Evaluation of Novel Substituted Furopyridines as Inhibitors of Protein Kinases Related to Tau Pathology in Alzheimer´s Disease.

BACKGROUND: Alzheimer´s disease (AD) is characterized by a progressive neuronal degeneration caused by two pathological hallmarks, hyperphosphorylated tau protein aggregated into tau filaments and amyloid precursor protein derived beta amyloid peptides aggregated into extracellular amyloid plaques. All attempts so far to find effective drugs failed in clinical trials. AD is a multifactorial disease, so that selective drugs to target one AD-relevant structure alone may not be sufficient. OBJECTIVE: We built novel furopyridines with various substitution patterns to evaluate them as protein kinases inhibitors of enzymes related to tau pathology. METHODS: Furopyridine derivatives were synthesized and purified using column chromatography. The protein kinase inhibitory properties were determined in ATP-competition assays with determined affinity constants for the most active compounds. RESULTS: The compounds were prepared in simple two-component reactions of substituted 1,4- dihydropyridines and respective quinones to obtain various substitutions of the molecular furopyridine scaffold. The substituent effects on the determined kinase inhibitory properties of cdk1, cdk2, Fyn, JNK3 and gsk-3ß are discussed. CONCLUSION: Various 3-substitutions were found most sensitive for the protein kinase inhibition depending on the length, nature and a substituent positioning within. We identified compounds as inhibitors of several kinases as a tool to potentially combat the disease progress in a multitargeting approach.

Drug Development of Small-Molecule Inhibitors of AD-Relevant Kinases as Novel Perspective Multitargeted Approach.

So far monotargeted therapies in Alzheimers disease (AD) led to insufficient results. Slight improvements in the AD symptomatics have been limited to patients in the early stage of the disease. So multitargeting approaches have been started addressing amyloid plaques as preferred primary target structures beside acetylcholine esterase inhibition. Various protein kinases have been discussed to make a contribution to the progression of AD. So protein kinases are promising target structures for a perspective multitargeting. We identified substituted smallmolecule protein kinase inhibitors of the tricyclic benzofuropyridine type which showed partly nanomolar affinities to AD-relevant glycogen synthase kinase (gsk) 3ß, extracellular-signal regulated kinase (ERK) 2 and C-Jun-N-terminal kinase (JNK) 3. Substituent-dependent effects on the respective kinase inhibitions are discussed and inhibitor binding modes to those kinases are presented based on enzyme docking studies. Inhibitor effects on the tau protein target structure are shown for first compounds in cellular studies to prove the enzyme conditioned effects.

The impact of the induction of multidrug resistance transporters in therapies by used drugs: recent studies.

Multidrug resistance (MDR) against groups of therapeutic drugs emerged to a central problem in the treatment of various diseases, i.e. cancer and infectious diseases like HIV or malaria. ABC transporters namely P-glycoprotein (P-gp) and various multidrug resistance associated proteins (MRPs) mainly contribute to the MDR phenomenon in cancer treatment and HIV therapy. Their cellular expression in respective cells like cancer cells lowers the intracellular drug concentrations and thus reasons the cellular resistance. The induction of such efflux pumps occurs during the therapy with drugs which will be affected by the MDR phenomenon as a consequence of the induction. In this review studies which report such drugs-caused inductions will be viewed. The review will cover the literature of recent years and attract attention to this important question in drug resistance. Finally, the discussion will suggest possible strategies to overcome the problem, i. e. by using non-inducing drugs.

Antitumor activity of terpenoids against classical and atypical multidrug resistant cancer cells.

Nineteen terpenoids, including macrocyclic diterpenes, diterpenic lactones and other polycyclic diterpenes, steroids and a triterpene isolated from the methanolic extracts of Euphorbia species, were evaluated for their potential antineoplastic activity in various human cancer cell lines that were derived from three tumor entities: gastric (EPG85-257), pancreatic (EPP85-181) and colon (HT-29) carcinomas. Furthermore, different multidrug-resistant variants of these cancer cell lines with over-expression of MDR1/P-gp or no MDR1/P-gp expression were also investigated. In parental drug-sensitive cell lines, the tested compounds showed a moderate/weak antiproliferative effect or were inactive. Most of them were found more effective in drug-resistant cells than in the parental, drug-sensitive ones, and some of them showed high antineoplastic efficacy in classical or atypical drug-resistant cells. The most active compounds were the lathyrane diterpenes latilagascenes C and D, and the diterpenic lactones 3beta-acetoxy-helioscopinolide B and helioscopinolide E which exhibited high antineoplastic activities against the drug-resistant subline EPG85-257RDB derived from gastric carcinoma. In addition, the macrocyclic lathyrane diterpene jolkinol B was found to be highly effective in the multidrug-resistant variant HT-29RNOV.

Physicochemical characteristics of novel P-glycoprotein inhibitors of the cage dimeric 1,4-dihydropyridine type.

