Pyrazolone-Protein Interaction Enables Long-Term Retention Staining and Facile Artificial Biorecognition on Cell Membranes.

Cell membrane genetic engineering has been utilized to confer cell membranes with functionalities for diagnostic and therapeutic purposes but concerns over cost and variable modification results. Although nongenetic chemical modification and phospholipid insertion strategies are more convenient, they still face bottlenecks in either biosafety or stability of the modifications. Herein, we show that pyrazolone-bearing molecules can bind to proteins with high stability, which is mainly contributed to by the multiple interactions between pyrazolone and basic amino acids. This new binding model offers a simple and versatile noncovalent approach for cell membrane functionalization. By binding to cell membrane proteins, pyrazolone-bearing dyes enabled precise cell tracking in vitro (>96 h) and in vivo (>21 days) without interfering with the protein function or causing cell death. Furthermore, the convenient anchor of pyrazolone-bearing biotin on cell membranes rendered the biorecognition to avidin, showing the potential for artificially creating cell targetability.

Pyrazolone compounds could inhibit CES1 and ameliorates fat accumulation during adipocyte differentiation.

Carboxylesterase 1 (CES1), a member of the serine hydrolase superfamily, is involved in a wide range of xenobiotic and endogenous substances metabolic reactions in mammals. The inhibition of CES1 could not only alter the metabolism and disposition of related drugs, but also be benefit for treatment of metabolic disorders, such as obesity and fatty liver disease. In the present study, we aim to develop potential inhibitors of CES1 and reveal the preferred inhibitor structure from a series of synthetic pyrazolones (compounds 1-27). By in vitro high-throughput screening method, we found compounds 25 and 27 had non-competitive inhibition on CES1-mediated N-alkylated d-luciferin methyl ester (NLMe) hydrolysis, while compound 26 competitively inhibited CES1-mediated NLMe hydrolysis. Additionally, Compounds 25, 26 and 27 can inhibit CES1-mediated fluorescent probe hydrolysis in live HepG2 cells with effect. Besides, compounds 25, 26 and 27 could effectively inhibit the accumulation of lipid droplets in mouse adipocytes cells. These data not only provided study basis for the design of newly CES1 inhibitors. The present study not only provided the basis for the development of lead compounds for novel CES1 inhibitors with better performance, but also offered a new direction for the explore of candidate compounds for the treatment of hyperlipidemia and related diseases.

Activity of pyridyl-pyrazolone derivatives against Trypanosoma cruzi.

New affordable drugs are needed for the treatment of infection with the protozoan parasite Trypanosoma cruzi responsible for the Chagas disease (CD). Only two old drugs are currently available, nifurtimox and benznidazole (Bz) but they exhibit unwanted side effects and display a weak activity in the late chronic phase of the disease. In this context, we evaluated the activity of a series of aryl-pyrazolone derivatives against T cruzi, using both bloodstream trypomastigote and intracellular amastigote forms of the parasite. The test compounds originate from a series of anticancer agents targeting the immune checkpoint ligand PD-L1 and bear an analogy with known anti-trypanosomal pyrazolones. A first group of 6 phenyl-pyrazolones was tested, revealing the activity of a single pyridyl-pyrazolone derivative. Then a second group of 8 compounds with a common pyridyl-pyrazolone core was evaluated. The in vitro testing process led to the identification of two non-cytotoxic and highly potent molecules against the intracellular form of T. cruzi, with an activity comparable to Bz. Moreover, one compound revealed an activity largely superior to that of Bz against bloodstream trypomastigotes, while being non-cytotoxic (selectivity index >1000). Unfortunately, the compound showed little activity in vivo, most likely due to its very limited plasma stability. However, the study opens novel perspectives for the design of new anti-trypanosomal products and the mechanism of action of the compounds is discussed.

The P300 acetyltransferase inhibitor C646 promotes membrane translocation of insulin receptor protein substrate and interaction with the insulin receptor.

