Synthesis and evaluation against hepatitis C virus of 7-deaza analogues of 2'-C-methyl-6-O-methyl guanosine nucleoside and L-Alanine ester phosphoramidates.

7-Deazapurines are known to possess broad antiviral activity, however the 2'-C-methylguanosine analogue displays poor cell permeation and limited phosphorylation, thus is not an efficient inhibitor of hepatitis C virus (HCV) replication. We previously reported the 6-O-methyl entity as a prodrug moiety to increase liphophilicity of guanine nucleosides and the ProTide approach applied to 2'-C-methyl-6-O-methylguanosine has lead to potent HCV inhibitors now in clinical trials. In this Letter, we report the synthesis and biological evaluation of 2'-C-methyl-6-O-methyl-7-deaza guanosine and ProTide derivatives. In contrast to prior studies, removal of the N-7 of the nucleobase entirely negates anti-HCV activity compared to the 2'-C-methyl-6-O-methylguanosine analogues. To understand better this significant loss of activity, enzymatic assays and molecular modeling were carried out and suggested 2'-C-methyl-6-O-methyl-7-deaza guanosine and related ProTides do not act as efficient prodrugs of the free nucleotide, in marked contrast to the case of the parent guanine analogue.

Phosphorodiamidates as a promising new phosphate prodrug motif for antiviral drug discovery: application to anti-HCV agents.

We herein report phosphorodiamidates as a significant new phosphate prodrug motif. Sixty-seven phosphorodiamidates are reported of two 6-O-alkyl 2'-C-methyl guanosines, with significant variation in the diamidate structure. Both symmetrical and asymmetric phosphorodiamidates are reported, derived from various esterified amino acids, both d and l, and also from various simple amines. All of the compounds were evaluated versus hepatitis C virus in replicon assay, and nanomolar activity levels were observed. Many compounds were noncytotoxic at 100 µM, leading to high antiviral selectivities. The agents are stable in acidic, neutral, and moderately basic media and in selected biological media but show efficient processing by carboxypeptidases and efficiently yield the free nucleoside monophosphate in cells. On the basis of in vitro data, eight leads were selected for additional in vivo evaluation, with the intent of selecting one candidate for progression toward clinical studies. This phosphorodiamidate prodrug method may have broad application outside of HCV and antivirals as it offers many of the advantages of phosphoramidate ProTides but without the chirality issues present in most cases.

Dual pro-drugs of 2'-C-methyl guanosine monophosphate as potent and selective inhibitors of hepatitis C virus.

We have previously reported the power of combining a 5'-phosphoramidate ProTide, phosphate pro-drug, motif with a 6-methoxy purine pro-drug entity to generate highly potent anti-HCV agents, leading to agents in clinical trial. We herein extend this work with the disclosure that a variety of alternative 6-substituents are tolerated. Several compounds exceed the potency of the prior 6-methoxy leads, and in almost every case the ProTide is several orders of magnitude more potent than the parent nucleoside. We also demonstrate that these agents act as pro-drugs of 2'-C-methyl guanosine monophosphate. We have also reported the novel use of hepatocyte cell lysate as an ex vivo model for ProTide metabolism.

Protection of retinal cells from ischemia by a novel gap junction inhibitor.

Retinal cells which become ischemic will pass apoptotic signal to adjacent cells, resulting in the spread of damage. This occurs through open gap junctions. A class of novel drugs, based on primaquine (PQ), was tested for binding to connexin 43 using simulated docking studies. A novel drug has been synthesized and tested for inhibition of gap junction activity using R28 neuro-retinal cells in culture. Four drugs were initially compared to mefloquine, a known gap junction inhibitor. The drug with optimal inhibitory activity, PQ1, was tested for inhibition and was found to inhibit dye transfer by 70% at 10 microM. Retinal ischemia was produced in R28 cells using cobalt chloride as a chemical agent. This resulted in activation of caspase-3 which was prevented by PQ1, the gap junction inhibitor. Results demonstrate that novel gap junction inhibitors may provide a means to prevent retinal damage during ischemia.

Combination of nanogel polyethylene glycol-polyethylenimine and 6(hydroxymethyl)-1,4-anthracenedione as an anticancer nanomedicine.

