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Aim: Mammalian DNA Ligases play pivotal role in processes such as DNA replication, recombination, and repair, which qualifies them as potent therapeutic targets to eradicate cancer cells. Recently, we have identified a small molecule inhibitor, SCR7 and its oxidized form SCR7-pyrazine (2-mercapto-6,7-diphenylpteridin-4-ol) (SCR7-P), which can inhibit nonhomologous end-joining (NHEJ) in a Ligase IV-dependent manner. In the present study, we describe a water-soluble version of ligase inhibitor, sodium salt of SCR7-P (Na-SCR7-P) and its anti-tumor effects. Materials and Methods: Water soluble version of SCR7-P was synthesised. To study the inhibitory effect of Na-SCR7-P on ligases, we did in vitro DNA end joining assays using double strand DNA substrates. For this, different concentrations of Na-SCR7-P was used along with purified ligases or cell-free extracts. Further, cytotoxicity induced by Na-SCR7-P was evaluated through trypan blue exclusion assay, JC-1 assay and cell cycle analysis. Anti-tumor activity of Na-SCR7-P was investigated in Swiss albino mice and its off-target effects were studied by conducting kidney and liver test and histological evaluation. Further, the anti-angiogenic effect of the compound was studied using in ovo chorioallantoic membrane assay. Results: Na-SCR7-P inhibited NHEJ in a Ligase IV-dependent manner. However, unlike SCR7 and SCR7-P, it blocked joining catalyzed by all three ligases in vitro, making it an ideal cancer therapeutic agent, as it may target multiple DNA transaction processes within the cancer cells. Na-SCR7-P decreased mitochondrial membrane potential (MMP) leading to cell death in cancer cells. Importantly, the administration of Na-SCR7-P led to a significant reduction in tumor growth from 12th day of treatment, and its impact was significantly higher than previously described SCR7, which targets Ligase IV within cells. Antitumor activity of Na-SCR7-P in mice resulted in enhanced lifespan, with minimal side effects. In addition, the in ovo chorioallantoic membrane assay revealed potent antiangiogenic property of Na-SCR7-P. Conclusion: Our results suggest that Na-SCR7-P can target NHEJ and other DNA repair pathways by disrupting Ligase mediated joining and can potentially be used as a strategy for cancer treatment, owing to its water solubility.

Abstract Background Post-translational modifications (PTMs) of histones and other proteins are perturbed in tumours. For example, reduced levels of acetylated H4K16 and trimethylated H4K20 are associated with high tumour grade and poor survival in breast cancer. Drug-like molecules that can reprogram selected histone PTMs in tumour cells are therefore of interest as potential cancer chemopreventive agents. In this study we assessed the effects of the phytocompounds garcinol and curcumin on histone and p53 modification in cancer cells, focussing on the breast tumour cell line MCF7. Methods Cell viability/proliferation assays, cell cycle analysis by flow cytometry, immunodetection of specific histone and p53 acetylation marks, western blotting, siRNA and RT-qPCR. Results Although treatment with curcumin, garcinol or the garcinol derivative LTK-14 hampered MCF7 cell proliferation, differential effects of these compounds on histone modifications were observed. Garcinol treatment resulted in a strong reduction in H3K18 acetylation, which is required for S phase progression. Similar effects of garcinol on H3K18 acetylation were observed in the osteosarcoma cells lines U2OS and SaOS2. In contrast, global levels of acetylated H4K16 and trimethylated H4K20 in MCF7 cells were elevated after garcinol treatment. This was accompanied by upregulation of DNA damage signalling markers such as γH2A.X, H3K56Ac, p53 and TIP60. In contrast, exposure of MCF7 cells to curcumin resulted in increased global levels of acetylated H3K18 and H4K16, and was less effective in inducing DNA damage markers. In addition to its effects on histone modifications, garcinol was found to block CBP/p300-mediated acetylation of the C-terminal activation domain of p53, but resulted in enhanced acetylation of p53K120, and accumulation of p53 in the cytoplasmic compartment. Finally, we show that the elevation of H4K20Me3 levels by garcinol correlated with increased expression of SUV420H2, and was prevented by siRNA targeting of SUV420H2. Conclusion In summary, although garcinol and curcumin can both inhibit histone acetyltransferase activities, our results show that these compounds have differential effects on cancer cells in culture. Garcinol treatment alters expression of chromatin modifying enzymes in MCF7 cells, resulting in reprogramming of key histone and p53 PTMs and growth arrest, underscoring its potential as a cancer chemopreventive agent.

