2,7-Dihydro-3H-pyridazino[5,4,3-kl]acridin-3-one derivatives, novel type of cytotoxic agents active on multidrug-resistant cell lines. Synthesis and biological evaluation

We have earlier postulated that the presence of a pyridazone ring fused with an anthracenedione moiety resulted in the analog's ability to overcome multidrug resistance of tumor cells [J. Med. Chem.1999, 42, 3494]. High cytotoxic activity of obtained anthrapyridazones [Bioorg. Med. Chem.2003, 11, 561] toward the resistant cell lines, prompted us to synthesize the similarly modified acridine compounds. A series of pyridazinoacridin-3-one derivatives (2b-h) were prepared from the reaction of 9-oxo-9,10-dihydroacridine-1-carboxylate with POCl(3), followed by addition of the appropriate (alkylamino)alkylhydrazines. In vitro cytotoxic activity toward sensitive and resistant leukemia cell lines: L1210, K562, K562/DX, HL-60, HL-60/VINC, and HL-60/DX, with various type of multidrug resistance (MDR and MRP) was determined. The compounds studied exhibited in comparison to the reference cytostatics (DX, MIT) desirable very low resistance indexes (RI). Variations have been observed depending upon the substituent and the type of drug exporting pump. The cytotoxic activities of examined compounds, as well as of model anthrapyridazone derivative PDZ, were lower than those of reference drugs (DX, MIT) due to their diminished affinity to DNA.     

Characterization of an amsacrine-resistant line of human leukemia cells. Evidence for a drug-resistant form of topoisomerase II

HL-60/AMSA is a human leukemia cell line that is 100 times more resistant to the cytotoxic actions of the antineoplastic, topoisomerase II-reactive DNA intercalating acridine derivative amsacrine (m-AMSA) than is its parent HL-60 line. HL-60/AMSA cells are minimally resistant to etoposide, a topoisomerase II-reactive drug that does not intercalate. Previously we showed that HL-60 topoisomerase II activity in cells, nuclei, or nuclear extracts was sensitive to m-AMSA and etoposide, while HL-60/AMSA topoisomerase II was resistant to m-AMSA but sensitive to etoposide. Now we show that purified topoisomerase II from the two cell lines exhibits the same drug sensitivity or resistance as that in the nuclear extracts although the magnitude of the m-AMSA resistance of HL-60/AMSA topoisomerase II in vitro is not as great as the resistance of the intact HL-60/AMSA cells. In addition HL-60/AMSA cells are cross-resistant to topoisomerase II-reactive intercalators from the anthracycline and ellipticine families and the pattern of sensitivity or resistance to the cytotoxic actions of the various topoisomerase II-reactive drugs is paralleled by topoisomerase II-reactive drug-induced DNA cleavage and protein cross-link production in cells and the production of drug-induced, topoisomerase II-mediated DNA cleavage and protein cross-linking in isolated biochemical systems. In addition to its lowered sensitivity to intercalators, HL-60/AMSA differed from HL-60 in 1) the susceptibility of its topoisomerase II to stimulation of DNA topoisomerase II complex formation by ATP, 2) the catalytic activity of its topoisomerase II in an ionic environment chosen to reproduce the environment found within the living cell, and 3) the observed restriction enzyme pattern on a Southern blot probed with a cDNA for human topoisomerase II. These data indicate that an m-AMSA-resistant form of topoisomerase II contributes to the resistance of HL-60/AMSA to m-AMSA and to other topoisomerase II-reactive DNA intercalating agents. The drug resistance is associated with additional biochemical and molecular alterations that may be important determinants of cellular sensitivity or resistance to topoisomerase II-reactive drugs.     

