RESUMO
Replacing one of the morpholine groups of the phosphatidylinositol 3-kinase (PI3K) inhibitor ZSTK474 with a variety of sulfonamide-linked solubilizing substituents produced a new class of active and potent PI3Kα inhibitors, with several derivatives demonstrating high PI3Kα enzyme potency and good cellular potency in two human derived cell lines. The overall results suggest a preference for linear and somewhat flexible solubilizing functions. From this series, compound 16, also known as SN32976, was selected for advanced preclinical evaluation.
Assuntos
Fosfatidilinositol 3-Quinases/química , Inibidores de Fosfoinositídeo-3 Quinase/síntese química , Sulfonamidas/química , Triazinas/química , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Concentração Inibidora 50 , Camundongos , Neoplasias/tratamento farmacológico , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase/uso terapêutico , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/metabolismo , Relação Estrutura-Atividade , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Transplante HeterólogoRESUMO
Replacement of one of the morpholine groups of the phosphatidylinositol 3-kinase (PI3K) inhibitor ZSTK474 (1) with sulfonamide containing substituents produced a new class of active and potent PI3Kα inhibitors. Solubility issues prevented all but the 6-amino derivative 17 from being evaluated in vivo, but the clear activity of this compound demonstrated that this class of PI3K inhibitor shows great promise.
Assuntos
Inibidores de Fosfoinositídeo-3 Quinase , Sulfonamidas/síntese química , Sulfonamidas/farmacologia , Triazinas/química , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , Humanos , Estrutura Molecular , Solubilidade , Sulfonamidas/química , Triazinas/farmacologiaRESUMO
The emergence of extensively drug-resistant strains of Mycobacterium tuberculosis (Mtb) highlights the need for new therapeutics to treat tuberculosis. We are attempting to fast-track a targeted approach to drug design by generating analogues of a validated hit from molecular library screening that shares its chemical scaffold with a current therapeutic, the anti-arthritic drug Lobenzarit (LBZ). Our target, anthranilate phosphoribosyltransferase (AnPRT), is an enzyme from the tryptophan biosynthetic pathway in Mtb. A bifurcated hydrogen bond was found to be a key feature of the LBZ-like chemical scaffold and critical for enzyme inhibition. We have determined crystal structures of compounds in complex with the enzyme that indicate that the bifurcated hydrogen bond assists in orientating compounds in the correct conformation to interact with key residues in the substrate-binding tunnel of Mtb-AnPRT. Characterising the inhibitory potency of the hit and its analogues in different ways proved useful, due to the multiple substrates and substrate binding sites of this enzyme. Binding in a site other than the catalytic site was found to be associated with partial inhibition. An analogue, 2-(2-5-methylcarboxyphenylamino)-3-methylbenzoic acid, that bound at the catalytic site and caused complete, rather than partial, inhibition of enzyme activity was found. Therefore, we designed and synthesised an extended version of the scaffold on the basis of this observation. The resultant compound, 2,6-bis-(2-carboxyphenylamino)benzoate, is a 40-fold more potent inhibitor of the enzyme than the original hit and provides direction for further structure-based drug design.
Assuntos
Antituberculosos/química , Mycobacterium tuberculosis/enzimologia , Triptofano/biossíntese , ortoaminobenzoatos/química , Antranilato Fosforribosiltransferase/antagonistas & inibidores , Antranilato Fosforribosiltransferase/metabolismo , Antituberculosos/metabolismo , Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Relação Estrutura-Atividade , ortoaminobenzoatos/metabolismo , ortoaminobenzoatos/farmacologiaRESUMO
A series of 4-anilinoquinoline derivatives related to the known inhibitor SGI-1027, containing side chains of varying pK(a), were prepared by acid-catalysed coupling of the pre-formed side chains with 4-chloroquinolines. The compounds were evaluated for their ability to reduce the level of DNMT1 protein in HCT116 human colon carcinoma cells by Western blotting. With a very strongly basic N-methylpyridinium side chain, only NHCO-linked compounds were effective, whereas less strongly basic ((diaminomethylene)hydrazono)ethyl or 3-methylpyrimidine-2,4-diamine side chains allowed both NHCO- and CONH-linked compounds to show activity. In contrast, the pK(a) of the quinoline unit had little apparent influence on activity.
