lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cn1ncc2cc(C(N)=O)c(O[C@H]3CC4(C[C@H](NC(=O)c5nnc(-c6ccc(C(F)(F)F)cc6)s5)C4)C3)nc21
InChIKey: CLSCWYTXNGHVMH-PGUYCLEYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH) (SEQ ID NO. 1). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity. Compounds were tested in dose-response format, and the inhibition of kinase activity was calculated at each concentration of compound. The inhibition data were fit using a curve-fitting program to determine the IC50; i.e., the concentration of compound required to inhibit 50% of kinase activity.
Measurement: IC50 = 14.5 nM (pChEMBL 7.840000152587891)
Descriptors: MW 557.56, ALogP 3.72, HBA 9, HBD 2, PSA 137.91, QED 0.37
Compound (SMILES): CN(CCc1ccccc1)C(=O)NC[C@H](Cc1ccc(O)cc1)N(C)C
InChIKey: TWGBZXRWCWPPRH-IBGZPJMESA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41145
Assay: Radioligand Binding Assay: Each compound was initially tested at 20 μM and was incubated with 3H-DPN at a concentration equal to the Kd (0.4 nM) of the radioligand in μOR containing Sf9 insect cell membranes. The reaction contained 40 fmol of μOR and was incubated in a buffer of 20 mM HEPES pH 7.5, 100 mM sodium chloride, and 0.1% bovine serum albumin for 1 hour at 25° C. To separate free from bound radioligand, reactions were rapidly filtered over Whatman GF/B filters with the aid of a Brandel harvester and 3H-DPN counts were measured by liquid scintillation. Compounds with more than 25% of 3H-DPN radioactivity were further tested in full dose-response to determine the affinity (Ki) in HEK293 membranes. Subsequently, the 15 analogs were tested in full dose-response for affinity at the μOR and the κOR by the National Institutes of Mental Health Psychoactive Drug Screen Program (PDSP), as were the affinities of compounds 12, PZM21, and their stereoisomers at the μOR, δOR, κOR and nociception receptor.
Measurement: Ki = 160 nM (pChEMBL 6.800000190734863)
Descriptors: MW 355.48, ALogP 2.75, HBA 3, HBD 2, PSA 55.81, QED 0.77
Compound (SMILES): COc1nc2ccc([C@](O)(c3ccc(C)nc3C)c3cnnn3C)cc2c(Cl)c1CN1CCC(F)(F)CC1
InChIKey: LIVLLUXPXNXFRM-MHZLTWQESA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: RORgammat ThermoFluor Assay: For the RORγt construct used in the ThermoFluor® assay, numbering for the nucleotide sequences was based on the reference sequence for human RORγt, transcript variant 2, NCBI Accession: NM_001001523.1 (SEQ ID NO:1). Nucleotides 850-1635 (SEQ ID NO:2) coding for the wild type human RORγt ligand binding domain (RORγt LBD) were cloned into the pHIS1 vector, a modified pET E. coli expression vector (Accelagen, San Diego), containing an in-frame N-terminal His-tag and a TurboTEV protease cleavage site (ENLYFQG, SEQ ID NO:3) upstream of the cloned insert sequence. The amino acid sequence for the RORγt construct used in the Thermofluor® assay is shown as SEQ ID NO:4.ThermoFluor® experiments were carried out using instruments owned by Janssen Research and Discovery, L.L.C. through its acquisition of 3-Dimensional Pharmaceuticals, Inc. 1,8-ANS (Invitrogen) was used as a fluorescent dye. Protein and compound solutions are dispensed into black 384-well polypropylene PCR microplates (Abgene) and overlayed with silicone oil (1 μL, Fluka, type DC 200) to prevent evaporation.Bar-coded assay plates are robotically loaded onto a thermostatically controlled PCR-type thermal block and then heated at a typical ramp-rate of 1° C./min for all experiments. Fluorescence was measured by continuous illumination with UV light (Hamamatsu LC6) supplied via fiber optic and filtered through a band-pass filter (380-400 nm; >6 OD cutoff). Fluorescence emission of the entire 384-well plate was detected by measuring light intensity using a CCD camera (Sensys, Roper Scientific) filtered to detect 500±25 nm, resulting in simultaneous and independent readings of all 384 wells. Images were collected at each temperature, and the sum of the pixel intensity in a given area of the assay plate was recorded versus temperature. Reference wells contained RORγt without compounds, and the assay conditions were as follows:0.065 mg/mL RORγt60 M 1,8-ANS100 mM Hepes, pH 7.010 mM NaCl2.5 mM GSH0.002% Tween-20Project compounds were arranged in a pre-dosed mother plate (Greiner Bio-one) wherein compounds are serially diluted in 100% DMSO by 1:2 from a high concentration of 10 mM over 12 columns within a series (column 12 is a reference well containing DMSO, no compound). The compounds were robotically dispensed directly into assay plates (1x=46 nL) using a Hummingbird capillary liquid handling instrument (Digilab). Following compound dispense, protein and dye in buffer was added to achieve the final assay volume of 3 μL, followed by 1 μL of silicone oil.
