3,5-dibenzoyl-1,4-dihydropyridines: synthesis and MDR reversal in tumor cells

Fifteen 4-phenyl-3,5-dibenzoyl-1,4-dihydropyridines (BzDHPs) (1-15) substituted at the 4-phenyl ring were synthesized and compared to their cytotoxic activity and multidrug resistance (MDR)-reversing activity in in vitro assay systems. Among them, 2-CF3 (5) (IC50=8.7 microM), 2-Cl (11) (IC50=7.0 microM) and 3-Cl (12) (IC50=7.0 microM) derivatives showed the highest cytotoxic activity against human oral squamous carcinoma (HSC-2) cells. The activity of P-glycoprotein (Pgp) response for MDR in tumor cells was reduced by some of derivatives (3, 4, 8, 12), verapamil (VP) and nifedipine (NP). These data suggest that 3,5-dibenzoyl-4-(3-chlorophenyl)-1,4-dihydro-2,6-dimethylpyridine (12) can be recommended as a new drug candidate for MDR cancer treatment.     

Modification of swelling–contraction–aggregation processes in rat muscle mitochondria by the 1,4-dihydropyridines,cerebrocrast and glutapyrone,themselves and in the presence of azidothymidine

The influence of the 1,4-dihydropyridines (DHPs), water-soluble glutapyrone available as sodium, potassium and ammonium salts of 2-(2,6-dimethyl-3,5-diethoxycarbonyl-1,4-DHP-4-carboxamide)glutaric acid, from one side, and a lipophylic cerebrocrast, 2-propoxyethyl 2,6-dimethyl-4-(2-difluoromethoxyphenyl)-1,4-DHP-3,5-dicarboxylate, from the other side, on partially damaged mitochondria of the Wistar rat hindlimb muscle was also studied. The following tests were made: (1) rates of endogenous respiration and substrate (succinate) oxidation and oxidative phosphorylation; (2) rates and amplitudes of high-amplitude swelling and contraction after the addition of ATP, ADP and succinate to the previously swollen mitochondria and (3) rate of reversible self-aggregation of mitochondria isolated in salt media after ATP-induced contraction without and in the presence of azidothymidine (AZT). Cerebrocrast (10–100 μM ) partially normalized the endogenous respiration rate and slightly augmented the respiration rate after the addition of succinate and to lesser extent ADP. Cerebrocrast in a concentration-dependent manner (2·5–50 μM ) increased (two-fold at 20–50 μM ) the active contraction amplitude of swollen mitochondria, induced by single or repeated additions of ATP. The influence of cerebrocrast on the ADP- and succinate-induced contractions was less obvious. Unlike cerebrocrast glutapyrone caused a reduction of the ATP-induced contraction amplitude (two-fold at 0·5–5·0 mM ), not impairing the mitochondrial contraction ability in response to ATP or succinate. Pre-exposure to 2·5 mM glutapyrone resulted in at least a 10-fold inhibition of the reversible aggregation rate in the presence of 99 and 198 μM AZT. The results suggest the usefulness of further study of cerebrocrast and glutapyrone in preventing AZT-induced and some other mitochondrial myopathies. © 1997 John Wiley & Sons, Ltd.     

Hantzsch 1,4-dihydropyridines containing a diazen-1-ium-1,2-diolate nitric oxide donor moiety to study calcium channel antagonist structure-activity relationships and nitric oxide release

