Synthesis and library construction of privileged tetra-substituted Δ5-2-oxopiperazine as β-turn structure mimetics

Article abstract of DOI:10.1021/co400128a

In this study, we developed an efficient and practical procedure for the synthesis of tetra-substituted Δ⁵-2-oxopiperazine that mimics the bioactive β-turn structural motif of proteins. This synthetic route is robust and modular enough to accommodate four different substituents to obtain a high level of molecular diversity without any deterioration in stereochemical enrichment of the natural and unnatural amino acids. Through the in silico studies, including a distance calculation of side chains and a conformational overlapping of our model compound with a native β-turn structure, we successfully demonstrated the conformational similarity of tetra-substituted Δ⁵-2-oxopiperazine to the β-turn motif. For the library construction in a high-throughput manner, the fluorous tag technology was adopted with the use of a solution-phase parallel synthesis platform. A 140-membered pilot library of tetra-substituted Δ⁵-2- oxopiperazines was achieved with an average purity of 90% without further purification.

Full text of DOI:10.1021/co400128a

Research Article
pubs.acs.org/acscombsci
Synthesis and Library Construction of Privileged Tetra-Substituted
Δ5‑2-Oxopiperazine as β‑Turn Structure Mimetics
Jonghoon Kim,† Won Seok Lee,‡ Jaeyoung Koo,‡ Jeongae Lee,‡ and Seung Bum Park*,†,‡
†Department of Chemistry and ‡Department of Biophysics and Chemical Biology, Seoul National University, Seoul 151−747, Korea
S
* Supporting Information
ABSTRACT: In this study, we developed an efficient and practical procedure for the synthesis of tetra-substituted Δ5-2-
oxopiperazine that mimics the bioactive β-turn structural motif of proteins. This synthetic route is robust and modular enough to
accommodate four different substituents to obtain a high level of molecular diversity without any deterioration in stereochemical
enrichment of the natural and unnatural amino acids. Through the in silico studies, including a distance calculation of side chains
and a conformational overlapping of our model compound with a native β-turn structure, we successfully demonstrated the
conformational similarity of tetra-substituted Δ5-2-oxopiperazine to the β-turn motif. For the library construction in a high-
throughput manner, the fluorous tag technology was adopted with the use of a solution-phase parallel synthesis platform. A 140-
membered pilot library of tetra-substituted Δ5-2-oxopiperazines was achieved with an average purity of 90% without further
purification.
KEYWORDS: Δ5-2-oxopiperazine, synthesis, library construction, mimetics
oriented synthesis (DOS) strategy, which aims to provide an
efficient generation of complex and diverse compound libraries
that contain a large number of structurally diverse molecular
frameworks.5 Our group has been focused on the development
of divergent synthetic routes for the systematic construction of
libraries of drug-like polyheterocycles embedded with privileged
substructures, including benzopyran,6 benzodiazepine,7 pyr-
azole,8 pyrazolopyridine,9 and tetrahydroindazolone.10 We
named this approach a privileged-substructure-based diversity-
oriented synthesis (pDOS).11 As a continuation of our previous
efforts in pDOS and molecular diversity, we aimed to develop a
new robust route for the synthesis of tetra-substituted Δ5-2-
oxopiperazine.
INTRODUCTION
■
Since the completion of the Human Genome Project, the
development of novel small-molecule modulators toward
various gene products has been a key research field of interest.
Their great potential for use in the elucidation of gene
functions and the associated control of gene products, as well as
being prospective new therapeutic agents, make them hugely
valuable.1 In particular, the newly emerging interdisciplinary
research area of chemical biology has been focused on the
identification of novel bioactive small-molecules that can
specifically modulate biological phenomena, such as protein−
protein interactions and the associated signaling pathways, in
order to elucidate and gain control over their mechanism of
action.2 Historically, the majority of small-molecule modulators
and potential therapeutic agents have been identified through
biological screening using a library of natural products.3
However, the development of high-throughput screening
technology has led to a huge demand for collections of novel
drug-like and natural product-like small molecules with
maximized molecular diversity to the field of chemical biology
and the pharmaceutical industry.4 To address this unmet need,
the synthetic chemistry community has embraced the diversity-
Oxopiperazine is an important pharmacophore that mimics
the β-turn motif, which is one of the three main secondary
structures found in proteins and peptides (α-helix, β-turn, or β-
strand).12 In fact, a number of biologically interesting peptides,
specifically observed in protein−protein interactions, contain
Received: September 30, 2013
Revised: November 7, 2013
Published: November 11, 2013
© 2013 American Chemical Society
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the β-turn motif in important recognition segments.13,14
Therefore, oxopiperazine can serve as an attractive molecular
framework that mimics the key interaction residues and
uniquely displays the recognition motif in three-dimensional
space.15 As shown in Figure 1, Δ5-2-oxopiperazine moieties are
synthesize tetra-substituted Δ5-2-oxopiperazine [Figure
1(VI)], which can mimic the β-turn structure, the most
frequently observed turn structure in protein−protein inter-
actions. The major difference between β-turns and γ-turns is
the number of amino acid side chains on the molecular frame:
Compared to γ-turns that consist of three amino acid side
chains, β-turns contain an additional side chain.21 Therefore,
the introduction of additional substituents on the Δ5-2-
oxopiperazine structural motif would be essential for mimicking
β-turn structure along with additional diversification of its core
skeleton.