Nitric oxide releasing acridone carboxamide derivatives as reverters of doxorubicin resistance in MCF7/Dx cancer cells

A series of nitric oxide donating acridone derivatives are synthesized and evaluated for in vitro cytotoxic activity against different sensitive and resistant cancer cell lines MCF7/Wt, MCF7/Mr (BCRP overexpression) and MCF7/Dx (P-gp expression). The results showed that NO-donating acridones are potent against both the sensitive and resistant cells. Structure activity relationship indicate that the nitric oxide donating moiety connected through a butyl chain at N¹⁰ position as well as morpholino moiety linkage through an amide bridge on the acridone ring system at C-2 position, are required to exert a good cytotoxic effect. Further, good correlations were observed when cytotoxic properties were compared with in vitro nitric oxide release rate, nitric oxide donating group potentiated the cytotoxic effect of the acridone derivatives. Exogenous release of nitric oxide by NO donating acridones enhanced the accumulation of doxorubicin in MCF7/Dx cell lines when it was coadministered with doxorubicin, which inhibited the efflux process of doxorubicin. In summary, a nitric oxide donating group can potentiate the anti-MDR property of acridones.     

Synthesis and hypoxic-cytotoxic activity of some 3-amino-1,2,4-benzotriazine-1,4-dioxide derivatives

A series of 3-amino-1,2,4-benzotriazine-1,4-dioxide derivatives 1 have been synthesized and evaluated for their cytotoxic activity in vitro against human leukemia cell lines: Molt-4, K562, HL60, human liver cancer cell Hep-G2, human prostate cancer cell PC-3 in hypoxia. Most of the compounds showed more potent activity than TPZ. Compounds 1i and 1m displayed encouraging superior activity against Molt-4 and HL-60 cell lines. Three potential derivatives received the test of the activity in hypoxia and in normoxia against Molt-4 and HL-60 cell lines and showed obvious hypoxia selectivity. Further mechanism study revealed that the cytotoxic activities of compounds 1i and 1k in Molt-4 cells might be mediated by modulation of p53 protein expression and mitochondrial membrane potential (DeltaPsi(m)).     

New derivatives of 11-methyl-6-[2-(dimethylamino)ethyl]-6H-indolo[2,3-b]quinoline as cytotoxic DNA topoisomerase II inhibitors

Novel indolo[2,3-b]quinoline derivatives substituted at N-6 and C-2 or C-9 positions with (dimethylamino)ethyl chains linked to heteroaromatic core by ether, amide or amine bonds, were manufactured and evaluated in vitro for their cytotoxic activity against several cell lines of different origin including multidrug resistant sublines and tested for their ability to influence the cell cycle and inhibit topoisomerase II activity. It was found, that all compounds show cytotoxic activity against cell lines tested, including multidrug resistant LoVo/DX, MES-SA/DX5 and HL-60 sublines. The tested compounds induce the G(2)M phase cell cycle arrest in Jurkat cells, and inhibit topoisomerase II activity.     

Evidence for the involvement of carbon-centered radicals in the induction of apoptotic cell death by artemisinin compounds

Artemisinin and its derivatives are currently recommended as first-line antimalarials in regions where Plasmodium falciparum is resistant to traditional drugs. The cytotoxic activity of these endoperoxides toward rapidly dividing human carcinoma cells and cell lines has been reported, and it is hypothesized that activation of the endoperoxide bridge by an iron(II) species, to form C-centered radicals, is essential for cytotoxicity. The studies described here have utilized artemisinin derivatives, dihydroartemisinin, 10beta-(p-bromophenoxy)dihydroartemisinin, and 10beta-(p-fluorophenoxy)dihydroartemisinin, to determine the chemistry of endoperoxide bridge activation to reactive intermediates responsible for initiating cell death and to elucidate the molecular mechanism of cell death. These studies have demonstrated the selective cytotoxic activity of the endoperoxides toward leukemia cell lines (HL-60 and Jurkat) over quiescent peripheral blood mononuclear cells. Deoxy-10beta-(p-fluorophenoxy)dihydroartemisinin, which lacks the endoperoxide bridge, was 50- and 130-fold less active in HL-60 and Jurkat cells, respectively, confirming the importance of this functional group for cytotoxicity. We have shown that chemical activation is responsible for cytotoxicity by using liquid chromatography-mass spectrometry analysis to monitor endoperoxide activation by measurement of a stable rearrangement product of endoperoxide-derived radicals, which was formed in sensitive HL-60 cells but not in insensitive peripheral blood mononuclear cells. In HL-60 cells the endoperoxides induce caspase-dependent apoptotic cell death characterized by concentration- and time-dependent mitochondrial membrane depolarization, activation of caspases-3 and -7, sub-G(0)/G(1) DNA formation, and attenuation by benzyloxycarbonyl-VAD-fluoromethyl ketone, a caspase inhibitor. Overall, these results indicate that endoperoxide-induced cell death is a consequence of activation of the endoperoxide bridge to radical species, which triggers caspase-dependent apoptosis.     

