Solid-phase synthesis of arylpiperazine derivatives and implementatio of the distributed drug discovery (d3) project in the search for cns agents

Article abstract of DOI:10.3390/molecules16054104

We have successfully implemented the concept of Distributed Drug Discovery (D3) in the search for CNS agents. Herein, we demonstrate, for the first time, student engagement from different sites around the globe in the development of new biologically activ

Full text of DOI:10.3390/molecules16054104

Molecules 2011, 16, 4104-4121; doi:10.3390/molecules16054104
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Solid-Phase Synthesis of Arylpiperazine Derivatives and
Implementation of the Distributed Drug Discovery (D3)
Project in the Search for CNS Agents
Paweł Zajdel 1,*, Joanna Król 1, Katarzyna Grychowska 1, Maciej Pawłowski 1, Gilles Subra 2,
Gaël Nomezine 2, Jean Martinez 2, Grzegorz Satała 3, Andrzej J. Bojarski 3, Ziniu Zhou 4,
Martin J. O’Donnell 4 and William L. Scott 4,*
1
Department of Medicinal Chemistry, Jagiellonian University Medical College, 9 Medyczna Street,
Kraków 30-688, Poland
2
Équipe Aminoacides Peptides et Protéines, Institut des Biomolécules Max Mousseron IBMM,
UMR CNRS 5247, Faculté de Pharmacie, Université Montpellier I et II, 15 avenue Charles Flahault,
Montpellier 34060, France
3
Department of Medicinal Chemistry, Institute of Pharmacology, Polish Academy of Sciences,
12 Smętna Street, Kraków 31-343, Poland
4
Department of Chemistry and Chemical Biology, Indiana University-Purdue University
Indianapolis, Indianapolis, IN 46202, USA
* Authors to whom correspondence should be addressed; E-Mails: mfzajdel@cyf-kr.edu.pl (P.Z.);
wscott@iupui.edu (W.L.S.).
Received: 13 April 2011; in revised form: 14 May 2011 / Accepted: 16 May 2011 /
Published: 19 May 2011
Abstract: We have successfully implemented the concept of Distributed Drug Discovery
(D3) in the search for CNS agents. Herein, we demonstrate, for the first time, student
engagement from different sites around the globe in the development of new biologically
active compounds. As an outcome we have synthesized a 24-membered library of
arylpiperazine derivatives targeted to 5-HT1A and 5-HT2A receptors. The synthesis was
simultaneously performed on BAL-MBHA-PS resin in Poland and the United States, and
on BAL-PS-SynPhase Lanterns in France. The D3 project strategy opens the possibility of
obtaining potent 5-HT1A/5-HT2A agents in a distributed fashion. While the biological
testing is still centralized, this combination of distributed synthesis with screening will
enable a D3 network of students world-wide to participate, as part of their education, in the
synthesis and testing of this class of biologically active compounds.

Molecules 2011, 16
Keywords: Distributed Drug Discovery (D3); combinatorial chemistry; solid-phase synthesis;
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SynPhase Lanterns; long-chain arylpiperazines; succinimides; 5-HT1A; 5-HT2A receptor affinity
Abbreviations: CHCl3: chloroform; DCM: dichloromethane; DIC: N,N’-diisopropylcarbodiimide;
DIEA: diisopropylethylamine; DMF: dimethylformamide; HBTU: O-(1H-benzotriazol-1-
yl)-1,1,3,3-tetramethyluronium
TFA: trifluoroacetic acid.
hexafluorophosphate;
SOCl2:
thionyl
chloride;
1. Introduction
Arylpiperazines are valuable investigative tools in neuropsychopharmacology and have the
potential to be leads in the development of new therapeutic agents. Flibanserin (Figure 1) is an
excellent example of a classic long-chain arylpiperazine with an interesting new application. This
meta-trifluorophenylpiperazine derivative, classified as a 5-HT1A agonist and 5-HT2A antagonist, had
initially been developed as an antidepressant [1,2]. Later it was found that it exerts potential
antipsychotic effects. It has also been investigated as a drug for women with decreased sexual desire [3].
Figure 1. Chemical structure of flibanserin.
N
H
N
N
O
N
CF3
Previously we demonstrated that a member of an arylpiperazine library containing a cyclized
N-acylated aspartic acid (PZ-68, Figure 2), was a partial agonist of 5-HT1A receptors and a 5-HT2A
antagonist, and showed in vivo antidepressant-like activity at doses of 5–20 mg/kg [4].
Figure 2. Chemical structure of PZ-68.
O
O
N
N
N
O
H
O
N
It is well known [5-7] that the affinity profile, functional activity and therapeutic potential (mostly
antipsychotic, antidepressant, and/or anxiolytic) of arylpiperazine derivatives depends on the
substitution pattern of the phenyl ring, linker length, and the terminal substructures. Accordingly, we
designed a new series of meta-trifluoromethyl phenylpiperazine analogs of the most interesting
previous sets containing N-acyl-3-aminopyrrolidine-2,5-diones and N-acylprolinamides. These
molecules involved conformational restriction of a peptide unit in two ways: introduction of a cyclic

Molecules 2011, 16
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amino acid (scaffold A, Figure 3), or an imide (scaffold B, Figure 3, resulting from an internal
cyclization of aspartic acid). Based on our previous results [8], the structural modifications also varied
both the linker length (three and four methylene groups) between the amide and the basic nitrogen
atom of the arylpiperazine fragment, and the R2 acyl-substituents.
Figure 3. General structure of the target compounds A and B obtained via modification of
the peptide unit 1.
Peptide
unit
B
A
O
R
N
R1
N
H
N
n
O
N
CF3
imide
cyclization
(route B)
cyclic amino acid
(route A)
O
O
R1
O
HN
N
N
N
R1
H
O
O
This work provided an opportunity to further enable the concept of Distributed Drug Discovery
(D3) [9-11] by combining education, synthesis and biological screening. The chemistry required for
SAR evaluation can be reproducibly carried out at distant sites, using simple and inexpensive
equipment. Adding biological testing, even if centralized, with distributed synthesis engages students
in the D3 network in these two key disciplines of drug discovery.
2. Results
2.1. Library Synthesis
A parallel solid-phase synthesis was carried out on two types of solid supports: BAL linker
functionalized p-methylbenzhydrylamine (MBHA) polystyrene resin (1a) and BAL linker functionalized
polystyrene SynPhase Lanterns (1b, Scheme 1).
The solid-phase chemistry on MBHA resin was adapted from the protocol reported on SynPhase
Lanterns [9]. The separate, multiple syntheses on the resin were performed manually by using a
Bill-Board set [10-13]. This equipment keeps the solid-phase reactions organized in a grid and
simplifies repeated cycles of reactions, washing, cleavage, and the final solvent evaporation step. One
compound was synthesized in each of the reaction vessels, which were organized on a solvent resistant
2 × 3 grid.
The syntheses began with the preparation of 4-(3-trifluoromethylphenyl)-1-piperazinylpropyl- and
butylamine (2{8} and 2{9}, Figure 4).

