lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC(C)(NC(=O)c1cccc2c1OCCNC2)c1cccc2cnccc12
InChIKey: DNKZMADVSUZJJP-UHFFFAOYSA-N
Target: Somatostatin receptor type 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P31391
Assay: cAMP ASSAY: The activation of the SSTR4 receptor (Gi coupled) causes an inhibition of intracellular cAMP after stimulation with Forskolin, which can be quantifiable by use of a suitable assay Kit and an adequate plate reader. This technique is used to characterize pharmacological effects of the SSTR4 receptor agonists by use of hSSTR4 expressing H4 cells.Compounds are dissolved and diluted in DMSO. The final test solution contains 1 DMSO. The cAMP standard (Lance cAMP 384 Kit; PerkinElmer, Cat #AD0264) is prepared in assay buffer (HBSS with 0.1% BSA, 5 mM HEPES, 0.5 M IBMX, pH 7.4) containing 1% DMSO and the cAMP standard curve is included at least on one plate. Cells are centrifuged and suspended in assay buffer (incl. 1:100 diluted Alexa antibody).For the assay 5 μl of a cell suspension (approximately 5000 cells/well) incl. Alexa antibody (diluted 1:100) are added into a 384 well MTP microtitre plate excepting one row or column (depending on the plate layout), which is reserved for the standard curve. Then 2 μl of compound sample is added as concentration response curve (e.g. 1e-5 M to 6e-10 M), usually in triplicates. Each assay contains incubations with vehicle controls instead of compound as controls for non-inhibited cAMP generation (100% CTL; high values) and incubations with 1 μM Somatosatin as controls for full inhibition and background (0% CTL; low values). After approximately 10-15 min incubation time 3 μl Forskolin (dissolved in DMSO, final conc. 15 μM) is added. Then the plates are shaken briefly and incubated for 60 min at room temperature. After 60 min 10 μl of the detection mix is added into all wells followed by an additional incubation period of 1 h. The plates are read in a suitable plate reader.The analysis of the data is based on the ratio of the time-resolved fluorescence measurements of donor and acceptor fluorophore (Ex: 320 nm; Em1: 665 nm; Em2: 615 nm; ratio 665/615). From this ratio, cAMP concentrations are calculated from standard curve and the EC50 is estimated by least square curve fit program.
Measurement: EC50 = 423 nM (pChEMBL 6.369999885559082)
Descriptors: MW 361.44, ALogP 3.38, HBA 4, HBD 2, PSA 63.25, QED 0.75
Compound (SMILES): C=CC(=O)N1CC[C@H](n2nc(C#Cc3cc(OC)cc(OC)c3)c(C(N)=O)c2NCC(F)(F)F)C1
InChIKey: JXSFGLJKCOYKCU-HNNXBMFYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: FGFR1 Activity Inhibition Test: The reaction buffer contains the following components: 5-fold diluted enzymatic buffer/kinase 5x(Cisbio, Cat No. 62EZBFDD)(its main component is 50nM HEPES, pH 7.0), 5mM MgCl2 and 1 mM DTT, a human recombinant FGFR1 catalytic domain protein (amino acid 308-731) which was purified by the company, a0.6ng/uL kinase solution diluted by the reaction buffer; a substrate reaction solution containng 400 nM biotinylated tyrosine kinase substrate diluted by the reaction buffer (Cisbio, Cat. No. 62TKOPEC) and 40 uM ATP; a test solution containng 0.125 ng/uL Eu3+ labeled cage antibody (Cisbio, Cat. No 61T66KLB) diluted by a test buffer (Cisbio, Cat No 62SDBRDF), and 25nM streptavidin labeled XL665 (Cisbio, Cat. No. 610SAXLB).
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 491.47, ALogP 2.33, HBA 7, HBD 2, PSA 111.71, QED 0.45
Compound (SMILES): CC(C)(C)c1ccc2nc(CCc3cnc4ccccc4n3)[nH]c2c1
InChIKey: VGZXGYNVAYDDBO-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Fluorescence Polarization Assay: The activities of the compounds were determined using the Molecular Devices IMAP PDE Fluorescence Polarization assay using recombinant human PDE-10 enzyme expressed in a baculoviral system. Briefly, 10 μL of a compound (0.2 nM-20 μM) was added to either a 96-well half area black plate or a 384-well black plate along with 10 μL of Fluorescein-labeled cAMP/cGMP substrate as per manufacturer's instructions and 10 μL of PDE enzyme (activity 0.1 U). Following a 40-minute incubation at 37° C., 60 μL of IMAP binding reagent was added. The plate was then read on a Perkin Elmer Victor (480-535 nm). The data was analyzed using Prism Software (GraphPad Inc, San Diego, Calif.).
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 330.44, ALogP 4.59, HBA 3, HBD 1, PSA 54.46, QED 0.6
Compound (SMILES): CC(C)n1cc(C(=O)Nc2ccc(-c3cc(C4CCN(CC(=O)N(C)C)CC4)n4ncnc(N)c34)cc2)c(=O)n(-c2ccccc2)c1=O
InChIKey: BXTLIILQMFIVCN-UHFFFAOYSA-N
Target: Tyrosine-protein kinase receptor UFO (SINGLE PROTEIN), Homo sapiens
UniProt: P30530
Assay: TAM Enzymatic Assay: The kinase assay buffer contained 50 mM HEPES, pH7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% NP-40 and 2 mM DTT. 0.1 ul test compounds dissolved in DMSO were transferred from compound plates to white 384-well assay plates (Greiner LLIMITRAC plates). The final concentration of DMSO was 1.25%. Enzyme solutions of 5.1 nM phosphor-Axl, or 0.0625 nM c-Mer (Carna Biosciences, 08-108), or 0.366 nM Tyro3 (Life Technologies, PR7480A) were prepared in assay buffer. A 1 mM stock solution of peptide substrate Biotin-EQEDEPEGDYFEWLE-amide SEQ ID NO: 1 (Quality Controlled Biochemicals, MA) dissolved in DMSO was diluted to 1 uM in assay buffer containing 2000 uM ATP. 4 ul enzyme solution (or assay buffer for the enzyme blank) was added to the appropriate wells in each plate, and then 4 ul/well substrate solution was added to initiate the reaction. The plate was protected from light and incubated at room temperature for 60 min. The reaction was stopped by adding 4 ul detection solution containing 50 mM Tris-HCl, pH7.8, 150 mM NaCl, 0.05% BSA, 45 mM EDTA, 180 nM SA-APC (Perkin Elmer, CR130-100) and 3 nM Eu-W1024 anti-phosphotyrosine PY20 (Perkin Elmer, AD0067). The plate was incubated for 1 h at room temperature, and HTRF (homogenous time resolved fluorescence) signal was measured on a PHERAstar FS plate reader (BMG labtech). Percentage of inhibition was calculated for each concentration and IC50 value was generated from curve fitting with GraphPad Prism software.
