lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): C[C@](CN)(Cc1cccnc1)c1ccc2ccccc2c1
InChIKey: ZZLLQFDJKBAKCE-LJQANCHMSA-N
Target: Sodium-dependent dopamine transporter (SINGLE PROTEIN), Homo sapiens
UniProt: Q01959
Assay: Uptake Assay Protocol: DAT: This protocol was designed to measure inhibition of uptake by the human dopamine transporter. The reagents were human DAT (HEK293F) cells, GBR 12909 (Sigma), nomifensine, neurotransmitter transporter uptake assay kit (Molecular Devices), freestyle 293 expression medium (Invitrogen), 10× Hank's Balanced Salt Solution (HBSS; Invitrogen), 1 M HEPES (Mediatech), Biocoat poly-D-lysine 96-well, black, clear plates (Becton, Dickinson), and 500 pt polypropylene U-bottom 96-well plates (Fisher). The Assay Buffer (AB) was 1×HBSS and 0.02 M HEPES.The HEK293F cells were transfected with the human dopamine transporter and frozen in 1 mL aliquots at about 1E+07 cells/mL. On the day of the experiment, the cells were removed from −80° C. or liquid nitrogen and thawed in a room temperature water bath. The cells were dilute to about 1-2E+06 with Freestyle medium. A 1 mL sample (1:2 dilution) was prepared, and the cells were counted. The cells were spun at 1100 rpm for 5 minutes, and the medium was aspirated off. The cells were resuspend in medium at 1.5E+06 cells/mL for about 60,000 cells per well. 40 μL of cells were dispensed per well in the Biocoat plates. The plates were spun at 1100 rpm for 1 minute to improve homogeneity of the cell layer and were incubated at 37° C. for a minimum of 3 hours.12 μL of test compound (10 mM) in DMSO was added to the wells, and nomifensine was used as a control. GBR 12909 (final assay concentration of 10 μM) was used for background signal. The neurotransmitter transporter dye was prepared in AB prior to use.
Measurement: Ki = 89.5 nM (pChEMBL 7.050000190734863)
Descriptors: MW 276.38, ALogP 3.69, HBA 2, HBD 1, PSA 38.91, QED 0.79
Compound (SMILES): Cc1ccc(S(=O)(=O)C23CCN(C(=O)C45CCC(C(=O)O)(CC4)CC5)C2COc2cc(Br)ccc23)cc1
InChIKey: WSRCYQFNPFFCMX-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Binding Assay : The binding of potential ligands to RORγ is measured by competition with [3H]25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA). The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromatography. 15 μg/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 μM to 0.28 nM, for 10 min at rt in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% glycerol (Sigma Cat # G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat # RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined.
Measurement: IC50 = 182 nM (pChEMBL 6.739999771118164)
Descriptors: MW 588.52, ALogP 4.85, HBA 5, HBD 1, PSA 100.98, QED 0.55
Compound (SMILES): O=C(c1nc(NCc2ccc(C(F)(F)F)nc2)nc2ccsc12)N1CCCC1
InChIKey: LKMAMBXOZKJUQS-UHFFFAOYSA-N
Target: Adenosine receptor A2a (SINGLE PROTEIN), Homo sapiens
UniProt: P29274
Assay: Activity Assay: Compounds (Adenosine Receptor modulators) and activity assay.
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 407.42, ALogP 3.95, HBA 6, HBD 1, PSA 71.01, QED 0.71
Compound (SMILES): COCCCCCCC1CCc2cc([C@H]3CC[C@](N)(COP(=O)(O)O)C3)ccc2C1
InChIKey: HNZSBKMAUGOJAY-AMHZRHSASA-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 < 10 nM (pChEMBL 8)
Descriptors: MW 439.53, ALogP 4.46, HBA 4, HBD 3, PSA 102.01, QED 0.35
Compound (SMILES): CCOc1cc(-c2ccc(N3CCC(CN4CCNCC4)(NC(=O)OC(C)C)CC3)nc2)c2c(C#N)cnn2c1
InChIKey: GCQPEQWXXOPTJC-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of Formula I were screened for their ability to inhibit wildtype and V804M mutant RET kinase using CisBio's HTRF KinEASE-TK assay technology. Briefly, N-terminal GST tagged recombinant human RET cytoplasmic domain (aa 658-end) from Eurofins (0.25 nM RET; Catalog No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Catalog No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Catalog No. 62TK0PEC) and 1 mM ATP along with test compound in a buffer consisting of 25 mM HEPES pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO in a volume of 8 μL. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 30-minute incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.25 nM Sa-XL665 and 1×TK-ab-Cryptate in HTRF detection buffer (all from CisBio, part of Cat. No. 62TK0PEC). After a 1 hour incubation at 22° C., the extent of reaction was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using pre-quenched control reactions. The POC values were fit to a 4 parameter logistic curve, and the IC50 is defined as the concentration of inhibitor at which the POC equals 50 for the fitted curve.
