Regioselective construction and screening of 1,3-disubstituted tetrahydroindazolones in enantiomerically pure pairs

Article abstract of DOI:10.1021/co200150d

In this paper, we describe a regioselective synthetic pathway for enantiopure 1,3-disubstituted tetrahydroindazolone derivatives via the condensation of 2-acylcyclohexane-1,3-dione with various alkyl- and arylhydrazines using the steric effects of a Boc-p

Full text of DOI:10.1021/co200150d

Research Article
pubs.acs.org/acscombsci
Regioselective Construction and Screening of 1,3-Disubstituted
Tetrahydroindazolones in Enantiomerically Pure Pairs
Heebum Song,† Hanjae Lee,† Jonghoon Kim,† 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 paper, we describe a regioselective
synthetic pathway for enantiopure 1,3-disubstituted tetrahy-
droindazolone derivatives via the condensation of 2-
acylcyclohexane-1,3-dione with various alkyl- and arylhydra-
zines using the steric effects of a Boc-protected pyrrolidine
ring. This synthetic method has a broad scope for substrate
generality for various hydrazines with excellent regioselectivity.
To maximize the molecular diversity, further diversifications of
1,3-disubstituted tetrahydroindazolones were pursued by
systematic N-modification of the secondary amine of the
pyrrolidine ring using solution-phase parallel synthesis with
polymer-supported reagents. A library containing a total of 272
drug-like tetrahydroindazolones, including 85 enantiomeric pairs, was constructed; the average purity, without further
purification, was 95%.
KEYWORDS: tetrahydroindazolone, regioselective synthesis, solution-phase parallel synthesis, diversity-oriented synthesis,
stereochemical diversity
collection of drug-like polyheterocycles has been subjected to
various biological evaluations and yielded novel small-molecule
modulators such as agonists of the estrogen-related receptor
γ,7a an osteogenic agent,7b inhibitors of RANKL-induced
osteoclastogenesis,7c an activator of AMP-activated protein
kinase,7d and antagonist of androgen receptors.7e
INTRODUCTION
■
One of the great challenges in the field of chemical biology is
the identification of novel small-molecule modulators that can
specifically control the functions of gene products and elucidate
the associated signaling pathways.1 In addition, small-molecule
modulators can serve as perturbing agents in biomedical
research to pinpoint the control of complex functions of
biopolymers; this can help in the development of potential
therapeutic agents.2 For the efficient and systematic identi-
fication of novel small-molecule modulators, there is a great
demand for novel and structurally diverse chemical entities.3
The construction of drug-like small molecules with wide ranges
of biological activities has therefore become an essential
element of chemical biology and drug discovery.4 To address
this issue, we have been focusing on the development of
divergent and robust synthetic pathways, using diversity-
oriented synthesis (DOS) strategies, for the systematic
construction of a library of drug-like small molecules by
creative reconstruction of polyheterocycles embedded with
privileged substructures including benzopyrans, pyrroles,
carbohybrids, and acetal-fused pyranopyrones; we named this
approach a privileged-substructure-based diversity-oriented
synthesis (pDOS).5 In addition, we have constructed various
drug-like polyheterocycles, such as diazabicycles,6a tetrahydro-
β-carbolines,6b oxopiperazines,6c and benzodiazepines,6d by
developing simple and robust synthetic transformations from
common cyclic iminium intermediates using a divergent
approach; this provides the maximum molecular diversity
with the minimum number of linear synthetic steps. Our
As a continuation of our efforts to construct novel molecular
frameworks, we aimed to develop a practical and regioselective
synthetic route to tetrahydroindazolone, a privileged structural
motif that is frequently found in bioactive natural products and
therapeutic agents.8 As shown in Figure 1, indazole and
indazolone derivatives are attractive structural motifs because of
their various biological activities such as anti-inflammatory,
antibacterial, and anticancer activities.9 In particular, tetrahy-
droindazolone was recently recognized as an important
pharmacophore because of its excellent biological activity as a
potent inhibitor of heat-shock protein 90 (HSP90) and is
currently in phase III clinical trials.8,10 Despite the proven
importance of tetrahydroindazolone derivatives in biomedical
research, a robust and efficient synthesis of such compounds
remains a challenge because of the difficulties of regiochemical
control. The general procedure for regioselective synthesis of
tetrahydroindazolones is a simple condensation of 2-acylcyclo-
hexane-1,3-diones with substituted arylhydrazines. This is
because of the inherent differences between the nucleophilic-
Received: September 9, 2011
Revised: November 5, 2011
Published: November 22, 2011
© 2011 American Chemical Society
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Figure 1. Biologically active compounds containing indazole, indazolone, tetrahydroindazolone, or N-acyl pyrrolidine moieties.
