Non-amide-based combinatorial libraries derived from N-Boc-iminodiacetic acid: Solution-phase synthesis of piperazinone libraries with activity against LEF-1/β-catenin-mediated transcription

Article abstract of DOI:10.1002/1522-2675(20000809)83:8<1825::AID-HLCA1825>3.0.CO;2-4

The development of a solution-phase approach to the rapid, parallel synthesis of highly functionalized piperazinones in only four steps starting from N-Boc-iminodiacetic acid is detailed. The efforts represent the extension of the solution-phase synthesis of combinatorial libraries from N-Boc-iminodiacetic acid to non-amide-based libraries where simple liquid-liquid extractions are employed to purify all reaction products. This methodology was applied to the synthesis of a diverse 150-member library with substituents in three positions of the piperazinone core. Screening results from a luciferase reporter assay indicate that a number of library members are novel repressors of LEF-1/β-catenin-mediated transcription, and may be effective agents against colorectal tumors. Two secondary libraries (100 members each) designed from these lead structures were synthesized and screened, providing additional active agents and insight into key structure-activity relationships in the series. These compounds represent only the second class of small molecules which repress transcription of reporter genes containing LEF-1 responsive elements, and the first group not based on DNA minor-groove-binding agents.

Full text of DOI:10.1002/1522-2675(20000809)83:8<1825::AID-HLCA1825>3.0.CO;2-4

Helvetica Chimica Acta ± Vol. 83 (2000)
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Non-Amide-Based Combinatorial Libraries Derived from
N-Boc-Iminodiacetic Acid: Solution-Phase Synthesis of Piperazinone
Libraries with Activity Against LEF-1/b-Catenin-Mediated Transcription
by Dale L. Boger*a)1), Joel Goldberga), Shigeki Satoha), Yves Ambroisea), Steven B. Cohenb),
and Peter K. Vogtb)
a) Department of Chemistry and The Skaggs Institute for Chemical Biology,
b) Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey
Pines Road, La Jolla, California 92037, USA
Dedicated to Albert Eschenmoser on the occasion of his 75th birthday
The development of a solution-phase approach to the rapid, parallel synthesis of highly functionalized
piperazinones in only four steps starting from N-Boc-iminodiacetic acid is detailed. The efforts represent the
extension of the solution-phase synthesis of combinatorial libraries from N-Boc-iminodiacetic acid to non-
amide-based libraries where simple liquid-liquid extractions are employed to purify all reaction products. This
methodology was applied to the synthesis of a diverse 150-member library with substituents in three positions of
the piperazinone core. Screening results from a luciferase reporter assay indicate that a number of library
members are novel repressors of LEF-1/b-catenin-mediated transcription, and may be effective agents against
colorectal tumors. Two secondary libraries (100 members each) designed from these lead structures were
synthesized and screened, providing additional active agents and insight into key structure-activity relationships
in the series. These compounds represent only the second class of small molecules which repress transcription of
reporter genes containing LEF-1 responsive elements, and the first group not based on DNA minor-groove-
binding agents.
Introduction. ± Combinatorial libraries have emerged as a leading source of
compounds for biological screens. Originally described for peptides and related
oligomers [1][2], combinatorial chemistry has focused on the generation of libraries of
small organic molecules [3] structurally related to traditional pharmaceuticals [4]. In
recent efforts, we have described the development of multi-step solution-phase
techniques for the preparation of combinatorial libraries, whose technically non-
demanding nature and unlimited scale provide attractive alternatives to more
traditional solid-phase methods. In these studies, templates such as N-Boc-iminodi-
acetic acid anhydride (1) and related anhydrides have been exploited through
sequential amide couplings for the rapid parallel synthesis of individual compounds
and combinatorial mixtures [5][6]. Recently, we extended this approach by introducing
diversity through the formation of C,C bonds via olefin metathesis [7], Stille [8] and
biaryl [9] coupling reactions. In all cases, reaction and workup conditions were designed
that permitted the isolation and purification of products through simple liquid-liquid or
liquid-solid extractions.
1
)
Fax: (858) 784 7550; email: boger@scripps.edu.

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Helvetica Chimica Acta ± Vol. 83 (2000)
Herein, we describe an important extension of this solution-phase methodology to
the preparation of non-amide-based libraries from N-Boc-iminodiacetic acid with the
large scale, parallel synthesis of a prototypical 150-member library of 4-acyl-1,6-
dialkyl-piperazin-2-ones 2. Combinatorial piperazinone (ꢀketopiperazineꢁ) libraries
have previously been described, although with different substitution patterns [10].
Substituted piperazinones have been used in the generation of conformationally
restrained peptidomimetics [11], and several have been identified that have potent
activity in a variety of biological assays [12]. Screening results have revealed that a
number of compounds from our initial piperazinone library and subsequent secondary
libraries are active in a luciferase reporter assay measuring transcription from
lymphoid-enhancer binding factor (LEF-1) responsive elements. A majority of
colorectal tumors contain adenomatous polyposis coli (APC) mutations, which result
in nuclear accumulation of b-catenin, which binds to and activates transcription factors
including LEF-1 [13]. The resulting upregulated and aberrant gene expression is a key
step in the development of colorectal adenomas, and the discovery of compounds that
can disrupt LEF-1/b-catenin-mediated transcription may lead to novel therapeutics for
colon cancer.
Developmental Studies. The route to the piperazinone core was anticipated to
begin with introduction of the R1 substituent by addition of a carbon nucleophile to the
iminodiacetic acid template2), resulting in keto acid 3 (Scheme 1). The direct addition
of organometallic reagents to anhydride 1 was first investigated with several classes of
reagents including BuLi, Et2Zn, a-bromoacetate with Zn/Cu couple in DMF [15] and
malonic acid/Et3N [16]. By and large, the efforts were unsuccessful, giving complex
mixtures containing undesired side-products (Scheme 2).
Scheme 1. Two Routes Explored for the Synthesis of a 4-Acyl-1,6-dialkyl Piperazinone Library from N-Boc-
Iminodiacetic Acid Anhydride
The approach was improved by introducing the Weinreb amide 5 [17] as an
intermediate (Scheme 3). Addition of N-methoxy-N-methylamine hydrochloride to 1
2
)
N-Boc-Iminodiacetic acid anhydride (1) is typically generated in situ from the readily available dicarboxylic
acid (see [5][6]). Alternatively, it can be isolated and stored for subsequent reactions (cf. [14]).

Helvetica Chimica Acta ± Vol. 83 (2000)
1827
Scheme 2
Scheme 3
in the presence of EtN(i-Pr)2 provided 5. The R1 substituent could then be introduced
by addition of a Grignard reagent. The developmental model reaction with EtMgBr
required reaction temperatures of 0 ± 258 for more than 1 h to achieve complete
conversion, but no by-products from over-addition were observed. Isolation and
purification of the keto acid product 6 was accomplished by a phase-switch extractive
workup. After quenching, the reaction mixture was partitioned between Et2O and 2m
aqueous NaOH. The Et2O phase was discarded, removing neutral organic impurities,
and the desired keto-acid product was then liberated from the aqueous phase by
acidification (HCl) and extraction into Et2O. The isolated yield was 80% and the
1
product 6 was pure by TLC and H-NMR analysis.
The second substituent (R2) in our developmental system was introduced by
reductive amination of the ketone. Compound 6 was found to be an excellent

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Helvetica Chimica Acta ± Vol. 83 (2000)
substrate3), and reaction with 4-methoxybenzylamine in the presence of sodium
triacetoxyborohydride (NaBH(OAc)3)4) and AcOH proceeded smoothly, yielding the
crude amino acid 7 after evaporation of the solvent. This unpurified product was
directly cyclized by treatment with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide
hydrochloride (EDCI) to form the N-Boc-piperazinone 8. Although obtained in good
yield (92%), the product 8 was contaminated with neutral by-products that
necessitated purification by flash chromatography. Alternative reaction conditions
were developed that facilitated product isolation and purification by simple liquid-
liquid extractions. The major contaminant in product 8 was N-(4-methoxybenzyl)acet-
amide, derived from the EDCI coupling of unreacted amine and AcOH remaining
from the reductive amination step. Since we planned to perform this sequence
(reductive amination and cyclization) without purification of the amino-acid inter-
mediate, we developed conditions to ensure that any unreacted starting materials or
reagents from the reductive amination would not lead to inseparable by-products under
the conditions of the cyclization step. Several reaction stoichiometries were inves-
tigated; the optimal one had a ratio of amine (0.65 equiv.) to keto acid (1 equiv.) with a
slight excess of reducing and coupling agents. When 6 was submitted to these conditions
without purification of intermediate 7, a simple acid/base extractive workup after the
cyclization reaction provided piperazinone 8 (92% yield, >95% pure), identical (TLC,
1H-NMR) to material purified by flash chromatography.
The third subunit (R3) in our developmental system was incorporated by a
sequential deprotection/acylation sequence of the Boc-protected secondary amine.
Thus, treatment of 8 with HCl/AcOEt followed by coupling with 4-bromobenzoic acid
effected by EDCI afforded the final product 9 in 89% yield and excellent purity
(1H-NMR) after extractive workup.
Additional methods for the modification of the piperazinone secondary amine were
explored for introduction of the final (R3) substituent. Two alkylation reactions were
developed based on piperazinone 10, prepared from 6 using 3-(trifluoromethyl)benzyl-
amine under the above conditions. Thus, N-Boc deprotection (HCl/AcOEt) and
treatment of the resulting crude HCl salt with PhCH2Br (1.05 equiv.) and EtN(i-Pr)2
(1.3 equiv.) in DMFat 258 for 17 h provided the crude reaction product. After acid/base
extractive workup, the desired N-alkylated product 11 was isolated in 96% yield and
excellent purity (1H-NMR, >95% pure) (Scheme 4). Reductive-amination conditions
were also investigated. N-Boc deprotection (HCl/AcOEt) followed by treatment of the
free amine with 4-methoxybenzaldehyde (1.05 equiv.), NaBH3CN (2.6 equiv.), and
AcOH (2.0 equiv.) in 1,2-dichloroethane at 258 for 40 h provided 12. In this case, a
small amount (<20%) of 4-methoxybenzyl alcohol by-product remained after the
extractive workup. These model reactions demonstrated that alkylation of the
piperazinone secondary amine is a useful alternative to amide couplings for the
3
)
The 4-methylphenyl ketone (synthesized analogously from 5 by reaction with toluoyl magnesium bromide)
failed to react with 4-methoxybenzylamine in the presence of NaBH3CN or NaB(OAc)3H presumably
because of steric and/or electronic factors. We, therefore, limited our library to alkyl substituents at the R1
position.
NaBH3CN was also effective for this transformation, although the reactions were more sluggish. Since the
cyanoborohydride reagent itself and the resulting aqueous waste from the reaction workup are toxic,
NaBH(OAc)3 [18] was adopted for the library synthesis.
4
)

