RESUMEN
In this study we investigated the in vitro time dependence of radiosensitisation, pharmacokinetics and metabolism of NU7026, a novel inhibitor of the DNA repair enzyme DNA-dependent protein kinase (DNA-PK). At a dose of 10 muM, which is nontoxic to cells per se, a minimum NU7026 exposure of 4 h in combination with 3 Gy radiation is required for a significant radiosensitisation effect in CH1 human ovarian cancer cells. Following intravenous administration to mice at 5 mg kg(-1), NU7026 underwent rapid plasma clearance (0.108 l h(-1)) and this was largely attributed to extensive metabolism. Bioavailability following interperitoneal (i.p.) and p.o. administration at 20 mg kg(-1) was 20 and 15%, respectively. Investigation of NU7026 metabolism profiles in plasma and urine indicated that the compound undergoes multiple hydroxylations. A glucuronide conjugate of a bis-hydroxylated metabolite represented the major excretion product in urine. Identification of the major oxidation site as C-2 of the morpholine ring was confirmed by the fact that the plasma clearance of NU7107 (an analogue of NU7026 methylated at C-2 and C-6 of the morpholine ring) was four-fold slower than that of NU7026. The pharmacokinetic simulations performed predict that NU7026 will have to be administered four times per day at 100 mg kg(-1) i.p. in order to obtain the drug exposure required for radiosensitisation.
Asunto(s)
Cromonas/metabolismo , Cromonas/farmacocinética , Proteína Quinasa Activada por ADN/antagonistas & inhibidores , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacocinética , Morfolinas/metabolismo , Morfolinas/farmacocinética , Neoplasias Ováricas/metabolismo , Animales , Disponibilidad Biológica , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Evaluación Preclínica de Medicamentos , Femenino , Rayos gamma , Humanos , Ratones , Ratones Endogámicos BALB C , Neoplasias Ováricas/radioterapia , Tolerancia a Radiación , Ensayo de Tumor de Célula MadreRESUMEN
Values for reaction-kinetic parameters of electrophiles can be used to predict mutagenic potency. One approach employs the Swain-Scott relationship for comparative kinetic studies of electrophilic agents reacting with nucleophiles. In this way glycidamide (GA), the putatively mutagenic/carcinogenic metabolite of acrylamide, was assessed by determining the rates of reaction with different nucleophiles. The rate constants (kNu) were determined using the "supernucleophile" cob(I)alamin [Cbl(I)] as an analytical tool. The Swain-Scott parameters for GA were compared with those of ethylene oxide (EO). The substrate constants, s values, for GA and for EO were found to be 1.0 and 0.93, respectively. The reaction rates at low values of nucleophilic strength (n=1-3), corresponding to oxygens in DNA, were determined to be 2-3.5 times higher for GA compared to EO. GA was also more reactive than EO towards other nucleophiles (n=0-6.4). The mutagenic potency of GA was determined in Chinese hamster ovary cells (hprt mutations in CHO-AA8 cells per dose unit with gamma-radiation as reference standard). The potency of GA was estimated to be about three mutations per 10(5) cells and mMh corresponding to about 40 rad-equ./mMh. A preliminary comparison of the mutagenic potency (per mMh and as rad-equivalents) of GA and EO shows an approximately seven times higher potency for GA. A higher mutagenic potency of GA compared to EO is compatible with expectation from reaction-kinetic data of the two compounds. The data confirmed that GA is not a strong mutagen, which is in line with what is expected for simple oxiranes. The present study shows the value of cob(I)alamin for the determination of reaction-kinetic parameters and their use for prediction of mutagenic potency.
