Arylpiperazine-containing pyrimidine 4-carboxamide derivatives targeting serotonin 5-HT2A, 5-HT2C, and the serotonin transporter as a potential antidepressant

Article abstract of DOI:10.1016/j.bmcl.2010.09.081

Pyrimidine usually has good pharmacokinetic properties as a drug substance and considerable efforts have been devoted to develop pyrimidine derivatives into drug candidates. Arylpiperazine-containing pyrimidine 4-carboxamide derivatives were synthesized and evaluated for binding to serotonin receptors and transporter. Pyrimidine derivatives showed good antidepressant activity in FST (forced swimming test) animal model and also displayed no appreciable inhibitory activity against hERG channel blocking assay. Herein SAR studies of pyrimidine derivatives targeting serotonin receptors and transporter will be disclosed.

Full text of DOI:10.1016/j.bmcl.2010.09.081

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6439–6442
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Arylpiperazine-containing pyrimidine 4-carboxamide derivatives targeting
serotonin 5-HT2A, 5-HT2C, and the serotonin transporter as a
potential antidepressant
Jong Yup Kim a,b, Deukjoon Kim b, , Suk Youn Kang a, Woo-Kyu Park c, Hyun Jung Kim a, Myung Eun Jung a,
⇑
Eun-Jung Son a, Ae Nim Pae d, Jeongmin Kim a, Jinhwa Lee a,
⇑
a Research Center, Green Cross Corporation, Yongin 446-770, Republic of Korea
b The Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, San 56-1, Shinrim-Dong, Kwanak-Ku, Seoul 151-742, Republic of Korea
c Pharmacology Research Center, Korea Research Institute of Chemical Technology, 100 Jang-Dong, Yuseong-Gu, Daejeon 305-343, Republic of Korea
d 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:
Received 30 May 2010
Revised 30 August 2010
Accepted 14 September 2010
Available online 19 September 2010
Pyrimidine usually has good pharmacokinetic properties as a drug substance and considerable efforts
have been devoted to develop pyrimidine derivatives into drug candidates. Arylpiperazine-containing
pyrimidine 4-carboxamide derivatives were synthesized and evaluated for binding to serotonin receptors
and transporter. Pyrimidine derivatives showed good antidepressant activity in FST (forced swimming
test) animal model and also displayed no appreciable inhibitory activity against hERG channel blocking
assay. Herein SAR studies of pyrimidine derivatives targeting serotonin receptors and transporter will be
disclosed.
Keywords:
Antidepressant
Serotonin
Ó 2010 Elsevier Ltd. All rights reserved.
Piperazine
Pyrimidine
Major depressive disorder is a mental disorder characterized by
an all-encompassing low mood accompanied by low self-esteem
and loss of interest or pleasure in normally enjoyable activities.
There is a growing awareness that patients with depressive disor-
ders often also suffer cognitive impairment,1 and some studies
indicate that these deficits may persist even after remission.2,3
The monoaminergic hypothesis of depression assumes that depres-
sion is caused by the dysfunction of the serotonin (5-HT, SER), nor-
adrenaline (NE) and/or dopamine (DA) neurotransmitter systems.4
This hypothesis has been used to explain the efficacy of existing
antidepressant therapies. Among these available therapies, selec-
tive serotonin reuptake inhibitors (SSRIs) and more recently com-
bined serotonin- and noradrenaline reuptake inhibitors (SNRIs)
have become the standard treatment for depression.5
In recent years, SARI (serotonin antagonist/reuptake inhibitor)
drugs that block both the serotonin 5-HT2 receptors and the sero-
tonin transporter have been developed.6 YM-992 1,7 LY367265 2,8
Nefazodone 3,9 and aripiprazole 4 are the typical examples of that
series (Fig. 1). Unlike most SSRIs, nefazodone is reported to have no
negative effects on libido or sexual functioning. Nefazodone’s
claimed advantages over other antidepressants include reduced
possibility of disturbed sleep or sexual dysfunction, and ability to
treat some patients who did not respond to other antidepressant
drugs.10–13 In this regard, there are still urgent medical needs on
F
F
N
HN
O
N
N
S
O
NH
O
O
YM992, 1
LY367265, 2
O
N
N
N
N
O
N
Cl
Cl
nefazodone, 3
H
O
N
O
N
Cl
N
⇑
Corresponding authors. Tel.: +82 2 880 7850; fax: +82 2 880 0649 (D.K.), tel.:
+82 31 260 9892; fax: +82 31 260 9870 (J.L.).
aripiprazole, 4
Figure 1. SARI drugs (serotonin antagonist/reuptake inhibitor).
E-mail addresses: deukjoon@snu.ac.kr (D. Kim), jinhwalee@greencross.com (J.
Lee).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.081