Polyethylene glycol-polyethylenimine (PEG-PEI) nanogels have been used to deliver nucleic acids and oligonucleotides into cells. First, we synthesized PEG-PEI nanogels with methylene proton ratios (CH2O:CH2N) in PEG-PEI ranging from approximately 6.8:1 to 4:1 and less, as shown by 1H NMR spectra. We first synthesized various nanogels with varying ratios of CH2O:CH2N (methylene proton) in PEG-PEI as shown by 1H NMR spectra and tested their cytotoxicity using a rodent pancreatic adenocarcinoma cell line (Pan 02). We showed that the nanogel PEG-PEI with methylene proton ratio of 4:1 was strongly cytotoxic to Pan 02 cells in vitro, while the nanogel with the methylene proton ratio of 6.8:1 was not toxic. We incorporated a novel anti-cancer drug, 6-(hydroxymethyl)-1,4-anthracenedione (AQ) analogue (AQ10) into nontoxic nanogel PEG-PEI and tested the effect of AQ10 loaded nanogel PEG-PEI (AQ10-nanogel PEG-PEI) and AQ10 dissolved in DMSO on Pan 02 cell growth. The size of this AQ10-nanogel PEG-PEI was characterized using atomic force microscopy (AFM). Our studies showed that the AQ10-nanogel PEG-PEI is readily taken up by Pan 02 cells. Growth attenuation of Pan 02 cells treated with AQ10-nanogel PEG-PEI was three to four times that of cells treated with AQ10 dissolved in DMSO. These results suggest that PEG-PEI, usually used to deliver nucleic acids into cells, can also be used to deliver an insoluble small molecule anticancer drug, AQ10.

Synthesis and anti-breast cancer activities of substituted quinolines.

Promising anti-breast cancer agents derived from substituted quinolines were discovered. The quinolines were readily synthesized in a large scale from a sequence of reactions starting from 4-acetamidoanisole. The Michael addition product was isolated as the reaction intermediate in the ring closing reaction of 4-amino-5-nitro-2-(3-trifluoromethylphenyloxy)anisole with methyl vinyl ketone leading to 6-methoxy-4-methyl-8-nitro-5-(3-trifluoromethylphenyloxy)quinoline (14). The amino function of 8-amino-6-methoxy-4-methyl-5-(3-trifluoromethylphenyloxy)quinoline, prepared from 14, was connected to various side chains via alkylation with N-(3-iodopropyl)phthalimide, Michael addition with acrylonitrile, and reductive amination with various heterocycle carboxaldehydes, such as imidazole-4-carboxaldehyde, thiophene-2-carboxaldehyde, and 2-furaldehyde. Effects of the substituted quinolines on cell viability of T47D breast cancer cells using trypan blue exclusion assay were examined. The results showed that the IC(50) value of 6-methoxy-8-[(2-furanylmethyl)amino]-4-methyl-5-(3-trifluoromethylphenyloxy)quinoline is 16+/-3nM, the lowest IC(50) out of all the quinolines tested. IC(50) values of three other quinolines are in the nanomolar range, a desirable range for pharmacological testing.

Total syntheses of (+/-)-ovalicin, C4(S *)-isomer, and its C5-analogs and anti-trypanosomal activities.

Total syntheses of (+/-)-ovalicin, its C4(S( *))-isomer 44, and C5-side chain intermediate 46 were accomplished via an intramolecular Heck reaction of (Z)-3-(tert-butyldimethylsilyloxy)-1-iodo-1,6-heptadiene and a catalytic amount of palladium acetate. Subsequent epoxidation, dihydroxylation, methylation, and oxidation led to (3S( *),5R( *),6R( *))-5-methoxy-6-(tert-butyldimethylsilyloxy)-1-oxaspiro[2.5]octan-4-one (2), a reported intermediate. The addition of a side chain with cis-1-lithio-1,5-dimethyl-1,4-hexadiene (27) followed by oxidation afforded (+/-)-ovalicin. The functional group manipulation afforded a number of regio- and stereoisomers, which allow the synthesis of analogs for bioevaluation. The structure of 44 was firmly established via a single-crystal X-ray analysis. The stereochemistry at C4 generated from the addition reactions of alkenyllithium with ketones 2, 40, and 45 is dictated by C6-alkoxy functionality. Anti-trypanosomal activities of various ovalicin analogs and synthetic intermediates were evaluated, and C5-side chain analog, 46, shows the strongest activity. Compound 44 shows antiproliferative effect against HL-60 tumor cells in vitro. Compounds 46 and a precursor, (3S( *),4R( *),5R( *),6R( *))-5-methoxy-4-[(E)-(1',5'-dimethylhexa-1',4'-dienyl)]-6-(tert-butyldimethylsilyloxy)-1-oxaspiro[2.5]octan-4-ol (28), may be explored for the development of anti-parasitic drugs.

