Certain natural products, Ditamycin and Netropsin are considered as models of designing new DNA binding agents. A variety of DNA binding ligands were synthesized and accordingly characterized by different bioassays. In the series of azo-polyamide, it showed slight DNA binding affinity but has the properties of DNA photo-cleavage and recognition of mixed sequence. The carbohydrate-azo-polyamide series show the properties of DNA photo-cleavage and more effective in vitro experiment. In the quinoline series proves the attenuation effect for G-quadruplex stabilization which provides a novel strategy for development of G-quadruplex binding ligands.

A series of combilexin-like monoamidines has been synthesized by linking an intercalative unit with the DNA minor groove binder DB 818 via “Click chemistry.” DB 818 is a dicationic minor groove binder that has shown strong binding affinity to AT sequences. The aim was to synthesize novel classes of DNA minor groove binders that are combilexin-like – minor groove binder / intercalator hybrid – as potential unique DNA binding agents and therapeutics against African Sleeping Sickness. Additionally, a series of novel benzo[d]1,3,2-diazaboroles DAPI derivatives were also synthesized and investigated. These boron compounds have the potential to be strong DNA minor groove binders because of their lower pKa and act as potential chromophores for Boron Neutron Capture Therapy.

A series of combilexin-like monoamidines has been synthesized by linking an intercalative unit with the DNA minor groove binder DB 818 via “Click chemistry.” DB 818 is a dicationic minor groove binder that has shown strong binding affinity to AT sequences. The aim was to synthesize novel classes of DNA minor groove binders that are combilexin-like – minor groove binder / intercalator hybrid – as potential unique DNA binding agents and therapeutics against African Sleeping Sickness. Additionally, a series of novel benzo[d]1,3,2-diazaboroles DAPI derivatives were also synthesized and investigated. These boron compounds ave the potential to be strong DNA minor groove binders because of their lower pKa and act as potential chromophores for Boron Neutron Capture Therapy.

Natural and synthetic heterocyclic cations that bind to the DNA minor groove have demonstrated effectiveness as therapeutic agents for cancer, parasitic and viral diseases, as well as powerful probes for use to extend our fundamental understanding of DNA molecular recognition. Crystal and NMR structures with a variety of minor groove binding compounds have shed light on the structural varieties of these systems, the important solvent molecules in the complexes, and the induced fit effects for binding of both DNA and the bound small molecule. Topics of specific importance in DNA recognition are the development of a greater variety of cell-permeable minor groove agents that have increased DNA binding sequence selectivity. In this dissertation, the structural and energetic basis of the interaction between DNA and minor groove binders has been systematically investigated. A set of powerful and complementary biophysical methods have been used: gel electrophoresis with ligation ladder assay, circular dichroism, mass spectrometry, surface plasmon resonance and isothermal titration calorimetry have been applied to determine the binding stoichiometry, binding affinity, kinetics and thermodynamics, and also the structural influence that minor groove binders can have on DNA. The results of several minor groove complexes clearly show that based on DNA sequences, minor groove binders can have multiple binding modes and consequently affect the geometry of DNA minor groove and the overall DNA curvature in distinct manners. In addition, the binding enthalpy of a minor groove binder is essentially salt concentration and binding mode independent. Besides the investigation of DNA-minor groove binder complex, the binding and inhibition of transcription factor PU.1 has also been studied. The highly positive charged PU.1 targets DNA by inserting an α-helix in the major groove of the 5’-GGAA-3’ site, and displays a strong salt concentration dependency. A set of minor groove binders have been rationally designed based on the high-affinity DNA sequence for PU.1 to target the flanking sequences of the 5’-GGAA-3’ site. They display a structure-related PU.1 inhibition efficacy. This work demonstrates that minor groove binders are capable of modulating PU.1 by targeting the opposite groove and supports future efforts to develop agents for other transcription factors.

