ASP-9521 is a selective, potent and orally active indole-based AKR1C3 inhibitor with an IC50 of 11 nM for human AKR1C3.

AKR1C3:11 nM

方法：将H9c2细胞在ASP9521（25μM）存在下预孵育3小时。然后，在接下来的 24 小时内添加 DOX/DNR。使用 MTT 测定法测定细胞的活力。
结果：用DOX处理的H9c2细胞的存活率为79%，而用ASP9521预孵育的H9c2细胞的存活率为93%。ASP9521几乎完全保护了心肌细胞免受DOX的毒性活性。[2]
方法： ASP9521（0.1， 1， 10， 25， 50， 100 μM）和 DNR （0.05–1 μM）处理人肺癌细胞系 A549细胞，计算并比较了单独使用或与 ASP9521 (25 μM) 组合使用的 DNR 的 IC50。
结果：获得的 IC50 值为 0.442 μM（单独使用DNR ）和 0.379 μM（与 ASP9521 组合使用）。[2]

ASP9521 以浓度依赖性方式抑制重组人或食蟹猴 AKR1C3 将 AD 转化为 T（IC50，人：11 nM；IC50，猴：49 nM）;ASP9521 对 AKR1C3 的选择性比对同工型 AKR1C2 的选择性高 100 倍以上;在 CWR22R 异种移植中，单次口服 ASP9521（3 mg/kg）可抑制 AD 诱导的肿瘤内 T 产生，并且这种抑制作用持续 24 小时;口服后，ASP9521 迅速从血浆中消除，而其肿瘤内浓度仍然很高;口服（1 mg/kg）ASP9521 在大鼠、狗和猴子中的生物利用度分别为 35%、78% 和 58%。[1]

LNCaP-AKR1C3 cells stably expressing human AKR1C3 are seeded in 96-well plates at 10000 cells/100 μL/well in RPMI-1640 medium supplemented with heat-inactivated charcoal-dextran-stripped FBS (1 % for the PSA expression assay and T measurement and 5 % for the cell proliferation assay). After 24 h incubation, AD is added to each well with or without ASP-9521 (0.3-100 nM). The cell culture media are collected 24 h after administration of AD to measure T concentration and 6 days after administration of AD to measure cell proliferation using Cell-Titer Glo assay.

ASP-9521 is prepared in 0.5 % methyl cellulose.Mice carrying HEK293 or HEK293-AKR1C3 tumors with similar sizes are selected and randomly divided into 5 groups (N=3 for each group). All groups are treated with ASP-9521 (single oral administration; 3 mg/kg). Plasma (from the central vein) and tumor tissues are collected at 0.25, 0.5, 1, 2 and 4 h after administration of ASP-9521, and ASP-9521 concentrations are determined using the HPLCMS/MS method.