RESULTS AND DISCUSSION
■
Herein, we report the development of an efficient synthetic
pathway for tetra-substituted Δ5-2-oxopiperazine as a β-turn
mimetic, and the subsequent high-throughput construction of a
drug-like small molecule library based on this molecular
framework. In our previous report, we successfully demon-
strated the practical solid-phase parallel synthesis of trisub-
stituted Δ5-2-oxopiperazine via sequential transformations in a
single operation: acidolytic cleavage of masked aldehydes from
solid supports, subsequent cyclic iminium formation, and
double bond rearrangement.19,20 This original solid-phase
synthetic procedure allowed the construction of a pilot library
containing a trisubstituted Δ5-2-oxopiperazine core skeleton,
which mimics the dipeptidic γ-turn structure. For the expansion
of molecular diversity, we then aimed to achieve a robust
synthesis of tetra-substituted Δ5-2-oxopiperazine, which can
mimic a tripeptidic β-turn structure instead of a γ-turn.
However, it is inherently difficult to introduce additional
substituents on Δ5-2-oxopiperazine moieties using our existing
methods involving the solid-phase synthetic platform.
Figure 1. Bioactive small molecules with a Δ5-2-oxopiperazine moiety:
(I, II) Bradykinin receptor antagonists, (III) GGTI-2410, (IV) GGTI-
2421: R = Boc, GGTI-2422: R = H, (V) Chondrogenamine
(chondrogenesis inducer), (VI) our tetra-substituted Δ5-2-oxopiper-
azine molecules.
frequently observed in bioactive small molecules. For example,
Bradykinin receptor antagonists (I, II) mimic the peptide−
receptor interaction as anti-inflammatory drugs.16 Geranylger-
anyltransferase I (GGTase-I) inhibitor (III) and farnesyltrans-
ferase (FTase) inhibitor (IV) mimic the hotspot of peptide−
enzyme interaction. In addition, there are numerous other
bioactive compounds embedded with Δ5-2-oxopiperazine.17
The principal synthetic procedure for Δ5-2-oxopiperazine is
through formation of iminium ions from aldehydes or their
synthons with amines via intramolecular cyclization and
subsequent olefin migration or reduction.18,19 However, the
previously reported methods have a number of limitations that
prevent their application to the synthesis of diverse drug-like
compounds, including difficulty in controlling the stereo-
chemistry due to the requirement of strongly acidic conditions.
To tackle these problems, we previously developed a novel
synthetic method for Δ5-2-oxopiperazine derivatives using mild
acidic conditions. High-throughput construction of trisubsti-
tuted Δ5-2-oxopiperazines such as V as γ-turn mimetics was
achieved using a solid-phase parallel synthesis platform (Figure
1).19,20 The resulting trisubstituted Δ5-2-oxopiperazine library
was subjected to various cell-based high-throughput assays,
such as cell proliferation assay, reporter gene assay, image-based
phenotypic assay, and stem-cell differentiation assay. Through
these screenings, we identified chondrogenamine [Figure
1(V)], a novel small-molecule modulator containing Δ5-2-
oxopiperazine that induces chondrogenic differentiation of
human bone-marrow-derived mesenchymal stem cells.20 This
result emphasizes the importance of the development of new
synthetic pathways for drug-like small molecules that mimic the
structural motifs of gene products. As a continuation of our
efforts in maximizing molecular diversity, we aimed to
As illustrated in Scheme 1, the original synthetic procedure
for the Δ5-2-oxopiperazine moiety was modified to accom-
Scheme 1. Synthetic Procedure for Tetra-Substituted Δ5-2-
a
Oxopiperazines with a Diversification Point
a
Reagents and conditions: (a) 2,2-dimethoxyacetaldehyde, Na2SO4,
DCM, r.t, 3 h. (b) R2-MgX, THF, r.t, 1 h. (c) EDC, DCM, r.t. (d)
Neat formic acid, r.t, 1 h. (e) Morpholine, EDC, DMAP, DCM, r.t.