ities of the two nitrogens in arylhydrazines,8,11 but this is not
the case with alkylhydrazines.12 To address this problem, we
recently reported a regioselective pathway for the synthesis of
complementary regioisomers of 1,2- and 1,3-disubstituted
tetrahydroindazolones by the sequential protection and
deprotection of substituted hydrazines.13 We also reported a
practical solid-phase synthetic pathway for 1,3-disubstituted
one which does not depend on the native nucleophilicities of
the substituted hydrazines and which has broad scope for
substrate generality. First, we tested the regioisomeric ratios of
simple condensations between substituted hydrazines 1 and
various 2-acylcyclohexane-1,3-diones 2. As shown in Figure 2,
tetrahydroindazolones.14
Herein, we report a practical pathway for the regioselective
synthesis of 1,3-substituted tetrahydroindazolones with broad
substrate scopes including alkylhydrazines as well as arylhy-
drazines. In this study, we achieved regioselectivity by the
introduction of bulky N-Boc-pyrrolidine residues on 2-
acylcyclohexane-1,3-done; this provides sufficient steric hin-
drance to give regioselectivity, irrespective of the substitutents
on the hydrazines. In addition, the introduction of an N-Boc-
pyrrolidine moiety on tetrahydroindazolone might enhance
possible interactions with biopolymers because pyrrolidine is
frequently observed in various bioactive small molecules such as
antihyperglycemic agents15a,b and angiotensin-converting en-
zyme (ACE) inhibitors15c (Figure 1). Finally, we aimed to
maximize the molecular diversity through the systematic N-
modification on the pyrrolidine moiety and preparation of
enantiomeric pairs (R/S) of tetrahydroindazolone derivatives,
by using either L- or D-proline, for the study of stereochemical
diversity.
Figure 2. Regioisomeric ratio [regioisomeric ratios were determined
by H NMR spectroscopic analysis of samples after purification via
silica-gel flash column chromatography] of tetrahydroindazolones via
conventional condensation of various hydrazines 1 and 2-acylcyclo-
hexane-1,3-diones 2.
1
conventional heating in protic solvents allows the formation of
disubstituted tetrahydroindazolones in different regioisomeric
1
ratios; this was monitored by H and one-dimensional nuclear
Overhauser effect (1D NOE) NMR. However, poor
regioselectivity was observed in the case of alkylhydrazines.
For example, the condensation of methylhydrazine with 2-
acetylcyclohexane-1,3-dione provides an almost 1:1 mixture
(53:47) of 1,3- and 1,2-disubstituted regioisomers. In this
experiment, we paid particular attention to the linear
correlation of the steric hindrance at the R2 position on the
2-acylcyclohexane-1,3-diones 2 with regioselectivity for 1,3-
disubstituted tetrahydroindazolones.13 For a cyclohexyl moiety
at the R2 position on 2, we only observed single regioisomers,
except with the smallest possible substituent at the R1 position
on the substituted hydrazine, that is, methylhydrazine, in which
the 1,3-isomer was preferred over the 1,2-isomer (86:14). We
therefore believed that we could control the regioselectivity for
1,3-disubstituted tetrahydroindazolones, irrespective of the
substituents at the R1 position on the alkyl- or arylhydrazines,
RESULT AND DISCUSSION
■
Most regioselective syntheses of tetrahydroindazolone deriva-
tives are achieved by simple condensation of 2-acylcyclohexane-
1,3-diones with substituted hydrazines through the different
nucleophilicities of the two nitrogens on the substituted
hydrazines.8,10,11 In the case of arylhydrazines, the internal
nitrogen atom is much less nucleophilic than the external one
as a result of conjugation of the lone-pair electrons on the
internal nitrogen in the aromatic ring system; this leads to the
regioselective synthesis of 1,3-disubustituted tetrahydroindazo-
lones. In the case of alkylhydrazines, however, regiochemical
control in the synthesis of 1,3-disubstituted tetrahydroindazo-
lones is quite difficult to achieve because the nucleophilicities of
the two nitrogens on alkylhydrazines are indistinguishable.
