lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CNc1cc2nc(N[C@@H](C)c3cncc(F)c3)nc(C(=O)N3CCCC3)c2s1
InChIKey: DPRHOTSBCAIYFC-NSHDSACASA-N
Target: Adenosine receptor A3 (SINGLE PROTEIN), Homo sapiens
UniProt: P0DMS8
Assay: Activity Assay: Compounds (Adenosine Receptor modulators) and activity assay.
Measurement: IC50 = 75 nM (pChEMBL 7.119999885559082)
Descriptors: MW 400.48, ALogP 3.68, HBA 7, HBD 2, PSA 83.04, QED 0.68
Compound (SMILES): Cc1cc(Nc2cc(OC3COC3)nc(N(C)[C@@H]3C[C@H]4CCC[C@@H](C3)N4C(=O)CC3CC3)n2)n[nH]1
InChIKey: MGHOQDJXGMZXRB-REPLKXPHSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Biochemical JAK and Tyk2 Kinase Assays: 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 or discretely 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 1 h. 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 1 h 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.
Measurement: Ki < 0.1 nM (pChEMBL 10)
Descriptors: MW 481.6, ALogP 3.18, HBA 8, HBD 2, PSA 108.5, QED 0.59
Compound (SMILES): Cc1cc(N2C(=O)c3cc(-c4cccc(=O)n4C)n(C4CC4)c3C2c2ccc(Cl)cc2)cn(C)c1=O
InChIKey: XSOFZNARGSJIQN-UHFFFAOYSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: Assay for CREB protein: All assays were performed in 384-well microtiter plates. Each assay plate contained 8-point serial dilutions for 40 test compounds, plus 16 high- and 16 low controls. Liquid handling and incubation steps were done on an Innovadyne Nanodrop Express equipped with a robotic arm (Thermo CatX, Perkin Elmer/Caliper Twister II) and an incubator (Liconic STX40, Thermo Cytomat 2C450). The assay plates were prepared by addition of 50 nl per well of compound solution in 90% DMSO HummingBird nanodispenser (Zinsser Analytic). The assay was started by stepwise addition of 4.5 uL per well of bromo domain protein (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 45 nM His-Brd2(60-472) or 45 nM His-Brd3(20-477) or 45 nM His-Brd4(44-477) all proteins produced in-house) and 4.5 uL per well of peptide solution (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 60 nM acetyl-histone H4 (AcK 5, 8, 12, 16) (Biosyntan GmbH)). Reactions were incubated at 30° C. for 35 minutes. Subsequently 4.5 uL per well detection mix (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 3 nM Eu-labeled anti-His6 antibody, 21 nM streptavidin-allophycocyanin) were added. After 35 minutes incubation at 30° C., plates were measured in a Perkin Elmer EnVision multilabel reader.
Measurement: IC50 = 180 nM (pChEMBL 6.75)
Descriptors: MW 500.99, ALogP 4.6, HBA 6, HBD 0, PSA 69.24, QED 0.41
Compound (SMILES): CCC(CC)n1cc(-c2nc(-c3cnn(C[C@H](CO)N(C)C)c3)cn3nccc23)cn1
InChIKey: XLLHHLZZSBVTBI-LJQANCHMSA-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 422.54, ALogP 2.74, HBA 9, HBD 1, PSA 89.3, QED 0.45
Compound (SMILES): CO[C@@H](C)c1ncccc1-c1c2c3cc(ccc3n1CC(F)(F)F)-c1csc(n1)C[C@H](NC(=O)CCOC1CN(C(=O)C#CC(C)(C)N3CCC3)C1)C(=O)N1CCC[C@H](N1)C(=O)OCC(C)(C)C2
InChIKey: PBSXBQXBEZCQGL-UOPCVZPCSA-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 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 961.12, ALogP 5.85, HBA 13, HBD 2, PSA 160.46, QED 0.15
Compound (SMILES): C=CC(=O)N1CCN(C(=O)c2cc(Sc3cnc(Nc4ncc(C)cc4C)s3)c(C)cc2C)CC1
