Crystal data for 77e: C26H19ClF5N7O3S∙ H2O, MW = 658.00; crystal size, 0.20 x 0.13 x 0.10 mm;
colorless, block; triclinic, space group P-1, a = 9.15655(17) Å, b = 11.1562(2) Å, c = 13.4175(3) Å, α = 78.463(6)°, β = 89.913(7)°, γ = 84.381(6)°, V = 1336.25(6) Å3, Z = 2, Dx = 1.635 g/cm3, T = 100 K, μ = 2.767 mm-1, λ = 1.54187 Å, R1 = 0.038, wR2 = 0.105.
All measurements were made on a Rigaku R-AXIS RAPID-191R diffractometer using graphite monochromated Cu-K radiation. The structure was solved by direct methods with SHELXS-97100 and was refined using full-matrix least-squares on F2 with SHELXL-97.100 All non-H atoms were refined with anisotropic displacement parameters.
CCDC 1061355 for compound 77e contains the supplementary crystallographic data for this paper.
These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/Community/Requestastructure/Pages/DataRequest.aspx?.
Estimation of LogD at pH 7.4
LogD7.4, which is a partition coefficient between 1-octanol and aqueous buffer pH 7.4, of the compounds was measured on the chromatographic procedure whose condition was developed based on a published method.101 The instruments were Waters Alliance 2795 HPLC system with 2996 UV-vis detector (Milford, MA, USA).
Biology
In vitro inhibitory activity against MGAT2. Protein preparation. The full-length coding sequences of human MGAT2 and mouse MGAT2 are identical to NCBI accession numbers NM_02509841 and NM_177448,102 respectively. All genes of interests were subcloned with FLAG tag in the N-terminal region into pcDNA3.3 vector (Life Technologies, Carlsbad, CA). The expression vector was transfected into FreeStyle293 cells (Life Technologies, Carlsbad, CA) for human and mouse MGAT2 expression.
After 2-days-culture, cells were homogenized in ice-cold 20 mM Tris-HCl buffer (pH 7.5 at 25 °C) containing 1 mM EDTA and 300 mM sucrose. Total membrane fractions were isolated by ultracentrifugation. Pellets were re-suspended in the same buffer for each homogenization and stored at
−80°C. The protein concentration was determined with the BCA Protein Assay Kit (Pierce Biotechnology, Inc., IL) according to the instruction manual.
RapidFire/MS assay. The test compounds were dissolved in 5 μL of an assay buffer, which consisted of 100 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 200 mM sucrose, 0.01% Tween 20, 2 mM DTT, 0.01% BSA, and 5% DMSO and incubated with 10 μL of 0.6 μg/mL MGAT2 enzyme for 60 min. The reaction was started with the addition of 5 μL of 13C×18 oleoyl-CoA (Sigma-Aldrich, St. Lois, Missouri) and 2-oleoyl-glycerol (Sigma-Aldrich, St. Lois, Missouri) at 20 μM each. After incubation at rt for 30 min, the reaction was stopped with acetonitrile containing 0.88% formic acid and 1.3 μM 1,2-dioleoyl-glycerol (Sigma-Aldrich, St. Lois, Missouri) as an internal standard. High-throughput online solid phase extraction was performed using a RapidFire® 300 (Agilent Technologies, Santa Clara, CA). Mass spectrometric analysis was performed using an API-4000TM triple quadrupole mass spectrometer (AB SCIEX, Framingham, MA) in positive SRM mode. The SRM transitions for 13C×18 dioleoylglycerol as a reaction product and dioleoylglycerol were set as 656.6/339.2 and 638.5/339.2, respectively. Analytical data was acquired using Analyst software version 1.5.0 (AB SCIEX, Framingham, MA) and 656.6/339.2 was divided by 638.5/339.2 for calibration. The IC50 values for test compounds were calculated using XLfit software (IDBS, London, UK).
In vitro inhibitory activity against MGAT3, DGAT1, DGAT2, and ACAT1. Protein preparation.
The full-length coding sequences of human MGAT3 is identical to NCBI accession number NM_178176.103 The gene of interests was subcloned with FLAG tag in the N-terminal region into pcDNA3.3 vector (Life Technologies, Carlsbad, CA). The expression vector was transfected into FreeStyle293 cells (Life Technologies, Carlsbad, CA) for human MGAT3 expression. After 2-days-culture, cells were homogenized in ice-cold 20 mM Tris-HCl buffer (pH 7.5 at 25 °C) containing 1 mM EDTA and 300 mM sucrose. The full-length coding sequences of human DGAT1, DGAT2, and ACAT1 are identical to NCBI accession numbers AB057815,104 NM_032564,105 and NM_003101,106 respectively. All genes of interests were subcloned with FLAG tag in the N-terminal region to make expression vectors. Recombinant baculoviruses were prepared according to the procedure of the Bac-to-Bac baculovirus expression system (Life Technologies, Carlsbad, CA). After infection with recombinant baculoviruses, Sf-21 insect cells were homogenized in an ice-cold Buffer (100 mM sucrose, 50 mM KCl, and 40 mM KH2PO4/K2HPO4 at pH 7.4). Total membrane fractions were isolated by ultracentrifugation. Pellets were re-suspended in the same buffer for each homogenization and stored at
−80 °C. The protein concentration was determined with the BCA Protein Assay Kit (Pierce Biotechnology, Inc., IL) according to the instruction manual.
Thin Layer Chromatography (TLC) assay. The assays with human MGAT3, human ACAT1, human DGAT1, and human DGAT2 were performed by TLC assay. For human MGAT3 assay, the reaction was run in an assay buffer, which consisted of 100 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 200 mM sucrose, 2 mM DTT, 0.01% BSA, 0.01% Tween 20, and 5% DMSO, supplemented with 5 μM [1-14C]-oleoyl-CoA (PerkinElmer, Waltham, MA), 5 μM 2-oleoyl-glycerol, and 30 μg/mL MGAT3 enzyme for 30 min at rt.
For human ACAT1 assay, 5 μM [1-14C]-oleoyl-CoA and 100 μM cholesterol were reacted with 30 μg/mL human ACAT1 enzyme for 40 min at 32 °C. For human DGAT1 and DGAT2 assays, 5 μM [1-14C]-oleoyl-CoA and 40 μM 1,2-dioleoyl-glycerol were reacted with 30 μg/mL human DGAT1 and DGAT2 enzyme, respectively, for 40 min at 32 °C. The modified Bligh & Dyer method was applied to the sample preparations. Organic phase was applied onto a silica TLC plate (Merck KGaA, Darmstadt, Germany) and separated with a solvent system of hexane, ethyl ether, and acetic acid. The radioactivities incorporated into lipids were measured with Typhoon FLA 7000 (GE Healthcare UK Ltd, Buckinghamshire, England) and analyzed with Image Quant TL (GE Healthcare UK Ltd, Buckinghamshire, England).
Oral fat tolerance test (OFTT). Male C57BL/6J mice (20–25 g, Charles River Laboratories Japan, Inc.) were used in the OFTT study. The animals were fed with standard chow and tap water ad libitum, maintained at 23 ± 3 °C with a constant humidity of 40–70%, and acclimated with a cycle of 12 h of light and 12 h of darkness. All animal experiments were performed according to the guidelines of the Institutional Animal Care and Use Committee in Takeda Pharmaceutical Company Ltd. Overnight fasted mice were orally treated with a single dose of a test compound. At 6 h after the treatment of the test compound, mice were orally given 8 mL/kg olive oil or water. To inhibit the hydrolysis of plasma TG by
lipoprotein lipase (LPL), mice were treated with a LPL inhibitor, Pluronic F127 (Poloxamer 407, i.p., 500 mg/kg) 30 min prior to olive oil load. Blood samples were collected via tail vein at 0, 2, and 4 h after the olive oil load. Plasma fractions were obtained by centrifugation at 11,100 G for 5 min at 4 °C. Plasma TG concentration was determined using a 7180 biochemistry automatic analyzer (Hitachi high-Tech, Japan).
Chylomicron TG (CM/TG), a triacylglycerol derived from small intestine, was calculated by subtracting the plasma TG concentration of water-treated group from the plasma TG concentration of each olive oil-treated group.
Evaluation of metabolism and pharmacokinetics
In vitro metabolic clearance in human and mouse hepatic microsomes. Human and mouse liver microsomes were purchased from Xenotech, LLC (Lenexa, KS). An incubation mixture consisted of microsomal protein in 50 mM KH2PO4–K2HPO4 phosphate buffer (pH 7.4) and 1 µM test compound. The concentration of microsomal protein was 0.2 mg/mL. An NADPH-generating system containing 5 mM MgCl2, 5 mM glucose-6-phosphate, 0.5 mM β-NADP+, and 1.5 units/mL glucose-6-phosphate dehydrogenase was added to the incubation mixture to initiate the enzyme reaction. The reaction was terminated 15 and 30 min after the initiation of the reaction by mixing the reaction mixture with acetonitrile, followed by centrifugation. The supernatant was subjected to LC/MS/MS analysis. The metabolic velocity was calculated as the slope of the concentration-time plot.
Pharmacokinetic analysis in mouse cassette dosing. Test compounds were administered intravenously (0.1 mg/kg) or orally (1 mg/kg, suspended in 0.5% methylcellulose aqueous solution) by cassette dosing to non-fasted mice. After administration, blood samples were collected and centrifuged to obtain the plasma fraction. The plasma samples were deproteinized by mixing with acetonitrile followed by centrifugation. The compound concentrations in the supernatant were measured by LC/MS/MS.
Evaluation of time-dependent inhibition (TDI) of CYP3A4 (single-point assay)
Human liver microsomes were purchased from Xenotech, LLC (Lenexa, KS). A mixture of a test compound (30 µM) and microsomes in phosphate buffer (pH 7.4) was preincubated at 37 °C in the presence of an NADPH-generating system containing MgCl2, glucose-6-phosphate, β-NADP+, and glucose-6-phosphate dehydrogenase. After preincubation, enzymatic activity of CYP3A4 in the incubation mixture was determined by measuring 6β-hydroxytestosterone in the reaction with testosterone by UPLC. The activity (% of control) for each preincubation time was calculated to the following: {(activity with test compound)/(activity with DMSO)} ×100. The remaining activity (%
remaining) after preincubation was calculated to the following: {activity with preincubation (% of control)}/{activity without preincubation (% of control)} ×100.
Evaluation of reversible inhibition of CYP isoforms
Human liver microsomes were purchased from Xenotech, LLC (Lenexa, KS). Inhibition activity of a test compound of each CYP isoform (1A2/2C8/2C9/2D6/3A4) was evaluated by incubating its specific substrate (tacrine/paclitaxel/tolbutamide/dextromethorphan/midazolam) with 0.1 mg/mL human microsomes in the presence of 10 μM test compound. The incubation mixture was allowed to stand for 10 min at 37 °C, and then the incubation was terminated by addition of acetonitrile/water. After centrifugation, the supernatant was subjected to LC/MS/MS analysis to measure the peak of each specific metabolite (1-hydroxytacrine/6α-hydroxypaclitaxel/4-hydroxytolbutamide/dextrorphan/1'-hydroxymida- zolam).
Evaluation of phototoxic potential
BALB/c 3T3 cells were cultured at 37 C, 5% CO2 in DMEM supplemented with 10% fetal bovine serum, 50 IU/ml penicillin and 50 mg/ml streptomycin. Cells were seeded at 2.5 × 103 cells/well in 384-well white plate, and cultured in DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 IU/ml penicillin, and 50 µg/ml streptomycin for 1 day. Two 384-well plates per test compound (6.25 μM) in Earle's Balanced Salt Solution (EBSS) supplemented with 1 mM HEPES were preincubated for 1 h. One of the two plates was irradiated (+UV) for 60 min with 1.4–1.7 mW/cm2 (5–6 J/cm2 ), whereas the other plate was kept in the dark (−UV). In both plates the treatment medium was replaced by the culture medium, and after another 24 h of culture the cell viability was determined by the cellular ATP content. The cellular ATP content was measured by Celltiter-GloTM assay kit (Promega) according to the manufacture's instruction. ATP content was calculated to the following. ATP content (% of control) = (RLU of test compound / RLU of 1% DMSO) × 100.
