Sarcomelicope alkaloids as leads for the discovery of new antitumor acronycine derivatives

The acridone alkaloid acronycine first isolated from Acronychia baueri Schott (Rutaceae) in 1948, was later shown to exhibit a promising activity against a broad spectrum of solid tumors. Nevertheless, clinical trials only gave poor results, probably due to the moderate potency and low water solubility of this alkaloid. Early studies on structure-activity relationships in the series concluded that the 1,2-double bond was an essential structural requirement to observe cytotoxic activity. It is the reason why the isolation in our laboratory of the unstable acronycine epoxide from several New-Caledonian Sarcomelicope species led to the hypothesis of bioactivation of acronycine by transformation of the 1,2-double bond into the corresponding oxirane in vivo. Consequently, there was interest in the search for acronycine derivatives modified in the pyran ring and having improved stability, but a similar reactivity toward nucleophilic agents as acronycine epoxide. Accordingly, we synthesized a series of cis- and trans-1,2-dihydroxy-1,2-dihydroacronycine diesters which exhibited interesting antitumor properties with a broadened spectrum of activity and increased potency when compared with acronycine. (±)-Cis-1,2-diacetoxy-1,2-dihydroacronycine was of particular interest, due to its marked activity in vivo against the resistant solid tumor C 38 colon carcinoma. The demonstration that acronycine should interact with DNA by some noncovalent process able to stabilize the double helix against thermal denaturation prompted us to develop benzo[b]acronycine analogues possessing an additional aromatic ring linearly fused on the natural alkaloid basic skeleton. When tested against a panel of cancer cell lines in vitro, cis-1,2-dihydroxy-1,2-dihydrobenzo[b]acronycine diesters exhibited cytotoxic activities within the same range of potency as the most active drugs currently used in cancer chemotherapy. In vivo, cis-1,2-diacetoxy-1,2-dihydrobenzo[b]acronycine (S23906-1), selected for further preclinical development, demonstated a marked antitumor activity in human orthotopic models of lung, ovarian and colon cancers xenografted in nude mice. This revised version was published online in June 2006 with corrections to the Cover Date.     

Novel bifunctional alkylating agents, 5,10-dihydropyrrolo[1,2-b]isoquinoline derivatives, synthesis and biological activity

A series of linear pyrrolo[1,2-b]isoquinoline derivatives was synthesized for antitumor evaluation. The preliminary antitumor studies reveal that both bis(hydroxymethyl) and their bis(alkylcarbamate) derivatives show significant antitumor activity in inhibiting various human tumor cell growth in vitro. 1,2-Bis(hydroxymethyl)-3-methyl-5,10-dihydropyrrolo[1,2-b]isoquinoline (20a) was selected for antitumor studies in animal models. The results show that this agent can induce complete tumor remission or significant suppression in nude mice bearing human breast (MX-1) xenograft and ovarian (SK-OV-3) xenografts, respectively. Alkaline agarose gel shifting assay showed that 20a is able to cross-link with DNA. Studies on the cell cycle inhibition revealed that this agent induces cell arrest at G2/M phase. The results warrant further antitumor investigation against other human tumor growth in animal models.     

Novel acenaphtho[1,2-b]pyrrole-carboxylic acid family: Synthesis, cytotoxicity, DNA-binding and cell cycle evaluation

A family of 8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carboxylic acid derivatives were synthesized as a result of our efforts to modify a series of acenaphthopyrrole aromatic-heterocycle compounds that proved to be potent anticancer drugs. Among the derivatives, 3d (3-(dimethylamino-propylamino)-8-oxo-8H-acenaphtho-[1,2-b]pyrrole-9-carboxylic acid) and 3g (3-piperidine-8-oxo-8H-acenaphtho-[1,2-b]pyrrole-9-carboxylic acid) showed potential anticancer activity and different action mechanism from our previously reported compounds. UV–vis absorption, circular dichroism and viscosity measurement indicated that effect of both compounds on the advanced DNA conformation was different, although they could bind to DNA in the same way. Cell cycle analysis showed that 3d could induce S-phase arrest followed by apoptosis, while 3g induced apoptosis. The results seem to imply that different action mechanism could contribute to the dissimilitude of biological activities toward 3d and 3g.     

