Arylpiperazine-containing pyrrole 3-carboxamide derivatives targeting serotonin 5-HT2A, 5-HT2C, and the serotonin transporter as a potential antidepressant

Article abstract of DOI:10.1016/j.bmcl.2010.01.093

Arylpiperzine-containing pyrrole 3-carboxamide derivatives were synthesized and evaluated as novel antidepressant compounds. The various analogues were efficiently prepared and bio-assayed for binding to 5-HT_2A, 5-HT_2C receptor, and

Full text of DOI:10.1016/j.bmcl.2010.01.093

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1705–1711
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Arylpiperazine-containing pyrrole 3-carboxamide derivatives targeting
serotonin 5-HT2A, 5-HT2C, and the serotonin transporter as a potential
antidepressant
Suk Youn Kang a, Eun-Jung Park a, Woo-Kyu Park b, Hyun Jung Kim a, Daeyoung Jeong b, Myung Eun Jung a,
Kwang-Seop Song a, Suk Ho Lee a, Hee Jeong Seo a, Min Ju Kim a, MinWoo Lee a, Ho-Kyun Han a,
Eun-Jung Son a, Ae Nim Pae c, Jeongmin Kim a, Jinhwa Lee a,
*
a Central Research Laboratories, Green Cross Corporation, 303 Bojeong-Dong, Giheung-Gu, Yongin 446-770, Republic of Korea
b Pharmacology Research Center, Korea Research Institute of Chemical Technology (KRICT), 100 Jang-Dong, Yuseong-Gu, Daejeon 305-343, Republic of Korea
c Biochemical Research Center, Korea Institute of Science and Technology, 39-1 Hawolgok-Dong, Seongbuk-Gu, Seoul 136-791, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Arylpiperzine-containing pyrrole 3-carboxamide derivatives were synthesized and evaluated as novel
antidepressant compounds. The various analogues were efficiently prepared and bio-assayed for binding
to 5-HT2A, 5-HT2C receptor, and 5-HT transporter. Based on their in vitro and in vivo activities as well as
selectivity over other neurotransmitter receptors and PK profiles, 33 and 34 were identified as lead com-
pounds. Consequently, this pyrrole series of compounds appears to be promising enough to warrant fur-
ther investigation.
Received 8 December 2009
Revised 7 January 2010
Accepted 8 January 2010
Available online 21 January 2010
Keywords:
Depression
Antidepressant
Serotonin
Piperazine
Pyrrole
Ó 2010 Elsevier Ltd. All rights reserved.
Depression, especially major depression is among the serious
psychiatric disorders.1,2 Symptoms of depression include sadness,
loss of interest or pleasure in activities that were once enjoyed,
change in appetite or weight, difficulty sleeping or oversleeping,
physical slowing or agitation, energy loss, feeling of worthlessness
or inappropriate quilt, difficulty thinking or concentrating, and
recurrent thoughts of death or suicide. And depression is estimated
to affect 7–8% of the world population.3 Since the synaptic actions of
monoamine neurotransmitters such as norepinephrine (NE) and
serotonin (SER, 5-HT) were known as important drug target of psy-
chiatric diseases, an increasing number of treatment options have
become more available over the past two decades for individuals
with major depression disorder.4,5 Tricyclic antidepressants (TCAs)
and nonselective monoamine oxidase inhibitors (MAOIs) were
developed as first generation of antidepressant. Although they are
highly effective in treating depression, they also have side effects,
for example dry mouth, blurred vision, urinary retention, postural
hypotension, and insomnia.6,7 The first class of psychotropic drugs
to be rationally designed, selective serotonin reuptake inhibitors
(SSRIs) have been the most widely prescribed antidepressants since
1980s.8 Although SSRIs such as fluoxetine, sertraline, paroxetine,
and citalopram have achieved great success in treating depression,
they also have some troublesome effects including sedation, anxi-
ety, headache, tremor, and sexual dysfunction (especially anorgas-
mia) and generally effective only less than two-third patients.
