RESUMEN
The drug combination N-(phosphonacetyl)-L-aspartic acid (PALA), methylmercaptopurine riboside (MMPR) and 6-aminonicotinamide (6AN), referred to as PMA, induces regressions of advanced CD8F1 murine mammary carcinomas in vivo. We demonstrated that CD8F1 tumor regressions were preceded by the appearance of apoptotic bodies, as observed by microscopic examination of morphology and TUNEL endlabeling, and fragmentation of DNA into nucleosomal "ladder" patterns. These indications of apoptosis were present as early as 6 h after simultaneous administration of MMPR and 6AN and further increased by over fivefold during the next 3 to 6 h, then remained at 7 to 12.8% (0.6 to 2.4% in saline-treated controls) of the cell population for at least 24 h after MMPR + 6AN administration. The 5'-phosphate derivative of MMRP, MMPR-5P, which inhibits de novo purine biosynthesis, was present at a "steady-state" level, and significant (40%) depletion of ATP had occurred by 3 h and both of these events preceded the onset of apoptosis. In addition, MMPR-5P was retained in CD8F1 tumors at a high level over a prolonged period (> 96 h) even as tumors were undergoing regression. The prolonged presence of MMPR-5P was important for optimal chemotherapeutic effect, since treatment with iodotubercidin (IodoT), an inhibitor of MMPR/adenosine kinase, 6 h after MMPR+6AN administration prevented the prolonged accumulation of MMPR-5P and reversed the regression of CD8F1 tumors. In addition, compared to the PMA-treated group, there was a significant restoration of ATP levels after treatment with IodoT. In individual PMA-treated CD8F1 tumors the degree of ATP depletion was found to correlate with the degree of tumor shrinkage at 24 h, after tumors had sufficient time to respond to treatment. These results define the time-course of drug-induced apoptosis in CD8F1 tumors, show that ATP depletion occurs prior to apoptosis and demonstrate that prolonged retention of MMPR-5P is associated with optimal chemotherapy. Collectively, these results suggest that the depletion of ATP by PMA treatment may be a component of the biochemical apoptotic cascade in the CD8F1 tumor.
Asunto(s)
Adenosina Trifosfato/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Apoptosis/efectos de los fármacos , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/metabolismo , 6-Aminonicotinamida/administración & dosificación , Animales , Antimetabolitos Antineoplásicos/administración & dosificación , Ácido Aspártico/administración & dosificación , Ácido Aspártico/análogos & derivados , Femenino , Mercaptopurina/administración & dosificación , Mercaptopurina/análogos & derivados , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , Ácido Fosfonoacético/administración & dosificación , Ácido Fosfonoacético/análogos & derivados , Factores de TiempoRESUMEN
Paclitaxel alone is active against the CD8F1 murine spontaneous mammary cancer, and when administered following an ATP-depleting combination of N-(phosphonacetyl)-L-aspartate (PALA) + 6-methylmercaptopurine riboside (MMPR) + 6-aminonicotinamide (6-AN) (PMA) produced significantly enhanced partial tumor regressions over that produced by either paclitaxel alone at the maximal tolerated dose (MTD), or by the PMA drug combination alone, against advanced, first passage spontaneous murine breast tumors. The anticancer activity of paclitaxel is due to enhancement and stabilization of microtubule polymerization. Pertinently, microtubule disassembly (an ATP-dependent process) is known to sharply decrease in the presence of ATP depletion. Thus, the dramatic therapeutic enhancement observed with paclitaxel in combination with PMA is in agreement with biochemical expectations, since PMA has been shown to deplete ATP in CD8F1 tumor cells. The augmented therapeutic results were obtained with approximately one-third the MTD of paclitaxel as a single agent and suggest the potential clinical benefit of more effective treatment with lesser amounts of drug.
