Novel selective PDE4 inhibitors. 2. Synthesis and structure-activity relationships of 4-aryl-substituted cis-tetra- and cis-hexahydrophthalazinones.

A series of 4-aryl-substituted cis-4a,5,8,8a-tetra- and cis-4a,5,6,7,8,8a-hexahydro-2H-phthalazin-1-ones with high inhibitory activity toward cAMP-specific phosphodiesterase (PDE4) was synthesized. To study structure-activity relationships various substituents were introduced to the 2-, 3-, and 4-positions of the 4-phenyl ring. Substitution at the 4-position of the phenyl ring was restricted to a methoxy group, probably due to unfavorable steric interactions of larger groups with the binding site. The introduction of many alkoxy substituents including distinct ring systems and functional groups was allowed to the 3-position. It was found that in general the cis-4a,5,8,8a-tetrahydro-2H-phthalazin-1-ones are more potent than their hexahydrophthalic counterparts, the best activity residing in (4-imidazol-1-yl-phenoxy)butoxy analogue 16o (pIC(50) = 9.7).

In vitro and in vivo uroselectivity of B8805-033, an antagonist with high affinity at prostatic alpha1A- vs. alpha1B- and alpha1D-adrenoceptors.

We have investigated the pharmacological properties of B8805-033 [(+/-)- 1,3,5-trimethyl-6-[[3-[4-((2,3-dihydro-2-hydroxymethyl)-1,4-benzodioxin-5-yl)-1-piperazinyl]propyl] amino]-2,4(1H,3H)-pyrimidinedione], a new alpha1A-adrenoceptor (AR) selective antagonist. In radioligand binding studies, B8805-033 was 150- to 1200-fold selective for alpha1A-ARs (pKi rat cerebral cortex 8.70, cloned human receptor 7.71) relative to alpha1B-ARs (pKi rat cerebral cortex 5.60, rat liver 5.39, cloned human receptor 5.16) and alpha1D-ARs (pKi cloned human receptor 5.49). B8805-033 inhibited noradrenaline (NA) induced contractions mediated by alpha1A-ARs in rat vas deferens and rabbit and human prostate (pA2 7.62-8.40) much more potently than those mediated by alpha1B-ARs in guinea pig and mouse spleen or by alpha1D-ARs in rat aorta and pulmonary artery (pA2 5.21-5.52). With the exception of a high agonist affinity at 5-HT1A receptors (pKi 9.74 in pig cortex, pD2 6.82 for contraction of rabbit basilar artery) and a moderate to low affinity at histamine H1-receptors (pA2 6.74) and beta1-ARs (pA2 5.75), B8805-033 did not interact with a number of other neurotransmitter receptors (pKi or pA2<5.0). From the i.v. doses of B8805-033 to either inhibit the urethral pressure response to NA by 50% (29 nmol/kg) or to evoke a fall in diastolic blood pressure by 25% (1.54 micromol/kg) in anaesthetized dogs, an urethral/ vascular selectivity ratio of 52 was obtained, far exceeding that found for the nearly unselective prazosin (ratio 1.8). We conclude that B8805-033 is a highly alpha1A-AR selective antagonist, which may potentially be useful as pharmacological tool to investigate alpha1-AR heterogeneity and in the treatment of benign prostatic hyperplasia.

The inhibitory potency and selectivity of arginine substrate site nitric-oxide synthase inhibitors is solely determined by their affinity toward the different isoenzymes.

