ATPase family AAA domain-containing protein 2 (ATAD2) (IC50 = 166 nM)

In the TR-FRET test, BAY-850's IC50 of 166 nM indicates its competitiveness with the monoacetylated histone H4 N-terminal peptide for binding to ATAD2 BD. With an IC50 of 157 nM and a KD of 115 nM, BAY-850 substitutes tetraacetylated peptide. The remarkable isoform specificity of BAY-850 implies a distinct mechanism of action compared to traditional BD inhibitors [1].

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BAY-850 competed with the binding of a monoacetylated histone H4 N-terminal peptide to ATAD2 BD with an IC50 of 166 nM measured in TR-FRET assay (Figure 2A). Under similar conditions, the compound displaced a tetra-acetylated H4 peptide with an IC50 of 22 nM. A similar shift between the two assays was observed for all related compounds tested (Supporting Information Figure 4A). This was consistent with the fact that the K12 monoacetylated peptide had previously been shown to have higher binding efficacy in this assay.4 In orthogonal binding competition assays such as Alphascreen and BROMOscan, BAY-850 displaced the tetra-acetylated peptide with an IC50 of 157 nM and a KD of 115 nM, respectively, confirming that the effects shown by the compound were independent of the readout technology used to measure them (Figure 2A). Furthermore, the compound inhibited tetra-acetylated H4 peptide binding to a larger ATAD2 construct including the ATPase domain (Supporting Information Figure 4D). The negative control compound, BAY-460, was inactive at the same concentrations in all assays where it was tested[1].

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Next, we set out to confirm the biochemical activity of BAY-850 by detecting direct binding of the compound to ATAD2 with biophysical techniques. First, protein-observed NMR spectroscopy confirmed specific ATAD2 engagement by a close congener of BAY-850 (Supporting Information Figure 3). In a microscale thermophoresis (MST) assay, BAY-850 exhibited dose-dependent, saturatable effects on the ATAD2 hydration shell (Figure 2B), from which a KD of 85 nM was calculated. This value is in agreement with the potencies described above, a feature that could be shown for all compounds profiled with MST (Supporting Information Figure 4B). Furthermore, in a thermal shift assay (TSA) BAY-850 -unlike BAY-460- increased the melting temperature of ATAD2 in a dose-dependent fashion (Figure 2C). This assay also showed a trend to stronger thermal stabilization with increasing biochemical potencies of members of the same chemical series (Supporting Information Figure 4C). The selectivity profile of BAY-850 and BAY-460 was initially investigated in the BROMOscan panel, where the first compound exclusively hit ATAD2 but -surprisingly- not the closely related ATAD2B, while the latter did not show significant effects at the same concentrations (Figure 2D and Supporting Information Figure 5A). These results were confirmed in a TSA panel, where the effect of BAY-850 on the thermal stability of ATAD2 was not observed for other members of the BD family when the compound was tested at 10 μM (Figure 2E and Supporting Information Figure 5B). In addition, BAY-850 showed no inhibitory activity in a panel of 354 kinases and had only modest effects at high concentrations on a few GPCRs (Supporting Information Tables 2 and 3)[1].