Molecules 2011, 16
Scheme 1. Synthetic route to N-acyl-3-aminopyrrolidine-2,5-diones and N-acylprolinamides:
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(i) amine diversity reagents 2{8, 9} [14], NaBH3CN, 1% AcOH/DMF, rt, 24 h; (ii) DIC,
DMF, rt, 12 h, 2×; (iii) 20% piperidine/DMF (iv) Diversity reagents 6{1–6}, HBTU, DIEA,
DMF, RT, 3 h (series a) or 2 h (series b); (v) TFA/CHCl3/SOCl2, 35 °C, 12 h (series a) or
40 °C, 10 h (series b); (vi) TFA/CH2Cl2, RT, 1.5 h (series a) or 1 h (series b). Synthesis of
series a was carried out on BAL-MBHA-PS resin, while series b was synthesized on
BAL-PS-Lantern. Chemset numbering (Figures 4 and 5) corresponds to the system applied
in the historical libraries [9,14,15].
i
O
N
CF3
H
N
CF3
N
N
H2N
N
H
n
n
1a - BAL -MBHA-PS resin
1b - BAL-PS-Lantern
3a, 3b
2{8, 9}
ii, Fmoc-L-Asp(OtBu)OH
ii, Fmoc-L-Pro-OH
tBu
O
O
N
H
N
CF3
N
CF3
N
N
N
N
N
n
n
Fmoc
O
O
O
4a, 4b
Fmoc
5a, 5b
iii,
iii,
iv, R1CO2H (6{1-6})
iv, R1CO2H (6{1-6})
tBu
O
N
CF3
H
H
N
CF3
N
N
N
n
N
N
N
n
7a, 7b
N
O
O
R1
O
O
O
O
O
O
8a, 8b
R1
v
vi
O
H
N
N
CF3
N
CF3
N
N
n
10
N
N
n
R1
9
R1
n = 1, 2
R1 = Ac, Cpt, Chx, Bzl, Nrbo, Ada
Figure 4. Diversity reagent 2. Amines 2{8-9} [14] used in the current project. Amines
2{2–5} were used to synthesize historical compounds [9,15] presented for comparison in
the radioligand binding experiments.
N
Cl
N
Cl
N
N
N
N
H2N
H2N
H2N
H2N
2
2{3}
2{2}
2{4}
N
N
O
O
2
2{5}
N
CF3
N
CF3
N
N
H2N
H2N
2
2{8}
2{9}

Molecules 2011, 16
4108
These amines were attached to the resin by a one-pot reductive amination to give the support-bound
secondary amine products 3. Fmoc-protected amino acids were then coupled to the support by a
symmetric anhydride method using DIC in DMF to give chemsets 4 and 5. Following Fmoc removal,
the amines were coupled with carboxylic acids (diversity reagents 6, Figure 5) in the presence of
HBTU under basic conditions. The final pyrrolidine-2,5-dione derivatives 9 were obtained in a one-pot
cleavage-cyclization step using a mixture of TFA/CHCl3/SOCl2, while prolinamides 10 were obtained
directly after treatment with TFA/CH2Cl2.
Figure 5. Diversity of carboxylic acids 6{1–6}.
O
O
O
O
O
O
OH
OH
OH
OH
OH
H3C
OH
66{6}
6{1}
6{2}
6{3}
6{4}
6{5}
In parallel to solid-phase chemistry on the resin we have performed syntheses of selected library
representatives on SynPhase Lanterns. This modular solid-support consists of soluble polymers grafted
onto a rigid unreactive base polymer. As a tagging system for SynPhase Lanterns, we have used
colored cogs and spindles (corresponding to the building block). At each step the Lanterns were
manually sorted and pooled into separate vials containing respective reagents. Cleavage of the final
products was performed in glass vials. The syntheses were conducted in standard laboratory glassware
according to the protocol previously reported [9]. The crude purities of selected compounds
synthesized in parallel on resin and Lanterns were determined and compared after removal of the
cleavage cocktail (Table 1).
Table 1. Comparison of the analytical data for selected library members obtained from
either resin or Lanterns.
Purity a
Lantern Resin
Purity a
Lantern Resin
Compound
Compound
9{8,3}
9{8,4}
9{8,5}
9{9,3}
9{9,4}
9{9,6}
74
70
77
68
64
75
61
52
59
62
49
56
10{8,3}
10{8,4}
10{8,5}
10{9,3}
10{9,4}
10{9,6}
96
94
96
97
98
98
93
93
87
97
89
86
a
Percent purity of the crude product was calculated on the peak area integration during HPLC
analysis of cleaved compounds at a sum of wavelengths between 200 to 270 nm.
All the compounds synthesized on the BAL-MBHA-PS resin were purified on silica gel (now using
the reactor vessels to contain the silica for column chromatography) using DCM/MeOH (9/1 or 9/0.7, v/v)
mixture. The percent purity was based on the peak area integration during HPLC analysis of cleaved
compounds at a sum of wavelengths between 200 to 270 nm (Table 2).

Molecules 2011, 16
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Table 2. Analytical data of the library.
Rt
Compound
(min)
MW [M + H]+
Rt
(min)
MW
calc.
[M + H]+
found
Purity a
Compd
Puritya
calc.
found
9{8,1}
1.35
97
426.2
427.2
10{8,1} 1.34
99
426.2
427.3
9{8,2}
9{8,3}
9{8,4}
9{8,5}
9{8,6}
9{9,1}
9{9,2}
9{9,3}
9{9,4}
9{9,5}
1.60
1.66
1.60
1.78
1.85
1.38
1.60
1.67
1.62
1.78
1.85
95
95
95
95
99
97
99
99
95
96
98
480.2
494.2
488.2
520.2
546.3
440.2
494.2
508.2
502.2
534.3
560.3
481.4
495.1
489.3
521.6
547.4
441.5
495.5
509.3
503.4
535.4
561.4
10{8,2} 1.62
10{8,3} 1.69
10{8,4} 1.58
10{8,5} 1.82
10{8,6} 1.90
10{9,1} 1.37
10{9,2} 1.60
10{9,3} 1.68
10{9,4} 1.58
10{9,5} 1.80
10{9,6} 1.86
99
96
96
96
97
93
96
97
97
98
96
480.3
494.3
488.2
520.3
546.3
440.2
494.3
508.3
502.2
534.3
560.3
481.0
495.2
489.8
521.5
547.4
441.5
495.1
509.3
503.3
535.6
561.1
9{9,6}
a
Percent purity of the purified product was calculated on the peak area integration during HPLC
analysis of cleaved compounds at a sum of wavelengths between 200 to 270 nm.
2.2. Biological Evaluation
The affinity for serotonin 5-HT1A and 5-HT2A receptors of 13 selected library representatives was
measured in vitro (Table 3, column 1) on the basis of the screening protocol described previously [9].
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 3) [8].
Table 3. Radioligand binding data of synthesized compounds for 5-HT1A and 5-HT2A.
Ki [nM] a
5-HT1A 5-HT2A
Ki [nM]
Ki [nM]
5-HT1A 5-HT2A
Compound
m-CF3 set
9{8,1}
Compound
m-Cl set b
9{2,1} c
9{2,3} c
9{3,2}
9{3,3}
9{3,4}
9{3,5}
9{3,6}
10{2,4}
10{2,6}
10{3,2} c
10{3,4}
10{3,5}
10{3,6}
Compd
o-OCH3 set b
9{4,1} d
9{4,3} d
9{5,2}
5-HT1A 5-HT2A
129
170
24
19
18
15
21
122
42
26
19
10
8
164
276
79
70
61
NT e
360
28
NT
92
47
480
230
23
19
18
9
NT
NT
480
183
410
47
9{8,3}
9{9,2}
9{9,3}
9{9,4}
9{9,5}
9{9,6}
26
52
6.5
20
9{5,3}
9{5,4}
9{5,5}
9{5,6}
10{4,4}
10{4,6}
10{5,2}
10{5,4}
10{5,5}
10{5,6}
30
39
21
47
4.2
25
4
173
NT
NT
NT
NT
547
503
10{8,4} f
10{8,6} f
10{9,2} f
10{9,4} f
10{9,5} f
898
1651
191
145
82
NT
75
NT
78
875
360
NT
128
35
NT
NT
NT
NT
14
3
37
10{9,6} f
699
13
140
a
Estimated Ki values; as the reference drugs buspirone (Ki(5-HT1A) = 36 nM) and clozapine
b
c
(Ki(5-HT2A) = 6.05 nM) we used; Data taken from references [4] if not otherwise stated; Data
taken from reference [9]; d Data taken from reference [15]; e NT = Not tested; f Proline amides from
chemset 10 correspond to chemset 17 from historical libraries [9,15].

