The influence of an ethylene spacer on the 5-HT1A and 5-HT2A receptor affinity of arylpiperazine derivatives of amides with N-acylated amino acids and 3-differently substituted pyrrolidine-2,5-diones

Article abstract of DOI:10.1016/j.ejmech.2008.05.021

A library of ethylene analogs of the previously described arylpiperazines with N-acylated amino acids was synthesized on SynPhase Lanterns and the library representatives were evaluated for their 5-HT_1A and 5-HT_2A receptor affinities

Full text of DOI:10.1016/j.ejmech.2008.05.021

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 44 (2009) 800e808
http://www.elsevier.com/locate/ejmech
Original article
The influence of an ethylene spacer on the 5-HT1A and 5-HT2A receptor
affinity of arylpiperazine derivatives of amides with N-acylated amino
acids and 3-differently substituted pyrrolidine-2,5-diones
b
b
c
c
Pawe1 Zajdel a, , Gilles Subra , Pascal Verdie , Andrzej J. Bojarski , Beata Duszynska ,
*
´
Katarzyna Basista a, Jolanta Obniska a, Jean Martinez b, Maciej Paw1owski a
a Department of Medicinal Chemistry, Jagiellonian University Medical College, 9 Medyczna Street, 30-688 Krako´w, Poland
Equipe Aminoacides Peptides et Prote´ines, Institut des Biomole´cules Max Mousseron IBMM, UMR CNRS 5247, Faculte´ de Pharmacie,
b
´
Universite´ Montpellier I et II, 15 Avenue Charles Flahault, 34060 Montpellier, France
c Department of Medicinal Chemistry, Institute of Pharmacology, Polish Academy of Sciences, 12 Sme˛tna Street, 31-343 Krako´w, Poland
Received 11 March 2008; received in revised form 21 May 2008; accepted 22 May 2008
Available online 7 July 2008
Abstract
A library of ethylene analogs of the previously described arylpiperazines with N-acylated amino acids was synthesized on SynPhaseÔ Lan-
terns and the library representatives were evaluated for their 5-HT1A and 5-HT2A receptor affinities. The obtained results were compared with
those reported for compounds containing propylene and a butylene spacer and they revealed that 5-HT1A receptor affinity decreased proportion-
ally with the length of the alkyl chain. Furthermore, the synthesized 3-cycloalkyl derivatives containing two methylene group spacers (20, 21)
showed that the 3-position of pyrrolidine-2,5-dione preferred substituents of hydrophobic character.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Long-chain arylpiperazines; Succinimides; Spirosuccinimides; Pyrrolidine-2,5-dione; 2-Azaspiro[4.5]decane-1,3-dione; Pyrrolidine-5-one-carboxa-
mide; Prolinamides; Solid-phase synthesis; 5-HT1A/5-HT2A receptor ligands
1. Introduction
a general premise of combinatorial chemistry has evolved by
changing into the preparation of biological target-oriented and
rationally designed compound libraries [1]. Nowadays there is
a demand for a discrete compound library of high diversity.
Moreover, to provide reproducibility of biological data, library
members are often purified using automated or semi-automated
chromatography systems. Recent bibliography surveys of com-
binatorial library synthesis reveal researchers’ constant interest
in this technique which may ease the efforts of medicinal chem-
ists to evaluate structureeactivity relationships [2].
Apart from the increasing interest in discovering ligands
acting through high-end serotonin receptor subtypes (5-HT6,
5-HT7), compounds modulating 5-HT1A and/or 5-HT2A recep-
tors’ activity still remain an important group with a high po-
tential for the treatment of CNS disorders, including anxiety
[3], depression, and schizophrenia [4] and they provide a pro-
spective field for drug development [5e7].
Combinatorial chemistry has become an integral part of drug
discovery projects in many pharmaceutical companies in the
mid-1990s. Its introduction was a consequence of a growing
number of new pharmacological targets, and at the same time
a response to the rising demand for novel hit identification.
Combinatorial chemistry combined with solid-phase methodol-
ogies allowed quick generation of a vast number of closely
related compounds. A high complexity of first-generation
combinatorial libraries, followed by tedious deconvolution
and re-synthesis of individual library members resulted in the
identification of the so-called ‘‘false positives’’ and contributed
to the high attrition rate of several research projects. Since then,
* Corresponding author. Tel.: þ48 12 657 05 60; fax: þ48 12 657 02 62.
