Synthesis,topoisomerase I and II inhibitory activity,cytotoxicity, and structure–activity relationship study of hydroxylated 2,4-diphenyl-6-aryl pyridines

A new series of 2,4-diphenyl-6-aryl pyridines containing hydroxyl group(s) at the ortho, meta, or para position of the phenyl ring were synthesized, and evaluated for topoisomerase I and II inhibitory activity and cytotoxicity against several human cancer cell lines for the development of novel anticancer agents. Structure–activity relationship study revealed that the substitution of hydroxyl group(s) increased topoisomerase I and II inhibitory activity in the order of meta > para > ortho position. Substitution of hydroxyl group on the para position showed better cytotoxicity.     

Synthesis of novel disulfide and sulfone hybrid scaffolds as potent β-glucuronidase inhibitor

Novel series of disulfide and sulfone hybrid analogs (1 −2 0) were synthesized and characterized through EI-MS and ¹H NMR and evaluated for β-glucuronidase inhibitory potential. All synthesized analogs except 13 and 15 showed excellent β-glucuronidase inhibitory potential with IC₅₀ value ranging in between 2.20–88.16 μM as compared to standard d-saccharic acid 1,4 lactone (48.4 ± 1.25 μM). Analogs 19, 16, 4, 1, 17, 6, 10, 3, 18, 2, 11, 14 and 5 showed many fold potent activity against β-glucuronidase inhibitor. Structure activity relationship showed that substitution of electron withdrawing groups at ortho as well as para position on phenyl ring increase potency. Electron withdrawing groups at meta position on phenyl ring showed slightly low potency as compared to ortho and para position. The binding interactions were confirmed through molecular docking studies.     

Pyrrolidinone-bearing methylated and halogenated benzenesulfonamides as inhibitors of carbonic anhydrases

Two series of benzenesulfonamides bearing methyl groups at ortho/ortho or meta/ortho positions and a pyrrolidinone moiety at para position were synthesized and tested as inhibitors of the twelve catalytically active human carbonic anhydrase (CA) isoforms. Observed binding affinities were determined by fluorescent thermal shift assay and intrinsic binding affinities representing the binding of benzenesulfonamide anion to the Zn(II)-bound water form of CA were calculated. Introduction of dimethyl groups into benzenesulfonamide ring decreased the binding affinity to almost all CA isoforms, but gained in selectivity towards one CA isoform. A chloro group at the meta position of 2,6-dimethylbenzenesulfonamide derivatives did not influence the binding to CA I, but it increased the affinity to all other CAs, especially, CA VII and CA XIII (up to 500 fold). The compounds may be used for further development of CA inhibitors with higher selectivity to particular CA isoforms.     

Diaryl-1,2,4-oxadiazole antioxidants: Synthesis and properties of inhibiting the oxidation of DNA and scavenging radicals

Six 1,2,4-oxadiazole derivatives were prepared in order to compare their abilities to protect DNA against radical-mediated oxidation and to scavenge radicals. These derivatives had a structure based on disubstituted 1,2,4-oxadiazole, in which a vanillin group (A ring) and a substituted benzene group (B ring) were the substituents. The functional group at B ring was assigned as ortho- or meta-hydroxylbenzene group, ortho-chlorobenzene group, no group contained, and pyridine group or vanillin group at B ring. It was found that the compound with two vanillin groups attaching to oxadiazole can trap 2.05 radicals in protecting DNA against 2,2′-azobis(2-amidinopropane hydrochloride) (AAPH)-induced oxidation, and the compound with an ortho-hydroxylbenzene group at B ring can trap 1.78 radicals. The compound with an ortho-chlorobenzene group at B ring exhibited the highest ability to inhibit ^·OH-induced oxidation of DNA, while the compound with a meta-hydroxylbenzene group at B ring inhibited Cu²⁺/glutathione (GSH)-induced oxidation of DNA efficiently. The ortho- and para-hydroxylbenzene groups at B ring made the compounds possess the highest rate constant (k) in scavenging 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) cationic radical (ABTS^+.) and 2,2′-diphenyl-1-picrylhydrazyl radical (DPPH). Therefore, only a few hydroxyl groups can markedly enhance the activity of the core-branched antioxidant, which may be a novel structural feature in designing antioxidant.     

