lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC(=O)c1c(C)c2cnc(N[C@@H]3CCOC[C@H]3O)nc2n([C@H]2CCC[C@]2(C)O)c1=O
InChIKey: VXZRTFJMWFQYND-PTDBIEIZSA-N
Target: CDK6/cyclin D3 (PROTEIN COMPLEX), Homo sapiens
UniProt: P30281
Assay: Chelation-Enhanced Fluorescence (CHEF) Assay: CDK4/Cyclin D1 and CDK6/Cylcin D3: The Chelation-Enhanced Fluorescence (CHEF) monitors the phosphorylation state in real time where the level of fluorescence is directly proportional to the amount of phosphorylated substrate. CHEF exploits a synthetic α-amino acid with a side chain bearing an 8-hydroxyquinoline derivative (sulfonamido-oxine, Sox) which, upon coordination to Mg(II), relays information on the phosphorylation state of a proximal serine, threonine or tyrosine residue in peptide-based kinase substrates. Phosphorylation of a specific peptide (Catalog # AQT0258) from AssayQuant Technologies results in an increase in fluorescence at the excitation and emission wavelengths of 360 nm Ex/485 nm Em.Examples along with DMSO (negative) and Palbociclib (positive) controls were added to 384-well plates at 100-fold their final concentrations followed by the addition of 10 nM CDK4/Cyclin D1 (LJIC-2007F1) or 10 nM CDK6/Cyclin D3 (LJIC-2009H2) for a 20 minute pre-incubation in assay buffer containing 40 mM HEPES, 1 mM Dithiothreito (DTT, 10 mM MgCl2, 1% Glycerol, 0.1% BSA. Enzyme reactions were initiated by the addition of AssayQuant Technologies peptide and ATP substrates (10 μM CHEF peptide (Catalog # AQT0258), 2 mM ATP) and allowed to proceed for 2 hours followed by fluorescence read of the reaction. Ki determinations were made from a plot of the fractional velocity as a function of inhibitor concentration fit to the Morrison equation with the enzyme concentration as a variable.
Measurement: Ki = 7.7 nM (pChEMBL 8.109999656677246)
Descriptors: MW 416.48, ALogP 1.34, HBA 9, HBD 3, PSA 126.57, QED 0.64
Compound (SMILES): N#CCC1(n2ccc(-c3nc(-c4ccc(N)nc4)cc4ncccc34)n2)CC(C#N)C1
InChIKey: CRDHRCLRNDFCTL-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: JAK Caliper Enzyme Assay at 1mM ATP: Compounds were added to a 384-well plate. Reaction mixtures contained 10 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% BSA, 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20% to 30% phosphorylation. The assays were stopped with 15 μL of 180 mM HEPES, pH 7.4, 20 mM EDTA, and 0.2% Coating Reagent 3. The assay plates were placed on a Caliper Life Science LC3000 instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 2056 nM (pChEMBL 5.690000057220459)
Descriptors: MW 406.45, ALogP 3.68, HBA 8, HBD 1, PSA 130.09, QED 0.55
Compound (SMILES): CN(C)C(=O)N[C@@H]1CCCN(c2cnc(C(N)=O)c(Nc3ccc(C4(C(=O)N(C)C)CC4)cc3)n2)C1
InChIKey: WWFYYEUUXFNAFM-GOSISDBHSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: HotSpot kinase Assay: The degree of inhibition of a panel of kinases is determined using the in vitro HotSpot kinase assay (purified enzymes, 33P-ATP, an appropriate substrate and 1 uM ATP).
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 494.6, ALogP 1.68, HBA 7, HBD 3, PSA 136.79, QED 0.53
Compound (SMILES): CCCCNc1ncc(-c2ccc(N3CCCC3)nn2)c(N[C@H]2CC[C@H](O)CC2)n1
InChIKey: IGOHZEZLKPSIDE-QAQDUYKDSA-N
Target: Growth arrest-specific protein 6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14393
Assay: Activity Assay: Briefly, activity assays were performed in a 384 well, polypropylene microplate in a final volume of 50 μL of 50 mM Hepes, Ph 7.4 containing 10 mM MgCl2, 1.0 mM DTT, 0.01% Triton X-100, 0.1% Bovine Serum Albumin (BSA), containing 1.0 μM fluorescent substrate and ATP at the Km for each enzyme. All reactions were terminated by addition of 20 μL of 70 mM EDTA. After a 180 min incubation, phosphorylated and unphosphorylated substrate peptides were separated in buffer supplemented with 1×CR-8 on a LabChip EZ Reader equipped with a 12-sipper chip. Data were analyzed using EZ Reader software. Assay conditions for MCE assays
Measurement: IC50 = 377 nM (pChEMBL 6.420000076293945)
Descriptors: MW 411.55, ALogP 3.46, HBA 8, HBD 3, PSA 99.09, QED 0.57
Compound (SMILES): Cc1ncsc1-c1ccc(CNC(=O)[C@@H]2C[C@@H](O)CN2C(=O)[C@H](C(C)C)N2Cc3ccccc3C2=O)c(OCCOCCOCC#Cc2cccc3c2C(=O)N([C@@H](C(=O)Nc2nccs2)c2cc(F)ccc2O)C3)c1
InChIKey: NJLCDOIUFBCJLJ-RYXNNJGZSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: TR-FRET Assay for ERBB Kinases: All compounds and PROTACs were serially diluted in three-fold increments using 100% DMSO, followed by an intermediate 10-fold dilution using Buffer A (50 mM HEPES, pH 7.5, 50 mM NaCl, 10 mM MgCl2, 1 mM DTT, and 0.1% Pluronic F-68). Two microliters of serially diluted compound or PROTAC were then transferred to black 384-well Proxiplates (PerkinElmer, #6008260) using an Integra Viaflo96. Next, 10 uL of protein kinase in Buffer A was added to each well of the assay plate and pre-incubated with compound for 10 minutes. Kinase reactions were then initiated by addition of 5 uL substrate mix containing 3 mM ATP and 30 uM fluorescein-labeled Poly-GluTyr (Thermo Fisher, #PV3610) in Buffer A and allowed to proceed for 10 minutes at room temperature. Reactions were quenched by addition of a 5 uL mixture containing 5 nM LanthaScreen® Tb-pY20 Antibody (Thermo Fisher, #PV3552) and 40 mM EDTA in Buffer A. Assay plates were then read using a Synergy2 (Biotek Instruments, Winooski, Vt.) via excitation thru a 340/20 nm bandpass filter and emission collected thru 490/10 nm (donor) and 520/25 nm (acceptor) bandpass filters. The final kinase concentrations used for each 15 uL reaction were as follows: 0.2 nM EGFR Exon20NPG (SignalChem, #E10-132GG), 0.1 nM wild type EGFR (BPS Bioscience, #40187), 0.3 nM EGFR L858R/T790M/C797S (BPS Bioscience, #40351), 0.1 nM EGFR L858R (BPS Bioscience, #40189), 0.4 nM L858R/T790M (BPS Bioscience, #40350), InM EGFR Dell9 (SignalChem, #E10-122JG), 10 nM EGFR Dell9 T790M (SignalChem, #E10-122KG), 0.3 nM Her2 (BPS Bioscience, #40230), 15 nM Her2 InsYVMA (SignalChem, #E27-13BG).