Assuntos
Compostos de Anilina/síntese química , Antineoplásicos/síntese química , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Proteínas de Neoplasias/antagonistas & inibidores , Quinolinas/síntese química , Relação Estrutura-Atividade , Compostos de Anilina/química , Antineoplásicos/química , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferases/genética , Expressão Gênica/efeitos dos fármacos , Células HCT116 , Humanos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Quinolinas/químicaRESUMO
A series of aliphatic esters of the non-opioid anaesthetic/analgesic ketamine were prepared and their properties as shorter-acting analogues of ketamine itself were explored in an infused rat model, measuring the time after infusion to recover from both the anaesthetic (righting reflex) and analgesic (response to stimulus) effects. The potency of the esters as sedatives was not significantly related to chain length, but Me, Et and i-Pr esters were the more dose potent (up to twofold less than ketamine), whereas n-Pr esters were less potent (from 2- to 6-fold less than ketamine). For the Me, Et and i-Pr esters recovery from anaesthesia was 10-15-fold faster than from ketamine itself, and for the n-Pr esters it was 20-25-fold faster than from ketamine. A new dimethylamino ketamine derivative (homoketamine) had ketamine-like sedative effects but was slightly less potent than, but ester analogues of homoketamine had very weak sedative effects.
Assuntos
Analgésicos/química , Ketamina/química , Analgésicos/síntese química , Analgésicos/farmacologia , Animais , Ésteres , Feminino , Frequência Cardíaca/efeitos dos fármacos , Ketamina/síntese química , Ketamina/farmacologia , Ratos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
Using a scaffold-hopping approach, imidazo[1,2-a]pyridine analogues of the ZSTK474 (benzimidazole) class of phosphatidylinositol 3-kinase (PI3K) inhibitors have been synthesized for biological evaluation. Compounds were prepared using a heteroaryl Heck reaction procedure, involving the palladium-catalysed coupling of 2-(difluoromethyl)imidazo[1,2-a]pyridines with chloro, iodo or trifluoromethanesulfonyloxy (trifloxy) substituted 1,3,5-triazines or pyrimidines, with the iodo intermediates being preferred in terms of higher yields and milder reaction conditions. The new compounds maintain the PI3K isoform selectivity of their benzimidazole analogues, but in general show less potency.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Piridinas/síntese química , Piridinas/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Humanos , Estrutura Molecular , Fosfatidilinositol 3-Quinases/metabolismo , Piridinas/química , Relação Estrutura-AtividadeRESUMO
Multiple therapeutic agents have been developed to target the phosphatidylinositol 3-kinase (PI3K) signaling pathway, which is frequently dysregulated in cancer promoting tumor growth and survival. These include pan PI3K inhibitors, which target class Ia PI3K isoforms and have largely shown limited single agent activity with narrow therapeutic windows in clinical trials. Here, we characterize SN32976, a novel pan PI3K inhibitor, for its biochemical potency against PI3K isoforms and mTOR, kinase selectivity, cellular activity, pharmacokinetics, pharmacodynamics and antitumor efficacy relative to five clinically-evaluated pan PI3K inhibitors: buparlisib, dactolisib, pictilisib, omipalisib and ZSTK474. SN32976 potently inhibited PI3K isoforms and mTOR, displaying preferential activity for PI3Kα and sparing of PI3Kδ relative to the other inhibitors, while showing less off-target activity than the clinical inhibitors in a panel of 442 kinases. The major metabolites of SN32976 were also potent PI3K inhibitors with similar selectivity for PI3Kα as the parent compound. SN32976 compared favorably with the clinically-evaluated PI3K inhibitors in cellular assays, inhibiting pAKT expression and cell proliferation at nM concentrations, and in animal models, inducing a greater extent and duration of pAKT inhibition in tumors than pictilisib, dactolisib and omipalisib at similarly tolerated dose levels and inhibiting tumor growth to a greater extent than dactolisib and ZSTK474 and with similar efficacy to pictilisib and omipalisib. These results suggest that SN32976 is a promising clinical candidate for cancer therapy with enhanced kinase selectivity and preferential inhibition of PI3Kα compared to first generation pan PI3K inhibitors, while retaining comparable anticancer activity.