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The unprecedented isoform selectivity of BAY-850 suggested a different mode of action to those exhibited by canonical BD inhibitors. This interpretation was reinforced by isothermal calorimetry (ITC) analysis (Supporting Information Figure 4E), in which weak heat signals for the interaction could only be recorded at 37 °C, and the stoichiometry was uncharacteristic when compared to orthosteric ligands.1 A closer examination of BAY-850’s dose titration in TSA revealed that ATAD2 melting curves transitioned from a monophasic to a biphasic shape with increasing concentrations of the compound, suggesting the appearance of a new protein species with a differentiated melting profile upon saturation with BAY-850 (Figure 2C). These observations prompted us to conduct an in-depth investigation of the BAY-850 binding mode using nondenaturing or native mass spectrometry (MS), a well-established method to investigate protein–ligand interactions,10 which delivers information about the multimeric character and the conformation of a protein by its charge-state distribution in the mass spectrum. The native mass spectrum of tag-free ATAD2 is dominated by two signals derived from monomeric ATAD2 with charge states +6 and +7, respectively, two minor signals derived from dimeric ATAD2 with charge states +9 and +10, respectively, and signals of partially unfolded ATAD2 in the m/z range between 1100 and 2000 (Figure 3A). Next, we acquired the native mass spectrum of ATAD2 incubated with BAY-850. To our surprise, the spectrum was dominated by signals derived from a complex where one BAY-850 molecule binds to an ATAD2 dimer (Figure 3B), with no binding observed for monomeric ATAD2. In contrast, the inactive control BAY-460 showed no binding to monomeric or dimeric ATAD2. The dimer-inducing effect of BAY-850 is concentration-dependent, suggesting a specific process (Figure 3C and Supporting Information Figure 6A). These observations were confirmed by analytical size exclusion chromatography (SEC) analysis, where we observed a compound-induced shift from the ATAD2-monomer to ATAD2-dimer form in the elution profile (Figure 3D). Importantly, BAY-850 was not able to induce further multimerization of GST dimers when the experiments were performed with GST-tagged ATAD2 (Figure 3D, inset), confirming that this effect originates from an interaction between the compound and ATAD2 and not with the GST tag. Competition native MS experiments showed weak binding of a single monoacetylated histone H4 peptide molecule to the inhibitor-bound ATAD2 dimer. In contrast, in the absence of BAY-850, clear 1:1 binding of the same peptide to monomeric ATAD2 and 2:2 binding to dimeric ATAD2 was observed (Supporting Information Figure 6B)[1].

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Knockdown experiments have suggested that the survival of some cancer cell lines may depend on ATAD2. Therefore, we investigated the antiproliferative effects of BAY-850, BAY-460, and several related compounds with a broad range of biochemical activities on three cancer cell lines. We observed a weak correlation between the biochemical potency and growth inhibition, with GI50’s in the single-digit micromolar range for BAY-850 and similarly potent compounds, while BAY-460 showed no efficacy up to the highest concentrations tested (Figure 4D). Since growth inhibition occurred only at high compound concentrations, and without particular discrimination between cell lines, we went on to compare activities of BAY-850 and BAY-460 in normal epithelial and cancer cells. Here again, a clear differentiation between both compounds was observed, but BAY-850 was similarly active on the nontransformed and cancer cells (Figure 4E). Thus, we concluded that the cytotoxic effects displayed by BAY-850 cannot be unmistakably linked to ATAD2 BD inhibition. Further evidence for a disconnect between the observed growth inhibition and the inhibition of ATAD2 BD was provided by gene expression studies, in which BAY-850 treatment did not affect the expression of some of the previously identified ATAD2 target genes (Supporting Information Figure 7). Consistently, other recently published ATAD2 BD inhibitors also failed to demonstrate significant effects on target gene expression and cancer cell survival below 20 μM despite engaging ATAD2 BD in living cells. Potential dispensability of the BD in multidomain containing proteins was recently observed for SMARCA2/4 proteins, where discrepancies between the knockdown of the entire protein and a specific inhibition of their BD function were observed. In line with these reports, there is no convincing domain-specific genetic validation of ATAD2 BD in cancer cell survival thus far and, at least in the leukemia-domain-focused CRISPR/Cas9 screen, revealed no dependencies on the BD of ATAD2 [1].

Time-resolved fluorescence resonance energy transfer (TR-FRET) binding competition assay [1]
Assays were performed at room temperature (RT) in 384-well low volume black microtiter plates in a final volume of 5 µL. Test compounds were serially diluted in DMSO (3.5- fold, 12-points, 0-20 µM) using a Precision liquid handling robot and 50 nl were dispensed onto the plates at 100X the test concentration with a Hummingbird capillary dispenser. Next, 2 µl of 10 nM GST-ATAD2 BD [in assay buffer 50 mM Hepes pH 7.5, 100 mM NaCl, 50 mM KF, 0.25 mM CHAPS, 0.05% bovine serum albumin (BSA) and 1 mM dithiotreitol (DTT)] were added with a Multidrop and the plates were incubated for 15 min. Finally, 3 µl of 50 nM of C-terminal biotinylated synthetic acetylated peptides derived from histone H4 a.a.1-25 (K12 mono-acetylated or K5,8,12,16 tetraacetylated) and detection reagents (10 nM anti-6His-XL665), 2.5 nM streptavidin Eu, both in assay buffer) were dispensed and further incubated for at least 1 h. TRFRET signals corresponding to the number of protein-peptide complexes in equilibrium were acquired either with Viewlux or Pherastar microtiter plate readers. The normalized ratios of the fluorescence emission at 665 nm and at 620-622 nm after excitation at 330-350 nm, at increasing compound concentrations were used to calculate IC50 values with the Screener Software by regression analysis based on a fourparameter equation [minimum, maximum, IC50, Hill; Y = Max + (Min - Max) / (1 + (X/IC50)^Hill]. [1]