Asunto(s)
Compuestos Epoxi/química , Modelos Biológicos , Mutágenos/química , Mutación , Animales , Línea Celular , Cricetinae , Cricetulus , Relación Dosis-Respuesta a Droga , Compuestos Epoxi/toxicidad , Óxido de Etileno/química , Óxido de Etileno/toxicidad , Cinética , Mutágenos/toxicidad , Vitamina B 12/químicaRESUMEN
Isoprene (2-methylbuta-1,3-diene) is a multi-site carcinogen in rodents. To evaluate the role of the diepoxide metabolite (1,2:3,4-diepoxy-2-methylbutane) in carcinogenesis, measurements of in vivo doses of the diepoxide are needed. The in vivo dose may be inferred from levels of reaction products with hemoglobin (Hb adducts). This report presents in vitro studies of the adduct formation by the diepoxide of isoprene with valinamide and oligopeptides as model compounds of N-terminal valines in hemoglobin (Hb). In the reaction with valinamide it was shown that isoprene diepoxide forms as the main product a ring-closed adduct, which is a pyrrolidine derivative [N,N-(2,3-dihydroxy-2-methyl-1,4-butadiyl)valinamide, MPyr-Val]. The analysis was performed by gas chromatography/mass spectrometry (GC/MS) (EI and PICI) after acetylation. The ring-closed adduct was also identified by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) as the main product in the reaction between isoprene diepoxide and standard hepta- or (2H8)octapeptides, corresponding to the N-terminal peptides of the alpha-chains in mouse and rat Hb. These peptides, alkylated with isoprene diepoxide, to be used as internal standards and calibration standards for quantification of MPyr-adduct levels in vitro and in vivo, were analyzed with respect to the degree of MPyr-alkylation by two independent methods, amino acid analysis and HPLC-UV; similar results were obtained using these methods. A method for measurement of Hb adducts as modified peptides, used earlier to measure a similar adduct to N-terminal valines in Hb from the diepoxide of 1,3-butadiene, has in the present work been tested for application to isoprene diepoxide. The method is based on tryptic degradation of globin and LC/ESI-MS analysis of N-terminal Pyr-heptapeptides of the Hb alpha-chain enriched by HPLC. MPyr-adduct levels in isoprene diepoxide alkylated hemolysate from mouse erythrocytes incubated with different concentrations of isoprene diepoxide (2 and 10 mM) for 1 h were quantified. The adduct level was about 50 nmol/g alpha-chain Hb per mM x h. From the adduct levels the rate constant of isoprene diepoxide for reaction with N-terminal valine was calculated to be about 1.6 times faster than for diepoxybutane.
Asunto(s)
Butadienos/análisis , Eritrocitos/metabolismo , Cromatografía de Gases y Espectrometría de Masas/métodos , Hemiterpenos/análisis , Hemoglobinas/análisis , Pentanos/análisis , Valina/análisis , Alquilación , Animales , Butadienos/química , Células Cultivadas , Compuestos Epoxi/análisis , Compuestos Epoxi/química , Hemiterpenos/química , Hemoglobinas/química , Ratones , Pentanos/química , Unión Proteica , Ratas , Especificidad de la Especie , Valina/químicaRESUMEN
Chloroprene (1) was metabolized by liver microsomes from Sprague-Dawley rats, Fischer 344 rats, B6C3F1 mice, and humans to the monoepoxides, (1-chloro-ethenyl)oxirane (5a/5b), and 2-chloro-2-ethenyloxirane (4a/4b). The formation of 4a/4b was inferred from the identification of their degradation products. With male Sprague-Dawley and Fischer 344 rat liver microsomes, there was a ca. 3:2 preference for the formation of (R)-(1-chloroethenyl)oxirane (5a) compared to the (S)-enantiomer (5b). A smaller but distinct enantioselectivity in the formation of (S)-(1-chloro-ethenyl)oxirane occurred with liver microsomes from male mouse (R:S, 0.90:1) or male human (R:S, 0.86:1). 2-Chloro-2-ethenyloxirane was very unstable in the presence of the microsomal mixture and was rapidly converted to 1-hydroxybut-3-en-2-one (11) and 1-chlorobut-3-en-2-one (12). An additional rearrangement pathway of 2-chloro-2-ethenyloxirane gave rise to 2-chlorobut-3-en-1-al (14) and 2-chlorobut-2-en-1-al (15). Further reductive metabolism of these metabolites occurred to form 1-hydroxybutan-2-one (17) and 1-chlorobutan-2-one (18). In the absence of an epoxide hydrolase inhibitor, the microsomal incubations converted (1-chloroethenyl)oxirane to 3-chlorobut-3-ene-1,2-diol (21a/21b). When microsomal incubations were supplemented with glutathione, 1-hydroxybut-3-en-2-one was not detected because of its rapid conjugation with this thiol scavenger.
Asunto(s)
Cloropreno/metabolismo , Compuestos Epoxi/química , Animales , Cloropreno/química , Femenino , Cromatografía de Gases y Espectrometría de Masas , Humanos , Masculino , Ratones , Ratones Endogámicos , Microsomas Hepáticos/efectos de los fármacos , Ratas , Ratas Endogámicas F344 , Ratas Sprague-Dawley , Valores de Referencia , EstereoisomerismoRESUMEN
The first reported synthesis of the DNA-PK inhibitor 3-cyano-6-hydrazonomethyl-5-(4-pyridyl)pyrid-[1H]-2-one (OK-1035) is described. The structure of OK-1035 was validated by X-ray crystallography. An IC(50) value of 100 microM was determined for inhibition of DNA-PK, and this is approximately 12-fold higher than that reported previously.