Inhibition of P300 acetyltransferase activity by specific inhibitor C646 has been shown to improve insulin signaling. However, the underlying molecular mechanism of this improvement remains unclear. In this study, we analyzed P300 levels of obese patients and found that they were significantly increased in liver hepatocytes. In addition, large amounts of P300 appeared in the cytoplasm. Inhibition of P300 acetyltransferase activity by C646 drastically increased tyrosine phosphorylation of the insulin receptor protein substrates (IRS1/2) without affecting the tyrosine phosphorylation of the beta subunit of the insulin receptor (IRß) in hepatocytes in the absence of insulin. Since IRS1/2 requires membrane translocation and binding to inositol compounds for normal functions, we also examined the role of acetylation on binding to phosphatidylinositol(4,5)P2 and found that IRS1/2 acetylation by P300 reduced this binding. In contrast, we show that inhibition of IRS1/2 acetylation by C646 facilitates IRS1/2 membrane translocation. Intriguingly, we demonstrate that C646 activates IRß's tyrosine kinase activity and directly promotes IRß interaction with IRS1/2, leading to the tyrosine phosphorylation of IRS1/2 and subsequent activation of insulin signaling even in the absence of insulin. In conclusion, these data reveal the unique effects of C646 in activating insulin signaling in patients with obesity and diabetes.

Potent and selective carbonic anhydrase inhibition activities of pyrazolones bearing benzenesulfonamides.

This research introduces a series of fourteen 4-aryl-hydrazonopyrazolone sulfonamide derivatives, denoted as 3(a-g) and 4(a-g), which encompass various aromatic substitutions. The aim was to assess the inhibitory potential of these compounds against four significant isoforms, including the cytosolic isoforms hCA I and II, as well as the tumor-associated membrane-bound isoforms hCA IX and XII. Most of the tested compounds exhibited substantial inhibition against the tumor-associated isoform hCA IX, with Ki values spanning from 1.1 to 158.2 nM. Notably, compounds 3e and 3g showed particularly strong inhibitory activity against the tumor-associated membrane-bound isoforms, hCA IX and XII, while maintaining a high selectivity ratio over cytosolic off-target isoforms hCA I and II. This selectivity is vital due to the potential of hCA IX and hCA XII as drug targets for hypoxic tumors. In an effort to create novel analogs that exhibit enhanced carbonic anhydrase inhibitory activity and specificity, we investigated the structure-activity relationships of these compounds and provided a concise interpretation of our findings. Consequently, these compounds merit consideration for subsequent medicinal and pharmacological research, holding potential for developing novel therapeutic agents targeting specific isoforms in hypoxic tumors.

Enhancement by pyrazolones of colistin efficacy against mcr-1-expressing E. coli: an in silico and in vitro investigation.

Owing to the emergence of antibiotic resistance, the polymyxin colistin has been recently revived to treat acute, multidrug-resistant Gram-negative bacterial infections. Positively charged colistin binds to negatively charged lipids and damages the outer membrane of Gram-negative bacteria. However, the MCR-1 protein, encoded by the mobile colistin resistance (mcr) gene, is involved in bacterial colistin resistance by catalysing phosphoethanolamine (PEA) transfer onto lipid A, neutralising its negative charge, and thereby reducing its interaction with colistin. Our preliminary results showed that treatment with a reference pyrazolone compound significantly reduced colistin minimal inhibitory concentrations in Escherichia coli expressing mcr-1 mediated colistin resistance (Hanpaibool et al. in ACS Omega, 2023). A docking-MD combination was used in an ensemble-based docking approach to identify further pyrazolone compounds as candidate MCR-1 inhibitors. Docking simulations revealed that 13/28 of the pyrazolone compounds tested are predicted to have lower binding free energies than the reference compound. Four of these were chosen for in vitro testing, with the results demonstrating that all the compounds tested could lower colistin MICs in an E. coli strain carrying the mcr-1 gene. Docking of pyrazolones into the MCR-1 active site reveals residues that are implicated in ligand-protein interactions, particularly E246, T285, H395, H466, and H478, which are located in the MCR-1 active site and which participate in interactions with MCR-1 in ≥ 8/10 of the lowest energy complexes. This study establishes pyrazolone-induced colistin susceptibility in E. coli carrying the mcr-1 gene, providing a method for the development of novel treatments against colistin-resistant bacteria.

NOX1/NADPH Oxidase Promotes Synaptic Facilitation Induced by Repeated D2 Receptor Stimulation: Involvement in Behavioral Repetition.

