Chemistry
The synthetic scheme of the compounds 6a–d is depicted in Chart 1. Starting from ethyl 2-chloro-5-iodonicotinate (2),8) the alkylnyl part was introduced at the 5-position of the pyridine ring by the Sonogashira reaction. Ester hydrolysis and amidation of the 3-position of the pyridine ring yielded compound 5. Among the reagents tested for amidation, (benzotriazole-1-yloxy)tris(dimethylamino) phosphonium hexafluorophosphate (BOP reagent) gave the best yield. Alkyl amination at the 2-position of the pyridine ring was achieved by heating the mixure of compound 5, the corresponding amine and organic base in ethanol, yielding the final comopunds 6a and b. By using similar procedure, sulfonamide-type compounds 6c and d were synthesized from intermidiate 8, which was obtained by sequential introduction of substituents at 3- and 5-position of 2-chloro-5-iodonicotinic acid.
Chart 1
(a) 3-Methyl butyn-3-ol, PdCl2(PPh3)2, CuI, Et3N, 60°C, 64%; (b) 1 M NaOH aq., MeOH, 50°C, 100%; (c) 3-(difluoromethyl)-4-methyl-benzylamine, BOP reagent, DIPEA, DMF, 0°C, 94%; (d) imidazo[1,2-b]pyridazin-2-ylmethaneamine, Et3N, EtOH, 100°C, 86%; (e) (2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methanamine, DIPEA, EtOH, 160°C (microwave irradiation), 30%; (f) 3-(difluoromethyl)-4-methyl-benzylamine, BOP reagent, DIPEA, DMF, 0°C, 76%; (g) PdCl2(PPh3)2, CuI, Et3N, 60°C, 100%; (h) (2,2-difluorobenzo[d][1,3]dioxol-5-yl)methanamine, Et3N, N-methylpyrrolidone, 150°C (microwave irradiation), 29%; (i) 2-(aminomethyl)-4-methoxypyridine, Et3N, N-methylpyrrolidone 150°C (microwave irradiation), 69%.
Preparation of the compounds 18a and b is described in Chart 2. Because the key intermediate 14 was not commercially available, 14 was prepared from 3-methylpicolinaldehyde. Protection of the aldehyde followed by N-oxidation gave compound 10. The cyano group was introduced by a modified Reissert–Henze reaction.11) In this condition, excess reagent was necessary, and yield was relatively low (28%) because of the generation of 2-cyano-3-methyl pyridine as a byproduct. Deprotection and difluorination of the aldehyde gave compound 13, and the cyano group was reduced to yield 14. Although the protection–deprotection process seems to be unnecessary if the difluorination of the aldehyde was achieved first, the Reissert–Henze-type reaction did not proceed when using the difluorinated intermediate. For the preparation of the sulfonamide-type compounds 18a and b, a method similar to the preparation of compound 6a was adopted, except that alkylamination at the 2-position of the pyridine ring preceded the deprotection and amidation at the 3-position. The total yield was not affected by the order of these reactions, and these methods were applicable to various substitutions with acceptable yield (>28%).
Chart 2
(a) Ethylene glycol, p-TsCl, toluene, reflux, 73%; (b) m-CPBA, CHCl3, rt, 65%; (c) trimethylsilyl cyanide, dimethylcarbamoyl chloride, CH2Cl2, rt, 28%; (d) 2 N HCl aq., 1,4-dioxane, 60°C, 77%; (e) deoxofluor, CHCl3, rt, 73%; (f) Pd–C, H2, 4 M HCl in dioxane, MeOH, rt, 89%; (g) PdCl2(PPh3)2, CuI, Et3N, 60°C, 65%; (h) 2-(aminomethyl)-4-methoxypyridine, Et3N, N-methylpyrrolidone, 150°C (microwave irradiation), 64%; (i) 1 M aq. NaOH, MeOH, 50°C, 71%; (j) 14, BOP reagent, DIPEA, DMF, rt, 100%; (k) (2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methanamine, DIPEA, EtOH, 155°C (microwave irradiation), 84%; (l) 4 M aq. NaOH, MeOH, 50°C, 71%; (m) 14, BOP reagent, DIPEA, DMF, rt, 53%.
MBI activity was evaluated by comparing the activity of CYP3A4 enzyme after 30-min preincubation with the test compounds and with or without reduced nicotinamide adenine dinucleotide phosphate (NADPH). If the compound itself inhibits CYP3A4, the enzyme activity is diminished both with and without NADPH in the preincubation media. If the metabolite of the compound has an MBI activity, the enzyme activity is not affected in the absence of NADPH but is diminished in the presence of NADPH in the incubation media. MBI activity is indicated by the percentage of CYP3A4 activity with or without NADPH, confirming that the compound itself does not have inhibitory activity. No compound showed CYP3A4 inhibitory activity alone and 10 µM of compound 1 in the presence of NADPH inhibited the enzyme activity by 80%. Log P is calculated by the S+log P method12) and lipophilic ligand efficiency (LLE) is calculated as p(binding IC50)−(S+log P). The LLE was invented as an indicator of drug-likeness13) based on the concept that the rate of compound-related abortion in clinical trial is proportional to the lipophilicity of the compound, and the balance between the potency and lipophilicity of the compound is important considering the clinical dose and possibility of drug-related adverse events. The appropriate LLE for a drug candidate is said to be 6 or above, although it depends on the target molecule and therapeutic indication. Chemical structures and all in vitro data14) are listed in Table 1.