Additionally, delayed onset of action (2–6 weeks) is less than desir-
able in the treatment of depression.9
In recent years, the novel antidepressant model was studied to
prepare compounds with dual or multiple activities.10 Because
numerous side effects are associated with non-selective binding at
post-synaptic 5-HT receptors, it has been proposed that addition
of a 5-HT receptor antagonist component could increase synaptic
5-HT levels, eventually achieve rapid onset time.11–13 With those
approaches, various compounds have been proposed and developed
as potential antidepressants, with dual activity at SERT while bind-
ing antagonistically to the 5-HT2A receptor. For example, Eli Lilly’s
LY367265, 1 exhibits excellent binding affinities (Ki = 2.3 nM for
SERT; Ki = 0.81 nM for 5-HT2A).14 Yamanouchi has also discovered
YM-35992, 2 as an antidepressant with moderate affinities for
SERT/5-HT2A (Ki = 21 nM and 86 nM, respectively).15 Bristol–Myers
Squibb’s Nefazodone, 3 has been described as having a similar mode
of action with an improved side effect profile.16 This compound had
advantages over other antidepressants including reduced possibil-
ity to disturbed sleep or sexual dysfunction, and ability to treat
some patients who did not respond to other antidepressant drugs.
* Corresponding author. Tel.: +82 31 260 9892; fax: +82 31 260 9020.
E-mail address: jinhwalee@greencross.com (J. Lee).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.093

1706
S. Y. Kang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1705–1711
However, Bristol–Myers Squibb discontinued the sale on 2004 with
adverse hepatic events including liver failure.17 More recently, ari-
piprazole, 4, which was approved for the treatment of atypical anti-
psychotics, was also used together with other medications to treat
major depressive disorder in adults. Unlike other FDA-approved
atypical antipsychotics targeting D2 receptor, aripiprazole appears
a 5-HT1A partial agonist, 5-HT2A antagonist, and 5-HT2C partial ago-
nist. In addition, it has moderate affinity for the serotonin trans-
porter. However, it has numerous side effects including headache,
nausea, constipation, anxiety, restlessness, insomnia, nervousness,
and so on. In this regard, there are still urgent medical needs on
the development of novel drugs with better developability charac-
teristics: improved pharmacologic properties and reduced side ef-
fects. Herein, we wish to describe the design, synthesis, and
biological evaluation of novel arylpiperazine-containing pyrrole 3-
carboxamide derivatives targeting serotonin 5-HT2A, 5-HT2C, and
the serotonin transporter as a potential antidepressant (see Fig. 1).
From the literature, common structure of antidepressants hav-
ing dual activities appeared to exhibit combination of two distinct
structural motifs. Aryl piperazine elements and heterocyclic motifs
were connected to each other using a linker frequently to serve as
SERT and 5-HT receptor inhibitors. With this in mind, the general
structure of our target compounds is shown in Figure 2. We envi-
sioned that novel target compounds A can be readily prepared by
typical amide coupling with acid and amine at the final stage. Pyr-
role 3-carboxylic acid B and arylpiperazinyl alkyl amine C can be
used as acid and amine partners, respectively.
Preparation of pyrrole derivatives, particularly consisting of
5-phenyl and 3-carboxylic acid, started from alkylation of ethyl
acetoacetate (5) with 2-bromo-1-phenylethanone (6) using so-
dium hydride to produce alkylated acetoacetate 7 (Scheme 1).18
Cyclization of 7 to ethyl 2-methyl-5-phenyl-1H-pyrrole-3-carbox-
ylate (8) was accomplished by heating ammonium acetate in acetic
acid at 80 °C. Various alkyl groups were substituted at NH of pyr-
F
F
N
HN
O
N
S
O
N
O
HN
O
O
LY367265, 1
YM-35992, 2
H
N
O
O
N
N
N
N
N
O
N
Cl
Cl
N
Cl
aripiprazole, 4
nefazodone, 3
Figure 1. Chemical structures of representative antidepressant compounds.
O
R2
O
N
OH
R2
N
N
+
N
n
N
N
n
H
Me
H2N
R1
(n = 1,2,3)
N
R1
(n = 1,2,3)
Me
B
C
A
Figure 2. Preparation of target compounds using peptide bond formation.