Measurement: IC50 = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 543.02, ALogP 4.55, HBA 8, HBD 1, PSA 89.19, QED 0.38
Compound (SMILES): Cc1cc(S(=O)(=O)Nc2cscn2)c(F)cc1N(C)C1CCN([C@H](C)c2ccccc2)CC1
InChIKey: SUUGGLCRMKIDCF-GOSISDBHSA-N
Target: Sodium channel protein type 1 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P35498
Assay: Sodium Influx Assay (In Vitro Assay): This sodium influx assay employs the use of the cell permeable, sodium sensitive dye ANG2 to quantify sodium ion influx through sodium channels which are maintained in an open state by use of sodium channel modulators. This high throughput sodium influx assay allows for rapid profiling and characterization of sodium channel blockers.In general, Trex HEK293 cells were stably transfected with an inducible expression vector containing the full-length cDNA coding for the desired human sodium channel α-subunit and with an expression vector containing full length cDNA coding for the β1-subunit. Sodium channel expressing cell lines were induced with tetracycline (1 μg/mL) and plated on 384-well PDL-coated plates at a density of 25K-30K cells/well in culture media (DMEM, containing 10% FBS and 1% L-glutamine). After overnight incubation (37° C., 5% CO2), culture media was removed and cells were loaded with 5 uM ANG2 dye for 1-1.5 h in Buffer 1 (155 mM NMDG, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, adjusted with Tris to pH 7.4). Access dye was removed and cells were incubated with test compounds for 1 hr in buffer 1 containing sodium channel modulator(s) at room temperature. Hamamatsu FDSS ρCell was used to perform a 1:1 addition of Na/K challenge buffer (140 mM NaCl, 20 mM HEPES, 1 mM CaCl2, 15 mM KCl, 1 mM MgCl2, 10 mM glucose, adjusted with Tris to pH 7.4) and simultaneously read plates at excitation wavelength of 530 nm and emission wavelength set at 558 nm. Percent inhibition of sodium ion influx was calculated for each test compound at each test concentration to determine the IC50 values.
Measurement: IC50 = 14584 nM (pChEMBL 4.840000152587891)
Descriptors: MW 488.65, ALogP 5.05, HBA 6, HBD 1, PSA 65.54, QED 0.5
Compound (SMILES): COc1cc(-c2cc3c(N4CCC(c5nc(C6CC6)c(-c6ccccc6)[nH]5)CC4)ncnc3[nH]2)ccn1
InChIKey: PQXYHRGRKOHJTG-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Biochemical Assay: The c-kit mutant D816V (or kinase domain thereof) is an active kinase in AlphaScreen. IC50 values are determined with respect to inhibition of c-Kit mutant D816V kinase activity, where inhibition of phosphorylation of a peptide substrate is measured as a function of compound concentration. Compounds to be tested were dissolved in DMSO to a concentration of 30 mM. These were diluted 30 ml into 130 ml of DMSO (4 mM) and 1 μl was added to an assay plate. These were then serially diluted 1:3 (50 μl to 100 ml DMSO) for a total of 8 points. Plates were prepared such that each kinase reaction is 30 ml in 1× kinase buffer (25 mM HEPES, pH 7.2, 8 mM MgCl2, 2 mM MnCl2, 50 mM NaCl, 0.01% Brij, 1 mM DTT, 0.01% BSA), 5% DMSO and 10 μM ATP. Substrate was 30 nM biotin-(E4Y)10 (EMD Millipore, Cat#12-440). C-kit mutant D816V kinase was at 0.75 ng per sample. After incubation of the kinase reaction for 30 minutes at room temperature, 5 μl of donor beads (Streptavidin coated beads (Perkin Elmer Life Science) final concentration 7.5 μg/ml) in stop buffer (25 mM Hepes pH 7.5, 100 mM EDTA, 0.01% BSA) was added, the sample was mixed and incubated for 30 minutes at room temperature before adding 5 ml of acceptor beads (PY30 coated beads (Perkin Elmer Life Science) final concentration 7.5 μg/ml) in stop buffer.
Measurement: Ki = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 491.6, ALogP 5.68, HBA 6, HBD 2, PSA 95.61, QED 0.32
Compound (SMILES): CCN1CC2(CCN(Cc3cccnc3)CC2)OC2(CC2)C1=O
InChIKey: HSRCEFZNEYPBBP-UHFFFAOYSA-N
Target: Sigma non-opioid intracellular receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99720
Assay: Radioligand Assay: To investigate binding properties of test compounds to human σ1 receptor, transfected HEK-293 membranes and [3H](+) -pentazocine (Perkin Elmer, NET-1056), as the radioligand, were used. The assay was carried out with 7 μg of membrane suspension, 5 nM of [3H](+)-pentazocine in either absence or presence of either buffer or 10 μM Haloperidol for total and non-specific binding, respectively. Binding buffer contained Tris-HCl 50 mM at pH 8. Plates were incubated at 37° C. for 120 minutes. After the incubation period, the reaction mix was then transferred to Multiscreen HTS, FC plates (Millipore), filtered and plates were washed 3 times with ice-cold 10 mM Tris-HCL (pH 7.4). Filters were dried and counted at approximately 40% efficiency in a MicroBeta scintillation counter (Perkin-Elmer) using EcoScint liquid scintillation cocktail Results: As this invention is aimed at providing a compound or a chemically related series of compounds which act as ligands of the σ1 receptor it is a very preferred embodiment in which the compounds are selected which act as ligands of the σ1 receptor and especially compounds which have a binding expressed as Ki which is preferably <1000 nM, more preferably <500 nM, even more preferably <100 nM.
