RESUMO
Selective recognition of DNA folding is central to multiple biological and pharmacological applications aimed at precise targeting of distinct genomic regions. Here, we focused on the recognition of physiologically relevant G-quadruplex (G-4) structures by bis-phenanthroline (bis-Phen) ligands containing two Phen moieties covalently linked through an amine or thioether bond. The transition metal ions Mn(2+), Ni(2+), Cu(2+), and the biologically relevant Mg(2+) and Zn(2+) efficiently form 1 : 1 bis-Phen complexes characterised by a large planar structure fit to successfully recognise G-quartet arrangements.Interestingly, metal ion complexation dramatically affects ligand-stabilising effects on G-quadruplex, the melting temperature of the folded structure being increased up to 30 degrees C at ligand concentrations as low as 1 microM in the presence of Ni(2+) and Cu(2+). In addition, the test complexes were able to induce G-4 formation from essentially unfolded G-rich sequences even in the absence of K(+) ions as shown by gel shift and circular dichroism experiments. In line with their G-4 stabilising properties bis-Phen complexes are effective inhibitors of telomerase activity, Ni(II) complexes being effective in the sub-micromolar range. This is combined with lack of unselective DNA-damaging activity and short-term cellular toxicity, which makes the novel compounds (above all their Ni(II) complexes) interesting antiproliferative drug leads.
Assuntos
Quadruplex G , Compostos Organometálicos/química , Fenantrolinas/química , Dicroísmo Circular , DNA/efeitos dos fármacos , DNA/metabolismo , Clivagem do DNA/efeitos dos fármacos , Humanos , Ligantes , Metais/química , Estrutura Molecular , Compostos Organometálicos/síntese química , Fenantrolinas/síntese química , Fenantrolinas/farmacologia , Telomerase/antagonistas & inibidores , Telomerase/química , Telomerase/metabolismoRESUMO
The physicochemical and DNA-binding properties of anticancer 9-aza-anthrapyrazoles (9-aza-APs) were investigated and compared with the carbocyclic analogs losoxantrone (LX) and mitoxantrone (MX). Unlike their carbocyclic counterparts, the tested 9-aza-APs do not undergo self-aggregation phenomena. The pyridine nitrogen at position 9, missing in the carbocyclic derivatives, is involved in protonation equilibria at physiological pH. In addition, 9-aza-APs are electrochemically reduced at a potential intermediate between LX and MX. These data fully agree with quantum mechanical calculations. Binding to nucleic acids was examined by spectroscopic, chiroptical, and DNase I footprinting techniques as a function of ionic strength and base composition. The 9-aza-APs exhibit prominent affinity for DNA, with an important electrostatic contribution to the binding free energy. A very remarkable sequence preference pattern dramatically favors GC steps in double-helical DNA, whereas the carbocyclic reference compounds show a substantially lower selectivity for GC. A common DNA complexation geometry, considerably differing from that of MX, characterizes all anthrapyrazoles. Hence, bioisosteric substitution and ring-hydroxy deletion play an important role in defining the physicochemical properties and in modulating the affinity of anthrapyrazoles for the nucleic acid, the geometry of the intercalation complex, and the sequence specific contacts along the DNA chain. Drug stimulation of topoisomerase II-mediated DNA cleavage is remarkably attenuated in the aza-bioisosteric derivatives, suggesting that other non-enzyme-mediated cytotoxic mechanism(s), possibly connected with free radical production, are responsible for efficient cell killing. The biophysical and biochemical properties exhibited by 9-aza-APs contribute to clarifying the peculiar pharmacological profile of this family of compounds.
