Methyl 3-((6-methoxy-1,4-dihydroindeno[1,2-c]pyrazol-3-yl)amino)benzoate (GN39482) as a tubulin polymerization inhibitor identified by morphobase and chemproteobase profiling methods

Article abstract of DOI:10.1021/acs.jmedchem.5b00035

A series of indenopyrazoles was synthesized from the corresponding indanones and phenyl isothiocyanates in two steps. Among the compounds synthesized, methyl 3-((6-methoxy-1,4-dihydroindeno[1,2-c]pyrazol-3-yl)amino)benzoate 6m (GN39482) was found to posse

Full text of DOI:10.1021/acs.jmedchem.5b00035

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Article
Methyl 3-((6-methoxy-1,4-dihydroindeno[1,2-c]pyrazol-3-yl)amino)
benzoate (GN39482) as a Tubulin Polymerization Inhibitor
Identified by MorphoBase and ChemProteoBase Profiling Methods
Hidemitsu Minegishi, Yushi Futamura, Shinji Fukashiro, Makoto
Muroi, Makoto Kawatani, Hiroyuki Osada, and Hiroyuki Nakamura
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 04 May 2015
Downloaded from http://pubs.acs.org on May 4, 2015
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Methyl
3-((6-methoxy-1,4-dihydroindeno[1,2-c]pyrazol-3-yl)amino)
benzoate (GN39482) as a Tubulin Polymerization Inhibitor Identified
by MorphoBase and ChemProteoBase Profiling Methods
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,2)
3)
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3)
3)
Hidemitsu Minegishi, Yushi Futamura, Shinji Fukashiro, Makoto Muroi, Makoto Kawatani,
3
)
,1)
Hiroyuki Osada, and Hiroyuki Nakamura*
1
2
) Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsutaꢀcho, Midoriꢀku,
Yokohama 226ꢀ8503, Japan
) Department of Life Science, Faculty of Science, Gakushuin University, Mejiro, Toshimaꢀku,
Tokyo 171ꢀ8588, Japan
3) Chemical Biology Research Group, RIKEN Center for Sustainable Resource Center, Hirosawa,
Wako, Saitama 351ꢀ0198, Japan
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Abstract
A series of indenopyrazoles was synthesized from the corresponding indanones and phenyl
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isothiocyanates
in
two
steps.
Among
the
compounds
synthesized,
methyl
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ꢀ((6ꢀmethoxyꢀ1,4ꢀdihydroindeno[1,2ꢀc]pyrazolꢀ3ꢀyl)amino)benzoate 6m (GN39482) was found to
possess a promising antiproliferative activity toward human cancer cells without affecting any
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antiꢀmicrobial and antiꢀmalarial activities at 100 nM. Both a methoxy group at R position and a
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methoxycarbonyl group at R position of the anilinoquinazoline framework are essential for the high
cell growth inhibition. Both MorphoBase and ChemProteoBase profiling analyses suggested that
compound 6m was classified as a tubulin inhibitor. Indeed, compound 6m inhibited the acetylated
tubulin accumulation and the microtubule formation and induced G2/M cell cycle arrest in HeLa
cells, revealing that a promising antiproliferative activity of compound 6m toward human cancer
cells is probably caused by the tubulin polymerization inhibition.
Keywords
Indenopyrazole, antiproliferative activity, tubulin polymerization inhibitor, MorphoBase profiling
analysis, ChemProteoBase profiling analysis
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Introduction
Indenopyrazole, a novel pseudoazulenic system containing two fused fiveꢀmembered rings, was first
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synthesized by Boyd in 1965. Although indenopyrazoles were reported by Lemke and coworkers as
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potential antipsychotic agents in 1978, other biological activities have not been reported until 1998.
Nugiel and coworkers reported indenopyrazoles as cyclin dependent kinase (CDK) inhibitors. After
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their finding, various indenopyrazoleꢀbased CDK inhibitors have been reported. Galemmo and
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coworkers reported plateletꢀderived growth factor receptor (PDGFR) tyrosine kinase inhibitors, and
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Tao and coworkers reported checkpoint kinase 1 (Chk1) inhibitors.
We also found
indenopyrazoleꢀcontaining inhibitions of epidermal growth factor receptor (EGFR) and vascular
endothelial growth factor receptor (VEGFR)ꢀ2 tyrosine kinases by in silico highꢀthroughꢀput
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screening (Figure 1). Thus, indenopyrazole has been paid attention as one of the attractive
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frameworks for development of kinase inhibitors in medicinal chemistry.
Recently much attention has been focused on a hypoxia inducible factor (HIF)ꢀ1 inhibitor as a
target for anticancer agents because HIFꢀ1 is a transcription factor that controls the expression on
genes such as vascular endothelial growth factor (VEGF), erythropoietin (EPO) and insulin like
11ꢀ13
growth factor.
VEGF and EPO are key factors in tumor growth and pathological angiogenesis.
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The overexpression of HIFꢀ1α has been observed in various cancers,
including brain, breast,
cervical, esophageal, and ovarian cancers correlated with treatment failure and mortality, as a result
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of intratumoral hypoxia and genetic alterations affecting key oncogenes and tumor suppressor genes.
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Therefore, drugs targeting HIFꢀ1 could represent a novel approach to cancer therapy.
Recently,
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we reported indenopyrazole 1 (GN44028) as a new HIFꢀ1 inhibitor. In the structure activity
relationship (SAR) study based on 1, indenopyrazole 2 was found to possess unexpectedly high cell
growth inhibition toward several cancer cell lines.
(
Figure 1)
In this paper, we performed the further SAR study based on cytotoxic compound 2, and discovered
more potent cell growth inhibitors than 2. Furthermore, we clarified that their promising
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antiproliferative activity has been caused by inhibiting tubulin polymerization using MorphoBase
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and ChemProteoBase
profiling methods.
Results and Discussion
Chemistry
The synthesis of phenyl isothiocyanates is shown in Scheme 1. Anilines 3a-d, which were prepared
from corresponding 2ꢀ or 3ꢀhydroxyl anilines and 3ꢀaminobenzoic acid, and aniline 3e were treated
with phenyl chlorothionoformate in THF to give thiocarbamate intermediates, which reacted with
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triethylamine (TEA) and Cl SiH to afford phenyl isothiocyanates 4a-e in high yields.
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(Scheme 1)
Scheme 2 shows the formation of indenopyrazoles from the corresponding indanones 5aꢀc and
phenyl isothiocyanates 4aꢀj including commercially available starting materials (5c and 4fꢀj).
Indanones 5a and 5b were prepared form 5ꢀhydroxylindanone with TBS or ethyl protections,
respectively. Deprotonation of αꢀproton of carbonyls in indanones 5aꢀc was carried out using lithium
hexamethyldisilazide (LiHMDS) and the resulting enolates reacted with phenyl isothiocyanates 4 to
give the thioamide intermediates, that underwent condensation with hydrazine monohydrate to form
indenopyrazoles 6 in moderate yields (37ꢀ60%). Deprotection of a TBS group in 6b, 6d and 6f was
carried out by treatment with TBAF in THF to give indenopyrazoles 6lꢀm in moderate to high yields.
Methyl ester 6m was hydrolyzed using LiOH to give indenopyrazole 6n in 76% yield (Scheme 2).
