Synthesis and biological evaluation of glucagon-like peptide-1 receptor agonists

Abstract

In this study, a series of fused-heterocyclic derivatives were systematically designed and synthesized using an efficient route, and evaluated in terms of GLP-1R agonist activity. We employed short synthetic steps and reactions that are tolerant of the presence of various functional groups and suitable for parallel operations to enable the rapid generation of libraries of diverse and structurally complex small molecules. Of the compounds synthesized, 3-(8-chloro-6-(trifluoromethyl)imidazo[1,2-a] pyridin-2-yl)phenyl methanesulfonate (8e) was the most potent agonist with an EC₅₀ of 7.89 μM, and thus is the compound with the greatest potential for application. These findings represent a valuable starting point for the design and discovery of small-molecule GLP-1R agonists that can be administered orally.

Introduction

Type 2 diabetes mellitus (DM2), a state of hormonal disruption and incretin deficiency, is increasingly becoming a worldwide epidemic (Kwak and Ha 2013). Current drugs utilized in the treatment of DM2 have well-established shortcomings: (1) increasing body weight and (2) increasing loss of β-cell function (Whitehouse 1997; Giugliano et al. 2009). However, the recent emergence of incretin-based therapies, which focus on glucagon-like peptide-1 (GLP-1), has attracted much interest.

GLP-1 is a peptide hormone of 30 amino acid residues. As a peptide, it has a very short half-life (<2 min) (Deacon et al. 1995). Such a short half-life has limited the utility of native GLP-1 in the treatment of DM2. The effort to identify GLP-1 analogues has resulted in the development of the drugs exenatide (Sennik et al. 2011; Buse et al. 2004) and liraglutide (Sjöholm 2010; Hribal and Sesti 2010). However, the requirement for injection limits the clinical utility of these peptide drugs. Therefore, orally active, small-molecule agonists of the GLP-1 receptor (GLP-1R) are highly sought after (Murphy and Bloom 2007).

Figure 1 shows synthetic small molecule agonists reported by several groups (Teng et al. 2000; Wang et al. 2009; Teng et al. 2007; Kopin 2004; Gong et al. 2010). Compound 6b, characterized by a novel imidazopyridine hit core, was identified from a library of 10,000 heterocyclic small molecules (Gong et al. 2010). As a small and drug-like active molecule, it represents an interesting starting point for the development of novel drugs. Therefore, we selected this compound as a model. In an effort to move away from the labile ester group of the phenol, we planned a synthetic pathway of new derivatives of imidazo[1,2-α]pyridine-based molecules (Fig. 2). To evaluate the structure–activity relationship, we designed and synthesized a series of heterocyclic derivatives containing a ring-junction nitrogen using a three-dimensional (3D) pharmacophore model reported previously (Gong et al. 2010) (Fig. 2). For the first stage, only combinations of five- and six-membered rings are considered, including imidazo[1,5-α]pyridine, imidazo[1,2-α]pyrimidine and imidazo[1,2-α]pyrazine. We employed short synthetic steps and reactions that are tolerant of the presence of various functional groups and suitable for parallel operations to enable the rapid generation of libraries of diverse, structurally complex, small molecules.

Fig. 1

Known ago-allosteric modulators of GLP-1R

Fig. 2

Structures of synthesized compounds. a Synthesized imidazo[1,2-a]pyridine-based molecules. b Other synthesized heterocycle-series compounds

Materials and methods

Chemistry

All the chemicals used in synthesis were supplied by Aldrichand TCI, and were used without further purification. All solvents were purified and stored in a dry condition. Reaction progress was determined by thin-layer chromatography (TLC) on Merck TLC Silica gel 60 F245 plates. Column chromatography was carried out using a silica gel 60 (63–200 mesh, Merck). NMR spectra were recorded on Agilent 400 instruments operating at 400 MHz for ¹H and 100 MHz for ¹³C, and Agilent 500 instruments operating at 500 MHz for ¹H and 125 MHz for ¹³C. Chemical shifts are expressed as parts per million (ppm) with tetramethylsilane as the internal standard. MS spectra were recorded on an Agilent G6530A Q-TOF.

General synthetic procedure for (6a–b)

To a stirred solution of bromomethylketone 3 (1.21 g, 4.7 mmol) and 2-amino-5-trifuoromethylpyridine 4 (0.61 g, 4.7 mmol) or 2-amino-3-chloro-5-trifluoromethylpyridine 5 (0.92 g, 4.7 mmol) in EtOH (50 mL) was added NaHCO₃ (0.31 g, 4.7 mmol) at room temperature. The reaction mixture was heated to reflux and monitored by TLC (hexane/ethyl acetate: 2/1) until completion. After removing EtOH, the residue was extracted with ethyl acetate and water. The combined organic phases were washed with water, 1 N HCl, and brine, dried, and filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (ethyl acetate/hexane = 10–20 %, Rf = 0.23).

3-(6-(Trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)phenyl acetate (6a)

Pale yellow solid; Yield: 64 %;¹H NMR (500 MHz, CDCl₃): δ 2.33 (s, 3H), 7.10 (d, J = 10.1 Hz, 1H), 7.32 (d, J = 11.9 Hz, 1H), 7.45 (t, J = 10.0 Hz, 1H), 7.70–7.73 (m, 2H), 7.80 (d, J = 9.7 Hz, 1H), 7.94(s, 1H), 8.49 (s, 1H);¹³C NMR (125 MHz, CDCl₃); δ 21.1, 109.8, 117.0, 117.3, 118.0, 119.5, 121.1, 121.8, 123.6, 124.8, 129.9, 134.2, 145.2, 146.2, 151.2, 169.6; EI-HRMS calculated for (C₁₆H₁₁F₃N₂O₂+H)⁺ 321.0851, found 321.0860.