Measurement: Ki < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 315.42, ALogP 1.83, HBA 4, HBD 0, PSA 45.67, QED 0.85
Compound (SMILES): CN1C(=O)[C@@H](NC(=O)c2nn(Cc3ncccn3)cc2F)COc2ccccc21
InChIKey: VMCCHFDTVZAUCV-ZDUSSCGKSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: Fluorescent Polarization Binding (FP Binding) assay: Assay was performed in polystyrene low volume 384-well black plate, at Room Temperature (RT) in a final volume of 10.1 μl/well using 10 nM of GST-hRIPK1 (8-327) enzyme and 5 nM of fluorescent labeled ligand (14-(2-{[3-({2-{[4-(cyanomethyl)phenyl]amino}-6-[(5-cyclopropyl-1H-pyrazol-3-yl)amino]-4-pyrimidinyl}amino) propyl]amino}-2-oxoethyl)-16,16,18,18-tetramethyl-6,7,7a,8a,9,10,16,18-octahydrobenzo[2″,3″]indolizino[8″,7″:5′,6]pyrano[3′,2′:3,4]pyrido[1,2-a]indol-5-ium-2-sulfonate.Test Compounds were serially diluted in DMSO at 100 fold final concentrations in the assay (1% DMSO final). In each well of a 384-well Plate were dispensed 0.1 μL of compound solution (or DMSO for controls) followed by 5 μL of GST-hRIPK1 (8-327) at twice the final concentrations in assay buffer (50 mM HEPES pH 7.5, 10 mM NaCl, 50 mM MgCl2, 0.02% CHAPS, 0.5 mM DTT and 0.01% Pluronic F127). For negative control the enzyme addition was replaced by assay buffer only.
Measurement: IC50 = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 396.38, ALogP 1.01, HBA 7, HBD 1, PSA 102.24, QED 0.71
Compound (SMILES): Oc1ccc2c(c1)CCCC(c1cnc3[nH]ccc3c1)=C2c1ccc(O[C@H]2CCN(CCCF)C2)cc1
InChIKey: OZEYEMPNKONANE-MHZLTWQESA-N
Target: Estrogen receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P03372
Assay: Estrogen Receptor Coactivator Assay: It is a competition assay, where binding of a test compound to a complex comprised of (i) His6-ERα298-554 protein representing ERα ligand-binding domain, (ii) Tb-labeled His6 antibody, (iii) a fluorescein-labeled PGC1α coactivator peptide (EAEEPSLLKKLLLAPANTQ), and (iv) estradiol, results in a decrease of the TR-FRET signal due to dissociation of the coactivator peptide. His6-ERα298-554 proteins were expressed as WT or D538G or Y537S mutants in E. coli and purified by affinity chromatography. The assay works in a homogeneous mix-and-read format. In a typical experiment, a 4 μL mixture of 0.5 nM His6-ERα298-554, 0.5 nM Tb-labeled His6 antibody, 250 nM PGC1a peptide, and 3 nM estradiol in 100 mM potassium phosphate, pH 7.4, 0.01% Tween-20, 0.02% NaN3, 5 mM DTT, was added to 40 nL test compound in DMSO and incubated overnight at room temperature. The TR-FRET 520:495 nm emission ratio was calculated and used to determine the IC50 value from a dose response curve fit to the 4-parameter logistic equation. The antagonist activity with respect to estrogen receptors in this test is given by the concentration which inhibits 50% of the estrogen receptor activity (or IC50) in nM.
Measurement: IC50 = 45 nM (pChEMBL 7.349999904632568)
Descriptors: MW 497.61, ALogP 6.38, HBA 4, HBD 2, PSA 61.38, QED 0.31
Compound (SMILES): N#Cc1ccc(N2CCN(Cc3ccc4c5c(c(=O)[nH]c4c3)CCCN5)CC2)nn1
InChIKey: XUFWBGKENCGHSD-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TNK1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13470
Assay: Inhibition Assay: The tankyrase-1 or tankyrase-2 enzyme inhibitory activities of the compounds of the present invention were assayed in the following manner by use of a kit (cat. 80573 or 80578) purchased from BPS Bioscience.The 96-well plate provided in BPS Bioscience kit was coated with histone and incubated at 4° C. for 16 hours. Then, the plate was washed four times with PBST (7.5 mM Na2HPO4, 2.5 mM NaH2PO4, 145 mM NaCl, 0.05% Tween 20, pH 7.4), and blocking buffer (provided in BPS Bioscience kit) was added thereto in order to block nonspecific reaction, and was then incubated at 25° C. for 1 hour. After incubation for 1 hour, the plate was washed four times with PBST, and varying concentrations of each of the compounds of the Examples were added to a reaction solution containing tankyrase-1 enzyme (40 ng/well) or tankyrase-2 enzyme (15 ng/well) and an assay mixture, and allowed to react at 25° C. for 1 hour. After 1 hour, each well was washed four times with PBST, and in order to measure the level of ribosylation by PARP enzyme, streptavidin-linked peroxidase (Strep-HRP, 1:50 dilution) was added and allowed to react at 25° C. for 30 minutes. The plate was washed four times with PBST, and then finally an HRP chemiluminescent substrate was added and allowed to react. The level of histone ribosylation formed by each enzyme was quantified using Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTek Instruments, Inc., USA).
Measurement: IC50 = 2.77 nM (pChEMBL 8.5600004196167)
Descriptors: MW 401.47, ALogP 1.87, HBA 7, HBD 2, PSA 100.94, QED 0.69
Compound (SMILES): OC1CCc2cncc(-c3ccc(C(F)(F)F)c(F)c3)c21
InChIKey: SXPIIQUKRKSQRQ-UHFFFAOYSA-N
Target: Cytochrome P450 11B2, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P19099
Assay: Enzymatic Activity Assay: The expression plasmids contains the ORF for either human/cyno CYP11B1 or CYP11B2 under the control of a suitable promoter (CMV-promoter) and a suitable resistance marker (neomycin). Using standard techniques the expression plasmid is transfected into G-402 cells and these cells are then selected for expressing the given resistance markers. Individual cell-clones are then selected and assessed for displaying the desired enzymatic activity using 11-Deoxycorticosterone (Cyp11B2) or 11-Deoxycortisol (Cyp11B1) as a substrate.G-402 cells expressing CYP11 constructs were established as described above and maintained in McCoy's 5a Medium Modified, ATCC Catalog No. 30-2007 containing 10% FCS and 400 μg/ml G418 (Geneticin) at 37° C. under an atmosphere of 5% CO2/95% air. Cellular enzyme assays were performed in DMEM/F12 medium containing 2.5% charcoal treated FCS and appropriate concentration of substrate (0.3-10 uM 11-Deoxycorticosterone, 11-Deoxycortisol or Corticosterone). For assaying enzymatic activity, cells were plated onto 96 well plates and incubated for 16 h. An aliquot of the supernatant is then transferred and analyzed for the concentration of the expected product (Aldosterone for CYP11B2; Cortisol for CYP11B1). The concentrations of these steroids can be determined using HTRF assays from CisBio analyzing either Aldosterone or Cortisol.