Physicochemical characteristics of two structurally different cage dimeric 1,4-dihydropyridines HX (1) and CC (2) have been determined and compared to their P-glycoprotein inhibiting properties. While the weakly basic compound (1) showed pH-dependent apparent partition coefficients (log D), the neutral compound (2) proved to have almost identical log D values at varying pH-values. The subsequent determination of partition coefficients (log P) resulted in comparably low log P values revealing a less lipophilic compound character. Determined significantly differing P-glycoprotein (P-gp) inhibitory properties indicated that the lipophilicity of the compounds does not play a decisive role for the P-gp activity.

Synthesis and biological evaluation of first N-alkyl syn dimeric 4-aryl-1,4-dihydropyridines as competitive HIV-1 protease inhibitors.

A first series of novel N-alkyl substituted syn dimeric 4-aryl-1,4-dihydropyridines 12--17 have been synthesised and evaluated as HIV-1 protease inhibitors in in vitro assays. While the N-methyl derivatives 12 and 13 were almost inactive, with IC(50)-values of about 225 microM, the N-benzyl compounds with varied ester groups all exhibited stronger activities, with IC(50)-values of 11--12 microM for the presently best compounds 16 and 17 with ethyl ester functions. The type of HIV-1 protease inhibition of the novel inhibitors was characterised as competitive. With the increase of observed activity from N-methyl derivatives to N-benzyl compounds the binding mode may correspond to that of cyclic ureas with hydrophobic interactions of the four aromatic residues to the S1/S1' and S2/S2' regions of HIV-1 protease.

Formation of novel photodimers from 4-aryl-1,4-dihydropyridines.

N-substituted 3-alkoxycarbonyl-4-aryl-1.4-dihydropyridines have been photochemically investigated for the first time. In contrast to reports of analogous 3,5-dialkoxycarbony] derivatives, they are unreactive in the solid state with shortest distances of potentially reacting double bonds of 6.883(3) A for one derivative examined by x-ray crystal structure analysis. Solution irradiation with unfiltered light (lambda > or = 270 nm) led to novel diazatetrakishomocubanes in 30-50% yields. Diazatetrakishomocubanes were also obtained by irradiation with filtered light (lambda > 313 nm) besides head-to-tail connected syn-dimers. The irradiation of the syn-dimers with unfiltered light led to centrosymmetric cage dimers accompanied by some dimer fragmentation. Formation of the homocubanes via intermediate biradicals is supported by the available data.

Bioanalysis of syn dimeric HIV-1 protease inhibitor N-benzyl 4-aryl-1,4-dihydropyridine H19: metabolic and cytotoxic properties in Hep G2 cells.

Syn dimeric N-benzyl 4-aryl-1,4-dihydropyridine H19 is a nonpeptidic HIV-1 protease inhibitor of the dihydroxyethylene type representing novel C2-symmetric inhibitors. Great interest was focussed on the extent of metabolism of these novel inhibitory structures as their functional groups are similar to certain peptidic and non-peptidic HIV-1 protease inhibitors with poor bioavailability due to extensive metabolism. Thus, early characterization of metabolic and toxic properties decisively determines the future prospects of those novel HIV-1 protease inhibitors. Both metabolism and toxicity were evaluated in Hep G2 monolayers. While no phase-I metabolites were found the extent of conjugation in phase-II of biotransformation was poor. Moreover, cytotoxic evaluation of protein and DNA decrease and, furthermore, of membrane toxicity characterized the novel inhibitors as non-toxic. Consequently, the favourable poor metabolism and non-toxic properties encourage further development of these novel HIV-1 protease inhibitors.

The First Functionalized 6,12-Diazatetrakishomocubanes.

The photodimerization of asymmetric 4-aryl-1,4-dihydropyridines results, totally unexpectedly, in the new 6,12-diazatetrakishomocubanes 1. This is in contrast to the previously observed [2+2] photocycloaddition reactions of symmetrical 4-aryl-1,4-dihydropyridines

First bioanalytical evaluation of nonpeptidic cage dimeric HIV-1 protease inhibitor N-benzyl 4-aryl-1,4-dihydropyridine H17: biotransformation and toxicity on Hep G2 cells.

Cage dimeric N-benzyl 4-aryl-1,4-dihydropyridine H17 is a moderate inhibitor of HIV-1 protease. As representative of an innovative and promising class of nonpeptidic HIV-1 protease inhibitors H17 was selected for the characterization of the biochemical profile of the cage dimers concerning metabolic and toxic aspects. In the first bioanalytical evaluation of H17 on Hep G2 monolayers no phase-I metabolites were found and the extent of conjugation on phase-II of biotransformation was poor due to steric hindrance of the hydroxymethylene groups. H17 was found to be nearly non-toxic. A slight noticeable influence on cell proliferation, however, did not result from apoptotic activities. Thus, first biochemical evaluation of H17 practically suggests no decrease of an in-vivo bioavailability by metabolization.

Cage dimeric N-acyl- and N-acyloxy-4-aryl-1,4-dihydropyridines as first representatives of a novel class of HIV-1 protease inhibitors.

