ERK5 (IC50 = 35 nM)

BAY-885 shows strong ERK5 kinase and transcriptional inhibition in the SN12C-MEF2 reporter cell line (IC50 = 115 nM/IC90 = 691 nM) but no effects on the SN12C-CMV-luc reporter control cell line (IC50 > 30 M), ruling out potential effects as a general transcription or translation inhibitor.[1]

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The combination of the best residues and properties lead to the identification of 41 (BAY-885): a potent and selective in vitro ERK5 probe molecule (Figure 4). The synthesis of 41 is outlined in Scheme 2. 4,7-Dichloropyrido[3,2-d]pyrimidine 43 is converted to 44 via a Suzuki reaction followed by a Buchwald amination to provide 45. Cleavage of the Boc protecting group followed by hydrogenation of the double bond and amide formation gives rise to 41 (BAY-885). As a control compound for pharmacological experiments we also provide the negative ERK5 probe 42 (BAY-693, Table 6).
Furthermore, compound 41/BAY-885 was tested at 1 μM concentration against 358 kinases in the commercial Eurofins kinase panel with only Fer (r), EphB3 (h), and EphA5 (h) kinases being inhibited at 62%, 58%, and 43%, respectively. For other kinases, inhibition was ≤20%, thus demonstrating that compound 41 (BAY-885) is a highly selective ERK5 inhibitor (Table S2). In contrast to XMD8–92, compound 41 showed no binding to BRD4 up to 20 μM.

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The ERK5 probe 41 (BAY-885) showed potent ERK5 kinase and transcriptional inhibition in the SN12C-MEF2 reporter cell line (IC50 = 115 nM/IC90 = 691 nM) and had no effects on a reporter control cell line with constitutive luciferase expression (SN12C-CMV-luc, IC50 > 30 μM), thereby ruling out potential effects as a general inhibitor of transcription or translation. To further address the potential of ERK5 as a therapeutic target in oncology, we tested the impact of compound 41 (BAY-885) on the proliferation of cells with ERK5 genomic amplification (SN12C, SNU-449, MFM-223) or with constitutively active ERK5 signaling (BT-474, SK-BR-3). Importantly, despite its high potency, compound 41 (BAY-885) failed to inhibit the proliferation of all these cell lines. These results are in strong contrast with literature data suggesting that ERK5 is an oncogenic driver in tumors with dysregulated ERK5 signaling. A possible explanation lies on the different methodology used, with the mentioned studies employing RNAi technology to silence the ERK5 protein, while our results are based on ERK5 kinase inhibition with a small molecule.

Biochemical ERK5 Inhibition Assay [1]
Recombinant fusion protein of N-terminal Glutathion-S-Transferase (GST) and a fragment of human ERK5 (amino acids 1–398 of accession number NP_002740.2]), expressed in E. coli, purified via affinity chromatography using Glutathion Sepharose and subsequently activated with His-tagged MAP2K5, was used as kinase. As substrate for the kinase reaction biotinylated peptide biotin-Ahx-PPGDYSTTPGGTLFSTTPGGTRI (C-terminus in amide form) was used. For the assay, 50 nL of a 100-fold concentrated solution of the test compound in DMSO was pipetted into either a black low volume 384-well microtiter plate or a black 1536-well microtiter plate, 2 μL of a solution of ERK5 in aqueous assay buffer [50 mM Hepes pH 7.0, 15 mM MgCl2, 1 mM dithiothreitol, 0.5 mM EGTA, 0.05% (w/v) bovine γ-globulin were added, and the mixture was incubated for 15 min at 22 °C to allow prebinding of the test compounds to the enzyme before the start of the kinase reaction. Then the kinase reaction was started by the addition of 3 μL of a solution of adenosine-triphosphate (ATP, 417 μM → the final conc. in the 5 μL assay volume is 250 μM) and substrate (1.67 μM → final conc. in the 5 μL assay volume is 1 μM) in assay buffer, and the resulting mixture was incubated for a reaction time of 60 min at 22 °C. The concentration of ERK5 was adjusted depending of the activity of the enzyme lot and was chosen appropriate to have the assay in the linear range, a typical concentration was 0.5 μg/mL. The reaction was stopped by the addition of 3 μL of a solution of TR-FRET detection reagents (0.33 μM streptavidine-XL665 and 1.67 nM anti-4E-BP1 (pT46) antibody and 1.67 nM LANCE EU-W1024 labeled antirabbit IgG antibody in an aqueous EDTA-solution (83.3 mM EDTA, 0.2% (w/v) bovine serum albumin in 50 mM HEPES, pH 7.5). The resulting mixture was incubated 1 h at 22 °C to allow the formation of complex between the phosphorylated biotinylated peptide and the detection reagents. Subsequently, the amount of phosphorylated substrate was evaluated by measurement of the resonance energy transfer from the Eu-chelate to the streptavidine-XL. Therefore, the fluorescence emissions at 620 and 665 nm after excitation at 350 nm was measured in a TR-FRET reader, e.g., a Pherastar FS or a Viewlux. The ratio of the emissions at 665 nm and at 622 nm was taken as the measure for the amount of phosphorylated substrate. The data were normalized (enzyme reaction without inhibitor = 0% inhibition; all other assay components but no enzyme = 100% inhibition). Usually the test compounds were tested on the same microtiter plate in 11 different concentrations in the range of 20 μM to 0.07 nM (20 μM, 5.7 μM, 1.6 μM, 0.47 μM, 0.13 μM, 38 nM, 11 nM, 3.1 nM, 0.9 nM, 0.25 nM, and 0.07 nM, the dilution series prepared separately before the assay on the level of the 100-fold concentrated solutions in DMSO by serial dilutions; exact concentrations may vary depending pipettors used) in duplicate values for each concentration, and IC50 values were calculated using Genedata Screener software.