Measurement: IC50 = 195 nM (pChEMBL 6.710000038146973)
Descriptors: MW 546.68, ALogP 3.05, HBA 10, HBD 2, PSA 120.05, QED 0.44
Compound (SMILES): CN1CCn2nc(NC(=O)c3cccc(CNC(=O)c4ccc(-c5ccncc5)cc4)c3)cc2C1
InChIKey: QYGFMWMXWCEAQQ-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: Kinase Inhibition Assay: The following assay protocol is for measuring the phosphorylation of a peptide substrate (FAM-KKLRRTLSVA-OH wherein FAM is carboxyfluorescein). The peptide is >98% purity by Capillary Electrophoresis. The peptide is phosphorylated by the protein kinase ROCK1 or ROCK2. The ROCK1 or ROCK2 enzyme, substrate, and cofactors (ATP and Mg2+) are combined in a well of a microtiter plate and incubated for 3 hours at 25° C. in the presence or absence of an inhibitor compound. At the end of the incubation, the reaction is quenched by the addition of an EDTA-containing buffer. The substrate and product are separated and quantified electrophoretically using the microfluidic-based LABCHIP® 3000 Drug Discovery System from Caliper Life Sciences (Hopkinton, Mass.).The components of the assay mixture are:100 mM HEPES, pH 7.50.1% BSA0.01% Triton X-1001 mM DTT10 mM MgCl210 μM Sodium Orthovanadate10 LM Beta-Glycerophosphate5 μM ATP (for ROCK1) or 7 μM ATP (for ROCK2)1% DMSO (from compound)1.25 μM FAM-KKLRRTLSVA-OH3 nM ROCK1 or 2.5 nM ROCK2 enzymeSubstrate and product peptides present in each sample are separated electrophoretically using the LABCHIP® 3000 capillary electrophoresis instrument. As substrate and product peptides are separated two peaks of fluorescence are observed. Change in the relative fluorescence intensity of the substrate and product peaks is the parameter measured reflecting enzyme activity. Capillary electrophoregramms (RDA acquisition files) are analyzed using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Mass.). The kinase activity in each sample is determined as the product to sum ratio (PSR): P/(S+P), where P is the peak height of the product peptide and S is the peak height of the substrate peptide. For each compound, enzyme activity is measured at various concentrations (12 concentrations of compound spaced by 3× dilution intervals).
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 466.55, ALogP 3.57, HBA 6, HBD 2, PSA 92.15, QED 0.45
Compound (SMILES): Cc1[nH]c(-c2cc(-c3cncc(S(C)(=O)=O)c3)ccn2)nc1C(=O)N(C)C
InChIKey: YENKSCPLMCWHFP-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: PI3Kδ Scintillation Proximity Assay: The kinase reaction was conducted in polystyrene 384-well Greiner Bio-one white plate from Thermo Fisher Scientific in a final volume of 25 μL. Inhibitors were first diluted serially in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 2%. The PI3Kδ assay was carried out at room temperature in 20 mM MOPS, pH 6.7, 10 mM MgCl2, 5 mM DTT and CHAPS 0.03%. Reactions were initiated by the addition of ATP, the final reaction mixture consisted of 20 μM PIP2, 2 μM ATP, 0.5 μCi [γ-33P] ATP, 3.4 nM PI3Kδ. Reactions were incubated for 120 min and terminated by the addition of 40 μL SPA beads suspended in quench buffer: 163 mM potassium phosphate pH 7.8, 20% glycerol, 25 mM EDTA. The final concentration of SPA beads is 1.0 mg/mL. After the plate sealing, plates were shaken overnight at room temperature and centrifuged at 1500 rpm for 10 min, the radioactivity of the product was determined by scintillation counting on Topcount (Perkin-Elmer). IC50 determination was performed by fitting the curve of percent of the solvent control activity versus the log of the inhibitor concentration using the GraphPad Prism 6.0 software.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 385.45, ALogP 1.95, HBA 6, HBD 1, PSA 108.91, QED 0.74
Compound (SMILES): CCn1cc(C(=O)Nc2ccc(Oc3ccnc4[nH]nc(N[C@@H](C)COC)c34)c(F)c2)c(=O)n(-c2ccc(F)cc2)c1=O
InChIKey: HILCAESKDJTLRO-INIZCTEOSA-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 = 3100 nM (pChEMBL 5.510000228881836)