Asunto(s)
Proteínas de Unión al ADN , Inhibidores Enzimáticos/farmacología , Hidrazonas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Piridonas/farmacología , Cromonas/farmacología , Cristalografía por Rayos X , Proteína Quinasa Activada por ADN , Inhibidores Enzimáticos/química , Hidrazonas/química , Modelos Moleculares , Morfolinas/farmacología , Piridonas/químicaRESUMEN
The interconversion of (S)-glutamate and (2S,3S)-3-methylaspartate catalyzed by B(12)-dependent glutamate mutase is discussed using results from high-level ab initio molecular orbital calculations. Evidence is presented regarding the possible role of coenzyme-B(12) in substrate activation and product formation via radical generation. Calculated electron paramagnetic resonance parameters support experimental evidence for the involvement of substrate-derived radicals and will hopefully aid the future detection of other important radical intermediates. The height of the rearrangement barrier for a fragmentation-recombination pathway, calculated with a model that includes neutral amino and carboxylic acid substituents in the migrating glycyl group, supports recent experimental evidence for the interconversion of (S)-glutamate and (2S,3S)-3-methylaspartate through such a pathway. Our calculations suggest that the enzyme may facilitate the rearrangement of (S)-glutamate through (partial) proton-transfer processes that control the protonation state of substituents in the migrating group.
Asunto(s)
Ácido Aspártico/análogos & derivados , Ácido Aspártico/metabolismo , Ácido Glutámico/metabolismo , Transferasas Intramoleculares/metabolismo , Ácido Aspártico/química , Catálisis , Clostridium/enzimología , Cobamidas/metabolismo , Desoxiadenosinas/química , Espectroscopía de Resonancia por Spin del Electrón , Ácido Glutámico/química , Glicina/química , Hidrógeno/química , Transferasas Intramoleculares/química , Metilmalonil-CoA Mutasa/metabolismo , Modelos Químicos , Estructura Molecular , Oxidación-Reducción , Propilaminas/metabolismo , Estereoisomerismo , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The activity of antimetabolite inhibitors of de novo deoxyribonucleotide biosynthesis can be compromised by the salvage of extracellular preformed nucleosides and nucleobases. Dipyridamole (DP) is a nucleoside transport inhibitor that has been used clinically in an attempt to increase antimetabolite activity; however, DP binds tightly to the serum protein alpha1-acid glycoprotein (AGP) thereby rendering this therapeutic strategy largely ineffective. Four novel DP analogues (NU3076, NU3084, NU3108, and NU3121) have been developed with substitutions at the 2,6- and 4,8-positions of the pyrimidopyrimidine ring. The novel DP analogues inhibit thymidine (dThd) uptake into L1210 cells in vitro (NU3076 IC(50), 0.25 microM; NU3084 IC(50), 0.27 microM; NU3108 IC(50), 0.31 microM; NU3121 IC(50), 0.26 microM; and DP IC(50), 0.37 microM), but, unlike DP, their activity remains largely unaffected in the presence of 5 mg/ml AGP. The four DP analogues inhibit dThd and hypoxanthine rescue from Alimta (multitargeted antifolate)-induced growth inhibition in A549 and COR L23 human lung carcinoma cell lines in the presence of 2.5 mg/ml AGP, whereas the activity of DP is completely abolished. i.p. administration of 10 mg/kg NU3108, NU3121, and DP produced peak plasma concentrations of 4.4, 2.1, and 6.7 microM, respectively, and levels were sustained above 1 microM for approximately 45 min (DP) and 120 min (NU3108 and NU3121). [3H]thymidine incorporation into COR L23 xenografts grown in CD1 nude mice was reduced by 64% (NU3108), 44% (NU3121), and 65% (DP) 2 h after administration of the nucleoside transport inhibitors. In conclusion, two novel DP analogues (NU3108 and NU3121) have been identified that do not bind to AGP and that display superior pharmacokinetic profiles in comparison to DP and inhibit [3H]thymidine incorporation into human tumor xenografts in vivo.
Asunto(s)
Proteínas Portadoras/antagonistas & inhibidores , Dipiridamol/farmacología , Antagonistas del Ácido Fólico/farmacología , Guanina/análogos & derivados , Proteínas de la Membrana/antagonistas & inhibidores , Animales , Proteínas Portadoras/metabolismo , División Celular/efectos de los fármacos , Dipiridamol/química , Dipiridamol/farmacocinética , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Femenino , Glutamatos/farmacología , Guanina/farmacología , Humanos , Hipoxantina/farmacología , Hígado/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Proteínas de Transporte de Nucleósidos , Orosomucoide/farmacología , Pemetrexed , Tetrahidrofolatos/farmacología , Timidina/metabolismo , Factores de Tiempo , Tritio , Células Tumorales CultivadasRESUMEN
Ab initio molecular orbital theory is used to investigate the coenzyme B(12)-dependent reactions catalyzed by diol dehydratase. The key step in such reactions is believed to be a 1,2-hydroxyl migration, which occurs within free-radical intermediates. The barrier for this migration, if unassisted, is calculated to be too high to be consistent with the observed reaction rate. However, we find that "pushing" the migrating hydroxyl, through interaction with a suitable acid, is able to provide significant catalysis. This is denoted retro-push catalysis, the retro prefix signifying that the motion of the migrating group is in the direction opposite to the electron motion. Similarly, the "pulling" of the migrating group, through interaction of the spectator hydroxyl with an appropriate base, is found to substantially reduce the rearrangement barrier. Importantly, the combination of these two effects results in a barrier reduction that is notably greater than additive. This synergistic interplay of the push and the pull provides an attractive means of catalysis. Our proposed retro-push--pull mechanism leads to results that are consistent with isotope-labeling experiments, with experimental rate data, and with the crystal structure of the enzyme.