A different strategy is therefore needed for the robust
synthesis of 1,3-disubstituted tetrahydroindazolones, that is,
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Scheme 1. Regioselective Synthetic Pathway for 1,3-Disubstituted Tetrahydroindazolones Using Hydrazines 1 and 2-
(Hydroxy(N-Boc-pyrrolidin-2-yl)methylene)-5,5-dimethylcyclohexane-1,3-dione 3
using cyclohexyl or equivalent substituents at the R2 position
on the 2-acylcyclohexane-1,3-diones. Bulkier substituents at the
R1 position on alkylhydrazines also allow some regioselectivity
for the synthesis of 1,3-disubstituted tetrahydroindazolones.
However, this regioselectivity is not high enough except for the
limited cases such as cyclopentylhydrazines or alkylhydrazines
with equivalent substituents, which makes this approach
impractical for high throughput synthesis. In the case of
arylhydrazines, excellent regioselectivity was obtained, irre-
spective of the R2 substituents on the 2-acylcyclohexanes-1,3-
diones, as expected (see full data set in the Supporting
Information).
These experiments clearly showed that the condensation of
substituted hydrazines 1 with 2-acylcyclohexane-1,3-diones 2 is
strongly influenced by steric hindrance at the R2 position of the
2-acylcyclohexane-1,3-diones. On the basis of these observa-
tions, we designed a regioselective synthetic pathway for 1,3-
disubstituted tetrahydroindazolone derivatives by introducing a
bulky substituent, namely, an N-Boc-protected pyrrolidine ring,
at the R2 position on 2-acylcyclohexane-1,3-diones 2 (Scheme
1). As well as achieving regioselectivity, N-Boc-protected
pyrrolidines introduced into 1,3-disubstituted tetrahydroinda-
zolones can be used for further N-modification, after Boc
deprotection, to maximize the molecular diversity of the
resulting small-molecule library. In addition, we might expect
an improvement in the possibility of interactions with
biopolymers and the subsequent discovery of bioactive small
molecules because N-acylpyrrolidine moieties are also recog-
nized as privileged substructures and are frequently observed in
many bioactive natural products and therapeutic agents.16
The introduction of N-Boc-protected pyrrolidine on 2-
acylcyclohexane-1,3-diones was achieved by 4,4-
dimethylaminopyridine(DMAP)-mediated coupling of 5,5-
dimethylcyclohexane-1,3-dione with Boc-protected L-proline
in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide (EDC). We then validated the regioselectivity
of this synthetic strategy using 2-(hydroxy(N-Boc-pyrrolidin-2-
yl)methylene)-5,5-dimethylcyclohexane-1,3-dione 3 and benzyl
hydrazine 1d. The simple condensation of 3 at 60 °C in EtOH
solution allows the regioselective formation of 1,3-disubstituted
tetrahydroindazolones 4, and was consistent for various
alkylhydrazines, including n-hexyl (4a), 2-methoxyethyl (4b),
1-acetylpiperidin-4-yl (4c), and 2-pyridylmethyl (4e), 3-
pyridylmethyl (4f), 2-amino-2-oxoethyl (4g), cyclopentyl
(4h), as well as for arylhydraiznes (4i−4k in Scheme 1). The
regiochemistry was confirmed by 1D NOE experiments after
Boc deprotection in 20% trifluoroacetic acid (TFA); this
demonstrated the clear correlations between protons at the C7
and C1′ positions in the 1,3-disubstituted tetrahydroindazo-
lones 6 (see Supporting Information). We concluded that this
regioselective strategy can be widely applied and has a broad
scope for substrate generality at the R1 position of hydrazines
for the synthesis of 1,3-disubstituted tetrahydroindazolones.
