Molecular docking studies and synthesis of novel bisbenzimidazole derivatives as inhibitors of α-glucosidase.

A series of bisbenzimidazole derivatives starting from o-phenylenediamine and 4-nitro-o-phenylenediamine were prepared with oxalic acid. Most of the reactions were conducted using both the microwave and conventional methods to compare yields and reaction times. The operational simplicity, environmental friendly conditions and high yield in a significantly short reaction time were the major benefits. All substances' inhibitory activities against α-glucosidase were evaluated. The results may suggest a significant role for the nature of bisbenzimidazole compounds in their inhibitory action against α-glucosidase. They showed different range of α-glucosidase inhibitory potential with IC50 value ranging between 0.44±0.04 and 6.69±0.01µM when compared to the standard acarbose (IC50, 13.34±1.26µM). This has described a new class of α-glucosidase inhibitors. Molecular docking studies were done for all compounds to identify important binding modes responsible for inhibition activity of α-glucosidase.

DNA specific fluorescent symmetric dimeric bisbenzimidazoles DBP(n): the synthesis, spectral properties, and biological activity.

A series of new fluorescent symmetric dimeric bisbenzimidazoles DBP(n) bearing bisbenzimidazole fragments joined by oligomethylene linkers with a central 1,4-piperazine residue were synthesized. The complex formation of DBP(n) in the DNA minor groove was demonstrated. The DBP(n) at micromolar concentrations inhibit in vitro eukaryotic DNA topoisomerase I and prokaryotic DNA methyltransferase (MTase) M.SssI. The DBP(n) were soluble well in aqueous solutions and could penetrate cell and nuclear membranes and stain DNA in live cells. The DBP(n) displayed a moderate effect on the reactivation of gene expression.

Fluorine-modified bisbenzimide derivative as a molecular probe for bimodal and simultaneous detection of DNAs by (19)F NMR and fluorescence.

3,5-Bis(trifluoromethyl)benzene modified bisbenzimide H 33258 was synthesized as a (19)F magnetic resonance-based DNA detection probe. The chemical shift and fluorescence of the probe were significantly changed by the addition of hairpin DNAs having an AATT sequence. The probe enables (19)F NMR/fluorescence bimodal detection of the model DNA double strands simultaneously.

Newly synthesized bis-benzimidazole derivatives exerting anti-tumor activity through induction of apoptosis and autophagy.

In this study, a new series of bis-benzimidazole derivatives were designed and synthesized. Most of these new compounds showed significant anti-tumor activity in vitro compared to Hoechst 33258. Among them, the most potent compound 8 had the IC(50) values of 0.56µM for HL60 (Human promyelocytic leukemia cells) tumor cell line and 0.58µM for U937 (Human leukemic monocyte lymphoma cells) tumor cell line. Subsequent toxicity study on human peripheral blood mononuclear cells (PBMC) showed that compound 8 exhibited less toxicity than 5-FU. We also found that apoptosis and autophagy were simultaneously induced by compound 8 in HL60 cells, and inhibition of autophagy by 3-MA decreased compound 8-induced apoptosis, indicating that they acted in synergy to exert tumor cell death.

[DNA sequence-specific ligands: XIV. Synthesis of fluorescent biologicaly active dimeric bisbenzimidazoles-DB(3, 4, 5, 7, 11)].

Five fluorescent symmetric dimeric bisbenzimidazoles DB(n) have been synthesized containing four 2,6-substited benzimidazole fragments and differ in length of oligomethylene linker (n=3, 4, 5, 7, 11) between the two bisbenzimidazole blocks. The ability of these dimeric bisbenzimidazoles to form complexes with double-stranded DNA (dsDNA) was shown by spectral methods. Upon binding to dsDNA DB(n) are localized in the minor groove. The DNA-methyltransferase Dnmt3a inhibition data are demonstrate the site-specific binding of dimeric bisbenzimidazoles DB(3) and DB(11) with oligonucleotide duplex.

Dimeric bisbenzimidazoles inhibit the DNA methylation catalyzed by the murine Dnmt3a catalytic domain.

When located in the DNA minor groove, dimeric bisbenzimidazoles DB(n) effectively inhibited in vitro the Dnmt3a catalytic domain (IC50 5-77 µM). The lowest IC50 value was observed for compound DB(11) with an 11-unit methylene linker joining the bisbenzimidazole fragments. Increased time of incubation of DNA with DB(n) as well as the presence of AT-clusters in DNA enhances the inhibitory effect.