Repetitive behavior is a widely observed neuropsychiatric symptom. Abnormal dopaminergic signaling in the striatum is one of the factors associated with behavioral repetition; however, the molecular mechanisms underlying the induction of repetitive behavior remain unclear. Here, we demonstrated that the NOX1 isoform of the superoxide-producing enzyme NADPH oxidase regulated repetitive behavior in mice by facilitating excitatory synaptic inputs in the central striatum (CS). In male C57Bl/6J mice, repeated stimulation of D2 receptors induced abnormal behavioral repetition and perseverative behavior. Nox1 deficiency or acute pharmacological inhibition of NOX1 significantly shortened repeated D2 receptor stimulation-induced repetitive behavior without affecting motor responses to a single D2 receptor stimulation. Among brain regions, Nox1 showed enriched expression in the striatum, and repeated dopamine D2 receptor stimulation further increased Nox1 expression levels in the CS, but not in the dorsal striatum. Electrophysiological analyses revealed that repeated D2 receptor stimulation facilitated excitatory inputs in the CS indirect pathway medium spiny neurons (iMSNs), and this effect was suppressed by the genetic deletion or pharmacological inhibition of NOX1. Nox1 deficiency potentiated protein tyrosine phosphatase activity and attenuated the accumulation of activated Src kinase, which is required for the synaptic potentiation in CS iMSNs. Inhibition of NOX1 or ß-arrestin in the CS was sufficient to ameliorate repetitive behavior. Striatal-specific Nox1 knockdown also ameliorated repetitive and perseverative behavior. Collectively, these results indicate that NOX1 acts as an enhancer of synaptic facilitation in CS iMSNs and plays a key role in the molecular link between abnormal dopamine signaling and behavioral repetition and perseveration.SIGNIFICANCE STATEMENT Behavioral repetition is a form of compulsivity, which is one of the core symptoms of psychiatric disorders, such as obsessive-compulsive disorder. Perseveration is also a hallmark of such disorders. Both clinical and animal studies suggest important roles of abnormal dopaminergic signaling and striatal hyperactivity in compulsivity; however, the precise molecular link between them remains unclear. Here, we demonstrated the contribution of NOX1 to behavioral repetition induced by repeated stimulation of D2 receptors. Repeated stimulation of D2 receptors upregulated Nox1 mRNA in a striatal subregion-specific manner. The upregulated NOX1 promoted striatal synaptic facilitation in iMSNs by enhancing phosphorylation signaling. These results provide a novel mechanism for D2 receptor-mediated excitatory synaptic facilitation and indicate the therapeutic potential of NOX1 inhibition in compulsivity.

Blockade of mTORC1-NOX signaling pathway inhibits TGF-ß1-mediated senescence-like structural alterations of the retinal pigment epithelium.

The retinal pigment epithelium (RPE) undergoes characteristic structural changes and epithelial-mesenchymal transition (EMT) during normal aging, which are exacerbated in age-related macular degeneration (AMD). Although the pathogenic mechanisms of aging and AMD remain unclear, transforming growth factor-ß1 (TGF-ß1) is known to induce oxidative stress, morphometric changes, and EMT as a senescence-promoting factor. In this study, we examined whether intravitreal injection of TGF-ß1 into the mouse eye elicits senescence-like morphological alterations in the RPE and if this can be prevented by suppressing mammalian target of rapamycin complex 1 (mTORC1) or NADPH oxidase (NOX) signaling. We verified that intravitreal TGF-ß1-induced stress fiber formation and EMT in RPE cells, along with age-associated morphometric changes, including increased variation in cell size and reduced cell density. In RPE cells, exogenous TGF-ß1 increased endogenous expression of TGF-ß1 and upregulated Smad3-ERK1/2-mTORC1 signaling, increasing reactive oxygen species (ROS) production and EMT. We demonstrated that inhibition of the mTORC1-NOX4 pathway by pretreatment with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an activator of AMP-dependent protein kinase, or GKT137831, a NOX1/4 inhibitor, decreased ROS generation, prevented stress fiber formation, attenuated EMT, and improved the regularity of the RPE structure in vitro and in vivo. These results suggest that intravitreal TGF-ß1 injection could be used as a screening model to investigate the aging-related structural and functional changes to the RPE. Furthermore, the regulation of TGF-ß-mTORC1-NOX signaling could be a potential therapeutic target for reducing pathogenic alterations in aged RPE and AMD.

Recent advances in the applications of pyrazolone derivatives in enantioselective synthesis.