We have investigated various nitric oxide (NO) synthase inhibitors for their affinity and selectivity toward the three human isoenzymes in radioligand binding experiments. Therefore, we developed the new radioligand [(3)H]2-amino-4-picoline to measure binding of these compounds to the three human NO synthase (NOS) isoenzymes. Aminopicoline is a potent and nonselective inhibitor of all three isoforms. [(3)H]2-amino-4-picoline bound saturably and with high affinity to human NOSs. Affinity constants (K(D) values) of 59, 111, and 136 nM were obtained for the inducible, neuronal, and endothelial NOS isoforms (iNOS, nNOS, eNOS). Binding of [(3)H]2-amino-4-picoline was competitive with the substrate arginine. From all the inhibitors tested, AMT (2-amino-5, 6-dihydro-6-methyl-4H-1,3-thiazine hydrochloride) showed the highest affinity and no selectivity. L-NIL [L-N(6)-(1-Iminoethyl)lysine hydrochloride] and aminoguanidine were moderately iNOS-selective while L-NA (N(G)-nitro-L-arginine) and L-NAME (N(G)-nitro-L-arginine methyl ester hydrochloride) showed selectivity toward the constitutive isoforms. High iNOS versus eNOS selectivity was found for 1400W, whereas several isothiourea derivatives and 1400W displayed moderate n- versus eNOS selectivity. To relate the affinity of these compounds to their inhibitory potency, we measured the inhibitory potency under almost identical conditions using a new microtiter plate assay. The inhibitory potency of selective and nonselective NOS inhibitors was almost exactly mirrored by their affinity toward the different isoenzymes. Highly significant correlations were obtained between the potency of enzyme inhibition and the inhibition of [(3)H]2-amino-4-picoline binding for all three isoenzymes. These data show that the potency and selectivity of NOS inhibitors are solely determined by their affinity toward the different isoforms. Furthermore, these data identify the new radioligand [(3)H]2-amino-4-picoline as a very useful radiolabel for the investigation of the substrate binding site of all three isoforms.

Localization of the 1,4-dihydropyridine drug acceptor of P-glycoprotein to a cytoplasmic domain using a permanently charged derivative N-methyl dexniguldipine.

INTRODUCTION: P-glycoprotein (P-gp) is a 170 kDa ATPase which can transport a wide range of natural product cytotoxic drugs out of cells, thus conferring the multidrug resistance (MDR) phenotype. METHODS: In this paper we used the 1,4-dihydropyridine (1,4-DHP) MDR-reversing agent dexniguldipine (DN), and a derivative with a quaternary nitrogen which is permanently charged, N-methyl-DN, to explore the sidedness of block of [3H]-vinblastine transport by P-gp. RESULTS: In cytotoxicity assays, 1 microM DN sensitized MCF7 ADR cells, causing a 13-fold decrease in the EC50 of vinblastine from 400 +/- 80 nM to 30 +/- 25 nM. In marked contrast, N-methyl-DN was without effect. In intact MCF7 ADR cells, DN reversed the [3H]vinblastine uptake deficit with an EC50 of 445 +/- 100 nM, again, N-methyl-DN was inactive. In photoaffinity labelling studies using the arylazide [3H]-B9209-005 in whole cells, DN potently inhibited incorporation of the photoaffinity label into P-gp whilst N-methyl-DN was without effect. However, in photoaffinity labelling studies in membrane fragments, both DN and N-methyl-DN potently inhibited [3H]-B9209-005 photoaffinity labelling of P-gp. Furthermore, in membrane fragments [3H]-vinblastine binding to P-glycoprotein was potently inhibited by both N-methyl-DN (Ki 10.7 +/- 4.9 nM) and DN (Ki 11.2 +/- 3.8 nM), and both N-methyl-DN and DN blocked ATP-dependent [3H]-vinblastine transport into inside-out vesicles. Thus, in intact cells the permanently charged 1,4-dihydropyridine, N-methyl-DN is unable to reverse the MDR phenotype or photoaffinity labelling of P-gp. However, in cell fragments and inside-out vesicles, N-methyl-DN binds avidly to P-gp and this binding blocks [3H]-vinblastine transport. CONCLUSION: These data are consistent with the hypothesis that 1,4-DHPs block [3H]-vinblastine binding, and thereby transport by P-gp, by acting at a domain accessible only from the cytoplasm.

Rhodamine 123 efflux modulation in the presence of low or high serum from CD56+ hematopoietic cells or CD34+ leukemic blasts by B9309-068, a newly designed pyridine derivative.