Assuntos
Antraquinonas/farmacologia , Antineoplásicos/farmacologia , DNA/efeitos dos fármacos , Mitoxantrona/farmacologia , Pirazóis/farmacologia , Pirazolonas , Animais , Sequência de Bases , Sítios de Ligação , Bovinos , Clostridium perfringens/genética , DNA/metabolismo , Pegada de DNA , DNA Topoisomerases Tipo II/metabolismo , DNA Viral/efeitos dos fármacos , DNA Viral/metabolismo , Desoxirribonuclease I/metabolismo , Fluorometria , Substâncias Intercalantes , Micrococcus/genética , Modelos Moleculares , Dados de Sequência Molecular , Oxirredução , Plasmídeos/genética , Timo/químicaRESUMO
The synthesis of 1- and 2-substituted aza-benzothiopyranoindazoles has been accomplished. The comparisons of the in vitro antitumor activities of the 2-substituted analogues with the benzothiopyranoindazole chemotypes indicate that the positioning of the nitrogen atom at C-9 (9-aza analogue 4d) leads to a substrate with potent antitumor activity. The 1-substituted aza-benzothiopyranoindazoles, in comparison with the corresponding 2-substituted analogues, exhibit a much lower potency.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Indazóis/síntese química , Indazóis/farmacologia , Animais , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Espectroscopia de Ressonância Magnética , Camundongos , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
The DNA-binding properties of a series of 2-aza-anthracenedione (benz[g]isoquinoline-5,10-dione) derivatives bearing two 3-dimethylaminopropylamino side chains at different (6,9, 7,9 and 8,9) positions of the planar ring system have been investigated. The affinity for the nucleic acid is dramatically affected by the substitution pattern, the 6,9-regioisomer being substantially more effective than the 7,9- or the 8,9-congeners. This cannot be ascribed to different binding mechanisms, as all compounds are shown to intercalate into the double helix. Instead, the geometry of intercalation into DNA and the site specificity are extensively affected by the substitution pattern. The site preference is CA (or AC) for the 6,9-regioisomer, whereas it is TA (or AT) for the 8,9-congener, the 7,9-analogue lying in between. Molecular modeling studies are in agreement with the experimental results. Although the 6,9-regioisomer was remarkably cytotoxic, it stimulated topoisomerase II-mediated cleavage of DNA very poorly. Hence, a different mechanism of DNA damage is probably operating in 2-aza-anthracenediones as the main cell-killing event. Changes in affinity for DNA, intercalation geometry and sequence specificity can explain the different cytotoxic responses exhibited by the test drugs.
Assuntos
Antraquinonas/metabolismo , DNA/metabolismo , Animais , Antraquinonas/química , Antraquinonas/farmacologia , Compostos Aza/química , Compostos Aza/metabolismo , Compostos Aza/farmacologia , Bovinos , DNA/química , DNA Super-Helicoidal/metabolismo , Humanos , Substâncias Intercalantes/química , Substâncias Intercalantes/metabolismo , Substâncias Intercalantes/farmacologia , Modelos Moleculares , Espectrofotometria , Estereoisomerismo , Relação Estrutura-Atividade , Especificidade por Substrato , Inibidores da Topoisomerase IIRESUMO
The synthesis and antitumor evaluation of 2, 5-disubstituted-indazolo[4,3-gh]isoquinolin-6(2H)-ones (9-aza-APs) are described. The key intermediates in the synthesis are benz[g]isoquinoline-5,10-diones which are substituted at positions 6 and 9 with groups of different nucleofugacity for SNAr displacements. The initial displacement of fluoride by a substituted hydrazine leads to the pyrazole analogues. Substitution of the remaining leaving group by an amine or BOC-protected amines leads to the 9-aza-APs 12. These analogues were converted into their maleate or hydrochloride salts 13. In two cases, namely, 13x and 13z, sidearm buildup was also employed in the synthetic pathway. In vitro evaluation of 9-aza-APs against the human colon tumor cell line LoVo uncovered for most of the compounds a cytotoxic potency lower than that of DuP-941 or mitoxantrone and comparable to that of doxorubicin. Only analogues 13c, 13n, and 13ff were as cytotoxic as DuP-941. Interestingly, while DuP-941 was highly cross-resistant in the LoVo cell line resistant to doxorubicin (LoVo/Dx), the 9-aza-APs carrying a distal lipophilic tertiary amine moiety in both chains were capable of overcoming the MDR resistance induced in this cell line. The 9-aza-APs show outstanding in vivo antitumor activity against both systemic P388 murine leukemia and MX-1 human mammary carcinoma transplanted in nude mice. At their optimal dosages, congeners 13a-c, 13f, 13n, 13q, 13x, and 13dd were highly effective against P388 leukemia with T/C% of 200-381, while the T/C% value of DuP-941 was 147. In the MX-1 tumor model, 24 compounds elicited percentages of tumor weight inhibitions (TWI) ranging from 50% to 99%. Congeners 13d, 13k, 13l, 13x, 13z, and 13ee emerged as the most effective ones, with TWI% 96, simliar to that of DuP-941 (TWI% = 95). On the basis of their efficacy profile in additional experimental tumors and lack of cardiotoxicity in preclinical models, two congeners have surfaced as potential clinical candidates.