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modate various R2 substituents, such as 4-methoxybenzyl (a),
benzyl (b), 4-methoxyphenyl (c), and cyclopentyl (d), via the
reductive alkylation of imine intermediates with Grignard
reagents as R2 substituents. The imine intermediate was formed
with 4-methoxybenzyl amine (1) and 2,2-dimethoxyaldehyde in
anhydrous dichloromethane (DCM) solution at room temper-
ature and the subsequent reductive alkylation with various
Grignard reagents allowed the formation of secondary amines
2a−2d in moderate to excellent yields. To confirm the
robustness of this synthetic route, we pursued the synthesis
of representative compounds 5a−5d with fixed substituents,
such that R1 and R3 were 4-methoxybenzyl and R4 was a
morpholinocarbonyl 3-nitrophenyl moiety. Compound 3{1}
was formed via nucleophilic aromatic substitution of free amino
acids with electron-deficient tert-butyl-4-fluoro-3-nitrobenzoate
and coupled with compound 2 via an amide bond using 1-ethyl-
3-(3-(dimethylamino)propyl)carbodiimide (EDC) and 4-dime-
thylaminopyridine (DMAP) to form intermediate A. However,
unexpectedly, significant amounts of unidentified byproducts
were observed under the standard conditions for amide bond
formation. After extensive screening with various coupling
reagents, temperatures, and bases, intermediate A was obtained
with high efficiency by using the optimized conditions of EDC
in the absence of a base and a catalyst in dichloromethane
(DCM) at room temperature for a prolonged reaction time
(data not shown). Following this, intermediate A was subjected
to neat formic acid without purification to obtain compound 4
through a series of transformations: acidolysis of acetal to yield
aldehyde, iminium ion formation by reaction of resulting
aldehyde with a secondary amine, acid-catalyzed rearrangement
of an iminium ion to enamine to yield the Δ5-2-oxopiperazine
core skeleton, and in situ removal of the tert-butyl protecting
group. These four sequential chemical transformations were
carried out in one pot with high efficiency. It is worth
mentioning that the electron-withdrawing nitro group on the
aryl moiety at the R4 position plays an important role in the
efficient rearrangement of the iminium ion to enamine. In
addition, this nitro moiety can serve as a potential functional
handle for further diversification after its reduction to amine.
Using this solution-phase synthetic route, we successfully
prepared representative tetra-substituted Δ5-2-oxopiperazines
5a−5d in good to excellent yields, which confirms the
robustness of this route. In this pilot study, we focused on
the diversification of carboxylic acid group in 4a−4d through
amide coupling with amines, such as morpholine, to maximize
the molecular diversity.
Figure 2. (A) Schematic diagram of a secondary structure of β-turn
motif. (B) Distances between substituents in the energy-minimized
conformer of tetra-substituted Δ5-2-oxopiperazine 5a. (C) β-turn
mimicry by tetra-substituent Δ5-2-oxopiperazine 5a. (D) Overlay of
compound 5a with β-turn backbone motif (Tyr-Gly-Leu in sky blue)
in gonadotropin-releasing hormone (GnRH, PDB: 1YY1) [PreAD-
MET, Vconf Interface, Discovery studio 3.0].
turn crystal structure of gonadotropin-releasing hormone
(GnRH), one of the peptides containing a β-turn structural
motif in its biologically active conformation (PDB ID of
GnRH: 1YY1).22 As shown in Figure 2D, 5a overlapped well
with the β-turn backbone structure of GnRH, which consists of
tyrosine-glycine-leucine (in sky blue), with excellent conforma-
tional similarity. As a result, it was successfully demonstrated
that the introduction of new substituents at the R2 position
transformed the original trisubstituted Δ5-2-oxopiperazines as
γ-turn mimetics to tetra-substituted Δ5-2-oxopiperazines as β-
turn mimetics.
For the practical construction of a tetra-substituted Δ5-2-
oxopiperazine compound library, a high-throughput synthesis
platform was sought. As previously mentioned, the solid-phase
parallel synthesis route cannot be applied for the synthesis of
tetra-substituted Δ5-2-oxopiperazines due to intrinsic difficulty
in introducing an additional substituent through reductive
alkylation of secondary amine with aldehyde. Therefore, a
fluorous tag-based high-throughput strategy using solution-
phase parallel synthesis was employed. A 4-hydroxybenzyl
moiety was selected as a biased R1 substituent not only because
it mimics the tyrosine side chain in the β-turn structure but also
because a perfluorosilane tag can be easily attached through a
phenolic hydroxyl group. As shown in Scheme 2, library
synthesis was initiated by the preparation of fluorous tagged
secondary amines 7a−7d with various R2 substituents. After the
imine formation from 4-hydroxybenzylamine 6 with 2,2-
dimethoxyacetaldehyde in DCM solution, various Grignard
reagents were added to the reaction mixture for the
introduction of four different R2 substituents, including 4-
methoxybenzyl (a), benzyl (b), 4-methoxyphenyl (c), and
cyclopentyl (d) moieties, via reductive alkylation. The resulting
secondary amines containing a phenolic moiety were selectively
protected with the fluorous tag under the anhydrous DCM
solution in the presence of 2,6-lutidine after the acid-catalyzed
activation of diisopropyl-(1H,1H,2H,2H-perfluorododecyl)-
silane in DCM.
