BCL6

The Cl- and F-substituted pyridine-containing compound CCT374705 had the best balance of properties, including increased solubility and microsomal clearance similar to CCT374284[1].

We chose to progress CCT374705 to an efficacy study using a Karpas 422 xenograft model, the cell line in which CCT374705 had the most antiproliferative effect. The goal of this work was to study the effect of BCL6 inhibition in vivo. To assess this, we wanted to ensure complete coverage of our inhibitor (>IC90) for the entirety of the study. To achieve this high level of sustained exposure, the study was conducted with a twice-daily 50 mg/kg oral dosing regimen. We were able to confirm target engagement with BCL6 by measuring an increase in ARID3A mRNA expression. PK/PD analysis showed that free concentrations of CCT374705 remained well above the free IC90 for over 12 h post dose. However, similarly to our previously reported efficacy results with our BCL6 degrader CCT373566, only moderate in vivo efficacy was observed. Having optimized the in vivo pharmacokinetic profile of CCT374705, this compound is a suitable probe to examine the role and function of BCL6 in diseases within mice models[1].

TR-FRET assay [1]
Assays were performed in a 384-well black Proxiplate (Perkin Elmer) containing 1 nM Trx-6xHis-BCL6 (in house-produced, human BCL6 BTB domain covering amino-acid sequence 5-129), 300 nM BCOR-AF633 peptide (RSEIISTAPSSWVVPGP-Cys-AlexaFluor 633-amide) and 0.5 nM anti-6xHis-Terbium cryptate, in assay buffer (25 mM Hepes pH8, 100 mM NaCl, 0.05% Tween20, 0.5 mM TCEP, 0.05% bovine serum albumin). Test compounds in DMSO or DMSO alone were added to the wells using an ECHO550 acoustic dispenser to give the appropriate test concentration in 0.7% v/v DMSO final. After 2 hours incubation at room temperature the plate was read on an Envision plate reader with 337 nm laser excitation, a first emission filter APC 665 nm and a second emission filter Europium 615 nm, or alternatively on a Pherastar FSX plate reader equipped with 337 nm laser excitation filter, a first emission filter at 620 nm and a second emission filter at 665 nm. The % inhibition at each concentration was calculated by normalising FRET ratio to the appropriate high (DMSO with all reagents) and low (DMSO without BCL6) controls. IC50 values were determined using GraphPad Prism 6.0 or Dotmatics software by fitting the normalised data to a sigmoidal four-parameter logistic fit equation.

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NanoBRET Assay [1]
A cellular nano-Bioluminescence Resonance Energy Transfer (nanoBRET) assay was used to detect inhibition of the BCL6-SMRT (also called NCOR2) corepressor protein-protein interaction. DNA encoding full length BCL6 and SMRT were inserted into pFC32K.NanoLuc and pFC14K.HaloTag vectors to produce C-terminal tagged fusion proteins BCL6-nanoLuc and SMRT-HaloTag, respectively. HEK293T cells were plated (5x105 ) in T75 tissue culture flask and bulk transfected 48 hours later with Fugene 6 reagent and 18 µg total DNA plasmids encoding BCL6-nanoLuc as donor and SMRT-HaloTag as acceptor, at a donor:acceptor DNA ratio of 1:25. At 24 hr post-transfection, HEK293T cells were collected and stored in liquid nitrogen in 90% FBS and 10% DMSO. At the time of assay, compounds (100nL/well) and NanoBRET 618 ligand (10nL of 1mg/ml stock solution per well) were dispensed in a dry 384-well NUNC white assay plate using Echo550 acoustic dispensing. Frozen transfected HEK293T cells were thawed, centrifuged and freezing medium was replaced by phenol red-free OptiMEM+4% FBS. The cell density was adjusted to 3x105 cells/ml and 20 µL (6000 cells) were plated in each well containing test compounds (0.0125-50 µM) in DMSO or DMSO alone and 0.5 µg/ml NanoBRET 618 fluorescence ligand, in 0.55% v/v DMSO final concentration. Cells were incubated for 6 hr at 37 °C / 5% CO2 then NanoBRET furimazine substrate was added to give a final concentration of 10 µM. After a short centrifugation the plates were read on an Envision plate reader equipped with a LUM/D600 Dual mirror, Lum 450/40 nm bandpass and D605 nm longpass filters, with a 0.2 sec reading to determine the BRET ratio. Alternatively, plates were read on Pherastar FSX equipped with BRET module LP610 nm (1st emission filter) / 450-80 nm (2nd emission filter). The % inhibition at each test concentration was calculated by normalising the BRET ratio to the appropriate high and low controls. The compound IC50s were determined using Graphpad Prism 6.0 or Dotmatics software by fitting the normalised data to a sigmoidal four-parameter logistic fit equation.

Cells were seeded in 96-well culture plates at a density of 2500 cells/well in RPMI-1640 medium supplemented with 10% FBS. Compounds were initially dispensed into 96-well U-bottom plates using an Echo 550 acoustic dispenser, then diluted in RPMI-1640 medium and transferred onto the cells. Cells were treated with 8 compound concentrations in duplicate, ranging from 1.07 nM to 10 µM, in a final DMSO concentration of 0.1% and final volume of 100 µl. Cells were incubated with compound for 14 days, with medium changes at days 3, 7 and 10 carried out as follows: fresh 96-well cell culture plates were prepared containing 100 µl medium plus compound at the assay concentrations (white plates were used on day 10 to optimise luminescence measurement). Assay plates containing cells were vortexed to mix and cell density in one control well was counted using a Coulter Z2 cell counter. The volume of medium containing 2500 cells in the control well was calculated and this volume of cells was transferred from every well of the assay plates to the corresponding well of the fresh plates containing compound. After 14 days, CellTiter Glo reagen was added to the medium in each well of the assay plate at a ratio of 1:2, mixed on a plate shaker, then incubated at room temperature for 10 minutes. Luminescence was measured using an Envision plate reader and the relative luminescence at each compound concentration, compared to DMSO alone, was calculated. GI50 were determined using a 4-parameter curve fit in Dotmatics [1].