After establishing the regioselective synthetic pathway for
1,3-disubstituted tetrahydroindazolones with a broad scope for
substituted hydrazines, we focused on further diversification of
tetrahydroindazolones as key intermediates for maximizing
molecular diversity. As shown in Scheme 2, the introduction of
Scheme 2. Further Diversification Strategy for 1,3-
Disubstituted Tetrahydroindazolones
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a
Table 1. Building Blocks and Purities of 7{R/S,R1,R2} Produced via N-modification (I): Amide Formation with Activated
Esters on HOBt Resin Using Solution-Phase Parallel Synthesis
a
Purities were obtained by PDA-based LC/MS analysis of final products without further purification.
various substituents at the R1 position can be achieved using
various hydrazines, including alkyl- and arylhydrazines. The
resulting tetrahydroindazolone derivatives were further diversi-
fied through N-modification at the pyrrolidine secondary amine
as follows: (i) amide formation with various carboxylic acids,
(ii) urea or thiourea formation with isocyanates or
isothiocyanates, and (iii) sulfonamide formation with sulfonyl
chlorides (Scheme 2). For convenient and efficient N-
modification, we adopted solution-phase parallel synthesis
using polymer-supported reagents.
First, amide formation was achieved by direct incubation of
the free secondary amine on the pyrrolidine moiety of
tetrahydroindazolones 6 in dicholoroethane (DCE) for 24 h
at room temperature with activated esters on an HOBt-6-
carboxaminomethyl polystyrene support generated by treat-
ment of carboxylic acid in the presence of N,N′-diisopropyl-
carbodiimide (DIC), DMAP, and diisopropylethylamine
(DIPEA) in dimethylformamide (DMF) for 4 h at room
temperature. As shown in Table 1, we successfully generated
128 tetrahydroindazolone derivatives 7 via pyrrolidine mod-
ification of 16 different tetrahydroindazolones 6, with amide
formation using eight different carboxylic acids that included
alkyl, aryl, acrylic, and heterocyclic substitutents. The resulting
compounds 7 had excellent purities without further purifica-
tion; the overall purity was 95.6% (Table 1).
supported scavengers to remove excess N-modification
reagents. The 1,3-disubstituted tetrahydroindazolone inter-
mediates 6 were treated with an excess of isocyanates or
isothiocyanates and sulfonyl chlorides in DCE containing
triethylamine (TEA) at room temperature; this provided the
desired ureas or thioureas and sulfonamides, respectively.
Excess N-modification reagents were then removed by tris-(2-
aminoethyl)amine (TAEA) polystyrene scavenger resin in DCE
for 24 h at room temperature. Initially, we pursued the N-
modification of pyrrolidines with isocyanates or isothiocyanates
in the absence of an organic base; this required a higher
temperature (80 °C) and a longer time (12−24 h) to achieve
full conversion of the starting materials. These initial conditions
with an elevated temperature gave moderate yields, and an
undesired aza-Cope rearrangement was observed in the case of
allyl isothiocyanate (data not shown); this led us to N-
modification in the presence of an organic base under much
milder conditions (2.5 h at room temperature). As shown in
Table 2, we successfully generated 80 tetrahydroindazolone
derivatives 8 with urea or thiourea moieties by modification
with three different isocyates and two different isothiocyanates,
respectively, with an overall purity of 95.5%.
In the case of sulfonamide formation, we successfully
generated 64 1,3-disubstituted tetrahydroindazolones 9 with
sulfonamide moieties, with an overall purity of 93.7%, without
further purification, by treatment of four different sulfonyl
chlorides in the presence of TEA at room temperature,
Urea/thiourea formation and sulfonamide formation were
also pursued by solution-phase parallel synthesis using polymer-
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a
Table 2. Building Blocks and Purities of 8{R/S,R1,R2} Produced via N-modification (II): Urea/Thiourea Formation with
Isocyanates/Isothiocyanates and Subsequent Scavenging with Trisamine Resin
a
Purities were obtained by PDA-based LC/MS analysis of final products without further purification.
followed by removal of excess reagents via incubation with
TAEA polystyrene resins for 24 h at room temperature (Table
3).
