RESUMEN
The synthesis of 16a'-homo-leurosidine was achieved through enantioselective generation of a ring D'-seco-precursor 33 (without requirement of a chiral auxiliary). Its cyclization provided the N(b')-quaternary salt 35 with a configuration corresponding to the atropisomeric form 8a rather than 8b of the target product. On debenzylation, the amine 8a was obtained and found not to isomerize thermally to the anticipated atropisomer 8b (in contrast to its lower homologue, with its formation of natural leurosidine). However, on protonation, a 1:1 mixture of atropisomers of 16a'-homo-leurosidine was obtained. A synthesis of 16a'-homo-vinblastine provided two atropisomers 5a and 5b for the free base at equilibrium (1:2.3 at room temperature in CDCl(3)), with a shift to the major conformer 5b with increasing solvent acidity or decreasing temperature. The synthesis was achieved through a stereoselective inversion of the tertiary hydroxyl function in the enantioselectively generated C-20' progenitor 39.
Asunto(s)
Alcaloides/síntesis química , Antineoplásicos Fitogénicos/síntesis química , Vinblastina/síntesis química , Alcaloides de la Vinca , Alcaloides/química , Alcaloides/farmacología , Animales , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/farmacología , Ratones , Conformación Molecular , Células Tumorales Cultivadas , Vinblastina/análogos & derivados , Vinblastina/química , Vinblastina/farmacologíaRESUMEN
The objective of this study was to determine the effects of nitric oxide (NO) on lymphocyte proliferation and cytokine release. Bronchoalveolar lavage (BAL) cells served as the source of NO and were obtained from rats treated with a single, intratracheal dose of bleomycin (3.6 mg/kg). At the time of sacrifice, the spleens were removed and the lymphocytes separated. Co-cultures containing BAL cells, lymphocytes and concanavalin-A were established and incubated at 37 degrees C for 24 hours at which time proliferation, nitrite concentration and interleukin-2 (IL-2) production were measured. At ratios from 5:1 to 1:4 (BAL:lymphocyte) there was a significant reduction in lymphocyte proliferation. There was a significant, negative correlation between NO concentration and thymidine incorporation which was reversed when the NO synthase inhibitor NG-monomethyl-L-arginine (NMA) was added to the co-cultures. Despite marked inhibition of spleen lymphocyte proliferation by NO, released by BAL cells, there was no corresponding reduction in IL-2 production. These data demonstrate that macrophages, activated in vivo, produce NO which regulates lymphocyte growth but not necessarily functions such as the secretion of the cytokine IL-2. Further, the ability of IL-2-dependent CTLL-2 cells to proliferate in the presence of excess IL-2 was also inhibited by BAL cells, confirming that NO inhibits lymphocyte growth.
Asunto(s)
Interleucina-2/metabolismo , Activación de Linfocitos/efectos de los fármacos , Linfocitos/efectos de los fármacos , Óxido Nítrico/farmacología , Aminoácido Oxidorreductasas/antagonistas & inhibidores , Animales , Arginina/análogos & derivados , Arginina/farmacología , División Celular/efectos de los fármacos , Células Cultivadas , Concanavalina A/farmacología , Interleucina-1/biosíntesis , Linfocitos/metabolismo , Masculino , Óxido Nítrico Sintasa , Ratas , Ratas Sprague-Dawley , Bazo/citología , Bazo/inmunología , omega-N-MetilargininaRESUMEN
Alveolar macrophages, taken from rats treated with a single intratracheal dose of bleomycin, release reactive nitrogen intermediates in the form of nitric oxide which are cytostatic to murine leukemia L1210 cells. When cultured in the presence of erythrocytes the cytostatic activity of alveolar macrophages was inhibited which corresponded with an increase in nitrosylated hemoglobin content when compared with erythrocytes cultured alone. These results suggest that erythrocytes inhibit alveolar macrophage cytostatic activity by preventing reactive nitrogen intermediates from reaching target cells because the hemoglobin serves as a sink for reactive nitrogen intermediates in the form of nitric oxide.