O
O
O
O
OEt
OEt
Me
O
O
a
b
Br
O
+
OEt
N
H
5
6
7
8
O
O
OEt
Me
OH
Me
c
d
N
R
N
R
9
10 (R = H, Me, Et, nPr, etc)
Scheme 1. Reagents and conditions: (a) NaH, THF; (b) NH4OAc, AcOH, 80 °C; (c) NaH, RI, DMF; and (d) NaOH, EtOH, reflux.

S. Y. Kang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1705–1711
1707
role under the conditions of sodium hydride and the corresponding
alkyl iodides in DMF. Hydrolysis of pyrrole ester 9 using sodium
hydroxide in refluxed ethanol afforded the corresponding acids 10.
Meanwhile, direct cyclization of acetoacetate 7 to N-substituted
pyrrole compounds was also accomplished using various amines
instead of ammonium acetate to produce the corresponding pyr-
role compounds 9 as shown in Scheme 2.
As shown in Scheme 3, preparation of aminoalkyl-arylpiper-
azine 14 started from the corresponding bromoalkyl-phthalimide
11 and arylpiperazine 12.19 Treatment of bromoalkyl-phthalimide
11 with arylpiperazine hydrochloride 12 in the presence of potas-
sium carbonate in DMF at room temperature afforded N-protected
amine 13. Compound 13 was then treated with hydrazine in etha-
nol at 80 °C to give amine 14. For the sake of convenience of han-
dling on scale, liquid amine 14 was transformed to hydrochloride
salt form 14a with 4 N HCl in dioxane.
phenyl)piperazin-1-yl)propan-1-amine dihydrochloride (14a) in
hand, typical amide coupling was conducted under conditions
involving EDCI, HOBT, and NMM in methylene chloride to produce
amide 15. As reaction was completed, purification was performed
using preparative reverse-phase HPLC with acetonitrile and water
containing 0.2% TFA or not. The neutral form of product 15 was
converted to HCl salt form 15a to increase the overall solubility
for biological evaluation.
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.20,23
The initial work was focused on exploration of the central pyr-
role moiety and substitution of arylpiperazine group (Table 1). N-
Alkylated pyrrole derivatives and 3-chlorophenyl, 2,3-dimethyl-
phenyl, and 2,3-dichlorophenyl piperazine were connected with
propyl-carboxamide as a linker. The binding affinities of prepared
compounds against the 5-HT2A, 5-HT2C, and SERT are shown in
Finally, coupling reaction of acid 10a and aminoalkyl-arylpiper-
azine 14a was conducted as shown in Scheme 4. With 2-methyl-5-
phenyl-1H-pyrrole-3-carboxylic acid (10a) and 3-(4-(3-chloro-
O
O
O
O
OH
OEt
OEt
b
a
O
Me
N
Me
N
R
R
10
7
9
R = isobutyl, cyclohexylmethyl, benzyl, 4-fluorophenyl
Scheme 2. Reagents and conditions: (a) RNH2, p-TsOH, EtOH, 80 °C and (b) NaOH, EtOH, reflux.
O
O
N
N
a
b
Br
N
n
R
N
n
HN
HCl
N
O
13
O
R
11
12
2HCl
c
N
N
N
N
H2N
H2N
n
n
R
R
14a
14
Scheme 3. Reagents and conditions: (a) 12, K2CO3, DMF, rt; (b) NH2NH2ꢀH2O, EtOH, rt; and (c) 4 N HCl in dioxane.
O
O
H2N
N
N
N
OH
Me
Ph
N
H
a
H
N
H
+
N
H
2HCl
Me
Ph
N
Cl
N
H
14a
15
Cl
10a
O
b
N
H
Ph
N
H
N
H
HCl
Me
15a
Cl
Scheme 4. Reagents and conditions: (a) EDCI, HOBT, NMM, CH2Cl2 or DMF and (b) HCl, MeOH, 0 °C.