Measurement: IC50 = 1400 nM (pChEMBL 5.849999904632568)
Descriptors: MW 1056.21
Compound (SMILES): Cc1cc(Nc2nccc(N3C[C@H]4CC[C@@H](C3)N4C(=O)[C@@H]3CC3(F)F)n2)cnc1C(N)=O
InChIKey: MUZOPNUVNZDWFF-ZNMIVQPWSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Caliper Enzyme Assay: Compounds were added to a 384-well plate. Reaction mixtures contained 10 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% BSA, 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20% to 30% phosphorylation. The assays were stopped with 15 μL of 180 mM HEPES, pH 7.4, 20 mM EDTA, and 0.2% Coating Reagent 3. The assay plates were placed on a Caliper Life Science LC3000 instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 443.46, ALogP 1.86, HBA 7, HBD 2, PSA 117.34, QED 0.72
Compound (SMILES): CC[C@H](NC(=O)[C@H](C)NC)C(=O)N1C[C@@H](F)C[C@H]1Cc1c(-c2nc3cc(F)ccc3n2C[C@@H]2C[C@H](F)CN2C(=O)[C@@H](NC(=O)[C@H](C)NC)C2CCCCC2)[nH]c2cc(F)ccc12
InChIKey: APTRBTUTSFUIJT-IAECAYPDSA-N
Target: Tumor necrosis factor (SINGLE PROTEIN), Homo sapiens
UniProt: P01375
Assay: TNF-alpha inducing NF-kappaB reporter: First day:Dilution of Compounds:  1) tested compounds were added into DMSO to give parent liquor (10 mM);  2) reference compound Birinapant (32 μL, 10 mM) was added into DMSO (128 μL) to give a solution (2 mM);  3) 0.5 μL reference compound Birinapant (2 mM) and tested compounds (10 mM) were taken by Echo and added into Greiner 96 well black cell culture plate respectively. The initial concentration of the reference compound and tested compound was 10 μM and 50 μM, respectively. Then, the reference compound and tested compounds were diluted at 9 points in series by 5-fold; and every plate was repeated for 3 times.  4) the reference compound WXFL2012A001 (Birinapant) (final concentration: 10 μM) as HPE and DMSO (final concentration: 0.5%) as ZPE.Second DayTNF-α Inducing1) TNF-α (100 μg/mL) was diluted into 20 ng/mL with 0.1% FBS medium and transferredto the third plate with 100 μL per well2) Removing medium of the first 96 well black cell culture plate (compound activity testing plate) after 24 h, replacing with fresh medium containing the compound and 20 ng/mL TNF-α in the third plate3) Placing cell culture plate into cell incubator (37° C., 5% CO2) and incubatingfor 6 h4) after 6 h, compound's activity in the first plate was tested according to Bright-Glo (Promega) manual method, luciferase signal in each well was tested by Envision plate reader5) compound's cytotoxicity in the second plate was tested according to ATPlite 1Step (Perkin Elmer) manual method, luciferase signal in each well was tested by Envision plate reader6) analyzing with software to obtain EC50 of compound.
Measurement: EC50 < 10 nM (pChEMBL 8)
Descriptors: MW 866.02, ALogP 5.06, HBA 8, HBD 5, PSA 156.49, QED 0.1
Compound (SMILES): COc1cc(CO)cc(S(=O)(=O)N2CCc3cccc(CCc4ccc(C(=O)O)cc4)c32)c1
InChIKey: GNQFVWHBHKWCJW-UHFFFAOYSA-N
Target: Prostaglandin E2 receptor EP2 subtype (SINGLE PROTEIN), Homo sapiens
UniProt: P43116
Assay: Radioligand Binding Assay: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4° C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2a (5 nM) were performed in a 100 μl volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell harvester. The filters were washed 3 times with ice-cold buffer and oven dried for one hour.[3H] PGE2 (specific activity 180 Ci mmol) was used as the radioligand for EP receptors. [3H] 17-phenyl PGF2a was employed for FP receptor binding studies. Binding studies employing EP1, EP2, EP4 and FP receptors were performed in duplicate in at least three separate experiments. A 200 μl assay volume was used. Incubations were for 60 min at 25° C. and were terminated by the addition of 4 ml of ice-cold 50 mM TRIS-HCl, followed by rapid filtration through Whatman GF/B filters and three additional 4 ml washes in a cell harvester (Brandel).
