lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1cnc(C(=O)N2CC3CC(Oc4cnc(C(F)(F)F)cn4)C2C3)c(-n2nccn2)c1
InChIKey: YLFJSQKDYGUXAW-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P56719
Assay: Receptor Radioligand Binding Assay: Human Orexin 2 Receptor:HEK293 stably expressing human orexin 2 receptor (Genebank accession number NM_001526) were grown to confluency in DMEM (Hyclone, cat # SH30022), 10% FBS, 1× Pen/Strep, 1× NaPyruvate, 10 mM HEPES, 600 ug/ml G418 media on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1× with Calcium and Magnesium, Cat # SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2K×G, 5 min at 4° C.), the supernatant was aspirated and the pellets frozen and stored at −80° C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL. Each cell pellet from a 15 cm plate was resuspended in 10 mL, stored on ice, and homogenized for 45 sec just prior to addition to the reactions. Competition binding experiments in 96 well polypropylene plates were performed using [3H]-EMPA (Moraveck Corporation, specific activity=29.6 Ci/mmol), diluted to a 5 nM concentration in PBS (2 nM final concentration). Compounds were solubilized in 100% DMSO (Acros Organics, Cat. #61042-1000) and tested over a range of 7 concentration (from 0.1 nM to 10 μM). The final concentration of DMSO in the reactions is equal to or less than 0.1%. Total and nonspecific binding was determined in the absence and presence of 10 μM almorexant. The total volume of each reaction is 200 μL (20 μL of diluted compounds, 80 μL of [3H]-EMPA diluted in PBS and 100 μL of the cell suspension). Reactions were run for 60 min at room temperature and terminated by filtration through GF/C filter plates (PerkinElmer, Cat. #6005174) presoaked in 0.3% polyethylenimine using the cell harvester (PerkinElmer Filtermate). The plates were washed 3 times by aspirating 30 ml PBS through the plates. Plates were dried in 55° C. oven for 60 min, scintillation fluid was added, and the radioactivity was counted on a Topcount (Packard).
Measurement: Ki > 10000 nM (pChEMBL 5)
Descriptors: MW 445.41, ALogP 2.46, HBA 8, HBD 0, PSA 98.92, QED 0.61
Compound (SMILES): O=C(c1ccccc1-n1nccn1)N1CC2CC(Oc3ccc(C(F)(F)F)cn3)C1C2
InChIKey: PSWAVZZVHYHQBT-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Human Orexin 1 Receptor Ca2+ Mobilization Assay: CHO cells stably transfected with the human orexin 1 receptor (Genebank accession number NM_001526) were grown to confluency in DMEM/F12, 10% FBS, 1×pen-strep, 400 μg/ml G418. Cells were seeded on to 384-well Packard viewplates at a density of 10,000 cells/well and incubated overnight at 37° C., 5% CO2. The cells were dye-loaded with BD Calcium Assay kit (BD, cat #640178) in HBSS (Gibco, cat #14025-092) with 2.5 mM probenecid and incubated at 37° C., 5% CO2 for 45 min. Cells were pre-incubated with compounds (diluted in DMEM/F-12) for 15-30 minutes before agonist (orexin A, 10 nM) stimulation. Ligand-induced Ca2+ release was measured using a Fluorometric Imaging Plate Reader (FLIPR, Molecular Devices, Sunnyvale, Calif.). Functional responses were measured as peak fluorescence intensity minus basal. The concentration of agonist that produced a half-maximal response is represented by the EC50 value. Antagonistic potency values were converted to apparent pKB values using a modified Cheng-Prusoff correction. Apparent pKB=−log IC50/1+[conc agonist/EC50].
Measurement: Ki = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 429.4, ALogP 3.36, HBA 6, HBD 0, PSA 73.14, QED 0.64
Compound (SMILES): CCOc1ncc2ccccc2c1C(=O)N1CC2CC(Oc3ccc(C(F)(F)F)cn3)C1C2
InChIKey: WORAHSUSUFAEIQ-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Radioligand Binding Assay: Human Embryonic Kidney 293 cells (HEK293) stably expressing rat orexin 1 receptor (Genebank accession number NM 001525) or Chinese ovary cells (CHO) stably expressing human orexin 1 receptor (Genebank accession number NM 001526) were grown to confluency in DMEM (Hyclone, cat # SH30022), 10% FBS, 1x Pen/Strep, 1x sodium pyruvate, 10 mM HEPES, 600 ug/mL G418 and DMEM/F12 (Gibco, Cat #11039), 10% FBS, 1x Pen/Strep, 600 ug/mL G418 media, respectively on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1x with Calcium and Magnesium, Cat # SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2Kx G, 5 min at 4 C.), the supernatant was aspirated and the pellets frozen and stored at -80 C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL.
Measurement: Ki = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 457.45, ALogP 4.73, HBA 5, HBD 0, PSA 64.55, QED 0.55
Compound (SMILES): Cc1cnc(C(=O)N2CC3CC(Oc4ccc(C(F)(F)F)cn4)C2C3)c(-n2nccn2)c1
InChIKey: OAQRBSBYWFGTPG-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P56718
Assay: Radioligand Binding Assay: Human Embryonic Kidney 293 cells (HEK293) stably expressing rat orexin 1 receptor (Genebank accession number NM 001525) or Chinese ovary cells (CHO) stably expressing human orexin 1 receptor (Genebank accession number NM 001526) were grown to confluency in DMEM (Hyclone, cat # SH30022), 10% FBS, 1x Pen/Strep, 1x sodium pyruvate, 10 mM HEPES, 600 ug/mL G418 and DMEM/F12 (Gibco, Cat #11039), 10% FBS, 1x Pen/Strep, 600 ug/mL G418 media, respectively on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1x with Calcium and Magnesium, Cat # SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2Kx G, 5 min at 4 C.), the supernatant was aspirated and the pellets frozen and stored at -80 C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL.