Effect of Molecular Characteristics on Cellular Uptake,Subcellular Localization,and Phototoxicity of Zn(II) N-Alkylpyridylporphyrins

Tetra-cationic Zn(II) meso-tetrakis(N-alkylpyridinium-2 (or -3 or -4)-yl)porphyrins (ZnPs) with progressively increased lipophilicity were synthesized to investigate how the tri-dimensional shape and lipophilicity of the photosensitizer (PS) affect cellular uptake, subcellular distribution, and photodynamic efficacy. The effect of the tri-dimensional shape of the molecule was studied by shifting the N-alkyl substituent attached to the pyridyl nitrogen from ortho to meta and para positions. Progressive increase of lipophilicity from shorter hydrophilic (methyl) to longer amphiphilic (hexyl) alkyl chains increased the phototoxicity of the ZnP PSs. PS efficacy was also increased for all derivatives when the alkyl substituents were shifted from ortho to meta, and from meta to para positions. Both cellular uptake and subcellular distribution of the PSs were affected by the lipophilicity and the position of the alkyl chains on the periphery of the porphyrin ring. Whereas the hydrophilic ZnPs demonstrated mostly lysosomal distribution, the amphiphilic hexyl derivatives were associated with mitochondria, endoplasmic reticulum, and plasma membrane. A comparison of hexyl isomers revealed that cellular uptake and partition into membranes followed the order para > meta > ortho. Varying the position and length of the alkyl substituents affects (i) the exposure of cationic charges for electrostatic interactions with anionic biomolecules and (ii) the lipophilicity of the molecule. The charge, lipophilicity, and the tri-dimensional shape of the PS are the major factors that determine cellular uptake, subcellular distribution, and as a consequence, the phototoxicity of the PSs.     

Biotransformation of organic sulfides: Part. 10. Formation of chiral ortho- and meta-substituted benzyl methyl sulfoxides by biotransformation using Helminthosporium species NRRL 4671

Benzyl methyl sulfides substituted with methyl, chloro, cyano, bromo, methoxy, nitro and amino groups in the ortho or meta positions of the aromatic ring have been converted to (S) sulfoxides by biotransformation using the fungal biocatalyst Helminthosporium species NRRL 4671. The enantiomeric excesses for meta-substituted examples were high in those cases where the substituent was of a polar nature, and comparable to those observed for the corresponding para-substituted substrates. With one exception (o-amino), the ortho-substituted examples gave sulfoxides of lower enantiomeric purity. The role of a suitably located polar substituent on an aryl ring of the substrate in ensuring a high enantiomeric excess in sulfoxidation by Helminthosporium species has been confirmed by the biotransformations of 4-(methylthiomethyl)benzyl alcohol and 2-(4-nitrophenyl) ethyl methyl sulfide, which give sulfoxides of much higher optical purity than those obtained from the corresponding unsubstituted substrates.     

Synthesis and biological evaluation of 2-phenol-4-chlorophenyl-6-aryl pyridines as topoisomerase II inhibitors and cytotoxic agents

A new series of 2-phenol-4-chlorophenyl-6-aryl pyridines were designed, synthesized, and evaluated for topoisomerase (topo) I and II inhibitory activities as well as cytotoxic activity against four different human cancer cell lines such as HCT15, T47D, DU145, and Hela. Most of the tested compounds exhibited stronger topo II inhibitory activity at 100 μM as compared to etoposide. All the compounds, except 39, did not show topo I inhibitory activity. Interestingly, compounds that showed better topo II inhibition than etoposide have ortho- or para-chlorophenyl at 4-position of central pyridine, and none of the compounds possess meta-chlorophenyl. SAR study revealed the importance of ortho- or para-chlorophenyl at 4-position of the central pyridine for selective topo II inhibitory activity. Similarly, all compounds possessing meta- or para-hydroxyphenyl moieties showed moderate to significant cytotoxic effects. Particularly, compounds 27–37, and 39 which showed excellent cytotoxicity (IC₅₀ = 0.68–1.25 μM) against T47D breast cancer cells suggest the importance of meta- or para-hydroxyphenyl moiety at 2-position of the central pyridine for the design of anticancer agents with related scaffolds.     

