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Design, synthesis and anti-diabetic activity of chromen-2-one derivatives
⁎Corresponding author. shubhangiss@rediffmail.com (Shubhangi S. Soman)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Abstract
DPP-IV inhibitors have been immersed as promising pathway to treat Type 2 diabetes. Here we have reported designing of coumarin derivatives as DPP-IV inhibitors. Designed compounds have been studied for their binding with DPP-IV enzyme through molecular docking followed by synthesis. All synthesized compounds have been fully characterized and screened for DPP-IV inhibition activity. Two compounds showed very good inhibition at 10 μM concentration.
Keywords
Coumarin derivatives
Molecular docking
Antidiabetic activity
DPP-IV inhibition
1 Introduction
Diabetes is chronic disease and the number of people with diabetes has risen from 108 million in 1982 to 422 million in 2014 (WHO, 2016). Out of two types of diabetes, type 1 diabetes is due to lack of insulin Type 2 diabetes which is due to body’s ineffective use of insulin. Type 2 diabetes mellitus (T2DM) is a metabolic disease characterized by hyperglycemia or high blood sugar level. It is largely the result of excess body weight and physical inactivity (Abdullah et al., 2010). When it is not controlled or treated properly, it can lead to blindness, kidney failure, heart attacks, stroke and lower limb amputation into patients (Ripsin et al., 2009). Diabetes can be managed by healthy diet, regular physical activity and maintaining a normal body weight. It can be treated through various pathways by controlling blood glucose level (Bennett et al., 2011). Main disadvantage of these pathways is side effects such as weight gain, hypoglycemia and joint pains. Current treatment for type 2 diabetes is based upon increasing insulin availability, improving sensitivity to insulin, delaying the delivery of insulin and absorption of carbohydrates from gastrointestinal tract or increasing urinary glucose excretion (Sena et al., 2010). Recently, dipeptidyl peptidase IV (DPP-IV) inhibitors have been immerged as quite promising pathway to treat Type 2 diabetes. Inhibition of DPP-IV leads to increased half-life of endogenous incretins such as GLP-1, as active sites of enzyme which cleaves the N-terminal dipeptide with l-proline and l-alanine are blocked by inhibitors (Hansen et al., 1999). Different classes of DPP-IV have been reported and marketed to treat Type 2 Diabetes such as Vildagliptin and Sitagliptin (Villhauer et al., 2003; Kim et al., 2005) (Fig. 1). Recently, Omarigliptin has been reported as potent DPP-IV inhibitors with dose only once in a week for treatment of diabetes (Biftu et al., 2014) (Fig. 1).
Coumarin is one of the interesting heterocycle found in nature. Several coumarin derivatives have been reported with various activities such as anti-inflammatory, anticoagulant, anti-oxidant, anticancer, antifungal, and neuroprotective agents (Sandhu et al., 2014; Barot et al., 2015; Pisani et al., 2016). Synthetic coumarin derivatives have also been reported to treat diabetes by different pathways such as activation of phosphatidylinositol-3-kinase and α-glucosidase inhibitor (Dwivedi et al., 2008; Anand et al., 2011). The coumarin based drug dicumarol is in market since long time with minimum side effects. The pharmacological properties and application of coumarins depend on type and position of substitution on coumarin rings. There are only few reports on synthetic coumarin derivatives as DPP-IV inhibitors to treat Type 2 diabetes. In continuation to our work on coumarin containing DPP-IV inhibitors, we have designed compounds 10, 14 and 15 based on our earlier coumarin derivatives as DPP-IV inhibitors 4 and 5 (Fig. 2). Compounds 4 and 5 have shown very good DPP-IV inhibition at micromolar concentration (Sharma and Soman, 2015, 2016). Molecular docking is a tool available to chemist to check whether the designed molecule is efficient in binding with receptor. So we have designed the molecules having glycine linkage between substituted amine and coumarin and studied its molecular docking with DPP-IV enzyme. Designed molecules were compared with Vildagliptin as they have same active pharmacophore. Compounds 10a–h, 14a–h and 15g–h were synthesized and screened for DPP-IV inhibition activity.