Measurement: Ki = 18 nM (pChEMBL 7.75)
Descriptors: MW 467.54, ALogP 3.42, HBA 5, HBD 2, PSA 104.14, QED 0.53
Compound (SMILES): Cc1cc(Nc2cc3ncccc3c(N[C@@H]3C[C@H]4CCC[C@@H](C3)N4Cc3cccc(O)c3)n2)n[nH]1
InChIKey: WSFQMCDKLICTAR-MZADTFQBSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Biochemical JAK Assay: A panel of four LanthaScreen JAK biochemical assays (JAK1, 2, 3 and Tyk2) were carried in a common kinase reaction buffer (50 mM HEPES, pH 7.5, 0.01% Brij-35, 10 mM MgCl2, and 1 mM EGTA). Recombinant GST-tagged JAK enzymes and a GFP-tagged STAT1 peptide substrate were obtained from Life Technologies.Serially diluted compounds were pre-incubated with each of the four JAK enzymes and the substrate in white 384-well microplates (Corning) at ambient temperature for lh. ATP was subsequently added to initiate the kinase reactions in 10 μL total volume, with 1% DMSO. The final enzyme concentrations for JAK1, 2, 3 and Tyk2 are 4.2 nM, 0.1 nM, 1 nM, and 0.25 nM respectively; the corresponding Km ATP concentrations used are 25 μM, 3 μM, 1.6 μM, and 10 μM; while the substrate concentration is 200 nM for all four assays. Kinase reactions were allowed to proceed for 1 hour at ambient temperature before a 10 μL preparation of EDTA (10 mM final concentration) and Tb-anti-pSTAT1 (pTyr701) antibody (Life Technologies, 2 nM final concentration) in TR-FRET dilution buffer (Life Technologies) was added. The plates were allowed to incubate at ambient temperature for lh before being read on the EnVision reader (Perkin Elmer). Emission ratio signals (520 nm/495 nm) were recorded and utilized to calculate the percent inhibition values based on DMSO and background controls.For dose-response analysis, percent inhibition data were plotted vs. compound concentrations, and IC50 values were determined from a 4-parameter robust fit model with the Prism software (GraphPad Software). Results were expressed as pIC50 (negative logarithm of IC50) and subsequently converted to pKi (negative logarithm of dissociation constant, Ki) using the Cheng-Prusoff equation.Test compounds having a higher pKi value in each of the four JAK assays show greater inhibition of JAK activity. Compounds of the invention tested in this assay typically exhibited pKi values between about 7 and about 10.3
Measurement: Ki = 0.55 nM (pChEMBL 9.260000228881836)
Descriptors: MW 469.59, ALogP 5.11, HBA 7, HBD 4, PSA 101.99, QED 0.31
Compound (SMILES): COC(=O)c1cnc(N)c(-c2cc3cc(NC(=O)Nc4cc(C(F)(F)F)ccc4F)ccc3s2)c1
InChIKey: LSCATNNDFBVKMR-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: VEGFR2 Cellular Assay: Automated FLIPR (Fluorometric Imaging Plate Reader) technology was used to screen for inhibitors of VEGF induced increases in intracellular calcium levels in fluorescent dye loaded endothelial cells. HUVEC (human umbilical vein endothelial cells) (Clonetics) were seeded in 384-well fibronectin coated black-walled plates overnight @ 37° C./5%CO2. Cells were loaded with calcium indicator Fluo-4 for 45 minutes at 37° C. Cells were washed 2 times (Elx405, Biotek Instruments) to remove extracellular dye. For screening, cells were pre-incubated with test agents for 30 minutes, at a single concentration (10 uM) or at concentrations ranging from 0.0001 to 10.0 uM followed by VEGF165 stimulation (10 ng/mL). Changes in fluorescence at 516 nm were measured simultaneously in all 384 wells using a cooled CCD camera. Data were generated by determining max-min fluorescence levels for unstimulated, stimulated, and drug treated samples. IC50 values for test compounds were calculated from % inhibition of VEGF stimulated responses in the absence of inhibitor.
Measurement: IC50 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 504.47, ALogP 6.13, HBA 6, HBD 3, PSA 106.34, QED 0.23
Compound (SMILES): Cc1c(-c2[nH]c3ccc(C4CCC(N5CC6(CCOC6)C5)CC4)nc3c2C(C)C)cn2ncnc2c1C
InChIKey: HSWFDNXIUMOTQR-UHFFFAOYSA-N
Target: Toll-like receptor 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR97
Assay: TLR7/8/9 Inhibition Reporter Assays: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 4.8 nM (pChEMBL 8.319999694824219)
Descriptors: MW 498.68, ALogP 5.76, HBA 6, HBD 1, PSA 71.34, QED 0.39
Compound (SMILES): CCc1cc(O)c(F)cc1-c1ccc2c(-c3nc4c([nH]3)CCN(C(=O)N3CCOCC3)C4)n[nH]c2c1
InChIKey: DJUAJWMTCKJXRM-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Biochemical Assay: The biochemical JAK family enzyme binding assays carried out at DiscoveRx.
Measurement: Kd = 0.11 nM (pChEMBL 9.960000038146973)
Descriptors: MW 490.54, ALogP 3.84, HBA 5, HBD 3, PSA 110.37, QED 0.4
Compound (SMILES): COc1cc(OC)c(F)c(N2Cc3cnc(-c4cncc(CN5CCOCC5)c4)cc3C3(CC3)C2=O)c1F
InChIKey: MDUMQYWNCWDCFN-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P22455
Assay: FGFR Enzymatic Assay 1: FGFR1, FGFR2, and FGFR4 are measured under equivalent conditions with the following changes in enzyme and ATP concentrations: FGFR1, 0.02 nM and 210 uM respectively, FGFR2, 0.01 nM and 100 uM, respectively, and FGFR4, 0.04 nM and 600 uM respectively. The enzymes were purchased from Millipore or Invitrogen. GraphPad prism3 was used to analyze the data. The IC50 values were derived by fitting the data to the equation for a sigmoidal dose-response with a variable slope. Y=Bottom+(Top−Bottom)/(1+10^((Log IC50−X)*HillSlope)) where X is the logarithm of concentration and Y is the response. Compounds having an IC50 of 1 μM or less are considered active. The compounds of the invention were found to be selective inhibitors of FGFR3 and/or FGFR4 according to the FGFR Enzymatic Assays. Compounds of Formula (I′) and (I) and all the compounds as described herein have been tested and exhibit an IC50 of less than 1 μM. Table 1 provides IC50 data for compounds of the invention assayed in the FGFR Enzymatic Assay after dilution in assay buffer, added to the plate and pre-incubated for 4 hours.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 522.55, ALogP 3.85, HBA 7, HBD 0, PSA 77.02, QED 0.49
Compound (SMILES): Nc1nc2cc(-c3ccc4ncc(C(=O)N5CC[C@H](n6cccn6)C5)n4c3)ccc2o1
