CCT251236

HSP72 ( IC50 = 19 nM )

CCT251236（0-100 nM；24 小时）抑制 HSF1 介导的 HSP72 诱导，pIC50=7.73 ± 0.07 (IC50=19 nM)，证明体外特性的适当平衡，同时保持优异的细胞活性。根据细胞测定中的游离分数，SK-OV-3 细胞中的游离 GI50 为 1.1 nM[1]。
CCT251236（0-100 nM；24 小时）抑制 HSF1 介导的热- SK-OV-3 细胞中休克蛋白 HSP72 和 HSP27 以浓度依赖性方式产生[1]。
CCT251236（0-100 nM；24 小时）抑制 17-AAG 对 HSPA1A mRNA 的剂量依赖性诱导用 250 nM 17-AAG 预处理 6 小时后[1]。

在无肿瘤的免疫活性 BALB/c 小鼠中，CCT251236（口服给药；5 或 20 mg/kg）的游离 Cav0-24h 值分别为 2.0 nM 和 1.2 nM [2]。
CCT251236（口服给药；20 mg/kg） /kg；33天）对小鼠表现出明确的治疗效果，基于最终肿瘤体积，肿瘤生长抑制（%TGI）为70%。 33 天后，与对照组相比，平均肿瘤重量减少了 64%。此外，肿瘤中含有浓度高达 940 nM 的 CCT251236，证明了该化合物的碱性和广泛的分布体积[2]。

蛋白质结合[1]
使用快速平衡透析法测量蛋白质结合。血浆取自雌性BALB/c和Ncr-Foxn1nu小鼠，并储存在-20°c下。细胞培养基为添加了10%FCS、2 mmol/L L-谷氨酰胺和1 x非必需氨基酸的DMEM。透析前，将红板、缓冲液、血浆和介质溶液加热至37°C。将DMSO中的试验化合物/化合物26（CCT251236）加入稀释的血浆（在100 mM磷酸盐缓冲液中稀释10倍）或细胞培养基中，使其浓度为5 PM，用于透析，含1%DMSO。将300PL的加标稀释血浆或培养基添加到RED板的供体侧，并将500PL的100mM磷酸盐缓冲液添加到受体孔中。用S70透气盖密封板，在37°C下摇动4小时进行透析。

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皮林表面等离子体共振（SPR）[1]
所有表面等离子体共振（SPR）实验都是在增强到T200灵敏度的Biacore T100上进行的。胺偶联化学用于将pirin蛋白固定在研究级CM5传感器芯片上。固定化步骤中使用的运行缓冲液由1 x磷酸盐缓冲盐水（10 S74 mM NaHPO4/NaH2PO4，pH 7.4，2.7 mM KCl，137 mM NaCl）组成，使用100 mM N-羟基琥珀酰亚胺和400 mM 1-乙基-3-（3-二甲基氨基丙基）-碳二亚胺的1:1混合物将芯片表面活化10分钟。在pH 5.5的10mM醋酸盐缓冲液中以约5µg/mL的浓度注射Pirin，并加入5µM三苯基化合物a，为活性位点赖氨酸提供保护。实时监测反应，当达到~5000RU的目标固定水平时停止反应。最后，通过在pH 8.5下注射1M乙醇胺7分钟来堵塞表面。在所有上述程序中，流速保持在10µL/min。流动池1保持不变，用作参考表面。

细胞系：SK-OV-3 细胞浓度：0 nM； 10 nM； 100 nM 孵育时间：24 小时 结果：10 nM 剂量抑制 HSP72 和 HSP27 表达。

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体外细胞活力测定[1]
CellTiter Blue活力（CTB）测定提供了一种均匀的荧光法来估算活细胞的数量。它使用深蓝色指示染料刃天青来测量细胞的代谢能力，这是细胞存活率的指标。活细胞能够将刃天青还原为高度荧光的resorufin（粉红色）。简而言之，将细胞（约6 x 103个细胞/mL）接种到384孔板中，孵育24小时。使用ECHO 550液体处理器加入化合物（浓度范围），然后在37℃下放置96小时。向每个孔中加入滴定蓝试剂，并在37℃放置3-4小时。使用Envision机器测量荧光。50%生长抑制浓度（GI50）是通过使用非线性回归将数据无限制地拟合到剂量反应曲线上来确定的。每种浓度测试两次。

Athymic mice with SK-OV-3 cells
20 mg/kg; 33 days
Oral adminstation

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Mouse Pharmacokinetics[1]
Female BALB/c and Ncr-Foxn1nu mice were adapted to laboratory conditions for at least 1 week prior to dosing and were S69 allowed food and water ad libitum. Compound 26 (CCT251236) was administered iv or po (0.1 mL/10g) in either 10 % DMSO, 5 % Tween 20 in saline (BALB/c PK) or 10 % DMSO in 25 % w/v hydroxypropyl beta cyclodextrin in 50 mM sodium citrate buffer (Ncr-Foxn1nu PK). Blood samples were collected from the tail vein (20 µL) at 8 time points over 24 h post dosing and spotted onto Whatman FTA-DMPK B cards (VWR) together with a calibration curve and quality controls spiked in control blood. Cards were allowed to dry at room temperature for at least 2 h. 6 mm discs were punched from the cards and extracted with 200 µL methanol containing 500 nM olomoucine as an internal standard. Following centrifugation, extracts were analyzed by multiple reaction monitoring of precursor and product ions by LC-ESIMS/MS on a QTRAP 4000 using a short gradient consisting of 0.1 % formic acid and methanol on a Phenomenex KinetexTM C18, 5 cm x 2.6 µm, 2.1 mm i.d UHPLC column. Pharmacokinetic parameters were derived from noncompartmental analysis using Phoenix (model 200 and 201) Pharsight WinNonlin® version 6.1/6.3.

