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Biothiols, characterized by their unique sulfhydryl (-SH) groups, possess excellent antioxidant properties, effectively neutralizing the damage to cellular structures caused by reactive oxygen species (ROS) in living organisms. Additionally, lysosomes play a crucial role in decomposing damaged biomolecules through the action of their internal enzymes, regulating the cellular redox state, and mitigating oxidative stress. To facilitate rapid monitoring of intracellular biothiols, particularly within lysosomes, we constructed a lysosome-targeted biothiol fluorescent probe, PHL-DNP, in this study. PHL-DNP exhibited excellent photophysical properties in an aqueous test system, including strong fluorescence enhancement response, excellent selectivity, and low detection limits (Cys 16.5 nM, Hcy 16.8 nM, GSH 21.3 nM, Cap 26.6 nM). These attributes enabled easy and efficient qualification of Cys on test strips and accurate determination of the effective content of captopril tablets. Notably, PHL-DNP demonstrated low cytotoxicity and precise lysosomal targeting. Through bioimaging, PHL-DNP not only monitored changes in biothiol levels under oxidative stress but also assessed biothiols in complex biological systems such as live HeLa cells, zebrafish, tumor tissue sections, and radish roots. This provides a promising tool for quantitative analysis of biothiols, disease marker detection, and drug testing. [ABSTRACT FROM AUTHOR]

Alzheimer's disease is a complex and progressive form of dementia. Its treatment relies on the behavioral and cognitive symptoms of an individual. Inhibiting acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) is a primary strategy used for treating Alzheimer's disease. Pyrazoline is a well-recognized nitrogen-containing five-membered heterocyclic skeleton. The unique structure of pyrazoline gives it a spatial configuration that leads to the multiple substitution pattern and advanced pharmacological properties. In an attempt to identify potent acetylcholine (AChE) and butyrylcholinesterase (BChE) inhibitors, new pyrazoline derivatives (IIIa–IIId) were synthesized using conventional method. The synthesised compounds were purified by column chromatography and were analysed using LCMS, 1HNMR, 13CNMR, and Mass Spectroscopic HR-MS techniques. The derivatives underwent initial screening for in-vitro antioxidant potential. The most potent compound, IIIa, showed IC50 values of 22.15 and 25.09 nM against AChE and BChE, respectively. Additionally, in-silico screening with AutoDock tools indicated that IIIa had promising binding affinities to the targets, with a binding energy (− 8.9 kcal/mol) comparable to donepezil (− 10.6 kcal/mol). The binding pattern of IIIa to AChE's active site justified its in-vitro inhibitory activity. These findings suggest that compound IIIa has potential as a new therapeutic agent for Alzheimer's disease and is suitable for pre-clinical evaluation. [ABSTRACT FROM AUTHOR]

A new fluorescent sensor (E)-3-(1-acetyl-5-styryl-4,5-dihydro-1H -pyrazol-3-yl)-4-hydroxy-6-methyl-2H-pyran-2-one (DRS) for Al3+ detection was developed, and the optical properties of DRS were measured in H2O/DMSO (pH = 5.5, 99:1, V/V) solution. DRS showed notable fluorescence enhancement in the presence of Al3+ and had good selectivity and sensitivity to Al3+. Compared to the probes designed by other previous research groups, a lower detection limit (9.13 × 10−9 M) and a shorter response time (14 s) were found. Furthermore, the Job's plot showed that the stoichiometric ratio among DRS and Al3+ was 1:1, and 1H NMR, IR and DFT calculations showed that the sensing mechanism of the probe DRS for detecting Al3+ was chelation-enhanced fluorescence. DRS has also been applied to Al3+ ion detection in actual water samples, drugs, and HeLa cells, indicating that it has potential application value in the biological process of detecting Al3+ ion. [ABSTRACT FROM AUTHOR]

Abstract Organic compounds with pyrazole cores have a variety of uses, notably in the pharmaceutical and agrochemical sectors. The interest in creating pyrazole compounds, examining their many features, and looking for potential uses is growing. Our work has concert with synthesis of chalcones and pyrazolines, then finally pyrazoline-aniline derivatives and evaluation their anti-inflammatory, antibacterial and antifungal activities