Assuntos
Antineoplásicos/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Glucose/metabolismo , Humanos , Masculino , Camundongos , Fosforilação , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacocinética , Ratos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Heterocyclic phenazinecarboxamides were prepared by condensation of aminoheterocycles and 2-halo-3-nitrobenzoic acids, followed by reductive ring closure and amidation. They showed similar inhibition of paired cell lines that underexpressed topo II or overexpressed P-glycoprotein, indicating a non topo II mechanism of cytotoxicity and indifference to P-glycoprotein mediated multidrug resistance. Compounds with a fused five-membered heterocyclic ring were generally less potent than the pyrido[4,3-a]phenazines. A 4-methoxypyrido[4,3-a]phenazine (IC(50)s 2.5-26 nM) gave modest (ca. 5 day) growth delays in H69/P xenografts with oral dosing.
Assuntos
Amidas/síntese química , Antineoplásicos/síntese química , Inibidores Enzimáticos/síntese química , Compostos Heterocíclicos de 4 ou mais Anéis/síntese química , Fenazinas/síntese química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Amidas/química , Amidas/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Compostos Heterocíclicos de 4 ou mais Anéis/química , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Substâncias Intercalantes/síntese química , Substâncias Intercalantes/química , Substâncias Intercalantes/farmacologia , Camundongos , Transplante de Neoplasias , Fenazinas/química , Fenazinas/farmacologia , Relação Estrutura-Atividade , Inibidores da Topoisomerase II , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
Tirapazamine (TPZ, 1,2,4-benzotriazin-3-amine 1,4-dioxide) is a bioreductive hypoxic cytotoxin currently in Phase II/III clinical trials in combination with radiotherapy and with cisplatin-based chemotherapy. As part of a program to develop TPZ analogues with improved solubility/potency and therapeutic indices, we synthesized 34 1,2,4-benzotriazin-3-amine 1,4-dioxides (BTO) to examine structure-activity relationships (SAR) for ring substitution. The electronic, hydrophobic, and steric parameters of substituents at the 5-, 6-, 7-, and 8-positions were systematically varied, and the aqueous solubility and one-electron reduction potentials [E(1)] of the analogues were determined. For each compound, we determined cell killing of mouse SCCVII tumor cells in vitro under aerobic and hypoxic conditions by clonogenic survival and determined their relative hypoxic toxicity (RHT; relative to TPZ) and hypoxic cytotoxicity ratio (HCR). A subset of compounds was independently evaluated using a 96-well SRB proliferation assay, the data from which correlated well with that derived by the clonogenic endpoint. Most substituents, except 5- and 8-dimethylamino and 8-diethylamino, gave analogues less soluble than TPZ. E(1) values ranged from -240 mV through -670 mV (with TPZ having a value of -456 mV) and correlated well with the electronic parameter sigma for substituents at the 5-, 6-, 7-, and 8-positions. Aerobic cytotoxic potency showed a strong positive correlation with E(1) (i.e., electron-withdrawing substituents increased aerobic toxicity). Hypoxic cytotoxicity also generally increased with increasing E(1), with a maximum (RHT up to 3.9-fold) seen in halo- and trifluoromethyl-substituted BTO derivatives having E(1) between ca. -370 to -400 mV. Analogues with high HCRs (>50) all had E(1)s in the range -450 to -510 mV (weakly electron-donating substituents) with the exception of the 8-CF(3) analogue, which had an HCR of 112 against SCCVII despite a high E(1) of -372 mV). The results suggest that ring-A substituents in BTO analogues can be used to predictably vary one-electron reduction potentials and also provide a much better definition than previously of the optimum range of these reduction potentials for a desirable biological activity profile (high HCR, RHT, and solubility).