3-(8-Chloro-6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)phenyl acetate (6b)

Pale yellow solid; Yield: 29 %;¹H NMR (500 MHz, CDCl₃): δ 2.34 (s, 3H), 7.10 (dd, J = 10.1, 2.1 Hz, 1H), 7.41–7.47 (m, 2H), 7.76 (t, J = 2.2 Hz, 1H), 7.81 (d, J = 10.2 Hz, 1H), 7.98 (s, 1H), 8.43 (s, 1H);¹³C NMR (125 MHz, CDCl₃): δ 21.2,111.2, 119.62, 119.65, 119.68, 122.0, 123.3, 123.4, 123.7, 124.3, 129.8, 134.0, 142.7, 147.0, 151.1, 169.6; EI-HRMS calculated for (C₁₆H₁₀ClF₃N₂O₂+H)⁺ 355.0461, found 355.0470.

General synthetic procedure for (8a and 8d)

To a mixture of 7a (99 mg, 0.36 mmol) or 7b (113 mg, 0.36 mmol) and K₂CO₃ (250 mg, 1.81 mmol) in acetone (10 mL) was added 1-chloroacetone (1 mL, 34.83 mmol) at room temperature. The reaction mixture was heated to reflux for 6 h. After removing acetone and 1-chloroacetone, the residue was extracted with ethyl acetate and water. The combined organic phases were washed with water and brine, dried, and filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (ethyl acetate/hexane = 10–20 %, Rf = 0.25).

General synthetic procedure for (8b and 8e)

To a solution of 7a (98 mg, 0.35 mmol) or 7b (121 mg, 0.45 mmol) in pyridine (5 mL) was added methanesulfonyl chloride (66 mg, 0.60 mmol) dropwise with stirring overnight in an ice bath. The reaction mixture was quenched with water in an ice bath and extracted with ethyl acetate (3 × 30 mL). The combined organic phases were washed with water, 1 N HCl, and brine, dried, and filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (hexane/ethyl acetate = 4/1, Rf = 0.25).

General synthetic procedure for (8c and 8f)

To a solution of 7a (90 mg, 0.32 mmol) or 7b (100 mg, 0.32 mmol) in pyridine (10 mL) was added toluenesulfonyl chloride (80 mg, 0.42 mmol) dropwise in an ice bath. After stirring for 2 h at room temperature, the reaction mixture was quenched with water in an ice bath and then extracted with ethyl acetate (3 × 30 mL). The combined organic phases were washed with water, 1 N HCl, and brine, dried, and filtered and concentrated. The residue was purified by silica gel column chromatography (hexane/ethyl acetate = 8/1, Rf = 0.23).

1-(3-(6-(Trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)phenoxy)propan-2-one (8a)

Pale yellow solid; Yield: 63 %;¹H NMR (500 MHz, CDCl₃): δ 2.32 (s, 3H), 4.64 (s, 2H), 6.91 (d, J = 10.1 Hz, 1H), 7.32–7.40 (m, 2H), 7.55–7.57 (m, 2H), 7.73 (d, J = 11.8 Hz, 1H), 7.94 (s, 1H), 8.50 (s, 1H);¹³C NMR (125 MHz, CDCl₃): δ 26.7, 73.0, 109.6, 112.2, 114.8, 118.1, 119.6, 120.7, 122.4, 124.6, 126.7, 130.1, 134.6, 145.2, 147.1, 158.2, 205.5; EI-HRMS calculated for (C₁₇H₁₃F₃N₂O₂+H)⁺ 335.1007, found 335.1019.

3-(6-(Trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)phenyl methanesulfonate (8b)

Pale yellow solid; Yield: 44 %;¹H NMR (500 MHz, CDCl₃): δ 3.20 (s, 3H), 7.29–7.36 (m, 2H), 7.50 (t, J = 9.9 Hz, 1H), 7.72 (d, J = 11.7 Hz, 1H), 7.89–7.92 (m, 2H), 7.98 (s, 1H), 8.51 (s, 1H);¹³C NMR (125 MHz, CDCl₃): δ 37.6, 109.9, 118.3, 119.7, 121.09, 121.11, 122.0, 124.77, 124.81, 125.0, 130.5, 135.3, 145.3, 145.9, 149.8; EI-HRMS calculated for (C₁₅H₁₁F₃N₂O₃S + Na)⁺ 379.0340, found 379.0360.

3-(6-(Trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)phenyl 4-methylbenzenesulfonate (8c)

Pale yellow solid; Yield: 64 %;¹H NMR (500 MHz, CDCl₃): δ 2.43 (s, 3H), 6.91 (d, J = 8.2 Hz, 1H), 7.30–7.35 (m, 4H), 7.62 (s, 1H), 7.67 (m, 1H), 7.75 (d, J = 7.4 Hz, 2H), 7.84–7.89 (m, 2H), 8.48(s, 1H);¹³C NMR (125 MHz, CDCl₃): δ 21.7, 109.8, 118.2, 120.2, 120.9, 122.0, 124.8, 128.5, 129.8, 130.0, 132.2, 134.9, 145.3, 145.5, 146.1, 150.1; EI-HRMS calculated for (C₂₁H₁₅F₃N₂O₃S + Na)⁺ 455.0653, found 455.0656.

1-(3-(8-Chloro-6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)phenoxy)propan-2-one (8d)

Pale yellow solid; Yield: 50 %;¹H NMR (500 MHz, CDCl₃): δ 2.32 (s, 3H), 4.64 (s, 2H), 6.89 (dd, J = 8.2, 2.7 Hz, 1H), 7.35 (t, J = 7.9 Hz, 1H), 7.40 (s, 1H), 7.54–7.57 (m, 2H), 7.97 (s, 1H), 8.43 (s, 1H);¹³C NMR (125 MHz, CDCl₃): δ 26.8, 73.0, 111.3, 112.6, 115.0, 116.6, 116.9, 119.6, 119.7, 123.3, 124.3, 130.1, 134.1, 142.7, 147.6, 158.1, 205.5; EI-HRMS calculated for (C₁₇H₁₂ClF₃N₂O₂+H)⁺ 369.0618, found 369.0669.

