In order to investigate the effect on combinations of aromatic antibiotics used in chemotherapy, the hetero-association of the antitumour antibiotics actinomycin D (AMD) with daunomycin (DAU) or novatrone (NOV) has been studied by the methods of 1D- and 2D 500 MHz 1H-NMR spectroscopy and molecular mechanics calculations. The experimental concentration and temperature dependences of the proton chemical shifts of mixtures of the aromatic drugs have been analyzed in terms of a modified statistical–thermodynamical model of hetero-association to give the equilibrium reaction constants, the thermodynamical parameters (ΔH, ΔS) of hetero-association of AMD with DAU or NOV and the limiting values of proton chemical shifts of the molecules in the hetero-complexes. The most favorable averaged structures of the 1:1 DAU–AMD and NOV-AMD hetero-association complexes have been determined using both the limiting values of proton chemical shifts of the molecules and molecular mechanics methods (X-PLOR software). The results show that intermolecular complexes between DAU–AMD and NOV–AMD are mainly stabilized by stacking interactions of the aromatic chromophores, although the DAU–AMD hetero-complex has additional stabilization, which may be explained by an intermolecular hydrogen bond between a carbonyl group of ring C of DAU and the NH group of D–Val of the pentapeptide side chain ring of AMD. The relative content of each type of molecular complex in the mixed solution has been calculated at different values of the ratio (r) of the initial concentrations of DAU and AMD. It is found that the contributions of hetero-complexes to the general equilibrium in solution are predominant at quite different values of r, viz. at r>12 for AMD with NOV and at r>2 for AMD with DAU, compared to r>0.3 for the DAU–NOV system observed previously. It is concluded that anticancer drugs have quite different affinities for formation of hetero-complexes with other aromatic antibiotics in aqueous solution, which may need to be taken into consideration for their use in combination chemotherapy.

The molecular mechanism of the combined action of antibiotic and vitamin was studied by NMR spectroscopy. The heteroassociation of the antitumor antibiotic actinomycin D and flavin mononucleotide was investigated as a function of concentration and temperature by 500 MHz 1H NMR spectroscopy. The equilibrium association constant, the thermodynamic parameters (deltaH, deltaS) of heteroassociation of actinomycin D with flavin mononucleotide, and the limiting values of proton chemical shifts in the heterocomplex were determined from the concentration and temperature dependences of proton chemical shifts of molecules. The most favorable structure of the 1:1 actinomycin D-flavin mononucleotide heteroassociation complex was determined using both the molecular mechanics methods (X-PLOR software) and the limiting values of proton chemical shifts of the molecules. In the calculated structure, the planes of the chromophores of actinomycin D and flavin mononucleotide molecules in the 1:1 heterocomplex are parallel and separated from each other by a distance of about 0.34 nm. At the same time, there is a probability of formation of intermolecular hydrogen bonds in the calculated structure of 1:1 actinomycin D-flavin mononucleotide complex. The analysis of the results obtained suggests that aromatic molecules of vitamins, e.g., flavin mononucleotide, can form energetically favorable heterocomplexes with aromatic antitumor antibiotics in aqueous solution, modulating thereby the efficacy of their medical and biological action.

Stochastic cooperative (STOCH-C) and non-cooperative (STOCH-NC) models have been developed for NMR analysis of the hetero-association of aromatic compounds in solution, in order to take into account all physically meaningful association reactions of molecules in which there are no limitations on the lengths of the aggregates and complexes. These algorithmical approaches are compared with previously published basic (BASE) and generalized (GEN) analytical statistical thermodynamical models of hetero-association of biologically active aromatic molecules using the same sets of published NMR data measured under the same solution conditions (0.1M phosphate buffer, pD=7.1, T=298K). It is shown that, within experimental errors, the BASE analytical model may be used to describe molecular systems characterized by relatively small contributions of hetero-association reactions, whereas the GEN model may be applied to hetero-association reactions of any aromatic compound with different self-association properties. The STOCH-C computational algorithm enabled the effect on hetero-association of the interactions of molecules with different cooperativity parameters of self-association to be estimated for the first time and it is proposed that the algorithm for the stochastic models has great potential for detailed investigation and understanding of the interactions of aromatic molecules in solution.