Measurement: EC50 = 69 nM (pChEMBL 7.159999847412109)
Descriptors: MW 297.25, ALogP 3.89, HBA 2, HBD 1, PSA 33.12, QED 0.81
Compound (SMILES): COc1c(F)cccc1NC1CCN(c2ccc(-c3cn[nH]c3)c(OC(F)F)c2)C1=O
InChIKey: HMMYMYFVOCXDIC-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro Assays: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH (SEQ ID NO: 1)). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity. Compounds were tested in dose-response format, and the inhibition of kinase activity was calculated at each concentration of compound. The inhibition data were fit using a curve-fitting program to determine the IC50; i.e., the concentration of compound required to inhibit 50% of kinase activity.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 432.4, ALogP 4.04, HBA 5, HBD 2, PSA 79.48, QED 0.59
Compound (SMILES): Cc1cc(Nc2ccc(F)c(C[C@@]3(C(=O)O)CCN(Cc4cc(Cl)c(F)c(Cl)c4)[C@H](C)C3)n2)n[nH]1
InChIKey: HOGWQTOSZMFOAJ-JBEBIEQOSA-N
Target: Aurora kinase B (SINGLE PROTEIN), Homo sapiens
UniProt: Q96GD4
Assay: Evaluation of Aurora a and Aurora B Inhibitory EffectKinase Assay: Experimental Methods:(1) Prepare a 1× kinase buffer [50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), PH 7.5, 0.01% Brij-35, 10 mM MgCl2, 2 mM dithiothreitol (DTT)].(2) Prepare compound:The testing compound was dissolved with DMSO. The testing compound was diluted up to 100× final concentration in a 384-well plate. Transfer 250 nL of the compound dilution to a 384-well assay plate using Echo 550. 100% DMSO of 250 nL was added to the negative control well and the positive control well.(3) Prepare a 2.5× enzyme solution using the above 1× kinase buffer.(4) Add 10 μL of the 2.5× enzyme solution to the compound well and the positive control well of the 384-well assay plate. Add 10 μL of the 1× kinase buffer to the negative control well.(5) The 384-well plate was centrifuged at 1000 rpm for 30 seconds and incubated at room temperature for 10 min.(6) A mixture of ATP and Kinase substrate 21 with a final concentration of 25/15× was prepared by the 1× kinase buffer.(7) Add 15 μL of the mixture of 25/15×ATP and kinase substrate solution 21 to start reaction.(8) The 384-well plate was centrifuged at 1000 rpm for 30 seconds and incubated at room temperature.(9) 30 μL of the stop and detection buffer was added to stop the kinase reaction and centrifuged at 1000 rpm for 30 seconds.(10) Collect data on Caliper EZ ReaderII.Data Analysis:
Measurement: IC50 = 557 nM (pChEMBL 6.25)
Descriptors: MW 524.4, ALogP 5.74, HBA 5, HBD 3, PSA 94.14, QED 0.34
Compound (SMILES): O=C(c1ccccc1Cl)N1CCN(Cc2c(-c3ccc(Cl)cc3)nc3ccccn23)CC1
InChIKey: WBKYNZJRUXTLGA-UHFFFAOYSA-N
Target: Potassium channel subfamily K member 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NPC2
Assay: Inhibition In Vito Assay: The investigations on the inhibition of the recombinant TASK-1 and TASK-3 channels were carried out using stably transfected CHO cells. Here, the compounds according to the invention were tested with administration of 40 mM of potassium chloride in the presence of a voltage-sensitive dye for high-throughput screening of potassium channel from Molecular Devices FLIPR Application Note: Measuring membrane potential using the FLIPR membrane potential assay kit on Fluorometric Imaging Plate Reader (FLIPR) systems. The activity of the test substances was determined as their ability to inhibit a depolarization induced in the recombinant cells by 40 mM potassium chloride.