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Biochemical BRD4 BD1 and BD2 Binding Inhibition Assays [1]
To test inhibitor affinity toward bromodomains BD 1 and 2 of Bromodomain containing 4 (BRD4), a biochemical TR-FRET based assay was used, similarly to that described previously. In this assay, a fluorescence signal is produced through the binding of N-terminal His6-tagged human BRD4 BD1 or BD2 to synthetic, tetra-acetylated peptides derived from human histone 4. BD1 (amino acids 44–168) and BD2 (amino acids 333–460) proteins were expressed in E. coli, purified via affinity chromatography/IMAC and size exclusion chromatography. Acetylated, biotinylated peptides (peptide for BD1 interaction: SGRGK(Ac)GGK(Ac)GLGK(Ac)GGAK(Ac)RHGSGSK-Btn; peptide for BD2 interaction: SGRGK(Ac)GGK(Ac)GLGK(Ac)GGAK(Ac)RHRKVLRDNGSGSK-Btn) were used. The extent of inhibitor binding to BRD4, i.e., displacement of the biotinylated peptides, is monitored by the degree of signal decrease. The experiments were performed in 384-well black small-volume microtiter plates , where 50 nL of a 100-fold concentrated solution of a test compound (see final conc. below) in DMSO was predispensed. BRD4 BD1 (final conc. in assay 10 nM) in 2 μL of assay buffer composed of 50 mM HEPES (pH 7.5), 50 mM NaCl, 0.25 mM CHAPS, and 0.05% bovine serum albumin was added to the test compound. After 10 min preincubation at room temperature, 3 μL of a detection solution containing biotinylated peptide for BD1 interaction (final conc. 50 nM), anti-6His-XL665 (final conc. 10 nM), streptavidin Eu3+ chelate, and potassium fluoride (KF; final conc. 50 mM) in assay buffer were added to the plate. After incubation for 3 h at 4 °C, the plate was measured in a PheraStar reader using the homogeneous time-resolved fluorescence (HTRF) module (excitation, 337 nm; emission, 620 and 665 nm). The BRD4 BD2 (final conc. in assay 100 nM) assay was performed similarly, except for the following modifications: conditions assay buffer (50 mM HEPES (pH 7.5), 100 mM NaCl, 0.25 mM CHAPS, and 0.05% BSA) and detection solution (3 μL, containing biotinylated peptide for BD2 interaction (final conc. in assay 50 nM), anti-6His-XL665 (final conc. 50 nM), streptavidin Eu3+ chelate (final conc. 8.6 nM), and KF (final conc. 50 mM) in assay buffer). The ratio of the emissions at 665 and 620 nm were calculated and normalized to neutral (DMSO instead of test compound = 0% binding inhibition) and inhibitor control (all assay components except BRD4 BD1/BD2 = 100% binding inhibition). The compounds were tested at 11 different concentrations in the range of 20 μM to 0.07 nM (20 μM, 5.7 μM, 1.6 μM, 0.47 μM, 0.13 μM, 38 nM, 11 nM, 3.1 nM, 0.9 nM, 0.25 nM, and 0.07 nM) in duplicates for each concentration, and IC50 values were calculated using the Genedata Screener software.