Assuntos
Antineoplásicos/síntese química , Óxidos N-Cíclicos/química , Triazinas/química , Triazinas/síntese química , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Divisão Celular/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Óxidos N-Cíclicos/síntese química , Óxidos N-Cíclicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Concentração Inibidora 50 , Camundongos , Oxirredução , Solubilidade , Relação Estrutura-Atividade , Tirapazamina , Triazinas/farmacologia , Células Tumorais CultivadasRESUMO
Tirapazamine (TPZ, 1,2,4-benzotriazin-3-amine 1,4-dioxide) is a bioreductive hypoxia-selective cytotoxin, currently in phase II/III clinical trials in combination with radiotherapy and with cisplatin-based chemotherapy. We have prepared a series of 1,2,4-benzotriazine 1,4-dioxide (BTO) analogues of TPZ where a DNA-targeting chromophore is attached at the 3-position via a flexible linker. DNA binding affinity was modified through variation of the chromophore or the pK(a) of the linker chain. The association constants (K(DNA)) for calf thymus DNA ranged from 1 x 10(2) to 5.6 x 10(5) M(-1) (ionic strength of 0.01 M). DNA binding affinity was dependent on the presence of a positive charge, either in the linker chain or in the chromophore, and (for a series of 4-acridine carboxamide chromophore analogues) correlated strongly with linker chain pK(a). The efficacy of these BTOs in killing aerobic and hypoxic mouse SCCVII tumor cells in vitro was determined by clonogenic survival. Cytotoxicity was measured as the concentration required to reduce plating efficiency to 10% of controls (C(10)), and the hypoxic cytotoxicity ratio (HCR) for each BTO was calculated as C(10)(aerobic)/C(10)(hypoxic). BTOs bearing a positive charge showed increased hypoxic cytotoxicity (1.5-56-fold) compared to TPZ and mostly modest HCRs (8-51), but some excellent (>167 and 400) values. There was a strong correlation between K(DNA) and hypoxic cytotoxicity but no correlation between K(DNA) and HCR. Cytotoxicity in HT-29 human colon carcinoma cells, determined using IC(50) assays, showed similar relationships with a correlation between K(DNA) and hypoxic cytotoxicity but no correlation between K(DNA) and HCR. In this cell line, a higher proportion of compounds (7 of 11) had HCRs greater than or equal to that of TPZ. The data confirm that DNA targeting is a useful concept for increasing potency while maintaining hypoxic selectivity and provide a direction for the further development of DNA-targeted analogues of TPZ.
Assuntos
Antineoplásicos/síntese química , DNA/química , Triazinas/síntese química , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Divisão Celular/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Concentração Inibidora 50 , Camundongos , Relação Estrutura-Atividade , Tirapazamina , Triazinas/química , Triazinas/farmacologiaRESUMO
A range of 4-substituted derivatives of the pan class I PI 3-kinase inhibitor 2-(difluoromethyl)-1-[4,6-di-(4-morpholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazole (ZSTK474) were prepared in a search for more soluble analogs. 4-Aminoalkoxy substituents provided the most potent derivatives, with the 4-O(CH2)3NMe2 analog (compound 14) being identified as displaying the best overall activity in combination with good aqueous solubility (25 mg/mL for the hydrochloride salt). This compound was tested in a U87MG xenograft model, but displayed less potency than ZSTK474 as a result of an unfavorable pharmacokinetic profile.
