A comparative study of the antimutagenic effects of antioxidants on chemical mutagenesis in Drosophila melanogaster

The 1,4-dihydropyridine derivative 2,6-dimethyl-3,5-diethoxycarbonyl-4-(Na carboxylate)-1,4-dihydropyridine (1,4-DHP) was studied for antimutagenic effects in the dominant lethal test and in the sex-linked recessive lethal test of Drosophila melanogaster. The observed effects were compared with those of the radioprotectors cysteine and cysteamine and with those of the phenolic antioxidant butylated hydroxytoluene (BHT). In a wide range of concentrations, including low ones, 1,4-DHP reduces the frequency of EMS-induced genetic damage (point mutations and chromosome breakage). A reduction of the mutation rate induced by EMS in adults could be observed independently of the developmental stages (larvae or imago) pretreated with 1,4-DHP. The protective effect of this new antimutagen against the alkylating agent depended on both the 1,4-DHP dose and the level of the EMS-induced mutation rate. The effect of 1,4-DHP was more pronounced than that of the studied radioprotectors. It is concluded that dihydropyridine-type compounds are able to protect eukaryote germs cells from genetic damage produced by direct-acting mutagens such as EMS.     

Synthesis and structure--activity analysis of novel dihydropyridine derivatives to overcome multidrug resistance

The structure activity relationships were studied on newly synthesized 1,4-dihydropyridine derivatives possessing a 1-pentyl group at the 4-position, and 3-pyridylpropylester was found to be one of the effective fragments for overcoming P-glycoprotein mediated multidrug-resistance (MDR) in cultured human cancer cells, in vitro. 3-Pyridylpropylester was also found to be one of the effective fragments for increasing the life span of P-glycoprotein overexpressing MDR P388 leukemia-bearing mice, in vivo. All compounds had weak calcium antagonistic activities, but there appeared no relationship between MDR reversing effect and calcium antagonistic activity.     

Action of antioxidants on membrane-toxic effects of anticholinesterase agents

It was found therapeutic-preventive effectiveness of antioxidants (1,4-dihydropyridine derivatives) at poisoning with malathion insecticide. The effect of 1,4-dihydropyridine derivatives can be attributed to a prevention of lipid peroxidation. Antioxidants do not affect the toxicity of 0,0-dimethyl-0-2,2-dichlorvinylphosphate. Thus, antioxidants are pathogenetic drugs for treatment of poisonings with cholinesterase inhibitors.     

Effects of nifedipine and riodipine on biochemical processes in intact erythrocytes

Cardiovascular drugs nifedipine and riodipine (1,4-dihydropyridine derivatives) were studied as to their effect on some biochemical characteristics of erythrocytes. Both compounds failed to effect 2,3-diphosphoglycerate concentration in intact erythrocytes, but slightly decreased the level of adenosinetriphosphate. The drugs inhibited the process of acid hemolysis, this inhibition being particularly manifest in the case of nifedipine.     

Chemoenzymatic synthesis of enantiopure 1,4-dihydropyridine derivatives

1,4-Dihydropyridines possess a broad range of biological activities, such as the ability to control the influx of calcium into cells, as well as neuroprotective, antineurodegenerative, cognition and memory enhancing, anti-inflammatory, antiviral and many other properties. Chirality plays an important role in the biological activity of 1,4-dihydropyridines. The chemoenzymatic synthesis of 1,4-dihydropyridine derivatives in enantiopure form as the key intermediates for the synthesis of enantiopure drugs and chiral analogues of symmetrical drugs has become an advantageous alternative to the other synthetic methods. Hydrolytic enzymes, as efficient chemo-, regio- and stereoselective biocatalysts have been successfully applied for the asymmetrisation or kinetic resolution of various 1,4-dihydropyridine derivatives. Several synthetic strategies to overcome the inactivity of hydrolytic enzymes towards 1,4-dihydropyridine carboxylic acids have been developed during the last decade, often based on the introduction of a spacer between an enzymatically labile group and the 1,4-DHP nucleus. Good to excellent enantioselectivities can be obtained by careful optimisation of the reaction temperature and the organic (co)solvent used in the enzymatic transformations.     