3-(8-Chloro-6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)phenyl methanesulfonate (8e)

Pale yellow solid;Yield: 45 %;¹H NMR (500 MHz, CDCl₃): δ 3.20 (s, 3H), 7.31 (d, J = 10.2 Hz, 1H), 7.43 (s, 1H), 7.50 (t, J = 10.0 Hz, 1H), 7.91 (m, 1H), 7.94 (d, J = 9.8 Hz, 1H), 8.03 (s, 1H), 8.45(s, 1H);¹³C NMR (125 MHz, CDCl₃): δ 37.7, 111.5, 116.9, 117.2, 120.0, 122.2, 123.38, 123.43, 124.5, 125.3, 130.4, 134.8, 142.8, 146.4, 149.7; EI-HRMS calculated for (C₁₅H₁₀ClF₃N₂O₃S+H)⁺ 391.0131, found 391.0135.

3-(8-Chloro-6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)phenyl 4-methylbenzene sulfonate (8f)

Pale yellow solid; Yield: 38 %;¹H NMR (500 MHz, CDCl₃): δ 2.43 (s, 3H), 6.91 (d, J = 8.2 Hz, 1H), 7.27–7.33 (m, 3H), 7.39 (m, 1H), 7.61 (s, 1H), 7.74 (d, J = 8.2 Hz, 2H), 7.87 (d, J = 7.8 Hz, 1H), 7.94 (s, 1H), 8.48(s, 1H);¹³C NMR (125 MHz, CDCl₃): δ 21.7, 111.5, 119.8, 120.3, 122.2, 123.5, 124.3, 125.1, 126.9, 128.5, 130.0, 132.3, 134.4, 138.6, 145.6, 145.8, 146.5, 149.8, 150.0. EI-HRMS calculated for (C₂₁H₁₄ClF₃N₂O₃S + Na)⁺ 489.0263, found 489.0274.

General synthetic procedure for (12a–c)

Pyridium bromide perbromide (1.79 g, 5.60 mmol) was added to a solution of 10a–c (0.9 g, 5.08 mmol) in AcOH (100 mL) with stirring for 3 h at room temperature. The reaction mixture was poured into ice-cold water and then extracted with ethyl acetate (3 × 50 mL). The combined organic phases were washed with saturated aqueous NaHCO₃, water, and brine, dried, and filtered and concentrated in vacuo to give crude 11a–c as a yellow oil (1.29 g, 98 %). The resulting crude 11a–c could be used without further purification. To a stirred solution of bromomethylketone 11a–c (1.30 g, 5.1 mmol) and aminopyridine 4 (0.82 g, 5.1 mmol) in EtOH (80 mL) was added NaHCO₃ (0.43 g, 5.1 mmol) at room temperature. The reaction mixture was heated to reflux for 8 h. After removing EtOH, the residue was extracted with ethyl acetate and water. The combined organic phases were washed with water, 1 N HCl, and brine, dried, and filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (12a–b, hexane/ethyl acetate = 1/1, Rf = 0.24; 12c, hexane/ethyl acetate = 4/1, Rf = 0.22).

N-(3-(6-(Trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)phenyl)acetamide(12a)

Pale yellow solid;Yield: 39 %;¹H NMR (500 MHz, CDCl₃): δ 2.19 (s, 3H), 7.31 (d, J = 11.6 Hz, 1H), 7.39 (t, J = 9.8 Hz, 1H), 7.51 (s, NH), 7.60 (d, J = 9.8 Hz, 1H), 7.68 (d, J = 11.1 Hz, 2H), 7.93 (s, 1H), 8.07 (s, 1H), 8.47(s, 1H);¹³C NMR (125 MHz, CDCl₃): δ 24.7, 109.6, 117.4, 118.1, 119.9, 120.7, 122.0, 124.6, 124.7, 129.6, 133.7, 138.5, 145.2, 147.1, 168.5; EI-HRMS calculated for (C₁₆H₁₂F₃N₃O + Na)⁺ 342.0830, found 342.0835.

N-(3-(6-(Trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)phenyl)methanesulfonamide (12b)

Pale yellow solid;Yield: 20 %;¹H NMR (500 MHz, CDCl₃): δ 3.05 (s, 3H), 7.30 (dt, J = 7.0, 1.3 Hz, 1H), 7.34 (dd, J = 9.5, 1.8 Hz, 1H), 7.40 (t, J = 7.9 Hz, 1H), 7.69–7.74 (m, 3H), 7.82 (t, J = 1.9 Hz, 1H), 7.99 (s, 1H), 8.51(s, 1H);¹³C NMR (125 MHz, CDCl₃): δ 39.4, 110.0, 118.0, 118.5, 120.7, 121.3, 123.0, 124.8, 124.9, 130.2, 130.3, 134.2, 137.6, 145.2, 146.2; EI-HRMS calculated for (C₁₅H₁₂F₃N₃O₂S+H)⁺ 356.0681, found 356.0705.

4-Methyl-N-(3-(6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)phenyl)benzene sulfonamide (12c)

Pale yellow solid;Yield: 21 %;¹H NMR (500 MHz, CDCl₃): δ 2.30 (s, 3H), 7.14–7.19 (m, 3H), 7.26–7.29 (m, 2H), 7.63 (dd, J = 7.7, 0.9 Hz, 1H), 7.66–7.70 (m, 4H), 7.85 (brs, NH), 7.89 (s, 1H), 8.45(s, 1H);¹³C NMR (125 MHz, CDCl₃): δ 21.5, 109.9, 118.0, 118.8, 120.7, 121.0, 122.7, 124.7, 127.3, 129.7, 129.9, 134.0, 136.0, 137.5, 143.9, 145.2, 146.5; EI-HRMS calculated for (C₂₁H₁₆ClF₃N₃O₂S+H)⁺ 432.0994, found 432.1020.

3-(8-Chloro-6-(trifluoromethyl)imidazo[1,5-a]pyridin-3-yl)phenyl acetate (19)

To a solution of 18 (300 mg, 0.80 mmol) in benzene (10 mL) was added POCl₃ (1.2 mL, 13.04 mmol) dropwise at room temperature. The reaction mixture was heated to reflux for 6 h. After cooling to room temperature, the mixture was poured into iced-water and then extracted with ethyl acetate (3 × 50 mL). The combined organic phases were washed with water and brine, dried, and filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (hexane/ethyl acetate = 12/1, Rf = 0.25). White solid; Yield: 90 %;¹H NMR (400 MHz, CDCl₃): δ 2.35 (s, 3H), 6.92 (s, 1H), 7.25 (d, J = 8.6 Hz, 1H), 7.54–7.61 (m, 3H), 7.77 (s, 1H), 8.51(s, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 21.1, 113.9, 117.6, 117.9, 119.6, 121.5, 121.8, 122.5, 122.9, 124.9, 125.3, 126.5, 126.8, 129.7, 130.1, 130.5, 151.3, 169.2; EI-HRMS calculated for (C₁₆H₁₀ClF₃N₂O₂+H)⁺ 355.0461, found 355.0474.

