A375 melanoma cells are sensitized to cisplatin-induced toxicity by a synthetic nitro-flavone derivative 2-(4-Nitrophenyl)-4H-chromen-4-one through inhibition of PARP1.

BACKGROUND: Cisplatin has been extensively used in therapeutics for its broad-spectrum anticancer activity and frequently used for the treatment of solid tumors. However, it presents several side-effects and several cancers develop resistance. Combination therapy of cisplatin with poly (ADP-ribose) polymerase 1 (PARP1) inhibitors has been effective in increasing its efficacy at lower doses. METHODS AND RESULTS: In this work, we have shown that the nitro-flavone derivative, 2-(4-Nitrophenyl)-4H-chromen-4-one (4NCO), can improve the sensitivity of cancer cells to cisplatin through inhibition of PARP1. The effect of 4NCO on cisplatin toxicity was studied through combination therapy in both exponential and density inhibited A375 melanoma cells. Combination index (CI) was determined from isobologram analysis. The mechanism of cell killing was assessed by lactate dehydrogenase (LDH) assay. Temporal nicotinamide adenine dinucleotide (NAD+) assay was done to show the inhibition of PARP1. We also performed in silico molecular modeling studies to know the binding mode of 4NCO to a modeled PARP1-DNA complex containing cisplatin-crosslinked adduct. The results from both in silico and in cellulo studies confirmed that PARP1 inhibition by 4NCO was most effective in sensitizing A375 melanoma cells to cisplatin. Isobologram analysis revealed that 4NCO reduced cell viability both in exponential and density inhibited A375 cells synergistically. The combination led to cell death through apoptosis. CONCLUSION: The synthetic nitro-flavone derivative 4NCO effectively inhibited the important nuclear DNA repair enzyme PARP1 and therefore, could complement the DNA-damaging anticancer drug cisplatin in A375 cells and thus, could act as a potential adjuvant to cisplatin in melanoma therapy.

Synthesis of Cyclic Megamolecules.

This paper describes the synthesis of giant cyclic molecules having diameters of 10-20 nm. The molecules are prepared through the reactions of a fusion protein building block with small molecule linkers that are terminated in irreversible inhibitors of enzyme domains present in the fusion. This building block has N-terminal cutinase and C-terminal SnapTag domains that react irreversibly with p-nitrophenyl phosphonate (pNPP) and benzylguanine (BG) groups, respectively. We use a bis-BG and a BG-pNPP linker to join these fusion proteins into linear structures that can then react with a bis-pNPP linker that joins the ends into a cyclic product. The last step can occur intramolecularly, to give the macrocycle, or intermolecularly with another equivalent of linker, to give a linear product. Because these are coupled first- and second-order processes, an analysis of product yields from reactions performed at a range of linker concentrations gives rate constants for cyclization. We determined these to be 9.7 × 10-3 s-1, 2.3 × 10-3 s-1, and 8.1 × 10-4 s-1 for the dimer, tetramer, and hexamer, respectively. This work demonstrates an efficient route to cyclic macromolecules having nanoscale dimensions and provides new scaffolds that can be generated using the megamolecule approach.

Design, synthesis and biological evaluation of novel N-nitrophenyl derivatives based on the structure of acetohydroxyacid synthase.

Acetohydroxyacid synthase (AHAS, EC: 2.2.1.6) is a target for the development of novel herbicides. Two series of N-nitrophenyl derivatives, type-A and type-B, were designed and synthesized based on the active site of the AHAS structure. All the structures of newly prepared compounds were thorough characterized by IR, and 1H NMR spectrums. The IC50 values of all synthesized target compounds against AHAS enzyme and EC50 values for herbicidal activity against Brassica campestris L., Amaranthus mangostanus L. and Sorghum sudanense were determined. The bioactive assay results showed that the type-B compounds exhibited highly improved inhibitory activity against the AHAS enzyme and the tested plants comparing to type-A compounds. The IC50 values of most type-B compounds against the AHAS enzyme were between 25-177µM. The EC50 values of several type-B compounds against Sorghum sudanense reached 5.0mg/L. The differences in the biological activity between type-A and type-B compounds were attributed to two structural features - the orthogonal bend at the N-nitro amides group and the common plane structure of another phenyl with chain bridge. With the structure of the target compounds and the IC50 values for AHAS enzyme, a statistically significant CoMFA model with high predict abilities (q2=0.606, r2=0.982, N=4, SEE=0.058, F=280.255) was obtained, and its reliability was verified. The model will provide a theoretical basis for the further structural optimization.