Cytotoxic activity of acridin-3,6-diyl dithiourea hydrochlorides in human leukemia line HL-60 and resistant subline HL-60/ADR

A series of acridin-3,6-diyl dithiourea hydrochloride derivatives (alkyl-AcrDTU) was prepared and tested against sensitive and drug resistant leukemia cell lines for their cytotoxic/cytostatic activity. The products (ethyl-, n-propyl-, n-butyl-, n-pentyl-AcrDTU) showed high DNA binding affinity via intercalation (K = 7.6 ? 2.9 × 10⁵ M^?1). All derivatives inhibited proliferation of HL-60 cells and its resistant subline HL-60/ADR, unexpectedly the resistant subline was more sensitive than the parental one (IC₅₀ = 3.5 μM, 48-treatment of HL-60/ADR with pentyl-AcrDTU). Cytotoxicity of tested compounds was associated with their DNA-binding properties and the level of intracellular thiols has been changed in the presence of AcrDTU.     

Cross-resistance of an amsacrine-resistant human leukemia line to topoisomerase II reactive DNA intercalating agents. Evidence for two topoisomerase II directed drug actions.

HL-60/AMSA is a human leukemia cell line that is 50-100-fold more resistant than its drug-sensitive HL-60 parent line to the cytotoxic actions of the DNA intercalator amsacrine (m-AMSA). HL-60/AMSA topoisomerase II is also resistant to the inhibitory actions of m-AMSA. HL-60/AMSA cells and topoisomerase II are cross-resistant to anthracycline and ellipticine intercalators but relatively sensitive to the nonintercalating topoisomerase II reactive epipodophyllotoxin etoposide. We now demonstrate that HL-60/AMSA and its topoisomerase II are cross-resistant to the DNA intercalators mitoxantrone and amonafide, thus strongly indicating that HL-60/AMSA and its topoisomerase II are resistant to topoisomerase II reactive intercalators but not to nonintercalators. At high concentrations, mitoxantrone and amonafide were also found to inhibit their own, m-AMSA's, and etoposide's abilities to stabilize topoisomerase II-DNA complexes. This appears to be due to the ability of these concentrations of mitoxantrone and amonafide to inhibit topoisomerase II mediated DNA strand passage at a point in the topoisomerization cycle prior to the acquisition of the enzyme-DNA configuration that yields DNA cleavage and topoisomerase II-DNA cross-links. In addition, amonafide can inhibit the cytotoxic actions of m-AMSA and etoposide. Taken together, these results suggest that the cytotoxicity of m-AMSA and etoposide is initiated primarily by the stabilization of the topoisomerase II-DNA complex. Other topoisomerase II reactive drugs may inhibit the enzyme at other steps in the topoisomerization cycle, particularly at elevated concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of 7-oxo-7H-naphtho[1,2,3-de]quinoline derivatives as potential anticancer agents active on multidrug resistant cell lines

Following our earlier finding that tetracyclic anthraquinone analogs with a fused pyridone ring exhibit cytotoxic activity toward multidrug resistant tumor cells, a series of new potential antitumor agents, 7-oxo-7H-naphtho[1,2,3-de]quinoline derivatives (3, 6-8, 10-12, 14, 15, and 18), bearing one or two basic side chains and various substituents at the pyridone ring, have been synthesized. The compounds have been obtained from 1-amino-4-chloroanthraquinone or 1-aminoanthraquinone by cyclization with diethyl malonate and the subsequent reactions of the key intermediates 2, 4, and 17. The compounds exhibited cytotoxic activity toward sensitive human leukemia cell line HL-60 and against its resistant sublines HL-60/VINC (MDR1 type) and HL-60/DX (MRP1 type).     