One- and two-dimensional 1 HNMR spectroscopy (500 MHz) was used to study the heteroassociation of caffeine (CAF), a hydrotropic agent, with flavin mononucleotide (FMN) in an aqueous solution in order to gain insights intothe molecular mechanism of solubilization in the FMN-CAF system. The equilibrium constants, induced proton chemical shifts and thermochemical parameters (AH and AS) for the heteroassociation of the molecules were determined from the concentration and temperature dependences of the proton chemical shifts for interacting CAF and FMN molecules. An analysis of the results obtained demonstrated that CAF-FMN heteroassociates are formed due to the stacking-interaction between the aromatic chromophores of these molecules. The most probable structure of the 1 : 1 CAF-FMN complex was determined from molecular dynamics simulations with the use of the X-PLOR program and an analysis of the induced proton chemical shifts for the molecules under study. Calculations of the relative content of homo- and heterocomplexes at various values of the ratio r of the concentrations CAF and FMN demonstrated that, at r > 10, the contribution from the CAF-FMN heterocomplexes is predominant in the equilibrium distribution of associates in aqueous solutions. It was concluded that the formation of stack-type intermolecular associates of hydrotropic agents (caffeine and nicotinamide) with bioactive compounds is the governing factor in the solubilization of such compounds.

One- and two-dimensional (HNMR)-H-1 spectroscopy (500 MHz) was used to study the heteroassociation of caffeine (CAF), a hydrotropic agent, with flavin mononucleotide (FMN) in an aqueous solution in order to gain insights into the molecular mechanism of solubilization in the FMN-CAF system. The equilibrium constants, induced proton chemical shifts and thermochemical parameters (Delta H and AS) for the heteroassociation of the molecules were determined from the concentration and temperature dependences of the proton chemical shifts for interacting CAF and FMN molecules. An analysis of the results obtained demonstrated that CAF-FMN heteroassociates are formed due to the stacking-interaction between the aromatic chromophores of these molecules. The most probable structure of the 1 : 1 CAF-FMN complex was determined from molecular dynamics simulations with the use of the X-PLOR program and an analysis of the induced proton chemical shifts for the molecules under study. Calculations of the relative content of homo- and heterocomplexes at various values of the ratio r of the concentrations CAF and FMN demonstrated that, at r > 10, the contribution from the CAF-FMN heterocomplexes is predominant in the equilibrium distribution of associates in aqueous solutions. It was concluded that the formation of stack-type intermolecular associates of hydrotropic agents (caffeine and nicotinamide) with bioactive compounds is the governing factor in the solubilization of such compounds.

NMR spectroscopy has been used to elucidate the molecular mechanism of solubilization action of hydrotropic agents nicotinamide (NA) and caffeine (CAF). Hetero-association of NA with riboflavine-mononucleotide (FMN) and CAF with low soluble in aqueous solution synthetic analogue of antibiotic actinomycin D, actinocyl-bis-(3-dimethylaminopropyl) amine (Actill), has been investigated by 500 MHz 1H NMR spectroscopy. Concentration and temperature dependences of proton chemical shifts have been analysed in terms of a statistical-thermodynamic model of indefinite self- and heteroassociation of aromatic molecules. The obtained results enable to conclude that NA-FMN and CAF-Actill intermolecular complexes are mainly stabilized by the stacking interactions of the aromatic chromophores. Hetero-association of the investigated molecules plays an important role in solubilization of aromatic drugs by hydrotropic agents nicotinamide and caffeine.

