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NXP-800 (also known as CCT361814; CCT-361814) is a potent ERK dimerization inhibitor and heat shock factor 1 (HSF1) inhibitor with anticancer effects.
| Targets |
HSF1 (heat shock factor 1)
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|---|---|
| ln Vitro |
NXP800 (Example 169) (IC50=0.056 μM) reduces the viability of U20S cells.
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| ln Vivo |
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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| Enzyme Assay |
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]. |
| Cell Assay |
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]. |
| Animal Protocol |
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 |
| ADME/Pharmacokinetics |
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). |
| References |
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| Additional Infomation |
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]
|
| Molecular Formula |
C32H32FN5O4
|
|---|---|
| Molecular Weight |
569.63
|
| Exact Mass |
569.243
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| Elemental Analysis |
C, 67.47; H, 5.66; F, 3.34; N, 12.29; O, 11.23
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| CAS # |
1693734-80-3
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| Related CAS # |
1693734-80-3;
|
| PubChem CID |
117996795
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| Appearance |
Light yellow to green yellow solid powder
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| LogP |
3.7
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
7
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| Heavy Atom Count |
42
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| Complexity |
919
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| Defined Atom Stereocenter Count |
0
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| SMILES |
N1C2C(=CC(C(NC3=CC(NC(C4=CC=C5OCCOC5=C4)=O)=CC=C3F)=O)=CC=2)C=CC=1CN1CCN(CC)CC1
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| InChi Key |
UBALMDIKIGDHJW-UHFFFAOYSA-N
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| InChi Code |
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)
|
| Chemical Name |
N-[5-(2,3-dihydro-1,4-benzodioxine-6-carbonylamino)-2-fluorophenyl]-2-[(4-ethylpiperazin-1-yl)methyl]quinoline-6-carboxamide
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| Synonyms |
CCT-361814; NPX800; CCT 361814; SCHEMBL16621389; NXP-800; BDBM610359; CCT361814; NPX 800; NPX-800
|
| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~100 mg/mL (~175.55 mM)
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|---|---|
| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.7555 mL | 8.7776 mL | 17.5553 mL | |
| 5 mM | 0.3511 mL | 1.7555 mL | 3.5111 mL | |
| 10 mM | 0.1756 mL | 0.8778 mL | 1.7555 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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