InChIKey: CECVIWNNFCTJPO-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: In Vitro Assay: The potency of inhibition is measured by binding affinity.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 507.69, ALogP 5.14, HBA 7, HBD 1, PSA 78.43, QED 0.47
Compound (SMILES): N[C@H]1CCCC[C@H]1C(=O)NCc1nc2cc(C(F)(F)F)cc(C(F)(F)F)c2[nH]1
InChIKey: WNUICAKVAIZFRY-KOLCDFICSA-N
Target: Voltage-dependent T-type calcium channel subunit alpha-1H (SINGLE PROTEIN), Homo sapiens
UniProt: O95180
Assay: High-Throughput Cav3.1 T-Type Fluorescent Assay: Cells were plated in 384-well, clear-bottom, black-walled, poly-D-lysine coated plates (Becton Dickinson, Franklin Lake, N.J.) 2 days prior to use in the FLIPR assay. 100 μL of cells (2.0×106 cell/mL) containing doxycyline (Sigma-Aldrich, 1.5 μg/mL; to induce channel expression) were added to each well using a Multidrop (Thermo Scientific, Waltham, Mass.) and were maintained in 5% CO2 incubator at 37° C. On the morning of the assay, cells were transferred to a 5% CO2 incubator at 29° C. Cells were washed with a wash buffer containing (in mM): 118 NaCl, 18.4 HEPES, 11.7 D-glucose, 0.05 CaCl2, 0.5 MgSO4, 1 KCl, and 1.2 KH2PO4, with the pH adjusted to 7.2 with NaOH. 4.4 μM of the fluorescent indicator dye, Fluo-4 (Invitrogen), prepared in pluronic acid (Sigma-Aldrich), were loaded into the wells and incubated for 45 minutes at 29° C. in 5% CO2. Cells were then rinsed with the following low Ca2+ buffer (in mM): 0.34 Na2HPO4, 4.2 NaHCO3, 0.44 KH2PO4, 0.41 MgSO4, 0.49 MgCl2-6H2O, 20 HEPES, 5.5 D-Glucose, 137 NaCl, 5.3 KCl, and 0.001 CaCl2, with 0.1% BSA and the pH adjusted to 7.2 with NaOH. Concentration-dependent response curves were generated from 5 mM stock solutions prepared in DMSO (Sigma-Aldrich) and diluted in the buffer containing low Ca2+ and incubated for 20 minutes at 29° C. in 5% CO2 Calcium entry was evoked with an addition of (in mM): 0.34 Na2HPO4, 4.2 NaHCO3, 0.44 KH2PO4, 0.41 MgSO4, 0.49 MgCl2-6H2O, 20 HEPES, 5.5 D-Glucose, 137 NaCl, 5.3 KCl, and 6 CaCl2, with 0.1% BSA and the pH adjusted to 7.2 with NaOH. A change in the Fluo-4 fluorescence signal was assessed using FLIPRTETRA¿ instrument (Molecular Devices, Sunnyvale, Calif.) for 3 minutes following the elevation of extracellular KCl using an illumination wavelength of 470-495 nm with emissions recorded at 515-575 nm.
Measurement: IC50 = 7200 nM (pChEMBL 5.139999866485596)
Descriptors: MW 408.35, ALogP 3.73, HBA 3, HBD 3, PSA 83.8, QED 0.68
Compound (SMILES): CCCCNc1ncc(-c2ncc(C)s2)c(N[C@H]2CC[C@H](O)CC2)n1
InChIKey: BGFQHBDQBASGDN-HDJSIYSDSA-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 = 6.9 nM (pChEMBL 8.15999984741211)
Descriptors: MW 361.52, ALogP 3.84, HBA 7, HBD 3, PSA 82.96, QED 0.65
Compound (SMILES): Nc1nc2ccc(-c3ccc4nnc(C(=O)N5CCCC5)n4c3)cc2o1
InChIKey: UPMPVYAKWYGRFP-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 < 10000 nM (pChEMBL 5)
Descriptors: MW 348.37, ALogP 2.36, HBA 7, HBD 1, PSA 102.55, QED 0.6
Compound (SMILES): C[C@H](Nc1nc(C(=O)N2CC(NC(=O)C(C)(C)C)C2)c2sccc2n1)c1cncc(F)c1
InChIKey: NANBKGMCXHTIDJ-LBPRGKRZSA-N
Target: Adenosine receptor A3 (SINGLE PROTEIN), Homo sapiens
UniProt: P0DMS8
Assay: Activity Assay: Compounds (Adenosine Receptor modulators) and activity assay.