Molecules 2011, 16
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3. Discussion
The D3 program, developed at IUPUI to engage students in the discovery of new biologically active
chemical entities, provided a framework to create a small international working group. The ultimate
goal of the D3 project is to discover compounds for neglected diseases prevalent in developing
countries. We wanted to demonstrate the successful application of the D3 process in the design,
synthesis and biological evaluation, against 5-HT receptors, of this small library of arylpiperazine
derivatives. The library design was based on our previously successful introduction of N-acylated
amino acids fragments into the arylpiperazine moiety yielding potent 5-HT1A and 5-HT2A receptor
ligands [8,9,15,16]. For the educational purpose, in the current project we chose a subset of analogs of
the most interesting sets containing N-acyl-3-aminopyrrolidine-2,5-diones and N-acylprolinamides.
We wished to enable students to further continue the structural modifications in the arylpiperazine
fragment and differentiate the length of the alkyl spacer.
The library synthesis strategy was based on the previously developed solid-phase methodology
performed on SynPhase Lanterns [9]. For the purpose of the D3 project we adapted the Lantern
protocol to MBHA resin. Synthesis of the library on the resin beads was performed in Krakow and
Indianapolis, while the modular Lantern solid supports were used in Montpellier. Average overall
yields of the crude products, calculated on the basis of the initial loading of the solid support, were
50% to 69% and 35% to 58% for the resin and Lanterns, respectively. The crude purities of the
compounds differed slightly among the laboratories. For the identified library members synthesized on
the Lanterns the average purity was 76%, while for compounds obtained from the resin the average
purity was 69%. Generally, Lanterns provided products with higher crude purities than the resin (Table 1).
Within the series, the compounds bearing a proline residue (chemset 10) were always of higher purity.
This is likely a function of the simple cleavage from the linker involving TFA.
To fulfill the quality standards for the combinatorial libraries and demonstrate compatibility with
the D3 process, all 24 members were purified using the synthesis vessels now filled with silica gel as
simple and inexpensive chromatography columns (Table 2). This enabled us to perform a distributed
synthesis and to obtain replicated results by students from three sites around the globe: Jagiellonian
University Medical College (Poland), University Montpellier, Biomolecules Institute Max Mousseron
(France) and Indiana University-Purdue University Indianapolis (USA).
For the next stage of the project we selected 13 library representatives and tested them for their
affinity for 5-HT1A and 5-HT2A receptors. Biological evaluation was also performed by students in the
Polish Academy of Science. This is the first presentation of our synthetic cross-boundary efforts
followed by biological evaluation of the new compounds within D3 project. The investigated
compounds displayed low-to-good affinity for 5-HT2A receptors (Ki = 1651 to 30 nM) and good-to-high
affinity for 5-HT1A receptors (Ki = 170 to 8 nM) in in vitro binding experiments. Generally, compounds
containing a four methylene spacer were more potent 5-HT1A agents than their propylene counterparts,
e.g., 9{8,3} vs. 9{9,3} and 10{8,4} vs. 10{9,4}.
To continue the structure-activity relationship studies we compared the binding data of the new
compounds (Table 3, column 1) with the historical data for the meta-chloro [4,9] and ortho-methoxy [4,15]
analogs (Table 3, column 2 and 3). Replacement of the chlorine atom in the meta position of the
phenyl ring with the more bulky trifluoromethyl substituent increased affinity of the new compounds

Molecules 2011, 16
4111
for 5-HT1A receptors. Interestingly, the 5-HT1A receptor affinity of the compounds bearing a meta-CF3
substituent was comparable to the affinity of ortho-OCH3 derivatives (well-known 5-HT1A ligands).
On the other hand, this modification decreased affinity of the new compounds for 5-HT2A receptors in
comparison to their meta-chloro analogs. In opposite to ortho-OCH3 substituted pyrrolidine-2,5-dione
derivatives, their meta-substituted analogs (Cl and CF3) presented limited dependence of volume of
cycloalkylcarbonyl fragment on 5-HT1A receptor affinity; on the other hand, 5-HT1A receptor affinity
of meta-CF3 prolinamides was clearly dependent on the kind of cycloalkyl substituent. It is worth
noting that compounds containing the norborn-2-yl moiety in the cycloalkyl fragment were always the
most potent 5-HT2A agents.
4. Experimental
4.1. General Methods
Solution and solid-phase organic transformations, Lanterns and resin washes were carried out at
ambient temperature, unless indicated otherwise. Organic solvents (from Acros Organics, Aldrich)
were of reagent grade and were used without purification. PS-BAL linker SynPhase Lanterns with
38-μmol loading were purchased from Mimotopes Pty, Australia. 4-Methylbenzhydrylamine
hydrochloride resin (PL-MBHA·HCl, 1.6 mmol/g, 75–150 μm) was purchased from Polymer
Laboratories. 4-(4-Formyl-3,5-dimethoxyphenoxy) butyric acid (BAL linker) was purchased from
NovaBiochem. All the Fmoc amino acids and HBTU reagent were purchased from NovaBiochem and
from Iris Chemicals. All other reagents were from Aldrich.
Purity of the synthesized compounds was confirmed by TLC performed on Merck silica gel 60 F254
aluminium sheets (Merck, Darmstadt, Germany). Spots were detected by their absorption under UV
light (λ = 254 nm). In all the cases HPLC technique was used as a routine.
Analytical HPLC were run on a Waters Alliance HPLC instrument, equipped with a Chromolith
SpeedROD column (4.6 × 50 mm). Standard conditions were eluent system A (water/0.1% TFA),
system B (acetonitrile/0.1% TFA). A flow rate of 5 mL/min and a gradient of (0–100)% B over 3 min
were used. Detection was performed on a PDA detector. Retention times (tR) are given in minutes.
Column chromatography was performed on silica gel (irregular particles 40–63 μm) filled synthesis
reactors. The yields of the final compounds, after chromatographic purification, were calculated on the
basis of the initial loading of the starting resins and are the overall yields of all reaction steps starting
from these resins.
1H-NMR and 13C-NMR spectra were recorded at 500 MHz (Bruker Avance III 500) or 300 MHz
(Varian BB 200) spectrometer using TMS (0.00 ppm) and chloroform-d1, or chloroform-d1 mixed with
methanol-d4 (2%–10%); J values are in Hertz (Hz), and splitting patterns are designated as follows:
s (singlet), d (doublet), t (triplet), m (multiplet).
Electrospray ionization mass spectrometry was conducted using a PESciex API III triple stage
quadrupole mass spectrometer or Waters Alliance 2690 HPLC, coupled to a Micromass (Manchester,
UK) Platform II spectrometer (electrospray ionization mode) operated in either positive-ion or
negative-ion detection mode. Samples were prepared in acetonitrile/water (50/50 v/v), containing a
0.1% TFA. All the analyses were carried out using a C18 Xterra MS, 2.1 × 30 mm column.