3-(8-Chloro-6-(trifluoromethyl)imidazo[1,5-a]pyridin-3-yl)phenyl cyclohexane carboxylate (21a)

To a solution of 19 (257 mg, 0.72 mmol) in THF (20 mL) was added a solution of NaOH (50 mg, 1.25 mmol) in water (10 mL) with stirring for 3 h at room temperature. After removing THF, the resulting mixture was extracted with ethyl acetate. The combined organic phases were washed with water and brine, dried, and filtered and concentrated in vacuo. The resulting crude 20 could be used without further purification. Cyclohexanecarboxylic chloride (28 mg, 0.19 mmol) was added to a solution of 20 (50 mg, 0.16 mmol), TEA (19 mg, 0.19 mmol), and DMAP (4 mg, 0.03 mmol) in anhydrous CH₂Cl₂ (20 mL) slowly in an ice bath. After stirring for 3 h at room temperature, the reaction mixture was poured into ice water and then extracted with CH₂Cl₂ (3 × 20 mL). The combined organic phases were washed with 1 N HCl, water, and brine, dried, and filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (hexane/ethyl acetate = 20/1, Rf = 0.23) to afford 21a as a pale yellow solid (64 mg, 94 %). ¹H NMR (400 MHz, CDCl₃): δ 1.25–1.39 (m, 4H), 1.57–1.65 (m, 2H), 1.81–1.84 (m, 2H), 2.06–2.09 (m, 2H), 2.58 (t, J = 10.1 Hz, 1H), 6.92 (s, 1H), 7.24 (m, 1H), 7.52–7.59 (m, 3H), 7.78 (s, 1H), 8.52(s, 1H);¹³C NMR (100 MHz, CDCl₃): δ 25.3, 25.6, 28.8, 43.1, 113.9, 117.7, 119.6, 121.4; EI-HRMS calculated for (C₂₁H₁₈ClF₃N₂O₂ + Na)⁺ 445.0907, found 445.0907.

1-(3-(8-Chloro-6-(trifluoromethyl)imidazo[1,5-a]pyridin-3-yl)phenoxy)propan-2-one (21b)

Using the same method as for the preparation of 8a, starting with 20 (73 mg, 0.23 mmol), 1-chloroacetone (0.5 mL, 17.41 mmol) and K₂CO₃ (161 mg, 1.17 mmol), 21b was generated as a pale yellow solid (30 mg, 35 %). ¹H NMR (400 MHz, CDCl₃): δ 2.30 (s, 3H), 4.66 (s, 2H), 6.91 (s, 1H), 7.06 (d, J = 7.9 Hz, 1H), 7.28 (s, 1H), 7.35 (d, J = 7.5 Hz, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.76 (s, 1H), 8.48 (s, 1H);¹³C NMR (100 MHz, CDCl₃): δ 26.6, 73.0, 113.9, 114.6, 116.5, 117.5, 117.9, 119.6, 120.9, 121.4, 122.1, 124.1, 126.5, 129.6, 130.2, 130.6, 141.3, 158.5, 204.6; EI-HRMS calculated for (C₁₇H₁₂ClF₃N₃O₂+H)⁺ 369.0618, found 369.0670.

General synthetic procedure for (27a–d)

POCl₃ (0.3 mL, 3.40 mmol) was added to a mixture of 24 or 26a–c (0.17 mmol) and pyridine (0.93 mL, 11.60 mmol) in anhydrous dichloroethane (14 mL) at room temperature. The reaction mixture was heated to reflux for 7 h. After cooling to room temperature, the reaction mixture was concentrated, filtered, and extracted with ethyl acetate. The combined organic phases were washed with 1 N HCl, water, and brine, dried, and filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (hexane/ethyl acetate = 2/1, Rf = 0.24).

N-(3-(8-Chloro-6-(trifluoromethyl)imidazo[1,5-a]pyridin-3-yl)phenyl)acetamide (27b)

Pale yellow solid; Yield: 96 %;¹H NMR (400 MHz, CDCl₃): δ 2.17 (s, 3H), 6.90 (s, 1H), 7.44–7.48 (m, 2H), 7.63 (d, J = 6.7 Hz, 1H), 7.74 (s, 1H), 7.96 (s, 1H), 8.16 (s, 1H), 8.53(s, 1H);¹³C NMR (100 MHz, CDCl₃): δ 24.5, 113.9, 117.5, 117.9, 119.7, 119.8, 121.1, 121.5, 122.1, 123.6, 124.2, 126.4, 129.2, 129.6, 130.0, 139.1, 141.5, 168.9; EI-HRMS calculated for (C₁₆H₁₁ClF₃N₃O + Na)⁺ 376.0440, found 376.0447.

N-(3-(8-Chloro-6-(trifluoromethyl)imidazo[1,5-a]pyridin-3-yl)phenyl)cyclohexane carboxamide (27c)

Pale yellow solid; Yield: 32 %;¹H NMR (400 MHz, CDCl₃): δ 1.28–1.35 (m, 2H), 1.50–1.59 (m, 2H), 1.71 (m, 2H), 1.83–1.85 (m, 2H), 1.94–1.97 (m, 2H), 2.26 (t, J = 11.6 Hz, 1H), 6.90 (s, 1H), 7.44–7.52 (m, 2H), 7.55 (s, 1H), 7.71 (d, J = 7.6 Hz, 1H), 7.75 (s, 1H), 7.95 (s, 1H), 8.54 (s, 1H);¹³C NMR (100 MHz, CDCl₃): δ 25.6, 29.6, 46.5, 113.8, 117.4, 117.8, 119.7, 119.8, 121.0, 122.2, 123.3, 126.3, 129.4, 129.6, 130.0, 139.1, 141.5, 174.6; EI-HRMS calculated for (C₂₁H₁₉ClF₃N₃O + Na)⁺ 444.1066, found 444.1075.