P-Postlabeling high-performance liquid chromatographic improvements to characterize DNA adduct stereoisomers from benzo[a]pyrene and benzo[c]phenanthrene, and to separate DNA adducts from 7,12-dimethylbenz[a]anthracene

Single compounds can generate complex DNA adduct patterns by reactions through different pathways, with different target nucleotides and through different configurations of the products. DNA adduct analysis by ³²P-HPLC was improved by adding an isocratic plateau in an otherwise linear gradient, thereby enhancing resolution of predictable retention time intervals. This enhanced ³²P-HPLC technique was used to analyze and at least partly resolve 14 out of 16 available benzo[c]phenanthrene deoxyadenosine and deoxyguanosine adduct standards, 8 out of 8 available benzo[a]pyrene deoxyadenosine and deoxyguanosine adduct standards, and 51 peaks from 7,12-dimethylbenz[a]anthracene-calf thymus DNA reaction products. The same type of gradient modifications could be used to enhance resolution in analyses of other complex DNA adduct mixtures, e.g., in vivo in humans.     

Determination of albumin adducts of (+)-anti-benzo[a]pyrene-diol-epoxide using an high-performance liquid chromatographic column switching technique for sample preparation and gas chromatography–mass spectrometry for the final detection

A novel method has been developed for the determination of (+)-anti-benzo[a]pyrene-diol-epoxide [(+)-anti-BPDE] albumin adducts in the low-picogram range. Blood from rats and humans was investigated for the validation of the method. Instead of the usual acid hydrolysis we used alkaline conditions for the cleavage of the esters formed with asparagic or glutamic acid residues of albumin. Alkaline hydrolysis gave rise to benzo[a]pyrene-r-7,t-8,t-9,c-10-tetrahydrotetrol (BT I-1) which was separated from the matrix by HPLC with a column switching technique. The analytes were collected by an automated fraction collector and after silylation determined with GC–MS using negative chemical ionization. Adduct concentrations were calculated by the internal standard method. Benzo[a]pyrene-r-7,t-8,c-9,c-10-tetrahydrotetrol (BT-II-2) was used as an internal standard because of its similar physicochemical properties and its absence from human samples. To determine the recovery of the analytical procedure benzo[a]pyrene-r-7,t-8,t-9,t-10-tetrahydrotetrol (BT I-2) was added at the end of the sample clean-up. Single ion recording mode was applied for the detection of the analyte and the standards using the abundant fragment ion m/z 284 for quantitation of the three tetrols. The mean recovery of the internal standard BT II-2 was about 50%. The limit of detection was 0.15 pg per injection corresponding to 0.01 fmol/mg albumin. Regression coefficients of the calibration curves were r²=0.99 and r²=0.98 for BT I-1 concentration ranges of 4–400 ng/l and 4–40 ng/l, respectively. The mean coefficient of variation for duplicate analyses of human albumin samples was found to be 22%.     

Synthesis and antiproliferative evaluation of 6-aryl-11-iminoindeno[1,2-c]quinoline derivatives

A number of 6-aryl-11-iminoindeno[1,2-c]quinoline derivatives were synthesized and evaluated for their antiproliferative activities. Among them, (E)-6-{4-[3-(dimethylamino)propoxy]phenyl}-2-fluoro-9-hydroxy-11H-indeno[1,2-c]quinolin-11-one O-3-(dimethylamino)propyl oxime (23a) was the most active, exhibited GI₅₀ values of 0.64, 0.39, 0.55, 0.67, and 0.65 μM against the growth of Hep G2, Hep 3B, A549, H1299, and MDA-MB-231, respectively. Compound 23a inhibited the growth of hepatoma cell lines in a dose- and time-dependent manner. The proportion of cells was decreased in the G1 and accumulated in G2/M phase after 12 h treatment of 23a, while the hypodiploid (sub-G0/G1 phase) cells increased. Further investigations have shown that 23a induced cell cycle arrest at G2/M phase and induce apoptosis via activation of p53, Bax, and caspase-8 which consequently cause cell death.     