1708
S. Y. Kang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1705–1711
Table 1
Competition binding assays at 5-HT2A and 5-HT2C receptor and the serotonin transporter to arylpipierazinyl pyrrole 3-carboxamide derivatives (IC50 values, unit:nM)
O
NH
N
N
Me
N
R
R'
R''
R
R0, R00
H, CI
Me, Me
CI, CI
5-HT2C
No.
5-HT2A
5-HT2C
SERT
No.
5-HT2A
5-HT2C
SERT
No.
5-HT2A
SERT
H
Me
Et
nPr
15
16
17
18
19
20
21
22
23
245
92
92
191
333
441
22.9
179
1067
280
160
192
273
1602
448
443
1436
611
92
11.1
25.8
93
110
110
80.2
235
213
24
25
26
27
28
29
30
31
32
300
57
75
129
897
133
6.9
224
1233
615
133
139
225
1298
417
162
297
638
77
11.8
35.9
76
350
92
53.6
14.3
352
33
34
35
36
37
38
39
40
41
300
46.2
376
324
406
67.1
4.1
599
12
66
628
342
795
466
220
1034
137
61.7
182
420
381
298
266
125
412
iBu
N-Piperidinyl
4-Fluorophenyl
Benzyl
354
4383
Cyclohexylmethyl
Table 2
Binding affinities for 5-HT2A, 5-HT2C receptor, and serotonin transporter of arylpipierazinyl pyrrole 3-carboxamide derivatives (IC50 values, unit:nM)
O
N
R'
N
n
R'
N
H
N
R
Me
R0 = Cl
R
n = 1
n = 2
n = 3
No.
5-HT2A
5-HT2C
SERT
No.
5-HT2A
5-HT2C
SERT
137
61.7
420
No.
5-HT2A
5-HT2C
SERT
H
Me
nPr
42
43
44
40
175
408
37
156
310
87
57
32
33
34
36
300
46.2
324
599
12
628
45
46
28
104
32
20.9
93
76
R0 = Me
R
n = 2
n = 3
No.
5-HT2A
5-HT2C
SERT
No.
5-HT2A
5-HT2C
SERT
H
Me
nPr
24
25
27
300
57
129
615
13
225
77
11.8
76
47
48
49
10
61
792
63
61
149
21
149
76
Figure 3. Effects of drugs on immobility in forced swimming test on mice. Drugs (100 and 50 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 S.E.M.
Table 1. Because these compounds would interact with multiple
targets, it is not easy to evaluate prepared compounds following
SARI (serotonin antagonist and reuptake inhibitor) mechanism.
Thus, it is more reasonable to compare not only binding affinities
against specific receptors, but also overall binding tendency of
our compounds against the 5-HT2A, 5-HT2C, and SERT. Initially,
most compounds tested displayed IC50 <1
lM, implying that this
series of arylpiperazinyl pyrrole 3-carboxamide might hold prom-

S. Y. Kang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1705–1711
1709
ises as a potential antidepressant. Although 1H-pyrrole-3-carbox-
amides (15, 24, and 33) have moderate in vitro activities, substitu-
tion of NH in pyrrole with small alkyl such as methyl (16, 25, and
34), ethyl (17, 26, and 35), and n-propyl (18, 27, and 36) increased
binding affinities against 5-HT2A, 5-HT2C, and SERT. But the binding
affinities were reduced when bulkier alkyl groups (i-butyl 19, 28,
37; cyclohexylmethyl 23, 32, 41; benzyl 22, 31, 40) were installed
at the NH of pyrrole. Similar phenomenon was observed if a het-
eroatom was included as in N-piperidinyl 20, 29, 38. Particularly,
4-fluorophenyl group responded selectively to 5-HT2A receptor
(21, 30, and 39). Also, it showed little difference in binding affinity
between 3-chlorophenylpiperazinyl compounds and 2,3-dimethyl-
phenyl piperzinyl compounds. Therefore, it is safe to say that such
SAR could be observed to only short alkylated (N-Me, N-Et, and N-
n-propyl) pyrrole compounds.
