4.1. Compound synthesis
4.1.1. General
All commercially available reagents and solvents were used as-received. All reactions were carried out using oven-dried flasks or glassware, and mixtures were stirred with stirring bars and concentrated using a standard rotary evaporator unless otherwise noted. Procedures for preparation of all intermediates 2 and 3 were described previously [15]. The 1H NMR spectra were recorded by a JEOL JNM-ECP400 spectrometer operating at 400 MHz in DMSO-d6 at 25°C with tetramethylsilane as the internal standard. The data are reported as follows: chemical shift in ppm (δ), integration, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br = broad singlet, m = multiplet), and coupling constant (Hz). LC/MS spectra were determined on a Waters ZMD2000 equipped with a Waters 2690 injector and a PDA detector operating at 210-400 nm and interfaced with a Micromass ZMD mass spectrometer.
4.1.2. Representative procedure for preparation of pyrrolidine carbonitrile 4; (S)-1-(2-(4-(Isoindolin-2-yl)-2-methyl-4-oxobutan-2-ylamino)acetyl)pyrrolidine-2-carbonitrile HCl Salt (4a)
A solution of (S)-1-(2-chloroacetyl)pyrrolidine-2-carbonitrile (467 mg, 2.70 mmol) in acetone (5.0 mL) was added drop-by-drop to an ice-cooled stirred suspension of 3a (550 mg, 2.50 mmol), K2CO3 (370 mg, 2.70 mmol), and NaI (200 mg, 1.30 mmol) in acetone (20 mL). The reaction mixture was stirred at room temperature overnight. The resulting mixture was filtered to remove insoluble materials, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (CH2Cl2/MeOH = 20/1) to give 540 mg (61%) of 4a of the free base. To an ice-cooled solution of 4a of the free base (250 mg, 0.70 mmol), 1,4-dioxane (5.0 mL) was added 4N-HCl/1,4-dioxane (180 μL, 0.72 mmol). The reaction mixture was stirred at 0°C for 1 h and then evaporated to yield the title compound (240 mg, Y. 88%). 1H NMR 1.38 (6H, s), 2.00-2.22 (4H, m), 2.85-2.90 (2H, m), 3.30-4.10 (4H, m), 4.69 (2H, s), 4.87 (2H, s), 4.80-4.85 (1H, m), 7.25-7.40 (4H, m); MS m/z 355 (M+H)+.
4.1.3. (S)-1-(2-(2-Methyl-4-(5-methylisoindolin-2-yl)-4-oxobutan-2-ylamino)acetyl)pyrrolidine-2-carbonitrile HCl Salt (4b)
Colorless solid (92%). 1H NMR 1.41 (6H, s), 1.99-2.11(2H, m), 2.18-2.24 (2H, m), 2,32 (3H, s), 2.88-2.98 (2H, m), 3.21-3.39 (2H, m), 3.50-3.57 (1H, m), 3.68-3.72 (1H, m), 4.06-4.10 (1H, m), 4.66 (2H, s), 4.86 (2H, sm), 7.13-7.28 (3H, m), 9.29 (2H, brs); MS m/z 369 (M+H)+.
4.1.4. (S)-1-(2-(4-(5-Fluoroisoindolin-2-yl)-2-methyl-4-oxobutan-2-ylamino)acetyl)pyrrolidine-2-carbonitrile HCl Salt (4c)
Colorless solid (31%). 1H NMR 1.40 (6H, s), 2.02-2.08(2H, m), 2.19-2.22 (2H, m), 2.88-2.89 (2H, m), 3.50-3.69 (2H, m), 4.04-4.07 (2H, m), 4.67-4.70 (2H, m), 4.85-4.89 (3H, m), 7.16 (1H, t, J = 9.2 Hz), 7.24 (1H, t, J = 9.2 Hz), 7.37-7.44 (1H, m), 9.10 (2H, brs); MS m/z 373 (M+H)+.
4.1.5. (S)-1-(2-(4-(5-Bromoisoindolin-2-yl)-2-methyl-4-oxobutan-2-ylamino)acetyl)pyrrolidine-2-carbonitrile HCl Salt (4d)
Colorless solid (81%). 1H NMR 1.35 (6H, s), 1.95-2.05 (2H, m), 2.12-2.18 (2H, m), 2.83 (2H, s) 3.70-4.05 (4H, m), 4.62-4.72 (2H, m), 4.78-4.84 (3H, m), 7.29-7.60 (3H, m), 8.21 (2H, brs); MS m/z 423 (M+H)+.