6440
J. Y. Kim et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6439–6442
R3
cyanopyrimidine 8. An acid form 9 was transformed from the
intermediate 8 using sodium hydroxide, followed by acidification,
as shown in Scheme 1.
R2
Cl
O
O
NH
acid
NH2
c
a, b
R2
O
+
R1
N
N
As shown in Scheme 2, preparation of aminoalkyl-arylpiper-
azine 13 started from the corresponding bromoalkyl-phthalimide
10 and arylpiperazine 11.14 Treatment of bromoalkyl-phthalimide
10 with arylpiperazine hydrochloride 11 in the presence of potas-
sium carbonate in DMF at room temperature afforded N-protected
amine 12. Compound 12 was then treated with hydrazine in etha-
nol to give amine 13. For the sake of convenience of handling on
scale, liquid amine 13 was transformed to hydrochloride salt form
13a with 4 N HCl in dioxane.
R3
R1
7
5
R3
6
R3
O
R2
CN
R2
d
OH
N
N
N
N
R1
R1
Finally, coupling reaction of acid 9 and aminoalkyl-arylpiper-
azine 13a was conducted as shown in Scheme 3. With pyrimi-
8
9
dine-4-carboxylic
acid
9
and
aminoalkyl-arylpiperazine
Scheme 1. Reagents and conditions: (a) NaOEt, EtOH, 100 °C; (b) POCl3, 110 °C; (c)
Zn(CN)2, Pd(PPh3)4, THF, microwave reaction 165 °C, 30 min; and (d) NaOH, EtOH,
water, reflux.
dihydrochloride 13a in hand, typical amide coupling was con-
ducted under conditions involving EDCI, HOBT, and NMM in meth-
ylene chloride or DMF to produce amide 14. As reaction was
completed, purification was performed using preparative reverse-
phase HPLC with 0.1% TFA mixture of acetonitrile and water solu-
tion. The neutral form of product 14 was converted to HCl salt form
14a 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, were evaluated by displacement binding using
[3H]Ketanserin, [3H]Mesulergine, and [3H]Imipramine, respec-
tively, as radioligands.15,16
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 pyrimi-
dine 4-carboxamide derivatives targeting serotonin 5-HT2A, 5-
HT2C, and the serotonin transporter as a potential antidepressant.
The carboxylic acid derivative 9 was prepared by a conventional
method, for example, by reacting an amidine acid salt 5 with a
keto-ester derivative 6 using sodium ethoxide, followed by chlori-
nation using POCl3 to produce a corresponding 4-chloropyrimidine
7. Subsequent reaction of the resulting 4-chloropyrimidine 7 with
zinc cyanide in the presence of Pd(PPh3)4 gave an intermediate 4-
The work was focused on exploration of the substitution group
of pyrimidine moiety. 2-Methylpyrimidine derivatives and 2,3-
dichlorophenyl- or 2,3-dimethylphenylpiperazine were connected
with propyl-carboxamide as a linker.
O
O
Br
a
N
N
b
N
N
n
n
X
X
HN
HCl
N
O
10
O
12
X
X
11
2 HCl
H2N
c
N
N
N
N
H2N
n
n
X
X
X
X
13
13a
Scheme 2. Reagents and conditions: (a) 11, K2CO3, DMF, rt; (b) NH2NH2ÁH2O, EtOH, rt; and (c) 4 N HCl in dioxane.
R3
O
R2
2 HCl
H2N
a
OH
N
N
+
N
N
n
X
X
R1
9
13a
R3
O
N
X
R3
O
N
X
b
R2
N
X
R2
N
X
N
N
H
n
n
H
HCl
N
N
N
N
R1
R1
14
14a
Scheme 3. Reagents and conditions: (a) EDCI, HOBt, NMM, DCM or DMF, rt; and (b) HCl, MeOH, 0 °C.