(
Scheme 2)
Biology
Synthesized indenopyrazoles 6a, 6c, 6e, 6g-q were tested for antiproliferative activity toward three
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human cancer cell lines, HeLa (human cervical carcinoma), PC3 (human prostate cancer) and
HCT116 (human colon cancer), and a normal cell line, HEK293 (human embryonic kidney) using
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MTT assay. We first examined the effect of indenopyrazoles substituted at R position on
antiproliferative activity and the results are summarized in Table 1. Compound 6a, which has an
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ethoxy group at R position, exhibited antiproliferative activity with IC50 values between 20.4 and
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0.2 nM. Although compound 6c containing a hydroxyl group instead of an ethoxy group as the R
group did not show a significant antiproliferative activity at 100 nM, compound 2 containing a
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methoxy group as the R group possessed an antiproliferative activity more potently than compound
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a (the IC50 values of compound 2 toward human cancer cell lines: 8.9 ~ 35.6 nM). Furthermore,
compound 6p, which has no substituent on the anilinoquinazoline framework resulted in no
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significant inhibition at 100 nM, revealing that an alkoxy group at the R position is essential for
antiproliferative activity toward the human cancer cells induced by indenopyrazoles.
(Table 1)
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We next examined the effect of substituents at the R position of 6ꢀmethoxyanilinoquinazolines on
the antiproliferative activity. The results are summarized in Table 2. A substituent at 2’ (6e and 6h) or
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’ (6j) position resulted in weak inhibitory effects on antiproliferative activity toward HeLa, PC3,
HCT116, and HEK293 cells, whereas 3’ꢀsubstituted anilinoquinazolines 6g and 6i exhibited
significant inhibition of antiproliferative activity with IC50 values between 2.8 and 10.9 nM toward
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human cancer cells. Interestingly, 6g did not show significant inhibition toward HEK293 cells at 100
nM. Although 3’,4’ꢀdimethoxyꢀsubstituted 6ꢀmethoxyanilinoquinazoline 6k showed weak inhibitory
effects similar to compound 6j, 3’,5’ꢀdimethoxyꢀsubstituted 6ꢀmethoxyanilinoquinazoline 6l
exhibited potent inhibition of antiproliferative activity toward HeLa, PC3, HCT116, and HEK293
cells with IC50 = 3.2, 7.0, 7.4, and 4.6 nM, respectively. The best results were obtained in the case of
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compound 6m (GN39482), which has a methoxycarbonyl group at the R position of
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ꢀmethoxyanilinoquinazoline and the IC50 values toward HeLa, PC3, HCT116, and HEK293 cells
were 2.47, 2.64, 2.7 and 2.2 nM, respectively. The carboxylic acid derivative 6n did not show
significant inhibition probably due to the difficulty of cellꢀmembrane penetration. A bulky
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substituent such as an isobutyl group (6o) at the R position of 6ꢀmethoxyanilinoquinazoline also
diminished inhibitory effects on the antiproliferative activity. Needless to say, a 6ꢀmethoxy group of
anilinoquinazolines is essential for the significant inhibitory potency (6p vs. 6q; 6i vs. 6r).
(Table 2)
Because compound 6m was found to possess a promising antiproliferative activity toward human
cancer cells, we next examined antimicrobial activities against Staphylococcus aureus 209,
Escherichia coli HO141, Candida albicans JCM1542, Aspergillus fumigatus Af293, and
Magnaporte oryzae kitaꢀ1 and antiꢀmalarial activity against Plasmodium falciparum 3D7 by
compound 6m. Recently, antimicrobacterial activities, such as an antitubercular activity, of
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indenopyrazoles have been reported.
Interestingly, compound 6m did not display any
antimicrobial and antiꢀmalarial activities at 100 nM (data not shown). These results indicate that
compound 6m possesses selective inhibition toward human cancer cells. We also investigated the
effect of compound 6m on inhibitory activity for EGFR, KDR, PDGFRβ, CDK2/CycE1,
CDK4/CycD3 and CHK1 kinases to confirm whether 6m has a potential to inhibit these kinases that
have been reported as targets of indenopyrazoles in Figure 1. In vitro kinase inhibition assays
revealed that compound 6m showed inhibitory activities toward KDR, PDGFRβ, and CDK2/CycE1
at 10 µM (>82% inhibition). However significant inhibition of compound 6m toward these kinases
was not observed at 0.1 µM (See Table S2 in the supporting information).
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To clarify the action mechanism of compound 6m in the antiproliferation of human cancer cell
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lines, we next analyzed MorphoBase profiling
and ChemProteoBase profiling
of compound
6m. The results of MorphoBase profiling were summarized in Figure 2. Following treatment with
compound 6m, the nuclei were stained with Hoechst33342 and the resulting morphological changes
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of src ꢀNRK and HeLa cells were segmented and quantified by the IN Cell Analyzer. The
phenotypic multiparameters generated were compared with the reference dataset—a compilation of
the morphological features induced by 225 compounds—and subjected to statistical analyses, i.e.,
principal component analysis (PCA), probability scores, and ranking of the top 15 nearest neighbors.
Regarding the projection of PCA scores, the phenotypic response of compound 6m was visible in the
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cloud of tubulin inhibitors in the PC1ꢀPC2 scatter plots. Based on the similarity analysis, compound
6m was also predicted to perturb microtubule dynamics (Scoretubulin = 1.68) and the typical tubulin
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inhibitors,
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ꢀchloroꢀ5ꢀ(2,5ꢀdimethoxyphenyl)ꢀ1,3ꢀdihydroꢀ2Hꢀbenzofuro[3,2ꢀe][1,4]diazepinꢀ2ꢀone (NPD8617),
(
E)ꢀ1ꢀ(6,7ꢀdimethoxyꢀ3,4ꢀdihydroisoquinolinꢀ2(1H)ꢀyl)ꢀ3ꢀ(furanꢀ2ꢀyl)propꢀ2ꢀenꢀ1ꢀone (NPD6689),
vinblastine, and methyl 6ꢀ(furanꢀ2ꢀyl)ꢀ3ꢀmethylꢀ4ꢀoxoꢀ4,5,6,7ꢀtetrahydrobenzofuranꢀ2ꢀcarboxylate
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(
NPD8969), were listed among most of its 15 closest neighbors. These data indicate that the
putative molecular target of 6m is the microtubule system.
(
Figure 2)
To verify the predictions, compound 6m was subjected to ChemProteoBase profiling. The
proteomic variation of 296 spots that matched on all gel images was quantified, cosine similarities of
compounds in database against compound 6m was calculated and hierarchical cluster analysis was
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performed as previously described. As is expected by the MorphoBase profiling, tubulin inhibitors
were the most similar compounds to 6m among 87 compounds in the ChemProteoBase (Figure 3). In
hierarchical cluster analysis with 41 standard compounds, compound 6m was clustered in the cluster
with tubulin inhibitors and a TOP2 catalytic inhibitor, which is a different cluster of other type of
inhibitors in the database, such as VꢀATPase inhibitors and proteasome inhibitors (Figure 3).
(
Figure 3)
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Since tubulins have been suggested as one of the target proteins of compound 6m by both
MorphoBase profiling and ChemProteoBase profiling analyses, we next examined effect of
compound 6m on tubulin polymerization in vitro. Vinblastine (10 µM) and paclitaxel (2 µM) were
used as positive controls, respectively. As shown in Figure 4, compound 6m inhibited tubulin
polymerization in a concentrationꢀdependent manner in a range of 3ꢀ30 µM. It is known that tubulin
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inhibitors induced G2/M cell cycle arrest. Therefore, we examined the effect of compound 6m on
the cell cycle in HeLa cells. HeLa cells were treated with compounds at various concentrations for
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4 h and stained with propidium iodide (PI) and RNase A for 30 min. The analysis of the DNA
content of the cells was performed by flow cytometry. As shown in Figure 5, compound 6m arrested
cell cycles at G2/M stage similar to colchicine known as a tubulin polymerization inhibitor. It should
be noted that cell cycles were not affected by compound 6p which was used as a negative control.
