lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1nc(C(=O)N2C[C@H](Oc3nccc(C#N)c3C)CC[C@H]2C)c(-c2ccc(F)cc2)s1
InChIKey: KIYVTVFULTUXJQ-JLTOFOAXSA-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 = 1196 nM (pChEMBL 5.920000076293945)
Descriptors: MW 450.54, ALogP 4.9, HBA 6, HBD 0, PSA 79.11, QED 0.56
Compound (SMILES): C=CC(=O)N1CC[C@]2(C1)C[C@H](n1c(NC(=O)c3ccc(-c4ccnc(CO)c4)s3)nc3ccccc31)C2
InChIKey: CQYOCHGVYYCAJH-ANXHRGMGSA-N
Target: Tyrosine-protein kinase Tec (SINGLE PROTEIN), Homo sapiens
UniProt: P42680
Assay: Kinase Inhibition Assay: The in vitro kinase assays were performed at Nanosyn (Santa Clara, Calif.) utilizing microfluidic detection technology. The test compounds were serially pre-diluted in DMSO and added, by the acoustic dispensing (Labcyte 550), directly to 384 well assay plates into 10 uL of a buffer with enzyme (ITK, TXK, BTK or TEC) comprising: 100 mM HEPES, pH7.5, 5 mM MgCl2, 0.1% bovine serum albumin, 1 mM DTT, 0.01% Triton X-100 and the enzyme. Final DMSO concentration was maintained at 1% in all samples, including the controls. The reactions were initiated by addition of ATP (1 mM final concentration) and the fluorescently labeled peptide substrate to a final concentration of 1 uM, and incubated for 3 hours at 25° C. Following incubation, the reactions were quenched by addition of 40 μL of termination buffer (100 mM HEPES, pH7.5, 0.01% Triton X-100, 50 mM EDTA). Terminated plates were analyzed using Caliper LabChip 3000 microfluidic electrophoresis instrument (Caliper Life Sciences/Perkin Elmer). The enzymatic modification of the peptide substrate (phosphorylation) results in a change of net charge enabling electrophoretic separation of product from substrate. As substrate and product are separated by electrophoresis, two peaks of fluorescence are observed. Change in the relative fluorescence intensity of the substrate and product peaks was the parameter measured, reflecting enzyme activity. In the presence of inhibitor, the ratio between product and substrate is altered: signal of the product decreases, while the signal of the substrate increases. Activity in each test sample was determined as the product to sum ratio (PSR): P/(S+P), where P is the peak height of the product and S is the peak height of the FAM-cAMP substrate. For each compound, enzyme activity was measured at 12 concentrations spaced by 3× dilution intervals. Negative control samples (0%-inhibition in the absence of inhibitor, DMSO only) and positive control samples (100%-inhibition, in the absence of enzyme or in the presence of control inhibitor) were assembled in replicates of four and were used to calculate %-inhibition values in the presence of compounds. Percent inhibition (Pinh) was determined using the following equation: Pinh=(PSR0%−PSRinh)/(PSR0%−PSR100%)*100, where PSRinh is the product sum ratio in the presence of inhibitor, PSR0% is the product sum ratio in the absence of inhibitor and PSR100% is the product sum ratio in 100%-inhibition control samples. To determine IC50 values, the inhibition curves (Pinh versus inhibitor concentration) were fitted by 4 parameter sigmoid dose-response model using XLfit software (IDBS).
Measurement: IC50 = 0.16 nM (pChEMBL 9.800000190734863)
Descriptors: MW 513.62, ALogP 4.64, HBA 7, HBD 2, PSA 100.35, QED 0.37
Compound (SMILES): CC(C)N1C[C@H]2C[C@@]2(c2ccc(-c3cnc(N)c(C(=O)N[C@H]4CC[C@H](O)CC4)c3)cc2)C1
InChIKey: HZFKGEGMOFIQKL-BILGYAHESA-N
Target: Activin receptor type-1-like (SINGLE PROTEIN), Homo sapiens
UniProt: P37023
Assay: Caliper Assay: In the applied method, this separation takes place inside a chip that contains a complex capillary system for simultaneous analysis of 12 samples (12-sipper chip, Caliper Technologies Corp., Mountain View, USA). In order to allow the detection and quantification of the peptides in the capillary system, the peptides carry a fluorescent label (fluorescein). With this label the peptides can be quantified by fluorescence intensity through the instruments laser and detection system (LC3000, Caliper Life Sciences).The assays were performed in 384-well, low volume microtiter assay plates in a final reaction volume of 9 ul. Dose-response curves were generated by incubating 10 nM of each kinase together with 2 μm of the fluorescently labeled substrate peptide 5-Fluo-Ahx-KKYQAEEN-T-YDEYENKK-amid (10 mM stock solution in DMSO) in 50 mM Hepes pH 7.5, 0.02% Tween 20, 0.02% BSA, 1 mM DTT, 10 μm Na3VO4, 10 mM β-Glycerolphosphate, 1 mM MgCl2, 12 mM MnCl2 (ALK1 and ALK6 7 mM) and 15 μm ATP for 60 min at 30° C. in the presence or absence of compound diluted in DMSO.Kinase reaction were terminated by adding 15 ul STOP buffer (100 mM HEPES pH 7.5, 5% DMSO, 0.1% Caliper coating reagent, 10 mM EDTA, and 0.015% Brij35.Plates with terminated kinase reactions were transferred to the Caliper LC3000 workstation (Caliper Technologies Corp., Mountain View, USA) for reading. The relative amount of phosphorylated peptide r, was calculated using the heights of the substrate peak, s, and the product peak, p: r=p/(p+s).