Table 1. GhrelinR Binding Affinity, Inverse Agonist IC
50, Calculated Log
P, LLE Score, and MBI Activity for Selected Compounds Varying R
1, R
2, and X
 |
|---|
| Compd. | R1 | R2 | X | Binding IC50 (nM) | Inverse agonist IC50 (nM)a) | S+log Pb) | LLEc) | MBId) |
|---|
| Rat | Human |
|---|
| 1 |  | OH | CH | 0.28 | 0.44 | 5.4 | 4.67 | 5.69 | 80% |
| 6c |  | NHSO2CH3 | CH | 55 | No data | n.d. | 6.23 | 1.03e) | 10% |
| 6a |  | OH | CH | 5.5 | No data | 34 | 4.69 | 3.57e) | 40% |
| 18b | NHSO2CH3 | N | 2.0 | 1.4 | 0.51 | 4.48 | 4.38 | 20% |
| 6b |  | OH | CH | 6.6 | 4.9 | 2.7 | 3.39 | 4.92 | 50% |
| 6d |  | NHSO2CH3 | CH | 0.35 | 0.41 | 0.36 | 3.48 | 5.92 | 50% |
| 18a | NHSO2CH3 | N | 0.39 | 0.38 | 0.26 | 2.97 | 6.45 | 16% |
All values indicate the mean of at least two independent measurements. a) All compounds showed the same efficacy (>95% block). b) Calculated using ADME predictor (TM). c) Ligand-lipophilicity efficiency=p(human binding IC50)−(S+log P). d) Mechanism-based inactivation of CYP3A4 at 10 µM. e) Rat binding IC50 was used for the calculation.
At first, introduction of fluorine atoms to the position of carbene generation, i.e., at 2-position of 1,3-benzodioxol (6c) reduces MBI activity substantially (10%). Based on the same idea, dihydrobenzodioxin derivatives (6a, 18b) were tested, and reduced MBI activities were observed. In addition to the substitution at the 2-position of the pyridine ring, the substitution at the 5-position affects MBI activity to some extent and the sulfonamide-type substitution is preferable to propargyl alcohol-type substitution. Despite the improvement of the MBI activity, the binding affinities of these compounds were lower than that of compound 1, and lipophilicity was not improved, resulting in lower LLE. We, therefore, sought for better structures at the 2-position of the pyridine ring, which would improve lipophilicity and MBI activity while maintaining the main activity. Compounds with an imidazo[1,2-b]pyridazin ring showed good lipophilicity and moderate main and MBI activities. Compounds with a 4-methoxypyridine showed good main activity, lipophilicity and MBI activity. Comparing 6d and 18a, MBI activity was improved by substituting the pyridine ring for the benzene ring at 3-position. Lower molecular lipophilicity of 18a is related to lower MBI activity because the affinity to the binding pocket of CYP3A4 is proportional to the lipophilicity of the compound.
ConclusionStarting from compound 1, lipophilicity and MBI activity were improved by structural optimization. The final compound 18a showed low MBI activity and high LLE (6.45) and, therefore, might be a good anti-obesity or anti-diabetes drug candidate.
ExperimentalProton nuclear magnetic resonance spectra (1H-NMR) and carbon nuclear magnetic resonance spectra (13C-NMR) were recorded on Brucker ARX-400 or Brucker Avance III (400 MHz) spectrometer in the indicated solvent. Chemical shifts (δ) are reported in parts per million relative to the internal standard tetramethylsilane. High-resolution mass spectra (HR-MS) and fast atom bombardment (FAB) mass spectra were recorded on JEOL JMS-700 mass spectrometer. Electro-spray ionization (ESI) mass spectra were recorded on Agilent G1956A MSD spectrometer system. Purity was analyzed by Agilent 1100 HPLC system. HPLC conditions utilized are as follows; column: YMC-Pack Pro C18, 4.6 mm×100 mm, mobile phase: solvent A=MeCN–H2O–trifluoroacetic acid (5 : 95 : 0.1), solvent B=MeCN–trifluoroacetic acid (100 : 0.09), gradient: 10 to 100% solvent B in solvent A for 8 min (method A) or 0 to 98% solvent B in solvent A for 4 min (method B), UV detector: 210 nm, flow rate: 1 mL/min. Retention times are in minutes and purity is calculated as % total area. Other chemical reagents and solvents were purchased from Aldrich, Tokyo Kasei Kogyo, Wako Pure Chemical Industries, Ltd., Kanto Kagaku or Nacalai Tesque and used without purification. Flash column chromatography was performed using Merck Silica gel 60 (230–400 mesh) or Purif-Pack® SI 30 µm supplied by Shoko Scientific. Preparative TLC was performed using silica gel GF Uniplate (20×20 cm, 1000 microns) supplied by Analtech Inc.
Ethyl 2-Chloro-5-(3-hydroxy-3-methylbut-1-yn-1-yl)nicotinate (3)To a solution of compound 28) (2.0 g, 6.42 mmol) in triethylamine (20 mL) were added 3-methyl-1-butyn-3-ol (0.65 g, 7.7 mmol), PdCl2(PPh3)2 (0.135 g, 0.19 mmol) and copper(I) iodide (0.037 g, 0.19 mmol). The reaction mixture was heated at 60°C for 6 h under Ar atmosphere. The mixture was filtered through celite® and the filtrate was concentrated. The residue was purified by silica gel chromatography (ethyl acetate (EtOAc)–n-hexane; 20 : 80) to yield 3 as a pale yellow oil (1.1 g, 64%).