Promising inhibitory activities of the parasite multiplication were obtained upon evaluation of in vivo antimalarial activities of new pyrazolylpyrazoline derivatives against Plasmodium berghei infected mice. Further evaluation of 5b and 6a against chloroquine-resistant strain (RKL9) of P. falciparum showed higher potency than chloroquine. In vitro antileishmanial activity testing against Leishmania aethiopica promastigote and amastigote forms indicated that 5b, 6a and 7b possessed promising activity compared to miltefosine and amphotericin B deoxycholate. Moreover, antileishmanial activity reversal of the active compounds via folic and folinic acids showed comparable results to the positive control trimethoprim, indicating an antifolate mechanism via targeting leishmanial DHFR and PTR1. The compounds were non-toxic at 125, 250 and 500 mg/kg. In addition, docking of the most active compound against putative malarial target Pf-DHFR-TS and leishmanial PTR1 rationalised the observed activities. Molecular dynamics simulations confirmed a stable and high potential binding of 7a against leishmanial PTR1.

Promising inhibitory activities of the parasite multiplication were obtained upon evaluation of in vivo anti-malarial activities of new pyrazolylpyrazoline derivatives against Plasmodium berghei infected mice. Further evaluation of 5b and 6a against chloroquine-resistant strain (RKL9) of P. falciparum showed higher potency than chloroquine. In vitro antileishmanial activity testing against Leishmania aethiopica promastigote and amastigote forms indicated that 5b, 6a and 7b possessed promising activity compared to miltefosine and amphotericin B deoxycholate. Moreover, antileishmanial activity reversal of the active compounds via folic and folinic acids showed comparable results to the positive control trimethoprim, indicating an antifolate mechanism via targeting leishmanial DHFR and PTR1. The compounds were nontoxic at 125, 250 and 500mg/kg. In addition, docking of the most active compound against putative malarial target Pf-DHFR-TS and leishmanial PTR1 rationalised the observed activities. Molecular dynamics simulations confirmed a stable and high potential binding of 7a against leishmanial PTR1. [ABSTRACT FROM AUTHOR]

A series of new thiophene-containing triaryl pyrazoline derivatives, 3a–3t, were synthesized and evaluated regarding PI3K inhibition activity and anti-tumor potency based on a trial of introducing significant moieties, including pyrazoline and thiophene, and simplifying the parallel ring structures. Most of the tested compounds indicated potent PI3K inhibitory potency, with this series of compounds showing more potency for PI3Kγ than PI3Kα. The top hit 3s seemed more potent than the positive control LY294002 on inhibiting PI3Kγ (IC50 values: 0.066 μM versus 0.777 μM) and more selective from PI3Kα (Index values: 645 versus 1.74). It could be inferred that the combination of para- and meta-, as well as the modification of the electron-donating moieties, led to the improvement in potency. The anti-proliferation inhibitory activity and the enzymatic inhibition potency indicated consistent tendencies. The top hit 3s could inhibit the phosphorylation of Akt by inhibiting PI3K through the PI3K-Akt-mTOR pathway. The molecular docking simulation indicated that the binding pattern of 3s into PI3Kγ was preferable than that of PI3Kα, with more hydrogen bond, more π-involved interactions, and fewer π-sulfur interactions. The information in this work is referable for the further development of selective inhibitors for specific isoforms of PI3K. [ABSTRACT FROM AUTHOR]

A series of new thiophene-containing triaryl pyrazoline derivatives, 3a–3t, were synthesized and evaluated regarding PI3K inhibition activity and anti-tumor potency based on a trial of introducing significant moieties, including pyrazoline and thiophene, and simplifying the parallel ring structures. Most of the tested compounds indicated potent PI3K inhibitory potency, with this series of compounds showing more potency for PI3Kγ than PI3Kα. The top hit 3s seemed more potent than the positive control LY294002 on inhibiting PI3Kγ (IC50 values: 0.066 μM versus 0.777 μM) and more selective from PI3Kα (Index values: 645 versus 1.74). It could be inferred that the combination of para- and meta-, as well as the modification of the electron-donating moieties, led to the improvement in potency. The anti-proliferation inhibitory activity and the enzymatic inhibition potency indicated consistent tendencies. The top hit 3s could inhibit the phosphorylation of Akt by inhibiting PI3K through the PI3K-Akt-mTOR pathway. The molecular docking simulation indicated that the binding pattern of 3s into PI3Kγ was preferable than that of PI3Kα, with more hydrogen bond, more π-involved interactions, and fewer π-sulfur interactions. The information in this work is referable for the further development of selective inhibitors for specific isoforms of PI3K.

