Subtle Structural Changes of Dyes Lead to Distinctly Different Fluorescent Behaviors in Cellular Context: The Role of G-Quadruplex DNA Interaction Using Coumarin-Quinazolinone Conjugates as a Case Study.

Fluorogenic organic materials have gained tremendous attention due to their unique properties. However, only a few of them are suitable for bioimaging. Their different behaviors in organic and cellular environments hinder their application in bioimaging. Thus understanding the photoluminescent behaviors of organic materials in a cellular context is particularly important for their rational design. Herein, we describe two coumarin-quinazolinone conjugates: CQ and MeCQ. The high structure similarity makes them possess similar physical and photophysical properties, including bright fluorescence ascribed to the monomer forms in organic solvents and aggregation-caused quenching (ACQ) effect due to self-assembly aggregation in aqueous solution. However, they behave quite differently in cellular context: that is, CQ exhibits bright fluorescence in living cells, while the fluorescence of MeCQ is almost undetectable. The different performance between CQ and MeCQ in living cells is attributed to their different scenario in G-quadruplex (G4) DNA interaction. CQ selectively binds with G4 DNA to recover its fluorescence via aggregation-disaggregation switching in living cells, while MeCQ remained in the aggregate form due to its poor interplay with G4 DNA. Furthermore, CQ is applied as a two-photon fluorescent dye, and its photoswitchable fluorescence capability is exploited for super-resolution imaging of the specific mitochondrial structure in living cells via the STORM technique.

Antioxidant, Cytotoxic, Genotoxic, and DNA-Protective Potential of 2,3-Substituted Quinazolinones: Structure-Activity Relationship Study.

The evaluation of antioxidant compounds that counteract the mutagenic effects caused by the direct action of reactive oxygen species on DNA molecule is of considerable interest. Therefore, a series of 2,3-substituted quinazolinone derivatives (Q1-Q8) were investigated by different assays, and the relationship between their biological properties and chemical structure was examined. Genotoxicity and the potential DNA-protective effects of Q1-Q8 were evaluated by comet assay and DNA topology assay. Antioxidant activity was examined by DPPH-radical-scavenging, reducing-power, and total antioxidant status (TAS) assays. The cytotoxic effect of compounds was assessed in human renal epithelial cells (TH-1) and renal carcinoma cells (Caki-1) by MTT assay. Analysis of the structure-activity relationship disclosed significant differences in the activity depending on the substitution pattern. Derivatives Q5-Q8, bearing electron-donating moieties, were the most potent members of this series. Compounds were not genotoxic and considerably decreased the levels of DNA lesions induced by oxidants (H2O2, Fe2+ ions). Furthermore, compounds exhibited higher cytotoxicity in Caki-1 compared to that in TH-1 cells. Substantial antioxidant effect and DNA-protectivity along with the absence of genotoxicity suggested that the studied quinazolinones might represent potential model structures for the development of pharmacologically active agents.

Synthesis and biofilm inhibition studies of 2-(2-amino-6-arylpyrimidin-4-yl)quinazolin-4(3H)-ones.

Synthesis of novel 4(3H)-quinazolinonyl aminopyrimidine derivatives has been achieved via quinazolinonyl enones which in turn were obtained from 2-acyl-4(3H)-quinazolinone. They have been assayed for biofilm inhibition against Gram-positive (methicillin-resistant Staphylococcus aureus (MRSA)) and Gram-negative bacteria (Acinetobacter baumannii). The analogues with 2,4,6-trimethoxy phenyl, 4-methylthio phenyl, and 3-bromo phenyl substituents (5h, 5j & 5k) have been shown to inhibit biofilm formation efficiently in MRSA with IC50 values of 20.7-22.4 µM). The analogues 5h and 5j have demonstrated low toxicity in human cells in vitro and can be investigated further as leads.

PI3K/AKT inhibitors aggravate death receptor-mediated hepatocyte apoptosis and liver injury.