第二章に関する実験 Chemistry
General method. Melting points were determined in open capillary tubes on a Büchi melting point apparatus B545 and are uncorrected. 1H NMR and 13C NMR spectra were recorded on Bruker Avance 300, Bruker Avance III 300, or Bruker Avance III 400 spectrometer. Chemical shifts are given in parts per million (ppm) downfield from tetramethysilane (δ) as the internal standard in deuterated solvent and coupling constants (J) are in Hertz (Hz). Data of 1H NMR are reported as follows: chemical shift, integration, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet of doublet, bs = broad singlet), and coupling constants. All solvents and reagents were obtained from commercial suppliers and used without further purification. Thin-layer chromatography (TLC) was performed on Merck silica gel plates 60F254. Column chromatography was performed on silica gel 60 (0.063–0.200 or 0.040–0.063 mm, E. Merck), basic silica gel (Chromatorex NH, 100–200 mesh, Fuji Silysia Chemical Ltd.) or Purif-Pack (Si or NH, Moritex Corporation). LC–MS analysis was performed
on a Waters, Agilent, or Shimadzu Liquid Chromatography–Mass Spectrometer System, operating in APCI (+ or −) or ESI (+ or −) ionization mode. Analytes were eluted using a linear gradient of 0.05%
TFA containing water/acetonitrile or 5 mM ammonium acetate containing water/acetonitrile mobile phase.
Determination of chemical purity by HPLC (detection at 220 nm) was conducted using a Shimadzu Liquid Chromatography System with 0.05% TFA containing water/acetonitrile mobile phase. Elemental analyses and high-resolution mass spectroscopy (HRMS) measurements (ESI-MS) were performed by Takeda Analytical Research Laboratories, Ltd. Yields are not optimized.
2-[1-(tert-Butoxycarbonyl)piperidin-4-yl]propyl 5-(methylsulfonyl)-2,3-dihydro-1H-indole-1-car- boxylate (87a). A mixture of 5-(methylsulfonyl)-2,3-dihydro-1H-indole (85, 150 mg, 0.760 mmol), tert- butyl 4-(2-((methylsulfonyl)oxy)ethyl)piperidine-1-carboxylate (86, 468 mg, 1.52 mmol), potassium carbonate (210 mg, 1.52 mmol), and sodium iodide (114 mg, 0.765 mmol) in DMF (10 mL) was stirred at 60 °C for 24 h. The reaction was quenched with water and extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and concentrated under reduced pressure. Purification by silica gel column chromatography (hexane/AcOEt = 100/0 to 50/50) gave the title compound as a white solid (146 mg, 43%). MS (ESI/APCI) m/z 353 [M − Boc + 2H]+. 1H NMR (300 MHz, CDCl3) δ 1.10–1.26 (2H, m), 1.39–1.78 (14H, m), 2.61–2.78 (2H, m), 3.03 (3H, s), 3.20 (2H, t, J = 8.5 Hz), 4.01–4.18 (4H, m), 4.25–
4.38 (2H, m), 7.71 (1H, s), 7.74–7.82 (1H, m), 7.98 (1H, bs). 13C NMR (100.6 MHz, CDCl3) δ 27.0, 28.5, 32.0, 33.2, 35.4, 43.7, 44.8, 48.1, 63.8, 79.4, 114.6, 123.9, 128.2, 134.1, 153.1, 154.8. Mp 126–129 °C.
Anal. Calcd for C22H32N2O6S: C, 58.39; H, 7.13; N, 6.19. Found: C, 58.38; H, 7.02; N, 6.10.
2-[1-(tert-Butoxycarbonyl)piperidin-4-yl]propyl 5-(methylsulfonyl)-2,3-dihydro-1H-indole-1-car- boxylate (87b). Compound 87b was prepared from compound 85 and compound 88a in a manner similar to that described for compound 102g. White powder. Yield 50%. MS (ESI/APCI) m/z 367 [M − Boc + 2H]+. 1H NMR (300 MHz, CDCl3) δ 1.00 (3H, d, J = 6.8 Hz), 1.15–1.37 (2H, m), 1.38–1.56 (10H, m), 1.60–1.71 (2H, m), 1.73–1.89 (1H, m), 2.58–2.74 (2H, m), 3.03 (3H, s), 3.21 (2H, t, J = 8.9 Hz), 4.03–
4.33 (6H, m), 7.71 (1H, m), 7.77 (1H, dd, J = 8.3, 1.5 Hz), 7.97 (1H, bs). 13C NMR (100.6 MHz, CDCl3) δ 14.0, 27.0, 28.5, 29.7, 37.3, 38.6, 44.2, 44.8, 48.0, 68.9, 79.4, 114.6, 124.0, 128.2, 134.1, 153.1, 154.8.
Mp 144–146 °C. Anal. Calcd for C23H34N2O6S: C, 59.21; H, 7.34; N, 6.00. Found: C, 59.15; H, 7.46; N, 5.96.
1-{1-[(Benzyloxy)carbonyl]piperidin-4-yl}propan-2-yl 5-(methylsulfonyl)-2,3-dihydro-1H-indole- 1-carboxylate (90). Compound 90 was prepared from compound 85 and compound 89 in a manner similar to that described for compound 102g. Colorless oil. Yield 22%. 1H NMR (300 MHz, CDCl3) δ 1.05–1.27 (3H, m), 1.34 (2H, d, J = 6.1 Hz), 1.41–1.61 (2H, m), 1.62–1.85 (3H, m), 2.64–2.85 (2H, m), 3.01–3.07 (3H, m), 3.14–3.28 (2H, m), 4.02–4.29 (4H, m), 5.12 (3H, s), 7.28–7.42 (5H, m), 7.70 (1H, s), 7.77 (1H, dd, J = 8.5, 1.7 Hz), 7.87–8.09 (1H, m).
1-[1-(tert-Butoxycarbonyl)piperidin-4-yl]propan-2-yl 5-(methylsulfonyl)-2,3-dihydro-1H-indole- 1-carboxylate (87c). A mixture of compound 90 (110 mg, 0.220 mmol), 20% Pd(OH)2 on carbon (40 mg),
and di-tert-butyl dicarbonate (131 mg, 0.600 mmol) in THF (10 mL) was stirred at room temperature under H2 atmosphere for 24 h. After the catalyst was removed by filtration, the filtrate was concentrated under reduced pressure. Purification by silica gel column chromatography (hexane/AcOEt = 100/0 to 70/30) gave the title compound as a colorless oil (76 mg, 74%). MS (ESI/APCI) m/z 367 [M − Boc + 2H]+. 1H NMR (300 MHz, CDCl3) δ 1.02–1.21 (2H, m), 1.34 (3H, d, J = 6.4 Hz), 1.41–1.50 (9H, m), 1.52–1.81 (5H, m), 2.67 (2H, t, J = 12.8 Hz), 3.03 (3H, s), 3.20 (2H, t, J = 8.9 Hz), 3.97–4.17 (4H, m), 4.96–5.23 (1H, m), 7.70 (1H, s), 7.74–7.82 (1H, m), 7.88–8.11 (1H, m). 13C NMR (100.6 MHz, CDCl3) δ 21.0, 26.9, 28.5, 32.0, 32.5, 32.9, 43.1, 43.9, 44.9, 48.1, 70.9, 79.4, 114.7, 124.0, 128.2, 134.0, 152.8, 154.8. Anal. Calcd for C23H34N2O6S: C, 59.21; H, 7.34; N, 6.00. Found: C, 59.28; H, 7.43; N, 5.90.
4-Nitrophenyl 5-(methylsulfonyl)-2,3-dihydro-1H-indole-1-carboxylate (91). To a mixture of compound 85 (8.50 g, 43.1 mmol) and 4-nitrophenyl chloroformate (8.69 g, 43.1 mmol) in THF (120 mL) was added pyridine (3.50 mL, 43.1 mmol) at 0 °C. The mixture was stirred at room temperature under N2 atmosphere for 3 h. The solid was collected and washed with AcOEt to give the title compound as a pale yellow solid (14.6 g, 94%). 1H NMR (300 MHz, DMSO-d6) δ 3.18 (3H, s), 3.24–3.46 (2H, m), 4.19–4.43 (2H, m), 7.52–8.45 (7H, m).
2-[1-(tert-Butoxycarbonyl)piperidin-4-yl]-2-oxoethyl 5-(methylsulfonyl)-2,3-dihydro-1H-indole- 1-carboxylate (92). To a mixture of compound 91 (1.50 g, 4.14 mmol) and compound 88b (1.01 g, 4.14 mmol) in DMF (20 mL) was added Cs2CO3 (1.35 g, 4.14 mmol) at room temperature. The mixture was stirred at 60 °C under Ar atmosphere for 2 h. The mixture was quenched with water at room temperature and extracted with AcOEt. The organic layer was separated, washed with water and brine, dried over MgSO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane/AcOEt = 95/5 to 50/50) to give the title compound as a pale yellow solid (425 mg, 22%). MS (ESI/APCI) m/z 367 [M − Boc + 2H]+. 1H NMR (300 MHz, DMSO-d6) δ 1.25–1.38 (2H, m), 1.39 (9H, s), 1.75–1.87 (2H, m), 2.66–2.91 (3H, m), 3.15 (3H, s), 3.22 (2H, t, J = 8.9 Hz), 3.84–4.21 (4H, m), 5.05 (2H, s), 7.60–7.88 (3H, m).
2-[1-(tert-Butoxycarbonyl)piperidin-4-yl]-2-hydroxyethyl 5-(methylsulfonyl)-2,3-dihydro-1H-in- dole-1-carboxylate (87d). To a solution of compound 92 (100 mg, 0.214 mmol) in EtOH (1 mL) and THF (1 mL) was added NaBH4 (8.1 mg, 0.21 mmol) at 0 °C. The mixture was stirred at room temperature under Ar atmosphere for 1 h. The mixture was quenched with water at room temperature and extracted with AcOEt. The organic layer was separated, washed with water and brine, dried over MgSO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane/AcOEt = 95/5 to 50/50) to give the title compound as a pale yellow amorphous solid (39 mg, 39%). MS (ESI/APCI) m/z 369 [M − Boc + 2H]+. 1H NMR (300 MHz, DMSO-d6) δ 1.05–1.29 (2H, m), 1.39 (9H, s), 1.49–1.80 (3H, m), 2.57–2.78 (2H, m), 3.10–3.24 (5H, m), 3.45–3.60 (1H, m), 3.87–4.31 (6H, m), 4.97 (1H, d, J = 5.3 Hz), 7.69–7.92 (3H, m). 13C NMR (100.6 MHz, CDCl3) δ 27.0, 27.5, 28.1, 28.5, 39.5, 43.8, 44.8, 48.1, 68.6, 73.6, 79.5, 114.9, 124.0, 128.2, 134.5, 153.3, 154.8. Mp 144–146 °C.