Measurement: IC50 > 100 nM (pChEMBL 7)
Descriptors: MW 649.76, ALogP 3.39, HBA 11, HBD 2, PSA 152.86, QED 0.26
Compound (SMILES): CC(C)Nc1c(-c2cscn2)nnc2cc(-c3ccc(S(C)(=O)=O)cc3)ccc12
InChIKey: BTULEQGTMYPOON-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: In Vitro Kinase Activities (LRRK2 TR-FRET Peptide Assay): Compounds as described herein (compounds of Formula I, e.g., compounds of the above Examples) are tested for their in vitro kinase activities using various LRRK2 (including LRRK2 G2019S mutant) assays. For example, assays were performed in a total volume of 20 μL using the same kinase reaction buffer (50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM EGTA, 2 mM DTT and 0.01% Tween-20) for wild-type or G2019S mutant LRRK2. Serially diluted compounds (1% DMSO as co-solvent) were pre-incubated with recombinant GST-LRRK2 (wild-type, or G2019S mutant, Invitrogen) for 15 minutes at room temperature in 384-well Corning black plates. Mixtures of ATP and biotin-LRRKtide substrate (biotin-RLGRDKYKTLRQIRQ) (SEQ ID NO: 1) were added to the wells at a final concentration of 100 μM ATP and 100 nM substrate, with final kinase concentration of 10 nM. The kinase reactions were carried out at room temperature for 60 minutes, then the reaction was stopped with the addition of 10 μL/well of stop/detection buffer (25 mM HEPES pH 7.5, 66 mM EDTA, 0.8 M KF and 0.1% BSA) that contains streptavidin-XL665 (12.5 nM) and europium-conjugated phospho-specific antibody (2nM). The plates were read 1 hour later and the time-resolved fluorescence (665 nm to 615 nm ratio) measured using an Envision reader. The inhibition of each well was calculated using the control and background readings for that plate. IC50 values were determined from dose-response curves using eight concentrations from the serial dilution of the test compounds.Many compounds of the Examples are demonstrated to be inhibitors of LRRK2, with most of the compounds tested typically measuring an IC50 below 1 μM for both LRRK2 and G2019S mutant LRRK2 kinase activity.
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 424.55, ALogP 4.64, HBA 7, HBD 1, PSA 84.84, QED 0.5
Compound (SMILES): CS(=O)(=O)N1CCC(Nc2ncc(C(F)(F)F)c(-c3cn(-c4ccc(CN5CC6CCC5CO6)cc4Cl)cn3)n2)CC1
InChIKey: VTMMHSLYFZLWKO-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 2/cyclin E1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24864
Assay: HTRF Enzyme Activity Assay: CDK2/Cyclin E1 enzyme activity assays utilize full-length human CDK2 co-expressed as N-terminal GST-tagged protein with FLAG-Cyclin E1 in a baculovirus expression system (Carna Product Number 04-165). Assays were conducted in white 384-well polystyrene plates in a final reaction volume of 8 μL. CDK2/Cyclin E1 (0.25 nM) was incubated with the compounds of the Examples (40 nL serially diluted in DMSO) in the presence of ATP (50 μM or 1 mM) and 50 nM ULight-labeled eIF4E-binding protein 1 (THR37/46) peptide (PerkinElmer) in assay buffer (containing 50 mM HEPES pH 7.5, 1 mM EGTA, 10 mM MgCl2, 2 mM DTT, 0.05 mg/mL BSA, and 0.01% Tween 20) for 60 minutes at room temperature. The reactions were stopped by the addition of EDTA and Europium-labeled anti-phospho-4E-BP1 antibody (PerkinElmer), for a final concentration of 15 mM and 1.5 nM, respectively. HTRF signals were read after 1 hour at room temperature on a PHERAstar FS plate reader (BMG Labtech). Data was analyzed with IDBS XLFit and GraphPad Prism 5.0 software using a three or four parameter dose response curve to determine IC50 for each compound.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 626.11, ALogP 4.2, HBA 9, HBD 1, PSA 105.48, QED 0.42
Compound (SMILES): Cc1ccc(C(F)(F)F)cc1C1CC(O)c2nc3ccc(-c4ccc(C(C)(C)O)cc4)nc3n21
InChIKey: JBBNVIMXSSPSFU-UHFFFAOYSA-N
Target: Tumor necrosis factor (SINGLE PROTEIN), Homo sapiens
UniProt: P01375
Assay: TNF induced HEK-Blue assay: Test compounds serially diluted in DMSO were plated in an assay plate (Labcyte, Cat. #LP-0200) at final concentrations ranging from 0.004 μM to 25 μM. TNFα (final concentration 0.5 ng/ml) or CD40L (final concentration 30 ng/ml) in assay buffer [DMEM, 4.5 g/l glucose (Gibco, Cat. 21063-029), 10% FBS (Sigma, F4135), 1% Penicillin-Streptomycin (Gibco, Cat. 15140-122), 1% Anti-Anti (Gibco, Cat. 15240-112) and 2 mM L-glutamine (Gibco, Cat. 25030-081)] was then added to the assay plate. After a 30 minute pre-incubation at 37° C. and 5% CO2, HEK-Blue-CD40L cells (InvivoGen, Cat. Code hkb-cd40) containing a NF-κB-driven secreted alkaline phosphatase reporter gene were seeded into the assay plate at a density of 20,000 cells per well. This plate was then incubated for 18 h at 37° C. and 5% CO2. Secreted alkaline phosphatase expression was measured using QUANTI-Blue (InvivoGen, Cat. Code rep-qb1) according to manufacturer's specifications and the assay plate was read on a PerkinElmer Envision at 620 nm.Inhibition data for the test compound over a range of concentrations was plotted as percentage inhibition of the test compound (100%=maximum inhibition). IC50 values were determined after correcting for background [(sample read−mean of low control)/(mean of high control−mean of low control)] where by the low control is DMSO without stimulation and high control is DMSO with stimulation. The IC50 is defined as the concentration of test compound which produces 50% inhibition and was quantified using the 4 parameter logistic equation to fit the data.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 467.49, ALogP 5.68, HBA 5, HBD 2, PSA 71.17, QED 0.41