(Figure 4)
(Figure 5)
We next examined the western blotting analysis of acetylated tubulins and the immunofluorescence
of microtubules to confirm whether compound 6m inhibits tubulin polymerization in cells. It is
known that acetylation of Lys 40 in αꢀtubulin is necessary process for microtubule formation.
Therefore, effects of compound 6m on acetylated tubulin accumulation and microtubule dynamics
were examined by western blot analysis and immunofluorescence with the specific tubulin antibody
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using HeLa cells, respectively. The results are shown in Figure 6. Colchicine, a positive control,
arrested acetylated tubulin accumulation in a range of 30ꢀ100 nM. Interestingly, compound 6m
inhibited the acetylated tubulin accumulation in a similar manner to colchicine (Figure 6a). We also
examined the effects of compounds 6o, 2, and 6q on acetylated tubulin accumulation. Compound 6o,
which exhibited a 10ꢀfold weaker proliferative activity than compound 6m, inhibited the acetylated
tubulin accumulation at 300 nM. However, compounds 2 and 6q did not display significant
inhibition of the acetylated tubulin accumulation even at 1000 nM.
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Figure 6b shows immunofluorescence analysis of microtubule formation using the tubulinꢀspecific
antibody. Colchicine inhibited microtubule formation, whereas paclitaxel did not affect the
microtubule formation at 100 nM. It is known that paclitaxel stabilizes microtubule formation and
arrests tubulin depolymerization process. Compound 6m exhibited microtubule formation similar to
colchicine at 30 nM. Compound 6o, which inhibited acetylated tubulin accumulation at 300 nM, also
exhibited microtubule formation at the same concentration (300 nM). However, compound 2, which
did not affect acetylated tubulin accumulation at 1,000 nM, showed no effect on microtubule
formation at the same concentration (1,000 nM). These results suggest that compound 6m inhibits
antiproliferative activity toward human cancer cells probably by inhibiting tubulin polymerization
process in the cells. Compound 6o also inhibited acetylated tubulin accumulation and microtubule
formation at tenꢀfold higher concentration (300 nM) than compound 6m and this is reflected in the
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antiproliferative effect by compounds 6m and 6o. Indeed, their IC50 values toward HeLa cells are
2.47 and 19.95 nM, respectively.
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(Figure 6)
Conclusion
We developed compound 6m as a potent cell growth inhibitor by SAR based on the structure of
1
2
indenopyrazole 2. Both a methoxy group at R position and a methoxycarbonyl group at R position
of the anilinoquinazoline framework are essential for the high cell growth inhibition. Compound
6
m was found to possess a promising antiproliferative activity toward human cancer cells without
affecting any antiꢀmicrobial and antiꢀmalarial activities at 100 nM. We clarified the action
mechanism of cell growth inhibition induced by compound 6m using MorphoBase and
ChemProteoBase profiling methods. MorphoBase profiling analysis revealed that the phenotypic
response of compound 6m was visible in the cloud of tubulin inhibitors in the PC1ꢀPC2 scatter plots,
predicting to perturb microtubule dynamics (Scoretubulin = 1.68). ChemProteoBase profiling analysis
also suggested that tubulin inhibitors were the most similar compounds to 6m among 87 compounds
in the database. Indeed, compound 6m inhibited tubulin polymerization in
a
concentrationꢀdependent manner and induced G2/M cell cycle arrest in HeLa cells. Furthermore,
compound 6m inhibited the acetylated tubulin accumulation and the microtubule formation in a
similar manner to colchicine in HeLa cells, revealing that a promising antiproliferative activity of
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compound 6m toward human cancer cells is probably caused by the tubulin polymerization
inhibition. Although indenopyrazoles have been reported as central nervous system agents, kinase
inhibitors, and antibacterial agents, this is the first to observe tubulin polymerization inhibition by
indenopyrazoles. The current findings suggest that the indenopyrazole framework is a possible
candidate for development of biologically active molecules in the pharmaceutical drug design.
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Experimental section
General Information
Analytical thin layer chromatography (TLC) was performed on a glass plates of silica gel 60 GF254
(
Merck), which were visualized by the quenching of UV fluorescence (254 nm), and/or by an
4
aqueous alkaline KMnO solution followed by heating. Column chromatography was conducted on
silica gel (Merck Kieselgel 70ꢀ230 mesh). Most commercially supplied chemicals were used without
1
13
further purification. H and C NMR spectra were recorded on a Bruker biospin AVANCE II (400
MHz) or a VARIAN UNITYꢀINOVA 400 (400 MHz) spectrometer. The chemical shifts are reported
in δ units relative to internal tetramethylsilane. IR spectra were recorded on a JASCO FT/IRꢀ4100
spectrometer. Liquid chromatogram mass spectrometer was recorded on Shimadzu LCMSꢀ2010EV.
Highꢀresolution mass spectra (ESI) were recorded on a Bruker Daltonics micro TOFꢀ15 focus. Purity
of all final compounds was determined by HPLC analysis with a Hiber LiChrosorb Si60 5 ꢁm
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(CicaꢀMERCK) with UV detection at 254 nm or elemental analysis performed by a CE instrument
EA1110 CHNSꢀO automatic elemental analyser, with the purity all being higher than 95%.
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tert-Butyl(2-isothiocyanatophenoxy)dimethylsilane (4a)
A
mixture of 2ꢀ((tertꢀbutyldimethylsilyl)oxy)aniline (0.208 g, 0.93 mmol) and phenyl
chlorothionoformate (0.065 mL, 0.47 mmol) in THF (5 mL) was stirred 2h at room temperature. The
reaction was quenched with water and the mixture was extracted with EtOAc. The organic layer was
2 4
washed with water and brine, dried over anhydrous Na SO , and concentrated. To the residue
3
dissolved in toluene (5 mL) were added TEA (0.13 mL, 0.9 mmol) and Cl SiH (0.09 mL, 0.9 mmol)
2
under N and the mixture was stirred for 2 h at room temperature. The reaction was quenched with
water and the precipitate was filtered off. The mixture was extracted with EtOAc and the organic
2 4
layer was washed with water and brine, dried over anhydrous Na SO , and concentrated. The residue
was
purified
by
column
chromatography
with
hexane
to
give
1
tertꢀbutyl(2ꢀisothiocyanatophenoxy)dimethylsilane (4a) as a colorless oil, quantitatively. H NMR
1
3
(
(
400 MHz, CDCl
3
): δ 7.16ꢀ7.11 (2H, m), 6.92ꢀ6.87 (2H, m), 1.03 (9H, s), 0.29 (6H, s); CNMR
100 MHz, CDCl
3
): δ 152.1, 136.6, 128.1, 126.3, 123.0, 121.5, 120.0, 25.7, 18.4, ꢀ4.2; IR (NaCl)
ꢀ1
3
734, 2930, 2858, 2360, 2076, 1507, 1490, 1472, 1456, 1289, 1255, 1225, 1109 cm ; HRMS (ESI,
+
positive) m/z calcd. for C13
H
19NOSSi [M+Na] : 288.0849, found: 288.0848.