New benzo[g]isoquinoline-5,10-diones and dihydrothieno [2,3-b]naphtho-4,9-dione derivatives: synthesis and biological evaluation as potential antitumoral agents

Novel antitumoral agents with quinonic structure were synthesized and evaluated for their in vitro cytotoxic activities. This study examines the cytotoxic activities of several aryl benzo[g]isoquinoline-5,10-dione derivatives and a number of aminoacyl dihydrothieno[2,3-b]naphtho-4,9-dione (DTNQ) derivatives containing amino acids in position 3 of the ring system. Compound 6 showed remarkable cytotoxic activity at submicromolar concentration not only against several human leukaemia and solid tumour cell lines, but also toward sensitive and resistant human cell lines.     

DNA topoisomerase I as a site of action for 10-hydroxycamptothecin in human promyelocytic leukemia cells

We investigated the antiproliferative effect of 10-hydroxycamptothecin (HCPT), an alkaloid isolated from Camptotheca acuminata, on the human promyelocytic leukemia cell line, HL-60, and a 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA)-resistant mutant, HL-60/m-AMSA. Using trypan blue dye exclusion and colony formation, doses of HCPT ranging from 0.01 to 1 microM progressively inhibited growth in both cell lines in a concentration-dependent manner. A minimal cross-resistance, approximately five-fold, between the wild-type and resistant cells was observed. Using the technique of alkaline elution, HCPT produced DNA single-strand breaks and protein-associated DNA strand cleavage in HL-60 and HL-60/m-AMSA cells. Quantitative analysis of drug-induced protein-DNA complexes was performed using sodium dodecyl sulfate-potassium chloride precipitation. In both cell lines, a good correlation with HCPT-induced cytotoxicity was observed. Similar results were achieved in wild-type cells treated with m-AMSA. Enzyme activity was measured in nuclei isolated from HL-60 and HL-60/m-AMSA cells, and in each case HCPT inhibited topoisomerase I activity to the same extent. The data suggest that the principle mechanisms for HCPT-induced cytotoxicity in HL-60 and HL-60/m-AMSA cells are inhibition of DNA topoisomerase I and production of protein-associated DNA strand breaks.     

Synthesis, characterization and biological activity of lanthanum(III) complexes containing 2-methylene-1,10-phenanthroline units bridged by aliphatic diamines

Two novel lanthanum(III) complexes containing 2-methylene-1,10-phenanthroline units bridged by aliphatic diamines were synthesized and characterized by elemental analysis, IR, NMR, thermal analysis and conductance measurements. They have been assayed for anticancer activity in vitro against HL-60 (human leukocytoma) cells, PC-3MIE8 (human prostate carcinoma) cells, BGC-823 (human stomach carcinoma) cells, MDA-MB-435 (human galactophore carcinoma) cells, Bel-7402 (human liver carcinoma) cells, and Hela (human cervix carcinoma) cells. The results show that the two complexes exhibit good cytotoxic activities against different cell lines in general, especially more effective than cisplatin against Bel-7402, BGC-823 and MDA-MB-435 cell lines. DNA-binding studies indicate that, besides the intercalation, the complexes bind to DNA by the other interaction(s), which might be responsible for the production of more compact DNA, coinciding with more A-like feature of DNA as suggested by CD spectra.     

