Rosowsky Andre, Forsch Ronald A, Wright Joel E
Dana-Farber Cancer Institute and Department of Biological Chemistry, Harvard Medical School, Boston, MA 02115, USA.
J Med Chem. 2004 Dec 30;47(27):6958-63. doi: 10.1021/jm040122s.
Heretofore unknown analogues of aminopterin (AMT) and methotrexate (MTX) in which free rotation of the amide bond between the phenyl ring and amino acid side chain is prevented by a CH(2) bridge were synthesized and tested for in vitro antifolate activity. The K(i) of the AMT analogue (9) against human dihydrofolate reductase (DHFR) was 34 pM, whereas that of the MTX analogue (10) was 2100 pM. Both compounds were less potent than the parent drugs. However, although the difference between AMT and MTX was <2-fold, the difference between 9 and 10 was 62-fold, suggesting that the effect of N(10)-methyl substitution is amplified in the bridged compounds. The K(i) values of 9 and 10 as inhibitors of [(3)H]MTX influx into CCRF-CEM human leukemia cells via the reduced folate carrier (RFC) were 0.28 and 1.1 muM, respectively. The corresponding K(i) and K(t) values determined earlier for AMT and MTX were 5.4 and 4.7 muM, respectively. Thus, in contrast to its unfavorable effect on DHFR binding, the CH(2) bridge increased RFC binding. In a 72 h growth assay with CCRF-CEM cells, the IC(50) values of 9 and 10 were 5.1 and 140 nM, respectively, a 27-fold difference that was qualitatively consistent with the observed combination of weaker DHFR binding and stronger RFC binding. Although rotationally restricted inhibitors of other enzymes of folate pathway enzymes have been described previously, 9 and 10 are the first reported examples of DHFR inhibitors of this type.
合成了氨甲蝶呤（AMT）和甲氨蝶呤（MTX）的此前未知类似物，其中苯环与氨基酸侧链之间的酰胺键通过-CH₂-桥被阻止自由旋转，并对其进行了体外抗叶酸活性测试。AMT类似物（9）对人二氢叶酸还原酶（DHFR）的抑制常数（Kᵢ）为34 pM，而MTX类似物（10）的Kᵢ为2100 pM。这两种化合物的活性均低于母体药物。然而，尽管AMT和MTX之间的差异小于2倍，但9和10之间的差异为62倍，这表明N(10)-甲基取代的影响在桥连化合物中被放大。9和10作为[(³)H]MTX通过还原型叶酸载体（RFC）流入CCRF-CEM人白血病细胞的抑制剂，其Kᵢ值分别为0.28和1.1 μM。此前测定的AMT和MTX相应的Kᵢ和Kₜ值分别为5.4和4.7 μM。因此，与对DHFR结合的不利影响相反，-CH₂-桥增加了RFC结合。在对CCRF-CEM细胞进行的72小时生长试验中，9和10的半数抑制浓度（IC₅₀）值分别为5.1和140 nM，相差27倍，这在定性上与观察到的较弱的DHFR结合和较强的RFC结合的组合一致。尽管此前已描述了叶酸途径其他酶的旋转受限抑制剂，但9和10是此类DHFR抑制剂的首个报道实例。