InChIKey: XXTBEQCPJJICGS-INIZCTEOSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Inhibition Assay of PI3K: An exemplary system is PI 3-Kinase (human) HTRF Assay from Upstate. The assay can be carried out according to the procedures suggested by the manufacturer. Briefly, the assay is a time resolved FRET assay that indirectly measures PIP3 product formed by the activity of a PI3-K. The kinase reaction is performed in a microtitre plate (e.g., a 384 well microtitre plate). The total reaction volume is approximately 20 ul per well. In the first step, each well receives 2 ul of test compound in 20% dimethylsulphoxide resulting in a 2% DMSO final concentration. Next, approximately 14.5 ul of a kinase/PIP2 mixture (diluted in 1× reaction buffer) is added per well for a final concentration of 0.25-0.3 ug/ml kinase and 10 μM PIP2. The plate is sealed and incubated for 15 minutes at room temperature. To start the reaction, 3.5 ul of ATP (diluted in 1× reaction buffer) is added per well for a final concentration of 10 μM ATP. The plate is sealed and incubated for 1 hour at room temperature. The reaction is stopped by adding 5 ul of Stop Solution per well and then 5 ul of Detection Mix is added per well. The plate is sealed, incubated for 1 hour at room temperature, and then read on an appropriate plate reader.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 413.44, ALogP 3.01, HBA 8, HBD 1, PSA 107.48, QED 0.49
Compound (SMILES): CN1CCCc2ccc(CN3CCCC34CCN(C(=O)OC(C(F)(F)F)C(F)(F)F)CC4)cc21
InChIKey: PQJZDZZCXRXGHG-UHFFFAOYSA-N
Target: Monoacylglycerol lipase ABHD6 (SINGLE PROTEIN), Mus musculus
UniProt: Q8R2Y0
Assay: In Vitro Competitive Activity-Based Protein Profiling Assay: MAGL, ABHD6 and FAAH: Proteomes (mouse brain membrane fraction or cell lysates) (50 μL, 1.0 mg/mL total protein concentration) were preincubated with varying concentrations of inhibitors at 37° C. After 30 min, FP-Rh (1.0 μL, 50 μM in DMSO) was added and the mixture was incubated for another 30 min at 37° C. Reactions were quenched with SDS loading buffer (50 μL-4X) and run on SDS-PAGE. Following gel imaging, serine hydrolase activity was determined by measuring fluorescent intensity of gel bands corresponding to MAGL, ABHD6 and FAAH using ImageJ 1.43 u software.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 493.49, ALogP 5.13, HBA 4, HBD 0, PSA 36.02, QED 0.55
Compound (SMILES): C#Cc1cc(Nc2nccc(Oc3ccc(NC(=O)Nc4cc(C(C)(C)C)nn4-c4ccc(C)cc4)c4ccccc34)n2)cc(C(=O)NCCOCCOC)c1
InChIKey: YOSOEKMQFOYUKT-UHFFFAOYSA-N
Target: Glycogen synthase kinase-3 alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P49840
Assay: GSK 3alpha Enzyme Inhibition Assay (method 2): The inhibitory activities of compounds of the invention against the GSK 3α enzyme isoform (Invitrogen), are evaluated by determining the level of activation/phosphorylation of the target peptide. The GSK3-α protein (500 ng/mL, 2.5 μL) is mixed with the test compound (2.5 μL at either 4 μg/mL, 0.4 μg/mL, 0.04 μg/mL, or 0.004 μg/mL) for 105 minutes at RT. The FRET peptide (8 μM, 2.5 μL), which is a phosphorylation target for GSK3α, and ATP (40 μM, 2.5 μL) are then added to the enzyme/compound mixture and the resulting mixture incubated for 1 hr. Development reagent (protease, 5 μL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan® Flash, ThermoFisher Scientific).In all cases, the site-specific protease cleaves non-phosphorylated peptide only and eliminates the FRET signal. Phosphorylation levels of each reaction are calculated using the ratio of coumarin emission (donor) over fluorescein emission (acceptor), for which high ratios indicate high phosphorylation and low ratios indicate low phosphorylation levels. The percentage inhibition of each reaction is calculated relative to non-inhibited control and the 50% inhibitory concentration (IC50 value) is then calculated from the concentration-response curve.
Measurement: IC50 = 2890 nM (pChEMBL 5.539999961853027)
Descriptors: MW 752.88, ALogP 7.98, HBA 10, HBD 4, PSA 153.55, QED 0.06
Compound (SMILES): CCCCNc1ncc2c(-c3ccc(S(=O)(=O)C4CCOCC4)cc3)cn(C3CCC(O)CC3)c2n1
InChIKey: IEXXROGGKUBLPS-UHFFFAOYSA-N
Target: Receptor-type tyrosine-protein kinase FLT3 (SINGLE PROTEIN), Homo sapiens
UniProt: P36888
Assay: Inhibition Assay: Inhibition constants of MerTK, Flt3, Tyro3 and Axl kinase activity by an active compound as described herein is determined at the Km for ATP using a microfluidic capillary electrophoresis (MCE) assay in which phosphorylated and unphosphorylated substrate peptides were separated and analyzed using a LabChip EZ Reader.Briefly, activity assays were performed in a 384 well, polypropylene microplate in a final volume of 50 μl, of 50 mM Hepes, Ph 7.4 containing 10 mM MgCl2, 1.0 mM DTT, 0.01% Triton X-100, 0.1% Bovine Serum Albumin (BSA), containing 1.0 μM fluorescent substrate and ATP at the Km for each enzyme. All reactions were terminated by addition of 20 μL of 70 mM EDTA. After a 180 mM incubation, phosphorylated and unphosphorylated substrate peptides were separated in buffer supplemented with 1×CR-8 on a LabChip EZ Reader equipped with a 12-sipper chip. Data were analyzed using EZ Reader software. Assay conditions for MCE assays
Measurement: IC50 = 2.8 nM (pChEMBL 8.550000190734863)
Descriptors: MW 512.68, ALogP 4.74, HBA 8, HBD 2, PSA 106.34, QED 0.42
Compound (SMILES): C[C@H](O)c1nc2ccc3[nH]ccc3c2n1C1CCC(C#N)CC1
InChIKey: UDGMZRVCKLNKHN-HIFPTAJRSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Enzymatic Inhibition Assay: Substrate (NH2-KGGEEEEYFELVKK-CO2), internal standard peptide (NH2-SWGAIETDKEYYTVKD-CO2) and product peptide (for standard curve only) (NH2-KGGEEEEY-Pi-FELVKK-CO2), were purchased from AnaSpec (Fremont, Calif., USA). JAK1-JH1JH2 (574-1154 with a His-GST Tag and a C-terminal tev (ENLYFQ-G) cleavage site), JAK3-JH1JH2 (512-1124 with a GST Tag and a C-terminal tev (ENLYFQ-G) cleavage site), and Tyk2-JH1JH2 (8H_tev_580-1182-C936A-C1142A with a C-terminal tev (ENLYFQ-G) cleavage site) were purified internally. JAK2-JH1JH2 (532-1132 with a GST tag and C-terminal tev (ENLYFQ-G) cleavage site), was purchased from Invitrogen. LC/MS grade water and acetonitrile (ACN), were purchased from HoneyWell, Burdick & Jackson (Muskegon, Mich., USA). Dimethylsulfoxide 99.8% (DMSO) and trifluoroacetic acid 99.5% (TFA) were purchased from EMD Chemical (Gibbstown, N.J., USA). Adenosine triphosphate (ATP), 4-morpholinepropanesulfonic acid (MOPS), magnesium chloride (MgCl2), ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT), formic acid >95% (FA) and Tween-20 were purchased from Sigma (St Louis, Mo., USA). 384-well polypropylene plates, Cat #781280 were purchased from Greiner (Monroe, N.C.), RapidFire™ cartridge A C4 Column (Agilent Technologies, Santa Clara, Calif.).