1H-NMR (400 MHz, chloroform-d3 (CDCl3)) δ: 8.50 (1H, d, J=2.4 Hz), 8.15 (1H, d, J=2.4 Hz), 4.43 (2H, q, J=7.2 Hz), 1.63 (6H, s), 1.42 (3H, t, J=7.2 Hz). MS (ESI) m/z: 268.0 (M+H)+. HPLC purity >99% (4.05 min, method B).
2-Chloro-5-(3-hydroxy-3-methylbut-1-yn-1-yl)nicotinic Acid (4)A mixture of compound 3 (1.22 g, 4.56 mmol) and 1 M aqueous NaOH (9.11 mL) in methanol (MeOH) (12 mL) was stirred at 50°C for 1 h. After cooling, the solution was concentrated in vacuo, neutralized with 1 M aqueous hydrochloric acid (HCl), and extracted with EtOAc. The organic phase was dried over sodium sulfate (Na2SO4) and concentrated to give 4 as a pale yellow solid (1.1 g, 100%).
1H-NMR (400 MHz, CDCl3) δ: 8.49 (1H, d, J=2.4 Hz), 8.21 (1H, d, J=2.4 Hz), 1.56 (6H, s). MS (ESI) m/z: 240.0 (M+H)+. HPLC purity 95.6% (3.19 min, method B).
2-Chloro-N-(3-(difluoromethyl)-4-methylbenzyl)-5-(3-hydroxy-3-methylbut-1-yn-1-yl)nicotinamide (5)To a solution of compound 4 (800 mg, 3.34 mmol), 3-(difluoromethyl)-4-methyl-benzylamine8) (762 mg, 3.67 mmol), and N,N-diisopropylethylamine (DIPEA) (1.75 mL) in N,N-dimethylformamide (DMF) (16 mL) was added BOP reagent (1.62 g, 3.67 mmol) at 0°C. The mixture was stirred at 0°C for 2 h and diluted with a mixture of EtOAc (100 mL) and n-hexane (50 mL). The organic solution was washed with water and brine, dried over Na2SO4, and concentrated in vacuo. The residue was purified by silica gel column chromatography (EtOAc–n-hexane; 20 : 80) to yield 5 (1.27 g, 97%) as a white solid.
1H-NMR (400 MHz, CDCl3) δ: 8.45 (1H, d, J=2.4 Hz), 8.21 (1H, d, J=2.4 Hz), 7.50 (1H, s), 7.37 (1H, d, J=7.6 Hz), 7.21 (1H, d, J=7.6 Hz), 6.75 (1H, t, J=55.4 Hz), 6.72 (1H, br s), 4.66 (2H, d, J=6.0 Hz), 2.43 (3H, s), 2.05 (1H, s), 1.62 (6H, s). MS (ESI) m/z: 393.0 (M+H)+. HPLC purity 92.9% (4.35 min, method B).
N-(3-(Difluoromethyl)-4-methylbenzyl)-5-(3-hydroxy-3-methylbut-1-yn-1-yl)-2-((imidazo[1,2-b]pyridazin-2-ylmethyl)amino)nicotinamide (6b)A mixture of compound 5 (50 mg, 0.127 mmol), imidazo[1,2-b]pyridazin-2-ylmethanamine (28 mg, 0.191 mmol), and triethylamine (0.177 mL, 1.27 mmol) in EtOH (0.4 mL) was placed into sealed tube and stirred at 100°C for 96 h. The mixture was cooled and concentrated in vacuo. The residue was purified by preparative TLC (MeOH–CHCl3; 3 : 97) to yield 6b (55.1 mg, 86%) as a pale yellow solid.
1H-NMR (400 MHz, CDCl3) δ: 8.22 (1H, t, J=5.2 Hz), 8.28 (1H, d, J=2.0 Hz), 8.25 (1H, dd, J=4.4, 1.6 Hz), 7.92 (1H, s), 7.85 (1H, dd, J=8.8, 1.6 Hz), 7.61 (1H, d, J=2.0 Hz), 7.45 (1H, s), 7.33 (1H, d, J=8.0 Hz), 7.22 (1H, d, J=7.6 Hz), 6.97 (1H, dd, J=9.2, 4.4 Hz), 6.60 (1H, t, J=55.2 Hz), 6.51 (1H, br s), 4.92 (2H, d, J=5.2 Hz), 4.56 (2H, d, J=5.6 Hz), 2.42 (3H, s), 2.21 (1H, s), 1.60 (6H, s). 13C-NMR (100 MHz, CDCl3) δ: 167.3, 156.6, 154.7, 145.4, 142.7, 138.8, 137.8, 135.8 (t, J=4.2 Hz), 135.8, 132.7 (t, J=20.9 Hz), 131.7 (t, J=2.1 Hz), 130.1 (t, J=7.4 Hz), 125.4, 125.0, 116.6, 114.3 (t, J=238 Hz), 114.2, 109.3, 106.4, 94.5, 79.1, 65.6, 43.4, 39.8, 31.5, 18.2. MS (ESI) m/z: 505.1 (M+H)+. FAB-MS m/z: 505.2141 (Calcd for C27H27F2N6O2+: 505.2164). HPLC purity 99.7% (4.55 min, method A).
N-(3-(Difluoromethyl)-4-methylbenzyl)-2-(((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)amino)-5-(3-hydroxy-3-methylbut-1-yn-1-yl)nicotinamide (6a)A mixture of compound 5 (50 mg, 0.127 mmol), (2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methanamine (40 mg, 0.242 mmol), and DIPEA (0.1 mL, 0.127 mmol) in EtOH (0.6 mL) was placed into sealed tube and stirred at 160°C under microwave irradiation for 1 h. The mixture was cooled and purified by silica gel column chromatography (EtOAc–n-hexane; 22 : 67) to yield 6a (20 mg, 30%) as a colorless solid.