Anal. Calcd for C22H32N2O7S: C, 56.39; H, 6.88; N, 5.98. Found: C, 56.22; H, 6.96; N, 5.75.
tert-Butyl 4-(1-ethoxy-1-oxopropan-2-ylidene)piperidine-1-carboxylate (94). To a mixture of triethyl 2-phosphonopropionate (13.9 mL, 65.0 mmol) in THF (80 mL) was added portionwise sodium hydride (60% oil dispersion, 2.60 g, 65.0 mmol) at 0 °C, and the mixture was stirred under N2 atmosphere for 1 h. A solution of tert-butyl 4-oxopiperidine-1-carboxylate (9.96 g, 50.0 mmol) in THF (25 mL) was added to the mixture, and the resulting mixture was stirred at room temperature for 1 h. The reaction was quenched by addition of water and diluted with saturated aqueous NH4Cl solution. The mixture was extracted with AcOEt, washed with brine, and dried over Na2SO4. After removal of the solvent, the residue was purified by silica gel column chromatography (hexane/AcOEt = 95/5 to 60/40) to give the title compound as a colorless oil (13.1 g, 92%). 1H NMR (300 MHz, CDCl3) δ 1.30 (3H, t, J = 7.2 Hz), 1.47 (9H, s), 1.88 (3H, s), 2.35 (2H, t, J = 5.9 Hz), 2.63 (2H, t, J = 5.9 Hz), 3.38–3.53 (4H, m), 4.20 (2H, q, J = 7.2 Hz).
tert-Butyl 4-(1-hydroxypropan-2-yl)piperidine-1-carboxylate (88a). A mixture of compound 94 (15.5 g, 54.7 mmol) and 10% Pd on carbon (3.0 g) in MeOH (200 mL) was stirred under H2 atmosphere (1 atm, balloon) at room temperature for 24 h. The catalyst was removed by filtration, and the filtrate was concentrated under reduced pressure to give a colorless oil. The oil was dissolved in EtOH (280 mL), and CaCl2 (12.1 g, 109 mmol) was added. NaBH4 (9.19 g, 219 mmol) was added portionwise to the mixture at 0 °C, and the resulting mixture was stirred at room temperature for 23 h. The reaction mixture was cooled to 0 °C and diluted with water. The mixture was carefully treated with 10% aqueous citric acid solution and extracted with AcOEt. The organic layer was washed with brine and dried over Na2SO4. After removal of the solvent, the residue was purified by silica gel column chromatography (hexane/AcOEt = 80/20 to 30/70) to give the title compound as a colorless oil (11.7 g, 88% over 2 steps). 1H NMR (300 MHz, CDCl3) δ 0.91 (3H, d, J = 6.8 Hz), 1.09–1.34 (3H, m), 1.39–1.65 (13H, m), 2.56–2.77 (2H, m), 3.38–3.69 (2H, m), 4.00–4.26 (2H, m).
Benzyl 4-(2-hydroxypropyl)piperidine-1-carboxylate (89). To a solution of benzyl 4-(2-hydroxy- ethyl)piperidine-1-carboxylate (1.41 g, 5.36 mmol) and triethylamine (3.00 mL, 21.5 mmol) in DMSO (30 mL) was added sulfur trioxide-pyridine complex (1.72 g, 10.8 mmol). After stirring at room temperature for 3 h, the reaction was quenched with saturated aqueous NaHCO3 solution and extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and concentrated under reduced pressure. The residue was dissolved in THF (30 mL), and methyl magnesium bromide (1.0 M in THF, 6.0 mL, 6.0 mmmol) was added at 0 °C. The resulting mixture was stirred at 0 °C for 4 h and quenched with water. The mixture was extracted with AcOEt, washed with brine, dried over MgSO4, and concentrated under reduced pressure. Purification by silica gel column chromatography (hexane/AcOEt = 100/0 to 50/50) gave the title compound as a colorless oil (816 mg, 55% over 2 steps). 1H NMR (300 MHz, CDCl3) δ 1.01–1.51 (8H, m), 1.61–1.84 (3H, m), 2.78 (2H, t, J = 12.1 Hz), 3.84–4.02 (1H, m), 4.08–4.30 (2H, m), 5.12 (2H, s), 7.27–7.45 (5H, m).
4-Fluoro-2,3-dihydro-1H-indole (99a). Compound 99a was prepared from 4-fluoro-1H-indole (98a) in a manner similar to that described for compound 99f. Yield 98%. 1H NMR (400 MHz, CDCl3) δ 3.07 (2H, t, J = 8.4 Hz), 3.61 (2H, t, J = 8.4 Hz), 6.38–6.42 (2H, m), 6.94–6.97 (1H, m).
4-Fluoro-5-(methylsulfanyl)-2,3-dihydro-1H-indole (100a). Compound 100a was prepared from compound 99a in a manner similar to that described for compound 100f. Yield 35%. 1H NMR (400 MHz, CDCl3) δ 2.37 (3H, s), 3.08 (2H, t, J = 8.8 Hz), 3.63 (2H, t, J = 8.8 Hz), 6.34 (1H, d, J = 8.0 Hz), 7.10 (1H, t, J = 7.6 Hz).
4-Fluoro-5-(methylsulfonyl)-2,3-dihydro-1H-indole hydrochloride (101a). Compound 101a was prepared from compound 100a in a manner similar to that described for compound 101f. White solid.
Yield 55% from 100a. MS (ESI) m/z 216 [M + H]+. 1H NMR (400 MHz, CDCl3) δ 3.05 (2H, t, J = 8.8 Hz). 3.15 (3H, s), 3.65 (2H, t, J = 8.8 Hz), 6.34 (1H, d, J = 8.4 Hz), 7.38 (1H, t, J = 8.0 Hz).
4-Chloro-2,3-dihydro-1H-indole (99b). Compound 99b was prepared from 4-chloro-1H-indole (98b) in a manner similar to that described for compound 99f. Yield 99%. 1H NMR (400 MHz, CDCl3) δ 3.08 (2H, t, J = 8.4 Hz), 3.60 (2H, t, J = 8.4 Hz), 6.49 (1H, d, J = 8.0 Hz), 6.66 (1H, d, J = 7.6 Hz), 6.93 (1H, t, J = 7.6 Hz).
4-Chloro-5-(methylsulfanyl)-2,3-dihydro-1H-indole (100b). Compound 100b was prepared from compound 99b in a manner similar to that described for compound 100f. Yield 23%. 1H NMR (400 MHz, CDCl3) δ 2.39 (3H, s), 3.09 (2H, t, J = 8.4 Hz), 3.62 (2H, t, J = 8.4 Hz), 6.47 (1H, d, J = 8.0 Hz), 7.09 (1H, d, J = 8.4 Hz).
4-Chloro-5-(methylsulfonyl)-2,3-dihydro-1H-indole hydrochloride (101b). Compound 101b was prepared from compound 100b in a manner similar to that described for compound 101f. White solid.
Yield 74% from 100b. MS (ESI) m/z 232 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 3.05 (2H, t, J = 8.8 Hz), 3.19 (3H, s), 3.65 (2H, t, J = 8.8 Hz), 6.45 (1H, d, J = 8.0 Hz), 7.58 (1H, d, J = 8.0 Hz).
4-Methyl-5-(methylsulfanyl)-2,3-dihydro-1H-indole (100c). Compound 100c was prepared from 4-methyl-1H-indole (98c) in a manner similar to that described for compounds 99f and 100f. Yield 12%
from 98c. 1H NMR (400 MHz, CDCl3) δ 2.33 (3H, s), 2.34 (3H, s), 2.98 (2H, t, J = 8.4 Hz), 3.58 (2H, t, J
= 8.4 Hz), 6.47 (1H, d, J = 8.0 Hz), 7.10 (1H, d, J = 8.0 Hz).
4-Methyl-5-(methylsulfonyl)-2,3-dihydro-1H-indole hydrochloride (101c). Compound 101c was prepared from compound 100c in a manner similar to that described for compound 101f. Yellow solid.
Yield 47% from 100c. MS (ESI) m/z 212 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 2.46 (3H, s), 3.03 (2H, t, J = 8.8 Hz). 3.11 (3H, s), 3.63 (2H, t, J = 8.8 Hz), 6.69 (1H, d, J = 8.4 Hz), 6.78 (2H, m), 7.62 (1H, d, J = 8.8 Hz).
6-Fluoro-2,3-dihydro-1H-indole (99d). Compound 99d was prepared from 6-fluoro-1H-indole (98d) in a manner similar to that described for compound 99f. White solid. Yield 100%. 1H NMR (400 MHz, CDCl3) δ 2.96 (2H, t, J = 8.4 Hz), 3.58 (2H, t, J = 8.4 Hz), 3.81 (1H, bs), 6.29–6.37 (2H, m), 6.96–6.99 (1H, m).
6-Fluoro-5-(methylsulfonyl)-2,3-dihydro-1H-indole hydrochloride (101d). Compound 101d was prepared from compound 99d in a manner similar to that described for compounds 100f and 101f. White solid. Yield 13% from 99d. MS (ESI) m/z 216 [M + H]+. 1H NMR (400 MHz, CDCl3) δ 2.97 (2H, t, J = 8.8 Hz). 3.13 (3H, s), 3.60 (2H, t, J = 8.8 Hz), 6.33 (1H, d, J = 8.0 Hz), 7.31 (1H, d, J = 7.6 Hz).
6-Methoxy-2,3-dihydro-1H-indole (99e). Compound 99e was prepared from 6-methoxy-1H-indole (98e) in a manner similar to that described for compound 99f. Yield 100%. 1H NMR (400 MHz, CDCl3) δ 2.96 (2H, t, J = 8.4 Hz), 3.55 (2H, t, J = 8.4 Hz), 3.75 (3H, s), 6.24–6.26 (2H, m), 6.98 (1H, d, J = 8.8 Hz).
6-Methoxy-5-(methylsulfanyl)-2,3-dihydro-1H-indole (100e). Compound 100e was prepared from compound 99e in a manner similar to that described for compound 100f. Yield 12%. 1H NMR (400 MHz, CDCl3) δ 2.35 (3H, s), 2.97 (2H, t, J = 8.4 Hz), 3.57 (2H, t, J = 8.4 Hz), 3.83 (3H, s), 6.26 (1H, s), 7.10 (1H, s).
6-Methoxy-5-(methylsulfonyl)-2,3-dihydro-1H-indole hydrochloride (101e). Compound 101e was prepared from compound 100e in a manner similar to that described for compound 101f. Yellow solid.
Yield 74% from 100e. MS (ESI) m/z 228 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 2.92 (2H, t, J = 8.4 Hz), 3.07 (3H, s), 3.56 (2H, t, J = 8.8 Hz), 3.83 (3H, s), 6.26 (1H, s), 7.34 (1H, s).
7-Fluoro-2,3-dihydro-1H-indole (99f). 7-Fluoro-1H-indole (98f, 20.0 g, 148 mmol) was dissolved in acetic acid (60 mL) and sodium cyanoborohydride (18.7 g, 296 mmol) was added in portions. The mixture was stirred for 2 h and then poured into 1500 mL of 2 M aqueous NaOH solution. The mixture was extracted with CH2Cl2. The organic layers were combined, washed with brine, dried over MgSO4, and concentrated under reduced pressure to give the title compound (20.0 g, 98%). 1H NMR (400 MHz, CDCl3) δ 3.08 (2H, t, J = 8.4 Hz), 3.62 (2H, t, J = 8.4 Hz), 6.62–6.66 (1H, m), 6.78–6.83 (1H, m), 6.90 (1H, dd, J = 7.6, 0.4 Hz).
7-Fluoro-5-(methylsulfanyl)-2,3-dihydro-1H-indole (100f). To a stirred solution of potassium thiocyanate (14.0 g, 145 mmol) in MeOH (150 mL) was added bromine (3.80 mL, 72.3 mmol) dropwise at 0 °C under N2 atmosphere. After the mixture was stirred for 15 min, a solution of compound 99f (9.00 g, 65.7 mmol) in MeOH (100 mL) was added, and the resulting mixture was stirred at room temperature for 3 h. A solution of potassium hydroxide (18.4 g, 329 mmol) in water (120 mL) was added slowly to the mixture followed by stirring at 43 °C for further 30 min. The reaction mixture was cooled to 10 °C, and then iodomethane (4.10 mL, 65.7 mmol) was added. The resulting mixture was stirred at room temperature for 30 min. After the mixture was concentrated under reduced pressure, the residue was partitioned between CH2Cl2 and water. The organic layer was separated, dried, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether/CH2Cl2
= 2/1 to 1/1) to give the title compound (2.80 g, 23%). 1H NMR (400 MHz, CDCl3) δ 2.42 (3H, s), 3.05 (2H, t, J = 8.4 Hz), 3.62 (2H, t, J = 8.4 Hz), 6.85 (1H, d, J = 10.8 Hz), 6.93 (1H, s).