Spectrum of the Reductive Dehalogenation Activity of Desulfitobacterium frappieri PCP-1

Desulfitobacterium frappieri PCP-1 was induced for ortho- and para-dechlorinating activities by different chlorophenols. Dehalogenation rates ranging from 25 to 1,158 nmol/min/mg of cell protein were observed according to the chlorophenol tested and the position of the chlorine removed. D. frappieri shows a broad substrate specificity; in addition to tetrachloroethylene and pentachloropyridine, strain PCP-1 can dehalogenate at ortho, meta, and para positions a large variety of aromatic molecules with substituted hydroxyl or amino groups. Reactions of O demethylation and reduction of nitro to amino substituents on aromatic molecules were also observed.     

Effect of Sulfate and Organic Carbon Supplements on Reductive Dehalogenation of Chloroanilines in Anaerobic Aquifer Slurries

When di-, tri-, and tetrachloroaniline were incubated in methanogenic groundwater slurries, they were reductively dehalogenated by the aquifer microbiota. 2,3,4-Trichloroaniline was metabolized by two pathways. Primary dehalogenation occurred at either the meta or ortho position of this substrate to form 2,4- and 3,4-dichloroaniline, respectively. The latter chemical could be stoichiometrically converted to 3-chloroaniline. 2,3,4,5-Tetrachloroaniline was degraded by the sequential removal of halogens from the para and then the ortho position to form 3,5-dichloroaniline. An additional pathway was observed with this substrate when the aquifer slurries were amended with butyrate. That is, halogens could be removed from both the meta and ortho positions of tetrachloroaniline. The amendment of sulfate to methanogenic aquifer slurries slowed the rate of 2,3,4,5-tetrachloroaniline degradation and increased the amount of substrate channeled through the additional pathway. The reported intermediates or end products are identified by their chromatographic mobility and mass-spectral profiles.     

Inhibition of LPS-stimulated ROS production by fluorinated and hydroxylated chalcones in RAW 264.7 macrophages with structure-activity relationship study

Based on the importance of the previous fluorinated and/or hydroxylated chalcones studies, thirty-six compounds were designed as phenyl or hydroxyphenyl bearing fluoro, trifluoromethyl or trifluoromethoxy phenyl propenones and synthesized by applying modified Claisen-Schmidt condensation reaction as a single step. Inhibitory effects of the synthesized compounds on ROS production stimulated by LPS in RAW 264.7 macrophage were evaluated. Structure-activity relationship (SAR) study revealed that the compounds possessing para-hydroxyphenyl group combined with meta-fluoro or meta-trifluoromethyl phenyl group, and meta/para-hydroxyphenyl group combined with ortho-trifluoromethoxyphenyl group have an essential role in inhibiting the LPS-stimulated ROS production in RAW 264.7 macrophages. The most significant inhibitory effect on LPS-stimulated ROS production in RAW 264.7 macrophages was observed in compound 30 that possessed para-hydroxyphenyl group along with ortho-trifluoromethoxyphenyl group.     

Relative hepatotoxicity of ortho and meta mono substituted thiobenzamides in the rat

Thiobenzamide is known to be hepatotoxic in the rat and the relative hepatotoxicity of para-substituted thiobenzamides has previously been shown to depend strictly on the electronic character of the para substituent. We have now extended this study to include ortho and meta monosubstituted thiobenzamides. Among the meta-substituted compounds, hepatotoxicity varies in strict accordance with the electronic character of the substituent, whereas the ortho-substituted compounds show no toxicity at comparable doses regardless of the nature of the substituent. Explanations for these substituent effects are provided in terms of the chemical reactivity of the compounds and their corresponding S-oxide and S,S-dioxide metabolites.     