Asunto(s)
Antineoplásicos Fitogénicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Paclitaxel/uso terapéutico , 6-Aminonicotinamida/administración & dosificación , Animales , Ácido Aspártico/administración & dosificación , Ácido Aspártico/análogos & derivados , Femenino , Metiltioinosina/administración & dosificación , Ratones , Ácido Fosfonoacético/administración & dosificación , Ácido Fosfonoacético/análogos & derivados , Inducción de RemisiónRESUMEN
Treatment with a combination (PMA) of (N-phosphonacetyl)-L-aspartic acid (PALA), methylmercaptopurine riboside (MMPR), and 6-aminonicotinamide (6AN) induced partial regressions of CD8F1 murine mammary tumors and provided for tumor growth inhibition without regression of Colon 38 tumors. HPLC-nucleotide pool analysis of CD8 mammary tumors obtained at various times after treatment with PMA revealed that MMPR-5'-phosphate, which inhibits de novo purine nucleotide biosynthesis, was constant at levels of approximately 2.5 nmol/mg protein for 72 hr after treatment. In contrast, the MMPR-5'-phosphate levels of C38 tumors decreased from 24-hr levels at 1.5 nmol/mg protein with a half-time of about 24 hr. Treatment of CD8 tumor-bearing mice with iodotubercidin, a potent inhibitor of adenosine/MMPR kinase, at various times after PMA, reversed both the accumulation of high levels of MMPR-5'-phosphate and the number of partial tumor regressions. These data demonstrate that a cycle of MMPR rephosphorylation is active in the CD8 mammary tumor and suggest that this recycling of MMPR is important for the optimal effect of PMA treatment.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Metiltioinosina/metabolismo , 6-Aminonicotinamida/administración & dosificación , Adenosina Quinasa/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Animales , Ácido Aspártico/administración & dosificación , Ácido Aspártico/análogos & derivados , Neoplasias Mamarias Experimentales/metabolismo , Metiltioinosina/administración & dosificación , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , Ácido Fosfonoacético/administración & dosificación , Ácido Fosfonoacético/análogos & derivadosRESUMEN
A three-drug combination, PMA, consisting of (phosphonacetyl)-L-aspartic acid + 6-methylmercaptopurine riboside + 5-aminonicotinamide, preceding either 5-fluorouracil (5-FU) or adriamycin (Adr), produced tumor-regressing activity in a murine advanced breast tumor model not attainable with either 5-FU or Adr as single agents, or with any lesser combination of these drugs administered at maximally tolerated doses. Marked tumor-regressing activity was further increased significantly by using 5-FU and Adr together in conjunction with the modulatory biochemical conditioning (particularly ATP depletion) provided by pretreatment with PMA.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Animales , Antibióticos Antineoplásicos/administración & dosificación , Antimetabolitos Antineoplásicos/administración & dosificación , Ácido Aspártico/administración & dosificación , Ácido Aspártico/análogos & derivados , Doxorrubicina/administración & dosificación , Fluorouracilo/administración & dosificación , Neoplasias Mamarias Animales/tratamiento farmacológico , Mercaptopurina/administración & dosificación , Mercaptopurina/análogos & derivados , Ratones , Niacinamida/administración & dosificación , Ácido Fosfonoacético/administración & dosificación , Ácido Fosfonoacético/análogos & derivadosRESUMEN
DNA-damaging agents, e.g. Adriamycin (ADR), are reported to cause tumor regression by induction of apoptosis. A reduction in the intracellular content of ATP is part of the biochemical cascade of events that ultimately results in programmed death of the cell, or apoptosis. A chemotherapeutic three-drug combination (PMA) consisting of N-(phosphonacetyl)-L-aspartate (PALA) + 6-methylmercaptopurine riboside (MMPR) + 6-aminonicotinamide (6AN) significantly lowers levels of ATP in CD8F1 murine breast tumors in vivo and produces tumor regression by apoptosis. Addition of the DNA-damaging antitumor agent ADR to PMA was found to further significantly deplete ATP in CD8F1 murine breast tumors in vivo with a concomitant significant increase in the number of tumor regressions. The correlative biochemical and therapeutic results are consistent with, and support, the hypothesis that ATP depletion is a significant factor and, therefore, is a worthy therapeutic target in the production of apoptosis.