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tert-Butyl(3-isothiocyanatophenoxy)dimethylsilane (4b)
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This compound was prepared from 3ꢀ((tertꢀbutyldimethylsilyl)oxy)aniline (0.929 g, 4.2 mmol),
3
chlorothionoformate (0.29 mL, 2.1 mmol), TEA (0.47 mL, 3.4 mmol), and Cl SiH (0.34 mL, 3.4
mmol) using the procedure described for 4a. Purification by column chromatography
(
Hexane:EtOAc = 100:1 v/v) gave tertꢀbutyl(3ꢀisothiocyanatophenoxy)dimethylsilane (4b) as a
1
colorless oil, quantitatively. H NMR (400 MHz, CDCl
3
): δ 7.18 (1H, dd, J = 8.0, 8.4 Hz), 6.84 (1H,
13
d, J = 8.0 Hz), 6.76 (1H, d, J = 8.0 Hz), 6.69 (1H, s), 0.98 (9H, s), 0.21 (6H, s); CNMR (100 MHz,
3
CDCl ): δ 156.5, 135.1, 131.9, 130.1, 119.5, 118.9, 117.4, 25.6, 18.1, ꢀ4.2; IR (NaCl) 3734, 2955,
ꢀ1
2
930, 2858, 2360, 2112, 1592, 1486, 1472, 1294, 1254, 1184, 1153, 1006 cm ; HRMS (ESI,
+
positive) m/z calcd. for C13H19NOSSi [M+H] : 266.1029, found: 266.1036.
1-Isothiocyanato-3,5-dimethoxybenzene (4c)
This compound was prepared from 3,5ꢀdimethoxyaniline (0.153 g,
chlorothionoformate (0.07 mL, 0.5 mmol), TEA (0.11 mL, 0.77 mmol), and Cl
1 mmol), phenyl
3
SiH (0.08 mL, 0.77
mmol) using the procedure described for 4a. Purification by column chromatography
(
(
hexane:EtOAc = 80:1 v/v) gave 1ꢀisothiocyanatoꢀ3,5ꢀdimethoxybenzene (4c) as a white solid
1
0.0896 g, 0.49 mmol, 96%). mp: 48ꢀ49 ºC; H NMR (400 MHz, CDCl
3
): δ 6.37 (3H, s), 3.77 (3H,
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6
1
3
3
s); CNMR (100 MHz, CDCl ): δ 161.1, 135.4, 132.5, 104.0, 100.3, 55.5; IR (NaCl) 3099, 3006,
ꢀ1
2967, 2941, 2840, 2360, 2147, 1598, 1458, 1422, 1353, 1304, 1208, 1192, 1156, 1062, 1006 cm ;
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2
3
4
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7
8
9
0
+
HRMS (ESI, positive) m/z calcd. for C
9
H
9
NO
2
S [M+H] : 196.0427, found: 196.0432.
Methyl 3-isothiocyanatobenzoate (4d)
This compound was prepared from methyl 3ꢀaminobenzoate (0.151 g, 1 mmol), chlorothionoformate
(
3
0.07 mL, 0.5 mmol), TEA (0.17 mL, 1.2 mmol), and Cl SiH (0.12 mL, 1.2 mmol) using the
procedure described for 4a. Purification by column chromatography (hexane:EtOAc = 50:1 v/v)
1
gave methyl 3ꢀisothiocyanatobenzoate (4d) as a colorless oil, quantitatively. H NMR (400 MHz,
1
3
3
CDCl ): δ 7.91 (1H, d, J = 7.2 Hz), 7.89 (1H, s), 7.46ꢀ7.38 (2H, m), 3.94 (3H, s); CNMR (100
3
MHz, CDCl ): δ 165.3, 137.1, 131.7, 129.6, 128.1, 126.6, 52.4; IR (NaCl) 3734, 2955, 2360, 2090,
ꢀ1
1727, 1582, 1443, 1292, 1224, 1104, 1077, 1000 cm ; HRMS (ESI, positive) m/z calcd. for
+
C
9
H
7
NO
2
S [M+H] : 194.0270, found: 194.0266.
1
-Isobutoxy-3-isothiocyanatobenzene (4e)
This compound was prepared from 3ꢀisobutoxyaniline (0.151 g, 1 mmol), chlorothionoformate (0.05
3
mL, 0.38 mmol), TEA (0.15 mL, 1.1 mmol), and Cl SiH (0.11 mL, 1.1 mmol) using the procedure
described for 4a. Purification by column chromatography (hexane:EtOAc = 100:1 v/v) gave
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1ꢀisobutoxyꢀ3ꢀisothiocyanatobenzene (4e) as a light yellow oil, quantitatively. H NMR (400 MHz,
3
CDCl ): δ 7.22 (1H, dd, J = 8.0, 8.0 Hz), 6.83ꢀ6.79 (2H, m), 6.74 (1H, s), 3.70 (2H, d, J = 6.4 Hz),
10
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1
3
3
2.13ꢀ2.03 (1H, m), 1.02 (6H, d, J = 6.4 Hz); CNMR (100 MHz, CDCl ): δ 160.0, 135.3, 132.0,
1
1
2
30.1, 117.8, 114.2, 111.7, 74.6, 28.2, 19.2; IR (NaCl) 2959, 2872, 2100, 1600, 1488, 1470, 1394,
ꢀ1
+
313, 1287, 1267, 1185, 1155, 1038 cm ; HRMS (ESI, positive) m/z calcd. for C11H13NOS [M+H] :
08.0791, found: 208.0796.
6
-Ethoxy-N-phenyl-1,4-dihydroindeno[1,2-c]pyrazol-3-amine (6a)
To a mixture of 5ꢀethoxyꢀ2,3ꢀdihydroꢀ1Hꢀindenꢀ1ꢀone (0.103 g, 0.58 mmol) and phenyl
isothiocyanate (0.07 mL, 0.58 mmol) in THF was added LiHMDS, which was prepared from 1.6 M
hexane solution of nꢀBuLi (0.36 mL, 0.58 mmol) and HMDS (0.12 mL, 0.58 mmol) with stirring at
0 ºC for 30 min, dropwose at room temperature and the mixture was stirred for 12 h. Hydrazine (0.03
mL, 0.58 mmol) and acetic acid (0.03 mL) were added and the reaction mixture was refluxed for 24
h. The reaction was quenched with water and the mixture was extracted with EtOAc. The organic
3 2 4
layer was washed with aqueous NaHCO , water, and brine, dried over anhydrous Na SO , and
2 2
concentrated. The residue was purified by column chromatography with CH Cl to give
6
0
ꢀethoxyꢀNꢀphenylꢀ1,4ꢀdihydroindeno[1,2ꢀc]pyrazolꢀ3ꢀamine (6a) as a yellow solid (0.0825 g,
1
.28 mmol, 48%). mp: 74ꢀ75 ºC; H NMR (400 MHz, CDCl
3
): δ 7.38 (1H, d, J = 8.0 Hz), 7.21 (2H,
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2
2
2
2
2
2
2
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dd, J = 7.6, 8.4 Hz), 6.94 (1H, s), 6.90ꢀ6.85 (3H, m), 6.72 (1H, d, J = 8.4 Hz), 4.00 (2H, q, J = 7.2
13
3
Hz), 3.33 (2H, s), 1.41 (3H, t, J = 7.2 Hz); CNMR (100 MHz, CDCl ): δ 158.2, 155.3, 150.7, 143.1,
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2
3
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7
8
9
0
1
2
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7
8
9
0
141.8, 129.2, 126.2, 120.1, 120.0, 116.1, 112.8, 112.4, 110.9, 63.6, 30.4, 14.9; IR (NaCl) 3734, 3053,
ꢀ1
2
979, 2900, 2360, 1600, 1540, 1508, 1497, 1464, 1394, 1309, 1241, 1111, 1043 cm ; HRMS (ESI,
+
positive) m/z calcd. for C18
H
17
N
3
O [M+H] : 292.1444, found: 292.1445; HPLC purity: 99.7%,
2 2
retention time: 6.96 min, The eluents were mixture of MeOH and CH Cl (1:4).