Pyrazolones and pyrazoles, featuring nitrogen-nitrogen bonds, are two of the most important classes of heterocycles, owing to their widespread occurrence in medicinal chemistry and functional materials. The last decade has witnessed a rapid increase in the construction of chiral pyrazolone and pyrazole derivatives, with the application of pyrazolone derivatives as powerful synthons. Since our last review in 2018, a large number of new achievements has emerged in this area, requiring a timely update. Thus, this review summarizes these elegant achievements based on the multiple reactive sites of different pyrazolone synthons. In addition, important mechanisms and interesting biological investigations relating to the corresponding products are also discussed.

Conventional and microwave-assisted synthesis, anticancer evaluation, 99mTc-coupling and In-vivo study of some novel pyrazolone derivatives.

New pyrazolone derivatives were successfully synthesized by both microwave-assisted and conventional techniques. Compound 3 (3-(3-Methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)-3-oxopropanehydrazide) displayed remarkable anti-cancer activity (IC50 obtained at 8.71 and 10.63 µM for HCT-116 and MCF-7, respectively. Moreover, biodistribution study using radiolabeling approach revealed a remarked uptake of [99mTc]Tc-compound 3 complex into tumour induced in mice. The biodistribution showed high accumulation in tumour tissues with T/NT of 6.92 after 60 min post injection. As a result of these promising data, the newly synthesized compounds especially compound 3 affords a potential radio-carrier that could be used as a tumour marker and can be used for cancer therapy after further preclinical studies.

Synthesis, Biological Evaluation, Migratory Inhibition and Docking Study of Indenopyrazolones as Potential Anticancer Agents.

Some bioactive derivatives of indeno[1,2-c]pyrazolones were synthesized through the reaction of phenylhydrazine, different aldehydes and indan-1,2,3-trione at room temperature in acetonitrile. Analytical and spectroscopic studies have confirmed the structural characteristics of the synthesized compounds. In addition, the target compounds were screened for the in-vitro antiproliferative properties against the B16F10 melanoma cancer cell lines by the standard MTT assay. The effect on inflammatory marker cyclooxygenase 2 and matrix metalloproteinase 2, 9 was also checked to determine the anti-inflammatory and anti-cell migratory properties of these compounds. The final compounds were also tested for their tyrosinase inhibitory activity. Among all compounds, screened for anticancer activity, three compounds 4e, 4f and 4h reduced the cell proliferation significantly comparable to that of the positive standard drug erlotinib (IC50 =418.9±1.54 µM) with IC50 values ranging from 20.72-29.35 µM. The compounds 4c-4h decreased the COX-2 expression whereas the MMP 2, 9 expressions were significantly reduced by 4a, 4b and 4h. This was confirmed by molecular docking studies, as 4e, 4f and 4h displayed good interactions with the active site of BRAF protein. The compounds 4b, 4f and 4h exhibited moderate tyrosinase inhibition effect as compared to α-MSH. Collectively, compound 4h can be considered as a candidate for further optimization in the development of anticancer therapies based on the results of biological investigations in this study.

Discovery of pyrazolones as novel carboxylesterase 2 inhibitors that potently inhibit the adipogenesis in cells.

Carboxylesterase 2 (CES2) is one of the most important Phase I drug metabolizing enzymes in the carboxylesterase family. It plays crucial roles in the bioavailability of oral ester prodrugs and the therapeutic effect of some anticancer drugs such as irinotecan (CPT11) and capecitabine. In addition to the well-known roles of CES2 in xenobiotic metabolism, the enzyme also participates in endogenous metabolism and the production of lipids. In this study, we synthesized a series of pyrazolones and assayed their inhibitory effects against CES2 in vitro. Structure-activity relationship analysis of these pyrazolones reveals that the introduction of 4-methylphenyl unit (R1), 4-methylbenzyl (R2) and cyclohexyl (R3) moieties are beneficial for CES2 inhibition. Guided by these SARs results, 1-cyclohexyl-4-(4-methylbenzyl)-3-p-tolyl-1H- pyrazol-5(4H)-one (27) was designed and synthesized. Further investigations demonstrated that the compound 27 exhibited stronger CES2 inhibition activity with a lower IC50 value (0.13 µM). The inhibition kinetic study demonstrated that compound 27 inhibited the hydrolysis of CES2-fluorescein diacetate (FD) through non-competitive inhibition. In addition, the molecular docking showed that the core of pyrazolone, the cyclohexane moiety, 4-methylbenzyl and 4-methylphenyl groups in compound 27 all played important roles with the amino acid residues of CSE2. Also, compound 27 could inhibit adipocyte adipogenesis induced by mouse preadipocytes. In brief, we designed and synthesized a novel pyrazolone compound with a strong inhibitory ability on CES2 and could inhibit the adipogenesis induced by mouse preadipocytes, which can be served as a promising lead compound for the development of more potent pyrazolone-type CES2 inhibitors, and also used as a potential tool for exploring the biological functions of CES2 in human being.