A group of racemic 3-isopropyl 5-[(2-piperazin-1-yl)ethyl] 1,4-dihydro-2,6-dimethyl-4-(pyridyl)-3,5-pyridinedicarboxylates (12a-c), 3-isopropyl 5-{2-[4-nitrosopiperazinyl]ethyl} 1,4-dihydro-2,6-dimethyl-4-(pyridyl)-3,5-pyridinedicarboxylates (14a-c) and 3-isopropyl 5-{2-[(O(2)-acetoxymethyldiazen-1-ium-1,2-diolate)(N,N-dialkylamino or 4-piperazin-1-yl)]ethyl} 1,4-dihydro-2,6-dimethyl-4-(pyridyl)-3,5-pyridinedicarboxylates (22-30) were prepared using modified Hantzsch reactions. This group of compounds (12a-c, 14a-c, and 22-30) exhibited less potent calcium channel antagonist activity (IC(50)=0.11 to 3.35muM range) than the reference drug nifedipine (IC(50)=0.01 microM). The point of attachment of the isomeric C-4 substituent was a determinant of calcium channel antagonist activity providing the potency profile 2-pyridyl3-pyridyl4-pyridyl. The N-nitrosopiperazinyl compounds (14a-c) did not release nitric oxide. The prodrugs 22-30 that have a C-5 2-[(O(2)-acetoxymethyldiazen-1-ium-1,2-diolate)(N,N-dialkylamino or 4-piperazin-1-yl)]ethyl ester substituent, upon incubation with guinea pig serum, undergo consecutive cleavage of the O(2)-acetoxymethyl moiety to give a nitric oxide donor diazenium-1-ium-1,2-diolate species that subsequently releases nitric oxide. The extent of nitric oxide released from the diazen-1-ium-1,2-diolate group is dependent upon the nature of the amino functionality attached directly to the diazen-1-ium N-1 position where the nitric oxide release profile is 1,4-piperazinyl>N-Et>N-(n-Bu)>N-Me upon exposure to guinea pig serum esterase(s). The results from this study suggest this class of hybrid calcium channel antagonist/nitric oxide donor prodrugs should release the vasodilator nitric oxide in vivo, preferentially in the vascular endothelium, to enhance the smooth muscle calcium channel antagonist effect to produce a combined synergistic antihypertensive effect.     

Synthesis and biological evaluation of some novel N-aryl-1,4-dihydropyridines as potential antitubercular agents

1,4-Dihydropyridines are the emerging class of antitubercular agent. Recently, studies have revealed that 1,4-dihydropyridine-3,5-dicarbamoyl derivatives with lipophilic groups have demonstrated excellent antitubercular activity. We have synthesized new N-aryl-1,4-dihydropyridines bearing carbethoxy and acetyl group at C-3 and C-5 of the DHP ring. In addition, 1H-pyrazole ring is substituted at C-4 position. The lowest minimum inhibitory concentration value, 0.02 μg/mL, was found for diethyl 1-(2-chlorophenyl)-1,4-dihydro-2,6-dimethyl-4-(1,3-diphenyl-1H-pyrazol-4-yl)pyridine-3,5-dicarboxylate 4e making it more potent than first line antitubercular drug isoniazid. In addition, this compound exhibited relatively low cytotoxicity.     

Synthesis and Anti-Tubercular (Tb) Evaluation of Bis[4-Ethylidineamino[1,2,4]Triazole-3-Thiol] Tethered by 1,4-Dihydropyridine

Russian Journal of Bioorganic Chemistry - A new series of 5,5'-(2,6-dimethyl-4-phenyl-1,4-dihydropyridine-3,5-diyl)bis[4-(ethylideneamino)-4H-[1,2,4]-triazole-3-thiol) was synthesized via...     

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.     

Induction of a sodium ion influx by progesterone in human spermatozoa

In human spermatozoa, progesterone (P(4)) induces a depolarization of the plasma membrane, a rapid calcium (Ca(2+)) influx, and a chloride efflux. The sodium ion (Na(+)) was partly responsible for the P(4)-induced depolarizing effect but was not required for calcium influx. We used fluorescent probes for spectrofluorometry to investigate whether P(4) induced a Na(+) influx and whether voltage-operated channels were involved in Na(+) and/or Ca(2+) entries. We found that 10 microM P(4) significantly increased intracellular Na(+) concentration from 17.8 +/- 2.0 mM to 27.2 +/- 1. 6 mM (P < 0.001). Prior incubation of spermatozoa with 10 microM flunarizine, a Na(+) and Ca(2+) voltage-dependent channel blocker, inhibited the sodium influx induced by 10 microM P(4) by 84.6 +/- 15.4%. The Ca(2+) influx induced by 10 microM P(4) was also significantly inhibited in a Na(+)-containing medium by 10 microM flunarizine or 10 microM pimozide (P < 0.01). In contrast, flunarizine had no inhibitory effect on the Ca(2+) influx induced by 10 microM P(4) in spermatozoa incubated in Na(+)-depleted medium. The P(4)-promoted acrosome reaction (AR) was significantly higher when spermatozoa were incubated in Na(+)-containing medium as compared to Na(+)-depleted medium. These data demonstrate that P(4) stimulates a Na(+) influx that could be involved in the AR completion. They also suggest that voltage-dependent Na(+) and Ca(2+) channels are implicated in P(4)-mediated signaling pathway in human spermatozoa.     