4.1.6. (S)-1-(2-(4-(5-Chloroisoindolin-2-yl)-2-methyl-4-oxobutan-2-ylamino)acetyl)pyrrolidine-2-carbonitrile HCl salt (4e)
Colorless solid (38%). 1H NMR 1.65 (6H, s), 2.20-2.35 (4H, m), 2.90-3.35 (2H, m), 3.70-4.40 (4H, m), 4.75-5.00 (5H, m), 7.20-7.30 (3H, m); MS m/z 389 (M+H)+.
4.1.7. (S)-1-(2-(2-Methyl-4-oxo-4-(5-(trifluoromethyl)isoindolin-2-yl)butan-2-ylamino)acetyl)pyrrolidine-2-carbonitrile HCl salt (4f)
Colorless solid (37%). 1H NMR 1.41 (6H, s), 1.98-2.09 (2H, m), 2.18-2.25 (2H, m), 2.92 (2H, d, J = 3.3 Hz), 3.50-3.54 (1H, m), 3.67-3.72 (1H, m), 4.00-4.13 (2H, m), 4.78 (2H, s), 4.87 (1H, dd, J = 3.3 and 7.3 Hz), 4.97 (2H, s), 7.59-7.64 (1H, m), 7.69 (1H, d, J = 8.1 Hz), 7.76-7.80 (1H, m), 9.19 (2H, brs); MS m/z 423 (M+H)+.
4.1.8. (S)-1-(2-(4-(4-Fluoroisoindolin-2-yl)-2-methyl-4-oxobutan-2-ylamino)acetyl)pyrrolidine-2-carbonitrile HCl salt (4g)
Colorless solid (23%). 1H NMR 1.40 (6H, s), 2.01-2.09(2H, m), 2.18-2.25 (2H, m), 2.92-2.94 (2H, m), 3.51-3.53 (1H, m), 3.66-3.72 (1H, m), 4.00-4.13 (2H, m), 4.75 (2H, s), 4.85-4.87 (1H, m), 4.97 (1H, s), 7.16 (1H, t, J = 8.8 Hz), 7.21-7.25 (1H, m), 7.37-7.43 (1H, m), 9.18 (2H, brs); MS m/z 373 (M+H)+.
4.1.9. (S)-1-(2-(2-Methyl-4-(4-methylisoindolin-2-yl)-4-oxobutan-2-ylamino)acetyl)pyrrolidine-2-carbonitrile HCl salt (4h)
Colorless solid (33%). 1H NMR 1.41 (6H, s), 2.03-2.11(2H, m), 2.17-2.24 (2H, m), 2.26 (3H, s), 2.95 (2H, d, J = 8.9 Hz), (2H, m), 3.40-3.66 (2H, m), 3.97-4.10 (2H, m), 4.25-4.31 (1H, m), 4.68 (2H, s), 4.88 (2H, s), 7.11-7.25 (3H, m), 9.29 (2H, brs); MS m/z 369 (M+H)+.
4.1.10. (S)-1-(2-(4-(4,7-Dichloroisoindolin-2-yl)-2-methyl-4-oxobutan-2-ylamino)acetyl)pyrrolidine-2-carbonitrile HCl salt (4i)
Colorless solid (69%). 1H NMR 1.41 (6H, s), 2.03-2.10 (2H, m), 2.19-2.25 (2H, m), 2.94-2.97 (2H, m), 3.67-3.72 (2H, m), 4.03-4.14 (2H, m), 4.77 (2H, s), 4.86(1H, dd, J = 4.4 and 7.3 Hz), 5.00 (2H, s), 7.48 (2H, s), 9.18 (2H, brs); MS m/z 423 (M+H)+.
4.1.11. (S)-1-(2-(4-(5,6-Dichloroisoindolin-2-yl)-2-methyl-4-oxobutan-2-ylamino)acetyl)pyrrolidine-2-carbonitrile HCl salt (4j)
Colorless solid (10%). 1H NMR 1.65 (6H, s), 2.20-2.35 (4H, m), 2.90-3.35 (2H, m), 3.70-4.40 (4H, m), 4.80-4.95 (5H, m), 7.37-7.44 (2H, m); MS m/z 423 (M+H)+.