E-mail address: mfzajdel@cyf-kr.edu.pl (P. Zajdel).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.05.021

P. Zajdel et al. / European Journal of Medicinal Chemistry 44 (2009) 800e808
801
We previously reported on the successful application of
combinatorial chemistry techniques for generating the ratio-
nally designed libraries of 5-HT1A and 5-HT2A receptor
ligands, namely arylpiperazine derivatives containing N-acyl-
ated amino acid fragments [8,9]. Structural modification
comprised variations in the type of amide/imide moiety
(3-aminopyrrolidine-2,5-dione, pyrrolidine-5-one-2-carboxa-
mide, and asparaginyl, pipecolyl, prolyl amides), the nature
of the N-acyl fragment (alkyl, cycloalkyl, aryl), the length of
an alkyl spacer (propylene, butylene) and finally the kind
of substituents at the 4-phenylpiperazine fragment (o-OCH3,
m-Cl) (Fig. 1).
of 3-amino-pyrrolidine-2,5-dione, pyrrolidine-2-oxo-carboxa-
mide, prolinamide derivatives which contained two methylene
group alkyl spacers between the imide/amide part and the ba-
sic nitrogen atom of the arylpiperazine fragment. In the second
part, we discussed the influence of the kind of substituent at
the 3-position of pyrrolidine-2,5-dione moiety on serotonin re-
ceptor affinity.
2. Chemistry
2.1. Library synthesis
The library representatives tested showed diversified affinity
for 5-HT1A receptors ranging from 4 nM to 4000 nM, and dis-
played high-to-low affinity for 5-HT2A receptors (1e2130 nM)
(Table 1). Structureeactivity relationship studies showed that
the length of a tetramethylene spacer between the amide/imide
core and arylpiperazine fragment was optimal to obtain potent
5-HT1A and 5-HT2A receptor ligands. It was finally found that
the optimized lead structure (Ie9{5,3}) e pyrrolidine-2,5-
dion-3-yl-cyclohexanecarboxamide, a pre- and postsynaptic
5-HT1A agonists e demonstrated distinct activity in some
models of anxiety and depression (Fig. 2) [10].
In the course of developing 5-HT1A/5-HT2A receptor li-
gands our research group also described some derivatives of
pyrrolidine-2,5-dione with spirocycloalkyl moiety at the 3-po-
sition e 2-azaspiro[4.4]nonane- and [4.5]decane-1,3-diones
[11,12]. Linkage of these imide cores using a methylene, eth-
ylene and propylene spacer with 4-arylpiperazine moieties
yielded compounds which displayed moderate-to-high affinity
for 5-HT1A receptors (Ki ¼ 3.1e370 nM), and low-to-high af-
finity for 5-HT2A (Ki ¼ 32e1370 nM). Interestingly, the recep-
tor affinity depended highly upon the length of an alkyl spacer,
the highest affinity for both receptor subtypes being observed
for ethylene analogs.
The library synthesis was carried out on solid support e
SynPhaseÔ Lanterns (Mimotopes, Pty). This modular type
of support consists of a plastic core grafted with polyamide
surface functionalized with a BAL linker [13]. To manually
manage the library construction, a split-and-pool approach
was chosen. The Lanterns were equipped with colored cogs
and spindles (corresponding to building blocks) producing a vi-
sual tagging system. The library was constructed as outlined in
Schemes 1 and 2.
The structure of the final products was encoded according
to the nomenclature defining the assignment of the residues
of the library members based on the American Chemical So-
ciety guidelines, where x{y,z} represents a compound having
the general structure x (presented in scheme), where y and z
indicate the particular residues from the separately defined
list of diversity reagents a, and diversity reagents b,
respectively.
The workflow began upon the preparation of substituted 4-
(2-methoxyphenyl)-1-piperazinyl-ethylamine and 4-(3-chloro-
phenyl)-1-piperazinyl-ethylamine (diversity reagent 2, Fig. 3),
according to the procedure described elsewhere [14]. The
substituted ethylamines were attached to a solid support by re-
ductive amination to give the support-bound secondary prod-
uct 3. Lanterns were pooled together and washed with
a 10% AcOH and subsequently according to the classic wash-
ing protocol. Fmoc amino acids were then selectively coupled
Based on the above findings, in the present research we
conducted a solid-phase synthesis and preliminary biological
investigation of a series of the previously described analogs
O
H
N
N
*
O
O
R
ArP
O
R1
Amide
N
*
N
O
O
N
R
O
N
N
n
N
*
R
1 = H, o-OCH3, m-Cl
O
O
spacer
n = 1, 2
*
R
O
R
NH2
R = methyl, cyclopentyl, cyclohexyl,
phenyl, norborn-2-ylmethyl, adamantyl
O
H
N
*
O
O
R
N
R
*
O
O
Fig. 1. General structure and chemical modifications of the investigated compounds.