Measurement: IC50 = 60 nM (pChEMBL 7.21999979019165)
Descriptors: MW 465.38, ALogP 5.27, HBA 4, HBD 0, PSA 40.85, QED 0.41
Compound (SMILES): O=C(NC1CC1)c1nc2cccnn2c1-c1ccc(Cl)c(F)c1
InChIKey: VPDYRGHPOFWFHI-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4D (SINGLE PROTEIN), Homo sapiens
UniProt: Q08499
Assay: The PDE4A3, PDE4B1, PDE4C1 and PDE4D3 assays: 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 = 6.84 nM (pChEMBL 8.15999984741211)
Descriptors: MW 330.75, ALogP 3.08, HBA 4, HBD 1, PSA 59.29, QED 0.8
Compound (SMILES): CCn1c(-c2cccnc2C(C)OC)c2c3cc(ccc31)-c1cccc(c1)C[C@H](NC(=O)[C@@H](COC1CN(C(=O)C#C[C@@H]3OCCCN3C)C1)C(C)C)C(=O)N1CCC[C@H](N1)C(=O)OCC(C)(C)C2
InChIKey: XBJBUJASAIYCKU-XTGOPNKVSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
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 944.19, ALogP 5.93, HBA 12, HBD 2, PSA 156.8, QED 0.14
Compound (SMILES): OCCc1ccc(Nc2nc(-c3cccc(Cl)c3)nc3c2SCC3)cc1
InChIKey: FYFIVVYQYPPOTE-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4D (SINGLE PROTEIN), Homo sapiens
UniProt: Q08499
Assay: Kinetic Assay: Assays are performed in 96-well plates in a total volume of 200 μl/well. Compounds are dissolved in dimethylsulfoxide (DMSO) and added to plates in a volume of 10 μl followed by addition of 165 μl of assay mix. Plates are pre-incubated at 25° C. for 15 min and the reactions are initiated by the addition of 25 μl of cAMP followed by thorough mixing. Reaction rates are measured by monitoring the decrease in fluorescence using excitation at 355 nm and emission at 460 nm for a period of 10 min in a fluorescence plate reader. Initial rates (slopes) are determined from linear portions of the progress curves. Final concentrations of assay components are as follows: 50 mM Tris, pH 8, 10 mM MgCl2, 50 mM KCl, 2% DMSO, 5 mM tris(2-carboxyethyl)phosphine (TCEP), 0.4 mM phosphenolpyruvate (PEP), 0.01 mM NADH, 0.04 mM adenosine triphosphate (ATP), 0.004 mM cAMP, 7.5 units myokinase from yeast, 1.6 units pyruvate kinase, 2 units lactate dehydrogenase, and either 0.5 nM human PDE4D7 or 10 nM human PDE4B1. All data are percent normalized relative to controls and are presented as percent inhibition. An inhibitory concentration 50% (IC50) value is calculated by fitting of a sigmoidal dose response curve. Human PDE4D7 contained a mutation of serine 54 to aspartic acid to mimic activation by cAMP-dependent protein kinase A (PKA). Human PDE4B1 contained a corresponding mutation of serine 133 to aspartic acid to also mimic PKA activation.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 383.9, ALogP 4.72, HBA 5, HBD 2, PSA 58.04, QED 0.67
Compound (SMILES): O=C(Nc1ccc2ncc(-c3cc4ccccc4o3)n2n1)C(O)c1ccccc1
InChIKey: QXPFEBPNDCKVSY-UHFFFAOYSA-N
Target: MAP kinase-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9BUB5
Assay: Kinase High ATP Assay: For the assay 50 nL of a 100 fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384 well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2 μL of a solution of MKNK1 in aqueous assay buffer [50 mM HEPES pH 7.5, 5 mM magnesium chloride, 1.0 mM dithiothreitol, 0.005% (v/v) Nonidet-P40 (Sigma)] was added and the mixture was incubated for 15 min at 22° C. to allow pre-binding of the test compounds to the enzyme before the start of the kinase reaction. Then the kinase reaction was started by the addition of 3 μL of a solution of adenosine-tri-phosphate (ATP, 3.3 mM=>final conc. in the 5 μL assay volume is 2 mM) and substrate (0.1 μM=>final conc. in the 5 μL assay volume is 0.06 μM) in assay buffer and the resulting mixture was incubated for a reaction time of 30 min at 22° C. The concentration of MKNK1 was adjusted depending of the activity of the enzyme lot and was chosen appropriate to have the assay in the linear range, typical concentrations were in the range of 0.003 μg/mL. The reaction was stopped by the addition of 5 μL of a solution of TR-FRET detection reagents (5 nM streptavidine-XL665 [Cisbio Bioassays, Codolet, France] and 1 nM anti-ribosomal protein S6 (pSer236)-antibody from Invitrogen [#44921G] and 1 nM LANCE EU-W1024 labeled ProteinG [Perkin-Elmer, product no. AD0071]) in an aqueous EDTA-solution (100 mM EDTA, 0.1% (w/v) bovine serum albumin in 50 mM HEPES pH 7.5). The resulting mixture was incubated for 1 h at 22° C. to allow the formation of complex between the phosphorylated biotinylated peptide and the detection reagents.
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 384.4, ALogP 3.81, HBA 6, HBD 2, PSA 92.66, QED 0.49
Compound (SMILES): Cc1oc(C[C@H]2CCCCN2C(=O)c2ccc(F)cc2-n2nccn2)nc1C(C)(C)O
InChIKey: KFGRQNJZKWEXFD-MRXNPFEDSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P56718
Assay: FLIPR Assay: 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 pM Fluo-4AM ester, 0.02% pluronic acid, and 1% BSA.
Measurement: IC50 = 39 nM (pChEMBL 7.409999847412109)
Descriptors: MW 427.48, ALogP 3.17, HBA 7, HBD 1, PSA 97.28, QED 0.67
Compound (SMILES): Cc1nc(S(=O)(=O)Nc2ccon2)c(F)cc1NCc1ccccc1CN(C)C(C)C
InChIKey: OODKKTOJIQYMDN-UHFFFAOYSA-N
Target: Sodium channel protein type 1 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P35498
Assay: Sodium Influx Assay (In Vitro Assay): This sodium influx assay employs the use of the cell permeable, sodium sensitive dye ANG2 to quantify sodium ion influx through sodium channels which are maintained in an open state by use of sodium channel modulators. This high throughput sodium influx assay allows for rapid profiling and characterization of sodium channel blockers.In general, Trex HEK293 cells were stably transfected with an inducible expression vector containing the full-length cDNA coding for the desired human sodium channel α-subunit and with an expression vector containing full length cDNA coding for the β1-subunit. Sodium channel expressing cell lines were induced with tetracycline (1 μg/mL) and plated on 384-well PDL-coated plates at a density of 25K-30K cells/well in culture media (DMEM, containing 10% FBS and 1% L-glutamine). After overnight incubation (37° C., 5% CO2), culture media was removed and cells were loaded with 5 uM ANG2 dye for 1-1.5h in Buffer 1 (155 mM NMDG, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, adjusted with Tris to pH 7.4). Access dye was removed and cells were incubated with test compounds for 1 hr in buffer 1 containing sodium channel modulator(s) at room temperature. Hamamatsu FDSS μCell was used to perform a 1:1 addition of Na/K challenge buffer (140 mM NaCl, 20 mM HEPES, 1 mM CaC2, 15 mM KCl, 1 mM MgCl2, 10 mM glucose, adjusted with Tris to pH 7.4) and simultaneously read plates at excitation wavelength of 530 nm and emission wavelength set at 558 nm. Percent inhibition of sodium ion influx was calculated for each test compound at each test concentration to determine the IC50 values.