Synthesis of new 4-[2-(alkylamino)ethylthio]pyrrolo[1,2-a]quinoxaline and 5-[2-(alkylamino)ethylthio]pyrrolo[1,2-a]thieno[3,2-e]pyrazine derivatives,as potential bacterial multidrug resistance pump inhibitors

The synthesis of new 4-[2-(alkylamino)ethylthio]pyrrolo[1,2-a]quinoxaline derivatives 1a-l is described in five or six steps starting from various substituted nitroanilines 2a-e. The bioisostere 5-[2-(alkylamino)ethylthio]pyrrolo[1,2-a]thieno[3,2-e]pyrazine 1m was also prepared. The new derivatives were evaluated as efflux pump inhibitors (EPIs) in a model targeting the NorA system of Staphylococcus aureus. The antibiotic susceptibility of two strains overproducing NorA, SA-1199B and SA-1, was determined alone and in combination with the neo-synthesised compounds by the agar diffusion method and MIC determination, in comparison with reserpine and omeprazole taken as reference EPIs. A preliminary structure-activity relationship study firstly allowed to clarify the influence of the substituents at positions 7 and/or 8 of the pyrrolo[1,2-a]quinoxaline nucleus. Methoxy substituted compounds, 1b and 1g, were more potent EPIs than the unsubstituted compounds (1a and 1f), followed by chlorinated derivatives (1c-d and 1h). Moreover, the replacement of the N,N-diethylamino group (compounds 1a-e) by a bioisostere such as pyrrolidine (compounds 1f-h) enhanced the EPI activity, in contrast with the replacement by a piperidine moiety (compounds 1i-k). Finally, the pyrrolo[1,2-a]thieno[3,2-e]pyrazine compound 1m exhibited a higher EPI activity than its pyrrolo[1,2-a]quinoxaline analogue 1a, opening the way to further pharmacomodulation.     

Discovery of imidazo[1,2-b]pyridazines as IKKβ inhibitors. Part 3: exploration of effective compounds in arthritis models

We have discovered imidazo[1,2-b]pyridazine derivatives that show suppressive activity of inflammation in arthritis models. We optimized the substructures of imidazo[1,2-b]pyridazine derivatives to combine potent IKKβ inhibitory activity, TNFα inhibitory activity invivo and excellent pharmacokinetics. The compound we have acquired, which had both potent activities and good pharmacokinetic profiles based on improved physicochemical properties, demonstrated efficacy on collagen-induced arthritis models in mice and rats.     

Degradation of benz[<Emphasis Type="Italic">a</Emphasis>]anthracene by <Emphasis Type="Italic">Mycobacterium vanbaalenii</Emphasis> strain PYR-1

Cultures of Mycobacterium vanbaalenii strain PYR-1 grown in mineral salts medium and nutrients in the presence of benz[a]anthracene metabolized 15% of the added benz[a]anthracene after 12days of incubation. Neutral and acidic ethyl acetate extractable metabolites were isolated and characterized by high performance liquid chromatography (HPLC) and uv–visible absorption, gas chromatography/mass (GC/MS) and nuclear magnetic resonance (NMR) spectral analysis. Trimethylsilylation of the metabolitesfollowed by GC/MS analysis facilitated identification of metabolites. The characterization of metabolites indicated that M. vanbaalenii initiated attack of benz[a]anthracene at the C-1,2-, C-5,6-, C-7,12- and C-10,11-positions to form dihydroxylated and methoxylated intermediates. The major site of enzymatic attack was in the C-10, C-11 positions. Subsequent ortho- and meta-cleavage of each of the aromatic rings led to the accumulation of novel ring-fission metabolites in the medium. The major metabolites identified were 3-hydrobenzo[f]isobenzofuran-1-one (3.2%), 6-hydrofuran[3,4-g]chromene-2,8-dione (1.3%), benzo[g]chromene-2-one (1.7%), naphtho[2,1-g]chromen-10-one (48.1%), 10-hydroxy-11-methoxybenz[a]anthracene (9.3%), and 10,11-dimethoxybenz[a]anthracene (36.4%). Enzymatic attack at the C-7 and C-12 positions resulted in the formation of benz[a]anthracene-7,12-dione, 1-(2-hydroxybenzoyl)-2-naphthoic acid, and 1-benzoyl-2-naphthoic acid. A phenyl-naphthyl metabolite, 3-(2-carboxylphenyl)-2-naphthoic acid, was formed when M. vanbaalenii was incubated with benz[a]anthracene cis-5,6-dihydrodiol, indicating ortho-cleavage of 5,6-dihydroxybenz[a]anthracene. A minor amount of 5,6-dimethoxybenz[a]anthracene was also formed. The data extend and propose novel pathways for the bacterial metabolism of benz[a]anthracene.     