Compound (SMILES): CC(C)c1c(-c2cn(CCS(C)(=O)=O)c3ncccc23)[nH]c2ccc(C3CCC(N4CC5(COC5)C4)CC3)nc12
InChIKey: NAPSKFBBBOUWJV-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: TLR7/8/9 Inhibition Reporter Assays: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 561.75, ALogP 5.11, HBA 7, HBD 1, PSA 93.11, QED 0.34
Compound (SMILES): CN1CCCC1CCOC(=O)Nc1ccc(C(F)(F)F)cc1-c1ccc(F)cc1
InChIKey: BEYBEVJMGRCLRP-UHFFFAOYSA-N
Target: Muscarinic acetylcholine receptor M3 (SINGLE PROTEIN), Homo sapiens
UniProt: P20309
Assay: Binding Assay: Cell membrane proteins (Perkin Elmer) wherein human muscarinic M3 receptor was overexpressed, [3H]-methyl scopolamine and test compounds in various concentration were cultured in 0.2 ml of Tris-HCl buffer at 25° C. for 120 minutes. The same was filtered under suction through glass fiber filter (Whatman GF/B), and then the filter was washed 5 times with 1 ml of Tris-HCl buffer. The radioactivity of [3H]-methyl scopolamine adsorbed on the filter was measured by a liquid scintillation counter. Non-specific binding was evaluated under existence of 5 μM of atropine. Affinity of the compound of the present invention to muscarinic M3 receptor was calculated as the dissociation constant (Ki), which can be calculated from concentration (IC50) of test compounds inhibiting 50% of binding of [3H]-methyl scopolamine (i.e. labeled ligand) according to Cheng and Prusoff [Cheng and Prusoff, Biochem. Pharmacol., 22, 3099, 1973]. In following Table, compounds having stronger binding affinity to human muscarinic M3 receptor have lower dissociation constant (Ki).
Measurement: Ki = 258 nM (pChEMBL 6.590000152587891)
Descriptors: MW 410.41, ALogP 5.54, HBA 3, HBD 1, PSA 41.57, QED 0.66
Compound (SMILES): CC1(NS(=O)(=O)c2ccc(-c3c(Cl)cc(Nc4n[nH]c(N)n4)cc3C(F)(F)F)cc2)CN(C(=O)OC(C)(C)C)C1
InChIKey: XOZBTNIZQOSNEW-UHFFFAOYSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1a (isolate H) (HCV)
UniProt: P27958
Assay: Pseudotyped HCV particle (HCVpp) reporter assay: Plasmids expressing HCV E1 and E2 envelope proteins of GT1a H77 strain (Proc Natl Acad Sci USA 1997 94:8738-43) or GT1b Con1 strain (Science 1999 285:110-3) were constructed by cloning the nucleic acids encoding the last 60 amino acids of HCV core protein and all of the HCV E1 and E2 proteins into pcDNA3.1(+) vector. Plasmid pVSV-G expressing the glycoprotein G of the vesicular stomatitis virus (VSV G) is from Clontech (cat #631530). The HIV packaging construct expressing the firefly luciferase reporter gene was modified based on the envelope defective pNL.4.3.Luc-Râ¿¿.Eâ¿¿ vector (Virology 1995 206:935-44) by further deleting part of the HIV envelope protein.
Measurement: EC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 602.04, ALogP 4.76, HBA 8, HBD 4, PSA 155.33, QED 0.32
Compound (SMILES): Cc1cc2c(cc1C#N)c(-c1cccc3c1CC(C)(C)CN3)cn2[C@H]1CC[C@H](O)CC1
InChIKey: WGOSMGXZXLHDLF-MXVIHJGJSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: FL-LSD1 LC-MS Assay: Representative compounds of the present invention were serially and separately diluted 3-fold in DMSO to obtain a total of twelve concentrations. Then the test compounds at each concentration (100 nL of each) were transferred into a 384-well Perkin Elmer ProxiPlate 384 plus plates by Mosquito Solutions (5 μL) of 0.8 nM, the full-length LSD1 and 0.5 μM FAD in reaction buffer (40 mM Tris-HCl, 0.01% Triton-×100.10 mM KCl, 1 mM DTT) were added into the wells and then incubated with the test compound for 30 min. A 5 μL solution of 1 μM of the peptide substrate H3K4me1 (histone H3[1-21]-biotin) in reaction buffer was added to each initiate reaction. The final components in the reaction solution include 0.4 nM FL-LSD1, 0.25 μM FAD, and 0.5 μM H3K4me1 peptide with varying concentration of the compounds. A positive control consisted of the enzyme, 0.25 μM FAD and 0.5 μM substrate in the absence of the test compound, and a negative control consisted of 0.5 μM substrate only. Each reaction was incubated at room temperature for 60 min, and then stopped by the addition of 3 μL quench solution (2.5% TFA with 320 nM d4-SAH). The reaction mixture was centrifuged (Eppendorf centrifuge 5810, Rotor A-4-62) for 1 min at 2000 rpm and read on an API 4000 triple quadrupole mass spec with Turbulon Spray (Applied Biosystem) coupled with Prominence UFLC (Shimadzu).
