Anagliptin; Uses, Contra indications, Dosage, Side effects, Drug Interaction

Compounds

AnagliptinKa to N, Oka M, Murase T, et al. Discovery and pharmacological characterization of N-[2-({2-[(2S)-2-cyanopyrrolidine-1-yl]-2-oxoethyl}amino)-2-methylpropyl]-2-methylpyrazolo[1,5-a]pyrimidine-6-caroxamide hydrochloride (anagliptin hydrochloride salt) as a potent and selective DPP-IV inhibitor. Bioorg Med Chem 2011;19:7221–7sitagliptinKim D, Wang L, Beconi M, et al. (2R)-4-Oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine: a potent, orally active dipeptidyl peptidase IV inhibitor for the treatment of type 2 insulin? is low or not working well. সহজ বাংলা: রক্তে চিনি বেশি থাকার রোগ।" data-rx-term="diabetes" data-rx-definition="Diabetes is a condition where blood sugar stays too high because insulin is low or not working well. সহজ বাংলা: রক্তে চিনি বেশি থাকার রোগ।">diabetes?. J Med Chem 2005;48:141–51, vildagliptinVillhauer EB, Brinkman JA, Naderi GB, et al. 1-[[(3-Hydroxy-1-adamantyl)amino]acetyl]-2-cyano-(S)-pyrrolidine: a potent selective, and orally bioavailable dipeptidyl peptidase IV inhibitor with antihyperglycemic properties. J Med Chem 2003;46:2774–89and alogliptinFeng J, Zhang Z, Wallace MB, et al. Discovery of alogliptin: a potent, selective, bioavailable, and efficacious inhibitor of dipeptidyl peptidase IV. J Med Chem 2007;50:2297–300were prepared by known methods, respectively.

Crystallographic study of anagliptin

A colourless single crystal (0.3 × 0.3 × 0.08 mm³) was obtained by standing at room temperature after heating to dissolve in ethyl acetate. The data were collected by the ω scan method with 0.3° in scan width and 10 s in exposure time at room temperature, using a SMART 1000 two-dimensional X-ray? diffractometer (Bruker; Mo Kα, 50 kV, 40 mA) with a 0.5-mmϕ collimator. The structure was solved by the direct method using the SHELXS-97 program (Göttingen, Germany), and refined by the full-matrix least-squares method using the SHELXL-97 program. The crystal structure data has been deposited to the Cambridge Crystallographic Data Centre (CCDC 1003533).

Expression and purification of DPP-4

Human DPP-4 (hDPP-4) was expressed in the Silkworm–Baculovirus System produced by ProCube (Sysmex Corporation; . The hDPP-4 gene encoding Ser39-Pro766 was amplified by PCR from the synthesised full-length hDPP-4 gene template with optimised codon usage for Bombyx mori. The PCR was run using a forward primer containing a BglII site and a cleavage site for HRV 3C protease (5′-GGAAGATCTCTGGAAGTTCTGTTCCAGGGGCCCTCAAGGAAGACTTATACATT-3′) and a reverse primer containing an EcoRV site (5′-GGCGATATCTTATGGTAAACTGAAACATTG-3′). The PCR product was inserted between BglII and EcoRV in the multiple cloning site of the pM24 vector (Sysmex Corporation) based on the vector pUC19 for B. mori nucleopolyhedrovirus (BmNPV). The vector was designed to express a fusion protein containing a DDDDK-tag and HRV3C protease recognition site at the N-terminal fragment of hDPP-4. This transfer vector was co-transfected with BmNPV DNA into a B. mori-derived cell line (BmN). After 7 days of incubation, the recombinant baculovirus was injected into the body of silkworm pupae. The pupae were harvested 6 days after infection?, frozen and homogenised with PBS buffer (pH 7.4) containing a protease inhibitor cocktail. The homogenised solution was centrifuged to separate the supernatant and precipitate using an ultracentrifuge (100 000 g) for 1 h at 4 °C. The supernatant containing DDDDK-tag fusion hDPP-4 was applied to DDDDK-tagged Protein PURIFICATION GEL (MBL Co., Ltd.), and the fusion protein was eluted with DDDDK peptide containing PBS buffer (pH 7.4). The eluted sample was treated with HRV 3C protease (Wako Pure Chemical Industries, Ltd.) for 16 h at 4 °C to remove the N-terminal DDDDK-tag from the recombinant fusion protein and then applied to a Glutathione Sepharose 4B column (GE Healthcare) to remove the HRV 3C protease. The flow-through fraction was collected and rapidly purified by TOYOSCREEN SuperQ (TOSOH Corporation), which is an ion exchange column with a linear gradient of 0–300 mM NaCl in Tris–HCl buffer (pH 8.0). Finally, hDDP4 was further purified by gel filtration chromatography using a Superdex 200 column (GE Healthcare) with 20 mM Tris–HCl (pH 8.0) buffer containing 100 mM NaCl. The purified hDPP-4 was concentrated using an Apollo spin concentrator (Orbital Biosciences, LLC) to 10 mg/mL for the crystallisation sample.