Chemoenzymatic synthesis of enantiopure 1,4-dihydropyridine derivatives

1,4-Dihydropyridines possess a broad range of biological activities, such as the ability to control the influx of calcium into cells, as well as neuroprotective, antineurodegenerative, cognition and memory enhancing, anti-inflammatory, antiviral and many other properties. Chirality plays an important role in the biological activity of 1,4-dihydropyridines. The chemoenzymatic synthesis of 1,4-dihydropyridine derivatives in enantiopure form as the key intermediates for the synthesis of enantiopure drugs and chiral analogues of symmetrical drugs has become an advantageous alternative to the other synthetic methods. Hydrolytic enzymes, as efficient chemo-, regio- and stereoselective biocatalysts have been successfully applied for the asymmetrisation or kinetic resolution of various 1,4-dihydropyridine derivatives. Several synthetic strategies to overcome the inactivity of hydrolytic enzymes towards 1,4-dihydropyridine carboxylic acids have been developed during the last decade, often based on the introduction of a spacer between an enzymatically labile group and the 1,4-DHP nucleus. Good to excellent enantioselectivities can be obtained by careful optimisation of the reaction temperature and the organic (co)solvent used in the enzymatic transformations.     

Blockers of Ca2+ channels in the plasmalemma of perfused Characeae cells

Ionic currents in the plasmalemma of perfused Nitella syncarpa cells identified as currents through Ca²⁺ channels were registered for the first time. The effect of 1,4-dihydropyridine derivatives (nifedipine, nitredipine, riodipine) and phenylalkylamines (verapamil, D600) as well as the agonist CGP-28392 on the Ca²⁺ channels in the plasmelemma of perfused cells of Nitellopsis obtusa and Nitella syncarpa have been studied. A blocking effect of 1,4-dihydropyridine derivatives and phenylalkylamines on the plasmalemma Ca²⁺ channels has been detected. Phenylalkylamines have been found to block both inward and outward Ca²⁺ currents. The activating effect of the agonist CGP-28392 on the Ca²⁺ channels of plasmalemma has been shown.     

Rapid and efficient oxidation of Hantzsch 1,4-dihydropyridines with sodium periodate catalyzed by manganese (III) Schiff base complexes

Rapid and efficient oxidation of Hantzsch 1,4-dihydropyridine with sodium periodate is reported. The Mn(III)-salophen/NaIO4 catalytic system converts 1,4-dihydropyridines to their corresponding pyridine derivatives at room temperature in a 1:1, CH3CN/H2O mixture. The ability of various Schiff base complexes in the oxidation of 1,4-dihydropyridine was also investigated.     

Synthesis,Anticancer Evaluation and in Silico Studies of 1,4-Dihydropyridines

An efficient 1,4-dihydropyridine synthesis under mild conditions has been developed. Numerous substrates were tested, with yields of 1,4-dihydropridines ranging from good to excellent and a wide range of functional group tolerance. A549, HT-29, and HepG2 cancer cells were used to investigate the anticancer efficacy of each of the produced compounds. Additionally, in-silico docking studies were conducted to understand the structure-based features of the anticancer mechanism with the cancer medication target of Adenosine A2A receptor as well as the molecular level interactions of the compounds.     

Subcellular distribution of the 1,4-dihydropyridine receptor in rabbit skeletal muscle in situ: an immunofluorescence and immunocolloidal gold- labeling study