Novel cisplatin-type platinum complexes and their cytotoxic activity

A series of novel cisplatin-type platinum complexes, formulated as [PtA2(OCOCH2OR)2] (A2 = two monoamines or one diamine, R is an alkyl group), were designed, characteristic of alkoxyacetate as carboxylato ligands. The pertinent compounds were prepared and characterized by elementary analyses, IR, 1H NMR, and ESI-MS spectra. The cytotoxic activities of compound 1a in vitro toward HL-60 human leukemia and BEL-7402 human hepatocellular carcinoma cell lines were pioneeringly studied. Then, compounds 1b-3d were evaluated for their in vitro cytotoxicity against Ramos human lymphoma, 3AO human ovarian carcinoma, and A549 human non-small cell lung cancer cell lines. Most of them showed better cytotoxic activity than carboplatin against above selected cell lines.     

Platinum complexes of diaminocarboxylic acids and their ethyl ester derivatives: the effect of the chelate ring size on antitumor activity and interactions with GMP and DNA

A number of new Pt(II) complexes is described having the general formula PtCl(2)(LL), where LL is a chelating diamine ligand. Ligands LL were chosen as D,L-2,3-diaminopropionic acid and its ethyl ester, and D,L-2,4-diaminobutyric acid and its ethyl ester. The compounds were characterized using analytical and spectroscopic methods. The influence of the size of the chelate ring and its functionalization on the biological properties was studied. It was demonstrated by circular dichroism (CD) that the effects on the secondary structure of DNA induced by the four complexes are different. The interaction takes place at the N7 position of the purine bases, as shown by NMR studies. The platinum complexes of 2,3-diaminopropionic acid and 2,4-diaminobutyric acid are able to form intrastrand adducts with DNA and to distort the double helix by changing the base stacking. The ethyl ester derivatives uncoil the DNA from the B form to the C form. The interactions with 5'-GMP and DNA were compared with their antitumor activity. The platinum complexes of diaminocarboxylic acids exhibit cytotoxic activity in the A431, HeLa, and HL-60 cell lines in a dose- and time-dependent manner.     

Cellular resistance against troxacitabine in human cell lines and pediatric patient acute myeloid leukemia blast cells

Troxacitabine is a cytotoxic deoxycytidine analogue with an unnatural L-configuration, which is activated by deoxycytidine kinase (dCK). The configuration is responsible for differences in the uptake and metabolism of troxacitabine compared to other deoxynucleoside analogues. To determine whether troxacitabine has an advantage over other nucleoside analogues several cell lines resistant to cladribine and gemcitabine were exposed to troxacitabine, while blast cells from pediatric leukemia patients were tested for cross-resistance with other deoxynucleoside analogues. The gemcitabine resistant AG6000 (IC50: >3000 nM), and the cladribine resistant CEM (IC50: 150 nM) and HL-60 (IC50: >3000 nM) cell lines, all with no or decreased dCK expression, were less sensitive to troxacitabine than their wild type counterparts (IC50; A2780: 410, CEM: 71 and HL-60: 158 nM). dCK protein expression in CEM was higher than in HL-60, which, in turn, was higher than in A2780. Catalytically inactive p53 seems to increase the sensitivity to troxacitabine. The patient samples showed a large range of sensitivity to troxacitabine, similar to other deoxynucleoside analogues. Cross-resistance with all other deoxynucleoside analogues was observed.     

Synthesis and cytotoxic activity of novel derivatives of 4'-demethylepipodophyllotoxin

Nine novel 4beta-N-substituted-5-FU-4'-demethylepipodophyllotoxin derivatives were synthesized and evaluated as potential antitumor agents. All of the target compounds showed more significant cytotoxic activity against HL-60 and A-549 in vitro than VP-16 and 5-FU. Among them, 4beta-N-substituted-phenylalanine 5-Fu pentyl ester-4'-demethylepipodophyllotoxin (9 g) was found to exhibit most potent cytotoxic activity against HL-60 and A-549 cell (IC50 is 0.04 and <0.01 microM, respectively).     