Asunto(s)
Adenosina Trifosfato/análisis , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Mamarias Animales/tratamiento farmacológico , Neoplasias Mamarias Animales/patología , 6-Aminonicotinamida/administración & dosificación , Animales , Apoptosis , Ácido Aspártico/administración & dosificación , Ácido Aspártico/análogos & derivados , Doxorrubicina/administración & dosificación , Neoplasias Mamarias Animales/metabolismo , Metiltioinosina/administración & dosificación , Ratones , Ratones Endogámicos , Trasplante de Neoplasias , Ácido Fosfonoacético/administración & dosificación , Ácido Fosfonoacético/análogos & derivados , Factores de TiempoRESUMEN
A quadruple drug combination--consisting of a triple-drug combination of N-(phosphonacetyl)-L-aspartate (PALA) + 6-methylmercaptopurine riboside (MMPR) + 6-amino-nicotinamide (6-AN), designed to primarily deplete cellular energy in tumor cells, + Adriamycin (Adria)--yielded significantly enhanced anticancer activity (i.e., tumor regressions) over that produced by either Adria alone at maximum tolerated dose (MTD) or by the triple-drug combination, against large, spontaneous, autochthonous murine breast tumors. The adenosine triphosphate (ATP)-depleting triple-drug combination administered prior to Adria resulted in a 100% tumor regression rate (12% complete regression; 88% partial regression) of spontaneous tumors. Histological examination of treated tumors demonstrated that the treatment-induced mechanism of cancer cell death was by apoptosis. The augmented therapeutic results (100% tumor regressions) were obtained with approximately one-half the MTD of Adria as a single agent and suggest the potential clinical benefit of longer, more effective, and safer treatment by low doses of Adria when combined with the triple-drug combination. Two likely mechanisms of action are discussed: (1) prevention of DNA repair; (2) complementary disruption of biochemical pathways by both the triple-drug combination and the biochemical cascade of apoptosis that is induced by a DNA-damaging anticancer agents such as Adria.
Asunto(s)
Doxorrubicina/administración & dosificación , Neoplasias Mamarias Experimentales/tratamiento farmacológico , 6-Aminonicotinamida/administración & dosificación , Animales , Apoptosis , Ácido Aspártico/administración & dosificación , Ácido Aspártico/análogos & derivados , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Metabolismo Energético , Neoplasias Mamarias Experimentales/metabolismo , Metiltioinosina/administración & dosificación , Ácido Fosfonoacético/administración & dosificación , Ácido Fosfonoacético/análogos & derivadosRESUMEN
Molecular modeling techniques and a knowledge of thymidylate synthase protein structure have assisted in the development of several potent new inhibitors of thymidylate synthase, the enzyme mediating de novo formation of thymidylate for use in DNA synthesis. Information on several new and specific thymidylate synthase inhibitors, including ICI-D1694, 1843U89, AG-331, and AG-337, is presented. The effects of thymidylate synthase inhibition on the induction of thymidylate synthase protein synthesis have raised the question of whether thymidylate synthase inhibition alone will be sufficient to provide a desirable clinical effect. Formation of a thymidylate synthase-inhibitor complex prevents posttranscriptional regulation of thymidylate synthase synthesis, allowing for increased thymidylate synthase synthesis and the possibility of drug resistance. Therefore, recent efforts have also focused on characterizing this increase in thymidylate synthase protein induced by inhibitors of thymidylate synthase and on devising combination drug strategies that may prevent the induction of thymidylate synthase protein synthesis in addition to inhibiting thymidylate synthase activity.
Asunto(s)
Timidilato Sintasa/antagonistas & inhibidores , Animales , Neoplasias del Colon/tratamiento farmacológico , Fluorouracilo/farmacología , HumanosRESUMEN
The combination of N-(phosphonacetyl)-L-aspartate, 6-methylmercaptopurine, and 6-aminonicotinamide has been shown to be an effective antineoplastic regimen and also to enhance the effects of other chemotherapeutic agents. The mechanism of action of this combination of drugs is not known definitively, but one possible mechanism is biochemical modulation of energy metabolism and inhibition of production of tumor ATP. Tumor-bearing mice were treated with N-(phosphonacetyl)-L-aspartate, followed 17 h later by 6-methylmercaptopurine and 6-aminonicotinamide. 