Synthesis and Characterization of New Pyrano[2,3-c]pyrazole Derivatives as 3-Hydroxyflavone Analogues.

In this paper, an efficient synthetic route from pyrazole-chalcones to novel 6-aryl-5-hydroxy-2-phenylpyrano[2,3-c]pyrazol-4(2H)-ones as 3-hydroxyflavone analogues is described. The methylation of 5-hydroxy-2,6-phenylpyrano[2,3-c]pyrazol-4(2H)-one with methyl iodide in the presence of a base yielded a compound containing a 5-methoxy group, while the analogous reaction of 5-hydroxy-2-phenyl-6-(pyridin-4-yl)pyrano[2,3-c]pyrazol-4(2H)-one led to the zwitterionic 6-(N-methylpyridinium)pyrano[2,3-c]pyrazol derivative. The treatment of 5-hydroxy-2,6-phenylpyrano[2,3-c]pyrazol-4(2H)-one with triflic anhydride afforded a 5-trifloylsubstituted compound, which was further used in carbon-carbon bond forming Pd-catalyzed coupling reactions to yield 5-(hetero)aryl- and 5-carbo-functionalized pyrano[2,3-c]pyrazoles. The excited-state intramolecular proton transfer (ESIPT) reaction of 5-hydroxypyrano[2,3-c]pyrazoles from the 5-hydroxy moiety to the carbonyl group in polar protic, polar aprotic, and nonpolar solvents was observed, resulting in well-resolved two-band fluorescence. The structures of the novel heterocyclic compounds were confirmed by 1H-, 13C-, 15N-, and 19F-NMR spectroscopy, HRMS, and single-crystal X-ray diffraction data.

Synthesis of N-aryl-2,6-diphenyl-2H-pyrazolo[4,3-c]pyridin-7-amines and their photodynamic properties in the human skin melanoma cell line G361.

A small series of N-aryl-2,6-diphenyl-2H-pyrazolo[4,3-c]pyridin-7-amines was synthesized from easily accessible 1-phenyl-1H-pyrazol-3-ol via 7-iodo-2,6-diphenyl-2H-pyrazolo[4,3-c]pyridine and 7-iodo-4-methyl-2,6-diphenyl-2H-pyrazolo[4,3-c]pyridine intermediates and their subsequent use in palladium catalyzed Buchwald-Hartwig cross-coupling reaction with various anilines. Majority of the compounds were not significantly cytotoxic to melanoma G361 cells in the dark up to 10 µM concentration, but their activity could be increased by irradiation with visible blue light (414 nm). The most active compound 10 possessed EC50 values of 3.5, 1.6 and 0.9 µM in cells irradiated with 1, 5 and 10 J/cm2, respectively. The treatment caused generation of reactive oxygen species in cells and extensive DNA damage, documented by the comet assay and by detection of phosphorylated histone H2A.X, followed by apoptotic cell death. Our results suggest that N-aryl-2,6-diphenyl-2H-pyrazolo[4,3-c]pyridin-7-amines could serve as a potential source of photosensitizing compounds with anticancer activities.

Convenient Synthesis of N-Heterocycle-Fused Tetrahydro-1,4-diazepinones.

A general approach towards the synthesis of tetrahydro-4H-pyrazolo[1,5-a][1,4]diazepin-4-one, tetrahydro[1,4]diazepino[1,2-a]indol-1-one and tetrahydro-1H-benzo[4,5]imidazo[1,2-a][1,4]diazepin-1-one derivatives was introduced. A regioselective strategy was developed for synthesizing ethyl 1-(oxiran-2-ylmethyl)-1H-pyrazole-5-carboxylates from easily accessible 3(5)-aryl- or methyl-1H-pyrazole-5(3)-carboxylates. Obtained intermediates were further treated with amines resulting in oxirane ring-opening and direct cyclisation-yielding target pyrazolo[1,5-a][1,4]diazepin-4-ones. A straightforward two-step synthetic approach was applied to expand the current study and successfully functionalize ethyl 1H-indole- and ethyl 1H-benzo[d]imidazole-2-carboxylates. The structures of fused heterocyclic compounds were confirmed by 1H, 13C, and 15N-NMR spectroscopy and HRMS investigation.

Synthesis and Characterization of Novel Heterocyclic Chalcones from 1-Phenyl-1H-pyrazol-3-ol.

An efficient synthetic route to construct diverse pyrazole-based chalcones from 1-phenyl-1H-pyrazol-3-ols bearing a formyl or acetyl group on the C4 position of pyrazole ring, employing a base-catalysed Claisen-Schmidt condensation reaction, is described. Isomeric chalcones were further reacted with N-hydroxy-4-toluenesulfonamide and regioselective formation of 3,5-disubstituted 1,2-oxazoles was established. The novel pyrazole-chalcones and 1,2-oxazoles were characterized by an in-depth analysis of NMR spectral data, which were obtained through a combination of standard and advanced NMR spectroscopy techniques.

Convenient Synthesis of Pyrazolo[4',3':5,6]pyrano[4,3-c][1,2]oxazoles via Intramolecular Nitrile Oxide Cycloaddition.