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AlphaScreen® binding competition assays[1]
Compound binding was determined by the displacement of a tetra-acetylated biotinylated peptide from a Hexa His tagged ATAD2 BD or a FLAG tagged ATAD2 (342 - 1390) protein using AlphaScreen® Histidine (Nickel Chelate) or FLAG (M2) Detection Kits. Compound was dispensed from DMSO stocks into assay plates using an Echo 525 Liquid Handler. Assays were performed at 25°C in a volume of 20 µl in a buffer that was 25 mM HEPES pH7.4, 100mM NaCl, 0.1% BSA, 0.05% CHAPS with 25 nM peptide (SGRGK(ac)GGK(ac)GLGK(ac)GGAK(ac)RHRK(biotin)-acid), 100 nM ATAD2, 0.2 % DMSO. Peptide and protein were pre-mixed, added to the compound plate and incubated for 30 minutes after which AlphaScreen® beads were added to a final concentration of 6 µg/ml. Assay plates were incubated for 60 minutes then luminescence was measured using a Pherastar FS.

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ADP Glo® ATPase activity assay [1]
ATPase assays were conducted using the ADP-Glo® kit according to manufacturer’s instructions. Compound and ATP was dispensed into assay plates using an Echo 525 Liquid Handler (Labcyte). Assays were performed at 25°C in a volume of 4 µl in a buffer that was 20 mM HEPES pH7.4, 50 mM NaCl, 0.5 mM TCEP, 1 mM MgCl2 and 0.1% BSA. Reactions were initiated by the addition of 100 nM ATAD2 protein (342 - 1390) and incubated for three hours. Reactions were then terminated by the addition of 4 µl of the ADP Glo® reagent, to deplete unhydrolysed ATP, which was then incubated for one hour at 25 °C. Luciferase/luciferin reactions were then initiated by the addition of 4 µl ADP-Glo® detection reagent which then added incubated at 25 °C for one hour after which assay plates were read on BMG Pherastar FSX.

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Microscale Thermophoresis (MST) [1]
For experiments with fluorescence labeled protein, GST-ATAD2 BD was labeled using the RED-NHS kit according to the manufacturer’s instructions. Alternatively, Alexa Fluor 647 was covalently attached by NHS coupling to the N-terminal primary amine groups of the protein. Experiments were performed on the Monolith NT.115 Instrument using the RED detector. When Nano Temper proprietary labeling was chosen, a protein concentration of 20 nM and a buffer containing 25 mM HEPES pH7.4, 150 mM NaCl, 0.05% Tween 20 and 0.5 mM TCEP were used. For experiments using Alexa Fluor 647 labeling, the concentration of GST-ATAD2 BD was 50 nM, and the buffer 50 mM Hepes pH 7.5, supplemented with 150 mM NaCl and 0.05% Pluronic. Compounds were screened in a twelve point threefold serial dilution beginning at 100 μM compound concentration down to 0.5 nM either manually or using an Echo 525 Liquid Handler. After 30 minutes incubation, the protein and compound samples were centrifuged for 5 min at 14000 rpm and then loaded into Monolith™ NT.115 Series MST Premium Coated Capillaries. The MST experiment was performed using LED Power of 20% or 40 %, and MST Power of 40% or 80% (depending on whether Nanotemper proprietary- or Alexa Fluor 647 labeling were used) with thermophoresis occurring over 30 seconds. KD values were determined with the MO.Affinity Analysis Software v2.1 from Nanotemper. Experiments with unmodified protein were performed on the Monolith NT.Automated Instrument using the Label free detector with a GST-ATAD2 BD concentration of 500 nM. Compounds were screened in a twelve point two-fold serial dilution beginning at 500 μM 26 compound concentration down to 244 nM. The assay was performed in buffer containing 150 mM NaCl, 50 mM HEPES 7.5, 0.05% Pluronic. After 30 minutes incubation, the samples were loaded into Monolith™ NT.Automated Zero Background MST Premium Coated Capillary Chips and the MST experiment was performed using the Monolith NT.Automated (LED Power 50 %, MST Power 80%). KD values were determined with the MO.Affinity Analysis Software v2.1 from Nanotemper.