Leishmania donovani: Oral therapy with glycosyl 1,4-dihydropyridine analogue showing apoptosis like phenotypes targeting pteridine reductase 1 in intracellular amastigotes

Glycosyl 1,4-dihydropyridine analogue (2,6-dimethyl-4-(3-O-benzyl-1,2-O-isopropylidene-β-l-threo pentofuranos-4-yl)-1-phenyl-1,4-dihydro-pyridine-3,5-dicarboxylic acid diethyl ester) synthesized in our laboratory, inhibited Leishmania donovani infection in vitro and in hamsters (Mesocricetus auratus) when administered orally. This analogue is nontoxic, cell-permeable and orally effective. This glycosyl dihydropyridine analogue functioned through arrest of cells in sub-G0/G1-phase, triggering mitochondrial membrane depolarization-mediated programmed cell death of the intracellular amastigotes.     

Use of a carrier for quantitation of a new dihydropyridine calcium antagonist (OPC-13340) in human plasma by highly sensitive gas chromatography with negative-ion chemical ionization mass spectrometry

A sensitive gas chromatographic—mass spectrometric method for the quantitation of (±)-methyl 3-phenyl-2(E)-propenyl 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylate (OPC-13340, I), a new dihydropyridine calcium antagonist with a potent and long-acting antihypertensive and antianginal effect, was developed in order to elucidate its pharmacokinetics. Dihydropyridine calcium antagonists have been usually quantified by this technique in the negative-ion chemical ionization mode. However, direct application of this method to quantify trace amounts of I in biological fluids completely failed, owing to its adsorption on the column and oxidation of its dihydropyridine ring. Human plasma containing I and (±)-[²H₅]methyl 3-phenyl-2(E)-propenyl 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylate (II), the internal standard, was extracted with n-hexane—diethyl ether under weakly basic conditions (pH 8). In order to prevent adsorption of the compounds on the column, (±)-[²H₃]ethyl 3-phenyl-2(E)-propenyl 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylate (III), an analogue of I, was added to the extracts as a carrier. In addition, this carrier was also effective in preventing the oxidation of I. The quantitation limit of I in human plasma by this method was found to be less than 30 pg/ml. Thus, the method is sufficiently sensitive to study the pharmacokinetics of I in humans.     

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.     

Enantioselective syntheses and calcium channel modulating effects of (+)- and (−)-3-isopropyl 5-(4-methylphenethyl) 1,4-dihydro-2,6-dimethyl-4-(2-pyridyl)-3,5-pyridinedicarboxylates

The (+)- and (?)-enantiomers of 3-isopropyl 5-(4-methylphenethyl) 1,4-dihydro-2,6-dimethyl-4-(2-pyridyl)-3,5-pyridinedicarboxylate were synthesized using an efficient highly enantioselective (ee ≥ 96%) variant of the Hantzsch dihydropyridine synthesis. The key step in this procedure involved the asymmetric Michael addition of a metalated chiral aminocrotonate, derived from D -valine or L -valine, respectively, to the Knoevenagel acceptor (Z)-2-isopropoxycarbonyl-1-(2-pyridyl)-but-1-en-3-one. Both enantiomers exhibited a dual cardioselective partial calcium channel agonist (positive inotropic)/smooth muscle selective calcium channel antagonist effect. The relative in vitro smooth muscle calcium channel antagonist activities of the (?):(+) enantiomers was 26:1. In contrast, the (+)-enantiomer exhibited a greater in vitro positive inotropic effect on guinea pig left atrium where the contractile force was maximally increased by 14.8% at a concentration of 1.63 × 10^?8 M. © 1994 Wiley-Liss, Inc.     

Synthesis and biological evaluation of 1,4-dihydropyridine calcium channel modulators having a diazen-1-ium-1,2-diolate nitric oxide donor moiety for the potential treatment of congestive heart failure