The newly designed pyridine derivative B9309-068 and a series of structurally different compounds were tested for their ability to modulate rhodamine 123 (RHO) efflux from CD56+ hematopoietic cells in the presence of either 10% fetal calf serum or undiluted human AB serum. Furthermore, efflux modulation was investigated on CD34+ blast populations obtained from four patients with relapsed state AML. Target cells were specified throughout by labeling with peridinine chlorophyll protein (PerCP)-conjugated monoclonal antibodies, allowing clear differentiation from RHO emission spectrum by flow cytometry. In the presence of low serum each compound efficiently modulated RHO efflux without significant differences in the range of final concentrations (1.0-3.0 microM). At 0.1 microM, however, RHO efflux was differentially modulated following the series GF120918 approximately B9309-068 > PSC 833 > DNIG approximately DVER. With CD56+ cells in the presence of undiluted human AB serum at a final modulator concentration of 0.1 microM, all chemosensitizers tested were found to be inefficient. At final concentrations of 0.3 microM or higher, distinct RHO efflux modulation was found with the following efficacies: B9309-068 approximately GF120918 > PSC 833 >> DVER approximately DNIG. The efficacies seen in undiluted human AB serum at 3.0 microM were comparable to those seen on CD56+ cells at final modulator concentrations of 0.1 microM in low serum. Our results identify the pyridine derivative B9309-068 as a promising compound for modulating P-glycoprotein-mediated drug resistance under conditions resembling the clinical setting. Nonetheless, modulation potencies of a series of structurally very different chemosensitizers was revealed to be substantially diminished at high serum concentrations in vitro.

Modulation of P-glycoprotein mediated drug accumulation in multidrug resistant CCRF VCR-1000 cells by chemosensitisers.

P-glycoprotein (PGP) mediated transport of cytostatic drugs out of resistant cancer cells is a major cause of experimental and probably also of clinical multidrug resistance, which often leads to treatment failure during chemotherapy. The broad substrate specificity of PGP strongly restricts effective chemotherapy and diminishes the patients' prognosis. Inhibition of PGP's pumping function by chemosensitisers is one way to restore cellular responsiveness to otherwise ineffective cytostatics. Clinical trials with several chemosensitisers are under way. To date, it is not clear whether a certain chemosensitiser potentiates the action of different cytostatic drugs, transported by PGP equally well, or whether the chemosensitising potency is dependent on the cytostatic drugs used. Therefore, we compared the effects of five potent chemosensitisers on cellular accumulation using [3H]daunomycin, [3H]vincristine and rhodamine-123 as substrates for PGP. The acridonecarboxamide derivative GF 120918 was the most potent compound and a half-maximal effect was seen at concentrations ranging from 5 nM for rhodamine-123 accumulation to 14 and 19 nM for [3H]vincristine or [3H]daunomycin accumulation, respectively. The new chemosensitiser B9203-016 was slightly less effective than GF 120918 in all three test systems. Dexniguldipine was of intermediate potency with half-maximal effects at concentrations between 62 and 194 nM. The cyclic undecapeptide SDZ PSC 833 showed somewhat lower potency ranging from 151 to 331 nM. Cyclosporin A was less potent than SDZ PSC 833. Furthermore, enhancement of drug accumulation produced by each chemosensitiser was similar, regardless of which PGP substrate was measured, that is, the rank order of potency to increase accumulation was the same in each of the assays used. Our data point to similar, if not identical, mechanisms of drug transport by PGP and inhibition of drug transport by chemosensitisers at least for the substrates rhodamine-123, vincristine and daunomycin.

Reversible labeling of a chemosensitizer binding domain of p-glycoprotein with a novel 1,4-dihydropyridine drug transport inhibitor.

A photoreactive dihydropyridine (DHP), BZDC-DHP (2,6-dimethyl-4-(2-(trifluoromethyl)-phenyl)-1,4-dihydropyridine-3,5- dicarboxylic acid (2-[3-(4-benzoylphenyl)propionylamino]ethyl) ester ethyl ester), and its tritiated derivative were synthesized as novel probes for human p-glycoprotein (p-gp). (-)-[3H]BZDC-DHP specifically photolabeled p-gp in membranes of multidrug-resistant CCRF-ADR5000 cells. In reversible labeling experiments a saturable, vinblastine-sensitive and high-affinity (Kd = 16.3 nM, Bmax = 58 pmol/mg of protein, k(+1) = 0.031 nM-1 min-1, k(-1) = 0.172 min-1) binding component was present in CCRF-ADR5000 membranes but absent in the sensitive parent cell line. Binding was inhibited by cytotoxics and known chemosensitizers with a p-gp characteristic pharmacological profile. For eight chemosensitizers tested, the potency for binding inhibition correlated (r > 0.94) with the potency for drug transport inhibition (measured using rhodamine 123 accumulation). The DHP niguldipine and a structurally related pyrimidine stereoselectively stimulated reversible (-)-[3H]BZDC-DHP binding, suggesting that more than one DHP molecule can bind to p-gp at the same time. Our data demonstrate that DHPs label multiple chemosensitizer domains on p-gp, distinct from the vinblastine interaction site. (-)-[3H]BZDC-DHP represents a valuable tool to characterize the molecular organization of chemosensitizer binding domains on p-gp by both reversible binding and photoinduced covalent modification. It provides a novel simple screening assay for p-gp active drugs.