Measurement: IC50 = 185 nM (pChEMBL 6.730000019073486)
Descriptors: MW 413.57, ALogP 5.96, HBA 4, HBD 2, PSA 60.98, QED 0.54
Compound (SMILES): CC(C)N(C)C(=O)c1nc(C(=O)NCC(C)(C)O)sc1-c1ccc(C(O)(C(F)(F)F)C(F)(F)F)c(Cl)c1Cl
InChIKey: VPAAXHQWKPTSME-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: ThermoFluor Assay: Project 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 (1×=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: Kd = 6.1 nM (pChEMBL 8.210000038146973)
Descriptors: MW 610.4, ALogP 5.41, HBA 6, HBD 3, PSA 102.76, QED 0.36
Compound (SMILES): Cc1cnc(NC(=O)c2ccc(-n3c(=O)n(-c4cccc(N(C)C(=O)/C=C/CN(C)C)c4)c4ncnc(N)c43)cc2)s1
InChIKey: WIWXQCBIUNESPC-RMKNXTFCSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: In Vitro Inhibitory Assay: Human Btk kinase (Genbank accession # NP_000052) was purified from insect cells as a full-length construct containing a N-terminal 6×-His tag. Btk kinase activity was determined using a radiometric filter binding assay. Measurements are performed in a low μL reaction volume 384-well assay plates. BTK enzyme (8 nM final in reaction), inhibitor (at requested doses), and 0.2 mg/mL peptide substrate (Poly-Glu-Tyr, 4:1 ratio) are incubated in a reaction buffer composed of 20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO for 15 min. followed by addition of 1 μM ATP to start the assay. Kinase reactions are carried out for 120 min. at RT. The reaction was stopped by spotting of reaction sample onto P81 cationic exchange paper (Whatman). Unbound phosphate was removed by extensive washing of filters in 0.75% Phosphoric acid. After subtraction of background derived from control reactions containing inactive enzyme (via addition of saturating EDTA), kinase activity data for each dose of compound tested was expressed as the percent of remaining kinase activity in test samples compared to vehicle (dimethyl sulfoxide) reactions.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 583.68, ALogP 3.25, HBA 11, HBD 2, PSA 144.27, QED 0.27
Compound (SMILES): CC(C)C[C@H](N)[C@](O)(Cc1cc(SCCC2CC2)ccn1)C(=O)O
InChIKey: DDVJLKHYPSRVSO-FUHWJXTLSA-N
Target: Leucyl-cystinyl aminopeptidase (SINGLE PROTEIN), Rattus norvegicus
UniProt: P97629
Assay: Inhibition of IRAP Assay: Rat epididymal fat pads were homogenized and subjected to ultracentrifugation at 100,000×g for 30 minutes to obtain microsomes containing IRAP. The microsomes (with a total protein content of 55 g/well) were mixed with a solvent (dimethyl sulfoxide; hereinafter, abbreviated as DMSO (final concentration: 0.1%)) or with each test compound (common ratio: 3; maximum concentration: 10 μM). AVP was then added to the solution to a final concentration of 25 μM, and the resulting solution was allowed to react for one hour at 37° C. An aqueous trifluoroacetic acid (hereinafter, abbreviated as TFA) solution was then added to the solution (final concentration: 1%) to stop the enzymatic reaction. Residual AVP was then determined by mass spectrometry (MALDI-MS). Based on the results, IC50 values (nM), i.e. concentrations required for 50% inhibition of decrease in AVP level in the solvent control group, of the individual test compounds were calculated by the logistic regression to evaluate inhibition of IRAP activity. As comparative examples, similar tests were performed with the compounds of the Reference Examples 1 and 2 described below: (2S,3S)-3-amino-2-hydroxy-5-methyl-2-({4-[(trans-4-methylcyclohexyl)oxy]pyridin-2-yl}methyl)hexanoic acid dihydrochloride; and (2S,3R)-3-amino-2-hydroxy-5-methyl-2-({4-[(trans-4-methylcyclohexyl)oxy]pyridin-2-yl}methyl)hexanoic acid hydrochloride, respectively.
Measurement: IC50 = 3.2 nM (pChEMBL 8.489999771118164)
Descriptors: MW 352.5, ALogP 2.71, HBA 5, HBD 3, PSA 96.44, QED 0.56
Compound (SMILES): COC(=O)c1ccnc2cc(C3(O)CCCCC3)[nH]c12
InChIKey: VTOAKTBHCSDFGQ-UHFFFAOYSA-N
Target: Lysine-specific demethylase 5A (SINGLE PROTEIN), Homo sapiens
UniProt: P29375
Assay: Enzymatic Assay: The enzymatic assay of Jarid1A activity is based upon Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) detection. The ability of test compounds to inhibit the activity of Jarid1A was determined in 384-well plate format under the following reaction conditions: 1 nM Jarid1A, 300 nM H3K4me3-biotin labeled peptide (Anaspec cat #64357), 2 μM alpha-ketoglutaric acid in assay buffer of 50 mM HEPES, pH7.3, 0.005% Brij35, 0.5 mM TCEP, 0.2 mg/ml BSA, 50 μM sodium L-ascorbate, and 2 μM ammonium iron(II) sulfate. Reaction product was determined quantitatively by TR-FRET after the addition of detection reagent Phycolink Streptavidin-allophycocyanin (Prozyme) and Europium-anti-mono- or di-methylated histone H3 lysine 4 (H3K4me1-2) antibody (PerkinElmer) in the presence of 5 mM EDTA in LANCE detection buffer (PerkinElmer) at a final concentration of 25 nM and 1 nM, respectively.The assay reaction was initiated by the following: 2 μl of the mixture of 900 nM H3K4me3-biotin labeled peptide and 6 μM alpha-ketoglutaric acid with 2 μl of 11-point serial diluted inhibitor in 3% DMSO was added to each well of plate, followed by the addition of 2 μl of 3 nM Jarid1A to initiate the reaction.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 274.32, ALogP 2.5, HBA 4, HBD 2, PSA 75.21, QED 0.82
Compound (SMILES): CC(C)c1nc(-c2ccc(O)c(Nc3ncnn4ccc(CN5CCC(N)CC5)c34)c2)no1
InChIKey: AOLOWAUIBIHZCK-UHFFFAOYSA-N
