HSP72 ( IC50 = 19 nM )

CCT251236 (0-100 nM; 24 hours) inhibits HSF1-mediated HSP72 induction with a pIC50=7.73 ± 0.07 (IC50=19 nM), demonstrating the appropriate balance of in vitro properties while maintaining excellent cellular activity. Based on the free fraction in the cell assay, the free GI50 in SK-OV-3 cells is 1.1 nM[1].
CCT251236 (0-100 nM; 24 hours) inhibits the expression of the HSF1-mediated heat-shock proteins, HSP72 and HSP27, in a concentration-dependent manner in SK-OV-3 cells[1].
CCT251236 (0-100 nM; 24 hours) inhibits the dose-dependent induction of HSPA1A mRNA by 17-AAG after being pre-treated with 250 nM 17-AAG for 6 hours[1].

CCT251236 (oral adminstation; 5 or 20 mg/kg) has a free Cav0-24h value of 2.0 nM and 1.2 nM, respectively, in immunocompetent BALB/c mice without tumors [2].
CCT251236 (oral adminstation; 20 mg/kg; 33 days) exhibits a definite therapeutic efficacy in mice with a tumor growth inhibition (%TGI) of 70% based on final tumor volumes. The mean tumor weights decreased by 64% after 33 days compared to the control group. Furthermore, tumors containing CCT251236 at concentrations as high as 940 nM demonstrate the compound's basicity and broad volume of distribution[2].

Protein Binding [1]
Protein binding was measured using Rapid Equilibrium Dialysis. Plasma was obtained from female BALB/c and Ncr-Foxn1nu mice and stored at -20°C. Cell culture media was DMEM supplemented with 10 % FCS, 2 mmol/L of Lglutamine, and 1 x non-essential amino acids. The RED plate, buffer, plasma and media solutions were heated to 37 °C before dialysis. Test compound/Compound 26 (CCT251236) in DMSO was spiked into either diluted plasma (10-fold dilution in 100 mM phosphate buffer) or cell culture media as appropriate resulting in a concentration of 5 PM for dialysis, containing 1 % DMSO. 300 PL of spiked diluted plasma or media was added to the donor side of the RED plate and 500 PL of 100 mM phosphate buffer was added to the receiver well. The plate was sealed with a gas- S70 permeable lid and dialysis was performed by shaking for 4 h at 37 °C.

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Pirin Surface Plasmon Resonance (SPR)[1]
All surface plasmon resonance (SPR) experiments were carried out on a Biacore T100 enhanced to T200 sensitivity. Amine coupling chemistry was used to immobilize the pirin protein on a research grade CM5 sensor chip. The running buffer used in the immobilization step consisted of 1 x phosphate buffered saline (10 S74 mM NaHPO4/NaH2PO4, pH 7.4, 2.7 mM KCl, 137 mM NaCl) and the chip’s surface was activated for 10 min using a 1:1 mixture of 100 mM N-hydroxysuccinimide and 400 mM 1- ethyl-3-(3-dimethylaminopropyl)-carbodiimide. Pirin was injected at a concentration of ~5 µg/mL in a 10 mM acetate buffer pH 5.5 with 5 µM triphenyl compound A added to provide protection for the active site lysines. The reaction was monitored in real time and stopped when a target immobilization level of ~5000RU was obtained. Finally, the surface was blocked via an injection of 1 M ethanolamine at pH 8.5 for 7 min. The flow rate was maintained at 10 µL/min for all the above procedures. Flow cell one was left unmodified and used as the reference surface.

Cell Line: SK-OV-3 cells Concentration: 0 nM; 10 nM; 100 nM Incubation Time: 24 hours Result: Inhibited HSP72 and HSP27 expression at the dose of 10 nM.

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In vitro cell viability assay [1]
The CellTiter Blue viability (CTB) 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 (at a range of concentrations) were added using the ECHO 550 liquid handler and then left at 37 o C for 96 h. Titre blue reagent was added to each well and left at 37 o C 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.

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

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.52 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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