Author(s): Ravani Ananda Roopa [sup.1], Kempegowda Mantelingu [sup.2], Mridula Guin [sup.3], Sridhar Bilgumba Thimmaiah [sup.4] Author Affiliations: (1) Department of Chemistry, Pooja Bhagavat Memorial Mahajana Education Centre, , 570016, Mysuru, [...], In this study, a novel and efficient bienzymatic method for the quantification of uric acid in serum and urine samples was developed. This method is based on the bienzymatic reaction of uricase and peroxidase in the presence of substrates pyrocatechol and 3-methyl-2-benzothiazolinone hydrazone to produce a red colored product. Under optimized conditions, a linearity of uric acid assay was obtained in the ranges of 4 to 384 [micro]M and 2 to 256 [micro]M by kinetic method and fixed time method, respectively. The low limit of detection and limit of quantification were found to be 0.5 and 1.6 [micro]M, respectively. The developed assay was used to quantify uric acid in human serum and urine samples. The present method has good recovery range of 98.3-101.8% and accuracy range of 92.0-101.4%. Hence, the proposed method could be successfully adopted for the quantification of uric acid in clinical laboratories.

We herein present a novel and an unexpected method for the synthesis of α-ketothioesters by the reaction of pyrrolidine with α-oxodithioesters followed by treatment with methyl iodide in methanol. One of the synthetic applications of α-ketothioesters for the synthesis of 2-acylbenzimidazoles by acid-catalyzed cyclocondensation with benzene-1,2-diamines is described. The probable mechanisms of formation of α-ketothioesters and 2-acylbenzimidazoles are proposed. [ABSTRACT FROM AUTHOR]

The microwave‐assisted synthesis, crystallographic studies, in‐silico docking and the evaluation of in‐vitro biological activities of 2‐(3,4‐dimethoxy)‐6‐(substituted phenyl)‐ imidazo[2,1‐b][1,3,4]thiadiazole‐5‐carbaldehydes is represented here. Single‐crystal X‐ray diffraction experiments were used to identify the crystal structure of the 6‐(4‐chlorophenyl)‐2‐(3,4‐dimethoxybenzyl)imidazo[2,1‐b][1,3,4]thiadiazole‐5‐carbaldehyde. Using fingerprint plots, Hirshfeld surface analysis was used to confirm the contributions of distinct intermolecular interactions in the development of the crystal packing. Further, density functional theory calculations were employed to calculate the molecule's electronic properties. The chemically active regions on the molecule are identified by analysing the molecular electrostatic potential map. in‐silico studies revealed that the molecules can be effectively used as antibacterial agents by taking the 3q82 receptor with chloramphenicol as a standard. As compared to the chloramphenicol standard, in‐vitro tests showed that molecules 4 a, 4 b, 4 c, and 4 f had a zone of inhibition that ranged from 9 to 11 mm against both Gram‐positive (Bacillus subtilis) and Gram‐negative (Escherichia coli) bacteria. The minimum inhibitory concentration was between 40 to 70 μg/ml. These compounds′ Hydroxyl free radical scavenging activity was between 40 to 85 %. Whereas, DPPH radical scavenging activity was between 40 and 70 %. Percentage hemolysis or cell protective activity of the compounds was ineffective at 20–40 %. [ABSTRACT FROM AUTHOR]

