Study of the bisintercalation of the antitumor drug ditercalinium by 31P-NMR

Bisintercalation of ditercalinium, a potent antitumoral 7H-pyriodo[4,3-c]carbazole rigid dimer, into the self-complementary tetranucleotides d(CpGpCpG)₂, d(m⁵CpGpm⁵CpG) and the self-complementary hexanucleotide d(CpGpApTpCpG)₂ was investigated by 162-MHz ³¹P-nmr. The slow exchange, on the nmr time scale, observed between the free and complexed nucleotides allows identification of the phosphorus signals in the complexes through two-dimensional chemical exchange spectroscopy. Differences in ³¹P chemical shifts upon intercalation are discussed in relation to the complex geometry and nature of the drug.     

Bisintercalation of ditercalinium into a d[CpGpCpG]2 minihelix: structure and dynamics aspects--a 400-MHz 1H-NMR study

The structure of the complex formed in aqueous solution at pH 5.5 between ditercalinium, a potent antitumoral 7H-pyrido[4,3-c]carbazole rigid dimer, and the self-complementary tetranucleotide d[CpGpCpG], was investigated by 400-MHz ¹H-nmr. For a 1:2.5 drug-to-helix ratio, the dimer was only found in bound form, whereas free and complexed tetranucleotide were in slow exchange. This allowed unambiguous assignment of the protons in the complex through exchange polarization transfer measurements. The tetranucleotide existed as a right-handed double helix in the complex. The strong upfield shifts measured on most aromatic protons on both drug and nucleobases as well as on DNA imino protons were consistent with bisintercalation of the dimer. According to the negative nuclear Overhauser effects generated to protons on the convex edge of the bound drug rings by saturation of sugar protons, it was concluded that ditercalinium was intercalated with its rigid bis-ethyl bispiperidine spacer fitting the major groove of the helix. Difference in antitumor activity of various pyridocarbazole dimers is discussed in relation to the binding kinetics and the complex geometry determined in this study.     

1H-NMR studies of a monointercalating drug into a d(CpGpApTpCpG)2 minihelix

The structure of the complex formed between the 7H-pyridocarbazole monomer [[(2-piperidyl)-2,1-ethane-yl] [10-methoxy-7H-pyrido[4,3-c]carbazolium] dimethane sulfonate] and the autocomplementary hexanucleotide d(CpGpApTpCpG)2 in aqueous solution is analyzed by 270- and 400-MHz 1H-nmr. The large upfield shifts observed for both the drug and the self-complementary hexanucleotide protons provide evidence for intercalated complexes. The observation of intermolecular nuclear Overhauser effects between drug and the hexanucleotide protons gives a privileged orientation of the drug in the intercalation site with the quaternarizing ethyl piperidine chain protruding in the major groove. Moreover, the data suggest an intercalation based on the neighbor exclusion site principle in the three alternating sequences.     

Substituted 7H-pyrido[4,3-c]carbazoles with potent anti-HIV activity

Several substituted 7H-pyrido[4,3-c]carbazoles were synthesized from the natural product mukonal and tested for inhibition of HIV replication in H9 lymphocytes. 5-Methoxy-7-methyl-7H-pyrido[4,3-c]carbazole (7) had an EC50 value of 0.0054 microgram/mL and the highest therapeutic index (TI = 503) in the series.     

1H-NMR studies of a monointercalating drug into a d[CpGpCpG]2 minihelix

The structure of the complex formed between the 7H-pyridocarbazole monomer [{(2-piperidyl)-2,1-ethane-yl} {10-methoxy-7H-pyrido[4,3-c]carbazolium} dimethane sulfonate] and the autocom-plementary tetranucleotide d(CpGpCpG)₂ in aqueous solution is analyzed by 270-MHz and 400-MHz ¹H-nmr. The strong upfield shifts observed on most aromatic resonances of both the drug and the nucleotide are interpreted as the result of intercalation of the 7H-pyridocarbazole monomer in the base-paired minihelix of d(CpGpCpG). The observation of intermolecular negative nuclear Overhauser effects induced in some drug resonances by irradiation of sugar protons confirms this conclusion. A privileged orientation of the drug in the intercalation site with the quaternizing ethyl piperidine chain protruding in the major groove is proposed.     