Measurement: IC50 = 0.2 nM (pChEMBL 9.699999809265137)
Descriptors: MW 308.39, ALogP 3.83, HBA 4, HBD 2, PSA 77.63, QED 0.76
Compound (SMILES): Cc1cc(Nc2cc3ncccc3c(N[C@@H]3C[C@H]4CC[C@@H](C3)N4S(=O)(=O)N3CCC3)n2)n[nH]1
InChIKey: RKLSKEZFFCOVQP-OSYLJGHBSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Biochemical JAK Assay: A panel of four LanthaScreen JAK biochemical assays (JAK1, 2, 3 and Tyk2) were carried in a common kinase reaction buffer (50 mM HEPES, pH 7.5, 0.01% Brij-35, 10 mM MgCl2, and 1 mM EGTA). Recombinant GST-tagged JAK enzymes and a GFP-tagged STAT1 peptide substrate were obtained from Life Technologies.Serially diluted compounds were pre-incubated with each of the four JAK enzymes and the substrate in white 384-well microplates (Corning) at ambient temperature for lh. ATP was subsequently added to initiate the kinase reactions in 10 μL total volume, with 1% DMSO. The final enzyme concentrations for JAK1, 2, 3 and Tyk2 are 4.2 nM, 0.1 nM, 1 nM, and 0.25 nM respectively; the corresponding Km ATP concentrations used are 25 μM, 3 μM, 1.6 μM, and 10 μM; while the substrate concentration is 200 nM for all four assays. Kinase reactions were allowed to proceed for 1 hour at ambient temperature before a 10 μL preparation of EDTA (10 mM final concentration) and Tb-anti-pSTAT1 (pTyr701) antibody (Life Technologies, 2 nM final concentration) in TR-FRET dilution buffer (Life Technologies) was added. The plates were allowed to incubate at ambient temperature for lh before being read on the EnVision reader (Perkin Elmer). Emission ratio signals (520 nm/495 nm) were recorded and utilized to calculate the percent inhibition values based on DMSO and background controls.For dose-response analysis, percent inhibition data were plotted vs. compound concentrations, and IC50 values were determined from a 4-parameter robust fit model with the Prism software (GraphPad Software). Results were expressed as pIC50 (negative logarithm of IC50) and subsequently converted to pKi (negative logarithm of dissociation constant, Ki) using the Cheng-Prusoff equation.Test compounds having a higher pKi value in each of the four JAK assays show greater inhibition of JAK activity. Compounds of the invention tested in this assay typically exhibited pKi values between about 7 and about 10.3
Measurement: Ki = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 468.59, ALogP 2.76, HBA 7, HBD 3, PSA 119.14, QED 0.51
Compound (SMILES): Cc1cc2cnc(NC3CCN(S(=O)(=O)C4CCS(=O)(=O)C4)CC3)nc2n([C@@H]2CCC[C@@]2(C)O)c1=O
InChIKey: UXXXHEOMIMPLKB-DLCOBIQGSA-N
Target: CDK6/cyclin D1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24385
Assay: CDK6/Cyclin D1 Mobility Shift Assay: The purpose of the CDK6/Cyclin D1 assay is to evaluate the inhibition (% inhibition, Kiapp and Ki values) in the presence of small molecule inhibitors by using a fluorescence based microfluidic mobility shift assay. CDK6/Cyclin D1 catalyzes the production of ADP from ATP that accompanies the phosphoryl transfer to the substrate peptide 5-FAM-Dyrktide (5-FAM-RRRFRPASPLRGPPK) (SEQ ID NO:2). The mobility shift assay electrophoretically separates the fluorescently labeled peptides (substrate and phosphorylated product) following the kinase reaction. Both substrate and product are measured and the ratio of these values is used to generate % conversion of substrate to product by the LabChip EZ Reader. Typical reaction solutions contained 2% DMSO (± inhibitor), 10 mM MgCl2, 1 mM DTT, 2 mM ATP, 0.005% Tween 20 (TW-20), 3 μM 5-FAM-Dyrktide, 3 nM (active sites) CDK6/Cyclin D1 in 40 mM HEPES buffer at pH 7.5.Inhibitor Ki determinations for non-phosphorylated CDK6/CyclinD1 (LJIC-2003A2/1865) were initiated with the addition of ATP (50 μL final reaction volume), following a twelve minute pre-incubation of enzyme and inhibitor at 22° C. in the reaction mix. The reaction was stopped after 35 minutes by the addition of 50 μL of 25 mM EDTA. Ki determinations were made from a plot of the fractional velocity as a function of inhibitor concentration fit to the Morrison equation with the enzyme concentration as a variable.