Descriptors: MW 591.58, ALogP 4.06, HBA 10, HBD 3, PSA 145.16, QED 0.22
Compound (SMILES): N#Cc1cnc2ccc(-c3c(-c4ccc(F)c(Cl)c4)ncn3C3CC(F)C3)nn12
InChIKey: QTSUEIBSKWPOLS-UHFFFAOYSA-N
Target: TGF-beta receptor type-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P37173
Assay: Biological Assay: Assays for the compounds reported below were conducted in 1536-well plates and 2 mL reactions are prepared from addition of HIS-TGF-βR1 T204D or HIS-TGF-βR2 WT, anti-HIS detection antibody, a labeled small molecule probe (Kd=<100 nM; koff=<0.001 s−1) and test compounds in assay buffer (20 mM HEPES pH 7.4, 10 mM MgCl2, 0.015% Brij35, 4 mM DTT, and 0.05 mg/ml BSA). The reaction is incubated for 1 hour at room temperature and the HTRF signal was measured on an Envision plate reader (Ex: 340 nm; Em: 520 nm/495 nm). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay are 1 nM HIS-TGF-βR1 T204D or HIS-TGF-βR2 WT, 0.2 nM anti-HIS detection antibody, labeled small molecule prode (at Kd) and 0.5% DMSO. Dose response curves were generated to determine the concentration required inhibiting 50% of kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 3954 nM (pChEMBL 5.400000095367432)
Descriptors: MW 410.82, ALogP 4.6, HBA 6, HBD 0, PSA 71.8, QED 0.49
Compound (SMILES): C[C@@H](Oc1cc(-c2ccc(N3CCCS3(=O)=O)cc2)cc2nn(C)cc12)[C@H]1CNC(=O)C1
InChIKey: WITCHRZHWIURGZ-CRAIPNDOSA-N
Target: Aurora kinase B (SINGLE PROTEIN), Homo sapiens
UniProt: Q96GD4
Assay: Aurora B Kinase Assay: Recombinant human Aurora B (amino acids 1-344, clone number DU1773, Molecular weight 40.2 kDa, University of Dundee) was expressed as a fusion protein with an N-terminal His tag, affinity-purified and deep-frozen at a concentration of approx. 0.25-0.5 mg/ml in storage buffer (50 mM Tris-HCl pH 8; 25 mM Na-β-glycerophosphat; 0.1 mM EGTA; 150 mM NaCl; 0.03% Brij-35; 1 mM DTT and 10% glycerol) at −80° C. until use.The activity of the Aurora B kinase protein was determined using the ADP Glo® Luminescence Kinase test (Promega; V9103X). In this homogeneous test the amount of ADP remaining after the kinase reaction is quantified by a luciferin-luciferase reaction using luminescence. The luminescence signal obtained correlates with the amount of ADP still present and thus correlates with the activity of the protein kinase.
Measurement: IC50 > 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 454.55, ALogP 2.68, HBA 6, HBD 1, PSA 93.53, QED 0.64
Compound (SMILES): Cc1cccc([C@H]2C[C@@H]2COc2nc(C)ncc2-c2cn(C)nc2C(C)O)n1
InChIKey: REEQFVBJGGGWDC-CZVYVAOFSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Fluorescence Polarization Assay: The fluorescence polarization assay for cyclic nucleotide phosphodiesterases was performed using an IMAP® FP kit supplied by Molecular Devices, Sunnyvale, Calif. (product # R8139). IMAP® technology has been applied previously to phosphodiesterase assays (Huang, W., et al., J. Biomol Screen, 2002, 7: 215). Assays were performed at room temperature in 384-well microtiter plates with an incubation volume of 20.2 μL. Solutions of test compounds were prepared in DMSO and serially diluted with DMSO to yield 8 μL of each of 10 solutions differing by 3-fold in concentration, at 32 serial dilutions per plate. 100% inhibition is determined using a known PDE10 inhibitor, which can be any compound that is present at 5,000 times its Ki value in the assay described as follows, such as papaverine (see Siuciak, et al. Neuropharmacology (2006) 51:386-396; Becker, et al. Behav Brain Res (2008) 186(2):155-60;
Measurement: Ki = 2.28 nM (pChEMBL 8.640000343322754)
Descriptors: MW 379.46, ALogP 3.12, HBA 7, HBD 1, PSA 85.95, QED 0.71
Compound (SMILES): C[C@H](Nc1nc(C(=O)N2CC(O)C2)c2sccc2n1)c1ccccn1
InChIKey: RDGREPAJDVPVQC-JTQLQIEISA-N
Target: Adenosine receptor A2a (SINGLE PROTEIN), Homo sapiens
UniProt: P29274
Assay: Activity Assay: Compounds (Adenosine Receptor modulators) and activity assay.