Measurement: IC50 = 75 nM (pChEMBL 7.119999885559082)
Descriptors: MW 456.55, ALogP 3.39, HBA 7, HBD 2, PSA 100.11, QED 0.61
Compound (SMILES): COc1cccc(CN2CCc3cccc(CCc4ccc(C(=O)O)s4)c32)c1
InChIKey: LSFZELNYLNCJMQ-UHFFFAOYSA-N
Target: Prostaglandin E2 receptor EP4 subtype (SINGLE PROTEIN), Homo sapiens
UniProt: P35408
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 = 1486 nM (pChEMBL 5.829999923706055)
Descriptors: MW 393.51, ALogP 4.8, HBA 4, HBD 1, PSA 49.77, QED 0.63
Compound (SMILES): N#CC[C@@H](C1CC1)n1nc(Nc2ccc(C(N3CCSCC3)C(F)(F)F)cc2)c2c(=O)[nH]ccc21
InChIKey: CQPQTZCYNUNXLH-YMXDCFFPSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Jak Biochemical HTRF Assay Protocol: The ability of compounds to inhibit the activity of JAK1, JAK2, JAK3, and Tyk2 was measured using a recombinant purified GST-tagged catalytic domain for each enzyme (Invitrogen JAK1 #M4290, JAK2 #M4290, JAK3 #M4290, Tyk2 #M4290) in an HTRF format biochemical assay. The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 μL of 1.11× enzyme and 1.11× substrate in 1× assay buffer (Invitrogen kinase buffer #PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 μL of 10×ATP in 1× assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 μL of 2× stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction. Plates were shaken and centrifuged and then incubated 60 minutes at room temperature and then read on a Perkin Elmer Envision (λex=337 nm, λem=665 and 615 nm, TRF delay time=20 μs). HTRF signal=10,000*665 nm reading/615 nm reading. After normalization to untreated controls, the percent inhibition of the HTRF signal at each compound concentration was calculated.
Measurement: IC50 = 770 nM (pChEMBL 6.110000133514404)
Descriptors: MW 502.57, ALogP 4.99, HBA 7, HBD 2, PSA 89.74, QED 0.47
Compound (SMILES): CN1CCN(c2ccc(C(=O)Nc3nc(-c4ccc(F)cc4)ccc3N)cn2)CC1
InChIKey: TUVFSLMAKAPSFZ-UHFFFAOYSA-N
Target: Histone deacetylase 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9BY41
Assay: Histone Deacetylase Assay: The inhibitory activities of compounds of present invention were determined using biochemical HDAC assays (Reaction Biology Corp. biochemical assay services). Compound with indicated doses was tested in the biochemical assays of HDAC 1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC 8, HDAC9, HDAC10, and HDAC11 enzyme.Compounds were tested in singlicate 10-dose IC50 mode with 3-fold serial dilution starting at 10 μM against 11 HDACs. HDAC reference compounds Trichostatin A (TSA) and TMP269 were tested in a 10-dose IC50 with 3-fold serial dilution starting at 10 μM.Substrate for HDAC1,2,3,6,10: Fluorogenic peptide from p53 residues 379-382 (RHKK(Ac)AMC). Substrate for HDAC4,5,7,9, and 11: Fluorogenic HDAC Class2a Substrate (Trifluoroacetyl Lysine). Substrate for HDAC 8: Fluorogenic peptide from p53 residues 379-382 (RHK(Ac)K(Ac)AMC).General Reaction Procedure: (Standard IC50 determination)    a. 2× enzyme was added to wells of reaction plate except to No Enzyme (No En) control wells. Add buffer in No En wells.    b. Compounds to be tested in 100% DMSO were added to the enzyme mixture by Acoustic technology (Echo550; nanoliter range). The mixture was spinned down and preincubated.    c. 2× Substrate Mixture (Fluorogenic HDAC Substrate and co-factor (500 μM of Nicotinamide adenine dinucleotide (NAD<+>) in all Sirt assay) were added to all reaction wells to initiate the reaction. The plates were spinned and shaken.    d. The plates were incubated for 1-2 hr. at 30° C. with seal.    e. Developer with Trichostatin A (or TMP269 or NAD<+>) was added to stop the reaction and to generate fluorescent color.    f. Fluorescence was read (excitatory, 360; emission, 460) using the EnVision Multilabel Plate Reader (Perkin Elmer)    g. Endpoint reading was taken for analysis after the development reaches plateau.Data Analysis: The percentages of enzyme activity (relative to DMSO controls) and IC50 values were calculated using the GraphPad Prism 4 program based on a sigmoidal dose-response equation. The blank (DMSO) value was entered as 1.00E-12 of concentration for curve fitting.