Helvetica Chimica Acta ± Vol. 83 (2000)
1829
introduction of the final (R3) diversity subunit, although we limited our first
prototypical library synthesis to the latter.
Scheme 4
A briefly explored alternative proceeded through the symmetrical piperazine-2,6-
dione 4 (Scheme 1). Reaction conditions were developed for the cyclization of
monoamide 13, obtained from the reaction of 4-methoxybenzylamine with 1, to give
imide 14 (Scheme 5). Several dehydrating reagents were investigated, including
(CF3CO)2O, Ac2O, and EDCI. The most effective reagent was 1,1'-carbonyldiimid-
azole (CDI; 4 equiv.), which provided 14 in 90% yield and excellent purity after a
simple acid/base extractive workup. However, the yield of the addition of Grignard and
organolithium reagents to 14 to provide the desired product (e.g., 15) was typically
<30%. In addition, initial attempts at subsequent cyclization, either directly via
intramolecular reductive amination, or by reduction to alcohol 17 and subsequent
Mitsunobu cyclization with Ph3P and diethyl azodicarboxylate (DEAD) to form
piperazinone 16 were not encouraging.
Library Synthesis. The reaction sequence and conditions optimized in our
developmental system were applied to the synthesis of a 150-member piperazinone
library. From commercially available materials, three organomagnesium reagents were
selected for the R1 subunit (A1 ± A3), five primary amines for the R2 subunit (B1 ± B5),
and ten aromatic carboxylic acids were chosen for the R3 position (C1 ± C10; see
Fig. 1). Compound 5 was used as the starting point for the library synthesis, and
addition of ethyl, benzyl, and phenethyl Grignard reagents provided large quantities
(> 8 g) of 6, 18, and 19, respectively, in excellent purity after the phase-switch
extractive workup (Scheme 6).
Compounds 6, 18, and 19 were each divided into five equal portions and submitted
to the reductive amination/intramolecular cyclization sequence with amines B1 ± B5.
This afforded the desired N-Boc-protected piperazinones (8, 10, 20 ± 32) in excellent

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Helvetica Chimica Acta ± Vol. 83 (2000)
Scheme 5
yields (average 97%) after acid/base extractive workup5). Each of 8, 10, 20 ± 32 were
divided into ten equal portions and deprotected (HCl/AcOEt). The resulting crude
HCl salts were directly coupled (EDCI) with each of the ten carboxylic acids C1 ± C10.
The products were isolated by acid/base liquid-liquid extractions, which effectively
removed all reagents, unreacted starting materials, and reaction by-products, providing
the library of 150 individual compounds (33). The yields for these reactions were good-
to-excellent (Table 1), and 30 representative samples were characterized by 1H-NMR,
HR-MS, and IR (matrixcharacterization). In each case, the desired acylated
piperazinone was identified in excellent purity.
Repression of Transcription from LEF-1 Response Elements. ± The amount of each
isolated product (typically 80 ± 100 mg) is ideal for depository libraries, which are tested
5
)
To establish the integrity of intermediates 8, 10, and 20 ± 32, portions of the crude material of one series
(23 ± 27) were purified by flash chromatography. The purity of the intermediates was determined from the
ratio of the masses of the materials isolated from the flash column to the masses of the crude reaction
products. By this method, an average purity of 86% was established.

Helvetica Chimica Acta ± Vol. 83 (2000)
1831
Fig. 1. Structures of R1 subunits A1 ± A3, R2 subunits B1 ± B5, and R3 subunits C1 ± C10
in several biological screens over many years, and our initial 150-member piperazinone
library is currently being examined in a number of assays that target protein-protein
interactions. Several leads were identified from the screening of this library against
LEF-1/b-catenin-mediated transcription in a colon-cancer cell line (SW480). In a
luciferase assay based on a reporter containing 4 copies of the LEF-1 binding site
upstream from the c-fos promoter (known as TOPFLASH) [19], several compounds
(10 mm concentration) significantly repressed transcription (Table 2). Sixteen selected
compounds were then retested by repeating the assay with TOPFLASH and a similar
reporter where the LEF-1 binding sites in front of the promoter were mutated and
inactive (FOPFLASH [19], Fig. 2). Several of these agents (e.g., 33: A1B1C8,
A1B2C3, A1B2C8, A1B3C6, A1B3C9, and A3B4C6, see Table 3) inhibited tran-
scription as much as five-fold and displayed an approximately twofold greater
repression of transcription in TOPFLASH than in the FOPFLASH system, demon-
strating their selectivity. These agents represent, to the best of our knowledge, only the
second class of small-molecule compounds that repress transcription from LEF-1

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Helvetica Chimica Acta ± Vol. 83 (2000)
Scheme 6
Table 1. Yields [%] for the Final Coupling Step for Library 33
33
C1
C2
C3
C4
C5
C6
C7
81
C8
C9
C10
A1B1
A1B2
A1B3
A1B4
A1B5
A2B1
A2B2
A2B3
A2B4
A2B5
A3B1
A3B2
A3B3
A3B4
A3B5
76
83
91
91
77
83
79
95
59
78
84
84
86
85
72
79
77
93
87
77
82
88
89
59
77
83
80
83
85
86
82
77
77
77
81
94
80
87
65
75
78
74
77
80
80
71
73
79
79
67
79
80
81
66
76
70
71
75
70
74
71
76
82
84
69
83
80
87
69
71
77
76
82
79
75
61
85
91
80
77
82
82
89
62
76
84
84
85
83
82
71
72
74
74
71
90
49
86
69
69
73
67
73
73
66
71
71
86
81
74
91
79
93
68
76
74
69
79
77
73
74
81
93
87
80
98
90
93
56
87
87
90
88
87
88
78
85
83
85
100
70
87
59
62
57
81
81
81
86
Average yield 80%

Helvetica Chimica Acta ± Vol. 83 (2000)
1833
responsive elements and the first class that is not based on DNA minor-groove-binding
agents6).
Fig. 2. Schematic representation of LEF-1 activation of transcription. a) b-Catenin binds to LEF-1 and activates
transcription from LEF-1 responsive elements. b) In both the TOPFLASH and FOPFLASH reporter systems,
expression of the luciferase gene (gray box) is driven by the c-fos minimal promoter (black box). TOPFLASH
contains four copies of the LEF-1 binding site (CCTTTGATC) upstream from the promoter that augment
transcription, whereas FOPFLASH contains mutated, inactive LEF-1 binding sites (CCTTTGGCC). The level
of transcription is determined by measuring luciferase activity (luminescence).
Table 2. Fold Repression of Transcription of a Luciferase Reporter with LEF-1 Binding Sites in the Promoter
Region (TOPFLASH)a)
33
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
A1B1
A1B2
A1B3
A1B4
A1B5
A2B1
A2B2
A2B3
A2B4
A2B5
A3B1
A3B2
A3B3
A3B4
A3B5
1.14
1.72
1.89
1.33
0.72
1.00
1.32
1.67
2.04
1.10
1.15
1.52
1.11
1.37
1.10
1.49
1.92
1.92
1.75
1.20
1.04
1.32
1.03
1.20
1.82
1.28
1.61
1.16
1.64
1.19
2.13
3.70
2.94
2.17
0.63
1.20
1.41
1.54
2.50
1.56
0.84
1.12
1.25
1.89
1.35
1.06
1.14
1.96
1.10
0.83
0.85
1.03
0.90
1.04
1.54
1.27
1.14
1.61
1.64
1.02
1.19
1.52
1.82
1.08
0.89
1.33
1.41
1.05
1.45
0.97
1.02
1.14
1.54
1.92
1.23
1.64
2.63
2.94
2.08
1.32
0.72
0.97
1.61
1.82
1.45
1.27
1.02
1.23
3.57
1.14
2.00
1.75
1.45
1.09
1.25
1.61
0.86
1.08
1.39
1.33
1.30
1.02
0.88
0.68
1.10
3.85
3.45
2.50
1.85
1.45
1.33
1.28
1.11
1.67
2.22
1.39
1.10
1.10
1.23
1.19
1.52
4.76
4.17
2.00
1.15
1.19
2.17
1.56
1.49
1.47
1.30
1.59
1.47
1.04
1.47
1.61
2.38
2.00
1.69
0.79
1.89
1.27
1.56
1.89
2.44
1.47
1.20
1.52
1.61
0.68
a) All compounds were tested at 10 mm concentration. Inhibition of transcription was determined by luciferase
activity.
6
)
Recently, heterocyclic polyamides that bind to the minor groove of DNA have been shown to repress
transcription of genes with LEF-1 responsive elements in the promoter [20].

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Table 3. Ratio of Fold Repression of Transcription with LEF-1 (TOPFLASH) and Mutated, Inactive LEF-1
(FOPFLASH) Binding Sites in the Luciferase Promoter Regiona)
TOPFLASH/
FOPFLASH
TOPFLASH/
FOPFLASH
33
Repression ratio
33
Repression ratio
A1B1C8
A1B2C3
A1B2C8
A1B2C9
A1B3C3
A1B3C6
A1B3C9
A1B4C2
2.00
2.16
1.67
1.50
1.44
1.88
2.07
1.38
A1B4C6
A2B1C10
A2B2C9
A2B4C1
A2B4C3
A2B5C8
A2B5C10
A3B4C6
1.07
1.23
1.40
1.17
1.00
1.06
1.26
2.22
a) All compounds were tested at 10 mm concentration. Inhibition of transcription was determined by luciferase
activity. See text and Fig. 2 for explanation of TOPFLASH and FOPFLASH reporter systems.
Secondary Library Synthesis and Screening. The screening results in Tables 2 and 3
reveal that the majority of active compounds from the library contain the ethyl subunit
(A1) in the R1 position, and that 4-methyl- and 4-chlorobenzylamines (B2 and B3)
were the most active among the R2 substitutions. Since each of these subunits was
tested only in combination with a limited number of R3 subunits (C1 ± C10) in the initial
library, secondary libraries containing A1B2 and A1B3 were prepared containing
increased diversity at position R3. The synthesis of these libraries required only one step
from archived samples of compounds 20 and 21, following the same synthetic strategy
as in the original 150-member library. Each of 20 and 21 was subjected to N-Boc
deprotection (HCl/AcOEt), and reacted with C11 ± C110 (20) or C111 ± C210 (21) to
provide two 100-member libraries 33, A1B2C11 ± 110, and 33, A1B3C111 ± C210
(Figs. 3 and 4, and Scheme 7). Each library product was purified by acid/base liquid-
liquid extractions, providing the final library members in ꢀ90% purity in good to
excellent conversions (Table 4). As in the initial library synthesis, the majority of the
new R3 subunits coupled to 20 and 21 were carboxylic acids. However, sulfonyl
chlorides, chloroformates, and isocyanates were also included, producing sulfonamide,
carbamate, and urea linkages, respectively, in the final library products. The inclusion of
the latter functional groups demonstrates the diversity of chemistry achievable by
solution-phase techniques.
Results from the screening of 33, A1B2C11 ± C110, and 33, A1B3C111 ± C210, in
the TOPFLASH reporter assay are presented in Figs. 3 and 4. Although no increase in
repression over the original library was observed for these compounds, the nine most
active in the series were tested in the FOPFLASH assay (Table 5), where they
Table 4. Average Yields for the Synthesis of the Secondary Libraries (33, A1B2C11 ± 110 and 33, A1B2C111 ±
210) from N-Boc-Piperazinones 20 and 21
RCO2H
ROCOCl
RSO2Cl
RNCO
Reagent type
(C11 ± C95)
(C111 ± C196)
(C96 ± C100)
(C197 ± C201)
(C101 ± C105)
(C202 ± C206)
(C106 ± C110)
(C207 ± C210)
20 (A1B2)
21 (A1B3)
54%
70%
80%
90%
66%
91%
86%
95%

Helvetica Chimica Acta ± Vol. 83 (2000)
1835
Fig. 3. Structures of the 100 R3 subunits (C11 ± C110) coupled to 20. Results of the TOPFLASH reporter assay
(fold repression of transcription) are given in parentheses.
displayed selectivity for repressing LEF-1-mediated transcription (TOPFLASH/
FOPFLASH transcription ratios of 1.6 ± 2.3). Analysis of the screening results (Figs. 3
and 4) for entire set of compounds also reveals insight into structure-activity
relationships (SAR) for the R3 substitutions and provides several general observations:
± Carboxamide > carbamate > sulfonamide > urea linkages
± A hydrophobic or aromatic group is required

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Helvetica Chimica Acta ± Vol. 83 (2000)
Fig. 4. Structures of the 100 R3 subunits (C111 ± C210) coupled to 21. Results of the TOPFLASH reporter assay
(fold repression of transcription) are given in parentheses.
± For aromatic substituents: phenyl > naphthyl > anthracyl, fluorenyl
± Aryl substitution: NO2, Cl, Br, OH > I, Me, OMe > F, Acetyl
± For phenyl substitutions: para > meta > ortho; No. of substitutions: 1 > 2 > 3, 4
± Spacer length between the piperazinone N(4) and phenyl ring: 3 atoms
(cinnamyl) > 2 atoms (phenylacetyl) > 1 atom (benzoyl)
± Hydrophilic subunits should be avoided.