Assuntos
Antineoplásicos/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Triazinas/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Células HCT116 , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Estrutura Molecular , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Transdução de Sinais/efeitos dos fármacos , Solubilidade , Relação Estrutura-Atividade , Triazinas/síntese química , Triazinas/químicaRESUMO
A structure-activity relationship (SAR) study of the pan class I PI 3-kinase inhibitor 2-(difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazole (ZSTK474) identified substitution at the 4 and 6 positions of the benzimidazole ring as having significant effects on the potency of substituted derivatives. The 6-amino-4-methoxy analogue displayed a greater than 1000-fold potency enhancement over the corresponding 6-aza-4-methoxy analogue against all three class Ia PI 3-kinase enzymes (p110α, p110ß, and p110δ) and also displayed significant potency against two mutant forms of the p110α isoform (H1047R and E545K). This compound was also evaluated in vivo against a U87MG human glioblastoma tumor xenograft model in Rag1(-/-) mice, and at a dose of 50 mg/kg given by ip injection at a qd × 10 dosing schedule it dramatically reduced cancer growth by 81% compared to untreated controls.
Assuntos
Antineoplásicos/síntese química , Benzimidazóis/síntese química , Inibidores de Fosfoinositídeo-3 Quinase , Triazinas/síntese química , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Benzimidazóis/farmacocinética , Benzimidazóis/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Camundongos , Camundongos Knockout , Modelos Moleculares , Mutação , Transplante de Neoplasias , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Transdução de Sinais/efeitos dos fármacos , Solubilidade , Relação Estrutura-Atividade , Transplante Heterólogo , Triazinas/farmacocinética , Triazinas/farmacologiaRESUMO
Reactivation of silenced tumor suppressor genes by 5-azacytidine (Vidaza) and its congener 5-aza-2'-deoxycytidine (decitabine) has provided an alternate approach to cancer therapy. We have shown previously that these drugs selectively and rapidly induce degradation of the maintenance DNA methyltransferase (DNMT) 1 by a proteasomal pathway. Because the toxicity of these compounds is largely due to their incorporation into DNA, it is critical to explore novel, nonnucleoside compounds that can effectively reactivate the silenced genes. Here, we report that a quinoline-based compound, designated SGI-1027, inhibits the activity of DNMT1, DNMT3A, and DNMT3B as well M. SssI with comparable IC(50) (6-13 micromol/L) by competing with S-adenosylmethionine in the methylation reaction. Treatment of different cancer cell lines with SGI-1027 resulted in selective degradation of DNMT1 with minimal or no effects on DNMT3A and DNMT3B. At a concentration of 2.5 to 5 micromol/L (similar to that of decitabine), complete degradation of DNMT1 protein was achieved within 24 h without significantly affecting its mRNA level. MG132 blocked SGI-1027-induced depletion of DNMT1, indicating the involvement of proteasomal pathway. Prolonged treatment of RKO cells with SGI-1027 led to demethylation and reexpression of the silenced tumor suppressor genes P16, MLH1, and TIMP3. Further, this compound did not exhibit significant toxicity in a rat hepatoma (H4IIE) cell line. This study provides a novel class of DNA hypomethylating agents that have the potential for use in epigenetic cancer therapy.
Assuntos
Azacitidina/toxicidade , Metilação de DNA/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Genes Supressores de Tumor/efeitos dos fármacos , Quinolinas/toxicidade , Animais , Neoplasias da Mama , Carcinoma Hepatocelular , Linhagem Celular Tumoral , Neoplasias do Colo , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferases/efeitos dos fármacos , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/isolamento & purificação , DNA Metiltransferase 3A , Metilases de Modificação do DNA/metabolismo , Feminino , Células HeLa , Humanos , Neoplasias Hepáticas , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , DNA Metiltransferase 3BRESUMO
A series of phenazine-1-carboxamides were prepared, including variations in both chromophore substituents and the nature of the cationic side chain. The novel side-chain analogues were prepared from the corresponding phenazine-1-carboxylic acids via Schmidt conversion to the 1-amines and from the corresponding 1-halides. Structure-cytotoxicity relationships for these compounds in a panel of tumor cell lines showed that there is very limited scope for variation of the structure of the 1-carboxamide side chain, consistent with the recent structural model of how tricyclic carboxamides bind to DNA. There was generally little difference in IC(50)s between parent and P-glycoprotein expressing cell lines, suggesting that most of the compounds are not affected by the presence of this efflux pump.