Assuntos
Antineoplásicos/síntese química , Indazóis/síntese química , Isoquinolinas/síntese química , Adenocarcinoma/patologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Neoplasias do Colo/patologia , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Indazóis/química , Indazóis/farmacologia , Concentração Inibidora 50 , Isoquinolinas/química , Isoquinolinas/farmacologia , Leucemia P388/tratamento farmacológico , Leucemia P388/patologia , Masculino , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Nus , Transplante de Neoplasias , Células Tumorais CultivadasRESUMO
The physicochemical, cytotoxic and pharmacological properties of 2,3-diaza-anthracenedione derivatives were examined to gain insight into the structure-activity relationships in this class of compounds. Spectrophotometric, chiroptical and voltammetric measurements were performed, along with cell cytotoxicity, alkaline elution, topoisomerase II-mediated DNA cleavage and cellular drug-uptake determination. In comparison with classic anthracenediones such as mitoxantrone and ametantrone, the aza derivatives were characterized by less negative reduction potentials, lower affinity for DNA and modified geometry of intercalation. The biological effects of the new compounds were also profoundly affected by bioisosteric N for C replacement. Stimulation of topoisomerase II-mediated DNA cleavage was not observed, whereas other mechanisms of cell cytotoxicity, possibly involving oxidative DNA damage appeared to be operative. The inability to generate protein-associated strand breaks could be explained by an unfavorable orientation of the drug in the intercalation complex rather than by a reduced binding to DNA. Geometry of drug intercalation may have a critical influence on the formation of the ternary complex. In turn, the onset of a different DNA-damaging pathway is likely to be related to easy redox cycling of the 2,3-diaza-substituted anthracenedione derivatives, which could produce radical species to a remarkably greater extent than could the carbocyclic parent drugs.
Assuntos
Antraquinonas/farmacologia , Antineoplásicos/farmacologia , Inibidores da Topoisomerase II , Antraquinonas/química , Antraquinonas/metabolismo , DNA/metabolismo , Dano ao DNA , Humanos , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Aza-anthracenediones are a new class of anti-cancer drugs, which demonstrate promising in vitro and in vivo activity. Our laboratory has synthesized a variety of structural analogs in which we determined previously that the positioning of the nitrogen within the backbone, as well as sidearm modification, results in dramatic differences in the potency of cytotoxicity. We reported previously that although DNA reactivity appears to be a necessary component for mediating cell death, it is not sufficient for predicting cytotoxicity of the aza-anthracenediones. We have chosen three aza-anthracenediones (BBR 2828, BBR 2778 and BBR 2378) to investigate the importance of DNA strand breaks and/or protein-concealed DNA breaks induced by aza-anthracenediones. We determined in the present study that, while all three drugs cause DNA breaks as determined by alkaline and neutral elution, as well as KCl-SDS precipitation, these breaks do not correlate directly with their potency as cytotoxic compounds. Further, we found significant differences in the types of DNA breaks induced by these drugs. Finally, we report that the persistence of protein-DNA complexes induced by all three drugs was similar and, therefore, cannot account for differences in the potency of cytotoxicity of the aza-anthracenediones. Thus, we postulate that, while the total number of drug-induced protein-concealed DNA breaks is an important indicator of drug toxicity, it is possible that the actual nature of the breaks may differ among the aza-anthracenedione congeners, and it is these differences in the actual proteins present in the DNA breaks that differentiate between aza-anthracenediones.