Helvetica Chimica Acta ± Vol. 83 (2000)
1837
Scheme 7. Synthesis of Two Secondary Piperazinone Libraries Based on Initial Screening Results. See text and
Table 4 for details.
Table 5. Biological Activity of the Most Effective Compounds from the Secondary Librariesa)
33
Fold repression on TOPFLASH
TOPFLASH/FOPFLASH repression ratio
A1B2C13
A1B2C44
A1B2C49
A1B2C50
A1B2C57
A1B3C144
A1B3C146
A1B3C148
A1B3C150
2.7
2.9
2.3
3.0
2.5
3.0
2.2
2.7
2.7
1.8
1.9
2.0
2.3
1.6
1.8
1.7
1.7
1.6
a) All compounds were tested at 10 mm concentration. Inhibition of transcription was determined by luciferase
activity. See text and Fig. 2 for an explanation of TOPFLASH and FOPFLASH reporter systems.
The syntheses of additional secondary libraries to further explore the effects of
increased diversity at positions R1 and R2 are in progress. Studies are also underway to
further define the structural features contributing to transcription inhibition, to
determine the agentsꢁ cellular targets, and to measure their effect on the growth of
colorectal tumors. These results will be disclosed in due course.
Conclusions. ± A 150-member library of non-amide-based 4-acyl-1,6-dialkylpiper-
azinones was synthesized from N-Boc-iminodiacetic acid by solution-phase techniques.
Simple liquid-liquid extractions provided pure reaction intermediates and final
products in quantities sufficient (80 ± 100 mg) for screening in a variety of biological
assays, thus extending the solution-phase combinatorial methodology beyond amide-
bond formation. Members of this library were found to have activity against LEF-1/b-

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Helvetica Chimica Acta ± Vol. 83 (2000)
catenin-mediated transcription. Two secondary libraries (100 compounds each) based
on further diversification of position 4 of the 1,6-dialkylpiperazinone core (R3)
provided additional active compounds and revealed structural requirements for the
observed biological activity. Exploration of these novel leads as well as the results from
subsequent syntheses and screens will be forthcoming.
Experimental Part
General. IR Spectra were obtained on KBr plates (film); in cmÀ1 1H-NMR Spectra were recorded at
.
400 MHz; chemical shifts d in ppm, coupling constants J in Hz. HR-MS was performed by the fast-atom-
bombardment (FAB) technique with a 3-nitrobenzyl alcohol (NBA)/NaI matrix.
N-[(tert-Butoxy)carbonyl]-N-[(methoxy)(methyl)carbamoylmethyl]glycine (5). A soln. of N-Boc-imino-
diacetic acid (1; 90 g, 390 mmol) in DMF (600 ml) was treated with EDCI (89 g, 460 mmol) in several portions
at 08 and then warmed to 258, and stirred for 1 h. The mixture was cooled to 08 and treated dropwise with a
premixed soln. of N-methoxymethylamine hydrochloride (45 g, 460 mmol) and EtN(i-Pr)2 (80 ml, 460 mmol) in
DMF (200 ml), and stirred for an additional 10 h at 258. The mixture was poured into a flask containing ice
(1.0 kg) and 10% aq. HCl (1.0 l) and extracted wtih AcOEt (2 Â 1.0 l). The org. phases were combined and
washed with 10% aq. HCl (2 Â 1.0 l) and sat. aq. NaCl soln. (1.0 l), dried (Na2SO4), and evaporated in vacuo.
The resulting solid was washed with Et2O (250 ml) and filtered to afford 5 (42.02 g, 39%). White powder: M.p.
92 ± 948. IR: 2998, 2943, 2707, 2605, 1754, 1702, 1625, 1461, 1446, 1390, 1369, 1246, 1159, 1133, 1000, 969, 933, 897,
856, 795, 769. 1H-NMR ((D6)DMSO): 4.18 (s, 1 H); 4.14 (s, 1 H); 3.90 (s, 1 H); 3.87 (s, 1 H); 3.69 (s, 1.5 H); 3.67
‡
(s, 1.5 H); 3.10 (s, 1.5 H); 3.09 (s, 1.5 H); 1.35 (s, 4.5 H); 1.34 (s, 4.5 H). HR-MS: 277.1398 ([M ‡ H] ,
‡
C9H15NO6 ; calc. 277.1400).
General Procedure for the First Diversification: N-[(tert-Butoxy)carbonyl]-N-(2-oxobutyl)glycine (6). A
soln. of 5 (9.96 g, 36.1 mmol) in THF (200 ml) was cooled to 08 and treated with EtMgBr (1.0m soln. in THF,
79 ml, 79 mmol) dropwise via syringe. The mixture was stirred at 258 for 1 h, and then cooled to 08 and quenched
by the slow addition of sat. aq. NH4Cl (100 ml) soln. and H2O (100 ml). After stirring for 3 h at 258, Et2O
(500 ml) was added, and the mixture was extracted with 2m aq. NaOH (300 ml). The aq. layer was acidified by
the addition of conc. aq. HCl (30 ml) at 08 and then twice extracted with Et2O (1000 ml and 500 ml). The org.
phases were combined, washed with sat. aq. NaCl soln. (300 ml), dried (Na2SO4), and evaporated to give 6
(8.24 g, 93%). Pale yellow oil. IR: 2979, 2940, 1708, 1698, 1650, 1642, 1632, 1478, 1454, 1394, 1369, 1251, 1165,
1042, 901, 877, 858, 778. 1H-NMR ((D6)DMSO): 4.06 (s, 1 H); 4.03 (s, 1 H); 3.87 (s, 1 H); 3.83 (s, 1 H); 2.40
(q, J ˆ 7.2, 1 H); 2.38 (q, J ˆ 7.2, 1 H); 1.35 (s, 4.5 H); 1.32 (s, 4.5 H); 0.94 (t, J ˆ 7.2, 1.5 H); 0.92 (t, J ˆ 7.2,
‡
‡
1.5 H). HR-MS: 268.1298 ([M ‡ Na] , C11H19NO5 ; calc. 268.1290).
N-[(tert-Butoxy)carbonyl]-N-(2-oxo-3-phenylpropyl)glycine (18). Yield 89%. IR: 2978, 2937, 1726, 1700,
1496, 1475, 1449, 1396, 1370, 1250, 1156, 1067, 962, 905, 858, 758, 701, 659. 1H-NMR (CDCl3): 7.37 ± 7.24 (m, 3 H);
7.23 ± 7.16 (m, 2 H); 4.20 (m, 1.33 H); 4.10 (m, 0.67 H); 3.95 (s, 0.67 H); 3.82 (s, 1.33 H); 3.80 (s, 1.33 H); 3.71
(s, 0.67 H); 1.42 (s, 6 H); 1.33 (s, 3 H). HR-MS: 308.1506 ([M ‡ H] , C16H21NO5 ; calc. 308.1498).
N-[(tert-Butoxy)carbonyl]-N-(2-oxo-4-phenylbutyl)glycine (19). Yield 79%. IR: 2974, 2923, 1728, 1697,
1456, 1390, 1369, 1246, 1154, 907, 851, 749, 703. 1H-NMR (CDCl3): 7.31 ± 7.09 (m, 5 H); 4.13 ± 3.75 (m, 4 H);
‡
‡
‡
‡
2.99 ± 2.66 (m, 4 H); 1.44 (s, 6 H); 1.35 (s, 3 H). HR-MS: 344.1485 ([M ‡ Na] , C17H23NO5 ; calc. 344.1474).
General Procedure for the Second Diversification and Cyclization to the N-Boc-Piperazinone: 4-[(tert-
Butoxy)carbonyl]-6-ethyl-1-[(4-methoxyphenyl)methyl]piperazin-2-one (8).
A stirred soln. of 6 (1.70 g,
6.92 mmol) in 1,2-dichloroethane (35 ml) was treated with 4-methoxybenzylamine (617 mg, 4.50 mmol),
AcOH (0.40 ml, 6.9 mmol), and NaBH(OAc)3 (1.91 g, 8.99 mmol), and the resulting suspension was stirred at
258 for 16 h. The solvent was removed under reduced pressure, and the residue was dissolved in DMF (30 ml)
and treated with EDCI (1.33 g, 6.92 mmol), and stirred at 258 for 17 h. The mixture was extracted with AcOEt
(150 ml) and washed with 10% aq. HCl (4 Â 30 ml), sat. aq. NaHCO3 (4 Â 30 ml), and sat. aq. NaCl soln.
(30 ml), dried (Mg2SO4), and evaporated to provide 8 (1.43 g, 92%). Pale yellow oil. IR: 2974, 1690, 1658, 1642,
1510, 1478, 1461, 1425, 1414, 1366, 1324, 1292, 1244, 1175, 1132, 1037, 994, 893. 1H-NMR (CDCl3): 7.15 (d, J ˆ 8.5,
2 H); 6.83 (d, J ˆ 8.5, 2 H); 5.30 (d, J ˆ 14.6, 1 H); 4.52 ± 4.25 (m, 1 H); 4.15 ± 3.97 (m, 1 H); 3.94 ± 3.81 (m, 2 H);
3.78 (s, 3 H); 3.14 ± 2.83 (m, 2 H); 1.54-1.36 (m, 2 H); 1.44 (s, 9 H); 0.90 (t, J ˆ 7.6, 3 H). HR-MS: 349.2120
‡
‡
([M ‡ H] , C19H28N2NO4 ; calc. 349.2127).