Chemoenzymatic synthesis of para-nitrophenol (pNP)-tagged α2-8-sialosides and high-throughput substrate specificity studies of α2-8-sialidases.

para-Nitrophenol (pNP)-tagged α2-8-linked sialosides containing different sialic acid forms were chemoenzymatically synthesized using an efficient one-pot three-enzyme α2-8-sialylation system. The resulting compounds allowed high-throughput substrate specificity studies of the α2-8-sialidase activity of a recombinant human cytosolic sialidase hNEU2 and various bacterial sialidases. The sialoside substrate profiles obtained can be used to guide the selection of suitable sialidases for sialylglycan analysis and for cell and tissue surface glycan modification. They can also be used to guide sialidase inhibitor design.

Synthesis and aromatase inhibitory evaluation of 4-N-nitrophenyl substituted amino-4H-1,2,4-triazole derivatives.

In this paper, 13 4-N-nitrophenyl substituted amino-4H-1,2,4-triazole derivatives were synthesized and their aromatase inhibitory activities were measured. The results show that the substitution of the groups on benzyl group can further improve their bioactivity and the compound with Cl on the para position of benzyl has the highest bioactivity (IC50=9.02nM). A QSAR model was constructed from the 13 compounds with genetic function approximation using DS 2.1 package. This model can explain 90.09% of the variance (R(2)Adj), while it can predict 84.95% of the variance (R(2)cv) with the confidence interval of 95%.

Discovery of potent anti-tuberculosis agents targeting leucyl-tRNA synthetase.

Tuberculosis is a serious infectious disease caused by human pathogen bacteria Mycobacterium tuberculosis. Bacterial drug resistance is a very significant medical problem nowadays and development of novel antibiotics with different mechanisms of action is an important goal of modern medical science. Leucyl-tRNA synthetase (LeuRS) has been recently clinically validated as antimicrobial target. Here we report the discovery of small-molecule inhibitors of M. tuberculosis LeuRS. Using receptor-based virtual screening we have identified six inhibitors of M. tuberculosis LeuRS from two different chemical classes. The most active compound 4-{[4-(4-Bromo-phenyl)-thiazol-2-yl]hydrazonomethyl}-2-methoxy-6-nitro-phenol (1) inhibits LeuRS with IC50 of 6µM. A series of derivatives has been synthesized and evaluated in vitro toward M. tuberculosis LeuRS. It was revealed that the most active compound 2,6-Dibromo-4-{[4-(4-nitro-phenyl)-thiazol-2-yl]-hydrazonomethyl}-phenol inhibits LeuRS with IC50 of 2.27µM. All active compounds were tested for antimicrobial effect against M. tuberculosis H37Rv. The compound 1 seems to have the best cell permeability and inhibits growth of pathogenic bacteria with IC50=10.01µM and IC90=13.53µM.

[SEARCHING FOR NEW SYNTHETIC DIURETICS].

This study was aimed at estimation of the diuretic and saluretic activity of 4-nitrophenyl-O-ß-D-glucopyranoside and its aglicon 4-nitrophenol in rats. Test animals daily received 4-nitrophenyl-O-ß-D-glucopyranoside (group 1) and 4-nitrophenol (group 2) intragastrically in 2 mL of distilled water in a dose of 18 µmol/kg from 1st to 7th day and 54 µmol/kg from 8th to 14th days. During the experiment, the most pronounced diuretic activity was observed for 4-nitrophenyl-O-ß-D-glucopyranoside in a dose of 54 µmol/kg, which increased the diuresis in rats 2.5 times as compared to the control value.

Synthesis of novel bisindolylmethane Schiff bases and their antibacterial activity.

In an effort to develop new antibacterial drugs, some novel bisindolylmethane derivatives containing Schiff base moieties were prepared and screened for their antibacterial activity. The synthesis of the bisindolylmethane Schiff base derivatives 3-26 was carried out in three steps. First, the nitro group of 3,3'-((4-nitrophenyl)-methylene)bis(1H-indole) (1) was reduced to give the amino substituted bisindolylmethane 2 without affecting the unsaturation of the bisindolylmethane moiety using nickel boride in situ generated. Reduction of compound 1 using various catalysts showed that combination of sodium borohydride and nickel acetate provides the highest yield for compound 2. Bisindolylmethane Schiff base derivatives were synthesized by coupling various benzaldehydes with amino substituted bisindolylmethane 2. All synthesized compounds were characterized by various spectroscopic methods. The bisindolylmethane Schiff base derivatives were evaluated against selected Gram-positive and Gram-negative bacterial strains. Derivatives having halogen and nitro substituent display weak to moderate antibacterial activity against Salmonella typhi, S. paratyphi A and S. paratyphi B.