The subcellular distribution of the 1,4-dihydropyridine receptor was determined in rabbit skeletal muscle in situ by immunofluorescence and immunoelectron microscopy. Longitudinal and transverse cryosections (5-8 microns) of rabbit gracilis muscle were labeled with monoclonal antibodies specific against either the alpha 1-subunit (170,000-D polypeptide) or the beta-subunit (52,000-D polypeptide) of the 1,4-dihydropyridine receptor by immunofluorescence labeling. In longitudinal sections, specific labeling was present only near the interface between the A- and I-band regions of the sarcomeres. In transverse sections, specific labeling showed a hexagonal staining pattern within each myofiber however, the relative staining intensity of the type II (fast) fibers was judged to be three- to fourfold higher than that of the type I (slow) fibers. Specific immunofluorescence labeling of the sarcolemma was not observed in either longitudinal or transverse sections. These results are consistent with the idea that the alpha 1-subunit and the beta-subunit of the purified 1,4-dihydropyridine receptor are densely distributed in the transverse tubular membrane. Immunoelectron microscopical localization with a monoclonal antibody to the alpha 1-subunit of the 1,4-dihydropyridine receptor showed that the 1,4-dihydropyridine receptor is densely distributed in the transverse tubular membrane. Approximately half of these were distributed in close proximity to the junctional region between the transverse tubules and the terminal cisternae. Specific labeling was also present in discrete foci in the subsarcolemmal region of the myofibers. The size and the nonrandom distribution of these foci in the subsarcolemmal region support the possibility that they correspond to invaginations from the sarcolemma called caveolae. In conclusion, our results demonstrate that the 1,4-dihydropyridine receptor in skeletal muscle is localized to the transverse tubular membrane and discrete foci in the subsarcolemmal region, possibly caveolae but absent from the lateral portion of the sarcolemma.     

Binding of A 1,4-dihydropyridine calcium channel activator, (-) S Bay K 8644, to cardiac preparations

The 1,4-dihydropyridine Ca2+ channel activator, (-) [3H]Bay K 8644, binds to cardiac membranes and polarized [5 mM K+] and depolarized [50 mM K+] cardiac cells. Binding to microsomal membranes at 25 degrees C indicates a single set of binding sites, KD = 2.9 x 10(-9) M and a site density, 337 fmoles/mg protein, not different from that measured by antagonist 1,4-dihydropyridines. Binding to neonatal rat myocytes at 37 degrees C was independent of membrane potential with a KD value of 5 x 10(-8)M and a site density, 63 fmoles/mg protein, not significantly different from that measured by PN 200 110. These results indicate that 1,4-dihydropyridine activators and antagonists label the same number of binding sites in cardiac tissue, but that activator binding to intact myocytes is voltage-independent.     

Antibodies reveal the cytolocalization and subunit structure of the 1,4-dihydropyridine component of the neuronal Ca2+ channel

Rabbit brain synaptosomes bind the 1,4-dihydropyridine derivative (+)[3H]-PN 200-110 with an equilibrium dissociation constant of 0.04 nM and a maximal binding capacity of 400 fmol/mg of protein. Using polyclonal antibodies raised against the different components of the skeletal muscle 1,4-dihydropyridine receptor, we have demonstrated that the brain and muscle receptors share the same subunit composition comprising a large polypeptide chain of Mr 140,000 associated by disulfide bridges with a smaller peptide of Mr 32,000. These antibodies have been used in immunofluorescence staining of brain sections. They reveal a distribution of the Ca2+ channel protein similar to that of 1,4-dihydropyridine binding sites with (+)[3H]PN 200-110 by the autoradiographic technique.     

Discovery and evaluation of selective N-type calcium channel blockers: 6-unsubstituted-1,4-dihydropyridine-5-carboxylic acid derivatives

A structure-activity relationship study of 6-unsubstituted-1,4-dihydropyridine and 2,6-unsubstituted-1,4-dihydropyridine derivatives was conducted in an attempt to discover N-type calcium channel blockers that were highly selective over L-type calcium channel blockers. Among the tested compounds, (+)-4-(3,5-dichloro-4-methoxy-phenyl)-1,4-dihydro-pyridine-3,5-dicarboxylic acid 3-cinnamyl ester was found to be an effective and selective N-type calcium channel blocker with oral analgesic potential.     