31P nuclear magnetic resonance spectroscopic studies demonstrated a significant depletion of high energy phosphates at 10 h post-6-methylmercaptopurine and 6-aminonicotinamide. The addition of radiation at this time was shown to induce a significantly longer tumor growth delay and a greater number of regressions (including durable complete regressions) than either chemotherapy or radiation alone. The combination of chemotherapy and radiation was found to be supra-additive compared to the antineoplastic effects of either modality administered separately, without a measurable increase in host toxicity.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Experimentales/terapia , 6-Aminonicotinamida/administración & dosificación , Animales , Ácido Aspártico/administración & dosificación , Ácido Aspártico/análogos & derivados , Terapia Combinada , Femenino , Mercaptopurina/administración & dosificación , Mercaptopurina/análogos & derivados , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , Trasplante de Neoplasias , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/radioterapia , Ácido Fosfonoacético/administración & dosificación , Ácido Fosfonoacético/análogos & derivadosRESUMEN
Cytosolic 5'-nucleotidase has been implicated in the phosphorylation of certain nucleosides of therapeutic interest. In vitro, IMP and GMP serve as the optimal phosphate donors for this nucleoside phosphotransferase reaction. Existing assays for nucleoside phosphorylation effected by 5'-nucleotidase require a radiolabeled nucleoside as the phosphate acceptor and separation of the substrate-nucleoside from product-nucleotide has been accomplished either by a filter binding method or HPLC. However, detection of the phosphorylation of unlabeled nucleoside by HPLC is difficult since the ultraviolet absorbance of the phosphate donor, IMP, frequently obscures the absorbance of newly formed nucleotide. The use of ribavirin 5'-phosphate (RMP, 1,2,4-triazole-3-carboxamide riboside 5-monophosphate) as the phosphate donor obviates this difficulty since this triazole heterocycle does not significantly absorb at the wavelengths used to detect most nucleoside analogs. Using this procedure, a 5'-nucleotidase activity from the 100,000 x g supernatant fraction of human T-lymphoblasts deficient in adenosine kinase, hypoxanthine-guanine phosphoribosyltransferase, and deoxycytidine kinase, was characterized with regard to structure-activity relationships for certain inosine and guanosine analogs.
Asunto(s)
5'-Nucleotidasa/metabolismo , Nucleósidos/metabolismo , Adenosina Trifosfato/farmacología , Radioisótopos de Carbono , Células Cultivadas , Guanosina/análogos & derivados , Guanosina/metabolismo , Humanos , Inosina/metabolismo , Inosina Monofosfato/metabolismo , Nucleósidos/análisis , Fosforilación , Estimulación Química , Linfocitos T/citología , Linfocitos T/enzimologíaRESUMEN
This report describes a highly active chemotherapeutic drug combination, consisting of N-(phosphonacetyl)-L-aspartate plus 6-methylmercaptopurine riboside plus 6-aminonicotinamide plus 5-fluorouracil, in CD8F1 mice bearing spontaneous, autochthonous, breast tumors or first-passage advanced transplants of these spontaneous tumors. The combination and sequence of administration of these drugs were selected on the basis of known potentiating biochemical interactions. High performance liquid chromatography and nuclear magnetic resonance spectroscopy measurements of biochemical changes resulting from treatment with N-(phosphonacetyl)-L-aspartate plus 6-methylmercaptopurine riboside plus 6-aminonicotinamide indicated a severe depletion of cellular energy levels in the treated tumors. 6-Aminonicotinamide produced a severe block of the pentose shunt, and 5-fluorouracil severely inhibited both thymidylate synthase and thymidine kinase in the treated tumors. This quadruple drug combination, administered on a 10-11-day schedule, produced an impressive partial tumor regression rate of 67% of large, spontaneous, autochthonous, murine breast tumors and a tumor regression rate of 74% of first-passage transplants of the spontaneous breast tumors.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Fluorouracilo/uso terapéutico , Neoplasias Mamarias Experimentales/tratamiento farmacológico , 6-Aminonicotinamida/administración & dosificación , Animales , Antineoplásicos/administración & dosificación , Ácido Aspártico/administración & dosificación , Ácido Aspártico/análogos & derivados , Femenino , Fluorouracilo/administración & dosificación , Neoplasias Mamarias Experimentales/metabolismo , Metiltioinosina/administración & dosificación , Ratones , Ratones Endogámicos , Trasplante de Neoplasias , Ácido Orótico/metabolismo , Ácido Fosfonoacético/administración & dosificación , Ácido Fosfonoacético/análogos & derivados , Fosforribosil Pirofosfato/metabolismoRESUMEN