A group of racemic 4-aryl(heteroaryl)-1,4-dihydro-2,6-dimethyl-3-nitropyridines possessing nitric oxide donor O(2)-acetoxymethyl-1-(N-ethyl-N-methylamino, or 4-ethylpiperazin-1-yl)diazen-1-ium-1,2-diolate, C-5 ester substituents were synthesized by coupling the respective 4-aryl(heteroaryl)-1,4-dihydro-2,6-dimethyl-3-nitropyridine-5-carboxylic acids with either O(2)-acetoxymethyl-1-[N-(2-methylsulfonyloxyethyl)-N-methylamino]diazen-1-ium-1,2-diolate, or O(2)-acetoxymethyl-1-[4-(2-methylsulfonyloxyethyl)piperazin-1-yl]diazen-1-ium-1,2-diolate. Compounds having a C-4 2-pyridyl, 4-pyridyl, 2-trifluoromethylphenyl, or benzofurazan-4-yl substituent exhibited more potent smooth muscle calcium channel antagonist activity (IC(50)'s in the 0.37-1.09 microM range) than related analogs having a C-4 3-pyridyl substituent (IC(50)'s=3.03-9.14 microM range) relative to the reference drug nifedipine (IC(50)=9.13 nM). The point of attachment of C-4 isomeric pyridyl substituents was a determinant of smooth muscle calcium channel antagonist activity where the relative potency profile was 4-pyridyl>2-pyridyl>3-pyridyl. Replacement of the C-5 methyl ester substituent of methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)pyridine-5-carboxylate (Bay K 8644) by an O(2)-acetoxymethyl-1-(N-ethyl-N-methylamino)diazen-1-ium-1,2-diolate, or O(2)-acetoxymethyl-1-(4-ethylpiperazin-1-yl)diazen-1-ium-1,2-diolate, C-5 ester substituent provided compounds, which exhibited a lower, yet respectable, cardiac positive inotropic effect (IC(50)'s=4.82 and 4.05 microM, respectively) relative to the reference drug Bay K 8644 (IC(50)=0.30 microM). All compounds released nitric oxide upon incubation with either phosphate buffer at pH7, or porcine liver esterase. However, the percentage nitric oxide released was up to 3-fold higher (76%) when these O(2)-acetoxymethyl-1-(alkylamino)diazen-1-ium-1,2-diolates were incubated with guinea pig serum. These results suggest that *NO would be released in vivo, upon cleavage by nonspecific serum esterases, preferentially in the vascular endothelium where it may enhance smooth muscle calcium channel antagonist activity.     

Cytochrome P-450-catalyzed hydroxylation and carboxylic acid ester cleavage of Hantzsch pyridine esters

Cytochrome P-450 (P-450)-catalyzed oxidation of 2,6-dimethyl-4-phenyl-3,5-pyridinedicarboxylic acid diethyl ester gives rise to 2,6-dimethyl-4-phenyl-3,5-pyridinedicarboxylic acid monoethyl ester and to 2-hydroxymethyl-6-methyl-4-phenyl-3,5-pyridinedicarboxylic acid diethyl ester, identified in this work. A pyridine hydroxymethyl diester of the sort of the latter compound is novel; under acidic or dehydrating conditions the diester is readily converted to a cyclic lactone (2-hydroxymethyl-6-methyl-4-phenyl-3,5-pyridinedicarboxylic acid 5-ethyl ester lactone). 2,6-Dimethyl-4-phenyl-3,5-pyridinedicarboxylic acid monoethyl ester was not hydroxylated to form this hydroxymethyl compound or lactone, but 1,4-dihydro-2-hydroxymethyl-4-phenyl-6-methyl-3,5-pyridinedicarboxyli c acid diethyl ester was enzymatically oxidized to give both products. The rates of oxidative carboxylic ester cleavage and methyl hydroxylation varied among individual forms of P-450 tested. Experiments with 2H and 3H labels were used to estimate an intrinsic kinetic deuterium isotope effect of 15 for ethyl ester cleavage by rat liver P-450PB-B in a reconstituted system. Rat liver microsomal systems showed kinetic deuterium and tritium isotope effects of 8 and 11, respectively, and this deuterium isotope effect was not attenuated in either intra- or intermolecular competitive experiments. When deuterium was present in the ethyl (ester) groups, increases in the rate of 2-methyl hydroxylation were observed in rat liver microsomes and with purified P-450 beta NF-B (but not with P-450PB-B). Deuteration of the methyl groups gave rise to kinetic isotope effects of 7-11, but no increases were seen in the rates of ester cleavage. These studies and those on rates of substrate disappearance indicate that isotopically sensitive branching (metabolic switching) observed in these systems is not necessarily bidirectional.     

Kinetics of Na(+)-dependent conformational changes of rabbit kidney Na+,K(+)-ATPase.