Antitumor triptycene analogs directly interact with isolated mitochondria to rapidly trigger markers of permeability transition.

BACKGROUND: Substituted triptycenes (TT code number), which block nucleoside transport, macromolecule syntheses and DNA topoisomerase activities, induce cytochrome c release and apoptotic DNA fragmentation, inhibit the proliferation of drug-sensitive and -resistant tumor cells in the nM range in vitro and rapidly trigger the collapse of mitochondrial transmembrane potential in cell and cell-free systems. Because mitochondrial permeability transition (MPT) requires more than depolarization, antitumor TTs were tested for their ability to directly trigger specific markers of MPT in isolated mitochondria. MATERIALS AND METHODS: Large amplitude swelling and Ca2+ release were assayed in isolated mitochondria to demonstrate TT-induced MPT. RESULTS: Antitumor TTs interact with isolated mitochondria in a concentration- and time-dependent manner to rapidly cause large amplitude swelling and Ca2+ release in relation with their antiproliferative activities in L1210, HL-60 and LL/2 tumor cells in vitro. The ability of 4-10 uM TT15, TT16 and TT24 to maximally induce mitochondrial swelling and Ca2+ release within 20 min is similar to that of classic MPT inducers, such as 5 microg/ml alamethicin, 200 microM atractyloside, 5 microM phenylarsine oxide, 100 microM arsenic trioxide and a 100 microM Ca2+ overload. TT15 requires a priming concentration of 20 microM Ca2+ to trigger mitochondrial swelling and Ca2+ release and these 0.1 microM ruthenium red-sensitive MPT events are abolished by 1 microM cyclosporin A, 2 mM ADP and 20 microM bongkrekic acid, which block components of the permeability transition pore (PTP), and by 50-100 microM of various ubiquinones, which interact with the quinone binding site of the PTP and raise the Ca2+ load required for PTP opening. CONCLUSION: Antitumor TTs that trigger MPT in isolated mitochondria might interact with components of the PTP to boost its Ca2+-sensitive transition from the closed to the open state and might be valuable to develop mitochondriotoxic drugs that directly activate early components of apoptosis.

Novel substituted 1,4-anthracenediones with antitumor activity directly induce permeability transition in isolated mitochondria.

Synthetic analogs of 1,4-anthraquinone (AQ code number), which block nucleoside transport, decrease DNA, RNA and protein syntheses, trigger cytochrome c release without caspase activation, induce apoptotic DNA fragmentation and inhibit the proliferation of wild-type and multidrug resistant tumor cells in the nM range in vitro, rapidly cause the collapse of mitochondrial transmembrane potential in cell and cell-free systems. Because mitochondrial permeability transition (MPT) requires more than depolarization to occur, antitumor AQs were tested for their ability to directly trigger specific markers of MPT in isolated mitochondria. In contrast to a spectrum of conventional anticancer drugs that are inactive, various AQs interact with isolated mitochondria in a concentration- and time-dependent manner to rapidly cause large amplitude swelling and Ca2+ release in relation with their effectiveness against L1210, HL-60 and LL/2 tumor cells in vitro. Indeed, the lead antitumor AQ8, AQ9 and AQ17 are also the most effective inducers of MPT in isolated mitochondria, whereas all AQ derivatives devoid of anti-proliferative activity also fail to trigger mitochondrial swelling and Ca2+ release. Moreover, the ability of 4 microM AQ17 to maximally induce mitochondrial swelling and Ca2+ release within 15 min is similar to that of classic MPT-inducing agents, such as 5 microg/ml alamethicin, 200 microM atractyloside, 5 microM phenylarsine oxide, 100 microM arsenic trioxide and a 100 microM Ca2+ overload. Interestingly, AQ17 requires a priming concentration of 20 microM Ca2+ to trigger mitochondrial swelling and Ca2+ release and these 0.1 microM ruthenium red-sensitive MPT events are abolished by 1 microM cyclosporin A, 2 mM ADP and 20 microM bongkrekic acid, which block components of the permeability transition pore (PTP), and also inhibited by 50-100 microM of various ubiquinones, which interact with the quinone binding site of the PTP and raise the Ca2+ load required for PTP opening. Hence, antitumor AQs that target isolated mitochondria and trigger MPT might directly interact with components of the PTP to induce conformational changes that increase its Ca2+ sensitivity and transition from the closed to the open state.