Measurement: Ki = 0.19 nM (pChEMBL 9.720000267028809)
Descriptors: MW 539.68, ALogP 0.97, HBA 10, HBD 2, PSA 151.56, QED 0.57
Compound (SMILES): CN1CCN(CC(=O)Nc2cc(C(=O)Nc3nnc(-c4ccccc4)s3)ccc2OC(F)F)CC1
InChIKey: RZAYXPOLDBZMMO-UHFFFAOYSA-N
Target: Protein Wnt-3a (SINGLE PROTEIN), Homo sapiens
UniProt: P56704
Assay: HEK FOP Assay: HEK293 cells were cotransfected with the FOP control vector and pcDNA3. The FOP vector is identical to the TOP construct, but it contains instead of functional TCF elements a randomized, non-functional sequence
Measurement: IC50 > 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 502.55, ALogP 3.24, HBA 8, HBD 2, PSA 99.69, QED 0.49
Compound (SMILES): Cc1c(C#N)ccnc1O[C@@H]1CC[C@@H](C)N(C(=O)c2cccc(F)c2-n2nccn2)C1
InChIKey: NVFLDPIJWMCIKG-RHSMWYFYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: FLIPR Ca2+ Flux Assay: The utility of the compounds in accordance with the present invention as orexin receptor OX1R and/or OX2R antagonists may be readily determined without undue experimentation by methodology well known in the art, including the FLIPR Ca2+ Flux Assay (Okumura et al., Biochem. Biophys. Res. Comm. 280:976-981, 2001). In a typical experiment the OX1 and OX2 receptor antagonistic activity of the compounds of the present invention was determined in accordance with the following experimental method. For intracellular calcium measurements, Chinese hamster ovary (CHO) cells expressing the rat orexin-1 receptor or the human orexin-2 receptor, are grown in Iscove's modified DMEM containing 2 mM L-glutamine, 0.5 g/ml G418, 1% hypoxanthine-thymidine supplement, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat-inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into Becton-Dickinson black 384-well clear bottom sterile plates coated with poly-D-lysine. All reagents were from GIBCO-Invitrogen Corp. The seeded plates are incubated overnight at 37° C. and 5% CO2. Ala-6,12 human orexin-A as the agonist is prepared as a 1 mM stock solution in 1% bovine serum albumin (BSA) and diluted in assay buffer (HBSS containing 20 mM HEPES, 0.1% BSA and 2.5 mM probenecid, pH7.4) for use in the assay at a final concentration of 70 pM. Test compounds are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates, first in DMSO, then assay buffer. On the day of the assay, cells are washed 3 times with 100 μl assay buffer and then incubated for 60 min (37° C., 5% CO2) in 60 μl assay buffer containing 1 μM Fluo-4AM ester, 0.02% pluronic acid, and 1% BSA. The dye loading solution is then aspirated and cells are washed 3 times with 100 μl assay buffer. 30 μl of that same buffer is left in each well. Within the Fluorescent Imaging Plate Reader (FLIPR, Molecular Devices), test compounds are added to the plate in a volume of 25 μl, incubated for 5 min and finally 25 μl of agonist is added. Fluorescence is measured for each well at 1 second intervals for 5 minutes and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 70 pM Ala-6,12 orexin-A with buffer in place of antagonist. For each antagonist, IC50 value (the concentration of compound needed to inhibit 50% of the agonist response) is determined. Alternatively, compound potency can be assessed by a radioligand binding assay (described in Bergman et. al. Bioorg. Med. Chem. Lett. 2008, 18, 1425-1430) in which the inhibition constant (Ki) is determined in membranes prepared from CHO cells expressing either the OX1 or OX2 receptor. The intrinsic orexin receptor antagonist activity of a compound which may be used in the present invention may be determined by these assays.
Measurement: IC50 = 927 nM (pChEMBL 6.03000020980835)
Descriptors: MW 420.45, ALogP 3.05, HBA 7, HBD 0, PSA 96.93, QED 0.64
Compound (SMILES): CCCCCC[C@H]1COc2cc([C@@H]3CC[C@](N)(COP(=O)(O)O)C3)ccc2C1
InChIKey: MJHBSCYJBMENCH-OZOXKJRCSA-N
Target: Sphingosine 1-phosphate receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99500
Assay: [35S] GTPgammaS Binding Assays: Compounds were loaded in a 384 Falcon v-bottom plate (0.5 ul/well in a 11 point, 3-fold dilution). Membranes prepared from S1P1/CHO cells or EDG3-Gal5-bla HEK293T cells (EDG3 equivalent S1P3) were added to the compound plate (40 ul/well, final protein 3 ug/well) with MULTIDROP. [35S] GTP (1250 Ci/mmol, Perkin Elmer) was diluted in assay buffer: 20 mM HEPES, pH7.5, 10 mM MgCl2, 150 mM NaCl, 1 mM EGTA (ethylene glycol tetraacetic acid), 1 mM DTT (Dithiothreitol), 10 uM GDP, 0.1% fatty acid free BSA, and 10 ug/ml Saponin to 0.4 nM. 40 ul of the [35S] GTP solution was added to the compound plate with a final concentration of 0.2 nM. The reaction was kept at room temperature for 45 min. At the end of incubation, all the mixtures in the compound plate were transferred to Millipore 384-well FB filter plates via the VELOCITY11 Vprep liquid handler. The filter plate was washed with water 4 times by using the manifold Embla plate washer and dried at 60° C. for 45 min. MicroScint 20 scintillation fluid (30 ul) was added to each well for counting on the Packard TOPCOUNT. EC50 is defined as the agonist concentration that corresponds to 50% of the Ymax (maximal response) obtained for each individual compound tested.