Two-step error-prone bypass of the (+)- and (−)-trans-anti-BPDE-N-dG adducts by human DNA polymerases η and κ

Benzo[a]pyrene is a polycyclic aromatic hydrocarbon (PAH) associated with potent carcinogenic activity. Mutagenesis induced by benzo[a]pyrene DNA adducts is believed to involve error-prone translesion synthesis opposite the lesion. However, the DNA polymerase involved in this process has not been clearly defined in eukaryotes. Here, we provide biochemical evidence suggesting a role for DNA polymerase η (Polη) in mutagenesis induced by benzo[a]pyrene DNA adducts in cells. Purified human Polη predominantly inserted an A opposite a template (+)- and (−)-trans-anti-BPDE-N²-dG, two important DNA adducts of benzo[a]pyrene. Both lesions also dramatically elevated G and T mis-insertion error rates of human Polη. Error-prone nucleotide insertion by human Polη was more efficient opposite the (+)-trans-anti-BPDE-N²-dG adduct than opposite the (−)-trans-anti-BPDE-N²-dG. However, translesion synthesis by human Polη largely stopped opposite the lesion and at one nucleotide downstream of the lesion (+1 extension). The limited extension synthesis of human Polη from opposite the lesion was strongly affected by the stereochemistry of the trans-anti-BPDE-N²-dG adducts, the nucleotide opposite the lesion, and the sequence context 5′ to the lesion. By combining the nucleotide insertion activity of human Polη and the extension synthesis activity of human Polκ, effective error-prone lesion bypass was achieved in vitro in response to the (+)- and (−)-trans-anti-BPDE-N²-dG DNA adducts.     

Inhibition of the benzo[<Emphasis Type="Italic">a</Emphasis>]pyrene-induced toxicity by allyl sulfides in human epidermal keratinocytes

Diallyl sulfide (DAS) and diallyl disulfide (DADS) at 25 g ml^–1decreased the benzo[a]pyrene (B[a]P)-induced colony growth inhibition of human epidermal keratinocytes. DAS and DADS decreased B[a]P-DNA and B[a]P-protein adducts by 65% and 49–55%, respectively. The B[a]P-induced ethoxyresorufin O-deethylase activity, a marker enzyme for cytochrome P450 1, was decreased from 3 to 1.7–1.9 nmol min^–1 mg^–1 microsomal protein by DAS and DADS treatments. The activity of glutathione S-transferase, a detoxifying enzyme for B[a]P, but was decreased by DADS, but was unaffected by DAS.     

Synthesis,antitumor screening and cell cycle analysis of novel benzothieno[3,2-b]pyran derivatives

Abstract

Three series of benzothiophene derivatives were designed and synthesized as cytotoxic agents. The compounds were subjected to in vitro antitumor screening at the National Cancer Institute (NCI), Bethesda, MD. The results of the single dose screening indicated that only the benzothieno[3,2-b]pyran series 3a–f exhibited potent and broad spectrum cytotoxic activity and was subjected to five dose cytotoxic screening. The most active compound in this study was 2-amino-6-bromo-4-(4-nitrophenyl)-4H-[1]benzothieno[3,2-b]pyran-3-carbonitrile (3e) with MG-MID GI₅₀, TGI, and LC₅₀ values of 0.11, 7.94 and 42.66?μM, respectively. Compound 3e exhibited broad spectrum anticancer activity against a panel of 59 cell lines. To elucidate the underlying mechanism of compound 3e cytotoxic activity, we examined its effect on cell cycle progression and its ability to induce apoptosis using human colon adenocarcinoma cell line (HCT-116). The effect of compound 3e on the cell cycle progression indicated that exposure of HCT-116 cells to compound 3e for 24 and 48?h, induced a significant disruption in the cell cycle profile including time dependent decrease in cell population at G1 phase with concomitant increase in pre-G and G2/M cell population. Moreover, compound 3e induced time dependent increase in the percentage of early and late apoptotic and necrotic cell population. In conclusion, we were able to successfully design a new series of benzothieno[3,2-b]pyran derivatives with potent cytotoxic activity and their mechanism of cytotoxicity was examined.     