J. Y. Kim et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6439–6442
6441
The binding affinities of prepared compounds against the 5-
120
100
80
60
40
20
0
HT2A, 5-HT2C, and SERT are shown in Table 1. Because these com-
pounds would interact with multiple targets, it is not easy to eval-
uate prepared compounds following SARI (serotonin antagonist
and reuptake inhibitor) mechanism. Initially, most compounds
tested displayed IC50 <1 lM, implying that this series of arylpiper-
azinyl pyrimidine 4-carboxamide might hold promises as a poten-
tial antidepressant. The substituted R2 group of pyrimidine showed
a tendency of preference for bulky group. Also, it showed consider-
able difference in binding affinity between 2,3-dichlorophenylpip-
erazinyl
compounds
and
2,3-dimethylphenylpiperazinyl
compounds. In order to improve binding affinity against 5-HT2A
,
5-HT2C receptors, and serotonin transporter, contraction of linker
size was undertaken. For comparison, dimethylphenylpiperazinyl
derivatives were synthesized and screened as shown in Table 2.
In the case of 5-H pyrimidine (R3 = H), 5-HT2A, and 5-HT2C receptor
binding affinities were increased when the length of the linker was
shorten from C3 to C2. On the contrary, SERT binding affinities
were decreased at the same conditions. In the case of 5-OMe
pyrimidine (R3 = OMe), the effect of the linker size on the binding
affinities was not obvious (see Table 2). To evaluate antidepressant
activity of the interesting compounds, immobility in forced swim-
ming test (FST) on mice were measured.17,18 Zoloft (sertraline) was
used as a reference compound for comparison. The results are
shown in Figure 2.
Dose (25 mg/kg, po)
Figure 2. Effects of drugs on immobility in forced swimming test on mice. Drugs
(25 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.
Compared with Zoloft, dichlorophenylpiperazine compounds
with C3 linker size (15a, 15b) showed more potent in vivo efficacy
in animal model. In the case of 2-SMe series (R1 = SMe), the C2 lin-
ker size compounds (16d,19 16e) appeared to be more efficacious
than C3 linker size compound (16c). 5-OMe substituted com-
pounds (R3 = OMe) also showed a similar trend of results in immo-
bility test.
To distinguish antidepressant effect from hyperactivity of ani-
mal, spontaneous locomotor activity tests were conducted in mice.
As a result, 15a compound showed hyperactivity, while 16g com-
pound showed hypoactivity. Therefore, the efficacy in immobility
of 15a compound was caused by hyperactivity of animal, that is,
false positive result. The results of the other compounds fell within
the normal range (Fig. 3).
Table 1
Binding affinities for 5-HT2A, 5-HT2C receptor, and serotonin transporter of arylpip-
erazinyl pyrimidine 4-carboxamide derivatives
O
R2
N
H
N
X
HCl
N
N
N
X
Compounds such as trazodone or nefazodone, are reported to be
metabolized into meta-chlorophenylpiperazine (m-CPP), a seroto-
nin receptor agonist with high affinity to 5-HT2A and even higher
to 5-HT2C receptor. This compound has itself anxiogenic-like and
hypolocomotor effects. Furthermore, SSRIs through the increase
of extracellular 5-HT concentration, activate at least 5-HT2C recep-
tor to induce anxiogenic-like and hypolocomotion effects which
could be antagonised by subtype-selective 5-HT2C receptor antag-
onists.13 Thus, compound 16d should be further studied for in vivo
metabolism. Off target activity of selected compounds was briefly
evaluated. The activity of 16d in the hERG potassium channel assay
15
Compound
R2
X
IC50 (nM)
5-HT2C
5-HT2A
SERT
15a
15b
15c
15d
15e
Me
iPr
Ph
iPr
tBu
Cl
Cl
Cl
Me
Me
166
113
35
490
458
610
227
30
1548
575
289
644
588
1213
730
Table 2
was determined to be greater than 10
pound 16d also showed no appreciable inhibition against CYP1A2,
CYP2D6, CYP2C9, and CYP3A4, showing IC50 >20 M.
lM IC50 (Table 3). Com-
Binding affinities for 5-HT2A, 5-HT2C receptor, and serotonin transporter of arylpipi-
erazinyl pyrimidine 4-carboxamide derivatives
l
In summary, we investigated a series of arylpiperazine contain-
ing pyrimidine 4-carboxamide derivatives for the treatment of
depressive disorders. As an approach to overcome side effects of
R3
R1
O
N
X
N
X
N
H
n
HCl
N
N
25000
20000
15000
10000
5000
16
Compound
R1
R3
X
n
IC50 (nM)
5-HT2C
5-HT2A
SERT
16a
16b
16c
16d
16e
16f
16g
16h
16i
tBu
tBu
SMe
SMe
SMe
cPr
Me
Me
Me
H
H
H
H
H
H
Me
Me
Me
Me
Cl
Me
Me
Me
Cl
2
1
2
1
1
2
2
1
1
274
69
54
47
55
256
744
842
263
2688
494
1170
148
537
6374
394
1014
881
347
892
2096
646
0
81
15a 15b 15d 15e 16a 16b 16c 16d 16e 16f 16g 16h 16i
1178
2049
1266
135
OMe
OMe
OMe
Dose (25 mpk)
Figure 3. Locomotor activities of the mice treated with compounds were counted
for 30 min by activity analyzer. Data were expressed as mean SEM of 6–7 mice.