Measurement: IC50 = 30000 nM (pChEMBL 4.519999980926514)
Descriptors: MW 447.54, ALogP 3.77, HBA 7, HBD 2, PSA 100.36, QED 0.51
Compound (SMILES): Cc1ccc(-n2ccc(C)n2)c(C(=O)N2CCC[C@@H](C)[C@H]2CNc2nnc(C(F)(F)F)s2)c1
InChIKey: KOTYWCBPAIQSPY-RDTXWAMCSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Cell-based Functional Assay: Measurement of [Ca2+]i using a FLIPR: CHO-OX1 or CHO-OX2 cells are seeded into black-walled clear-base 384-well plates (Costar) at a density of 20,000 cells per well in F12-K medium supplemented with 10% FBS and selection antibiotic (500 ug/ml G418 or 15 ug/ml Blasticidin) and cultured overnight. The cells are incubated with equal volume of calcium4 loading buffer (Molecular Devices, Inc.) containing 2.5 mM probenecid at 37° C. for 30 min, followed by putative OX1 or OX2 receptor antagonists (dose-range 0.1 nM-10 μM) for another 30 min.
Measurement: IC50 > 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 478.54, ALogP 4.71, HBA 7, HBD 1, PSA 75.94, QED 0.57
Compound (SMILES): C[C@H]1CN(c2ccncc2NC(=O)c2ccc3sc(-c4ccccc4)cc3n2)C[C@@H](N)[C@@H]1O
InChIKey: FOGCGQFPKUSEQD-KPNPRUKOSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
Assay: Enzyme Assay: Pim-2 kinase assay—20 μL reactions were run in white 384 well polystyrene plates dotted with 0.8 μL compound/DMSO in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 5 mM MgCl2, 0.01% BSA, 5 mM DTT), containing 0.05 μM Fluorescein-labeled CREBtide peptide substrate (Invitrogen PV3508), 1 mM ATP, and 1 nM enzyme (Invitrogen PV3649) for 2 h at 25° C. Reactions were stopped by addition of 10 μL TR-FRET Dilution Buffer (Invitrogen PV3574) with 30 mM EDTA and 1.5 nM LanthaScreen Tb-CREB pSer133 antibody (Invitrogen PV3566). After 30 min. incubation at room temperature, plates were read on a PHERAstar FS plate reader (BMG Labtech).
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 459.58, ALogP 3.75, HBA 7, HBD 3, PSA 104.37, QED 0.43
Compound (SMILES): CNCc1cc(OCc2cc(Cl)ccc2F)n(CCC(C)(C)CO)n1
InChIKey: JFNIBJVZOJNQSF-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 2C (SINGLE PROTEIN), Homo sapiens
UniProt: P28335
Assay: Receptor Binding Assay: 5-HT2C: A solution containing 50 μL of [H]-mesulergine (GE Healthcare) diluted with 50 mmol/L Tris-HCl (pH=7.4) (the final concentration: about 2 nM), 149 μL of 5-HT2C/CHO cell membrane sample (20 μg/well for the amount of protein), and 1 μL of a solution of a test compound dissolved in DMSO or a solvent (DMSO) was reacted at 37° C. for 30 minutes, and then filtered promptly by aspiration under lower pressure with a glass fiber filter coated with a 1% aqueous solution of bovine serum albumin. The filtered substance on the glass fiber filter was washed with 250 μL of 50 mmol/L Tris-HCl (pH=7.4) twice, and then transferred to an ACS-II (Amersham) 4-mL-glass vial. The radioactivity of the filtered substance remained on the filter was measured by liquid scintillation counter. The radioactivity value measured by liquid scintillation counter was deemed to be a receptor binding activity, and the binding inhibition was calculated from the total binding (TB), non-specific binding (NSB), and specific binding (SB) of a test compound according to the following equations.
Measurement: Ki = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 369.87, ALogP 3.38, HBA 5, HBD 2, PSA 59.31, QED 0.71
Compound (SMILES): Cc1cc(N2CC(CC#N)(n3cc(-c4ncnc5[nH]ccc45)cn3)C2)ccc1NC(=O)N1CCCCC1
InChIKey: UVNSRQZWEXBKEQ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: In vitro JAK Kinase Assay: The catalytic domains of human JAK1 (a.a. 837-1142), Jak2 (a.a. 828-1132) and Jak3 (a.a. 781-1124) with an N-terminal His tag were expressed using baculovirus in insect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3 was assayed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by homogenous time resolved fluorescence (HTRF). IC50s of compounds were measured for each kinase in the 40 microL reactions that contain the enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%) BSA. For the 1 mM IC50 measurements, ATP concentration in the reactions was 1 mM. Reactions were carried out at room temperature for 1 hr and then stopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, Mass.). Binding to the Europium labeled antibody took place for 40 minutes and HTRF signal was measured on a Fusion plate reader (Perkin Elmer, Boston, Mass.)