3-Arylisoquinolines as novel topoisomerase I inhibitors

Topoisomerase I (topo I) is an essential enzyme for vital cellular processes. Inhibition of topo I activities is lethal and leads to cell death, thus establishing topo I as a promising target for cancer treatment. Camptothecin, a natural alkaloid, inhibits topo I. Topotecan and irinotecan, synthetic derivatives of camptothecin, are the most potent anticancer drugs in clinical use. However, several limitations of camptothecins such as solubility, toxicity, stability, resistance and the required high drug dose have encouraged the development of non-camptothecin topo I inhibitors. Natural alkaloid benzo[c]phenanthridines and synthetic indenoisoquinolines have been extensively studied as alternatives to camptothecin. Interestingly, these non-camptothecin topo I inhibitors share a common 3-arylisoquinoline scaffold. This review will describe the development of novel indeno[1,2-c]isoquinolines, isoindolo[2,1-b]isoquinolines, 12-oxobenzo[c]phenanthridines and benz[b]oxepines with a 3-arylisoquinoline nucleus as topo I inhibitors.     

The synthesis of 2-nitroaryl-1,2,3,4-tetrahydroisoquinolines, nitro-substituted 5,6-dihydrobenzimidazo[2,1-a]isoquinoline N-oxides and related heterocycles as potential bioreducible substrates for the enzymes NAD(P)H: quinone oxidoreductase 1 and E. coli nitroreductase

A series of 2-nitroaryl-1,2,3,4-tetrahydroisoquinolines 10 and nitro-substituted 5,6-dihydrobenzimidazo[2,1-a]isoquinoline N-oxides 11 have been synthesised and evaluated as potential bioreducible substrates for the enzymes NAD(P)H: quinone oxidoreductase 1 (NQO1) and Escherichia coli nitroreductase (NR). Also prepared and evaluated were 2-(3,5-dinitropyridin-2-yl)-1,2,3,4-tetrahydroisoquinoline 12 and 5,6-dihydro-10-nitropyrido[3″,2″:4′,5′]imidazo[2′,1′-a]isoquinoline 12-oxide 13. Both compounds 10b and 13 were reduced faster by human NQO1 than by CB-1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide].     

Synthesis, crystal structure, antitumor activity and DNA-binding study on the Mn(II) complex of 2H-5-hydroxy-1,2,5-oxadiazo[3,4-f]1,10-phenanthroline

The complex [Mn(L)(NO₃)₂(H₂O)₂] (1) (L=2H-5-hydroxy-1,2,5-oxadiazo[3,4-f]1,10-phenanthroline) was synthesized and characterized by elemental analysis, IR and UV. The crystal and molecular structure of 1 was determined by single-crystal X-ray diffraction; crystal data: light yellow, monoclinic, space group P2₁/n, Z=4, a=7.432(2) Å, b=9.582(3) Å, c=23.445(7) Å, β=90.519(5)°. The Mn atom in 1 is hexa-coordinated in a distorted octahedral arrangement by two N atoms of the ligand L and four O atoms of two water molecules and two nitrate anions. Biological tests in vitro showed that 1 has significant antitumor activity against HL-60, KB, Hela and BGC-823 cells. The interaction of 1 with calf thymus DNA was investigated by absorption titration, thermal denaturation and viscosity measurements. The results suggest that 1 binds with DNA by intercalating via the ligand L.     