Indiana University-Purdue University Indianapolis (IUPUI) / The DNA minor groove continues to be an important biological target in the development of anticancer, antiviral, and antimicrobial compounds. Among agents that target the minor groove, studies of well-established benzimidazole-based DNA binders such as Hoechst 33258 have made it clear that the benzimidazole-amidine portion of these molecules promotes an efficient, site-selective DNA association. Building on the beneficial attributes of existing benzimidazole-based DNA binding agents, a series of benzimidazole-amino acid conjugates was synthesized to investigate their DNA recognition and binding properties. In this series of compounds, the benzimidazole-amidine moiety was utilized as a core DNA “anchoring” element accompanied by different amino acids to provide structural diversity that may influence DNA binding affinity and site-selectivity. Single amino acid conjugates of benzimidazole-amidines were synthesized, as well as a series of conjugates containing 20 dipeptides with the general structure Xaa-Gly. These conjugates were synthesized through a solid-phase synthetic route building from a resin-bound amino acid (or dipeptide). The synthetic steps involved: (1) the coupling of 4-formylbenzoic acid to the resin-bound amino acid (via diisopropylcarbodiimide and hydroxybenzotriazole); followed by (2) introduction of a 3,4-diaminobenzamidoxime in the presence of 1,4-benzoquinone to construct the benzimidazole ring; and, finally, (3) reduction of the resin-bound amidoxime functionality to an amidine via treatment with 1M SnCl2•2H2O in DMF before cleavage of final product from the resin. The synthetic route developed and employed was simple and straightforward except for the final reduction that proved to be very arduous. All target compounds were obtained in good yield (based upon weight), averaging 73% mono-amino acid and 78% di-amino acid final compound upon cleavage from resin. Ultimately, the DNA binding activities of the amino acid-benzimidazole-amidine conjugates were analyzed using a fluorescent intercalator displacement (FID) assay and calf thymus DNA as a substrate. The relative DNA binding affinities of both the mono- and di-amino acid-benzimidazole-amidine conjugates were generally weaker than that of netropsin and distamycin with the dipeptide conjugates showing stronger binding affinities than the mono-amino acid conjugates. The dipeptide conjugates containing amino acids with positively charged side chains, Lys-Gly-BI-(+) and Arg-Gly-BI-(+), showed the strongest DNA binding affinities amongst all our synthesized conjugates.

Computer-aided drug discovery involves the application of computer science and programming to solve chemical and biological problems. Specifically, the QSAR (Quantitative Structure Activity Relationships) methodology is used in drug development to provide a rational basis of drug synthesis, rather than a trial and error approach. Molecular dynamics (MD) studies focus on investigating the details of drug-target interactions to elucidate various biophysical characteristics of interest. Infectious diseases like Trypanosoma brucei rhodesiense (TBR) and P. falciparum (malaria) are responsible for millions of deaths annually around the globe. This necessitates an immediate need to design and develop new drugs that efficiently battle these diseases. As a part of the initiatives to improve drug efficacy QSAR studies accomplished the formulation of chemical hypothesis to assist development of drugs against TBR. Results show that CoMSIA 3D QSAR models, with a Pearson’s correlation coefficient of 0.95, predict a compound with meta nitrogens on the phenyl groups, in the combinatorial space based on a biphenyl-furan diamidine design template, to have higher activity against TBR relative to the existing compound set within the same space. Molecular dynamics study, conducted on a linear benzimidazole-biphenyl diamidine that has non-classical structural similarity to earlier known paradigms of minor groove binders, gave insights into the unique water mediated interactions between the DNA minor groove and this ligand. Earlier experiments suggested the interfacial water molecules near the terminal ends of the ligand to be responsible for the exceptianlly high binding constant of the ligand. Results from MD studies show two other modes of binding. The first conformation has a single water molecule with a residency time of 6ns (average) that is closer to the central part of the ligand, which stabilizes the structure in addition to the terminal water. The second conformation that was detected had the ligand completely away from the floor of the minor groove, and hydrogen bonded to the sugar oxygens.