We also carried out an investigation into whether the
representative tetra-substituted Δ5-2-oxopiperazine 5a was
conformationally similar to the β-turn structure. As shown in
Figure 2A, the β-turn structure consists of four amino acid
residues, designated as i, i + 1, i + 2, and i + 3. One of the
important features of the β-turn structure is that the distance
between the Cαi and the Cαi+3 atoms is less than 7 Å.21 The
energy-minimized structure of compound 5a was virtually
constructed, and the distances between each of substituents of
compound 5a were calculated. As shown in Figure 2B, the
largest distance gap between substituents was found to be 5.704
Å, which is consistent with the important characteristic of the
β-turn structure. In addition, the orientations of individual
substituents in 5a were well aligned with those of the side
chains in the β-turn structure (Figure 2C). For further
confirmation, the energy-minimized 3D structure of tetra-
substituted Δ5-2-oxopiperazine 5a was overlapped with the β-
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rather than neat formic acid because of the poor solubility of
intermediate B in the neat formic acid. Under this acidic
environment, intermediate B can be successfully converted to
the desired tetra-substituted Δ5-2-oxopiperazine 8 through a
series of chemical transformations: acidolytic formation of an
aldehyde moiety, iminium ion formation, rearrangement of the
iminium ion to Δ5-2-oxopiperazine, and the in situ removal of
the tert-butyl protecting group. After removal of the fluorous
tag by treatment with hydrofluoric acid (HF)/pyridine and
subsequent quenching with ethoxytrimethylsilane (TMSOEt),
purities of 20 final compounds 9{a−d,1−5} were assessed
using a PDA-equipped LC/MS without any further purification
(average purities: 95%). Five representative compounds,
9{a,1}, 9{a,2}, 9{a,3}, 9{c,5}, and 9{d,1}, were fully
characterized using 1H and 13C NMR spectroscopy along
with LC/MS analysis (see Table 1 and the Supporting
Information).
Scheme 2. General Synthetic Scheme for Tetra-Substituted
Δ5-2-Oxopiperazines Using Fluorous Tag-Based Solution-
Phase Parallel Synthesis, and the Purity of Compounds 9{a−
ab
,
d,1−5}
Table 1. Purity and Mass Conformation of Representative
Compounds 9{a−d,1−5}
a
Overall percentage yields were calculated after removal of fluorous
b
tag and F-SPE. Purities were obtained using PDA-based LC/MS
analysis of crude final compounds after removal of fluorous tag.
a
Reagents and conditions: (a) 2,2-dimethoxyacetaldehyde, Na2SO4,
DCM, r.t, 3 h. (b) R2-MgX, THF, r.t, 1 h. (c) Diisopropyl-
(1H,1H,2H,2H-perfluorododecyl)silane, p-TfOH, DCM, 0 °C to r.t,
15 h. (d) 2,6-Lutidine, DCM, r.t, 3 h. (e) EDC, DCE, 50 °C. (f)
Formic acid, DCM, r.t, 1 h. (g) HF/pyridine/THF (5:5:90), r.t, 2 h.
The resulting fluorous tagged tetra-substituted Δ5-2-
oxopiperazines 8 containing a free carboxylic acid were further
diversified with various amines, including diethylamine, 1-
methylpiperazine, morpholine, N-ethyl-2-methoxyethylamine,
2-methylpropylamine, and (S)-(tetrahydrofuran-2-yl)-
methanamine, as R5 substituents through HATU-based amide
coupling in N,N-dimethylformamide (DMF) containing N,N-
diisopropylethylamine (DIPEA). As shown in Scheme 3,
intermediate C with the fluorous tag can be easily purified by
simple solid-phase extraction, and the final compounds
10{R2,R3,R5} were obtained after removal of fluorous tag
using HF/pyridine without any further purification. Represen-
tative tetra-substituted Δ5-2-oxopiperazines, 10{a,2,3}, 10-
{b,3,6}, 10{c,3,1}, 10{c,5,6}, and 10{d,1,3}, were fully
characterized using 1H and 13C NMR spectroscopy along
with LC/MS analysis (see Table 2 and the Supporting
Information). Using this fluorous tag-based high-throughput
synthesis platform, we efficiently constructed a 120-member
drug-like library of tetra-substituted Δ5-2-oxopiperazines on a
scale of approximately 10 mg each. The purities of all library
members were measured using LC/MS equipped with a PDA
detector without further purification, and their average purity
was found to be 89% after five chemical transformations.