Helvetica Chimica Acta ± Vol. 83 (2000)
1839
4-[(tert-Butoxy)carbonyl]-6-ethyl-1-{[3-(trifluoromethyl)phenyl]methyl}piperazin-2-one (10). Yield 100%.
1
IR: 2974, 2932, 1695, 1653, 1637, 1419, 1366, 1324, 1292, 1244, 1164, 1127, 1074, 1000, 893, 803, 766. H-NMR
(CDCl3): 7.57 ± 7.49 (m, 1 H); 7.48 ± 7.38 (m, H); 5.33 (d, J ˆ 15.1, 1 H); 4.64 ± 4.27 (m, 1 H); 4.21 ± 3.78
(m, 2 H); 4.00 (d, J ˆ 15.1, 1 H); 1.59 ± 1.37 (m, 2 H); 1.45 (s, 9 H); 0.95 (t, J ˆ 7.6). HR-MS: 387.1904 ([M ‡
‡
‡
H] , C19H25F3N2O3 ; calc. 387.1896).
4-[(tert-Butoxy)carbonyl]-6-ethyl-1-[(4-methylphenyl)methyl]piperazin-2-one (20). Yield 91%, based on
the amine. IR: 2974, 2932, 1695, 1653, 1462, 1419, 1366, 1324, 1286, 1244, 1164, 1138, 994, 893. 1H-NMR (CDCl3):
7.10 (s, 4 H); 5.34 (d, J ˆ 14.8, 1 H); 4.54 ± 4.27 (m, 1 H); 4.16 ± 3.95 (m, 1 H); 3.94 ± 3.81 (m, 1 H); 3.82 (d, J ˆ
14.8, 1 H); 3.13 ± 2.87 (m, 2 H); 2.31 (s, 3 H); 1.56 ± 1.39 (m, 2 H); 1.44 (s, 9 H); 0.90 (t, J ˆ 7.6, 3 H). HR-MS:
‡
‡
333.2170 ([M ‡ H] , C19H28N2O3 ; calc. 333.2178).
4-[(tert-Butoxy)carbonyl]-1-[(4-chlorophenyl)methyl]-6-ethylpiperazin-2-one (21). Yield 95%, based on
the amine. IR: 2974, 2931, 2878, 1690, 1648, 1494, 1462, 1419, 1409, 1366, 1324, 1276, 1244, 1159, 1138, 1090, 1021,
994, 899. 1H-NMR (CDCl3): 7.27 (d, J ˆ 8.4, 2 H); 7.15 (d, J ˆ 8.4, 2 H); 5.26 (d, J ˆ 14.8, 1 H); 4.54 ± 4.27
(m, 1 H); 4.18 ± 4.00 (m, 1 H); 3.96 ± 3.77 (m, 1 H); 3.90 (d, J ˆ 14.8, 1 H); 3.14 ± 2.88 (m, 2 H); 1.56 ± 1.35
‡
‡
(m, 2 H); 1.45 (s,9 H); 0.91 (t, J ˆ 7.3, 3 H). HR-MS: 353.1624 ([M ‡ H] , C18H25ClN2O3 ; calc. 353.1632).
4-[(tert-Butoxy)carbonyl]-6-ethyl-1-[(furan-2-yl)methyl]piperazin-2-one (22). Yield 88%, based on the
amine. IR: 2963, 2932, 2878, 1695, 1653, 1462, 1419, 1393, 1366, 1324, 1276, 1249, 1228, 1170, 1132, 1090, 1005,
989, 899, 856, 766, 734. 1H-NMR (CDCl3): 7.33 (br. s, 1 H); 6.30 (dd, J ˆ 3, 2, 1 H); 6.26 (d, J ˆ 3, 1 H); 5.12
(d, J ˆ 15.4, 1 H); 4.49 ± 4.21 (m, 1 H); 4.17 ± 4.00 (m, 1 H); 4.06 (d, J ˆ 15.4, 1 H); 3.91 ± 3.71 (m, 1 H); 3.29 ±
‡
2.95 (m, 2 H); 1.54 ± 1.36 (m, 2 H); 1.44 (s, 9 H); 0.93 (t, J ˆ 7.3, 3 H). HR-MS: 309.1808 ([M ‡ H] ,
‡
C16H24N2O4 ; calc. 309.1814).
4-[(tert-Butoxy)carbonyl]-1-[(4-methoxyphenyl)methyl]-6-(phenylmethyl)piperazin-2-one (23). Yield 93%,
based on the amine. IR: 2976, 2932, 1693, 1649, 1557, 1540, 1509, 1474, 1456, 1417, 1364, 1325, 1241, 1167, 1127,
1031, 960, 882. 1H-NMR (CDCl3): 7.33 ± 7.07 (m, 7 H); 6.85 (d, J ˆ 11.4, 2 H); 5.35 (d, J ˆ 14.6, 1 H); 4.49 ± 4.29
(m, 1 H); 4.10 ± 3.71 (m, 2 H); 3.88 (d, J ˆ 15, 1 H); 3.77 (s, 3 H); 3.50 (m, 1 H); 2.96 ± 2.66 (m, 2 H); 2.71
‡
‡
(dd, J ˆ 13.5, 10, 1 H); 1.53 ± 1.40 (m, 9 H). HR-MS: 411.2272 ([M ‡ H] , C24H30N2O4 ; calc. 411.2284).
4-[(tert-Butoxy)carbonyl]-1-[(4-methylphenyl)methyl]-6-(phenylmethyl)piperazin-2-one
(24).
Yield
100%, based on the amine. IR: 2974, 2930, 1697, 1649, 1474, 1456, 1417, 1364, 1325, 1285, 1246, 1167, 1132,
1031, 960, 925, 882, 750, 737, 702. 1H-NMR (CDCl3): 7.33 ± 7.07 (m, 9 H); 5.38 (d, J ˆ 14.6, 1 H); 4.48 ± 4.28
(m, 1 H); 4.11 ± 3.78 (m, 3 H); 3.34 (m, 1 H); 2.96 ± 2.75 (m, 2 H); 2.71 (dd, J ˆ 13.2, 9.7, 1 H); 2.31 (s, 3 H);
‡
‡
1.54 ± 1.38 (m, 9 H). HR-MS: 395.2342 ([M ‡ H] , C24H30N2O3 ; calc. 395.2335).
4-[(tert-Butoxy)carbonyl]-1-[(4-chlorophenyl)methyl]-6-(phenylmethyl)piperazin-2-one (25). Yield 100%,
based on the amine. IR: 2976, 2928, 1697, 1654, 1491, 1472, 1457, 1419, 1405, 1366, 1323, 1270, 1242, 1165, 1131,
1088, 1064, 1016, 964, 925, 877, 849. 1H-NMR (CDCl3): 7.36 ± 7.08 (m, 9 H); 5.35 (d, J ˆ 15.1, 1 H); 4.52 ± 4.30
(m, 1 H); 4.14 ± 4.00 (m, 0.6 H); 3.97 ± 3.73 (m, 2.4 H); 3.34 (m, 1 H); 3.00 ± 2.76 (m, 2 H); 2.75 (dd, J ˆ 13.5, 9.7,
‡
‡
1 H); 1.55 ± 1.41 (m, 9 H). HR-MS: 415.1775 ([M ‡ H] , C23H27ClN2O3 ; calc. 415.1788).
4-[(tert-Butoxy)carbonyl]-6-(phenylmethyl)1-{[3-(trifluoromethyl)phenyl]methyl}piperazin-2-one (26).
Yield 100%, based on the amine. IR: 2976, 2928, 1692, 1654, 1452, 1414, 1390, 1366, 1328, 1242, 1160, 1122,
1093, 1074, 1021, 964, 877, 700. 1H-NMR (CDCl3): 7.60 ± 7.38 (m, 4 H); 7.36 ± 7.08 (m, 5 H); 5.42 (d, J ˆ 15.1,
1 H); 4.53 ± 4.30 (m, 1 H); 4.16 ± 4.01 (m, 0.8 H); 4.00 ± 3.81 (m, 2.2 H); 3.33 (m, 1 H); 3.01 ± 2.80 (m, 2 H); 2.77
‡
‡
(dd, J ˆ 13.5, 9.4, 1 H); 1.53 ± 1.38 (br. s, 9 H). HR-MS: 471.1886 ([M ‡ Na] , C24H27F3N2O3 ; calc. 471.1871).
4-[(tert-Butoxy)carbonyl]-1-[(furan-2-yl)methyl]-6-(phenylmethyl)piperazin-2-one (27). Yield 100%,
based on the amine. IR: 2975, 2929, 1700, 1653, 1558, 1540, 1506, 1472, 1457, 1419, 1369, 1324, 1272, 1247,
1166, 1128, 1078, 1009, 962, 935, 883, 743, 700, 668. 1H-NMR (CDCl3): 7.43 ± 7.09 (m, 6 H); 6.39 ± 6.25 (m, 2 H);
5.15 (d, J ˆ 15.6, 1 H); 4.48 ± 4.25 (m, 1 H); 4.19 ± 3.98 (m, 1.6 H); 3.96 ± 3.77 (m, 1.4 H); 3.58 ± 3.43 (m, 1 H);
‡
3.02 ± 2.80 (m, 2 H); 2.70 (dd, J ˆ 13, 10, 1 H); 1.56 ± 1.40 (br. s, 9 H). HR-MS: 371.1979 ([M ‡ H] ,
‡
C21H26N2O4 ; calc. 371.1971).
4-[(tert-Butoxy)carbonyl]-1-[(4-methoxyphenyl)methyl]-6-(2-phenylethyl)piperazin-2-one (28). Yield 100%,
based on the amine. IR: 2971, 2930, 1696, 1650, 1557, 1506, 1450, 1419, 1239, 1168, 1127, 1070, 1034, 701. 1H-NMR
(CDCl3): 7.34 ± 7.17 (m, 3 H); 7.15 ± 7.08 (m, 2 H); 6.95 (d, J ˆ 8.4, 2 H); 6.75 (d, J ˆ 8.4, 2 H); 5.22 (d, J ˆ 14.6,
1 H); 4.52 ± 4.04 (m, 2 H); 3.91 ± 3.79 (m, 1 H); 3.76 (s, 3 H); 3.64 (d, J ˆ 14.6, 1 H); 3.20 ± 2.72 (m, 3 H); 2.53 ±
‡
‡
2.38 (m, 1 H); 1.93 ± 1.72 (s, 2 H); 1.47 (s, 9 H). HR-MS: 425.2446 ([M ‡ H] , C25H32N2O4 ; calc. 425.2440).
4-[(tert-Butoxy)carbonyl]-1-[(4-methylphenyl)methyl]-6-(2-phenylethyl)piperazin-2-one (29). Yield 100%,
based on the amine. IR: 2923, 2862, 1692, 1646, 1451, 1415, 1241, 1164, 1128, 749, 697. 1H-NMR (CDCl3): 7.34 ±
7.08 (m, 5 H); 7.06 (d, J ˆ 7.6, 2 H); 6.93 (d, J ˆ 7.6, 2 H); 5.24 (d, J ˆ 14.6, 1 H); 4.54 ± 4.03 (m, 2 H); 3.94 ± 3.78