1-(3-(8-Chloro-6-(trifluoromethyl)imidazo[1,5-a]pyridin-3-yl)phenyl)pyrrolidine-2,5-dione (27d)

Pale yellow solid; Yield: 28 %;¹H NMR (400 MHz, CDCl₃): δ 2.95 (s, 4H), 6.93 (s, 1H), 7.52 (d, J = 7.9 Hz, 1H), 7.69 (t, J = 7.8 Hz, 1H), 7.76–7.82 (m, 3H), 8.63 (s, 1H);¹³C NMR (100 MHz, CDCl₃): δ 28.4, 114.0, 117.7, 118.0, 119.7, 121.5, 122.6, 124.2, 125.7, 126.4, 127.1, 128.2, 129.8, 130.3, 132.7, 140.7, 175.8; EI-HRMS calculated for (C₁₈H₁₁ClF₃N₃O₂+H)⁺ 394.0570, found 394.0608.

tert-Butyl (3-(8-chloro-6-(trifluoromethyl)imidazo[1,5-a]pyridin-3-yl)phenyl) carbamate (27a)

Pale yellow solid;Yield: 13 %;¹H NMR (400 MHz, CDCl₃): δ 1.53 (s, 9H), 6.71 (s, 1H), 6.90 (s, 1H), 7.41–7.48 (m, 3H), 7.76 (s, 1H), 7.88 (s, 1H), 8.58 (s, 1H);¹³C NMR (100 MHz, CDCl₃): δ 28.2, 113.7, 117.3, 117.6, 118.3, 119.7, 119.9, 122.3, 122.5, 124.2, 126.3, 129.5, 130.0, 139.4, 141.6, 152.6; EI-HRMS calculated for (C₁₉H₁₇ClF₃N₃O₂ + Na)⁺ 434.0859, found 434.0865.

N-(3-(8-Chloro-6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)phenyl)acetamide (33a)

Acetic anhydride (20 mg, 0.20 mmol) was added to a mixture of 32 (50 mg, 0.16 mmol) and DMAP (3 mg, 0.02 mmol) in anhydrous CH₂Cl₂ (10 mL) with stirring for 1 h at room temperature. After removing the solvent, the residue was extracted with ethyl acetate and water. The combined organic phases were washed with 1 N HCl, saturated Na₂CO₃, water, and brine, dried, and filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (ethyl acetate/hexane = 33–50 %, Rf = 0.23). Pale yellow solid; Yield: 50 %;¹H NMR (400 MHz, CDCl₃): δ 2.19 (s, 3H), 7.35–7.39 (m, 2H), 7.62–7.66 (m, 2H), 7.72 (s, 1H), 7.94 (s, 1H), 8.06 (s, 1H), 8.39 (s, 1H);¹³C NMR (100 MHz, CDCl₃): δ 24.6, 111.3, 116.6, 117.6, 119.6, 120.3, 121.5, 122.2, 123.4, 124.2, 129.5, 133.1, 138.5, 142.6, 147.5, 168.8; EI-HRMS calculated for (C₁₆H₁₁ClF₃N₃O + Na)⁺ 376.0440, found 376.0453.

General synthetic procedure for (33b–c)

Cyclohexanecarboxylic chloride (28 mg, 0.19 mmol) or toluenesulfonyl chloride (39 mg, 0.21 mmol) was added to a solution of 32 (50 mg, 0.16 mmol), TEA (19 mg, 0.19 mmol), and DMAP (4 mg, 0.03 mmol) in anhydrous CH₂Cl₂ (10 mL) slowly in an ice bath. After stirring for 3 h at room temperature, the reaction mixture was poured into ice water and then extracted with CH₂Cl₂ (3 × 20 mL). The combined organic phases were washed with 1 N HCl, water, and brine, dried, and filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (hexane/ethyl acetate = 9/1, Rf = 0.22).

N-(3-(8-Chloro-6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)phenyl)cyclohexane carboxamide (33b)

Pale yellow solid; Yield: 54 %; ¹H NMR (400 MHz, CDCl₃): δ 1.28–1.33 (m, 2H), 1.51–1.60 (m, 2H), 1.72 (m, 2H), 1.84–1.86 (m, 2H), 1.96–1.99 (m, 2H), 2.25 (m, 1H), 7.35–7.39 (m, 2H), 7.48 (s, 1H), 7.66 (d, J = 7.0 Hz, 2H),7.98 (s, 1H), 8.13 (s, 1H), 8.40 (s, 1H);¹³C NMR (100 MHz, CDCl₃): δ 25.6, 25.7, 29.7, 46.6, 111.3, 116.6, 116.9, 117.5, 119.6, 120.2, 122.0, 123.3, 124.2, 129.5, 133.0, 138.7, 142.7, 147.6, 174.7; EI-HRMS calculated for (C₂₁H₁₉ClF₃N₃O+H)⁺ 422.12470, found 422.12446.

N-(3-(8-Chloro-6-(trifluoromethyl)imidazo[1,2-a]pyridin-2-y l)phenyl)-4-toluenesulfonamide (33c)

Pale yellow solid;Yield: 68 %;¹H NMR (400 MHz, CDCl₃): δ 2.46 (s, 3H), 7.00 (d, J = 7.5 Hz, 1H), 7.33–7.35 (m, 3H), 7.40 (s, 1H), 7.58 (s, 1H), 7.83–7.87 (m, 4H), 8.10 (d, J = 7.4 Hz, 1H), 8.40 (s, 1H);¹³C NMR (100 MHz, CDCl₃): δ 21.7, 111.3, 116.6, 117.0, 119.7, 123.3, 124.4, 128.2, 128.5, 128.6, 129.4, 129.6, 129.7, 129.8, 131.5, 133.9, 134.9, 136.5, 142.7, 145.1, 146.6; EI-HRMS calculated for (C₂₁H₁₅ClF₃N₃O₂S+H)⁺ 466.06038, found 466.07034.