Complexation of anthracycline antibiotic daunomycin (DAU) with self-complementary deoxyhexanucleotide d(GCATGC) in aqueous solution has been investigated by one-dimensional and two-dimensional homonuclear H-1 NMR spectroscopy (TOCSY and NOESY) and heteronuclear H-1-P-31 NMR spectroscopy (HMBC). Quantitative determination of parameters of oligonucleotide self-association and its complexation with DAU was based on the analysis of the dependences of proton chemical shifts on concentration and temperature. Experimental results were analysed in terms of the equilibrium reaction constants, limiting proton chemical shifts and thermodynamical parameters (enthalpies DeltaH, entropies DeltaS) of the formation of hexamer duplex and different drug-DNA complexes. The most favourable structures of the single-stranded form of d(GCATGC) and the intercalated DAU-hexamer complex have been determined using X-PLOR software taking into consideration both intra- and intermolecular NOE contacts.

The self-association of self-complementary deoxyhexanucleotide d(GCATGC) was investigated in aqueous salt solution. Homonuclear 1H NMR correlation spectroscopy (2D-TOCSY and 2D-NOESY) was used for complete assignments of nonexchangeable protons of the hexamer. The equilibrium reaction constants and thermodynamical parameters of duplex d(GCATGC)2 formation were determined from experimental concentration and temperature dependences of proton chemical shifts of the deoxyhexanucleotide. Distinctive features of the concentration dependences in the range of small concentrations at relatively low temperatures of solution enable one to assume that one single-stranded hexamer sequence forms a compact structure (similar to a hairpin) in aqueous solution. A possible spatial hairpin structure of the hexamer was proposed. Comparative analysis of the experimental and theoretical (using the "nearest neighbor" model) thermodynamical parameters of duplex formation was made.

The complexation of antitumour antibiotics novatrone (NOV) and daunomycin (DAU) in aqueous solution has been studied by one- and two-dimensional 1H-NMR spectroscopy (500 MHz) in order to elucidate the probable molecular mechanism of the action of aromatic antitumour drugs in combination chemotherapy. The equilibrium reaction constants, thermodynamical parameters (ΔH, ΔS) of hetero-association of NOV with DAU and the limiting values of proton chemical shifts of the molecules in the hetero-complexes have been determined from the experimental concentration and temperature dependences of proton chemical shifts of the aromatic molecules. The most favourable structure of the 1:1 NOV–DAU hetero-association complex has been determined using both the molecular mechanics methods (X-PLOR software) and the limiting values of proton chemical shifts of the molecules. The obtained results have shown that intermolecular complexes between NOV and DAU molecules are mainly stabilized by stacking interactions of the aromatic chromophores. It is likely that there is an additional stabilization of the NOV–DAU hetero-complexes by intermolecular hydrogen bonds. It is concluded that aromatic molecules of antibiotics may form energetically stable hetero-association complexes in aqueous solution and hence effect their medical–biological (and probably toxic) activity.

A stochastic model for the NMR analysis of the heteroassociation of two aromatic compounds was developed, which takes into account all physically possible reactions of association of molecules in solution. Expressions for calculating the experimentally observed proton chemical shift were obtained in the general form, and an algorithm for calculating the parameters of heteroassociation using the stochastic model was proposed. The effects of limitations of the basic and general models, as compared with the stochastic model, on the model parameters of the heteroassociation of various biologically active aromatic molecules was analyzed. It was shown that the basic model can be used with a sufficient degree of accuracy for systems with a relatively small contribution of heteroassociation reactions to the total dynamic equilibrium in solution, whereas the general model describes satisfactorily the parameters of heteroassociation practically for all systems studied.