6
-((tert-Butyldimethylsilyl)oxy)-N-phenyl-1,4-dihydroindeno[1,2-c]pyrazol-3-amine (6b)
This compound was prepared from 5ꢀ((tertꢀbutyldimethylsilyl)oxy)ꢀ2,3ꢀdihydroꢀ1Hꢀindenꢀ1ꢀone (3)
(
0.249 g, 0.95 mmol) and phenyl isothiocyanate (0.11 mL, 0.95 mmol)using the procedure described
for 6a. Purification by column chromatography (hexane:EtOAc
=
2:1 v/v) gave
6ꢀ((tertꢀbutyldimethylsilyl)oxy)ꢀNꢀphenylꢀ1,4ꢀdihydroindeno[1,2ꢀc]pyrazolꢀ3ꢀamine (6b) as a light
1
brown solid (0.197 g, 0.52 mmol, 55%). mp: 93ꢀ94 ºC; H NMR (400 MHz, CDCl
3
): δ 7.42 (1H, d, J
=
8.4 Hz), 7.28ꢀ7.25 (3H, m), 6.99 (2H, d, J = 8.0 Hz), 6.94 (1H, s), 6.91 (1H, dd, J = 7.6, 7.2 Hz),
1
3
6
.78 (1H, d, J = 8.0 Hz), 6.01 (1H, bs), 3.41 (2H, s), 0.99 (9H, s), 0.21 (6H, s); CNMR (100 MHz,
3
CDCl ): δ 155.3, 154.9, 150.7, 143.1, 141.9, 129.2, 126.8, 120.08, 120.05, 118.5, 117.9, 116.1, 111.4,
3
1
0.2, 25.7, 18.2, ꢀ4.4; IR (NaCl) 3181, 3051, 2955, 2929, 2896, 2858, 2360, 1600, 1530, 1497, 1463,
ꢀ1
444, 1418, 1396, 1281, 1250, 1086 cm ; HRMS (ESI, positive) m/z calcd. for C22
H
27
N
3
OSi
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+
[M+H] : 378.1996, found: 378.1994.
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3-(Phenylamino)-1,4-dihydroindeno[1,2-c]pyrazol-6-ol (6c)
To a solution of 6b (0.17 g, 0.45 mmol) in MeOH (10 mL) was added TBAF (1M solution of THF,
0
.81 mL, 0.81 mmol) and the mixture was stirred 2 h at room temperature. The reaction mixture was
4
extracted with EtOAc and the organic layer was washed with aqueous NH Cl, water, and brine, dried
over anhydrous Na
2
SO
4
, and concentrated. The residue was purified by column chromatography
(
CH Cl :MeOH = 50:1 v/v) to give 3ꢀ(phenylamino)ꢀ1,4ꢀdihydroindeno[1,2ꢀc]pyrazolꢀ6ꢀol (6c) as a
2 2
1
light yellow solid (0.114 g, 0.43 mmol, 96%). mp: 150ꢀ151 ºC; H NMR (400 MHz, CD
3
OD): δ 7.41
(
1H, d, J = 8.4 Hz), 7.21 (2H, dd, J = 7.6, 7.6 Hz), 7.00 (2H, d, J = 7.6 Hz), 6.91 (1H, s), 6.80 (1H,
13
dd, J = 7.2, 7.6 Hz), 6.75 (1H, d, J = 8.4 Hz), 3.38 (2H, s), 3.34 (1H, s); CNMR (100 MHz,
3
CD OD): δ 156.6, 155.6, 151.0, 143.7, 141.6, 128.7, 125.3, 119.6, 119.3, 115.4, 113.4, 113.0, 110.3,
ꢀ1
29.3; IR (KBr) 3376, 3052, 2896, 2810, 2362, 1620, 1600, 1528, 1497, 1449, 1243, 1104, 1082 cm ;
+
HRMS (ESI, positive) m/z calcd. for C16
13
H N
3
O [M+H] : 264.1131, found: 264.1132; HPLC purity:
9
2 2
8.3%, retention time: 7.69 min (MeOH:CH Cl = 1:4).
N-(2-((tert-butyldimethylsilyl)oxy)phenyl)-6-methoxy-1,4-dihydroindeno[1,2-c]pyrazol-3-amin
e (6d)
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This compound was prepared from 5ꢀmethoxyꢀ1ꢀindanone (0.078 g, 0.48 mmol) and
tertꢀbutyl(2ꢀisothiocyanatophenoxy)dimethylsilane (4a) (0.127 g, 0.48 mmol) using the procedure
described for 6a. Purification by column chromatography with (hexane:EtOAc = 5:1 v/v) gave
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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8
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Nꢀ(2ꢀ((tertꢀbutyldimethylsilyl)oxy)phenyl)ꢀ6ꢀmethoxyꢀ1,4ꢀdihydroindeno[1,2ꢀc]pyrazolꢀ3ꢀamine
1
(
6d) as a yellow solid (0.0565 g, 0.14 mmol, 29%). mp: 72ꢀ73 ºC; H NMR (400 MHz, CDCl ): δ
3
7
8
.51 (1H, d, J = 8.0 Hz), 7.36 (1H, d, J = 6.8 Hz), 7.04 (1H, s), 6.95ꢀ6.83 (3H, m), 6.76 (1H, dd, J =
13
.0, 7.2 Hz), 6.26 (1H, bs), 3.85 (3H, s), 3.48 (2H, s), 1.06 (9H, s), 0.28 (6H, s); CNMR (100 MHz,
3
CDCl ): δ 158.9, 153.9, 150.7, 143.2, 142.7, 134.9, 126.2, 121.9, 120.0, 119.5, 118.1, 115.2, 112.2,
112.1, 111.9, 55.5, 29.8, 25.9, 18.3, ꢀ4.3; IR (NaCl) 3423, 3063, 2929, 2857, 2360, 1598, 1537, 1495,
ꢀ1
1
29 3 2
460, 1283, 1250, 1106, 1082, 1035 cm ; HRMS (ESI, positive) m/z calcd. for C23H N O Si
+
[M+H] : 408.2102, found: 408.2105;
2-((6-Methoxy-1,4-dihydroindeno[1,2-c]pyrazol-3-yl)amino)phenol (6e)
This
compound
was
prepared
from
Nꢀ(2ꢀ((tertꢀbutyldimethylsilyl)oxy)phenyl)ꢀ6ꢀmethoxyꢀ1,4ꢀdihydroindeno[1,2ꢀc]pyrazolꢀ3ꢀamine
(
6d) (0.057 g, 0.14 mmol) and TBAF (1M in THF; 0.25 mL, 0.25 mmol) using the procedure
described for 6c. Purification by column chromatography with (CH Cl :MeOH = 50:1 v/v) gave
2
2
2
ꢀ((6ꢀmethoxyꢀ1,4ꢀdihydroindeno[1,2ꢀc]pyrazolꢀ3ꢀyl)amino)phenol (6e) as a dark green solid
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5
6
7
8
9
1
(0.0195 g, 0.066 mmol, 47%). mp: 196ꢀ197 ºC; H NMR (400 MHz, CD
3
OD): δ 7.50 (1H, d, J = 8.0
Hz), 7.10 (1H, d, J = 7.6 Hz), 7.05 (1H, s), 6.88 (1H, d, J = 8.4 Hz), 6.84ꢀ6.72 (3H, m), 3.81 (3H, s),
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
3
3
3.44 (2H, s); CNMR (100 MHz, CD OD): δ 159.3, 155.5, 150.8, 145.6, 142.0, 131.7, 126.3, 120.1,
1
19.6, 119.4, 115.6, 114.4, 112.1, 111.6, 109.8, 54.5, 29.5; IR (KBr) 3368, 3241, 2830, 2682, 2499,
ꢀ1
1
589, 1510, 1471, 1400, 1310, 1286, 1246, 1112, 1090, 1038 cm ; HRMS (ESI, positive) m/z calcd.