Temperature-dependent regioselectivity of nucleophilic aromatic photosubstitution. Evidence that activation energy controls reactivity.

Irradiation (λ > 330 nm) of 2-chloro-4-nitroanisole (1) at 25 °C in aqueous NaOH forms three substitution photoproducts: 2-methoxy-5-nitrophenol (2), 2-chloro-4-nitrophenol (3), and 3-chloro-4-methoxyphenol (4), in chemical yields of 69.2%, 14.3%, and 16.5%. The activation energies for the elementary steps from the triplet state at 25 °C were determined to be 1.8, 2.4, and 2.7 kcal/mol, respectively. The chemical yields of each of the three products were determined for exhaustive irradiations at 0, 35, and 70 °C. The variation with temperature of the experimental yields is reproduced almost exactly by the yields calculated with the Arrhenius equation. This indicates that activation energy is the fundamental property related to regioselectivity in nucleophilic aromatic photosubstitution of the S(N)2 Ar* type. The many methods proposed for predicting regioselectivity in reactions of this type have had limited success and have not been related to activation energy.

Selective capture and collection of live target cells using a photoreactive silicon wafer device modified with antibodies via a photocleavable linker.

A device for the capture and recollection of live target cells is described. The platform was a silicon (Si) wafer modified with an anti-HEL antibody (anti-HEL-IgG, HEL = hen egg lysozyme) through a photocleavable 3-amino-3-(2-nitrophenyl)propionic acid (ANP) linker. The modification processes of the Si wafer surface were monitored by Fourier transform infrared spectroscopy-attenuated total reflection (FTIR-ATR) and fast-scanning atomic force microscopy (FS-AFM). The attachment of IgG and its release reaction on the Si surface via the photochemical cleavage of the ANP linker were observed directly by FS-AFM. The results of an enzyme-linked immunosorbent assay (ELISA) indicated that the photorelease of the complex of anti-HEL-IgG with the secondary antibody-alkaline phosphatase hybrid (secondary IgG-AP) from the Si surface occurs with minimum damage. Furthermore, it was possible to collect SP2/O cells selectively that express HEL on their cell membranes (SP2/O-HEL) on the Si wafer device. Photochemical cleavage of the ANP linker facilitated the effective release of living SP2/O cells whose viability was verified by staining experiments using tripan blue. Moreover, it was possible to reculture the recovered cells. This methodology represents an effective strategy for isolating intact target cells in the biological and medicinal sciences and related fields.

Chemoselective amide formation using O-(4-nitrophenyl)hydroxylamines and pyruvic acid derivatives.

A series of O-(4-nitrophenyl)hydroxylamines were synthesized from their respective oximes using a pulsed addition of excess NaBH(3)CN at pH 3 in 65-75% yield. Steric hindrance near the oxime functional group played a key role in both the ease by which the oxime could be reduced and the subsequent reactivity of the respective hydroxylamine. Reaction of the respective hydroxylamines with pyruvic acid derivatives generated the desired amides in good yields. A comparison of phenethylamine systems bearing different leaving groups revealed significant differences in the rates of these systems and suggested that the leaving group ability of the N-OR substituent plays an important role in determining their reactivity with pyruvic acid. Competition experiments (in 68% DMSO/phosphate buffered saline) using 1 equiv of N-phenethyl-O-(4-nitrophenyl)hydroxylamine and 2 equiv of pyruvic acid in the presence of other nucleophiles such as glycine, cysteine, phenol, hexanoic acid, and lysine demonstrated that significant chemoselectivity is present in this reaction. The results suggest that this chemoselective reaction can occur in the presence of excess α-amino acids, phenols, acids, thiols, and amines.

Kinetic and theoretical studies on alkaline ethanolysis of 4-nitrophenyl salicylate: effect of alkali metal ions on reactivity and mechanism.