1840
Helvetica Chimica Acta ± Vol. 83 (2000)
(m, 1 H); 3.69 (d, J ˆ 14.6, 1 H); 3.21 ± 2.67 (m, 3 H); 2.53 ± 2.39 (m, 1 H); 2.31 (s, 3 H); 1.94 ± 1.74 (m, 2 H);
‡
‡
1.48 (s, 9 H). HR-MS: 409.2497 ([M ‡ H] , C25H32N2O3 ; calc. 409.2491).
4-[(tert-Butoxy)carbonyl]-1-[(4-chlorophenyl)methyl]-6-(2-phenylethyl)piperazin-2-one (30). Yield 100%,
based on the amine. IR: 2973, 2930, 1693, 1649, 1417, 1363, 1239, 1163, 1126, 1088, 753, 699. 1H-NMR (CDCl3):
7.35 ± 7.19 (m, 3 H); 7.18 (d, J ˆ 8.4, 2 H); 7.15 ± 7.08 (m, 2 H); 6.92 (d, J ˆ 8.4, 2 H); 5.20 (d, J ˆ 14.6, 1 H); 4.54 ±
4.05 (m, 2 H); 3.94 ± 3.76 (m, 1 H); 3.65 (d, J ˆ 14.6, 1 H); 3.17 ± 2.74 (m, 3 H); 2.55 ± 2.38 (m, 1 H); 1.94 ± 1.79
‡
‡
(m, 2 H); 1.47 (s, 9 H). HR-MS: 429.1936 ([M ‡ H] , C24H29ClN2O3 ; calc. 429.1945).
4-[(tert-Butoxy)carbonyl]-6-(2-phenylethyl)-1-{[(3-(trifluoromethyl)phenyl]methyl}piperazin-2-one (31).
Yield 100%, based on the amine. IR: 2965, 2922, 1689, 1654, 1645, 1452, 1417, 1325, 1241, 1162, 1123, 1070,
1
698. H-NMR (CDCl3): 7.49 (br. d, J ˆ 7.6, 1 H); 7.36 (t, J ˆ 7.6, 1 H); 7.33 ± 7.15 (m, 5 H); 7.15 ± 7.07 (m, 2 H);
5.25 (d, J ˆ 15.1, 1 H); 4.57 ± 4.08 (m, 2 H); 3.96 ± 3.81 (m, 1 H); 3.78 (d, J ˆ 15.1, 1 H); 3.18 ± 2.66 (m, 3 H);
‡
‡
2.54 ± 2.34 (m, 1 H); 1.97 ± 1.80 (m, 2 H); 1.48 (s, 9 H). HR-MS: 485.2016 ([M ‡ Na] , C25H29F3N2O3 ; calc.
485.2028).
4-[(tert-Butoxy)carbonyl]-1-[(furan-2-yl)methyl]-6-(2-phenylethyl)piperazin-2-one (32). Yield 100%,
based on the amine. IR: 2965, 2922, 1689, 1654, 1417, 1364, 1320, 1246, 1162, 1123, 1009, 746. 1H-NMR
(CDCl3): 7.36 ± 7.11 (m, 6 H); 6.30 ± 6.24 (m, 1 H); 6.10 (d, J ˆ 3.0, 1 H); 5.05 (d, J ˆ 15.4, 1 H); 4.50 ± 4.08
(m, 2 H); 4.02 (d, J ˆ 15.4, 1 H); 3.91 ± 3.74 (m, 1 H); 3.36 ± 2.94 (m, 2 H); 2.88 ± 2.72 (m, 1 H); 2.58 ± 2.46
‡
‡
(m, 1 H); 1.94 ± 1.71 (m, 2 H); 1.47 (s, 9 H). HR-MS: 385.2119 ([M ‡ H] , C22H28N2O4 ; calc. 385.2127).
4-Benzyl-6-ethyl-1-{[3-(trifluoromethyl)phenyl]methyl}piperazin-2-one (11).
A soln. of 10 (10.5 mg,
0.027 mmol) in 3.2m HCl/AcOEt (0.5 ml) was stirred at 258 for 1 h before solvent and excess acid were
removed under a stream of N2. The residue was dried under high vacuum for 5 h, and treated with a soln. of
EtN(i-Pr)2 in DMF (0.1m, 0.35 ml, 0.035 mmol) and a soln. of PhCH2Br in DMF (0.1m, 0.29 ml, 0.029 mmol) and
stirred at 258 for 17 h. The mixture was extracted with AcOEt (10 ml) and washed with H2O (2 Â 5 ml) and sat.
aq. NaCl soln. (5 ml), dried (MgSO4), and evaporated to afford 11 (9.8 mg, 96%). 1H-NMR (CDCl3): 7.58 ± 7.18
(m, 9 H); 5.29 (d, J ˆ 15.4, 1 H); 4.05 (d, J ˆ 15.4, 1 H); 3.64 (d, J ˆ 13.0, 1 H); 3.48 (d, J ˆ 16.5, 1 H); 3.44
(d, J ˆ 13.0, 1 H); 3.07 (d, J ˆ 16.5, 1 H); 3.03 (m, 1 H); 2.75 (br. d, J ˆ 11.9, 1 H); 2.37 (dd, J ˆ 11.9, 3.8, 1 H);
1.84 (m, 1 H); 1.63 (m, 1 H); 0.70 (t, J ˆ 7.3, 3 H). IR: 2961, 2930, 1648, 1463, 1448, 1427, 1327, 1158, 1121, 1074,
‡
‡
916, 800, 747, 694. HR-MS: 377.1844 ([M ‡ H] , C21H23F3N2O ; calc. 377.1841).
6-Ethyl-4-(4-methoxybenzyl)-1-{[3-(trifluoromethyl)phenyl]methyl}piperazin-2-one (12). A soln. of 10
(7.5 mg, 0.020 mmol) in 3.2m HCl/AcOEt (0.5 ml) was stirred at 258 for 1 h before the solvent and excess acid
were removed under a stream of N2. The resulting residue was dissolved in 1,2-dichloroethane (2.0 ml) and
treated with a soln. of 4-methoxybenzaldehyde in 1,2-dichloroethane (0.1m, 0.23 ml, 0.023 mmol), AcOH
(2.3 ml, 0.040 mmol), and NaBH(OAc)3 (11 mg, 0.053 mmol), and stirred at 258 for 40 h. The mixture was
extracted with AcOEt (10 ml) and washed with sat. aq. NaHCO3 (3 Â 5 ml) and sat. aq. NaCl soln. (5 ml), dried
(MgSO4), and concentrated to afford 12 (8.0 mg, 100%). 1H-NMR (CDCl3): 7.57 ± 7.37 (m, 4 H); 7.20 (d, J ˆ 8.4,
2 H); 6.84 (d, J ˆ 8.4, 2 H); 5.27 (d, J ˆ 15.4, 1 H); 4.04 (d, J ˆ 15.4, 1 H); 3.78 (s, 3 H); 3.55 (d, J ˆ 12.7, 1 H);
3.44 (d, J ˆ 16.5, 1 H); 3.35 (d, J ˆ 12.7, 1 H); 3.06 (d, J ˆ 16.5, 1 H); 2.99 (m, 1 H); 2.72 (br. d, J ˆ 11.6, 1 H);
2.33 (dd, J ˆ 11.6, 3.8, 1 H); 1.80 (m, 1 H); 1.60 (m, 1 H); 0.69 (t, J ˆ 7.3, 3 H). IR: 2919, 1649, 1508, 1461, 1326,
‡
‡
1243, 1161, 1114, 1007, 1032, 814, 703. HR-MS: 407.1942 ([M ‡ H] , C22H25F3N2O2 ; calc. 407.1946).
2-([(tert-Butoxy)carbonyl]{2-[(4-methoxybenzyl)amino]-2-oxoethyl}amino)acetic Acid (13). A soln. of N-
Boc-iminodiacetic acid (1, 500 mg, 2.15 mmol) in DMF (6.5 ml) was treated with EDCI (433 mg, 2.25 mmol)
and stirred at 258 for 1 h. 4-Methoxybenzylamine (300 mg, 2.15 mmol) was added, and the mixture stirred for an
additional 20 h, and then poured into a separatory funnel containing 10% aq. HCl (50 ml) and extracted with
AcOEt (100 ml). The org. phase was washed with 10% aq. HCl (2 Â 50 ml) and sat. aq. NaCl soln. (50 ml), dried
(MgSO4), and concentrated to afford 13 (704 mg, 93%). Amorphous solid: 1H-NMR (CDCl3): 7.41 (br. s, 1 H);
7.23 ± 7.14 (m, 2 H); 6.91 ± 6.78 (m, 2 H); 6.54 (m, 1 H); 4.46 ± 4.35 (m, 2 H); 4.03 ± 3.91 (m, 3 H); 3.88 (br. s,
1 H); 3.78, 3.78 (s, 2 H); 3.77 (s, 1 H); 1.43 (s, 6 H); 1.33 (s, 3 H).
4-[(tert-Butoxy)carbonyl]-1-[(4-methoxyphenyl)methyl]piperazine-2,6-dione (14). A soln. of 13 (253 mg,
0.73 mmol) in THF (5.0 ml) was treated with 1,1'-carbonyldiimidazole (CDI; 472 mg, 2.90 mmol) and stirred at
258 for 18 h. The solvent was removed in vacuo, and the residue was dissolved in AcOEt (30 ml) and washed
sequentially with 10% aq. HCl (2 Â 10 ml), sat. aq. NaCl (10 ml), sat. aq. NaHCO3 (2 Â 10 ml), and sat. aq. NaCl
(10 ml) solns. The org. phase was dried (MgSO4) and concentrated to afford 14 (214 mg, 90%). 1H-NMR
(CDCl3): 7.34 (d, J ˆ 8.8, 2 H); 6.81 (d, J ˆ 8.8, 2 H); 4.87 (s, 2 H); 4.30 (s, 4 H); 3.76 (s, 3 H); 1.44 (s, 9 H). HR-
‡
‡
MS: 357.1438 ([M ‡ Na] , C17H22N2O5 ; calc. 357.1426).
2-{[(tert-Butoxy)carbonyl][2-(4-methylphenyl)-2-oxoethyl]amino}-N-(4-methoxybenzyl)acetamide (15).
A soln. of 14 (161 mg, 0.48 mmol) in THF (3.2 ml) was cooled to 08 and treated with 4-toluoylmagnesium