4.1.12. (S)-1-(2-(4-(4-Methoxy-6-methylisoindolin-2-yl)-2-methyl-4-oxobutan-2-ylamino)acetyl)pyrrolidine-2-carbonitrile HCl salt (4k)
Colorless solid (77%). 1H NMR 1.55-1.70 (6H, m), 2.20-2.35 (4H, m), 2.37 (3H, s), 2.80-3.40 (2H, m), 3.60-4.45 (7H, m), 4.65-4.90 (5H, m), 6.55-6.75 (2H, m); MS m/z 399 (M+H)+.
4.1.13. (S)-1-(2-(4-(5-(Hydroxymethyl)isoindolin-2-yl)-2-methyl-4-oxobutan-2-ylamino)acetyl)pyrrolidine-2-carbonitrile HCl salt (4l)
Colorless solid (81%). 1H NMR 1.41 (6H, s), 2.0-2.25 (4H, m), 2.92 (2H, m), 3.5-4.1 (4H, m), 4.5-4.9 (7H, m), 7.2-7.4 (3H, m), 9.28 (2H, brs); MS m/z 385 (M+H)+.
4.2. Biological evaluation
4.2.1. In vitro assay for DPP-IV inhibition
Inhibition of DPP-IV activity was determined by measuring the rate of hydrolysis of a surrogate substrate, H-Gly-Pro-7-amino-4-methylcoumarin (H-Gly-Pro-AMC). Human recombinant DPP-IV was purchased from R&D Systems, Minneapolis, MN. 10 μL of appropriately diluted solutions of the test compounds in water was added to 96-well microtiter plates, followed by the addition of 40 μL of DPP-IV diluted in assay buffer (25 mM HEPES, 140 mM NaC1, 0.1 mg/mL BSA, pH 7.8). After a 30-min preincubation at room temperature, the reaction was initiated by the addition of 50 μL of the assay buffer containing 0.2 mM H-Gly-Pro-AMC. After incubation at room temperature for 20 min, the reaction was stopped by the addition of 100 μL of 25% aqueous acetic acid and fluorescence was measured using an excitation wavelength of 390 nm and an emission wavelength of 460 nm. A standard curve for AMC was generated by adding 0.2-20 μmol of AMC to buffer solutions containing 12.5% aqueous acetic acid. The inhibitory rate relative to the control without inhibitor was calculated and IC50 values were determined by nonlinear regression (GraphPad Prism 4, ver. 4.03 software).
4.2.2 In vitro assays for inhibition of DPP-8 and DPP-9
Human DPP-8 and DPP-9 were expressed in baculovirus-infected Sf9 insect cells and purified using His-tagged protein purification resins. Inhibition of DPP-8 and -9 activities was determined as described above. 10 μL of appropriately diluted aqueous solutions of the test compounds was added to 96-well microtiter plates, followed by the addition of 50 μL of 1.0 mM H-Gly-Pro-AMC in buffer solution (50 mM HEPES, 0.1 mg/mL BSA, pH 8.0). The reaction was initiated by the addition of 40 μL of the enzyme solution diluted in the assay buffer. After incubation at room temperature for 30 min, the reaction was stopped by the addition of 100 μL of 25% aqueous acetic acid and fluorescence was measured using an excitation wavelength of 390 nm and an emission wavelength of 460 nm.
4.2.3. In vivo assay methods
All procedures were approved by the Sanwa Kagaku Kenkyusho Institutional Animal Care and Use Committee. 7-week old Wistar/ST rats were housed under standard conditions and allowed free access to water and a commercial diet for at least 5 days. The rats were fasted overnight prior to dosing and then received 4b orally at doses of 0.1-1 mg/kg or vehicle as a 5 mL/kg aqueous solution 30 min before glucose challenge. After an oral glucose challenge (5 mL/kg of an aqueous solution of 20% glucose), blood samples were collected from the tail vein of each animal into heparin-containing tubes at serial time points for 2 h. Plasma was prepared immediately, frozen, and stored at -20°C prior to analysis.