+
15
for C17H N
3
O
2
[M+H] : 294.1237, found: 294.1241; HPLC purity: 98.8%, retention time: 10.65
2 2
min (MeOH:CH Cl = 1:1).
N-(3-((tert-butyldimethylsilyl)oxy)phenyl)-6-methoxy-1,4-dihydroindeno[1,2-c]pyrazol-3-amin
e (6f)
This compound was prepared from 5ꢀmethoxyꢀ1ꢀindanone (0.224 g, 1.38 mmol) and
tertꢀbutyl(3ꢀisothiocyanatophenoxy)dimethylsilane (4b) (0.366 g, 1.38 mmol) using the procedure
described for 6a. Purification by column chromatography with (hexane:EtOAc = 2:1 v/v) gave
Nꢀ(3ꢀ((tertꢀbutyldimethylsilyl)oxy)phenyl)ꢀ6ꢀmethoxyꢀ1,4ꢀdihydroindeno[1,2ꢀc]pyrazolꢀ3ꢀamine
1
(
6f) as a yellow solid (0.386 g, 0.946 mmol, 68%). mp: 136ꢀ137 ºC; H NMR (400 MHz, CDCl ):
3
δ7.50 (1H, d, J = 8.0 Hz), 7.10 (1H, dd, J = 8.0, 8.0 Hz), 7.03 (1H, s), 6.87 (1H, d, J = 6.8 Hz), 6.61
(
1H, d, J = 6.8 Hz), 6.52 (1H, s), 6.40 (1H, d, J = 8.0 Hz), 5.94 (1H, bs), 3.85 (3H, s), 3.47 (2H, s),
13
0
.97 (9H, s), 0.19 (6H, s); CNMR (100 MHz, CDCl
3
): δ 158.9, 156.7, 155.1, 150.7, 144.4, 141.8,
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
129.8, 126.3, 120.1, 112.1, 111.9, 111.8, 109.4, 108.0, 55.4, 30.4, 25.7, 18.1, ꢀ4.4; IR (NaCl) 3063,
ꢀ1
2954, 2929, 2858, 2360, 1600, 1524, 1490, 1471, 1310, 1283, 1250, 1181, 1155, 1083, 1035 cm ;
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
HRMS (ESI, positive) m/z calcd. for C23
H
29
N
3
O
2
Si [M+H] : 408.2102, found: 408.2104;
3
-((6-Methoxy-1,4-dihydroindeno[1,2-c]pyrazol-3-yl)amino)phenol (6g)
This
compound
was
prepared
from
Nꢀ(3ꢀ((tertꢀbutyldimethylsilyl)oxy)phenyl)ꢀ6ꢀmethoxyꢀ1,4ꢀdihydroindeno[1,2ꢀc]pyrazolꢀ3ꢀamine
(
6f) (0.386 g, 0.94 mmol) and TBAF (1M in THF; 1.69 mL, 1.69 mmol) using the procedure
described for 6c. Purification by column chromatography with (CH Cl :MeOH = 50:1 v/v) gave
2
2
3
ꢀ((6ꢀmethoxyꢀ1,4ꢀdihydroindeno[1,2ꢀc]pyrazolꢀ3ꢀyl)amino)phenol (6g) as
a
white solid,
1
quantitatively. mp: 201ꢀ202 ºC; H NMR (400 MHz,CD
3
OD): δ 7.52 (1H, d, J = 8.0 Hz), 7.09 (1H,
s), 7.05 (1H, dd, J = 8.0, 8.0 Hz), 6.90 (1H, d, J = 8.4 Hz), 6.52ꢀ6.49 (2H, m), 6.31 (1H, d, J = 8.0
13
3
Hz), 3.83 (3H, s), 3.47 (2H, s); CNMR (100 MHz, CD OD): δ 159.3, 157.9, 155.5, 150.9, 145.1,
141.2, 129.5, 126.4, 119.4, 112.1, 111.6, 110.7, 107.2, 106.4, 102.3, 54.6, 29.5; IR (KBr) 3402, 3347,
ꢀ1
3
058, 2944, 2900, 2826, 2703, 1615, 1600, 1543, 1463, 1267, 1250, 1157, 1108, 1075, 1039 cm ;
+
HRMS (ESI, positive) m/z calcd. for C17
H
15
3
N O
2
[M+H] : 294.1237, found: 294.1245; HPLC
2 2
purity: 99.9%, retention time: 10.65 min (MeOH:CH Cl = 1:1).
6
-Methoxy-N-(2-methoxyphenyl)-1,4-dihydroindeno[1,2-c]pyrazol-3-amine (6h)
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9
This compound was prepared from 5ꢀmethoxyꢀ1ꢀindanone (0.08 g, 0.5 mmol) and
1ꢀisothiocyanatoꢀ2ꢀmethoxybenzene (0.082 g, 0.5 mmol) using the procedure described for 6a.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Purification by column chromatography with (hexane:EtOAc
=
2:1 v/v) gave
6
ꢀmethoxyꢀNꢀ(2ꢀmethoxyphenyl)ꢀ1,4ꢀdihydroindeno[1,2ꢀc]pyrazolꢀ3ꢀamine (6h) as a light yellow
1
3
solid (0.0615 g, 0.2 mmol, 40%). mp: 182ꢀ183 ºC; H NMR (400 MHz, CDCl ): δ 7.55 (1H, d, J =
8
.4 Hz), 7.06 (1H, s), 6.97ꢀ6.86 (5H, m), 6.39 (1H, bs), 3.94 (3H, s), 3.87 (3H, s), 3.53 (2H, s);
13
3
CNMR (100 MHz, CDCl ): δ159.0, 155.0, 150.7, 147.3, 141.6, 133.0, 126.5, 121.1, 120.0, 119.5,
1
1
3
14.3, 112.4, 112.0, 112.0, 110.2, 55.6, 55.5, 30.1; IR (NaCl) 3064, 2933, 2834, 2360, 1600, 1532,
ꢀ1
+
17 3 2
461, 1310, 1283, 1245, 1112, 1030 cm ; HRMS (ESI, positive) m/z calcd. for C18H N O [M+H] :
08.1394, found: 308.1399; HPLC purity: 99.9%, retention time: 6.91 min, The eluents were
2 2
mixture of MeOH and CH Cl (1:9).