2 Materials and methods
2.1 Chemistry
Reagent grade chemicals and solvents were purchased from commercial supplier and used after purification. TLC was performed on silica gel F254 plates (Merck). Acme’s silica gel (60–120 mesh) was used for column chromatographic purification. All reactions were carried out in nitrogen atmosphere. Melting points are uncorrected and were measured in open capillary tubes, using a Rolex melting point apparatus. IR spectra were recorded as KBr pellets on Perkin Elmer RX 1 spectrometer. 1H NMR and 13C NMR spectral data were recorded on Advance Bruker 400 spectrometer (400 MHz) with CDCl3 or DMSO-d6 as solvent and TMS as internal standard. J values are in Hz. Mass spectra were determined by ESI-MS, using a Shimadzu LCMS 2020 apparatus. Elemental analyses were recorded on Thermo Finnigan Flash 11–12 series EA. All reactions were carried out under nitrogen atmosphere. Compounds 1-(bromomethyl)-3H-benzo[f]chromen-3-one 9 (Dey and Sankaranarayan, 1934), 3-acetyl-7-hydroxy-2H-chromen-2-one 11 (Shah and Shah, 1954) and ethyl 7-hydroxy-2-oxo-2H-chromene-3-carboxylate 12 (Bigi et al., 1999) were prepared using literature methods.
2.1.1 Preparation of substituted glycinamide derivatives (6)
A mixture of Boc-glycine (1.0 mmol), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (1.5 mmol) (EDCI), 1-hydroxybenzotriazole (1.0 mmol) (HOBt), triethylamine (2.0 mmol) and amine (1° and 2°) (1.10 mmol) in dichloromethane (50 mL) (DCM) was stirred at room temperature for 16 h. The reaction was monitored using TLC. On completion of the reaction, it was washed with water (2 × 20 mL), brine (1 × 10 mL), dried over anhydrous sodium sulfate and the solvent evaporated under reduced pressure to give the crude product which was then purified by column chromatography using silica gel as stationary phase and DCM:MeOH (95:5) as eluent to yield desired N-Boc glycinamide 6, as white solid.
2.1.2 Boc deprotection of glycinamide derivatives
Compound 6 was deprotected by stirring in 10% trifluoroacetic acid (TFA) in DCM. On completion of the reaction after monitored by TLC, the solvent was evaporated under reduced pressure. Compound was dissolved in DCM and concentrated under reduced pressure to remove to give compound 7 as trifluoroacetic acid salt and was directly used for next step without any purification.
2.1.3 General procedure for the preparation of compounds 10a–g
4-Bromomethylnaphthopyrone 9 (0.5 g, 1.730 mmol) dissolved in DMF (20 mL) and substituted glycinamide 7 (1.1 eq), along with base triethylamine (1.5 eq) was added to it. The resulting mixture was stirred at room temperature for 16 h and then poured into cold water. The aqueous layer thus obtained was extracted using ethyl acetate and/or dichloromethane (checked by TLC) and solvent evaporated to give crude product. The product thus, obtained was purified by column chromatography using Pet ether: ethyl acetate.
Representative characterization data for compounds 10a, 10b and 10h. Data for compounds 10c–g are provided in supporting information.
2.1.3.1 N-(4-methylphenyl)-2-{[(3-oxo-3H-benzo[f]chromen-1-yl)methyl]amino}acetamide (10a)
Yield: 45%; M.P.: 176–178 °C; IR (KBr): 3325, 3074, 2890, 1726, 1688, 1549, 1519, 1448, 1402, 1337, 1209, 1142, 999, 873, 814, 743 cm−1; 1H NMR (400 MHz, CDCl3): 2.30 (s, 3H), 3.62 (s, 2H), 4.51 (s, 2H), 6.82 (s, 1H), 7.09 (d, J = 8.4 Hz, 2H), 7.29 (d, J = 8.4 Hz, 2H), 7.50 (d, J = 9.2 Hz, 1H), 7.59 (t, J = 7.2 Hz, 1H), 7.67–7.71 (m, 1H), 7.96 (dd, J = 0.8, 8.0 Hz, 1H), 8.01 (d, J = 9.2 Hz, 1H), 8.39 (d, J = 8.4 Hz, 1H), 8.75 (s, 1H); 13C NMR (100 MHz, CDCl3): 20.88, 52.52, 54.53, 113.18, 114.74, 117.99, 119.48, 125.02, 125.76, 128.44, 129.29, 129.49, 129.99, 131.42, 134.08, 134.14, 134.59, 154.51, 155.07, 160.41, 168.46; MASS: 372.90 [M+H]+; Anal. Calc. for C23H20N2O3; C, 74.18; H, 5.41; N, 7.52; found: C, 74.38; H, 5.61; N, 7.32%.