1H- and 31P-NMR studies of ditercalinium binding to a d(GCGC)2 and d(CCTATAGG)2 minihelices: a sequence specificity study

The structures of the complexes formed in aqueous solution between ditercalinium, a bis-intercalating drug, and both the self-complementary tetranucleotide d(GCGC)2 and octanucleotide d(CCTATAGG)2, have been investigated by 400-MHz 1H-nmr and 162-MHz 31P-nmr. All the nonexchangeable protons, as well as the exchangeable imino protons and the phosphorus signals, have been assigned. Both oligonucleotides have been shown to adopt a right-handed B-DNA type structure. The addition of ditercalinium to the oligonucleotides lead to the formation of complexes in slow exchange at the nmr time scale with the free helices. At all drug-to-helix ratios studied, the ditercalinium was found in the bound form, whereas free and complexed oligonucleotides were in slow exchange, allowing resonance assignments through two-dimensional chemical exchange experiments. for d(GCGC)2 the strong upfield shifts induced on all aromatic protons of both the bases and the drug by complexation with ditercalinium suggest an interaction by intercalation of the two rings. However, the loss of twofold symmetry upon binding, as well as the chemical shift variation of the drug proton signals of one of the chromophores with temperature and concentration, favor a model in which the drug-nucleotide complexes have one ring of the drug intercalated and the other stacked on top of the external base pair. The intermolecular contacts between drug protons and nucleotide protons give a defined geometry for complexation that is consistent with the proposed model. In contrast, with d(CCTATAGG)2 several drug-nucleotide complexes were formed and a large increase in line broadening was observed at high drug-to-DNA ratios, precluding a detailed analysis of these complexes. However, the large upfield shift in the imino proton resonances together with the shielding of the ditercalinium ring protons favor a model with bis-intercalation of ditercalinium. This model is supported by the downfield shift of at least 4 out of 14 phosphorus signals. The results are compared with those obtained on ditercalinium binding to the homologous sequences d(CGCG)2 and d(TTCGCGAA)2, and discussed in terms of sequence specificity.     

Bisintercalation of ditercalinium into a d(CpGpApTpCpG)2 minihelix: a 1H- and 31P-NMR study

The structure of the complex formed in aqueous solution between ditercalinium, a bisintercalating drug, and the self-complementary hexanucleotide d(CpGpApTpCpG)2 is investigated by 400-MHz 1H-nmr and 162-MHz 31P-nmr. Whatever the drug to helix ratio, ditercalinium occurred in the bound form, whereas free and complexed hexanucleotide are in slow exchange. This allows unambiguous resonance assignment through two-dimensional chemical exchange experiments. The strong upfield shifts measured on most aromatic protons on both drug and bases as well as on DNA imino protons are consistent with bisintercalation of the dimer. Nuclear Overhauser effects observed between drug and nucleotide protons give a defined geometry for complexation, and suggest a DNA conformational change upon drug binding.     

Intercalative binding of ditercalinium to d(CpGpCpG)2: a theoretical study

The structure of the complex formed between ditercalinium, 2,2'-[4,4'-bipiperidine-1,1'-bis-(ethane-1,2-diyl)]bis(10-me thoxy-7H- pyrido[4,3-c]carbazolium) tetramethane sulfonate (NSC 366241), and the self-complementary tetranucleotide duplex d(CpGpCpG)2 has been investigated by means of a novel theoretical approach for modelling the conformational flexibility of nucleic acids. The methodology used is the JUMNA procedure, a molecular mechanics systematics capable of evaluating the internal energy and the interaction energy of a complex formed from a large number of fragments. In the best energy-minimized structures, the piperidinium chains of ditercalinium are located in the major groove of the right-handed oligonucleotide. Calculations show a distortion of the base-paired d(CpGpCpG)2 minihelix consisting of lateral dislocation of one base pair with respect to another along an axis parallel to the long axis; strong propeller twist and tilt of the end base pairs; a collective motion of all base pairs with respect to the helical axis towards the drug; and an overwinding at the exclusion site. The proposed structure of the complex is in good agreement with reported proton NMR data, supporting the feasibility of such model.     