Measurement: IC50 > 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 406.47, ALogP 2.87, HBA 6, HBD 2, PSA 87.38, QED 0.69
Compound (SMILES): OCCn1cc(-c2cnc(Nc3cccc(Cl)c3Cl)nc2Nc2ccc3c(c2)CNCC3)cn1
InChIKey: OOZMTBCGVZLMGL-UHFFFAOYSA-N
Target: Tyrosine-protein kinase receptor TYRO3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06418
Assay: Verification on Inhibitory Activities of TAM Receptor Inhibiting Compounds on Tyro 3, Axl, and Mer: For evaluation of inhibitory activities of the compounds according to the present invention on Tyro 3, Axl, and Mer, the following test was carried out.The specific test method followed the method provided by Cisbio. The compounds of the examples were prepared at various concentrations, followed by addition of Tyro 3, Axl, or Mer and substrate peptides, and then ATP was added to initiate a reaction. After 1 hour, the reaction was stopped by addition of a solution containing EDTA. Thereafter, the amount of phosphorylated peptides was measured. Here, the phosphorylated peptides were treated with an europium (Eu)-labeled antibody recognizing phosphorylated peptides, and then after 1 hour, excited by irradiation of the light of a wavelength of 320 or 340 nm using an Envision Reader. The amount of light emitted at 665 nm was measured to investigate the degrees of inhibition of TAM receptors. The TAM receptor inhibitory activities (IC50 values) of the respective compounds were analyzed using the GraphPad Prism program, and tabulated in Tables 1 to 3 below. LDC1267 (CAS no. 1361030-48-9, Calbiochem), which is a TAM receptor inhibitor compound, was used as a positive control.
Measurement: IC50 = 0.01 nM (pChEMBL 11)
Descriptors: MW 496.4, ALogP 4.77, HBA 8, HBD 4, PSA 99.92, QED 0.29
Compound (SMILES): C[C@@H]1CN(CCCNC(=O)c2nc3c(N4CCOCC4)nc(-c4cnn(C)c4)nc3[nH]2)C[C@H](C)O1
InChIKey: RCSKGQVEJHQING-IYBDPMFKSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: In Vitro Enzyme PI3K Assay: The Kinase-Glo luminescent kinase assay kit (from Promega) was used to measure kinase activity. In this assay, amount of ATP remaining in solution following a kinase reaction is measured. The effect of compounds on PI3Kδ inhibition was carried out by adding 2.29 μg/ml of recombinant PI3K δ enzyme (Proteros, Germany) to reaction mixture containing assay buffer (50 mM HEPES, pH 7.4, 50 mM NaCl, 0.05% CHAPS) supplemented with 10 mM MgCl2, 5 mM DTT, 60 μM Phosphatidyl inositol bisphosphate (PIP2) and 10 μM ATP in the absence or presence of different concentrations of the compounds in a final volume of 15 μl/well, in a 384 well plate. The reaction mixture was incubated for 2 hours at room temperature. At the end of incubation period the equal volume of Kinase-Glo plus (Promega, V3772), was added per well and the luminescence was measured after incubating for 10 minutes at room temperature in dark. Results were calculated by measuring the luminescence units of test samples to blanks containing no enzyme.In certain embodiments, the compounds showed IC50 of less than 1000 nM, in another embodiment, the IC50 values range from about 100 nM to 500 nM, and in yet another embodiment, it is even less than 30 nM for PI3Kδ as shown in Table 3 and 4.The compounds of the present invention were tested for their selectivity for PI3Kδ over PI3Kα, PI3Kβ and PI3Kγ following the above assay using specific recombinant enzymes (Proteros, Germany) for each kinase. Assay condition of kinase assay (PI3Kα, PI3Kβ, PI3Kγ and PI3Kδ) was as follows. Enzyme: 2.29 μg/mL; ATP: 10 μM; PIP2 substrate: 60 μM and Reaction Time: 2 hours.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 483.58, ALogP 0.82, HBA 10, HBD 2, PSA 126.32, QED 0.47
Compound (SMILES): CC(C)(OCc1cc(-c2cccc(OCC3CC3)c2)n(Cc2ccccc2Cl)n1)C(=O)O
InChIKey: JKSRDKKNSRKQBO-UHFFFAOYSA-N
Target: Monocarboxylate transporter 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P53985
Assay: MCT1-Mediated Lactate Transport in BT20 Breast Cancer Cells: MCT1 activity may be measured using BT-20 breast cancer cells that express high native levels of MCT1, but do not express MCT4 and are known to those with skill in the art. Preparation of BCECF loaded cells are as described for Assay 1. Lactate transport assay is as described for Assay 1, except 10 mM L-lactate (rather than 50 mM) is added.