2.1.3.2 N-(4-chlorophenyl)-2-{[(3-oxo-3H-benzo[f]chromen-1-yl)methyl]amino}acetamide (10b)
Yield: 53%; M.P.: 182–184 °C; IR (KBr): 3322, 3277, 3075, 2845, 1712, 1675, 1590, 1553, 1512, 1398, 1304, 1207, 1092, 1020, 820, 747 cm−1; 1H NMR (400 MHz, CDCl3): 3.63 (s, 2H), 4.52 (s, 2H), 6.79 (s, 1H), 7.22 (d, J = 8.8 Hz, 2H), 7.31 (d, J = 8.8 Hz, 2H), 7.49 (d, J = 8.8 Hz, 1H), 7.61 (t, J = 7.6 Hz, 1H), 7.68 (t, J = 8.0 Hz, 1H), 7.96–8.02 (m, 2H), 8.40 (d, J = 8.8 Hz, 1H), 8.86 (s, 1H); 13C NMR (100 MHz, CDCl3): 52.41, 54.65, 113.09, 115.06, 118.03, 120.59, 124.94, 125.79, 128.45, 128.97, 129.25, 129.34, 130.06, 131.45, 134.19, 135.70, 154.19, 155.14, 160.30, 168.66; MASS: 393.25 [M+H]+; Anal. Calc. for C22H17ClN2O3; C, 67.26; H, 4.36; N, 7.13; found: C, 67.46; H, 4.55; N, 7.31%.
2.1.3.3 1-({[2-(morpholin-4-yl)-2-oxoethyl]amino}methyl)-3H-benzo[f]chromen-3-one (10h)
Yield: 56%; M.P.: 114–116 °C; IR (KBr): 3556, 3492, 3080, 2871, 2811, 1695, 1649, 1549, 1429, 1276, 1241, 1107, 859, 825, 744 cm−1; 1H NMR (400 MHz, CDCl3): 3.38 (t, J = 4.8 Hz, 2H), 3.52 (s, 2H), 3.66–3.70 (m, 6H), 4.42 (s, 2H), 6.93 (s, 1H), 7.46 (d, J = 8.8 Hz, 1H), 7.55 (t, J = 7.6 Hz, 1H), 7.55 (dt, J = 1.2, 7.6 Hz, 1H), 7.91 (d, J = 7.6 Hz, 1H), 7.96 (d, J = 8.8 Hz, 1H), 8.45 (d, J = 8.8 Hz, 1H); 13C NMR (100 MHz, CDCl3): 42.20, 44.78, 49.57, 54.02, 66.38, 66.79, 113.90, 114.45, 117.80, 125.54, 125.72, 128.11, 129.53, 129.65, 131.29, 133.68, 154.84, 155.69, 160.86, 169.21; MASS: 352.90 [M+H]+; Anal. Calc. for C20H20N2O4; C, 68.17; H, 5.72; N, 7.95; found: C, 68.30; H, 5.83; N, 7.87%.
2.1.4 Preparation of N-substituted chloroacetamide derivatives (13)
To an ice-cold solution of substituted amines (20 mmol) in dichloromethane (DCM) (25 mL) was added triethylamine (TEA) (20.2 mmol) and stirred for 5–10 min. To this chloroacetyl chloride (20 mmol) was added dropwise over a period of 10 min under cooling. The resulting solution was stirred at 0–5 °C for 30 min and at room temperature for 24 h. The reaction mixture was diluted with water and extracted with DCM (2 × 30 mL). The organic layers were combined, washed with HCl solution (0.5 N, 15 mL), dried over anhydrous Na2SO4, filtered and evaporated on a rotavapor to give compound 13. The substituted chloroacetamide 13 thus obtained, was used directly for next step without any purification.