Asunto(s)
Cobamidas/metabolismo , Propanodiol Deshidratasa/metabolismo , Catálisis , Cobamidas/química , Transporte de Electrón , Modelos Químicos , Propanodiol Deshidratasa/químicaRESUMEN
Isoprene (2-methylbuta-1,3-diene) is a large-scale petrochemical used principally in the manufacture of synthetic rubbers. It is also produced by plants and trees and is the major endogenous hydrocarbon formed by mammals, probably from mevalonic acid. Isoprene is metabolised by mammals in processes that involve epoxidation by cytochrome P450-dependent monooxygenases to the isomeric mono-epoxides, (1-methylethenyl)-oxirane and 2-ethenyl-2-methyloxirane. Further metabolism of the mono-epoxides to mutagenic isoprene di-epoxides, (2, 2')-2-methylbioxiranes, can also occur. The oxidations to the mono- and di-epoxides occur enantioselectively and diastereoselectively. The mono-epoxides are hydrolysed enantioselectively to vicinal diols under catalysis by epoxide hydrolase. 2-Ethenyl-2-methyloxirane is also readily hydrolysed non-enzymatically. Because of the stereochemical possibilities for metabolites, the metabolism of isoprene is complex. The metabolism of isoprene by liver microsomes in vitro from a range of species including rat, mouse and human shows significant differences between species, strains and gender in respect of the diastereoselectivity and enantioselectivity of the metabolic oxidation and hydrolysis reactions. The impact of the extra methyl in isoprene on di-epoxide reactivity also appears to be critically important for the resulting biological effects. Isoprene di-epoxides may exhibit a lower cross-linking potential in vivo compared to butadiene di-epoxides. Differences in metabolism and reactivity of metabolites may be factors contributing to the significant differences in toxicological response to isoprene observed between species.
Asunto(s)
Butadienos/metabolismo , Butadienos/toxicidad , Hemiterpenos , Pentanos , Animales , Butadienos/química , Carcinógenos/toxicidad , Compuestos Epoxi/química , Compuestos Epoxi/metabolismo , Compuestos Epoxi/toxicidad , Femenino , Humanos , Masculino , Ratones , Microsomas Hepáticos/metabolismo , Oxidación-Reducción , Ratas , Especificidad de la Especie , EstereoisomerismoRESUMEN
The nuclear enzyme poly(ADP-ribose) polymerase (PARP) facilitates the repair of DNA strand breaks and is implicated in the resistance of cancer cells to certain DNA-damaging agents. Inhibitors of PARP have clinical potential as resistance-modifying agents capable of potentiating radiotherapy and the cytotoxicity of some forms of cancer chemotherapy. The preclinical development of 2-aryl-1H-benzimidazole-4-carboxamides as resistance-modifying agents in cancer chemotherapy is described. 1H-Benzimidazole-4-carboxamides, particularly 2-aryl derivatives, are identified as a class of potent PARP inhibitors. Derivatives of 2-phenyl-1H-benzimidazole-4-carboxamide (23, K(i) = 15 nM), in which the phenyl ring contains substituents, have been synthesized. Many of these derivatives exhibit K(i) values for PARP inhibition < 10 nM, with 2-(4-hydroxymethylphenyl)-1H-benzimidazole-4-carboxamide (78, K(i) = 1.6 nM) being one of the most potent. Insight into structure-activity relationships (SAR) for 2-aryl-1H-benzimidazole-4-carboxamides has been enhanced by studying the complex formed between 2-(3-methoxyphenyl)-1H-benzimidazole-4-carboxamide (44, K(i) = 6 nM) and the catalytic domain of chicken PARP. Important hydrogen-bonding and hydrophobic interactions with the protein have been identified for this inhibitor. 2-(4-Hydroxyphenyl)-1H-benzimidazole-4-carboxamide (45, K(i) = 6 nM) potentiates the cytotoxicity of both temozolomide and topotecan against A2780 cells in vitro (by 2.8- and 2.9-fold, respectively).