Measurement: IC50 = 93 nM (pChEMBL 7.03000020980835)
Descriptors: MW 434.58, ALogP 3.35, HBA 5, HBD 3, PSA 91.48, QED 0.67
Compound (SMILES): CNC(=O)C(C)N1CC[C@@]2(S(=O)(=O)c3ccc(F)cc3)c3ccc(C(F)(C(F)(F)F)C(F)(F)F)cc3CC[C@@H]12
InChIKey: VZUREBOIBVKHIF-FRPDIZBGSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Binding Assay : The binding of potential ligands to RORγ is measured by competition with [3H]25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA). The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromatography. 15 μg/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 μM to 0.28 nM, for 10 min at rt in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% glycerol (Sigma Cat # G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat # RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined.
Measurement: IC50 = 99 nM (pChEMBL 7)
Descriptors: MW 584.53, ALogP 4.94, HBA 4, HBD 1, PSA 66.48, QED 0.4
Compound (SMILES): CC(CO)(CO)Nc1n[nH]c2nccc(Oc3ccc(NC(=O)c4ccnn(-c5ccc(F)cc5)c4=O)cc3F)c12
InChIKey: CTPHZJUGWFVHAY-UHFFFAOYSA-N
Target: Hepatocyte growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08581
Assay: Enzyme Assay: The affinity of compound binding to wild type and mutant human MET kinases is measured using Invitrogen's LanthaScreen Eu Kinase Binding technology. Briefly, GST-tagged recombinant human MET kinase domain from Signal Chem (see Table 8 below for concentration in assay) is incubated with 50 nM Alexa-Fluor® Tracer 236 (Invitrogen Cat No. PR9078A) and 2 nM Europium-anti-GST (Invitrogen Cat. No. A151 6) along with test compound in a buffer consisting of 25 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, 1 mM DTT, and 2% DMSO. Compounds are typically prepared in a three-fold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 60-minute incubation at 22° C., the reaction is measured using a PerkinElmer EnVision multimode plate reader via TR-FRET dual wavelength detection, and the percent of control (POC) calculated using a ratiometric emission factor. 100 POC is determined using no test compounds and 0 POC is determined using a concentration of control compound that completely inhibits the enzyme
Measurement: IC50 = 3.4 nM (pChEMBL 8.470000267028809)
Descriptors: MW 563.52, ALogP 2.98, HBA 10, HBD 5, PSA 167.28, QED 0.18
Compound (SMILES): C[C@H](Nc1nc(C(=O)N2CC[C@@H](O)C2)c2sc(C(F)(F)F)cc2n1)c1cncc(F)c1
InChIKey: BRHDNQMATGJYIO-JOYOIKCWSA-N
Target: Adenosine receptor A3 (SINGLE PROTEIN), Homo sapiens
UniProt: P0DMS8
Assay: Activity Assay: Compounds (Adenosine Receptor modulators) and activity assay.
Measurement: IC50 > 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 455.44, ALogP 3.62, HBA 7, HBD 2, PSA 91.24, QED 0.58
Compound (SMILES): COc1cccc(OC)c1-n1c(NS(=O)(=O)[C@@H](C)[C@H](C)c2ncc(Cl)cn2)nnc1[C@@H]1CO[C@@H](C)C1
InChIKey: PZPKWNCTGJSYPV-VGWMRTNUSA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: [35S]GTPgammaS Binding: The human APJ receptor was cloned by polymerase chain reaction and the gene encoding the receptor was subcloned in pFLAG-CMV-3 expression vector (Sigma, Saint Louis, Mo. USA) in-house at Amgen. A GTPγS binding assay was performed on membranes prepared from CHO cells stably expressing human APJ receptor. The optimum experimental conditions for the concentrations of GDP, MgCl2, and NaCl in the assay buffer were initially determined. The assay was performed in assay buffer [20 mM HEPES, pH 7.5, 5 mM MgCl2, and 0.1% (w/v) BSA with 200 mM NaCl, 3 μM GDP] and membranes expressing human APJ receptor/well along with WGA PS beads. The reaction was initiated by addition of 0.2 nM [35S]GTPγS (Perkin Elmer Life and Analytical Sciences, Waltham USA) in the absence or presence of various ligands and incubated at RT for 90 min. Nonspecific binding was determined in the presence of 100 μM GTPγS and was always less than 0.2% of total binding. All the results presented are means of several independent experiments and analyzed by non-linear regression methods using commercially available program Prism (GraphPad, San Diego, Calif.).