Measurement: Ki = 32 nM (pChEMBL 7.5)
Descriptors: MW 444.42, ALogP 3.07, HBA 7, HBD 0, PSA 86.03, QED 0.61
Compound (SMILES): CCOc1ccc2cccnc2c1C(=O)N1CC2CC(Oc3ccc(C(F)(F)F)cn3)C1C2
InChIKey: TWCLRHNSMBRHHR-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Radioligand Binding Assay: Human Embryonic Kidney 293 cells (HEK293) stably expressing rat orexin 1 receptor (Genebank accession number NM 001525) or Chinese ovary cells (CHO) stably expressing human orexin 1 receptor (Genebank accession number NM 001526) were grown to confluency in DMEM (Hyclone, cat # SH30022), 10% FBS, 1x Pen/Strep, 1x sodium pyruvate, 10 mM HEPES, 600 ug/mL G418 and DMEM/F12 (Gibco, Cat #11039), 10% FBS, 1x Pen/Strep, 600 ug/mL G418 media, respectively on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1x with Calcium and Magnesium, Cat # SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2Kx G, 5 min at 4 C.), the supernatant was aspirated and the pellets frozen and stored at -80 C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL.
Measurement: Ki = 27 nM (pChEMBL 7.570000171661377)
Descriptors: MW 457.45, ALogP 4.73, HBA 5, HBD 0, PSA 64.55, QED 0.55
Compound (SMILES): O=C(c1cccc(F)c1-c1ncccn1)N1CC2CC(Oc3ccc(C(F)(F)F)cn3)C1C2
InChIKey: HUKWIAXQBOHZIX-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Human Orexin 1 Receptor Ca2+ Mobilization Assay: CHO cells stably transfected with the human orexin 1 receptor (Genebank accession number NM_001526) were grown to confluency in DMEM/F12, 10% FBS, 1×pen-strep, 400 μg/ml G418. Cells were seeded on to 384-well Packard viewplates at a density of 10,000 cells/well and incubated overnight at 37° C., 5% CO2. The cells were dye-loaded with BD Calcium Assay kit (BD, cat #640178) in HBSS (Gibco, cat #14025-092) with 2.5 mM probenecid and incubated at 37° C., 5% CO2 for 45 min. Cells were pre-incubated with compounds (diluted in DMEM/F-12) for 15-30 minutes before agonist (orexin A, 10 nM) stimulation. Ligand-induced Ca2+ release was measured using a Fluorometric Imaging Plate Reader (FLIPR, Molecular Devices, Sunnyvale, Calif.). Functional responses were measured as peak fluorescence intensity minus basal. The concentration of agonist that produced a half-maximal response is represented by the EC50 value. Antagonistic potency values were converted to apparent pKB values using a modified Cheng-Prusoff correction. Apparent pKB=−log IC50/1+[conc agonist/EC50].
Measurement: Ki = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 458.42, ALogP 4.38, HBA 5, HBD 0, PSA 68.21, QED 0.54
Compound (SMILES): COc1ccc(C(=O)N2CC3CC(Oc4ccc(C(F)(F)F)cn4)C2C3)c(-c2ncccn2)c1
InChIKey: DSTQNGFYZOHDRW-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Radioligand Binding Assay: HEK293 stably expressing human orexin 2 receptor (Genebank accession number NM 001526) were grown to confluency in DMEM (Hyclone, cat # SH30022), 10% FBS, 1x Pen/Strep, 1x NaPyruvate, 10 mM HEPES, 600 ug/ml G418 media on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1x with Calcium and Magnesium, Cat # SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2Kx G, 5 min at 4x C.), the supernatant was aspirated and the pellets frozen and stored at -80 C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL. Each cell pellet from a 15 cm plate was resuspended in 10 mL, stored on ice, and homogenized for 45 sec just prior to addition to the reactions. Competition binding experiments in 96 well polypropylene plates were performed using [3H]-EMPA (Moraveck Corporation, specific activity=29.6 Ci/mmol).
Measurement: Ki > 10000 nM (pChEMBL 5)
Descriptors: MW 470.45, ALogP 4.25, HBA 6, HBD 0, PSA 77.44, QED 0.56
Compound (SMILES): COc1ccc(C(=O)N2CC3CC(Oc4ccc(C(F)(F)F)cn4)C2C3)c(-n2nccn2)c1
InChIKey: JAACBBVXXQKQLD-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Radioligand Binding Assay: Human Embryonic Kidney 293 cells (HEK293) stably expressing rat orexin 1 receptor (Genebank accession number NM 001525) or Chinese ovary cells (CHO) stably expressing human orexin 1 receptor (Genebank accession number NM 001526) were grown to confluency in DMEM (Hyclone, cat # SH30022), 10% FBS, 1x Pen/Strep, 1x sodium pyruvate, 10 mM HEPES, 600 ug/mL G418 and DMEM/F12 (Gibco, Cat #11039), 10% FBS, 1x Pen/Strep, 600 ug/mL G418 media, respectively on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1x with Calcium and Magnesium, Cat # SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2Kx G, 5 min at 4 C.), the supernatant was aspirated and the pellets frozen and stored at -80 C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL.