Target: AP2-associated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q2M2I8
Assay: Kinase Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 μl prepared from 15 μl additions of enzyme and substrates (fluoresceinated peptide (5-FAM)-Aha-KEEQSQITSQVTGQIGWR-NH2 and ATP) and test compounds in assay buffer (10 mM Tris-HCL pH 7.4, 10 mM MgCl2, 0.01% Tween-20 and 1.0 mM DTT). The reactions were initiated by the combination of bacterially expressed, GST-Xa-hAAK1 with substrates and test compounds. The reactions were incubated at room temperature for 3 hours and terminated by adding 60 μl of 35 mM EDTA buffer to each sample. The reactions were analyzed on the Caliper LabChip 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to EDTA quenched control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assays are ATP, 22 μM; (5-FAM)-Aha-KEEQSQITSQVTGQIGWR-NH2, 1.5 μM; GST-Xa-hAAK1, 3.5 nM; and DMSO, 1.6%. 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.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 448.53, ALogP 3.28, HBA 10, HBD 3, PSA 130.63, QED 0.38
Compound (SMILES): C=CC(=O)N1CCN(CCCc2cn3c(n2)c(-c2c(Cl)c(OC)cc(OC)c2Cl)cc2cnc(NCC)nc23)CC1
InChIKey: VPVHSKPAISNXDR-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: Inhibitory Activities Assay: (1) Preparation of 1×Kinase buffer; (2) Preparation of the gradient concentrations of the compounds: the concentration of the compounds to be tested started from 10 μM, which was 3-fold diluted to prepare 10 dilutions, and then tested in duplicated wells. The compounds were 100-fold serially diluted into 10 dilutions with different final concentrations in 96-well plates, each of which was further diluted with 1×Kinase buffer to prepare an intermediate dilution having a concentration of 5 times the final concentration; (3) adding 5 μL of each of the prepared compound solution into the compound wells of 384-well plates, and each concentration set single well; adding 5 μL of 5% DMSO into negative control wells and positive control wells respectively; (4) preparing a kinase solution having a concentration of 2.5 times the final concentration with 1×kinase buffer; (5) adding 10 μL of kinase solution at a concentration of 2.5 times the final concentration into the compound wells and the positive control wells respectively; and adding 10 μL of 1×Kinase buffer into the negative control wells; (6) centrifuging at 1000 rpm for 30 seconds, and incubating at room temperature for 10 minutes after shaking and mixing evenly; (7) preparing a mixed solution of ATP and Kinase substrate 22 having a concentration of 2.5 times the final concentration with 1×Kinase Buffer; (8) adding 10 μL of the mixed solution of ATP and substrate at a concentration of 2.5 times the final concentration to initiate the reaction; (9) Centrifuging the 384-well plates at 1000 rpm for 30 seconds, and incubating at 28° C. for corresponding time respectively after shaking and mixing; (10) adding 30 μL of stop detection solution to stop the kinase reaction, centrifuging at 1000 rpm for 30 seconds, and shaking and mixing the mixture evenly; (11) reading the conversion rate by Caliper Ezreader II. The dose-effect curve was fitted by the log (inhibitor) vs. response-variable slope of GraphPad Prism 5 with the log value of concentration as the X axis and the percentage inhibition rate as the Y axis, then the IC50 value of each compound on the enzyme activity was obtained.
Measurement: IC50 < 20 nM (pChEMBL 7.698999881744385)
Descriptors: MW 598.54, ALogP 4.96, HBA 9, HBD 1, PSA 97.12, QED 0.26
Compound (SMILES): NC1=N[C@](CF)(c2cc(NC(=O)c3ccn(C(F)F)n3)ccc2F)C(F)(F)CC1
InChIKey: YABHBFKTFCFJMS-MRXNPFEDSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE1 Binding Assay: The binding assay was performed as SPA-based assay using a biotinylated form of human BACE1 recombinantly expressed and subsequently purified from Freestyle HEK293 cells. The binding assay was run in a 50 mM sodium acetate buffer, pH 4.5 containing 50 mM NaCl and 0.03% Tween-20 in white clear bottom 384 plates (Corning #3653). 10 nM (final concentration) radioligand ([3H]-N-((1S,2R)-1-benzyl-3-cyclopropylamino-2-hydroxy-propyl)-5-(methanesulfonyl-methyl-amino)-N-((R)-1-phenyl-ethyl)-isophthalamide) (TRQ11569 purchased from GE Healthcare) was mixed with test compound at a given concentration, 6 nM (final concentration) human BACE1 and 25 μg Streptavidin coated PVT core SPA beads (RPNQ0007, GE Healthcare Life Sciences) in a total volume of 40 μl. Several concentrations of each test compound were tested in the assay for IC50 determination. The plates were incubated for one hour at room temperature and counted in a Wallac Trilux counter. Total and non-specific binding were determined using buffer and 1 μM (final concentration) of the high affinity BACE1 reference inhibitor (S)-6-[3-chloro-5-(5-prop-1-ynyl-pyridin-3-yl)-thiophen-2-yl]-2-imino-3,6-dimethyl-tetrahydro-pyrimidin-4-one, respectively. For each test compound, a IC50 value (the concentration mediating 50% inhibition of the specific binding of the radioligand) was determined from concentration-response curve and used to calculate the Ki from the equation Ki=IC50/(1+L/Kd), where L and Kd are the final concentration of the radioligand used in the assay and the dissociation constant of the radioligand, respectively. The Kd of the radioligand was determined from saturation binding experiments.