General synthetic procedure for (37a–c)

A mixture of 5-bromopyrimidin-2-amine 35–36 (222 mg, 1.27 mmol) and bromoacetone 3 (257 mg, 1.0 mmol) in dioxane (10 mL) with or without NaHCO₃ (84 mg, 1.00 mmol) was stirred until reflux for 7 h. After cooling to room temperature, ethyl acetate was added, washed with water and brine, dried over calcium oxide, and filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (gradient eluent: ethyl acetate/hexane = 20–50 %, Rf = 0.21).

3-(6-Bromoimidazo[1,2-a]pyrimidin-2-yl)phenyl acetate (37a)

White solid;Yield: 11 %; ¹H NMR (400 MHz, CDCl₃): δ 2.33 (s, 3H), 7.10 (d, J = 7.9 Hz, 1H), 7.45 (t, J = 7.9 Hz, 1H), 7.75 (s, 1H), 7.77 (s, 1H), 7.84 (d, J = 7.6 Hz, 1H), 8.51 (s, 1H), 8.55 (s, 1H);¹³C NMR (100 MHz, CDCl₃): δ 21.2, 104.8, 106.6, 119.6, 122.1, 123.7, 129.8, 132.5, 134.2, 150.7, 151.2, 158.6, 169.5; EI-HRMS calculated for (C₁₄H₁₀BrN₃O₂+H)⁺ 332.00346, found 332.00372.

3-(Imidazo[1,2-a]pyrimidin-2-yl)phenyl acetate (37b)

Pale yellow solid; Yield: 21 %;¹H NMR (400 MHz, CDCl₃): δ 2.32 (s, 3H), 6.82 (t, J = 5.6 Hz, 1H), 7.07 (d, J = 7.7 Hz, 1H), 7.42 (t, J = 7.8 Hz, 1H), 7.76 (m, 2H), 7.83 (d, J = 7.7 Hz, 1H), 8.39 (d, J = 6.3 Hz, 1H), 8.49 (s, 1H);¹³C NMR (100 MHz, CDCl₃): δ 21.2, 106.5, 108.9, 119.5, 121.7, 123.6, 129.7, 133.1, 134.7, 146.2, 148.6, 150.1, 151.2, 169.5; EI-HRMS calculated for (C₁₄H_{11 N3}O₂+H)⁺ 254.09295, found 254.09322.

3-(Imidazo[1,2-a]pyrazin-2-yl)phenyl acetate (37c)

Pale yellow solid;Yield: 11 %;¹H NMR (400 MHz, CDCl₃): δ 2.34 (s, 3H), 7.12 (d, J = 7.6 Hz, 1H), 7.47 (t, J = 7.5 Hz, 1H), 7.74 (s, 1H), 7.83 (d, J = 7.3 Hz, 1H), 7.89 (s, 1H), 7.95 (s, 1H), 8.07 (s, 1H), 9.10 (s, 1H);¹³C NMR (100 MHz, CDCl₃): δ 21.1, 109.4, 118.6, 119.6, 122.0, 123.7, 129.8, 129.9, 134.4, 140.9, 143.8, 146.8, 151.2, 169.4; EI-HRMS calculated for (C₁₄H_{11 N3}O₂+H)⁺ 254.09295, found 254.09288.

2-(3-Nitrophenyl)imidazo[1,2-a]pyrimidine (37d)

A mixture of 2-aminopyrazine 34 (195 mg, 2.05 mmol) and bromoacetone 29 (660 mg, 2.70 mmol) in ethanol (20 mL) was stirred until reflux for 3 h, After cooling to room temperature, the mixture was concentrated. The residue was dissolved in ethyl acetate, washed with 1 N HCl, water, and brine, dried over calcium oxide, and filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (methanol/methylene chloride = 0–1 %, Rf = 0.21) to afford 37d as a yellow solid (24 mg, 5 %). ¹H NMR (400 MHz, CDCl₃): δ 6.95 (t, J = 1.7 Hz, 1H), 7.65 (t, J = 8.0 Hz, 1H), 7.97 (s, 1H), 8.21 (d, J = 7.9 Hz, 1H), 8.45 (d, J = 7.6 Hz, 1H), 8.50 (d, J = 5.9 Hz, 1H), 8.61 (d, J = 1.2 Hz, 1H), 8.80 (s, 1H);¹³C NMR (100 MHz, CDCl₃): δ 107.1, 108.8, 109.4, 113.6, 113.9, 120.9, 123.1, 129.9, 132.2, 133.3, 134.9, 150.8; EI-HRMS calculated for (C₁₂H₈N₄O₂+H)⁺ 241.07255, found 241.07288.

2-(3-Nitrophenyl)imidazo[1,2-a]pyrazine (37e)

A mixture of 2-aminopyrazine 36 (95 mg, 1.0 mmol) and bromoacetone 29 (488 mg, 2.0 mmol) in ethanol (10 mL) was stirred until reflux for 3 h. After cooling to room temperature, the mixture was concentrated. Then the residue was dissolved in ethyl acetate and washed with water. The combined aqueous phase was extracted with ethyl acetate. The combined organic phase was washed with brine, dried over calcium oxide, and filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (methylene chloride/methanol = 100/1, Rf = 0.21) to afford 37e as a yellow solid (40 mg, 17 %). ¹H NMR (400 MHz, CDCl₃): δ 7.68 (t, J = 7.6 Hz, 1H), 7.97 (s, 1H), 8.11 (s, 1H), 8.13 (s, 1H), 8.24 (d, J = 7.3 Hz, 1H), 8.37 (d, J = 6.7 Hz, 1H), 8.80 (s, 1H), 9.18 (s, 1H);¹³C NMR (100 MHz,CDCl₃): δ 102.6, 110.0, 114.3, 117.1, 118.9, 121.2, 123.4, 129.7, 130.1, 132.2, 134.6, 144.1; EI-HRMS calculated for (C₁₂H₈N₄O₂+H)⁺ 241.07255, found 241.07260.