Assuntos
Antraquinonas/farmacologia , Antineoplásicos/farmacologia , Dano ao DNA , Isoquinolinas/farmacologia , Animais , Divisão Celular/efeitos dos fármacos , DNA de Cadeia Simples/efeitos dos fármacos , DNA de Cadeia Simples/metabolismo , Resistência a Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Leucemia L1210 , Mitoxantrona/farmacologia , Proteínas/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
The cytotoxic activity of mitoxantrone and related anthracenediones has been ascribed to the ability of these compounds to interfere with DNA topoisomerase II function, resulting in DNA cleavage stimulation. The molecular details of enzyme inhibition by these intercalating agents remain to be defined. In an attempt to identify the structural determinants for optimal activity, the molecular and cellular effects of a series of heteroanalogues bearing different side-chains were examined in relation to the physico-chemical and DNA binding properties of these compounds. The results indicated that substitution of a pyridine ring for the dihydroxyphenylene ring in the planar chromophore caused a marked reduction of cytotoxic activity and of the ability to stimulate topoisomerase II-mediated DNA damage in intact cells and with simian virus 40 DNA in vitro. Although all tested derivatives were shown to intercalate into DNA, their DNA binding affinities were appreciably lower than that of mitoxantrone. The behavior of 2-aza derivatives more closely resembled that of ametantrone, suggesting that the potency of agents of this class is influenced more by the presence of hydroxyl groups than by the phenylene ring. The observation that a dramatic reduction (or loss) of the ability of aza derivatives to stimulate DNA cleavage is associated with a marked reduction of cytotoxic potency supports a primary role of topoisomerase II-mediated effects in the mechanism of action of the effective agents of this class. Because appreciable cytotoxic activity and significant in vivo antitumor efficacy are retained by compounds inactive (or poorly active) in inhibition of topoisomerase II, these results are consistent with multiple effects of anthracenediones at the cellular level.
Assuntos
Antineoplásicos/farmacologia , Óxidos N-Cíclicos/farmacologia , DNA Topoisomerases Tipo II/metabolismo , DNA/metabolismo , Isoquinolinas/farmacologia , Antineoplásicos/química , Óxidos N-Cíclicos/química , DNA de Cadeia Simples/efeitos dos fármacos , DNA Viral/metabolismo , Humanos , Hidrólise , Isoquinolinas/química , Oxirredução , Ligação Proteica , Vírus 40 dos Símios/genética , Estereoisomerismo , Termodinâmica , Células Tumorais CultivadasRESUMO
Doxorubicin and mitoxantrone are carboxyclic anti-cancer drugs that interact with DNA through intercalation. Our laboratory has synthesized a new series of anti-tumor agents, the aza-anthracenediones, which are structurally related to mitoxantrone but contain a heterocyclic, rather than a carbocyclic, chromophore. Both the in vivo and in vitro anti-tumor activities of these compounds were exquisitely sensitive to the positioning of the nitrogen atom within the heterocyclic backbone. Compounds having a 2-aza were 30- to 100-fold more potent than the 1-aza or the di-aza compounds against L1210 cells in vitro. When tested in vivo, the 2-aza-anthracenediones had marked anti-tumor activity, in some cases curative, whereas the 1-aza-anthracenediones had but minimal antitumor activity. To define the importance of the aza positioning on DNA reactivity, spectral shift and gel mobility assays were used. The spectral shift assay suggested that the 2-aza compounds reacted with DNA solely through intercalation whereas the 1-aza-anthracenediones, and mitoxantrone all reacted with DNA through intercalative and non-intercalative processes. The affinity of DNA binding was five to seven times greater for the 2-aza compounds compared to the 1-aza or the di-aza derivatives. The retardation of supercoiled pBR322 DNA mobility in agarose gel electrophoresis further suggested an intercalative type of DNA interaction. Differences in DNA interaction appear related to but can not completely account for differences in cytotoxicity of the aza anthracenediones.
