lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): c1ccc(CNC[C@H]2CC[C@H](c3nnn4cnc5[nH]ccc5c34)CC2)cc1
InChIKey: ZKPHIGUGQLUGBW-QAQDUYKDSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 360.46, ALogP 3.67, HBA 5, HBD 2, PSA 70.9, QED 0.57
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)c3cc(CO)nc(C(F)(F)F)n3)CC2)C1
InChIKey: MSTHLKLQMFFCQQ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: In vitro JAK Kinase Assay: The catalytic domains of human JAK1 (a.a. 837-1142), Jak2 (a.a. 828-1132) and Jak3 (a.a. 781-1124) with an N-terminal His tag were expressed using baculovirus in insect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3 was assayed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by homogenous time resolved fluorescence (HTRF). IC50s of compounds were measured for each kinase in the 40 microL reactions that contain the enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%) BSA. For the 1 mM IC50 measurements, ATP concentration in the reactions was 1 mM. Reactions were carried out at room temperature for 1 hr and then stopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, Mass.). Binding to the Europium labeled antibody took place for 40 minutes and HTRF signal was measured on a Fusion plate reader (Perkin Elmer, Boston, Mass.)
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 566.55, ALogP 2.35, HBA 10, HBD 2, PSA 152.74, QED 0.36
Compound (SMILES): COc1ccc(C(=O)N(C)C2(c3ccccc3)CCC(c3ccccc3)(N(C)C)CC2)cc1
InChIKey: IPZJSBVNBXSDFB-UHFFFAOYSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Binding Assay: The affinity to the human μ-opiate receptor was determined in a homogeneous preparation in microtiter plates. For this, dilution series of the respective compound to be tested were incubated for 90 minutes at room temperature with a receptor membrane preparation (15-40 mg of protein per 250 μl of incubation batch) of CHO-K 1 cells, which express the human μ-opiate receptor (RB-HOM receptor membrane preparation of NEN, Zaventem, Belgium), in the presence of 1 nmol/l of the radioactive ligand [3H'-naloxone (NET719, NEN, Zaventem, Belgium) and of 1 mg WGA-SPA beads (wheat germ agglutinin SPA beads from Amersham/Pharmacia, Freiburg, Germany) in a total volume of 250 μl. 50 mmol/l of tris-HCl supplemented by 0.05% by wt. of sodium azide and 0.06% by wt. of bovine serum albumin was used as incubation buffer. 25 μmol/l of naloxone were additionally added to determine the non-specific bond. After the ninety-minute incubation time had ended, the microtiter plates were centrifuged for 20 minutes at 1000 g and the radioactivity measured in a β-counter (Microbeta-Trilux, PerkinElmer Wallac, Freiburg, Germany).
Measurement: Ki = 1.2 nM (pChEMBL 8.920000076293945)
Descriptors: MW 442.6, ALogP 5.69, HBA 3, HBD 0, PSA 32.78, QED 0.49
Compound (SMILES): CC(N)(CSCCNC(=N)N)C(=O)O.O=C(O)C(F)(F)F.O=C(O)C(F)(F)F
InChIKey: OSWKVCNKIGCJLV-UHFFFAOYSA-N
Target: Nitric oxide synthase, inducible (SINGLE PROTEIN), Homo sapiens
UniProt: P35228
Assay: Inhibition of human iNOS expressed in Sf9 insect cells assessed as reduction in conversion of L-[2,3-3H]-arginine to L-[2,3-3H]-citrulline incubated for 15 mins by liquid scintillation counting method
Measurement: IC50 = 7200 nM (pChEMBL 5.139999866485596)
Descriptors: MW 220.3, ALogP -1, HBA 4, HBD 5, PSA 125.22, QED 0.22
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)Nc3ccccc3OC(F)(F)F)CC2)C1
InChIKey: YGGIBMIELOSOBZ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: In vitro JAK Kinase Assay: The catalytic domains of human JAK1 (a.a. 837-1142), Jak2 (a.a. 828-1132) and Jak3 (a.a. 781-1124) with an N-terminal His tag were expressed using baculovirus in insect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3 was assayed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by homogenous time resolved fluorescence (HTRF). IC50s of compounds were measured for each kinase in the 40 microL reactions that contain the enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%) BSA. For the 1 mM IC50 measurements, ATP concentration in the reactions was 1 mM. Reactions were carried out at room temperature for 1 hr and then stopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, Mass.). Binding to the Europium labeled antibody took place for 40 minutes and HTRF signal was measured on a Fusion plate reader (Perkin Elmer, Boston, Mass.)