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 355.42, ALogP 2.08, HBA 7, HBD 2, PSA 91.24, QED 0.74
Compound (SMILES): CC(=O)Nc1nc2ccc(-c3nn(Cc4cc5ccccc5nc4N4CCOCC4)c4ncnc(N)c34)cc2s1
InChIKey: QJSVSMODOMCUGC-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Inhibition Assay: Class I PI3-Ks can be either purchased (p110α/p85α, p110β/p85α, p110δ/p85α from Upstate, and p110γ from Sigma) or expressed as previously described (Knight et al., 2004). IC50 values are measured using either a standard TLC assay for lipid kinase activity (described below) or a high-throughput membrane capture assay. Kinase reactions are performed by preparing a reaction mixture containing kinase, inhibitor (2% DMSO final concentration), buffer (25 mM HEPES, pH 7.4, 10 mM MgCl2), and freshly sonicated phosphatidylinositol (100 μg/ml). Reactions are initiated by the addition of ATP containing 10 μCi of γ-32P-ATP to a final concentration 10 or 100 μM and allowed to proceed for 5 minutes at room temperature. For TLC analysis, reactions are then terminated by the addition of 105 l 1N HCl followed by 160 μl CHCl3:MeOH (1:1). The biphasic mixture is vortexed, briefly centrifuged, and the organic phase is transferred to a new tube using a gel loading pipette tip precoated with CHCl3. This extract is spotted on TLC plates and developed for 3-4 hours in a 65:35 solution of n-propanol:1M acetic acid. The TLC plates are then dried, exposed to a phosphorimager screen (Storm, Amersham), and quantitated. For each compound, kinase activity is measured at 10-12 inhibitor concentrations representing two-fold dilutions from the highest concentration tested (typically, 200 μM). For compounds showing significant activity, IC50 determinations are repeated two to four times, and the reported value is the average of these independent measurements.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 551.64, ALogP 4.08, HBA 11, HBD 2, PSA 136.97, QED 0.32
Compound (SMILES): N=C(N)c1ccc(CNC(=O)[C@@H]2CCN2C(=O)[C@H](N)c2ccccc2)cc1
InChIKey: DSAGIBSRHSMFSJ-DLBZAZTESA-N
Target: Prothrombin (SINGLE PROTEIN), Homo sapiens
UniProt: P00734
Assay: Enzymatic Assay for Thrombin: The thrombin assay utilizes a fluorogenic peptide substrate (Boc-VPR-AMC (R&D Systems) and was run at room temperature in an assay buffer containing 20 mM Hepes, pH 7.4, 140 mM NaCl and 0.1% Tween 20. Assay parameters were adjusted such that the assay was linear with respect to time, enzyme and substrate concentrations. Under these optimized assays conditions, IC50values were equivalent to Ki values, except in a few cases of tight binding inhibitors. Cases of tight binding or possible slow binding inhibitors were handled by the methods described in Copeland R. A. (2013) Evaluation of Enzyme Inhibitors in Drug Discovery. 2nd Ed. John Wiley and Sons, Inc., Chapters 5-7.
Measurement: Ki < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 365.44, ALogP 0.89, HBA 4, HBD 4, PSA 125.3, QED 0.45
Compound (SMILES): Cc1ccc(S(=O)(=O)N(C)C23CCC(C#N)(C2)C3)cc1-c1cnc2c(N)ncnn12
InChIKey: QOQFIKHAUWRKHY-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Scintillation Proximity Assay: PI3Kδ:[γ-33P]ATP (10 mCi/mL) and Wheat Germ Agglutinin (WGA) YSi SPA Scintillation Beads was purchased from Perkin-Elmer (Waltham, Mass.). Lipid kinase substrate, D-myo-Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)D (+)-sn-1,2-di-O-octanoylglyceryl, 3-O-phospho linked (PIP2), CAS 204858-53-7, was purchased from Echelon Biosciences (Salt Lake City, Utah). PI3Kδ (p110δ/p85α) Recombinant Human Protein was purchased from Eurofins (St Charles, Mo.). ATP, MgCl2, DTT, EDTA, MOPS and CHAPS were purchased from Sigma-Aldrich (St. Louis, Mo.).The kinase reaction was conducted in polystyrene 384-well Greiner Bio-one white plate from Thermo Fisher Scientific in a final volume of 25 μL. Inhibitors were first diluted serially in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 2%. The PI3Kδ assay was carried out at room temperature in 20 mM MOPS, pH 6.7, 10 mM MgCl2, 5 mM DTT and CHAPS 0.03%. Reactions were initiated by the addition of ATP, the final reaction mixture consisted of 20 μM PIP2, 2 μM ATP, 0.5 μCi [γ-33P] ATP, 3.4 nM PI3Kδ. Reactions were incubated for 120 min and terminated by the addition of 40 μL SPA beads suspended in quench buffer: 163 mM potassium phosphate pH 7.8, 20% glycerol, 25 mM EDTA. The final concentration of SPA beads is 1.0 mg/mL. After the plate sealing, plates were shaken overnight at room temperature and centrifuged at 1500 rpm for 10 min, the radioactivity of the product was determined by scintillation counting on Topcount (Perkin-Elmer).