Measurement: Ki = 109 nM (pChEMBL 6.960000038146973)
Descriptors: MW 459.43, ALogP 3.37, HBA 7, HBD 0, PSA 82.37, QED 0.58
Compound (SMILES): COc1cccc(-n2nccn2)c1C(=O)N1CC2CC(Oc3ccc(C(F)(F)F)cn3)C1C2
InChIKey: XSMDMCQWMREAHT-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Radioligand Binding Assay: HEK293 stably expressing human orexin 2 receptor (Genebank accession number NM 001526) were grown to confluency in DMEM (Hyclone, cat # SH30022), 10% FBS, 1x Pen/Strep, 1x NaPyruvate, 10 mM HEPES, 600 ug/ml G418 media on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1x with Calcium and Magnesium, Cat # SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2Kx G, 5 min at 4x C.), the supernatant was aspirated and the pellets frozen and stored at -80 C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL. Each cell pellet from a 15 cm plate was resuspended in 10 mL, stored on ice, and homogenized for 45 sec just prior to addition to the reactions. Competition binding experiments in 96 well polypropylene plates were performed using [3H]-EMPA (Moraveck Corporation, specific activity=29.6 Ci/mmol).
Measurement: Ki = 229 nM (pChEMBL 6.639999866485596)
Descriptors: MW 459.43, ALogP 3.37, HBA 7, HBD 0, PSA 82.37, QED 0.58
Compound (SMILES): O=C(c1cccc(F)c1-n1nccn1)N1CC2CC(Oc3ccc(C(F)(F)F)cn3)C1C2
InChIKey: VEJWAVKRPRJLDD-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Radioligand Binding Assay: HEK293 stably expressing human orexin 2 receptor (Genebank accession number NM 001526) were grown to confluency in DMEM (Hyclone, cat # SH30022), 10% FBS, 1x Pen/Strep, 1x NaPyruvate, 10 mM HEPES, 600 ug/ml G418 media on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1x with Calcium and Magnesium, Cat # SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2Kx G, 5 min at 4x C.), the supernatant was aspirated and the pellets frozen and stored at -80 C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL. Each cell pellet from a 15 cm plate was resuspended in 10 mL, stored on ice, and homogenized for 45 sec just prior to addition to the reactions. Competition binding experiments in 96 well polypropylene plates were performed using [3H]-EMPA (Moraveck Corporation, specific activity=29.6 Ci/mmol).
Measurement: Ki = 1000 nM (pChEMBL 6)
Descriptors: MW 447.39, ALogP 3.5, HBA 6, HBD 0, PSA 73.14, QED 0.57
Compound (SMILES): Cc1cccc(C(=O)N2CC3CC(Oc4ccc(C(F)(F)F)cn4)C2C3)c1-n1nccn1
InChIKey: VMCVDMDKIAUUAU-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Radioligand Binding Assay: HEK293 stably expressing human orexin 2 receptor (Genebank accession number NM 001526) were grown to confluency in DMEM (Hyclone, cat # SH30022), 10% FBS, 1x Pen/Strep, 1x NaPyruvate, 10 mM HEPES, 600 ug/ml G418 media on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1x with Calcium and Magnesium, Cat # SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2Kx G, 5 min at 4x C.), the supernatant was aspirated and the pellets frozen and stored at -80 C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL. Each cell pellet from a 15 cm plate was resuspended in 10 mL, stored on ice, and homogenized for 45 sec just prior to addition to the reactions. Competition binding experiments in 96 well polypropylene plates were performed using [3H]-EMPA (Moraveck Corporation, specific activity=29.6 Ci/mmol).
Measurement: Ki = 234 nM (pChEMBL 6.630000114440918)
Descriptors: MW 443.43, ALogP 3.67, HBA 6, HBD 0, PSA 73.14, QED 0.62
Compound (SMILES): O=C(c1c(F)cccc1-n1nccn1)N1CC2CC(Oc3ccc(C(F)(F)F)cn3)C1C2
InChIKey: SGBSAQGVDFRKJX-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P56719
Assay: Receptor Radioligand Binding Assay: Human Orexin 2 Receptor:HEK293 stably expressing human orexin 2 receptor (Genebank accession number NM_001526) were grown to confluency in DMEM (Hyclone, cat # SH30022), 10% FBS, 1× Pen/Strep, 1× NaPyruvate, 10 mM HEPES, 600 ug/ml G418 media on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1× with Calcium and Magnesium, Cat # SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2K×G, 5 min at 4° C.), the supernatant was aspirated and the pellets frozen and stored at −80° C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL. Each cell pellet from a 15 cm plate was resuspended in 10 mL, stored on ice, and homogenized for 45 sec just prior to addition to the reactions. Competition binding experiments in 96 well polypropylene plates were performed using [3H]-EMPA (Moraveck Corporation, specific activity=29.6 Ci/mmol), diluted to a 5 nM concentration in PBS (2 nM final concentration). Compounds were solubilized in 100% DMSO (Acros Organics, Cat. #61042-1000) and tested over a range of 7 concentration (from 0.1 nM to 10 μM). The final concentration of DMSO in the reactions is equal to or less than 0.1%. Total and nonspecific binding was determined in the absence and presence of 10 μM almorexant. The total volume of each reaction is 200 μL (20 μL of diluted compounds, 80 μL of [3H]-EMPA diluted in PBS and 100 μL of the cell suspension). Reactions were run for 60 min at room temperature and terminated by filtration through GF/C filter plates (PerkinElmer, Cat. #6005174) presoaked in 0.3% polyethylenimine using the cell harvester (PerkinElmer Filtermate). The plates were washed 3 times by aspirating 30 ml PBS through the plates. Plates were dried in 55° C. oven for 60 min, scintillation fluid was added, and the radioactivity was counted on a Topcount (Packard).