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 565.56, ALogP 4.34, HBA 8, HBD 2, PSA 127.99, QED 0.36
Compound (SMILES): Cc1cccc2cc([C@H](C)Nc3ncnc4[nH]c(Cl)nc34)n(C3CC3)c(=O)c12
InChIKey: ZYCRCLGCVUXALL-NSHDSACASA-N
Target: PI3-kinase p110-alpha/p85-alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: P42336
Assay: Inhibition of N-terminal His6-tagged recombinant full-length human p110alpha/untagged recombinant full length human p85alpha expressed in baculovirus infected Sf21 cells using phosphatidylinositol as substrate in presence of gamma-32P-ATP after 5 mins by thin layer chromatographic method
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 394.87, ALogP 4.14, HBA 6, HBD 2, PSA 88.49, QED 0.51
Compound (SMILES): COc1ccc(C(=O)C2CCN(CC(=O)NCc3nc4c(c(=O)[nH]3)COCC4)CC2)cc1C
InChIKey: MMWOCAKTGVKRBA-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UGN5
Assay: Biochemical Assay: The autoparsylation activity of the TNKS 1/2 or PARP1/2 enzymes was measured by the liquid chromatography-mass spectrometry (LC/MS) detection of nicotinamide as readout. Compound activity in inhibiting the TNKS and PARP autoparsylation was evaluated by IC50 measurements. In the compound screening assays, the reaction is composed of 5 μL of compound in 8-point serial dilutions with concentrations ranging from 0.0086 to 18.75 μM, 20 nM of purified enzyme, and 250 μM of β-NAD+ in the 1× Assay Buffer. After 60 min incubation at room temperature, the reactions were quenched by the addition of 10 μL of 5× quenching solution (20% formic acid and 500 nM [d]-nicotinamide in water). For the background control wells, 10 μL of the 5× quenching solution per well was added prior to the addition of β-NAD+.
Measurement: IC50 = 4260 nM (pChEMBL 5.369999885559082)
Descriptors: MW 454.53, ALogP 1.37, HBA 7, HBD 2, PSA 113.62, QED 0.61
Compound (SMILES): COc1cc(N2CCOCC2)ccc1Nc1nccc(-c2c(-c3cccc(C(=O)Nc4c(F)cccc4F)c3)nc3ccccn23)n1
InChIKey: MZIRRPYGJKSMOV-UHFFFAOYSA-N
Target: Receptor tyrosine-protein kinase erbB-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P04626
Assay: Inhibition of recombinant human full length ErbB2 expressed in baculovirus expression system assessed as decrease in transfer of gamma-phosphate from ATP onto tyrosine residue of biotinylated peptide substrate preincubated for 1 hr followed by substrate addition measured after 90 mins by HTRF method
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 633.66, ALogP 6.58, HBA 9, HBD 2, PSA 105.91, QED 0.19
Compound (SMILES): COC(=O)c1cnc(N2CCN(c3nnc(-c4ccc(C(F)(F)F)cc4)c(C)c3C)C[C@H]2C)cn1
InChIKey: XQENYCWPJSUPCZ-CQSZACIVSA-N
Target: Protein smoothened (SINGLE PROTEIN), Homo sapiens
UniProt: Q99835
Assay: Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 486.5, ALogP 4.07, HBA 8, HBD 0, PSA 84.34, QED 0.51
Compound (SMILES): O=C(OCC1c2ncn(-c3ccccc3)c2CCN1C(=O)c1cccc(C(F)(F)F)c1Cl)c1cccc(C(F)(F)F)c1Cl
InChIKey: KEBGPPGXKDCYFN-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Ca2+ flux 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 = 3388 nM (pChEMBL 5.46999979019165)
Descriptors: MW 642.38, ALogP 7.81, HBA 5, HBD 0, PSA 64.43, QED 0.16
Compound (SMILES): c1ccc(CCNC2CCC(c3nnn4cnc5[nH]ccc5c34)CC2)cc1
InChIKey: RAYNZHXRGJCCNG-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 870 nM (pChEMBL 6.059999942779541)
Descriptors: MW 360.46, ALogP 3.46, HBA 5, HBD 2, PSA 70.9, QED 0.57
Compound (SMILES): CN1CN([C@H]2CC[C@H](CNCC(F)F)CC2)c2c(cnc3[nH]ccc23)C1=O
InChIKey: MKIDOWPCPNPANG-JOCQHMNTSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Enzyme Assay: The inhibitory activity of the compounds of the present invention against JAK was measured.The respective enzymes (JAK1, JAK2, JAK3 and Tyk2) were purchased from Carna Biosciences, Inc.As a substrate of the enzymes (hereinafter referred to as substrate), LANCE Ultra ULight-JAK-1 (Tyr1023) Peptide (manufactured by PerkinElmer Inc.) was used.As an antibody for detecting phosphorylation of the substrate, LANCE Ultra Europium-anti-phospho tyrosine antibody (PT66) (manufactured by PerkinElmer Inc.) was used.The other reagents were purchased from the following.Adenosine triphosphate (ATP): Sigma-Aldrich4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES): DOJINDO LABORATORIESGlycol ether diamine tetraacetic acid (EGTA): DOJINDO LABORATORIES Magnesium chloride (MgCl2): Wako Pure Chemical Industries, Ltd.Dithiothreitol (DTT): Wako Pure Chemical Industries, Ltd.Tween 20: Sigma-AldrichEthylenediaminetetraacetic acid (EDTA): DOJINDO LABORATORIESThe compounds of the present invention, the enzymes (JAK1, JAK2, JAK3 and Tyk2), the substrate and ATP were used for assay as diluted with assay buffer.Â  As the assay buffer, one having the following composition was used.Â  HEPES (pH7.5): 50 mM EGTA: 1 mM MgCl2: 10 mM DTT: 2 mM Tween 20: 0.01% (wt/wt). The dilute concentration and the addition amount on a well plate as described hereinafter were adjusted so that the following final concentrations were achieved on the well plate.