Molecules 2011, 16
4112
A flow rate of 500 μL/min and a gradient of (0–100)% 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 100–500 μL/min. Nitrogen was used for both the nebulizing 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.
4.2. General Procedures for Manual Solid-Phase Reactions
Manual solid-phase organic syntheses at ambient temperature were carried out in several types of
reaction vessels: 50 mL peptide synthesis reaction vessels with coarse porosity fritted glass support
and supplied with a GL thread and a Teflon lined PBT screw cap (ChemGlass, CG-1860-03) were used
for large scale (up to 3.7 mmol) reactions. Small scale reactions (typically 50 μmol) were performed in
3.5 mL fritted glass reaction vessels equipped with polypropylene screw caps with Teflon faced silicon
septa on the Bill-Board set [13], which was designed by one of us (WLS) as inexpensive equipment
to simplify and expedite multiple, manual solid-phase syntheses. The Bill-Board reaction vessel
components are available from Chem-Glass: IUP-0305-270H for 3.5 mL reaction vessel; IUP-0305-280H
(polypropylene screw cap); CV-4080-0013 (Teflon faced silicone septa).
For agitation purpose, the large scale reactions in the peptide synthesizers were placed on an orbital
shaker Roto Mix while motor rotators were used for small scale reactions. Resin-bound intermediates
were air-dried after the final CH2Cl2 washes, unless they needed to be re-weighed in which case
overnight drying under high vacuum (≤2 mm) or under low vacuum over 24–36 h in a vacuum
desiccator was carried out. During wash of the resin with solvents in the reaction vessels, at least 3 min
of gravity draining was used for large scale whereas at least 30 seconds is needed for small scale
followed by air-push.
BAL linker polystyrene SynPhase Lanterns with 38-μmol loading, and colored tagging system were
from Mimotopes (Clayton, Australia) [17]. 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 visual tagging system. Solid-phase manipulations involving SynPhase
Lanterns were carried out in a glass reaction vessels capped with silicone caps. As a standard the Lanterns
were washed with respective solvents by immersing them in DMF (3 × 5 min), MeOH (1 × 5 min), and
CH2Cl2 (3 × 5 min), respectively. A single 200-mL standard Schott flask equipped with a drilled
topper was used. The Lanterns were allowed to air-dry for 15 min after the last CH2Cl2 washing.
4.3. Solid-Phase Synthesis on BAL-MBHA-PS Resin
Preparation of BAL-MBHA-PS Resin (1a, Scheme 1): MBHA HCl-PS resin (0.469 g, 0.75 mmol,
1.6 mmol/g) loaded in a 25 mL peptide synthesis reaction vessel was sequentially washed with DMF
(3 × 8 mL), CH2Cl2 (2 × 8 mL), 10% DIEA/ CH2Cl2 (5 × 8 mL) and CH2Cl2 (2 × 8 mL). A solution of
4-(4-formyl-3,5-dimethoxyphenoxy)butyric acid (0.805 g, 3.00 mmol, 4 equiv.), HBTU (1.138 g,
3.00 mmol, 4 equiv.) and DIEA (1.05 mL, 6.00 mmol, 8 equiv.) in anhydrous DMF prepared 5 min
before was then added to the washed resin. The reaction vessel was put on the orbital shaker and
allowed to shake at room temperature for 18 h. The completion of the reaction was monitored by

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Chloranil test. The resultant orange yellow MBHA-BAL-PS resin (1a) was washed with DMF (3 × 8 mL),
CH2Cl2 (4 × 8 mL), and dried under low vacuum for 24 h.
4.3.1. Procedure for preparation of amine-bound Resin 3a, 3b via reductive amination
The resin (0.9 g, 1.44 mmol) was divided into two reactors containing a suspension of sodium
cyanoborohydride ([NaBH3CN] = 0.23 g, 3.6 mmol, 5 equiv.) and the amine ([Diversity reagent 2,
2{8,9}] = 3.6 mmol, 5 equiv., Figure 1), in a 1% acetic acid in 20 mL of DMF. The reactors were
shaken on for 24 hours at room temperature. Then the resin was drained. The resin was first washed
with 10% AcOH in DMF (1 × 5 mL) then with DMF (3 × 8 mL) and CH2Cl2 (4 × 8 mL), and dried
under low vacuum.
4.3.2. Coupling of amine resin 3a, 3b with N-α-Fmoc-Asp(OtBu)-OH and N-α-Fmoc-proline
Two DMF solutions (24 mL) containing a Fmoc-protected amino acid and DIC each, were freshly
prepared in a standard Schott flask before acylation ([Fmoc-AA-OH] = 3.6 mmol, 5 equiv, [DIC] = 1.8 mmol,
2.5 equiv.), and the mixture was left for 15 min to form an active anhydride. Then 2 mL of the
preactivated solutions were added to the resin and shaken for 12 h at a room temperature. The resin
was drained and was washed following with DMF (3 × 8 mL) and CH2Cl2 (4 × 8 mL), and dried under
low vacuum. The acylation was repeated one more time for 12 h.
4.3.3. Standard Fmoc-deprotection protocol
The Fmoc-deprotection step was carried out by treating the resin with a mixture of piperidine and
DMF (20/80; v/v) for 3 min. Then the resin was drained and the piperidine treatment was repeated for
next 15 minutes. After removal of the deprotection solution, the Fmoc-deprotected resins were washed
with DMF (3 × 8 mL) and CH2Cl2 (4 × 8 mL), and dried under low vacuum for 12 h.
4.3.4. Acylation with carboxylic acids using HBTU
The resins 4, 5 (31mg, 0.05 mmol) were swelled in DCM (5 mL) for 20 min, and then washed with
DCM/DMF (80/20; v/v). A solution of respective carboxylic acid [R2-COOH] = 0.25 mmol, 5 equiv.
(Figure 5: Diversity reagent 6{1–6}) was then added to the reactor, followed by HBTU (0.25 mmol,
5 equiv.) and DIEA (0.5 mmol, 10 equiv.). The solutions were added to the resin and the reactors were
shaken for 3 h at room temperature. The resins were drained, and were washed with DMF (3 × 8 mL),
CH2Cl2 (4 × 8 mL), and dried under low vacuum for 24 h. The procedure described above was repeated.
4.3.5. Succinimide derivatives formation via cleavage and intramolecular ring closure
Supported chemsets 7a, 7b were placed in glass vials containing a 2 mL mixture of TFA/CHCl3/
SOCl2 (50/50/1.5, v/v/v). The reaction was allowed to stand for 12 h at 35 °C. Then the solutions were
filtered through the cotton via a Pasteur pipette to the evaporation vials fitted on Bill-Board. The
“cocktail” was then evaporated by nitrogen flow used with Bill-Board evaporator.