Measurement: EC50 = 1.9 nM (pChEMBL 8.720000267028809)
Descriptors: MW 537.04, ALogP 3.55, HBA 10, HBD 1, PSA 130.35, QED 0.44
Compound (SMILES): Cn1cnc(-c2cc(Cl)c(F)c(C(=O)NNS(=O)(=O)CC3CN(C(N)=O)C3)c2)c1
InChIKey: XLVQFBDMXUFRDN-UHFFFAOYSA-N
Target: Histone acetyltransferase KAT8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H7Z6
Assay: KAT-8 Enzyme Assay: Enzyme assay buffer was 50 mM Tris pH 8.0, 0.002% Tween20, 0.005% bovine skin gelatin, and 1 mM dithiothreitol (DTT). For determination of IC50values, compounds were serially diluted with 2% (v/v) DMSO in the final reaction, pre-incubating each dilution of each compound with 40 μL of assay buffer containing KAT-8 enzyme (12.5 nM final concentration). 10 μL of assay buffer containing 1 μM peptide substrate and 5 μM acetyl coenzyme A (final concentrations) was added. Reactions (50 μL total) were then carried out at 25° C. for 90 minutes. Reactions were terminated by the addition of 0.5% formic acid (final concentration), and a sample of each reaction was analyzed by SAMDI Tech, Inc. (Chicago, Ill.) using self-assembled monolayer desorption/ionization time-of-flight mass spectrometry (Mrksich, M. (2008) Mass spectrometry of self-assembled monolayers: a new tool for molecular surface science. ACS Nano 2, 7-18).
Measurement: IC50 = 30000 nM (pChEMBL 4.519999980926514)
Descriptors: MW 444.88, ALogP 0.45, HBA 6, HBD 3, PSA 139.42, QED 0.56
Compound (SMILES): Cc1cc(CN2CCC[C@H]2C)ccc1-c1cnc(N)c(C(=O)N[C@H]2CC[C@](C)(O)CC2)c1
InChIKey: DYPKOAOCIPJRSB-KAWPREARSA-N
Target: Bone morphogenetic protein receptor type-1A (SINGLE PROTEIN), Homo sapiens
UniProt: P36894
Assay: ADPGlo Assay: The assays were performed in 384-well, low volume microtiter assay plates in a final reaction volume of 6 ul. Dose-response curves were generated by incubating 10 nM of each kinase in 50 mM Hepes pH 7.5, 0.02% Tween 20, 0.02% BSA, 1 mM DTT, 10 μm Na3VO4, 10 mM β-Glycerolphosphate, 1 mM MgCl2, 12 mM MnCl2 and 15 μm ATP for 60 min at 32° C. in the presence or absence of compound diluted in DMSO. The amount of generated ADP is a measure of kinase activity and is quantified using the ADP-Glo™ Kinase Assay (Promega) according to manufacturer's instructions. ADP is converted to ATP by adding 3 ul of ADP-Glo™ Reagent and incubation at 32° C. for 60 min. ATP is subsequently converted into a bioluminescent signal by adding 6 ul luciferase assay reagents (Kinase detection buffer+Kinase Detection Substrate (Promega)) and further incubation at 32° C. for 60 min. For the measurement of luminescence a PHERAstar™ Multilabel Reader was used at a measurement interval time of 0.1 second (optical module for luminescence measurements in the 230 nm to 750 nm wavelength range).
Measurement: IC50 = 15100 nM (pChEMBL 4.820000171661377)
Descriptors: MW 436.6, ALogP 4.05, HBA 5, HBD 3, PSA 91.48, QED 0.66
Compound (SMILES): O=C(NS(=O)(=O)c1ccc(NC[C@H]2COCCO2)c([N+](=O)[O-])c1)c1ccc(-c2ccc(N3CCC[C@@H]3c3ccccc3C3CC3)cc2)cc1Oc1cnc2[nH]ccc2c1
InChIKey: JLSMYNRDIKMILB-ROUMXBAISA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07817
Assay: Bcl-2/Bcl-X TR-FRET Assay: Compounds disclosed herein were tested for blocking of Bcl-2/Bcl-X protein with its ligand in an assay based on Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) methodology. Recombinant human 0.05 nM Bcl-2/0.03 nM Bcl-X protein was pre-incubated with a serial dilution of compounds disclosed herein (maximum concentration is 0.1 μM for Bcl-2 assay, and 10 μM for Bcl-xl assay, 3-fold serially diluted, 10 points; or maximum concentration is 0.02 μM for Bcl-2 assay, and 2 μM for Bcl-xl assay, 3-fold serially diluted, 10 points) at room temperature for 0.5 hour in an assay buffer containing 20 mM potassium phosphate buffer, pH 7.5, 50 mM NaCl, 1 mM EDTA, 0.05% Tween-20, 0.01% BSA. Then the FITC labeled Bak peptide Ac-GQVGRQLAIIGDK(FITC)INR-amide (0.5 nM for Bcl-2, 0.3 nM for Bcl-xl) and MAb Anti 6His Tb cryptate Gold were added to plate and further incubated at room temperature for 1 hour. The TR-FRET signals (337 nm-520 nm-490 nm) were read on BMG PHERAstar FSX instrument. The inhibition percentage of Bcl-2/Bcl-X interaction with its ligand in presence of increasing concentrations of compounds was calculated based on the TR-FRET signals. The IC50 for each compound was derived from fitting the data to the four-parameter logistic equation by Graphpad Prism software. To improve the assay sensitivity and test more potent compounds in the present application, the bcl-2 concentration was reduced in method B.