Measurement: IC50 = 84 nM (pChEMBL 7.079999923706055)
Descriptors: MW 377.44, ALogP 2.83, HBA 4, HBD 2, PSA 64.26, QED 0.84
Compound (SMILES): O=C(O)c1cnc2c(N3CCN(CCc4ccc(OCCCN5CCCCCC5)cc4)CC3)cccc2c1
InChIKey: SYDQPIHOSLJNHM-UHFFFAOYSA-N
Target: Alpha-1B adrenergic receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35368
Assay: Antagonist activity at adrenergic alpha1B receptor (unknown origin) expressed in Rat1 cells assessed as inhibition of phenylephrine-induced Ca2+ flux preincubated for 30 mins followed by phenylephrine addition by Fluo-4-AM-dye based FLIPR assay
Measurement: Ki > 1995.26 nM (pChEMBL 5.699999809265137)
Descriptors: MW 516.69, ALogP 4.94, HBA 6, HBD 1, PSA 69.14, QED 0.38
Compound (SMILES): C[C@H](Nc1ncnc(N)c1C#Cc1ccc(F)cn1)c1nc2c(F)cccc2c(=O)n1-c1ccccc1
InChIKey: JNUOOWAUQJYVBW-INIZCTEOSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2xKm ATP (100-300 uM), 10 uM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, 1% (v/v) DMSO at the following concentrations for each isoform: PI3K alpha, beta, delta, and gamma at 50 picomolar (pM) and PI3Kgamma at 2 nanomolar (nM). After an assay reaction time of 30 minutes at 25° C., reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 us delay and 500 us read window).
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 495.49, ALogP 4, HBA 8, HBD 2, PSA 111.61, QED 0.36
Compound (SMILES): CN[C@@H](C)C(=O)Nc1ccc(-c2c(-c3cnc(C)nc3)nc3cc(C)ccn23)c(C#Cc2ccc3c(C)nccc3c2)n1
InChIKey: OOOHUPBTSUEVGE-QFIPXVFZSA-N
Target: Cytochrome P450 2C8 (SINGLE PROTEIN), Homo sapiens
UniProt: P10632
Assay: Inhibition Assays: The final incubation volume contains TRIS buffer (0.1 M), MgCl2 (5 mM), a certain concentration of human liver microsomes dependent on the P450 isoenzyme measured (P450 2C9, P450 3A4: 0.1 mg/ml; P450 2D6: 0.2 mg/ml; P450 2C19: 0.5 mg/ml; P450 2C8: 0.05 mg/ml) and a certain concentration of the individual substrate for each isoenzyme (P450 2C9: Diclofenac 10 μM; P450 3A4: Midazolam 5 μM; P450 2D6: Dextromethorphan 5 μM; P450 2C19: S-Mephenytoin 70 μM; P450 2C8: Amodiaquine 1 μM).
Measurement: IC50 > 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 566.67, ALogP 5.27, HBA 8, HBD 2, PSA 109.99, QED 0.27
Compound (SMILES): Cc1cccc2cc([C@H](C)Nc3ncnc4[nH]cnc34)n(C3CCC3)c(=O)c12
InChIKey: OVBKKAQIDXMNIF-ZDUSSCGKSA-N
Target: PI3K p110 beta/p85 alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: P27986
Assay: Inhibition of N-terminal His6-tagged recombinant full-length human p110beta/untagged recombinant full length p85alpha expressed in baculovirus infected Sf21 cells using phosphatidylinositol as substrate in presence of gamma-32P-ATP after 5 mins by thin layer chromatographic method
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 374.45, ALogP 3.87, HBA 6, HBD 2, PSA 88.49, QED 0.57
Compound (SMILES): O=C[C@H]1CC[C@H](c2nnn3cnc4[nH]ccc4c23)CC1
InChIKey: TWVLDWHLQMIHJW-MGCOHNPYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 269.31, ALogP 2.08, HBA 5, HBD 1, PSA 75.94, QED 0.72
Compound (SMILES): COc1cc(OC)cc(N(C[C@@H](O)CO)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: DDNOUEXAVDQPMR-GOSISDBHSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of VEGFR2 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in presence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 435.48, ALogP 2.54, HBA 9, HBD 2, PSA 105.76, QED 0.44
Compound (SMILES): CC/C=C/C(=O)Nc1cccc(-c2cnc3[nH]nc(C)c3c2)c1
InChIKey: LJXVSFRUMNKAHQ-XBXARRHUSA-N
Target: Tyrosine-protein kinase ITK/TSK (SINGLE PROTEIN), Homo sapiens
UniProt: Q08881
Assay: Biochemical Inhibition Assay: 1H-pyrrolo[2,3-b]pyridine, 1H-pyrazolo[3,4-b]pyridine and 5H-pyrrolo[2,3-b]pyrazine series of compounds exhibited over >100 fold selective against Janus and TEC family of kinases except for the BMX and TXK it had activity of 30 and 45 nM respectively.