Two strategies for modulation of 5-fluorouracil (FUra) activity were compared in vivo in advanced murine CD8F1 breast tumors with regard to three parameters: chemotherapeutic activity, inhibition of thymidylate synthase (TSase) activity, and incorporation of FUra into RNA, (FU)RNA. Inhibition of TSase by FUra was modulated by leucovorin (LV), and the incorporation of FUra into RNA was increased by the administration of otherwise lethal doses of FUra followed by uridine "rescue". Thymidylate synthase activity was inhibited substantially (49%) by low-dose FUra at 25 mg/kg, but was not further enhanced (48%) by repeated daily treatments at the same dose (FUra25 x 4). Inhibition of TSase was somewhat enhanced (55%) by the addition of LV to FUra25 x 4, and a greater therapeutic effect was obtained with FUra25 x 4 + LV over FUra25 x 4 alone. FUra as a single agent at the maximum tolerated weekly dose of 100 mg/kg inhibited TSase activity 66-73%. This inhibition was further enhanced slightly by the addition of LV (71-82%), and its therapeutic efficacy was greater than with FUra25 x 4 with or without LV. However, in contrast to low dose FUra25 x 4, the antitumor effect of FUra100 was not enhanced by LV. (FU)RNA increased with FUra dose from 0.4 (FUra25) to 2.2 nmol/mg DNA (FUra100). At a very-high-dose of FUra (200-225 mg/kg) followed by uridine "rescue", TSase inhibition was not further enhanced, but both (FU)RNA (4.8 nmol/mg DNA) and the therapeutic efficacy were increased. Since TSase could not be further inhibited at doses above FUra100, the increased chemotherapeutic efficacy correlated with increased (FU)RNA.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Fluorouracilo/uso terapéutico , Leucovorina/farmacología , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Uridina/metabolismo , Animales , Peso Corporal/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Fluorouracilo/administración & dosificación , Fluorouracilo/metabolismo , Leucovorina/administración & dosificación , Neoplasias Mamarias Experimentales/patología , Ratones , ARN/metabolismo , Timidilato Sintasa/antagonistas & inhibidores , Factores de TiempoRESUMEN
The effects of 5-fluorouracil (FUra) treatment on thymidine kinase (TKase) activity were examined in vivo in CD8F1 mice bearing first generation CD8F1 mouse mammary tumors. TKase activity was not affected by low dose FUra25 (25 mg/kg), a dose which substantially inhibited thymidylate synthase (TSase), but was severely inhibited 24 hr following treatment with FUra100, a weekly maximally tolerated dose, as judged by activity measurements and labeling of DNA with [3H]thymidine. The amount of (FU)RNA was increased markedly with increasing FUra dose from 0.4 nmol/mg DNA at FUra25 to 2.2 nmol/mg DNA at FUra100. At FUra100, TKase activity gradually declined over 24 hr to less than 10% of the control value, remained low for a further 48 hr, and then was gradually restored to control levels by 168 hr. The loss of TKase activity followed the incorporation of FUra into RNA which peaked at 4-5 hr. TKase activity was not restored by removal of endogenous inhibitors but was restored by treatment with uridine. TKase activity was not inhibited by therapeutic levels of methotrexate (300 mg/kg). TKase from murine colon 38 carcinoma was also severely inhibited, but the activity from colon 26 was only partially (50%) inhibited. Ornithine decarboxylase was also inhibited by FUra100 treatment in the CD8F1 tumor. These results demonstrate that certain short-lived, proliferation-related enzymes are affected by FUra doses higher than those required for TSase inhibition, and this effect appears to correlate with incorporation of FUra into RNA. Thus, in some tumors high doses of FUra can inhibit salvage as well as de novo synthesis of thymidylate providing an increased block of DNA synthesis and increased therapeutic advantage.
Asunto(s)
Fluorouracilo/uso terapéutico , Neoplasias Mamarias Experimentales/tratamiento farmacológico , ARN/metabolismo , Timidina Quinasa/metabolismo , Animales , ADN/biosíntesis , Relación Dosis-Respuesta a Droga , Fluorouracilo/metabolismo , Neoplasias Mamarias Experimentales/metabolismo , Metotrexato/farmacología , Ratones , Ornitina Descarboxilasa/metabolismo , Timidina Quinasa/antagonistas & inhibidores , Timidilato Sintasa/antagonistas & inhibidores , Tritio , Uridina/farmacologíaRESUMEN
Two triazole nucleosides, 1 (3-beta-D-ribofuranosyl-1,2,4-triazole-5-carboxamide) and 2 (2-beta-D-ribofuranosyl-1,2,3-triazole-4,5-dicarboxamide), and a pyrazole nucleoside, 3 (1-beta-D-ribofuranosylpyrazole-3,4-dicarboxamide), were found to inhibit pyrimidine nucleotide biosynthesis in the human myeloid leukemia cell line, K562. Cells treated with these inhibitors released orotate in quantities of 8-35 nmol/10(5) cells/day. Treatment with these compounds caused the K562 cells to accumulate in the S phase of the cell cycle and induced the cells to synthesize hemoglobin.