Asunto(s)
Bencimidazoles/química , Bencimidazoles/síntesis química , Dacarbazina/análogos & derivados , Inhibidores Enzimáticos/síntesis química , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Antineoplásicos/farmacología , Bencimidazoles/farmacología , Cristalografía por Rayos X , Dacarbazina/farmacología , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Modelos Moleculares , Relación Estructura-Actividad , Temozolomida , Topotecan/farmacología , Células Tumorales CultivadasRESUMEN
A series of O(6)-allyl- and O(6)-(2-oxoalkyl)guanines were synthesized and evaluated, in comparison with the corresponding O(6)-alkylguanines, as potential inhibitors of the DNA-repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT). Simple O(6)-alkyl- and O(6)-cycloalkylguanines were weak AGT inactivators compared with O(6)-allylguanine (IC(50) = 8.5 +/- 0.6 microM) with IC(50) values ranging from 100 to 1000 microM. The introduction of substituents at C-2 of the allyl group of O(6)-allylguanine reduced activity compared with the parent compound, while analogous compounds in the O(6)-(2-oxoalkyl)guanine series exhibited very poor activity (150-1000 microM). O(6)-Cycloalkenylguanines proved to be excellent AGT inactivators, with 1-cyclobutenylmethylguanine (IC(50) = 0.55 +/- 0.02 microM) and 1-cyclopentenylmethylguanine (IC(50) = 0.39 +/- 0.04 microM) exhibiting potency approaching that of the benchmark AGT inhibitor O(6)-benzylguanine (IC(50) = 0.18 +/- 0.02 microM). 1-Cyclopentenylmethylguanine also inactivated AGT in intact HT29 human colorectal carcinoma cells (IC(50) = 0.20 +/- 0.07 microM) and potentiated the cytotoxicity of the monomethylating antitumor agent Temozolomide by approximately 3- and 10-fold, respectively, in the HT29 and Colo205 tumor cell lines. The observation that four mutant AGT enzymes resistant to O(6)-benzylguanine also proved strongly cross-resistant to 1-cyclopentenylmethylguanine indicates that the O(6)-substituent of each compound makes similar binding interactions within the active site of AGT.
Asunto(s)
Antineoplásicos Alquilantes/farmacología , Dacarbazina/análogos & derivados , Dacarbazina/farmacología , Inhibidores Enzimáticos/síntesis química , Guanina/síntesis química , O(6)-Metilguanina-ADN Metiltransferasa/antagonistas & inhibidores , Extractos Celulares , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Guanina/análogos & derivados , Guanina/química , Guanina/farmacología , Humanos , Mutación , O(6)-Metilguanina-ADN Metiltransferasa/genética , Relación Estructura-Actividad , Temozolomida , Células Tumorales CultivadasRESUMEN
Substituted guanines and pyrimidines were tested as inhibitors of cyclin B1/CDK1 and cyclin A3/CDK2 and soaked into crystals of monomeric CDK2. O6-Cyclohexylmethylguanine (NU2058) was a competitive inhibitor of CDK1 and CDK2 with respect to ATP (Ki values: CDK1, 5 +/- 1 microM; CDK2, 12 +/- 3 microM) and formed a triplet of hydrogen bonds (i.e., NH-9 to Glu 81, N-3 to Leu 83, and 2-NH2 to Leu 83). The triplet of hydrogen bonding and CDK inhibition was reproduced by 2,6-diamino-4-cyclohexylmethyloxy-5-nitrosopyrimidine (NU6027, Ki values: CDK1, 2.5 +/- 0.4 microM; CDK2, 1.3 +/- 0.2 microM). Against human tumor cells, NU2058 and NU6027 were growth inhibitory in vitro (mean GI50 values of 13 +/- 7 microM and 10 +/- 6 microM, respectively), with a pattern of sensitivity distinct from flavopiridol and olomoucine. These CDK inhibition and chemosensitivity data indicate that the distinct mode of binding of NU2058 and NU6027 has direct consequences for enzyme and cell growth inhibition.
Asunto(s)
Antineoplásicos/síntesis química , Proteína Quinasa CDC2/antagonistas & inhibidores , Quinasas CDC2-CDC28 , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Inhibidores Enzimáticos/síntesis química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Purinas/síntesis química , Pirimidinas/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Proteína Quinasa CDC2/química , Cristalografía por Rayos X , Quinasa 2 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/química , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Proteínas Serina-Treonina Quinasas/química , Purinas/química , Purinas/farmacología , Pirimidinas/química , Pirimidinas/farmacología , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
The supernucleophilic cobalt compound, cob(I)alamin, has been kinetically characterized with respect to its ability to bring about transalkylation of adducts to DNA phosphates (phosphotriesters). The reactivity of cob(I)alamin toward different phosphotriesters (model compounds and methylated DNA), as well as its specificity toward DNA-phosphate adducts, has been investigated. Through nucleophilic displacement on the alkyl by cob(I)alamin, the alkyl groups (methyl and ethyl) were transferred from phosphotriesters within minutes at room temperature. In contrast, methylated nucleosides (base adducts) were stable in the presence of cob(I)alamin.