Measurement: IC50 = 54.5 nM (pChEMBL 7.260000228881836)
Descriptors: MW 454.95, ALogP 5.42, HBA 5, HBD 1, PSA 73.58, QED 0.44
Compound (SMILES): CCCCCOC[C@H]1CCc2cc([C@H]3CC[C@](N)(COP(=O)(O)O)C3)ccc2C1
InChIKey: FGTVMGUXDDFJPX-BULFRSBZSA-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 425.51, ALogP 4.07, HBA 4, HBD 3, PSA 102.01, QED 0.39
Compound (SMILES): CCOc1ccc(Nc2nc(N3CCC[C@@H](NC(=O)c4ccc(OC)cc4)C3)cnc2C(N)=O)cc1
InChIKey: LOSAVORYRNGPML-LJQANCHMSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
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 > 10000 nM (pChEMBL 5)
Descriptors: MW 490.56, ALogP 3.13, HBA 8, HBD 3, PSA 131.7, QED 0.42
Compound (SMILES): O=C(c1ccc(Nc2ncc3cc4n(c3n2)C2(CCCCC2)C(=O)NN4)cc1)N1CCOCC1
InChIKey: CEIJSVLEARLWRF-UHFFFAOYSA-N
Target: CDK6/cyclin D3 (PROTEIN COMPLEX), Homo sapiens
UniProt: P30281
Assay: CDK Inhibition In Vitro Assay: Selected compounds disclosed herein were tested in kinase assays by Nanosyn (Santa Clara, Calif.) to determine their inhibitory effect on these CDKs. The assays were performed using microfluidic kinase detection technology (Caliper Assay Platform). The compounds were tested in 12-point dose-response format in singlicate at Km for ATP. Specifics of each assay are as described below:CDK1/Cyclin B1: Enzyme concentration: 0.08 nM; ATP concentration: 40 μM; Incubation time: 3 hr. CDK2/Cyclin A: Enzyme concentration: 0.1 nM; ATP concentration: 50 μM; Incubation time: 3 hr. CDK2/Cyclin E: Enzyme concentration: 0.15 nM; ATP concentration: 100 μM; Incubation time: 3 hr. CDK4/Cyclin D1: Enzyme concentration: 1 nM; ATP concentration: 200 μM; Incubation time: 3 hr. CDK6/Cyclin D3: Enzyme concentration: 2 nM; ATP concentration: 300 μM; Incubation time: 3 hr. CDK9/Cyclin T1: Enzyme concentration: 5 nM; ATP concentration: 10 μM; Incubation time: 17 hr.
Measurement: IC50 = 137 nM (pChEMBL 6.860000133514404)
Descriptors: MW 461.53, ALogP 2.76, HBA 8, HBD 3, PSA 113.41, QED 0.55
Compound (SMILES): COCCOCCOCCOc1cc(Nc2nccc(Oc3ccc(NC(=O)Nc4cc(C(C)(C)C)cc(-c5cnn[nH]5)c4OC)c4ccccc34)n2)cc(OC)c1
InChIKey: XWCPBCVFAOFEEK-UHFFFAOYSA-N
Target: Glycogen synthase kinase-3 alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P49840
Assay: Enzyme Inhibition Assay: Method 2: This method follows the same steps as Method 1 above, but utilises a shorter period of mixing of the test compound (105 minutes instead of 2 hours) with the GSK3-α protein. In addition, the concentrations of test compound employed are either 10 ug/mL, 1 ug/mL, 0.1 ug/mL, or 0.01 ug/mL. 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 I phosphorylation of the target peptide. The GSK3-α protein (500 ng/mL, 2.5 uL) is mixed with the test compound (2.5 uL at either 4 ug/mL, 0.4 ug/mL, 0.04 ug/mL, or 0.004 ug/mL) for 2 hr at RT. The FRET peptide (8 uM, 2.5 uL), which is a phosphorylation target for GSK3α, and ATP (40 uM, 2.5 uL) are then added to the enzyme\compound mixture and the resulting mixture incubated for 1 hr. Development reagent (protease, 5 uL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan Flash, ThermoFisher Scientific).
Measurement: IC50 = 3968 nM (pChEMBL 5.400000095367432)
Descriptors: MW 792.89, ALogP 7.97, HBA 13, HBD 4, PSA 185.12, QED 0.06
Compound (SMILES): COc1cccc(-c2nnc(NS(=O)(=O)[C@@H](C)[C@@H](C)n3cc(C)cn3)n2-c2c(OC)cccc2OC)n1
InChIKey: OJMMCIHTIJPORO-SJORKVTESA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: [35S]GTPγS 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 M 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 M 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.) to obtain EC50.