Symmetrical bisbenzimidazoles with benzenediyl spacer: the role of the shape of the ligand on the stabilization and structural alterations in telomeric G-quadruplex DNA and telomerase inhibition.

The extremities of chromosomes end in a G-rich single-stranded overhang that has been implicated in the onset of the replicate senescence. The repeated sequence forming a G-overhang is able to adopt a four-stranded DNA structure called G-quadruplex, which is a poor substrate for the enzyme telomerase. Small molecule based ligands that selectively stabilize the telomeric G-quadruplex DNA, induce telomere shortening eventually leading to cell death. Herein, we have investigated the G-quadruplex DNA interaction with two isomeric bisbenzimidazole-based compounds that differ in terms of shape (V-shaped angular vs linear). While the linear isomer induced some stabilization of the intramolecular G-quadruplex structure generated in the presence of Na(+), the other, having V-shaped central planar core, caused a dramatic structural alteration of the latter, above a threshold concentration. This transition was evident from the pronounced changes observed in the circular dichroism spectra and from the gel mobility shift assay involving the G-quadruplex DNA. Notably, this angular isomer could also induce the G-quadruplex formation in the absence of any added cation. The ligand-quadruplex complexes were investigated by computational molecular modeling, providing further information on structure-activity relationships. Finally, TRAP (telomerase repeat amplification protocol) experiments demonstrated that the angular isomer is selective toward the inhibition of telomerase activity.

Design, synthesis, and anti-tumor evaluation of novel symmetrical bis-benzimidazoles.

A novel symmetrical bis-benzimidazole was designed as DNA minor groove binder. Molecular modeling study showed that it could dock into the minor groove of DNA. Several derivatives were synthesized and confirmed by IR, MS, and (1)H NMR. All these novel compounds were screened for cytotoxic activity on SKOV-3, HeLa, and BGC-823 cell lines in vitro. Some compounds showed IC(50)s in the single-digit micromolar range for cytotoxicity in several tumor cell lines.

Novel bisbenzimidazoles with antileishmanial effectiveness.

A small library of 2,2'-[(alpha,omega-alkanediylbis(oxyphenylene)]bis-1H-benzimidazoles has been prepared and screened in vitro against Pneumocystis carinii, Trypanosoma brucei rhodesiense, and Leishmania donovani. Among the six tested compounds two derivatives emerged as promising hits characterized by IC(50) values lower than that determined for pentamidine against L. donovani.

Triple helix stabilization by covalently linked DNA-bisbenzimidazole conjugate synthesized by maleimide-thiol coupling chemistry.

Tethering of BBZPNH2, an analogue of the Hoechst 33258, with a 14 nucleotide long DNA sequence with the help of succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), a heterobifunctional crosslinking reagent, using DMF/ water as solvent yields a conjugate which effectively stabilizes the triple helix. The above conjugate was hybridized with 26 bp long double stranded (ds) DNA having 14 bp long polypurine-polypyrimidine stretch to form a pyrimidine motif triple helix. The above conjugate increases the thermal stability of both the transitions, that is, triple helix to double helix by 12 degrees C and double helix to single strand transition by 16 degrees C for the triple helix formed with conjugated TFO over the triple helix made from non-conjugated TFO. Fluorescence and circular dichroism spectra recorded at different temperatures confirm the presence of minor groove binding bisbenzimidazole in the AT-rich minor groove of dsDNA even after the major groove bound TFO separates out.

DNA sequence recognition by Hoechst 33258 conjugates of hairpin pyrrole/imidazole polyamides.

A series of hairpin pyrrole/imidazole polyamides linked to a Hoechst 33258 (Ht) analogue (5-7) were synthesized on solid-phase by adopting an Fmoc technique using a series of PyBOP/HOBt mediated coupling reactions. The dsDNA binding properties of Ht-polyamides 5-7 were determined by thermal denaturation experiments. Hairpin Ht-polyamides 5-7 bound to dsDNA sequences 16 and 18 show DeltaTm values that are 14-18 degrees higher than linear Ht-polyamides bound to the same sequences. All three Ht-polyamides were found to be selective for their 9-bp match dsDNA sequences, supporting a relative stronger interaction of an Im/Py anti-parallel dimer with an appropriately positioned G/Cbp rather than sequences containing only A/Tbps. In addition, Ht-polyamides 5 and 7 showed a 20-fold preference for a properly placed G/Cbp over a C/Gbp, while 6 showed a 10-fold preference.