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X-ray Structures of 1 and 35 in Complex with Erk5 [1]
Protein was produced as described previously and concentrated to 12 mg/mL in 50 mM HEPES pH 6.5, 150 mM NaCl, 10% glycerol, and 2 mM DTT. Prior to crystallization, the protein solution was supplemented with either compound 1 or compound 35 (final concentration of 1 mM) and incubated for 3 h on ice. Samples were then clarified by centrifugation (5 min, 13 000g, 277 K). Complex crystals were grown using the hanging-drop method by mixing 0.75 μL of protein and with 0.5 μL of reservoir solution (Compound 1: 11% PEG 4000, 100 mM MgCl2, 160 mM sodium formate, 100 mM MES, pH 6.75, and 100 mM Tris, pH 8.5; compound 35: 15% PEG 4000, 100 mM MgCl2, 180 mM sodium formate, 100 mM MES, pH 6.5, and 100 mM Tris, pH 8.5. Crystals were harvested and cryoprotected by brief immersion in a cryoprotection solution consisting of mother liquor supplemented with 30% glycerol and then flash frozen in liquid nitrogen. Data were collected under cryogenic conditions at the synchrotron facility at the SLS in Villigen, Switzerland. The structure was solved by molecular replacement using PDB 4IC8 as a search model. The structure was refined using REFMAC5 within the CCP4 suite. (24) Statistics for the final modes are given in Table S3.

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In Vitro Metabolic Stability in Human Liver Microsomes [1]
The in vitro metabolic stability of test compounds was determined by incubating them at 1 μM in a suspension of liver microsomes in 100 mM phosphate buffer, pH 7.4 (NaH2PO4·H2O + Na2HPO4·2H2O) and at a protein concentration of 0.5 mg/mL at 37 °C. The microsomes were activated by adding a cofactor mix containing 8 mM Glukose-6-Phosphat, 4 mM MgCl2, 0.5 mM NADP, and 1 IU/mL G-6-P-dehydrogenase in phosphate buffer, pH 7.4. The metabolic assay was started shortly afterward by adding the test compound to the incubation at a final volume of 1 mL. Organic solvent in the incubations was limited to ≤0.01% dimethyl sulfoxide (DMSO) and ≤1% acetonitrile. During incubation, the microsomal suspensions were continuously shaken at 580 rpm, and aliquots were taken at 2, 8, 16, 30, 45, and 60 min, to which equal volumes of cold methanol were immediately added. Samples were frozen at −20 °C overnight and subsequently centrifuged for 15 min at 3000 rpm, and the supernatant was analyzed with an Agilent 1200 HPLC-system with LC–MS/MS detection. The half-life of a test compound was determined from the concentration–time plot. From the half-life the intrinsic clearances and the hepatic in vivo blood clearance (CL) and maximal oral bioavailability (Fmax) were calculated using the “well stirred” liver model together with the additional parameters liver blood flow, specific liver weight, and microsomal protein content. The following parameter values were used: liver blood flow 1.32 L/h/kg, specific liver weight 21 g/kg, and microsomal protein content 40 mg/g.

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Inhibition of CYP450 Metabolism [1]
The inhibitory potency of the test compounds toward cytochrome P450-dependent metabolic pathways was determined in human liver microsomes by applying individual CYP isoform-selective standard probes (CYP1A2 phenacetin, CYP2C8 amodiaquine, CYP2C9 diclofenac, CYP2D6 dextromethorphan, CYP3A4 midazolam). Reference inhibitors were included as positive controls. Incubation conditions (protein and substrate concentration, incubation time) were optimized with regard to linearity of metabolite formation. Assays were processed in 96-well plates at 37 °C by using a Genesis Workstation. After protein precipitation, the metabolite formation was quantified by LC–MS/MS analysis followed by inhibition evaluation and IC50 calculation.