Interaction of cytostatics and chemosensitizers with the dexniguldipine binding site on P-glycoprotein.

The interaction of cytostatics and chemosensitizers with the dexniguldipine binding site of P-glycoprotein was investigated in photoaffinity labeling experiments. A tritiated azidoderivative of the chemosensitizer dexniguldipine with dihydropyridine structure, [3H]B9209-005, was used to irreversibly label P-glycoprotein. The apparent affinity of cytostatics and chemosensitizers to this binding site was estimated from labeling experiments in the presence of increasing concentrations of compounds. From the cytostatics tested, the vinca alkaloids and taxol showed the highest affinity, anthracyclins possessed moderate affinity while methotrexate, ara C and camptothecin, cytostatics not involved in P-glycoprotein-mediated multidrug resistance, were almost inactive. The chemosensitizers GF 120918, cyclosporin A and SDZ PSC-833 inhibited photoincorporation with the highest potency. Steep dose-inhibition curves were obtained with the cyclic peptides and S9788, indicating that these compounds may bind allosterically to a separate binding site. Compounds with dihydropyridine structure with or without chemosensitizing potency were also tested and some structure-activity relationships could be derived from the data. Our data show that inhibition of photoaffinity labeling by [3H]B9209-005 is a valuable and reliable system for measuring the interaction with and potency of chemosensitizing compounds at P-glycoprotein. Furthermore, data obtained in this test system are well suited to investigate structure-activity relationships for chemosensitizers at P-glycoprotein. In addition cytostatics underlying P-glycoprotein-mediated multidrug resistance can be identified.

B9209-005, an azido derivative of the chemosensitizer dexniguldipine-HCl, photolabels P-glycoprotein.

P-glycoprotein is an energy-dependent drug extrusion pump for a variety of anticancer drugs and is involved in the development of multidrug resistance in cancer. Dexniguldipine-HCl is a potent chemosensitizer for P-glycoprotein-mediated multidrug resistance in vitro, and clinical phase I/II trials are underway. To investigate the mechanisms of chemosensitization and to identify the binding sites for dexniguldipine-HCl on target proteins involved in chemosensitization, [3H]B9209-005, an azido derivative of dexniguldipine-HCl, was synthesized and used as a photoaffinity ligand. In two models of multidrug resistance reversal, i.e., sensitization to vincristine and modulation of rhodamine-123 uptake, B9209-005 and dexniguldipine-HCl showed identical biological activities. Photoaffinity labeling experiments with [3H]B9209-005 in cell membranes from multidrug-resistant CCRF ADR-5000 cells, in comparison with labeling experiments with [3H]azidopine (an established photoaffinity ligand for P-glycoprotein), showed that [3H]B9209-005 labeled two proteins, with apparent molecular masses of 170 and 95 kDa. The pharmacological specificity of labeling was demonstrated by inhibition of photoincorporation by several cytostatic drugs transported by P-glycoprotein, as well as by chemosensitizers. Immunoprecipitation of the labeled proteins with the P-glycoprotein-specific monoclonal antibody C 219 and with a site-directed polyclonal antibody to the amino-terminal sequence of P-glycoprotein (amino acids 389-406) identified these proteins as intact P-glycoprotein and the amino-terminal fragment thereof. No specific labeling was obtained in the drug-sensitive parent cell line CCRF-CEM, which is devoid of significant P-glycoprotein expression. Maximal labeling of 17 pmol of the 170-kDa protein/mg of crude membrane protein was obtained. The affinity of [3H]B9209-005 for binding to and photoincorporation into P-glycoprotein was 5-fold greater than that of [3H]azidopine, and photoincorporation of [3H]B9209-005 showed a different photoincorporation pattern, compared with [3H]azidopine, in that the latter compound was incorporated specifically into the carboxyl-terminal 55-kDa fragment of P-glycoprotein. In contrast to [3H]azidopine, no specific labeling of this fragment was obtained with [3H]B9209-005, indicating different binding sites for or different photoincorporation of the two dihydropyridine ligands. Because B9209-005 carries the photoreactive azido group in the dihydropyridine moiety, whereas the azido group of azidopine is located in the side chain, these results suggest that the dihydropyridine moiety of the two compounds probably interacts with the amino-terminal part of P-glycoprotein, whereas the side chains react preferentially with the carboxyl-terminal 55-kDa fragment.(ABSTRACT TRUNCATED AT 400 WORDS)