Measurement: IC50 = 61 nM (pChEMBL 7.210000038146973)
Descriptors: MW 814.92, ALogP 8.1, HBA 11, HBD 3, PSA 178.02, QED 0.08
Compound (SMILES): CC(C)(COc1cc(-c2ccc(N3CC4CC(C3)N4C(=O)C(F)(F)F)nc2)c2c(C#N)cnn2c1)OC(=O)C(F)(F)F
InChIKey: NUWAIBOSTDBFLB-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of Formula I were screened for their ability to inhibit wildtype and V804M mutant RET kinase using CisBio's HTRF® KinEASE¿-TK assay technology. Briefly, N-terminal GST tagged recombinant human RET cytoplasmic domain (aa 658-end) from Eurofins (0.25 nM RET; Catalog No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Catalog No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Catalog No. 62TK0PEC) and 1 mM ATP along with test compound in a buffer consisting of 25 mM HEPES pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO in a volume of 8 μL. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 30-minute incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.25 nM Sa-XL665 and 1×TK-ab-Cryptate in HTRF detection buffer (all from CisBio, part of Cat. No. 62TK0PEC). After a 1 hour incubation at 22° C., the extent of reaction was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using pre-quenched control reactions. The POC values were fit to a 4 parameter logistic curve, and the IC50 is defined as the concentration of inhibitor at which the POC equals 50 for the fitted curve.
Measurement: IC50 = 733 nM (pChEMBL 6.130000114440918)
Descriptors: MW 596.49, ALogP 3.88, HBA 9, HBD 0, PSA 113.06, QED 0.31
Compound (SMILES): CCCCNc1ncc(-c2ccc(CNC3C(C)(C)C3(C)C)cn2)c(N[C@H]2CC[C@H](O)CC2)n1
InChIKey: YAUQWMBRTAHSFI-MXVIHJGJSA-N
Target: Receptor-type tyrosine-protein kinase FLT3 (SINGLE PROTEIN), Homo sapiens
UniProt: P36888
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 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 466.67, ALogP 4.99, HBA 7, HBD 4, PSA 94.99, QED 0.37
Compound (SMILES): COC(=O)c1cnc2cc(Nc3cc(C)[nH]n3)nc(N[C@@H]3C[C@H]4CC[C@@H](C3)N4CCF)c2c1
InChIKey: SBOXBAGRWUVPBR-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 453.52, ALogP 3.57, HBA 8, HBD 3, PSA 108.06, QED 0.47
Compound (SMILES): C[C@H](O)C(=O)N1C[C@H]2C[C@H](N(C)c3ncnc4[nH]ccc34)C[C@H]2C1
InChIKey: IJKKBQHULDYNJO-WUHRBBMRSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: In Vitro Kinase Assay: In vitro kinase assays described below can be used to determine the activity of a test compound for inhibiting the activity of JAK1 kinase. The test compounds were dissolved in dimethyl sulfoxide and diluted with water to a serial concentration gradient as required in the experiment. JAK1 substrates (Cell Signaling Technology, Catalog Number: 1305s) and ATP (2 mM) solution were diluted with water to obtain a final concentration of 20 uM ATP and 1.2 uM substrate solution. The appropriate amount of JAK1 kinase (Invitrogen, Catalog Number: pv4774) was mixed with 4x buffer (prepared by user, and comprising 50 mM HEPES, pH 7.3, 125 mM NaCl, 24 mM MgCl2, 1.25 mM DTT) to a final concentration of 8 ng/uL. To each well of a microplate [DELFIA Streptavidin-coated clear plate (Perkin Elmer, Catalog Number: AAAND-0005)]17.5 uL of ATP/substrate mixture, 5 uL of an aqueous solution of a test compound (5 uL of pure water only were added to the control and blank), and 7.5 uL of the kinase solution prepared above (4x buffer only was added to the control) were added. Each well was mixed sufficiently, then incubated at room temperature (27° C.) for 50 minutes, washed with wash buffer, and dried three times, then HRP conjugated antibody was added [Phospho-Tyrosine Mouse mAb (P-Tyr-100) (HRP Conjugate, Cell Signaling Technology, Catalog Number: 5465)], and incubated for 1 hour. The microplate was washed with wash buffer and dried three times, and then TMB (Sigma, Catalog Number: T4444) was added, and incubated for 5 to 15 minutes to allow for color change. Stop solution (1 N sulfuric acid solution) was added to stop the reaction. Absorbance was measured on a Novostar microplate reader at a wavelength of 450 nm. IC50 values of test compounds was calculated from the data of the test compounds in inhibiting the activity of JAK1 kinase at different concentrations.
Measurement: IC50 = 0.2 nM (pChEMBL 9.699999809265137)
Descriptors: MW 329.4, ALogP 1.01, HBA 5, HBD 2, PSA 85.35, QED 0.88
Compound (SMILES): COc1cc(OC)c(F)c(N(C[C@H]2CCC(=O)N2)c2ncc3ncc(-c4cnn(C)c4)nc3n2)c1
InChIKey: LUJNIROONWQIJG-CQSZACIVSA-N
Target: Fibroblast growth factor receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P22455
Assay: FGFR4 (Enzymatic Assay): In a final reaction volume of 30 μL, FGFR4 (h) (60 ng/ml) was incubated with 50 mM HEPES pH 7.5, 6 mM MnCl2, 1 mM DTT, 0.1 mM Na3VO4, 0.01% Triton-X-100, 500 nM Btn-Flt3 and 5 μM ATP in the presence of compound (1% DMSO final). After incubation for 60 minutes at room temperature the reaction was stopped with 2.27 nM EU-anti P-Tyr, 7 mM EDTA, 31.25 nM SA-XL-665 and 0.02% BSA which was present for 60 minutes at room temperature. Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) signal (ex340 nm. Em 620 nm, em 655 nm) was measured afterwards and results are expressed in RFU (Relative Fluorescence Units). In this assay, the inhibitory effect of different compound concentrations (range 10 μM to 0.1 nM) was determined and used to calculate an IC50 (M) and pIC50 (−log IC50) value.
