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 (Example 169) (IC50=0.056 μM) reduces the viability of U20S cells.

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CCT361814/NXP800 demonstrated potent antiproliferative activity against a panel of nine genetically diverse human ovarian cancer cell lines (geometric mean pGI50 >7.3). The free GI50 against SK-OV-3 cells was 3.7 nM (fu,a = 0.43) [2].
In a surrogate biomarker assay, CCT361814/NXP800 blocked HSP72 induction by the HSP90 inhibitor 17-AAG in SK-OV-3 cells with an IC50 of 94 nM (pIC50 = 7.03 ± 0.07, n=40), confirming HSF1 pathway antagonism [2].
Gene expression profiling in cancer cell lines and tumor xenografts treated with CCT361814/NXP800 showed inhibition of a heat integrated stress response gene signature and activation of a related integrated stress response signature. Specifically, treatment increased CHAC1 mRNA expression and decreased HSPA1A mRNA expression [2].
CCT361814/NXP800 showed low potential for P-glycoprotein (P-gp)-mediated efflux, as indicated by a low CH1^doxR/CH1^WT efflux ratio in a cell-based assay [2].

Pharmacokinetic analysis [2] Species pathway Dose (mg/kg) Tmax (h) AUClast (ng·h/mL) Cltb (mL/min/kg) t1/2 (h) F (%) AUCu0-t ( h·nM) Free Cav0-24h (nM) Clu (mL/min/kg) Rat Oral/IV 5/1 6.0 2600 (Oral) 24 (iv) 3.1 45 (Oral) 86 3.7 730 (iv) Dog Oral/IV 2.5/0.5 2.0 250 (Oral) 21 (IV) 1.4 9.1 (PO) 35 1.9 150 (IV)

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In an immunodeficient athymic mouse model bearing established SK-OV-3 human ovarian cancer solid tumor xenografts, oral administration of CCT361814/NXP800 at 35 mg/kg once daily for 20 days resulted in significant antitumor activity. Tumor growth inhibition (TGI) was 120% relative to control, with 8 out of 10 tumors showing regression. The T/C value based on final mean tumor weights was 37% of control (p = 0.0008) [2].
A single-dose PK/PD study in the same model (50 mg/kg po) showed that free tumor concentrations of CCT361814/NXP800 exceeded the in vitro free GI50 for 21 hours. Induction of the PD biomarker CHAC1 protein and reduction of HSPA1A mRNA in tumors correlated with the tumor-free drug concentration, confirming on-target pathway modulation [2].

CH1doxR/CH1wt MDR Assay [2]
The antiproliferative activity of compounds such as NXP-800 (CCT361814) was assessed in CH1wt and CH1doxR cells using the same method described previously using the CellTiter-Blue viability assay. The rescue of the antiproliferative activity in the CH1doxR cell line was confirmed by treating the cells with 2 µM (R)-(+)-verapamil monohydrochloride hydrate (http://www.sigmaaldrich.com/catalog/product/sigma/v106?lang=en®ion=GB, February 2017) and the bisamide analogue. The geometric mean pGI50 values (pGI50=-log GI50 (M)) of at least n=3 biological repeats in the CH1wt and CH1doxR cells for each were then compared using a Student’s t-test with Welch’s correction; when p<0.05 the compound considered to be an MDR substrate (GraphPad Prism 7.01). The ratio of geometric mean GI50s in CH1doxR and CH1wt was defined as the MDR ratio[2].

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A competitive binding assay against 87 potentially high-risk off-target proteins (Cerep panel) was performed. CCT361814/NXP800 showed adenosine A2A receptor antagonism with an IC50 of 2.0 µM, which is approximately 100-fold higher than its efficacious free concentrations [2].
Cytochrome P450 (CYP) inhibition was assessed. CCT361814/NXP800 showed no significant CYP inhibition (IC50 > 10 µM) [2].
hERG channel inhibition was evaluated. CCT361814/NXP800 showed no hERG liability (IC50 > 30 µM) [2].