Structures of m-iodo Hoechst-DNA complexes in crystals with reduced solvent content: implications for minor groove binder drug design

The DNA photosensitisers m-iodo Hoechst and m-iodo, p-methoxy Hoechst have been co-crystallised with the oligonucleotide d(CGCGAATTCGCG)₂ and their crystal structures determined. The crystals were then subjected to slow dehydration, which reduced their solvent contents from 40 (normal) to 30 (partially dehydrated) and then 20% (fully dehydrated) and caused a reduction in cell volume from 68 000 to 60 000 then 51 000 ų. The dehydration resulted in a dramatic enhancement of diffraction resolution from ~2.6 to beyond 1.5 Å. Crystal structures have also been determined for the partially and fully dehydrated states. The fully dehydrated crystals consist of an infinite polymeric network, in which neighbouring dodecamer duplexes are crosslinked through phosphate oxygens via direct bonding to bridging magnesium cations. This unique three-dimensional structure for DNA is described in detail in the following companion paper. The present paper details evidence from the sequence of crystal structures that the DNA is able to breathe locally, allowing the ligand to leave the minor groove, re-orient in the surrounding solvent medium and then re-enter the groove in a different orientation and location. The rearrangement of the minor groove binding ligands during the dehydration process mimics the binding behaviour of these ligands in solution and in vivo. We also present details of the DNA–ligand interactions that are consistent with a hydrogen atom abstraction mechanism for photocleavage of DNA.     

DNA sequence-dependent positioning of the linker histone in a nucleosome: A single-pair FRET study

Linker histones (LHs) bind to nucleosomes with their globular domain (gH) positioned in either an on- or an off-dyad binding mode. Here, we study the effect of the linker DNA (L-DNA) sequence on the binding of a full-length LH, Xenopus laevis H1.0b, to a Widom 601 nucleosome core particle (NCP) flanked by two 40 bp long L-DNA arms, by single-pair FRET spectroscopy. We varied the sequence of the 11 bp of L-DNA adjoining the NCP on either side, making the sequence either A-tract, purely GC, or mixed with 64% AT. The labeled gH consistently exhibited higher FRET efficiency with the labeled L-DNA containing the A-tract than that with the pure-GC stretch, even when the stretches were swapped. However, it did not exhibit higher FRET efficiency with the L-DNA containing 64% AT-rich mixed DNA when compared to the pure-GC stretch. We explain our observations with a model that shows that the gH binds on dyad and that two arginines mediate recognition of the A-tract via its characteristically narrow minor groove. To investigate whether this on-dyad minor groove-based recognition was distinct from previously identified off-dyad major groove-based recognition, a nucleosome was designed with A-tracts on both the L-DNA arms. One A-tract was complementary to thymine and the other to deoxyuridine. The major groove of the thymine-tract was lined with methyl groups that were absent from the major groove of the deoxyuridine tract. The gH exhibited similar FRET for both these A-tracts, suggesting that it does not interact with the thymine methyl groups exposed on the major groove. Our observations thus complement previous studies that suggest that different LH isoforms may employ different ways of recognizing AT-rich DNA and A-tracts. This adaptability may enable the LH to universally compact scaffold-associated regions and constitutive heterochromatin, which are rich in such sequences.     