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 306.37, ALogP 3.84, HBA 3, HBD 2, PSA 70.67, QED 0.72
Compound (SMILES): N#CCC1(n2cc(-c3ccnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)c3ccc(O)c(F)c3)CC2)C1
InChIKey: FSMLEFFQVXGPGK-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Inhibition of human N-terminal His-tagged JAK1 catalytic domain (837 to 1142 residues) expressed in baculovirus infected insect cells assessed as decrease in phosphorylation of biotinylated peptide substrate after 1 hr in presence of 1 mM ATP by HTRF method
Measurement: IC50 <= 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 499.55, ALogP 3.5, HBA 7, HBD 2, PSA 114.07, QED 0.43
Compound (SMILES): Brc1ccc(CN2CCC(c3nnn4cnc5[nH]ccc5c34)CC2)s1
InChIKey: ZHGLVRFKUKDKKV-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 417.34, ALogP 3.81, HBA 6, HBD 1, PSA 62.11, QED 0.55
Compound (SMILES): CN1CCN(Cc2ccc(-c3cn4c5nc(ncc35)NCCCCCCCCC(O)CCCC4)cc2)CC1
InChIKey: IKQPJOFHAZFMNC-UHFFFAOYSA-N
Target: Tyrosine-protein kinase receptor UFO (SINGLE PROTEIN), Homo sapiens
UniProt: P30530
Assay: Inhibition of Axl (unknown origin) by microfluidic capillary electrophoresis assay
Measurement: IC50 = 9.3 nM (pChEMBL 8.029999732971191)
Descriptors: MW 518.75, ALogP 5.53, HBA 7, HBD 2, PSA 69.45, QED 0.47
Compound (SMILES): COc1cc(OC)cc(N(CCO)c2ccc3ncc(-c4cnn(C5CCN(C)CC5)c4)nc3c2)c1
InChIKey: IMRAJPPXPOXARE-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of VEGFR2 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in presence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 = 9549.93 nM (pChEMBL 5.019999980926514)
Descriptors: MW 488.59, ALogP 3.91, HBA 9, HBD 1, PSA 88.77, QED 0.4
Compound (SMILES): COc1cc2c(Nc3ccc(Cl)c(Cl)c3)ncnc2cc1OCc1csc(C2CCCCN2C)n1
InChIKey: BQCYIIVLNTWNSU-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of recombinant human N-terminal GST-fused KDR (D807 to V1356 residues) expressed in baculovirus infected Escherichia coli or insect Sf9 cells using poly-EY as substrate after 240 mins by luciferase coupled chemiluminescence assay
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 530.48, ALogP 6.88, HBA 8, HBD 1, PSA 72.4, QED 0.28
Compound (SMILES): NC/C(=C\F)COc1ccc(C(N)=O)cc1
InChIKey: CUIDJRZBPVHOGV-VMPITWQZSA-N
Target: Amine oxidase [copper-containing] 3 (SINGLE PROTEIN), Rattus norvegicus
UniProt: O08590
Assay: Recombinant SSAO/VAP-1 Inhibition Assay in Rat Fat: Abdominal fat from BALB/c mice, Wistar or Sprague Dawley rats, which are tissues enriched with SSAO/VAP-1-were surgically removed. For every gram of animal abdominal fat tissue, 1 mL of 0.1 M NaPO4 buffer (pH 7.4) was added. Tissues were homogenized using IKA Ultra-Turrax T 10 homogenizer for 3 min, homogenate was centrifuged for 15 min at 3000×g. The middle layer (clear supernatant) was removed without disturbing the top layer (high fat content) or the debris on the bottom of the tube. SSAO/VAP-1 activity was determined by checking the fluorescent signal. Km/Vmax values were determined and the fat homogenate was aliquoted and stored at −80 °C until assays were performed. Assay was performed in a similar fashion as for human SSAO/VAP-1 (Example 5) except, the substrate (benzylamine) concentrations used for mouse fat homogenate and rat fat homogenate were 80 μM and 30 μM respectively: Briefly, in a standard 96 well plate assay 50 μL of purified human SSAO/VAP-1 (0.25 μg/mL) in 0.1 M NaPO4 buffer (pH 7.4) was added into each well. Test compounds were dissolved in DMSO and tested in a Concentration Response Curve (CRC) with 4-9 data points, typically in the micromolar or nanomolar range after incubation with human SSAO/VAP-1 for 30 min at 37 °C. After 30 min incubation, 50 μL of the reaction mixture containing 600 μM benzylamine (Sigma Aldrich), 120 μM Amplex Red (Sigma Aldrich) and 1.5 U/mL horseradish peroxidase (Sigma Aldrich) prepared in 0.1 M NaPO4 buffer (pH 7.4) were added to the corresponding well. The fluorescence unit (RFU) was read every 2.5 min for 30 min at 37 °C. excitation 565 nm and emission 590 (Optima; BMG labtech). The slope of the kinetics for each well was calculated using MARS data analysis software (BMG labtech) and this value was used to deduce the IC50 value (Dotmatics).