1H-NMR (400 MHz, CDCl3) δ: 8.60 (1H, br s), 8.28 (1H, d, J=2.0 Hz), 7.58 (1H, d, J=2.4 Hz), 7.45 (1H, br s), 7.34 (1H, d, J=8.8 Hz), 7.31 (1H, s), 7.23 (1H, d, J=8.8 Hz), 6.87 (1H, d, J=1.2 Hz), 6.82 (1H, d, J=1.2 Hz), 6.81 (1H, s), 6.61 (1H, t, J=55.2 Hz), 6.27 (1H, br s), 4.60 (2H, d, J=5.2 Hz), 4.56 (2H, d, J=5.6 Hz), 4.24 (4H, s), 2.43 (3H, s), 1.94 (1H, s), 1.59 (6H, s). 13C-NMR (100 MHz, CDCl3) δ: 167.3, 156.7, 154.9, 143.5, 142.7, 137.6, 135.9 (t, J=4.3 Hz), 135.7, 132.8 (t, J=20.9 Hz), 132.5, 131.7, 130.1 (t, J=2.1 Hz), 125.4 (t, J=7.4 Hz), 120.7, 117.3, 116.6, 114.1 (t, J=238 Hz), 108.9, 106.0, 94.3, 79.2, 65.7, 64.4, 64.3, 44.4, 43.5, 31.6, 18.2. MS (ESI) m/z: 522.1 (M+H)+. FAB-MS m/z: 522.2230 (Calcd for C29H30F2N3O4+: 522.2204). HPLC purity 95.1% (6.14 min, method A).
2-Chloro-N-(3-(difluoromethyl)-4-methylbenzyl)-5-iodonicotinamide (7)To a solution of 2-chloro-5-iodonicotinic acid (1 g, 3.38 mmol), 3-(difluoromethyl)-4-methyl-benzylamine (876 mg, 4.22 mmol), and DIPEA (1.8 mL) in DMF (10 mL) cooled at 0°C was added BOP reagent (1.65 g, 3.73 mmol). The mixture was stirred at 0°C for 2 h and at room temperature for 1 h and diluted with a mixture of EtOAc (100 mL) and n-hexane (50 mL). The organic solution was washed with water, saturated aqueous NaHCO3 and brine, dried over Na2SO4, and concentrated in vacuo. 2-Propanol (15 mL) was added to the residual solid, and the resulting suspension was stirred at room temperature for 1 h. The precipitates were collected by filtration to yield 7 (1.11 g, 76%) as pale yellow solid.
1H-NMR (400 MHz, DMSO-d6) δ: 9.17 (1H, t, J=6.0 Hz), 8.73 (1H, d, J=2.3 Hz), 8.29 (1H, d, J=2.3 Hz), 7.52 (1H, br s), 7.41 (1H, br d, J=7.7 Hz), 7.15 (1H, t, J=55.0 Hz), 7.29 (1H, br d, J=7.7 Hz), 4.46 (2H, d, J=6.0 Hz), 2.37 (3H, s). MS (ESI) m/z: 436.9 (M+H)+. HPLC purity 94.8% (4.37 min, method B).
2-Chloro-N-(3-(difluoromethyl)-4-methylbenzyl)-5-(3-methyl-3-(methylsulfonamido)but-1-yn-1-yl)nicotinamide (8)To a solution of compound 7 (584 mg, 1.34 mmol) in triethylamine (6 mL) were added N-(2-methylbut-3-yn-2-yl)methanesulfonamide (237 mg, 1.47 mmol), PdCl2(PPh3)2 (29 mg, 0.041 mmol) and copper(I) iodide (8 mg, 0.042 mmol), and the reaction mixture was heated at 60°C for 2.5 h under Ar atmosphere. The mixture was concentrated in vacuo, and the residue was diluted with EtOAc (50 mL) and n-hexane (25 mL). The mixture was washed with water, saturated aqueous NH3, water and brine, dried over Na2SO4, and concentrated in vacuo. The residue was purified by silica gel chromatography (MeOH–CHCl3; 3 : 97 to 10 : 90) to yield 8 as a pale yellow amorphous (629 mg, 100%).
1H-NMR (400 MHz, CDCl3) δ: 8.47 (1H, d, J=2.3 Hz), 8.13 (1H, d, J=2.3 Hz), 7.51 (1H, br s), 7.38 (1H, br d, J=7.7 Hz), 7.24 (1H, d, J=8.2 Hz), 6.76 (2H, t, J=57.5 Hz), 6.74 (1H, br t, J=4.5 Hz), 4.67 (2H, d, J=5.8 Hz), 4.52 (1H, s), 3.13 (3H, s), 2.43 (3H, s), 1.73 (6H, s). MS (ESI) m/z: 470.0 (M+H)+. HPLC purity 98.7% (4.14 min, method B).
2-(((2,2-Difluorobenzo[d][1,3]dioxol-5-yl)methyl)amino)-N-(3-(difluoromethyl)-4-methylbenzyl)-5-(3-methyl-3-(methylsulfonamido)but-1-yn-1-yl)nicotinamide (6c)A mixture of compound 8 (60 mg, 0.131 mmol), (2,2-difluorobenzo[d][1,3]dioxol-5-yl)methanamine (59 mg, 0.264 mmol), and triethylamine (0.2 mL, 1.44 mmol) in N-methylpyrrolidone (0.4 mL) was placed into sealed tube and stirred at 150°C under microwave irradiation for 3 h. The mixture was diluted with EtOAc (20 mL) and n-hexane (10 mL), washed with water and brine, dried over Na2SO4, and concentrated in vacuo. The residue was purified by silica gel column chromatography (EtOAc–n-hexane; 36 : 64 to 57 : 43) and preparative TLC (MeOH–CHCl3; 1 : 40) to yield 6c (23 mg, 29%) as colorless solid.