Assuntos
Antraquinonas/uso terapêutico , Antineoplásicos/farmacologia , Substâncias Intercalantes , Leucemia L1210/tratamento farmacológico , Animais , Antraquinonas/química , Antraquinonas/farmacologia , Sítios de Ligação , DNA de Neoplasias/metabolismo , Compostos Heterocíclicos , Leucemia L1210/mortalidade , Masculino , Camundongos , Mitoxantrona/metabolismo , Mitoxantrona/uso terapêutico , Células Tumorais CultivadasRESUMO
Synthetic routes to aza and diaza bioisosteres related to the anthracene-9,10-dione, mitoxantrone, have been developed. The antitumor properties of these chemotypes are compared with those exhibited by the corresponding carbocyclic analogues. The sensitivity of the expressed cytotoxicities on the position(s) of the nitrogen atom(s) are discussed in terms of potential cellular targets. Several analogues show potential for clinical evaluations.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Mitoxantrona/análogos & derivados , Animais , Humanos , Mitoxantrona/síntese química , Mitoxantrona/farmacologia , Células Tumorais CultivadasRESUMO
Synthetic procedures have been developed which lead to the 2-aza congeners 3 and several related N-oxides 4. The analogues 3 exhibited a wide range of in vitro cytotoxicity against L1210 leukemia, the human colon adenocarcinoma cell line LoVo, and the doxorubicin resistant LoVo/DX cell line. Selected analogues of 3 showed significant P388 antileukemic activity in mice with 3c exhibiting high activity. This activity was also retained in the related N-oxide 4a. These heterocyclic bioisosteric models are representative of the first anthracene-9,10-diones which display antileukemic activity comparable to mitoxantrone.
Assuntos
Antraquinonas/síntese química , Antraquinonas/uso terapêutico , Antineoplásicos/síntese química , Isoquinolinas/síntese química , Isoquinolinas/uso terapêutico , Células Tumorais Cultivadas/efeitos dos fármacos , Adenocarcinoma/tratamento farmacológico , Animais , Antineoplásicos/uso terapêutico , Neoplasias do Colo/tratamento farmacológico , Humanos , Leucemia L1210/tratamento farmacológico , Leucemia P388/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos DBA , Mitoxantrona/análogos & derivados , Mitoxantrona/uso terapêutico , Relação Estrutura-AtividadeRESUMO
Mitoxantrone, a DNA intercalator, is an effective antitumor drug known to interfere with topoisomerase II function through stimulation of enzyme-mediated DNA cleavage. To clarify the drug structural requirements for stimulation of topoisomerase II DNA cleavage, the cytotoxic activity and molecular effects of mitoxantrone, ametantrone, and a new derivative (BBR2577), bearing a modification on one of the side chains, were examined in relation to their DNA binding affinities and modes of drug-DNA interaction. The results showed a good correlation between cytotoxicity and topoisomerase II DNA cleavage. The modification of one side chain did not influence the cytotoxic potency or the ability of the drug to stimulate DNA cleavage. In contrast, removal of the hydroxyl substituents in the planar aromatic moiety (ametantrone) markedly affected the efficacy of the drug. Ametantrone showed a markedly lower capacity, compared with the other two compounds, to induce cleavable complexes both in intact cells and in SV40 DNA, which suggests a critical role of these substituents in the formation of the ternary topoisomerase II-DNA-drug complex. The poor efficacy of ametantrone is likely due to low stability of the ternary complex. This is possibly related to a different orientation of the drug chromophore intercalated into DNA, compared with those of mitoxantrone and BBR2577. The DNA cleavage efficiencies of the tested drugs at low concentrations correlated with the DNA binding affinity. Identical DNA cleavage patterns were observed with the three compounds, which suggests that all tested drugs share a similar specificity for interaction with sites recognized by the enzyme.