2.1.5 General procedure for the synthesis of compounds 14a–g and 15g–h
To a solution of compound 11/12 (1.0 eq) in dry N,N-dimethylformamide (DMF) (15 mL) was added compound 13 (1.2 eq). To this anhydrous K2CO3 (1.5 eq) was added followed by pinch of KI. The resulting mixture was heated in water bath at 70–80 °C 12–18 h. The completion of reaction was checked by TLC using Pet. ether: ethyl acetate (1:1). The reaction mixture was poured in ice cold water. The solid separated out was filtered, washed with water and dried. When there was no solid separated out in water, the aqueous layer was extracted using DCM (3 × 20 mL). The organic layers were combined, washed with brine, dried over anhydrous Na2SO4, filtered and concentrated to give an oily residue. The residue was triturated in Pet. ether to give compound as solid. The crude compound was purified by column chromatography over silica gel using Pet. ether: ethyl acetate (40:60–20:80) to obtain pure compound as solid.
Representative characterization data for compounds 14a, 14b and 15g. Data for compounds 14c–g and 15h are provided in supporting information.
2.1.5.1 2-[(3-Acetyl-2-oxo-2H-chromen-7-yl)oxy]-N-(4-methylphenyl)acetamide (14a)
Pale yellow solid, Yield: 91%; M.P.: 210–212 °C; IR (KBr): 3374, 3053, 2919, 1727, 1681, 1613, 1597, 1372, 1204, 1052, 820, 770 cm−1; 1H NMR (400 MHz, CDCl3): δ 2.35 (s, 3H), 2.73 (s, 3H), 4.72 (s, 2H), 6.96 (d, J = 2.2 Hz, 1H), 7.04 (dd, J = 2.2, 8.8 Hz, 1H), 7.19 (d, J = 8.4 Hz, 2H), 7.48 (d, J = 8.4 Hz, 2H), 7.66 (d, J = 8.8 Hz, 1H), 8.10 (s, 1H), 8.52 (s, 1H); 13C NMR (100 MHz, CDCl3): δ ppm 20.95, 30.62, 67.71, 102.04, 113.29, 113.33, 120.35, 121.92, 129.71, 132.03, 133.84, 135.06, 147.31, 157.37, 159.27, 161.86, 164.37, 195.34; Anal. Calc. for C20H14NO5; C, 68.37; H, 4.88; N, 3.99; found: C, 68.22; H, 4.90; N, 3.72%; ESI-MS: 352.0 [M+H]+.
2.1.5.2 2-[(3-Acetyl-2-oxo-2H-chromen-7-yl)oxy]-N-(4-chlorophenyl)acetamide (14b)
Pale yellow solid, Yield: 79%; M.P.: 216–218 °C; IR (KBr): 3364, 2925, 2355, 1728, 1682, 1615, 1593, 1531, 1454, 1372, 1260, 1203, 1127, 1056, 1010, 831, 770 cm−1; 1H NMR (400 MHz, DMSO-d6): δ 2.56 (s, 3H), 4.91 (s, 2H), 7.10–7.12 (m, 2H), 7.38 (d, J = 6.8 Hz, 2H), 7.64 (d, J = 9.2 Hz, 2H), 7.91 (d, J = 9.2 Hz, 1H), 8.64 (s, 1H), 10.33 (s, 1H); 13C NMR (100 MHz, DMSO-d6): δ ppm 30.55, 67.75, 101.60, 112.75, 114.19, 121.23, 121.65, 127.83, 129.18, 132.70, 137.71, 147.95, 157.25, 159.27, 163.68, 166.21, 195.25; Anal. Calc. for C19H14ClNO5; C, 61.38; H, 3.80; N, 3.77; found: C, 61.16; H, 4.02; N, 3.94%; ESI-MS: 394.00 [M+Na]+.