Biology

In vitro GLP-1R activation assay (Chen et al. 2007)

CHO-K1 cells (4 × 10⁶/100 mm dish) were transiently transfected with the pCMV6-GLP-1R (Origene #SC124060) and pCRE-Luc (Promega #631911) plasmids using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). After 24 h incubation at 37 °C, cells were seeded into 96-well culture plates (2 × 10⁴/well), and further incubated at 37 °C overnight. At the time of assay, GLP-1 (7–37) (Sigma, St. Louis, MO) or test compounds in DMSO were added to the plate. After 8 h incubation, cells were lysed and luciferase activity quantified using the Steady-Glo luciferase assay system (Promega #E2550).

Data were analyzed in Excel and EC₅₀ values were determined graphically from dose–response curves in OriginPro.

Results and discussion

Chemistry

The general synthetic pathway yielding the novel derivatives 6a, 6b, and 8a–8f is outlined in Scheme 1. 3-Hydroxy acetophenone 1 was converted into 3-acetoxy acetophenone 2 by acetylation. Treatment of substituted acetophenone 2 with bromine in the presence of AlCl₃ in Et₂O (Bunders et al. 2010) afforded á-bromomethylketone 3. Subsequently, 3 and the substituted 2-aminopyridines 4, 5 were allowed to react in the presence of sodium bicarbonate in refluxing ethanol (Fookes et al. 2008), resulting in the generation of compounds 6a and 6b. The deacetylation of compounds 6a and 6b with sodium hydroxide afforded compounds 7a and 7b, respectively. The akylation or acylation of 7a and 7b furnished the target compounds 8a–8f.

Scheme 1

Reagents and conditions: (a) acetic anhydride, DMAP, anhydrous CH₂Cl₂, RT; (b) Br₂, AlCl₃, Et₂O, ice bath; (c) NaHCO₃, EtOH, reflux; (d) NaOH, THF/H₂O, RT; (e) R¹Cl, K₂CO₃, acetone, reflux or R²Cl, pyridine, 0 °C or RT

The derivatives 12a–12c were readily prepared in three steps, as illustrated in Scheme 2. 3-Aminoacetophenone 9 was first acylated with acyl chloride or sulfonyl chloride, as appropriate, to produce compounds 10a–10c. Subsequent bromination of 10a–10c with PBB (pyridinium bromide perbromide) in acetic acid (Yu et al. 2008) afforded compounds 11a–11c, which were then cyclized with 5-trifluoro-2-aminopyridine in refluxing ethanol to give the desired derivatives 12a–12c.

Scheme 2

Reagents and conditions: (a) acetic anhydride, DMAP, anhydrous CH₂Cl₂, RT or R³Cl, pyridine, 0 °C or RT; (b) pyridium bromide perbromide, AcOH, RT; (c) NaHCO₃, EtOH, reflux

Next we synthesized the imidazo[1,5-α]pyridine derivatives 19, 21a–21b, and 27a–27d, as detailed in Scheme 3. The intermediate 2-aminomethylpyridine 15, prepared from 2,3-dichloro-5-triflouropyridine 14 in two steps using a method reported elsewhere (Stolting 2004), was treated with 3-acetoxybenzoinc acid 17, followed by cyclization in the presence of POCl₃ in refluxing benzene (Bower and Ramage 1955) to give intermediate 19. Then, deacylation of 19 and subsequent acylation or alkylation of the resulting compound 20 with appropriate acyl chloride or 1-chloroacetone resulted in the generation of the desired compounds 21a–21b. For the synthesis of derivatives 27a–27d, the first step is the amidation of intermediate 15 with benzoic acid 23 in the presence of DCC and DMAP in dichloromethane, resulting in the generation of compound 24. Then, deprotection of 24 with trifluoroacetic acid (Mu 2001) followed by acylation of the resulting compound 25 afforded the compounds 26a–26c. Finally, the amides 24 and 26a–26c were cyclized in the presence of POCl₃ and pyridine in refluxing dichloroethane (Cookson et al. 1986), resulting in the generation of the target derivatives 27a–27d, respectively.

Scheme 3

Reagents and conditions: (a) CH₃NO₂, KOH, DMSO, RT; (b) SnCl₂, conc. HCl, EtOH, reflux; (c) acetic anhydride, reflux; (d) DCC, DMAP, anhydrous CH₂Cl₂, RT; (e) POCl₃, benzene, reflux; (f) NaOH, THF/H₂O, RT; (g) R⁴Cl, TEA, DMAP, anhydrous CH₂Cl₂, RT, or R⁵Cl, K₂CO₃, acetone, reflux. (h) di-tert-butyl dicarbonate, TEA, dioxane/H₂O; (i) DCC, DMAP, anhydrous CH₂Cl₂, RT; (j) CF₃COOH, anhydrous CH₂Cl₂, RT; (k) acetic anhydride, DMAP, anhydrous CH₂Cl₂, RT or R⁶Cl, TEA, DMAP, anhydrous CH₂Cl₂, RT or succimyl chloride, K₂CO₃, CH₃CN, reflux; (l) POCl₃, pyridine, dichloroethane, reflux

Scheme 4 describes the synthesis of derivatives 33a–33c. The formation of á-diazoketone intermediate 30 was achieved from á-bromomethylketone 28 by treatment with N,N′-ditosylhydrazine and DBU (Toma et al. 2007). Subsequent coupling of á-diazoketone with 3-chloro-5-trifluoro-2-aminopyridine in the presence of 10 mol % Cu(OTf)₂ in dichloroethane (DCE) (Yadav et al. 2007) afforded substituted 2-arylimidazo[1,2-α]pyridine 31. Reduction of 31 with stannous chloride in a refluxing mixture of ethanol and concentrated hydrochloride (Denora et al. 2008) resulted in the generation of compound 32. Finally, the acylation of 32 with the corresponding acyl chloride afforded derivatives 33a–33c.

Scheme 4

Reagents and conditions: (a) pyridium bromide perbromide, AcOH, reflux; (b) TsNHNHTs, DBU, THF, RT; (c) 10 mol %Cu(OTf)₂, dichloroethane, 80 °C; (d) SnCl₂, conc. HCl, EtOH, reflux; (e) acetic anhydride, DMAP, anhydrous CH₂Cl₂, RT or R⁸Cl, TEA, DMAP, anhydrous CH₂Cl₂, RT

The syntheses of compounds 37a–37e are detailed in Scheme 5. The preparation of intermediate 35 was achieved by bromination of 2-aminopyrimidine 34 with NBS in refluxing acetonitrile. The subsequent cyclization of 35, 2-anmonoprimidine 34, and 2-aminopyrazine 36 with intermediate 3 yielded the target derivatives 37a–37c, respectively. The cyclization of 2-anmonoprimidine 34 and 2-aminopyrazine 36 with intermediate 29 yielded the target derivatives 37d–37e, respectively.

