HSF1 (heat shock factor 1)

NXP800（实施例169）（IC50=0.056μM）降低U20S细胞的活力。

药代动力学分析 [2] 物种途径 剂量 (mg/kg) Tmax (h) AUClast (ng·h/mL) Cltb (mL/min/kg) t1/2 (h) F (%) AUCu0-t ( h·nM) ) 游离 Cav0-24h (nM) Clu (mL/min/kg) 大鼠 口服/IV 5/1 6.0 2600（口服） 24 (iv) 3.1 45（口服） 86 3.7 730 (iv) 狗 口服/IV 2.5/0.5 2.0 250（口头） 21（IV） 1.4 9.1（PO） 35 1.9 150（IV）

CH1doxR/CH1wt多药耐药性检测[2]
使用之前使用CellTiter Blue活力测定法描述的相同方法，在CH1wt和CH1doxR细胞中评估了化合物如NXP-800（CCT361814）的抗增殖活性。通过用2µM（R）-（+）-维拉帕米单盐酸盐水合物处理细胞，证实了CH1doxR细胞系抗增殖活性的挽救(http://www.sigmaaldrich.com/catalog/product/sigma/v106?lang=en®ion=GB，2017年2月）和双酰胺类似物。然后使用Student t检验和Welch校正比较CH1wt和CH1doxR细胞中每个细胞中至少n=3个生物重复的几何平均pGI50值（pGI50=-log GI50（M））；当p<0.05时，该化合物被认为是MDR底物（GraphPad Prism 7.01）。CH1doxR和CH1wt中几何平均GI50s的比率被定义为MDR比率[2]。

体外细胞活力测定[2]
CellTiter Blue活性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，并孵育24小时。使用ECHO 550液体处理器加入不同浓度的化合物（例如NXP-800（CCT361814）），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℃上放置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[2]。

In vivo Studies [2]
Compound 22 (NXP-800 (CCT361814)) 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, NCr athymic mice (n=2 per cohort) were administered 50 mg/kg or 100 mg/kg of compound 22 (NXP-800 (CCT361814)) orally every day for five days. Mice were monitored for signs of distress and body weights were measured daily until full recovery was observed. Dosing at 100 mg/kg of compound 22 (NXP-800 (CCT361814)) was not tolerated and, therefore, was terminated at day 4. 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=20). Dosing commenced when tumors were well established (~5-6 mm diameter). Tumor volumes were determined as previously described. On study termination, blood samples were taken, and plasma was separated and stored at -80 C. [2]
CHAC1 Western Blot and MSD Assays Tumors were snap frozen in liquid nitrogen and stored at -80 oC until processed. Tumors were lysed in 50 mM Tris-HCl (pH 7.4), 1 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 mM NaF, 1 mM sodium vanadate (activated), 10 µg/mL Nα-tosyl-L-lysine chloromethyl ketone hydrochloride, 5 µM fenvalerate, 5 µM bpVphen, 1 mM phenylmethanesulfonyl fluoride, 1:100 protease cocktail and 1:50 of phosphatases inhibitor 2 and 3. Protein concentration was determined by Direct Detect® Infrared Spectrometer. Each lysate was separated by SDS-PAGE, electrotransferred onto PVDF membranes, blocked with 5% milk and probed with specific primary antibody CHAC1 (1:100 dilution) and horseradish peroxidase-conjugated secondary (1:1000) antibody. Signal was detected with enhanced chemiluminescence reagent. Glyceraldehyde-3-phosphate dehydrogenase (1:20000 dilution) was used as the loading control. All reagents were purch

The mouse in vivo CLu for compound 22/NXP-800 (CCT361814) was consistent with the predicted value from the MHeps assay and comparable to methyl analogue 16 (Table 2, entry 1). Despite the decreased lipophilicity, the CH1doxR/CH1WT-predicted P-gp-mediated efflux ratio was low and fluorobisamide 22 displayed good mouse oral bioavailability (42%) from moderate total blood clearance (CLtb = 10 mL/min/kg, extraction ratio = 11%, Fmax = 89%).22 Owing to these favorable data, fluorobisamide 22 was selected for evaluation of its in vivo efficacy against established SK-OV-3 human ovarian cancer solid tumor xenografts in athymic immunodeficient mice (Table 5).[1]
The fluorine MMP, compound 22/NXP-800 (CCT361814), pleasingly displayed the desired reduction in lipophilicity (Table 3, entry 6), which correlated with reduced in vitro MLM (15 μL/min/mg) and mouse hepatocyte CLint; while maintaining excellent antiproliferative activity (free GI50 = 3.7 nM, fua = 0.43; Table S4)39 and acceptable KS (50 μM). Fluorobisamide 22 was therefore submitted for an in vivo mouse PK study (Table 4, entry 1).

[1]. Fused 1,4-dihydrodioxin derivatives as inhibitors of heat shock transcription factor 1. WO2015049535A1.

[2]. HSF1 Pathway Inhibitor Clinical Candidate (CCT361814/NXP800) Developed from a Phenotypic Screen as a Potential Treatment for Refractory Ovarian Cancer and Other Malignancies. J Med Chem. 2023 Apr 27;66(8):5907-5936.

CCT251236 1, a potent chemical probe, was previously developed from a cell-based phenotypic high-throughput screen (HTS) to discover inhibitors of transcription mediated by HSF1, a transcription factor that supports malignancy. Owing to its activity against models of refractory human ovarian cancer, 1 was progressed into lead optimization. The reduction of P-glycoprotein efflux became a focus of early compound optimization; central ring halogen substitution was demonstrated by matched molecular pair analysis to be an effective strategy to mitigate this liability. Further multiparameter optimization led to the design of the clinical candidate, CCT361814/NXP800 22, a potent and orally bioavailable fluorobisamide, which caused tumor regression in a human ovarian adenocarcinoma xenograft model with on-pathway biomarker modulation and a clean in vitro safety profile. Following its favorable dose prediction to human, 22 has now progressed to phase 1 clinical trial as a potential future treatment for refractory ovarian cancer and other malignancies.[2]

C32H32FN5O4

569.63

569.243

C, 67.47; H, 5.66; F, 3.34; N, 12.29; O, 11.23

1693734-80-3

1693734-80-3;

117996795

Light yellow to green yellow solid powder

3.7

96

2

8

7

42

919

0

N1C2C(=CC(C(NC3=CC(NC(C4=CC=C5OCCOC5=C4)=O)=CC=C3F)=O)=CC=2)C=CC=1CN1CCN(CC)CC1

UBALMDIKIGDHJW-UHFFFAOYSA-N

InChI=1S/C32H32FN5O4/c1-2-37-11-13-38(14-12-37)20-25-6-3-21-17-22(4-9-27(21)34-25)32(40)36-28-19-24(7-8-26(28)33)35-31(39)23-5-10-29-30(18-23)42-16-15-41-29/h3-10,17-19H,2,11-16,20H2,1H3,(H,35,39)(H,36,40)

N-[5-(2,3-dihydro-1,4-benzodioxine-6-carbonylamino)-2-fluorophenyl]-2-[(4-ethylpiperazin-1-yl)methyl]quinoline-6-carboxamide

CCT-361814; NPX800; CCT 361814; SCHEMBL16621389; NXP-800; BDBM610359; CCT361814; NPX 800; NPX-800

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 : ~100 mg/mL (~175.55 mM)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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。