Measurement: Ki = 25 nM (pChEMBL 7.599999904632568)
Descriptors: MW 419.33, ALogP 3.62, HBA 5, HBD 2, PSA 85.3, QED 0.73
Compound (SMILES): C[C@H](O)C(=O)N1CC[C@H](Oc2ccc(-c3ncnc(Nc4cnn(C5COC5)c4)n3)cc2C#N)[C@H](F)C1
InChIKey: MZKFOXRCMQMVAM-ZKYUUJBMSA-N
Target: Serine/threonine-protein kinase TBK1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UHD2
Assay: TBK1 and IKKE Biological Assays: Enzymatic activity of IKKε and TBK1 was measured using a homogeneous time resolved fluorescence resonance energy transfer (TR-FRET) assay that monitors enzyme dependent phosphorylation of a biotinylated serine/threonine peptide substrate. An increase in the amount of phosphorylated peptide results in an increase in TR-FRET signal. TBK1 and IKKε were expressed and purified as full length recombinant proteins. Detection reagents for the assay were purchased from Cisbio. TBK1 and IKKε enzymes were assayed under initial rate conditions in the presence of 2×Km ATP (40-80 μM) and 1 μM peptide, hepes (pH 7), 0.1 mM orthovanadate, 0.02% NaN3, 0.01% BSA, 10 mM MgCl2, 0.01% (v/v) tritonX, 1 mM dithiothreitol, 0.5% (v/v) DMSO at the following concentrations for each enzyme: TBK1 at 2.5 nM and IKKε at 0.3 nM. After an assay reaction time of 240 minutes at 25° C., reactions were terminated with EDTA.Amount of phosphorylated peptide was determined by the addition of 125 nM streptavidin XL665 and europium cryptate labeled anti-phospho monoclonal antibody and the resulting TR-FRET signal was recorded on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 μs delay and 200 μs read window). Data was normalized based on a positive (1 μM Staurosporine) and negative (DMSO) controls and IC50 values calculated from the fit of the dose-response curves to a four-parameter equation. All IC50 values represent geometric mean values of a minimum of four determinations.
Measurement: IC50 = 7000 nM (pChEMBL 5.159999847412109)
Descriptors: MW 508.51, ALogP 1.62, HBA 11, HBD 2, PSA 151.31, QED 0.48
Compound (SMILES): CNC(=O)c1ccc(N2CCN([C@@H](C)c3ccc4c(c3)OCO4)CC2)nc1
InChIKey: WSJYHYJSBKTVLP-AWEZNQCLSA-N
Target: Protein O-GlcNAcase (SINGLE PROTEIN), Homo sapiens
UniProt: O60502
Assay: Human O-GlcNAcase enzyme inhibition assay: 5 μl of the appropriate concentration of a solution of inhibitor in McIlvaine's Buffer (pH 6.5) in 2% DMSO (for a dose response curve calculation) is added into each well of a 384-well plate (Greiner, 781900). Then, 20 nM of His-Tagged hOGA and 10 μM of FL-GlcNAc (Fluorescein mono-beta-D-(2-deoxy-2-N-acetyl) glucopyranoside; Marker Gene Technologies Inc, M1485) were added to the 384-well plate for a final volume of 20 μl. After incubation for 60 min at room temperature, the reaction was terminated by the addition of 10 μL of stop buffer (200 mM glycine, pH 10.75). The level of fluorescence (λexc 485 nm; (λemm 520 nm) was read on a PHERAstar machine. The amount of fluorescence measured was plotted against the concentration of inhibitor to produce a sigmoidal dose response curve to calculate an IC50. All individual data was corrected by subtraction of the background (Thiamet 3 uM=100% inhibition) whilst 0.5% DMSO was considered as the control value (no inhibition).
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 368.44, ALogP 2.05, HBA 6, HBD 1, PSA 66.93, QED 0.89
Compound (SMILES): CCCN(CCC)C(=O)C1=Cc2ccc(-c3cncnc3)cc2N=C(N)C1
InChIKey: YUZNJNKXFWNHEG-UHFFFAOYSA-N
Target: Toll-like receptor 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR97
Assay: Pharmacological Test: For TLR8 and TLR7 activity testing, HEK-Blue human TLR8 or TLR7 cells (Invivogen, San Diego, Calif., USA) are used, respectively. These cells are designed for studying the stimulation of human TLR8 or TLR7 by monitoring the activation of NF-κB. A SEAP (secreted embryonic alkaline phosphatase) reporter gene is placed under the control of the IFN-b minimal promoter fused to five NF-κB and AP-1-binding sites. Therefore the reporter expression is regulated by the NF-κB promoter upon stimulation of human TLR8 or TLR7 for 20 hours. The cell culture supernatant SEAP reporter activity was determined using Quanti Blue kit (Invivogen, San Diego, Ca, USA) at a wavelength of 640 nm, a detection medium that turns purple/blue in the presence of alkaline phosphatase. EC50 values were determined using Activity Base analysis (ID Business Solution, Limited).
Measurement: EC50 = 39 nM (pChEMBL 7.409999847412109)
Descriptors: MW 363.47, ALogP 3.57, HBA 5, HBD 1, PSA 84.47, QED 0.85
Compound (SMILES): COc1cn(Cc2ccccc2)c(Nc2cc(Cl)ccc2C)nc1=O
InChIKey: CBPIGEZCHXXKKG-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 355.83, ALogP 4.01, HBA 5, HBD 1, PSA 56.15, QED 0.75
Compound (SMILES): C[C@H]1Cn2c(nnc2-c2csc(C(F)(F)F)n2)C(=O)N1Cc1cccc(C(F)(F)F)c1Cl.S
InChIKey: HOJYUDXNXJOINY-QRPNPIFTSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: P2X7 FLIPR Assay: 1321N1 cells expressing the recombinant human or rat P2X7 channel was cultured in HyQ DME/(HyClone/Dulbecco's Modified Eagle Medium) high glucose supplemented with 10% Fetal Bovine Serum (FBS) and appropriate selection marker. Cells were seeded at a density of 25000 cells/well (96-well clear bottom black walled plates) in 100 μl volume/well. On the day of the experiment, cell plates were washed with assay buffer, containing (in mM): 130 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 5 glucose; pH 7.40 and 300 mOs. After the wash, cells were loaded with the Calcium-4 dye (Molecular Device) and incubated in the dark for 60 minutes. Test compounds were prepared at 250× the test concentration in neat DMSO. Intermediate 96-well compound plates were prepared by transferring 1.2 μL of the compound into 300 μL of assay buffer. A further 3× dilution occurred when transferring 50 μL/well of the compound plate to 100 μL/well in the cell plate. Cells were incubated with test compounds and dye for 30 minutes. Calcium dye fluorescence was monitored in FLIPR as the cells were challenged by adding 50 μL/well of BzATP (final concentration is 250 μM BzATP (human and rat)). The fluorescence change was measured 180 seconds after adding the agonist. Peak fluorescence was plotted as a function of BzATP concentration using Origin 7 software.