In vitro cell viability assay [2]
The CellTiter-Blue viability assay provides a homogenous, fluorometric method for estimating the number of viable cells. It uses the dark blue indicator dye resazurin to measure the metabolic capacity of cells which is an indicator of cell viability. Viable cells are able to reduce resazurin into resorufin (pink), which is highly fluorescent. Briefly, cells (~6 x 103 cells/mL) were seeded into 384-well plates and were incubated for 24 h. Compounds (e.g. NXP-800 (CCT361814)at a range of concentrations) were added using the ECHO 550 liquid handler and then left at 37 oC for 96 h. Titer-Blue reagent was added to each well and left at 37 oC for 3-4 h. Fluorescence was measured using the Envision machine. The 50% growth inhibitory concentration (GI50) was determined by fitting the data to a dose-response curve without limits using non-linear regression. Each concentration was tested twice[2].

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Antiproliferative activity (GI50) was determined using the CellTiter-Blue cell viability assay. Cells were seeded in 96-well plates, treated with compound for 96 hours, and fluorescence was measured. GI50 values were estimated by fitting a log[inhibitor] vs. response-variable slope (four-parameter) model [2].
A cell-based assay utilizing matched pair ovarian cancer cell lines (CH1^WT and acquired doxorubicin-resistant CH1^doxR) was used as a surrogate for assessing P-gp-mediated efflux. The fold-difference in geometric mean GI50 values between CH1^doxR and CH1^WT cells was calculated. A significant difference (p < 0.05, Student's t-test) indicated P-gp substrate activity [2].
For the HSF1 pathway inhibition biomarker assay, SK-OV-3 cells were treated with the HSP90 inhibitor 17-AAG to induce HSF1 activity and subsequent HSP72 expression. Co-treatment with CCT361814/NXP800 blocked this induction. HSP72 protein levels were quantified using a cell-based ELISA [2].
CHAC1 protein induction was confirmed in SK-OV-3 cells treated with CCT361814/NXP800 (19 nM, 5x free GI50) via immunoblotting [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

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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].

In mice, the predicted values of CLI and MHeps for compound 22/NXP-800 (CCT361814) were consistent with those of methyl analog 16 (Table 2, entry 1). Despite reduced lipophilicity, fluorobisamide 22 showed good oral bioavailability (42%) in mice with a moderate total blood clearance (CLtb = 10 mL/min/kg, extraction rate = 11%, Fmax = 89%), as predicted by CH1doxR/CH1WT. Due to these favorable data, fluorobisamide 22 was selected to evaluate its in vivo efficacy against established SK-OV-3 human ovarian cancer xenografts in thymic immunodeficient mice (Table 5). ^[1] Fluorodiamide 22, compound 22/NXP-800 (CCT361814), satisfactorily showed the desired reduction in lipophilicity (Table 3, entry 6), which was associated with a reduction in MLM (15 μL/min/mg) and mouse hepatocyte CLint in vitro; while maintaining excellent antiproliferative activity (free GI50 = 3.7 nM, fua = 0.43; Table S4)39 and an acceptable KS value (50 μM), fluorodiamide 22 was submitted for in vivo mouse pharmacokinetic studies (Table 4, entry 1). In immunocompetent BALB/c mice, after intravenous administration of a dose of 5 mg/kg, the total blood clearance (CLtb) of CCT361814/NXP800 was 10 mL/min/kg, the terminal half-life (t1/2) was 4.0 h, and the volume of distribution (Vss) was not specified. After oral administration (5 mg/kg po), the AUC0-6h was 6000 hM, the Tmax was 2.0 h, and the oral bioavailability (F) was 42%. The free fraction of the drug in the blood (fub) was 0.012, resulting in a free clearance (CLu) of 830 mL/min/kg [2]. In Sprague-Dawley rats, after intravenous administration of 5 mg/kg, the CL_tb was 24 mL/min/kg, and the t_1/2 was 3.1 hours. After oral administration (5 mg/kg po), the AUC_0-∞ was 2600 hM, and the bioavailability was 45%. The f_ub was 0.033, and the CL_u was 730 mL/min/kg [2]. In beagle dogs, after intravenous administration of 0.5 mg/kg, the CL_tb was 21 mL/min/kg, and the t_1/2 was 1.4 hours. After oral administration (2.5 mg/kg), the AUC_0-∞ was 250 hnM, the T_max was 2.0 hours, and the bioavailability was 9.1%. The free clearance (f_ub) was 0.14, and the free clearance (CL_u) was 150 mL/min/kg [2].
CCT361814/NXP800 showed moderate passive permeability (AB = 7.7 x 10^-6 cm/s) and low efflux ratio (2.8) in Caco-2 cell assays [2].
The compound was highly soluble in simulated gastric fluid and moderately soluble in simulated intestinal fluid [2].
Its in vitro metabolic stability was evaluated in mouse, rat, and human liver microsomes and hepatocytes. The predicted in vivo free clearance in human hepatocytes was used for human dose estimation [2].