The resulting 272-membered tetrahydroinazolone library
with 85 enantiomeric pairs was subjected to biological
evaluation to seek potential activities based on their structures
and stereochemistries. In this context, our tetrahydroindazolone
derivatives were treated against HeLa human cervical cancer
cells. The compound-induced inhibition of cell proliferation
was measured by monitoring mitochondrial activity with 4-[3-
(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene
disulfonate (WST-1, cell proliferation assay reagent) upon
treatment with the compounds in the concentration range 100
nM to 20 μM for 72 h. As show in Figure 3, we observed
interesting patterns of antiproliferative activity toward HeLa
cells among the enantiomer sets. For example, the enantiomeric
pair, 8{R,1,e} and 8{S,1,e}, show similar antiproliferative
activities (IC50 = 6.64 μM and 6.71 μM, respectively). On the
other hand, a different enantiomeric pair, 8{R,1,d} and
8{S,1,d}, show totally different activities: compound 8{R,1,d}
shows some cytotoxicity toward HeLa cells (IC50 = 6.51 μM),
but the enantiomer 8{S,1,d} has no cytotoxic activity, even at
the highest treatment concentration (20 μM). This observation
indicates the importance of stereochemical diversity, and that
we might be missing much information regarding dependence
on compound chirality in various biological evaluations (see
Supporting Information for viability data of whole library
compounds).
It is worth mentioning that we constructed a 1,3-
disubstituted tetrahydroindazolone library containing a series
of enantiomeric pairs, synthesized as a single enantiomer,
because we believe that stereochemical diversity is relatively
unexplored among the diversity elements in the DOS approach,
and that the systematic construction of enantiomeric pairs
might provide information regarding the importance of
stereochemical outcomes in molecular interactions with various
biopolymers. In fact, most biopolymers, including proteins,
DNA, RNA, and polysaccharides, are in chiral environments
and they should have clear preferences for a particular
stereochemistry of their small-molecule partners. Once we
can access a systematic collection of enantiomeric pairs having
identical physical and chemical properties, we can explore the
importance of a single stereogenic center in biological
environments through various bioevaluations. There are
sporadic reports in the literature,17 but systematic studies are
still rare. To address this unmet need, we constructed a library
of enantiopure 1,3-disubstituted tetrahydroindazolones; the
library contained 40 enantiomeric pairs from 7{S,1,a} to
7{R,5,h}, 25 pairs from 8{S,1,a} to 8{R,5,e}, and 20 pairs from
9{S,1,a} to 9{R,5,d}. The (S) enantiomer sets were derived
from L-proline for the synthesis of 2-(hydroxy(N-Boc-
pyrrolidin-2-yl)methylene)-5,5-dimethylcyclohexane-1,3-dione
3, and the (R) enantiomer sets were derived from D-proline for
the synthesis of the key intermediates 3.
CONCLUSION
■
In this study, we developed a practical and robust strategy for
regioselective synthesis of 1,3-disubstituted tetrahydroindazo-
lone derivatives by introduction of steric hindrance using a Boc-
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a
Table 3. Building Blocks and Purities of 9{R/S,R1,R2} Produced via N-modification (III): Sulfonamide Formation Using
Sulfonyl Chlorides and Subsequent Scavenging with Trisamine Resin
a
Purities were obtained by PDA-based LC/MS analysis of final products without further purification.
Figure 3. Representative enantiomeric compound sets and their antiproliferative activities [cell proliferative data was measured by WST-1 assay upon
treatment with representative compounds against HeLa cell in does-dependent pattern for 72 h. All experiments were duplicated and mean value was
used] against HeLa human cervical cancer cells.
protected pyrrolidine ring on 2-acylcyclohexane-1,3-diones 2.