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4.3.6. Cleavage of the proline derivatives off the resin
A 500 μL of the TFA/DCM (80/20; v/v) was dispensed into glass vials containing the resin 8a, 8b.
Cleavage was carried out at room temperature for 90 min. The cleavage cocktail was removed from the
Bill-Board glass vials under nitrogen flow.
4.4. Analytical Data for Biologically Tested Library Members Synthesized on MBHA Resin
N-((S)-1-{3-[4-((3-trifluoromethyl)phenyl)piperazin-1-yl]propyl}pyrrolidin-2,5-dion-yl)acetamide [9{8,1}].
Yield: 13 mg (59% isolated yield) as a yellow oil following chromatographic purification over silica
1
gel with CH2Cl2/MeOH (9:1); initial LC/MS purity 73%, tR = 1.35 min. H-NMR (300 MHz, CDCl3):
δ 1.42–1.54 (m, 2H), 2.04 (s, 3H), 2.61–2.69 (dd, J = 18.5 Hz, J = 4.9 Hz, 1H), 2.89 (m, 2H), 3.06–3.19
(m, 1H), 3.24–3.39 (m, 4H), 3.56–3.75 (m, 6H), 4.83–4.88 (m, 1H), 7.05–7.09 (d, J = 8.2 Hz, 1H),
7.11 (s, 1H), 7.21–7.25 (d, J = 7.7 Hz, 1H), 7.38–7.44 (t, J = 7.9 Hz, 1H), 7.49–7.51 (d, J = 7.9 Hz,
1H). MS calcd for [M + H]+: C20H26N4O3F3 m/z 427.2, found 427.2.
N-((S)-1-{3-[4-((3-trifluoromethyl)phenyl)piperazin-1-yl]propyl}pyrrolidin-2,5-dion-yl)cyclohexanecarbox
amide [9{8,3}]. Yield: 15 mg (61% isolated yield) as a yellow oil following chromatographic
purification over silica gel with CH2Cl2/MeOH (90:7); initial LC/MS purity 61%, tR = 1.66 min. 1H-NMR
(300 MHz, CDCl3): δ 1.42–1.54 (cluster, 12H), 2.01–2.09 (m, 1H), 2.13–2.18 (m, 2H), 2.61–2.68 (dd,
J = 18.2 Hz, J = 4.9 Hz, 1H), 3.06–3.12 (m, 1H), 3.27–3.36 (m, 4H), 3.56–3.71 (m, 6H), 4.72–4.79 (m,
1H), 7.06–7.09 (d, J = 7.9 Hz, 1H), 7.12 (s, 1H), 7.14–7.16 (d, 1H), 7.20–7.23 (d, J = 7.7 Hz, 1H),
7.38–7.44 (t, J = 7.9 Hz, 1H). MS calcd for [M + H]+: C25H34N4O3F3 m/z 495.2, found 495.1.
N-((S)-1-{4-[4-((3-trifluoromethyl)phenyl)piperazin-1-yl]butyl}pyrrolidin-2,5-dion-yl)cyclopentanecarbox
amide [9{9,2}]. Yield: 17 mg (69% isolated yield) as a yellow oil following chromatographic
purification over silica gel with CH2Cl2/MeOH (95:5); initial LC/MS purity 51%, tR = 1.60 min. 1H-NMR
(500 MHz, CD3OD/CDCl3): δ 1.54–1.61 (m, 2H), 1.65–1.78 (m, 5H), 1.80–1.97 (m, 5H), 2.58–2.70 (m,
1H), 2.77 (dd, J = 17.9 Hz, J = 5.4 Hz, 1H), 2.99–3.21 (m, 4H), 3.36–3.79 (m, 9H), 4.59 (m, 1H), 7.08 (d,
J = 8.2 Hz, 1H), 7.12 (s, 1H), 7.21 (d, J = 7.6 Hz, 1H), 7.41 (t, J = 7.9 Hz, 1H), 7.62 (d, J = 7.2 Hz, 1H).
13C-NMR (500 MHz, CD3OD/CDCl3): δ 20.1, 24.3, 26.0, 30.3, 35.6, 37.5, 44.9, 46.5, 48.8, 51.7, 56.6,
113.7, 118.2, 119.9, 122.9, 124.0 (q, 1JCF = 272 Hz), 125.1, 130.1, 132.4, 149.7, 175.2, 176.9. HRMS
calcd for [M + H]+: C25H34N4O3F3 m/z 495.2578, found 495.2575.
N-((S)-1-{4-[4-((3-trifluoromethyl)phenyl)piperazin-1-yl]butyl}pyrrolidin-2,5-dion-3-yl)cyclohexane
carboxamide [9{9,3}]. Yield: 12 mg (46% isolated yield) as a yellow oil following chromatographic
purification over silica gel with CH2Cl2/MeOH (90:7); initial LC/MS purity 62%, tR = 1.67 min.
1H-NMR (300 MHz, CDCl3): δ 1.15–1.42 (cluster, 8H), 1.64–1.86 (m, 4H), 2.06–2.18 (m, 3H), 2,56 (br
s, 4H), 2.61–2.69 (dd, J = 18.2 Hz, J = 4.9 Hz, 1H), 2.99–3.12 (q, 1H), 3.27–3.35 (m, 2H), 3.56–3.75
(m, 6H), 4.65–4.73 (m, 1H), 7.06–7.08 (d, J = 8.2 Hz, 2H), 7.11 (s, 1H), 7.20–7.24 (m, 1H), 7.38–7.42
(t, J = 8.2 Hz, 1H). MS calcd for [M + H]+: C26H36N4O3F3 m/z 509.2, found 509.3.