Asunto(s)
Aductos de ADN/metabolismo , Vitamina B 12/metabolismo , Alquilación , Sensibilidad y EspecificidadRESUMEN
The synthesis and biological evaluation of potent 4,8-dibenzylaminopyrimidopyrimidine nucleoside transport inhibitors, with reduced binding to alpha1-acid glycoprotein, is reported.
Asunto(s)
Resistencia a Antineoplásicos , Nucleósidos/metabolismo , Orosomucoide/metabolismo , Pirimidinas/síntesis química , Animales , Transporte Biológico Activo/efectos de los fármacos , Fenómenos Químicos , Química Física , Humanos , Leucemia L1210/tratamiento farmacológico , Pirimidinas/farmacología , Relación Estructura-ActividadRESUMEN
Dipyridamole has been shown to enhance the in vitro activity of antimetabolite anticancer drugs through the inhibition of nucleoside transport. However, the clinical potential of dipyridamole has not been realized because of the avid binding of the drug to the plasma protein alpha1-acid glycoprotein (AGP). Dipyridamole analogues that retain potent nucleoside transport inhibitory activity in the presence of AGP are described and their ability to enhance the growth inhibitory and cytotoxic effects of thymidylate synthase (TS) inhibitors has been evaluated. Three dipyridamole analogues (NU3026, NU3059 and NU3060) were shown to enhance the growth inhibitory activity of the TS inhibitor CB3717 and block thymidine rescue in L1210 cells. The extent of potentiation at a fixed analogue concentration (10 microM) was related to the potency of inhibition of thymidine uptake. A further analogue, NU3076, was identified, which was more potent than dipyridamole with a Ki value for inhibition of thymidine uptake of 0.1 microM compared to 0.28 microM for dipyridamole. In marked contrast to dipyridamole, inhibition of thymidine uptake by NU3076 was not significantly affected by the presence of AGP (5 mg ml(-1)). NU3076 and dipyridamole produced equivalent potentiation of the cytotoxicity of the non-classical antifolate TS inhibitor, nolatrexed, in L1210 cells with both compounds significantly reducing the LC90, by > threefold in the absence of salvageable thymidine. Thymidine rescue of L1210 cells from nolatrexed cytotoxicity was partially blocked by both 1 microM NU3076 and 1 microM dipyridamole. NU3076 also caused a significant potentiation of FU cytotoxicity in L1210 cells. These studies demonstrate that nucleoside transport inhibition can be maintained in the absence of AGP binding with the dipyridamole pharmacophore and that such analogues can enhance the cytotoxicity of TS inhibitors.
Asunto(s)
Antineoplásicos/toxicidad , Dipiridamol/análogos & derivados , Dipiridamol/toxicidad , Inhibidores Enzimáticos/toxicidad , Ácido Fólico/análogos & derivados , Leucemia L1210/patología , Orosomucoide/metabolismo , Quinazolinas/toxicidad , Timidilato Sintasa/antagonistas & inhibidores , Animales , Transporte Biológico/efectos de los fármacos , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Dipiridamol/farmacocinética , Sinergismo Farmacológico , Ácido Fólico/toxicidad , Leucemia L1210/metabolismo , Ratones , Estructura Molecular , Unión Proteica , Relación Estructura-Actividad , Timidina/metabolismo , Células Tumorales CultivadasRESUMEN
We review the history of the toxicology of benzene and consider current exposure levels, the metabolism of benzene, reactions of the metabolites with biomolecules and possible mechanisms of carcinogenesis due to benzene. Epidemiological evidence indicates a relationship between exposure to benzene and the occurrence of acute non-lymphocytic leukaemia in humans. Working groups convened by IARC and other organizations have therefore judged that there is sufficient evidence for classifying benzene as a human carcinogen. Despite much research, including numerous studies in animals, the detailed mechanism of the carcinogenicity of benzene is unknown. The significant differences in the responses of rodents and humans to benzene are not understood. Benzene forms many metabolites, some of which are reactive towards biomolecules, but the metabolite(s) responsible for the induction of leukaemia is unknown. Candidate metabolites, either singly or in combination, include epoxides, oxepins, quinones and aldehydes, all of which are reactive towards proteins and DNA. Our studies on muconaldehydes and benzene oxide-oxepin are discussed in this context. The significance of DNA adduct formation in respect of human leukaemia is uncertain. The overall reactivity of benzene towards DNA has been shown to be very low in experimental animals, although dose-related reactivity of metabolites with DNA was observed. The lack of significant DNA reactivity is reflected in the lack of activity of benzene in short-term tests for genotoxicity; however, benzene causes oxidative stress, which can be detected as oxidative damage to DNA. Mechanisms other than DNA damage may play a role in benzene-related toxicity, e.g. reactions of benzene metabolites with essential enzymes such as topoisomerase II.