Asunto(s)
Leucemia Mieloide/metabolismo , Pirazoles/farmacología , Nucleótidos de Pirimidina/biosíntesis , Ribavirina/farmacología , Ribonucleósidos/farmacología , Adenosina Trifosfato/metabolismo , División Celular/efectos de los fármacos , Guanosina Trifosfato/metabolismo , Hemoglobinas/biosíntesis , Humanos , IMP Deshidrogenasa/antagonistas & inhibidores , Inosina Monofosfato/metabolismo , Interfase/efectos de los fármacos , Leucemia Mieloide/patología , Estructura Molecular , Ácido Orótico/metabolismo , Ribavirina/análogos & derivados , Células Tumorales Cultivadas , Uridina Trifosfato/biosíntesisRESUMEN
A rationale for the antitumor activity of 4-methoxy- and 4-amino-8-(beta-D-ribofuranosylamino)pyrimido-[5,4-d]pyrimidine (beta-MRPP and beta-ARPP, respectively) was studied by a molecular modeling method. Although these nucleoside analogues are structurally different from adenosine, they act as substrates for adenosine kinase. The molecular modeling method, which considered the three-dimensional structure and atom-based physicochemical properties of the nucleosides to quantify the molecular similarities, showed that certain low-energy conformations of the beta anomers of a series of nucleosides including beta-MRPP, beta-ARPP, and their 4-hydroxy, 4-amino-6-chloro, 4-methylthio-2,6-dichloro, 4,6-diamino, 4-dimethylamino, 4-methylamino, and 4-hydroxy-2,6-dichloro analogues have remarkable structural similarity to adenosine. The method also suggested that the selection of the reference compound adenosine in the structural comparison is of primary importance to gain insight into the observed antitumor activity. The success of the present method led to AM1 (Austin model 1) molecular orbital calculations and experimental studies indicating that the antitumor activity of the alpha anomer of ARPP is probably due to equilibration to the beta anomer. The AM1 calculation of the protonation energy of N5 of pyrimido[5,4-d]pyrimidines, which occupies the same position in space as the N1 of adenosine, gave a direct correlation between the basicity of the nitrogen with a lone pair of electrons and the observed antitumor activity.
Asunto(s)
Adenosina/análogos & derivados , Antineoplásicos , Nucleósidos de Pirimidina , Isomerismo , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Relación Estructura-ActividadRESUMEN
The basis for the antitumor activities of the exocyclic amino nucleosides 4-amino-(ARPP) and 4-methoxy-8-(D-ribofuranosylamino)pyrimido[5,4-d]pyrimidine (MRPP) was investigated. The primary target of these nucleosides appeared to be 5-phospho-alpha-D-ribofuranose-1-pyrophosphate (PRPP) synthetase. MRPP-5'-monophosphate was a competitive inhibitor (Ki = 40 microM) of the activation of this enzyme by the cofactor inorganic phosphate (K alpha = 2.2 mM). Consequently, ARPP and MRPP treatment of WI-L2 cultures rapidly inhibited both de novo pyrimidine and purine synthesis as well as the nucleotide salvage reactions dependent on PRPP, ARPP or MRPP treatment completely prevented [14C]bicarbonate incorporation into acid-soluble pyrimidine and purine nucleotides. The rate of salvage of [8-14C]hypoxanthine to form IMP was decreased by 85%. Treatment of cells with these agents caused a 50% reduction in the steady-state level of PRPP. When the capacity of the treated cells for sustained synthesis of PRPP was examined by adenine incorporation, the rate of adenine uptake was inhibited by greater than 50%. In vivo treatment of BDF1 mice with a single dose of ARPP (173 mg/kg) or MRPP (62 mg/kg) extended the mean life span of the mice, which had been inoculated intraperitoneally 1 day earlier with 1 x 10(6) L1210 murine leukemia cells, by 62 and 82% respectively. These studies indicate that MRPP and ARPP inhibit PRPP synthetase, and that PRPP synthetase may be a viable target in the development of certain antitumor agents.