Measurement: IC50 < 2000 nM (pChEMBL 5.698999881744385)
Descriptors: MW 423.5, ALogP 2.14, HBA 8, HBD 1, PSA 130.27, QED 0.68
Compound (SMILES): CN1CCN(c2ccc(Nc3ncc4ccn(-c5cccc(NS(=O)(=O)C(C)(C)C)c5)c4n3)cc2Cl)CC1
InChIKey: SAFSNEIJARKHHO-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: AlphaLisa Assay: 1. Add 20 μl of the diluted compound to each well of the 384-well opti plate2. Tap the plate gently3. Add 10 μl of the enzyme to each well of the opti plate4. Cover with plate sealer5. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.6. Incubate the plate at room temperature for 10 minutes7. Add 10 μl of the substrate8. Cover with plate sealer9. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.10. Cover with plate sealer and incubate at room temperature for 1 hour11. After incubation, transfer 10 μl of reaction mix to fresh wells of a 384-well plate.12. Add 10 μl acceptor bead from working stock.13. Cover with plate sealer14. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.15. Cover with plate sealer and incubate at room temperature for 30 minutes16. Add 10 μl donor bead from working stock (in dark).17. Cover with plate sealer18. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.19. Cover with plate sealer and incubate at room temperature for 15-30 minutes20. Read the plate in pherastar (alpha screen protocol)
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 554.12, ALogP 5.11, HBA 8, HBD 2, PSA 95.39, QED 0.34
Compound (SMILES): O=C1NCc2c(-c3cnc4cnccn34)ccc(Nc3ccc(S(=O)(=O)N4CCC4)cn3)c21
InChIKey: KIHFYZKQPBZYAH-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: Biochemical Enzyme Assay: HPK1 biochemical enzyme assay: HPK1 enzyme inhibition was measured using a microfluidic mobility shift assay. Reactions were performed in a 384-well plate, containing 1.5 nM HPK1 (Invitrogen), in assay buffer (Carna Biosciences; pH 7.4). Test compounds were titrated in ten point curves (top final assay concentration 3 μM), and preincubated with enzyme/substrate mix for 30 min prior to initiation of the reaction by addition of ATP (1 mM final concentration) and substrate (1 μM final concentration; Carna Biosciences) diluted in assay buffer supplemented by MgCl2 (final assay concentration of 5 mM). Following 60 min incubation at RT, the reaction was terminated by addition of 60 μl/well termination buffer (Carna Biosciences) and signal determination using a Caliper EZ Reader (Perkin Elmer, UK).
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 461.51, ALogP 2.17, HBA 8, HBD 2, PSA 121.59, QED 0.47
Compound (SMILES): Cc1cc(C)c(-c2cccnc2)cc1C(=O)Nc1cn2nc(-c3c(-c4ccc(F)cc4)nc(C)n3C)ccc2n1
InChIKey: FUQVYNBDVXCEPI-UHFFFAOYSA-N
Target: Casein kinase I isoform delta (SINGLE PROTEIN), Homo sapiens
UniProt: P48730
Assay: Kinase Assay: The kinase assay was performed in V-bottom 384-well plates. The final assay volume was 30 μl prepared from 15 μl additions of enzyme, substrates (fluoresceinated peptide FL-AHA-KRRRAL-PSER-VASLPGL-OH and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.4, 30 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was incubated at room temperature for 22 hours and terminated by adding 45 μl of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP®3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the unphosphorylated substrate and phosphorylated product. Inhibition data were calculated by comparison of the no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay were 200 pM CK1ε or CK1δ, 50 μM ATP, 1.5 μM FL-AHA-KRRRAL-PSER-VASLPGL-OH, and 1.6% DMSO. Dose response curves were generated to determine the concentration required to inhibit 50% of the kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations.
Measurement: IC50 = 0.463 nM (pChEMBL 9.329999923706055)
Descriptors: MW 531.6, ALogP 6.18, HBA 7, HBD 1, PSA 90, QED 0.29
Compound (SMILES): COc1ccc(Nc2nc(=O)cnn2Cc2ccccc2F)c(C)c1
InChIKey: OZWKCDBDBNVMIH-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Evaluation of a Human P2X7 Receptor Inhibitory Activity: Stably expressing cell line (1321N1 cell transfected with the human P2X7 receptor gene (GenBank accession number NM_002502.5 including T606C and G952A SNP)) was used. The cells were seeded in a 384-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (10% fetal bovine serum, 25 mM HEPES, 1% penicillin and streptomycin in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. After replacing with 20 μL of the HBSS buffer (20 mM HEPES, 55.6 mM D-glucose, 1×HBSS(−), pH7.4-7.5). 16 μL of 17.3 μM Yo-Pro solution in the HBSS buffer was added. The plate was placed in high-throughput cellular screening system FLIPR TETRA (Molecullar Devices, LLC.) and 15 μL of 130 μM BzATP solution in the HBSS buffer was added. Measurement, of fluorescence intensity by FLIPR TETRA was started. After eight minutes, 15 μL of DMSO solutions containing different concentrations of the compound of the present invention as prepared by dilution with the HBSS buffer were dispensed to each well through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 20 minutes. The maximum fluorescence intensity without the compound of the present invention is calculated as 0% inhibition and the maximum fluorescence intensity when the reference compound was added is calculated as 100% inhibition. Changing values of fluorescence intensity by the compound of the present invention were calculated by difference between maximum and minimum fluorescence intensity for 20 minutes.