Helvetica Chimica Acta ± Vol. 83 (2000)
1841
bromide (1.0m in Et2O, 0.72 ml, 0.72 mmol). The mixture was stirred for 2 h, warmed to 258 for 1 h, and then
cooled back to 08 and quenched by addition of sat. aq. NH4Cl (3 ml) soln. The mixture was extracted with
AcOEt (30 ml) and washed with H2O (2 Â 10 ml) and sat. aq. NaCl soln. (10 ml). The org. phase was dried
(MgSO4), concentrated under reduced pressure, and FC (SiO2; 0 ± 2% MeOH/CHCl3) afforded 15 (44 mg,
21%). Colorless oil: 1H-NMR (CDCl3): 8.71 (m, 1 H); 8.18 (m, 1 H); 7.80 (d, J ˆ 8.4, 2 H); 7.75 (d, J ˆ 8.1, 1 H);
7.38 ± 7.15 (m, 2 H); 6.84 (d, J ˆ 8.4, 2 H); 6.79 (d, J ˆ 8.1, 1 H); 4.68 (br. s, 2 H); 4.59 (br. s, 1 H); 4.48 ± 4.37
(m, 2 H); 4.01 (br. s, 1 H); 3.90 (br. s, 1 H); 3.77 (s, 2 H); 3.75 (s, 1 H); 2.41 (s, 1 H); 2.40 (s, 2 H); 1.39 (s, 6 H);
‡
‡
1.29 (s, 3 H). HR-MS: 427.2219 ([M ‡ H] , C24H30N2O5 ; calc. 427.2233).
2-{[(tert-Butoxy)carbonyl][2-hydroxy-2-(4-methylphenyl)ethyl]amino}-N-(4-methoxybenzyl)acetamide (17).
A soln. of 15 (41.7 mg, 0.098 mmol) in MeOH/THF 1:1 (1 ml) was cooled to 08, treated with NaBH4 (4.0 mg,
0.10 mmol), warmed to 258, and stirred for 1.5 h. The mixture was diluted with sat. aq. NaHCO3 soln. (10 ml)
and extracted with AcOEt (25 ml). The org. phase was washed with H2O (25 ml) and sat. aq. NaCl soln. (25 ml),
dried (MgSO4), and concentrated. FC (SiO2; 50% AcOEt/hexanes) provided 17 (25 mg, 60%). Viscous oil:
1H-NMR (CDCl3): 7.32 ± 7.18 (m, 2 H); 7.20 (d, J ˆ 8.4, 2 H); 7.14 (d, J ˆ 8.1, 2 H); 6.88 (d, J ˆ 8.4, 2 H); 6.77
(br. s, 0.5 H); 6.36 (br. s, 0.5 H); 5.00 ± 4.89 (m, 1 H); 4.50 ± 4.28 (m, 2 H); 4.10 ± 3.95 (m, 1 H); 3.92 ± 3.60
(m, 2 H); 3.77 (s, 3 H); 3.56 ± 3.27 (m, 1 H); 3.19 ± 3.04 (m, 0.5 H); 2.32 (s, 3 H); 1.47 (br. s, 4.5 H); 1.36 (br. s,
‡
‡
4.5 H). HR-MS: 429.2379 ([M ‡ H] , C24H32N2O5 ; calc. 427.2389).
General Procedure for the Final Coupling Reaction: 6-Ethyl-4-(4-methoxybenzoyl)-1-[(4-methoxyphenyl)-
methyl]piperazin-2-one (33, A1B1C1). A portion of 8 (80.3 mg, 0.276 mmol) was treated with 3.2m HCl/AcOEt
(1 ml) and stirred at 258 for 1 h before the solvent and excess acid were removed under a stream of N2, followed
by high vacuum for 4 h. The resulting residue was dissolved in DMF (1 ml) and treated with 4-methoxybenzoic
acid (41.9 mg, 0.276 mmol), EtN(i-Pr)2 (53 ml, 0.30 mmol), and EDCI (53 mg, 0.276 mmol), and stirred at 258
for 16 h. The mixture was extracted with AcOEt (30 ml) and washed with 10% aq. HCl (3 Â 10 ml), sat. aq.
NaHCO3 (3 Â 10 ml), and sat. aq. NaCl (10 ml) solns. Drying (MgSO4) and evaporation afforded 33, A1B1C1
(80 mg,76%). Colorless viscous oil. IR: 2963, 2926, 1639, 1606, 1507, 1423, 1241, 1170, 1030, 842. 1H-NMR
(CDCl3): 7.35 (d, J ˆ 8.6, 2 H); 7.16 (d, J ˆ 8.6, 2 H); 6.90 (d, J ˆ 8.6, 2 H); 6.84 (d, J ˆ 8.6, 2 H); 5.32 (d, J ˆ 14.6,
1 H); 4.91 ± 4.00 (m, 3 H); 3.82 (s, 3 H); 3.80 (d, J ˆ 14.6, 1 H); 3.32 ± 2.96 (m, 2 H); 1.77 ± 1.38 (m, 2 H); 1.14 ±
‡
‡
0.45 (m, 3 H). HR-MS: 383.1974 ([M ‡ H] , C22H26N2O4 ; calc. 383.1971).
4-(4-Bromobenzoyl)-6-ethyl-1-[(4-methoxyphenyl)methyl]piperazin-2-one (9). Yield 89%. 1H-NMR
(CDCl3): 7.83 (d, J ˆ 7.8, 1 H); 7.81 (d, J ˆ 7.3, 1 H); 7.26 (d, J ˆ 7.8, 1 H); 7.23 (d, J ˆ 7.3, 1 H); 4.81 (s, 1 H);
4.69 (s, 1 H); 4.15 (s, 1 H); 4.04 (s, 1 H); 3.713 (s, 1.5 H); 3.708 (s, 1.5 H); 2.40 (s, 1.5 H); 2.39 (s, 1.5 H); 1.43
‡
‡
(s, 4.5 H); 1.36 (s, 4.5 H). HR-MS: 431.0979 ([M ‡ H] , C21H23BrN2O3 ; calc. 431.0970).
6-Ethyl-4-(4-methylbenzoyl)-1-[(4-methylphenyl)methyl]piperazin-2-one (33, A1B2C2). IR: 2954, 2923,
1646, 1631, 1415, 1256, 831. 1H-NMR (CDCl3): 7.35 ± 7.05 (m, 8 H); 5.35 (d, J ˆ 14.6, 1 H); 5.03 ± 4.15 (m, 2 H);
4.07 (d, J ˆ 18.4, 1 H); 3.81 (d, J ˆ 14.6, 1 H); 3.45 ± 2.84 (m, 2 H); 2.36 (s, 3 H); 2.32 (s, 3 H); 1.85 ± 1.35
‡
‡
(m, 2 H); 1.16 ± 0.39 (m, 3 H). HR-MS: 351.2076 ([M ‡ H] , C22H26N2O2 ; calc. 351.2073).
4-(4-Chlorobenzoyl)-1-[(4-chlorophenyl)methyl]-6-ethylpiperazin-2-one (33, A1B3C3). IR: 2971, 2930,
1645, 1424, 1255, 1157, 1091, 1014. 1H-NMR (CDCl3): 7.40 (d, J ˆ 8.4, 2 H); 7.33 (d, J ˆ 8.4, 2 H); 7.28 (d, J ˆ 8.4,
2 H); 7.17 (d, J ˆ 8.4, 2 H); 5.28 (d, J ˆ 14.8, 1 H); 5.03 ± 4.01 (m, 3 H); 3.88 (d, J ˆ 14.8, 1 H); 3.84 ± 2.79
‡
‡
(m, 2 H); 1.87 ± 1.37 (m, 2 H); 1.22 ± 0.44 (m, 3 H). HR-MS: 391.0982 ([M ‡ H] , C20H20Cl2N2O2 ; calc.
391.0980).
6-Ethyl-4-[(1H-pyrrol-2-yl)carbonyl]-1-{[(3-(trifluoromethyl)phenyl]methyl}piperazin-2-one (33, A1B4C4).
IR: 3271, 2967, 2938, 1657, 1643, 1605, 1429, 1325, 1163, 1120, 1068. 1H-NMR (CDCl3): 9.56 (br. s, 1 H); 7.62 ±
7.35 (m, 4 H); 6.96 (br. s, 1 H); 6.66 (br. s, 1 H); 6.29 (m, 1 H); 5.35 (d, J ˆ 15.1, 1 H); 4.81 (d, J ˆ 17.6, 1 H);
4.75 ± 4.55 (m, 1 H); 4.48 ± 4.29 (m, 1 H); 4.05 (d, J ˆ 15.1, 1 H); 3.28 ± 3.09 (m, 2 H); 1.79 ± 1.43 (m, 2 H); 0.94
‡
‡
(t, J ˆ 7.0, 3 H). HR-MS: 380.1589 ([M ‡ H] , C19H20F3N3O2 ; calc. 380.1586).
6-Ethyl-4-[(furan-2-yl)carbonyl]-1-[(furan-2-yl)methyl]piperazin-2-one (33, A1B5C5). IR: 2965, 2930,
1658, 1641, 1483, 1426, 1277, 1189, 1013, 755. 1H-NMR (CDCl3): 7.50 (br. s, 1 H); 7.35 (d, J ˆ 1.1, 1 H); 7.11
(d, J ˆ 3.5, 1 H); 6.50 (dd, J ˆ 3.5, 1.9, 1 H); 6.32 (dd, J ˆ 3.0, 1.9, 1 H); 6.29 (d, J ˆ 3.2, 1 H); 5.15 (d, J ˆ 15.4,
1 H); 4.93 ± 4.70 (m, 1 H); 4.66 (br. d, J ˆ 13.2, 1 H); 4.47 ± 4.15 (m, 1 H); 4.10 (d, J ˆ 15.4, 1 H); 3.61 ± 2.95
‡
‡
(m, 2 H); 1.81 ± 1.42 (m, 2 H); 1.12 ± 0.71 (m, 3 H). HR-MS: 303.1339 ([M ‡ H] , C16H18N2O4 ; calc. 303.1345).
6-Ethyl-1-[(4-methoxyphenyl)methyl]-4-[(thiophen-2-yl)carbonyl]piperazin-2-one (33, A1B1C6). IR:
2965, 2950, 1649, 1619, 1513, 1426, 1241, 1176, 1031, 991, 847, 820, 733. 1H-NMR (CDCl3): 7.48 (dd, J ˆ 5.0,
0.8, 1 H); 7.35 (br. d, J ˆ 3.5, 1 H); 7.17 (d, J ˆ 8.6, 2 H); 7.05 (dd, J ˆ 5.0, 3.5, 1 H); 6.85 (d, J ˆ 8.6, 2 H); 5.32
(d, J ˆ 14.6, 1 H); 4.70 (d, J ˆ 18.1, 1 H); 4.60 ± 4.38 (m, 1 H); 4.36 ± 4.15 (m, 1 H); 3.83 (d, J ˆ 14.6, 1 H); 3.78