Cell surface saccharides are involved in a variety of essential biological events. Fluorescent sensors for saccharides can be used for detection, diagnosis, analysis and monitoring of pathological processes. The boronic acid functional group is known to bind strongly and reversibly to compounds with diol groups, which are commonly found on saccharides. Sensors that have been developed for the purpose of saccharide recognition have shown great potential. However, they are very hydrophobic and this lack of essential water-solubility makes them useful in biological applications. The first section of this dissertation details the process of developing water-soluble saccharide sensors that change fluorescent properties upon binding to saccharides. The second section of the dissertation focuses on the development of DNA-minor groove binders as antiparasitical agents. Parasitical diseases comprise some of the world’s largest health problems and yet current medication and treatments for these parasitical diseases are often difficult to administer, costly to the patients, and have disruptive side effects. Worse yet, these parasites are developing drug resistance, thus creating an urgent need for new treatments. Dicationic molecules constitute a class of antimicrobial drug candidates that possess high activity against various parasites. The second section details the development of a series of di-cationic agents that were then screened in in vitro activities against parasitical species.

The thesis entitled “Design and Synthesis of Benzimidazole Based Templates in Duplex and Quadruplex DNA Recognition and in Topoisomerase Inhibition” deals with the design and synthesis of several benzimidazole based molecules and their interaction with duplex and quadruplex DNA structures. It also elucidates the inhibition effect of the compounds on the activity of topoisomerase I enzyme of parasitic pathogen Leishmania donovani. The work has been divided into five chapters. Chapter 1: An Introduction to DNA and its Interaction with Small molecules. The first chapter provides an introduction to the double helical structure of DNA and the central dogma that suggests the flow of genetic information from DNA to RNA to protein. This chapter also presents an overview on the various types of small molecules that interact with duplex and quadruplex structures of DNA or interfere with the activity of DNA targeted enzymes like topoisomerase. This chapter describes the importance of such molecules as chemotherapeutic agents. Chapter 2 deals with three isomeric, symmetrical bisbenzimidazole derivatives bearing pyridine on the two termini. The syntheses, duplex DNA binding and computational structure analyses of the molecules have been divided into two sections. Chapter 2A: Novel Symmetrical Pyridine Derivatized Bisbenzimidazoles: Synthesis and Unique Metal Ion Mediated Tunable DNA Minor Groove Binding. The first chapter deals with the synthesis and double stranded (ds) DNA binding characteristics of the three bisbenzimidazole derivatives. Despite being positional isomers, their relative binding affinities towards ds-DNA varied considerably. Fluorescence, circular dichroism and temperature dependent UV-absorption spectroscopy have been employed to characterize ligand-DNA binding interaction. All spectroscopic studies revealed the strong A-T selective DNA binding affinities of the p- and m-pyridine derivatized molecules (p-pyben and m-pyben respectively) and indicated dramatically weak binding interaction of the ortho derivative (o-pyben) to ds-DNA. Additionally, unique transition metal ion mediated tunable DNA binding shown by o-pyben has been described in this chapter. While the ds-DNA binding characteristics of p- and m-pyben remained unaffected in presence of metal ions, that of o-pyben could be reversibly ‘switched off’ in the presence of divalent transition metal ions like Co2+, Ni2+, and Cu2+. Addition of EDTA reversed the effects and DNA binding was again observed. This interesting observation provides valuable insight into the DNA recognition property of these isomeric bisbenzimidazole derivatives. Figure 1. Molecular structures of pyridine derivatized symmetrical bisbenzimidazoles. Chapter 2B: Differential Binding of Positional Isomers of Symmetric Bisbenzimidazoles on DNA Minor-Groove: A Computational study. To explain the weak DNA binding affinity of o-pyben, compared to p- or m-pyben, detailed ab initio/DFT computational analyses