Measurement: IC50 < 100000 nM (pChEMBL 4)
Descriptors: MW 224.24, ALogP 0.98, HBA 3, HBD 2, PSA 78.34, QED 0.78
Compound (SMILES): O=C(N[C@H]1CC[C@H](CCN2CCN(c3nsc4ccccc34)CC2)CC1)c1cccnc1
InChIKey: RGWGDYMUCXDODX-XUTJKUGGSA-N
Target: 5-hydroxytryptamine receptor 2A (SINGLE PROTEIN), Homo sapiens
UniProt: P28223
Assay: Displacement of [3H]ketanserin from recombinant human 5-HT2A receptor expressed in HEK293 cell membranes after 60 mins by scintillation counting method
Measurement: Ki = 0.29 nM (pChEMBL 9.539999961853027)
Descriptors: MW 449.62, ALogP 4.19, HBA 6, HBD 1, PSA 61.36, QED 0.61
Compound (SMILES): CNC(=O)c1c(-c2ccc(F)cc2)oc2cc(N(C)S(C)(=O)=O)c(-c3ccc4c(n3)-c3cc5c(F)cccc5n3C(CN)O4)cc12
InChIKey: UYAKNXNEKVOJRR-UHFFFAOYSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1b (isolate Japanese) (HCV)
UniProt: P26662
Assay: ELISA-Based Assay: Measurement of inhibition by compounds was performed using the HCV replicon system. Several different replicons encoding different HCV genotypes or mutations were used. In addition, potency measurements were made using different formats of the replicon assay, including different ways of measurements and different plating formats. See Jan M. Vrolijk et al., A replicons-based bioassay for the measurement of interferons in patients with chronic hepatitis C, 110 J. VIROLOGICAL METHODS 201 (2003); Steven S. Carroll et al., Inhibition of Hepatitis C Virus RNA Replication by 2'-Modified Nucleoside Analogs, 278(14) J. BIOLOGICAL CHEMISTRY 11979 (2003). However, the underlying principles are common to all of these determinations, and are outlined below.Stable neomycin phosphotransferase encoding replicons-harboring cell lines were used, so all cell lines were maintained under G418 selection prior to the assay. Potency was determined using a cell ELISA assay with an antibody to the replicons encoded NS3/4a protease. See Caterina Trozzi et al., In Vitro Selection and Characterization of Hepatitis C Virus Serine Protease Variants Resistant to an Active-Site Peptide Inhibitor, 77(6) J. Virol. 3669 (2003).
Measurement: IC50 = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 643.67, ALogP 5.67, HBA 8, HBD 2, PSA 132.69, QED 0.24
Compound (SMILES): COC(=O)N1CCC(n2c(=O)n(C)c3cnc4ccc(-c5ccc(OC)nc5)cc4c32)CC1
InChIKey: NRCHETGDZOLUIQ-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase mTOR (SINGLE PROTEIN), Homo sapiens
UniProt: P42345
Assay: Cellular Assay: Assay d) MDA-MB-468 cells (human breast adenocarcinoma ##ATCC HTB 132) were seeded at 1500 cells/well in 40 μl of DMEM containing 10% FBS and 1% glutamine into Greiner 384 well black flat-bottomed plates. Cell plates were incubated for 18 h in a 37° C. incubator before dosing with compounds of formula (I) in 100% DMSO using acoustic dispensing. Compounds were dosed in a 12 point concentration range into a randomised plate map. Control wells were generated either by dosing of 100% DMSO (max signal) or addition of a reference compound (a PI3K-β inhibitor) that completely eliminated the pAKT signal (min control). Plates were incubated, at 37° C. for 2 h, cells were then fixed by the addition of 10 μl of a 3.7% formaldehyde solution. After 30 minutes the plates were washed with PBS using a Tecan PW384 plate washer. Wells were blocked and cells permeabilised with the addition of 40 μl of PBS containing 0.5% Tween20 and 1% Marvel™ (dried milk powder) and incubated for 60 minutes at r.t. The plates were washed with PBS containing 0.5% (v/v) Tween20 and 20 μl rabbit anti-phospho AKT Ser473 (Cell Signalling Technologies, #3787) in same PBS-Tween+1% Marvel™ was added and incubated overnight at 4° C. Plates were washed 3 times with PBS 0.05% Tween 20 using a Tecan PW384. 20 μl of secondary antibody Alexa Fluor 488 anti-Rabbit (Molecular Probes, #A11008) diluted in PBS+0.05% Tween20 containing 1% Marvel™ was added to each well and incubated for 1 h at r.t. Plates were washed three times as before then 20 μl PBS added to each well and plates sealed with a black plate sealer. The plates were read on an Acumen plate reader as soon as possible, measuring green fluorescence after excitation with 488 nm laser.
Measurement: IC50 = 72.9 nM (pChEMBL 7.139999866485596)
Descriptors: MW 447.5, ALogP 3.36, HBA 8, HBD 0, PSA 91.48, QED 0.48
Compound (SMILES): CC(=O)N1CCc2c(C(=O)N3CCC(c4ccc(F)c(F)c4C(F)(F)F)CC3)n[nH]c2C1
InChIKey: HAGSLCBZFRRBLS-UHFFFAOYSA-N
Target: Retinol-binding protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P02753
Assay: Scintillation Proximity Binding Assay: Untagged human RBP4 purified from urine of tubular proteinuria patients was purchased from Fitzgerald Industries International. It was biotinylated using the EZ-Link Sulfo-NHS-LC-Biotinylation kit from Pierce following the manufacturer's recommendations. Binding experiments were performed in 96-well plates (OptiPlate, PerkinElmer) in a final assay volume of 100 ul per well in SPA buffer (IX PBS, pH 7.4, 1 mM EDTA, 0.1% BSA, 0.5% CHAPS). The reaction mix contained 10 nM 3H-Retinol (48.7 Ci/mmol; PerkinElmer), 0.3 mg/well Streptavidin-PVT beads, 50 nM biotinylated RBP4 and a test compound. Nonspecific binding was determined in the presence of 20 uM of unlabeled retinol. The reaction mix was assembled in the dark under dim red light. The plates were sealed with clear tape (TopSeal-A: 96-well microplate, PerkinElmer), wrapped in the aluminum foil, and allowed to equilibrate 6 hours at room temperature followed by overnight incubation at +4° C. Radiocounts were measured using a TopCount NXT counter (Packard Instrument Company).