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Isothermal titration calorimetry (ITC) [1]
Experiments were carried out on a NanoITC microcalorimeter (TA Instruments) at 37 °C in 20 mM HEPES pH 7.5, 50 mM NaCl, 0.5 mM TCEP, 5% glycerol. ATAD2 protein solution was buffer exchanged by dialysis into the ITC buffer. The experiment was performed in reverse titration mode. Protein concentration in the syringe was 200 μM and the BAY-850 inhibitor concentration in the cell was 40 μM. Thermodynamic parameters were calculated using ΔG = ΔH - TΔS = -RTlnKB, where ΔG, ΔH and ΔS are the changes in free energy, enthalpy and entropy of binding respectively using a single binding site model.

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Thermal Shift Assay (TSA) / Differential Scanning Fluorimetry (DSF) [1]
Thermal melting experiments with GST-tagged and untagged ATAD2 BD and were carried out in 384-well plates using a ViiATM Real-Time PCR machine. Melting curves were obtained at a protein concentration of 5.3 µM in presence of environment sensitive fluorescence probe SYPRO Orange at a dilution of 1:625 from the supplied stock, using buffer containing 20 mM HEPES pH7.5; 200 mM NaCl; 10% Glycerol; 0.5 mM TCEP. For binding experiments, ligands in serial dilution (0.049 µM to 100 µM, 5-fold) were added to the mixture, as control 1% DMSO was used. Excitation and emission filters for the SYPRO 27 Orange dye were set to 465 and 590 nm, respectively and scans were measured from 25°C to 95 °C at a rate of 4 °C/min. To assess the bromodomain selectivity of BAY-850, thermal melting experiments were carried out using an Mx3005p machine. Proteins were buffered in 10 mM HEPES, pH 7.5, 500 mM NaCl and assayed in a 96-well plate at a final concentration of 2 μM in 20 μL volume. BAY-850 was added to obtain a final concentration of 10 μM and SYPRO Orange was added at a dilution of 1:1000 from the supplied stock. Excitation and emission filters for the SYPRO Orange dye were set to 465 and 590 nm, respectively. The temperature was raised with a step of 3°C per minute from 25 to 96°C, and fluorescence readings were taken at each time interval

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Native Mass Spectrometry [1]
ATAD2 samples were buffer exchanged by dialysis against 100 mM ammonium acetate (pH 6.8) and 1 mM DTT using Slide-A-Lyzer dialysis cassettes with a molecular weight cut-off of 10 kDa. The protein concentration after dialysis was determined by absorbance at 280 nm using a NanoDrop 2000 spectrophotometer. For the titration experiments ATAD2 protein samples were diluted to 10 µM with 100 mM ammonium acetate and incubated with ligand concentrations ranging from 1.25 to 50 µM with a final DMSO concentration of 1 %. The peptide competition experiments were performed by incubating 10 µM ATAD2 protein with 10 µM peptide and subsequently with increasing concentration of ligand (1.25 – 50 µM) at a final DMSO concentration of 1.2 %. 28 Nano-electrospray MS data were collected on a Waters SYNAPT G2-S quadrupole time-offlight mass spectrometer connected to a Triversa NanoMate chip-based nano-ESI-source operated at a chip nozzle voltage of 1.7 kV in the positive ion mode and a pressure of 2.7 bar at the back of the conductive pipet tip. The source temperature was kept at room temperature. All measurements were performed at three different cone voltages (50, 75 and 100 V) in order to find optimal conditions to obtain a narrow peak shape without disrupting the noncovalent protein-ligand interactions. Calibration of the instrument was performed using sodium iodide clusters (2 µg/µL in 2-propanol:water; 1:1) up to m/z 3500. In order to improve the signal-to-noise ratio mass spectra were accumulated for 1-2 min. The instrument was controlled and data were evaluated using MassLynx software version 4.1.