802
P. Zajdel et al. / European Journal of Medicinal Chemistry 44 (2009) 800e808
Table 1
Affinity data for 5-HT1A and 5-HT2A receptors of the library members tested and their respective propyl and butyl analogs reported elsewhere
Compound
Ki (nM)
Compound
Ki (nM)
Compound
Ki (nM)a
5-HT1A
5-HT2A
5-HT1A
5-HT2A
5-HT1A
5-HT2A
9{6,3}
9{6,4}
9{7,1}
9{7,3}
9{7,5}
9{7,6}
10{6,3}
10{7,3}
10{7,4}
17{6,4}
17{7,1}
17{7,3}
17{7,6}
17{6,2}
a
3775
10 020
7586
7440
497
1411
1742
5678
7343
12 417
4799
3258
14 936
8706
1302
8600
2326
1917
357
9{2,3}a
e
360
e
92
e
9{3,3}c
9{3,4}a
9{5,1}b
9{5,3}c
9{5,5}c
9{5,6}c
10{3,3}a
10{5,3}b
e
26 ꢂ 4
41
69
6.51 ꢂ 1
75
9{4,1}b
9{4,3}b
9{4,5}b
9{4,6}b
10{2,3}a
e
480
230
40
e
e
e
19 ꢂ 3
9 ꢂ 2
4 ꢂ 2
111
183 ꢂ 12
47 ꢂ 6
173 ꢂ 17
163
e
579
63
e
8518
1001
1507
1950
6600
3553
350
1730
e
800
e
17
e
10{4,4}d
e
17{4,1}d
e
390
e
e
e
78
e
128
e
17{3,4}a
e
580
e
3140
e
e
e
17{5,3}c
17{5,6}c
Buspironee
19 ꢂ 3
3 ꢂ 0.2
36
990 ꢂ 17
503 ꢂ 8
e
e
e
e
6.05
2013
Clozapinee
e
The estimated Ki (see Ref. [8]).
The estimated Ki (see Ref. [9]).
Ki from Ref. [10].
b
c
d
e
Personal communication.
Ki values reported in the literature: buspirone (rat hippocampus, [3H]-8-OH-DPAT) 3.8e56 nM; clozapine (rat cortex, [3H]-ketanserin) 2.6e12 nM
(see Ref. [18]).
to a secondary amine by a symmetric anhydride method using
diisopropylcarbodiimide (DIC) in dimethylformamide (DMF)
to obtain chemsets 4, 5, and 11 (Schemes 1 and 2). The effec-
tiveness of secondary amine acylation was checked by the
HPLC analysis, and by a colorimetric test performed on a slice
of the Lantern (the chloranil test). Then, all the Lanterns were
pooled together and treated with a solution of a 20% piperi-
dine in DMF. After washing, the Lanterns were sorted again
and reacted with six discrete proteogenic carboxylic acids
(Fig. 4) in the presence of HBTU and DIEA in DMF solutions,
to form the solid supported chemsets 7, 8, and 14. The conver-
sion of the representatives of chemsets 7 and 8 into pyrroli-
dine-2,5-diones (9) and pyrrolidine-5-one-2-carboxamides
(10) was achieved by the treatment with a mixture of TFA/
CHCl3/SOCl2 (50/50/1.5, v/v/v) at a temperature of 40 ꢀC
for 10 h. The reaction was carried out in small glass vials. Fi-
nally, cleavage of the Lanterns of chemset 14 with a mixture of
TFA/DCM at room temperature for 60 min afforded the de-
sired proline amides of chemset 17. Prolyl amides were
cleaved in individual polypropylene vials. After removal of
the cleavage cocktail under a nitrogen flow, the samples
were solubilized in an acetonitrile/water (50:50, v/v) mixture
containing a 0.1% TFA. An aliquot of each library member
was submitted to an LC/MS analysis and the remainder was
lyophilized.
Library members 10{6,6} and 10{7,6} containing the same
adamantyl group were obtained with low yields (ꢁ5%); the
main products cleaved from the support were N-unacylated
pyroglutamyl derivatives.
All library members were purified by using a reverse-phase
preparative LC/MS ESI automated system (Waters Micro-
mass). Then, the fractions containing the expected ions were
pooled together, freeze-dried, and submitted again to LC/MS
analysis. The purity percentage was based on the relative
peak absorbance at 214 nm on the UV spectra (Table 2).
The overall yields of the final products, calculated on the basis
of the initial loading of the Lanterns, were between 26% and
49%, with purities ranging from 78% to 100%. Finally, 14 pu-
rified compounds (HPLC purity over 81%) were submitted to
biological assays.
2.2. Synthesis of arylpiperazine derivatives of
pyrrolidine-2,5-diones
Compounds 18e21 were synthesized according to Scheme 3.
The starting 2-cyclohexane succinic acid was prepared as de-
scribed elsewhere [15]. A one-pot cyclization of the succinic de-
rivatives with the appropriate arylpiperazineeethylamines by
heating them at ca. 190e200 ꢀC for 1.5e2 h yielded final
imides.
O
H
N
N
N
O
O
N
O
compd I
Ki 5-HT1A 19 nM
Ki 5-HT2A = 183 nM
3. Biological evaluation
=
Biological characteristics, i.e. affinity for serotonin 5-HT1A
and 5-HT2A receptors of the 14 selected library representa-
tives, were measured in vitro on the basis of the screening
Fig. 2. Chemical structure of lead structure I and its affinity for 5-HT1A and 5-
HT2A receptors.