Assuntos
Fenazinas/química , Fenazinas/toxicidade , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/toxicidade , Cátions/química , Linhagem Celular Tumoral , Humanos , Ligação de Hidrogênio , Concentração Inibidora 50 , Metilação , Camundongos , Estrutura Molecular , Fenazinas/síntese química , Relação Estrutura-AtividadeRESUMO
Revealing the free radical mechanism by which the anticancer drug tirapazamine (3-amino-1,2,4-benzotriazine 1,4-dioxide) induces hypoxia-selective cytotoxicity, is seen as a way forward to develop clinically useful bioreductive drugs against chemo- and radiation-resistant hypoxic tumor cells. Our previous studies point to the formation of an active benzotriazinyl radical following the one-electron reduction of tirapazamine and its elimination of water from the initial reduction intermediate, and have suggested that this species is a cytotoxin. In this paper we have used pulse radiolysis to measure the one-electron reduction potentials of the benzotriazinyl radicals E(B*,H(+)/B) of 30 analogues of tirapazamine as well as the one-electron reduction potentials of their two-electron reduced metabolites, benzotriazine 1-oxides E(B/B*-). The redox dependencies of the back-oxidation of the one-electron reduced benzotriazine 1,4-dioxides by oxygen, their radical prototropic properties and water elimination reactions were found to be tracked in the main by the one-electron reduction potentials of the benzotriazine 1,4-dioxides E(A/A*-). Multiple regression analysis of published aerobic and hypoxic clonogenic cytotoxicity data for the SCCVII murine tumor cell line with the physical chemistry parameters measured in this study, revealed that hypoxic cytotoxicity is dependent on E(B*, H(+)/B) thus providing strong evidence that the benzotriazinyl radicals are the active cytotoxic species in hypoxia, while aerobic cytotoxicity is dependent on E(B/B*-). It is concluded that maximizing the differential ratio between these two controlling parameters, in combination with necessary pharmacological aspects, will lead to more efficacious anticancer bioreductive drugs.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Triazinas/química , Triazinas/farmacologia , Radicais Livres , Cinética , Espectroscopia de Ressonância Magnética , Oxirredução , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Symmetrical dimers of lipophilic intercalating chromophores linked by cation-containing chains have recently been shown to have broad-spectrum in vivo anticancer activity. We report the preparation and evaluation of a series of both symmetric and unsymmetric dimers of a variety of intercalating chromophores of varied DNA binding strength, including naphthalimides, acridines, phenazines, oxanthrenes and 2-phenylquinolines. The unsymmetrical dimers were prepared by sequential coupling of the chromophores to linkers with selectively protected primary terminal amines to ensure high yields and unequivocal product. Protection of the internal (secondary) amines as BOC derivatives was used to ensure complete structural specificity, and was also an aid to the purification of these very polar compounds. The growth inhibitory abilities (as IC(50) values) of the compounds in a range of cell lines showed that the nature of the linker chain was important, and independent of the nature of the chromophore, with compounds containing the dicationic linker [-(CH2)2NH(CH2)2NH(CH2)2-] being on average 30-fold more potent than the corresponding compounds containing the monocationic linker [-(CH2)3NMe(CH2)3-]. However, the chromophores also play a role in determining biological activity, with the cytotoxicities of symmetric and unsymmetric dicationic dimers correlating with the overall DNA binding abilities of the chromophores.