Measurement: EC50 = 0.22 nM (pChEMBL 9.65999984741211)
Descriptors: MW 527.61, ALogP 3.25, HBA 11, HBD 1, PSA 135.28, QED 0.33
Compound (SMILES): CC(C)OC(=O)N1Cc2cc(Cl)ccc2-n2c(C3CC4(C3)CN(c3cccc(C#N)n3)C4)nnc2C1C
InChIKey: VQZYMUWBUBIYHT-UHFFFAOYSA-N
Target: Vasopressin V1a receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P37288
Assay: Vasopressin V1A Receptor Antagonist Assay: The purpose of this assay was to determine the inhibitory effect of synthesized compounds on the Vasopressin V1a receptor. The assay as performed in Chinese Hamster Ovary (CHO) cells expressing the human Arginine Vasopressin Receptor 1a (AVPR1a). Arginine Vasopressin (AVP) evokes an increase in intracellular calcium in CHO-AVPR1a cells which is measured in a fluorescence assay on the FLIPRTETRA using calcium sensitive dyes. Test compounds were assessed for their ability to affect the magnitude of the response to AVP, with antagonists showing a concentration-dependent reduction in the AVP-mediated fluorescence. Compounds were tested in duplicate in a 10-point, 1:3 dilution series starting at a nominal concentration of 3 μM in the assay.CHO-AVPR1a cells were maintained in routine culture in T175 Flasks at 37° C., 5% CO2. The growth medium consists of Ham's F12 media supplemented with 10% v/v fetal bovine serum, 1× non-essential amino acids, and 0.4 mg/ml Geneticin G418.
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 518.02, ALogP 4.99, HBA 8, HBD 0, PSA 100.17, QED 0.48
Compound (SMILES): COc1ccc(C(O)c2cc(-c3ncnc4cc(N5CCOCC5)ccc34)c(F)cc2F)nn1
InChIKey: FWMURCSQJAVESQ-UHFFFAOYSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Patch Clamp Assay: The patch-clamp measurement was carried out at room temperature in whole-cell configuration on human embryonic kidney cells (HEK293) which have been transfected in a stable manner with the hERG gene.The whole-cell configurations were carried out using an automated patch clamp device (Patchliner, Nanion Technologies, Munich). This is a glass chip-based system with which automated whole-cell measurements on up to 8 cells simultaneously are possible. The glass chip has a hole of defined size to which the cell is transferred into the Gigaseal by application of a reduced pressure and brought into the whole-cell configuration. Buffer, cell suspension and test substances were added to microchannels of the chip using a Teflon-coated pipette. The cells were clamped to a holding potential of -80 mV. For measurement of substance-promoted inhibition of the Kv11.1 channel, the following voltage protocol was applied at 10-second intervals: 51 ms/-80 mV, 500 ms/+40 mV, 500 ms/-40 mV, 200 ms/-80 mV. The leakage current is subtracted by means of the P4 method. The cells were resuspended in extracellular buffer (EC) and applied to the chip. After the cell had been collected, the seal was improved by addition of a seal enhancer buffer. As soon as the whole-cell configuration had been reached, the seal enhancer buffer was washed out and replaced by extracellular buffer. The measurement started in EC for 1.5 min. DMSO (vehicle control, 0.1% of DMSO) was then applied, and the control current was recorded for 3 min. The test substance was subsequently added twice in the same concentration, and the potassium current was measured for 3.5 min in each case.