Proteomic analysis on multi-drug resistant cells HL-60/DOX of acute myeloblastic leukemia

Multi-drug resistance (MDR) is an important factor that causes treatment failure in acute leukemia. However, the full development mechanisms of MDR still await [corrected] investigation. The purpose of this study is to investigate differentially expressed proteins in the multi-drug resistant acute myeloblastic leukemia (AML) cell line HL-60/DOX and the drug sensitive cell line HL-60, and to identify new potential multi-drug resistant related molecules with the proteomic approach. Two-dimensional gel electrophoresis (2-DE) maps of the proteins, extracted from two AML cell lines, HL-60/DOX and HL-60, were established respectively. The extracted proteins were digested by enzymes and identified with the matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The data of the peptide mass fingerprinting (PMF) was matched with databases of proteomics available on the Internet. Results showed that 16 proteins were identified to be differentially expressed between HL-60/DOX and HL-60 cells. They involved the protein disulfide isomerase precursor (PDI), the proteasomes alpha1 and other proteins which are related to drug resistance or cell metabolism, but their functional significances are required further investigation. Nevertheless, it is clear that this proteomic approach for studing the biology and development of MDR is a prerequisite in leukemia.     

Potential new antitumor agents from an innovative combination of camphorato, a ramification of traditional Chinese medicine, with a platinum moiety

Eight new camphorato platinum complexes have been synthesized and evaluated for their in vitro cytotoxicity against HL-60 human leukemia, 3AO human ovarian carcinoma, BEL-7402 human hepatocarcinoma, and A549 human lung carcinoma cell lines. Most complexes showed good cytotoxic activity against the above-selected cell lines. Among the complexes, two compounds were assayed for their in vivo antitumor activity against LS-174T human colon carcinoma cells implanted in mice. One complex exhibited not only higher in vivo antitumor activity, but also less toxicity than oxaliplatin when it was administered intravenously at a dose of 6 mg/kg three times.     

Synthesis and biological evaluation of novel conjugates of podophyllotoxin and 5-FU as antineoplastic agents

A series of novel conjugates of podophyllotoxin and 5-FU were designed using association strategy and were synthesized by coupling 4′-demethylepipodophyllotoxin with 5-FU-N¹-alkyl amino acid ester. These derivatives have been evaluated for cytotoxicity in vitro against tumour cell lines (HL-60, K562, A-549 and AGS), and their octanol–water partition coefficients (log P) were also determined. As compared with VP-16, most compounds showed superior water solubility, as well as more potent inhibitions against these four tumour cell lines. Compound 21 showed interaction with calf thymus DNA, and it was relatively resistant to metabolism by human plasma.     

Digital morphonuclear analyses of sensitive versus resistant neoplastic cells to vinca-alkaloid, alkylating, and intercalating drugs

We tested 12 resistant cell lines in vitro in order to evaluate common morphonuclear characteristics induced by various cytotoxic drugs on cell lines of different origins. We used the MXT mouse mammary cancer and the neoplastic J82 and T24 human bladder cell lines, whose variants are either sensitive or resistant to a vinca alkaloid derivative (Navelbine, NVB), to an investigational alkylating agent (PE1001), and to Adriamycin (ADR). We tested cell population variants resistant to NVB + PE1001 + ADR. The level of chemoresistance was evaluated by a colorimetric assay assessing the 50% concentration-induced inhibition of cellular growth (IC50) brought about by each drug on the growth of each cell variant under study. We show that resistant neoplastic cell nuclei present common morphonuclear characteristics, independent of cell origin (neoplastic mouse mammary versus human bladder cells) and the drug used (vinca alkaloid, alkylating, and intercalating derivatives). Our results further indicate that the phenotype of resistant versus sensitive cells corresponds to cell nuclei populations with smaller nuclei and less nuclear DNA content and, as a consequence, a chromatin texture showing large pale areas with some hyperchromatic clumps.