1H-NMR (400 MHz, CDCl3) δ: 8.91 (1H, br s), 8.27 (1H, d, J=2.0 Hz), 7.63 (1H, d, J=2.0 Hz), 7.47 (1H, s), 7.34 (1H, d, J=8.8 Hz), 7.08 (1H, s), 7.05 (1H, d, J=8.0 Hz), 6.99 (1H, d, J=2.0 Hz), 6.62 (1H, t, J=54.4 Hz), 6.41 (1H, br s), 4.70 (2H, d, J=5.6 Hz), 4.57 (2H, d, J=5.6 Hz), 4.42 (1H, s), 3.13 (3H, s), 2.43 (3H, s), 1.69 (6H, s). 13C-NMR (100 MHz, CDCl3) δ: 167.2, 156.8, 154.6, 143.9, 142.8, 138.0, 135.8, 135.8 (t, J=4.4 Hz), 135.7, 132.7 (t, J=20.5 Hz), 131.6 (t, J=255 Hz), 131.6, 130.1 (t, J=2.1 Hz), 125.2 (t, J=7.4 Hz), 122.6, 114.1 (t, J=238 Hz), 109.2, 109.1, 109.0, 105.9, 91.7, 80.6, 50.8, 44.4, 43.4, 43.0, 31.2, 18.1. MS (ESI) m/z: 621.1 (M+H)+. FAB-MS m/z: 621.1776 (Calcd for C29H29F4N4O5S+: 621.1795). HPLC purity 98.1% (7.57 min, method A).
N-(3-(Difluoromethyl)-4-methylbenzyl)-2-(((4-methoxypyridin-2-yl)methyl)amino)-5-(3-methyl-3-(methylsulfonamido)but-1-yn-1-yl)nicotinamide (6d)A mixture of compound 8 (60 mg, 0.131 mmol), 2-(aminomethyl)-4-methoxypyridine (45 mg, 0.316 mmol), and triethylamine (0.2 mL, 1.44 mmol) in N-methylpyrrolidone (0.4 mL) was placed into sealed tube and stirred at 150°C under microwave irradiation for 3 h. The mixture was diluted with EtOAc (20 mL) and n-hexane (10 mL), washed with water and brine, dried over Na2SO4, and concentrated in vacuo. The residue was purified by NH-silica gel chromatography twice ((1) MeOH–CHCl3; 0 : 100 to 4 : 96; (2) EtOAc–n-Hex–MeOH=10 : 10 : 1) to yield 6d (50.5 mg, 69%) as a pale yellow amorphous.
1H-NMR (400 MHz, CDCl3) δ: 9.02 (1H, t, J=5.2 Hz), 8.40 (1H, d, J=5.2 Hz), 8.25 (1H, d, J=2.0 Hz), 7.63 (1H, d, J=2.4 Hz), 7.48 (1H, s), 7.36 (1H, d, J=8.0 Hz), 7.23 (1H, d, J=8.0 Hz), 6.81 (1H, d, J=2.4 Hz), 6.75 (1H, t, J=55.2 Hz), 6.70 (1H, dd, J=5.6, 2.4 Hz), 6.56 (1H, br s), 4.80 (1H, d, J=5.2 Hz), 4.61 (1H, d, J=5.6 Hz), 3.81 (3H, s), 3.13 (3H, s), 2.43 (3H, s), 1.68 (6H, s). 13C-NMR (100 MHz, CDCl3) δ: 167.2, 166.3, 160.0, 156.8, 154.6, 150.5, 137.9, 135.9, 135.8 (t, J=4.4 Hz), 132.7 (t, J=20.7 Hz), 131.6, 130.2 (t, J=2.2 Hz), 125.4 (t, J=7.4 Hz), 114.2 (t, J=238 Hz), 109.7, 108.2, 107.5, 105.6, 91.5, 80.8, 55.1, 50.8, 46.6, 43.4, 42.9, 31.3, 18.2. MS (ESI) m/z: 572.1 (M+H)+. FAB-MS m/z: 572.2131 (Calcd for C28H32F2N5O4S+: 572.2143). HPLC purity 98.6% (4.72 min, method A).
2-(1,3-Dioxolan-2-yl)-3-methylpyridine (9)A mixture of 3-methylpicolinaldehyde (2.0 g, 16.5 mmol), ethylene glycol (10 mL), and p-toluenesulfonic acid monohydrate (50 mg) in toluene (15 mL) was heated at reflux with Dean–Stark apparatus overnight. The mixture was concentrated in vacuo and the residue was purified by silica gel chromatography (EtOAc) to yield 9 (2.63 g, 97%) as a colorless oil.
1H-NMR (400 MHz, CDCl3) δ: 8.46 (1H, dd, J=4.6, 1.0 Hz), 7.48 (1H, dd, J=8.0, 0.8 Hz), 7.19 (1H, dd, J=8.0, 4.6 Hz), 6.00 (1H, s), 4.23–4.27 (2H, m), 4.06–4.10 (2H, m), 2.44 (3H, s). MS (ESI) m/z: 166.1 (M+H)+. HPLC purity 98.3% (1.98 min, method B).