6-Methoxy-N-(3-methoxyphenyl)-1,4-dihydroindeno[1,2-c]pyrazol-3-amine (6i)
This compound was prepared from 5ꢀmethoxyꢀ1ꢀindanone (0.1 g, 0.62 mmol) and 3ꢀmethoxyphenyl
isothiocyanate (0.087 mL, 0.62 mmol) using the procedure described for 6a. Purification by column
chromatography
with
(CH
2
Cl
2
:MeOH
=
30:1
v/v)
gave
6ꢀmethoxyꢀNꢀ
(
3ꢀmethoxyphenyl)ꢀ1,4ꢀdihydroindeno[1,2ꢀc]pyrazolꢀ3ꢀamine (6i) as a white solid (0.113 g, 0.37
1
mmol, 60%). mp: 153ꢀ154 ºC; H NMR (400 MHz, CDCl
3
): δ 7.50 (1H, d, J = 8.4 Hz), 7.19 (1H, dd,
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1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
J = 8.4, 8.4 Hz), 7.04 (1H, s), 6.88 (1H, d, J = 8.4 Hz), 6.60ꢀ6.59 (2H, m), 6.49 (1H, d, J = 7.6 Hz),
13
3
6.07 (1H, bs), 3.86 (3H, s), 3.79 (3H, s), 3.49 (2H, s); CNMR (100 MHz, CDCl ): δ 160.7, 158.9,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
155.0, 150.7, 144.5, 141.7, 130.0, 126.2, 120.0, 112.3, 111.9, 111.7, 108.6, 105.6, 101.7, 55.5, 55.2,
3
1
0.3; IR (NaCl) 3004, 2904, 2834, 2360, 1601, 1524, 1494, 1310, 1284, 1248, 1202, 1158, 1082,
ꢀ1
+
17 3 2
034 cm ; HRMS (ESI, positive) m/z calcd. for C18H N O [M+H] : 308.1394, found: 308.1389;
2 2
HPLC purity: 97.7%, retention time: 7.74 min (MeOH: CH Cl = 1:9).
6
-Methoxy-N-(4-methoxyphenyl)-1,4-dihydroindeno[1,2-c]pyrazol-3-amine (6j)
This compound was prepared from 5ꢀmethoxyꢀ1ꢀindanone (0.2 g, 0.1.23 mmol) and
4
ꢀmethoxyphenyl isothiocyanate (0.17 mL, 1.23 mmol) using the procedure described for 6a.
Purification by column chromatography with (hexane:EtOAc
=
1:1 v/v) gave
6ꢀmethoxyꢀNꢀ(4ꢀmethoxyphenyl)ꢀ1,4ꢀdihydroindeno[1,2ꢀc]pyrazolꢀ3ꢀamine (6j) as a white solid
1
(0.141 g, 0.46 mmol, 37%). mp: 137ꢀ138 ºC; H NMR (400 MHz, CDCl
3
): δ7.50 (1H, d, J = 8.4 Hz),
7
.05ꢀ7.03 (3H, m), 6.89ꢀ6.87 (3H, m), 5.81 (1H, bs), 3.86 (3H, s), 3.82 (3H, s), 3.40 (2H, s);
1
3
3
CNMR (100 MHz, CDCl ): δ 158.8, 155.5, 154.2, 150.8, 143.3, 136.5, 126.5, 120.1, 118.8, 114.5,
1
1
3
12.1, 111.8, 109.6, 55.6, 55.4, 30.3; IR (NaCl) 3182. 2903, 2832, 2360, 1615, 1509, 1466, 1310,
ꢀ1
+
282, 1240, 1179, 1082, 1034 cm ; HRMS (ESI, positive) m/z calcd. for C18
H
17
N
3
O
2
[M+H] :
08.1394, found: 308.1393; HPLC purity: 97.9%, retention time: 11.04 min (MeOH:CH
Cl
2 2
= 3:97).
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N-(3,4-dimethoxyphenyl)-6-methoxy-1,4-dihydroindeno[1,2-c]pyrazol-3-amine (6k)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
This compound was prepared from 5ꢀmethoxyꢀ1ꢀindanone (0.2 g, 0.123 mmol) and
3
,4ꢀdimethoxyphenyl isothiocyanate (0.24 g, 1.23 mmol) using the procedure described for 6a.
Purification by column chromatography with (CH
Cl
2 2
:MeOH
=
100:1 v/v) gave
Nꢀ(3,4ꢀdimethoxyphenyl)ꢀ6ꢀmethoxyꢀ1,4ꢀdihydroindeno[1,2ꢀc]pyrazolꢀ3ꢀamine (6k) as a light
1
brown solid (0.202 g, 0.6 mmol, 49%). mp: 102ꢀ103 ºC; H NMR (400 MHz, CDCl
3
): δ 7.51 (1H, d,
J = 8.4 Hz), 7.04 (1H, s), 6.89 (1H, d, J = 8.4 Hz), 6.83 (1H, d, J = 8.4 Hz), 6.71 (1H, s), 6.61 (1H, d,
13
J = 8.4 Hz), 5.85 (1H, bs), 3.88 (3H, s), 3.86 (6H, s), 3.42 (2H, s); CNMR (100 MHz, CDCl
3
): δ
1
58.9, 155.5, 150.7, 149.7, 143.6, 143.0, 137.0, 126.3, 120.0, 112.4, 112.2, 111.9, 109.9, 108.7,
102.6, 56.4, 55.7, 55.4, 30.3; IR (NaCl) 3337, 3014, 2934, 2833, 1612, 1511, 1466, 1310, 1282,
ꢀ1
+
1232, 1165, 1134, 1082, 1027 cm ; HRMS (ESI, positive) m/z calcd. for C19
H
19
N
3
O
3
[M+H] :
338.1499, found: 338.1503; HPLC purity: 96.0%, retention time: 8.28 min (MeOH:CH
2
Cl
2
= 1:9).
N-(3,5-dimethoxyphenyl)-6-methoxy-1,4-dihydroindeno[1,2-c]pyrazol-3-amine (6l)
This compound was prepared from 5ꢀmethoxyꢀ1ꢀindanone (0.08 g, 0.49 mmol) and
1
ꢀisothiocyanatoꢀ3,5ꢀdimethoxybenzene (2c) (0.0896 g, 0.49 mmol) using the procedure described
for 6a. Purification by column chromatography with hexane:EtOAc
=
1:1 v/v) gave
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1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Nꢀ(3,4ꢀdimethoxyphenyl)ꢀ6ꢀmethoxyꢀ1,4ꢀdihydroindeno[1,2ꢀc]pyrazolꢀ3ꢀamine (6l) as a light brown
1
solid (0.0827 g, 0.245 mmol, 50%). mp: 88ꢀ89 ºC; H NMR (400 MHz, CDCl
3
): 7.52 (1H, d, J = 8.4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Hz), 7.06 (1H, s), 6.90 (1H, d, J = 8.4 HZ), 6.20 (2H, s), 6.09 (1H, s), 6.00 (1H, bs), 3.87 (3H, s),
1
3
3
1
1
3
.78 (6H, s), 3.52 (2H, s); CNMR (100 MHz, CDCl ): δ161.6, 158.9, 155.3, 150.7, 145.1, 141.4,
26.2, 120.0, 112.3, 111.8, 111.6, 94.3, 92.6, 55.4, 55.2, 30.4; IR (NaCl) 3003, 2935, 2837, 2360,
ꢀ1
600, 1525, 1488, 1310, 1283, 1254, 1202, 1152, 1066, 1033 cm ; HRMS (ESI, positive) m/z calcd.
+
for C19
H
19
N
3
O
3
[M+H] : 338.1499, found: 338.1495; HPLC purity: 97.0%, retention time: 7.66 min
(
MeOH:CH
2
Cl
2
= 1:9).
Methyl 3-((6-methoxy-1,4-dihydroindeno[1,2-c]pyrazol-3-yl)amino)benzoate (6m)
This compound was prepared from 5ꢀmethoxyꢀ1ꢀindanone (0.1 g, 0.62 mmol) and methyl
3ꢀisothiocyanatobenzoate (4d) (0.119 g, 0.62 mmol) using the procedure described for 6a.