In immunocompetent BALB/c mice, after intravenous injection of 5 mg/kg CCT361814/NXP800, the total plasma clearance (CLtb) was 10 mL/min/kg, the terminal half-life (t1/2) was 4.0 h, and the volume of distribution (Vss) was not determined. After oral administration (5 mg/kg po), the AUC0-6h was 6000 hM, the Tmax was 2.0 h, and the oral bioavailability (F) was 42%. The free drug fraction in the blood (fub) was 0.012, resulting in a free clearance (CLu) of 830 mL/min/kg [2]. In Sprague-Dawley rats, after intravenous administration of 5 mg/kg, the CL_tb was 24 mL/min/kg, and the t_1/2 was 3.1 h. Oral administration (5 mg/kg po) resulted in an AUC_0-∞ of 2600 hM and a bioavailability of 45%. f_ub was 0.033, and CL_u was 730 mL/min/kg [2]. In beagle dogs, after intravenous administration of 0.5 mg/kg, the CL_tb was 21 mL/min/kg, and the t_1/2 was 1.4 h. After oral administration (2.5 mg/kg), the AUC_0-∞ was 250 hnM, the T_max was 2.0 h, and the bioavailability was 9.1%. The free clearance (f_ub) was 0.14, and the free clearance (CL_u) was 150 mL/min/kg [2].
CCT361814/NXP800 showed moderate passive permeability (AB = 7.7 x 10^-6 cm/s) and low efflux ratio (2.8) in Caco-2 cell assays [2].
The compound was highly soluble in simulated gastric fluid and moderately soluble in simulated intestinal fluid [2].
Its in vitro metabolic stability was evaluated in mouse, rat, and human liver microsomes and hepatocytes. The predicted in vivo free clearance in human hepatocytes was used for human dose estimation [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 is a potent chemical probe previously developed using cell-based phenotypic high-throughput screening (HTS) to discover HSF1-mediated transcriptional inhibitors. HSF1 is a transcription factor that promotes malignant tumorigenesis. Due to its activity in a refractory human ovarian cancer model, compound 1 was advanced to the lead compound optimization stage. Reducing P-glycoprotein efflux became a focus of early compound optimization; matching molecular pair analysis confirmed that central cyclic halogen substitution was an effective strategy to reduce this adverse effect. Further multi-parameter optimization ultimately designed the clinical candidate drug CCT361814/NXP800 22, a potent and orally bioavailable fluorobisamide compound that induced tumor regression in a human ovarian adenocarcinoma xenograft model and exhibited pathway biomarker regulation and good in vitro safety. Due to its promising dose prediction results in humans, compound 22 has now entered Phase I clinical trials and is expected to become a potential therapeutic agent for refractory ovarian cancer and other malignant tumors. [2]
CCT361814/NXP800 (also known as a derivative of CCT251236 or fluorobisamide 22) is a clinical candidate drug developed through phenotypic screening of HSF1 pathway inhibitors. It is intended to be a potential therapeutic for refractory ovarian cancer and other malignancies. [2]
This compound is believed to exert a “non-oncogene addiction” effect by inhibiting the HSF1 stress pathway, which plays an important role in tumorigenesis and development of cancers such as ovarian cancer. [2]
Although its molecular mechanism of action involves pyridine binding and HSF1 pathway inhibition, it is not fully elucidated. Transcriptomic analysis and chemical proteomics studies are underway [2].
Based on preclinical efficacy and pharmacokinetic data, allometric scaling was used to predict a human dose of CCT361814/NXP800 of less than 210 mg/person/day [2]. Following successful preclinical development, CCT361814/NXP800 entered a Phase I clinical trial in cancer patients in 2022 (NCT05226507) [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

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)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)