Scheme 5

Reagents and conditions: (a) NBS, CH₃CN, reflux; (b) dioxane, reflux or NaHCO₃, dioxane, reflux; (c) EtOH, reflux

Biology

The compounds prepared in this study were evaluated in terms of GLP-1R agonist activity using an in vitro activation efficacy assay in CHO-K1 cells (Chen et al. 2007), and the magnitude of the responses have been compared at two concentrations of compounds used. GLP-1 (7–37) was used as the positive control and DMSO (0.1 %) was used as the negative control. Induction values represent luciferase activities driven by CRE (cAMP response element). Compounds were grouped into three series according to fused-heterocyclic ring type.

In general, the first series of compounds, 6a–6b, 8a–8f, 12a–12c, and 33a–33c (Fig. 3), based on the imidazo[1,2-α]pyridine structure and containing various substituted groups, generated higher responses than those of the second series. Compound 6b is the model compound, in which replacement of the acetyl group with propanyl-2-one 8d, mesyl 8e, or tosyl 8f resulted in a significant increase in magnitude of the response, suggesting that the hydrogen-bond donor is preferred to be this region and that the length of linker affects binding to the ago-allosteric binding site of GLP-1R. To determine whether the chlorine in imidazo[1,2-α]pyridine is essential for its activity, compounds 6a and 8a–8c were synthesized. Compounds 8a and 8b showed good responses similar to that of compounds 8d and 8e at 10 μM. However, a loss of response was observed for compounds 6a and 8c. The bioisosteric replacement of the ester group (6a, 8b–8c) with an amide group (12a–12c) resulted in comparable activities; in particular, compound 12a exhibited a two fold enhanced response. On the contrary, compounds 33a and 33c exhibited decreased responses. However, compound 33b, which contains acyclohexanecarboxamide group, exhibited a moderate response. Further increases in concentration resulted in a significant drop in the responses of compounds 8a, 6a–6b, 8f, and 12a–12b to an about <1-fold increase, likely due to cytotoxicity at a high concentration (100 μM). Surprisingly, compound 8e, which generated the highest response at 10 μM, also exhibited the greatest response at a high concentration (100 μM).

Fig. 3

In vitro responses of compounds 6a–6b, 8a–8f, 12a–12c and 33a–33c on CHO-K1 cells at 10 and 100 μM. The cells were transfected with the pCMV6-GLP-1R and pCRE-Luc plasmids. The transfected and cultured cells were incubated with different compounds for 8 h, and luciferase activity quantified using the Steady-Glo luciferase assay system. Vertical and horizontal axes show the fold increases compared to the control and the synthesized compounds, respectively. Values shown are mean ± SD of three independent experiments. Significant difference from 0.1 % DMSO treated group: *p ≤ 0.05, **p ≤ 0.01 and ***p < 0.001

In the second series of compounds, 19, 21a–21b, and 27a–27d (Fig. 4), the imidazo[1,2-α]pyridine structure was changed to an imidazo[1,5-α]pyridine structure. Unfortunately, all derivatives exhibited low responses at a low concentration (10 μM). Compounds 21a–21b and 27b–27d showed moderate responses at 100 μM. Generally, both first- and second-series compounds with substituted ester groups showed higher responses than those with a substituted amide group.

Fig. 4

In vitro responses of compounds 19, 21a–21b, 27a–27d, and 37a–37e on CHO-K1 cells at 10 and 100 μM. Experimental details are described in Fig. 3. Vertical and horizontal axes show the fold increases compared to the control and the synthesized compounds, respectively. Values shown are mean ± SD of three independent experiments. Significant difference from 0.1 % DMSO treated group: *p ≤ 0.05, **p ≤ 0.01, and ***p < 0.001

Finally, a nitrogen atom was introduced into the six-membered fused-heterocyclic ring to evaluate the effect of electron density on activity (Fig. 4). The majority of the compounds 37a–37e thus generated showed loss of responses compared to the first series. We speculated that the loss of responses might be attributable to a decreased interaction between the π-electron and the receptor.

Over the half of the synthesized compounds, the effects did not appear concentration-dependent. However the effects of the compounds on coupling the GLP-1R to the signaling way may well be concentration-dependent, but the responses measured did not appear concentration-dependent due to cytotoxicity.

In addition, selected compounds 8a, 8b and 8e which showed >2.5-fold increases at 10 μM were assayed further to determine concentration–response curves (Fig. 5) and calculate EC₅₀ values (Table 1). Compound 8e, bearing chlorine substitution imidazo[1,2-α]pyridine ring and mesyl group of benzene ring, was found to be a potent GLP-1R agonist exhibiting an EC₅₀ of 7.89 μM. Compounds 8a and 8b, without chlorine substitution of imidazo[1,2-α]pyridine ring, were about threefold less potent, with EC₅₀ values of 20 μM and 17 μM, respectively (Table 1). Concentration–response curves of selected compounds are shown in Fig. 5. The concentration are in a range from 1 μM to 100 μM. Compounds 8b and 8e showed above 50 % response (8b, 52 %; 8e, 58 %) at their EC₅₀ values, while compound 8a showed lower response 43 % at EC₅₀ value (Fig. 5). Thus, compound 8e may serve as a GLP-1R agonist with potential for application.

Fig. 5

Concentration-response curves of agonists 8a, 8b and 8e of GLP-1R. Experimental procedures were performed as in Fig. 3. Vertical axes show the response percentage of GLP-1 response. Values shown are mean ± SD of three independent experiments. For determined EC₅₀ values see Table 1

Table 1 Potency of agonists 8a, 8b and 8e at GLP-1R

In conclusion, these new compounds, synthetic methodology developed and preliminary biological evaluation results could be helpful in further design and discovery of more potent GLP-1R agonists for the treatment of DM2.