Measurement: IC50 = 2.824 nM (pChEMBL 8.550000190734863)
Descriptors: MW 456.42, ALogP 3.63, HBA 3, HBD 1, PSA 69.3, QED 0.7
Compound (SMILES): O=C(O)CCC[C@H]1CC[C@@H]2[C@@H](/C=C/[C@@H](O)COc3ccccc3)[C@H](O)C[C@@H]2OC1
InChIKey: JBWVPQXSFRGWCV-HJGWJQNWSA-N
Target: Prostaglandin E2 receptor EP2 subtype (SINGLE PROTEIN), Homo sapiens
UniProt: P43116
Assay: Agonist activity at EP2 receptor (unknown origin)
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 404.5, ALogP 3.03, HBA 5, HBD 3, PSA 96.22, QED 0.55
Compound (SMILES): Cc1cn(-c2ccc(-c3cn(CC(=O)Nc4cccc(Cl)c4Cl)nn3)cc2)cn1
InChIKey: ULUGOYAEEFLQDR-UHFFFAOYSA-N
Target: Probable G-protein coupled receptor 142 (SINGLE PROTEIN), Homo sapiens
UniProt: Q7Z601
Assay: Agonist activity at human GPR142 expressed in CHO cells measured for 3 to 5 mins by Fluo-4AM dye-based FLIPR assay
Measurement: EC50 = 34 nM (pChEMBL 7.46999979019165)
Descriptors: MW 427.3, ALogP 4.38, HBA 6, HBD 1, PSA 77.63, QED 0.51
Compound (SMILES): CC#CC(=O)Nc1cccc(-c2cnc3[nH]cc(C)c3c2)n1
InChIKey: BVGQRQFYDJKNLY-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Biochemical Inhibition Assay: 1H-pyrrolo[2,3-b]pyridine, 1H-pyrazolo[3,4-b]pyridine and 5H-pyrrolo[2,3-b]pyrazine series of compounds exhibited over >100 fold selective against Janus and TEC family of kinases except for the BMX and TXK it had activity of 30 and 45 nM respectively.
Measurement: IC50 = 739 nM (pChEMBL 6.130000114440918)
Descriptors: MW 290.33, ALogP 2.9, HBA 3, HBD 2, PSA 70.67, QED 0.71
Compound (SMILES): O=C(O)C1CN(C[C@H]2CC[C@H](c3nnn4cnc5[nH]ccc5c34)CC2)C1
InChIKey: BYSKVWHEBHTGOZ-HAQNSBGRSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 400 nM (pChEMBL 6.400000095367432)
Descriptors: MW 354.41, ALogP 1.9, HBA 6, HBD 2, PSA 99.41, QED 0.74
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)c3ccc(C(F)(F)F)cc3F)CC2)C1
InChIKey: KBXFUEFOJIBZET-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 552.54, ALogP 4.21, HBA 7, HBD 1, PSA 106.73, QED 0.37
Compound (SMILES): Cc1ccc(NC(=O)c2cccc(C(F)(F)F)c2)cc1-c1cc(N2CCOCC2)nc(N2CCOCC2)n1
InChIKey: ZHNCJARBTUXWNB-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: In Vitro Enzyme Assay: Final assay conditions were 4 pM cRaf, 3 uM ATP, 10 nM biotin tagged MEK1 kinase dead protein substrate. Reactions were performed in Greiner384 well plates, catalog #784075. Reaction buffer was 50 mM Tris, pH 7.5, 50 mM NaCl, 10 mM MgCl2, 0.01% Tween-20, 0.05% BSA and 1 mM DTT. 5 ul of 2×cRaf plus MEK1 were dispensed into plates containing 0.25 ul of compound in 100% DMSO and incubated at room temperature for 30 minutes. Reactions were started by addition of 5 ul of 2×ATP. Reactions were run for two hours and terminated by addition of 10 ul of 2× antibody and Alphascreen bead mixture in Stop Buffer. Stop Buffer was 50 mM Tris, pH 7.5, 0.005% Tween-20, 25 mM EDTA.