2-(1,3-Dioxolan-2-yl)-3-methylpyridine-1-oxide (10)To a mixture of compound 9 (2.63 g, 15.9 mmol) in CHCl3 was added m-CPBA (4.19 g, 16.5 mmol) at 0°C. The mixture was stirred at room temperature for 3 h, diluted with CHCl3, and washed with sat. NaHCO3 and brine. The solvent was removed in vacuo and the residue was purified by silica gel column chromatography (EtOAc) to yield 10 (1.86 g, 65%) as a colorless solid.
1H-NMR (400 MHz, CDCl3) δ: 8.10 (1H, d, J=6.4 Hz), 7.12 (1H, dd, J=8.0, 6.4 Hz), 7.03 (1H, d, J=8.0 Hz), 6.78 (1H, s), 4.17–4.21 (2H, m), 4.05–4.09 (2H, m), 2.45 (3H, s). MS (ESI) m/z: 182.0 (M+H)+. HPLC purity 97.2% (2.47 min, method B).
6-(1,3-Dioxolan-2-yl)-5-methylpicolinonitrile (11)To a solution of compound 10 (1.86 g, 10.3 mmol) in CH2Cl2 were added trimethylsilyl cyanide (1.76 mL, 13.1 mmol) and N,N-dimethylcarbamoyl chloride (1.1 g, 10.3 mmol) at room temperature. The mixture was stirred at room temperature for 48 h and trimethylsilyl cyanide (1.76 mL, 13.1 mmol) and N,N-dimethylcarbamoyl chloride (1.1 g, 10.3 mmol) were added to the mixture because compound 10 was still exist. After 8 h of stirring, trimethylsilyl cyanide (1.76 mL, 13.1 mmol) and N,N-dimethylcarbamoyl chloride (1.1 g, 10.3 mmol) were added again and the mixture was stirred at room temperature overnight (the starting material was fully consumed at the period). The mixture was diluted with CHCl3, washed with water and brine, dried over Na2SO4, and concentrated in vacuo. The residue was purified by silica gel column chromatography (EtOAc–n-hexane; 33 : 67, and CHCl3 only to remove 2-cyano-3-methylpyridine) to yield 11 (543 mg, 28%) as a colorless solid.
1H-NMR (400 MHz, CDCl3) δ: 7.63 (1H, d, J=8.0 Hz), 7.58 (1H, d, J=8.0 Hz), 5.99 (1H, s), 4.22–4.26 (2H, m), 4.07–4.11 (2H, m), 2.52 (3H, s). MS (ESI) m/z: 191.1 (M+H)+. HPLC purity 98.0% (3.40 min, method B).
6-Formyl-5-methylpicolinonitrile (12)A mixture of compound 11 (2.7 g, 14.2 mmol) and 2 M aqueous HCl (40 mL) in 1,4-dioxane (40 mL) was stirred at 60°C for 6 h. The organic solvent was removed in vacuo and the mixture was made alkaline by sat. NaHCO3. The mixture was extracted with EtOAc and the organic phase was washed with brine, dried over Na2SO4, and concentrated in vacuo. The residue was purified by silica gel column chromatography (EtOAc–n-hexane; 33 : 67) to yield 12 (1.6 g, 77%) as a colorless solid.
1H-NMR (400 MHz, CDCl3) δ: 10.14 (1H, s), 7.79 (1H, d, J=8.0 Hz), 7.75 (1H, d, J=8.0 Hz), 2.75 (3H, s). MS (ESI) m/z: 147.1 (M+H)+. HPLC purity could not be determined due to peak broadening probably caused by hydration of aldehyde.
6-(Difluoromethyl)-5-methylpicolinonitrile (13)To a solution of compound 12 (173 mg, 1.18 mmol) in CHCl3 was added deoxofluor (0.87 mL, 4.7 mmol) at 0°C. The mixture was stirred at 0°C to room temperature overnight. The reaction was quenched with sat. NaHCO3 and the organic phase was separated. After removal of the solvent, the mixture was purified by silica gel column chromatography (n-hexane–EtOAc; 67 : 33) to yield 13 (146 mg, 73%) as a colorless solid.
1H-NMR (400 MHz, CDCl3) δ: 7.75 (1H, d, J=8.0 Hz), 7.69 (1H, d, J=8.0 Hz), 6.67 (1H, t, J=54 Hz), 2.61 (3H, s). MS (ESI) m/z: 169.1 (M+H)+. HPLC purity 97.0% (3.89 min, method B).
(6-(Difluoromethyl)-5-methylpyridin-2-yl)methanamine Hydrochloride (14)A mixture of compound 13 (1.42 g, 8.45 mmol), 5% palladium–carbon (Pd–C) (1.6 g) and 4 M HCl in 1,4-dioxane (7 mL) in MeOH (50 mL) was stirred under H2 atmosphere for 3 h. The mixture was filtered through celite® and the filtrate was concentrated in vacuo. The residue was stirred in EtOAc (10 mL) and the insoluble material was collected and dried to yield 14 (1.85 g, 89%) as a colorless solid.
1H-NMR (400 MHz, DMSO-d6) δ: 8.50 (2H, br s), 7.88 (1H, d, J=8.0 Hz), 7.62 (1H, d, J=8.0 Hz), 7.02 (1H, t, J=54 Hz), 4.15–4.20 (2H, m), 2.46 (3H, s). MS (ESI) m/z: 173.1 (M+H)+. HPLC purity 94.3% (2.57 min, method B).