Asunto(s)
Bleomicina/farmacología , Eritrocitos/fisiología , Hemoglobinas/metabolismo , Macrófagos Alveolares/fisiología , Óxido Nítrico/metabolismo , Animales , Arginina/análogos & derivados , Arginina/farmacología , Comunicación Celular , Células Cultivadas , Eritrocitos/citología , Inflamación , Leucemia L1210/patología , Macrófagos Alveolares/citología , Macrófagos Alveolares/efectos de los fármacos , Masculino , Ratones , Nitritos/metabolismo , Ratas , Ratas Endogámicas , Espectrofotometría , omega-N-MetilargininaRESUMEN
Bleomycin (BLM) is a useful anticancer agent sometimes associated with a diffuse pulmonary inflammation and fibrosis. Using an intratracheal model of BLM-induced pulmonary damage, we have further investigated alveolar macrophage (AM) activation following intratracheal BLM. From rats that had been treated with either a single, fibrogenic, intratracheal dose of BLM (BLM-AM) or a comparable volume of saline (C-AM), bronchoalveolar lavage fluid was collected, and AM were isolated using Percoll gradient centrifugation. Using a spectrophotometric assay, production of nitrites by AM was measured. C-AM released low levels of nitrites, whereas BLM-AM as well as C-AM activated in vitro with lipopolysaccharide released significant amounts of nitrites. The addition of N6-monomethylarginine, a substrate-specific inhibitor of the L-arginine-dependent effector mechanism in activated macrophages, reduced the amount of measurable nitrites released from both BLM-AM and activated C-AM. Similar results were observed when 12 x 10(6) RBC were added to the cocultures. In the presence of N6-monomethylarginine, BLM-AM had no effect on two consequences of BLM-AM-induced cytostatic activity, DNA synthesis inhibition and aconitase activity reduction in the L1210 target cell. These results suggest that reactive nitrogen intermediates measured as nitrites are important moieties in our in vivo model of macrophage activation. Further, the identification of this effector molecule presents possibilities for therapeutic and biochemical manipulations.
Asunto(s)
Bleomicina/toxicidad , Replicación del ADN/efectos de los fármacos , Pulmón/patología , Activación de Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Nitrilos/metabolismo , Nitritos/metabolismo , Aconitato Hidratasa/metabolismo , Animales , Arginina/análogos & derivados , Arginina/farmacología , Citotoxicidad Inmunológica , Cinética , Leucemia L1210/inmunología , Leucemia L1210/metabolismo , Pulmón/efectos de los fármacos , Macrófagos/efectos de los fármacos , Masculino , Ratones , Ratas , Ratas Endogámicas , Valores de Referencia , omega-N-MetilargininaRESUMEN
Bleomycin (BLM) has been successfully used to treat a number of human neoplasms. The main toxicity associated with BLM therapy is an acute pulmonary inflammation that can culminate in diffuse chronic fibrosis. The effect of BLM-induced pulmonary inflammation on the cytostatic activity of alveolar macrophages (AM) was investigated using AM obtained from rats that had been previously treated with BLM. Bronchoalveolar lavage fluid was collected at selected time intervals following a single fibrogenic dose of intratracheally administered BLM (3.6 mg/kg). AM obtained 12 to 72 h following intratracheal BLM (BLM-AM) caused cytostasis of murine leukemia L1210 cells in co-culture, whereas AM obtained from saline-treated controls were not cytostatic. These results indicate that the growth-inhibitory activity of the AM was related to the pulmonary inflammation. Cytostatic activity in control AM could be induced by in vitro exposure to lipopolysaccharide (5 micrograms). When RBC were added to the AM-L1210 co-culture, the cytostatic activity of the BLM-AM was abrogated. The fact that chemical treatment of the RBC with sodium nitrite and potassium cyanide or N-ethylmaleimide did not alter the ability of the RBC to abrogate AM cytostatic activity suggests that the RBC is not acting as a scavenger of oxygen radicals. In contrast, the addition of FeSO4 to the AM-L1210 co-culture mimicked the effect of RBC addition. Aconitase, an iron-sulfur-containing enzyme necessary for mitochondrial respiration, is decreased in L1210 cells that have been co-cultured with BLM-AM but not when the co-cultures also contain RBC. These results suggest that (a) pulmonary inflammation induces cytostatic activity in AM, (b) the alteration of iron homeostasis plays an important role in this cytostatic process, and (c) RBC can prevent this cytostatic activity.