The PI3K/AKT signaling pathway is one of the most frequently activated signaling networks in human cancers and has become a valuable target in anticancer therapy. However, accumulating reports suggest that adverse effects such as severe liver injury and inflammation may accompany treatment with pan-PI3K and pan-AKT inhibitors. Our prior work has demonstrated that activation of the PI3K/AKT pathway has a protective role in Fas- or TNFα-induced hepatocytic cell death and liver injury. We postulated that PI3K or AKT inhibitors may exacerbate liver damage via the death factor-mediated hepatocyte apoptosis. In this study we found that several drugs targeting PI3K/AKT either clinically used or in clinical trials sensitized hepatocytes to agonistic anti-Fas antibody- or TNFα-induced apoptosis and significantly shortened the survival of mice in in vivo liver damage models. The PI3K or AKT inhibitors promoted Fas aggregation, inhibited the expression of cellular FLICE-inhibitory protein S and L (FLIPL/S), and enhanced procaspase-8 activation. Conversely, cotreatment with the AKT specific activator SC79 reversed these effects. Taken together, these findings suggest that PI3K or AKT inhibitors may render hepatocytes hypersensitive to Fas- or TNFα-induced apoptosis and liver injury.

Novel quinazolin-4-one derivatives as potentiating agents of doxorubicin cytotoxicity.

We report the design, synthesis and biological evaluation of 17 novel 8-aryl-2-morpholino-3,4-dihydroquinazoline derivatives based on the standard model of DNA-PK and PI3K inhibitors. Novel compounds are sub-divided into two series where the second series of five derivatives was designed to have a better solubility profile over the first one. A combination of in vitro and in silico techniques suggested a plausible synergistic effect with doxorubicin of the most potent compound 14d on cell proliferation via DNA-PK and poly(ADP-ribose) polymerase-1 (PARP-1) inhibition, while alone having a negligible effect on cell proliferation.

Synthesis and evaluation of new 4-oxoquinazolin-3(4H)-yl)benzoic acid and benzamide derivatives as potent antibacterial agents effective against multidrug resistant Staphylococcus aureus.

Treatment of nosocomial and community acquired Staphylococcus aureus infections has become more challenging due to the egression of multi-drug resistance. This has spurred the need for rapid development of new therapeutic agents which can effectively negate the resistance mechanisms. In our current work, several new 4-oxoquinazolin-3(4H)-yl)benzoic acid and benzamide derivatives were synthesized and examined for their antimicrobial activity against ESKAP pathogen panel and pathogenic mycobacteria. In the primary screening, compounds 4a, 4b, 6'a, 6'b, 6'h, 6'i and 6'j were found to demonstrate selective and potent inhibitory activity against Staphylococcus aureus (MICs = 0.25-0.5 µg/mL). When tested against Vero cells, all the compounds were found to be non toxic possessing favourable selectivity index (SI > 10), which encouraged us for carrying out further studies. Compound 6'a (SI > 40) was tested against a number of multiple clinical strains of multi-drug resistant S. aureus and was found to exhibit potent activity, irrespective of the resistant status of the strain. Besides, compound 6'a also exhibited concentration dependent bactericidal activity and synergized with the FDA approved drugs tested. The interesting results obtained suggest the potential utility of the newly synthesized compounds for treatment of multidrug resistant S. aureus infections.

Graph theoretical analysis, in silico modeling, prediction of toxicity, metabolism and synthesis of novel 2-(methyl/phenyl)-3-(4-(5-substituted-1,3,4-oxadiazol-2-yl) phenyl) quinazolin-4(3H)-ones as NMDA receptor inhibitor.

Hit, Lead & Candidate Discovery A variety of novel 2-(methyl/phenyl)-3-(4-(5-substituted-1,3,4-oxadiazol-2-yl)phenyl) quinazolin-4(3H)-ones have been synthesized by treating 3-(4-(5-mercapto-1,3,4-oxadiazol-2-yl)phenyl)-2-(methyl/phenyl)-quinazolin-4(3H)-one with a variety of secondary amines. Graph theoretical analysis was used in identification of drug target that is, NMDAR (N-methyl-d-aspartate receptors). The observed reports of in silico modeling and ligand based toxicity, metabolism prediction studies were encouraging us to synthesize of title compounds and evaluate their antiepileptic effects. The title compounds were tested for its antiepileptic potency by MES and scPTZ model. Rotorod test is used to assess its neurotoxicity. In the preliminary test it was found that in MES test, analogs 6d, 6e, 6f, and 6l were potent; whereas in scPTZ test analogs 6d, 6e, 6f, and 6k displayed potent antiepileptic activity. Additionally these five derivatives were tested in rats orally at a dose of 30 mg/kg and found that compounds 2-methyl-3-(4-(5-morpholino-1,3,4-oxadiazol-2-yl)phenyl)quinazolin-4(3H)-one 6e and 2-methyl-3-(4-(5-(piperidin-1-yl)-1,3,4-oxadiazol-2-yl)phenyl)quinazolin-4(3H)-one 6f exhibited superior activity than reference Phenytoin. In MES test, these derivatives 6e and 6f showed activity at 30 mg/kg i.p. dose after 0.5 hr and 4.0 hr. In scPTZ test these derivatives 6e and 6f showed activity at 100 and 300 mg/kg i.p. dose after 0.5 hr and 4.0 hr, respectively.