1H and 31P nuclear magnetic resonance studies of the differences in DNA deformation induced by anti-tumoral 7H-pyrido[4,3-c]carbazole dimers

Ditercalinium (2,2'-[( 4,4'-bipiperidine]-1,1'-diyldi-2,1-ethane-diyl) bis-[10-methoxy-7H pyrido[4,3-c]carbazolium)tetramethane sulfonate (NSC 366241], a DNA bis-intercalating compound, is a potent anti-tumoral rigid dimer. Previous studies have shown that a reduced flexibility of the linking chain of such a dimer is essential for its biological activity. In order to understand, at the molecular level, the mechanism of action and the structure-activity relationships of this series of DNA intercalators, new dimers with additional methylene groups between the two piperidine rings have been synthesized. Addition of one methylene group in the chain preserved the activity, whereas addition of two methylene groups reduced the cytotoxicity, which finally disappeared when three methylene groups were inserted. Therefore, the study of the interaction of dimers bearing no (202), two (222) and three (232) methylene groups with the self-complementary hexanucleotide d(CGATCG)2 have been investigated by 1H and 31P nuclear magnetic resonance studies. The results reported here indicate that all dimers bis-intercalate into the minihelix. The intermolecular nuclear Overhauser effects (NOEs) between the dimers and the nucleotide lead to the conclusion that the three dimers intercalate with their rigid bis-ethyl bipiperidine chain fitting the major groove of the helix. Inter-residue nuclear Overhauser effects at the DNA level, as well as induced shifts, are discussed in relation to the conformational changes induced in DNA upon intercalation and to the different activity of the dimers.     

A theoretical investigation of the intercalative binding of 7-H pyrido[4.3C]carbazole chromophore into a d(CpG)2 minihelix

Theoretical computations are performed of the intercalative binding to a model d(CpG)2 minihelix of 7-H pyrido[4.3C]carbazole, the precursor of the antitumor bisintercalating drug ditercalinium. The conformations of the intercalation site are generated by the AGNAS procedure (algorithm to generate nucleic acid structures) of Miller and co-workers. The ligand-nucleotide interactions and the nucleotide conformational energies are computed with the SIBFA procedures (sum of interactions between fragments ab initio computed), which use formulas of empirical origin that reproduce ab initio SCF (self-consistent field) computations. Among the candidate intercalation sites most favored energetically, one has a pattern of conformational angles related to the one determined crystallographically by Sobell et al. in a series of x-ray structural studies of small intercalator-dinucleotide monophosphate complexes. Optimal values of the unwinding angle, found in the range of -12 degrees to -14 degrees, are consistent with available experimental data on DNA.     

Synthesis and biological evaluation of 2-thiopyrimidine derivatives

Various 2-thiopyrimidine derivatives have been synthesized by an efficient, one-pot reaction of functionalized amines with either 4-isothiocyanato-4-methyl-2-pentanone or 3-isothiocyanatobutanal. All the synthesized compounds were fully characterized by elemental analysis (CHN), FT-IR, (1)H NMR, and mass spectral data. One of the compounds, 7,7,8a-trimethyl-hexahydro-thiazolo[3,2-c]pyrimidine-5-thione (17) showed good anti-inflammatory (37.4% at 100 mg/kg p.o.) and analgesic activity (75% at 100 mg/kg p.o.). 7-(1-Mercapto-3,3,4a-trimethyl-4,4a,5,9b-tetrahydro-3H-pyrido[4,3-b]indol-7-yl)-3,3,4a-trimethyl-3,4,4a,5-tetrahydro-benzo[4,5]imidazo[1,2-c]pyrimidine-1-thiol (3) showed moderate activity against CDK-1 (IC(50)=5 microM). The other compounds showed moderate anti-inflammatory (5-20%), analgesic (25-75%) and protein kinase (CDK-5, GSK-3) inhibitory activities (IC(50)> 10 microM).     