New substituted pyrazolones and dipyrazolotriazines as promising tyrosyl-tRNA synthetase and peroxiredoxin-5 inhibitors: Design, synthesis, molecular docking and structure-activity relationship (SAR) analysis.

New substituted pyrazolone and dipyrazolotriazine derivatives have been synthesized, designed and well characterized as promising dual antimicrobial/antioxidant agents to overcome multidrug resistant bacteria (MDR), oxidative stress and their related diseases. Among all strains, S. aureus was found to be the most susceptible for all compounds except 10b and 12b. Out of the three investigated series, sulfonamide analogues 5a-c displayed excellent antibacterial activity with 5b (MIC = 7.61 µM) and 5a (MIC = 8.98 µM) displaying activity that exceeds the reference drug tetracycline (MIC = 11.77 µM). The same sulfonamide derivatives 5a-c demonstrates high ABTS scavenging capacity comparable to standard. Moreover, the structure-activity relationship (SAR) revealed that benzenesulfonamide is a crucial group for enhancing activity. Molecular docking studies of the potent analogues were performed by targeting the crystal structures of S. aureus tyrosyl-tRNA synthetase and human peroxiredoxin-5 enzymes and the obtained results supported well the in vitro data revealing stronger binding interactions. Pharmacokinetics prediction together with modeling outcomes suggests that our sulfonamide derivatives may serve as useful lead compounds for the treatment of infectious disease.

Structure-based modification of pyrazolone derivatives to inhibit mTORC1 by targeting the leucyl-tRNA synthetase-RagD interaction.

The enzyme leucyl-tRNA synthetase (LRS) and the amino acid leucine regulate the mechanistic target of rapamycin (mTOR) signaling pathway. Leucine-dependent mTORC1 activation depends on GTPase activating protein events mediated by LRS. In a prior study, compound BC-LI-0186 was discovered and shown to interfere with the mTORC1 signaling pathway by inhibiting the LRS-RagD interaction. However, BC-LI-0186 exhibited poor solubility and was metabolized by human liver microsomes. In this study, in silico physicochemical properties and metabolite analysis of BC-LI-0186 are used to investigate the addition of functional groups to improve solubility and microsomal stability. In vitro experiments demonstrated that 7b and 8a had improved chemical properties while still maintaining inhibitory activity against mTORC1. The results suggest a new strategy for the discovery of novel drug candidates and the treatment of diverse mTORC1-related diseases.

T-cell tumour exclusion and immunotherapy resistance: a role for CAF targeting.

Recent studies have highlighted a major role for cancer-associated fibroblasts (CAFs) in promoting immunotherapy resistance by excluding T cells from tumours. Recently, we showed that CAFs can be effectively targeted by inhibiting the enzyme NOX4; this 'normalises' CAFs and overcomes immunotherapy resistance. Here we discuss our study and other strategies for CAF targeting.

Efficacy of Novel Pyrazolone Phosphodiesterase Inhibitors in Experimental Mouse Models of Trypanosoma cruzi.

