mL). The obtained product was washed with diethyl ether
to provide the title compound (6.98 g, 73%) as a white solid. 1H NMR
(400 MHz, CD3OD) δ 7.22ꢀ7.18 (m, 2H), 7.08(dd, J = 7.2, 2.4Hz, 1H),
3.06ꢀ3.02 (m, 6H), 2.72ꢀ2.64 (m, 4H), 2.60 (t, J = 6.8 Hz, 2H),
1.90ꢀ1.84 (m, 2H). MS m/z: 288 (MH+ ꢀ 2HCl).
The binding affinity (IC50) of a compound for the receptor was
calculated by computerized nonlinear regression analysis (GraphPad
Prism Program, San Diego, USA) using 7ꢀ8 varied concentrations of
the compound run in duplicate tubes.
1-Amino-3-(4-(2,3-dichlorophenyl)piperazin-1-yl)propan-2-ol Dihy-
drochloride (36). To a stirred solution of N-(2,3-epoxypropyl)phthali-
mide (34, 10 g, 0.049 mol) in THF (100 mL) was added 1-(2,3-
dichlorophenyl)piperazine hydrochloride (31, 8.7 g, 0.033 mol) and
triethylamine (4.6 mL, 0.033 mol) at room temperature, and then the
resultant solution was heated at 80 °C overnight. The reaction was
quenched with H2O and extracted with CH2Cl2. The organic layer was
washed with brine, dried over MgSO4, filtered, and evaporated. The
solid residue was solidified with CH2Cl2 (20 mL)/diethyl ether
(200 mL), filtered, and dried in vacuo, which was used for the following
synthesis without further purification. To the prepared white solid
piperazine (35, 13 g, 0.030 mol) in EtOH was added hydrazine
monohydrate (20 mL), and the reaction solution was refluxed at
80 °C for 2 h. The reaction solution was cooled to room temperature
and evaporated. The oily crude compound was extracted with EtOAc/
H2O, and organic the layer was combined and evaporated. The pale-
yellow solid was tritulated with ether to afford pure targeted amine (36,
8.7 g, 95%) as white solid. 1H NMR (400 MHz, CD3OD) δ 7.31ꢀ7.27
(m, 2H), 7.19ꢀ7.15 (m, 1H), 4.40ꢀ4.36 (m, 1H), 3.42ꢀ3.39 (m, 4H),
3.37ꢀ3.32 (m, 4H), 3.24ꢀ2.93 (m, 4H). MS m/z: 304 (MH+ ꢀ 2HCl).
General Procedure (Synthesis of A). N-(3-(4-(2,3-Dichloro-
phenyl)piperazin-1-yl)propyl)-5-methyl-1,2-diphenyl-1H-imidazole-4-
carboxamide Dihydrochloride (43). To a mixture of lithium 5-methyl-
1,2-diphenyl-1H-imidazole-4-carboxylate (16, 300 mg, 1.06 mmol),
1-amino-3-(4-(2,3-dichlorophenyl)piperazin-1-yl)propan-2-ol dihydro-
For serotonin 5-HT2C binding, frozen membranes from stable CHO-
K1 cell line expressing the human recombinant 5-HT2C receptor
(PerkinElmer, 4 μg/well), [3H]Mesulergine (Amersham, 1.3 nM),
and test compounds were added into 50 mM Tris-HCl (pH 7.4) buffer
containing 0.1% ascorbic acid and 4 mM CaCl2. Nonspecific binding was
determined using 100 μM mianserin. The incubations were performed
for 60 min at 27 °C, and these were terminated by rapid filtration
through Filtermate A glass fiber filter presoaked in 0.5% PEI.