In order to improve binding affinity against 5-HT2A and 5-HT2C
receptors and the serotonin transporter, varying linker size around
key compounds was undertaken. Initially, dichlorophenylpiperazi-
nyl derivatives were synthesized and screened. In the case of
unsubstituted pyrrole 33, overall binding affinity was increased
when the length of linker shorten from C3 to C2 (33 vs 42). On
the contrary, methylated pyrrole displayed increased binding affin-
ity when linker was elongated (34, 43). In case of substitution of
NH in pyrrole with n-propyl, C2- and C3-linker compounds made
no clear difference, but increased binding affinity as linker was ex-
tended from C3 to C4 (36, 44, and 46). In the case of dimethylphe-
nylpiperazinyl group, binding affinity enhancement was observed
for unsubstituted pyrroles (24 vs 47). However, overall binding
affinity was decreased when NH in pyrrole was substituted with
alkyl group. (25 vs 48 and 27 vs 49) (see Table 2).
To evaluate antidepressant activity of the interesting com-
pounds, immobility in forced swimming test (FST) on mice was
measured.21,24 Prozac (fluoxetine) was used as a reference com-
pound for comparison. The results are shown in Figure 3. At
100 mg/kg, 3-chlorophenylpiperazine analogues 16, 17 show virtu-
ally no appreciable immobility effect. Compared with fluoxetine,
dimethylphenylpiperazine compounds 25, 26 display similar or a
little off activity at 50 mg/kg, and N-ethyl pyrrole 26 shows more
effective than N-methyl congener 25. Improved results are consis-
tently obtained with dichlorophenylpiperazine moieties. Substitu-
tion of NH in pyrrole with small alkyl groups such as methyl 34 or
ethyl 35 shows extremely potent in vivo efficacy on animal model
(less than 25% at 100 mg/kg). N-Methylpyrrole 34 is sixfold more
active than NH pyrrole 33, and fivefold more active than N-ethyl-
pyrrole compound 35. Its in vivo structure–activity relationship
appears to go well with in vitro assay results. N-Piperidinylpyrrole
38 displays a similar level of efficacy to NH pyrrole counterpart 33.
Figure 4. Effects of drugs on immobility in forced swimming test on mice. Drugs
(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 S.E.M.
Although 4-fluorophenylpyrrole derivative 39 shows good inhibi-
tory activity against 5-HT2A receptor (IC50 = 4.1 nM), it displays
only modest efficacy (58.3 8.4%). From the viewpoint of linker
size, propyl (C3-linker) shows better efficacy than C2 (42 vs 33)
and C4 (34 vs 45). In the case of dimethylphenylpiperazine ana-
logues, C4-linker appears to be more efficacious than C3-linker
compounds (16, 17 vs 47, 48). However, all these compounds dis-
play relatively low immobility effects.
Dose-dependent immobility test was also conducted. At 25 mg/
kg, compounds 33, 34, and 36 still showed good efficacy (immobil-
ity <50%). Compound 34 proved to be most potent in vivo efficacy
(immobility = 40.7 10.4%). When oral dose reduced from 25 mg/
kg to 10 mg/kg, all the compounds tested showed insufficient anti-
depressant activity. Either 34 or 36 demonstrated merely modest
in vivo efficacy (immobility ꢁ80%)25 (see Fig. 4).
Interesting compounds were further evaluated with the recep-
tor selectivity studies. The results were shown in Table 3. It was
demonstrated that binding affinities of these compounds against
5-HT2A, 5-HT2C and SERT were stronger than binding affinities
against any other neurotransmitter receptors except 5-HT1A. It is
well-known that inhibition of 5-HT1A receptor affects depressive
disorder. Inhibition of dopamine receptor was only marginal. Thus,
34 not only shows favorable binding affinities against 5-HT2A, 5-
HT2C receptors, and the serotonin transporter, but also provides
good selectivity profiles over other neurotransmitter receptors.