Ethyl 2-Chloro-5-(3-methyl-3-(methylsulfonamido)but-1-yn-1-yl)nicotinate (15)To a solution of compound 2 (3.25 g, 10.4 mmol) and N-(2-methylbut-3-yn-2-yl)methanesulfonamide (2.52 g, 15.6 mmol) in triethylamine (30 mL) were added PdCl2(PPh3)2 (0.22 g, 0.31 mmol) and copper (I) iodide (0.12 g, 0.63 mmol). The reaction mixture was heated at 60°C for 10 h under Ar atmosphere. The mixture was filtered through celite® and the filtrate was concentrated. The residue was purified by silica gel chromatography (EtOAc–n-hexane; 20 : 80) to yield 15 as a pale yellow oil (2.35 g, 65%).
1H-NMR (400 MHz, CDCl3) δ: 8.51 (1H, d, J=2.4 Hz), 8.14 (1H, d, J=2.4 Hz), 4.56 (1H, br s), 4.44 (2H, q, J=7.2 Hz), 3.14 (3H, s), 1.74 (6H, s), 1.42 (3H, t, J=7.2 Hz). MS (ESI) m/z: 345.0 (M+H)+. HPLC purity 99.0% (4.16 min, method B).
Ethyl 2-(((4-Methoxypyridin-2-yl)methyl)amino)-5-(3-methyl-3-(methylsulfonamido)but-1-yn-1-yl)nicotinate (16)A mixture of compound 15 (800 mg, 2.32 mmol), 2-(aminomethyl)-4-methoxypyridine (481 mg, 3.48 mmol), and triethylamine (1.5 mL) in N-methylpyrrolidone (3 mL) was heated at 150°C for 3 h in a Biotage microwave reactor then was cooled to room temperature. The mixture was diluted with EtOAc–hexane (150 mL, 2 : 1), washed with water (50 mL) and brine (50 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by NH-silica gel chromatography (elution with hexane–EtOAc–MeOH=49.5 : 49.5 : 1 to 48 : 48 : 4) to yield 16 (658 mg, 64%) as a pale yellow solid.
1H-NMR (400 MHz, CDCl3) δ: 8.98 (1H, br s), 8.43 (1H, d, J=6.0 Hz), 8.32 (1H, d, J=2.4 Hz), 8.17 (1H, d, J=2.4 Hz), 6.80 (1H, d, J=2.4 Hz), 6.72 (1H, dd, J=5.6, 2.4 Hz), 4.82 (2H, d, J=5.2 Hz), 4.48 (1H, br s), 4.38 (2H, q, J=7.2 Hz), 3.83 (3H, s), 3.17 (3H, s), 1.72 (6H, s), 1.41 (3H, t, J=7.2 Hz). MS m/z: (ESI) 447.1 (M+H)+. HPLC purity 98.3% (3.23 min, method B).
2-(((4-Methoxypyridin-2-yl)methyl)amino)-5-(3-methyl-3-(methylsulfonamido)but-1-yn-1-yl)nicotinic Acid (17)A mixture of compound 16 (650 mg, 1.46 mmol) and 1 M aqueous NaOH (2.91 mL) in MeOH (13 mL) was stirred at 50°C for 2 h. After cooling, the solution was concentrated in vacuo, neutralized with 1 N aqueous HCl. The insoluble material was filtrated, washed with water, and dried to yield 17 (432 mg, 71%) as a colorless solid.
1H-NMR (400 MHz, DMSO-d6) δ: 13.31 (1H, br s), 8.98 (1H, br s), 8.35 (1H, d, J=5.6 Hz), 8.29 (1H, d, J=2.4 Hz), 8.05 (1H, d, J=2.4 Hz), 7.45 (1H, s), 6.86–6.91 (2H, m), 4.73 (2H, d, J=5.2 Hz), 3.80 (3H, s), 3.06 (3H, s), 1.72 (6H, s). MS (ESI) m/z: 419.1 (M+H)+. HPLC purity 97.2% (2.93 min, method B).
N-((6-(Difluoromethyl)-5-methylpyridin-2-yl)methyl)-2-(((4-methoxypyridin-2-yl)methyl)amino)-5-(3-methyl-3-(methylsulfonamido)but-1-yn-1-yl)nicotinamide (18a)To a solution of compound 17 (30 mg, 0.072 mmol), compound 14 (21.1 mg, 0.086 mmol), and DIPEA (0.05 mL) in DMF (1 mL) was added BOP reagent (34.9 mg, 0.079 mmol) at room temperature. The mixture was stirred at room temperature for 2 h and diluted with a mixture of EtOAc–n-hexane (1 : 2, 10 mL). The organic solution was washed with water and brine, dried over Na2SO4, and concentrated in vacuo. The residue was purified by silica gel column chromatography (MeOH–CHCl3; 3 : 97) to yield 18a (41 mg, 100%) as a white solid.
1H-NMR (400 MHz, CDCl3) δ: 9.03 (1H, br s), 8.40 (1H, d, J=5.6 Hz), 8.27 (1H, d, J=2.0 Hz), 7.78 (1H, d, J=2.4 Hz), 7.61 (1H, d, J=8.0 Hz), 7.52 (1H, br s), 7.33 (1H, d, J=8.0 Hz), 6.81 (1H, d, J=2.0 Hz), 6.73 (1H, t, J=54.4 Hz), 6.70 (1H, dd, J=5.6, 2.4 Hz), 4.80 (2H, d, J=2.0 Hz), 4.70 (2H, d, J=0.8 Hz), 3.81 (3H, s), 3.17 (3H, s), 2.51 (3H, s), 1.71 (6H, s). 13C-NMR (100 MHz, CDCl3) δ: 167.2, 166.3, 160.1, 156.8, 154.5, 152.8, 150.6, 149.3 (t, J=25.3 Hz), 140.7, 138.2, 131.5, 123.7, 116.2 (t, J=240 Hz), 110.0, 108.2, 107.4, 105.8, 91.6, 80.9, 55.1, 50.8, 46.6, 43.9, 42.9, 31.3, 16.8. MS (ESI) m/z: 573.1 (M+H)+. FAB-MS m/z: 573.2098 (Calcd for C27H31F2N6O4S+: 573.2096). HPLC purity 95.7% (4.27 min, method A).