H-1 NMR spectroscopy at 500 Mhz has been used to determine the structures and thermodynamics in aqueous salt solution of the hetero-association of Daunomycin (DAU) with a series of phenanthridine dyes having different numbers of amino/azido groups in the chromophore, together with the self-association of the phenthridine dyes under the same solution conditions (0.1 M phosphate buffer, pD 7.1, 298 K). The NMR measurements have been analyzed using statistical-thermodynamical models of both self-association and hetero-association in which no limitation is set on the size of molecular stacks. In this work the magnitudes of the self-association parameters of Ethidium Bromide (EB) and its azido-derivatives, 8-azido-Ethidium Bromide (EMB) and 3,8-diazido-Ethidium Chloride (EDC), show a successive decrease with rnono- and di-substitition of the 3,8-amino groups of EB. A similar pattern is observed for the equilibrium constants for hetero-association of the phenanthridines with DAU. The thermodynamical and structural parameters of hetero-association of the phenanthridines with DAU are consistent with an intermolecular hydrogen bond between the 3,8 amino-groups of EB and the 9 MeCO group of DAU contributing to the stability of the hetero-complex in aqueous solution.

1D- and 2D H-1 NMR spectroscopy (500 MHz) has been used to study the self-association of a bifunctional intercalator, ethidium homodimer (EBH), in aqueous solution. A physical model describing the equilibrium of different associated forms of the homodimer in solution, including the most probable unfolded, folded conformations, dimer and trimer, has been developed. The magnitudes of equilibrium constants and thermodynamical characteristics of the complexation reactions have been obtained from analysis of the concentration and temperature dependences of proton chemical shifts of EBH molecules. Comparative analysis of the self-association parameters of ethidium homodimer and a monointercalator, ethidium bromide, enables to make some conclusions about structural features and energetic characteristics of aggregates of aromatic molecules in solution.

The structure–activity relations of a series of synthetic phenoxazone drugs with aminoalkyl side chains of variable length and different terminal groups were investigated by examining their biological activity and DNA complexation affinity. Biological activity was determined from their ability to induce apoptosis and cell cycle perturbations (activation of cell cycle checkpoints) using the human malignant MOLT‐3 cell line. The thermodynamic parameters of drug–DNA complexation were determined by differential scanning calorimetry. By comparing the activities of compounds with different terminal groups (amino, dimethylamino and diethylamino), we found that the existence of a terminal dimethylamino group in the alkylamino side chain is an important factor for anti‐tumour activity. Minor modifications in the dimethylaminoalkyl side chain (e.g. elongation by one methylene group) led to notable changes in both the anti‐tumour activity and DNA‐binding properties of the drug, providing unambiguous evidence of a marked structure–activity relation.

The self-association of a bifunctional intercalator (ethidium homodimer (EBH)) in an aqueous salt solution (0.1 mol/l NaCl) was studied using one- and two-dimensional H-1 NMR spectroscopies (500 MHz). A physical model of the equilibrium of the different associated homodimer forms in the solution was suggested. The most probable forms are open and folded conformations, dimers, and trimers. The chemical shifts for the protons of the EBH monomer and associates, as well as the equilibrium constants and thermodynamic parameters (enthalpy and entropy) of the self-association, were obtained from the concentration and temperature dependences of the apparent proton chemical shifts. A conclusion was made that the EBH dimers and trimers are stabilized by dispersion and hydrophobic interactions.

The self-association of a bis-intercalator, ethidium homodimer (EBH), and its hetero-association with phenanthridine dye, propidium iodide (PI), have been studied by 1D and 2D 1H NMR spectroscopy using the analysis of proton chemical shifts changes in aqueous solution as a function of concentration and temperature. Experimental results have shown that dynamic equilibrium in solution includes different conformational states of EBH molecules: folded (F) and unfolded (U) forms, a dimer form (F2) where an aromatic chromophore of one of EBH molecules is inserted (intercalated) between the linked chromophores of the other homodimer molecule and a trimer complex (F3) with two partitially intercalated aromatic chromophores between the chromophores of the folded EBH molecule. It has been found that EBH associates with propidium iodide forming 1:1 complex, where PI is inserted between the chromophores of the folded form, and 1:2 complex resulting from intercalation of PI into F2 EBH dimer. Thermodynamical parameters of EBH self-association and complexation between EBH and PI have been determined and conclusions about the nature of the physical forces responsible for the formation of intermolecular complexes have been made.