Table 3
Profiles of interesting compounds via competition binding assay at 5-HT1A, 5-HT2A, 5-HT2C, 5-HT6, 5-HT7, SERT, D2, D3 and D4. (D; Dopamine) (IC50 values, unit:nM)
Cl
N
H
N
N
Cl
R
N
Me
O
Compound
R
Unit: nM
Binding affinity
5-HT1A
5-HT2A
5-HT2c
5-HT6
5-HT7
SERT
D2
D3
334
915
1072
—
D4
33
34
36
38
39
H
Me
nPr
117
54
94
—
300
46
324
67.1
6.9
599
12
628
795
599
1412
713
1904
—
456
333
408
493
524
133
62
420
298
54
1071
2542
3736
—
1845
984
645
—
N-Piperidinyl
4-Fluorophenyl
—
—
—
—
—

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S. Y. Kang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1705–1711
Table 4
Pharmacokinetic parameters and brain/plasma ratio of compounds 33 and 34 after single oral (5 mg/kg) and i.v. (1 mg/kg) administration to rat
Compound
33a
34a
PK parameters
i.v. (1 mg/kg n = 2)
Oral (5 mg/kg, n = 3)
Plasma
Brain
i.v. (1 mg/kg n = 3)
Oral (5 mg/kg, n = 3)
Plasma
Brain
Cmax
Clast
Tmax (h)
(
l
g/mL)
0.690
0.004
—
1.756
20.945
20.945
0.245
0.001
1.167
2.521
3.868
12.783
0.19
0.10
2.00
1.97
0.09
0.06
2.00
3.42
0.454
0.008
—
2.822
18.703
18.703
0.118
0.001
0.750
3.137
23.150
4.630
0.07
0.04
1.00
3.71
0.03
0.02
2.00
4.28
(
lg/mL)
t1/2 (h)
AUCirrf (min
AUCirlf/Dose
AUCall (min
l
g/mL)
l
g/mL)
32.25
14.84
14.38
4.96
BA (%)
61.03
24.76
Brain/plasma (%)
46.01
34.47
a
HCl salt compounds were used.
Murray, C. J. L., Lopez, A. D., Eds.; Harvard University Press: Cambridge, MA,
1996; p 38.
3. WHO The World Health Report 2001, Mental Health: New Understanding, New
Hope; Fact Sheet No. 265.
With promising in vitro and in vivo results in hand, pharmaco-
kinetic properties of selected compounds 33, 34 were evaluated,
and these results are summarized in Table 4.22 After oral adminis-
4. 5-Hydroxyltryptamine Mechanisms in Primary Headaches; Olesen, J., Saxena, P.,
Eds., 1st ed.; Raven Press: New York, 1992.
5. Rang, H.; Dale, M.; Ritter; Pharmacol, J., 5th ed.; Churchhill Livingstone:
Edinburgh, 2003.
tration of a 5 mg/kg dose of 33 to rats, a Cmax of 0.245 lg/mL was
obtained at 1.167 h. The elimination half-life for 33 following oral
administration was 2.521 h in rats. Compound 33 showed rela-
tively good oral bioavailability (F = 61.03%) in rats. Meanwhile,
after oral administration of a 5 mg/kg dose of 34 to rats, a Cmax of
6. Blackwell, B. Drugs 1981, 29, 201.
7. Evrard, D. A.; Harrison, B. L. Annu. Rep. Med. Chem. 1999, 34, 1.
8. Blier, P.; Bergeron, R. J. Clin. Psychiatry 1998, 59, 16.
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Frazer, A. Neuropsychophamacology 2004, 29, 566. and references cited therein.
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12. Romeo, L.; Arigas, F. J. Neurochem. 1997, 68, 2593.
13. Berman, R. M.; Anand, A.; Cappiello, A.; Miller, H. L.; Xu, X. S.; Oren, D. A.;
Charney, D. S. Biol. Psychiatry 1999, 45, 1170.
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Eur. J. Pharm. 2000, 407, 39.
