HSF1 (heat shock factor 1)
Heat shock factor 1 (HSF1) pathway inhibitor. The compound binds to pirin (putative transcription factor regulator) with a K_D of 44 nM [2].

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

---

CCT361814/NXP800 对一组9种遗传背景不同的人卵巢癌细胞系表现出强效的抗增殖活性（几何平均pGI50 >7.3）。其对SK-OV-3细胞的游离GI50为3.7 nM (fu,a = 0.43) [2]。
在一项替代生物标志物实验中，CCT361814/NXP800 在SK-OV-3细胞中阻断了HSP90抑制剂17-AAG诱导的HSP72表达，IC50为94 nM (pIC50 = 7.03 ± 0.07, n=40)，证实了其对HSF1通路的拮抗作用 [2]。
在经CCT361814/NXP800处理的癌细胞系和肿瘤异种移植物中进行的基因表达谱分析显示，热整合应激反应基因特征受到抑制，而相关的整合应激反应特征被激活。具体来说，处理增加了CHAC1 mRNA的表达，降低了HSPA1A mRNA的表达 [2]。
基于细胞的实验表明，CCT361814/NXP800 的P-糖蛋白（P-gp）介导的外排风险较低，表现为较低的CH1^doxR/CH1^WT外排比 [2]。

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）

---

在携带已建立的SK-OV-3人卵巢癌实体瘤异种移植物的免疫缺陷无胸腺裸鼠模型中，以35 mg/kg的剂量每日一次口服给予CCT361814/NXP800，连续20天，显示出显著的抗肿瘤活性。相对于对照组，肿瘤生长抑制率（TGI）为120%，10个肿瘤中有8个出现消退。基于最终平均肿瘤重量的T/C值为对照组的37% (p = 0.0008) [2]。
在同一模型中进行单剂量PK/PD研究（50 mg/kg 口服）表明，CCT361814/NXP800的游离肿瘤浓度超过其体外游离GI50的时间达21小时。肿瘤中PD生物标志物CHAC1蛋白的诱导和HSPA1A mRNA的减少与肿瘤游离药物浓度相关，证实了其对靶点通路的调节作用 [2]。

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]。

---

针对87个潜在高风险脱靶蛋白（Cerep面板）进行了竞争性结合实验。CCT361814/NXP800 显示出对腺苷A2A受体的拮抗作用，IC50为2.0 µM，这约比其有效游离浓度高100倍 [2]。
评估了细胞色素P450（CYP）抑制活性。CCT361814/NXP800 未显示出显著的CYP抑制（IC50 > 10 µM） [2]。
评估了hERG通道抑制活性。CCT361814/NXP800 未显示出hERG毒性（IC50 > 30 µM） [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]。

---

使用CellTiter-Blue细胞活力测定法确定抗增殖活性（GI50）。将细胞接种在96孔板中，用化合物处理96小时，然后测量荧光强度。通过拟合log[抑制剂]与反应-可变斜率（四参数）模型来估算GI50值 [2]。
使用配对的卵巢癌细胞系（CH1^WT和获得性多柔比星耐药的CH1^doxR）建立基于细胞的实验，作为评估P-gp介导外排的替代方法。计算CH1^doxR和CH1^WT细胞之间几何平均GI50值的倍数差异。显著性差异（p < 0.05，学生t检验）表明具有P-gp底物活性 [2]。
对于HSF1通路抑制生物标志物实验，用HSP90抑制剂17-AAG处理SK-OV-3细胞以诱导HSF1活性和随后的HSP72表达。与CCT361814/NXP800共处理可阻断这种诱导作用。使用基于细胞的ELISA法定量HSP72蛋白水平 [2]。
通过免疫印迹法证实，在经CCT361814/NXP800处理（19 nM，5倍游离GI50）的SK-OV-3细胞中，CHAC1蛋白被诱导表达 [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

---

For efficacy studies in immunodeficient athymic mice, SK-OV-3 human ovarian cancer cells were implanted subcutaneously. When tumors reached a designated size, mice were randomized into groups. CCT361814/NXP800 was administered orally (po) once daily (qd) as a solution at 35 mg/kg for 20 days without dose breaks. Tumor volumes and body weights were monitored regularly [2].
For pharmacokinetic (PK) studies in BALB/c mice, CCT361814/NXP800 was administered both intravenously (iv) and orally (po). Blood samples were collected over a 24-hour period. PK parameters were derived from blood concentration-time curves using non-compartmental analysis [2].
Comparative PK studies were conducted in wild-type (CF1^WT) and P-gp knockout (CF1^PGK-KO) mice to assess the contribution of P-gp efflux to clearance [2].
A single-dose PK/PD study was performed in athymic mice bearing SK-OV-3 xenografts. Mice received a single oral dose of 50 mg/kg CCT361814/NXP800. Plasma and tumor samples were collected at multiple time points for drug concentration analysis and biomarker (CHAC1 protein by MSD assay, HSPA1A mRNA by qPCR) assessment [2].