Assuntos
Amidas/síntese química , Amidas/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , DNA Topoisomerases Tipo I/efeitos dos fármacos , Animais , Avaliação Pré-Clínica de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Espectroscopia de Ressonância Magnética , Células Tumorais CultivadasRESUMO
Tirapazamine (3-amino-1,2,4-benzotriazine 1,4-dioxide) is the lead bioreductive drug in clinical trials as an anticancer agent to kill refractory hypoxic cells of solid tumors. It has long been known that, upon metabolic one-electron reduction, tirapazamine induces lethal DNA double strand breaks in hypoxic cells. These strand breaks arise from radical damage to the ribose moiety of DNA, and in this pulse radiolysis and product analysis study we examine mechanistic aspects of the dual function of tirapazamine and analogues in producing radicals of sufficient power to oxidize 2-deoxyribose to form radicals, as well as the ability of the compounds to oxidize the resulting deoxyribose radicals to generate the strand breaks. Both the rate of oxidation of 2-deoxyribose and the radical yield increase with the one-electron reduction potentials of the putative benzotriazinyl radicals formed from the benzotriazine 1,4-dioxides. Subsequent oxidation of the 2-deoxyribose radicals by the benzotriazine 1,4-dioxides and 1-oxides proceeds through adduct formation followed by breakdown to form the radical anions of both species. The yield of the radical anions increases with increasing one-electron reduction potentials of the compounds. We have previously presented evidence that oxidizing benzotriazinyl radicals are formed following one-electron reduction of the benzotriazine 1,4-dioxides. The reactions reported in this work represent the kinetic basis of a short chain reaction leading to increased oxidation of 2-deoxyribose, a process which is dependent on the one-electron reduction potential of the benzotriazinyl radicals that are above a threshold value of ca. 1.24 V.
Assuntos
Antineoplásicos/química , Desoxirribose/química , Radicais Livres/química , Óxidos/química , Triazinas/química , Antineoplásicos/farmacologia , DNA/química , DNA/efeitos dos fármacos , Dano ao DNA , Modelos Químicos , Oxirredução , Radiólise de Impulso , Análise Espectral , Temperatura , Fatores de Tempo , Tirapazamina , Triazinas/farmacologiaRESUMO
The mechanism by which a benzotriazine 1,4-dioxide class of anticancer drugs produce oxidizing radicals following their one-electron reduction has been investigated using tirapazamine (3-amino-1,2,4-benzotriazine 1,4-dioxide, 1) and its 6-methoxy (6), 7-dimethylamino (7), and 8-methyl (8) analogues. By measuring the changes in absorption with pH, we found that the radical anions undergo protonation with radical pK(r) values of 6.19 +/- 0.05, 6.10 +/- 0.03, 6.45 +/- 0.04, and 6.60 +/- 0.04, respectively. The one-electron reduced species underwent a first-order reaction, with increased rate constants from 112 +/- 23 s(-)(1) for 1 to 777 +/- 12 s(-)(1)(6), 1120 +/- 29 s(-)(1) (7), and 825 +/- 89 s(-)(1) (8) at pH 7. No overall change in conductance was observed following the one-electron reduction of 6, and 8 at pH 4.5, consistent with the protonation of the radical anions, but a loss in conductance was seen for one-electron reduced 7 because of further protonation of the initially formed radical. This is assigned to the protonation of the dimethylamino group of the radical species, which has a pK(a) of 8.8 +/- 0.3. All conductance changes take place on a time-scale shorter than those of the above first-order reactions, which are not associated with the formation or loss of charged species. The absorption spectra present at the end of the unimolecular reactions were found to be similar to those formed immediately upon the one-electron oxidation of the respective substituted 3-amino-1,2,4-benzotriazine 1-oxides, and it is suggested that common benzotriazinyl radicals are formed by both routes. All these intermediate radicals underwent dismutation to produce final spectra matched by equal contributions of the parent compound and their respective substituted 3-amino-1,2,4-benzotriazine 1-oxides. By establishing redox equilibria between the intermediate radicals formed on the one-electron oxidation of the respective 3-amino-1,2,4-benzotriazine 1-oxides of the compounds and reference compounds, we found the one-electron reduction potential of the oxidizing radicals to range from 0.94 to 1.31 V. The benzotriazinyl radical of tirapazamine was found to oxidize dGMP and 2-deoxyribose with rate constants of (1.4 +/- 0.2) x 10(8) M(-)(1) s(-)(1) and (3.7 +/- 0.5) x 10(6) M(-)(1) s(-)(1), respectively.