Synthesis and evaluation of the antiproliferative activity of novel isoindolo[2,1-a]quinoxaline and indolo[1,2-a]quinoxaline derivatives

A novel series of isoindolo[2,1-a]quinoxaline and indolo[1,2-a]quinoxaline derivatives was synthesized and evaluated in vitro against various human cancer cell lines for antiproliferative activity. These new compounds displayed activity against leukemia and breast cancer cell lines in the 3- to 18-µM concentration range.     

2-Phenylimidazo[1,2-b]pyridazine derivatives highly active against Haemonchus contortus

A series of 2-phenylimidazo[1,2-b]pyridazine derivatives were synthesized and evaluated for their in vitro anthelmintic activity against Haemonchus contortus. The most active compounds had in vitro LD₉₉ values of 30 nM, which is comparable to that of the benchmark commercial nematocide, Ivermectin.     

Selective cleavage of 10-methyl benzo[b]naphtho[2,1-d]thiophene by recombinant Mycobacterium sp. strain

Recombinant Mycobacterium sp. strain MR65 carrying dszABCD genes was used for desulfurization of 10-methylbenzo[b]naphtho[2,1-d]thiophene (10-methyl BNT) in the hexadecane phase. The specific activity was 25% of that of dibenzothiophene (DBT). One of two major metabolites of 10-methyl BNT produced by strain MR65 was identified as 1-methoxy-2-(3-methylphenyl)naphthalene by ¹H and ¹³C NMR. The other major metabolite and two minor metabolites were determined as 1-hydroxy-2-(3-methylphenyl)naphthalene, 2-(2-methoxy-3-methylphenyl)naphthalene and 2-(2-hydroxy-3-methylphenyl)naphthalene, respectively, by HPLC and GC-MS. The production ratio of the two desulfurization metabolite isomers was 0.99:0.01, calculated on the basis of peak GC areas. These results indicated that the C-S bond adjacent to the naphthalene skeleton was selectively cleaved to form the two major compounds.     

Microbial metabolism and detoxification of 7,12-dimethylbenz[a]anthracene

Summary Six strains of fungi grown on Sabouraud dextrose broth in the presence of 7,12-dimethylbenz[a]anthracene (DMBA) were surveyed for their ability to metabolize DMBA. Experiments with [¹⁴C]DMBA indicated that the extent of formation of organic-soluble metabolites ranged from 6 to 28% after 5 days of incubation, depending on the organism tested. The yields of water-soluble metabolites also varied, and ranged from 1 to 33% after 5 days.Cunninghamella elegans ATCC 36112 andSyncephalastrum racemosum UT-70 exhibited the highest DMBA-metabolizing activity among the organisms surveyed.S. racemosum metabolized DMBA primarily to 7-hydroxymethyl-12-methylbenz[a]anthracene (7-OHM-12-MBA)_ and 7,12-dihydroxymethylbenz[a]anthracene (7,12-diOHMBA). Minor metabolites included 7-OHM-12-MBA-trans-5,6-, 8,9- and 10,11-dihydrodiols, and glucuronide and sulfate conjugates of phenolic derivatives of DMBA. In contrast, the major DMBA metabolites produced byC. elegans were water-soluble. The predominant organic-soluble metabolites produced byC. elegans included 7-OHM-12-MBA-trans-5,6-, 8,9- and 10,11-dihydrodiols. DMBA-trans-3,4-dihydrodiol was also detected. Circular dichroism spectral analysis revealed that the major enantiomer of the 7-OHM-12-MBA-trans-8,9-dihydrodiol formed by each organism has anS,S absolute configuration, while the major enantiomers of the 5,6-, 10,11- and 3,4-dihydrodiols had anR,R configuration. The mutagenic activity of extracts fromS. racemosum exposed to DMBA were determined inSalmonella typhimurium TA98. The mutagenicity of DMBA decreased by 36% over a period of 5 days as 33% of the compound was metabolized. Comparison of these results with previously reported results in mammalian systems suggests that there are similarities and differences between the fungal and mammalian oxidation of DMBA and that the overall balance of fungal metabolism is towards a detoxification rather than a bioactivation pathway.