Syntheses, molecular targets and antitumor activities of novel triptycene bisquinones and 1,4-anthracenedione analogs.

Novel substituted triptycene bisquinones and 1, 4-anthracenediones were synthesized and screened for their anti-cancer activities. A number of analogs were synthesized utilizing various synthetic transformations and found to elicit interesting antitumor effects. Analogs included water-soluble pro-drugs and ammonium salts. These potent antitumor drugs are DNA topoisomerase inhibitors that induce DNA strand breaks, inhibit DNA, RNA and protein syntheses and reduce tumor cell proliferation in the nanomolar range in vitro. They induce cytochrome c release, caspase-9, -3 and -8 activities, poly(ADP)-ribose polymerase-1 (PARP) cleavage, and internucleosomal DNA fragmentation by a mechanism which involves caspase-2 activation but not Fas signaling. Moreover, these drugs remain effective in multidrug-resistant tumor cells and have the advantage of blocking nucleoside transport and inducing a rapid loss of mitochondrial transmembrane potential. Based on their effects in tumor cells and isolated mitochondria, it is hypothesized that these drugs might, directly and indirectly, target components of the permeability transition pore to induce mitochondrial permeability transition and the release of proapoptotic factors. This review provides a summary of synthetic efforts and mechanistic endeavor.

A novel tricyclic pyrone compound ameliorates cell death associated with intracellular amyloid-beta oligomeric complexes.

The neurotoxicity of amyloid-beta protein (Abeta) is widely regarded as one of the fundamental causes of neurodegeneration in Alzheimer's disease (AD). This toxicity is related to Abeta aggregation into oligomers, protofibrils and fibrils. Recent studies suggest that intracellular Abeta, which causes profound toxicity, could be one of the primary therapeutic targets in AD. So far, no compounds targeting intracellular Abeta have been identified. We have investigated the toxicity induced by intracellular Abeta in a neuroblastoma MC65 line and found that it was closely related to intracellular accumulation of oligomeric complexes of Abeta (Abeta-OCs). We further identified a cell-permeable tricyclic pyrone named CP2 that ameliorates this toxicity and significantly reduces the levels of Abeta-OCs. In aqueous solution, CP2 attenuates Abeta oligomerization and prevents the oligomer-induced death of primary cortical neurons. CP2 analogs represent a new class of promising compounds for the amelioration of Abeta toxicities within both intracellular and extracellular sites.

Antitumor triptycene analogs induce a rapid collapse of mitochondrial transmembrane potential in HL-60 cells and isolated mitochondria.

