666-15 is a selective CREB inhibitor with an IC50 of 81 nM. 666-15 can effectively inhibit the growth of breast cancer.

CREB:81 nM (in HEK 293T cells)

方法：666-15浓度为62.5至500nM处理OA 样软骨细胞，研究 666-15 对 OA 样老年豚鼠软骨细胞的活力。
结果：p-CREB1蛋白水平显著降低，并显著提高老年豚鼠软骨细胞的细胞活力。[1]
方法：为了研究CREB抑制的生物学效应，将SySa细胞与浓度增加（0.13-5μM）的666-15一起孵育并进行在MTT测定。
结果：666-15以剂量依赖性方式显着抑制SySa细胞活力（IC50：0.36-1.72 μM）。[2]

方法：小鼠被随机分配到不同组别，包括正常组、假手术组、ACLT+溶剂对照组、以及两个不同剂量的666-15处理组（ACLT+666-15低剂量组，5 mg/kg；ACLT+666-15高剂量组，10 mg/kg,腹腔注射，每周一次，六周）。
结果：666-15治疗能缓解关节退变，降低软骨中降解标志物如CTX-II的水平，减轻滑膜炎症，且不影响小鼠体重，表明其安全性。[1]

HEK 293T cells in a 10 cm plate were transfected with pCRE-RLuc (6 μg) with Lipofectamine2000 following the manufacturer's instructions. Three hours after transfection, the cells were collected and replated into 96-well plates at ～10?000 cells/well. The cells were allowed to attach to the bottom of the plates overnight. The cells were then treated with different concentrations of different compounds for 30 min when forskolin (10 μM) was added to each well. The cells were incubated for further 5 h before cell lysis using 1× 30 μL Renilla luciferase lysis buffer. An amount of 5 μL of the lysate was combined with 30 μL of benzyl-coelenterazine solution in PBS (pH 7.4, 10 μg/mL). The protein concentration in each well was determined. The Renilla luciferase activity was normalized to protein content in each well and expressed as relative luciferase unit/μg protein (RLU/μg protein). The IC50 was derived from nonlinear regression analysis of the RLU/μg protein–concentration curve [1].

Each 6- to 8-week old BALB/c nude mouse was inoculated subcutaneously at the right flank with MDA-MB-468 cells (5 × 10^6) in 0.1 mL of HBSS with Matrigel (1:1) for tumor development. When the tumor volume reached approximately 100 mm3, the mice were randomized to be treated with either vehicle or 666-15 at 10 mg/kg. 3i was dissolved in 1% N-methylpyrrolidone (NMP), 5% Tween-80 in H2O. The dosing solution was prepared weekly. The mice were treated once a day for 5 consecutive days a week, and the treatment lasted for 5 weeks. During the treatment, the tumor size and body weight were measured 2–3 times a week. The tumors were measured in two dimensions using a digital caliper, and the volume was expressed in mm3 using the formula V = 0.5ab2, where a and b represent the long and short diameters of the tumor, respectively. The tumor volume was normalized to the initial tumor volume at the time of the first treatment [1].