Asunto(s)
Benceno/toxicidad , Carcinógenos/toxicidad , Neoplasias/inducido químicamente , Animales , Benceno/química , Benceno/metabolismo , Carcinógenos/química , Carcinógenos/metabolismo , Aductos de ADN/biosíntesis , Aductos de ADN/química , Humanos , Ratones , Modelos Químicos , Unión Proteica , Inhibidores de Topoisomerasa IIRESUMEN
Differences in the metabolism of 1,3-butadiene (Bd) in rats and mice may account for the observed species difference in carcinogenicity. Previous studies of the metabolic fate of Bd have identified epoxide formation as a key metabolic transformation which gives 1, 2-epoxy-3-butene (BMO), although some evidence of aldehyde metabolites is reported. In this study, male Sprague-Dawley rats and male B6C3F1 mice received single doses of [4-14C]BMO at 1, 5, 20, and 50 mg/kg of body weight (0.014, 0.071, 0.286, and 0.714 mmol/kg of body weight). Analysis of urinary metabolites indicated that both species preferentially metabolize BMO by direct reaction with GSH when given by ip administration. The excretion of (R)-2-(N-acetyl-L-cystein-S-yl)-1-hydroxybut-3-ene (IIa), 1-(N-acetyl-L-cystein-S-yl)-2-(S)-hydroxybut-3-ene (IIb), 1-(N-acetyl-L-cystein-S-yl)-2-(R)-hydroxybut-3-ene (IIc), and (S)-2-(N-acetyl-L-cystein-S-yl)-1-hydroxybut-3-ene (IId) accounted for 48-64% of urinary radioactivity in rats and 46-54% in mice. The metabolites originating from the R-stereoisomer of BMO (IIc and IId) predominated over those arising from the S-stereoisomer (IIa and IIb) in both species. IIc was formed preferentially in mice and IId in rats. The corresponding mercaptoacetic acids, S-(1-hydroxybut-3-en-2-yl)mercaptoacetic acid (IIf) and S-(2-hydroxybut-3-en-1-yl)mercaptoacetic acid (IIg), were identified only in mouse urine (ca. 20% of the recovered radioactivity). 4-(N-Acetyl-L-cystein-S-yl)-1,2-dihydroxybutane (Ia), a metabolite derived from hydrolysis of BMO, accounted for 10-17% of the radioactivity in rat and 6-10% in mouse urine. 4-(N-Acetyl-L-cystein-S-yl)-2-hydroxybutanoic acid (Ib), 3-(N-acetyl-L-cystein-S-yl)propan-1-ol (Ic), and 3-(N-acetyl-L-cystein-S-yl)propanoic acid (Id), also derived from the hydrolysis of BMO, were only present in the rat. Metabolites of 1,2,3,4-diepoxybutane (DEB) were not detected after administration of BMO in rat or mouse urine. This study showed both quantitative and qualitative differences in the metabolism of BMO with varying doses and between species. The data aid in the safety evaluation of Bd and contribute to the interpretation of mathematical models developed for quantitative risk assessment and extrapolation of animals to humans.
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Compuestos Epoxi/farmacocinética , Mutágenos/farmacocinética , Animales , Biotransformación , Cromatografía Líquida de Alta Presión , Compuestos Epoxi/orina , Heces/química , Cromatografía de Gases y Espectrometría de Masas , Masculino , Ratones , Ratones Endogámicos , Ratas , Ratas Sprague-Dawley , Distribución TisularRESUMEN
Clinical studies concerning the role of poly(ADP-ribose) polymerase (PARP) in the repair of drug- and radiation-induced DNA damage have been impeded by the poor solubility, lack of potency, and limited specificity of currently available inhibitors. A series of 2-alkyl- and 2-aryl-substituted 8-hydroxy-, 8-methoxy-, and 8-methylquinazolin-4(3H)-ones has been synthesized and evaluated for PARP inhibitory activity in permeabilized L1210 murine leukemia cells. 8-Methoxy- and 8-methylquinazolinones (14-34) were readily prepared by acylation of 3-substituted anthranilamides with the appropriate acid chloride, followed by base-catalyzed cyclization. The requisite 8-hydroxyquinazolinones (6, 35-39) were synthesized by demethylation of the corresponding 8-methoxyquinazolinones with BBr3. N-Methylation of 8-methoxy-2-methylquinazolinone (15) with MeI, followed by O-demethylation by BBr3, afforded the control N3-methylquinazolinones 42 and 43, respectively. In general, an 8-hydroxy or 8-methyl substituent enhanced inhibitory activity in comparison with an 8-methoxy group. 2-Phenylquinazolinones were marginally less potent than the corresponding 2-methylquinazolinones, but the introduction of an electron-withdrawing or electron-donating 4'-substituent on the 2-aryl ring invariably increased potency. This was particularly evident in the 8-methylquinazolinone series (IC50 values 0.13-0.27 microM), which are among the most potent PARP inhibitors reported to date. N3-Methylquinazolinones 42 and 43 were essentially devoid of activity (IC50 values > 100 microM). In studies with L1210 cells in vitro, a concentration of 200 microM 8-hydroxy-2-methylquinazolinone (6, NU1025) (IC50 value 0.40 microM) potentiated the cytotoxicity of the monomethylating agent 5-(3-methyltriazen-1-yl)imidazole-4-carboxamide and gamma-radiation 3.5- and 1.4-fold, respectively, at the 10% survival level.