Pyrazolones are heterocyclic compounds with interesting biological properties. Some derivatives inhibit phosphodiesterases (PDEs) and thereby increase the cellular concentration of cyclic AMP (cAMP), which plays a vital role in the control of metabolism in eukaryotic cells, including the protozoan Trypanosoma cruzi, the etiological agent of Chagas disease (CD), a major neglected tropical disease. In vitro phenotypic screening identified a 4-bromophenyl-dihydropyrazole dimer as an anti-T. cruzi hit and 17 novel pyrazolone analogues with variations on the phenyl ring were investigated in a panel of phenotypic laboratory models. Potent activity against the intracellular forms (Tulahuen and Y strains) was obtained with 50% effective concentration (EC50) values within the 0.17 to 3.3 µM range. Although most were not active against bloodstream trypomastigotes, an altered morphology and loss of infectivity were observed. Pretreatment of the mammalian host cells with pyrazolones did not interfere with infection and proliferation, showing that the drug activity was not the result of changes to host cell metabolism. The pyrazolone NPD-227 increased the intracellular cAMP levels and was able to sterilize T. cruzi-infected cell cultures. Thus, due to its high potency and selectivity in vitro, and its additive interaction with benznidazole (Bz), NPD-227 was next assessed in the acute mouse model. Oral dosing for 5 days of NPD-227 at 10 mg/kg + Bz at 10 mg/kg not only reduced parasitemia (>87%) but also protected against mortality (>83% survival), hence demonstrating superiority to the monotherapy schemes. These data support these pyrazolone molecules as potential novel therapeutic alternatives for Chagas disease.

The role of nucleus accumbens CREB attenuation in rescuing low voluntary running behavior in female rats.

Given the integral role of nucleus accumbens (NAc) cAMP response element binding protein (CREB) activity in motivational processes, the goal of the current study was to determine whether blunting chronic NAc CREB activity could rescue the low physical activity motivation of female, low voluntary running (LVR) rats. NAc CREB phosphorylation is elevated in these rats, a state previously attributed to deficits in reward valuation. It was recently shown that overexpression of the upstream CREB inhibitor, protein kinase inhibitor alpha (PKIα), increased LVR nightly running by ~threefold. Therefore, the current study addresses the extent to which NAc CREB attenuation influences female LVR and wild-type (WT) wheel-running behavior. Inducible reductions in NAc neuronal activity using Gi-coupled hM4Di DREADDs increased running behavior in LVR, but not in WT, rats. Similarly, site-directed pharmacological inhibition of NAc CREB activity significantly increased LVR nightly running distance and time by ~twofold, with no effect in WT rats. Finally, environmentally enriched LVR rats exhibit higher levels of running compared to socially isolated rats in what appeared to be a CREB-related manner. Considering the positive outcomes of upstream CREB modulation and environmental enrichment on LVR behavior, we believe that blunting NAc CREB activity has the neuromolecular potential to partially reverse low physical activity motivation, as exemplified by the LVR model. The positive physical activity outcome of early life enrichment adds translatable value to human childhood enrichment and highlights its importance on motivational processes later in life.

Discovery of pyrazolone spirocyclohexadienone derivatives with potent antitumor activity.

Starting from easy accessible pyrazoletetrahydropyran acetals, a series of new pyrazolone spirocyclohexadienone derivatives were synthesized and assayed for antitumor activity. Compound 10s was identified to possess good antitumor activity. It could induce MDA-MB-231 cancer cell apoptosis in a concentration dependent manner and arrest the cell cycle progression mainly at the G1 phase.

Pyrrolinone derivatives as a new class of P2X3 receptor antagonists. Part 3: Structure-activity relationships of pyrropyrazolone derivatives.

The P2X3 receptor is an attractive target for the treatment of pain and chronic coughing, and thus P2X3 antagonists have been developed as new therapeutic drugs. We previously reported selective P2X3 receptor antagonists by derivatization of hit compound 1. As a result, we identified hit compound 3, the structure of which was similar to hit compound 1. On the basis of SAR studies of hit compound 1, we modified hit compound 3 and compound 42 was identified as having analgesic efficacy by oral administration.

Design, synthesis, and evaluation of indeno[2,1-c]pyrazolones for use as inhibitors against hypoxia-inducible factor (HIF)-1 transcriptional activity.

HIF-1 is regarded as a promising target for the drugs used in cancer chemotherapy, and creating readily accessible templates for the development of synthetic drug candidates that could inhibit HIF-1 transcriptional activity is an important pursuit. In this study, indeno[2,1-c]pyrazolones were designed as readily available synthetic inhibitors of HIF-1 transcriptional activity. Nine compounds were synthesized in 4-5 steps from commercially available starting materials. In evaluations of the ability to inhibit the hypoxia-induced transcriptional activity of HIF-1, compound 3c showed a higher level compared with that of known inhibitor, YC-1. The compound 3c suppressed HIF-1α protein accumulation without affecting the levels of HIF-1α mRNA.