Measurement: Ki > 25000 nM (pChEMBL 4.6020002365112305)
Descriptors: MW 465.46, ALogP 3.29, HBA 8, HBD 1, PSA 93.49, QED 0.48
Compound (SMILES): O=C(O)CC(NC(=O)CNC(=O)c1cc(O)cc(NC2=NCC(O)CN2)c1)c1cc(Br)cc(Br)c1O
InChIKey: NFGCCSJOLREWKI-UHFFFAOYSA-N
Target: Integrin alpha-V/beta-3 (PROTEIN COMPLEX), Homo sapiens
UniProt: P06756
Assay: Solid Phase Receptor Assay (SPRA) for alphavbeta3 Function: Recombinant human vitronectin (R& D Systems, 2308-VN) diluted to 1 μg/mL in TBS+ buffer (25 mM Tris pH 7.4, 137 mM NaCl, 2.7 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2) was added to wells (50 μL/well) of a 96-well half-well transparent microtiter plate (Greiner 675061) and incubated overnight at 4° C. Wells were washed 3 times with 150 μL TBS+ and 150 μL of blocking buffer (TBS+ with 1% bovine serum albumin, Sigma A7906) were added. The plate was incubated for 1 hr at 37° C. and then washed 3× with TBS+ buffer. Recombinant human integrin αvβ3 (R&D Systems, 3050-AV) was diluted to 1 μg/mL in TBS+/0.1% bovine serum albumin. Compounds were diluted 1:100 into the integrin solution and then 50 μL added to empty wells of the washed vitronectin-coated plate according to a standard template with each sample repeated in triplicate. After incubation for two hours at room temperature, the plate was washed 3× with 150 μL of TBS+ buffer. To each well, 50 μL of biotinylated anti-αv antibody (R&D Systems, BAF1219) at 0.5 μg/mL in TBS+/0.1% BSA were added and the plate covered and incubated for 1 hr at room temperature. After washing the plate 3× with 150 μL of TBS+ buffer, 50 μL of streptavidin-conjugated horseradish peroxidase (R&D Systems, DY998) diluted in TBS+ blocking buffer were added to the wells and the plate incubated for 20 min at room temperature. The plate was washed 3× with TBS+ buffer followed by 50 μL of TMB substrate (Sigma, T4444) added to each well and the plate was incubated for 20 min at room temperature. Plates were read by colorimetric detection at 650 nm wavelength using a Tecan Safire II plate reader. Concentration-response curves were constructed by non-linear regression (best fit) analysis, and IC50 values were calculated for each compound.
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 629.26, ALogP 1.42, HBA 9, HBD 8, PSA 192.61, QED 0.21
Compound (SMILES): COc1cc(Nc2nccc(Oc3ccc(NC(=O)Nc4cc(C(C)(C)C)nn4-c4ccc(C)cc4)c4ccccc34)n2)cc(OCCCN2CCOCC2)c1
InChIKey: DMFLXIRKHPTWEU-UHFFFAOYSA-N
Target: Glycogen synthase kinase-3 alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P49840
Assay: Enzyme Inhibition Assay: 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).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.
Measurement: IC50 = 7258 nM (pChEMBL 5.139999866485596)
Descriptors: MW 756.91, ALogP 8.71, HBA 11, HBD 3, PSA 136.92, QED 0.1
Compound (SMILES): CN(C)C(=O)N[C@@H]1CCCN(c2cnc(C(N)=O)c(Nc3ccc(C4CCCCC4)cc3)n2)C1
InChIKey: PCNGXJBDBMUJQG-HXUWFJFHSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
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 465.6, ALogP 3.61, HBA 6, HBD 3, PSA 116.48, QED 0.6
Compound (SMILES): C=CC(=O)N1C[C@H](C)N(C(=O)N(C)[C@H](C(=O)N[C@H]2Cc3nc(cs3)-c3ccc4c(c3)c(c(-c3cccnc3[C@H](C)OC)n4CC)CC(C)(C)COC(=O)[C@@H]3CCCN(N3)C2=O)C(C)C)[C@@H](C)C1
InChIKey: ZNXTVNQRHCWVHW-SWDFTDOLSA-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 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 938.21, ALogP 6.39, HBA 12, HBD 2, PSA 171.54, QED 0.14
Compound (SMILES): COc1cc(Nc2cc(Oc3ccc(NC(=O)Nc4cc(C(C)(C)C)nn4-c4ccc(C)cc4)c4ccccc34)ccn2)cc(C(=O)NCCN2CCOCC2)c1
InChIKey: XXSPPIWQGKTKEQ-UHFFFAOYSA-N
Target: Glycogen synthase kinase-3 alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P49840
Assay: GSK 3alpha Enzyme Inhibition 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). 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 = 2860 nM (pChEMBL 5.539999961853027)
Descriptors: MW 768.92, ALogP 8.28, HBA 10, HBD 4, PSA 143.9, QED 0.1
Compound (SMILES): CNC[C@H](c1ccc2ccccc2c1)[C@H](OCc1cc(C)on1)c1ccccc1
InChIKey: BIAPMBIBJRGUDT-JWQCQUIFSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Activity Assay: HERG: The pre- and post-compound hERG current was evoked by a single voltage pulse consisting of a 20 s period holding at −70 mV, a 160 ms step to −60 mV (to obtain an estimate of leak), a 100 ms step back to −70 mV, a 1 s step to +40 mV, a 2 s step to −30 mV, and finally a 500 ms step to −70 mV. In between the pre- and post-compound voltage pulses, there was no clamping of the membrane potential. Currents were leak-subtracted based on the estimate of current evoked during the +10 mV step at the start of the voltage pulse protocol. The current signal was sampled at 2.5 k Hz. For each compound, the IC50 value was determined.
