A series of new cyclic phosphoramidate mustard-quinazoline conjugates were designed and synthesized based on the drug candidate EMB-3, a multi-target-directed ligand against tumor cells, and their anti-tumor activities were evaluated on breast cancer and lung cancer cells. Compound 6d exhibited the best anti-tumor performance with IC5o = 0.6 pM （8-fold of EMB-3） on BT474 breast tumor cells. Compound 6d inhibited epidermal growth factor receptor （EGFIL biomarker for NSCLC） and human epidermal growth factor 2 （HER2, biomarker for breast cancer） with IC50 of 18 nM and 78 nM, respectively. The preliminary pharmacokinetic study revealed that 6d was more stable than EMB-3 during the in vivo metabolism. A single dose per os （PO） administration of 6d in rat model （10 mg/kg） resulted in a moderate tl/2 of 1.7 h. These results indicated that compound 6d was a potential lead compound for the treatment of breast cancer.
As a synthesized antineoplastic organoselenium compound, ethaselen is known to induce apoptosis in tumor cells via dose-dependent thioredoxin reductase （TrxR） inhibition. Thioredoxin, the multifunctional biological substrate of TrxR, is then left in the oxidized state, which subsequently leads to intracellular accumulation of reactive oxygen species （ROS）, cell cycle arrest and/or apoptosis. However, the low dose effect of ethaselen remains largely unknown. Several subclones have been derived from HepG2 cells by using single cell or colony isolation. The low dose of ethaselen was defined as the drug concentration of retaining 〉90% HepG2 cells alive. The HepG2 cells were used as reference of its subclones （SM01, SM02 and SM03）, and the cell cycle transition, intracellular proteins change, colony formation and sphere growth were assayed in treatment of low dose ethaselen. HepG2 and its subclones differently responded to lethal dose of cisplatin or 5-fluorouracil. Low dose of ethaselen （1 μm） modulated the cell cycle transition at 12 h of treatment, but ceils were partially recovered at 24 h of treatment though some proteins were still affected. Low dose of ethaselen did not inhibit the small colony （diameter 〉 100 μm） formation and sphere growth of HepG2 and SM01. However, low dose of ethaselen could specifically inhibit the survival, large colony （diameter 〉500 μm） formation and sphere growth of SM03, although SM03 could be rapidly recovered from ethaselen-induced cell cycle check. HepG2 and its subclone cells could survive but respond differently to treatment of low dose ethaselen （1 μM）. Low dose of ethaselen could significantly inhibit a HepG2 subclone （SM03） in cell survival and colony growth.