Measurement: IC50 = 977 nM (pChEMBL 6.010000228881836)
Descriptors: MW 529.92
Compound (SMILES): CNC(=O)O[C@@H]1CCC[C@H]1[C@@](C#N)(c1ccccc1)C1CCN(CC2CN(c3ccc(S(=O)(=O)c4ccncc4)cc3)C2)CC1
InChIKey: GWDOGFQBFWTQSO-POYCELQWSA-N
Target: Menin (SINGLE PROTEIN), Homo sapiens
UniProt: O00255
Assay: Fluorescence Polarization (FP) Competitive Binding Assay: A fluorescence polarization (FP) competitive binding assay was used to determine the binding affinities of representative menin inhibitors. A FAM labeled fluorescent probe was designed and synthesized based on a MLL1 peptide (FAM-MM2). Equilibrium dissociation constant (Kd) value of FAM-MM2 to menin protein was determined from protein saturation experiments by monitoring the total fluorescence polarization of mixtures composed with the fluorescent probe at a fixed concentration and the protein with increasing concentrations up to full saturation. Serial dilutions of the protein were mixed with FAM-MM2 to a final volume of 200 μl in the assay buffer (PBS with 0.02% Bovine γ-Globulin and 4% DMSO. 0.01% Triton X-100 was added right before assays). Final FAM-MM2 concentration was 2 nM. Plates were incubated at room temperature for 30 minutes with gentle shaking to assure equilibrium. FP values in millipolarization units (mP) were measured using the Infinite M-1000 plate reader (Tecan U.S., Research Triangle Park, N.C.) in Microfluor 1 96-well, black, v-bottom plates (Thermo Scientific, Waltham, Mash.) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Kd value of FAM-MM2, which was calculated by fitting the sigmoidal dose-dependent FP increases as a function of protein concentrations using Graphpad Prism 6.0 software (Graphpad Software, San Diego, Calif.), is determined as 1.4 nM.
Measurement: IC50 = 62 nM (pChEMBL 7.210000038146973)
Descriptors: MW 627.81, ALogP 5.05, HBA 8, HBD 1, PSA 115.63, QED 0.35
Compound (SMILES): Cc1n[nH]c2ncc(-c3cncc(N4CCCC4=O)n3)cc12
InChIKey: KAXRCKDFKFCWIX-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LRRK2 Kinase Assay: Procedure: Enzyme was incubated with substrate peptide in reaction buffer in the presence and absence of test compounds or Staurosporine. All additions were done on ice, followed by the addition of ATP mix. Wells were uniformly mixed using an Eppendorff plate shaker and incubated at 30° C. for 20 min, and stopped by the addition of 5 μl of 3% phosphoric acid. Volume was increased to 100 μl by adding 0.8% phosphoric acid which was then transferred to PC filter mats (Millipore), pre-equilibrated with 70% ethanol and water. Plates were washed thrice with 100 μl 0.8% phosphoric acid and dried for an hour at 60° C. 100 μl scintillation fluid was added into each well and reading taken in Perkin Elmer TOPCOUNT beta counter. The data analysis was performed by averaging the duplicate top count readings for each standard, negative, positive control (enzyme control) and samples and subtracting the average negative control from each reading which results in corrected values. A validation EC50 curve was generated by plotting CPM for each Staurosporine concentration on y-axis against the Log concentration of Staurosporine (nM) on the x-axis followed by a best fit curve through the points.
Measurement: IC50 > 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 294.32, ALogP 1.85, HBA 5, HBD 1, PSA 87.66, QED 0.78
Compound (SMILES): Cn1nnc2cccc(C(=O)N[C@H]3CC[C@H](CCN4CCc5nc(OCC(C)(F)F)sc5CC4)CC3)c21
InChIKey: NQOMONLOFJNENB-IYARVYRRSA-N
Target: D(3) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35462
Assay: Binding Inhibition for Dopamine D3 Receptor: Buffer solution: 50 mM Tris-HCl (35409-45, Nacalai Tesque) (pH 7.4) containing 120 mM NaCl (31320-05, Nacalai Tesque), 1 mM MgCl.6HO (20909-55, Nacalai Tesque), 5 mM KCl (28514-75, Nacalai Tesque) and 2 mM CaCl) (067-31, NAKARAI CHEMICALS, LTD.) Radioligand: (final concentration) 2 nM [H]-Methylspiperone ([H���N-methyl-]-Methylspiperone, NET-856, 83.8 Ci/mmol, PerkinElmer)Non-specific ligand: (final concentration) 10 uM Butaclamol [(+)-Butaclamol Hydrochloride, D033, Sigma] SPA beads solution: SPA beads [WGA PVT SPA Scintillation Beads, RPNQ0001 (500 mg), RPNQ0060 (2 g), PerkinElmer] (0.2 mg/well) Incubation time and temperature: 120 min at 25° C.