2.1.5.3 Ethyl 2-oxo-7-[2-oxo-2-(piperidin-1-yl)ethoxy]-2H-chromene-3-carboxylate (15g)
Off white solid, Yield: 43%; M.P.: 128–130 °C; IR (KBr): 3052, 2993, 2937, 2848, 1756, 1659, 1603, 1556, 1502, 1445, 1431, 1380, 1292, 1204, 1181, 1112, 1051, 1035, 1010, 962, 862, 799 cm−1; 1H NMR (400 MHz, DMSO-d6): 1.30 (t, J = 7.2 Hz, 3H), 1.44 (br s, 2H), 1.57 (br s, 4H), 3.34–3.42 (m, 4H), 4.27 (q, J = 7.2 Hz, 2H), 5.02 (s, 2H), 6.99–7.00 (m, 2H), 7.82–7.85 (m, 1H), 8.73 (s, 1H); 13C NMR (100 MHz, DMSO-d6): 14.59, 24.37, 25.70, 26.30, 42.61, 45.39, 61.40, 66.61, 101.50, 112.02, 113.80, 114.19, 131.96, 149.66, 156.72, 157.20, 163.28, 164.25, 164.90; Anal. Calc. for C19H21NO6; C, 63.50; H, 5.89; N, 3.90; found: C, 63.23; H, 5.72; N, 3.63%; ESI-MS: 360.0 [M+H]+.
2.2 Biological activity screening
In vitro enzyme (DPP-IV) inhibitory activity was determined using fluorescence based assay. Gly-Pro-Aminomethylcoumarin (AMC) was used as a substrate to measure DPP-IV activity. Cleavage of the peptide bond by DPP-IV releases the free AMC group, resulting in fluorescence that is analyzed using an excitation wavelength of 350–360 nm and emission wavelength of 450–465 nm. Human recombinant DPP-IV enzyme procured from Enzo Life Science (batch no. BML-SE434-9091), substrate, H-Gly-Pro-AMC procured from Enzo life science (batch no. BML-P189-9091) and assay buffer, having pH. 7.8 were used in the assay DPP-IV activity which was measured by mixing reagents in 96-well plate (order of addition of reagents: assay buffer, enzyme, solvent/inhibitor and finally substrate). Both the enzyme and 96-well plate were incubated for 30 min and the resulting fluorescence was measured using Spectra Max Fluorometer (Molecular Devices, Sunnyvale, CA) by exciting at 360 nm and emission at 460 nm with the excitation filter at 360 nm and emission filter at 460 nm at sensitivity of 45.
3 Results and discussion
3.1 Docking studies
The chain A of protein DPP-IV (PDB ID: 3W2T) was selected for docking study as the only crystallized structure of human DPP-IV complexed with an approved drug Vildagliptin available in PDB (Berman et al., 2002; Nabeno et al., 2013). Molecular docking studies were performed using glide XP (extra precision) scoring function of maestro version 9.0 (Repasky et al., 2007). All designed molecules and cocrystal Vildagliptin (Fig. 3) were docked flexibly with the target protein, and the QikProp and toxicity prediction applications were used to find the absorption, distribution metabolism and excretion (ADME) property (QikProp, 2009) and the toxicity level of the designed molecules respectively.
The softwares PyMol, a powerful and comprehensive molecular visualization product for rendering and animating 3D molecular structure (Schrodinger, 2015) and LigPlots were used to visualize the docking results. A schematic 2D representation of protein-ligand complex was generated by LigPlot, a graphical system of automatically generating multiple 2D diagrams of ligand protein interactions from 3D coordinates. The diagrams portray the hydrogen bond interaction patterns and hydrophobic contacts between the ligands and the main chain and side chain elements of proteins. To evaluate the reliability of docking algorithm, the RMSD between docked Vildagliptin and the X-ray solved Vildagliptin which is present in the protein complex was calculated and it has the value of 0.027 Å, which shows the algorithm is accurate (Nicolotti et al., 2009; Totrov and Abagyan, 2008) (Fig. 3a).
The docking results showed many of the designed molecules have considerable good binding affinity with the target protein than the cocrystal approved drug Vildagliptin, which has ΔG = −4.610 kcal/mol (Table 1, Fig. 4) and the designed molecules 10a–c and 14a bind in the large pocket of the target protein and among those molecules 10a–c, 14a had the best docking score than the co-crystal approved drug Vildagliptin. On other hand, the designed compounds 10g–h fit well in smaller pocket of protein just like Vildagliptin. As compound 10h binds in small pocket like Vildagliptin (Fig. 4), it was superimposed with Vildagliptin (Fig. 3b). RMSD between 10h and the X-ray solved Vildagliptin which is present in the protein complex was calculated and it has the value of 0.246 Å.