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Helvetica Chimica Acta ± Vol. 83 (2000)
‡
(s, 3 H); 3.29 ± 3.06 (m, 2 H); 1.76 ± 1.44 (m, 2 H); 0.98 ± 0.76 (m, 3 H). HR-MS: 359.1425 ([M ‡ H] ,
‡
C19H22N2OS ; calc. 359.1429).
6-Ethyl-4-[(1H-indol-2-yl)carbonyl]-1-[(4-methylphenyl)methyl]piperazin-2-one (33, A1B2C7). IR: 3281,
2962, 2926, 1638, 1608, 1428, 1411, 1345, 1244, 1161, 1139, 1073, 981, 911, 805. 1H-NMR (CDCl3): 9.28 (br. s,
1 H); 7.66 (br. d, J ˆ 7.8, 1 H); 7.41 (d, J ˆ 8.4, 1 H); 7.19 ± 7.05 (m, 5 H); 6.91 (br. s, 1 H); 5.38 (d, J ˆ 14.6, 1 H);
5.00 ± 4.80 (m, 1 H); 4.79 ± 4.61 (m, 1 H); 4.60 ± 4.25 (m, 1 H); 3.88 (d, J ˆ 14.6, 1 H); 3.31 ± 3.12 (m, 2 H); 2.34
‡
‡
(s, 3 H); 1.79 ± 1.41 (m, 2 H); 1.08 ± 0.75 (m, 3 H). HR-MS: 376.2029 ([M ‡ H] , C23H25N3O2 ; calc. 376.2025).
4-[([1]Benzofuran-2-yl)carbonyl]-1-[(4-chlorophenyl)methyl]-6-ethylpiperazin-2-one (33, A1B3C8). IR:
2962, 2927, 2874, 2656, 2634, 1424, 1411, 1292, 1253, 1231, 1173, 1090, 1011, 914, 800, 739. 1H-NMR (CDCl3): 7.66
(d, J ˆ 7.8, 1 H); 7.51 (br. s, 1 H); 7.45 (s, 1 H); 7.42 (t, J ˆ 7.6, 1 H); 7.34 ± 7.15 (m, 5 H); 5.31 (br. d, J ˆ 14.6,
1 H); 5.13 ± 4.78 (d, J ˆ 13.0, 1 H); 4.64 ± 4.31 (m, 1 H); 3.95 (d, J ˆ 14.6, 1 H); 3.62 ± 2.96 (m, 2 H); 1.81 ± 1.49
‡
‡
(m, 2 H); 1.08 ± 0.79 (m, 3 H). HR-MS: 397.1316 ([M ‡ H] , C22H21ClN2O3 ; calc. 397.1319).
4-[([1]Benzothiophen-2-yl)carbonyl]-6-ethyl-1-{[3-(trifluoromethyl)phenyl]methyl}piperazine (33, A1B4C9).
IR: 2969, 2933, 1655, 1638, 1410, 1322, 1283, 1243, 1164, 1120, 1063, 993, 752, 704. 1H-NMR (CDCl3): 7.89 ± 7.72
(m, 2 H); 7.61 ± 7.30 (m, 7 H); 5.37 (d, J ˆ 15.1, 1 H); 4.79 (d, J ˆ 18.1, 1 H); 4.69 ± 4.19 (m, 2 H); 4.03 (d, J ˆ
‡
15.1, 1 H); 3.47 ± 3.04 (m, 2 H); 1.81 ± 1.45 (m, 2 H); 1.05 ± 0.67 (m, 3 H). HR-MS: 447.1341 ([M ‡ H] ,
‡
C23H21F3N2O2S ; calc. 447.1354).
6-Ethyl-1-[(furan-2-yl)methyl]-4-(3-phenylprop-2-enoyl)piperazin-2-one (33, A1B5C10). IR: 2969, 2933,
1
2872, 1649, 1610, 1427, 1357, 1331, 1278, 1225, 1199, 1155, 1068, 1006, 980, 765. H-NMR (CDCl3): 7.72 (d, J ˆ
15.1, 1 H); 7.51 (br. s, 1 H); 7.43 ± 7.28 (m, 4 H); 6.78 (d, J ˆ 15.1, 1 H); 6.36 ± 6.32 (m, 1 H); 6.30 (d, J ˆ 3.2, 1 H);
5.14 (d, J ˆ 15.1, 1 H); 4.72 (d, J ˆ 13.0, 1 H); 4.49 (d, J ˆ 17.6, 0.7 H); 4.21 (d, J ˆ 17.6, 1 H); 4.11 (d, J ˆ 15.1,
1 H); 3.97 (d, J ˆ 17.6, 0.3 H); 3.52 ± 3.40 (m, 0.3 H); 3.34 (m, 1 H); 3.04 (br. d, J ˆ 13.2, 0.7 H); 1.78 ± 1.35
‡
‡
(m, 2 H); 0.98 (br. t, J ˆ 7.3, 3 H). HR-MS: 339.1706 ([M ‡ H] , C20H22N2O3 ; calc. 339.1709).
6-Benzyl-4-(4-methoxybenzoyl)-[(4-methoxyphenyl)methyl]piperazin-2-one (33, A2B1C1). IR: 2925,
1
1655, 1607, 1423, 1296, 1243, 1173, 1023, 958, 844, 756, 699. H-NMR (CDCl3): 7.40 (d, J ˆ 8.6, 2 H); 7.35 ± 7.10
(m, 5 H); 7.16 (d, J ˆ 8.6, 2 H); 6.96 ± 6.80 (m, 2 H); 6.85 (d, J ˆ 8.6, 2 H); 5.36 (d, J ˆ 14.9, 1 H); 4.68 ± 4.16
(m, 1 H); 4.09 (d, J ˆ 18.3, 1 H); 3.85 (m, 1 H); 3.82 (s, 3 H); 3.78 (s, 3 H); 3.71 (m, 1 H); 3.49 ± 3.37 (m, 1 H);
‡
‡
3.25 ± 2.55 (m, 3 H). HR-MS: 445.2114 ([M ‡ H] , C27H28N2O4 ; calc. 445.2127).
6-Benzyl-4-(4-methylbenzoyl)-1-[(4-methylphenyl)methyl]piperazin-2-one (33, A2B2C2). IR: 2916, 1655,
1638, 1427, 1414, 1252, 1212, 1182, 1155, 1076, 1024, 826, 747, 704. 1H-NMR (CDCl3): 7.45 ± 6.90 (m, 13 H); 5.39
(d, J ˆ 14.6, 1 H); 5.01 ± 3.93 (m, 3 H); 3.90 ± 3.11 (m, 2 H); 3.10 ± 2.50 (m, 2 H); 2.36 (s, (3 H); 2.31 (s, 3 H).
‡
‡
HR-MS: 413.2217 ([M ‡ H] , C27H28N2O2 ; calc. 413.2229).
6-Benzyl-4-(4-chlorobenzoyl)-1-[(4-chlorophenyl)methyl]piperazin-2-one (33, A2B3C3). IR: 2927, 1655,
1638, 1427, 1410, 1256, 1155, 1090, 1011, 840, 730. 1H-NMR (CDCl3): 7.51 ± 6.98 (m, 13 H); 5.35 (d, J ˆ 15.1,
‡
1 H); 4.99 ± 3.95 (m, 3 H); 3.98 ± 3.15 (m, 2 H); 3.12 ± 2.44 (m, 3 H). HR-MS: 453.1127 ([M ‡ H] ,
‡
C25H22Cl2N2O2 ; calc. 453.1137).
6-Benzyl-4-[(1H-pyrrol-2-yl)carbonyl]-1-{[3-(trifluoromethyl)phenyl]methyl}piperazin-2-one (33, A2B4C4).
1
IR: 3275, 2915, 1655, 1642, 1427, 1326, 1164, 1124, 1072, 966. H-NMR (CDCl3): 9.53 (br. s, 0.4 H); 7.61 ± 6.94
(m, 11.4 H); 6.58 (br. s, 0.6 H); 6.28 (br. s, 0.6 H); 5.44 (d, J ˆ 15.1, 0.4 H); 5.39 (d, J ˆ 15.1, 0.6 H); 4.84 (d, J ˆ
18.1, 0.6 H); 4.70 ± 4.29 (m, 2.4 H); 4.03 (d, J ˆ 15.1, 0.4 H); 3.81 (d, J ˆ 15.1, 0.6 H); 3.46 (m, 0.6 H); 3.32
‡
‡
(m, 0.4 H); 3.20 ± 2.68 (m, 3 H). HR-MS: 442.1728 ([M ‡ H] , C24H22F3N3O2 ; calc. 442.1742).
6-Benzyl-3-[(furan-2-yl)carbonyl]-1-[(furan-2-yl)methyl]piperazin-2-one (33, A2B5C5). IR: 3117, 2924,
1659, 1642, 1480, 1427, 1352, 1326, 1273, 1225, 1190, 1146, 1059, 1010, 888. 1H-NMR (CDCl3): 7.59 ± 6.83
(m, 8 H); 6.49 (br. s, 1 H); 6.38 ± 6.26 (m, 2 H); 5.17 (d, J ˆ 15.4, 1 H); 4.87 (br. d, J ˆ 18.4, 1 H); 4.70 ± 4.00
‡
‡
(m, 2 H); 4.06 (d, J ˆ 15.4, 1 H); 3.63 (m, 1 H); 3.28 ± 2.53 (m, 3 H). HR-MS: 365.1508 ([M ‡ H] , C21H20N2O4
;
calc. 365.1501).
6-Benzyl-1-[(4-methoxyphenyl)methyl][(thiophen-2-yl)carbonyl]piperazin-2-one (33, A2B1C6). IR: 2933,
1651, 1611, 1510, 1428, 1361, 1248, 1171, 1028, 910, 853, 735. 1H-NMR (CDCl3): 7.49 (d, J ˆ 4.3, 1 H); 7.37 ± 6.94
(m, 9 H); 6.85 (d, J ˆ 8.6, 2 H); 5.35 (d, J ˆ 14.8, 1 H); 4.74 (br. d, J ˆ 18.4, 1 H); 4.56 ± 4.31 (m, 1 H); 4.24
(br. d, J ˆ 18.4, 1 H); 3.79 (s, 3 H); 3.75 (d, J ˆ 14.8, 1 H); 3.47 (m, 1 H); 3.21 ± 3.03 (m, 1 H); 2.90 (dd, J ˆ 13.5,
‡
‡
4.3, 1 H); 2.72 (m, 1 H). HR-MS: 421.1594 ([M ‡ H] , C24H24N2O3S ; calc. 421.1586).
6-Benzyl-4-[(1H-indol-2-yl)carbonyl]-1-[(4-methylphenyl)methyl]piperazin-2-one (33, A2B2C7). IR:
3278, 3014, 2921, 1650, 1614, 1516, 1424, 1336, 1316, 1244, 1127, 1085, 808, 747, 705. 1H-NMR (CDCl3): 9.34
(br. s, 1 H); 7.63 (br. s, 1 H); 7.43 (d, J ˆ 8.4, 1 H); 7.31 (t, J ˆ 8.4, 1 H); 7.29 ± 7.00 (m, 11 H); 5.41 (d, J ˆ 14.6,
1 H); 4.90 (br. d, J ˆ 17.8, 1 H); 4.74 ± 4.17 (m, 2 H); 3.78 (d, J ˆ 14.6, 1 H); 3.38 ± 2.98 (br. s, 1 H); 2.92 (dd, J ˆ
‡
‡
13.5, 4.0, 1 H); 2.84 ± 2.63 (m, 1 H); 2.33 (s, 3 H). HR-MS: 438.2188 ([M ‡ H] , C28H27N3O2 ; calc. 438.2182).