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Antineoplásicos/síntesis química , Reparación del ADN , Inhibidores Enzimáticos/síntesis química , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Quinazolinas/síntesis química , Alquilantes/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Dacarbazina/análogos & derivados , Dacarbazina/farmacología , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Rayos gamma , Leucemia L1210/patología , Ratones , Quinazolinas/química , Quinazolinas/farmacología , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
The ability of the potent poly(ADP-ribose) polymerase (PARP) inhibitor, NU1025 (8-hydroxy-2-methyl-quinazolin-4-[3H]one) to potentiate the cytotoxicity of a panel of mechanistically diverse anti-cancer agents was evaluated in L1210 cells. NU1025 enhanced the cytotoxicity of the DNA-methylating agent MTIC, gamma-irradiation and bleomycin 3.5-, 1.4- and 2-fold respectively. The cytotoxicities of the thymidylate synthase inhibitor, nolatrexed, and the cytotoxic nucleoside, gemcitabine, were not increased. Potentiation of MTIC cytotoxicity by a delayed exposure to NU1025 was equally effective as by a simultaneous exposure to NU1025, indicating that the effects of NU1025 were mediated by an inhibition of the cellular recovery. The recovery from potentially lethal gamma-irradiation damage cytotoxicity in plateau-phase cells was also inhibited by NU1025. Investigation of DNA strand breakage and repair in gamma-irradiated cells by alkaline elution demonstrated that NU1025 caused a marked retardation of DNA repair. A structurally different PARP inhibitor, NU1064 (2-methylbenzimidazole-4-carboxamide), also potentiated the cytotoxicity of MTIC, to a similar extent to NU1025. NU1064 potentiated a sublethal concentration of a DNA methylating agent in a concentration-dependent manner. Collectively, these data suggest that the most suitable cytotoxic agents for use in combination with PARP inhibitors are methylating agents, bleomycin and ionizing radiation, but not anti-metabolites.
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Amidas/farmacología , Antineoplásicos/toxicidad , Bencimidazoles/farmacología , Daño del ADN/efectos de los fármacos , Reparación del ADN , Inhibidores Enzimáticos/toxicidad , Rayos gamma , Poli(ADP-Ribosa) Polimerasas/metabolismo , Quinazolinas/farmacología , Animales , Antimetabolitos Antineoplásicos/farmacología , Antineoplásicos Alquilantes/farmacología , Bleomicina/farmacología , Muerte Celular , Metilación de ADN , Dacarbazina/análogos & derivados , Dacarbazina/farmacología , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Resistencia a Antineoplásicos , Sinergismo Farmacológico , Leucemia L1210/patología , Ratones , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Células Tumorales CultivadasRESUMEN
Bifunctional alkylating agents, such as those based on nitrogen mustard, form important parts of many anti-cancer chemotherapy protocols and are responsible for increased incidences of secondary tumors in successfully treated patients. These drugs generally form a majority of monofunctional DNA adducts, although the bifunctional adducts appear to be necessary for their powerful cytotoxic and antitumor effects. The relative importance of bifunctional as opposed to monofunctional adducts in the varied biological consequences of drug exposure has not been studied in detail, particularly in relation to the role and specificity of biochemical responses to therapy-related DNA damage. A simple method is described for the preparation of useful quantities of a pure monofunctional derivative of the nitrogen mustard-based drug melphalan. Monohydroxymelphalan was prepared by partial hydrolysis, purified by reversed phase chromatography, and characterized by MS, NMR, and HPLC. Contamination with melphalan was =0.2%. The heat labile DNA base adducts formed by monohydroxymelphalan were shown to contain undetectable levels of cross-linked species. The ratio of adenine to guanine adducts was 0.62, similar to the equivalent ratio for melphalan. The sequence-dependent pattern of alkylation of purified DNA was indistinguishable from that of melphalan, but required a higher dose to achieve comparable extents of reaction. The specificities of two monoclonal antibodies that recognize melphalan-DNA adducts were investigated using DNA alkylated with [3H]monohydroxymelphalan. Adducts on this DNA showed similar immunoreactivities to adducts formed by melphalan. This shows clearly that neither antibody was specific for cross-linked adducts and that it is therefore possible to quantify adducts formed by both monohydroxymelphalan and melphalan with high sensitivities. The availability of monohydroxymelphalan in addition to melphalan, together with sensitive immunoassays for adducts on extracted DNA and in individual cells, constitutes a useful system for investigating cellular responses to the DNA modifications formed by a clinically relevant drug.