Measurement: EC50 > 625 nM (pChEMBL 6.203999996185303)
Descriptors: MW 411.48, ALogP 4.28, HBA 4, HBD 3, PSA 102.01, QED 0.41
Compound (SMILES): Nc1nc2ccc(-c3cc(F)c4oc(N)nc4c3)cc2o1
InChIKey: VAEQLUVTTMTFAO-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Inhibition Assay of PI3K: An exemplary system is PI 3-Kinase (human) HTRF Assay from Upstate. The assay can be carried out according to the procedures suggested by the manufacturer. Briefly, the assay is a time resolved FRET assay that indirectly measures PIP3 product formed by the activity of a PI3-K. The kinase reaction is performed in a microtitre plate (e.g., a 384 well microtitre plate). The total reaction volume is approximately 20 ul per well. In the first step, each well receives 2 ul of test compound in 20% dimethylsulphoxide resulting in a 2% DMSO final concentration. Next, approximately 14.5 ul of a kinase/PIP2 mixture (diluted in 1× reaction buffer) is added per well for a final concentration of 0.25-0.3 ug/ml kinase and 10 μM PIP2. The plate is sealed and incubated for 15 minutes at room temperature. To start the reaction, 3.5 ul of ATP (diluted in 1× reaction buffer) is added per well for a final concentration of 10 μM ATP. The plate is sealed and incubated for 1 hour at room temperature. The reaction is stopped by adding 5 ul of Stop Solution per well and then 5 ul of Detection Mix is added per well. The plate is sealed, incubated for 1 hour at room temperature, and then read on an appropriate plate reader.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 284.25, ALogP 2.94, HBA 6, HBD 2, PSA 104.1, QED 0.56
Compound (SMILES): COCC[C@@H]1CCc2cc([C@H]3CC[C@](N)(COP(=O)(O)O)C3)ccc2C1
InChIKey: FWYZNCDCDHDYPQ-ZOCIIQOWSA-N
Target: Sphingosine 1-phosphate receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99500
Assay: [35S] GTPgammaS Binding Assays: Compounds were loaded in a 384 Falcon v-bottom plate (0.5 ul/well in a 11 point, 3-fold dilution). Membranes prepared from S1P1/CHO cells or EDG3-Gal5-bla HEK293T cells (EDG3 equivalent S1P3) were added to the compound plate (40 ul/well, final protein 3 ug/well) with MULTIDROP. [35S] GTP (1250 Ci/mmol, Perkin Elmer) was diluted in assay buffer: 20 mM HEPES, pH7.5, 10 mM MgCl2, 150 mM NaCl, 1 mM EGTA (ethylene glycol tetraacetic acid), 1 mM DTT (Dithiothreitol), 10 uM GDP, 0.1% fatty acid free BSA, and 10 ug/ml Saponin to 0.4 nM. 40 ul of the [35S] GTP solution was added to the compound plate with a final concentration of 0.2 nM. The reaction was kept at room temperature for 45 min. At the end of incubation, all the mixtures in the compound plate were transferred to Millipore 384-well FB filter plates via the VELOCITY11 Vprep liquid handler. The filter plate was washed with water 4 times by using the manifold Embla plate washer and dried at 60° C. for 45 min. MicroScint 20 scintillation fluid (30 ul) was added to each well for counting on the Packard TOPCOUNT. EC50 is defined as the agonist concentration that corresponds to 50% of the Ymax (maximal response) obtained for each individual compound tested.
Measurement: EC50 > 625 nM (pChEMBL 6.203999996185303)
Descriptors: MW 383.43, ALogP 2.9, HBA 4, HBD 3, PSA 102.01, QED 0.63
Compound (SMILES): Cc1ccc(N(C)c2ccc3c(c2)CCN[C@H]3CNc2cnccc2C(=O)O)cc1
InChIKey: BWZXDTADOZVXSQ-QHCPKHFHSA-N
Target: Lysine-specific demethylase 6B (SINGLE PROTEIN), Homo sapiens
UniProt: O15054
Assay: In Vitro Enzyme Inhibition Assay: JMJD3: The enzymatic assay of JMJD3 activity is based upon Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) detection. The ability of test compounds to inhibit the activity of JMJD3 was determined in 384-well plate format under the following reaction conditions: 5 nM JMJD3, 250 nM H3K27me3-biotin labeled peptide (Anaspec cat #64367), 0.4 to 2 μM o-ketoglutaric acid in assay buffer of 50 mM HEPES, pH7.3, 0.005% Brij35, 0.5 mM TCEP, 0.2 mg/ml BSA, 50 μM sodium L-ascorbate, and 5 μM ammonium iron(II) sulfate. Reaction product was determined quantitatively by TR-FRET after the addition of detection reagent Phycolink Streptavidin-allophycocyanin (Prozyme) and Europium-anti-H3K27me2 antibody (PerkinElmer) in the presence of 5 mM EDTA in LANCE detection buffer (PerkinElmer) at a final concentration of 50 nM and 1 nM, respectively.The assay reaction was initiated by the following: 2 μl of the mixture of 750 nM H3K27me3-biotin labeled peptide and 1.2 to 6 μM alpha-ketoglutaric acid with 2 μL of 11-point serial diluted inhibitor in 3% DMSO were added to each well of plate, followed by the addition of 2 μl of 15 nM JMJD3 to initiate the reaction. The reaction mixture was incubated at RT for 30 min, and terminated by the addition of 6 μL of 5 mM EDTA in LANCE detection buffer containing 100 nM Phycolink Streptavidin-allophycocyanin and 2 nM Europium-anti-H3K27me2 antibody. Plates were read by EnVisionMultilabel Reader in TR-FRET mode (excitation at 320 nm, emission at 615 nm and 665 nm) after 1 hr incubation at RT.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 402.5, ALogP 4.16, HBA 5, HBD 3, PSA 77.49, QED 0.57
Compound (SMILES): CCn1cc(-c2ccc3cc(C(=O)c4cnn(-c5ccc6[nH]c(C7CC7)nc6c5)c4N)[nH]c3c2)cn1
InChIKey: LGWVEHCWSZKUPK-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P22455
Assay: Determination of the Effects of the Compounds of the Present Invention on FGFR Kinase Activity: The following assay was used to determine the inhibition rate of the preferred compounds of the present invention to the kinase activity of the recombinant human FGFR protein in vitro conditions. This assay used Cisbio's HTRF®KinEASE-TK tyrosine kinase kit (Cat. No. 62TK0PEB), through determining the degree of phosphorylation of the biotinylated polypeptide substrate, the determination was carried out by time-resolved fluorescence energy resonance transfer method (TF-FRET). Human FGFR protein was purchased from Carna bioscience (Japan, Cat. No. FGFR1#08-133, FGFR2#08-134, FGFR3#08-135, FGFR4#08-136).For detailed assay, please refer to the kit instructions, the experimental procedure was summarized as follows: the compound of the present invention was first dissolved in DMSO, the solution was subjected to gradient dilution with the buffer solution provided in the kit so that the range of the final concentration of the compound to be tested in the reaction system was 10 μM to 0.1 nM, the final concentration of DMSO was 0.1%. The concentration of the tested adenosine triphosphate (ATP) was the pre-determined Km value corresponding to the ATP of each FGFR subtype. The compound was first incubated with a certain amount of FGFR protein at room temperature for 5 to 30 minutes, followed by the addition of ATP and the biotinylated polypeptide substrate to the reaction solution to initiate the phosphorylation reaction, and the mixture was incubated at room temperature for 50 minutes. Subsequently, an antiphosphorylated tyrosine antibody coupled with a compound containing europium-based element and streptavidin coupled with a modified allophycocyanin XL665 were added to the reaction and the mixture was incubated continuously for 1 hour at room temperature. After incubation, the fluorescence intensity of each well at emission wavelengths of 620 nM and 665 nM were read at the excitation wavelength of 304 nm in the TF-FRET mode of the microplate reader.