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 495.59, ALogP 4.28, HBA 7, HBD 2, PSA 118.76, QED 0.43
Compound (SMILES): CN(Cc1cc2c(nc1C=O)N(C(=O)Nc1cc(N(C)C)c(C#N)cn1)CCC2)C(=O)C1CCCO1
InChIKey: SSJUVDCYSLWUFA-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: Enzymologic Experiment of FGFR1: In this experiment, the inhibitory effect of the compounds on FGFR1 kinase activity was tested by a fluorescence resonance energy transfer (TR-FRET) method, and the half maximal inhibitory concentration (IC50) of the compounds on the FGFR1 kinase activity was determined. 1) 15 μL of FGFR1 enzymatic solution was added to a 384-well plate, and the final concentration of the enzyme was 0.15 nM. 2) 15 μL of diluted solution in gradient of the compound was added.3) 15 μL of a substrate mixture containing substrate polypeptide with a final concentration of 550 nM and ATP with a final concentration of 10200 μM was added. 4) The mixture was incubated for 0.5-3 hours at room temperature. 5) 10 μL of EDTA and a test solution comprising labeled antibody were added, and the plate was incubated for 1 hour at room temperature. 6) The fluorescence signal values of each plate were determined by a microplate reader at 665 nm. 7) The inhibition rates were calculated according to the fluorescence signal values.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 491.55, ALogP 2.35, HBA 8, HBD 1, PSA 131.76, QED 0.61
Compound (SMILES): Cn1cc(-c2c(-c3ccc(F)cc3)nc(N)n3nc(Cc4c(F)cccc4F)nc23)ccc1=O
InChIKey: KVTHXAIJSHWNRJ-UHFFFAOYSA-N
Target: Adenosine receptor A1 (SINGLE PROTEIN), Homo sapiens
UniProt: P30542
Assay: Binding Affinities to Different Adenosine Receptors: The compounds at different concentrations were incubate with hA1 membrane (from PerkinElmer) and [3H]-8-Cyclopentyl-1,3-dipropylxanthine (DPCPX) for 50 min at 25° C., meanwhile 100 μL 0.5% PEI solution was added into UNFILTER-96 GF/B filter plate for 60 min at 4° C., then UNIFILTER-96 GF/B filter plate was washed twice with 50 ml wash buffer, the membrane mix was transferred into UNIFILTER-96 GF/B filter plate, and the filter plate was washed 4 times before incubated at 55° C. for 10 min. At last, 40 L ULTIMA GOLD was added into each well, and CPM was read by TopCount.The compounds at different concentrations were incubate with hA2a membrane (from PerkinElmer) and [3H]-CGS21680 for 90 min at 25° C., meanwhile 100 μL 0.5% PEI solution was added into UNFILTER-96 GF/B filter plate for 60 min at 4° C., then UNIFILTER-96 GF/B filter plate was washed twice with 50 ml wash buffer, the membrane mix was transferred into UNIFILTER-96 GF/B filter plate, and the filter plate was washed 4 times before incubated at 55° C. for 10 min. At last, 40 L ULTIMA GOLD was added into each well, and CPM was read by TopCount.The compounds at different concentrations were incubate with hA2b membrane (from PerkinElmer) and [3H]-DPCPX for 60 min at 27° C., and the binding reactions were stopped by rapid filtration through 0.5% BSA coated UNIFILTER-96 GF/C plates using cell harvester. The filter plates were then washed three times with ice cold wash buffer, and dried at 37° C. for 120 min. At last, 50 L of scintillation cocktail was added into each well, and CPM was read by TopCount.The compounds at different concentrations were incubate with hA3 membrane (from PerkinElmer) and [1211]-AB-MECA for 60 min at 27° C., the binding reactions were stopped by rapid filtration through 0.50% BSA coated UNIFILTER-96 GF/C plates using cell harvester. The filter plates were then washed three times with ice cold wash buffer, and dried at 37° C. for 120 min. At last, 50 L of scintillation cocktail was added into each well, and CPM was read by TopCount.
Measurement: IC50 = 0.2 nM (pChEMBL 9.699999809265137)
Descriptors: MW 462.44, ALogP 3.75, HBA 7, HBD 1, PSA 91.1, QED 0.44
Compound (SMILES): C[C@H](Nc1nc(C(=O)N2CC[C@H](NC(=O)OC(C)(C)C)C2)c2sccc2n1)c1cccnc1
InChIKey: QXNIXSHLXYRDIJ-HOCLYGCPSA-N
Target: Adenosine receptor A2a (SINGLE PROTEIN), Homo sapiens
UniProt: P29274
Assay: Activity Assay: Compounds (Adenosine Receptor modulators) and activity assay.