Measurement: IC50 = 501 nM (pChEMBL 6.300000190734863)
Descriptors: MW 478.49, ALogP 2.39, HBA 10, HBD 1, PSA 120.18, QED 0.43
Compound (SMILES): COc1cc(Nc2cc(Oc3ccc(NC(=O)Nc4cc(C(C)(C)C)cc(P(C)(C)=O)c4OC)c4ccccc34)ccn2)cc(S(=O)(=O)N(C)CCCN2CCOCC2)c1
InChIKey: IEMKOBYQSRDPTC-UHFFFAOYSA-N
Target: Glycogen synthase kinase-3 alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P49840
Assay: Enzyme Inhibition Assay: Method 1: 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 2 hr 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).
Measurement: IC50 = 3939 nM (pChEMBL 5.409999847412109)
Descriptors: MW 859, ALogP 8.32, HBA 11, HBD 3, PSA 160.66, QED 0.09
Compound (SMILES): CCC(CC)n1cc(-c2nc(-c3cnn(C4CN(C(=O)NC)C4)c3)cn3nccc23)cn1
InChIKey: MVSWGWIKKZTFSP-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: JAK3 Inhibition Assay: Compounds of Formula I were screened for their ability to inhibit JAK3 using the general enzyme inhibition assay method, in which the assay mixture contained 1 mM ATP, 10 μM Omnia® Y7 peptide (Catalog # IVGN KNZ3071C, Invitrogen Corporation, Carlsbad, Calif.) and 2 nM JAK3 in a total volume of 25 μL. JAK3 was purchased from Invitrogen Corporation, Carlsbad, Calif. (catalog # IVGN PV4080).
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 433.52, ALogP 3.01, HBA 8, HBD 1, PSA 98.17, QED 0.5
Compound (SMILES): C=CC(=O)N1CC[C@H](n2nc(C#Cc3cc(OC)cc(OC)c3)c(C(N)=O)c2NCCN2CCCC2)C1
InChIKey: HUXAYMWWNBTISC-FQEVSTJZSA-N
Target: Fibroblast growth factor receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P22455
Assay: FGFR4 Activity Inhibition Test: The reaction buffer contains the following components: 5-fold diluted enzymatic buffer/kinase 5x(Cisbio, Cat No. 62EZBFDD)(its main component is 50nM HEPES, pH 7.0), 5mM MgCl2 and 1 mM DTT, a human recombinant FGFR4 catalytic domain protein (amino acid 460-802) which was purchased from Tsinghua University Protein Research Technology Center, a 0.5 kinase solution diluted by the reaction buffer; a substrate reaction solution containing 500 nM biotinylated tyrosine kinase substrate diluted by the reaction buffer (Cisbio, Cat. No. 62TKOPEC) and 90 uM ATP; a test solution containing 0.125 ng/uL Eu3+ labeled cage antibody (Cisbio, Cat. No 61T66KLB) diluted by a test buffer (Cisbio, Cat No 62SDBRDF), and 31.25nM streptavidin labeled XL665 (Cisbio, Cat. No. 610SAXLB).
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 506.61, ALogP 1.87, HBA 8, HBD 2, PSA 114.95, QED 0.39
Compound (SMILES): CC[C@H](NC(=O)[C@H](C)NC)C(=O)N1CC(F)(F)C[C@H]1Cc1c(-c2nc3cc(F)ccc3n2C[C@@H]2CC(F)(F)CN2C(=O)[C@@H](NC(=O)[C@H](C)NC)C(C)C)[nH]c2cc(F)ccc12
InChIKey: BVNKXOWYTHHDGP-AQQFEGBLSA-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 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 861.93, ALogP 4.73, HBA 8, HBD 5, PSA 156.49, QED 0.11
Compound (SMILES): O=C1c2cc(-c3cccnc3)c(OC3CCCC3)cc2CN1c1cccc(-c2nncn2C2CC2)n1
InChIKey: QXEUJUYSECCFGD-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99683
Assay: ASK1 Kinase Assay: The ASK1 enzymatic assay was run following Promega ASK1 Kinase Enzyme System (Cat #V3881). The kit provides the protocol, enzymes and all reagents necessary to run an assay. Firstly, the compounds, enzyme, substrate and ATP were diluted in provided assay buffer. The final concentration of the enzyme was 50 nM, substrate (Myelin basic protein) 1 μg/ml and ATP 10 μM. The compound and the enzyme were pre-incubated in a 384 well white solid bottom plate (Greiner, Cat#784075) for 10 minutes. After incubation, the substrate and ATP were added and incubated for further 60 minutes. After 60 minutes, ADP-Glo™ was added and plate was incubated for another 40 minutes. After 40 minutes, Kinase Detection Reagent was added and the plate was incubated for 45 minutes. After 45 minutes, plate was read on Perkin Elmer EnVision using luminescence read (0.5 seconds/well).