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Size Exclusion Chromatography (SEC) [1]
To assess ATAD2 bromodomain dimerisation by BAY-850, ATAD2 and GST-ATAD2 preparations were each equally separated into two samples; one of which was incubated with BAY-850 (50 mM DMSO stock solution) in a molar ratio of 1:1, while the other was incubated with the same amount of DMSO instead of compound (apo samples). The samples were then analysed by SEC using 16/600 HiLoad Superdex200 prep grade column and buffer B. Column calibration was performed using commercial gel filtration standards.

Permeability Assay. [1]
Caco2 cells purchased from the DSMZ were seeded at a density of 4.5 x 104 cells per well and grown for 15 d in DMEM with typical supplements. Cells were kept at 37 °C in a humidified 5% CO2 atmosphere. Before the permeation assay was run, the culture medium was replaced by a FCS-free Hepes carbonate transport buffer (pH 7.2). For assessment of monolayer integrity the transepithelial electrical re-sistance was measured. Test compounds were predissolved in DMSO and added either to the apical or basolateral compartment at a final concentration of 2 µM. Before and after 2 h incubation at 37 °C, samples were taken from both compartments. Analysis of compound content was conducted after precipitation with methanol by LC/MS/MS analysis. Permeability (Papp) was calculated in the apical to basolateral (A → B) and basolateral to apical (B → A) directions. The efflux ratio basolateral (B) to apical (A) was calculated by dividing Papp B-A by Papp A-B. Reference compounds were analyzed in parallel as assay control.

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Metabolic Stability in Hepatocytes. [1]
A hepatocyte cell suspension of the respective species was filtered through sterile gauze in 50 mL Falcon tubes and centrifuged at 50×g for 3 min at RT. The cell pellet was re-suspended in 30 mL WME and centrifuged through a Percoll® gradient twice at 100×g. The hepatocytes were washed again with WME and resuspended in medium containing 5% FCS. Liver cells were distributed in WME containing 5% FCS to glass vials at a density of 1.0 × 106 vital cells/mL. BAY-850 and BAY-460 were added at 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 33 were frozen at -20 °C overnight, then centrifuged for 15 min at 3000 rpm and the supernatant was analyzed by LC/MS/MS. The half-lives of BAY-850 and BAY-460 were determined from the concentration-time plot. The intrinsic clearances were calculated from the half-life.

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Metabolic Stability in Liver Microsomes. [1]
The in vitro metabolic stability was determined by incubating com-pounds at 1 µM concentration in a suspension of liver microsomes in 100 mM phosphate buffer, pH 7.4 (NaH2PO4 x H2O + Na2HPO4 x 2H2O) and at a protein concentration of 0.5 mg/mL at 37 °C. The microsomes were activated by adding a co-factor mix containing 8 mM glucose-6- phosphate, 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 afterwards by adding BAY-850 or BAY460 to the incubation in a final volume of 1 mL. 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, then centrifuged for 15 min at 3000 rpm and the supernatant was analyzed by LC/MS/MS. The half-lives of BAY-850 and BAY-460 were determined from the concentrationtime plot. The in vitro blood clearances were calculated from the half-life.

BAY-850 and BAY-460 have overall acceptable physicochemical and in vitro pharmacokinetics profiles with moderate permeability as determined using a Caco-2 assay (Supporting Information Table 4). These properties allowed for the determination of in-cell target engagement. We used fluorescence recovery after photobleaching (FRAP) assay to measure compound-related displacement of labeled full-length ATAD2 from chromatin in MCF7 cells (Figure 3A–C) with an optimized method we described earlier.4 Treatment with 1 μM of BAY-850 resulted in a decreased half recovery time (t1/2) of GFP-tagged full-length wild type ATAD2. This effect was not observed with BAY-460 and is comparable to the mutagenesis of ATAD2 BD (Figure 4B,C). This approach allowed us to demonstrate that BAY-850 is a cellularly active ATAD2 BD inhibitor with maximal on-target cellular activity at 1 μM. [1]

[1]. Isoform-Selective ATAD2 Chemical Probe with Novel Chemical Structure and Unusual Mode of Action. ACS Chem Biol. 2017 Nov 17;12(11):2730-2736.