Measurement: IC50 = 200 nM (pChEMBL 6.699999809265137)
Descriptors: MW 340.36, ALogP 2.89, HBA 6, HBD 1, PSA 69.04, QED 0.77
Compound (SMILES): Cc1cc(-c2[nH]c3ccc(C4CCN(C(=O)CCN5CCOCC5)CC4)cc3c2C(C)C)cn2ncnc12
InChIKey: AWGKDQVCUAMKAS-UHFFFAOYSA-N
Target: Toll-like receptor 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR97
Assay: TLR7/8/9 Inhibition Reporter Assay: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 31.3 nM (pChEMBL 7.5)
Descriptors: MW 514.67, ALogP 4.74, HBA 6, HBD 1, PSA 78.76, QED 0.4
Compound (SMILES): CN(C)[C@]1(c2ccccc2)CC[C@]2(CC1)CN(CC(=O)N1CCN(S(C)(=O)=O)CC1)C(=O)N2CC1CCC1
InChIKey: BNRBOILPUOEHRY-HNRBIFIRSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Human Mu-Opioid Peptide (hMOP) Receptor Binding Assay: The hMOP receptor binding assay was performed as homogeneous SPA-assay (scintillation proximity assay) using the assay buffer 50 mM TRIS-HCl (pH 7.4) supplemented with 0.052 mg/ml bovine serum albumin (Sigma-Aldrich Co., St. Louis, Mo.). The final assay volume (250 μl/well) included 1 nM of [N-allyl-2,3-3H]naloxone as ligand (PerkinElmer Life Sciences, Inc. Boston, Mass., USA), and either test compound in dilution series or 25 μM unlabelled naloxone for determination of unspecific binding. The test compound was diluted with 25% DMSO in H2O to yield a final 0.5% DMSO concentration, which also served as a respective vehicle control. The assay was started by adding wheat germ agglutinin coated SPA beads (GE Healthcare UK Ltd., Buckinghamshire, UK) which had been preloaded with hMOP receptor membranes (PerkinElmer Life Sciences, Inc. Boston, Mass., USA). After incubation for 90 minutes at RT and centrifugation for 20 minutes at 500 rpm the signal rate was measured by means of a 1450 Microbeta Trilux ß-counter (PerkinElmer Life Sciences/Wallac, Turku, Finland). Half-maximal inhibitory concentration (IC50) values reflecting 50% displacement of [3H]naloxone-specific receptor binding were calculated by nonlinear regression analysis and Ki values were calculated by using the Cheng-Prusoff equation, (Cheng and Prusoff, 1973).
Measurement: Ki = 49 nM (pChEMBL 7.309999942779541)
Descriptors: MW 545.75, ALogP 2.4, HBA 5, HBD 0, PSA 84.48, QED 0.53
Compound (SMILES): COc1ccccc1N1CCN(CCCCOc2ccc3c(c2)NC(=O)CO3)CC1
InChIKey: ZQIBMJPPTOOPCH-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 1A (SINGLE PROTEIN), Homo sapiens
UniProt: P08908
Assay: Binding Assay: Receptor Source: Human recombinant expressed in HEK-293 cells  Radioligand: [3H]-8-OH-DPAT (221 Ci/mmol)  Control Compound: 8-OH-DPAT  Incubation Conditions: The reactions were carried out in 50 mM TRIS-HCl (pH 7.4) containing 10 mM MgSO4, 0.5 mM EDTA and 0.1% Ascorbic acid at room temperature for 1 hour. The reaction was terminated by rapid vacuum filtration onto glass fiber filters. Radioactivity trapped onto the filters was determined and compared to control values in order to ascertain any interactions of test compounds with the cloned serotonin—5HT1A binding site (Literature Reference: Hoyer, D. et al. Eur. Journal Pharmacol. 1985, 118, 13-23; Schoeffter, P. and Hoyer, D. Naunyn-Schmiedeberg's Arch. Pharmac. 1989, 340, 135-138)
Measurement: IC50 = 0.97 nM (pChEMBL 9.010000228881836)
Descriptors: MW 411.5, ALogP 3.01, HBA 6, HBD 1, PSA 63.27, QED 0.67
Compound (SMILES): C=CC(=O)Nc1cc(N(C)CCOC)ccc1Nc1ncc2c(n1)N(C)C(=O)N(c1cc(OC)cc(OC)c1)C2
InChIKey: HZCHTZOQZVZMML-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P22455
Assay: Omnia Assay Protocol for Potency Assessment Against FGFR 4 Enzyme: A 10× stock solution of FGFR4-WT (PR4380C or P3054), (from Invitrogen, Carlsbad, Calif.) corresponding to method a in Table 10, was prepared as described below. Alternatively, a 10× stock solution of FGFR4-WT (F01-11G), (SignalChem, Richmond, BC) corresponding to method b in Table 10, was prepared as described below. A solution of 1.4×ATP (AS001A) and 5× Tyr-Sox conjugated peptide substrate (KNZ3101) were prepared in 1× kinase reaction buffer consisting of 20 mM Tris, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 5 mM P3-glycerophosphate, 5% glycerol (10× stock, KB002A) and 0.2 mM DTT (DS001A). 5 μL of FGFR4 was pipetted into a Corning (#3574) 384-well, white, non-binding surface microtiter plate (Corning, N.Y.), containing a 0.5 μL volume of 100% DMSO. The serially diluted compounds were prepared on a Tecan EVO100. A second addition of 10 μl of Tyr-Sox FGFR4 substrate was added to each well and the kinase reactions were started with the addition of 35 μL of 1.4×ATP. The reactions were monitored every 71 seconds for 240 minutes at λex360/λem485 in a Synergy plate reader from BioTek (Winooski, Vt.). At the conclusion of each assay, progress curves from each well were examined for linear reaction kinetics and fit statistics (R2, 95% confidence interval, absolute sum of squares). Initial velocity (0 minutes to −60 minutes) from each reaction was determined from the slope of a plot of relative fluorescence units vs time (seconds) and then plotted against inhibitor concentration to estimate IC50 from log[Inhibitor] vs Response (Variable Slope model in GraphPad Prism from GraphPad Software (San Diego, Calif.)).