At a density of 10,000 cells per well in 20 μL of culture medium, the cells are seeded in 384-well white plates on day 1. The HP D300 Digital Dispenser is used to dispense the test compounds, including BAY-885, on day 2, and they are then incubated at 37 °C for 16 hours.

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Caco2 Permeability Assay [1]
Cell culture: Caco-2 cells were seeded at a density of 2.5 × 105 cells per well on 24-well insert plates, 0.4 μm pore size, 0.3 cm2, and grown for 13–15 days in DMEM medium supplemented with 10% fetal calf serum (FCS), 1% GlutaMAX (100×), 100 U/mL penicillin, 100 μg/mL streptomycin, and 1% nonessential amino acids (100×). Cells were maintained at 37 °C in a humidified 5% CO2 atm. Medium was changed every 2–3 days. Evaluation of Caco-2 permeability in a bidirectional transport assay: The bidirectional transport assay was done in 24-well insert plates using a robotic system (Tecan). Before running the bidirectional transport assay, culture medium was replaced by transport medium (FCS-free HEPES-carbonate transport puffer, pH 7.2). For assessment of monolayer integrity, the transepithelial electrical resistance (TEER) was measured. Only monolayers with a TEER of at least 400 Ω cm2 were used. Test compounds were predissolved in DMSO and added either to the apical or basolateral compartment in final concentration of 2 μM. Evaluation was done in triplicates. Before and after 2 h of incubation at 37 °C, samples were taken from both compartments and analyzed after precipitation with methanol by LC–MS/MS. The apparent permeability coefficient (Papp) was calculated both for the apical to basolateral (A → B) and the basolateral to apical (B → A) direction using following equation: Papp = (Vr/P0)(1/S)(P2/t) where Vr is the volume of medium in the receiver chamber, P0 is the measured peak area of the test drug in the donor chamber at t = 0, S is the surface area of the monolayer, P2 is the measured peak area of the test drug in the acceptor chamber after 2 h of incubation, and t is the incubation time. The efflux ratio basolateral (B) to apical (A) was calculated by dividing Papp(B-A) by Papp(A-B). Cellular Luciferase Reporter Assay [1]
The SN12C-MEF2-luc reporter cell line has been generated by stably transducing SN12C cells with a MEF2-responsive transcription element upstream of a firefly luciferase gene and was used to determine the cellular activity of ERK5 inhibitors. In parallel, a SN12C-CMV-luc reporter cell line has been generated that recombinantly carries a CMV-promoter driven firefly luciferase gene, thereby constitutively expressing luciferase. The latter is used to detect false positive hits of SN12C-MEF2-luc reporter assay, being either toxic compounds, general inhibitors of the transcriptional or translational machinery, or inhibitors of luciferase activity. Generation of the poly- and selection of the monoclonal reporter cell lines was carried out at the NMI at the University of Tuebingen. These cell lines were grown in RPMI 1640 Medium without Phenol Red supplemented with 10% FCS and Glutamax. Additionally, for SN12C-CMV-luc, 0.4 μg/μL of Blasticidin was also present in the culture medium. All cells were grown at 37 °C in a humidified atmosphere with 5% CO2. On day 1, the cells were seeded in 384-well white plates at a density of 10,000 cells per well in 20 μL of culture medium. On day 2, the test compounds were added in serial dilutions using the HP D300 Digital Dispenser and incubated at 37 °C for 16 h. On day 3, EGF was added to every well (final concentration = 100 ng/mL), and the plates were incubated for additional 2 h at 37 °C. Then, 25 μL of ONE-Glo was added to each well, and the plates were incubated for 5 min at RT (shaking). The luminescence signal was read on PHERAStar. IC50s were calculated using the DRC Master Spreadsheet. Values obtained for cells treated with EGF and DMSO were defined as the maximum control, while values for cells treated with EGF and 10 μM of XMD8–92 (SN12C-MEF2-luc) or 1 μM Staurosporine (SN12C-CMV-luc) were defined as the minimum control (i.e., maximum inhibition).