As a result of this steric hindrance, we successfully controlled
the regioselectivity of 1,3-disubstituted tetrahydroindazolones
with a broad scope for substrate generality for substituted
hydrazines, including alkylhydrazines. The pyrrolidine moiety
can also serve as a privileged substructure in the resulting
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tetrahydroindazolone derivatives; this might increase the
probability of potential interactions with various biopolymers
for the discovery of novel bioactive small molecules. We
particularly focused on constructing a library of enantiopure
tetrahydroindazolone derivatives and the preparation of 85
enantiomer pairs, simply by the introduction of chiral
pyrrolidine from L-/D-proline. To maximize the molecular
diversity and process efficiency, we pursued the systematic N-
modification of pyrrolidine secondary amine using solution-
phase parallel synthesis in conjunction with polymer-supported
reagents. Finally, we constructed a 272-membered 1,3-
disubstituted tetrahydroindazolone library containing 85
enantiomeric pairs, without further purification, with an average
purity of 95.1%. In our preliminary biological evaluations, a
series of enantiopure tetrahydroindazolone derivatives showed
dramatic differences in their antiproliferative activity toward
HeLa cells, depending on the differences in their stereo-
chemistries; this emphasizes the importance of stereochemical
diversity in library construction using combinatorial chemistry
or diversity-oriented synthesis. Further biological evaluations of
the resulting tetrahydroindazolones will be reported in due
course.
chromatography provided the desired product 3 (68.4%) as a
mixture of rotamers (43:57).
General Synthetic Procedure for (S)-6,6-dimethyl-3-
(N-Boc-pyrrolidin-2-yl)-1,3-disubstituted Tetrahydoin-
dazolones (4). To a 0.1 M solution of (S)-tert-butyl-2-
((4,4-dimethyl-2,6-dioxocyclohexylidene)(hydroxy)methyl)-
pyrrolidine-1-carboxylate 3 (1.0 equiv.) in ethanol, hydrazine 1
(1.2 equiv.) was added, and the resulting mixture was stirred at
60 °C. After complete consumption of the starting materials,
the reaction mixture was concentrated under reduced pressure
and purified by silica-gel flash column chromatography to give
the desired products 4.
General Synthetic Procedure for (S)-6,6-dimethyl-3-
(pyrrolidin-2-yl)-1,3-disubstitued Tetrahydroindazolone
(6). 0.1 M solution of (S)-6,6-dimethyl-3-(N-Boc-pyrrolidin-2-
yl)-1,3-disubstituted tetrahydroindazolone 4 in 20% trifluoro-
acetic acid in DCM was stirred at room temperature. After
complete consumption of the starting materials, monitored by
TLC, the reaction was quenched by the addition of saturated
NaHCO3 solution and extracted three times with DCM. The
combined organic layer was dried over Na2SO4(s). The filtrate
was condensed under reduced pressure and purified by silica-
gel flash column chromatography to provide 6.
General Procedure for the Solution-Phase Parallel
Synthesis of Compounds 7, 8, and 9. A. Amide
Formation for 7{S,1,a}−7{S,11,h} (128 Compounds). PS-
HOBt resin (90 mg, loading level 1.1 g/mol) was loaded in
individual wells of a 96-deep-well filtration block. Eight
different carboxylic acids (3.5 equiv.) in 0.6 mL of a DMF
solution of N,N′-diisopropylcarbodiimide (7.0 equiv.), diiso-
propylethylamine (7.0 equiv.), and 4-dimethylaminopyridine
(0.2 equiv.) were added to the designated wells of the reaction
block. The reaction block was sealed and incubated for 4 h at
room temperature in a rotating oven (Robbins Scientific). The
resulting resin was thoroughly washed four times with
dichloromethane. Then, 16 Boc-deprotected tetrahydroindazo-
lone intermediates 6 (11 mg) in 0.6 mL of DMF were added to
the designated wells charged with individual activated esters on
PS-HOBt resin. The reaction block was sealed and incubated
for 24 h at room temperature in a rotating oven. The final
products were collected by removal of excess activated-ester-
bound PS-HOBt resins via filtration and condensing the filtrate
in vacuo using a GeneVac (Thermo Savant). The crude
products (7{S,1,a}−7{S,11,h}) were directly analyzed by LC/
MS without further purification.
