Hay Michael P, Pruijn Frederik B, Gamage Swarna A, Liyanage H D Sarath, Kovacs Mary S, Patterson Adam V, Wilson William R, Brown J Martin, Denny William A
Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand.
J Med Chem. 2004 Jan 15;47(2):475-88. doi: 10.1021/jm030399c.
Tirapazamine (TPZ, 1,2,4-benzotriazin-3-amine 1,4-dioxide) is a bioreductive hypoxia-selective cytotoxin, currently in phase II/III clinical trials in combination with radiotherapy and with cisplatin-based chemotherapy. We have prepared a series of 1,2,4-benzotriazine 1,4-dioxide (BTO) analogues of TPZ where a DNA-targeting chromophore is attached at the 3-position via a flexible linker. DNA binding affinity was modified through variation of the chromophore or the pK(a) of the linker chain. The association constants (K(DNA)) for calf thymus DNA ranged from 1 x 10(2) to 5.6 x 10(5) M(-1) (ionic strength of 0.01 M). DNA binding affinity was dependent on the presence of a positive charge, either in the linker chain or in the chromophore, and (for a series of 4-acridine carboxamide chromophore analogues) correlated strongly with linker chain pK(a). The efficacy of these BTOs in killing aerobic and hypoxic mouse SCCVII tumor cells in vitro was determined by clonogenic survival. Cytotoxicity was measured as the concentration required to reduce plating efficiency to 10% of controls (C(10)), and the hypoxic cytotoxicity ratio (HCR) for each BTO was calculated as C(10)(aerobic)/C(10)(hypoxic). BTOs bearing a positive charge showed increased hypoxic cytotoxicity (1.5-56-fold) compared to TPZ and mostly modest HCRs (8-51), but some excellent (>167 and 400) values. There was a strong correlation between K(DNA) and hypoxic cytotoxicity but no correlation between K(DNA) and HCR. Cytotoxicity in HT-29 human colon carcinoma cells, determined using IC(50) assays, showed similar relationships with a correlation between K(DNA) and hypoxic cytotoxicity but no correlation between K(DNA) and HCR. In this cell line, a higher proportion of compounds (7 of 11) had HCRs greater than or equal to that of TPZ. The data confirm that DNA targeting is a useful concept for increasing potency while maintaining hypoxic selectivity and provide a direction for the further development of DNA-targeted analogues of TPZ.
替拉扎明（TPZ，1,2,4-苯并三嗪-3-胺1,4-二氧化物）是一种生物还原型缺氧选择性细胞毒素，目前正处于与放疗以及基于顺铂的化疗联合使用的II/III期临床试验阶段。我们制备了一系列TPZ的1,2,4-苯并三嗪1,4-二氧化物（BTO）类似物，其中一个靶向DNA的发色团通过柔性连接子连接在3位。通过改变发色团或连接子链的pK(a)来修饰DNA结合亲和力。小牛胸腺DNA的结合常数（K(DNA)）范围为1×10²至5.6×10⁵ M⁻¹（离子强度为0.01 M）。DNA结合亲和力取决于连接子链或发色团中是否存在正电荷，并且（对于一系列4-吖啶甲酰胺发色团类似物）与连接子链pK(a)密切相关。这些BTOs在体外杀死需氧和缺氧小鼠SCCVII肿瘤细胞的功效通过克隆形成存活率来确定。细胞毒性以将接种效率降低至对照的10%所需的浓度（C(10)）来衡量，每个BTO的缺氧细胞毒性比（HCR）计算为C(10)(需氧)/C(10)(缺氧)。带有正电荷的BTOs与TPZ相比显示出增加的缺氧细胞毒性（1.5至56倍），并且大多数具有适度的HCRs（8至51），但也有一些优异的值（>167和400）。K(DNA)与缺氧细胞毒性之间存在强相关性，但K(DNA)与HCR之间无相关性。使用IC(50)测定法在HT-29人结肠癌细胞中测定的细胞毒性显示出类似的关系，即K(DNA)与缺氧细胞毒性之间存在相关性，但K(DNA)与HCR之间无相关性。在该细胞系中，更高比例的化合物（11种中的7种）的HCRs大于或等于TPZ的HCRs。数据证实，靶向DNA是在保持缺氧选择性的同时提高效力的有用概念，并为TPZ的靶向DNA类似物的进一步开发提供了方向。