Measurement: IC50 = 280 nM (pChEMBL 6.550000190734863)
Descriptors: MW 476.54, ALogP 4.8, HBA 7, HBD 3, PSA 123.2, QED 0.3
Compound (SMILES): C[C@@H]1[C@H](NC(=O)N(C)C)CCCN1c1cnc(C(N)=O)c(Nc2ccc(C3(C(F)(F)F)CC3)cc2)n1
InChIKey: UXYQVESVIKVEHT-RHSMWYFYSA-N
Target: Tyrosine-protein kinase Lck (SINGLE PROTEIN), Homo sapiens
UniProt: P06239
Assay: HotSpot kinase Assay: The degree of inhibition of a panel of kinases is determined using the in vitro HotSpot kinase assay (purified enzymes, 33P-ATP, an appropriate substrate and 1 uM ATP).
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 505.55, ALogP 3.54, HBA 6, HBD 3, PSA 116.48, QED 0.55
Compound (SMILES): O=C(Nc1nc2cccc(C3CCC(NC(=O)[C@H]4CC4(F)F)CC3)n2n1)C1CC1
InChIKey: JQKDWSJGLLIFBM-UXUKBGGZSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: In Vitro Activity Assay: TYK2 (h) was incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA, 250 μM GGMEDIYFEFMGGKKK, 10 mM magnesium acetate and [γ-33P]-ATP (activity and concentration were formulated as needed). Mg/ATP mixture was added to initiate the reaction. After incubating at room temperature for 40 minutes, the reaction mixture was added with 0.5% phosphoric acid to stop the reaction. Then 10 μL reaction was spotted on a P30 filter pad and washed with 0.425% phosphoric acid three times and methanol once over 4 minutes, dried, and counted scintillation.
Measurement: IC50 = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 403.43, ALogP 2.88, HBA 5, HBD 2, PSA 88.39, QED 0.8
Compound (SMILES): C[C@@H]1C2CC(C2)N(C(=O)c2cc(C#N)ccc2-n2nccn2)C1CNc1nc2cccnc2s1
InChIKey: IBFMPAYSGLIJHV-HDNVEZOSSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Scintillation Proximity Assay (SPA) Binding Assay: CHO cells stably transfected with human Orexin type 1 receptors (CHO-hOX1) or HEK-293 cells transiently transfected with human Orexin type 2 receptors (HEK-hOX2) were collected after 16 h induction with 5 mM sodium butyrate. The cell pellets were re-suspended, homogenized in 15 mM Tris/HCl pH=7.5, 1 mM EGTA, 0.3 mM EDTA, 2 mM MgCl2, protease inhibitors and centrifuged at 40,000 g (20 min, 4° C.). After re-suspension, homogenization and centrifugation as above, the final pellets were re-suspended in 75 mM Tris/HCl pH=7.5, 1 mM EGTA, 0.3 mM EDTA, 12.5 mM MgCl2, 250 mM Sucrose, protease inhibitors, divided into aliquots and frozen down at −80° C.Compounds of invention were serially diluted in neat DMSO at 100-fold concentrations (1% DMSO final in the assay) and 2 μl/well were plated into 96-well Isoplates (Perkin Elmer).
Measurement: Ki = 7762 nM (pChEMBL 5.110000133514404)
Descriptors: MW 470.56, ALogP 3.49, HBA 9, HBD 1, PSA 112.62, QED 0.47
Compound (SMILES): N#Cc1ccc(-c2c(C(=O)N3CCC3)nc3cccnn23)cc1F
InChIKey: COQGBMKNVJUVAB-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4A (SINGLE PROTEIN), Homo sapiens
UniProt: P27815
Assay: Scintillation Proximity Assay (SPA): The PDE4A3, PDE4B1, PDE4C1 and PDE4D3 assays use the Scintillation Proximity Assay (SPA) technology to measure the inhibition of human recombinant PDE4A1, PDE4B3, PDE4C1, and PDE4D3 enzyme activity by compounds in vitro. The PDE4A1, PDE4B3, PDE4C1, and PDE4D3 assays are run in parallel using identical parameters, except for the concentration of enzyme (80 pM PDE4A3, 40 pM PDE4B3, 40 pM PDE4C1 and 10 pM PDE4D). The assays are performed in a 384-well format with 50 uL assay buffer (50 mM TRIS pH7.5; 1.3 mM MgCl2; 0.01% Brij) containing enough PDE4A3, PDE4B1, PDE4C1, and PDE4D to convert 20% of substrate (1 μM cAMP consisting of 20 nM 3H-cAMP+980 uM cold cAMP) and a range of inhibitors. Reactions are incubated for 30 min at 25° C. The addition of 20 uL of 8 mg/ml yitrium silicate SPA beads (Perkin Elmer) stops the reaction. The plates are sealed (TopSeal, Perkin Elmer) and the beads are allowed to settle for 8 hrs, after which they are read on the Trilux Microbeta overnight.
Measurement: IC50 = 15.1 nM (pChEMBL 7.820000171661377)
Descriptors: MW 321.32, ALogP 2.25, HBA 5, HBD 0, PSA 74.29, QED 0.73