Pseudo-first-order rate constants (k(obsd)) for reactions of 4-nitrophenyl salicylate (7) with alkali metal ethoxides (EtOM, M = K, Na, and Li) in anhydrous ethanol have been measured spectrophotometrically. Interestingly, the k(obsd) value decreases significantly as the concentration of EtOM increases. Because the phenolic moiety of substrate 7 would be deprotonated and exist as an anionic form (i.e., 7(-)) under kinetic conditions, the ground-state stabilization of 7(-) through formation of a six-membered cyclic complex with M(+) (i.e., 8) is proposed to be responsible for the decreasing k(obsd) trend. The k(obsd) value at a given concentration of EtOK increases steeply upon addition of [18]crown-6 ether (18C6) up to [18C6]/[EtOK] = 1 in the reaction mixture and then remains relatively constant thereafter. In contrast, k(obsd) decreases upon addition of salts (e.g., LiClO(4) or KSCN) to the reaction mixture, which indicates that M(+) ions inhibit the reaction. However, in the presence of 18C6, the k(obsd) value is independent of the concentration of EtOK but remains constant, which indicates that the reaction proceeds through a unimolecular mechanism in the presence of the complexing agent. Although two conceivable unimolecular pathways (formation of ketene 9 and lactone 10) can account for the kinetic results, the reaction has been concluded to proceed via formation of ketene 9 as the reactive intermediate on the basis of theoretical calculations.

Di-tert-butylsilylene-directed alpha-selective synthesis of p-nitrophenyl T-antigen analogues.

Seven analogues of p-nitrophenyl T-antigen [Galbeta(1-->3)GalNAcalpha(1-->O)PNP] have been synthesized as potential substrates for elucidation of the substrate specificity of endo-alpha-N-acetylgalactosaminidase. These compounds, which are commercially unavailable, include: GlcNAcbeta(1-->3){GlcNAcbeta(1-->6)}GalNAcalpha(1-->O)PNP [core 4 type], GalNAcalpha(1-->3)GalNAcalpha(1-->O)PNP [core 5 type], GlcNAcbeta(1-->6)GalNAcalpha(1-->O)PNP [core 6 type], GalNAcalpha(1-->6)GalNAcalpha(1-->O)PNP [core 7 type], Galalpha(1-->3)GalNAcalpha(1-->O)PNP [core 8 type], Glcbeta(1-->3)GalNAcalpha(1-->O)PNP and GalNAcbeta(1-->3)GalNAcalpha(1-->O)PNP. The assembly of these synthetic probes was accomplished efficiently, based on di-tert-butylsilylene(DTBS)-directed alpha-galactosylation as a key reaction.

A novel approach for the design of a highly selective sulfate-ion-selective electrode.

A hydroxyl Schiff base molecule was synthesized from tetraethylenetetramine and 4-nitrosalicylaldehyde and explored as an ionophore with ionic liquid, cation excluder in PVC membranes showing remarkable sulfate selectivity; the optimal recognition site was predicted by density function theory (DFT) simulation.

Colon cancer releases alpha-tocopherol from its O-glycosides better than normal colon tissue.

BACKGROUND/AIMS: Free radicals, in a colon, may damage DNA, make difficult DNA repair and change course of post-translational modifications of regulatory proteins, which promote tumor initiation and progression. Therefore risk of colon cancer is closely related to diet and other lifestyle factors. Dietary antioxidants, such as vitamin E, should reduce the levels of harmful oxidation products. However vitamin E is not soluble in water, which decreases its bioavailability. As O-glycosides of alpha-tocopherol are better soluble in water and penetrate to tissues easier than free alpha-tocopherol, the aim of our work was to investigate the rate of release the free tocopherol from its O-glycosides in colon cancer, in comparison to human healthy colon tissue. METHODOLOGY: The activities of enzymes catalysing hydrolysis of alpha-tocopheryl glucoside (1a) and mannoside (1b) as well as p-nitrophenyl beta-glucoside (2a) and mannoside (2b) in cancer and healthy human colon tissues, were determined according to the modified method described by Zwierz et al. RESULTS: The alpha-tocopherol and p-nitrophenol were significantly better released from the respective glucosides and mannosides in cancer tissue than in "healthy" human colon tissues, with p = 0.000947 for la, p = 0.033024 for 1b; p = 0.0028 for 2a, and p = 0.0033 for 2b, respectively. CONCLUSION: Alpha-tocopherol and p-nitrophenol are released from the O-glycosides of glucose and mannose in significantly higher amount in colon cancer than in healthy tissues. The alpha-tocopherol O-glycosides can be considered as prodrugs in prevention and treatment of the colon cancer.

Structure of supramolecular assemblies formed by alpha,delta-tetramethylcucurbit[6]uril and 4-nitrophenol.