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0.118 lg/mL was obtained at 0.750 h. The elimination half-life
for 34 following oral administration was 3.137 h in rats. Compound
34 showed moderate oral bioavailability (F = 24.76%) in rats. Com-
pound 33 has slightly superior brain/plasma ratio (46.01%) to that
of compound 34 (34.47%). For many companies, B/P (%) >30 is used
as a minimum guideline for CNS discovery projects.26 From these
results, both compounds 33 and 34 showed good PK profile and
B/P ratio. Compound 34 was further evaluated for effects of CYP
inhibition. Compound 34 showed no appreciable inhibition against
CYP1A2, CYP2D6, and CYP3A4, showing IC50 > 20
lM, while 34 is a
moderate inhibitor against CYP2C9 (IC50 = 9.7
l
M).27
16. (a) Rush, A. J.; Armitage, R.; Gillin, J. C.; Yonkers, K. A.; Winokur, A.; Moldofsky,
H.; Vogel, G. W.; Kaplita, S. B.; Fleming, J. B.; Montplaisir, J.; Erman, M. K.;
Albala, B. J.; McQuade, R. D. Biol. Psychiatry 1998, 44, 3; (b) Avila, A.; Vardona,
X.; Martin-Baranera, M.; Maho, P.; Sastre, F.; Bello, J. J. Clin. Psychopharmacol.
2003, 23, 509.
17. (a) Greene, D.; Barbhaiya, R. H. Clin. Pharmacokinet. 1997, 33, 260; (b) Kent, J. M.
Lancet 2000, 355, 911.
18. Vanotti, E; D’alessio, R.; Tibolla, M.; Varasi, M.; Montagnoli, A.; Santocanale, C.;
Martina, K.; Menichincheri, M. WO2005/013986, 2005.
19. Robarge, M. J.; Husbans, S. M.; Kieltyka, A.; Brodbeck, R.; Thurkauf, An.;
Newman, A. H. J. Med. Chem. 2001, 44, 3175.
20. Park, W.-K.; Jeong, D.; Cho, H.; Lee, S. J.; Cha, M. Y.; Pae, A. N.; Choi, K. I.; Koh, H.
Y.; Kong, J. Y. Pharmacol., Biochem. Behav. 2005, 82, 361.
21. Porsolt, R. D.; Bertin, A.; Jalfre, M. Eur. J. Pharmacol. 1978, 51, 291.
22. Spectrum Data of representative compounds; 33 1H NMR (400 MHz, CDCl3) d
8.46 (br s, 1H), 7.53 (m, 1H), 7.39 (d, J = 7.6 Hz, 2H), 7.21–7.13 (m, 2H), 6.98 (t,
J = 8.0 Hz, 1H), 6.81 (d, J = 8.0 Hz, 1H), 6.62 (d, J = 2.4 Hz, 1H), 3.57–3.53 (m,
2H), 3.13 (br s, 4H), 2.71 (br s, 5H), 2.63 (s, 3H), 1.84–1.79 (m, 2H). MH+ 471.
Compound 34 1H NMR (400 MHz, CDCl3) d 7.39 (br s, 1H), 7.34–7.30 (m, 4H),
7.13 (dd, J = 8.0, 1.6 Hz, 1H), 7.00 (t, J = 8.4 Hz, 1H), 6.66 (dd, J = 8.0, 1.2 Hz, 1H),
6.36 (s, 1H), 3.53 (dd, J = 11.6, 5.6 Hz, 2H), 3.49 (s, 3H), 3.06 (br s, 4H), 2.68 (m,
5H), 2.65 (s, 3H), 2.61 (t, J = 5.6 Hz, 2H), 1.83–1.77 (m, 2H). MH+ 485.
23. For serotonin 5-HT2A receptor binding, an aliquot of frozen membrane from
CHO-K1 cell line expressing the human recombinant 5-HT2A receptor
(PerkinElmer Life and Analytical Sciences, Boston, USA) and [3H]Ketanserin
In summary, we investigated a series of arylpiperazine contain-
ing pyrrole 3-carboxamide derivatives for the treatment of depres-
sive disorders. As an approach to overcome side effects of known
antidepressants and reach unmet needs in the field of antidepres-
sants, novel pyrrole-based small molecules which would work as
5-HT receptor antagonist and reuptake inhibitor (SARI) were de-
signed and synthesized. Subsequent SAR studies were performed
via substitution of NH in pyrrole, variation of the linker size by dif-
ferent number of carbons, and modification of substituents on aryl
group. Based on the outcomes of in vitro SAR studies and forced
swimming test, 34 was identified as a lead compound for this anti-
depressant program. Its receptor selectivity and PK properties were
promising enough to warrant further studies around this pyrrole
scaffold.
