Design and Synthesis of a Piperazinylalkylisoxazole Library for Subtype Selective Dopamine Receptor Ligands

Article abstract of DOI:10.1016/S0960-894X(02)00179-8

A piperazinylbutylisoxazole library was designed, synthesized and screened for the binding affinities to dopamine D₂, D₃, and D₄ receptors. Several ligands were identified to possess high binding affinity and selectivity for the D₃ and D₄ receptors over the D₂ receptor. Compounds 6s and 6t showed Ki values of 2.6 nM and 3.9 nM for the D₃ receptor with 46- and 50-fold selectivity over te D₂ receptor, respectively.

Full text of DOI:10.1016/S0960-894X(02)00179-8

Bioorganic & Medicinal Chemistry Letters 12 (2002) 1327–1330
Design and Synthesis of a Piperazinylalkylisoxazole Library
for Subtype Selective Dopamine Receptor Ligands
Mi Young Cha,a Byung Chul Choi,a Kyung Ho Kang,b Ae Nim Pae,a
Kung Il Choi,a Yong Seo Cho,a Hun Yeong Koh,a,* Hee-Yoon Lee,b,*
Daeyoung Jungc and Jae Yang Kongc
aBiochemicals Research Center, Korea Institute of Science and Technology, Cheongrayang, Seoul 130-650, South Korea
bCMDS, Department of Chemistry (BK21), Korea Advanced Institute of Science and Technology, Daejon 305-701, South Korea
cPharmaceutical Screening Research Team, Korea Research Institute of Chemical Technology, Daejon 305-600, South Korea
Received 22 February 2002; accepted 23 March 2002
Abstract—A piperazinylbutylisoxazole libary was designed, synthesized and screened for the binding affinities to dopamine D2, D3,
and D4 receptors. Several ligands were identified to possess high binding affinity and selectivity for the D3 and D4 receptors over the
D2 receptor. Compounds 6s and 6t showed Ki values of 2.6 nM and 3.9 nM for the D3 receptor with 46- and 50-fold selectivity over
the D2 receptor, respectively. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
Design of the Library
Recently we have reported1 a simple and efficient way of
constructing libraries of heterocycles focused on the
potential GPCR(G-Protein Coupled Receptor) binding
capability.2 In a classical pharmacology two types of
dopamine receptors acting through G-proteins had been
defined: the D1 receptor which mediates the activation
of adenylyl cyclase3 and the D2 receptor which mediates
its inhibition.4 Today at least five dopamine receptors
were identified through molecular pharmacology and they
Based on SAR(structure–activity relationships) analysis
of lignads for various GPCR’s, especially dopamine D3
and D4 receptors antagonists and privileged structures
for GPCRs,12 the major aspects of the structural design
to generate potent and selective ligands were divided
into three parts: part A containing a biogenic amine,
part B with alkyl chain as a linker and part C as elec-
trostatically similar groups to carbonyl group or aro-
matic group at Q. Part A generally includes heterocyclic
rings such as piperazine, piperidine or pyrrolidine. Part
B serves as a linker between the biogenic amine and the
hydrophobic aromatic group will be alkyl chain with
varying length or conformationally rigid carbocyclic
group. The chain length presumably reflects relative
position of aromatic groups and the biogenic amine in
spatial arrangement for binding to receptors, and could
impose selectivity among receptors. The Q position of
part C will both rigidify the part of the linker chain and be
used as alternatives to bioisosteres such as amide,13 car-
bonyl,14 oxygen,15 secondary alcohol,16 and hetero-
cycles.17 The substituents, S1 and S2 occupy the
hydrophobic binding sites for aromatic residues. In prac-
tice, we selected a scaffold with an arylpiperazine and an
isoxazole connected by a linker for the library. We
designed a synthetic scheme of piperazinylalkylisoxazole
library by combination of diverse arylpiperazines with
5
were grouped into two subfamilies, the D1-like (D1 and
6
9
D5 ) and the D2-like group (D2,7 D38 and D4 ), based on
amino acid homology and similar pharmacologic prop-
erties. The D2-like receptors show high affinities for the
most of the drugs used to treat schizophrenia and Par-
kinson’s disease. Classical dopamine D2 antagonists are
effective in treating schizophrenia. However, they cause
severe extrapyramidal side-effects10 that are thought to
result from the blockade of D2 receptors in the striatum
of brain. The distribution of the D3 and D4 receptors in
the limbic areas of brain suggests that these receptors
may be particularly an attractive target for the design of
potent and selective antipsychotic drugs without causing
extrapyramidal side effects.11
*Corresponding author. Tel.: +82-42-869-2835; fax: +82-42-869-
8370; e-mail: leehy@kaist.ac.kr
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00179-8