A simple and efficient synthetic route to the novel 3a,4-dihydro-3H,7H- and 4H,7H-pyrazolo[4',3':5,6]pyrano[4,3-c][1,2]oxazole ring systems from 3-(prop-2-en-1-yloxy)- or 3-(prop-2-yn-1-yloxy)-1H-pyrazole-4-carbaldehyde oximes has been developed by employing the intramolecular nitrile oxide cycloaddition (INOC) reaction as the key step. The configuration of intermediate aldoximes was unambiguously determined using NOESY experimental data and comparison of the magnitudes of 1JCH coupling constants of the iminyl moiety, which were greater by approximately 13 Hz for the predominant syn isomer. The structures of the obtained heterocyclic products were confirmed by detailed 1H, 13C and 15N NMR spectroscopic experiments and HRMS measurements.

Synthesis and Characterization of Novel Methyl (3)5-(N-Boc-piperidinyl)-1H-pyrazole-4-carboxylates.

Series of methyl 3- and 5-(N-Boc-piperidinyl)-1H-pyrazole-4-carboxylates were developed and regioselectively synthesized as novel heterocyclic amino acids in their N-Boc protected ester form for achiral and chiral building blocks. In the first stage of the synthesis, piperidine-4-carboxylic and (R)- and (S)-piperidine-3-carboxylic acids were converted to the corresponding ß-keto esters, which were then treated with N,N-dimethylformamide dimethyl acetal. The subsequent reaction of ß-enamine diketones with various N-mono-substituted hydrazines afforded the target 5-(N-Boc-piperidinyl)-1H-pyrazole-4-carboxylates as major products, and tautomeric NH-pyrazoles prepared from hydrazine hydrate were further N-alkylated with alkyl halides to give 3-(N-Boc-piperidinyl)-1H-pyrazole-4-carboxylates. The structures of the novel heterocyclic compounds were confirmed by 1H-, 13C-, and 15N-NMR spectroscopy and HRMS investigation.

Synthesis and Antiproliferative Activity of 2,4,6,7-Tetrasubstituted-2H-pyrazolo[4,3-c]pyridines.

A library of 2,4,6,7-tetrasubstituted-2H-pyrazolo[4,3-c]pyridines was prepared from easily accessible 1-phenyl-3-(2-phenylethynyl)-1H-pyrazole-4-carbaldehyde via an iodine-mediated electrophilic cyclization of intermediate 4-(azidomethyl)-1-phenyl-3-(phenylethynyl)-1H-pyrazoles to 7-iodo-2,6-diphenyl-2H-pyrazolo[4,3-c]pyridines followed by Suzuki cross-couplings with various boronic acids and alkylation reactions. The compounds were evaluated for their antiproliferative activity against K562, MV4-11, and MCF-7 cancer cell lines. The most potent compounds displayed low micromolar GI50 values. 4-(2,6-Diphenyl-2H-pyrazolo[4,3-c]pyridin-7-yl)phenol proved to be the most active, induced poly(ADP-ribose) polymerase 1 (PARP-1) cleavage, activated the initiator enzyme of apoptotic cascade caspase 9, induced a fragmentation of microtubule-associated protein 1-light chain 3 (LC3), and reduced the expression levels of proliferating cell nuclear antigen (PCNA). The obtained results suggest a complex action of 4-(2,6-diphenyl-2H-pyrazolo[4,3-c]pyridin-7-yl)phenol that combines antiproliferative effects with the induction of cell death. Moreover, investigations of the fluorescence properties of the final compounds revealed 7-(4-methoxyphenyl)-2,6-diphenyl-2H-pyrazolo[4,3-c]pyridine as the most potent pH indicator that enables both fluorescence intensity-based and ratiometric pH sensing.

Synthesis and anthelmintic activity of benzopyrano[2,3-c]pyrazol-4(2H)-one derivatives.

A series of benzopyrano[2,3-c]pyrazol-4(2H)-one derivatives were synthesized from readily available 1-phenyl- and 1-methyl-1H-pyrazol-3-ols by sequentially employing O-acylation, Fries rearrangement and potassium carbonate-induced cyclization. The anthelmintic properties of the obtained compounds were investigated in vivo in a model nematode, Caenorhabditis elegans. Five compounds, namely 2-phenyl[1]benzopyrano[2,3-c]pyrazol-4(2H)-one 33 and its 7-fluoro, 7-chloro-, 7-bromo- and 8-fluoro-analogues, 36, 38, 40 and 43, respectively, altered the development of C. elegans. While the activities of 33 and 43 were rather modest, compounds 36, 38 and 40 inhibited the growth of the worms at concentrations of approximately 1-3 µM. At these concentrations, the compounds did not kill the worms, but they strongly inhibited their development, with the majority of larvae never progressing past the L1 stage. Moreover, testing in non-cancer human cell lines showed that, with exception of 7-bromo derivative 40, the active compounds have favourable toxicity profiles.

Synthesis of 2H-furo[2,3-c]pyrazole ring systems through silver(I) ion-mediated ring-closure reaction.