Acknowledgments
This Letter is dedicated to the memory of Mr. Young-Sup Huh,
Chairman, CEO, Green Cross Corporation (GCC). We appreciate
Dr. Chong-Hwan Chang for his leadership and dedication during
his tenure of office as Head of GCC R&D. Also we are grateful to
Dr. Eun Chul Huh for his leadership and supports during his
tenure as Head of GCC R&D Planning and Coordination. This
work was in part supported by Korea Ministry of Knowledge
Economy.
1 nM (PerkinElmer) were mixed in the presence of mianserin (20 lM) 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 Filtermat A glass fiber filter presoaked in 0.5% PEI. The
filter was covered with MeltiLex, sealed in a sample bag followed by drying in
the microwave oven, and counted by MicroBeta Plus (Wallac, Finland).
Competition binding studies were carried out with 5–6 varied concentrations
of the test compounds run in duplicate tubes, and isotherms from three assays
were calculated by computerized nonlinear regression analysis (GraphPad
Prism, GraphPad Software, Inc., CA, USA) to yield IC50 values. For 5-HT2C
binding, frozen membranes from stable CHO-K1 cell line expressing the human
recombinant 5-HT2C receptor (PerkinElmer) were used. [3H]Mesulergine
(1.4 nM), receptor membrane and test compound were added into 50 mM
Tris–HCl (pH 7.4) buffer containing 4 mM CaCl2 and 0.1% ascorbic acid.
References and notes
1. Greenberg, D. E.; Kessler, R. C.; Birnbaum, H. G.; Leong, S. A.; Lowe, S. W.;
Berglund, P. A.; Corey-Lisle, P. K. J. Clin. Psychiatry 2003, 64, 1465.
2. The Global Burden of Disease; A Comprehensive Assessment of Mortality and
Disability from Disease, Injuries and Risk Factors in 1990 and Projected to 2020;
Nonspecific binding was determined using 10 lM of methiothepin. The

S. Y. Kang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1705–1711
1711
incubations were performed for 60 min at 27 °C, and these were terminated by
rapid filtration through Filtermat A glass fiber filter presoaked in 0.5% PEI.
Human serotonin transporter expressed in HEK293 (PerkinElmer) was used for
serotonin transporter binding assays. For the binding, frozen membrane, 4 nM
[3H]Imipramine (PerkinElmer) and appropriate concentrations of test
compounds were added to 0.25 mL assay buffer of 50 mM Tris–HCl (pH 7.4)
containing 120 mM NaCl and 5 mM KCl. Incubations were carried out for
30 min at 27 °C, and these were terminated by rapid filtration through
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 compound (10, 25, 50, and 100 mg/kg) and fluoxetine (50
and 100 mg/kg) were suspended in 3%-Tween 80 solution, and administered
(i.p.) 30 min before the testing.
25. Considering that 36 shows only moderate binding affinities against 5-HT2A, 5-
HT2C and SERT, 34 is regarded as a lead compound for our antidepressant
program.
26. Kerns, E. H.; Di, D. Drug-like Properties: Concepts, Structure Design and
Methods: from ADME to Toxicity Optimization; Academic Press: Burlington,
MA, 2008.
Filtermat A glass fiber filter presoaked in 0.5% PEI. Imipramine (100
used as the nonspecific ligand.
l
M) was
24. The forced swimming test was performed according to the methods described
by Porsolt et al. (1978).21 Each mouse was placed in a 25-cm glass cylinder
(10 cm diameter) containing 15 cm of water maintained at 23 1 °C, and was
forced to swim for 10 min. Twenty-four hours later, the mouse was replaced
into the cylinder and the total duration of immobility was recorded during the
27. For comparison, fluoxetine showed relatively strong inhibition against CYP2C9,
CYP2D6, and CYP3A4, demonstrating IC50 = 18.4
IC50 = 5.8 M, respectively in in-house assay, whereas it turned out to be a
weak inhibitor against CYP1A2 (IC50 >20 M) in this experiment.
lM, IC50 = 0.74 lM,
l
l