A receptor of dihydropyridine blockaders of Ca2+ influx in human embryonic fibroblasts

The plasma membrane of human embryonic fibroblasts was shown to contain receptor binding sites for 1,4-dihydropyridine (DHP) Ca2+ entry blockers. In a subconfluent culture grown in serum medium the content of the DHP receptor amounted to 1.2 +/- 0.3 pmol per 10(6) cells. With progression to confluency this value decreased up to 0.28 +/- 0.08 pmol per 10(6) cells. The DHP binding capacity was shown to be affected by the presence of growth factors in culture medium. In a subconfluent culture of serum-deprived cells the content of DHP binding sites increased 1.9 fold, the steady-state level being achieved within 3 days in culture. The serum gradually reversed this process, and the DHP receptor density approached the initial level within 3 days.     

Homologous regulation of voltage-dependent calcium channels by 1,4-dihydropyridines

Chronic treatment of PC 12 cells with the 1,4-dihydropyridine Ca2+ channel antagonist nifedipine [5 x 10-8M/5 days] and the activator S Bay K 8644 [5 x 10-7 M/5 days] resulted in up- and down-regulation of 1,4-dihydropyridine binding site density by 29 and 24%, respectively, without change in affinity. These changes in binding site density represent functional changes as indicated by the corresponding changes in K+ depolarization-induced 45Ca2+ uptake and in whole cell currents carried by Ba2+ ions. This homologous regulation of voltage-dependent Ca2+ channels [VDCC] by potent and specific ligands parallels that observed for other classes of membrane receptors.     

Calcium channels: evidence for oligomeric nature by target size analysis.

Radiation inactivation was employed to measure the molecular size of calcium channels in guinea-pig skeletal muscle membranes, labelled by the potent 1,4-dihydropyridine calcium antagonist [3H]nimodipine. The molecular size was decreased when the membranes were preincubated and assayed with d-cis-diltiazem, a calcium channel blocker, which is structurally unrelated to the 1,4-dihydropyridines. d-cis-Diltiazem, which is a positive heterotropic regulator of 1,4-dihydropyridine calcium channel binding in vitro, reduced the molecular size from 178 000 to 111 500. 1-cis-Diltiazem, the diastereoisomer, which is devoid of calcium antagonistic action, did not decrease the molecular size of the 1,4-dihydropyridine binding site. Neither diastereoisomer affected the molecular size of the membrane-bound acetyl-cholinesterase, indicating that a stereospecific interaction with the calcium channel structure is the basis for these observations. It is concluded that this decrease in size is indicative of the oligomeric nature of the calcium channel and that calcium channel blockers, acting via different, but interacting drug receptor sites, induce different conformations of the channel structure, resulting in altered conductivity for ions.     

1,4-Dihydropyridine derivatives as calcium channel modulators: the role of 3-methoxy-flavone moiety

It was earlier recognized that calcium antagonists, and in particular 1,4-dihydropyridines, exhibited distinct cardiovascular profiles. In addition two different splice variants of the L-type calcium channel were found in vascular and cardiac tissues. In this study, novel substituted 1,4-dihydropyridines with a 3-methoxy-flavone moiety were synthesized and structural modifications of the substituents in the dihydropyridine ring of nifedipine were carried out in order to find tissue specific compounds. The negative inotropic, chronotropic and vasorelaxant effects were investigated on guinea-pig left, right atria and aortic strips, respectively. The introduction of an heteroaromatic ring in 4-position of the 1,4-dihydropyridine nucleus led to compounds selective for cardiac tissues. Moreover, different residues in the 1,4-dihydropyridine ring could modulate the chronotropic versus inotropic activity.     