Purification by column chromatography with (Hexane:EtOAc
= 2:1 v/v) gave methyl
3
ꢀ((6ꢀmethoxyꢀ1,4ꢀdihydroindeno[1,2ꢀc]pyrazolꢀ3ꢀyl)amino)benzoate (6m) as a white solid (0.0839
1
3
g, 0.25 mmol, 40%). mp: 147ꢀ148 ºC; H NMR (400 MHz, CDCl ): δ 7.64 (1H, s), 7.54 (1H, d, J =
7
.6 Hz), 7.44 (1H, d, J = 8.0 Hz), 7.29 (1H, dd, J = 8.0, 8.0 Hz), 7.16 (1H, d, J = 8.0 Hz), 7.00 (1H,
13
s), 6.82 (1H, s, J = 8.0 Hz), 6.39 (1H, bs), 3.88 (3H, s), 3.28 (3H, s), 3.42 (2H, s); CNMR (100
MHz, CDCl
3
): δ 167.3, 158.9, 154.8, 150.7, 143.3, 141.8, 130.9, 129.1, 125.9, 120.9, 120.1, 120.0,
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7
8
9
116.4, 112.2, 111.8, 111.2, 55.4, 52.1, 30.3; IR (NaCl) 3734, 2950, 2360, 1716, 1592, 1540, 1508,
ꢀ1
1489, 1472, 1284, 1249, 1107, 1034, 999, 752, 687 cm ; HRMS (ESI, positive) m/z calcd. for
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
+
C
19
H
17
N
3
O
3
[M+H] : 336.1343, found: 336.1340; HPLC purity: 98.3%, retention time: 7.72 min
(
2 2 17 3 3
MeOH:CH Cl = 1:9); Anal. Calcd for C19H N O : C: 68.05, H: 5.11, N: 12.53, O: 14.31, found,
C: 67.81, H: 5.15, N: 12.47.
3
-((6-Methoxy-1,4-dihydroindeno[1,2-c]pyrazol-3-yl)amino)benzoic acid (6n)
A mixture of methyl 3ꢀ((6ꢀmethoxyꢀ1,4ꢀdihydroindeno[1,2ꢀc]pyrazolꢀ3ꢀyl)amino)benzoate (6m)
.
(
0.084 g, 0.25 mmol) was dissolve in THF (5 mL) and a solution of LiOH H
2
O (0.021 g, 0.5 mmol)
2
in H O (1 mL) was added. The mixture was stirred overnight at room temperature. The reaction was
quenched with 1N HCl and the mixture was extracted with EtOAc. The organic layer was washed
2 4
with water and brine, dried over anhydrous Na SO , and concentrated. The residue was suspended in
2 2
CH Cl and filtrated to give 3ꢀ((6ꢀmethoxyꢀ1,4ꢀdihydroindeno[1,2ꢀc]pyrazolꢀ3ꢀyl)amino)benzoic
1
acid (6n) as a whit solid (0.062g, 0.19 mmol, 76%). mp: 239ꢀ240 ºC; H NMR (400 MHz, CD OD):
3
7
.65 (1H, s), 7.53 (1H, d, J = 8.0 Hz), 7.47 (1H, d, J = 7.6 Hz), 7.27 (1H, dd, J = 7.6, 8.0 Hz), 7.14
(
1H, dd, J = 2.0, 8.0 Hz), 7.10 (1H, s), 6.91 (1H, dd, J = 2.0, 8.4 Hz), 3.84 (3H, s), 3.50 (2H, s);
13
3
CNMR (100 MHz, CD OD): δ 173.3, 159.2, 155.6, 150.9, 143.5, 141.2, 137.0, 128.2, 126.4, 120.3,
1
19.4, 117.9, 116.2, 112.1, 111.5, 110.3, 54.5, 29.5; IR (KBr) 3274, 2896, 2833, 1551, 1471, 1391,
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ꢀ
1
+
15 3 3
1310, 1283, 1245, 1083, 1032 cm ; HRMS (ESI, positive) m/z calcd. for C18H N O [M+H] :
322.1186, found: 322.1182; HPLC purity: 100%, retention time: 8.16 min, (MeOH).
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N-(3-isobutoxyphenyl)-6-methoxy-1,4-dihydroindeno[1,2-c]pyrazol-3-amine (6o)
This compound was prepared from 5ꢀmethoxyꢀ1ꢀindanone (0.094 g, 0.58 mmol) and
1
ꢀisobutoxyꢀ3ꢀisothiocyanatobenzene (4e) (0.12 g, 0.58 mmol) using the procedure described for 6a.
Purification by column chromatography with (Hexane:EtOAc
=
2:1 v/v) gave
Nꢀ(3ꢀisobutoxyphenyl)ꢀ6ꢀmethoxyꢀ1,4ꢀdihydroindeno[1,2ꢀc]pyrazolꢀ3ꢀamine (6o) as a light yellow
1
solid (0.113 g, 0.32 mmol, 55%). mp: 76ꢀ77 ºC; H NMR (400 MHz, CDCl
3
): δ 7.50 (1H, d, J = 8.4
Hz), 7.17 (1H, dd, J = 8.0, 8.4 Hz), 7.04 (1H, s), 6.87 (1H, d, J = 8.4 Hz), 6.58ꢀ6.56 (2H, m), 6.48
(1H, d, J = 7.6 Hz), 6.07 (1H, bs), 3.86 (3H, s), 3.70 (2H, d, J = 6.4 HZ), 3.48 (2H, s), 2.21ꢀ2.02 (1H,
13
3
m), 1.03 (3H, s), 1.00 (3H, s); CNMR (100 MHz, CDCl ): δ160.4, 158.9, 155.3, 150.7, 144.4,
141.6, 129.9, 126.3, 120.1, 112.2, 111.9, 111.4,108.5, 106.3, 102.4, 74.3, 55.4, 30.4, 28.3, 19.3; IR
(
NaCl) 3162, 2957, 2360, 1598, 1524, 1494, 1469, 1394, 1310, 1283, 1247, 1192, 1156, 1082, 1034
ꢀ1
+
cm ; HRMS (ESI, positive) m/z calcd. for C21
H
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N
O
3 2
[M+H] : 350.1863, found: 350.1865; HPLC
purity: 95.7%, retention time: 9.53 min (MeOH:CH
2
Cl
2
= 3:97).
Cell culture
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The human cervical carcinoma cell line HeLa cells were obtained the Cell Resource Center for
Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University (Sendai,
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Japan). The cells were cultured under 5% CO at 37 ºC in RPMI 1640 medium (Wako pure Chemical,
Osaka, Japan) supplemented with 10% fetal bovine serum (FBS, HyClone, Logan, UT), 100 U/mL
penicillin and 100 ꢁg/mL streptomycin (Invitrogen, Carlsbad, CA). For subsequent experiments, the
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cells were seeded at a density of 2.5 x 10 cells/mL/well in a 12ꢀwell TC plate (Greiner Japan, Tokyo,
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Japan), and incubated at 37 ºC for 12h. Hypoxic condition was achieved by replacing cells to 1% O ,
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5% N
2
and 5% CO
2
in a multigas incubator (Astec, Fukuoka, Japan).
MTT assay
3
HeLa cells were plated on 96 wells plate (5 x 10 cells per well ) and incubated at 37 ºC for 6 h in
100 ꢁL medium. The cells were incubated for 72h with or without drugs. After incubation, MTT
solution in PBS (5 mg/mL) 10 ꢁL was added to each well, and incubated at 37 ºC for 2 h. After
removal of the supernatant, DMSO 100 ꢁL was added, and the absorbance was determined.
Anti-microbial activities
The following microorganisms were used as test strains in the assay; Staphylococcus aureus 209
(
S.a.), Escherichia coli HO141 (E. coli), Candida albicans JCM1542 (C.a.), Aspergillus fumigatus
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