Since synthetic analogs of triptycene (TT code number), such as bisquinones TT2 and TT13, can trigger cytochrome c release without caspase activation and retain their ability to induce apoptosis in multidrug-resistant (MDR) tumor cells, fluorescent probes of transmembrane potential have been used to determine whether these antitumor compounds might directly target mitochondria in cell and cell-free systems to cause the collapse of mitochondrial membrane potential ( downward arrow Deltapsim) that is linked to permeability transition pore (PTP) opening. Using JC-1 dye, the abilities of various TT analogs to induce the downward arrow Deltapsim in wild-type and MDR HL-60 cells are rapid (within 5-20 min), irreversible after drug removal, concentration dependent in the 0.64-25 microM range, and generally related to their antitumor activities in vitro. The downward arrow Deltapsim caused by TT2 and TT13, which are more potent than mitoxantrone, staurosporine and the reference depolarizing agent, carbonyl cyanide m-chlorophenylhydrazone (CCCP), in HL-60 cells, are not prevented by caspase-2 or -8 inhibitors, suggesting that activation of these apical caspases upstream of mitochondria is not involved in this process. Antitumor TT analogs (0.64-25 microM) also mimic the abilities of the known depolarizing agents, CCCP, alamethicin, gramicidin A and 100 microM CaCl(2), to directly induce within 20 min the downward arrow Deltapsim in isolated mitochondria prepared from mouse liver and loaded with rhodamine 123 dye. The fact that 20 microM Ca(2+), which is insufficient to trigger depolarization on its own, is required to reveal the depolarizing effect of TT2 in isolated mitochondria suggests that antitumor TT analogs might interact with the PTP to alter its conformation and increase its Ca(2+) sensitivity. Indeed, such Ca(2+)-dependent downward arrowDeltapsim of isolated mitochondria treated with 25 microM TT2 or 100 microM Ca(2+) are blocked by ruthenium red. Daunorubicin (DAU) is unable to mimic the rapid downward arrowDeltapsim caused by antitumor TT bisquinones within 5-40 min of treatment in HL-60 cells or isolated mitochondria. Moreover, the downward arrowDeltapsim caused by 25 microM TT2 or 100 microM Ca(2+) in isolated mitochondria are similarly blocked by cyclosporin A (CsA), bongkrekic acid and decylubiquinone, which prevent PTP opening, suggesting that, in contrast to DAU, antitumor TT analogs that directly target mitochondria to trigger the Ca(2+)-dependent and CsA-sensitive downward arrowDeltapsim, might induce PTP opening and the mitochondrial pathway of apoptosis even in the absence of nuclear signals.

Total syntheses of (+)-chloropuupehenone and (+)-chloropuupehenol and their analogues and evaluation of their bioactivities.

Tetracyclic pyrans (+)-chloropuupehenone (1) and (+)-chloropuupehenol (5) and its C8-R-isomer (+)-3 were synthesized via a one-pot condensation of 1-chloro-2-lithio-3,5,6-tris(tert-butyldimethylsilyloxy)benzene (8) with (4aS,8aS)-3,4,4a,5,6,7,8,8a-octahydro-2,5,5,8a-tetramethylnaphthalene-1-carboxaldehyde (7). The major condensation product, (4aS,6aR,12bS)-2H-9,10-bis(tert-butyldimethylsilyloxy)-11-chloro-1,3,4,4a,5,6,6a,12b-octahydro-4,4,6a,12b-tetramethyl-benzo[a]xanthene (4), after desilylation provided tetracyclic pyran (+)-(4aS,6aR,12bS)-2H-11-chloro-1,3,4,4a,5,6,6a,12b-octahydro-4,4,6a,12b-tetramethyl-benzo[a]xanthene-9,10-diol (3). At a dosage of 42 mg/rat over 8 h, pyran diol 3 inhibited the intestinal absorption of cholesterol by 71% in rats. Tetracyclic pyran 4 was also converted to o-quinone 28, which inhibited cholesteryl ester transfer protein (CETP) activity and L1210 leukemic cell viability with IC(50) values of 31 and 2.4 microM, respectively. Diol (+)-5 inhibited CETP activity with an IC(50) value of 16 microM. The minor condensation product, (4aS,6aS,12bS)-2H-9,10-bis(tert-butyldimethylsilyloxy)-11-chloro-1,3,4,4a,5,6,6a,12b-octahydro-4,4,6a,12b-tetramethyl-benzo[a]xanthene (6), was transformed into (+)-5 and (+)-1. A stepwise stereoselective synthesis of (+)-1 was also developed utilizing an oxyselenylation ring-closure reaction. The synthetic sequence also produced four biologically active naturally occurring drimanic sesquiterpenes, (+)-drimane-8alpha,11-diol (34), (-)-drimenol (38), (+)-albicanol (39), and (-)-albicanal (31) as intermediates.