A host-guest assembly, [(C(40)H(44)N(24)O(12)).(C(6)H(5)NO(3))(8).13(H(2)O)] (1), based on a partial substituted cucurbituril, alpha,delta-tetramethylcucurbit[6]uril (TMeQ[6]), and 4-nitrophenol was synthesized and structurally characterized by single-crystal X-ray diffraction. A combination of hydrogen-bonding between the latticed water molecule and the hydroxyl group of 4-nitrophenol, the hydroxyl group of 4-nitrophenol and the carbonyl groups lining the portals in additon, the C-H...pi interactions between the 4-nitrophenol molecules could be the driving forces of formation such an exclusion host-guest assembly.

Studies leading to potent, dual inhibitors of Bcl-2 and Bcl-xL.

Overexpression of the antiapototic proteins Bcl-2 and Bcl-xL provides a common mechanism through which cancer cells gain a survival advantage and become resistant to conventional chemotherapy. Inhibition of these prosurvival proteins is an attractive strategy for cancer therapy. We recently described the discovery of a selective Bcl-xL antagonist that potentiates the antitumor activity of chemotherapy and radiation. Here we describe the use of structure-guided design to exploit a deep hydrophobic binding pocket on the surface of these proteins to develop the first dual, subnanomolar inhibitors of Bcl-xL and Bcl-2. This study culminated in the identification of 2, which exhibited EC50 values of 8 nM and 30 nM in Bcl-2 and Bcl-xL dependent cells, respectively. Compound 2 demonstrated single agent efficacy against human follicular lymphoma cell lines that overexpress Bcl-2, and efficacy in a murine xenograft model of lymphoma when given both as a single agent and in combination with etoposide.

Fast and Facile Synthesis of 4-Nitrophenyl 2-Azidoethylcarbamate Derivatives from N-Fmoc-Protected α-Amino Acids as Activated Building Blocks for Urea Moiety-Containing Compound Library.

A fast and facile synthesis of a series of 4-nitrophenyl 2-azidoethylcarbamate derivatives as activated urea building blocks was developed. The N-Fmoc-protected 2-aminoethyl mesylates derived from various commercially available N-Fmoc-protected α-amino acids, including those having functionalized side chains with acid-labile protective groups, were directly transformed into 4-nitrophenyl 2-azidoethylcarbamate derivatives in 1 h via a one-pot two-step reaction. These urea building blocks were utilized for the preparation of a series of urea moiety-containing mitoxantrone-amino acid conjugates in 75-92% yields and parallel solution-phase synthesis of a urea compound library consisted of 30 members in 38-70% total yields.

Current perspectives in fragment-based lead discovery (FBLD).

It is over 20 years since the first fragment-based discovery projects were disclosed. The methods are now mature for most 'conventional' targets in drug discovery such as enzymes (kinases and proteases) but there has also been growing success on more challenging targets, such as disruption of protein-protein interactions. The main application is to identify tractable chemical startpoints that non-covalently modulate the activity of a biological molecule. In this essay, we overview current practice in the methods and discuss how they have had an impact in lead discovery - generating a large number of fragment-derived compounds that are in clinical trials and two medicines treating patients. In addition, we discuss some of the more recent applications of the methods in chemical biology - providing chemical tools to investigate biological molecules, mechanisms and systems.

Synthesis, spectral characterisation of 2-(5-methyl-1H-benzimidazol-2-yl)-4-bromo/nitro-phenols and their complexes with zinc(II) ion, and solvent effect on complexation.

2-(5-Methyl-1H-benzimidazol-2-yl)-4-bromo/nitro-phenols (HLBr and HLNO2) and their Zn(II) complexes with ZnX2 (X = Cl, I, NO3) were synthesized and characterized by elemental analysis, molar conductivity, IR, 1H and 13C NMR spectra. The OH proton appears near the NH protons in the 1H NMR spectra of the ligands because of the strong intramolecular hydrogen bonding between the OH hydrogen and the C=N nitrogen atoms. The complexation is investigated in ethanol and isopropanol and it is observed that isopropanol is a better solvent than ethanol for the complex forming. HLBr gives harder complexation reaction with Zn(II) according to HLNO2 because of the stronger intramolecular hydrogen bonding in HLBr, and the both ligands react easier with Zn(NO3)2 than ZnCl2 and ZnI2. The Zn(II) complexes of HLBr have 1:1 M:L ratio and ionic character, however, HLNO2 give a non-ionic complex that has 1:2 M:L ratio. In the complexes the phenolic hydrogen is eliminated and a chelate structure is formed.