P. Zajdel et al. / European Journal of Medicinal Chemistry 44 (2009) 800e808
803
O
i
MeO
O
O
O
=
N
H
H2
N
N
N
N
H
H
N
R1
2{6-7}
1 = BAL PA Lantern
H
OMe
O
N
3
R1
ii, Fmoc-Asp(OtBu)OH
ii, Fmoc-Glu(OtBu)OH
O
O
tBu
tBu
O
N
H
H
N
N
N
N
N
Fmoc
O
O
O
N
N
Fmoc
R1
R1
4
5
O
H
iii, iv
O
iii, iv
tBu
tBu
O
H
N
N
N
N
N
N
N
N
O
R2
O
O
O
O
N
N
O
R2
R1
R1
7
8
v
v
O
H
H
N
N
O
N
N
O
R2
N
N
O
R2
R1
R1
9
10
Scheme 1. Solid-phase synthesis routes for chemsets 9 and 10: (i) diversity reagent 2{6e7}, NaBH3CN, 1% AcOH/DMF, 60 ꢀC, 12 h; (ii) DIC, DMF, RT, 12 h;
(iii) 20% piperidine/DMF; (iv) diversity reagent 6{1e6}, HBTU, DIEA, DMF, RT, 2 h; (v) TFA/CHCl3/SOCl2, 40 ꢀC, 10 h.
protocol described previously [8]. At the same time, the two
well-known reference serotonin drugs buspirone and clozapine
were examined, the obtained results being consistent with our
previous data as well as with those reported in the literature
(Table 1). For the four new pyrrolidine-2,5-dione derivatives,
standard full binding experiments [16] were conducted and
the Ki constants were calculated from IC50 values according
to the ChengePrusoff equation [17] (Table 3).
O
i
H2
N
N
H
N
N
H
N
R1
2{6-7}
N
1 = BAL PA Lantern
3
R1
ii, Fmoc-Pro-OH
iii, iv
N
N
N
N
N
N
O
O
Fmoc
N
N
O
O
R2
R1
R1
11
14
vi
H
N
N
N
O
N
O
R2
R1
17
Scheme 2. Solid-phase synthesis routes for chemset 17: (i) Diversity reagent 2{6e7}, NaBH3CN, 1% AcOH/DMF, 60 ꢀC, 12 h; (ii) DIC, DMF, RT, 12 h; (iii) 20%
piperidine/DMF; (iv) diversity reagent 6{1e6}, HBTU, DIEA, DMF, RT, 2 h; (vi) TFA/DCM.

804
P. Zajdel et al. / European Journal of Medicinal Chemistry 44 (2009) 800e808
H2N
N
N
1
H2N
N
O
H2N
N
n
n
N
N
Cl
2 n = 1
3 n = 2
6 n = 0
4 n = 1
5 n = 2
7 n = 0
Fig. 3. Diverse primary aliphatic amines, 2{1e7}.
4. Results and discussion
were always more potent ligands than their propylene analogs.
Thus in the following phase of experimentation, we focused
our attention on evaluating the role of the kind of substituent
in the 3-position of pyrrolidine-2,5-diones on 5-HT1A receptor af-
finity. To this end, two unsubstituted (18, 19) and 3-cyclohexyl-
pyrrolidine-2,5-diones (20, 21), connected by two methylene
alkyl spacers with o-methoxy and m-chlorophenylpiperazines,
were synthesized. The affinity of 18 and 19 for 5-HT1A receptors
was 2087 nM and 3675 nM for o-OCH3 and m-Cl derivatives,
respectively (Table 3). Introduction of a cycloalkyl fragment
directly connected to imide yielded compounds 20 and 21
with significantly higher 5-HT1A and 5-HT2A receptor affinity
(Table 3). However, these receptor affinity values were lower
than the values reported for the respective spiro analogs e
compounds 22 and 23. The difference in 5-HT1A receptor af-
finity for the arylpiperazinyl-ethyl derivatives of 3-acylamino-,
3-cyclohexyl- and unsubstituted pyrrolidine-2,5-diones, and
azaspiro[4.5]decane-1,3-dione derivatives suggests that the
additional amide group in chemset 9 is unfavorable for the in-
teraction with both types of the serotonin receptors tested. It
visibly stems from the shortening of the length of a linker
and concurrent restriction of conformational freedom. As
a consequence, chemset 9 derivatives containing a short spacer
cannot occupy a suitable space in the receptor pocket to sup-
port both the H-bond interaction originating with the amide
bond and the hydrophobic interactions resulting from the pres-
ence of the cycloalkyl group (R2). It is noteworthy that the two
kinds of interactions mentioned above were advantageous to
the binding of compounds containing a propylene spacer or,
preferably, a butylene one. As has been mentioned in Section
1, the latter displayed a significantly higher 5-HT1A and 5-
HT2A receptor affinity (Table 1).Therefore it seems that in
the case of the ethylene spacer containing pyrrolidine-2,5-
diones, substituents in 3-position that induce hydrophobic in-
teractions are the only preferable ones for 5-HT1A and
5-HT2A receptor binding. Taking account of the data presented
above, the introduction of a spirocycloalkyl fragment inducing
A 36-member library of o-methoxy- and m-chlorophenylpi-
perazines, connected by an ethylene spacer to N-acylated
amino acid-derived amide fragments, was synthesized on
a solid support and the 14 selected library representatives
were evaluated for their 5-HT1A and 5-HT2A receptor affinities
(Table 1). The compounds were chosen for direct comparisons
with the previously reported ones.