Measurement: Ki = 650 nM (pChEMBL 6.190000057220459)
Descriptors: MW 447.39, ALogP 3.5, HBA 6, HBD 0, PSA 73.14, QED 0.57
Compound (SMILES): O=C(c1cc(F)ccc1-c1ncccn1)N1CC2CC(Oc3ccc(C(F)(F)F)cn3)C1C2
InChIKey: MSBDPMYPBOZFHI-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Human Orexin 2 Receptor Ca2+ Mobilization Assay: PFSK-1 cells endogenously expressing the human orexin 2 receptor were grown to confluency in RPMI1640 (Hyclone, cat #30027.02), 10% FBS, 1× pen-strep. Cells were seeded on to 384-well Packard viewplates at a density of 5,000 cells/well and incubated overnight at 37° C., 5% CO2. The cells were dye-loaded with BD Calcium Assay kit (BD, cat #640178) in HBSS (Gibco, cat #14025-092) with 2.5 mM probenecid and incubated at 37° C., 5% CO2 for 45 min. Cells were pre-incubated with compounds (diluted in DMEM/F-12) for 15-30 minutes before agonist (orexin B, 100 nM) stimulation. Ligand-induced Ca2+ release was measured using a Fluorometric Imaging Plate Reader (FLIPR, Molecular Devices, Sunnyvale, Calif.). Functional responses were measured as peak fluorescence intensity minus basal. The concentration of agonist that produced a half-maximal response is represented by the EC50 value. Antagonistic potency values were converted to apparent pKB values using a modified Cheng-Prusoff correction. Apparent pKB=−log IC50/1+[conc agonist/EC50].
Measurement: Ki = 458 nM (pChEMBL 6.340000152587891)
Descriptors: MW 458.42, ALogP 4.38, HBA 5, HBD 0, PSA 68.21, QED 0.54
Compound (SMILES): O=C(c1ccccc1-n1cnnc1)N1CC2CC(Oc3ccc(C(F)(F)F)cn3)C1C2
InChIKey: CTAUDMRXJCJSKS-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P56719
Assay: Receptor Radioligand Binding Assay: Human Orexin 2 Receptor:HEK293 stably expressing human orexin 2 receptor (Genebank accession number NM_001526) were grown to confluency in DMEM (Hyclone, cat # SH30022), 10% FBS, 1× Pen/Strep, 1× NaPyruvate, 10 mM HEPES, 600 ug/ml G418 media on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1× with Calcium and Magnesium, Cat # SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2K×G, 5 min at 4° C.), the supernatant was aspirated and the pellets frozen and stored at −80° C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL. Each cell pellet from a 15 cm plate was resuspended in 10 mL, stored on ice, and homogenized for 45 sec just prior to addition to the reactions. Competition binding experiments in 96 well polypropylene plates were performed using [3H]-EMPA (Moraveck Corporation, specific activity=29.6 Ci/mmol), diluted to a 5 nM concentration in PBS (2 nM final concentration). Compounds were solubilized in 100% DMSO (Acros Organics, Cat. #61042-1000) and tested over a range of 7 concentration (from 0.1 nM to 10 μM). The final concentration of DMSO in the reactions is equal to or less than 0.1%. Total and nonspecific binding was determined in the absence and presence of 10 μM almorexant. The total volume of each reaction is 200 μL (20 μL of diluted compounds, 80 μL of [3H]-EMPA diluted in PBS and 100 μL of the cell suspension). Reactions were run for 60 min at room temperature and terminated by filtration through GF/C filter plates (PerkinElmer, Cat. #6005174) presoaked in 0.3% polyethylenimine using the cell harvester (PerkinElmer Filtermate). The plates were washed 3 times by aspirating 30 ml PBS through the plates. Plates were dried in 55° C. oven for 60 min, scintillation fluid was added, and the radioactivity was counted on a Topcount (Packard).
Measurement: Ki > 10000 nM (pChEMBL 5)
Descriptors: MW 429.4, ALogP 3.36, HBA 6, HBD 0, PSA 73.14, QED 0.64
Compound (SMILES): Cc1ccc(-n2nccn2)c(C(=O)N2CC3CC(Oc4ccc(C(F)(F)F)cn4)C2C3)n1
InChIKey: DIVFYQCMVDWPOD-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Radioligand Binding Assay: Human Embryonic Kidney 293 cells (HEK293) stably expressing rat orexin 1 receptor (Genebank accession number NM 001525) or Chinese ovary cells (CHO) stably expressing human orexin 1 receptor (Genebank accession number NM 001526) were grown to confluency in DMEM (Hyclone, cat # SH30022), 10% FBS, 1x Pen/Strep, 1x sodium pyruvate, 10 mM HEPES, 600 ug/mL G418 and DMEM/F12 (Gibco, Cat #11039), 10% FBS, 1x Pen/Strep, 600 ug/mL G418 media, respectively on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1x with Calcium and Magnesium, Cat # SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2Kx G, 5 min at 4 C.), the supernatant was aspirated and the pellets frozen and stored at -80 C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL.
Measurement: Ki = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 444.42, ALogP 3.07, HBA 7, HBD 0, PSA 86.03, QED 0.61
Compound (SMILES): Cc1ccc(C(=O)N2CC3CC(Oc4ccc(C(F)(F)F)cn4)C2C3)c(-n2nccn2)n1
InChIKey: FTIPLKXQDISZMV-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Radioligand Binding Assay: Human Embryonic Kidney 293 cells (HEK293) stably expressing rat orexin 1 receptor (Genebank accession number NM 001525) or Chinese ovary cells (CHO) stably expressing human orexin 1 receptor (Genebank accession number NM 001526) were grown to confluency in DMEM (Hyclone, cat # SH30022), 10% FBS, 1x Pen/Strep, 1x sodium pyruvate, 10 mM HEPES, 600 ug/mL G418 and DMEM/F12 (Gibco, Cat #11039), 10% FBS, 1x Pen/Strep, 600 ug/mL G418 media, respectively on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1x with Calcium and Magnesium, Cat # SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2Kx G, 5 min at 4 C.), the supernatant was aspirated and the pellets frozen and stored at -80 C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL.