Measurement: IC50 = 4600 nM (pChEMBL 5.340000152587891)
Descriptors: MW 386.5, ALogP 5.4, HBA 4, HBD 1, PSA 47.29, QED 0.44
Compound (SMILES): Clc1ccc(CSc2nnc(CCc3c[nH]c4ccccc34)n2CCCc2c[nH]cn2)cc1Cl
InChIKey: RXNXMWQDQOQTDF-UHFFFAOYSA-N
Target: Somatostatin receptor type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P32745
Assay: Activity Assays: Measurement of forskolin stimulated inhibition of cAMP was performed via time-resolved fluorescence resonance energy transfer (TR-FRET) LANCE assay (AD0262, PerkinElmer Life Science, Inc., Boston Mass.). Recombinant Chinese hamster ovary (CHO-K1) cells expressing human somatostatin SSTR4 cells (ES-524-CF, PerkinElmer Life Science, Inc., Boston Mass.) were thawed (37° C.), resuspended in 10 mL Hanks' balanced salt solution no phenol red (HBSS, Invitrogen, Carlsbad Calif.), and then centrifuged (150×g, 5 minutes). Cellular pellet resuspended in stimulation buffer containing HBSS 1×, HEPES 5 mM, Protease free BSA 0.1% (PerkinElmer), and 3-Isobutyl-1-methylxanthine 0.5 mM (pH 7.4) and seeded in 96-well plates at 4000 cells/well. LANCE cAMP assay was performed per manufacture instruction with assessment of respective compound, somatostatin-28 (Sigma-Aldrich Co., St. Louis, Mo.) as control agonist, against 5 μM forskolin, performed in triplicate across concentration curve. Fluorescence signal was measured at 20 hours (excitation 340 nm and emission 665 nm, 400-μs delay) via TR-FRET (FLUOSTAR Omega-F, BMG Labtech, Inc., Cary, N.C.). Data was calculated via GraphPad PRISM-5 software.
Measurement: EC50 = 35 nM (pChEMBL 7.460000038146973)
Descriptors: MW 511.48, ALogP 6.5, HBA 5, HBD 2, PSA 75.18, QED 0.21
Compound (SMILES): N#CC[C@H]1CC[C@H](n2c(CNS(=O)(=O)c3ccc(C#N)cc3)nc3cnc4[nH]ccc4c32)CC1
InChIKey: BFWTVBLDMYXCPC-SAABIXHNSA-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 = 26.9 nM (pChEMBL 7.570000171661377)
Descriptors: MW 473.56, ALogP 3.91, HBA 7, HBD 2, PSA 140.25, QED 0.43
Compound (SMILES): CCN1CCC(COc2ccc(Nc3ncc(F)c(-c4cc(F)c5nc6n(c5c4)C(C)(C)CC6)n3)nc2)CC1
InChIKey: UVVGSZJKQAELIC-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00535
Assay: CDK5 Inhibition Assay: Test compounds (compound of the present invention, reference compound Abemaciclib, and comparative example compounds) were dissolved in DMSO at 10 mM. 45 μL of the test compound solution was transferred into a 384-well compound source plate (LABCYTE cat #P-05525) and serially diluted at 1:3 ratio to create a 12-point dilutions. The same volume of DMSO was adopted as high control. 20 nL of the test compound solution in DMSO were dispensed into a new 384-well assay plate by Echo 550. CDK5 protein (0.08 nM, CARNA BIOSCIENCE, cat #04-106), florescent labeled substrate FLPeptide29 (2 μM, PerkinElmer, cat #760429) was prepared in kinase assay buffer (100 mM HEPES (pH 7.5), 10 mM MgCl2, 0.05% Brij-35, 0.5 mM DTT and 0.1 mg/ml BSA). 15 μL of kinase assay buffer containing CDK5 protein and substrate was transferred to assay plate and incubate at RT for 30 minutes. Kinase assay buffer supplemented with substrate peptides was employed as low control to monitor the background. 40 M ATP was prepared in kinase assay buffer containing and 5 μL of ATP solution was added to each well to start the reaction. The assay plate was incubated at 25° C. for 90 minutes and the reaction was stopped by adding 40 μL of 0.5 M EDTA.
Measurement: IC50 = 705 nM (pChEMBL 6.150000095367432)
Descriptors: MW 533.63, ALogP 5.7, HBA 8, HBD 1, PSA 80.99, QED 0.33