7-Fluoro-5-(methylsulfonyl)-2,3-dihydro-1H-indole hydrochloride (101f). Step A. To a solution of
compound 100f (4.40 g, 24.0 mmol) in CH2Cl2 (30 mL) were added di-tert-butyl dicarbonate (6.60 g, 30.2 mmol) and triethylamine (4.30 mL, 30.2 mmol), and the resulting mixture was stirred at room temperature for 48 h. The mixture was treated with saturated aqueous NaHCO3 solution and extracted with AcOEt. The combined organic layer was successively washed with 1 M hydrochloric acid, saturated aqueous NaHCO3 solution and brine, dried over MgSO4, and concentrated to give tert-butyl 7-fluoro-5-(methylsulfanyl)-2,3-dihydro-1H-indole-1-carboxylate as an oil. The oil was dissolved in CH2Cl2 (60 mL) and mCPBA (ca. 65%, 8.8 g, 72 mmol) was added at 0 °C. After being stirred for 1 h, the reaction mixture was quenched with 10% aqueous Na2SO3 solution and extracted with CH2Cl2. The combined organic layer was washed with saturated aqueous Na2CO3 solution and brine, and dried over Na2SO4. After removal of the solvent, the residue was purified by silica gel column chromatography (hexane/AcOEt = 10/1 to 60/40) to give tert-butyl 7-fluoro-5-(methylsulfonyl)-2,3-dihydro-1H-indole-1- carboxylate as a white powder (4.10 g, 54% from 100f). 1H NMR (400 MHz, CDCl3) δ 1.53 (9H, s), 3.04 (3H, s), 3.15 (2H, t, J = 8.4 Hz), 4.14 (2H, t, J = 8.4 Hz), 7.26 (1H, s), 7.52 (1H, s).
Step B. To a solution of tert-butyl 7-fluoro-5-(methylsulfonyl)-2,3-dihydro-1H-indole-1-carboxylate (4.10 g) in AcOEt (20 mL) was added 5 N HCl in AcOEt (40 mL), and the mixture was stirred at room temperature for 3 h. The precipitated solid was collected by filtration and washed with AcOEt–iPr2O to afford the title compound as a white solid (101f, 3.10 g, 94%). MS (ESI) m/z 216 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 3.09 (2H, t, J = 8.8 Hz), 3.11 (3H, s), 3.64 (2H, t, J = 8.8 Hz), 7.37 (1H, d, J = 9.6 Hz), 7.38 (1H, s).
7-Chloro-2,3-dihydro-1H-indole (99g). Compound 99g was prepared from 7-chloro-1H-indole (98g) in a manner similar to that described for compound 99f. Yield 99%. 1H NMR (400 MHz, CDCl3) δ 3.11 (2H, t, J = 8.4 Hz), 3.62 (2H, t, J = 8.4 Hz), 6.62 (1H, t, J = 7.6 Hz), 6.98–7.02 (2H, m).
7-Chloro-5-(methylsulfanyl)-2,3-dihydro-1H-indole (100g). Compound 100g was prepared from compound 99g in a manner similar to that described for compound 100f. Yield 27%. 1H NMR (400 MHz, CDCl3) δ 2.41 (3H, s), 3.09 (2H, t, J = 8.4 Hz), 3.63 (2H, t, J = 8.4 Hz), 4.00 (1H, bs), 7.03 (1H, s), 7.05 (1H, s).
7-Chloro-5-(methylsulfonyl)-2,3-dihydro-1H-indole hydrochloride (101g). Compound 101g was prepared from compound 100g in a manner similar to that described for compound 101f. White solid.
Yield 55% from 100g. MS (ESI) m/z 232 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 3.11 (3H, s), 3.13 (2H, t, J = 8.8 Hz), 3.64 (2H, t, J = 8.8 Hz), 7.43 (1H, d, J = 1.2 Hz), 7.48 (1H, d, J = 1.6 Hz).
7-Methyl-2,3-dihydro-1H-indole (99h). Compound 99h was prepared from 7-methyl-1H-indole (98h) in a manner similar to that described for compound 99f. Yield 99%. 1H NMR (400 MHz, CDCl3) δ 2.15 (3H, s), 3.07 (2H, t, J = 8.4 Hz), 3.58 (2H, t, J = 8.4 Hz), 6.67 (1H, t, J = 7.2 Hz), 6.87 (1H, d, J = 7.2 Hz), 7.00 (1H, d, J = 7.2 Hz).
7-Methyl-5-(methylsulfanyl)-2,3-dihydro-1H-indole (100h). Compound 100h was prepared from compound 99h in a manner similar to that described for compound 100f. Yield 14%. 1H NMR (400 MHz,
CDCl3) δ 2.11 (3H, s), 2.42 (3H, s), 3.04 (2H, t, J = 8.4 Hz), 3.58 (2H, t, J = 8.4 Hz), 6.93 (1H, d, J = 0.8 Hz), 7.03 (1H, s).
7-Methyl-5-(methylsulfonyl)-2,3-dihydro-1H-indole hydrochloride (101h). Compound 101h was prepared from compound 100h in a manner similar to that described for compound 101f. White solid.
Yield 62% from 100h. MS (ESI) m/z 212 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 2.13–2.16 (3H, m), 3.05–3.09 (5H, m), 3.59-3.63 (2H, m), 7.37–7.41 (2H, m).
7-Methoxy-5-(methylsulfanyl)-2,3-dihydro-1H-indole (100i). Compound 100i was prepared from 7-methoxy-1H-indole (98i) in a manner similar to that described for compounds 99f and 100f. Yield 22%
from 98i. 1H NMR (400 MHz, CDCl3) δ 2.44 (3H, s), 3.03 (2H, t, J = 8.4 Hz), 3.58 (2H, t, J = 8.4 Hz), 3.81 (3H, s), 6.72 (1H, s), 6.84 (1H, s).
7-Methoxy-5-(methylsulfonyl)-2,3-dihydro-1H-indole hydrochloride (101i). Compound 101i was prepared from compound 100i in a manner similar to that described for compound 101f. Yellow solid.
Yield 48% from 100i. MS (ESI) m/z 228 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 3.12 (2H, t, J = 8.4 Hz), 3.15 (3H, s), 3.63 (2H, t, J = 8.4 Hz), 3.89 (3H, s), 7.27 (1H, d, J = 1.2 Hz), 7.35 (1H, s), 7.95 (2H, m).
2-[1-(tert-Butoxycarbonyl)piperidin-4-yl]ethyl 4-fluoro-5-(methylsulfonyl)-2,3-dihydro-1H-in- dole-1-carboxylate (102a). Compound 102a was prepared from compound 101a and tert-butyl 4-(2-hydroxyethyl)piperidine-1-carboxylate (88c) in a manner similar to that described for compound 102g. White solid. Yield 53%. MS (ESI/APCI) m/z 371 [M − Boc + 2H]+. 1H NMR (300 MHz, CDCl3) δ 1.09–1.26 (2H, m), 1.39–1.77 (14H, m), 2.62–2.78 (2H, m), 3.15–3.27 (5H, m), 4.00–4.21 (4H, m), 4.31 (2H, t, J = 6.4 Hz), 7.58–7.85 (2H, m). 13C NMR (100.6 MHz, CDCl3) δ 23.4, 28.5, 32.0, 33.2, 35.4, 43.9, 44.2, 48.7, 64.2, 79.4, 110.2 (d, J = 3.0 Hz), 121.9 (d, J = 14.1 Hz), 130.8, 152.7, 154.8, 155.7 (d, J = 252.5 Hz). Mp 152–154 °C. Anal. Calcd for C22H31FN2O6S: C, 56.15; H, 6.64; N, 5.95. Found: C, 56.30;
H, 6.69; N, 6.08.
2-[1-(tert-Butoxycarbonyl)piperidin-4-yl]ethyl 4-chloro-5-(methylsulfonyl)-2,3-dihydro-1H-in- dole-1-carboxylate (102b). Compound 102b was prepared from compound 101b and compound 88c in a manner similar to that described for compound 102g. White solid. Yield 91%. MS (ESI/APCI) m/z 431 [M − tBu + 2H]+. 1H NMR (300 MHz, CDCl3) δ 1.09–1.26 (2H, m), 1.40–1.76 (14H, m), 2.63–2.78 (2H, m), 3.17–3.26 (5H, m), 4.01–4.19 (4H, m), 4.32 (2H, t, J = 6.4 Hz), 7.84 (1H, bs), 8.00 (1H, d, J = 8.7 Hz). 13C NMR (100.6 MHz, CDCl3) δ 27.0, 28.5, 32.0, 33.2, 35.4, 43.2, 43.8, 47.9, 64.2, 79.4, 112.3, 128.7, 131.1, 132.0, 152.8, 154.8. Mp 145–147 °C. Anal. Calcd for C22H31ClN2O6S: C, 54.26; H, 6.42; N, 5.75. Found: C, 54.34; H, 6.46; N, 5.75.
2-[1-(tert-Butoxycarbonyl)piperidin-4-yl]ethyl 4-methyl-5-(methylsulfonyl)-2,3-dihydro-1H-in- dole-1-carboxylate (102c). Compound 102c was prepared from compound 101c and compound 88c in a manner similar to that described for compound 102g. White solid. Yield 51%. MS (ESI/APCI) m/z 367 [M − Boc + 2H]+. 1H NMR (300 MHz, CDCl3) δ 1.09–1.26 (2H, m), 1.46 (9H, s), 1.49–1.78 (5H, m),
2.56 (3H, s), 2.62–2.77 (2H, m), 3.04–3.15 (5H, m), 4.02–4.17 (4H, m), 4.24–4.37 (2H, m), 7.68–7.97 (2H, m). 13C NMR (100.6 MHz, CDCl3) δ 16.3, 26.5, 28.5, 32.0, 33.2, 35.4, 43.9, 44.2, 47.9, 63.9, 79.4, 111.9, 130.6, 132.5, 133.7, 153.0, 154.9. Mp 143–145 °C. Anal. Calcd for C23H34N2O6S: C, 59.21; H, 7.34; N, 6.00. Found: C, 59.23; H, 7.33; N, 6.22.
2-[1-(tert-Butoxycarbonyl)piperidin-4-yl]ethyl 6-fluoro-5-(methylsulfonyl)-2,3-dihydro-1H-in- dole-1-carboxylate (102d). Compound 102d was prepared from compound 101d and compound 88c in a manner similar to that described for compound 102g. White solid. Yield 81%. MS (ESI/APCI) m/z 415 [M − tBu + 2H]+. 1H NMR (300 MHz, CDCl3) δ 1.07–1.31 (2H, m), 1.38–1.85 (14H, m), 2.61–2.80 (2H, m), 3.09–3.25 (5H, m), 4.00–4.22 (4H, m), 4.22–4.41 (2H, m), 7.59–7.85 (2H, m). 13C NMR (100.6 MHz, CDCl3) δ 26.4, 28.5, 32.0, 33.2, 35.4, 43.8, 44.1, 48.7, 64.1, 79.4, 103.6 (d, J = 28.2 Hz), 121.3 (d, J = 16.1 Hz), 125.0, 152.8, 154.8, 160.2 (d, J = 251.5 Hz). Mp 138–140 °C. Anal. Calcd for C22H31FN2O6S:
C, 56.15; H, 6.64; N, 5.95. Found: C, 56.34; H, 6.74; N, 5.97.
2-[1-(tert-Butoxycarbonyl)piperidin-4-yl]ethyl 6-methoxy-5-(methylsulfonyl)-2,3-dihydro-1H-in- dole-1-carboxylate (102e). Compound 102e was prepared from compound 101e and compound 88c in a manner similar to that described for compound 102g. Colorless oil. Yield 56%. MS (ESI/APCI) m/z 383 [M − Boc + 2H]+. 1H NMR (300 MHz, CDCl3) δ 1.08–1.27 (2H, m), 1.42–1.80 (14H, m), 2.62–2.77 (2H, m), 3.11 (2H, t, J = 8.7 Hz), 3.18 (3H, s), 3.99 (3H, s), 4.02–4.19 (4H, m), 4.24–4.36 (2H, m), 7.61–7.77 (2H, m). 13C NMR (100.6 MHz, CDCl3) δ 26.3, 28.5, 32.0, 33.2, 35.4, 43.1, 43.8, 48.6, 56.5, 63.8, 79.4, 99.4, 121.8, 125.5, 153.1, 154.8, 158.2. HRMS (ESI) Calcd for C23H34N2O7S: m/z 505.1979 [M + Na]+. Found: 505.1957 [M + Na]+. Analytical HPLC 100% .