To visualize the molecular diversity and distinctive character-
istics of the β-turn mimicking tetra-substituted Δ5-2-oxopiper-
azine library, we performed in silico analysis of this 140-member
library along with the direct comparison with γ-turn mimicking
trisubstituted Δ5-2-oxopiperazines. Both tri- and tetra-sub-
b
Purities were obtained using PDA-based LC/MS analysis of crude
final compounds after removal of fluorous tag
Then, secondary amine intermediates containing a fluorous
tag (7a−7d) were coupled with compound 3 using EDC-based
amide coupling in 1,2-dichloroethane (DCE). Five different
benzoate-containing intermediates 3{1}−3{5} were prepared
from the diversification of tert-butyl-4-fluoro-3-nitrobenzoate
with natural or unnatural amino acids, such as L-methox-
yphenylalanine, L-valine, D-valine, L-4-fluorophenylalanine and
L-glutamate methyl ester, respectively. Inconsistent with our
above-mentioned solution-phase study, we observed the
significant deterioration of reaction yields in the amide coupling
of 7a−7d with 3, which is probably due to the low reactivities
of fluorous tagged secondary amines 7a−7d. Therefore, the
amide coupling reaction was pushed to completion by mild
heating to 50 °C for 14 h without a significant decrease in
yields. After the completion of the reaction, the desired
intermediate B was easily purified through fluorous solid-phase
extraction (F-SPE) without silica-gel flash column chromatog-
raphy. Intermediate B was then subjected to the key
transformation for the synthesis of the Δ5-2-oxopiperazine
core skeleton. Unlike in our earlier solution-phase study,
intermediate B was treated with 90% formic acid in DCM
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negativity, number of hydrogen bond acceptors and donors, etc.
Three principal components (Prin1, Prin2, and Prin3)
represent 99.7% of the total variance in molecular descriptors.
Prin1 factor, which explains 93.8% of the total variance, is
mainly constituted by hydrophobic surface area (SA)_MPEOE,
2D VDW surface, and molecular weight. Prin2 factor, which
explains 4.7% of the total variance, is influenced by molecular
weight, 2D_VDW_volume, and topological polar surface area.
Prin3 factor, accounting for 1.2% of the total variance, includes
topological polar surface area, hydrophobic SA_MPEOE, and
2D_ polar van der Waals surface area (VSA). As shown in
Figure 3A, the resulting principle component analysis (PCA)
clearly visualized that the library members of tetra-substituted
Δ5-2-oxopiperazine (yellow and green dots) are widely
distributed in the 3D chemical space distinct from the
representative collection of trisubstituted Δ5-2-oxopiperazine
(red dots). On the basis of this in silico analysis, we are
confident that the tetra-substituted Δ5-2-oxopiperazine scaffold
can significantly expand the molecular diversity of drug-like
oxopiperazine structures. Therefore, both scaffolds should be
extremely valuable in a complementary manner because they
expand the molecular diversity of privileged oxopiperazines in
their own ways and populated their molecular diversity in the
different region of 3D chemical space. It is also worth
mentioning that tetra- and trisubstituted Δ5-2-oxopiperazine
scaffolds are mimicking the complementary β-turn and γ-turn
motif, respectively, whose importance cannot be overstated in
the recognition events in biopolymers. In addition, we would
like to emphasize that the introduction of various R5
substituents successfully expanded the molecular diversity of
the resulting tetra-substituted Δ5-2-oxopiperazine library, which
was visualized in different colors (see Figure 3b).
Scheme 3. High Throughput Construction of 120-Member
Library of Tetra-Substituted Δ5-2-Oxopiperazine 10{a−d,1−
5,1−6} through Amide Coupling of Intermediate 8
Containing Benzoic Acid with Various Amines (R5) and the
a
Purity of All Final Compounds
CONCLUSION
■
In this study, we designed and synthesized tetra-substituted Δ5-
2-oxopiperazines that can mimic the β-turn structure, one of
the key secondary structural motifs involved in protein−protein
interactions. Through distance calculations and the alignment
of our model compound 5a with side chains of a biologically
active β-turn structure, we confirmed that the tetra-substituted
Δ5-2-oxopiperazine could acquire conformational similarity to
the β-turn structure through the introduction of new
substituents on the skeleton of a trisubstituted Δ5-2-
oxopiperazine, which mimics the γ-turn structure. Distinct
from our original synthesis of trisubstituted Δ5-2-oxopiperazine,
a fluorous tag-based solution-phase strategy was adopted as a
high-throughput parallel synthesis platform to ensure the
efficient synthesis and purification of desired compounds with
a simple F-SPE-based filtration. For the library construction,
the R1 substituent was fixed with 4-hydroxybenzyl moiety,
identical to the side chain of tyrosine, because the common
phenolic group can serve as a functional handle to attach the
perfluorosilane tag for fluorous solid-phase extraction. In
addition, the unsaturated oxopiperazine core skeleton was
decorated with R2 and R3 substituents using various Grignard
reagents and chiral amino acids, respectively. The mild and
robust procedure of our route allowed the synthesis of final
compounds as single enantiomers through maintaining the
enantiopurity from chiral amino acids. Therefore, molecular
diversity of the tetra-substituted Δ5-2-oxopiperazine library can
be further expanded, not only with building block diversity but
also with stereochemical diversity. In this pilot library synthesis,
3-nitrobenzoic acid was used as the sole R4 substituent, owing
a
Purities were obtained using PDA-based LC/MS analysis of crude
final compounds after removal of the fluorous tag.