Measurement: IC50 = 0.33 nM (pChEMBL 9.479999542236328)
Descriptors: MW 527.55, ALogP 4.4, HBA 7, HBD 1, PSA 79.82, QED 0.53
Compound (SMILES): NC/C(=C/F)COc1ccc(S(N)(=O)=O)cc1
InChIKey: GJRSZKOPZAJCGZ-YVMONPNESA-N
Target: Amine oxidase [copper-containing] 3 (SINGLE PROTEIN), Rattus norvegicus
UniProt: O08590
Assay: Recombinant SSAO/VAP-1 Inhibition Assay in Rat Fat: Abdominal fat from BALB/c mice, Wistar or Sprague Dawley rats, which are tissues enriched with SSAO/VAP-1-were surgically removed. For every gram of animal abdominal fat tissue, 1 mL of 0.1 M NaPO4 buffer (pH 7.4) was added. Tissues were homogenized using IKA Ultra-Turrax T 10 homogenizer for 3 min, homogenate was centrifuged for 15 min at 3000×g. The middle layer (clear supernatant) was removed without disturbing the top layer (high fat content) or the debris on the bottom of the tube. SSAO/VAP-1 activity was determined by checking the fluorescent signal. Km/Vmax values were determined and the fat homogenate was aliquoted and stored at −80 °C until assays were performed. Assay was performed in a similar fashion as for human SSAO/VAP-1 (Example 5) except, the substrate (benzylamine) concentrations used for mouse fat homogenate and rat fat homogenate were 80 μM and 30 μM respectively: Briefly, in a standard 96 well plate assay 50 μL of purified human SSAO/VAP-1 (0.25 μg/mL) in 0.1 M NaPO4 buffer (pH 7.4) was added into each well. Test compounds were dissolved in DMSO and tested in a Concentration Response Curve (CRC) with 4-9 data points, typically in the micromolar or nanomolar range after incubation with human SSAO/VAP-1 for 30 min at 37 °C. After 30 min incubation, 50 μL of the reaction mixture containing 600 μM benzylamine (Sigma Aldrich), 120 μM Amplex Red (Sigma Aldrich) and 1.5 U/mL horseradish peroxidase (Sigma Aldrich) prepared in 0.1 M NaPO4 buffer (pH 7.4) were added to the corresponding well. The fluorescence unit (RFU) was read every 2.5 min for 30 min at 37 °C. excitation 565 nm and emission 590 (Optima; BMG labtech). The slope of the kinetics for each well was calculated using MARS data analysis software (BMG labtech) and this value was used to deduce the IC50 value (Dotmatics).
Measurement: IC50 < 100000 nM (pChEMBL 4)
Descriptors: MW 260.29, ALogP 0.52, HBA 4, HBD 2, PSA 95.41, QED 0.81
Compound (SMILES): O=C(/C=C/CN1CCCC1)Nc1cc2c(Nc3ccc(Cc4ccccn4)cc3)ncnc2cn1
InChIKey: JLWZRGNJHZVVKD-FNORWQNLSA-N
Target: Receptor tyrosine-protein kinase erbB-4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15303
Assay: Irreversible inhibition of GST-tagged ERBB4 (unknown origin) (Gly-259 to Gly-690 residues) expressed in baculovirus infected Sf9 insect cells assessed as reduction in Glu/Tyr copolymer phosphorylation after 6 mins by ELISA
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 465.56, ALogP 4.34, HBA 7, HBD 2, PSA 95.93, QED 0.37
Compound (SMILES): C[C@H](Nc1ncnc(N)c1C#Cc1ccccn1)c1nc2cccc(Cl)c2c(=O)n1-c1cccc(C(N)=O)c1
InChIKey: PTQYEXPCYCYVAH-INIZCTEOSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2xKm ATP (100-300 uM), 10 uM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, 1% (v/v) DMSO at the following concentrations for each isoform: PI3K alpha, beta, delta, and gamma at 50 picomolar (pM) and PI3Kgamma at 2 nanomolar (nM). After an assay reaction time of 30 minutes at 25° C., reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 us delay and 500 us read window).
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 536.98, ALogP 3.48, HBA 9, HBD 3, PSA 154.7, QED 0.29
Compound (SMILES): Cc1ccc(NC(=O)c2cccc(C(F)(F)F)c2)cc1NC(=O)c1cccc2c(N)nccc12
InChIKey: JETCTCUSGZDEGL-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition Assay: In a similar manner to Experimental Example 1, the % inhibitory activity (concentration of the compound 1.0 μM) and the IC50 values of the inventive compound of Example 1 against various kinases were obtained by using Kinase Profiling Service (Invitrogen) according to the manufacturer's instructions.
Measurement: IC50 > 2500 nM (pChEMBL 5.6020002365112305)
Descriptors: MW 464.45, ALogP 5.65, HBA 4, HBD 3, PSA 97.11, QED 0.36
Compound (SMILES): C[C@H]1C[C@H]2CSC(N)=N[C@@]2(c2nc(NC(=O)c3ccc(Cl)cn3)cs2)CO1
InChIKey: UFKMIVRYLMOXNT-YMNVWFMLSA-N
Target: Beta-secretase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Z0
Assay: Inhibition of human BACE2 using biotin-GLTNIKTEEISEISYEVEFR-C[Oregon green]KK-OH as substrate after 3 hrs by fluorescence polarization assay
Measurement: IC50 = 21.6 nM (pChEMBL 7.670000076293945)
Descriptors: MW 423.95, ALogP 3.13, HBA 8, HBD 2, PSA 102.49, QED 0.79
Compound (SMILES): COc1cc(OC)cc(N(Cc2nnc(N)o2)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: OXFRISWNUYFISN-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of VEGFR2 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in presence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 458.48, ALogP 3.35, HBA 11, HBD 1, PSA 130.24, QED 0.39
Compound (SMILES): CCN1CCN(C)CC1COc1cc2ncnc(Nc3ccc(Cl)c(Cl)c3)c2cc1OC