2-[1-(tert-Butoxycarbonyl)piperidin-4-yl]ethyl 7-fluoro-5-(methylsulfonyl)-2,3-dihydro-1H-in- dole-1-carboxylate (102f). Compound 102f was prepared from compound 101f and compound 88c in a manner similar to that described for compound 102g. Colorless oil. Yield 87%. MS (ESI/APCI) m/z 415 [M − tBu + 2H]+. 1H NMR (300 MHz, CDCl3) δ 1.07–1.24 (2H, m), 1.41–1.77 (14H, m), 2.61–2.78 (2H, m), 3.05 (3H, s), 3.18 (2H, t, J = 8.5 Hz), 4.00–4.25 (4H, m), 4.29 (2H, t, J = 6.6 Hz), 7.52–7.61 (2H, m).
13C NMR (100.6 MHz, CDCl3) δ 28.5, 28.7, 32.0, 32.9, 35.3, 43.9, 44.7, 50.4, 64.6, 79.3, 116.6 (d, J = 25.2 Hz), 119.6 (d, J = 4.0 Hz), 134.2 (d, J = 9.1 Hz), 136.6 (d, J = 5.0 Hz), 138.1 (d, J = 3.0 Hz), 150.1 (d, J = 259.5 Hz), 152.9, 154.9. HRMS (ESI) Calcd for C22H31FN2O6S: m/z 493.1779 [M + Na]+. Found:
493.1769 [M + Na]+. Analytical HPLC 100% .
2-[1-(tert-Butoxycarbonyl)piperidin-4-yl]ethyl 7-chloro-5-(methylsulfonyl)-2,3-dihydro-1H-in- dole-1-carboxylate (102g). To a mixture of compound 88c (0.229 g, 1.00 mmol) and triphosgene (0.099 g, 0.33 mmol) in Et2O (10 mL) was added dropwise a solution of pyridine (0.081 mL, 1.0 mmol) in Et2O (5 mL) at −40 °C under N2 atmosphere. After addition, the mixture was allowed to warm to 0 °C followed by stirring for 3 h. The precipitated solid was removed by filtration, and the filtrate was concentrated. The residue was dissolved in THF (10 mL), and then compound 101g (0.179 g, 0.667 mmol) and N,N-diisopropylethylamine (0.348 mL, 2.00 mmol) were added. The resulting mixture was stirred at room
temperature for 19 h. The reaction mixture was diluted with AcOEt, successively washed with aqueous citric acid solution and brine, and dried over Na2SO4. After removal of the solvent, the residue was purified by silica gel column chromatography (NH, hexane/AcOEt = 80/20 to 70/30) to give the title compound as a colorless oil (0.180 g, 55%). MS (ESI/APCI) m/z 431 [M − tBu + 2H]+. 1H NMR (400 MHz, DMSO-d6) δ 1.07–1.25 (2H, m), 1.41–1.56 (10H, m), 1.63–1.75 (4H, m), 2.61–2.78 (2H, m), 3.06 (3H, s), 3.15 (2H, t, J = 8.0 Hz), 3.98–4.17 (2H, m), 4.21 (2H, t, J = 8.0 Hz), 4.30 (2H, t, J = 6.8 Hz), 7.65 (1H, d, J = 1.5 Hz), 7.82 (1H, d, J = 1.5 Hz). 13C NMR (100.6 MHz, CDCl3) δ 28.5, 29.5, 32.0, 33.0, 35.4, 43.9, 44.7, 51.5, 64.6, 79.4, 121.8, 124.2, 129.5, 137.3, 138.2, 145.0, 153.3, 154.8. HRMS (ESI) Calcd for C22H31ClN2O6S: m/z 509.1484 [M + Na]+. Found: 509.1469 [M + Na]+. Analytical HPLC 100%.
2-[1-(tert-Butoxycarbonyl)piperidin-4-yl]ethyl 7-methyl-5-(methylsulfonyl)-2,3-dihydro-1H-in- dole-1-carboxylate (102h). Compound 102h was prepared from compound 101h and compound 88c in a manner similar to that described for compound 102g. Colorless oil. Yield 100%. MS (ESI/APCI) m/z 411 [M − tBu + 2H]+. 1H NMR (300 MHz, CDCl3) δ 1.08–1.25 (2H, m), 1.43–1.75 (14H, m), 2.35 (3H, s), 2.61–2.77 (2H, m), 3.02 (3H, s), 3.09 (2H, t, J = 7.8 Hz), 3.98–4.21 (4H, m), 4.26 (2H, t, J = 6.8 Hz), 7.59 (1H, s), 7.62 (1H, s). 13C NMR (100.6 MHz, CDCl3) δ 20.5, 28.5, 29.1, 32.0, 33.2, 35.5, 43.9, 44.8, 50.9, 64.2, 79.4, 121.0, 128.7, 130.1, 135.6, 136.1, 146.4, 153.9, 154.9. Anal. Calcd for C23H34N2O6S·0.1hexane: C, 59.65; H, 7.51; N, 5.89. Found: C, 59.62; H, 7.51; N 6.05. HRMS (ESI) Calcd for C23H34N2O6S: m/z 489.2030 [M + Na]+. Found: 489.2017 [M + Na]+.
2-[1-(tert-Butoxycarbonyl)piperidin-4-yl]ethyl 7-methoxy-5-(methylsulfonyl)-2,3-dihydro-1H-in- dole-1-carboxylate (102i). Compound 102i was prepared from compound 101i and compound 88c in a manner similar to that described for compound 102g. Colorless amorphous solid. Yield 92%. MS (ESI/APCI) m/z 383 [M − Boc + 2H]+. 1H NMR (300 MHz, CDCl3) δ 1.07–1.24 (2H, m), 1.45 (9H, s), 1.49 -1.75 (5H, m), 2.61–2.76 (2H, m), 3.04 (3H, s), 3.09 (2H, t, J = 8.1 Hz), 3.93 (3H, s), 4.02–4.21 (4H, m), 4.25 (2H, t, J = 6.6 Hz), 7.35–7.43 (2H, m). 13C NMR (100.6 MHz, CDCl3) δ 28.5, 29.4, 32.0, 33.0, 35.5, 43.9, 44.8, 51.3, 56.4, 64.1, 79.4, 111.3, 116.4, 136.1, 137.0, 137.1, 149.5, 153.7, 154.8. HRMS (ESI) Calcd for C23H34N2O7S: m/z 505.1979 [M + Na]+. Found: 505.1962 [M + Na]+. Analytical HPLC 99.2%.
2-[1-(tert-Butoxycarbonyl)piperidin-4-yl]propyl 7-fluoro-5-(methylsulfonyl)-2,3-dihydro-1H-in- dole-1-carboxylate (102j). Compound 102j was prepared from compound 101f and compound 88a in a manner similar to that described for compound 102g. Colorless oil. Yield 64%. MS (ESI/APCI) m/z 385 [M − Boc + 2H]+. 1H NMR (300 MHz, CDCl3) δ 0.96 (3H, d, J = 7.2 Hz), 1.13–1.36 (2H, m), 1.40–1.86 (13H, m), 2.56–2.75 (2H, m), 3.05 (3H, s), 3.19 (2H, t, J = 8.3 Hz), 4.05–4.31 (6H, m), 7.51–7.65 (2H, m). 13C NMR (100.6 MHz, CDCl3) δ 13.8, 28.2, 28.5, 28.7, 29.7, 37.2, 38.2, 44.3, 44.7, 50.4, 69.7, 79.4, 116.6 (d, J = 24.1 Hz), 119.6, 124.1, 134.2 (d, J = 10.1 Hz), 136.6 (d, J = 6.0 Hz), 138.1, 150.1 (d, J = 259.5 Hz), 153.0, 154.8. HRMS (ESI) Calcd for C23H33FN2O6S: m/z 507.1936 [M + Na]+. Found:
507.1920 [M + Na]+. Analytical HPLC 98.3%.
2-(Piperidin-4-yl)propyl 7-fluoro-5-(methylsulfonyl)-2,3-dihydro-1H-indole-1-carboxylate (103).
Compound 102j (650 mg, 1.34 mmol) was dissolved in TFA (5 mL) at 0 °C. After stirring at room temperature for 1 h, the reaction mixture was diluted with water, basified with 8 M aqueous NaOH solution, and extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and concentrated under reduced pressure to give the title compound as a colorless oil (550 mg, quant.) 1H NMR (300 MHz, CDCl3) δ 0.98 (3H, d, J = 6.8 Hz), 1.35–1.56 (2H, m), 1.71–1.89 (2H, m), 2.61–2.79 (2H, m), 3.05 (3H, s), 3.07–3.37 (7H, m), 4.07–4.30 (4H, m), 7.48–7.66 (2H, m).
2-[1-(5-Ethylpyrimidin-2-yl)piperidin-4-yl]propyl 7-fluoro-5-(methylsulfonyl)-2,3-dihydro-1H- indole-1-carboxylate (104). A mixture of compound 103 (0.210 g, 0.546 mmol), 2-chloro-5-ethyl- pyrimidine (0.086 g, 0.600 mmol), and Cs2CO3 (0.407 g, 1.25 mmol) in NMP (1 mL) was stirred at 70 °C under N2 atmosphere for 22 h. The mixture was diluted with AcOEt, washed with water and brine, dried over Na2SO4, and concentrated. The residue was purified by silica gel column chromatography (NH, hexane/AcOEt = 70/30 to 40/60) and crystallized from heptane–AcOEt to give the title compound as a white solid (0.115 g, 43%). MS (ESI/APCI) m/z 491 [M + H]+. 1H NMR (300 MHz, CDCl3) δ 0.97 (3H, d, J = 6.8 Hz), 1.14–1.44 (5H, m), 1.53–1.89 (4H, m), 2.45 (2H, q, J = 7.5 Hz), 2.72–2.87 (2H, m), 3.04 (3H, s), 3.19 (2H, t, J = 8.5 Hz), 4.07–4.32 (4H, m), 4.70–4.83 (2H, m), 7.52–7.60 (2H, m), 8.16 (2H, s). 13C NMR (100.6 MHz, CDCl3) δ 13.9, 15.6, 22.7, 28.2, 27.8, 29.6, 37.3, 38.7, 44.5, 44.6, 44.7, 50.4, 69.9, 116.6 (d, J = 24.1 Hz), 119.6 (d, J = 3.0 Hz), 124.1, 134.2 (d, J = 10.2 Hz), 136.6 (d, J = 5.0 Hz), 138.1 (d, J = 3.0 Hz), 150.1 (d, J = 258.5 Hz), 153.0, 157.1, 160.8. Mp 87–90 °C. Anal. Calcd for C24H31FN4O4S:
C, 58.76; H, 6.37; N, 11.42. Found: C, 58.48; H, 6.43; N, 11.38.
tert-Butyl 4-{4-[5-(methylsulfonyl)-2,3-dihydro-1H-indol-1-yl]-4-oxobutyl}piperidine-1-carboxy-
late (106a). A mixture of compound 85 (150 mg, 0.760 mmol), 4-[1-(tert-butoxycarbonyl)piperidin-4-yl]- butanoic acid (105, 310 mg, 1.14 mmol), HATU (358 mg, 0.942 mmol), and triethylamine (0.320 mL, 2.30 mmol) in DMF (10 mL) was stirred at room temperature for 24 h. The reaction mixture was quenched with water and extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and concentrated under reduced pressure. Purification by silica gel column chromatography (hexane/AcOEt = 100/0 to 50/50) gave the title compound as a pale pink solid (170 mg, 50%). MS (ESI/APCI) m/z 351 [M − Boc + 2H]+. 1H NMR (300 MHz, CDCl3) δ 1.02–1.18 (2H, m), 1.30–1.39 (2H, m), 1.45 (10H, s), 1.63–1.86 (4H, m), 2.45 (2H, t, J = 6.8 Hz), 2.68 (2H, t, J = 12.4 Hz), 2.96–3.09 (3H, m), 3.27 (2H, t, J = 8.5 Hz), 3.95–4.24 (4H, m), 7.67–7.84 (2H, m), 8.38 (1H, d, J = 7.9 Hz). 13C NMR (100.6 MHz, CDCl3) δ 21.5, 27.6, 28.5, 32.1, 36.1, 36.2, 44.1, 44.8, 48.3, 79.2, 116.9, 123.7, 128.2, 132.3, 134.8, 147.6, 154.9, 171.9. Mp 128–131 °C. HRMS (ESI) Calcd for C23H34N2O5S: m/z 473.2081 [M + Na]+. Found: 473.2067 [M + Na]+. Analytical HPLC 100%.
tert-Butyl 4-[2-({[5-(methylsulfonyl)-2,3-dihydro-1H-indol-1-yl]carbonyl}amino)ethyl]piperi- dine-1-carboxylate (106b). To a solution of compound 85 (112 mg, 0.568 mmol) and triphosgene (203 mg, 0.684 mmol) in AcOEt (6 mL) was slowly added triethylamine (0.120 mL, 0.861 mmol) at 0 °C.