Objectives: Resistance to antibiotics is recognized as one of the biggest threats to human health worldwide. Frequent and unnecessary use of antibiotics has caused infectious agents to adapt to antibiotics and thus drugs have become less effective. The resistance to many antibiotics necessitates the discovery of new antibiotics. In this study, two new and 23 previously reported 2-pyrazoline derivatives and one hydrazone derivative were evaluated for their in vitro antibacterial and antifungal activities. Materials and Methods: For the determination of the minimum inhibitory concentration (MIC) values of compounds, microbroth dilution was used. Results: The antimicrobial activities of the compounds were found in a wide range with MIC values of 32-512 μg/mL. Conclusion: The synthesized compounds showed moderate antimicrobial activity compared with the standards. They can be used as lead molecules for the synthesis of more effective compounds. [ABSTRACT FROM AUTHOR]

Deoxyribonucleic acid (DNA) acts as the most important intracellular target for various drugs. Exploring the DNA binding interactions of small bioactive molecules offers a structural guideline for designing new drugs with higher clinical efficacy and enhanced selectivity. This study presents the facile synthesis of pyrazoline-derived compounds (4a)-(4f) by reacting substituted chalcones with hydrazine hydrate using formic acid. The structure elucidation of substituted pyrazoline compounds was carried out using 1 H-NMR, FT-IR and elemental analyses. While the crystal structures of two compounds (4a) and (4b) have been resolved by single-crystal X-ray diffraction (SC-XRD) analysis. Hirshfeld surface analysis also endorsed their greater molecular stability. Computational calculations at DFT/B3LYP/6-311++G(d,p) were executed to compare the structural properties (bond angle and bond length) and explore reactivity descriptors, frontier molecular orbitals (FMO), Mulliken atomic charges (MAC), molecular electrostatic potential (MEP) and electronic properties. All the compounds were evaluated for DNA binding interactions by UV-Vis spectrophotometric analysis. The results revealed that compounds (4a)-(4f) bind to DNA via non-covalent binding mode having binding constant values ranging from 1.22 × 10 3 to 6.81 × 10 4 M -1 . The negative values of Gibbs free energy also proved the interaction of studied compounds with DNA as a spontaneous process. The findings of molecular docking simulations depicted that these studied compounds showed significant binding interactions with DNA and these results were consistent with experimental findings. Compound (4b) was concluded as the most potent compound of the series with the highest binding constant (4.95 × 10 4 ) and strongest binding affinity (-8.48 kcal/mol).Communicated by Ramaswamy H. Sarma.

A series of new isolongifolanone pyrazoline derivatives were synthesized and characterized by 1H NMR and HRMS methods. The compounds were assessed for their antiproliferative activity against three cancer cell lines (MDA-MB-231, Hela, and HepG2 cells) in vitro. Most of the derivatives showed considerable cytotoxic activity to all three cancer cell lines. Among them, compound 400 exhibited excellent antiproliferative activity with IC50 values of 15.45, 18.52, and 34.4 μM for MDA-MB-231, Hela, and HepG2 cells, respectively. Further mechanistic studies indicated that compound 400 induced apoptosis in HepG2 cells by enhancing the accumulation of intracellular reactive oxygen species (ROS). In summary, we report the synthesis of a new pyrazoline derivative of isolongifolanone (compound 400) that potently induces apoptosis in HepG2 cells by enhancing intracellular ROS production. [ABSTRACT FROM AUTHOR]

A simple solvent‐free method for the regioselective synthesis of pyrazoline derivatives via the annulation reactions of α‐cyano‐α,β‐unsaturated ketones and β‐carboxylic ester‐α,β‐unsaturated ketone with Huisgen zwitterions was developed. Interestingly, the reaction substrate is identified as a critical factor to control the final products. This method features simplicity, high efficiency, environmentally benign, and broad substrate scope. The gram scale applicability also points towards its possible implementation in industrial use. [ABSTRACT FROM AUTHOR]

In this study, first, a series of chalcone compounds S1–S6 were synthesized from various acetophenone derivatives (acetophenone, p-methyl acetophenone, and p-methoxy acetophenone) and aromatic aldehyde derivatives (benzaldehyde, p-methyl benzaldehyde, and p-methoxy benzaldehyde) by the Claisen–Schmidt condensation reaction. These S1–S6 compounds were then used in the preparation of 4,5-dihydropyrazol-1-yl derivatives S7–S15. Finally, four new compounds S16–S19 were synthesized from compound (S7, S8, S9, and S12) and 2,4-dinitrophenylhydrazine. Therefore, three known and ten new heterocyclic compounds were synthesized and completely characterized using 1H NMR, 13C NMR, IR, and elemental analysis. [ABSTRACT FROM AUTHOR]