The leukotriene LTD4 receptor antagonist MK571 specifically modulates MRP associated multidrug resistance.

The multidrug resistant cell lines HL60/AR and GLC4/ADR show high overexpression of the gene encoding the multidrug resistance associated protein MRP compared to their drug sensitive parental counterparts. This and the virtual absence of mdr1/P-glycoprotein gene expression was proven by a complementary DNA polymerase chain reaction (cDNA-PCR) approach. Applying a 72-hour tetrazolium based colorimetric MTT-assay we demonstrate on both MDR sublines a dose-dependent modulation of drug resistances by the leukotriene LTD4 receptor antagonist MK571. A complete reversal of vincristine resistances was achieved at final MK571 concentrations of 30 microM (HL60/AR) or 50 microM (GLC4/ADR) which by itself did not disturb cellular proliferation. The drug resistance of a mdr1/P-gp overexpressing multidrug-resistant HL60 subline, in contrast, was not significantly affected by MK571. Similar effects were seen using the glutathione (GSH) synthesis inhibitor buthionine sulfoximine (BSO). Our results point to a relationship between MRP and a conjugate transporter and identify MK571 as a new tool structure for developing modulators specific for a MRP associated multidrug resistance.

Stereoselective inhibition of thromboxane-induced coronary vasoconstriction by 1,4-dihydropyridine calcium channel antagonists.

The biological activity of the (+)-S- and (-)-R-enantiomers of niguldipine, of the (-)-S- and (+)-R-enantiomers of felodipine and nitrendipine, and of rac-nisoldipine and rac-nimodipine was investigated in vitro and in vivo. Inhibition of coronary vasoconstriction due to the thromboxane A2 (TxA2)-mimetic U-46619 in guinea pig Langendorff hearts, displacement of (+)-[3H]isradipine from calcium channel binding sites of guinea pig skeletal muscle T-tubule membranes, and blood pressure reduction in spontaneously hypertensive rats were determined. The enantiomers were obtained by stereoselective synthesis. Cross-contamination was less than 0.5% for both S- and R-enantiomers of niguldipine and nitrendipine and less than 1% for those of felodipine. From the doses necessary for a 50% inhibition of coronary vasoconstriction, stereoselectivity ratios for (+)-(S)-/(-)-(R)-niguldipine, (-)-(S)-/(+)-(R)-felodipine, and (-)-(S)-/(+)-(R)-nitrendipine of 28, 13, and 7, respectively, were calculated. The potency ratio rac-nisoldipine/rac-nimodipine was 3.5. Ratios obtained from binding experiments and antihypertensive activity were (+)-(S)-/(-)-(R)-niguldipine = 45 and 35, (-)-(S)-/(+)-(R)-felodipine = 12 and 13, (-)-(S)-/(+)-(R)-nitrendipine = 8 and 8, and rac-nisoldipine/rac-nimodipine = 8 and 7, respectively. Highly significant correlations were found between the in vitro potency of the substances to prevent U-46619-induced coronary vasoconstriction and their affinity for calcium channel binding sites as well as their antihypertensive activity.(ABSTRACT TRUNCATED AT 250 WORDS)