The synthesis of a series of novel cage dimeric N-acyl and N-acyloxy-4-aryl-1,4-dihydropyridines starting either from solid-state synthetic ester dimers or from monomeric 4-aryl-1,4-dihydropyridines is presented. Their biological evaluation as novel HIV-1 protease inhibitors showed the most active compounds to be 5c and 5i with inhibitory activities of 52% (50 microM) and 49% (25 microM), respectively. Within each series of N-acyl- and N-acyloxy derivatives NCOBz and NBoc groups were found to be the best substituents. Although they exhibiting only moderate activities these cage dimers hold promise as a class of novel non-peptidic HIV-1 protease inhibitors.

Synthesis and biological evaluation of the first N-alkyl cage dimeric 4-aryl-1,4-dihydropyridines as novel nonpeptidic HIV-1 protease inhibitors.

A first series of novel NH and N-alkyl-substituted cage dimeric 4-aryl-1,4-dihydropyridines 3a-f has been synthesized and evaluated as HIV-1 protease inhibitors in in vitro assays. While the NH and N-methyl derivatives 3a,b,e,f were almost inactive with IC(50) values of about 200 microM, the N-Benzyl compounds exhibited stronger activity with an IC(50) value of 16.2 microM for the presently best compound 3c. The type of HIV-1 protease inhibition of these novel inhibitors was characterized as competitive. With the increase of observed activity from NH and N-methyl derivatives to N-benzyl compounds, respectively, the binding mode may correspond to that of cyclic and azacyclic ureas showing hydrophobic interactions of the four aromatic residues to the S1/S1' and S2/S2' regions of HIV-1 protease.

Solid-state photodimerization of 4-aryl-1,4-dihydropyridines studied by 13C CPMAS NMR spectroscopy.

13C CPMAS NMR spectroscopy has been applied to monitor the solid-state reaction of two different photodimerizing 4-phenyl-1,4-dihydropyridines yielding a cage dimer in one case and an anti-dimer in the other case. The spectra of the reacting monomers exhibit a magnetical inequivalence of chemically equivalent CO and C2/4 carbon atoms caused by a rotation of the pseudoaxially oriented 4-phenyl substituent out off the plane through N1, C3, C8 which could be determined by X-ray crystal structure analyses of the centrosymmetrically arranged monomers. The 13C CPMAS NMR monitoring of the cage dimer formation proves that the reaction takes place in two steps via a syn-dimer for which a non-symmetrical structure was derived from the spectrum. The non-symmetrical structure was confirmed by X-ray crystal structure analysis of one structurally related derivative. A centrosymmetric structure for both the finally formed cage dimer and the anti-dimer of the other monitored photoreaction was proved by their spectra with one set of signals for each half of the dimers. respectively. Thus, conformational properties of the molecules as well as the symmetry of the products can be directly derived from the 13C CPMAS NMR spectra.

Comparison of azacyclic urea A-98881 as HIV-1 protease inhibitor with cage dimeric N-benzyl 4-(4-methoxyphenyl)-1,4-dihydropyridine as representative of a novel class of HIV-1 protease inhibitors: a molecular modeling study.

The functional groups of cage dimeric N-alkyl substituted 3,5-bis(hydroxymethyl)-4-(4-methoxyphenyl)-1,4-dihydropyridines are similar to those of cyclic and azacyclic ureas that are potent inhibitors of HIV-1 protease of the dihydroxyethylene- and hydroxyethylene type, respectively. In the following study the conformity of common functional groups is investigated concerning their orientation in space as well as in the enzyme HIV-1 protease. Starting from X-ray crystal data of the centrosymmetric cage dimeric N-benzyl derivative with ester groups, the derivative with hydroxymethylene groups was built and a systematic conformational search was performed for the conformationally important torsion angles considering electrostatic and van der Waals interactions. From the huge number of conformations those comprising centrosymmetrical and C2-symmetrical energy minima were selected and minimized. The three remaining conformers were fitted to the azacyclic urea A-98881 selected from the HIV-1 protease enzyme-inhibitor complex using the centroids of the corresponding aromatic residues and additionally by the field fit option of the Advanced CoMFA module of SYBYL. Interestingly, the energetically most favourable one, which, additionally, possesses C2-symmetry like the active site cavity of HIV-1 protease, showed the best fit. Comparing the electrostatic potential (EP) of the latter with the EP of A-98881 the aromatic residues show excellent accordance. Slight differences in the extent of the EP were found in the areas of the hydroxymethylene groups of the cage dimer and the single hydroxy group as well as the urea carbonyl group of A-98881, respectively. In order to compare the binding possibilities to the enzyme HIV-1 protease for the cage dimer and A-98881, their interaction fields with certain probes (CH3 for alkyl, NHamide, and carbonyl, O- of COO-), representing the decisive functional groups of the active site, have been calculated using GRID and projected into the enzyme placing the structures according to the position of A-98881 in the enzyme-inhibitor complex. The strongest calculated fields of the O- probe were found near Asp 25 for both structures. Another respective conformity consists in the overlap of the fields for the NHamide probe near Ile 50 and 50' for the investigated cage dimer and A-98881.