of the inherent structural features of the three isomers were performed both in the gas-phase and in water. The study revealed the presence of intramolecular hydrogen bond existing in the opyben, between the benzimidazole proton (H3) and the pyridine nitrogen (N1). Additionally, potential energy scans for rotation about the bonds connecting the pyridine-benzimidazole and benzimidazole-benzimidazole fragments were performed. This revealed surprising conformational rigidity existing in the o- isomer that resisted any out-of-plane twisting of the pyridine-benzimidazole fragment. The presence of intramolecular H-bonding was further confirmed by experimental determination of pKa of the three isomers. The molecules being bisbenzimidazole derivatives bound to the minor groove of ds-DNA, the benzimidazole protons forming hydrogen bonded interactions with the DNA bases. However in the o- derivative, the intramolecular hydrogen bonding made the crucial benzimidazole protons unavailable for DNA binding thereby leading to its poor interaction with DNA. Chapter 3. Novel Series of Anthra[1,2-d]imidazole-6,11-dione Derivatives: Synthesis, DNA Binding and Inhibition of Topoisomerase I of Leishmania donovani This chapter describes the synthesis of nine imidazole fused anthraquinone derivatives and their interaction with double-stranded DNA, investigated by UV-visible absorption spectroscopy and viscometric titrations. Figure 2. Molecular structures of the imidazole fused anthraquinone derivatives. All the molecules showed intercalative mode of binding to double stranded DNA, though their relative binding affinities were different. Next their inhibitory effects on the catalytic activity of topoisomerase I enzyme of Leismania donovani were investigated. L. donovani is the causative agent for human visceral leishmaniasis; a fatal disease affecting liver and spleen. Five out of the nine derivatives tested, proved to be extremely efficient inhibitors of the enzyme. Of them, three showed greater inhibition potency than camptothecin, a well-established topoisomerase I inhibitor and the precursor for several clinically useful anti-tumor drugs. The molecules were shown to inhibit by the stabilization of enzyme-DNA cleavable complex, and the inhibition efficiency was found to be highly dependent on the pKa of the side-chain nitrogen. These results provide useful insights towards developing more potent inhibitors of the parasitic enzyme. As the compounds are synthetically facile, chemically stable and possess long shelf life, they should be attractive candidates for design of novel family of topoisomerase I inhibitor. Indeed the nature of amine based side chain and its pKa would hold the key in such design. Chapter 4 deals with a series of symmetrical bisbenzimidazole derivatives in which the benzimidazole units have been connected via different aromatic linkers. The syntheses, duplex DNA interaction, topoisomerase inhibition and quadruplex DNA stabilization shown by these four molecules have been divided into two sections. Chapter 4A. Synthesis, Duplex DNA Binding and Topoisomerase I Inhibition by Symmetrical Bisbenzimidazole Derivatives with Aromatic Linkers. This chapter describes the synthesis of four symmetrical bisbenzimidazole derivatives bearing aromatic linkers, phenyl, naphthyl or anthryl between the benzimidazole rings. Next their interaction with duplex DNA was investigated using fluorescence and temperature dependent UV absorption spectroscopy and viscometric titration techniques. Addition of DNA caused fluorescence enhancement of the molecules implying their interaction with duplex DNA. All the four molecules on binding to double helical DNA induced thermal stabilization of the latter. Viscometric titration of calf thymus DNA with the four compounds revealed a partial-intercalative mode of binding for the anthracene derivatized molecule 4. Next, their inhibitory effects on the catalytic activity of topoisomerase I enzyme were studied. The anthracene derivatized compound (4) showed high inhibition of the enzyme catalyzed relaxation of supercoiled plasmid DNA. Naphthalene derivatized compound (3) exhibited weak inhibition whereas the derivatives bearing 1,4- and 1,3-disubstitued benzene (1 and 2 respectively) units showed no inhibition. Figure 3. Molecular structures of the symmetrical bisbenzimidazole derivatives. Chapter 4B. Quadruplex DNA Stabilization by Symmetrical Bisbenzimidazole Derivatives with Aromatic