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Mouse Tolerability Study [1]
Sterile Solvent Preparation The formulation (10 % DMSO, 90 % of a 25 % (2-hydroxypropyl)-E-cyclodextrin in 50 mM citrate buffer pH 5) was prepared by dissolving of citric acid monohydrate (10.5 g) and trisodium citrate dehydrate (14.8 g) in sterile water (500 mL, respectively). The citric acid solution (87.5 mL) was then added to the sodium citrate dehydrate solution (163 mL) to generate the pH 5 citrate buffer. (2-hydroxypropyl)-E-cyclodextrin (50 g, average MW~1460) was then added to the pH 5 citrate buffer (150 mL) and the pH measured to be 5.07. Compound 26 (CCT251236) was dissolved in DMSO (20.6 mL) and then added to the 25 % (2- hydroxypropyl)-E-cyclodextrin in 50 mM citrate buffer pH 5 (185 mL). The mixture was then sonicated to give a clear solution. Unused formulation was stored for up to 1 week at 5 o C. Compound 26 (CCT251236) was dissolved in 10 % DMSO and diluted in 90 % sterile solvent (25 % w/v hydroxypropyl β-cyclodextrin in 50 mM sodium citrate buffer pH 5) such that mice received the dose required in 0.1 mL of final solution per 10 g body weight. Controls received an equal volume of vehicle only. For multi-dose tolerability studies, female NCr athymic mice (n=2) were administered 30 mg/kg of Compound 26 (CCT251236) orally by gavage every day for five days. Animals were monitored for adverse effects and body weights were measured daily until full recovery was observed.

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Mouse Efficacy Study [1]
For efficacy studies, SK-OV-3 cells (5 million per site) were injected s.c. in the flanks of 6- to 8-week-old female NCr athymic mice (n=16). Dosing commenced when tumors were well established (~5-6 mm diameter). Tumors were measured with Vernier calipers across two perpendicular diameters and volumes were calculated as previously described.10 On study termination, heparinized blood samples were collected, and plasma was separated and stored S72 at -80 o C. Tumors were excised, weighed and snap frozen at -80 o C for subsequent PK and PD analyses.

Analysis of the mouse PK data revealed that bisamide 26 (CCT251236) possessed low total blood clearance (10% hepatic blood flow) and moderate oral bioavailability, with a half-life sufficient to allow once-daily dosing. In vitro assessment of the plasma protein binding and the blood to plasma ratio revealed that 26 was highly bound to plasma proteins (∼99%); therefore, the unbound clearance was high, with a low free exposure from the 5 mg/kg oral dose, equivalent to a free Cav0–24h = 2.0 nM. The high unbound volume of distribution indicates that 26 readily binds to tissues, consistent with the basicity of the compound.[1]

[1]. Discovery of a Chemical Probe Bisamide (CCT251236): An OrallyBioavailable Efficacious Pirin Ligand from a Heat ShockTranscription Factor 1 (HSF1) Phenotypic Screen. J Med Chem. 2017 Jan 12;60(1):180-201.

Phenotypic screens, which focus on measuring and quantifying discrete cellular changes rather than affinity for individual recombinant proteins, have recently attracted renewed interest as an efficient strategy for drug discovery. In this article, we describe the discovery of a new chemical probe, bisamide (CCT251236), identified using an unbiased phenotypic screen to detect inhibitors of the HSF1 stress pathway. The chemical probe is orally bioavailable and displays efficacy in a human ovarian carcinoma xenograft model. By developing cell-based SAR and using chemical proteomics, we identified pirin as a high affinity molecular target, which was confirmed by SPR and crystallography.[1]

C32H32N4O5

552.620287895203

552.24

C, 69.55; H, 5.84; N, 10.14; O, 14.48

1693731-40-6

117996749

White to off-white solid powder

4.8

102

2

7

8

41

884

0

KLHOCHQJHXNKAS-UHFFFAOYSA-N

InChI=1S/C32H32N4O5/c1-21-4-8-25(33-31(37)24-6-10-28-29(19-24)40-17-16-39-28)20-27(21)35-32(38)23-5-9-26-22(18-23)7-11-30(34-26)41-15-14-36-12-2-3-13-36/h4-11,18-20H,2-3,12-17H2,1H3,(H,33,37)(H,35,38)

N-[5-(2,3-dihydro-1,4-benzodioxine-6-carbonylamino)-2-methylphenyl]-2-(2-pyrrolidin-1-ylethoxy)quinoline-6-carboxamide

CCT251236; CCT 251236; CCT251236; 1693731-40-6; N-(5-(2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamido)-2-methylphenyl)-2-(2-(pyrrolidin-1-yl)ethoxy)quinoline-6-carboxamide; CHEMBL4087666; N-[5-(2,3-dihydro-1,4-benzodioxine-6-amido)-2-methylphenyl]-2-[2-(pyrrolidin-1-yl)ethoxy]quinoline-6-carboxamide; N-[5-(2,3-dihydro-1,4-benzodioxine-6-carbonylamino)-2-methylphenyl]-2-(2-pyrrolidin-1-ylethoxy)quinoline-6-carboxamide; SCHEMBL16621340; CCT-251236

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 150 mg/mL (~271.4 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (4.52 mM) (饱和度未知) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80+，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。