stituted Δ5-2-oxopiperazine libraries were subjected to
computational calculation with 15 major molecular descriptors
by using PreADMET 2.0 [BMDRC, Seoul, Korea], such as
hydrophobic surface area, molecular weight, ALogP, topological
polar surface area, van der Waals (VDW) volume, electro-
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Table 2. Purity and Mass Conformation of Representative Compounds 10{a−d,1−5,1−6}
a
b
Overall percentage yields were calculated after removal of the fluorous tag and F-SPE. Purities were obtained using PDA-based LC/MS analysis of
crude final compounds after removal of the fluorous tag.
Figure 3. (A) Principle component analysis (PCA) of the 140-member compound collection and representative trisubstituted Δ5-2-
oxopiperazines.20 (B) 3-D visualization of chemical space of tetra-substituted Δ5-2-oxopiperazine library differentiated by R5 substituents.
[PreADMET, Vconf Interface, SAS 9.2, Spotfire Decision site].
1
to its electron withdrawing effect that accelerated the initial
nucleophilic aromatic substitution and the rearrangement of the
iminium to enamine for the formation of the unsaturated
oxopiperazine core skeletons. The molecular diversity of library
members can be expanded via the selective reduction of this
nitro group and subsequent amine modifications. Instead, in
this study, we successfully demonstrated the introduction of R5
substituents through amide coupling of benzoic acids. In
summary, we constructed a 140-membered tetra-substituted
Δ5-2-oxopiperazine pilot library as β-turn mimetics, without
further purification, with an average purity of 90% using a
robust and efficient synthetic method. The biological evaluation
of the resulting tetra-substituted Δ5-2-oxopiperazines will be
reported in due course.
ninhydrin followed by thermal visualization. H, 13C NMR
spectra were obtained using Bruker DRX-300 [Bruker Biospin,
Germany], Varian DD2MR400, and Varian Inova-500 [Varian
Assoc., USA]. Chemical shifts were reported in parts per
million (ppm) from tetramethylsilane (TMS) as an internal
standard or the residual solvent peak. Multiplicities were
indicated as follows: s (singlet), d (doublet), t (triplet), q
(quartet), m (multiplet), dd (doublet of doublet), dt (doublet
of triplet), td (triplet of doublets), and br s (broad singlet).
Coupling constants were reported in hertz. The purities of all
library members were determined using an LC/MS system
equipped with a reverse-phase column (C-18, 50 × 2.1 mm, 5
μm) and a photodiode array (PDA) detector using electronic
spray ionization (ESI).
General Synthetic Procedure for Compound 2. To a
solution of 4-methoxybenzyl amine in dichloromethane
(DCM), 2,2-dimethoxyacetaldehyde (2.0 equiv) and anhydrous
Na2SO4(s) (30 equiv) were added, and the reaction mixture
was stirred at room temperature for 3 h. After the completion
of imine formation monitored by TLC, the reaction mixture
was filtered and condensed under reduced pressure. The
residue was dissolved in anhydrous tetrahydrofuran (THF), and
Grignard reagent (3.0 equiv) was added to this solution at 0 °C
under an argon atmosphere. The reaction mixture was stirred at
room temperature for 1 h. After the completion of reaction
monitored by TLC, the reaction mixture was quenched by the
EXPERIMENTAL PROCEDURES
■
General Information. All commercially available reagents
and solvents were used without further purification unless
noted otherwise. All solvents were purchased from commercial
suppliers. Diisopropyl-(1H,1H,2H,2H-perfluorododecyl)silane
was purchased from Fluorous Technologies Inc. (Pittsburgh,
PA, USA). Analytical thin-layer chromatography (TLC) was
performed using 0.25 mm silica-gel-coated Kiselgel 60 F254
plates, and the components were visualized by observation
under UV light (254 and 365 nm) or by treating the plates with
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addition of methanol and concentrated in vacuo. The residue
was redissolved in DCM, and the organic layer was washed with
saturated NH4Cl(aq) and brine, dried over anhydrous
Na2SO4(s), filtered, and concentrated in vacuo. The resulting
mixture was purified with silica-gel flash column chromatog-
raphy to provide compounds 2. Spectroscopy data of
representative compounds 2a−2d are in the Supporting
Information.