Anticlastogenicity of two derivatives of 1,4-dihydroisonicotinic acid in mouse micronucleus test

Effects of two derivatives of 1,4-dihydroisonicotinic acid (1,4-DHINA) against the monofunctional alkylating agent ethyl methanesulfonate (EMS) were studied in the micronucleus test in (CBA x C57Bl/6(j)) mice. Adult males and pregnant females were treated with an antimutagen (i.p.) and 12h later they were exposed to EMS (i.p.). The frequencies of micronucleated (MN) polychromatic erythrocytes (PCEs) in mouse bone marrow and foetal liver were analysed 6, 12, 18, 24, 30, 36, 48 or 24, 48 and 72 h after the mutagen injection. In adults, the maximum number of MNPCEs was observed 36 or 24h after the EMS administration. In foetuses, which were treated in a maternal organism, such peak was found at 24h. Pre-treatment of mice with the antimutagens 2,6-dimethyl-3,5-diethoxycarbonyl-4-(Na carboxylate)-1,4-dihydropyridine (DHP) and glutapyrone (GP) decreased the yield of MNPCEs in male bone marrow. Having been observed at a peak of MN induction, the anticlastogenic effect of DHP (1/10 LD(50) or 340 mg/kg) reached 30%. DHP at the doses of 0.5-1mM/kg did not affect the EMS-induced frequency of MNPCEs in bone marrow, whereas GP inhibited it at the similar millimolar concentrations. Simultaneously with maternal bone marrow, foetal liver cells were analysed for MNs in the transplacental test. The anticlastogenic effect of DHP (1/10 LD(50)) was found to be more prolonged and higher in females than in males and to average 50%, but this antimutagen was not efficient in foetuses. Both antimutagens did not change the polychromatic/normochromatic erythrocyte (PCE/NCE) ratio as compared with EMS action.Results presented indicate a peak of EMS-induced micronucleated cells in mouse bone marrow 24 or 36 h and in foetal liver 24h after animal treatment. Two 1,4-DHINA derivatives exhibited anticlastogenic activity in adults, but not in foetuses.     

High affinity binding of a calcium channel antagonist to smooth and cardiac muscle

Specific binding of the Ca²⁺-channel antagonist nitrendipine, a close structural analog of nifedipine, has been measured in microsomal membrane fractions from guinea pig ileal longitudinal smooth muscle. The dissociation constant was 0.18 nanomole per liter and maximum binding was 1.14 picomoles per milligram of protein. Binding with very similar characteristics was found in a rat ventricle preparation. This high affinity binding was sensitive to displacement by a series of 1,4-dihydropyridine analogs of nifedipine with an activity sequence correlating well with that determined for inhibition of mechanical responses in the intact smooth muscle.     

Heparin binds with high affinity to voltage-dependent L-type Ca2+ channels. Evidence for an agonistic action

Heparin and related polyanions are a new class of compounds interacting with 1,4-dihydropyridine-sensitive L-type Ca2+ channels in a tissue-specific manner. Labeling of membrane-bound Ca2+ channels in rabbit skeletal muscle transverse tubules at the phenylalkylamine, benzothiazepine, and 1,4-dihydropyridine-selective domains was inhibited reversibly by a noncompetitive mechanism as shown by equilibrium saturation analysis and kinetic studies. (+)-cis-diltiazem but not (-)-cis-diltiazem reduced the inhibitory potency of heparin for 1,4-dihydropyridines. Antagonistic but not agonistic 1,4-dihydropyridines reversed heparin inhibition at the benzothiazepine site. Heparin forms a tight complex with the purified Ca2+ channel which is highly sensitive with respect to heparin inhibition (IC50 value: 0.05 microgram/ml) of 1,4-dihydropyridine binding. Reconstituted channel complexes have completely lost 1,4-dihydropyridine binding-inhibition by heparin and are not retained by lectin or heparin affinity columns. In whole cell patch clamp experiments with guinea-pig cardiac myocytes heparin increased the current through L-type Ca2+ channels when applied extracellulary. Synthetic peptides (representing putative heparin binding domains) which were derived from the rabbit skeletal muscle alpha 1-subunit reversed the inhibitory effects of heparin on 1,4-dihydropyridine receptors. Reversal for a peptide representing an extracellular domain occurred by an apparently competitive mechanism. It is suggested that heparin and related polyanions may interact with an evolutionary conserved cluster of basic amino acids in the large putative extracellular domain connecting the fifth and sixth putative transmembrane segment in the first motif of the ionic pore-forming alpha 1-subunit from skeletal muscle.