ATAD2 (ANCCA) is an epigenetic regulator and transcriptional cofactor, whose overexpression has been linked to the progress of various cancer types. Here, we report a DNA-encoded library screen leading to the discovery of BAY-850, a potent and isoform selective inhibitor that specifically induces ATAD2 bromodomain dimerization and prevents interactions with acetylated histones in vitro, as well as with chromatin in cells. These features qualify BAY-850 as a chemical probe to explore ATAD2 biology. [1]

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In conclusion, we have used affinity-mediated selection with a DNA-encoded library to discover a new chemical class of potent, isoform selective ATAD2 BD inhibitors. These features, along with sufficient solubility and permeability, make BAY-850 and its inactive companion BAY-460 (dosed at noncytotoxic concentrations) valuable tools to further investigate the biology of ATAD2. Furthermore, our results suggest that BAY-850’s mode of action might be linked to protein dimer induction, a feature which could represent a new approach for inhibiting protein–protein interactions.18 Using this compound, we have confirmed the dispensability of ATAD2 BD for gene expression regulation. These findings combined with the roles attributed to the ATAD2 gene in knockdown studies make ATAD2 particularly well suited for targeted protein degradation approaches. Our work lays the foundation for further compound development, e.g., toward PROTAC “warheads” with higher potency and improved PK properties, which may ultimately lead to in vivo probes and novel therapeutic agents.[1]

C38H44CLN5O3

654.240668296814

653.313

C, 69.76; H, 6.78; Cl, 5.42; N, 10.70; O, 7.34

2099142-76-2

2099142-77-3 (isomer); 2099142-76-2; 2561471-14-3 (HCl);

129196931

White to light yellow solid powder

5.9

4

7

13

47

1000

2

ClC1=CC(=C(C2=CC=C(CN[C@@H](C)C3C=CC(C)=CC=3)O2)C=C1C(N[C@@H](CC1C=CC(C#N)=CC=1)CNC1CCC(CC1)N)=O)OC

BSISGUIVBKDTQO-JLXKDNNHSA-N

InChI=1S/C38H44ClN5O3/c1-24-4-10-28(11-5-24)25(2)42-23-32-16-17-36(47-32)34-19-33(35(39)20-37(34)46-3)38(45)44-31(18-26-6-8-27(21-40)9-7-26)22-43-30-14-12-29(41)13-15-30/h4-11,16-17,19-20,25,29-31,42-43H,12-15,18,22-23,41H2,1-3H3,(H,44,45)/t25-,29?,30?,31-/m1/s1

N-[(2R)-1-[(4-Aminocyclohexyl)amino]-3-(4-cyanophenyl)propan-2-yl]-2-chloro-4-methoxy-5-[5-({[(1R)-1-(4-methylphenyl)ethyl]amino}methyl)-2-furyl]benzamide

BAY-850; BAY850; BAY-850; 2099142-76-2; CHEMBL4536031; N-[(2R)-1-[(4-aminocyclohexyl)amino]-3-(4-cyanophenyl)propan-2-yl]-2-chloro-4-methoxy-5-[5-[[[(1R)-1-(4-methylphenyl)ethyl]amino]methyl]furan-2-yl]benzamide; N-[(2R)-1-[(4-aminocyclohexyl)amino]-3-(4-cyanophenyl)propan-2-yl]-2-chloro-4-methoxy-5-[5-({[(1R)-1-(4-methylphenyl)ethyl]amino}methyl)furan-2-yl]benzamide; trans-BAY-850; SCHEMBL18930553; SCHEMBL21144877; BAY 850

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 : ~62.5 mg/mL (~95.53 mM)
H2O : < 0.1 mg/mL

Solubility in Formulation 1: ≥ 6.25 mg/mL (9.55 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 62.5 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: ≥ 6.25 mg/mL (9.55 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 62.5 mg/mL clear DMSO stock solution to 900 μL corn oil and mix evenly.

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