Measurement: Ki = 47 nM (pChEMBL 7.329999923706055)
Descriptors: MW 444.42, ALogP 3.07, HBA 7, HBD 0, PSA 86.03, QED 0.61
Compound (SMILES): O=C(c1ncccc1-n1nccn1)N1CC2CC(Oc3ccc(C(F)(F)F)cn3)C1C2
InChIKey: FLULXEHMBMNJCY-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P56719
Assay: Receptor Radioligand Binding Assay: Human Orexin 2 Receptor:HEK293 stably expressing human orexin 2 receptor (Genebank accession number NM_001526) were grown to confluency in DMEM (Hyclone, cat # SH30022), 10% FBS, 1× Pen/Strep, 1× NaPyruvate, 10 mM HEPES, 600 ug/ml G418 media on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1× with Calcium and Magnesium, Cat # SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2K×G, 5 min at 4° C.), the supernatant was aspirated and the pellets frozen and stored at −80° C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL. Each cell pellet from a 15 cm plate was resuspended in 10 mL, stored on ice, and homogenized for 45 sec just prior to addition to the reactions. Competition binding experiments in 96 well polypropylene plates were performed using [3H]-EMPA (Moraveck Corporation, specific activity=29.6 Ci/mmol), diluted to a 5 nM concentration in PBS (2 nM final concentration). Compounds were solubilized in 100% DMSO (Acros Organics, Cat. #61042-1000) and tested over a range of 7 concentration (from 0.1 nM to 10 μM). The final concentration of DMSO in the reactions is equal to or less than 0.1%. Total and nonspecific binding was determined in the absence and presence of 10 μM almorexant. The total volume of each reaction is 200 μL (20 μL of diluted compounds, 80 μL of [3H]-EMPA diluted in PBS and 100 μL of the cell suspension). Reactions were run for 60 min at room temperature and terminated by filtration through GF/C filter plates (PerkinElmer, Cat. #6005174) presoaked in 0.3% polyethylenimine using the cell harvester (PerkinElmer Filtermate). The plates were washed 3 times by aspirating 30 ml PBS through the plates. Plates were dried in 55° C. oven for 60 min, scintillation fluid was added, and the radioactivity was counted on a Topcount (Packard).
Measurement: Ki = 1700 nM (pChEMBL 5.769999980926514)
Descriptors: MW 430.39, ALogP 2.76, HBA 7, HBD 0, PSA 86.03, QED 0.63
Compound (SMILES): COc1c(F)cccc1C(=O)N1CC2CC(Oc3ccc(C(F)(F)F)cn3)C1C2
InChIKey: RYMOHHYAOSEKIU-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Radioligand Binding Assay: HEK293 stably expressing human orexin 2 receptor (Genebank accession number NM 001526) were grown to confluency in DMEM (Hyclone, cat # SH30022), 10% FBS, 1x Pen/Strep, 1x NaPyruvate, 10 mM HEPES, 600 ug/ml G418 media on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1x with Calcium and Magnesium, Cat # SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2Kx G, 5 min at 4x C.), the supernatant was aspirated and the pellets frozen and stored at -80 C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL. Each cell pellet from a 15 cm plate was resuspended in 10 mL, stored on ice, and homogenized for 45 sec just prior to addition to the reactions. Competition binding experiments in 96 well polypropylene plates were performed using [3H]-EMPA (Moraveck Corporation, specific activity=29.6 Ci/mmol).
Measurement: Ki > 10000 nM (pChEMBL 5)
Descriptors: MW 410.37, ALogP 3.93, HBA 4, HBD 0, PSA 51.66, QED 0.72
Compound (SMILES): Cc1cccc(C(=O)N2CC3CC(Oc4ccc(C(F)(F)F)cn4)C2C3)c1-c1ncco1
InChIKey: IIVBMGSJEAWDEA-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P56718
Assay: Radioligand Binding Assay: Human Embryonic Kidney 293 cells (HEK293) stably expressing rat orexin 1 receptor (Genebank accession number NM 001525) or Chinese ovary cells (CHO) stably expressing human orexin 1 receptor (Genebank accession number NM 001526) were grown to confluency in DMEM (Hyclone, cat # SH30022), 10% FBS, 1x Pen/Strep, 1x sodium pyruvate, 10 mM HEPES, 600 ug/mL G418 and DMEM/F12 (Gibco, Cat #11039), 10% FBS, 1x Pen/Strep, 600 ug/mL G418 media, respectively on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1x with Calcium and Magnesium, Cat # SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2Kx G, 5 min at 4 C.), the supernatant was aspirated and the pellets frozen and stored at -80 C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL.
Measurement: Ki = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 443.43, ALogP 4.75, HBA 5, HBD 0, PSA 68.46, QED 0.58
Compound (SMILES): O=C(c1cccc(F)c1-n1ccnn1)N1CC2CC(Oc3ccc(C(F)(F)F)cn3)C1C2
InChIKey: RNUWTSJVSRFNSZ-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Radioligand Binding Assay: Human Embryonic Kidney 293 cells (HEK293) stably expressing rat orexin 1 receptor (Genebank accession number NM 001525) or Chinese ovary cells (CHO) stably expressing human orexin 1 receptor (Genebank accession number NM 001526) were grown to confluency in DMEM (Hyclone, cat # SH30022), 10% FBS, 1x Pen/Strep, 1x sodium pyruvate, 10 mM HEPES, 600 ug/mL G418 and DMEM/F12 (Gibco, Cat #11039), 10% FBS, 1x Pen/Strep, 600 ug/mL G418 media, respectively on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1x with Calcium and Magnesium, Cat # SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2Kx G, 5 min at 4 C.), the supernatant was aspirated and the pellets frozen and stored at -80 C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL.