Linkers. The ability of the aforementioned molecules to stabilize G-quadruplex structures was investigated next. DNA quadruplex secondary structures are potential molecular targets for new generation chemotherapeutic drugs; hence there is an impetus in developing quadruplex targeting molecules. The Tetrahymena thermophilia telomeric sequence 5´-(T2G4)4-3´ was selected for the studies as it exhibits interesting structural polymorphism depending on whether quadruplex formation occurs in presence of Na+ or K+. Circular dichroism and fluorescence anisotropy techniques were used to study the interaction of these newly synthesized molecules with quadruplex DNA. Also thermal stabilization of quadruplex structure induced by the molecules was determined by temperature dependent UV absorption studies. The compounds 1, 3 and 4 stabilized Na+ induced quadruplex without causing any structural alterations of the latter. However, the m-phenyl linker bearing molecule 2, above a certain [ligand]/[DNA] concentration ratio, caused uniquestructural alteration of the Na+ induced quadruplex such that the CD-signature of the latter resembled that of a K+ induced quadruplex structure. This result was corroborated by quadruplex thermal melting data and fluorescence anisotropy. Interestingly this ligand was also able to induce secondary structure formation in randomly oriented ss-DNA, akin to K+ induced quadruplex structure, even in the absence of Na+ or K+. Chapter 5. Synthesis and DNA Binding of Novel Biscationic Dimers of Bisbenzimidazole Systems. This chapter describes the design, synthesis and ds-DNA binding properties of four dicationic dimers of bisbenzimidazoles. Targeting long base pair sequences in double helical DNA is a key issue in chemical biology and connecting different DNA binding modules by appropriate linkers is an attractive strategy for achieving the same. The precursor monomer unit was a bisbenzimidazole derivative and an analogue of Hoechst 33258. Two such moieties were connected via bisoxyethylenic or 6- or 3-methylenic or piperazinyl units to achieve linker of varying length, rigidity and hydrophilicity. To study the interaction of the dimers with duplex DNA, fluorescence and circular dichroism spectroscopy were used. Two of the dimers, (bbim-2ox-bbim and bbim-6met-bbim) bearing long flexible spacers, were able to target 13-AT base pairs long oligonucleotide sequences in a 1:1 binding mode with an affinity 8-10 times better than the precursor monomer or Hoechst 33258. Also thermal denaturation experiments showed high duplex stabilization induced by the same two dimers. All studies indicated a bidentate mode of binding where both the arms of the dimers participated in DNA binding. The molecules bearing the short and rigid linkers (bbim-3met-bbim and bbimpiper- bbim) on the other hand showed low binding affinity towards duplex DNA, as indicated by fluorescence, circular dichroism and thermal melting studies. The short linkers probably did not favor simultaneous binding of both the monomeric arms of the dimers to DNA minor groove. The work reported in this chapter indicates the strong influence of the length and nature of linker in determining drug/DNA binding affinity. Figure 4. Molecular structures of dicationic dimeric bisbenzimidazole derivatives.(Refer PDF File)

yes / CC-1065, the duocarmycins and yatakemycin are members of a family of ultrapotent antitumour antibiotics that have been the subject of extensive investigations due to their mode of action and potential in the design of new anticancer therapeutics. The natural products and their analogues exert their effects through a sequence selective alkylation of duplex DNA in the minor groove at the N3 of adenine. An understanding of their structure and its effect on biological activity has been derived through chemical synthesis and has also generated new potential lead compounds. These studies form the first section of the review. The desire to progress these compounds to clinic has also led to studies of bioconjugation and prodrug formation and this is discussed in the second section of the review. The combination of synthesis with key biological experiments is a powerful tool to define the requirements for the development of natural products as potential therapeutic agents. The studies described herein form an excellent paradigm for the study and development of other natural products. / EPSRC, Yorkshire Cancer Research, Big C Cancer Research, UCB Pharma