Binding of Hoechst 33258 and its Derivatives to DNA

Abstract

In the present work, we employed UV-VIS spectroscopy, fluorescence methods, and circular dichroism spectroscopy (CD) to study the interaction of dye Hoechst 33258, Hoechst 33342, and their derivatives to poly[d(AT)]·poly[d(AT)], poly(dA)·poly(dT), and DNA dodecamer with the sequence 5′-CGTATATATACG-3′. We identified three types of complexes formed by Hoechst 33258, Hoechst 33342, and methylproamine with DNA, corresponding to the binding of each drug in monomer, dimer, and tetramer forms. In a dimer complex, two dye molecules are sandwiched in the same place of the minor DNA groove. Our data show that Hoechst 33258, Hoechst 33342, and methylproamine also form complexes of the third type that reflects binding of dye associates (probably tetramers) to DNA. Substitution of a hydrogen atom in the ortho position of the phenyl ring by a methyl group has a little effect on binding of monomers to DNA. However it reduces strength of binding of tetramers to DNA. In contrast, a Hoechst derivative containing the ortho-isopropyl group in the phenyl ring exhibits a low affinity to poly(dA)·poly(dT) and poly[d(AT)]·poly[d(AT)] and binds to DNA only in the monomer form. This can be attributed to a sterical hindrance caused by the ortho-isopropyl group for side-by-side accommodation of two dye molecules in the minor groove. Our experiments show that mode of binding of Hoechst 33258 derivatives and their affinity for DNA depend on substituents in the ortho position of the phenyl ring of the dye molecule. A statistical mechanical treatment of binding of Hoechst 33258 and its derivatives to a polynucleotide lattice is described and used for determination of binding parameters of Hoechst 33258 and its derivatives to poly[d(AT)]·poly[d(AT)] and poly(dA)·poly(dT).     

Enzymatic decolorization of sulfonphthalein dyes

The white rot fungus (WRF) Pleurotus ostreatus produced manganese peroxidase (MnP) and manganese-independent peroxidase (MIP) activities during solid state fermentation of wheat straw, a natural lignocellulosic substrate. Most of the sulfonphthalein (SP) dyes were decolorized by MnP at pH 4.0. The higher K_m for meta-cresol purple (40 μM) and lower K_m for ortho-cresol red (26 μM) for MnP activities explained the preference for the position of methyl group at ortho than at meta on chromophore. Bromophenol blue decolorizing activity was higher at pH 3.5 and decreased as the concentration of Mn^II was increased. SP-decolorizing activity was associated not only with MnP but also with MIP. Additional bromine group along with the methyl group on SP chromophores decreases the rate of decolorization. Bromination of sulfonphthalein chromophore makes them the poorer substrate for MnP. This is evident from the higher K_m for bromocresol green (117 μM) when compared to bromocresol purple (36 μM) and bromophenol blue (78 μM). The order of preference for the SP dyes as substrate for the MnP-catalyzed decolorizing activity is phenol red > ortho-cresol red > meta-cresol purple > bromophenol red > bromocresol purple > bromophenol blue > bromocresol green and the order of preference for the SP dyes as substrate for the MIP-catalyzed decolorizing activity is bromocresol green > bromophenol blue > bromocresol purple > bromophenol red > meta-cresol purple > ortho-cresol red > phenol red. Inhibition of PR decolorizing activity by NaN₃ provided the evidence of decolorizing activity as an oxidative process.     

A positively charged amino acid at position 129 in nitrilase from Rhodococcus rhodochrous ATCC 33278 is an essential residue for the activity with meta-substituted benzonitriles

A positively charged amino acid (Arg, Lys, or His) at position 129 in Rhodococcus rhodochrous ATCC 33278 nitrilase is essential for the activity of aromatic nitriles. The wild-type enzyme containing Arg129 was active only for meta- and para-substituted benzonitriles with a methyl or amino group, but the R129K and R129H mutant enzymes were active only for meta-substituted benzonitriles. The lack of activity of the mutants for para-substituted benzonitriles may be attributable to steric hindrance between the para-substituent and the side chain of Lys or His.     

Microbial Reductive Dechlorination of Aroclor 1260 in Baltimore Harbor Sediment Microcosms Is Catalyzed by Three Phylotypes within the Phylum Chloroflexi