After stirring at 0 °C for 1 h, saturated aqueous NaHCO3 solution was added to the mixture. The mixture was extracted with AcOEt. The extract was washed with brine, dried over MgSO4, and concentrated under reduced pressure. The residue was dissolved in THF (6 mL), and then tert-butyl 4-(2-aminoethyl)- piperidine-1-carboxylate (107, 130 mg, 0.567 mmol) and triethylamine (0.120 mL, 0.861 mmol) were added at 0 °C. The mixture was allowed to warm to room temperature followed by stirring for 20 h. The reaction mixture was quenched with water and extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and concentrated under reduced pressure. Purification by silica gel column chromatography gave the title compound as a white solid (228 mg, 89%). MS (ESI/APCI) m/z 352 [M − Boc + 2H]+. 1H NMR (300 MHz, CDCl3) δ 1.08–1.22 (2H, m), 1.45 (9H, s), 1.50–1.57 (3H, m), 1.70 (2H, d, J = 12.8 Hz), 2.60–2.76 (2H, m), 3.02 (3H, s), 3.26 (2H, t, J = 8.7 Hz), 3.32–3.45 (2H, m), 3.91–4.19 (4H, m), 4.64 (1H, t, J = 5.5 Hz), 7.67 (1H, s), 7.74 (1H, dd, J = 8.7, 1.9 Hz), 8.10 (1H, d, J = 8.7 Hz). 13C NMR (100.6 MHz, CDCl3) δ 27.3, 28.5, 32.0, 33.8, 36.9, 38.2, 43.8, 44.9, 47.5, 79.4, 114.8, 123.6, 128.3, 131.1, 132.9, 148.8, 154.6, 154.9. Mp 162–163 °C. Anal. Calcd for C22H33N3O5S·0.1AcOEt: C, 58.44; H, 7.40; N, 9.13. Found: C, 58.57; H, 7.48; N 8.87.
tert-Butyl 4-{4-[5-(methylsulfonyl)-2,3-dihydro-1H-indol-1-yl]butyl}piperidine-1-carboxylate (106c). To a stirred solution of compound 85 (882 mg, 4.47 mmol) in NMP (5 mL) was added sodium hydride (60% oil dispersion, 360 mg, 9.00 mmol) at 0 °C. After the mixture was stirred at 0 °C for 30 min, tert-butyl 4-{4-[(methylsulfonyl)oxy]butyl}piperidine-1-carboxylate (108, 1.50 g, 4.47 mmol) was added to the mixture at 0 °C. The resulting mixture was allowed to warm to room temperature followed by stirring overnight. The reaction mixture was quenched with water and extracted with AcOEt. The combined organic layer was washed with water and brine, dried over MgSO4, and concentrated under reduced pressure. The residual oil was purified by silica gel column chromatography (NH, hexane/AcOEt
= 95/5 to 75/25) to give the title compound as a colorless oil (321 mg, 16%). MS (ESI/APCI) m/z 337 [M
− Boc + 2H]+. 1H NMR (300 MHz, CDCl3) δ 0.88–1.19 (2H, m), 1.19–1.43 (5H, m), 1.41–1.51 (9H, m), 1.49–1.75 (4H, m), 2.66 (2H, t, J = 12.1 Hz), 2.99 (3H, s), 3.00–3.10 (2H, m), 3.16 (2H, t, J = 7.3 Hz), 3.27–3.63 (2H, m), 3.74–4.34 (2H, m), 6.35 (1H, d, J = 8.3 Hz), 7.48 (1H, d, J = 1.9 Hz), 7.60 (1H, dd, J
= 8.3, 1.9 Hz). 13C NMR (100.6 MHz, CDCl3) δ 24.2, 27.3, 27.6, 28.5, 32.2, 36.0, 36.3, 44.1, 45.1, 47.4, 52.3, 79.2, 104.3, 123.3, 126.7, 129.1, 130.2, 154.9, 156.4. Anal. Calcd for C23H36N2O4S: C, 63.27; H, 8.31; N, 6.42. Found: C, 63.06; H, 8.41; N, 6.24.
tert-Butyl 4-(3-bromophenoxy)piperidine-1-carboxylate (111a). A mixture of 3-bromophenol (109a, 8.00 g, 46.2 mmol), tert-butyl 4-{[(4-methylphenyl)sulfonyl]oxy}piperidine-1-carboxylate (110, 23.0 g, 64.7 mmol), and potassium carbonate (13.8 g, 100 mmol) in DMF (200 mL) was stirred at 80 °C for 16 h.
The mixture was partitioned between with AcOEt and water. The organic layer was washed with brine, dried over MgSO4, and concentrated. The residue was purified by silica gel column chromatography (NH, hexane/AcOEt = 100/0 to 90/10) to give the title compound as an oil (14.2 g, 86%). 1H NMR (300 MHz, CDCl3) δ 1.47 (9H, s), 1.64–1.81 (2H, m), 1.84–1.99 (2H, m), 3.27–3.41 (2H, m), 3.62–3.75 (2H, m),
4.39–4.50 (1H, m), 6.79–6.88 (1H, m), 7.03–7.20 (3H, m).
tert-Butyl 4-{3-[5-(methylsulfonyl)-2,3-dihydro-1H-indol-1-yl]phenoxy}piperidine-1-carboxylate (114a). A mixture of compound 111a (7.60 g, 21.3 mmol), compound 85 (3.00 g, 15.2 mmol), Pd2(dba)3
(641 mg, 0.700 mmol), Xantphos (810 mg, 1.40 mmol), and Cs2CO3 (9.90 g, 30.4 mmol) in toluene (130 mL) was stirred at 105 °C under Ar atmosphere for 16 h. The mixture was partitioned between with AcOEt and water. The organic layer was washed with brine, dried over MgSO4, and concentrated. The residue was purified by silica gel column chromatography (hexane/AcOEt = 75/25 to 60/40) to give the title compound as a solid (1.06 g, 15%). 1H NMR (300 MHz, CDCl3) δ 1.47 (9H, s), 1.70–1.85 (2H, m), 1.87–2.01 (2H, m), 3.02 (3H, s), 3.19 (2H, t, J = 8.7 Hz), 3.28–3.45 (2H, m), 3.61–3.80 (2H, m), 4.07 (2H, t, J = 8.7 Hz), 4.42–4.57 (1H, m), 6.60–6.69 (1H, m), 6.76–6.82 (1H, m), 6.82–6.92 (1H, m), 7.07 (1H, d, J = 9.0 Hz), 7.20–7.36 (1H, m), 7.56–7.71 (2H, m).
Propan-2-yl 4-{3-[5-(methylsulfonyl)-2,3-dihydro-1H-indol-1-yl]phenoxy}piperidine-1-carboxy- late (115a). A mixture of compound 114a (30 mg, 0.063 mmol) and TFA (3 mL) was stirred at room temperature for 30 min. After the mixture was concentrated under reduced pressure, the residue was dissolved in THF (10 mL). Triethylamine (0.044 mL, 0.32 mmol) and isopropyl chloroformate (0.013 mL, 0.10 mmol) were added to the mixture at room temperature followed by stirring at room temperature for 2 h. The reaction mixture was diluted with AcOEt, washed with water and brine, and dried over Na2SO4. After removal of the solvent, the residue was purified by silica gel column chromatography (hexane/AcOEt = 50/50 to 30/70) to give the title compound as a colorless oil (27 mg, 93% over 2 steps).
MS (ESI/APCI) m/z 459 [M + H]+. 1H NMR (300 MHz, DMSO-d6) δ 1.19 (6H, d, J = 6.4 Hz), 1.48–1.62 (2H, m), 1.86–1.97 (2H, m), 3.09 (3H, s), 3.12–3.36 (4H, m), 3.63–3.74 (2H, m), 4.06 (2H, t, J = 8.5 Hz), 4.56–4.66 (1H, m), 4.72–4.84 (1H, m), 6.72 (1H, dd, J = 8.1, 2.1 Hz), 6.84 (1H, t, J = 2.3 Hz), 6.91 (1H, dd, J = 7.9, 1.9 Hz), 7.09 (1H, d, J = 8.3 Hz), 7.30 (1H, t, J = 8.1 Hz), 7.55–7.64 (2H, m). 13C NMR (100.6 MHz, CDCl3) δ 22.3, 27.4, 30.5, 40.6, 45.0, 52.9, 68.7, 72.2, 107.2, 107.8, 110.2, 112.1, 124.0, 128.6, 129.3, 130.3, 132.0, 143.8, 151.8, 155.3, 158.2. HRMS (ESI) Calcd for C24H30N2O5S: m/z 459.1948 [M + H]+. Found: 459.1957 [M + H]+. Analytical HPLC 98.9%.
tert-Butyl 4-[(2-chloropyridin-4-yl)oxy]piperidine-1-carboxylate (111b). Compound 111b was prepared from compound 109b in a manner similar to that described for compound 111a. Oil. Yield 88%.
1H NMR (300 MHz, CDCl3) δ 1.47 (9H, s), 1.66–1.84 (2H, m), 1.87–2.03 (2H, m), 3.29–3.51 (2H, m), 3.59–3.77 (2H, m), 4.50–4.63 (1H, m), 6.74 (1H, dd, J = 6.0, 2.3 Hz), 6.83 (1H, d, J = 2.3 Hz), 8.19 (1H, d, J = 5.7 Hz).
Propan-2-yl 4-({2-[5-(methylsulfonyl)-2,3-dihydro-1H-indol-1-yl]pyridin-4-yl}oxy)piperidine-1- carboxylate (115b). Step A. A mixture of compound 111b (4.76 g, 15.2 mmol), compound 85 (3.00 g, 15.2 mmol), Pd2(dba)3 (641 mg, 0.700 mmol), Xantphos (810 mg, 1.40 mmol), and Cs2CO3 (9.90 g, 30.4 mmol) in toluene (130 mL) was stirred at 105 °C under Ar atmosphere for 16 h. The mixture was partitioned between AcOEt and water. The organic layer was washed with brine, dried over MgSO4, and
concentrated. The residue was purified by silica gel column chromatography (hexane/AcOEt = 75/25 to 60/40) to give tert-butyl 4-({2-[5-(methylsulfonyl)-2,3-dihydro-1H-indol-1-yl]pyridin-4-yl}oxy)piperi- dine-1-carboxylate (114b) as a crude material (oil, 7.20 g). The oil was dissolved in a mixed solvent of AcOEt (50 mL) and MeOH (50 mL), and then 4 M HCl in AcOEt (20 mL) was added. The resulting mixture was stirred at room temperature for 16 h. After the mixture was concentrated under reduced pressure, the residue was diluted with AcOEt and basified with 8 M aqueous NaOH solution. The organic layer was separated, washed with water and brine, and dried over MgSO4. After removal of the solvent, the residue was purified by silica gel column chromatography (NH, hexane/AcOEt = 50/50 to 0/100) to give 5-(methylsulfonyl)-1-[4-(piperidin-4-yloxy)pyridin-2-yl]-2,3-dihydro-1H-indole as a solid (1.84 g, 32% over 2 steps). 1H NMR (300 MHz, CDCl3) δ 1.39–1.61 (2H, m), 1.84–2.11 (2H, m), 2.53–2.67 (2H, m), 2.85–3.04 (2H, m), 3.12 (3H, s), 3.17–3.46 (3H, m), 4.10 (2H, t, J = 8.9 Hz), 4.52–4.70 (1H, m), 6.38 (1H, d, J = 1.9 Hz), 6.63 (1H, dd, J = 5.8, 2.1 Hz), 7.57–7.73 (2H, m), 8.17 (1H, d, J = 6.0 Hz), 8.43 (1H, d, J = 9.0 Hz). Anal. Calcd for C19H23N3O3S: C, 61.10; H, 6.21; N, 11.25. Found: C, 61.21; H, 6.42; N, 11.02.