Abstract Taking into consideration of the cytotoxicity and topo-IIα inhibitory activity of pyrazoline derivatives (1–3) against HCT15 cells, and known topo-IIα inhibitor, etoposide, respectively, the compounds were biotransformed in rat liver microsomes. LC-MS/MS and MALDI mass spectrometric techniques has been used for analysis. All three compounds were biotransformed into demethylated metabolites. Among three compounds, compounds 1 and 2 were biotransformed into mono-hydroxylated metabolites and compound 3 biotransformed into reduced and epoxidized metabolites. Reduced and reduced along with demethylation metabolites were identified from MALDI Orbitrap spectrometric analysis. Without NADPH or microsomes no compounds (1–3) were generated metabolites, it shows CYP450 enzymes involvement in the presence of NADPH in the metabolisms. Highlights • Metabolic profiling of three newly potential topoisomerase inhibitors in RLMs. • Liquid chromatography ion trap tandem mass spectrometry/MALDI were used. • Pyrazolines biotransformed into various metabolites are depicted. • Structures were elucidated by MS2 fragmentation pattern/MADLI data. • It is the first time to report pyrazolines metabolic study. [ABSTRACT FROM AUTHOR]

A variety of fluorescence-active fluorinated pyrazolines 13–33 was synthesized in good yields through cyclocondensation reaction of propenones 1–9 with aryl hydrazines 10–12. Some of the synthesized compounds provided promising fluorescence properties with quantum yield (Φ) higher than that of quinine sulfate (standard reference). Quantitative structure–property relationship studies were undertaken supporting the exhibited fluorescence properties and estimating the parameters governing properties. Five synthesized fluorescence-active pyrazolines (13, 15, 18, 19 and 23) with variable Φ were selected for treating two types of paper sheets (Fabriano and Bible paper). These investigated fluorescence compounds, especially compounds 19 and 23, provide improvements in strength properties of paper sheets. Based on the observed performance they can be used as markers in security documents.

Pyrazoline are widely being studied due to their potential applications in chemical field. Herein, five pyrazolines compounds were synthesized and characterized spectroscopically using nuclear magnetic resonance techniques ( 1 H NMR & 13 C NMR) to determine the structures of molecules along-with UV–Visible and infrared (FT-IR) studies for additional spectroscopic support in characterization of entitle synthesized molecules. Unit cells, specific space groups, bond lengths, bond angles and hydrogen bonding interactions were determined by the x-ray diffraction studies. Further, computational study of compounds with B3LYP/6–311 + G(d,p) level were carried out to explore optimized geometry, spectroscopic data for FT-IR, frontier molecular orbitals (FMOs) and non-linear optical (NLO) parameters. While, UV–Vis spectral were performed by TD-DFT/B3LYP/6–311 + G(d,p) level. The experimental results of spectroscopic and single crystal studies were compared and found in good agreement with the computational. The global reactivity parameters have been calculated with the help of the energy of FMOs. The order for the total first and second order hyperpolarizabilities of 1–5 is found in the following orders: 1  >  4  >  3  >  5  >  2 and 1  >  4  >  5  >  2  >  3 respectively. Overall, greater NLO response than urea molecule prove that investigated molecules are excellent candidate for NLO applications. [ABSTRACT FROM AUTHOR]

A series of ferrocenyl ketones were synthesized in reaction with ferrocene and corresponding substituted acryloyl chlorides, following previously described procedure. Synthesized products have conjugated enone system, which is suitable for further transformations. In a reaction with hydrazine in acidic medium (acetic acid) new pyrazoline derivatives were obtained. Their antimicrobial properties have been tested. Synthesized pyrazoline derivatives demonstrated expressed in vitro antimicrobial activity towards 12 strains of microorganisms inhibiting all tested bacteria and fungi. The most potent compound in all cases was sorbyl derivative; for bacteria activity was very close to streptomycin, and for fungi in one case the same as ketoconazole. It is established that this compound can be a new, potential antimicrobial agent with minimum inhibitory concentrations from 0.039 to 0.312 mg/mL. One of the starting compounds and two products were crystal substances, suitable for the single crystal X-ray diffraction analysis, which confirmed undoubtedly their structures. [ABSTRACT FROM AUTHOR]