Asunto(s)
Antineoplásicos/farmacología , Fosfotransferasas/antagonistas & inhibidores , Nucleósidos de Pirimidina/farmacología , Ribosa-Fosfato Pirofosfoquinasa/antagonistas & inhibidores , Adenosina Desaminasa/farmacología , Adenosina Quinasa/farmacología , Animales , Femenino , Ratones , NAD/metabolismo , Nucleótidos/biosíntesis , Fosforribosil Pirofosfato/análisis , Fosforribosil Pirofosfato/biosíntesisRESUMEN
A novel and direct synthesis of the antiviral and antitumor agent 4-amino-8-(beta-D-ribofuranosylamino)pyrimido[5,4-d]pyrimidine (ARPP, 8) and its alpha-anomer (11) has been developed. Treatment of 2,4,6,8-tetrachloropyrimido[5,4-d]pyrimidine (1) with 2,3-O-isopropylidene-D-ribofuranosylamine gave an anomeric mixture of 2,4,6-trichloro-8-(2,3-O-isopropylidene-beta- and -alpha-D-ribofuranosylamino)pyrimido[5,4-d]pyrimidines (3 and 4) in a ratio of 1.0:0.7. A nucleophilic displacement of the 4-chloro group of 3 and 4 with NH3 furnished 4-amino-2,6-dichloro-8-[(2,3-O-isopropylidene-beta-D-ribofuranosyl)amino ] pyrimido[5,4-d]pyrimidine (6) and its alpha-anomer (9), respectively. Catalytic hydrogenation of 6 and 9, followed by deisopropylidenation gave ARPP (8) and the alpha-anomer 11, respectively. Similarly, 3 and 4 have been transformed to 4-methoxy-8-(beta-D-ribofuranosylamino)pyrimido-[5,4-d]pyrimidine (MRPP, 14) and its alpha-anomer (17). Application of this procedure to 3 with NH2Me or NHMe2 resulted in the synthesis of 4-(methylamino)- and 4-(dimethylamino)-8-(beta-D-ribofuranosylamino)pyrimido [5,4-d]pyrimidine (24 and 27, respectively). A synthesis of 8-(beta-D-ribofuranosylamino)pyrimido[5,4-d]pyrimidin-4(3H)-one (21) has also been accomplished from 3 in three steps. Selective hydrogenation of 6 furnished 4-amino-6-chloro-8-[(2,3-O-isopropylidene-beta-D-ribofuranosyl)amino] pyrimido[5,4-d]pyrimidine (36), the structure of which was established by single-crystal X-ray diffraction analysis. Deisopropylidenation of 36 gave 6-chloro-ARPP (37). Extended treatment of 36 with NH3 furnished 4,6-diamino-8-[(2,3-O-isopropylidene-beta-D-ribofuranosyl)amino]pyrimido [5,4-d]pyrimidine (34), which on deisopropylidenation gave 6-amino-ARPP (35). An unambiguous synthesis of 34 and 36 has also been accomplished by the reaction of 4,6,8-trichloropyrimido[5,4-d]pyrimidine (28) with 2, followed by the treatment with NH3. Nucleophilic displacement studies with 1, 6, and 28 indicated the reactivity of the halogens in these compounds is in the order of 8 greater than 4 greater than 6 greater than 2. The structures of 3 and 9 have been assigned on the basis of 1H NMR data and further confirmed by single-crystal X-ray diffraction analysis. The exocyclic aminonucleosides synthesized during this study were tested for their activity against several RNA and DNA viruses in vitro and against L1210, WI-L2, and LoVo/L in cell culture. The effect of these compounds on the de novo nucleic acid biosynthesis has been studied. Compound 14 (MRPP) exhibited enhanced activity against L1210 in vivo, when compared to ARPP (8).