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In Vitro Metabolic Stability in Rat Hepatocytes [1]
Hepatocytes from Han/Wistar rats were isolated via a two-step perfusion method. After perfusion, the liver was carefully removed from the rat: the liver capsule was opened, and the hepatocytes were gently shaken out into a Petri dish with ice-cold Williams’ medium E (WME). The resulting cell suspension was filtered through sterile gaze in 50 mL falcon tubes and centrifuged at 50g for 3 min at room temperature. The cell pellet was resuspended in 30 mL of WME and centrifuged twice through a Percoll gradient at 100g. The hepatocytes were washed again with WME and resuspended in medium containing 5% FCS. Cell viability was determined by trypan blue exclusion. For the metabolic stability assay, liver cells were distributed in WME containing 5% FCS to glass vials at a density of 1.0 × 106 vital cells/mL. The test compound was added to a final concentration of 1 μM. During incubation, the hepatocyte suspensions were continuously shaken at 580 rpm, and aliquots were taken at 2, 8, 16, 30, 45, and 90 min, to which equal volumes of cold methanol were immediately added. Samples were frozen at −20 °C overnight, subsequently centrifuged for 15 min at 3000 rpm, and the supernatant was analyzed with an Agilent 1200 HPLC-system with LC–MS/MS detection. The half-life of a test compound was determined from the concentration–time plot. From the half-life, the intrinsic clearances, the hepatic in vivo blood clearance (CL), and the maximal oral bioavailability (Fmax) were calculated using the “well stirred” liver model together with the additional parameters of liver blood flow, specific liver weight, and amount of liver cells in vivo and in vitro. The following parameter values were used: liver blood flow 4.2 L/h/kg, specific liver weight 32 g/kg, liver cells in vivo 1.1 × 108 cells/g liver, and liver cells in vitro 1.0 × 106/mL.

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Automated hERG K+ Current Voltage-Clamp Assay [1]
The hERG K+ current assay is based on a recombinant HEK293 cell line with stable expression of the KCNH2(HERG) gene. The cells were cultured using a humidified incubator (37 °C, 5% CO2) and a standard culture medium (MEM with Earle’s salts and l-glutamine, 10% noninactivated fetal calf serum, 0.1 mmol/L nonessential amino acids, 1 mmol/L Na-pyruvate, penicillin/streptomycin (50 μg/mL each), 0.4 mg/mL Geneticin). Approximately 0.5–8 h following cell dissociation, the cells are investigated by means of the “whole-cell voltage-clamp” technique in an automated 8-channel system with PatchControlHT software (Nanion) to control the Patchliner system and to handle data aquisition and analysis. Voltage-clamp control was provided by two EPC 10 quadro amplifiers under control of the PatchMasterPro software and with NPC-16 medium resistance (∼2 MΩ) chips serving as planar substrate at room temperature (22–24 °C). NPC-16 chips are filled with intra- and extracellular solution (intracellular solution (in mmol/L): NaCl 10, KCl 50, KF 60, EGTA 20, HEPES 10, pH 7.2 (KOH); extracellular solution (in mmol/L): NaCl 140, KCl 4, CaCl2 2, MgCl2 1, glucose 5, HEPES 10, pH 7.4 (NaOH)) and with cell suspension. After formation of a GΩ-seal and entering whole-cell mode (including several automated quality control steps), the cell membrane is clamped to the holding potential (−80 mV). Following an activating clamp step (+20 mV, 1000 ms), exclusively hERG-mediated inward tail currents are elicited by hyperpolarizing voltage steps from +20 mV to −120 mV (duration 500 ms); this clamp protocol is repeated every 12 s. (27) After an initial stabilization phase (5–6 min), test compounds were added either as single concentration (10 μmol/L) or in ascending concentrations (0.1, 1, and 10 μmol/L; 5–6 min per concentration), followed by several washout steps. Effects of test compounds are quantified by analyzing the amplitude of the hERG-mediated inward tail currents (in percent of predrug control) as a function of test compound concentration. Mean concentration–response data were fitted with a standard sigmoidal 4-parameter logistic equation of the form: Y = bottom + (top – bottom)/(1 + exp((Log IC50 – X) × Hill Slope)), where Y is the current inhibition (in % of predrug control), X is the logarithm of drug concentration, and IC50 is the drug concentration producing half-maximal current inhibition, and using the following constraints: top = 100%, bottom = 0%. No curve fitting was performed in cases with an obvious lack of a concentration-dependent current inhibition and/or a too small effect size (approximately ≤20%).