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N-((S)-1-{4-[4-((3-trifluoromethyl)phenyl)piperazin-1-yl]butyl}pyrrolidin-2,5-dion-yl)benzamide [9{9,4}].
Yield: 11 mg (44% isolated yield) as a yellow oil following chromatographic purification over silica
1
gel with CH2Cl2/MeOH (92:8): initial LC/MS purity 40%, tR = 1.62 min. H-NMR (500 MHz,
CD3OD/CDCl3): δ 1.68–1.80 (m, 1H), 1.86–1.99 (m, 3H), 2.98 (dd, J = 17.9 Hz, J = 5.8 Hz, 1H),
3.07–3.24 (m, 4H), 3.38–3.73 (m, 9H), 4.85 (m, 1H), 7.03 (d, J = 8.4 Hz, 1H), 7.06 (s, 1H), 7.21 (d,
J = 7.6 Hz, 1H), 7.37–7.44 (m, 3H), 7.51 (t, J = 7.4 Hz, 1H), 7.88 (d, J = 7.5 Hz, 2H), 8.42 (d, J = 7.5 Hz,
1H). 13C-NMR (500 MHz, CD3OD/CDCl3): δ 19.9, 24.1, 35.3, 46.4, 50.1, 51.6, 56.4, 118.1, 119.8,
1
124.1 (q, JCF = 272 Hz), 127.3, 128.5, 129.9, 132.4, 149.6, 175.1. HRMS calcd for [M + H]+:
C26H30N4O3F3 m/z 503.2265, found 503.2242.
N-((S)-1-{4-[4-((3-trifluoromethyl)phenyl)piperazin-1-yl]butyl}pyrrolidin-2,5-dion-3-yl)-2-(bicyclo[2.2.1]
heptan-2-yl)acetamide [9{9,5}]. Yield: 15 mg (57% isolated yield) following chromatographic
purification over silica gel with CH2Cl2/MeOH (90:10): initial LC/MS purity 59%, tR = 1.78 min.
1H-NMR (CDCl3) δ 1.07–1.28 (m, 6H), 1.47–1.52 (m, 2H) 1.60 (m, 1H), 1.80–2.01 (m, 4H), 2.06–2.09
(m, 1H), 2.17–2.23 (m, 2H), 2.67–2.76 (s, 1H), 2.97–3.13 (m, 6H), 3.23–3.37 (m, 4H), 3.61–3.69 (m,
4H), 4.65–4.82 (m, 1H,) 7.06–7.12 (m, 2H), 7.20–7.26 (t, 1H), 7.34–7.43 (m, 2H). MS calcd for [M + H]+
C29H37N4O3F3 m/z 535.3, found 535.4.
N-((S)-1-{4-[4-((3-trifluoromethyl)phenyl)piperazin-1-yl]butyl}pyrrolidin-2,5-dion-yl)-1-adamatane
carboxamide [9{9,6}]. Yield: 22 mg (78% isolated yield) as a yellow oil following chromatographic
purification over silica gel with CH2Cl2/OH (95:5): Initial LC/MS purity 56%, tR = 1.85 min. 1H-NMR
(500 MHz, CD3OD/CDCl3): δ 1.61–1.68 (m, 3H), 1.69–1.77 (m, 4H), 1.78–1.95 (m, 9H), 2.01 (s, 3H),
2.78 (dd, J = 17.8 Hz, J = 5.5 Hz, 1H), 2.99 (dd, J = 17.8 Hz, J = 9.1 Hz, 1H), 3.04–3.25 (m, 3H),
3.30–3.77 (m, 9H), 4.41 (m, 1H), 7.07 (d, J = 8.5 Hz, 1H), 7.11 (s, 1H), 7.19 (d, J = 7.7 Hz, 1H), 7.40
(t, J = 7.9 Hz, 1H). 13C-NMR (500 MHz, CD3OD/CDCl3): δ 19.9, 24.2, 27.9, 35.1, 36.2, 37.5, 38.8,
1
40.4, 46.4, 48.9, 51.4, 56.2, 113.5, 117.9, 119.7, 122.8, 123.9 (q, JCF = 272 Hz), 124.9, 129.9, 131.7,
149.7, 175.1, 176.7, 179.1. HRMS calcd for [M + H]+: C30H40N4O3F3 m/z 561.3047, found 561.3024.
1-(Benzoyl)-N-{3-[4-((3-trifluoromethyl)phenyl)piperazin-1-yl]propyl}-L-prolinamide [10{8,4}]. Yield
14.5 mg (61% isolated yield) following chromatographic purification over silica gel with
CH2Cl2/MeOH (92:8): Initial LC/MS purity 93%, tR = 1.58 min. 1H-NMR (300 MHz, CDCl3): δ 1.25–1.28
(m, 3H), 1.77–1.85 (m, 2H), 2.02–2.09 (m, 2H), 2.59–2.62 (m, 6H), 3.25 (m, 4H), 3.38–3.40 (m, 2H),
3.60 (m, 1H), 4.65 (t, J = 7.5 Hz, 1H), 7.02–7.08 (m, 4H), 7.34 (m, 1H), 7.40–7.42 (d, 4H), 7.53 (br s,
1H). MS calcd for [M + H]+: C26H32N4O2F3 m/z 489.2, found 489.6.
1-(Adamantylcarbonyl)-N-{3-[4-((3-trifluoromethyl)phenyl)piperazin-1-yl]propyl}-L-prolinamide [10{8,6}].
Yield: 13 mg (47% isolated yield) following chromatographic purification over silica gel with
CH2Cl2/MeOH (92:8): Initial LC/MS purity 86%, tR = 1.90 min. 1H-NMR (300 MHz, CDCl3): δ 1.25–1.28
(m, 4H), 1.67–1.72 (m, 8H), 1.82–1.95 (m, 2H), 1.99–2.15 (m, 4H), 2.17–2.18 (m, 1H), 2.59 (br s, 4H),
2.65 (br s, 6H), 3.26–3.35 (m, 4H), 3.72–3.83 (m, 2H), 4.56–4.58 (m, 1H), 7.04–7.10 (m, 3H), 7.31–
7.32 (t, 1H), 7.34–7.37 (m, 1H). MS calcd for [M + H]+: C30H42N4O2F3 m/z 547.3, found 547.4.