Fused pyrazole ring systems are common structural motifs of numerous pharmaceutically important compounds. Nevertheless, access to derivatives of the aromatic 2H-furo[2,3-c]pyrazole ring system is still quite limited, and their chemistry and functional properties remain largely underexplored. The current study investigates routes to construct this system from easily accessible starting materials using metal-catalyzed reactions. A simple and efficient procedure to access the 2H-furo[2,3-c]pyrazole ring system was developed by employing the silver(I) ion-mediated ring-closure reaction of 4-alkynyl-3-hydroxy-1-phenyl-1H-pyrazoles as a key step. The required intermediate hydroxyalkynyl substrates for this reaction were prepared by a Pd-catalyzed coupling of 4-iodo-1-phenyl-1H-pyrazol-3-ol with ethyne derivatives. The structures of the obtained target compounds were unequivocally confirmed by detailed 1H, 13C and 15N NMR spectroscopic experiments, HRMS and a single-crystal X-ray diffraction analyses. This silver(I)-mediated 5-endo-dig cyclization of readily available 4-alkynyl-3-hydroxy-1H-pyrazoles can be used as an efficient method to access many novel 2,5-disubstituted 2H-furo[2,3-c]pyrazoles.

On the Tautomerism of N-Substituted Pyrazolones: 1,2-Dihydro-3H-pyrazol-3-ones versus 1H-Pyrazol-3-ols.

The tautomerism of 1-phenyl-1,2-dihydro-3H-pyrazol-3-One was investigated. An X-ray crystal structure analysis exhibits dimers of 1-phenyl-1H-pyrazol-3-ol units. Comparison of NMR (nuclear magnetic resonance) spectra in liquid state (¹H, 13C, 15N) with those of "fixed" derivatives, as well as with the corresponding solid state NMR spectra reveal this compound to exist predominantly as 1H-pyrazol-3-ol molecule pairs in nonpolar solvents like CDCl3 or C6D6, whereas in DMSO-d6 the corresponding monomers are at hand. Moreover, the NMR data of different related 1H-pyrazol-3-ol derivatives are presented.

Pyrazole-based lamellarin O analogues: synthesis, biological evaluation and structure-activity relationships.

A library of pyrazole-based lamellarin O analogues was synthesized from easily accessible 3(5)-aryl-1H-pyrazole-5(3)-carboxylates which were subsequently modified by bromination, N-alkylation and Pd-catalysed Suzuki cross-coupling reactions. Synthesized ethyl and methyl 3,4-diaryl-1-(2-aryl-2-oxoethyl)-1H-pyrazole-5-carboxylates were evaluated for their physicochemical property profiles and in vitro cytotoxicity against three human colorectal cancer cell lines HCT116, HT29, and SW480. The most active compounds inhibited cell proliferation in a low micromolar range. Selected ethyl 3,4-diaryl-1-(2-aryl-2-oxoethyl)-1H-pyrazole-5-carboxylates were further investigated for their mode of action. Results of combined viability staining via Calcein AM/Hoechst/PI and fluorescence-activated cell sorting data indicated that cell death was triggered in a non-necrotic manner mediated by mainly G2/M-phase arrest.

Dipyrazolo[1,5-a:4',3'-c]pyridines - a new heterocyclic system accessed via multicomponent reaction.

The synthesis of dipyrazolo[1,5-a:4',3'-c]pyridines is described. Easily obtainable 5-alkynylpyrazole-4-carbaldehydes, p-toluenesulfonyl hydrazide, and an aldehyde or ketone containing an α-hydrogen atom were reacted in a silver triflate catalyzed multicomponent reaction affording new tricyclic compounds with a dipyrazolo[1,5-a:4',3'-c]pyridine core. Detailed NMR spectroscopic investigations ((1)H, (13)C and (15)N) were undertaken with all obtained compounds.

Synthesis and anti-mitotic activity of 2,4- or 2,6-disubstituted- and 2,4,6-trisubstituted-2H-pyrazolo[4,3-c]pyridines.

An efficient synthetic route for the synthesis of 2H-pyrazolo[4,3-c]pyridines, primarily varying by the substituents at the 2-, 4- and 6-positions, is described here. A Sonogashira-type cross-coupling reaction was employed to yield 3-alkynyl-1H-pyrazole-4-carbaldehydes, ethanones and propanones from the corresponding 1H-pyrazol-3-yl trifluoromethanesulfonates. Subsequent treatment of the coupling products with dry ammonia afforded a versatile library of 2H-pyrazolo[4,3-c]pyridines, which were then evaluated for their cytotoxicity against K562 and MCF-7 cancer cell lines. The most potent of these compounds displayed low micromolar GI50 values in both cell lines. Active compounds induced dose-dependent cell-cycle arrest in mitosis, as shown by flow cytometric analysis of DNA content and phosphorylation of histone H3 at serine-10. Moreover, biochemical assays revealed increased activities of caspases-3/7 in treated cells, specific fragmentation of PARP-1, and phosphorylation of Bcl-2, collectively confirming apoptosis as the mechanism of cell death.