| 10ab | 10bb | 10cb | 10gc | 10hc | 14ab | Vildagliptinc | |
|---|---|---|---|---|---|---|---|
| Glide XP Docking Score | −5.061 | −5.307 | −5.550 | −4.369 | −4.696 | −4.719 | −4.610 |
| Interacting amino acid residues through Hydrogen bonda | Glu206, Tyr547, Tyr662 | Glu205, Glu206, Ser209 | Glu206, Tyr547, Gln553, Tyr585 | Lys122, Gln123, Tyr238 | Lys122, Gln123, Tyr238 | Lys122, Asn710 | Tyr238, Asp737 |
The docking results with the interacting pattern including interacting amino acid residues showed that 10a–c and 14a bind in the large pocket of the target protein while Vildagliptin fit well in the smaller pocket of proteins DPP-IV (Fig. 4). On other hand, the designed compounds 10g–h fit well in smaller pocket of protein just like Vildagliptin, thus confirmed our designing strategy is correct (Fig. 4). Interestingly, designed compounds 10a–c and 10g–h showed binding in different pockets of DPP-IV enzyme. These docking results prompted us to synthesize all designed compounds and to study their DPP-IV inhibition activity. Detailed docking results and LigPlots are given in supporting information.
3.2 Chemistry
The Boc-glycine on reaction with different amines in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 1-hydroxybenzotriazole (HOBt) gave various amides 6. Compound 6 was deprotected using trifluoroacetic acid in dichloromethane to give compound 7 which was used as such without purification for next step. On the other hand, ethyl acetoacetate was brominated using Br2 to give 4-bromo-3-oxobutanoate 8 as red oil (Sousa et al., 2012).
Compound 8 on Pechmann reaction with β-naphthol in conc. H2SO4 gave 1-(bromomethyl)-3H-benzo[f]chromen-3-one 9 (Dey and Sankaranarayan, 1934). 1H NMR for compound 9 showed presence of singlet at δ 4.90 for two protons indicated presence of —CH2Br group and all other aromatic protons appeared in the range of δ 6.6–8.5 thereby confirming the formation of 9. This compound 9 was used to carry out substitution reaction with compound 7 using triethylamine in DMF to form substituted aminomethylnaphthopyrone derivatives 10a–h (Scheme 1).
The IR spectrum of compound 10a exhibited strong band at 3325 cm−1 for the —NH stretching, another strong band at 1726 cm−1 for lactone carbonyl group of coumarin ring and 1688 cm−1 for amide carbonyl group. In the 1H NMR spectrum of 10a, all aromatic protons were observed at δ 8.39–6.82 and amide —NH at δ 8.75 as a singlet. The two methylene protons were observed as a singlet at δ 4.51 and 3.62 and methyl group at δ 2.30. In the 13C NMR spectrum, the lactone carbonyl carbon of coumarin ring was observed at δ 160, amide carbonyl carbon at δ 168, all aromatic carbons at δ 155–113, two methylene carbons at δ 54 and 52, and methyl carbon at δ 20. In the ESI-MS spectrum of 10a a peak at m/z 372.90 for [M+H]+ confirmed its formation. The structures of all substituted aminomethylnaphthopyrones 10a–h were confirmed by different analytical techniques such as 1H NMR, 13C NMR, IR, and ESI-MS. For compound 10h, single crystal was developed and was studied its structure by X-ray single crystal analysis (CCDC No. 1500813) (Fig. 5) and used for docking (Fig. 3b).
In general, the IR spectra of compounds 10a–g exhibited one strong band in the range of 1724–1711 cm−1 for the lactone carbonyl group of coumarin ring and amide carbonyl group at 1693–1622 cm−1. In the 1H NMR spectra of 10a–g, peak for the methylene protons was observed in the range of δ ∼ 5.01–3.91 depending on the effect of different amine substitution on it. All these new chemical entities were subjected to in vitro studies.