Dacomitinib-induced diarrhea: Targeting chloride secretion with crofelemer.

Dacomitinib, an irreversible small-molecule pan-ErbB TKI, has a high incidence of diarrhea, which has been suggested to be due to chloride secretory mechanisms. Based on this hypothesis, crofelemer, an antisecretory agent may be an effective intervention. T84 monolayers were treated with 1 µM dacomitinib and 10 µM crofelemer, and mounted into Ussing chambers for electrogenic ion analysis. Crofelemer attenuated increases in chloride secretion in cells treated with dacomitinib. Albino Wistar rats (n = 48) were treated with 7.5 mg/kg dacomitinib and/or 25 mg/kg crofelemer via oral gavage for 21 days. Crofelemer significantly worsened dacomitinib-induced diarrhea (p = 0.0003), and did not attenuate weight loss (p < 0.0001). Sections of the ileum and colon were mounted into Ussing chambers, and secretory processes analyzed. This indicated that crofelemer lost its anti-secretory action in the presence of dacomitinib in this model. Mass spectrometry revealed that crofelemer did not change serum concentration of dacomitinib. Serum FITC dextran levels indicated that crofelemer was unable to attenuate dacomitinib-induced barrier dysfunction. Tight junction proteins were visualized with immunofluorescence. Qualitative analysis showed dacomitinib induced proteolysis of ZO-1 and occludin, and internalization of claudin-1, which was not attenuated by crofelemer. Detailed histopathological analysis showed that crofelemer was unable to attenuate dacomitinib-induced ileal damage. Crofelemer worsened dacomitinib-induced diarrhea, suggesting that antisecretory drug therapy may be ineffective in this setting.

Dacomitinib-induced diarrhoea is associated with altered gastrointestinal permeability and disruption in ileal histology in rats.

Dacomitinib-an irreversible pan-ErbB tyrosine kinase inhibitor (TKI)-causes diarrhoea in 75% of patients. Dacomitinib-induced diarrhoea has not previously been investigated and the mechanisms remain poorly understood. The present study aimed to develop an in-vitro and in-vivo model of dacomitinib-induced diarrhoea to investigate underlying mechanisms. T84 cells were treated with 1-4 µM dacomitinib and resistance and viability were measured using transepithelial electrical resistance (TEER) and XTT assays. Rats were treated with 7.5 mg/kg dacomitinib daily via oral gavage for 7 or 21 days (n = 6/group). Weights, and diarrhoea incidence were recorded daily. Rats were administered FITC-dextran 2 hr before cull, and serum levels of FITC-dextran were measured and serum biochemistry analysis was conducted. Detailed histopathological analysis was conducted throughout the gastrointestinal tract. Gastrointestinal expression of ErbB1, ErbB2 and ErbB4 was analysed using RT-PCR. The ileum and the colon were analysed using multiplex for expression of various cytokines. T84 cells treated with dacomitinib showed no alteration in TEER or cell viability. Rats treated with dacomitinib developed severe diarrhoea, and had significantly lower weight gain. Further, dacomitinib treatment led to severe histopathological injury localised to the ileum. This damage coincided with increased levels of MCP1 in the ileum, and preferential expression of ErbB1 in this region compared to all other regions. This study showed dacomitinib induces severe ileal damage accompanied by increased MCP1 expression, and gastrointestinal permeability in rats. The histological changes were most pronounced in the ileum, which was also the region with the highest relative expression of ErbB1.