The kinetics of Na(+)-dependent partial reactions of the Na+,K(+)-ATPase from rabbit kidney were investigated via the stopped-flow technique, using the fluorescent labels N-(4-sulfobutyl)-4-(4-(p-(dipentylamino)phenyl)butadienyl)py ridinium inner salt (RH421) and 5-iodoacetamidofluorescein (5-IAF). When covalently labeled 5-IAF enzyme is mixed with ATP, the two labels give almost identical kinetic responses. Under the chosen experimental conditions two exponential time functions are necessary to fit the data. The dominant fast phase, 1/tau 1 approximately 155 s-1 for 5-IAF-labeled enzyme and 1/tau 1 approximately 200 s-1 for native enzyme (saturating [ATP] and [Na+], pH 7.4 and 24 degrees C), is attributed to phosphorylation of the enzyme and a subsequent conformational change (E1ATP(Na+)3-->E2P(Na+)3 + ADP). The smaller amplitude slow phase, 1/tau 2 = 30-45 s-1, is attributed to the relaxation of the dephosphorylation/rephosphorylation equilibrium in the absence of K+ ions (E2P<==>E2). The Na+ concentration dependence of 1/tau 1 showed half-saturation at a Na+ concentration of 6-8 mM, with positive cooperatively involved in the occupation of the Na+ binding sites. The apparent dissociation constant of the high-affinity ATP-binding site determined from the ATP concentration dependence of 1/tau 1 was 8.0 (+/- 0.7) microM. It was found that P3-1-(2-nitrophenyl)ethyl ATP, tripropylammonium salt (NPE-caged ATP), at concentrations in the hundreds of micromolar range, significantly decreases the value of 1/tau 1, observed. This, as well as the biexponential nature of the kinetic traces, can account for previously reported discrepancies in the rates of the reactions investigated.     

In vitro effects of sodium fluoride and sodium dichromate on dynamic properties of human erythrocyte membrane

Sodium fluoride (NaF) and sodium dichromate (Na2Cr2O7) are two different toxic compounds which are used as a dental caries prophylactic and as an oxidising agent in various industrial areas, respectively. However, accidental fluoride and chromate poisoning is not a rare occurrence, even death may result from cardiac or respiratory failure. In the present work, alterations produced by NaF, Na2Cr2O7 and temperature changes in the molecular dynamics of the human erythrocyte membrane were studied, in vitro, by the spin-labelling ESR technique. Human intact erythrocyte cells spin labelled with 5- and 16-doxyl stearic acids (5-DSA and 16-DSA) and treated with 40 microM NaF and 5 microM Na2Cr2O7 at 37 degrees C were used to quantify membrane fluidity. This was performed by measuring the changes in the order parameter (S), correlation time (tau) and phase transition temperature using recorded electron spin resonance (ESR) spectra. Experimental results show that 5 microM Na2Cr2O7 and 40 microM NaF do not produce any significant effects on the order parameter of 5-DSA spin label while they cause appreciable changes in the correlation time of the same label. As for 16-DSA, while Na2Cr2O7 does not produce any measurable effect on the order parameter of this label, NaF does in a certain extent. Although weak, the effects of both compounds on the correlation time of 16-DSA are found to be well above the experimental error limits. Change in temperature was observed to alter significantly S and tau parameters which show biphasic character in the temperature range of 5-50 degrees C. Activation energies of the hydrocarbon chains above and below transition temperatures were also determined for untreated and NaF or Na2Cr2O7 treated erythrocyte cells and the effect of NaF and Na2Cr2O7 on these energies and transition temperatures were discussed.     

High-performance liquid chromatographic determination of the polar metabolites of nifedipine in plasma, blood and urine

A relatively simple reversed-phase high-performance liquid chromatographic method for the determination of the polar metabolites of nifedipine in biological fluids is described. After conversion of 2-hydroxymethyl-6-methyl-4-(2-nitrophenyl)pyridine-3,5-dicarboxylic acid 5-methyl ester (IV) into 5,7-dihydro-2-methyl-4-(2-nitrophenyl)-5-oxofuro[3,4-b]pyridine-3-carboxylic acid methyl ester (V) by heating under acidic conditions, V was extracted with n-pentane—dichloromethane (7:3) and analysed on a C₁₈ column with ultraviolet detection. Subsequently, 2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylic acid monomethyl ester (III) was extracted with chloroform and analysed on the same system. Limits of determination in blood were 0.1 μg/ml for III and 0.05 μg/ml for IV and V; these limits were two to ten times higher for urine. This inter-assay variability was always less than 7.5%.     