Measurement: Ki < 200 nM (pChEMBL 6.698999881744385)
Descriptors: MW 478.56, ALogP 5.22, HBA 7, HBD 0, PSA 86.03, QED 0.38
Compound (SMILES): C=CC(=O)Nc1cc(Nc2ncc(N(C)C(=O)C(C)C)c(-c3cn(C)c4ccccc34)n2)c(OC)cc1N(C)CCN(C)C
InChIKey: IOKVRNZOKGJSMC-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: HER2 Exon 20 YVMA Insertion Mutation: A compound's ability in selectively inhibiting Her2 exon 20 YVMA insertion mutations can be assessed using Ba/F3 cells, a murine pro-B cell line, which have been transduced with Her2 Exon20 YVMA insertions. An expression vector, pLVX-IRES puro (Clontech) coding for human EGFR exon 20 insertions YVMA (A775_G776ins YVMA), was transfected into HEK293 cells by the Trans-Lentiviral ORF Packaging System (Thermo Scientific), to produce virus encoding EGFR exon 20 insertions. Ba/F3 (DSMZ) cells maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 200 μM L-glutamine/200 μg/mL penicillin/200 μg/mL streptomycin (Life Technology) and 10 ng/mL IL-3 (R&D system), were infected by EGFR Exon20 virus and subsequently selected by puromycin (Life Technology) selection and IL-3 depletion. Ba/F3 cells expressing Her2 Exon20 YVMA insertions (named Ba/F3-Her2 Exon20 YVMA) can proliferate in the absence of IL-3. The anti-proliferative activity of compounds was determined as follows: BaF3-Her2 Exon20 YVMA cells seeded in 96 well plates (2500 cells/well) were treated with test compound (dissolved in DMSO) at a series of concentrations (4-fold dilution, top concentration: 10,000 nM). The plates were incubated for 72 h in a 37° C. incubator with 5% CO2, and the number of viable cells in each well were measured indirectly by CellTiter 96® Aqueous One Solution Cell Proliferation Assay (Promega; this assay is a colorimetric method for determining the number of viable cells through measurement of their metabolic activity by detection of enzymatic conversion of tetrazolium salts into blue formazan derivatives). Reagent (20 μL) was added into each well, and the plates were returned to the incubator for 2 h. The absorbance in each well was then measured at 490 nm using an Envision plate reader (Perkin Elmer). IC50 values were calculated by determining the concentration of compound required to decrease the MTS signal by 50% comparing to the DMSO control in best-fit curves using Microsoft XLfit software or Accelrys Pipeline Pilot.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 598.75, ALogP 5.13, HBA 9, HBD 2, PSA 107.86, QED 0.22
Compound (SMILES): COC(=O)c1cc2c(cc1C)/C(=C(/Nc1ccc(C(=O)NOCCN3CCNC(=O)C3)cc1)c1ccccc1)C(=O)N2
InChIKey: QXWNZKXHVHCPOG-DQSJHHFOSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: VEGFR2 Enzyme Inhibition: The inhibitory activities of compounds of the invention against VEGFR2 (VEGFR2 Kinase Enzyme System: Promega), were evaluated by mixing the VEGFR2 protein (1.56 ng/mL, 2 μL), substrate (Poly (4:1 Glu4, Tyr1), 100 ng/mL, 2 μL) with the test compound (2 μL at either 3 μM, 0.67 μM, 0.15 μM, 0.033 μM, 0.0073 μM, 0.0016 μM, 0.0036 μM or 0.00008 μM) for 90 min at 25° C. The kinase reaction was initiated by adding ATP (50 μM, 2 μL) and the mixture was incubated for 1 hr at 25° C. ADP-Glo reagent was added for 40 min (8 μL) then development reagent (16 μL) was added for 40 min prior to detection in a microplate reader (EnVision® Multilabel Reader, Perkin Elmer).