The compounds synthesized within the present project in-
cluded structural analogs of the previously presented modifica-
tions in the amide fragment. The developed chemistry was
amenable to the diversification of amino acid-derived small
cyclic fragments: 3-acylaminopyrrolidine-2,5-dione and N-
acyl-pyrrolidine-2-oxo-carboxamide, which resulted from the
cyclization of aspartic and glutamic acid, respectively. Fur-
thermore, prolinamides were also introduced as deoxo-analogs
of pyroglutamyl derivatives.
In comparison with the previously reported libraries [8,9],
the pronounced decrease in the 5-HT1A and 5-HT2A receptor
affinity of the tested compounds resulted from the shortening
of the length of an alkyl spacer (Table 1). It has to be stressed
that, despite their lower activity, some general and well-known
trends were observed for arylpiperazine ligands. Differences in
the receptor binding profile resulted mainly from the substitu-
tion pattern in the phenylpiperazine fragment (o-methoxy de-
rivatives bind preferentially to 5-HT1A receptors) and the
molecular volume of the cycloalkyl moiety. In agreement
with our previous study, the substituents with a high molecular
bulk were always the most potent in the series; adamantane-
carboxamides had up to 10e12-fold higher affinity for 5-
HT1A sites than cyclohexanecarboxamides.
The study showed that the length of an alkyl chain played
an important role in the affinity for 5-HT1A and 5-HT2A recep-
tors (Table 1). However, these findings disagree with the re-
sults obtained previously with a group of azaspiranes [11].
Among others, compounds containing an ethylene spacer
O
O
O
O
O
O
OH
OH
H3C
OH
OH
OH
OH
1
2
3
4
5
6
Fig. 4. Diverse carboxylic acids, 6{1e6}.

P. Zajdel et al. / European Journal of Medicinal Chemistry 44 (2009) 800e808
805
Table 2
Analytical data for the library members
carboxamido one. And last but not least, the substitution of
pyrrolidin-2,5-dione with a spiro fragment seems to be the
most preferential for 5-HT1A/5-HT2A receptor binding.
Compound Purity MW
a
[M þ H]þ Compound Purity MW
[M þ H]þ
a
%
%
calc. found
calc. found
9{6,1}
9{6,2}
9{6,3}
9{6,4}
9{6,5}
9{6,6}
9{7,1}
9{7,2}
9{7,3}
9{7,4}
9{7,5}
9{7,6}
10{6,1}
10{6,2}
10{6,3}
10{6,4}
10{6,5}
10{6,6}
a
94
378.60 379.10
432.64 433.08
446.65 447.11
440.65 441.10
472.67 473.15
498.69 499.10
374.15 375.10
428.19 429.00
442.20 443.30
436.20 437.14
468.22 469.15
494.24 495.11
392.61 393.00
446.65 447.11
460.66 461.15
454.66 455.05
486.68 487.13
512.70 e
10{7,1}
10{7,2}
10{7,3}
10{7,4}
10{7,5}
10{7,6}
17{6,1}
17{6,2}
17{6,3}
17{6,4}
17{6,5}
17{6,6}
17{7,1}
17{7,2}
17{7,3}
17{7,4}
17{7,5}
17{7,6}
90
88
93
79
83
388.16 389.07
442.20 443.20
456.21 457.15
450.21 451.11
482.23 483.19
508.25 e
378.62 379.23
432.66 433.20
446.67 447.17
440.67 441.12
472.69 473.18
498.71 499.20
374.17 375.20
428.62 429.22
442.22 443.20
436.22 437.16
468.24 469.21
494.26 495.26
6. Experimental
98
97
6.1. Materials
80
85
All the solvents were obtained from Acros, and were used
without purification. PAeBAL linker polyamide SynPhaseÔ
Lanterns with 18 mmol loading, and colored tags were pro-
vided from Mimotopes. All the Fmoc amino acids and
HBTU reagent were purchased from Senn Chemicals. All
other reagents were from Aldrich.