Measurement: Ki = 86 nM (pChEMBL 7.070000171661377)
Descriptors: MW 447.39, ALogP 3.5, HBA 6, HBD 0, PSA 73.14, QED 0.57
Compound (SMILES): Cc1ccc(C(=O)N2CC3CC(Oc4ccc(C(F)(F)F)cn4)C2C3)c(-n2ccnn2)n1
InChIKey: ICOFMFLOEJHBPA-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Radioligand Binding Assay: HEK293 stably expressing human orexin 2 receptor (Genebank accession number NM 001526) were grown to confluency in DMEM (Hyclone, cat # SH30022), 10% FBS, 1x Pen/Strep, 1x NaPyruvate, 10 mM HEPES, 600 ug/ml G418 media on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1x with Calcium and Magnesium, Cat # SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2Kx G, 5 min at 4x C.), the supernatant was aspirated and the pellets frozen and stored at -80 C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL. Each cell pellet from a 15 cm plate was resuspended in 10 mL, stored on ice, and homogenized for 45 sec just prior to addition to the reactions. Competition binding experiments in 96 well polypropylene plates were performed using [3H]-EMPA (Moraveck Corporation, specific activity=29.6 Ci/mmol).
Measurement: Ki = 1100 nM (pChEMBL 5.960000038146973)
Descriptors: MW 444.42, ALogP 3.07, HBA 7, HBD 0, PSA 86.03, QED 0.61
Compound (SMILES): CCOc1ccc(C)nc1C(=O)N1CC2CC(Oc3ccc(C(F)(F)F)cn3)C1C2
InChIKey: HBQVLSLDECXOPX-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Radioligand Binding Assay: HEK293 stably expressing human orexin 2 receptor (Genebank accession number NM 001526) were grown to confluency in DMEM (Hyclone, cat # SH30022), 10% FBS, 1x Pen/Strep, 1x NaPyruvate, 10 mM HEPES, 600 ug/ml G418 media on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1x with Calcium and Magnesium, Cat # SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2Kx G, 5 min at 4x C.), the supernatant was aspirated and the pellets frozen and stored at -80 C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL. Each cell pellet from a 15 cm plate was resuspended in 10 mL, stored on ice, and homogenized for 45 sec just prior to addition to the reactions. Competition binding experiments in 96 well polypropylene plates were performed using [3H]-EMPA (Moraveck Corporation, specific activity=29.6 Ci/mmol).
Measurement: Ki = 1800 nM (pChEMBL 5.75)
Descriptors: MW 421.42, ALogP 3.88, HBA 5, HBD 0, PSA 64.55, QED 0.73
Compound (SMILES): O=C(c1c(F)cccc1-c1ncccn1)N1CC2CC(Oc3ccc(C(F)(F)F)cn3)C1C2
InChIKey: IGPUBGBQVDTOBH-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Radioligand Binding Assay: Human Embryonic Kidney 293 cells (HEK293) stably expressing rat orexin 1 receptor (Genebank accession number NM 001525) or Chinese ovary cells (CHO) stably expressing human orexin 1 receptor (Genebank accession number NM 001526) were grown to confluency in DMEM (Hyclone, cat # SH30022), 10% FBS, 1x Pen/Strep, 1x sodium pyruvate, 10 mM HEPES, 600 ug/mL G418 and DMEM/F12 (Gibco, Cat #11039), 10% FBS, 1x Pen/Strep, 600 ug/mL G418 media, respectively on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1x with Calcium and Magnesium, Cat # SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2Kx G, 5 min at 4 C.), the supernatant was aspirated and the pellets frozen and stored at -80 C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL.
Measurement: Ki = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 458.42, ALogP 4.38, HBA 5, HBD 0, PSA 68.21, QED 0.54
Compound (SMILES): COc1cccc(-c2ccn[nH]2)c1C(=O)N1CC2CC(Oc3ccc(C(F)(F)F)cn3)C1C2
InChIKey: CTMWEIJXXSCTJH-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P56718
Assay: Radioligand Binding Assay: Human Embryonic Kidney 293 cells (HEK293) stably expressing rat orexin 1 receptor (Genebank accession number NM_001525) or Chinese ovary cells (CHO) stably expressing human orexin 1 receptor (Genebank accession number NM_001526) were grown to confluency in DMEM (Hyclone, cat # SH30022), 10% FBS, 1× Pen/Strep, 1× sodium pyruvate, 10 mM HEPES, 600 μg/mL G418 and DMEM/F12 (Gibco, Cat #11039), 10% FBS, 1× Pen/Strep, 600 μg/mL G418 media, respectively on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1× with Calcium and Magnesium, Cat # SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2K×G, 5 min at 4° C.), the supernatant was aspirated and the pellets frozen and stored at −80° C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL. Each cell pellet from a 15 cm plate was resuspended in 10 mL, stored on ice, and homogenized for 45 sec prior to addition to the reactions. Competition binding experiments in 96 well polypropylene plates were performed using [3H]-(1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone) (Moraveck Corporation, specific activity=35.3 Ci/mmol), diluted to a 10 nM concentration in PBS (4 nM final). Compounds were solubilized in 100% DMSO (Acros Organics, Cat. #61042-1000) and tested over a range of 7 concentrations (from 0.1 nM to 10 μM). The final concentration of DMSO in the reactions is equal to or less than 0.1%. Total and nonspecific binding was determined in the absence and presence of 10 μM almorexant. The total volume of each reaction is 200 μL (20 μL of diluted compounds, 80 μL of [3H]-(1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone) diluted in PBS and 100 μL of the cell suspension). Reactions were run for 60 min at room temperature and terminated by filtration through GF/C filter plates (PerkinElmer, Cat. #6005174) presoaked in 0.3% polyethylenimine using the cell harvester (PerkinElmer Filtermate). The plates were washed 3 times by aspirating 30 ml PBS through the plates. Plates were dried in 55° C. oven for 60 min, scintillation fluid was added, and the radioactivity was counted on a Topcount (Packard).