Measurement of Binding Affinity for Serotonin Transporter. For
serotonin transporter binding assays, a reaction mixture with a final
volume of 0.25 mL was prepared by mixing a test compound, human
serotonin transporter membrane expressed in HEK-293 cells (Perkin-
Elmer, 5 μg/well), [3H]Imipramine (Perkin-Elmer, 2 nM), and 50 mM
Tris-HCl (pH 7.4) buffer containing 120 mM NaCl and 5 mM KCl. The
reaction mixture was incubated for 30 min at 27 °C and harvested
through Filtermate A glass fiber filter presoaked in 0.5% PEI with ice-
cold 50 mM Tris-HCl buffer (pH 7.4) containing 0.9% NaCl.
Measurement of Antidepressants Activity in Forced Swimming
Test. To evaluate the antidepressants activity of the compounds, the
inhibitory effects on immobility in forced swimming test in mice
were measured according to the methods described by Porsolt
et al.29
Each mouse was placed in a 25 cm glass cylinder (10 cm diameter)
containing 15 cm of water maintained at 22 ( 1 °C and was forced to
swim for 10 min. Twenty-four hours later, the mouse was replaced into
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the cylinder and the total duration of immobility was recorded during
the last 5 min of the 6 min testing period. Mice are judged immobile
when they float in an upright position and make only small movements
to keep their head above water. Test drugs were suspended in 3% Tween
80 solution and administered orally (po) 60 min before the testing.
Measurement of Spontaneous Locomotion Activity. Locomotor
activity was assessed with an animal activity monitoring apparatus
(EthoVision, Nolduls, Netherlands.). Fifty minutes after the adminis-
tration, mice were placed individually in 32 cm ꢁ 45 cm ꢁ 32 cm plastic
cages, to which they had not been previously exposed, under dim light
and sound-attenuated conditions. After an initial 10 min of familiariza-
tion period, locomotor activity was monitored for 30 min. The results of
the locomotor activity tests were expressed as mean ( SEM.
Pharmacokinetic Study. Male SpragueꢀDawley rats (260ꢀ280 g)
were purchased from Orient Bio Inc. (Gyeonggi-Do, Korea). Two days
before dosing, the femoral artery and vein (intravenous (iv) only) were
cannulated using polyethylene tube SP28 (Natsume, Tokyo, Japan). For
intravenous administration, prepared dosing solution was injected via the
femoral vein. The rats were fasted overnight before drug administration
and until 6 h after dosing. For the po experiment, rats (three in each
group) were given a single dose of 5 mg/kg, and heparinized samples of
blood (0.3 mL) were collected at 5, 15, 30, 60, 90, 120, 240, 360, 480, 600
min, and 24 h postdose. For the iv experiment, rats (three in each group)
were given a single 1 mg/kg dose, and blood samples were collected at 5,
10, 20, 40, 60, 90, 120, 240, 360, and 480 min postdose. Plasma was
harvested after centrifugation and stored frozen at ꢀ20 °C until analyzed.
The concentrations of compounds in plasma were determined by LC/
MS/MS (API3200). The results are shown as the maximum plasma
concentration (Cmax), the time to reach peak plasma concentration
(Tmax), terminal half-life (t1/2), and the area under the plasma concen-
trationꢀtime curve from zero to time infinity (AUC0ꢀ∞).
Strain TA 100 was chosen as the representative tester strain because of
its high spontaneous reversion rate. Spontaneous revertant numbers
were counted and plotted against the dose of the test chemical to
produce a survival curve for the his+ genotype.
The mutagenicity assay was performed by mixing one of the tester
strains which was cultured overnight with the test substance in the
presence and in the absence of S9 mixture condition, sodium phosphate
buffer added instead of S9 mixture both in negative and positive control in
a test tube. Then, incubating the mixture in water bath for 30 min at 37 °C
and after incubation, the mixture was mixed with top agar containing a
minimal amount of histidine and then poured onto the surface of a γ-ray
sterile Petri dish (Falcon, USA) containing 25 mL of solidified bottom
agar. The finished plates were incubated for 48 h at 37 °C, and revertant
colonies were counted later. Negative control plates containing no added
test chemical, but positive control plates containing appropriate amounts
of chemicals known to be active were included with each tester strain.