100
86
e
84
94
92
96
98
95
88
100
95
100
100
82
81
86
Purity of pyrrolidin-2,5-diones was confirmed by TLC per-
formed on Merck silica gel 60 F254 aluminium sheets (Merck,
Darmstadt, Germany), by using a mixture of DCM:methanol e
S1 (9:1, v/v). Spots were detected by their absorption under
UV light (l ¼ 254 nm). The chemical structures were con-
firmed by elemental and spectral analyses (1H NMR, MS).
1H NMR spectra were obtained using a Varian BB 200
(300 MHz) spectrometer; chemical shifts (d) are expressed in
ppm downfield from the internal TMS as a reference; J values
are in hertz (Hz), and splitting patterns are designated as fol-
lows: s (singlet), d (doublet), t (triplet), m (multiplet). Elemen-
tal analyses were carried out using an Elementar Vario EL III,
and were within ꢂ0.4% of the theoretical values.
85
78
93
100
99
79
96
81
e
Determined under monitoring HPLC at 214 nm.
orthogonal geometry of a non-pharmacophoric imide fragment
is responsible for the significant enhancement of the 5-HT1A
and 5-HT2A receptor affinity of compounds containing an eth-
ylene spacer.
The following abbreviations were used: CHCl3 e chloroform;
DCM e dichloromethane; DIC e N,N0-diisopropylcarbodii-
mide; DIEA e diisopropylethylamine; DMF e dimethylfor-
5. Conclusions
Summing up, we have synthesized a 36-member library of
arylpiperazines connected by an ethylene spacer with N-acyl-
ated amino acid-derived amide fragments. The surprisingly
low affinities of the library representatives for the 5-HT1A
and 5-HT2A receptors prompted us to determine the influence
of the length of an alkyl spacer and the nature of the substit-
uent at 3-position of the pyrrolidine-2,5-dione fragment on
receptor affinity. The subsequently synthesized pyrrolidine-
2,5-diones containing a flexible 3-cycloalkyl fragment and dis-
playing high 5-HT1A/5-HT2A affinity supported our hypothesis
that not only the length of an alkyl spacer, but above all the
type of substituent at 3-position influenced 5-HT1A and 5-
HT2A receptor affinity. The results of the present study seem
to suggest that in order to obtain high-affinity ligands for 5-
HT1A/5-HT2A receptors in a group of pyrrolidine-2,5-diones
containing an ethylene spacer, the imide core should rather
be substituted with a 3-cycloalkyl fragment than with a 3-
mamide;
HBTU
e
O-(1H-benzotriazol-1-yl)-1,1,3,
3-tetramethyluronium hexafluorophosphate; SOCl2 e thionyl
chloride; TFA e trifluoroacetic acid. The other abbreviations
used were recommended by the IUPAC-IUB Commission
(Eur. J. Biochem. 1984, 138, 9e37).
6.2. LC/MS analysis
Samples were prepared in acetonitrile/water (50:50 v/v),
containing a 0.1% TFA. The LC/MS system consisted of a Wa-
ters Alliance 2690 HPLC, coupled to a Micromass (Manches-
ter, UK) Platform II spectrometer (electrospray ionization
mode, ESIþ). All the analyses were carried out using a C18
Xterra MS, 21 ꢃ 3.0 mm column. A flow rate of 500 mL/min
and a gradient of 0e100% B over 5 min were used. Eluent
A: water/0.1% TFA; eluent B: acetonitrile/0.1% TFA. Positive
ion electrospray mass spectra were acquired at a solvent flow
rate of 100e500 mL/min. Nitrogen was used for both the neb-
ulizing gas and the drying gas. The data were obtained in
a scan mode ranging from 400 to 1400 m/z in 0.1 s intervals;
10 scans were summed up to get the final spectrum.
R1
N
R
O
O
O
i
N
N
OH
6.3. LC/MS purification
R
OH
O
18 - 23
Samples were prepared in acetonitrile/water (50/50 v/v)
mixture, containing 0.1% TFA. The LC/MS autopurification
system consisted of a binary pump Waters 2525, an injector/
Scheme 3. Synthesis of pyrrolidine-2,5-dione derivatives 18e23: (i) diversity
reagent 2{6e7}, 180e200 ꢀC, 3 h.

806
P. Zajdel et al. / European Journal of Medicinal Chemistry 44 (2009) 800e808
Table 3
Affinity data for 5-HT1A and 5-HT2A receptors of selected pyrrolidine-2,5-dione derivatives
R1
N
O
N
R
N
R'
O
Compound
R
R0
R1
Ki (nM)
5-HT1A
5-HT2A
9{7,3}
9{6,3}
18
(S )-Cyclohexanecarboxamide
H
H
H
H
H
H
o-OCH3
m-Cl
7440a
7343a
(S )-Cyclohexanecarboxamide
3775a
1411a
H
o-OCH3
m-Cl
2087 ꢂ 220
3675 ꢂ 412
24 ꢂ 3
17 570 ꢂ 1250
2120 ꢂ 185
868 ꢂ 73
137 ꢂ 9
19
20
H
Cyclohexyl
Cyclohexyl
(CH2)5
(CH2)5
o-OCH3
m-Cl
o-OCH3
m-Cl
21
227 ꢂ 15
5.7 ꢂ 0.8
4.3 ꢂ 0.7
22b
23c
a
254 ꢂ 12
15 ꢂ 0.5
The estimated Ki (see Ref. [8]).
b
c
Data taken from Ref. [11].