InChIKey: PQDLFPSEZJTCDS-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of recombinant human N-terminal GST-fused KDR (D807 to V1356 residues) expressed in baculovirus infected Escherichia coli or insect Sf9 cells using poly-EY as substrate after 240 mins by luciferase coupled chemiluminescence assay
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 476.41, ALogP 4.7, HBA 7, HBD 1, PSA 62.75, QED 0.53
Compound (SMILES): O=C(OC(C(F)(F)F)C(F)(F)F)N1CCN(Cc2cccc(Cl)c2N2CCOCC2)CC1
InChIKey: JLXNYQYXQCEKRB-UHFFFAOYSA-N
Target: Monoacylglycerol lipase ABHD6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9BV23
Assay: Inhibition of ABHD6 derived from human PC3 cell lysates preincubated for 30 mins followed by JW912 addition after 30 mins by gel-based ABPP assay
Measurement: IC50 = 3400 nM (pChEMBL 5.46999979019165)
Descriptors: MW 489.84, ALogP 3.92, HBA 5, HBD 0, PSA 45.25, QED 0.6
Compound (SMILES): COc1cc(OC)cc(N(CC(=O)NCCN)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1.Cl
InChIKey: LJMAHXISETVHHT-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of VEGFR2 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in presence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 461.53, ALogP 2.26, HBA 9, HBD 2, PSA 120.42, QED 0.39
Compound (SMILES): O=S(=O)(C[C@H]1CC[C@H](c2nnn3cnc4[nH]ccc4c23)CC1)C(F)(F)F
InChIKey: TVPUJUWGZFIFDI-MGCOHNPYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 370 nM (pChEMBL 6.429999828338623)
Descriptors: MW 387.39, ALogP 2.81, HBA 6, HBD 1, PSA 93.01, QED 0.75
Compound (SMILES): COc1cc(OC)cc(N(CC(=O)O)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1.Cl
InChIKey: URSLNMLJDNMYJS-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of VEGFR2 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in presence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 419.44, ALogP 3.27, HBA 8, HBD 1, PSA 102.6, QED 0.49
Compound (SMILES): CCCS(=O)(=O)Nc1ccc(F)c(C(=O)c2c[nH]c3ncc(-c4cncc(S(C)(=O)=O)c4)cc23)c1F
InChIKey: OEWMYLBBEIGHGH-UHFFFAOYSA-N
Target: Cytochrome P450 2C19 (SINGLE PROTEIN), Homo sapiens
UniProt: P33261
Assay: Inhibition of CYP2C19 (unknown origin) using CEC as substrate after 30 mins
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 534.57, ALogP 3.69, HBA 7, HBD 2, PSA 138.95, QED 0.33
Compound (SMILES): CNC[C@H](O)CN(c1cc(OC)cc(OC)c1)c1ccc2ncc(-c3cnn(C)c3)nc2c1
InChIKey: LCWWFMHHNQAPDZ-IBGZPJMESA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of VEGFR2 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in presence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 448.53, ALogP 2.77, HBA 9, HBD 2, PSA 97.56, QED 0.4
Compound (SMILES): O=C(c1cccc(C(F)(F)F)c1F)N1CCc2c(nnn2-c2ncccn2)C1
InChIKey: DPLVEZJPUODUHZ-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Ca2+ flux 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 = 22.4 nM (pChEMBL 7.650000095367432)
Descriptors: MW 392.32, ALogP 2.41, HBA 6, HBD 0, PSA 76.8, QED 0.63
Compound (SMILES): COc1cc(OC)cc(N(CCCN2[C@@H](C)CC[C@@H]2C)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1.Cl
InChIKey: NQEIXHXOMZOMCJ-GUTACTQSSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of VEGFR2 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in presence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 = 9549.93 nM (pChEMBL 5.019999980926514)
Descriptors: MW 500.65, ALogP 5.45, HBA 8, HBD 0, PSA 68.54, QED 0.3
Compound (SMILES): N#CCC1(n2ccc(-c3ncnc4[nH]ccc34)c2)CN(C2CCN(C(=O)c3ccc(F)c(F)c3)CC2)C1
InChIKey: JYTXMAGXSCRQTE-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: In vitro JAK Kinase Assay: The catalytic domains of human JAK1 (a.a. 837-1142), Jak2 (a.a. 828-1132) and Jak3 (a.a. 781-1124) with an N-terminal His tag were expressed using baculovirus in insect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3 was assayed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by homogenous time resolved fluorescence (HTRF). IC50s of compounds were measured for each kinase in the 40 microL reactions that contain the enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%) BSA. For the 1 mM IC50 measurements, ATP concentration in the reactions was 1 mM. Reactions were carried out at room temperature for 1 hr and then stopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, Mass.). Binding to the Europium labeled antibody took place for 40 minutes and HTRF signal was measured on a Fusion plate reader (Perkin Elmer, Boston, Mass.)
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 501.54, ALogP 3.93, HBA 6, HBD 1, PSA 93.84, QED 0.45
Compound (SMILES): CCn1nc(-c2cccc(-c3cccc(C(=O)NCCCCCCCN(C)CCO)c3)c2)cc(NC(=O)Nc2c(Cl)cncc2Cl)c1=O
InChIKey: OXMNWHWSPHHKPG-UHFFFAOYSA-N
Target: Adenosine receptor A2b (SINGLE PROTEIN), Homo sapiens
UniProt: P29275
Assay: Antagonist activity at adenosine A2b receptor (unknown origin)
Measurement: IC50 = 206 nM (pChEMBL 6.690000057220459)
Descriptors: MW 694.66, ALogP 6.55, HBA 8, HBD 4, PSA 141.48, QED 0.1