Step B. To a mixture of 5-(methylsulfonyl)-1-[4-(piperidin-4-yloxy)pyridin-2-yl]-2,3-dihydro-1H- indole (400 mg, 1.07 mmol) and triethylamine (0.279 mL, 2.00 mmol) in THF (15 mL) was added isopropyl chloroformate (184 mg, 1.50 mmol) at room temperature, and the mixture was stirred for 16 h.
The reaction mixture was diluted with AcOEt, washed with water and brine, and dried over MgSO4. The solvent was removed by evaporation to give the title compound as a solid (115b, 400 mg, 81%). MS (ESI/APCI) m/z 460 [M + H]+. 1H NMR (300 MHz, CDCl3) δ 1.19 (6H, d, J = 6.4 Hz), 1.47–1.70 (2H, m), 1.88–2.09 (2H, m), 3.12 (3H, s), 3.17–3.31 (4H, m), 3.61–3.80 (2H, m), 4.10 (2H, t, J = 8.9 Hz), 4.67–
4.91 (2H, m), 6.42 (1H, d, J = 1.9 Hz), 6.67 (1H, dd, J = 6.0, 1.9 Hz), 7.66 (2H, dd, J = 4.3, 2.4 Hz), 8.19 (1H, d, J = 5.7 Hz), 8.43 (1H, d, J = 9.0 Hz). 13C NMR (100.6 MHz, CDCl3) δ 22.3, 27.1, 30.3, 40.5, 45.0, 50.0, 68.8, 72.0, 96.7, 103.9, 113.4, 123.4, 128.3, 131.0, 132.3, 149.4, 149.8, 155.2, 156.6, 165.0. Mp 149–151 °C. Anal. Calcd for C23H29N3O5S·0.1H2O: C, 59.88; H, 6.38; N, 9.11. Found: C, 59.67; H, 6.47;
N, 9.04.
tert-Butyl 4-[(2-chloropyrimidin-4-yl)oxy]piperidine-1-carboxylate (111c). To a stirred solution of tert-butyl 4-hydroxypiperidine-1-carboxylate (113, 20.1 g, 100 mmol) in a mixed solvent of THF (100 mL) and DMF (100 mL) was added sodium hydride (60% oil dispersion, 4.00 g, 100 mmol) at 0 °C. After the mixture was stirred at room temperature for 30 min, the mixture was cooled to −78 °C and then 2,4-dichloropyrimidine (1.50 g, 4.47 mmol) was added to the mixture. The resulting mixture was allowed to warm to −50 °C followed by stirring for 2 h. The reaction mixture was quenched with water and extracted with AcOEt. The combined organic layer was washed with brine, dried over MgSO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane/AcOEt = 95/5) to give the title compound as a solid (5.00 g, 16%). 1H NMR (300 MHz, CDCl3) δ 1.47 (9H, s), 1.62–1.86 (2H, m), 1.91–2.13 (2H, m), 3.18–3.43 (2H, m), 3.62–3.90 (2H, m), 5.25–5.42
(1H, m), 6.64 (1H, d, J = 5.7 Hz), 8.29 (1H, d, J = 5.7 Hz).
tert-Butyl 4-({2-[5-(methylsulfonyl)-2,3-dihydro-1H-indol-1-yl]pyrimidin-4-yl}oxy)piperidine-1- carboxylate (114c). A mixture of compound 111c (4.76 g, 15.2 mmol), compound 85 (2.56 g, 13.0 mmol), and Cs2CO3 (6.52 g, 20.0 mmol) in NMP (150 mL) was stirred at 85 °C for 16 h. The mixture was partitioned between with AcOEt and water. The organic layer was washed with brine, dried over MgSO4, and concentrated. The residue was purified by silica gel column chromatography (hexane/AcOEt = 80/20 to 50/50) to give the title compound as a solid (2.10 g, 34%). 1H NMR (300 MHz, CDCl3) δ 1.48 (9H, s), 1.77–1.87 (2H, m), 2.01–2.08 (2H, m), 3.05 (3H, s), 3.24 (2H, t, J = 8.7 Hz), 3.29–3.38 (2H, m), 3.77–
3.82 (2H, m), 4.32 (2H, t, J = 8.7 Hz), 5.25–5.30 (1H, m), 6.27 (1H, d, J = 6.6 Hz), 7.70 (1H, s), 7.75–
7.78 (1H, m), 8.27 (1H, d, J = 6.6 Hz), 8.41 (1H, d, J = 8.7 Hz). Anal. Calcd for C23H30N4O5S: C, 58.21;
H, 6.37; N, 11.81. Found: C, 58.30; H, 6.26; N, 11.82.
Propan-2-yl 4-({2-[5-(methylsulfonyl)-2,3-dihydro-1H-indol-1-yl]pyrimidin-4-yl}oxy)piperidine- 1-carboxylate (115c). Compound 115c was prepared from 114c in a manner similar to that described for compound 115a. Solid. Yield 52% over 2 steps. MS (ESI/APCI) m/z 461 [M + H]+. 1H NMR (300 MHz, CDCl3) δ 1.27 (6H, d, J = 6.4 Hz), 1.72–1.94 (2H, m), 1.96–2.12 (2H, m), 3.05 (3H, s), 3.25 (2H, t, J = 8.7 Hz), 3.31–3.48 (2H, m), 3.73–3.91 (2H, m), 4.33 (2H, t, J = 8.9 Hz), 4.83–5.06 (1H, m), 5.22–5.42 (1H, m), 6.28 (1H, d, J = 5.7 Hz), 7.72 (1H, s), 7.74–7.85 (1H, m), 8.28 (1H, d, J = 5.7 Hz), 8.43 (1H, d, J
= 8.3 Hz). 13C NMR (100.6 MHz, CDCl3) δ 22.3, 26.7, 30.5, 41.0, 44.9, 49.4, 68.8, 71.4, 100.6, 114.5, 123.7, 128.0, 132.3, 133.5, 148.4, 155.2, 158.1, 158.6, 168.7. Mp 147–149 °C. Anal. Calcd for C22H28N4O5S: C, 57.37; H, 6.13; N, 12.17. Found: C, 57.48; H, 6.21; N, 11.96.
1-(6-Chloropyrimidin-4-yl)-5-(methylsulfonyl)-2,3-dihydro-1H-indole (116). A mixture of 4,6-di- chloropyrimidine (4.10 g, 27.5 mmol), compound 85 (5.00 g, 25.0 mmol), and EtOH (160 mL) was refluxed for 4 h. After the mixture was concentrated under reduced pressure, to the residue was added aqueous NaHCO3 solution. The precipitated solid was collected by filtration, washed with water, and dried under reduced pressure to give the title compound as a colorless solid (5.50 g, 71%). 1H NMR (300 MHz, DMSO-d6) δ 3.17 (3H, s), 3.24–3.37 (2H, m), 4.16 (2H, t, J = 8.7 Hz), 7.09 (1H, s), 7.73–7.85 (2H, m), 8.57 (1H, d, J = 9.4 Hz), 8.68 (1H, s).
tert-Butyl 4-({6-[5-(methylsulfonyl)-2,3-dihydro-1H-indol-1-yl]pyrimidin-4-yl}oxy)piperidine-1- carboxylate (117). To a stirred solution of compound 113 (20.8 g, 103 mmol) in THF (200 mL) was added sodium hydride (60% oil dispersion, 4.12 g, 103 mmol) at 0 °C. After the mixture was stirred at room temperature for 2 h, compound 116 (8.00 g, 25.8 mmol) was added to the mixture. The resulting mixture was stirred at 50 °C for 2 h. The reaction mixture was quenched with water and extracted with AcOEt. The organic layer was washed with brine, dried over MgSO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane/AcOEt = 80/20 to 50/50) to give the title compound as a solid (6.17 g, 50%). MS (ESI/APCI) m/z 475 [M + H]+. 1H NMR (300 MHz, CDCl3) δ 1.48 (9H, s), 1.65–1.84 (2H, m), 1.93–2.10 (2H, m), 3.04 (3H, s), 3.19–3.39 (4H, m),
3.70–3.89 (2H, m), 4.07 (2H, t, J = 8.9 Hz), 5.25–5.38 (1H, m), 5.98 (1H, s), 7.72 (1H, s), 7.78 (1H, dd, J
= 8.5, 2.1 Hz), 8.51 (1H, s), 8.57 (1H, d, J = 8.7 Hz).
5-(Methylsulfonyl)-1-[6-(piperidin-4-yloxy)pyrimidin-4-yl]-2,3-dihydro-1H-indole (118). To a mixture of compound 117 (6.17 g, 13.0 mmol), AcOEt (100 mL) and MeOH (100 mL) was added 4 M HCl in AcOEt (15 mL), and the mixture was stirred at room temperature for 16 h. After the mixture was concentrated under reduced pressure, the residue was diluted with AcOEt and basified with 1 M aqueous NaOH solution. The organic layer was separated, washed with water and brine, and dried over MgSO4. The solvent was removed by evaporation to give the title compound as a solid (4.56 g, 94%). This product was used for the next step without further purification. MS (ESI/APCI) m/z 375 [M + H]+.
Propan-2-yl 4-({6-[5-(methylsulfonyl)-2,3-dihydro-1H-indol-1-yl]pyrimidin-4-yl}oxy)piperidine- 1-carboxylate (119). Compound 119 was prepared from 118 in a manner similar to that described for compound 115a. Colorless solid. Yield 48%. MS (ESI/APCI) m/z 461 [M + H]+. 1H NMR (300 MHz, CDCl3) δ 1.26 (6H, t, J = 6.1 Hz), 1.61–1.84 (2H, m), 1.86–2.11 (2H, m), 2.89–3.12 (3H, m), 3.21–3.47 (4H, m), 3.74–3.93 (2H, m), 4.07 (2H, t, J = 8.7 Hz), 4.94 (1H, q, J = 6.2 Hz), 5.33 (1H, tt, J = 8.0, 3.9 Hz), 5.98 (1H, s), 7.72 (1H, s), 7.79 (1H, dd, J = 8.7, 1.9 Hz), 8.51 (1H, s), 8.57 (1H, s). 13C NMR (100.6 MHz, CDCl3) δ 22.3, 27.0, 30.7, 41.1, 44.9, 49.1, 68.7, 71.2, 90.1, 115.6, 123.7, 128.2, 132.7, 132.9, 148.6, 155.2, 157.3, 160.7, 169.5. Mp 188–189 °C. Anal. Calcd for C22H28N4O5S: C, 57.37; H, 6.13; N, 12.17. Found: C, 57.27; H, 6.15; N, 12.09.
1-(6-{[1-(5-Ethylpyrimidin-2-yl)piperidin-4-yl]oxy}pyrimidin-4-yl)-5-(methylsulfonyl)-2,3-dihydr o-1H-indole (120). Compound 120 was prepared from 118 in a manner similar to that described for compound 104. Colorless solid. Yield 82%. MS (ESI/APCI) m/z 481 [M + H]+. 1H NMR (300 MHz, CDCl3) δ 1.13 (3H, t, J = 7.6 Hz), 1.53–1.74 (2H, m), 1.95–2.14 (2H, m), 2.44 (2H, q, J = 7.6 Hz), 3.15 (3H, s), 3.24–3.31 (2H, m), 3.37–3.54 (2H, m), 4.09 (2H, t, J = 8.7 Hz), 4.20–4.38 (2H, m), 5.28–5.43 (1H, m), 6.23 (1H, s), 7.66–7.82 (2H, m), 8.26 (2H, s), 8.48–8.65 (2H, m). 13C NMR (100.6 MHz, CDCl3) δ 15.6, 22.7, 27.0, 30.7, 41.5, 44.9, 49.1, 72.0, 90.1, 115.6, 123.7, 124.5, 128.2, 132.6, 132.8, 148.6, 157.2, 157.3, 160.7, 160.8, 169.6. Mp 206–209 °C. Anal. Calcd for C24H28N6O3S: C, 59.98; H, 5.87; N, 17.49. Found: C, 59.81; H, 5.98; N, 17.21.