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M. Y. Cha et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1327–1330
the substituted phenylisoxazole aldehydes where n=3 or
n=4. (Fig. 1).
derivatives 5 using NaBH(OAc)3.19 Solutions of amines
5 and aldehydes 4 in CH2Cl2 was added NaBH(OAc)3
(3 equiv) and molecular sieve (3 beads). The reaction
mixtures were stirred for 5–12 h at room temperature.
After executing of the aqueous workup, reaction mix-
tures dissolved in 2 mL of diethyl ether followed by
treatment with 1M HCl solution in diethyl ether gave
HCl salts of the products 6 as solid. The solvent was
filtered off and the precipitant was washed with diethyl
ether, and dried in vacuo. All the compounds were
obtained in good yields (85–100%) and high purities
ranging from 85–96%. Purity and identity of the pro-
ducts were confirmed through 1H NMR, HPLC and
HRMS after the solid products were converted into the
corresponding free bases. A small library with well-
characterized 300 compounds was constructed by using
reductive amination, precipitation sequence (Scheme 2).
Chemistry
The synthetic strategy for obtaining a series of piper-
azinylalkylisoxazoles was adopted from the solution
phase combinatorial synthesis,1,18 where the amine
functionality in the scaffold provides particularly rapid
and easy purification of products. In practice, purifica-
tion was achieved by forming HCl salt of target mole-
cules without using chromatographic purification in the
last step.
The preparation of starting materials was outlined in
Scheme 1. The phenylalkylisoxazole aldehydes were pre-
pared from the substituted phenylaldehydes (1). Reaction
of hydroxylamine HCl with aldehydes 1 in ethanolic aqu-
eous solution (EtOH:H2O=2/1) gave the corresponding
oximes in 90–100% yields. Oximes 2 were treated with
NCS(N-chlorosuccinimide) and a catalytic amount of
pyridine in THF at 60 ꢀC under nitrogen atmosphere.
The reaction mixtures were stirred for 30 min at room
temperature and then 5-hexyn-1-ol (for n=4) was added
slowly. The reaction mixtures were treated with Et3N in
THF over 10 min. After executing the aqueous workup,
the alcohols 3 were obtained in 55–80% yields. Reaction
of alcohols 3 with PCC/SiO2 in CH2Cl2 gave aldehydes
4 in 45–85% yields (Scheme 1).
Results and Discussion
The generated phenylpiperazinylalkylisoxazole analogues
were evaluated in vitro for dopamine D2–D4 receptors
binding affinity by measuring their ability to displace
radioligands ([3H]spiperone for D2 and D4, [3H]YM-
09151–2 for D3) from the cloned human dopamine
receptors D2long, D3 and D4.2 which were expressed in
CHO cells, respectively. The affinity and selectivity of
this class of compounds for the dopamine receptors
were largely dependent on substitution pattern at
o-position on phenylpiperazinyl group and the length of
the alkyl chain linker. The extension of alkyl chain from
n=3 to n=4 affected the binding affinity greatly since
the affinities of compounds with a three atom tether
were very low and showed no selectivity among recep-
tors in the primary screening. Table 1 shows the binding
data of the selected compounds that showed good
activity and selectivity among dopamine D2, D3, and D4
receptors. With a methoxy group at o-position of phenyl
group of Ar1(Ar13), the binding affinity to dopamine D3
receptor increased (compounds 6k–6q). Extension of
methoxy group at o-position of phenyl group (Ar14) to
ethoxy group (compounds 6r–6u) gave the further
improvement of affinity for the D3 receptor (2.6–19 nM).
On the other hand, with a methyl group (compounds 6h–
Construction of solution phase combinatorial library was
described in Scheme 2. The combinatorial synthesis was
accomplished by the reductive amination of the prepared
aldehydes 4 with commercially available phenylpiperazine
Figure 1.
Scheme 1. Reagents and reaction conditions: (i) NH2OHꢁ HCl, Na2CO3, 60 ꢀC, 1 h, EtOH/H2O (2/1), 90–100%; (ii) pyridine (cat.), NCS, 60 ꢀC,
0.5 h, THF/4-pentyn-1-ol (for n=3) or 5-hexyn-1-ol (for n=4), Et3N, 50 ꢀC—rt, 2 h, 55–80%; (iii) PCC, silica gel, CH2Cl2, 5–12 h, 45–85%.
Scheme 2. Reagents and reaction conditions: (i) NaBH(OAc)3 (3 equiv), molecular sieves (3 beads), CH2Cl2, rt, 5–12 h; (ii) After executing aqueous
workup, the reaction mixture in 2 mL of diethyl ether was treated with ethereal HCl. The precipitant was washed with diethyl ether and dried, 85–
100%.