[Ligands with affinity to specific sequence of DNA base pairs. XII. The synthesis and cytological studies of dimeric Hoechst 33258 molecules].

We synthesized dimeric Hoechst dye molecules composed of two moieties of the Hoechst 33258 fluorescent dye phenolic hydroxy groups of which were tethered via pentamethylene, heptamethylene, or triethylene oxide linkers. A characteristic pattern of differential staining of chromosome preparations from human premonocytic leukemia HL60 cells was observed for all the three fluorescent dyes. The most contrast pattern was obtained for the bis-Hoechst analogue with the heptamethylene linker; its quality was comparable with the picture obtained in the case of chromosome staining with 4',6-diamidino-2-phenylindole. The ability to penetrate into the live human fibroblasts was studied for the three bis-Hoechst compounds. The fluorescence intensity of nuclei of live and fixed cells stained with the penta- and heptamethylene-linked bis-Hoechst analogues was found to differ only slightly, whereas the fluorescence of the nuclei of live cells stained with triethylene oxide-linked bis-Hoechst was considerably weaker than that of the fixed cells. The bis-Hoechst molecules are new promising fluorescent dyes that can both differentially stain chromosome preparations and penetrate through cell and nuclear membranes and effectively stain cell nuclei.

Synthesis, DNA binding, topoisomerase inhibition and cytotoxic properties of 2-chloroethylnitrosourea derivatives of hoechst 33258.

A number of novel 2-chloroethylnitrosourea derivatives of Hoechst 33258 were synthesized and examined for cytotoxicity in breast cancer cell cultures and for inhibition of topoisomerases I and II. Evaluation of the cytotoxicity of these compounds employing a MTT assay and inhibition of [3H]thymidine incorporation into DNA in both MDA-MB-231 and MCF-7 breast cancer cells demonstrated that these compounds were more active than Hoechst 33258. The DNA-binding ability of these compounds was evaluated by an ultrafiltration method using calf thymus DNA, poly(dA-dT)2 and poly(dG-dC)2, indicated that these compounds as well as Hoechst 33258 well interact with AT base pair compared with GC pair. Binding studies indicate that these compounds bind more tightly to double-stranded DNA than the parent compound Hoechst 33258. The degree to which these compounds inhibited cell growth breast cancer cells was generally consistent with their relative DNA binding affinity. Mechanistic studies revealed that these compounds act as topoisomerase I (topo I) or topoisomerase II (topo II) inhibitors in plasmid relaxation assays.

DNA sequence recognition in the minor groove by hairpin pyrrole polyamide-Hoechst 33258 analogue conjugate.

A hairpin pyrrole polyamide conjugated to a Hoechst 33258 (Ht) analogue, PyPyPy-gamma-PyPyPy-gamma-Ht, was synthesized on solid-phase by adaptation of an Fmoc technique using a series of PyBOP/HOBt mediated coupling reactions. Sequence selectivity and complex stabilities were characterized by spectrofluorometric titrations and thermal melting studies. The polyamide of the conjugate was observed to bind in a hairpin motif forming 1:1 conjugate:dsDNA complexes. The conjugate is able to recognize nine contiguous A/T bps, discriminating from the sequences containing fewer than nine contiguous A/T bps.

Aromatic analogues of DNA minor groove binders--synthesis and biological evaluation.

Nine carbocyclic analogues of mono- and bis-lexitropsins and two analogues of pentamidine with unsubstituted N-terminal amine group were synthesized. We have investigated the cytotoxic activity of new aromatic analogues of DNA binding ligands in MCF-7 breast cancer cells and assessed their ability to act as inhibitors of topoisomerase I and II. These studies indicate that aromatic analogues of bis-netropsin contain two identical units tethered by alkyloxyl chains are a potent catalytic inhibitor of both topoisomerases and exhibit moderate cytotoxicity in MCF-7 breast cancer cells.

Reaching into the major groove of B-DNA: synthesis and nucleic acid binding of a neomycin-hoechst 33258 conjugate.

Synthesis of a neomycin-Hoechst 33258 conjugate is reported. The conjugate combines the ligands known to selectively bind in the duplex and the triplex groove. The conjugate stabilizes DNA duplex over DNA triplex. The conjugate selectively stabilizes the DNA duplex (in the presence of salt), with as little as 2 muM of the ligand leading to a DeltaTm of 25 degrees C.