Two novel 1,3,5-trisubstituted pyrazoline derivatives—1-acetyl-3-(4-methoxyphenyl)-5-(6-methoxy-2-naphthyl)-pyrazoline (2a) and 1-(4-nitrophenyl)-3-(4-methoxyphenyl)-5-(6-methoxy-2-naphtyl)-pyrazoline (2b)—were synthesized and their structures were determined by single crystal X-ray crystallography. Both of the two crystals exhibit twisted structures due to the large dihedral angles between the pyrazolinyl ring and the aromatic ring at the 5-position (88.09° for 2a and 71.26° for 2b). The optical⁻physical properties of the two compounds were investigated. The fluorescent emission of 2b arises from the 1,3-disubstituted pyrazoline chromophores and exhibits a red shift in polar solvents and solid-state, which could be attributed to photo-induced intramolecular charge transfer (ICT) from N1 to C3 in the pyrazoline moiety and the intermolecular interactions within the crystal. The fluorescent emissions of 2a (λmax 358⁻364 nm) in solvents and solid-state both come from 6-methoxy-2-naphthyl chromophores, which are fairly insensitive to the solvent polarity.

This paper examines the cytotoxic effect of nine compounds with spiropyrazoline structures, and determines the reaction mechanism between diazomethane and selected benzylideneflavanones, their lipophilicity, and their binding ability to human serum albumin. The cytotoxic effect was determined on two human leukaemia cell lines (HL-60 and NALM-6) and melanoma WM-115 cells, as well as on normal human umbilical vein endothelial cells (HUVEC). The highest cytotoxicity was exhibited by compound B7 : it was found to have an IC 50 of less than 10 μM for all three cancer cell lines, with five to 12-fold lower sensitivity against normal cells (HUVEC). All the compounds exhibit comparable affinity energy in human serum albumin binding (from −8.1 to −8.6 kcal mol −1 ) but vary in their binding sites depending on the substituent. X-ray crystallography of two derivatives confirmed their synthetic pathway, and their structures were carefully examined. [ABSTRACT FROM AUTHOR]

Pyrazoline is an important 5-membered nitrogen heterocycle that has been extensively researched. Ten derivatives were synthesized and tested for antileukemic effects on 2 human acute leukemia cell lines, K562 and Jurkat. The most cytotoxic of these derivatives, compound 21, was chosen for investigation of cytotoxicity mechanisms. The results obtained with selectivity calculations revealed that compound 21 is more selective for acute leukemia (K562 and Jurkat cell lines) than for other tumor cell lines. Moreover, compound 21 was not cytotoxic to normal cell lines, indicating a potential use in clinical tests. Compound 21 caused a significant cell cycle arrest in the S-phase in Jurkat cells and increased the proportion of cells in the sub G0/G1 phase in both cell lines. Cells treated with compound 21 demonstrated morphological changes characteristic of apoptosis in the EB/AO assay, confirmed by externalization of phosphatidylserine by the annexin V - fluorescein isothiocyanate method and by DNA fragmentation. An investigation of cytotoxicity mechanisms suggests the involvement of an intrinsic apoptosis pathway due to mitochondrial damage and an increase in the ratio of mitochondrial Bax/Bcl2. Pyrazoline 21 obeyed Lipinski's 'rule of five' for drug-likeness. Based on these preliminary results, the antileukemic activity of compound 21 makes it a potential anticancer agent. [ABSTRACT FROM AUTHOR]

The title compound, C18H17ClN2O, crystallized with two independent molecules (the S and R enantiomers) in the asymmetric unit. The molecules are V-shaped with the two aromatic rings inclined to one another by 78.78 (11) and 81.23 (11)°. In the crystal, molecules are linked via C—H...O hydrogen bonds, forming chains along the c-axis direction. The chains are linked via C—H...π interactions, forming a three-dimensional framework. The crystal structure was refined as a two-component twin.