Design and synthesis of novel nitrogen mustard-evodiamine hybrids with selective antiproliferative activity.

A series of novel nitrogen mustard-evodiamine hybrids were synthesized and evaluated for their antitproliferative properties. The antiproliferative activities of 10a-d, 11a-d, and 12a-d against four different kinds of human cancer cell lines (PC-3, HepG2, THP-1 and HL-60) and human normal peripheral blood mononuclear cells (PBMC) were determined. The results showed that all the target hybrid compounds exhibited antiproliferative activities against tested human tumor cell lines to some extent and no antiproliferative activities (>200 µM) against human normal PBMC cells. The antiproliferative selectivity between tumorous and normal cells was very useful for further antitumor drug development. Among the target compounds, 12c showed the strongest cytotoxicity against two tumor cell lines (THP-1 and HL-60) with IC50 values of 4.05 µM and 0.50 µM, respectively, and selected for further mechanism study in HL-60 cells. The results showed that 12c could induce HL-60 cells apoptosis and arrest at G2 phase at low sub-micromolar concentrations via mitochondria-related pathways.

Synthesis of 4(3H)quinazolinimines with selective cytotoxic effect on human acute promyelocytic leukemia cells.

We synthesized a new family of six 4(3H)quinazolinimines based on the reaction between (E)-N-(2-cyanophenyl)benzimidoyl chloride and substituted anilines reaching the formation of their corresponding C2, N3-substituted quinazoliniminium chlorides. This method provides novel, direct and flexible access to diverse substituted 4(3H)quinazolinimines. New compounds obtained following the proposed synthesis were fully characterized and, including the thirteen 4(3H)quinazolinimines synthesized by this method and previously reported by us, were used to study its cytotoxic effect on neoplastic cell lines. The mechanism involved in cell toxicity was also studied. Results showed that these compounds were highly cytotoxic, in particular on Human Promyelocytic Leukemia cells (HL60) and Chronic Myelogenous Leukemia cells (K562) when compared with conventional antineoplastic drugs such as etoposide and cisplatin. The mechanism associated to cytotoxic effect was mainly apoptosis, which not was decreased by antioxidant addition, thereby suggesting that the compounds exert apoptotic death through a mechanism unrelated with oxidative stress.

Synthesis and biological evaluation of certain 3-substituted benzylideneamino-2-(4-nitrophenyl)quinazolin-4(3H)-one derivatives.

Certain new 3H-quinazolin-4-one Schiff's bases were synthesized and screened for their activities against ulcerative colitis "UC". Their activity against phospholipase A2 and protease enzymes was also investigated. Some compounds possessed remarkable effect with different potentials against acetic acid-induced colitis model in rats. Compound 14 (50 mg/kg) was more effective than dexamesathone (0.01 mg/kg). It produced 79.78% protection of control colitis; however, compound 13 produced 75.80% protection and was considered as effective as dexamesathone with 75.30% protection. The observed results could be explained partially by their anti-inflammatory activities which appear as phospholipase A2 (hGIIA) and/or through protease inhibitor potentials. However, all the compounds under test showed preferential inhibition towards hG-IIA type of PLA2 rather than DrG-IB with varying degrees. Interestingly, compounds 14, 13, 12 and 11 displayed excellent inhibitory activity against phospholipase A2 accompanied by protease inhibitory profile.

Synthesis and Anticonvulsant Activity Evaluation of 4-Phenyl-[1,2,4]triazolo[4,3-a]quinazolin-5(4H)-one and Its Derivatives.

A series of 4-(substituted-phenyl)-[1,2,4]triazolo[4,3-a]quinazolin-5(4H)-ones (6a-x) with triazole and other heterocyclic substituents (7-14) were synthesized and the compounds were evaluated for their anticonvulsant activity and neurotoxicity by maximal electroshock (MES) and rotarod neurotoxicity tests. Among the compounds studied, 6o and 6q showed wide margins of safety with protective indices (PIs) that were much higher than those of currently used drugs (PI6o > 25.5, PI6q > 26.0). Compounds 6o and 6q showed significant oral activity against MES-induced seizures in mice, with ED50 values of 88.02 and 94.6 mg/kg, respectively. The two compounds were also found to have potent activity against seizures that were induced by pentylenetetrazole and bicuculline.