Asunto(s)
Antineoplásicos/síntesis química , Antivirales/síntesis química , Nucleósidos de Pirimidina/síntesis química , Ribonucleósidos/síntesis química , Animales , Fenómenos Químicos , Química , Cristalografía , Virus ADN/efectos de los fármacos , Humanos , Modelos Moleculares , Nucleósidos de Pirimidina/farmacología , Virus ARN/efectos de los fármacos , Ribonucleósidos/farmacología , Relación Estructura-Actividad , Células Tumorales Cultivadas/efectos de los fármacosAsunto(s)
Inosina/análogos & derivados , Metiltioinosina/farmacología , Fosfotransferasas/antagonistas & inhibidores , Nucleótidos de Purina/metabolismo , Nucleósidos de Pirimidina/farmacología , Ribosa-Fosfato Pirofosfoquinasa/antagonistas & inhibidores , Linfocitos B/efectos de los fármacos , Linfocitos B/enzimología , Linfocitos B/metabolismo , Línea Celular , Relación Dosis-Respuesta a Droga , Eritrocitos/efectos de los fármacos , Eritrocitos/enzimología , Eritrocitos/metabolismo , Humanos , Nucleótidos de Purina/biosíntesis , Nucleótidos de Pirimidina/metabolismo , Ribosa-Fosfato Pirofosfoquinasa/metabolismoRESUMEN
The monophosphates of the exocyclic amino ribonucleosides, 4-amino- and 4-methoxy-8-(D-ribofuranosylamino)pyrimido[5,4-d]pyrimidine, are potent and specific inhibitors of human erythrocyte and B-lymphoblast PRPP synthetase. The inhibition by MRPP monophosphate is competitive (Ki = 35 microM with the PRPP synthetase cofactor, Pi (Km = 2 mM). The nucleosides are phosphorylated to the active metabolite by adenosine kinase and these nucleoside monophosphates accumulate in the cell. beta-ARPP is a substrate, albeit poor, for adenosine deaminase and solutions of the beta-anomer of this nucleoside and its monophosphate anomerize over time to give alpha- and beta-mixtures. beta-MRPP is more resistant to adenosine deaminase and anomerization of the nucleoside and its monophosphate is negligible. The effect of treatment of cells with the nucleosides is a time-dependent and nearly universal reduction in the nucleotide content which appears to result from a reduction in the availability of PRPP for dependent metabolic pathways. In studies with the WI-L2 lymphoblasts, some of these pathways, de novo and salvage (hypoxanthine and guanine) synthesis of purine nucleotides, are more sensitive to a restriction of PRPP availability than others, i.e. de novo pyrimidine synthesis. The nucleosides have shown promise as therapeutic agents in a mouse leukemia evaluation system but may also have future use in unravelling the complex regulation of PRPP synthetase and the dependent nucleotide synthesis pathways.
Asunto(s)
Fosfotransferasas/antagonistas & inhibidores , Ribonucleósidos/farmacología , Ribosa-Fosfato Pirofosfoquinasa/antagonistas & inhibidores , Animales , Antineoplásicos/farmacología , División Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Humanos , Leucemia L1210/tratamiento farmacológico , Ratones , Ribonucleósidos/síntesis química , Ribonucleósidos/uso terapéutico , Relación Estructura-Actividad , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Cyclization of activated nucleotide analogues by intramolecular phosphodiester-bond formation is likely to compete very effectively with template-directed condensation except in the cases of ribo- and arabinonucleotides. This could have excluded derivatives of most sugars from growing polyribonucleotide chains and thus reduced chain-termination in prebiotic polynucleotide synthesis.
Asunto(s)
Nucleótidos , Polirribonucleótidos/síntesis química , Química Orgánica , Fenómenos Químicos Orgánicos , Moldes GenéticosRESUMEN
The total synthesis of clitocine [6-amino-5-nitro-4-(beta-D-ribofuranosylamino)pyrimidine] (1), a nucleoside recently isolated from the mushroom Clitocybe inversa, has been accomplished. Glycosylation of 4,6-diamino-5-nitropyrimidine (4) with 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose afforded the protected nucleoside 6-amino-5-nitro-4-[(2,3,5-tri-O-benzoyl-beta-D-ribofuranosyl) amino]pyrimidine (5) in good yield exclusively as the beta-anomer. Deprotection of 5 with NaOMe/MeOH gave 1 as an 11.5:1 mixture of the beta- and alpha-anomers, respectively. Recrystallization from MeOH, followed by chromatography, afforded 1 containing less than 1% of its alpha-anomer. X-ray crystal data revealed a planar aglycon moiety in clitocine with each oxygen atom of the nitro group intramolecularly hydrogen bonded to the hydrogen atoms of the two adjacent amino functions. Clitocine inhibited L1210 cells in vitro with an ID50 of 3 X 10(-8) M. Clitocine was also found to be a substrate and inhibitor of adenosine kinase with a Ki value of 3 X 10(-6) M.