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1-(Cyclopentylcarbonyl)-N-{4-[4-((3-trifluoromethyl)phenyl)piperazin-1-yl]butyl}-L-prolinamide [10{9,2}].
Yield: 12 mg (49% isolated yield) as a yellow oil following chromatographic purification over silica
1
gel with CH2Cl2/MeOH (92:8): Initial LC/MS purity 83%, tR = 1.60 min. H-NMR (500 MHz, CDCl3):
δ 1.54–1.64 (m, 4H), 1.65–1.79 (m, 4H), 1.80–1.89 (m, 4H), 1.91–2.01 (m, 2H), 2.02–2.15 (m, 1H),
2.21–2.29 (m, 1H), 2.77–2.89 (m, 1H), 2.9–3.18 (m, 4H), 3.19–3.35 (m, 3H), 3.37–3.45 (m, 1H), 3.47–3.58
(m, 2H), 3.59–3.78 (m, 4H), 4.47 (dd, J = 8.0 Hz, J = 3.0 Hz, 1H), 7.06 (dd, J = 8.3 Hz, J = 2.3 Hz,
1H), 7.11 (m, 1H), 7.17–7.25 (m, 2H), 7.39 (t, J = 8.0 Hz, 1H). 13C-NMR (500 MHz, CDCl3): δ 20.7,
24.9, 26.0, 26.3, 28.2, 29.6, 30.1, 38.0, 42.9, 46.5, 47.5, 51.4, 56.5, 60.2, 113.5, 119.7, 122.9, 124.0 (q,
1JCF = 272 Hz), 125.1, 129.9, 131.8, 149.8, 172.4, 176.6. HRMS calcd for [M + H]+: C26H38N4O2F3 m/z
495.2941, found 495.2936.
1-(Benzoyl)-N-{4-[4-((3-trifluoromethyl)phenyl)piperazin-1-yl]butyl}-L-prolinamide [10{9,4}]. Yield:
14 mg (56% isolated yield) as a yellow oil following chromatographic purification over silica gel with
CH2Cl2/MeOH (92:8): Initial LC/MS purity 89%, tR = 1.58 min. 1H-NMR (500 MHz, CDCl3): δ 1.57–1.71
(m, 2H), 1.79–1.98 (m, 3H), 1.99–2.09 (m, 1H), 2.13–2.30 (m, 2H), 2.69–2.94 (m, 2H), 3.01–3.42 (m,
8H), 3.46–3.62 (m, 3H), 3.67–3.76 (m, 1H), 4.61 (t, J = 7.5 Hz, 1H), 6.88–6.99 (m, 2H), 7.17 (d,
J = 7.7 Hz, 1H), 7.30 (m, 1H), 7.33–7.47 (m, 4H), 7.61 (d, J = 6.9 Hz, 2H). 13C-NMR (500 MHz,
CDCl3): δ 20.4, 25.7, 25.9, 28.9, 38.0, 46.2, 50.8, 51.5, 56.5, 60.9, 113.3, 115.4, 117.7, 119.6, 122.9,
1
124.0 (q, JCF = 272 Hz), 125.1, 127.5, 128.4, 129.8, 130.6, 131.7, 135.9, 149.8, 162.3, 170.5, 172.3.
HRMS calcd for [M + H]+: C27H34N4O2F3 m/z 503.2628, found 503.2629.
1-((Bicyclo[2.2.1]heptan-2-yl)acetyl)-N-{4-[4-((3-trifluoromethyl)phenyl)piperazin-1-yl]butyl}-L-
prolinamide [10{9,5}]. Yield: 15 mg (59% isolated yield) following chromatographic purification over
1
silica gel with CH2Cl2/MeOH (90:10): Initial LC/MS purity 91%, tR = 1.80 min. H-NMR (300MHz,
CDCl3) δ (ppm) 1.01–1.42 (m, 3H), 1.25–1.32 (m, 5H), 1.79 (m, 1H), 1.93–1.98 (m, 3H), 2.10–2.35
(m, 4H), 2.44 (m, 2H), 2.62 (m, 4H), 3.25 (m, 6H), 3.36–3.45 (m, 3H), 4.54–4.57 (d, J = 7.9 Hz, 1H),
7.04–7.10 (m, 3H), 7.31–7.33 (m, 1H), 7.35–7.38 (d, 1H); MS calcd for [M + H]+ C28H38N4O3F3 m/z 535.3,
found 535.6.
1-(Adamantylcarbonyl)-N-{4-[4-((3-trifluoromethyl)phenyl)piperazin-1-yl]butyl}-L-prolinamide [10{9,6}].
Yield: 13 mg (46% isolated yield) as a yellow oil following chromatographic purification over silica
1
gel with CH2Cl2/MeOH (92:8): Initial LC/MS purity 86%, tR = 1.86 min. H-NMR (500 MHz, CDCl3):
δ 1.51–1.63 (m, 2H), 1.65–1.77 (m, 7H), 1.86–2.19 (m, 14H), 2.76–3.17 (m, 4H), 3.18–3.58 (m, 7H),
3.69–3.91 (m, 3H), 4.54 (m, 1H), 6.80 (s, 1H), 7.05 (dd, J = 8.3 Hz, J = 2.1 Hz, 1H), 7.10 (s, 1H), 7.15 (d,
J = 7.7 Hz, 1H), 7.37 (t, J = 8.0 Hz, 1H). 13C-NMR (500 MHz, CD3OD/CDCl3): δ 14.0, 20.9, 26.5,
1
28.1, 36.4, 38.1, 41.8, 47.1, 48.5, 51.8, 56.9, 60.4, 62.4, 112.9, 119.3, 122.9, 124.1 (q, JCF = 272 Hz),
125.1, 129.7, 131.6, 150.2, 173.1, 177.1. HRMS calcd for [M + H]+: C31H44N4O2F3 m/z 561.3411, found
561.3409.
4.5. Solid-Phase Synthesis on BAL-PS Lanterns
Reductive Amination Protocol. The Lanterns were divided into two groups and were placed in glass
vials containing a suspension of sodium cyanoborohydride ([NaBH3CN] = 100 mmol) and the amine

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([Diversity reagent 2] = 250 mmol, Figure 4) in a 1% acetic acid in 3 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 × 5 min) then with the standard washing
protocol, and after allowed to dry in the open air.
4.5.1. Secondary amine acylation protocol
Two DMF solutions (2 mL) containing a Fmoc-protected amino acid and DIC each, were freshly
prepared in a standard Schott flask before acylation ([Fmoc-AA-OH] = 200 mmol, [DIC] = 100 mmol),
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 temperature. The solution was decanted, and the
Lanterns were washed following the standard washing protocol. The acylation was repeated one time
more for 4 h.
4.5.2. Standard Fmoc-deprotection protocol
The Fmoc-deprotection step was carried out by immersing the Lanterns in a mixture of piperidine and
DMF (20/80, v/v) for 60 min. A 100-mL standard flask, equipped with a drilled topper was used. After
removal of the deprotection solution, the Lanterns were washed following the standard washing protocol.
4.5.3. Standard acylation with carboxylic acids
Six DMF solutions (2 mL), containing carboxylic acid (Figure 5: Diversity reagent 6), HBTU and
DIEA each, were freshly prepared in a standard Schott flask before coupling ([R2-CO2H] = 120 mmol;
[HBTU] = 120 mmol; [DIEA] = 240 mmol). The Lanterns were immersed for 2 h in the coupling
solution at a room temperature. The solution was decanted, and the Lanterns were washed following
the standard washing procedure. The procedure described above was repeated.
4.5.4. Cleavage/cyclization protocol for succinimide derivatives formation
Chemsets 7a and 7b were placed in glass vials containing a mixture of TFA/CHCl3/SOCl2 (1 mL,
50/50/1.5, v/v/v). The reaction was allowed to stand for 10 hours at 40 °C. Afterwards, the reaction
solution was removed under nitrogen flow. A 100 µL portion of acetonitrile/water (50/50, v/v)
containing a 0.1% TFA was poured into each tube to dissolve the samples. The samples were then
frozen at −80 °C and lyophilized.
4.5.5. Cleavage of the proline derivatives off the lantern
A 500 µL aliquot of TFA was dispensed into individual glass tubes. Cleavage was carried out for
60 min. The cleavage cocktail was removed from the tubes under nitrogen flow. A 100 µL portion of
acetonitrile/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 −80 °C and lyophilized.