Synthesis, DNA-binding activity and cytotoxicity of carbamate derivatives of Hoechst 33258 in breast cancer MCF-7 cells.

A series of carbamate derivatives of Hoechst 33258 was prepared as potential anticancer agents. These new compounds (1-4) were readily prepared in good yields by addition of chloroethyl, bromoethyl, chloropropyl or 4-(chloromethyl)phenyl isocyanates to Hoechst 33258. Their cytotoxic activity was evaluated on human breast cancer MCF-7. Compounds 1-4 were more cytotoxic than Hoechst 33258. In particular derivative 4, the most active of the series, is up to 3 times more potent than Hoechst 33258. The DNA-binding ability of these compounds was evaluated by an ultrafiltration method using calf thymus DNA. These data show that in broad terms the cytotoxic potency of 1-4 in cultured breast cancer MCF-7 cells increases, in accord with their increases in DNA affinity, as shown by the binding constant values.

Nuclease activity and binding characteristics of a cationic "manganese porphyrin-bis(benzimidazole) dye (Hoechst 33258)" conjugate.

To increase the binding affinity and/or the sequence selectivity of the chemical nuclease manganese-(III) tetrakis(4-N-methylpyridiniumyl)porphyrin, we synthesized a conjugate molecule by associating a tris(4-N-methylpyridiniumyl)metalloporphyrin motif to Hoechst 33258 (H33258), a DNA minor groove binding dye known for its selective affinity for A-T tracts. Selected double-stranded (ds) 35-mer oligodeoxyribonucleotides have been used to probe DNA chain breakages induced by the managanese derivative of the conjugate after activation by potassium monopersulfate. Gel electrophoresis analyses show that DNA cuts were generated by the metalloporphyrin moiety of the hybrid molecule, with the H33258 entity interacting in two different possible orientations, upstream or downstream, with its preferred affinity site inside the minor groove. Also studied was the cleavage of a ds 29-mer oligodesoxyribonucleotide containing two stretches of A.T basepair (bp) which clearly showed that the hybrid can occupy the binding region at least in four preferred ways. These cleavage experiments support the strong and selective interaction of the metalloporphyrin-dye hybrid with DNA and allow the estimate of 10 bp as an average size for the affinity site of an isolated conjugate molecule. Further studies by UV-visible spectroscopy, DNA melting temperature determinations, and DNase I footprinting showed, for higher concentrations of H33258 conjugate, a preferential interaction of only the H33258 moiety with DNA (estimated binding site size 6-7 bp) with the porphyrin entity pushed out of the groove and, for the highest concentrations, self-aggregation of the H33258 conjugate all along the DNA strand in a nonselective mode.

Enhanced DNA alkylation activities of Hoechst 33258 analogues designed for bioreductive activation.

A series of analogues of Hoechst 33258, designed to be subject to bioreductive activation, were synthesized, and interactions between these compounds and pBR322 DNA were investigated. Compounds containing a quinone group reacted with DNA via two possible pathways in the presence of reductants NADH or NADPH: radical cleavage and DNA alkylation. The corresponding dimethoxy compounds, which are not subject to reduction, showed very weak DNA binding ability. The strength of alkylation reaction of the quinone derivatives is related to leaving group ability. Furthermore, the quinone compounds preferentially alkylate DNA at 5'-CG and TG sequences rather than at the AT sites preferred as binding sites of Hoechst 33258.

Design, synthesis, DNA sequence preferential alkylation and biological evaluation of N-mustard derivatives of Hoechst 33258 analogues.

The design, synthesis and biological evaluation of a series of bis-benzimidazole analogues of Hoechst 33258 bearing nitrogen mustard moieties is described. The novel compounds show clear evidence of interstrand cross-linking of linear lambda DNA, in contrast to their distamycin nitrogen mustard counterparts. Interference of the cross-linking reaction by the minor groove-selective distamycin suggests that this process takes place in the minor groove of DNA. Sequence preferential alkylation is revealed by high-resolution polyacrylamide gel electrophoresis and autoradiography. Alkylation occurs predominantly at the 5'-A or 5'-G termini of mixed sequences, determined largely by the sequence-recognizing properties of the bis-benzimidazole carrier moiety. An analysis of the frequency of bases around the alkylation sites reveals marked individual base preferences, especially for A at -3 and +3 positions. All of the compounds tested showed cytotoxic properties against the human tumor cell line KB in culture.