Measurement: Ki = 25 nM (pChEMBL 7.599999904632568)
Descriptors: MW 569.62, ALogP 2.81, HBA 8, HBD 4, PSA 138.1, QED 0.13
Compound (SMILES): CCN(CCN1CCC(C2NOc3cc(F)ccc32)CC1)C(=O)C(O)CNC(C)=O
InChIKey: YGQYHFNCAZNGCT-UHFFFAOYSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Inhibitory Activity for hERG Channel: Extracellular and intracellular fluids used in the measurement were prepared as follows.Extracellular fluid: 2 mmol/L CaCl2, 1 mmol/L MgCl2, 10 mmol/L HEPES, 4 mmol/L KCl, 145 mmol/L NaCl, 10 mmol/L GlucoseIntracellular fluid: 10 mmol/L HEPES, 10 mmol/L EGTA, 20 mmol/L KCl, 130 mmol/L KFTest compound solution: A test compound was dissolved in DMSO to reach the concentration of 2 mmol/L or 20 mmol/L in order to prepare a test compound solution. The test compound solution was further diluted 200 times with the extracellular fluid, and serial-diluted with the extracellular fluid to prepare a test compound solution in each concentration which is necessary to calculate IC50 value of hERG inhibition.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 422.5, ALogP 0.82, HBA 6, HBD 3, PSA 94.14, QED 0.57
Compound (SMILES): CCCCOc1nc(N)c2nc(O)n(Cc3ccc4c(c3)Sc3cc(CNC(C)C)ccc3N4C)c2n1
InChIKey: VZAPQEUJSMDGBZ-UHFFFAOYSA-N
Target: Interleukin-6 (SINGLE PROTEIN), Homo sapiens
UniProt: P05231
Assay: Interleukin 6 Induction Assay: Compounds diluted in DMSO were transferred to individual wells of a Matrix Tech-nologies clear, V-bottom 384-well plate using ECHO acoustic liquid handling technology (25 nL per well). Human whole-blood samples (25 uL) were added to each well using a CyBio FeliX liquid handling instrument. The plate was shaken on a plate shaker for three min before incubating the reaction mixtures at 37° C. for 20 h. Basel RPMI 1640 medium (supplemented with L-glutamine) was then added to each well (25 uL per well) prior to liberating plasma from each sample by centrifugation (450×g, 5 min, ambient temperature). Treated plasma samples (3 uL) were subsequently transferred to individual wells of a white, shallow, 384-well ProxiPlate (Perkin Elmer) using the FeliX liquid handling instrument and their interleukin 6 levels were measured using AlphaLISA technology as described by the manufacturer, PerkinElmer. Data analyses software was used to determine compound EC50 values where the baseline was established using average DMSO values and 100% induction established using reference compound values at the highest concentration tested. EC50's can be determined with software such as Graphpad Prism.
Measurement: EC50 = 12.6 nM (pChEMBL 7.900000095367432)
Descriptors: MW 519.68, ALogP 5.07, HBA 10, HBD 3, PSA 114.35, QED 0.26
Compound (SMILES): CCCNC(=O)c1nc2cccnn2c1-c1cc(Cl)cc(Cl)c1
InChIKey: GXYHCJBIWBLNEK-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 = 15.6 nM (pChEMBL 7.809999942779541)
Descriptors: MW 349.22, ALogP 3.84, HBA 4, HBD 1, PSA 59.29, QED 0.78
Compound (SMILES): Cn1cc(-c2c(-c3nccs3)nc(N)n3nc(Cc4c(F)cccc4F)nc23)ccc1=O
InChIKey: VLEJDOHBWVIJDB-UHFFFAOYSA-N
Target: Adenosine receptor A1 (SINGLE PROTEIN), Homo sapiens
UniProt: P30542
Assay: FLIPR and cAMP Inhibition Assay: hADORA1/CHO (hA1 expressing) cells (Genscript) were plated at 1×104 cells/well into 384-well polystyrene plates one day before starting the experiment. On the day of experiment, the supernatant was discard and replaced with 40 L of dye (FLIPR calcium 5 Assay Kit) per well and the plates were incubated for 60 mins at 37° C. plus 5% CO2. Then testing compounds were added at different concentrations for FLIPR inhibition assay. After a 400 s incubation with compound, 10 M adenosine was added into the cells, and the signal was captured by FLIPR.hA2a/CHO, hA2b/CHO and mA2a/CHO (Genscript) were plated at 5×103 cells/well into 384-well polystyrene plates at the day of experiment. Compounds were pre-incubated with cells for 30 min at 37° C., 5% CO2. Then 10 M adenosine was added to the cells and incubated for 30 min at 37° C., 5% CO2. Detection reagent (CISBIO) was added and the plates were incubated for 60 min at room temperature. The signal was captured by Envision.
Measurement: IC50 = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 451.46, ALogP 3.06, HBA 9, HBD 1, PSA 103.99, QED 0.45
Compound (SMILES): CC(C)(C)C/C(=N/O)NCc1nc2cnc3[nH]ccc3c2n1[C@H]1CC[C@H](CC#N)CC1
InChIKey: ARMQCPPKWRFVLM-WKILWMFISA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Enzymatic Inhibition Assay: 11-point dosing series were made for each compound by serially diluting 1:3 or 1:4 in DMSO, with point 12 being a DMSO control. From the serial dilution plates, sample was transferred to a 384 wells assay plate (#781280, Greiner, Monroe, N.C.) using Labcyte Echo (Sunnyvale, Calif.), or Biosero ATS (San Diego, Calif.). The compounds were tested in duplicate. Column 12 was used for positive controls, and column 24 contained negative controls with no enzyme added. A compound from our internal collection, with inhibitory activity for JAK isoforms, was used as a reference compound. The final concentration of DMSO was ≤0.25% in a 20 μL reaction. Assay conditions for each of the proteins are summarized in Table 3. The enzyme reaction was initiated by the addition of 10 μL of 2× enzyme and ATP mixture to 10 μL of 2× substrate solution prepared in reaction buffer (50 mM MOPS pH 7.5, 10 mM MgCl2, 1 mM EDTA, 2 mM DTT, 0.002% Tween-20). The Tyk2 enzyme was pre-incubated with 2 mM ATP for 30 min prior to the reaction initiation. Immediately after the addition of the enzyme to the reaction mixture, the plate was centrifuged at 1000 rpm for 1 minute and incubated at 25° C. for 45 minutes for JAK 3 and 90 minutes for JAK1, JAK2 and Tyk2. The reaction was quenched by the addition of 20 μL of 0.5% TFA containing 0.15 μM of internal standard peptide using Multidrop Combi reagent dispenser (Thermo Scientific, Waltham, Mass.). Several wells in column 24 were typically used for the product standard curve. After the quench, the assay plate was centrifuged at 3000 rpm for 3 minutes and sealed with pierceable aluminum foil (Cat #06644-001, Agilent) using a PlateLoc (Agilent Technologies, Santa Clara, Calif.). The plates then were transferred on to the RapidFire for the MS analysis. Compound inhibition was assessed by a decrease of the phosphorylated product levels in sample wells compared to the non-inhibited enzyme reaction. The assay conditions for the above assays are shown in Table 3 and the results of Ex. 1-209 as tested in these assays are shown in Table 4.TABLE 3Assay conditions for JAK family enzyme assays* [p], [ATP], [Substrate], [IS],Enzyme nM μM μM nMJAK1-JH1JH2 8.0 12.5 200 100JAK2-JH1JH2 7.0 or 3.6 30 40 100JAK3-JH1JH2 2.0 150 40 100Tyk2-JH1JH2   25 or 14.7 50 200 100*Reaction buffer: 50 mM MOPS, pH 7.5 10 mM MgCl2, 1 mM EDTA, 2 mM DTT, 0.002% Tween-20; IS stands for internal standard peptide; p stands for phospho-peptide (product); Substrate stands for peptide.