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4.6. Analytical Data for 12 Library Members Synthesized on SynPhase Lanterns
N-((S)-1-{3-[4-((3-trifluoromethyl)phenyl)piperazin-1-yl]propyl}pyrrolidin-2,5-dion-yl)cyclohexanecarbox-
amide [9{8,3}]. Yield: 5.1 mg (47%) as a yellow oil following chromatographic purification over silica
gel with CH2Cl2/MeOH (90:10); initial LC/MS purity 74%, tR = 1.66 min. MS calcd for [M + H]+:
C25H34N4O3F3 m/z 495.2, found 495.1.
N-((S)-1-{3-[4-((3-trifluoromethyl)phenyl)piperazin-1-yl]propyl}pyrrolidin-2,5-dion-yl)benzamide [9{8,4}].
Yield: 3.8 mg (35%) as a yellow oil following chromatographic purification over silica gel
with CH2Cl2/MeOH (90:10); initial LC/MS purity 70%, tR = 1.60 min. MS calcd for [M + H]+:
C25H28N4O3F3 m/z 489.2, found 489.1.
N-((S)-1-{3-[4-((3-trifluoromethyl)phenyl)piperazin-1-yl]propyl}pyrrolidin-2,5-dion-yl)-2-(bicyclo[2.2.1]
heptan-2-yl)acetamide [9{8,5}]. Yield: 5.1 mg (45%) as a yellow oil following chromatographic
purification over silica gel with CH2Cl2/MeOH (90:10); initial LC/MS purity 77%, tR = 1.78 min.
MS calcd for [M + H]+: C27H36N4O3F3 m/z 521.2, found 521.4.
N-((S)-1-{4-[4-((3-trifluoromethyl)phenyl)piperazin-1-yl]butyl}pyrrolidin-2,5-dion-3-yl)cyclohexane-
carboxamide [9{9,3}]. Yield: 5.4 mg (48% isolated yield) as a yellow oil following chromatographic
purification over silica gel with CH2Cl2/MeOH (90:10); initial LC/MS purity 68%, tR = 1.67 min.
MS calcd for [M + H]+: C26H36N4O3F3 m/z 509.2, found 509.4.
N-((S)-1-{4-[4-((3-trifluoromethyl)phenyl)piperazin-1-yl]butyl}pyrrolidin-2,5-dion-yl)benzamide [9{9,4}].
Yield: 4.9 mg (44% isolated yield) as a yellow oil following chromatographic purification over silica
gel with CH2Cl2/MeOH (90:10); initial LC/MS purity 64%, tR = 1.62 min. MS calcd for [M + H]+:
C26H30N4O3F3 m/z 503.2, found 503.3.
N-((S)-1-{4-[4-((3-trifluoromethyl)phenyl)piperazin-1-yl]butyl}pyrrolidin-2,5-dion-yl)-1-adamatanecarbox
amide [9{9,6}]. Yield: 5.6 mg (46% isolated yield) as a yellow oil following chromatographic
purification over silica gel with CH2Cl2/MeOH (90:10); initial LC/MS purity 75%, tR = 1.85 min.
MS calcd for [M + H]+: C30H40N4O3F3 m/z 561.3, found 561.4.
1-(Cyclohexylcarbonyl)-N-{3-[4-((3-trifluoromethyl)phenyl)piperazin-1-yl]propyl}-L-prolinamide [10{8,3}].
Yield: 6.3 mg as a yellow oil (58% isolated yield) following chromatographic purification over silica
gel with CH2Cl2/MeOH (90:10); initial LC/MS purity 96%, tR = 1.69 min. MS calcd for [M + H]+:
C26H38N4O2F3 m/z 495.3, found 495.4.
1-(Benzoyl)-N-{3-[4-((3-trifluoromethyl)phenyl)piperazin-1-yl]propyl}-L-prolinamide [10{8,4}]. Yield: 5.1
mg as a yellow oil (47% isolated yield) following chromatographic purification over silica gel
with CH2Cl2/MeOH (90:10); initial LC/MS purity 94%, tR = 1.58 min. MS calcd for [M + H]+:
C26H32N4O2F3 m/z 489.2, found 489.4.
1-((Bicyclo[2.2.1]heptan-2-yl)acetyl)-N-{3-[4-((3-trifluoromethyl)phenyl)piperazin-1-yl]propyl}-L-
prolinamide [10{8,5}]. Yield: 6.3 mg as a yellow oil (55% isolated yield) following chromatographic

Molecules 2011, 16
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purification over silica gel with CH2Cl2/MeOH (90:10); initial LC/MS purity 96%, tR = 1.81 min;
MS calcd for [M + H]+ C28H40N4O2F3 m/z 521.3, found 521.5.
1-(Cyclohexylcarbonyl)-N-{4-[4-((3-trifluoromethyl)phenyl)piperazin-1-yl]butyl}-L-prolinamide [10{9,3}].
Yield: 5.5 mg as a yellow oil (49% isolated yield) following chromatographic purification over silica
gel with CH2Cl2/MeOH (90:10); initial LC/MS purity 97%, tR = 1.68 min. MS calcd for [M + H]+:
C27H40N4O2F3 m/z 509.3, found 509.2.
1-(Benzoyl)-N-{4-[4-((3-trifluoromethyl)phenyl)piperazin-1-yl]butyl}-L-prolinamide [10{9,4}]. Yield:
6.2 mg as a yellow oil (56% isolated yield) following chromatographic purification over silica gel
with CH2Cl2/MeOH (90:10); initial LC/MS purity 98%, tR = 1.57 min. MS calcd for [M + H]+:
C27H34N4O2F3 m/z 503.2, found 503.3.
1-(Adamantylcarbonyl)-N-{4-[4-((3-trifluoromethyl)phenyl)piperazin-1-yl]butyl}-L-prolinamide [10{9,6}].
Yield: 6.5 mg as a yellow oil (54% isolated yield) following chromatographic purification over silica
gel with CH2Cl2/MeOH (90:10); initial LC/MS purity 98%, tR = 1.85 min. MS calcd for [M + H]+:
C31H44N4O2F3 m/z 561.3, found 561.4.
Radioligand binding studies
The in vitro affinity for native serotonin 5-HT1A and 5-HT2A receptors was determined by inhibiting
[3H]-8-OH-DPAT (17 Ci/mmol; NEN Chemicals) and [3H]-ketanserin (88 Ci/mmol; NEN Chemicals)
binding to rat hippocampal and cortical membranes, respectively. Membrane preparation and a general
assay procedure were carried out according to the previously published protocols [18]. Two compound
concentrations were tested: 0.1 and 1 μM, each run in triplicate. The Ki values, estimated on the basis
of three independent binding experiments SEM ≤ 22%.
5. Conclusions
In summary, we have successfully adapted a previous synthetic protocol to enable the synthesis of
new chemical entities within the Distributed Drug Discovery (D3) project. As an outcome of student
involvement we have disclosed a 24-member library of novel arylpiperazines, obtained on BAL-PS-
SynPhase Lanterns and on BAL-MBHA-PS resin. A subset of these was then tested on 5-HT1A and
5-HT2A receptors as potential agents for the treatment of CNS-directed disorders. The biological results
indicated that introduction of the m-CF3 substituent in the phenylpiperazine fragments opens the
possibility of obtaining dual 5-HT1A/5-HT2A agents. While the biological testing is centralized, this
combination of distributed synthesis with screening will enable a D3 network of students worldwide
to participate, as part of their education, in the synthesis and testing of this class of biologically
active compounds.
Acknowledgements
This study was partially supported by the Polish Ministry of Science and Higher Education
(MNiSW), Grant Number N N405 378437. We also gratefully acknowledge funding by the National

Molecules 2011, 16
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Institutes of Health (R01 GM028193) and the National Science Foundation (MRI CHE-0619254).
Radioligand binding experiments were financially supported by the grant PNRF-103-AI-1/07 from
Norway through the Norwegian Financial Mechanism.
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