4.2.4 Inhibition of rat plasma DPP-IV ex vivo
Plasma DPP-IV activity was determined as described above. A 20 μL plasma sample was mixed with 5 μL of reaction buffer (140 mM NaCl, and 10 mM KCl, 25 mM Tris-HCl, pH 7.4, 1% bovine serum albumin) and 10 μL of buffer containing 60 μM H-Gly-Pro-AMC. After incubation at room temperature for 30 min, the reaction was stopped by the addition of 20 μL of 25% aqueous acetic acid and fluorescence was measured using an excitation wavelength of 360 nm and an emission wavelength of 460 nm.
4.2.5 Measurement of plasma glucose and insulin concentrations
Plasma glucose and insulin were determined with a glucometer (Glutest Pro; SKK, Japan) and a rat insulin ELISA kit (Shibayagi, Japan), respectively, according to the manufacturer's instructions. Statistical analyses were performed using Microsoft Excel. Individual comparisons among more than two experimental groups were assessed using ANOVA, with Fisher's Least Significant Difference post hoc test. Differences were considered significant at P values < 0.05. Analysis of dose-response data was performed by Dunnett's test.
4.2.6 Pharmacokinetics (PK) in rats
Sprague-Dawley (SD) rats were housed under standard conditions and allowed free access to water and a commercial diet. On the day before the experiment, rats were fasted overnight and for the first 12 h of the experiment. Compounds 4b were prepared in a saline/ethanol vehicle (50/50 v/v) at appropriate concentrations of 4b as an intravenous (iv) injection of 1 mL/kg via the femoral vein and as a suspension in 5% gum arabic solution for oral (po) administration. Blood samples were collected from the jugular vein of each animal with a heparinized syringe under diethyl ether anesthesia at serial time points for 24 h after drug administration. Plasma was obtained by centrifugation at 4°C and stored at -70°C until analysis. Protein precipitation was carried out by the addition of the internal standard solution (70% CH3CN with 0.2% acetic acid) to samples. The tubes underwent vigorous shaking and centrifugation for 5 min; then the supernatant was subjected to LC/MS/MS analysis. Peak areas were determined using Xcalibur® software (Thermo Electron Corporation, UK) and AUC values were calculated by the trapezoidal rule.
4.3. Metabolic stability
The incubation mixture containing 0.25 mg of rat or human liver microsomes was preincubated with an NADPH-generating system for 5 min at 37°C. The reaction was started by the addition of 5 μL of a DMSO solution containing the test compound (5 μM). At t = 0 and at two additional time points between 0 and 30 min, aliquots (100 μL) were removed and added to termination mixtures (CH3CN). Proteins were sedimented by centrifugation and an aliquot of the supernatant was analyzed by LC/MS/MS.
In determinations of the in vitrot
1/2, the analyte/ISTD peak area ratio was converted to percentage of drug remaining by assigning a value of 100% to the peak area ratio at t = 0. The slope of the regression line fitted to the log (percentage remaining) versus incubation time relationship (-k) was used in the conversion of raw data to the in vitrot
1/2 value. In vitro CLint was calculated using the following formula.
Enzyme induction was evaluated as follows: Hepatocytes isolated from male SD rats were maintained in culture for 1 day before treatment with the test compound or P-450 inducers. The cells were treated with the test compound (1, 10, 50 μM), β-naphthoflavone (10 μM, CYP1A inducer), phenobarbital (50 μM, CYP2B inducer), dexamethasone (10 μM, CYP3A inducer), or vehicle (0.1% DMSO final volume; used as negative control) for 2 days.
Induction of CYP1A, CYP2B, and CYP3A enzymes was determined based on measurements of 7-ethoxyresorufin O-dealkylation, 7-pentoxyresorufin O-dealkylation, and testosterone 6β-hydroxylation, respectively. Assays were started by the addition of Krebs-Henseleit buffer containing 8 μM 7-ethoxyresorufin, 10 μM 7-pentoxyresorufin, or 250 μM testosterone at a volume of 100 μL per well. After incubation at 37°C for 30 min, aliquots were removed and analyzed by fluorometry (an excitation wavelength of 538 nm and an emission wavelength of 590 nm) or LC/MS/MS to determine the quantities of metabolites formed. Any test compound causing a dose-dependent change equal to or greater than 10% of the positive control (see formula below) was considered an enzyme inducer.