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4-[([1]Benzofuran-2-yl)carbonyl]-6-benzyl-1-[(4-chlorophenyl)methyl]piperazin-2-one (33, A2B3C8).
IR: 3024, 2921, 1649, 1559, 1490, 1474, 1428, 1255, 1175, 1255, 1175, 1090, 1015, 963, 912, 803, 742, 701.
1H-NMR (CDCl3): 7.66 (d, J ˆ 7.6, 1 H); 7.61 ± 6.70 (m, 14 H); 5.38 (d, J ˆ 14.8, 1 H); 5.17 ± 4.80 (m, 1 H); 4.75 ±
‡
4.05 (m, 2 H); 3.90 ± 3.69 (m, 1 H); 3.59 ± 3.30 (m, 1 H); 3.15 ± 2.59 (m, 3 H). HR-MS: 459.1480 ([M ‡ H] ,
‡
C27H23ClN2O3 ; calc. 459.1475).
4-[([1]Benzothiophen-2-yl)carbonyl]-6-benzyl-1-{[3-(trifluoromethyl)phenyl]methyl}piperazin-2-one (33,
A2B4C9). IR: 3066, 2921, 1650, 1624, 1522, 1419, 1326, 1244, 1198, 1162, 1121, 1075, 752, 695. 1H-NMR (CDCl3):
7.86 (d, J ˆ 7.6, 1 H); 7.77 (d, J ˆ 6.7, 1 H); 7.60 ± 6.93 (m, 12 H); 5.43 (d, J ˆ 15.0, 1 H); 4.84 (br. d, J ˆ 18.4, 1 H);
4.65 ± 4.38 (m, 1 H); 4.33 (br. d, J ˆ 18.4, 1 H); 3.83 (d, J ˆ 15.0, 1 H); 3.48 (m, 1 H); 3.36 ± 3.15 (m, 1 H); 3.07 ±
‡
‡
2.69 (m, 2 H). HR-MS: 641.0498 ([M ‡ Cs] , C28H23F3N2O2S ; calc. 641.0487).
6-Benzyl-1-[(furan-2-yl)methyl]-4-(3-phenylprop-2-enoyl)piperazin-2-one (33, A2B5C10). IR: 3024, 2932,
1650, 1609, 1496, 1450, 1424, 1337, 1224, 1157, 1085, 1070, 1008, 972, 762, 736, 695. 1H-NMR (CDCl3): 7.76 (d, J ˆ
15.1, 1 H); 7.52 (br. s, 1 H); 7.44 ± 7.00 (m, 10 H); 6.77 (d, J ˆ 15.1, 0.5 H); 6.49 (d, J ˆ 15.1, 0.5 H); 6.41 ± 6.26
(m, 2 H); 5.18 (d, J ˆ 15.4, 1 H); 4.85 (br. d, J ˆ 18.4, 0.5 H); 4.68 (br. d, J ˆ 13.5, 0.5 H); 4.46 (br. d, J ˆ 17.6,
0.5 H); 4.29 ± 3.80 (m, 2.5 H); 3.78 ± 3.50 (m, 1 H); 3.42 ± 3.20 (m, 0.5 H); 3.11 ± 2.58 (m, 2.5 H). HR-MS:
‡
‡
401.1854 ([M ‡ H] , C25H24N2O3 ; calc. 401.1865).
4-(4-Methoxybenzoyl)-[(4-methoxyphenyl)methyl]-6-(2-phenylethyl)piperazin-2-one (33, A3B1C1). IR:
2932, 1640, 1609, 1511, 1455, 1419, 1301, 1244, 1172, 1116, 1070, 1029, 993, 844, 762, 700. 1H-NMR (CDCl3): 7.40
(d, J ˆ 8.6, 2 H); 7.34 ± 7.00 (m, 5 H); 6.95 (d, J ˆ 8.6, 2 H); 6.92 (d, J ˆ 8.6, 2 H); 6.76 (d, J ˆ 8.6, 2 H); 5.24
(d, J ˆ 14.6, 1 H); 5.02 ± 4.19 (m, 2 H); 4.16 ± 1.01 (m, 1 H); 3.83 (s, 3 H); 3.78 (s, 3 H); 3.64 (d, J ˆ 14.6, 1 H);
‡
‡
3.33 ± 2.28 (m, 4 H); 1.98 ± 1.71 (m, 2 H). HR-MS: 459.2276 ([M ‡ H] , C28H30N2O4 ; calc. 459.2284).
4-(4-Methylbenzoyl)-1-[(4-methylphenyl)methyl]-6-(2-phenylethyl)piperazin-2-one (33, A3B2C2). IR:
1
3024, 2921, 2870, 1642, 1422, 1256, 1073, 830, 750, 701. H-NMR (CDCl3): 7.39 ± 7.02 (m, 9 H); 7.05 (d, J ˆ 7.8,
2 H); 6.91 (d, J ˆ 7.8, 2 H); 5.25 (d, J ˆ 14.6, 1 H); 4.55 ± 3.98 (m, 3 H); 3.66 (d, J ˆ 14.6, 1 H); 3.33 ± 2.44
‡
(m, 2 H); 2.41 ± 2.28 (m, 2 H); 2.37 (s, 3 H); 2.30 (s, 3 H); 1.97 ± 1.74 (m, 2 H). HR-MS: 427.2377 ([M ‡ H] ,
‡
C28H30N2O2 ; calc. 427.2386).
4-(4-Chlorobenzoyl)-1-[(4-chlorophenyl)methyl]-6-(2-phenylethyl)piperazin-2-one (33, A3B3C3). IR:
2929, 1644, 1595, 1491, 1426, 1255, 1155, 1090, 1015, 840, 753, 701. 1H-NMR (CDCl3): 7.40 (d, J ˆ 8.4, 2 H);
7.38 ± 7.02 (m, 5 H); 7.35 (d, J ˆ 8.4, 2 H); 7.19 (d, J ˆ 8.4, 2 H); 6.91 (d, J ˆ 8.4, 2 H); 5.20 (d, J ˆ 14.6, 1 H);
4.96 ± 4.63 (m, 1 H); 4.41 ± 3.98 (m, 1 H); 4.06 (br. d, J ˆ 18.4, 1 H); 3.64 (d, J ˆ 14.6, 1 H); 3.39 ± 2.21 (m, 4 H);
‡
‡
1.97 ± 1.71 (m, 2 H). HR-MS: 467.1296 ([M ‡ H] , C26H24Cl2N2O2 ; calc. 467.1293).
6-(2-Phenylethyl)-4-[(1H-pyrrol-2-yl)carbonyl]-1-{[3-(trifluoromethyl)phenyl]methyl}piperazin-2-one (33,
A3B4C4). IR: 3270, 2932, 1650, 1604, 1450, 1429, 1321, 1162, 1121, 1075, 747, 700. 1H-NMR (CDCl3): 9.54 (br. s,
1 H); 7.52 (d, J ˆ 7.6, 1 H); 7.47 ± 7.07 (m, 6 H); 7.39 (t, J ˆ 7.6, 1 H); 7.11 (d, J ˆ 7.0, 1 H); 6.99 (br. s, 1 H); 6.67
(br. s, 1 H); 6.32 (m, 1 H); 5.25 (d, J ˆ 14.8, 1 H); 4.86 (d, J ˆ 17.8, 1 H); 4.81 (br. d, J ˆ 17.8, 1 H); 3.82 (d, J ˆ
‡
14.8, 1 H); 3.28 ± 3.01 (m, 2 H); 2.94 ± 2.43 (m, 2 H); 1.97 ± 1.81 (m, 2 H). HR-MS: 456.1910 ([M ‡ H] ,
‡
C25H24F3N3O2 ; calc. 456.1899).
4-[(Furan-2-yl)carbonyl]-1-[(furan-2-yl)methyl]-6-(2-phenylethyl)piperazin-2-one (33, A3B5C5). IR:
2921, 1650, 1624, 1473, 1422, 1273, 1186, 1063, 1006, 755. 1H-NMR (CDCl3): 7.52 (br. s, 1 H); 7.36 ± 6.95
(m, 7 H); 6.52 (dd, J ˆ 3.5, 1.9, 1 H); 6.27 (dd, J ˆ 3.2, 1.9, 1 H); 6.09 (d, J ˆ 3.2, 1 H); 5.07 (d, J ˆ 15.4, 1 H);
4.94 ± 4.64 (m, 2 H); 4.45 ± 4.16 (m, 1 H); 4.03 (d, J ˆ 15.4, 1 H); 3.42 ± 2.36 (m, 4 H); 1.95 ± 1.75 (m, 2 H). HR-
‡
‡
MS: 379.1664 ([M ‡ H] , C22H22N2O4 ; calc. 379.1658).
1-[(4-Methoxyphenyl)methyl]-6-(2-phenylethyl)-4-[(thiophen-2-yl)carbonyl]piperazin-2-one (33, A3B1C6).
IR: 2927, 1648, 1617, 1514, 1453, 1427, 1355, 1288, 1242, 1176, 1032, 980, 842, 739, 703. H-NMR (CDCl3): 7.51
1
(br. d, J ˆ 5.2, 1 H); 7.38 (br. d, J ˆ 3.2, 1 H); 7.34 ± 7.02 (m, 5 H); 7.09 (dd, J ˆ 4.9, 3.8, 1 H); 6.94 (d, J ˆ 8.6,
2 H); 6.77 (d, J ˆ 8.6, 2 H); 5.23 (d, J ˆ 14.6, 1 H); 4.82 ± 4.52 (m, 1 H); 4.73 (br. d, J ˆ 17.8, 1 H); 4.37 ± 4.17
(m, 1 H); 3.78 (s, 3 H); 3.66 (d, J ˆ 14.6, 1 H); 3.33 ± 2.98 (m, 2 H); 2.95 ± 2.30 (m, 2 H); 1.97 ± 1.80 (m, 2 H).
‡
‡
HR-MS: 435.1728 ([M ‡ H] , C25H26N2O3S ; calc. 435.1742).
4-[(1H-Indol-2-yl)carbonyl]-1-[(4-methylphenyl)methyl]-6-(2-phenylethyl)piperazin-2-one (33, A3B2C7).
IR: 3276, 2927, 1653, 1619, 1514, 1453, 1427, 1340, 1314, 1247, 1140, 1078, 970, 909, 806, 744, 703. 1H-NMR
(CDCl3): 9.26 (br. s, 1 H); 7.68 (d, J ˆ 7.8, 1 H); 7.43 (d, J ˆ 8.4, 1 H); 7.36 ± 7.01 (m, 9 H); 7.00 ± 6.83 (m, 1 H);
6.95 (d, J ˆ 7.8, 2 H); 5.28 (d, J ˆ 14.6, 1 H); 5.08 ± 4.65 (m, 2 H); 4.62 ± 4.15 (m, 1 H); 3.74 (d, J ˆ 14.6, 1 H);
‡
‡
3.38 ± 2.40 (m, 4 H); 2.32 (s, 3 H); 1.99 ± 1.77 (m, 2 H). HR-MS: 452.2343 ([M ‡ H] , C29H29N3O2 ; calc.
452.2338).
4-[([1]Benzofuran-2-yl)carbonyl]-1-[(4-chlorophenyl)methyl]-6-(2-phenylethyl)piperazin-2-one
A3B3C8). IR: 2925, 1651, 1636, 1423, 1251, 1178, 1090, 1017, 809, 747, 700. H-NMR (CDCl3): 7.68 (d, J ˆ 7.8,
(33,
1

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Helvetica Chimica Acta ± Vol. 83 (2000)
1 H); 7.47 (s, 1 H); 7.42 (br. t, J ˆ 7.3, 1 H); 7.36 ± 6.97 (m, 9 H); 6.92 (d, J ˆ 8.4, 2 H); 5.22 (m, 1 H); 5.12 ± 4.71
(m, 2 H); 4.57 ± 4.32 (m, 1 H); 3.68 (d, J ˆ 14.6, 1 H); 3.30 ± 2.31 (m, 4 H); 1.98 ± 1.79 (m, 2 H). HR-MS:
‡
‡
473.1623 ([M ‡ H] , C28H25ClN2O3 ; calc. 473.1632).
4-[([1]Benzothiophen-2-yl)carbonyl]-6-(2-phenylethyl)-1-{[3-(trifluoromethyl)phenyl]methyl}piperazin-2-
one (33, A3B4C9). IR: 2925, 1646, 1631, 1459, 1412, 1324, 1246, 1163, 1116, 1069, 747, 700. 1H-NMR (CDCl3):
7.91 ± 7.76 (m, 2 H); 7.57 (br. s, 1 H); 7.52 (br. d, J ˆ 7.8, 1 H); 7.48 ± 7.34 (m, 4 H); 7.33 ± 6.96 (m, 6 H); 5.26
(d, J ˆ 14.8, 1 H); 4.90 ± 4.50 (m, 1 H); 4.81 (d, J ˆ 18.1, 1 H); 4.44 ± 4.18 (m, 1 H); 3.79 (d, J ˆ 14.8, 1 H); 3.35 ±
‡
‡
3.01 (m, 2 H); 3.00 ± 2.29 (m, 2 H); 1.99 ± 1.84 (m, 2 H). HR-MS: 523.1651 ([M ‡ H] , C29H25F3N2O2S ; calc.
523.1667).
1-[(Furan-2-yl)methyl]-6-(2-phenylethyl)-4-(3-phenylprop-2-enoyl)piperazin-2-one (33, A3B5C10). IR:
2925, 1651, 1610, 1453, 1423, 1282, 1225, 1152, 1095, 1059, 1012, 971, 762, 700. 1H-NMR (CDCl3): 7.75 (d, J ˆ 15.1,
1 H); 7.59 ± 6.95 (m, 11 H); 6.85 ± 6.68 (m, 1 H); 6.34 ± 6.21 (m, 1 H); 6.19 ± 6.02 (m, 1 H); 5.16 ± 4.97 (m, 1 H);
4.92 ± 4.65 (m, 1 H); 4.62 ± 4.37 (m, 1 H); 4.32 ± 4.16 (m, 1 H); 4.15 ± 3.91 (m, 1 H); 3.55 ± 3.25 (m, 1 H); 3.11 ±
‡
‡
2.38 (m, 3 H); 1.98 ± 1.68 (m, 2 H). HR-MS: 415.2018 ([M ‡ H] , C26H26N2O3 ; calc. 415.2022).
The yields for all original library members (33, A1B1C1 ± A3B5C10, 150 compounds) are found in Table 1.
Secondary libraries (33, A1B2C11 ± A1B2C110, and 33, A1B3C111 ± A1B3C210) were prepared from 20
and 21 (on a 6 and 8 mmol scale, resp.) according to the same general procedure described above. The
deprotected secondary amines were coupled to carboxylic acids (C11 ± C95 and C111 ± C196) with EDCI,
EtN(i-Pr)2. The chloroformate esters (C96 ± C100 and C197 ± C201), sulfonyl chlorides (C101 ± C105 and
C202 ± C206), and isocyanates (C106 ± C110 and C207 ± C210) were reacted with the deprotected amine
hydrochloride salts of 20 and 21 with EtN(i-Pr)2. Average yields for these couplings are found in Table 4.
Representative samples were characterized (1H-NMR, HR-MS) and indicated the desired product in excellent
purity.
Luciferase Transcription Assays. SW480 Cells were plated at a density of 5 Â 108 cells/100 mm plate. After
24 h, the cells were transfected with DNA (TOPFLASH or FOPFLASH, 6 mg and bluescript, 6 mg) with
DOTAP according to the manufacturerꢁs (Boehringer Mannheim) protocol. After an additional 24 h, the cells
were seeded (8 Â 104 cells/well) in 96-well plates (total media volume was 100 ml/well), and treated with 1 ml of a
1 mm DMSO soln. of each test compound (10 mm final concentration). Controls contained 1 ml of DMSO only.
Luciferase activity was measured after 48 h, of incubation with the Steady Glo Luciferase Kit (Promega),
according to the manufacturerꢁs directions on a Berthold Lumat LB9501 luminometer.
We gratefully acknowledge the support of the National Institutes of Health (CA78045), Novartis, the award
of
a predoctoral fellowship (J.G., NDSEG-1995 ± 1998), and the sabbatical leave of S.S. (Fujisawa
Pharmaceutical Co., Ltd. 1998 ± 1999).
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Received May 2, 2000