3-Acetyl-7-hydroxy coumarin 11 and ethyl 3-carboxylate-7-hydroxy coumarin 12 were prepared by Knoevenagel condensation of resorcaldehyde with ethyl acetoacetate/diethyl malonate (Shah and Shah, 1954; Bigi et al., 1999). Various substituted amines on reaction with chloroacetyl chloride gave corresponding chloroacetamide derivatives 13 (Scheme 2) and these compounds were directly used for reaction with 7-hydroxy-3-substituted coumarins 11 and 12. These chloroacetamide derivatives 13 were reacted with 7-hydroxy-3-substituted coumarins 11–12 using anhydrous K2CO3 in DMF at 70–80 °C in the presence of catalytic amount of KI to give compounds 14a–g and 15g–h (Scheme 2). All these compounds were characterized by different analytical techniques such as 1H NMR, 13C NMR, IR, and ESI-MS.
3.3 Biological evaluation
Preliminary in vitro DPP-IV inhibition assay was performed to screen compounds 10a–h to study the effect of different amides at the P1 site on their inhibition potential at 100 μM concentration as shown in Table 2.
| Compd | —NR1R2 | Inhibition at 100 μMa | Compd | —NR1R2 | Inhibition at 100 μMa |
|---|---|---|---|---|---|
| 10a |
|
49.3% | 10e |
|
42.1% |
| 10b |
|
26.7% | 10f |
|
NA |
| 10c |
|
55.1% | 10g |
|
46.6% |
| 10d |
|
66.9% | 10h |
|
53.7% |
| Sitagliptin | 62.7% at 0.1 μM | Vildagliptin | 56.3% at 0.1 μM |
From the in vitro analysis as DPP-4 inhibitors, all tested compounds 10a–h showed inhibition in the range of 26.7–66.9% at 100 μM concentration (Table 2). Compounds 10a–h remained inactive at lower concentration such as 30 μM and 10 μM.
7-Substituted-3-acetyl coumarin derivatives showed interesting activities (Table 3). Compounds 14a with p-methyl group, showed 62.2% inhibition in in vitro assay at 100 μM concentration. Aromatic amide derivatives 14b–e remained poor at 100 μM concentration. Saturated amide derivatives, compound 14f with pyrrolidine and compound 14g with piperidine showed good activity with 84.5% and 65.7% inhibition respectively at 100 μM concentrations. Replacement of 3-acetyl group with ethyl 3-carboxylate group, resulted in enhancement of DPP-4 inhibition activity in compounds 15g and 15h. Compounds 15g and 15h showed DPP-4 inhibition at 10 μM concentration with 56.8% and 38.3% inhibition respectively (Table 3). Still it was less than Sitagliptin which showed 62.7% inhibition at 0.1 μM concentration.
| Compd | —NR1R2 | Inhibition at 100 μMa | Compd | —NR1R2 | Inhibition at 100 μMa |
|---|---|---|---|---|---|
| 14a |
|
62.2% | 14f |
|
84.5% |
| 14b |
|
NA | 14g |
|
65.7% |
| 14c |
|
4.7% | At 10 μM | ||
| 14d |
|
1.3% | 15g |
|
56.8% |
| 14e |
|
38.8% | 15h |
|
38.3% |
| Sitagliptin | 62.7% at 0.1 μM | Vildagliptin | 56.3% at 0.1 μM |
4 Conclusion
We have designed small peptide linkage coumarin derivatives for DPP-IV inhibition and studied their docking with known pockets of enzyme. Docking studies showed interesting results as compounds 10a–e with aromatic amines residue bind in big pocket of DPP-IV enzyme and 10f–h with cyclic secondary amines residue bind like Vildagliptin in small pocket of DPP-IV enzyme. So we have synthesized various coumarin derivatives, and characterized their structures. Although synthesized compounds show less activity than standard drug Vildagliptin and Sitagliptin, two of our compounds showed promising DPP-IV inhibition activity at 10 μM concentration. Interestingly, compound 15g showed very good activity with 56.8% inhibition at 10 μM.
Acknowledgments
One of the authors (RS) is thankful to Department of Science & Technology, Government of India for financial support vide reference no. SR/WOS-A/CS-1028/2014 under Women Scientist Scheme to carry out this work. The authors are thankful to The Head, Department of Chemistry Faculty of Science, The M. S. University of Baroda for providing laboratory facilities, Zydus Research Centre, Ahmedabad, for the activity and ESI-MS analyses and DST-PURSE for X-Ray crystallography facility.
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Appendix A
Supplementary material
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.arabjc.2016.11.011.
Appendix A
Supplementary material
Supplementary data 1
Supplementary data 1