Signal transducer and activator of transcription 3 (STAT3) and STAT5 are the transcription factors that have been studied extensively in relevance to the development of cancers in humans. Suppression of either STAT3 or STAT5‐mediated signaling events has been demonstrated to be effective in inducing cytotoxicity in cancer cells. Herein, new hybrids of triazolyl‐indolo‐quinoxaline are synthesized and examined for their effect on the activation of STAT3 and STAT5 pathways in gastric cancer (GC) cells. Among the newly synthesized compounds, 2,3‐difluoro‐6‐((1‐(3‐fluorophenyl)‐1H‐1,2,3‐triazol‐5‐yl)methyl)‐6H‐indolo[2,3‐b]quinoxaline (DTI) displayed selective cytotoxicity against GC cells over their normal counterpart. Flow cytometric analysis, annexin‐V‐fluorescein isothiocyanate staining, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, live and dead assay, and caspase activation experiments suggested DTI as a potent inducer of apoptosis. The mechanistic approach revealed that DTI imparts cytotoxicity via downregulating the phosphorylation of STAT3Y705 and STAT5Y694/699. DTI significantly reduced the nuclear pool of STAT3/STAT5 and reduced the DNA interaction ability of STAT3/STAT5 as evidenced by immunofluorescence and electrophoretic mobility shift assay. Further investigation revealed that inhibitory effects towards STAT proteins were mediated through the suppression of upstream kinases such as JAK1, JAK2, and Src. Treatment of GC cells with pervanadate counteracted the DTI‐driven STAT3/STAT5 inhibition suggesting the involvement of tyrosine phosphatase. Upon DTI exposure, there was a significant upregulation in the mRNA and protein expression of PTPεC, which is a negative regulator of the JAK‐STAT pathway. Knockdown of PTPεC suppressed the DTI‐induced STATs inhibition in GC cells. Taken together, triazolyl‐indolo‐quinoxaline is presented as a new inhibitor of the STAT3/STAT5 pathway in GC cells. [ABSTRACT FROM AUTHOR]

A wide range of functionalized di-substituted oxadiazoles (5a-5o) were formed by intramolecular cyclization of (Z)- N'-benzoylbenzohydrazonic hypoiodous thioanhydride, which was made possible by atom-efficient, environmentally friendly reactions that used the least amount of organic solvent. 2,5 disubstituted 1,3,4-oxadiazole (5a-5o) was prepared by the reaction of substituted di thioester with various aromatic benzo hydrazides in the presence of a mild base. Based on spectral and analytical data, the structures of all the substances have been determined. [ABSTRACT FROM AUTHOR]

Mercaptopyrimidine derivatives are heterocyclic compounds with potent biological activities including antiproliferative, antibacterial, and anti‐inflammatory properties. The present study describes the synthesis and characterization of several mercaptopyrimidine derivatives through condensation of 5,6‐diamino‐2‐mercaptopyrimidin‐4‐ol with various heterocyclic and aromatic aldehydes. Previous studies have shown that SCR7, synthesized from 5,6‐diamino‐2‐mercaptopyrimidin‐4‐ol, induced cytotoxicity by targeting cancer cells by primarily inhibiting DNA Ligase IV involved in nonhomologous end joining, one of the major DNA double‐strand break repair pathways. Inhibition of DNA repair pathways is considered as an important strategy for cancer therapy. Due to limitations of SCR7 in terms of IC50 in cancer cells, here we have designed, synthesized, and characterized potent derivatives of SCR7 using 5,6‐diamino‐2‐mercaptopyrimidin‐4‐ol as the starting material. Several synthesized imine compounds exhibited significant improvement in inhibition of end joining and cytotoxicity up to 27‐fold lower concentrations than SCR7. Among these, two compounds, SCR116 and SCR132, showed increased cancer cell death in a Ligase IV‐dependent manner. Treatment with the compounds also led to reduction in V(D)J recombination efficiency, cell cycle arrest at G2/M phase, accumulation of double‐strand breaks inside cells, and improved anti‐cancer potential when combined with γ‐radiation and radiomimetic drugs. Thus, we describe novel inhibitors of NHEJ with higher efficacy and potential, which can be developed as cancer therapeutics. [ABSTRACT FROM AUTHOR]

An expedient, efficient, economical, environmentally benign, and solvent free amidation protocol of benzhydrols with less reactive nitrogen nucleophiles assisted by propylphosphonic anhydride (T3P®) under microwave irradiation has been developed. The methodology has been deployed for a wide range of heterocycles and electron-withdrawing & electron-donating groups. The protocol resulted in good to excellent yields under the given conditions (26 examples, 68–93% yield). [ABSTRACT FROM AUTHOR]

A facile and conveniently simple new protocol is devised for the synthesis of 3,5-disubstituted and annulated isothiozoles under transition metal and catalyst free conditions utilizing easily available acetophenones, dithioesters and NH4OAc. This strategy involves C=O and C=S functionalization via sequential imine formation followed by intra molecular cyclization and aerial oxidation forming consecutive C−N and S−N bonds respectively in one pot. This protocol offers a low cost, user friendly, straight forward and widely applicable approach to 3,5-disubstituted isothiazoles. [ABSTRACT FROM AUTHOR]