Effects of oxidizing and reducing agents on ovine pulmonary artery responses to nitric oxide donors, sodium nitroprusside and 3-morpholino-sydnonimine

Nitrovasodilators-sodium nitroprusside (SNP; 10(-9)-10(-4) M) and 3-morpholino-sydnonimine (SIN-1; 10(-9)-10(-4) M) produced concentration-dependent relaxation of the fourth generation sheep pulmonary artery, preconstricted with 5-hydroxytryptamine (1 microM). Oxidizing agents [oxidized glutathione (GSSG, 1 mM) and CuSO4 (5 and 20 microM)] and reducing agents [dithiothreitol (DTT, 0.1 mM), ascorbic acid (1 mM) and reduced glutathione (GSH, 1 mM)] caused opposite effects on nitric oxide (NO)-induced vasodilation in the artery. Ascorbic acid and GSH potentiated the NO responses, while GSSG and CuSO4 inhibited relaxation caused by the nitrovasodilators. DTT, however, reduced the relaxant potency and efficacy of SNP and SIN-1. Pretreatment of the pulmonary artery strips with DTT (0.1 mM) inhibited SNP (10 microM)-induced Na(+)-K(+)-ATPase activity, while ascorbic acid (1 mM) and GSH (1 mM) had no effect either on basal or SNP (10 microM)-stimulated 86Rb uptake, an index of Na(+)-K(+)-ATPase activity, in ovine pulmonary artery. The results suggest that reducing agents like ascorbic acid may have beneficial effect in improving the vascular function under oxidative stress.     

Evaluation of anticancer effects of newly synthesized dihydropyridine derivatives in comparison to verapamil and doxorubicin on T47D parental and resistant cell lines in vitro

Failure of current anticancer drugs mandates screening for new compounds of synthetic or biological origin to be used in cancer therapy. Multidrug resistance (MDR) is one of the main obstacles in the chemotherapy of cancer. Efflux of cytotoxic agents mediated by P-glycoprotein (P-gp or MDR1) is believed to be an important mechanism of multidrug resistance. Therefore, we decided to investigate the antiproliferative effects of seven newly synthesized 1,4-dihydropyridine (DHP) derivatives in comparison to verapamil (VP) and doxorubicin (DOX) on human breast cancer T47D cells and its MDR1 overexpressed and moderately resistant cells (RS cells) using MTT cytotoxicity assay. We also examined the effects of these compounds on cytotoxicity of DOX in these two cell types. The cytotoxicity assays using MTT showed that most of the tested new DHP derivatives and VP at 10 μM concentration had varying levels of toxicity on both T47D and RS cells. The toxicity was mostly in the range of 10–25%. However, the cytotoxicity of these DHP derivatives, similar to VP, was significantly less than DOX when comparing IC₅₀ values. Furthermore, these compounds in general had relatively more cytotoxicity on T47D vs RS cells at 10-μM concentration. Among new DHPs, compounds 7a (3,5-dibenzoyl-4-(2-methylthiazol-4-yl)-1,4-dihydro-2,6-dimethylpyridine) and 7d (3,5-diacetyl-4-[2-(2-chlorophenyl)thiazol-4-yl)]-1,4-dihydro-2,6-dimethylpyridine) showed noticeable potentiation of DOX cytotoxicity (reduction of DOX IC₅₀) compared to DOX alone in both cells, particularly in RS cells. This effect was similar to that of VP, a known prototype of MDR1 reversal agent. In other words, compounds 7a and 7d resensitized RS cells to DOX or reversed their resistance. Results indicate that compound 7d exerts highest effect on RS cells. Therefore, these two newly synthesized DHP derivatives, compounds 7a and 7d, are promising as potential new MDR1 reversal agents and should be further studied on other highly resistant cells due to MDR1 overexpression and with further molecular investigation.     

Plasmids for biodegradation of 2,6-dimethylpyridine, 2,4-dimethylpyridine, and pyridine in strains of Arthrobacter]

Arthrobacter crysallopoietes strain KM-4 degrading 2,6-dimethylpyridine and strain KM-4a degrading both 2,6-dimethylpyridine and pyridine, Arthrobacter sp. KM-4b degrading 2,4-dimethylpyridine were isolated from soil. Arthrobacter crystallopoietes KM-4 and Arthrobacter sp. KM-4b contain 100 Md plasmids pBS320 and pBS323. Arthrobacter crystallopietes KM-4a harbours a 100 Md and 80 Md plasmids. Plasmid curing and conjugation transfer results confirm that these plasmids are involved in degradation of 2,6-dimethylpyridine, 2,4-dimethylpyridine and pyridine. A mutant with lost ability to degrade 2,6-dimethylpyridine was isolated during the growth of strain KM-4 rifR at 42 degrees C. Electrophoretic analysis of the plasmid from temperature sensitive mutant revealed the deletion the size of 26 Md from pBS320 plasmid.