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 547.62, ALogP 4.02, HBA 9, HBD 2, PSA 121.39, QED 0.34
Compound (SMILES): CC(=O)Nc1nc2cc(-c3cc(F)c4oc(N)nc4c3)ccc2s1
InChIKey: UMWWWWQECKEHLW-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: PI 3-Kinase HTRF Assay: 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 1x reaction buffer) is added per well for a final concentration of 0.25-0.3 ug/ml kinase and 10 uM PIP2. The plate is sealed and incubated for 15 minutes at room temperature. To start the reaction, 3.5 ul of ATP (diluted in 1x reaction buffer) is added per well for a final concentration of 10 uM 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 342.36, ALogP 3.78, HBA 6, HBD 2, PSA 94.04, QED 0.58
Compound (SMILES): N[C@]1(COP(=O)(O)O)CC[C@H](c2ccc3c(c2)CC[C@H](CSCCc2ccccc2)C3)C1
InChIKey: JJGLVCBKLNVALV-KSNOWIBYSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21453
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 < 10 nM (pChEMBL 8)
Descriptors: MW 475.59, ALogP 4.84, HBA 4, HBD 3, PSA 92.78, QED 0.35
Compound (SMILES): NC1CCN([C@@]2(N)CCCN(c3cccc4[nH]cc(-c5cncnc5)c34)C2)CC1
InChIKey: MMDWMUJGEZXOEW-QFIPXVFZSA-N
Target: Rho-associated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13464
Assay: Kinase Assay: MRCKα, MRCKβ, ROCK1 and ROCK2 assays were performed using an IMAP fluorescence polarization assay format (Molecular Devices Inc.). 8-12 nM of each kinase (Life Technologies) was incubated for 60 min at room temperature with 100 nM FAM-S6-ribosomal protein derived peptide (synthesized by Alta Biosciences, University of Birmingham UK) in the presence of 1 μM ATP and 0.5 mM MgCl2 in 20 mM Tris buffer (pH 7.4) containing 0.01% Tween-20 and 1 mM DTT (MRCKα and β); or 1 μM ATP, 10 mM MgCl2 in 20 mM Tris buffer (pH 7.5) containing 0.25 mM EGTA 0.01% Triton X-100 and 1 mM DTT (ROCK1 and ROCK2). Typically, dose response analyses were performed over concentration ranges from 0.005-100 μM. Reactions were stopped by adding 2 assay volumes of 0.25% (v/v) IMAP binding reagent in 1×IMAP binding buffer A (Molecular Devices). After 30 min incubation to allow binding reagent to bind phosphorylated peptide, fluorescence polarization was measured on a Tecan Saphire2 plate reader at excitation (470 nm) and emission (530 nm) wavelengths. Inhibition was calculated using no inhibitor and no enzyme controls as 0 and 100% inhibition, respectively.
Measurement: Ki = 4999 nM (pChEMBL 5.300000190734863)
Descriptors: MW 391.52, ALogP 2.3, HBA 6, HBD 3, PSA 100.09, QED 0.63
Compound (SMILES): COc1cccc(NC(=O)c2cccc3c2N[C@H](C)CC(=O)N3)c1
InChIKey: YTVHOTJONGZFRT-LLVKDONJSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: BRD4 AlphaLisa Binding Assay: His/Flag epitope tagged BRD4 BD142-168 was cloned, expressed, and purified to. BRD4 binding and inhibition was assessed by monitoring the engagement of biotinylated H4-tetraacetyl peptide (New England Peptide, NEP2069-1/13) with the target using the AlphaLisa technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate BRD4(BD1) (30 nM final) was combined with peptide (200 nM final) in 40 mM HEPES (pH 7.0), 40 mM NaCl, 1 mM DTT, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 1.2% DMSO) or compound dilution series in DMSO. After 20 minutes incubation at room temperature Alpha streptavidin donor beads and AlphaLisa anti-Flag acceptor beads were added to a final concentration of 10 ug/mL each. After three hours equilibration plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 5583 nM (pChEMBL 5.25)
Descriptors: MW 325.37, ALogP 3.09, HBA 4, HBD 3, PSA 79.46, QED 0.81
Compound (SMILES): N=C(/C(=N\S(=O)(=O)c1ccc(Cl)cc1)N1CC(c2ccccc2)C(c2ccc(Cl)cc2)=N1)c1ccc(Br)cc1
InChIKey: ORYSBPPSHNFGCU-PCJQJBIFSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Mus musculus
UniProt: P47746
Assay: Radioligand Displacement Assay: Measuring its tissue levels 1 hour after administration of 10 mg/kg to mice, plasma levels were comparable after oral or i.p. administration (indicating good oral bioavailability), and brain tissue level was <2% of plasma level, indicating low brain penetrance/peripheral selectivity. 1 h after oral administration of compound 2 at 10 mg/kg dose in mice, the metabolite generation in plasma was monitored by LC-MS/MS. As expected, this compound underwent in vivo metabolism to liberate the amidine moiety and a metabolite (structure 41V in the FIG. 1). Besides CB1R antagonism, both intact compound and its metabolically cleaved amidine moiety were able to inhibit iNOS activity about 48% and 37% at 1 μM concentration in lung homogenates from LPS-treated mouse (FIG. 4). Mice with diet-induced obesity (DIO) mice were orally treated for 14 days with the compound 2 (10 mg/kg/day).