M. Y. Cha et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1327–1330
Table 1. Binding affinity (Ki, nM)20 for n=4
1329
Entrya
Substituent
Receptor
D3
Selectivity
Ar1
Ar2
D2
D4
96
1697
774
76
117
541
346
413
465
683
33
50
257
34
21
198
88
51
28
12
34
233
58
1346
2314
2438
1118
2145
2243
603
42
D2/D3
D4/D3
6a
6b
6c
6d
6e
6f
Ar11
Ar11
Ar11
Ar11
Ar11
Ar11
Ar11
Ar12
Ar12
Ar12
Ar13
Ar13
Ar13
Ar13
Ar13
Ar13
Ar13
Ar14
Ar14
Ar14
Ar14
Ar15
Ar15
Ar16
Ar16
Ar16
Ar16
Ar16
Ar16
Ar16
Haloperidol
Ar21
Ar22
Ar23
Ar24
Ar25
Ar26
Ar27
Ar23
Ar25
Ar26
Ar21
Ar22
Ar23
Ar24
Ar25
Ar26
Ar27
Ar22
Ar23
Ar25
Ar26
Ar23
Ar27
Ar21
Ar22
Ar23
Ar24
Ar25
Ar26
Ar27
2530
1470
3388
2196
4136
3366
9724
1401
2108
6512
321
295
418
440
726
1727
964
95
119
194
341
5148
585
1461
1522
2618
3828
620
12
60
24
16
15
18
27
38
163
292
23
6
5.2
6.5
4.7
9.5
15
19
2.6
3.9
9
20
10
30
19
20
16
52
15
40
25
211
25
8
28
32
5
141
137
276
187
360
37
8
30
13
11
3
6g
6h
6i
6j
6k
6l
13
22
2
14
49
1.4
8
6m
6n
6o
6p
6q
6r
80
68
49
5
154
181
64
4
21
6
5
46
50
38
3
11
3
6s
6t
6u
6v
6w
6x
6y
6z
6aa
6bb
6cc
6dd
4
257
59
12
6
Figure 2.
49
80
130
239
12
32
12
0.72
45
121
122
70
41
150
15
1.7
the spacer that connected two parts generated an array
of activity and selectivity for the dopamine receptors.
Also, the isoxazole ring as an alternative of bioisosteres
play a important role in binding with receptors. With
the chain length of four-carbon tether, most of the
compounds showed good binding affinity and selectivity
at the desirable target receptors, the D3 and D4 recep-
tors over D2 receptor. Compounds 6s and 6t showed Ki
values of 2.6 and 3.9 nM for the D3 receptor with 46-
fold and 50-fold selectivity over the D2 receptor,
respectively. Further QSAR(Quantitative Structure and
Activity Relationships) studies of these series are in
progress.
486
491
18
aAll compounds gave satisfactory analytical and/or mass spectral data.
6j) at o-position of phenylpiperazine, the binding affi-
nities decreased (38–292 nM). o-Fluorophenylpiperazine
analogues (compounds 6v and 6w) also gave good
binding affinities (10 and 20 nM). Among phenylpiper-
azine analogues, compounds 6s and 6t showed high
binding affinity with Ki values of 2.6 and 3.9 nM,
respectively. Though diphenylmethylpiperazine analo-
gues (compounds 6x–6dd) showed rather lower binding
affinity (15–52 nM) than arylpiperazines for the D3,
compounds 6x to 6dd exhibited high selectivity for
dopamine D3 receptor over the D2 and D4 receptors
regardless of Ar2. For compounds 6z and 6aa, 130- to
239-fold higher selectivity of the D3 receptor over the
D2 receptor was observed. Compounds 6e, 6g and 6v
showed the high selectivity for the D3 over the D2
receptor (276-, 360- and 257-fold, respectively). Com-
pound 6x–6aa showed a good selectivity for the D3 over
the D4 receptor. Generally, high affinity appeared to be
guaranteed by 3,4-dimethoxyphenyl- (Ar23), 2,4-dime-
thoxyphenyl- (Ar24) and 2-thienyl- (Ar25) groups and
high selectivity was exerted either by o-alcoxy group or
an extra phenyl group (Table 1; Fig. 2 ).
Acknowledgements
This work was supported by the Korea Ministry of
Science and Technology.
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20. Assay protocol: Sf-9 membranes expressing either dopa-
mine hD2L, dopamine rD3 or hD4.2 receptors were purchased
from PerkinElmer Life Sciences (Boston, MA). Radioligands
used were [3H]Spiperone (D2 and D3 dopamine binding
assays, 1 nM), and [3H] YM-09151–2 (D4 dopamine binding
assays, 0.06 nM). [3H]Spiperone and [3H] YM-09151-2 bind-
ings were performed by the protocol provided by supplier of
Sf-9 membranes for both hD2L and rD3, and hD4.2 receptors,
respectively. Briefly, the buffer used in hD2L or rD3 receptor
binding assay was 50 mM Tris–HCl (pH 7.4), 10 mM MgCl2,
1 mM EDTA, or 50 mM Tris–HCl (pH 7.4), 5 mM MgCl2,
5 mM EDTA, 5 mM KCl, 1.5 mM CaCl2, 120 mM NaCl,
respectively. In [3H] YM-09151–2 receptor binding assays, the
buffer containing 50 mM Tris–HCl (pH 7.4), 5 mM MgCl2,
5 mM EDTA, 5 mM KCl and 1.5 mM CaCl2 was used. Non-
specific binding was determined with haloperidol (10 uM) and
clozapine (10 uM) for D2 and D3, and D4 receptors, respec-
tively. Competition binding studies were carried out with 8
concentrations of the test compound run in duplicate tubes,
and isotherms from three assays were calculated by compu-
terized nonlinear regression analysis (GraphPad Prism Pro-
gram, San Diego, CA) to yield inhibition constant (Ki)
values.