The phosphatidylinositol 3-kinases (PI3K) inhibitor GS-1101 synergistically potentiates histone deacetylase inhibitor-induced proliferation inhibition and apoptosis through the inactivation of PI3K and extracellular signal-regulated kinase pathways.

Previously, we showed that inhibition of the protein kinase C ß (PKCß)/AKT pathway augments engagement of the histone deacetylase inhibitor (HDI)-induced apoptosis in lymphoma cells. In the present study, we investigated the cytotoxicity and mechanisms of cell death induced by the delta isoform-specific phosphatidylinositide 3-kinase (PI3K) inhibitor, GS-1101, in combination with the HDI, panobinostat (LBH589) and suberoylanilide hydroxamic acid (SAHA). Lymphoma cell lines, primary non-Hodgkin Lymphoma (NHL) and chronic lymphocytic leukaemia (CLL) cells were simultaneously treated with the HDI, LBH589 and GS-1101. An interaction of the LBH589/GS-1101 combination was formally examined by using various concentrations of LBH589 and GS-1101. Combined treatment resulted in a synergistic inhibition of proliferation and showed synergistic effect on apoptotic induction in all tested cell lines and primary NHL and CLL cells. This study indicates that interference with PI3K signalling dramatically increases HDI-mediated apoptosis in malignant haematopoietic cells, possibly through both AKT-dependent or AKT- independent mechanisms. Moreover, the increase in HDI-related apoptosis observed in PI3K inhibitor-treated cells appears to be related to the disruption of the extracellular signal-regulated kinase (ERK) signalling pathway. This study provides a strong rational for testing the combination of PI3K inhibitors and HDI in the clinic.

Fluorinated quinazolinones as potential radiotracers for imaging kinesin spindle protein expression.

Anti-mitotic anti-cancer drugs offer a potential platform for developing new radiotracers for imaging proliferation markers associated with the mitosis-phase of the cell-cycle. One interesting target is kinesin spindle protein (KSP)-an ATP-dependent motor protein that plays a vital role in bipolar spindle formation. In this work we synthesised a range of new fluorinated-quinazolinone compounds based on the structure of the clinical candidate KSP inhibitor, ispinesib, and investigated their properties in vitro as potential anti-mitotic agents targeting KSP expression. Anti-proliferation (MTT and BrdU) assays combined with additional studies including fluorescence-assisted cell sorting (FACS) analysis of cell-cycle arrest confirmed the mechanism and potency of these biphenyl compounds in a range of human cancer cell lines. Additional studies using confocal fluorescence microscopy showed that these compounds induce M-phase arrest via monoaster spindle formation. Structural studies revealed that compound 20-(R) is the most potent fluorinated-quinazolinone inhibitor of KSP and represents a suitable lead candidate for further studies on designing (18)F-radiolabelled agents for positron-emission tomography (PET).

Synthesis and insecticidal activities of 2,3-dihydroquinazolin-4(1H)-one derivatives targeting calcium channel.

A series of compounds containing dihydroquinazolinone moiety was designed and synthesized. Amine bridge part was changed in comparison with known anthranilic diamides insecticides. Their insecticidal activities against oriental armyworm (Mythimna separata) indicated that most of the compounds showed moderate to high activities at the tested concentrations. In particular, compounds 5a and 5k showed 80% larvicidal activities against oriental armyworm at the concentration of 5mg/L. The present study also explored the possible effects of target compounds on the high voltage-gated calcium channel and the calcium channels in the endoplasmic reticulum in the central neurons isolated from the third instar larvae of Spodoptera exigua using whole-cell patch clamp and calcium imaging technique. The results showed that compound 5a activated the high voltage-gated calcium channel in the central neurons of S. exigua weakly. The peak currents only increased by 6% of the initial value at the end of the 10-min recording after treated with 0.22µM 5a, while chlorantraniliprole has an opposite effect. The effects of 5a on the intracellular calcium ion concentration ([Ca(2+)]i) in neurons were well investigated. The experimental results indicated that these novel compounds have different mechanism compared with chlorantraniliprole.

InCl3-catalysed synthesis of 2-aryl quinazolin-4(3H)-ones and 5-aryl pyrazolo[4,3-d]pyrimidin-7(6H)-ones and their evaluation as potential anticancer agents.