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).

---

In immunocompetent BALB/c mice, after a 5 mg/kg iv dose, CCT361814/NXP800 had a total blood clearance (CL_tb) of 10 mL/min/kg, a terminal half-life (t_1/2) of 4.0 hours, and a volume of distribution (V_ss) not explicitly stated. Oral administration (5 mg/kg po) resulted in an AUC_0-6h of 6000 hnM, a T_max of 2.0 hours, and an oral bioavailability (F) of 42%. The fraction unbound in blood (f_ub) was 0.012, leading to an unbound clearance (CL_u) of 830 mL/min/kg [2].
In Sprague-Dawley rats, after a 5 mg/kg iv dose, CL_tb was 24 mL/min/kg, t_1/2 was 3.1 hours. Oral administration (5 mg/kg po) resulted in an AUC_0-∞ of 2600 hnM and a bioavailability of 45%. The f_ub was 0.033, and CL_u was 730 mL/min/kg [2].
In beagle dogs, after a 0.5 mg/kg iv dose, CL_tb was 21 mL/min/kg, t_1/2 was 1.4 hours. Oral administration (2.5 mg/kg po) resulted in an AUC_0-∞ of 250 hnM, a T_max of 2.0 hours, and a bioavailability of 9.1%. The f_ub was 0.14, and CL_u was 150 mL/min/kg [2].
CCT361814/NXP800 exhibited moderate passive permeability in the Caco-2 assay (A-B = 7.7 x 10^-6 cm/s) with a low efflux ratio (2.8) [2].
The compound showed high solubility in simulated gastric fluid and modest solubility in simulated intestinal fluid [2].
In vitro metabolic stability was assessed in mouse, rat, and human liver microsomes and hepatocytes. The predicted in vivo unbound clearance from human hepatocytes was used for human dose projection [2].

In immunocompetent BALB/c mice, after a 5 mg/kg iv dose, CCT361814/NXP800 had a total blood clearance (CL_tb) of 10 mL/min/kg, a terminal half-life (t_1/2) of 4.0 hours, and a volume of distribution (V_ss) not explicitly stated. Oral administration (5 mg/kg po) resulted in an AUC_0-6h of 6000 hnM, a T_max of 2.0 hours, and an oral bioavailability (F) of 42%. The fraction unbound in blood (f_ub) was 0.012, leading to an unbound clearance (CL_u) of 830 mL/min/kg [2].
In Sprague-Dawley rats, after a 5 mg/kg iv dose, CL_tb was 24 mL/min/kg, t_1/2 was 3.1 hours. Oral administration (5 mg/kg po) resulted in an AUC_0-∞ of 2600 hnM and a bioavailability of 45%. The f_ub was 0.033, and CL_u was 730 mL/min/kg [2].
In beagle dogs, after a 0.5 mg/kg iv dose, CL_tb was 21 mL/min/kg, t_1/2 was 1.4 hours. Oral administration (2.5 mg/kg po) resulted in an AUC_0-∞ of 250 hnM, a T_max of 2.0 hours, and a bioavailability of 9.1%. The f_ub was 0.14, and CL_u was 150 mL/min/kg [2].
CCT361814/NXP800 exhibited moderate passive permeability in the Caco-2 assay (A-B = 7.7 x 10^-6 cm/s) with a low efflux ratio (2.8) [2].
The compound showed high solubility in simulated gastric fluid and modest solubility in simulated intestinal fluid [2].
In vitro metabolic stability was assessed in mouse, rat, and human liver microsomes and hepatocytes. The predicted in vivo unbound clearance from human hepatocytes was used for human dose projection [2].

[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]

---

CCT361814/NXP800 (also known as CCT251236 derivative or fluorobisamide 22) is a clinical candidate developed from a phenotypic screen for HSF1 pathway inhibitors. It is intended as a potential treatment for refractory ovarian cancer and other malignancies [2].
The compound is proposed to act via a "non-oncogene addiction" mechanism by inhibiting the HSF1 stress pathway, which is important for tumorigenesis and progression in cancers like ovarian cancer [2].
The molecular mechanism of action, while involving pirin binding and HSF1 pathway inhibition, is not fully elucidated. Transcriptional profiling and chemoproteomic studies are ongoing [2].
Based on preclinical efficacy and PK data, the predicted human dose for CCT361814/NXP800 was estimated to be less than 210 mg/person/day using allometric scaling [2].
Following successful preclinical development, CCT361814/NXP800 entered Phase 1 clinical trials (NCT05226507) in cancer patients in 2022 [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。

---

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。

View More

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)

---

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。

View More

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

---

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。

---

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。