The specific dechlorination pathways for Aroclor 1260 were determined in Baltimore Harbor sediment microcosms developed with the 11 most predominant congeners from this commercial mixture and their resulting dechlorination intermediates. Most of the polychlorinated biphenyl (PCB) congeners were dechlorinated in the meta position, and the major products were tetrachlorobiphenyls with unflanked chlorines. Using PCR primers specific for the 16S rRNA genes of known PCB-dehalogenating bacteria, we detected three phylotypes within the microbial community that had the capability to dechlorinate PCB congeners present in Aroclor 1260 and identified their selective activities. Phylotype DEH10, which has a high level of sequence identity to Dehalococcoides spp., removed the double-flanked chlorine in 234-substituted congeners and exhibited a preference for para-flanked meta-chlorines when no double-flanked chlorines were available. Phylotype SF1 had similarity to the o-17/DF-1 group of PCB-dechlorinating bacteria. Phylotype SF1 dechlorinated all of the 2345-substituted congeners, mostly in the double-flanked meta position and 2356-, 236-, and 235-substituted congeners in the ortho-flanked meta position, with a few exceptions. A phylotype with 100% sequence identity to PCB-dechlorinating bacterium o-17 was responsible for an ortho and a double-flanked meta dechlorination reaction. Most of the dechlorination pathways supported the growth of all three phylotypes based on competitive PCR enumeration assays, which indicates that PCB-impacted environments have the potential to sustain populations of these PCB-dechlorinating microorganisms. The results demonstrate that the variation in dechlorination patterns of congener mixtures typically observed at different PCB impacted sites can potentially be mediated by the synergistic activities of relatively few dechlorinating species.     

Interaction of procarbazine with calf thymus DNA—a biophysical and molecular docking study

Interaction of procarbazine (PCZ) with calf thymus DNA was studied using biophysical and molecular docking studies. Procarbazine was to interact with DNA with a binding constant of 6.52 × 10³ M⁻¹ as calculated using ultraviolet‐visible spectroscopy. To find out the binding mode, molecular docking was performed that predicted PCZ to interact with DNA through groove binding mode with binding affinity of −6.7 kcal/mole. To confirm the groove binding nature, different experiments were performed. Dye displacement assays confirmed the non‐intercalative binding mode. Procarbazine displaced Hoechst dye from the minor groove of DNA while it was unable to displace intercalating dyes. There was no increase in the viscosity of DNA solution in presence of PCZ. Also, negligible change in the secondary structure of DNA was observed in presence of PCZ as evident by circular dichroism spectra. Procarbazine caused decrease in the melting temperature of DNA possibly because of decrease in the stability of DNA caused by groove binding interaction of PCZ with DNA.     

The P1 phage replication protein RepA contacts an otherwise inaccessible thymine N3 proton by DNA distortion or base flipping

The RepA protein from bacteriophage P1 binds DNA to initiate replication. RepA covers one face of the DNA and the binding site has a completely conserved T that directly faces RepA from the minor groove at position +7. Although all four bases can be distinguished through contacts in the major groove of B-form DNA, contacts in the minor groove cannot easily distinguish between A and T bases. Therefore the 100% conservation at this position cannot be accounted for by direct contacts approaching into the minor groove of B-form DNA. RepA binding sites with modified base pairs at position +7 were used to investigate contacts with RepA. The data show that RepA contacts the N3 proton of T at position +7 and that the T=A hydrogen bonds are already broken in the DNA before RepA binds. To accommodate the N3 proton contact the T₊₇ /A_+7^′ base pair must be distorted. One possibility is that T₊₇ is flipped out of the helix. The energetics of the contact allows RepA to distinguish between all four bases, accounting for the observed high sequence conservation. After protein binding, base pair distortion or base flipping could initiate DNA melting as the second step in DNA replication.     

Synthesis, characterization and in vitro anti-cancer activity of N-(ferrocenyl)benzoyl tri- and tetrapeptide esters

N-ortho, N-meta and N-para-(ferrocenyl)benzoyl tri- and tetrapeptide esters (2-7) were prepared by coupling ortho, meta and para-ferrocenyl benzoic acids to the tri- and tetrapeptide ethyl esters of GlyGlyGly(OEt) and GlyGlyGlyGly(OEt) in the presence of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole. The compounds were characterized by a range of NMR spectroscopic techniques, mass spectrometry and cyclic voltammetry. The anti-proliferative effects of the ortho derivatives 2 and 5 were measured in vitro against H1299 lung cancer cells and both gave IC₅₀ values greater than 50 μM. Therefore, extending the length of the peptide chain had a negative effect on activity, relative to N-(ferrocenyl)benzoyl amino acid and dipeptide derivatives.