Measurement: Ki = 72 nM (pChEMBL 7.139999866485596)
Descriptors: MW 458.44, ALogP 4.18, HBA 5, HBD 1, PSA 80.34, QED 0.62
Compound (SMILES): COc1cccc(-c2ncccn2)c1C(=O)N1CC2CC(Oc3ccc(C(F)(F)F)cn3)C1C2
InChIKey: YREUWZWUXFUQNJ-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Radioligand Binding Assay: HEK293 stably expressing human orexin 2 receptor (Genebank accession number NM 001526) were grown to confluency in DMEM (Hyclone, cat # SH30022), 10% FBS, 1x Pen/Strep, 1x NaPyruvate, 10 mM HEPES, 600 ug/ml G418 media on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1x with Calcium and Magnesium, Cat # SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2Kx G, 5 min at 4x C.), the supernatant was aspirated and the pellets frozen and stored at -80 C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL. Each cell pellet from a 15 cm plate was resuspended in 10 mL, stored on ice, and homogenized for 45 sec just prior to addition to the reactions. Competition binding experiments in 96 well polypropylene plates were performed using [3H]-EMPA (Moraveck Corporation, specific activity=29.6 Ci/mmol).
Measurement: Ki = 340 nM (pChEMBL 6.46999979019165)
Descriptors: MW 470.45, ALogP 4.25, HBA 6, HBD 0, PSA 77.44, QED 0.56
Compound (SMILES): Cc1cnn(C)c1-c1ccccc1C(=O)N1CC2CC(Oc3ccc(C(F)(F)F)cn3)C1C2
InChIKey: JHIBIGDVURAFGJ-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P56719
Assay: Receptor Radioligand Binding Assay: Human Orexin 2 Receptor:HEK293 stably expressing human orexin 2 receptor (Genebank accession number NM_001526) were grown to confluency in DMEM (Hyclone, cat # SH30022), 10% FBS, 1× Pen/Strep, 1× NaPyruvate, 10 mM HEPES, 600 ug/ml G418 media on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1× with Calcium and Magnesium, Cat # SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2K×G, 5 min at 4° C.), the supernatant was aspirated and the pellets frozen and stored at −80° C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL. Each cell pellet from a 15 cm plate was resuspended in 10 mL, stored on ice, and homogenized for 45 sec just prior to addition to the reactions. Competition binding experiments in 96 well polypropylene plates were performed using [3H]-EMPA (Moraveck Corporation, specific activity=29.6 Ci/mmol), diluted to a 5 nM concentration in PBS (2 nM final concentration). Compounds were solubilized in 100% DMSO (Acros Organics, Cat. #61042-1000) and tested over a range of 7 concentration (from 0.1 nM to 10 μM). The final concentration of DMSO in the reactions is equal to or less than 0.1%. Total and nonspecific binding was determined in the absence and presence of 10 μM almorexant. The total volume of each reaction is 200 μL (20 μL of diluted compounds, 80 μL of [3H]-EMPA diluted in PBS and 100 μL of the cell suspension). Reactions were run for 60 min at room temperature and terminated by filtration through GF/C filter plates (PerkinElmer, Cat. #6005174) presoaked in 0.3% polyethylenimine using the cell harvester (PerkinElmer Filtermate). The plates were washed 3 times by aspirating 30 ml PBS through the plates. Plates were dried in 55° C. oven for 60 min, scintillation fluid was added, and the radioactivity was counted on a Topcount (Packard).
Measurement: Ki > 10000 nM (pChEMBL 5)
Descriptors: MW 456.47, ALogP 4.49, HBA 5, HBD 0, PSA 60.25, QED 0.58
Compound (SMILES): O=C(c1cccc2cc[nH]c12)N1CC2CC(Oc3ccc(C(F)(F)F)cn3)C1C2
InChIKey: BHDOEKBJNYFWML-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Radioligand Binding Assay: HEK293 stably expressing human orexin 2 receptor (Genebank accession number NM 001526) were grown to confluency in DMEM (Hyclone, cat # SH30022), 10% FBS, 1x Pen/Strep, 1x NaPyruvate, 10 mM HEPES, 600 ug/ml G418 media on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1x with Calcium and Magnesium, Cat # SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2Kx G, 5 min at 4x C.), the supernatant was aspirated and the pellets frozen and stored at -80 C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL. Each cell pellet from a 15 cm plate was resuspended in 10 mL, stored on ice, and homogenized for 45 sec just prior to addition to the reactions. Competition binding experiments in 96 well polypropylene plates were performed using [3H]-EMPA (Moraveck Corporation, specific activity=29.6 Ci/mmol).
Measurement: Ki = 5800 nM (pChEMBL 5.239999771118164)
Descriptors: MW 401.39, ALogP 4.26, HBA 3, HBD 1, PSA 58.22, QED 0.71
Compound (SMILES): O=C(c1ccc(F)cc1-n1nccn1)N1CC2CC(Oc3ccc(C(F)(F)F)cn3)C1C2
InChIKey: NOXUACCAWHVGCM-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Human Orexin 1 Receptor Ca2+ Mobilization Assay: CHO cells stably transfected with the human orexin 1 receptor (Genebank accession number NM_001526) were grown to confluency in DMEM/F12, 10% FBS, 1×pen-strep, 400 μg/ml G418. Cells were seeded on to 384-well Packard viewplates at a density of 10,000 cells/well and incubated overnight at 37° C., 5% CO2. The cells were dye-loaded with BD Calcium Assay kit (BD, cat #640178) in HBSS (Gibco, cat #14025-092) with 2.5 mM probenecid and incubated at 37° C., 5% CO2 for 45 min. Cells were pre-incubated with compounds (diluted in DMEM/F-12) for 15-30 minutes before agonist (orexin A, 10 nM) stimulation. Ligand-induced Ca2+ release was measured using a Fluorometric Imaging Plate Reader (FLIPR, Molecular Devices, Sunnyvale, Calif.). Functional responses were measured as peak fluorescence intensity minus basal. The concentration of agonist that produced a half-maximal response is represented by the EC50 value. Antagonistic potency values were converted to apparent pKB values using a modified Cheng-Prusoff correction. Apparent pKB=−log IC50/1+[conc agonist/EC50].