B. Urea and Thiourea Formation for 8{S,1,a}−8{S,11,e}
(80 Compounds). Sixteen different Boc-deprotected tetrahy-
droindazolone intermediates 6 (11 mg) and triethylamine (2
equiv.) were dissolved in 1,2-dichloroethane (0.3 mL) and
added to a 96-deep-well filtration block. Three isocyanates and
two isothiocyanates were added to the designated wells of the
reaction block. The reaction block was sealed and incubated for
2.5 h at room temperature in a rotating oven. After completion
of the reaction, tris(2-aminoethyl)amine polystyrene scavenger
resin (6.0 equiv., loading level ≥2.2 mmol/g) was added to
each well and incubated for an additional 24 h at room
temperature. After removal by filtration of the tris(2-
aminoethyl)amine polystyrene resin, the filtrate was condensed
in vacuo using a GeneVac. The crude products (8{S,1,a}−
8{S,11,e}) were directly analyzed by LC/MS without further
purification.
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. HOBt-6-carboxamiomethyl polystyrene (PS-HOBt)
and tris-(2-aminoethyl)-amine polystyrene (PS-trisamine) were
purchased from Novabiochem. Analytical thin-layer chroma-
tography (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 ninhydrin followed by thermal
visualization. 1H, 13C, and 1D NOE NMR spectra were
obtained using a Varian Inova-500 (Varian, Palo Alto, CA,
U.S.A.). Chemical shifts were reported in parts per million from
tetramethylsilane (TMS) as internal standard or the residual
1
1
solvent peak (CDCl3, H: 7.26, 13C: 77.23; (CD3)2SO, H:
2.50, 13C: 39.52). Multiplicities were indicated as follows: s
(singlet); d (doublet); t (triplet); q (quartet); m (multiplet);
dd (doublet of doublets); dt (doublet of triplets); td (triplet of
doublets); bs (broad singlet). Coupling constants were
reported in hertz. The purities of all the 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 electron spray
ionization (ESI).
tert-Butyl 2-((4,4-dimethyl-2,6-dioxocyclohexylidene)-
(hydroxy)methyl)pyrrolidine-1-carboxylate (3). To a sol-
ution of (2S)-1-(tert-butoxycarbonyl)-2-pyrrolidine carboxylic
acid (N-Boc-L-proline) or (2R)-1-(tert-butoxycarbonyl)-2-pyr-
rolidinecarboxylic acid (N-Boc-D-proline) (1.2 equiv.), 4-
dimethylaminopyridine (DMAP) (1.0 equiv.), and 1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide (EDC) (1.3 equiv.) in
anhydrous dichloromethane (DCM) (0.1 M), dimedone (1.0
equiv.) was added, and the reaction mixture was stirred for
several hours under an argon atmosphere. After complete
consumption of the starting materials, monitored by TLC, the
reaction mixture was diluted with ethyl acetate (EA) and
washed twice with saturated NH4Cl (aq). The combined
organic layer was dried over anhydrous Na2SO4, filtered, and
condensed under reduced pressure. Silica-gel flash column
C. Sulfonamide Formation for 9{S,1,a}−9{S,11,d} (64
Compounds). Sixteen different Boc-deprotected tetrahydroin-
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Research Article
R. J.; Bender, A.; Spring, D. R. Diversity-oriented synthesis; a spectrum
of approaches and results. Org. Biomol. Chem. 2008, 6, 1149−1158.
(e) Peuchmaur, M.; Y.-S. Wong., Y.-S. Expanding the chemical space
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dazolone intermediates 6 (11 mg) in a solution of 1,2-
dichloroethane (0.4 mL) containing triethylamine (1.5 equiv.)
were dispensed into a 96-deep-well filtration block. Four
different sulfonyl chlorides were added to the designated wells,
and the reaction block was sealed and incubated for 2 h at room
temperature in a rotating oven. After completion of the
reaction, tris(2-aminoethyl)amine polystyrene scavenger resin
(10.0 equiv., loading level ≥2.2 mmol/g) was added to each
well and incubated for an additional 24 h at room temperature.