A series of small molecules, theophylline acetohydrazide hydrazone derivatives were obtained via condensation of theophylline‐7‐acetohydrazide with different aromatic/heterocyclic aldehydes. The compounds were synthesized and characterized by using conventional methods. Further, the compounds and standard drugs were evaluated against Mycobacterium tuberculosis H37Rv strain, the activity obtained was varying depending on the functional group attached to theophylline acetohydrazide hydrazone compounds. Among these, Br‐substituted compounds showed more potent against M. tuberculosis with MIC 3.6–4 μM and better than the reference drugs used. The molecular docking studies have shown the possible binding modes of the compounds with protein (PDB ID: 4RHX); the compound 4h has shown highest glide score and binding energy. For all compounds, ADME properties were predicted. [ABSTRACT FROM AUTHOR]

Three title compounds, namely, 2-(4-chlorobenzyl)-5-[(1H-indol-3-yl)methyl]-6- phenylimidazo[2,1-b][1,3,4]thiadiazole, C26H19ClN4S, (I), 2-(4-chlorobenzyl)-6- (4-fluorophenyl)-5-[(1H-indol-3-yl)methyl]imidazo[2,1-b][1,3,4]thiadiazole, C26H18ClFN4S, (II), and 6-(4-bromophenyl)-2-(4-chlorobenzyl)-5-[(1H-indol-3- yl)methyl]imidazo[2,1-b][1,3,4]thiadiazole, C26H18BrClN4S, (III), have been prepared using a reductive condensation of indole with the corresponding 6-aryl-2-(4-chlorobenzyl)imidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehydes (aryl = phenyl, 4-fluorophenyl or 4-bromophenyl), and their crystal structures have been determined. The asymmetric unit of compound (I) consists of two independent molecules and one of the molecules exhibits disorder of the 4-chlorobenzyl substituent with occupancies 0.6289 (17) and 0.3711 (17). Each type of molecule forms a C(8) chain motif built from N--H ⋯ N hydrogen bonds, which for the fully ordered molecule is reinforced by C--H ⋯ π interactions. In compound (II), the chlorobenzyl unit is again disordered, with occupancies 0.822 (6) and 0.178 (6), and the molecules form C(8) chains similar to those in (I), reinforced by C--H ⋯ π interactions involving only the major disorder component. The chlorobenzyl unit in compound (III) is also disordered with occupancies of 0.839 (5) and 0.161 (5). The molecules are linked by a combination of one N--H ⋯ N hydrogen bond and four C--H⋯π interactions, forming a three-dimensional framework. [ABSTRACT FROM AUTHOR]

A vital approach for the synthesis of a range of novel thioxotriaza-spiro derivatives is described. These new heterocyclic systems are obtained via oxalic acid catalyzed reaction of α,β-unsaturated ketones in the presence of 5,6-diamino-2-mercaptopyrimidine-4-ols; thus, spiro rings are constructed in one step. Notably, this transformation involves condensation of an amino group followed by enamine reaction with alkenes and subsequent reaction promoted by oxalic acid to afford spiro compounds with excellent regioselectivity. [ABSTRACT FROM AUTHOR]

Novel and convenient methodology for the construction of N-substituted amide derivatives have been developed from nitriles and alcohols using propylphosphonic anhydride (T3P®). This methodology is an alternate approach to the synthesis of amides via Ritter reaction, which is one of the classical methods for the synthesis of N-substituted amides from nitriles and alcohols. In this approach, first T3P® activates the alcohol which is then attacked by nitrile to form N-substituted amides. This methodology can also apply for the synthesis of benzhydryl ether. This developed protocol is one of the novel applications of T3P®. [ABSTRACT FROM AUTHOR]

An efficient synthetic approach for the construction of 3,5‐disubstituted 1,3,4‐thiadiazoles from dithioesters, hydrazine hydrate and triethyl amine has been developed under transition‐metal free condition. This methodology efficiently constructs 3,5‐disubstituted 1,3,4‐thiadiazole in good to excellent yields with the advantages of wide functional group tolerance and operational simplicity. [ABSTRACT FROM AUTHOR]