General Synthetic Procedure for Compound 3. To a
solution of 4-fluoro-2-nitrobenzoic acid in DCM, isobutylene
(2.0 equiv) and H2SO4 (cat.) were added at −78 °C. The
reaction mixture was stirred at room temperature. After the
completion of reaction monitored by TLC, the reaction mixture
was diluted with DCM, washed with saturated NaHCO3(aq)
and brine, dried over anhydrous Na2SO4(s), filtered, and
concentrated in vacuo. The residue was purified with silica-gel
flash column chromatography to provide tert-butyl 4-fluoro-2-
nitrobenzoate. Then, to a solution of tert-butyl 4-fluoro-2-
nitrobenzoate in DMF, amino acid (1.5 equiv) and DIPEA (2.0
equiv) were added, and the reaction mixture was stirred at 50
°C. After the completion of reaction monitored by TLC, the
reaction mixture was concentrated in vacuo. The residue was
purified with silica-gel flash column chromatography to provide
compounds 3. Spectroscopy data of representative compounds
3{1}−3{5} are in the Supporting Information.
General Synthetic Procedure for Compound 4. To a
solution of compound 3 (1.3 equiv) and 1-ethyl-3-(3-
(dimethylamino)propyl)carbodiimide (EDC, 3 equiv) in
DCM, compound 2 was added. The reaction mixture was
stirred at room temperature. After the completion of the
reaction monitored by TLC, the reaction mixture was diluted
with DCM, washed with saturated NaHCO3(aq) and brine,
dried over anhydrous Na2SO4(s), filtered, and concentrated in
vacuo. Without any further purification, the resulting residue,
intermediate A, was treated with neat formic acid. After stirring
at room temperature for 1 h, the reaction mixture was
condensed under reduced pressure and purified with silica-gel
flash column chromatography to provide compounds 4.
Spectroscopy data of representative compounds 4a−4d are in
the Supporting Information.
General Synthetic Procedure for Compound 5. To a
solution of compound 4 in DCM, morpholine (3.0 equiv),
EDC (3.0 equiv), and DMAP (cat.) were added, and the
reaction mixture was stirred at room temperature. After the
reaction completion monitored by TLC, the reaction mixture
was diluted with DCM, washed with saturated NH4Cl(aq) and
brine, dried over anhydrous Na2SO4(s), filtered, and con-
centrated in vacuo. The residue was purified with silica-gel flash
column chromatography to provide compounds 5. Spectrosco-
py data of representative compounds 5a−5d are in the
Supporting Information.
concentrated in vacuo. The residue was diluted with DCM,
washed with saturated NH4Cl(aq) and brine, dried over
anhydrous Na2SO4(s), filtered, and concentrated in vacuo. For
the activation of the fluorous tag, to a solution of diisopropyl-
(1H,1H,2H,2H-perfluorododecyl)silane (1.5 equiv) in anhy-
drous DCM, trifluoromethanesulfonic acid (2.0 equiv) was
added at 0 °C under an argon atmosphere and stirred at room
temperature for 15 h. Then, to the solution of the activated
fluorous tag, the resulting amine (1.0 equiv) in anhydrous
DCM was added in the presence of 2,6-lutidine (4.0 equiv) and
stirred at room temperature for 2 h. After the completion of
reaction monitored by TLC, the reaction mixture was diluted
with DCM, and the organic layer was washed with saturated
NaHCO3(aq) and brine and dried over anhydrous Na2SO4(s).
The resulting mixture was filtered, concentrated in vacuo, and
purified with silica-gel flash column chromatography to provide
compounds 7. Spectroscopy data of representative compounds
7a−7d are in the Supporting Information.
General Synthetic Procedure for Compound 9. To a
solution of compound 3 (1.3 equiv) and EDC (3.0 equiv) in
1,2-dichloroethane (DCE), compound 7 was added at room
temperature in the absence of a base and catalyst. The reaction
mixture was stirred at 50 °C. After the completion of reaction
monitored by TLC, the reaction mixture was concentrated in
vacuo. The resulting residue was redissolved in a small amount
of DMF and purified by fluorous tag-based solid-phase
extraction (F-SPE) to provide intermediate A. To a solution
of intermediate A in DCM (5 mL), neat formic acid (45 mL)
was added. The reaction mixture was stirred at room
temperature for 1 h. After the completion of reaction
monitored by TLC, formic acid was removed under reduced
pressure. The residue was purified by F-SPE and subsequent
silica-gel flash column chromatography to provide compounds
8 with a fluorous tag. The fluorous tag in compound 8 was
removed by treatment with a HF/pyridine/THF (5/5/90)
solution (1.0 mL) for 2 h at room temperature, followed by the
addition of ethoxytrimethylsilane (TMSOEt, 1.0 mL) to
quench an excess amount of HF in solution. The resulting
reaction mixture was concentrated in vacuo using a GeneVac
centrifugal evaporator. The residue was dissolved in MeOH
(0.2 mL) and purified by F-SPE. The desired untagged product
9 was collected in the MeOH/H2O (4:1, v/v) and concentrated
in vacuo using a GeneVac centrifugal evaporator. After
lyophilization of the cleaved product in acetonitrile/water
(1:1, v/v), the final products 9 were obtained in powder and
analyzed by LC/MS. Spectroscopy data of representative final
compounds 9{a,1} 9{a,2}, 9{a,3}, 9{c,5}, and 9{d,1} are in the
Supporting Information.