Sodium azide (SA) and 2-aminoanthracene (2-AA) were used as positive
controlsubstances. Compounds weretested atseven concentrationpoints
in duplicate at a range of concentrations up to 2500 μg/plate. A response
was considered to be positive in our criteria if there was a dose-dependent
increase in revertants per plate, resulting in at least a doubling of the
background reversion rate for strains TA 100.
’ ASSOCIATED CONTENT
S
Supporting Information. Experimental procedures for
b
molecules not described in main paper text and H NMR, 13C
1
NMR, and HPLC spectrum data for compounds 57, 68, 70, 71,
100, 131, 143, and 145. This material is available free of charge
via the Internet at http://pubs.acs.org.
Measurement of hERG Inhibition. HEK293 cells stably expressing the
hERG potassium channel are a Homo sapien embryonic kidney epithelial
cell line transformed with the adenovirus 5 DNA. The parental HEK293
cell line was devoid of the IKr currents before transfection of the hERG
cDNAs. The HEK293 cells were cotransfected with the hERG cDNA and
G418-resistant gene incorporated into a modified pcDNA3 plasmid. The
stable transfectants were maintained under constant selection pressure
incorporated into the culture media. The culture media was Minimum
Essential Medium (MEM, including Earle’s Salts and L-glutamine)
supplemented with approximately 10% fetal bovine serum, 100 U/mL
of penicillin-g, 100 μg/mL streptomycin, 0.1 mM nonessential amino
acids (NEAA) solution, 1 mM sodium pyruvate, and 400 μg/mL G418.
Measurement of CYP. To evaluate the CYP of the compounds, we
used P450-Glo screening system. The systems include a membrane
preparation containing recombinant human cytochrome P450 (CYP)
enzyme, negative control membranes, a luminogenic substrate appro-
priate for the CYP enzyme, NADPH regeneration system, reaction
buffer, Luciferin detection reagent, and Luciferin-Free water. The
membranes are prepared from baculovirus-infected insect cells and
contain human CYP enzyme and P450 reductase (and cytochrome b5
for CYP2C9, 2C19, and 3A4). The negative control membranes are
devoid of CYP activity. The assays are ideal for testing the effects of
drugs and new chemical entities on CYP enzyme activities.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: +82-31-260-9892. Fax: +82-31-260-9020. E-mail: jinhwalee@
greencross.com.
’ ACKNOWLEDGMENT
We are grateful to Dr. Eun Chul Huh for his leadership and
supports as head of GCC R&D. This work was in part supported
by Korea Ministry of Knowledge Economy.
’ ABBREVIATIONS USED
SARI, serotonin antagonist and reuptake inhibitor; 5-HT, sero-
tonin or 5-Hydroxytryptamine; 5-HT2A/2C, serotonin receptor
subtype 2A/2C; CNS, central nervous system; FST, forced
swimming test; SSRIs, selective serotonin reuptake inhibitors;
SERT, serotonin transporter; DMF, N,N0-dimethylformamide;
EDCI, N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide; NMM,
N-methylmorpholine; HOBt, 1-hydroxybenzotriazole; CHO-K1
cells, Chinese hamster ovary-K1 cells; DMSO, dimethyl sulfox-
ide; SLA, spontaneous locomotor activity; SD rat, Spragueꢀ
Dawley rat; PK, pharmacokinetics; AUC, area under the plasma
concentration time curve; IC50, half-maximal inhibitory concen-
tration; BA, bioavailability
Measurement of Cytotoxicity. HT22 cells (mouse hippocampal
neuron, Salk Institute and KRIBB) were plated in a 96-well plates 3 ꢁ
103 cells/well for 18 h before treatment. 3-HM HBr were treated and
3
incubated for 48 h in growth media (DMEM with 10% FBS and 1%
penicillin streptomycin). MTT (3-(4,5-dimethylthazol-2-yl)-2,5-diphe-
nyltetrazolium bromide, Sigma) was treated for 4 h and detected with a
plate reader at a wavelength = 450 nm.
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