Two new pyrazoline derivatives, 3,5-bis(4-fluorophenyl)-4,5-dihydropyrazole-1-carboxamide (1) and 3,5-bis(4-fluorophenyl)-4,5-dihydropyrazole-1-carbothioamide (2), were synthesized by reacting 4,4'-difluoro chalcone with semicarbazide hydrochloride and thiosemicarbazide in ethanolic sodium hydroxide solution. Both the compounds were confirmed by single crystal X-ray diffraction data and supported by IR, NMR, and mass spectral data. In 1, crystal packing is stabilized by N–H…O hydrogen bonds and weak N–H...N, N–H…F and C–H…F intermolecular interactions. In 2, only weak N–H…F and N–H…S intermolecular interactions are observed. Crystal data: C16H13F2N3O, (1), Mr = 301.29, monoclinic, C2/c, a = 17.6219(6) Å, b = 10.8735(3) Å, c = 15.3216(5) Å, β = 102.864(3)°, V = 2862.11(16) Å3, Z = 8, T = 173 K, R(F) = 0.0511, wR(F2) = 0.1333; C16H13F2N3S, (2), Mr = 317.35, monoclinic, P21/c, a = 14.339(2) Å, b = 11.1478(17) Å, c = 9.541(2)(5) Å, β = 107.007(18)°, V = 1458.5(5) Å3, Z = 4, T = 173 K, R(F) = 0.0413, wR(F2) = 0.0959.

A series of a new chalcone derivatives (2a-i) containing benzyloxy moiety have been synthesized on the basis of base catalyzed claisen –Schmidt condensation in high yields from the reaction of the prepared starting material 3-(4-cholrobenzyloxy) paraldehyde (1)with different substituted acetophenones. The prepared chalcones were treated with hydrazine hydrate according to the Michael addition reaction to obtain new pyrazoline deivatives (3a-i). Finally the structures of the synthesized compounds were elucidated by using spectral methods such as; FT-IR, 1H-nm, 13C-nmr and 13C- Dept 135 spectra.

Design, synthesis and pharmacological activities of a group of 1,3,5-trisubstituted pyrazolines were reported. The chemical structures of the synthesized compounds have been assigned on the basis of IR, MS, 1 H NMR, and 13 C NMR spectral analyses. The synthesized 1,3,5-trisubstituted pyrazoline derivatives were evaluated in vivo for anti-inflammatory, analgesic activities and in vitro for COX-1/2 inhibition assay. Among the tested compounds, derivatives 4h , 6e , 7a , 7e , and 9 showed more potent anti-inflammatory and analgesic activities than the reference drug celecoxib. On the basis of their higher activities in the in vivo studies compared with celecoxib, the five compounds 4h , 6e , 7a , 7e and 9 were selected to test their inhibitory activities against ovine COX-1/2 using an in vitro cyclooxygenase inhibition assay. Docking study of compounds 7a , 7e and 9 into the COX-2 binding site revealed a similar binding mode to SC-558, a selective COX-2 inhibitor. [ABSTRACT FROM AUTHOR]

A series of pyrazoline derivatives ( 5 ) were synthesized in 92–96% yields from chalcones ( 3 ) and hydrazides ( 4 ). Subsequently, topo-I and IIα-mediated relaxation and antiproliferative activity assays were evaluated for 5 . Among the tested compounds, 5h had a very strong topo-I activity of 97% (Camptothecin, 74%) at concentration of 100 μM. Nevertheless, all the compounds 5a – 5i showed significant topo II inhibitory activity in the range of 90–94% (Etoposide, 96%) at the same concentration. Cytotoxic potential of these compounds was tested in a panel of three human tumor cell lines, HCT15, BT474 and T47D. All the compounds showed strong activity against HCT15 cell line with IC 50 at the range of 1.9–10.4 μM (Adriamycin, 23.0; Etoposide, 6.9; and Camptothecin, 7.1 μM). Moreover, compounds 5c , 5f and 5i were observed to have strong antiproliferative activity against BT474 cell lines. Since, compound 5d showed antiproliferative activity at a very low IC 50 thus 5d was then selected to study on their mode of action with diverse methods of ATP competition assay, ATPase assay and DNA-topo IIα cleavable complex assay and the results revealed that it functioned as a ATP-competitive human topoisomerase IIα catalytic inhibitor. Further evaluation of endogenous topo-mediated DNA relaxation in cells has been conducted to find that, 5d inhibited endogenous topo-mediated pBR322 plasmid relaxation is more efficient (78.0 ± 4.7% at 50 μM) than Etoposide (36.0 ± 1.7% at 50 μM). [ABSTRACT FROM AUTHOR]