Phenanthridine dyes have pronounced mutagenic activity due to their intercalative binding with double-helical DNA. Although the structural significance in the interacalation process of the phenanthridinium chromophore is well established, the role ofits side chains is still under discussion. The comparative analysis of complexation with DNA of phenanthridinium dyes - ethidium bormide (EB) and its two photosensitive analogues: 3-amino-8-azido-5-ethyl-6-phenyl phenanthridinium bormide (EMB) and 3,80diazido-5-ethyl-6-phenyl phenanthridinium chloride (EDC) has shown that they have different affinities of binding with nucleotide sequences in aqueous salt solution. In order to test the role of azido-groups in side chains of EB chromophore on the drug-DNA affinity, the self-association of EB and its two azido-analogues have been studied in this work by one- and two-dimensional 1H-NMR spectrosocpy. Self-association of the aromatic drug molecuels has been studied using concentration and temperature dependences of proton chemical shifts. The equilibrium reaction constants, cooperativity parameters, the limiting values of proton chemical shifts and thermodynamical parameters-enthalpy and entropy of drug self-association have been determined for all the molecular system studied.

500 MHz 1H NMR spectroscopy has been used to determine thermodynamic and structural information on the hetero-association of daunomycin (DAU) with the phenanthridine mutagenic dyes ethidium bromide (EB) and propidium iodide (PI). The NMR complexation data have been analysed by a statistical-thermodynamic model which takes into account indefinite association for both the self-association of the drugs and their hetero-association. The results have been used to estimate the effect of the side chains of the phenanthridines on the competitive binding between DAU and the mutagens with DNA. Knowledge of the equilibrium constants for self-association of the phenanthridines and DAU, their hetero-association and their complexation with a DNA fragment, the deoxytetranucleotide 5′-d(TpGpCpA), enabled the relative content of each of the EB-DAU, PI-DAU, EB-DAU-d(TGCA) and PI-DAU-d(TGCA) complexes to be calculated as a function of drug concentration in mixed solutions. The results provide some insight into the molecular basis of the action of combinations of biologically-active molecules. When intercalating drugs are used in combination, it is found that the decrease in binding of drug or mutagen with DNA is due both to formation of drug-mutagen hetero-association complexes in the mixed solution and to competition for the binding sites by the aromatic molecules; the relative importance of each process depends on the molecular properties of the drug or mutagen molecules being considered. Thus, the longer branched side chain of PI and the electrostatic contribution of the extra positive charge of the molecule compared with the ethyl group of EB results in lower affinity for self-association of PI molecules and their hetero-association with DAU, but increases the degree of binding of PI with DNA.

The complex formation of the antibiotic mitoxantrone (novantrone) with the deoxytetranucleotide 5'-d(TpGpCpA) in an aqueous salt solution was studied by one- and two-dimensional (2D-TOSCY and 2D-NOESY) H-1 NMR spectroscopy (500 MHz). Concentration and temperature dependence of proton chemical shifts of molecules were measured. On the basis of these data, the equilibrium constants of the reaction, the relative content of various complexes as a function of concentration and temperature, the limiting values of chemical shifts of novantrone in complexes, and the thermodynamic parameters DeltaH and DeltaS of complex formation of molecules were calculated. It was concluded that the attachment sites for novantrone are pyrimidine-purine nucleotide sequences, sites d(TG) and d(CA) of the tetranucleotide duplex. The analysis of the thermodynamic parameters of the complex formation suggests that intermolecular hydrogen bonds and electrostatic interactions of the aminoalkyl chains of novantrone with the duplex d(TpGpCpA)(2) play an important role in the stabilization of complexes 1:2 and 2:2. The results were compared with those obtained earlier for typical intercalators of ethidium bromide and daunomycin under identical experimental conditions.