Data taken from Ref. [12].
fraction collector Waters 2676, coupled to a Waters Micro-
mass ZQ spectrometer (electrospray ionization mode, ESIþ).
All the purifications were carried out using a Waters Symme-
try Shield C18 19 ꢃ 100 mm, 5-mm particle size, column. A
flow rate of 20 mL/min and a gradient of 0e60% B over
20 min were used. Eluent A: water/0.1% TFA; eluent B: ace-
tonitrile/0.1% TFA. Positive ion electrospray mass spectra
were acquired at a solvent flow rate of 204 mL/min. Nitrogen
was used for both the nebulizing gas and the drying gas.
The data were obtained in a scan mode ranging from 100 to
1000 m/z in 0.1 s intervals; 10 scans were summed up to get
the final spectrum. Collection control trigger was set on single
protonated and diprotonated ion with an MIT (minimum inten-
sity threshold) of 8 ꢃ 105.
6.6. Secondary amine acylation protocol
Three DMF solutions (20 mL) containing an Fmoc-pro-
tected amino acid and DIC each, were freshly prepared in
a
standard Schott flask before acylation ([Fmoc-AA-
OH] ¼ 200 mM, [DIC] ¼ 100 mM), and were left for 10 min
to form an active anhydride. Then the Lanterns were immersed
in a preactivated solution and left overnight at room tempera-
ture. The solution was decanted, and the Lanterns were
washed following the standard washing protocol. The acyla-
tion was repeated again for 4 h.
6.7. Standard Fmoc-deprotection protocol
TheFmoc-deprotectionstepwascarriedoutbyimmersingthe
Lanterns in a mixture of piperidine and DMF (20:80, v/v) for
60 min. A 200-mL standard flask, equipped with a drilled topper
was used. After removal of the deprotection solution, the Lan-
terns were washed following the standard washing protocol.
6.4. Standard reductive amination protocol
The Lanterns were divided into two groups and were placed
in glass vials containing a suspension of natrium cyanoborohy-
dride ([NaHB3CN] ¼ 100 mM) and the amine ([diversity re-
agent 2, 2{6e7}] ¼ 250 mM, Fig. 1), in a 1% acetic acid in
30 mL of DMF. The reaction mixture was allowed to stand
overnight at 60 ꢀC and was then removed via a drilled adapter.
The Lanterns were first washed with 10% AcOH in DMF
(1 ꢃ 5 min), then with the standard washing protocol, and after
that allowed to dry in the open air.
6.8. Standard coupling protocol
Six DMF solutions (25 mL), containing carboxylic acid
(Fig. 2: diversity reagent 6), HBTU and DIEA each, were
freshly prepared in a standard Schott flask before coupling
([R2eCOOH] ¼ 120 mM; [HBTU] ¼ 120 mM; [DIEA] ¼
240 mM). The Lanterns were immersed for 2 h in the coupling
solution at room temperature. The solution was decanted, and
the Lanterns were washed following the standard washing pro-
cedure. The procedure described above was repeated.
6.5. Standard washing protocol
Washing steps after reductive amination, coupling, or de-
protection steps were carried out by dipping the Lanterns in
DMF (3 ꢃ 5 min) and DCM (3 ꢃ 5 min), respectively. A sin-
gle 200-mL standard Schott flask, equipped with a drilled top-
per, was used. The Lanterns were allowed to air-dry for 15 min
after the last DCM washing.
6.9. Cleavage/cyclization protocol
Chemsets 7 and 8 were placed in glass vials containing a 1-
mL mixture of TFA/CHCl3/SOCl2 (50/50/1.5, v/v/v). The

P. Zajdel et al. / European Journal of Medicinal Chemistry 44 (2009) 800e808
807
1
reaction was allowed to stand for 10 h at 40 ꢀC. Afterwards,
the reaction solution was removed using a Jouan RC1010 vac-
uum centrifuge. A 100-mL portion of acetonitrile/water (50:50,
v/v) containing a 0.1% TFA was poured into each tube to dis-
solve the samples. The samples were then frozen at ꢄ80 ꢀC
and lyophilized. The procedure was repeated twice to com-
pletely remove the remaining volatile residues.