Compound 41 (BAY-885) exhibits favorable physicochemical properties such as high solubility in combination with reasonable lipophilicity as measured by LogD (Figure 4). Compound 41 is highly permeable in the Caco assay and shows no inhibition of activity of CYP enzymes up to 20 μM. Metabolic stability is low to moderate in rat hepatocytes and human liver microsomes, respectively. Compound 41 is chemically stable at different pH values.[1]

BAY-885 showed no inhibition of hERG up to 10 μM. [1]

[1]. Discovery and Characterization of the Potent and Highly Selective (Piperidin-4-yl)pyrido[3,2- d]pyrimidine Based in Vitro Probe BAY-885 for the Kinase ERK5. J Med Chem. 2019 Jan 24;62(2):928-940.

The availability of a chemical probe to study the role of a specific domain of a protein in a concentration- and time-dependent manner is of high value. Herein, we report the identification of a highly potent and selective ERK5 inhibitor BAY-885 by high-throughput screening and subsequent structure-based optimization. ERK5 is a key integrator of cellular signal transduction, and it has been shown to play a role in various cellular processes such as proliferation, differentiation, apoptosis, and cell survival. We could demonstrate that inhibition of ERK5 kinase and transcriptional activity with a small molecule did not translate into antiproliferative activity in different relevant cell models, which is in contrast to the results obtained by RNAi technology. [1]

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Our results are in agreement with the findings of Lin et al., which suggest that the kinase activity of ERK5 is dispensable for cancer cell growth, thus raising doubts as to the viability of ERK5 kinase as a therapeutic target for anticancer drug development. The availability of a potent and selective chemical probe, such as compound 41 (BAY-885), which was recently accepted as a donated chemical probe by the SGC, will significantly contribute to further understanding the biology of ERK5 signaling in cancer. Whether inhibition of ERK5 kinase activity can be compensated by other pathways remains to be shown by future works, e.g., by combination studies using ERK1/ERK2 inhibitor with ERK5 inhibitor as suggested by Cox et al. The lack of efficacy of an ERK5 kinase small molecule inhibitor contrasts with the antiproliferative effects of genetic depletion or deletion of ERK5, thus raising the question whether ERK5 can also act as a scaffolding protein. In this case, a PROTAC approach, leading to degradation of the whole ERK5 protein, would be appropriate to exploit the therapeutic potential of ERK5. [1]

C25H28F3N7O2

515.5

515.23

C, 58.24; H, 5.47; F, 11.06; N, 19.02; O, 6.21

2307249-33-6

2307249-33-6

134128280

White to light yellow solid powder

3.3

1

11

4

37

774

0

FC(OC1C=CC(=C(C=1)N)C(N1CCC(C2C3C(=CC(=CN=3)N3CCN(C)CC3)N=CN=2)CC1)=O)(F)F

QXURFIGBRGWPQD-UHFFFAOYSA-N

hI=1S/C25H28F3N7O2/c1-33-8-10-34(11-9-33)17-12-21-23(30-14-17)22(32-15-31-21)16-4-6-35(7-5-16)24(36)19-3-2-18(13-20(19)29)37-25(26,27)28/h2-3,12-16H,4-11,29H2,1H3

[2-amino-4-(trifluoromethoxy)phenyl]-[4-[7-(4-methylpiperazin-1-yl)pyrido[3,2-d]pyrimidin-4-yl]piperidin-1-yl]methanone

BAY 885; BAY885; [2-amino-4-(trifluoromethoxy)phenyl]-[4-[7-(4-methylpiperazin-1-yl)pyrido[3,2-d]pyrimidin-4-yl]piperidin-1-yl]methanone; compound 41 [PMID: 30563338]; compound 41 (PMID: 30563338); (2-amino-4-(trifluoromethoxy)phenyl)-(4-(7-(4-methylpiperazin-1-yl)pyrido(3,2-d)pyrimidin-4-yl)piperidin-1-yl)methanone; 2307249-33-6; CHEMBL4445670; BAY-885

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 16.67~30 mg/mL (32.3~58.2 mM)
Ethanol: ~8 mg/mL (~15.5 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.85 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 (4.85 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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.

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Solubility in Formulation 3: ≥ 1.67 mg/mL (3.24 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 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 16.7 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 4: ≥ 1.67 mg/mL (3.24 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 16.7 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 5: 0.5 mg/mL (0.97 mM) in 1% DMSO 99% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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.)