Measurement: IC50 = 6.7 nM (pChEMBL 8.170000076293945)
Descriptors: MW 421.55, ALogP 4.87, HBA 6, HBD 3, PSA 114.91, QED 0.24
Compound (SMILES): COCc1nn(Cc2ccc(N3CCC(F)(F)C3)nc2)cc1C(=O)NCc1c(-n2cnnn2)ccc(OC)c1F
InChIKey: HWYRMCCHOBIHJO-UHFFFAOYSA-N
Target: Coagulation factor XII (SINGLE PROTEIN), Homo sapiens
UniProt: P00748
Assay: Determination of the % Inhibition for FXIIa: Factor XIIa inhibitory activity in vitro was determined using standard published methods (see e.g. Shori et al., Biochem. Pharmacol., 1992, 43, 1209; Baeriswyl et al., ACS Chem. Biol., 2015, 10 (8) 1861; Bouckaert et al., European Journal of Medicinal Chemistry 110 (2016) 181). Human Factor XIIa (Enzyme Research Laboratories) was incubated at 25° C. with the fluorogenic substrate H-DPro-Phe-Arg-AFC and various concentrations of the test compound. Residual enzyme activity (initial rate of reaction) was determined by measuring the change in optical absorbance at 410 nm and the IC50 value for the test compound was determined.
Measurement: IC50 > 40000 nM (pChEMBL 4.3979997634887695)
Descriptors: MW 557.54, ALogP 2.37, HBA 11, HBD 1, PSA 125.11, QED 0.31
Compound (SMILES): C=CC(=O)N(C)C(=O)N(C)[C@H](C(=O)N[C@H]1Cc2nc(cs2)-c2ccc3c(c2)c(c(-c2cccnc2[C@H](C)OC)n3CC)CC(C)(C)COC(=O)[C@@H]2CCCN(N2)C1=O)C(C)C
InChIKey: QTYJFUOGJKBKCG-GOZBCIFISA-N
Target: GTPase NRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01111
Assay: Biochemical Assay: A variety of Ras proteins may be inhibited by compounds of the present invention (e.g., K-Ras, N-Ras, H-Ras, and mutants thereof at positions 12, 13 and 61, such as G12C, G12D, G12V, G12S, G13C, G13D, and Q61L, and others described herein). The purpose of this biochemical assay is to measure the ability of test compounds to covalently label nucleotide-loaded K-Ras isoforms. In assay buffer containing 12.5 mM HEPES pH 7.4, 75 mM NaCl, 1 mM MgCl2, 1 mM BME, 5 uM Cyclophilin A and 2 uM test compound, a 5 uM stock of GMP-PNP-loaded K-Ras (1-169) G12C is diluted 10-fold to yield a final concentration of 0.5 uM; with final sample volume being 100 uL. The sample is incubated at 25�� C. for a time period of up to 24 hours prior to quenching by the addition of 10 uL of 5% Formic Acid. Quenched samples are centrifuged at 15000 rpm for 15 minutes in a benchtop centrifuge before injecting a 10 uL aliquot onto a reverse phase C4 column and eluting into the mass spectrometer with an increasing acetonitrile gradient in the mobile phase. Analysis of raw data may be carried out using Waters MassLynx MS software, with % bound calculated from the deconvoluted protein peaks for labeled and unlabeled K-Ras.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 855.07, ALogP 5.92, HBA 12, HBD 2, PSA 168.3, QED 0.15
Compound (SMILES): CCCCNc1ncc2c(-c3ccc(S(=O)(=O)N4CCN(CC(F)(F)F)CC4)c(F)c3)cn([C@H]3CC[C@H](O)CC3)c2n1
InChIKey: BPFIPYIIVJLOML-MEMLXQNLSA-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 = 8.3 nM (pChEMBL 8.079999923706055)
Descriptors: MW 612.69, ALogP 4.79, HBA 8, HBD 2, PSA 103.59, QED 0.26