6442
J. Y. Kim et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6439–6442
8. Pullar, I. A.; Carney, S. L.; Colvin, E. M.; Lucaites, V. L.; Nelson, D. L.; Wedley, S.
Table 3
Eur. J. Pharmacol. 2000, 407, 39.
hERG channel binding assay
9. De Battista, C.; Sofuogula, M.; Schatzberg, A. F. Biol. Psychiatry 1998, 44, 341.
10. Greene, D. S.; Barbhaiya, R. H. Clin. Pharmacokinet. 1997, 33, 260.
11. Odagaki, Y.; Toyoshima, R.; Yamauchi, T. J. Psychopharmacol. 2005, 19, 235.
12. Ly, K. S.; Letavic, M. A.; Keith, J. M.; Miller, J. M.; Stocking, E. M.; Barbier, A. J.;
Bonaventure, P.; Lord, B.; Jiang, X.; Boggs, J. D.; Dvorak, L.; Miller, K. L.;
Nepomuceno, D.; Wilson, S. J.; Carruthers, N. I. Bioorg. Med. Chem. Lett. 2008, 18,
39.
Compound
IC50 (lM)
15a
15b
15e
16d
>10
5.4
5.9
>10
13. Bagdy, G.; Graf, M.; Anheuer, Z. E.; Modos, E. A.; Kantor, S. Int. J.
Neuropharmacol. 2001, 4, 399.
14. Robarge, M. J.; Husbands, S. M.; Kieltyka, A.; Brodbeck, R.; Thurkauf, A.;
Newman, A. H. J. Med. Chem. 2001, 44, 3175.
15. 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.
16. 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
known antidepressants and reach unmet needs in the field of anti-
depressants, novel pyrimidine-based small molecules which would
work as 5-HT receptor antagonist and reuptake inhibitor (SARI)
were designed and synthesized. Subsequent SAR studies were per-
formed via substitution of pyrimidine, variation of the linker size
by different number of carbons, and modification of aryl group.
Based on the outcomes of in vitro SAR studies, forced swimming
test, and locomotor activities, compound 16d was identified as a
lead compound in this series for this antidepressant program.20
Its receptor binding affinity and in vivo test results are promising
enough to warrant further studies around this pyrimidine scaffold.
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.
Acknowledgments
We are grateful to Dr. Eun Chul Huh for his leadership and sup-
ports as head of GCC R&D. This work was in part supported by Kor-
ea Ministry of Knowledge Economy.
Nonspecific binding was determined using 10 lM of methiothepin. The
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) were 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
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.09.081.
Filtermat A glass fiber filter presoaked in 0.5% PEI. Imipramine (100
used as the nonspecific ligand.
lM) was
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19. Spectrum data of representative compound; 16d 1H NMR (400 MHz, CDCl3) d
8.49 (br s, 1H), 7.62 (s, 1H), 7.09 (t, J = 7.6 Hz, 1H), 6.92–6.90 (m, 2H), 3.59 (q,
J = 6.0 Hz, 2H), 2.93 (t, J = 4.4 Hz, 4H), 2.70–2.67 (m, 6H), 2.64 (s, 3H), 2.55 (s,
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