Rebeccamycin analogues from indolo[2,3-c]carbazole

Glycosylated indolocarbazoles related to the antibiotic rebeccamycin represent an important series of antitumor drugs. In the course of structure-activity relationship studies, we report the synthesis of two new derivatives containing an indolo[2,3-c]carbazole chromophore instead of the conventional indolo[2,3-a]carbazole unit found in the natural metabolites. The N-methylated compound 8 containing one glucose residue behaves as a typical DNA intercalating agent, as judged from circular and electric linear dichroism measurements with purified DNA. In contrast, the bis-glycosylated derivative 7 containing a glucose residue on each indole nitrogen has lost its capacity to form stable complexes with DNA. DNA relaxation experiments reveal that the two drugs 7 and 8 have weak effects on human DNA topoisomerase I. The modified conformation of the indolocarbazole chromophore is detrimental to the stabilization of topoisomerase I-DNA complexes. The lack of potent topoisomerase I inhibition leads to decreased cytotoxicity but, however, we observed that the DNA-intercalating mono-glycosyl derivative 8 is about 5 times more cytoxic than the bis-glycosyl analogue 7. The study suggests that the naturally-occurring indolo[2,3-a]carbazole skeleton should be preserved to maintain the topoisomerase I inhibitory and cytotoxic activities.     

Preparation and pharmacological evaluation of the R- and S-enantiomers of 3-(2-butylamino)-4H- and 3-(3-methyl-2-butylamino)-4H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxide, two tissue selective ATP-sensitive potassium channel openers.

The preparation and the pharmacological evaluation of the R- and S-isomers of 3-(2-butylamino)-4H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxide (BPDZ 42) and 3-(3-methyl-2-butylamino)-4H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxide (BPDZ 44), two potassium channel openers, is described. Their optical purity was estimated by means of capillary electrophoresis (R- and S-BPDZ 42) and chiral HPLC (R- and S-BPDZ 44). The absolute configuration of each isomer of BPDZ 44 was deduced from crystallographic data. Pharmacological assays performed with the R- and S-isomers of BPDZ 44 revealed only slight differences in their activity on pancreatic B-cells but significant differences in their activity on vascular smooth muscle cells: the R-isomer being sixfold more potent than its corresponding S-isomer. The R-isomer of BPDZ 42 was shown to be more potent than its corresponding S-isomer on the endocrine pancreas. S-BPDZ 44 as well as R- and S-BPDZ 42 were found to exhibit tissue selectivity for the pancreatic versus the vascular smooth muscle tissue.     

Liver injury due to 3-amino-1-methyl-5h-pyrido [4,3-b] indole (Trp-P-2) and its prevention by miso

Trp-P-2(3-amino-1-methyl-5H-pyrido [4,3-b] indole) ingestion for 42 d by C3H/HeJJcl mice caused elevation of serum alanine transaminase (ALT) activity and several signs of liver injury. These alterations were not observed in mice fed the diet supplemented with 10% miso. This suggests a preventive effect of miso as to Trp-P-2 induced liver injury.     

Analysis of human plasma as an exposure level monitor for carcinogenic tryptophan pyrolysis products

A high-performance liquid chromatography method for detecting 3-amino-1,4- dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) in human plasma was developed. Plasma samples of 10 normal subjects were examined. Trp-P-1 and Trp-P-2, carcinogenic tryptophan pyrolysis products, were detected in all specimens, and the concentrations of Trp-P-1 and Trp-P-2 in plasma were 68.31 +/- 24.03 fmoles/ml (mean +/- S.D., n = 10) and 18.79 +/- 4.99 fmoles/ml, respectively. Our results suggest that plasma levels of carcinogenic tryptophan pyrolysis products may be useful indicators for estimating the exposure levels of the dietary carcinogens.     

Novel agents combining platelet activating factor (PAF) receptor antagonist with thromboxane synthase inhibitor (TxSI)

Compounds 1 or 2 which possess dual-acting PAF antagonist/TxSI in a previous paper were modified and evaluated for the dual-acting activity. It was found that several compounds were potent dual-acting PAF antagonist/TxSI in and ex vivo. 6-(2-Chlorophenyl)-3-[4-[(E/Z)-6-ethoxycarbonyl-1-(3-pyridyl)-1-hexenyl]phenylmethyl]-8,11-dimethyl-2,3,4,5-tetrahydro-8H-pyrido[4',3': 4,5]thieno[3,2-f]triazolo[4,3-a]diazepine (12) is excellent orally dual-acting PAF antagonist/TxSI.     