Measurement: Ki = 27 nM (pChEMBL 7.570000171661377)
Descriptors: MW 640.39, ALogP 7.41, HBA 4, HBD 1, PSA 85.95, QED 0.18
Compound (SMILES): COc1cnc(/C(F)=C/c2ccc(F)c([C@]3(C)CS(=O)(=O)C(C)(C)C(N)=N3)c2)cn1
InChIKey: ZSGMTRQKPXSNES-SLPYIPCOSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vitro BACE1 Cell-Based Assay: The cell-based assay measures inhibition or reduction of Aβ40 in conditioned medium of test compound treated cells expressing amyloid precursor protein. Cells stably expressing Amyloid Precursor Protein (APP) were plated at a density of 45K cells/well in 384 well plates (Corning/BioCoat 354663). The test compounds were then added to cells in 22-point dose response concentrations with the starting concentration being 62.5 μM. The compounds were diluted from stock solutions in DMSO and the final DMSO concentration of the test compounds on cells was 0.625%. The cells were cultivated overnight at 37° C. and 5% CO2 in DMEM supplemented with 10% FBS. After 24 h of incubation with the test compounds, the conditioned media was collected and the Aβ40 levels were determined using HTRF (Homogeneous Time Resolved Fluorescence). The IC50 of the compound was calculated from the percent of control or percent inhibition of Aβ 40 as a function of the concentration of the test compound.The HTRF to detect Aβ40 was performed in 384 well plates (Costar 3658). The antibody pair that were used to detect Aβ 40 from cell supernatants were ConfAb40 antibody (Amgen in-house) and biotinylated 6E10 (BIOLEGEND). As an alternative to ConfAb40, a commercially available antibody, Anti-beta Amyloid 1-40 antibody [BDI350] from Abcam, Cambridge, Mass. 02139-1517 (Product code: ab20068), may be used in this assay. The concentrations were 0.35 μg/mL of ConfAb40 antibody and 1.33 μg/mL of 6E10-biotinylated antibody, as well as 4.5 μg/mL of Streptavidin Allophycocyanin Conjugate (ThermoFisher Scientific) in HTRF Buffer (1M Hepes pH 7.5, 1M NaCl, 1% BSA, 0.5% Tween 20).The conditioned media was incubated with above antibodies and Streptavidin Allophycocyanin Conjugate for 30-60 minutes at 23° C. The final readout was performed on Envision from PerkinElmer.
Measurement: IC50 = 133 nM (pChEMBL 6.880000114440918)
Descriptors: MW 436.48, ALogP 2.87, HBA 7, HBD 1, PSA 107.53, QED 0.79
Compound (SMILES): N#CCC1(n2cc(-c3nccc4[nH]ccc34)cn2)CN(C2CCN(C(=O)c3cnc4ccccc4c3)CC2)C1
InChIKey: JYFACHILQIMFNN-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 < 10 nM (pChEMBL 8)
Descriptors: MW 516.61, ALogP 4.2, HBA 7, HBD 1, PSA 106.73, QED 0.38
Compound (SMILES): CNC(=O)c1cc(C(C)(C)C)cc(NC(=O)Nc2ccc(Oc3ccnc(Nc4cc(CO)cc(OC)c4)c3)c3ccccc23)c1OC
InChIKey: XEFBBQIBHYFSDW-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase Src (SINGLE PROTEIN), Homo sapiens
UniProt: P12931
Assay: Enzyme Inhibition Assay: The inhibitory activities of compounds of the invention against c-Src and Syk enzymes (Invitrogen), are evaluated in a similar fashion to that described hereinabove. The relevant enzyme (3000 ng/mL or 2000 ng/mL respectively, 2.5 μL) is incubated with the test compound (either 1 μg/mL, 0.1 μg/mL, 0.01 μg/mL, or 0.001 μg/mL, 2.5 μL each) for 2 hr at RT. The FRET peptides (8 μM, 2.5 μL), and appropriate ATP solutions (2.5 μL, 800 μM for c-Src, and 60 μM ATP for Syk) are then added to the enzymes/compound mixtures and the mixture incubated for 1 hr. Development reagent (protease, 5 μL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan® Flash, ThermoFisher Scientific).
Measurement: IC50 = 21 nM (pChEMBL 7.679999828338623)
Descriptors: MW 649.75, ALogP 7.58, HBA 8, HBD 5, PSA 143.07, QED 0.1