Measurement: Ki = 0.13 nM (pChEMBL 9.890000343322754)
Descriptors: MW 532.66, ALogP 4.24, HBA 8, HBD 1, PSA 85.17, QED 0.47
Compound (SMILES): O=C(c1ccc2nc(-c3ccccn3)oc2c1)N1CCN(c2nc3ccccc3c(=O)[nH]2)CC1
InChIKey: DUFMSWJTZFKBOV-UHFFFAOYSA-N
Target: Monoglyceride lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q99685
Assay: MAGL Inhibitory Activity: Compounds of the present invention are MAGL inhibitors. Thus, in one aspect, the present invention provides the use of compounds of Formula (I) or a pharmaceutically acceptable salt thereof as described herein for inhibiting MAGL in a mammal.In a further aspect, the present invention provides compounds of Formula (I) or a pharmaceutically acceptable salt thereof as described herein for use in a method of inhibiting MAGL in a mammal.In a further aspect, the present invention provides the use of compounds of Formula (I) or a pharmaceutically acceptable salt thereof as described herein for the preparation of a medicament for inhibiting MAGL in a mammal.In a further aspect, the present invention provides a method for inhibiting MAGL in a mammal, which method comprises administering an effective amount of a compound or a pharmaceutically acceptable salt thereof of Formula (I) as described herein to the mammal.Compounds were profiled for MAGL inhibitory activity by measuring the enzymatic activity of MAGL by following the hydrolysis of 4-nitorphenylacetate resulting in 4-nitrophenol, which absorbs at 405-412 nm (G. G. Muccioli, G. Labar, D. M. Lambert, Chem. Bio. Chem. 2008, 9, 2704-2710).The assay was carried out in 384 well assay plates (black with clear bottom, non-binding surface treated, Corning Ref. 3655) in a total volume of 40 μL. Compound dilutions were made in 100% DMSO (VWR Chemicals 23500.297) in a polypropylene plate in 3-fold dilution steps to give a final concentration range in the assay from 25 μM to 1.7 nM. 1 μL compound dilutions (100% DMSO) were added to 19 μL MAGL (recombinant wild-type) in assay buffer (50 mM TRIS (GIBCO, 15567-027), 1 mM EDTA (Fluka, 03690-100 ml)). The plate was shaked for 1 min at 2000 rpm (Variomag Teleshake) and then incubated for 15 min at RT. To start the reaction, 20 μL 4-Nitrophenlyacetate (Sigma N-8130) in assay buffer with 6% EtOH was added. The final concentrations in the assay were 1 nM MAGL and 300 μM 4-Nitrophenylacetate. After shaking (1 min, 2000 rpm) and 5 min incubation at RT, the absorbance at 405 nm was measured for a fist time (Molecular Devices, SpectraMax Paradigm). A second measurement was then done after incubation for 80 min at RT. From the two measurements, the slope was calculated by subtracting the first from the second measurement.
Measurement: IC50 = 2400 nM (pChEMBL 5.619999885559082)
Descriptors: MW 452.47, ALogP 3.09, HBA 7, HBD 1, PSA 108.22, QED 0.45
Compound (SMILES): CCOC(=O)N1CC=C(c2cccn3nc(Nc4ccc(C(=O)NCCc5cccnc5)cc4)nc23)CC1
InChIKey: PQPZUQSRVXSKCJ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 1.8 nM (pChEMBL 8.739999771118164)
Descriptors: MW 511.59, ALogP 4.09, HBA 8, HBD 2, PSA 113.75, QED 0.37
Compound (SMILES): Cc1nn(C)cc1-c1cc(C(=O)/N=c2\[nH]c3ccccc3n2C2CCCC2)ccn1
InChIKey: LIOWLWMKSLYHKL-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Biochemical EGFR Inhibition Assays (100 mM ATP): 5 μL of EGFR enzyme form in assay buffer (50 mM HEPES pH 7.3, 10 mM MgCl2, 1 mM EGTA, 0.01% Tween 20, 2 mM DTT) are dispensed into columns 1-23, than 5 μL of ATP and ULight-poly-GT substrate (PerkinElmer; TRF0100-M) mix in assay buffer is added to all wells (final assay concentration of the ULight-poly-GT substrate 200 nM). Each of the different EGFR enzyme form assays is available at high ATP levels (final assay concentration 100 μM). After 90 minutes incubation at room temperature 5 μL EDTA (final assay concentration 50 mM) and LANCE Eu-anti-P-Tyr (PT66) antibody (PerkinElmer, AD0069) (final assay concentration 6 nM) mix are added to stop the reaction and start the binding of the antibody. After additional 60 minutes incubation at room temperature the signal is measured in a PerkinElmer Envision HTS Multilabel Reader using the TR-FRET LANCE Ultra specs of PerkinElmer (used wavelengths: excitation 320 nm, emission 1 665 nm, emission 2 615 nm). Each plate contains 16 wells of a negative control (diluted DMSO instead of test compound; w EGFR enzyme form; column 23) and 16 wells of a positive control (diluted DMSO instead of test compound; w/o EGFR enzyme form; column 24). Negative and positive control values are used for normalization and IC50 values are calculated and analysed using a 4 parametric logistic model.
Measurement: IC50 = 14.2 nM (pChEMBL 7.849999904632568)
Descriptors: MW 400.49, ALogP 3.93, HBA 5, HBD 1, PSA 80.86, QED 0.57
Compound (SMILES): N#Cc1ccc(N2CCN(C(=O)CCc3nc4cc(F)ccc4c(=O)[nH]3)CC2)nc1
InChIKey: DDTJENLBLOWEGH-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: Scintillation Proximity Assay: The scintillation proximity assay (SPA) is designed to measure PARP activity using purified recombinant PARP-1 enzyme and is ideal for high throughput screening of small molecular inhibitors for drug discovery. Here, recombinant human PARP-1 or PARP-2 enzyme was incubated with substrate mix (NAD, 3H-NAD and biotinylated-NAD) and the [3H] and biotin-labeled ADP-ribose polymers were captured using Streptavidin-conjugated PVT SPA beads. In the absence of enzyme inhibition, 100% signal was obtained. Inhibitors are identified by a decrease in signal when PARP-1 or PARP-2 mediated poly-ADP ribose polymer formation is reduced.
Measurement: IC50 = 83 nM (pChEMBL 7.079999923706055)
Descriptors: MW 406.42, ALogP 1.61, HBA 6, HBD 1, PSA 105.98, QED 0.71