Detection of Trp-P-1 and Trp-P-2, carcinogenic tryptophan pyrolysis products, in dialysis fluid of patients with uremia

In order to estimate the exposure levels of mutagenic and carcinogenic heterocyclic amines in humans, we developed a high-performance liquid chromatography method to detect 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) in dialysis fluid of patients with uremia. Using this methods, dialysis fluid of 12 patients who had received hemodialysis treatment or continuous ambulatory peritoneal dialysis was examined. Trp-P-1 was detected in dialysate of all uremic patients (727 +/- 282 pmoles, n = 12). In patients who had been treated with continuous ambulatory peritoneal dialysis, the average amount of Trp-P-1 found in whole dialysate (6 l) per day was 710 +/- 203 pmoles (mean +/- S.D., n = 8). Moreover, Trp-P-2 could be detected in 5 out of 12 patients (206 +/- 85 pmoles, n = 5). These results indicate that patients with uremia are actually exposed to carcinogenic tryptophan pyrolysis products. The average exposure level of Trp-P-1 in uremic patients apparently exceeded 710 pmoles (150 ng) per day.     

1H NMR study of the structure of a pyridocarbazole dimer-d[CpGpCpG] complex

The structure of the complexes formed between a 7H-pyridocarbazole dimer (ditercalinium) or the corresponding monomer and d[CpGpCpG] is analyzed in aqueous solution by 270 MHz 1H NMR. In both cases the strong upfield shifts observed on most aromatic resonances are assigned to the formation of intercalated complexes. Bisintercalation of the dimer in the tetranucleotide minihelix is then observed at pH 5.5. The observation of intermolecular negative NOEs induced to some drug resonances by irradiation of sugar protons confirms these conclusions. The orientation of the ligand in the intercalation site is discussed.     

Interactions between the active metabolite of tryptophan pyrolysate mutagen, N-hydroxy-Trp-P-2, and lipids: the role of lipid peroxides in the conversion of N-hydroxy-Trp-P-2 to non-reactive forms

The interactions between lipids and the mutagenic active metabolite of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (N-hydroxy-Trp-P-2), were studied. Oleic acid showed an inhibitory effect on the formation of this active metabolite mainly by inhibition of hepatic microsomal oxidation systems. On the other hand, microsomal lipids from rat liver and commercial pig liver lecithin diminished the amount of N-hydroxy-Trp-P-2 without inhibiting the metabolism of Trp-P-2. The direct reaction of these lipids with N-hydroxy-Trp-P-2 was disclosed by experiments using N-hydroxy-Trp-P-2 and lipids without microsomes. Furthermore, the participation of lipid peroxides in this reaction was suggested by a linear relationship between the concentrations of the conjugated diene of lipids and the disappearance of N-hydroxy-Trp-P-2. When [3H]N-hydroxy-Trp-P-2 was incubated in the presence of pig liver lecithin, the polar products which were not formed in the incubation without lipids were newly detected by thin-layer chromatography (TLC) analysis.     

Inhibitory effect of dibenzoylmethane on mutagenicity of food-derived heterocyclic amine mutagens

Dibenzoylmethane (DBM), a structural analogue of curcumin (a bioactive phytochemical present in a widely used spice turmeric) was screened for its inhibitory effect against seven cooked food mutagens (heterocyclic amines): 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1), in both TA98 and TA100 strains of Salmonella typhimurium using Ames Salmonella/reversion assay in the presence of Aroclor1254-induced rat liver S9 homogenate. DBM has been reported to antagonize the mutagenicity of several chemical carcinogens in vitro and has recently been shown to be even more effective than curcumin in suppressing the 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors in rats. But there are no reports regarding its antimutagenic properties against cooked food mutagens. Results of the present investigations clearly indicate that dibenzoylmethane is a very potent antimutagenic agent, that could effectively inhibit mutagenicity induced by all the tested cooked food mutagens in both the frame shift (TA98) as well as the base pair mutation sensitive (TA100) strains of S. typhimurium. These highly potent inhibitory effects of dibenzoylmethane against heterocyclic amines observed in our preliminary investigations strongly warrant further studies of its efficacy as a cancer chemopreventive agent.