Measurement: IC50 = 75 nM (pChEMBL 7.119999885559082)
Descriptors: MW 468.58, ALogP 4, HBA 8, HBD 2, PSA 109.34, QED 0.58
Compound (SMILES): N[C@@H](Cc1ccc(-c2csc3c(O[C@H](c4ccc(Cl)cc4C4=CCCOC4)C(F)(F)F)ncnc23)cc1)C(=O)O
InChIKey: VVEXHDYBQYYFMG-WIOPSUGQSA-N
Target: Tryptophan 5-hydroxylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P17752
Assay: Inhibition of Tryptophan Hydroxylase Activity: In order to confirm the inhibitory effect of the compound represented by formula 1 according to the present invention on the tryptophan hydroxylase activity, it was measured using a TPH1 (tryptophan hydroxylase 1) inhibitor screening assay kit (BPS Bioscience, Catalog #72053). The TPH1 inhibitor screening assay kit was used according to the manufacturer's manual. The results are shown in table below.Particularly, the synthesized compound was dissolved in DMSO, which was added to a 96-well microplate (10 Figure US11407763-20220809-P00004/well), to which TPH1 enzyme was added (40 Figure US11407763-20220809-P00004/well). Then, TPH1 reaction solution was added to the microplate (50 Figure US11407763-20220809-P00004/well) and the microplate was shaded with aluminum foil. The microplate was transferred to 4° C. environment, shaken carefully and incubated for 4 hours. After adding a quench solution to the microplate (10 Figure US11407763-20220809-P00004/well), the TPH1 activity was measured by reading the degree of fluorescence color development with a Flexstation3 microplate reader. At this time, the excitation spectrum was 300 nm and the emission spectrum was 360 nm.
Measurement: IC50 = 371 nM (pChEMBL 6.429999828338623)
Descriptors: MW 590.02, ALogP 6.45, HBA 7, HBD 2, PSA 107.56, QED 0.24
Compound (SMILES): COc1ccccc1CCC1CCc2cc([C@H]3CC[C@](N)(COP(=O)(O)O)C3)ccc2C1
InChIKey: CDZQINDPHHFTHS-OYHZEJGOSA-N
Target: Sphingosine-1-phosphate lyase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O95470
Assay: Receptor [35S] GTPgammaS Binding Assays: (S1P1 GTPgammaS/S1P3 GTPgammaS): Compounds were loaded in a 384 Falcon v-bottom plate (0.5 l/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 l/well, final protein 3 μg/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 μM GDP, 0.1% fatty acid free BSA, and 10 μg/ml Saponin to 0.4 nM. 40 μl 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 μl) 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. The EC50 for Example 689 was determined to be 5.7 nM in the assay utilizing membranes prepared from S1P1/CHO cells. The EC50 for Example 689 was determined to be >2000 nM in the assay utilizing membranes prepared from EDG3-Gal5-bla HEK293T cells.A smaller value for GTPγS S1P1 EC50 value indicated greater activity for the compound in the GTPγS S1P1 binding assay. A larger value for the GTPγS S1P3 EC50 value indicated less activity in the GTPγS S1P3 binding assay. Example 689, which is the phosphate ester of Example 672, possessed activity as an agonist of S1P1 and is selective over S1P3. Example 697, which is the phosphate ester of Example 681, possessed activity as an agonist of S1P1 and is selective over S1P3. Thus the compounds of the present invention may be used in treating, preventing, or curing various S1P1 receptor-related conditions while reducing or minimizing the side effects due to S1P3 activity. The selectivity of the compounds of the present invention indicate their potential use in treating, preventing, or curing autoimmune and inflammatory diseases such as multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, lupus, psoriasis, or vascular diseases, while reducing or minimizing possible side effects due to S1P3 activity. Other potential uses of the compounds of the present invention include minimizing or reducing rejection of transplanted organs, while reducing or minimizing side effects due to S1P3 activity.
Measurement: EC50 = 0.6 nM (pChEMBL 9.220000267028809)
Descriptors: MW 459.52, ALogP 4.51, HBA 4, HBD 3, PSA 102.01, QED 0.5
Compound (SMILES): O=C1CN(CCCCOc2ccc(-c3ccn[nH]3)nc2)C(=O)N1
InChIKey: PJECQYXZOKGSSX-UHFFFAOYSA-N
Target: Cytochrome P450 4A11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q02928
Assay: Inhibition Test for Each Compound of the Present Invention Against 20-HETE Producing Enzymes (CYP4F2 and CYP4A11): In the CYP4F2 inhibition test, the reaction solution containing each compound [final concentration of 50 mM, KPO4 (pH 7.4), 2.5 μM luciferine derivative, and 1 mM NADPH] was added to an Escherichia coli membrane fraction (100 μg/mL protein) in which human CYP4F2 had been expressed. In the CYP4A11 inhibition test, the reaction solution containing each compound [final concentration of 100 mM, Tris-HCl (pH 7.5), 60 μM luciferine derivative, 1.3 mM NADP+, 3.3 mM Glucose 6-Phosphate, 3.3 mM MgCl2, and 0.4 U/mL Glucose 6-Phosphate dehydrogenase] was added to an Escherichia coli membrane fraction (100 μg/mL protein) in which human CYP4A11 had been expressed. Following this, the membrane fraction was left to stand at room temperature for 60 minutes to perform an enzymatic reaction. After the reaction, a luciferine detection reagent was added, and the luminescence value was measured using a plate reader. By using that value, the percent inhibition of 20-HETE producing enzyme (%) was calculated according to the equation described below, and the 50% inhibitory concentration (IC50 value) for each compound was calculated.
Measurement: IC50 = 750 nM (pChEMBL 6.119999885559082)
Descriptors: MW 315.33, ALogP 1.18, HBA 5, HBD 2, PSA 100.21, QED 0.59
Compound (SMILES): CCn1nc(C(=O)Nc2ccc(Oc3ccnc4[nH]nc(N[C@H](C)CO)c34)c(F)c2)c(=O)n(-c2ccc(F)c(F)c2)c1=O
InChIKey: UUOZMWBGIJQOQJ-CYBMUJFWSA-N
Target: Tyrosine-protein kinase Mer (SINGLE PROTEIN), Homo sapiens
UniProt: Q12866
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 = 37800 nM (pChEMBL 4.420000076293945)
Descriptors: MW 596.53, ALogP 2.94, HBA 11, HBD 4, PSA 169.05, QED 0.2