A convenient and practical methodology for the synthesis of 2-aryl quinazolin-4(3H)-ones by the condensation of o-aminobenzamides with aromatic aldehydes under mild conditions using catalytic InCl(3) with good yields and high selectivities. This method has been extended for the synthesis of 5-aryl pyrazolo[4,3-d]pyrimidin-7(6H)-ones which have potential applications in medicinal chemistry. Many of these compounds were evaluated for their anti-proliferative properties in vitro against four cancer cell lines and several compounds were found to be active. Further in vitro studies indicated that inhibition of sirtuins could be the possible mechanism of action of these molecules.

Quinazolinone exposure induces apoptosis and changes expressions of Fas/FasL and C-Flip in embryonic mice testicles.

Quinazolinones represent a class of sedative and anticancer drugs. Quinazolinones-based compounds have ability to suppress prostate tumor growth via apoptosis. Apoptosis is very common in embryos and adults of normal and injured mammalian testes. Effects a new derivative of quinazolinone (4(3H) quinazolinone-2-ethyl-2-phenyl ethyl (QEPE)), on the testis of Balb/C mice embryos were investigated. QEPE was able to reduce number of germ cells and diameter of seminiferous tubules. TUNEL assay analysis indicated that reduction correlated with an increase in the number of apoptotic cell. Furthermore, electron microscope observations confirmed typical apoptotic morphologies characterized by chromatin fragmentation. Finally, RT-PCR analysis showed QEPE increases the levels of Fas/Fasl and decreases C-Flip mRNAs in the testis of exposed embryos.

Synthesis, antiviral and cytotoxic investigation of 2-phenyl-3-substituted quinazolin-4(3H)-ones.

OBJECTIVES: A series of 3(benzylideneamino)-2-phenyl quinazoline-4(3H)-ones was synthesized by reaction of 3-amino-2-phenyl-3H-quinazoline-4-one with various carbonyl compounds. MATERIALS AND METHODS: Chemical structures of the synthesized compounds were confirmed by IR, 1H-NMR and mass spectral analysis. Title compounds were investigated for cytotoxicity and antiviral activity against herpes simplex virus-1 (KOS), herpes simplex virus-2 (G), vaccinia virus, vesicular stomatitis virus, herpes simplex virus-1 TK-KOS ACVr, para influenza-3 virus, reovirus-1, Sindbis virus, Coxsackie virus B4, Punta Toro virus, feline corona virus (FIPV), feline herpes virus, respiratory syncytial virus, influenza A H1 N1 subtype, influenza A H3N2 subtype, influenza B and vesicular stomatitis virus. RESULTS AND CONCLUSION: Compound 3d was found inhibit viral replication of para influenza-3virus, reovirus-1, Sindbis virus, Coxsackie virus B4, Punta Toro virus in Vero cell cultures.

Effects of quinazolinones on Balb/C mice embryonic livers.

Heterocyclic compounds such as quinazolinones have variety of biological and pharmacological properties (anticancer, antiinflammatory, antimicrobial, antimalaria, etc.). Effects of two new quinazolinones viz., 4(3H)-quinazolinone-2-propyl-2-phenylethyl (QPPE) and 4(3H)quinazolinone-2-ethyl-2-phenylethyl (QEPE) were investigated on Balb/C mice embryos livers-the major organ of metabolism and detoxification of drugs and toxins. Histological and pathological studies demonstrated QPPE and QEPE as producers of toxic metabolites after biotransformation, creating necrosis, fatty changes, increase in the number of band cells, hepatocytes' diameters and alkaline phosphatase, in addition to sinusoid dilation, hemorrhages and hyperemia. Transmission electron micrographs showed lipid droplets in hepatocytes' cytoplasm, necrosis, vacuolization, cytoplasm disintegration, disfigured and swollen mitochondria, irregular and abnormal nuclei, nuclei with heterochromatin, condensed chromatins, myelin figures and autophages in injured hepatocytes. In conclusion, QPPE and QEPE make toxic components after biotransformation injuring membranes and creating inflammatory reactions. They also disturb metabolism of lipids pathways and cause the appearances of lipid droplets in hepatocytes.