Assuntos
DNA Topoisomerases Tipo II/metabolismo , Substâncias Intercalantes/farmacologia , Mitoxantrona/análogos & derivados , Mitoxantrona/farmacologia , Animais , Carcinoma de Células Pequenas/genética , DNA/efeitos dos fármacos , DNA/metabolismo , Dano ao DNA , DNA Topoisomerases Tipo II/efeitos dos fármacos , DNA de Neoplasias/metabolismo , DNA Viral/metabolismo , Humanos , Substâncias Intercalantes/metabolismo , Neoplasias Pulmonares/genética , Camundongos , Mitoxantrona/metabolismo , Vírus 40 dos Símios/genética , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
The ipso bis displacements of fluoride from 1,4-difluoroanthracene-9,10-dione (3) and 1,4-difluoro-5,8-dihydroxyanthracene-9,10-dione (4) by excess of a diamine (or a monoamine) in pyridine at room temperature lead to the symmetrically substituted 1,4-bis-substituted analogues 5 and 6, respectively. The ipso monodisplacements of fluoride from 3 and 4 can be accomplished by treatment with less than 1 molar equiv of a diamine (or a monoamine) to yield 7 and 8, respectively. Treatment of 7 or 8 with a different diamine leads to the unsymmetrically substituted 1,4-bis[(aminoalkyl)amino]anthracene-9,10-diones 9 and 10, respectively. Many of the synthetic unsymmetrical analogues have been evaluated for their antitumor activity against L1210 in vitro and in vivo. Cross resistance of analogue 10a with mitoxantrone (2) and doxorubicin was evaluated against MDR lines in vitro against human colon carcinoma LOVO and its subline resistant to DOXO (LOVO/DOXO). Potential mechanisms for the observed cytotoxicity are presented and discussed.
Assuntos
Antraquinonas/síntese química , Antineoplásicos/síntese química , Mitoxantrona/análogos & derivados , Animais , Antraquinonas/uso terapêutico , Antineoplásicos/uso terapêutico , Fenômenos Químicos , Química , Neoplasias do Colo/tratamento farmacológico , Humanos , Leucemia L1210/tratamento farmacológico , Camundongos , Estrutura Molecular , Células Tumorais CultivadasRESUMO
A number of 5,8-bis[(aminoalkyl)amino]naphtho[2,3-b]thiophene-4, 9-diones have been synthesized and evaluated for antitumor activity against L1210 leukemia both in vitro and in vivo. Two of the congeners exhibited in vivo activities quite comparable to that of mitoxantrone.
Assuntos
Antineoplásicos/síntese química , Naftalenos/síntese química , Tiofenos/síntese química , Animais , Antineoplásicos/uso terapêutico , Fenômenos Químicos , Química , Leucemia L1210/tratamento farmacológico , Camundongos , Naftalenos/uso terapêutico , Relação Estrutura-Atividade , Tiofenos/uso terapêuticoRESUMO
A number of unsymmetrically substituted 1,4-bis[(aminoalkyl)amino]anthracene-9,10-diones have been synthesized and evaluated for their antitumor activity against L1210 in vitro and in vivo. The high activity of several compounds observed in vitro was not paralleled by comparable activity in vivo. The activities of the substituted 1,4-bis[(aminoalkyl)amino]anthracene-9,10-diones as inhibitors of cell growth were generally much higher than those of the related 1-[(aminoalkyl)amino]-4-methoxyanthracene-9,10-diones, and this correlated with the relative abilities of compounds of the two types to interact with calf thymus DNA.
Assuntos
Antracenos/síntese química , Antineoplásicos/uso terapêutico , Animais , Antineoplásicos/síntese química , Bovinos , Divisão Celular/efeitos dos fármacos , DNA/metabolismo , Leucemia L1210/tratamento farmacológico , CamundongosRESUMO
Several 5,8-bis[(aminoalkyl)amino]-1-azaanthracene-9,10-diones have been synthesized and evaluated for antitumor activity against L1210 leukemia both in vitro and in vivo. Comparisons are made to the corresponding carbocyclic analogues. One of the aza analogues showed modest in vivo activity.