Ethyl 2-(((2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)methyl)amino)-5-(3-methyl-3-(methylsulfonamido)but-1-yn-1-yl)nicotinate (19)A mixture of compound 15 (200 mg, 0.58 mmol), (2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methanamine (250 mg, 1.51 mmol), and DIEA (0.2 mL) in EtOH (1.5 mL) was heated at 155°C for 2.5 h in a Biotage microwave reactor then was cooled to room temperature. The mixture was purified by silica gel chromatography (hexane–EtOAc; 50 : 50) to yield 19 (230 mg, 84%).
1H-NMR (400 MHz, CDCl3) δ: 1H-NMR (chloroform-d): Shift 8.44 (1H, t, J=5.5 Hz), 8.33 (1H, d, J=2.3 Hz), 8.14 (1H, d, J=2.3 Hz), 6.80–6.87 (3H, m), 4.63 (2H, d, J=5.6 Hz), 4.46 (1H, br s), 4.32 (3H, q, J=7.2 Hz), 4.24 (4H, s), 3.17 (3H, s), 1.71 (6H, s), 1.38 (3H, t, J=7.2 Hz). MS m/z: (ESI) 474.1 (M+H)+. HPLC purity 97.1% (4.61 min, method B).
2-(((2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)methyl)amino)-5-(3-methyl-3-(methylsulfonamido)but-1-yn-1-yl)nicotinic Acid (20)A mixture of compound 19 (230 mg, 0.486 mmol) and 4 M aqueous NaOH (0.5 mL) in MeOH (2.5 mL) was stirred at 50°C for 2 h. MeOH was removed in vacuo and the mixture was acidified to pH 3 by 1 M aqueous HCl. The mixture was stirred with 0.5 mL of EtOAc and the solid was collected and dried to afford 20 (175 mg, 81%) as a pale yellow solid.
1H-NMR (400 MHz, DMSO-d6) δ: 8.61 (1H, t, J=5.8 Hz), 8.30 (1H, d, J=2.4 Hz), 8.03 (1H, d, J=2.4 Hz), 7.45 (1H, s), 6.74–6.87 (3H, m), 4.56 (2H, d, J=5.8 Hz), 4.20 (4H, s), 3.06 (3H, s), 1.57 (6H, s). MS m/z: (ESI) 446.1 (M+H)+. HPLC purity 95.7% (3.86 min, method B).
N-((6-(Difluoromethyl)-5-methylpyridin-2-yl)methyl)-2-(((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)amino)-5-(3-methyl-3-(methylsulfonamido)but-1-yn-1-yl)nicotinamide Hydrochloride (18b)A mixture of compound 20 (50 mg, 0.112 mmol), compound 14 (33.0 mg, 0.135 mmol), DIPEA (0.1 mL, 0.573 mmol) and BOP reagent (70 mg, 0.158 mmol) in DMF (3 mL) was stirred at room temperature for 2 h. The mixture was diluted with EtOAc and washed with water and brine. After evaporation, the residue was purified by silica gel chromatography (hexane–EtOAc; 50 : 50) to yield free base of 19 (60 mg). The free base (60 mg) was dissolved in EtOAc and treated with 4 M HCl in EtOAc and insoluble material was collected to give 18b (40 mg, 53%) as a pale yellow solid.
1H-NMR (400 MHz, DMSO-d6) δ: 9.34 (1H, t, J=6.0 Hz), 8.87 (1H, br s), 8.24 (1H, d, J=2.0 Hz), 8.12 (1H, d, J=2.4 Hz), 7.75 (1H, d, J=7.6 Hz), 7.48 (1H, s), 7.40 (1H, d, J=8.0 Hz), 6.97 (1H, t, J=54.4 Hz), 6.75–6.80 (3H, m), 4.51 (2H, s), 4.49 (2H, s), 4.42 (1H, s), 4.19 (3H, s), 3.08 (3H, s), 1.58 (6H, s). 13C-NMR (100 MHz, DMSO-d6) δ: 166.9, 155.8, 155.6, 152.8, 148.4 (t, J=23.5 Hz), 143.2, 142.4, 140.6, 139.1, 132.2, 130.3, 123.0, 120.3, 116.9, 116.1, 115.3 (t, J=238 Hz), 109.6, 105.3, 92.8, 79.5, 64.0, 63.9, 49.3, 44.1, 43.5, 42.2, 31.0, 16.1. MS (ESI) m/z: 600.2 (M+H)+. FAB-MS m/z: 600.2075 (Calcd for C29H32F2N5O5S+: 600.2092). HPLC purity 94.1% (4.27 min, method B).
Estimation of MBI ActivityA mixture of human liver microsomes (0.5 mg/mL) and 10 µM of test compound was pre-incubated in phosphate buffer (pH 7.4) for 30 min at 37°C with or without NADPH (1 mM). The mixture was next added to the assay buffer containing testosterone, a probe substrate of CYP3A4, and incubated for an additional 10 min at 37°C. The reaction was quenched with acetonitrile and then, the amount of testosterone metabolite was measured by LC-tandem mass spectrometry (LC-MS/MS) to determine the remaining CYP3A4 activity. The decrease in activity with/without NADPH was depicted as a percentage.