Measurement: Ki = 39 nM (pChEMBL 7.409999847412109)
Descriptors: MW 447.39, ALogP 3.5, HBA 6, HBD 0, PSA 73.14, QED 0.57
Compound (SMILES): O=C(c1cccc(F)c1Br)N1CC2CC(Oc3ccc(C(F)(F)F)cn3)C1C2
InChIKey: QSCWUOPMXMROKO-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Radioligand Binding Assay: Human Embryonic Kidney 293 cells (HEK293) stably expressing rat orexin 1 receptor (Genebank accession number NM 001525) or Chinese ovary cells (CHO) stably expressing human orexin 1 receptor (Genebank accession number NM 001526) were grown to confluency in DMEM (Hyclone, cat # SH30022), 10% FBS, 1x Pen/Strep, 1x sodium pyruvate, 10 mM HEPES, 600 ug/mL G418 and DMEM/F12 (Gibco, Cat #11039), 10% FBS, 1x Pen/Strep, 600 ug/mL G418 media, respectively on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1x with Calcium and Magnesium, Cat # SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2Kx G, 5 min at 4 C.), the supernatant was aspirated and the pellets frozen and stored at -80 C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL.
Measurement: Ki = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 459.24, ALogP 4.68, HBA 3, HBD 0, PSA 42.43, QED 0.63
Compound (SMILES): O=C(c1c(F)cccc1-c1ncccn1)N1CC2CC(Oc3ccc(Br)cn3)C1C2
InChIKey: DZJNFUJQJJPNEW-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Human Orexin 1 Receptor Ca2+ Mobilization Assay: CHO cells stably transfected with the human orexin 1 receptor (Genebank accession number NM_001526) were grown to confluency in DMEM/F12, 10% FBS, 1×pen-strep, 400 μg/ml G418. Cells were seeded on to 384-well Packard viewplates at a density of 10,000 cells/well and incubated overnight at 37° C., 5% CO2. The cells were dye-loaded with BD Calcium Assay kit (BD, cat #640178) in HBSS (Gibco, cat #14025-092) with 2.5 mM probenecid and incubated at 37° C., 5% CO2 for 45 min. Cells were pre-incubated with compounds (diluted in DMEM/F-12) for 15-30 minutes before agonist (orexin A, 10 nM) stimulation. Ligand-induced Ca2+ release was measured using a Fluorometric Imaging Plate Reader (FLIPR, Molecular Devices, Sunnyvale, Calif.). Functional responses were measured as peak fluorescence intensity minus basal. The concentration of agonist that produced a half-maximal response is represented by the EC50 value. Antagonistic potency values were converted to apparent pKB values using a modified Cheng-Prusoff correction. Apparent pKB=−log IC50/1+[conc agonist/EC50].
Measurement: Ki = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 469.31, ALogP 4.12, HBA 5, HBD 0, PSA 68.21, QED 0.58
Compound (SMILES): O=C(c1cccc(F)c1-c1ncccn1)N1CC2CC(Oc3ccc(Br)cn3)C1C2
InChIKey: WHPVXNLPYOHPJB-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P56719
Assay: Receptor Radioligand Binding Assay: Human Orexin 2 Receptor:HEK293 stably expressing human orexin 2 receptor (Genebank accession number NM_001526) were grown to confluency in DMEM (Hyclone, cat # SH30022), 10% FBS, 1× Pen/Strep, 1× NaPyruvate, 10 mM HEPES, 600 ug/ml G418 media on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1× with Calcium and Magnesium, Cat # SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2K×G, 5 min at 4° C.), the supernatant was aspirated and the pellets frozen and stored at −80° C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL. Each cell pellet from a 15 cm plate was resuspended in 10 mL, stored on ice, and homogenized for 45 sec just prior to addition to the reactions. Competition binding experiments in 96 well polypropylene plates were performed using [3H]-EMPA (Moraveck Corporation, specific activity=29.6 Ci/mmol), diluted to a 5 nM concentration in PBS (2 nM final concentration). Compounds were solubilized in 100% DMSO (Acros Organics, Cat. #61042-1000) and tested over a range of 7 concentration (from 0.1 nM to 10 μM). The final concentration of DMSO in the reactions is equal to or less than 0.1%. Total and nonspecific binding was determined in the absence and presence of 10 μM almorexant. The total volume of each reaction is 200 μL (20 μL of diluted compounds, 80 μL of [3H]-EMPA diluted in PBS and 100 μL of the cell suspension). Reactions were run for 60 min at room temperature and terminated by filtration through GF/C filter plates (PerkinElmer, Cat. #6005174) presoaked in 0.3% polyethylenimine using the cell harvester (PerkinElmer Filtermate). The plates were washed 3 times by aspirating 30 ml PBS through the plates. Plates were dried in 55° C. oven for 60 min, scintillation fluid was added, and the radioactivity was counted on a Topcount (Packard).
Measurement: Ki = 972 nM (pChEMBL 6.010000228881836)
Descriptors: MW 469.31, ALogP 4.12, HBA 5, HBD 0, PSA 68.21, QED 0.58