After removal by filtration of the tris(2-aminoethyl)amine
polystyrene resin, the filtrate was condensed in vacuo using a
GeneVac. The crude products (9{S,1,a}−9{S,11,d}) were
directly analyzed by LC/MS without further purification.
Cell Proliferation Assay. HeLa human cervical cancer
cells were cultured in RPMI 1640 media containing 10% fetal
bovine serum (FBS) and 1% antibiotic-antimycotic solution.
HeLa cells (2000 cells per well) in a 96-well plate were
incubated at 37 °C in an atmosphere of 5% CO2 and 95% air
for 72 h. The cells were treated with various concentrations of
individual compounds from 1,3-disubstituted tetrahydroinda-
zolone library using pin tool. After 72 h incubation, 10 μL of
EZ-Cytox cell viability assay kit solution (premixed WST-1 cell
proliferation assay reagent, Daeil lab service, Seoul, Korea) was
added to each well and incubated for additional 1 h. The
reduction of tetrazolium salts to formazan by the inhibition of
metabolic activity in mitochondria was quantified by measuring
the absorbance at 455 nm using Synergy HT multimode
microplate reader.
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tetra-aryl-substituted alkenes and their bioevaluation as a selective
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ASSOCIATED CONTENT
■
S
* Supporting Information
1H/13C NMR and 1D NOE spectra to confirm the
regioselectivity of compounds in Figure 2 and compounds
6a−6k, full characterization of representative compounds, and
LC/MS analysis and cell proliferative data of all library
members. 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.
(8) Barta, T. Te.; Veal, J. M.; Rice, J. W.; Partridge, J. M.; Fadden, R.
P.; Ma, W.; Jenks, M.; Geng, L.; Hanson, G. J.; Huang, K. J.; Barabasz,
A. F.; Foley, B. E.; Otto, J.; Hall, S. E. Discovery of benzamide
tetrahydro-4H-carbazol-4-ones as novel small molecule inhibitors of
Hsp90. Bioorg. Med. Chem. Lett. 2008, 18, 3517−3521.
■
Funding
(9) (a) Lien, J. C.; Lee, F. Y.; Huang, L. J.; Pan, S. L.; Guh, J. H.;
Teng, C. M.; Kuo, S. C. 1-benzyl-3-(5′hydroxymethyl-2′furyl)-indazole
(YC-1) derivatives as novel inhibitors against sodium nitroprusside-
induced apoptosis. J. Med. Chem. 2002, 45, 4947−4949. (b) Schindler,
This study was supported by (1) National Research Foundation
of Korea (NRF) grants (NRF-2009-0078236); (2) MarineBio
Program funded by Ministry of Land, Transport, and Maritime
Affairs (MLTM), Korea; and (3) the World Class University
program (R31-2010-000-10032-0). H.S., H.L., and J.K. are
grateful for the fellowships awarded by the BK21 Program and
the Seoul Science Fellowship.
R.; Fleischhauser, I.; Hofgen, N.; Sauer, W.; Egerland, U.; Poppe, H.;
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3,-ols with anti-inflammatory activity. Arch. Pharm. Pharm. Med. Chem.
1998, 331, 13−21. (c) Boehringer, M.; Boehm, H. J.; Bur, D.;
Gmuender, H.; Huber, W.; Klaus, W.; Kostrewa, D.; Kuehne, H.;
Luebbers, T.; Meunier-Keller, N.; Mueller, F. Novel inhibitors of DNA
gyrase: 3D structure based biased needle screening, hit validation by
biophysical methods, and 3D guided optimixation. A promising
alternative to random screening. J. Med. Chem. 2000, 43, 2664−2674.
(d) Cerecetto, H.; Gerpe, A.; Gonzalez, M.; Aran, V. J.; de Ocariz, C.
O. Pharmacological properties of inadazole derivatives: recent
developments. Mini-Rev. Med. Chem. 2005, 5, 869−878.
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