Crystallographic study of the DPP-4 complex with anagliptin

The complex was prepared by adding a 10-fold molar excess of anagliptin in a buffer (20 mM Tris–HCl, 0.1 M NaCl, pH 8.0) to the enzyme solution (20 mM Tris–HCl, 0.1 M NaCl, pH 8.0, concentration 9.6 mg/mL). The crystals of the complex were grown from a solution containing 18% PEG400, 15% PEG 1000 and 0.15 M Na/K phosphate (pH 6.5) at 293 K by the sitting-drop vapour-diffusion method. These crystallisation trials were conducted using the LIGHT method (laser irradiated growth technique)Adachi H, Takano K, Hosokawa Y, et al.Laser irradiated growth of protein crystal. Jpn J Appl Phys 2003;42:L798–800 to produce high-quality crystals. Before data collection, the crystal was cryo-cooled using a mixture of 25% glycerol, 18% PEG400, 15% PEG1000 and 0.15 M Na/K phosphate (pH 6.5). X-ray? diffraction data were collected on BL41XU at SPring-8 (Harima, Japan) at 100 K using a MX225HE (Rayonix, Evanston, IL) detector.

Measurements of in vitro activities

Inhibition of DPP-4 activity was determined by measuring the rate of hydrolysis of a surrogate substrate, Gly-Pro-4-methylcoumarin-7-amide (Gly-Pro-MCA; Peptide Institute, Osaka, Japan). Ten microliters of various concentrations of the test compound solutions and 45 μL of human recombinant DPP-4 (R&D Systems, Minneapolis, MN) with an assay buffer (25 mM HEPES, 140 mM NaC1, 0.1 mg/mL BSA, pH 7.8) were added to a black 96-well microplate and mixed. After a 30-min preincubation at room temperature, the enzymatic reaction was initiated by the addition of 45 μL of 0.4 mM Gly-Pro-MCA and allowed to react for 20 min. The reaction was stopped by the addition of 100 μL of 25% acetic acid solution, and the fluorescence intensity was measured at an excitation wavelength of 390 nm and a fluorescence wavelength of 460 nm. As standards, the fluorescence intensity of 7-amino-4-methylcoumarin (AMC) solutions plus 25% acetic acid solution was measured. The percent inhibition relative to the control without inhibitor was calculated and the IC₅₀ values were determined by a nonlinear regression model.

The levels of inhibition of the DPP8 and DPP9 activities were determined by the degradation of the fluorescent substrate Gly-Pro-MCA. Human DPP8 and DPP9 were expressed in baculovirus-infected Sf9 insect cells and purified using his-tagged protein purification resins. Inhibition of the DPP8 and DPP9 activities was determined as described above. Ten microliters of the test compounds and 50 μL of 1.0 mM Gly-Pro-MCA in buffer solution (50 mM HEPES, 0.1 mg/mL BSA, pH 8.0) were added to black 96-well microplates, and the reaction was initiated by the addition of 40 μL of the diluted enzyme solution for 20 min at room temperature. The reaction was stopped by the addition of 25% acetic acid solution and the fluorescence intensity was measured.

Inhibition of FAP activity was determined by the degradation of the fluorescent substrate Ala-Pro-7-amino-3-trifluoromethylcoumarine (Ala-Pro-AFC; Bachem AG, Switzerland). Human FAP was expressed in baculovirus-infected Sf9 insect cells and purified using his-tagged protein purification resins. The inhibition of FAP activities was measured as described above. Ten microliters of the test compound solutions and 50 μL of 1.0 mM Ala-Pro-AFC in buffer solution (50 mM Tris hydrochloride–150 mM sodium chloride, 0.1 mg/mL BSA, pH 8.0) were added to black 96-well microplates. The reaction was initiated by the addition of 40 μL of the diluted enzyme solution for 60 min at room temperature. The fluorescence intensity was measured at an excitation wavelength of 400 nm and a fluorescence wavelength of 505 nm. To establish the standards, various concentrations of AFC solutions were added to a 96-well black microplate, and the fluorescence intensities were measured.

Docking simulation

The initial ligand structure was prepared from the single-crystal structure of anagliptin determined in this paper. All ligands for docking simulation were provided from anagliptin analogues using the LigPrep software package, version 2.8 (Schrödinger, LLC, New York, NY, 2013). These ligand-calculation conditions adopted conventional values. The molecular-docking simulation of compounds against the DPP-4 crystal structure, PDB code 3QWH, was performed by Glide Glide version 6.1. New York (NY): Schrödinger, LLC; 2013 .

Crystal structure of anagliptin

The crystal structure of anagliptin was solved and refined by single-crystal X-ray? analysis. Anagliptin has a single asymmetric carbon on its pyrrolidine moiety. Since the pyrrolidine moiety is prepared from l-prolinamide without racemization, the stereochemistry must be 2S, like that of l-prolinamide. The structure having a 2S configuration was therefore refined; the final R index was 0.0454 and the absolute structure parameter χFlack HD. On enantiomorph-polarity estimation. Acta Crystallogr 1983;A39:876–81 was 0.0. Consequently, it was clarified that the configuration was 2S (space group: P3₂). For comparison, a 2R configuration (space group: P3₁) was modelled and refined. In this case the final R index was 0.0459 and the χ parameter was 1.1; these parameters showed that the 2R configuration was not correct. The crystallographic data and refinement are shown in Table 1 (CCDC 1003533).