Rosowsky A, Forsch R A, Galivan J, Susten S S, Freisheim J H
Mol Pharmacol. 1985 Jan;27(1):141-7.
Regiospecific syntheses of gamma- and alpha-conjugates of methotrexate and poly(L-lysine) are described. The alpha- and gamma-t-butyl esters, respectively, of methotrexate were coupled to poly(L-lysine) with diphenylphosphoryl azide in N,N-dimethylformamide, the ester-protecting group was cleaved with 15% hydrogen bromide in acetic acid, and small molecules were removed by dialysis. Poly(L-lysine) of Mr = 1,500-8,000 and 8,000-30,000 was used to prepare six different conjugates, which were characterized by ultraviolet absorbance measurement and quantitative amino acid analysis. The degree of substitution varied from one methotrexate per 4.7 lysines to one methotrexate per 10.2 lysines. Dihydrofolate reductase inhibition in a cell-free assay was observed with alpha- and gamma-conjugates, but the latter had the greater affinity (only 3-fold less than that of methotrexate itself). The binding of the conjugates exhibited a slight pH dependence, with affinity being greater at pH 7.2 than at pH 8.5 for both alpha- and gamma-conjugates. Toxicity to cultured rat hepatoma cells (H35) was also greater for the gamma-conjugates, and showed some dependence on the chain-length and degree of substitution of the poly(L-lysine) carrier. Cells resistant to methotrexate by virtue of a transport defect (H35R0.3 line) retained their sensitivity to the gamma-conjugate, but less so to the alpha-conjugate. There was also some retention of sensitivity in a more highly resistant cell line (H35R10) with impaired methotrexate transport and a concomitant increase in dihydrofolate reductase activity. gamma-Conjugation was likewise more favorable in cytotoxicity assays against L1210 murine leukemia cells, and there was partial retention of activity against highly methotrexate-resistant lines (L1210/R71 and L1210/R81) with a transport defect and/or an elevation of dihydrofolate reductase content. In antitumor assays against intraperitoneal L1210 leukemia in mice, a gamma-conjugate with Mr = 8,000-30,000 and one methotrexate per 5.5 lysines produced a 35-75% increase in lifespan when administered intraperitoneally at single doses equivalent to 10-20 mg/kg of methotrexate. A similar increase in lifespan with methotrexate alone on the single-dose regimen required 50-150 mg/kg. An alpha-conjugate of similar Mr and degree of substitution was inactive at nontoxic doses, as were other gamma-conjugates of lower Mr and/or degree of substitution.(ABSTRACT TRUNCATED AT 400 WORDS)
本文描述了甲氨蝶呤与聚（L - 赖氨酸）的γ - 和α - 共轭物的区域特异性合成。分别将甲氨蝶呤的α - 和γ - 叔丁酯与聚（L - 赖氨酸）在N，N - 二甲基甲酰胺中用叠氮基二苯基膦酰偶联，酯保护基团用15%的氢溴酸在乙酸中裂解，小分子通过透析除去。使用分子量为1500 - 8000和8000 - 30000的聚（L - 赖氨酸）制备了六种不同的共轭物，通过紫外吸光度测量和定量氨基酸分析对其进行表征。取代度从每4.7个赖氨酸一个甲氨蝶呤到每10.2个赖氨酸一个甲氨蝶呤不等。在无细胞试验中观察到α - 和γ - 共轭物对二氢叶酸还原酶有抑制作用，但后者具有更高的亲和力（仅比甲氨蝶呤本身低3倍）。共轭物的结合表现出轻微的pH依赖性，对于α - 和γ - 共轭物，pH 7.2时的亲和力均大于pH 8.5时的亲和力。γ - 共轭物对培养的大鼠肝癌细胞（H35）的毒性也更大，并且对聚（L - 赖氨酸）载体的链长和取代度有一定依赖性。因转运缺陷而对甲氨蝶呤耐药的细胞（H35R0.3系）对γ - 共轭物仍保持敏感性，但对α - 共轭物的敏感性较低。在甲氨蝶呤转运受损且二氢叶酸还原酶活性同时增加的更高耐药细胞系（H35R10）中也有一定的敏感性保留。在针对L1210小鼠白血病细胞的细胞毒性试验中，γ - 共轭同样更有利，并且对具有转运缺陷和/或二氢叶酸还原酶含量升高的高度耐甲氨蝶呤细胞系（L1210/R71和L1210/R81）仍有部分活性保留。在针对小鼠腹腔内L1210白血病的抗肿瘤试验中，分子量为8000 - 30000且每5.5个赖氨酸一个甲氨蝶呤的γ - 共轭物，以相当于10 - 20 mg/kg甲氨蝶呤的单剂量腹腔注射时，可使寿命延长35 - 75%。在单剂量方案中，单独使用甲氨蝶呤使寿命有类似延长则需要50 - 150 mg/kg。具有相似分子量和取代度的α - 共轭物在无毒剂量下无活性，其他分子量较低和/或取代度较低的γ - 共轭物也是如此。（摘要截于400字）