General Synthetic Procedure for Compound 10. To a
solution of compound 8, HATU (3.0 equiv), and DIPEA (2.0
equiv) in DMF, secondary or primary amine (5.0 equiv) was
added as an R5 substituent. The reaction mixture was stirred at
room temperature. After completion of the reaction monitored
by TLC, the reaction mixture was directly purified by F-SPE
without condensation to provide intermediates C. The fluorous
tag in intermediate C was removed by the treatment with HF/
pyridine/THF (5/5/90) solution (1.0 mL) for 2 h at room
temperature, followed by the addition of ethoxytrimethylsilane
(TMSOEt, 1.0 mL) to quench an excess amount of HF in
solution. The resulting reaction mixture was concentrated in
vacuo using a GeneVac centrifugal evaporator. The residue was
dissolved in MeOH (0.2 mL) and purified by F-SPE. The
desired untagged product 10 was collected in MeOH/H2O
General Synthetic Procedure for Compound 7. To a
solution of 4-(aminomethyl)phenol in DCM, 2,2-dimethox-
yacetaldehyde (2.0 equiv) and anhydrous Na2SO4(s) (30
equiv) were added, and the reaction mixture was stirred at
room temperature for 3 h. After the completion of reaction
monitored by TLC, the reaction mixture was stopped by
filtration and concentrated in vacuo. The residue was dissolved
in anhydrous THF, and Grignard reagent (3.0 equiv) was
added to this solution at 0 °C under argon atmosphere. The
reaction mixture was stirred at room temperature for 1 h. After
the completion of reaction monitored by TLC, the reaction
mixture was quenched by the addition of methanol and
30
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ACS Combinatorial Science
Research Article
Diversity-oriented synthesis; a spectrum of approaches and results.
Org. Biomol. Chem. 2008, 6, 1149−1158. (f) Schreiber, S. L. Target-
oriented and diversity-oriented organic synthesis in drug discovery.
Science 2000, 287, 1964−1968.
(4:1, v/v) and concentrated in vacuo using a GeneVac
centrifugal evaporator. After lyophilization of the cleaved
product in acetonitrile/water (1:1, v,v), the final products 10
weer obtained in powder and analyzed by LC/MS. Spectros-
copy data of representative final compounds 10{a,2,3}
10{b,3,6}, 10{c,3,1}, 10{c,5,6}, and 10{d,1,3} are in the
Supporting Information.
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diversity-oriented synthesis of novel scaffolds embedded with
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ASSOCIATED CONTENT
* Supporting Information
■
S
Characterization for intermediates and representative com-
pounds, tri-substituted Δ5-2-oxopiperazine as γ-turn mimetic,
principal component analysis (PCA) of Δ5-2-oxopiperazine
library, copies of 1H and 13C NMR spectra, PDA based LC/MS
data for representative compounds, and PDA based LC/MS
analysis data for library compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: sbpark@snu.ac.kr.
■
Funding
This study was supported by Bio & Medical Technology
Development Program (2012M3A9 C4048780), Basic Re-
search Laboratory (2010-0019766), and a Global Frontier
Project Grant (2011-0032150). J.K. is grateful for the BK21
Fellowship Program. W.S.L., J.K., and J.L. are grateful for the
WCU Fellowship Program.
(7) Lee, S. C.; Park, S. B. Novel application of Leuckart-Wallach
reaction for synthesis of tetrahydro-1,4-benzodiazepin-5-ones library.
Chem. Commun. 2007, 9, 825−835.
(8) Park, S.; Kim, J.; Koh, M.; Park, S. B. Efficient parallel synthesis of
privileged benzopyranylpyrazoles via regioselective condensation of β-
keto aldehydes with hydrazines. J. Comb. Chem. 2009, 11, 315−326.
(9) Lee, S.; Park, S. B. An efficient one-step synthesis of heteroiaryl
pyrazolo[3,4-b]pyridines via indole ring opening. Org. Lett. 2009, 11,
5214−5217.
Notes
The authors declare no competing financial interest.
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