Most of the tested heterosteroids showed remarkable anticancer activity against ERα-positive MCF-7 cancer cells. Estranes 3a,b containing the D-ring-fused pyrimidine moiety proved to be inactive, while their androstene analogs 3c–e were active with the IC50 values in the range of 12.0–21.6 μM. It is remarkable that the IC50 value for compound 3d containing the 3-OH group is higher than that for compound 3c possessing the 3-OAc protected group. The solubility of steroidal A-ring annulated pyrimidine 3f in DMSO is too low to perform the MTT assay.