In vivo pharmacokinetic studies[1]
Animals were adapted to laboratory conditions for at least 1 week prior to dosing and were allowed food and water ad libitum. Compounds were administered iv or po (mouse, 0.1 mL/10 g in 10% DMSO, 5% tween 20 in saline); blood samples were collected in heparinised capillaries from the tail vein at 7 or 8 time points over 6 or 24 h post dose and frozen on collection together with a standard curve and quality controls spiked in control blood. Samples were reconstituted in a water:MeOH mixture containing internal standard as previously described (Roberts et al, 2016). Following centrifugation, extracts were analyzed by multiple reaction monitoring of precursor and product ions by ESI-LCMS/MS on either a Waters Xevo TQ-S or Sciex QTrap6500 following gradient separation with 0.1% formic acid and methanol on a Phenomenex Kinetex C18 UPLC column (50 × 2.1 mm, 2.6 μM). Quantitation was carried out with an external calibration. Quality controls were included and were within 20% of nominal concentration. Pharmacokinetic parameters were derived from noncompartmental analysis using Phoenix Pharsight Non compartmental analysis (model 200 and 201) version 6.3. All parameters are calculated from timepoints up to 6 h.

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Formulation of CCT374705[1]
A solution formulation suitable for higher concentrations (> 5 mg/mL) of CCT374705 was developed by SEDA as described in Table S4. CCT374705 was dissolved in a pre-determined volume of DMSO and to this was added a pre-determined volume of Kolliphor HS15 at 40 °C. The solution was briefly vortexed before addition of a pre-determined volume of PEG400 at 40 °C. After a brief vortex a pre-determined volume of aqueous HPMC (1.25%, viscosity 40-60cp grade) was added. The formulation was vortexed and then sonicated at 40 oC for ~45- 60 mins. Complete vehicle was prepared in the same manner with no compound added. The formulation was stored at room temperature for 3-4 days maximum. Both compound and vehicle solutions were delivered orally (0.2 mL per 20 g mouse) using a gavage needle - mice were individually weighed and dosing volumes adjusted accordingly.

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In vivo pharmacodynamic (PK/PD) and efficacy studies [1]
Preparation of Karpas 422 xenograft model in mouse. Karpas 422B cells were prepared for injection at a final concentration of 5 x107 cells/mL, using serum–free RMPI-1640 and an equal volume of Matrigel®, both chilled to 4°C. Cells were delivered to female SCID mice at 107 per 200uL, subcutaneously, single site. At three weeks post injection tumour bearing mice (mean diameter 0.5 cm2 +/- 0.1) were randomly selected and assigned to a treated group and a control group (n = 10 per group). CCT374705 and vehicle were formulated as described in Supplementary experimental 5.2: CCT374705 (50 mg/kg) and vehicle (0.2 mL/20g body weight) were administered orally, twice per day, approximately 12 hours apart for 35 days.

[1]. Harnden AC, et al. Discovery of an In Vivo Chemical Probe for BCL6 Inhibition by Optimization of Tricyclic Quinolinones. J Med Chem. 2023 Apr 27;66(8):5892-5906.

B-cell lymphoma 6 (BCL6) is a transcriptional repressor and oncogenic driver of diffuse large B-cell lymphoma (DLBCL). Here, we report the optimization of our previously reported tricyclic quinolinone series for the inhibition of BCL6. We sought to improve the cellular potency and in vivo exposure of the non-degrading isomer, CCT373567, of our recently published degrader, CCT373566. The major limitation of our inhibitors was their high topological polar surface areas (TPSA), leading to increased efflux ratios. Reducing the molecular weight allowed us to remove polarity and decrease TPSA without considerably reducing solubility. Careful optimization of these properties, as guided by pharmacokinetic studies, led to the discovery of CCT374705, a potent inhibitor of BCL6 with a good in vivo profile. Modest in vivo efficacy was achieved in a lymphoma xenograft mouse model after oral dosing.[1]

C21H18CLF3N4O2

450.84

450.10703

2640647-90-9

156276733

Typically exists as white to off-white solids at room temperature

4.2

66.5Ų

MCRHRRIGPAVFNY-SFHVURJKSA-N

InChI=1S/C21H18ClF3N4O2/c1-29-14-5-4-11(27-13-6-7-26-19(23)15(13)22)8-12(14)16-17(20(29)30)31-9-21(24,25)18(28-16)10-2-3-10/h4-8,10,18,28H,2-3,9H2,1H3,(H,26,27)/t18-/m0/s1

(2S)-10-[(3-chloro-2-fluoropyridin-4-yl)amino]-2-cyclopropyl-3,3-difluoro-7-methyl-2,4-dihydro-1H-[1,4]oxazepino[2,3-c]quinolin-6-one

CCT374705; 2640647-90-9; SCHEMBL23248186

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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 (221.81 mM)

Solubility in Formulation 1: 2.5 mg/mL (5.55 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.55 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 900 μL of corn oil and mix evenly.

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