5-(Methylsulfonyl)-1-[6-({1-[3-(propan-2-yl)-1,2,4-oxadiazol-5-yl]piperidin-4-yl}oxy)pyrimidin- 4-yl]-2,3-dihydro-1H-indole (121). To a mixture of compound 118 (500 mg, 1.34 mmol), NaHCO3 (344 mg, 4.09 mmol), THF (30 mL), and water (10 mL) was added cyanogen bromide (191 mg, 1.80 mmol) at 0 °C, and the mixture was stirred at room temperature for 16 h. Aqueous NaHCO3 solution was added, and the mixture was extracted with a mixed solvent of AcOEt and THF. The organic layer was washed with brine, dried over MgSO4, and concentrated under reduced pressure. The residue was dissolved in AcOEt/THF (1:1, 200 mL), and then N-hydroxy-2-methylpropanimidamide (204 mg, 2.00 mmol) and zinc chloride (1 M Et2O solution, 2.0 mL, 2.0 mmol) were added. The resulting mixture was refluxed for 4 h. The precipitated solid was collected and washed with AcOEt. The solid was dissolved in a mixture of
EtOH (300 mL) and concentrated hydrochloric acid (1 mL) followed by stirring at 70 °C for 24 h. The reaction mixture was concentrated under reduced pressure and partitioned between AcOEt and aqueous NaHCO3 solution. The organic layer was washed with brine and dried over MgSO4. The solvent was removed by evaporation to give the title compound as a solid (560 mg, 86% from 118). MS (ESI/APCI) m/z 485 [M + H]+. 1H NMR (300 MHz, DMSO-d6) δ 1.19 (6H, d, J = 6.8 Hz), 1.64–1.88 (2H, m), 2.02–
2.21 (2H, m), 2.73–2.92 (1H, m), 3.15 (3H, s), 3.21–3.35 (2H, m), 3.42–3.59 (2H, m), 3.75–3.93 (2H, m), 4.09 (2H, t, J = 8.7 Hz), 5.25–5.47 (1H, m), 6.25 (1H, s), 7.68–7.81 (2H, m), 8.47–8.63 (2H, m). 13C NMR (100.6 MHz, CDCl3) δ 20.4, 27.0, 27.1, 30.1, 43.3, 44.9, 49.1, 69.9, 90.0, 115.7, 123.7, 128.2, 132.7, 133.0, 148.5, 157.3, 160.8, 169.3, 170.9, 175.9. Mp 187–189 °C. Anal. Calcd for C23H28N6O4S·0.2AcOEt: C, 56.92; H, 5.94; N, 16.73. Found: C, 56.74; H, 5.76; N, 16.72.
1-(4-Chloro-1,3,5-triazin-2-yl)-5-(methylsulfonyl)-2,3-dihydro-1H-indole (122). To a stirred solution of compound 85 (190 mg, 0.991 mmol) and 2,4-dichloro-1,3,5-triazine (165 mg, 1.10 mmol) in toluene (10 mL) was added 2,6-lutidine (0.150 mL, 1.29 mmol) at 0 °C. After being stirred at room temperature overnight, the mixture was quenched with water and extracted with AcOEt. The extract was washed with brine, dried over MgSO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane/AcOEt = 95/5 to 65/35) to give the title compound as colorless solid (135 mg, 39%). MS (ESI) m/z 311 [M + H]+. 1H NMR (300 MHz, CDCl3) δ 3.06 (3H, s,, 3.32 (2H, t, J = 8.7 Hz), 4.38 (2H, t, J = 8.3 Hz), 7.81 (1H, s), 7.88 (1H, t, J = 6.4 Hz), 8.56 (1H, dd, J = 14.7, 8.7 Hz) 8.66 (1H, d, J = 5.7 Hz).
Propan-2-yl 4-({4-[5-(methylsulfonyl)-2,3-dihydro-1H-indol-1-yl]-1,3,5-triazin-2-yl}oxy)piperi- dine-1-carboxylate (123). To a mixture of compound 122 (120 mg, 0.386 mmol) and isopropyl 4-hydroxypiperidine-1-carboxylate (95 mg, 0.51 mmol) in THF (5 mL) was added potassium tert-butoxide (60 mg, 0.54 mmol) at 0 °C. After being stirred at room temperature overnight, the mixture was poured into water and extracted with AcOEt. The extract was washed with brine, dried over MgSO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane/AcOEt = 100/1 to 4/1) to give the title compound as a colorless oil (110 mg, 62%). MS (ESI) m/z 462 [M + H]+. 1H NMR (300 MHz, CDCl3) δ 1.26 (6H, d, J = 6.0 Hz), 1.71–1.96 (2H, m), 1.94–2.21 (2H, m), 3.05 (3H, s), 3.17–3.47 (4H, m), 3.75–3.99 (2H, m), 4.36 (2H, d, J = 8.7 Hz), 4.94 (1H, dt, J = 12.4, 6.2 Hz), 5.28 (1H, dt, J = 7.8, 3.8 Hz), 6.29–6.75 (1H, m), 7.30–7.53 (1H, m), 7.61–8.23 (1H, m), 8.59 (1H, s). 13C NMR (100.6 MHz, CDCl3) δ 22.3, 26.7, 30.4, 41.0, 44.8, 49.0, 68.8, 73.3, 116.9, 124.0, 128.0, 134.1, 134.6, 146.9, 155.2, 164.1, 167.7, 169.5. Anal. Calcd for C21H27N5O5S: C, 54.65; H, 5.90; N, 15.17.
Found: C, 54.36; H, 5.87; N, 14.87.
tert-Butyl 4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)piperidine-1-carboxylate (125). To a mixture of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine (124, 6.40 g, 41.7 mmol), compound 113 (25.2 g, 125 mmol), and triphenylphosphine (21.9 g, 83.0 mmol) in THF (500 mL) was added dropwise diethyl azodicarboxylate (40% toluene solution, 36.3 g, 83.0 mmol) at 0 °C over 1 h. The mixture was allowed to
warm to room temperature followed by stirring for 10 h. After the reaction mixture was concentrated under reduced pressure, the residue was purified by silica gel column chromatography (hexane/AcOEt = 10/1 to 2/1) and crystallized from iPr2O/hexane to give the title compound as a solid (11.5 g, 82%). MS (ESI) m/z 337 [M + H]+. 1H NMR (300 MHz, CDCl3) δ 1.50 (9H, s), 1.84–2.11 (4H, m), 2.89–3.00 (2H, m), 4.26–4.41 (2H, m), 4.81–4.94 (1H, m), 6.64 (1H, d, J = 3.8 Hz), 7.30 (1H, d, J = 3.8 Hz), 8.63 (1H, s).
tert-Butyl 4-{4-[5-(methylsulfonyl)-2,3-dihydro-1H-indol-1-yl]-7H-pyrrolo[2,3-d]pyrimidin-7-yl}-
piperidine-1-carboxylate (126). To a mixture of compound 85 (97 mg, 0.49 mmol) and compound 125 (150 mg, 0.445 mmol) in DMF (5 mL) was added sodium hydride (60% oil dispersion, 39 mg, 0.98 mmol). After stirring at room temperature for 30 min, the mixture was stirred at 50 °C for 15 h. The reaction was cooled to room temperature followed by addition of water. The mixture was extracted with AcOEt, washed with brine, dried over MgSO4, and concentrated under reduced pressure. The residue was crystallized from AcOEt to give the title compound as a solid (163 mg, 74%). MS (ESI) m/z 498 [M + H]+.
1H NMR (300 MHz, DMSO-d6) δ 1.44 (9H, s), 1.83–2.01 (4H, m), 2.83–3.06 (2H, m), 3.16 (3H, s), 3.32–
3.40 (2H, m), 4.08–4.21 (2H, m), 4.62 (2H, t, J = 8.7 Hz), 4.76–4.92 (1H, m), 6.80 (1H, d, J = 3.8 Hz), 7.62 (1H, d, J = 3.8 Hz), 7.72–7.78 (2H, m), 8.45 (1H, s), 8.69 (1H, t, J = 9.1 Hz). 13C NMR (100.6 MHz, CDCl3) δ 27.9, 28.5, 32.4, 43.3, 45.0, 51.1, 51.7, 80.0, 101.0, 105.4, 116.3, 121.8, 123.6, 128.0, 132.6, 133.0, 149.2, 150.5, 151.2, 153.7, 154.7. Mp 238–240 °C. Anal. Calcd for C25H31N5O4S·0.1AcOEt: C, 60.24; H, 6.33; N, 13.83. Found: C, 60.42; H, 6.20; N, 13.54.
Estimation of LogD at pH 7.4
LogD7.4, which is a partion coefficient between 1-octanol and aqueous buffer pH 7.4, of the compounds was measured on the chromatographic procedure whose condition was developed based on a published method.101 The instruments were Waters Alliance 2795 HPLC system with 2996 UV-vis detector (Milford, MA, USA).
Solubility determination
Small volumes of the compound DMSO solutions were added to the aqueous buffer (pH 6.8). After incubation, precipitates were separated from by filtration through a filter plate. The filtrates were analyzed for compound in solution by HPLC analysis.
Biology
In vitro GPR119 agonist activity. GPR119 agonist activities were evaluated in the reporter gene assay using CHO cells stably co-expressing cyclic AMP response element (CRE)–luciferase reporter gene (Promega) and GPR119. Cells were seeded at 10,000 cells/well in Minimum essential medium (MEM) α containing 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 500 μg/mL
Geneticin in 384-well white opaque plates, and cultured at 37 °C under 5% CO2 with saturated humidity overnight. The cells were washed once with assay buffer (MEMα, 20 mmol/L HEPES, 0.1% bovine serum albumin, 100 U/mL penicillin, 100 μg/mL streptomycin), and incubated with various concentrations of test compounds diluted in assay buffer for 2 h. After removal of culture supernatant, cAMP-induced luciferase activities were measured with Steady-Glo reagent (Promega) and EnVision Multilabel Plate Reader (PerkinElmer). Agonist activities of test compounds on GPR119 were expressed as [(A−B)/(C−B)] × 100 (luciferase activities (A) in test compounds-treated cells, (B) in vehicle-treated cells, and (C) in cells treated with 10 μM N-[4-(methylsulfonyl)phenyl]-5-nitro-6-{4-[4-(trifluoro- methoxy)phenoxy]piperidin-1-yl}pyrimidin-4-amine107). EC50 values were obtained with XLfit software (ID Business Solutions).
Oral Glucose Tolerance Test (OGTT). The care and use of the animals and the experimental protocols used in this research were approved by the Experimental Animal Care and Use Committee of Takeda Pharmaceutical Company Limited. Male Wistar fatty (WF) rats were obtained from Takeda Rabics, Ltd. (Hikari, Japan). They were fed a commercial diet CE-2 (Clea Japan Co.) and tap water ad libitum. Male WF rats (19 weeks of age) were fasted overnight and orally given vehicle (0.5%
methylcellulose) or compounds. Sixty minutes later, all animals were received an oral glucose load (1 g/kg). Blood samples were collected from tail vein before drug administration (pre), and just before glucose load (time 0), and 10, 30, 60, and 120 min after glucose load. Plasma glucose and plasma insulin levels were measured by Autoanalyzer 7080 (Hitachi, Japan) and radioimmunoassay (Millipore, USA), respectively. Differences between two groups were analyzed by Aspin−Welch test or Student’s t-test.
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