Nonhomologous DNA end joining (NHEJ) is the major DNA double‐strand break (DSB) repair pathway in mammals. Previously, we have described a small molecule inhibitor, SCR7, which can inhibit NHEJ in a Ligase IV‐dependent manner. Administration of SCR7 within the cells resulted in the accumulation of DNA breaks, cell death, and inhibition of tumor growth in mice. In the present study, we report that parental SCR7, which is unstable, can be autocyclized into a stable form. Both parental SCR7 and cyclized SCR7 possess the same molecular weight (334.09) and molecular formula (C18H14N4OS), whereas its oxidized form, SCR7‐pyrazine, possesses a different molecular formula (C18H12N4OS), molecular weight (332.07), and structure. While cyclized form of SCR7 showed robust inhibition of NHEJ in vitro, both forms exhibited efficient cytotoxicity. Cyclized and oxidized forms of SCR7 inhibited DNA end joining catalyzed by Ligase IV, whereas their impact was minimal on Ligase III, Ligase I, and T4 DNA Ligase‐mediated joining. Importantly, both forms inhibited V(D)J recombination, although the effect was more pronounced for SCR7‐cyclized. Both forms blocked NHEJ in a Ligase IV‐dependent manner leading to the accumulation of DSBs within the cells. Although cytotoxicity due to SCR7‐cyclized was Ligase IV specific, the pyrazine form exhibited nonspecific cytotoxicity at higher concentrations in Ligase IV‐null cells. Finally, we demonstrate that both forms can potentiate the effect of radiation. Thus, we report that cyclized and oxidized forms of SCR7 can inhibit NHEJ in a Ligase IV‐dependent manner, although SCR7‐pyrazine is less specific to Ligase IV inside the cell. Nonhomologous DNA end joining (NHEJ) is the major DNA DSB repair pathway in mammals. We show that parental SCR7 gets autocyclized to stable SCR7‐cyclized, with the same molecular weight, molecular mass, and melting point, which upon oxidation, forms SCR7‐pyrazine having a different molecular weight, molecular mass, and melting point. Although both forms inhibit NHEJ in a Ligase IV‐dependent manner in vitro and induce cytotoxicity, SCR7‐pyrazine can be nonspecific inside the cell. [ABSTRACT FROM AUTHOR]

An innovative green synthesis of 3,5‐disubstituted 1,2,4‐oxadiazoles from dithioesters using water as a solvent. This approach becomes more interesting because it undergoes nucleophilic substitution–dehydration–intramolecular cyclization in a single step to give 3,5‐disubstituted 1,2,4‐oxadiazoles in good to excellent yields under transition‐metal free conditions and allows wide functional group tolerance. This green protocol leads to synthesis of a drug lead molecule, Ataluren and their analogues. [ABSTRACT FROM AUTHOR]

T3P–DMSO mediated desulfurative cyclization of in situ generated thioamides serves as an efficient and versatile method for the synthesis of imidazo[1,5-a]pyridines and [1,2,4]-triazolo[4,3-a]pyridines with good to excellent yields. Substrates such as 2-methylaminoquinoline and pyrazin-2-yl-methanamine also undergo the corresponding reactions at room temperature. This efficient protocol has several advantages such as mild conditions, short reaction time, operational simplicity and high yields. [ABSTRACT FROM AUTHOR]

A new approach for the synthesis of novel bis(imidazol-1-yl)vinyl-1,2,4-oxadiazoles has been developed. This method operates in a single step giving the products in good to excellent yields. Antiproliferative efficiency of the synthesized compounds was tested against HCT116 (colon cancer) and A549 (lung cancer) cell lines. Among them, compounds 3m (IC50, 9.7 μM) and 3h (IC50, 20.3 μM) were found to be the best antiproliferative agents. [ABSTRACT FROM AUTHOR]

Synthesis of polycyclic amines was achieved by benzoic acid catalysed reaction of 1-aryl THIQs and 1-aryl trypolines with o-allyl salicylaldehydes through the in situ generated azomethine ylide intermediates that undergo intramolecular [3+2]-cycloadditions with four new stereogenic centers in excellent diastereoselectivities under simple and mild conditions. [ABSTRACT FROM AUTHOR]

An efficient ligand- and catalyst-free intramolecular S-arylation leading to the direct synthesis of indalothiochromen-4-ones from simple dithioesters under mild conditions has been developed. This method is particularly noteworthy given its experimental simplicity, high generality, and good functional group toleration. [ABSTRACT FROM AUTHOR]