MeOH:9/1); Rf ¼ 0.57 (S1). H NMR (CDCl3) d: 1.02e1.30
(m, 4H, Chx), 1.45 (d, 1H, Chx, J ¼ 11.28 Hz), 1.65e1.78
(m, 5H, Chx), 1.90e1.97 (m, 1H, Chx), 2.48 (dd, 1H, imide,
J ¼ 17.44 Hz), 3.06e3.23 (m, 2H, imide), 3.46 (br s, 2H,
CH2CH2N(CH2)2), 3.55e3.60 (m, 2H, NCH2), 3.87e3.97
(m, 4H, N(CH2)2), 4.03 (s, 3H, OCH3), 4.37 (br s 2H,
(CH2)2N), 5.02 (t, 2H, (CH2)2N, J ¼ 12.05 Hz), 7.02e7.07
(m, 2H, Ph), 7.42e7.48 (m, 1H, Ph), 8.15 (d, 1H, Ph,
6.10. Cleavage protocol
J ¼ 7.69 Hz); 20$HCl
(C23H33N3O3$HCl) C, H, N.
Mp:
208e210 ꢀC.
Anal.
A 500 mL of the TFAwas dispensed into polypropylene tubes
of deep 96-well plate. Cleavage was carried out for 60 min. The
cleavage cocktail was removed from the tubes under nitrogen
flow. Some compounds were precipitated with dry diethyl ether,
centrifuged, and decanted one by one. A 100 mL portion of ace-
tonitrile/water (50:50, v/v) containing a 0.1% TFA was poured
into each tube to dissolve the sample. Then the samples were
frozen at ꢄ80 ꢀC and lyophilized. The procedure was repeated
twice to completely remove the remaining volatile residues.
6.11.4. N-[2-{4-(3-Chlorophenyl)-piperazin-1-yl}-ethyl]-3-
cyclohexyl-pyrrolidine-2,5-dione (21)
Obtained from 2-cyclohexyl succinic acid as a white pow-
der (61% yield); chromatography (SiO2, CH2Cl2/MeOH:9/1);
Rf ¼ 0.67 (S1). 1H NMR (CDCl3) d: 0.96e1.33 (m, 4H,
Chx), 1.43 (d, 1H, Chx, J ¼ 12.05 Hz), 1.64e1.78 (m, 5H,
Chx), 1.87e1.95 (m, 1H, Chx), 2.45 (dd, 1H, imide,
J ¼ 18.82 Hz), 2.91 (q, 2H, imide, J ¼ 8.60 Hz), 3.05e3.23
(m, 2H, CH2CH2N(CH2)2), 3.29e3.31 (d, 2H, NCH2,
J ¼ 5.39 Hz), 3.57e3.76 (m, 4H, N(CH2)2), 3.87e3.94 (m,
4H, (CH2)2N), 6.75e6.78 (m, 1H, Ph), 6.87e6.93 (m, 2H,
Ph), 7.16e7.22 (m, 1H, Ph); 21$HCl Mp: 240e243 ꢀC.
Anal. (C22H30ClN3O2$HCl) C, H, N.
6.11. General procedure for the preparation of
pyrrolidine-2,5-diones (18e21)
To a suspension of 2-cyclohexyl succinic acid or succinic
acid (0.005 mol) in 10 mL of water, the appropriately
substituted arylpiperazine ethylamine (0.005 mol) was gradu-
ally added. The mixture was heated in an oil bath and water
was simultaneously distilled off. When the water was com-
pletely removed, the reaction temperature was raised up to
180 ꢀC and maintained for 1.5 h. The crude products were pu-
rified by column chromatography.
6.12. Radioligand binding studies
The selected compounds were tested without further
purification. In vitro affinity for native serotonin 5-HT1A and
5-HT2A receptors was determined by inhibiting [3H]-8-OH-
DPAT (170 Ci/mmol; NEN Chemicals) and [3H]-ketanserin
(67 Ci/mmol; NEN Chemicals) binding to rat hippocampal
and cortical membranes, respectively. Membrane preparation
and general assay procedures were carried out according to
the previously published protocols [8,15]. In the screening
procedure two compound concentrations were tested: 0.1
and 1 mM, whereas in full binding experiments 7e9 sample
concentrations, each run in triplicate, were used to determine
inhibition constant (Ki).
6.11.1. N-[2-{4-(2-Methoxyphenyl)-piperazin-1-yl}-ethyl]-
pyrrolidine-2,5-dione (18)
Obtained from succinic acid as a white powder (64% yield);
chromatography (SiO2, CH2Cl2/MeOH:9/1); Rf ¼ 0.56 (S1). 1H
NMR (CDCl3) d (ppm): 2.59e2.63 (t, 2H, CH2CH2N(CH2)2),
2.69e2.71 (m, 8H, N(CH2)2, imide), 3.03 (br, 4H, (CH2)2N),
3.67e3.71 (t, 2H, (NCH2, J ¼ 6.41 Hz)), 3.85 (s, 3H, OCH3),
6.83e7.01 (m, 4H, Ph). ESI-MS: (M þ Hþ) 318.7; Mp:
104e105 ꢀC. Anal. (C27H23N3O3) C, H, N.
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