Optimisation of novel 4, 8-disubstituted dihydropyrimido[5,4-b][1,4]oxazine derivatives as potent GPR 119 agonists

Abstract

GPR119 is a promising target for discovery of anti-type 2 diabetes mellitus agents. We described the optimisation of a novel series of pyrimido[5,4-b][1,4]oxazine derivatives as GPR119 agonists. Most designed compounds exhibited good agonistic activities. Among them, compound 10 and 15 demonstrated the potent EC₅₀ values (13 and 12 nM, respectively) and strong inherent activities. Moreover, significant hypoglycaemic effect of compound 15 was observed by reducing the blood glucose AUC_0–2h at the dose of 30 mg/kg, which is stronger than Vildagliptin (23.4% reduction vs. 17.9% reduction).

Keywords:

• Optimisation
• pyrimidodihydrooxazine
• GPR 119 agonists
• type 2 diabetes mellitus

1. Introduction

Type 2 diabetes mellitus (T2DM) is a complex chronic disease characterised by metabolic disorder and hyperglycaemia due to insulin resistance, hepatic glucose overproduction and/or insufficient insulin secretion^1–3. Although there are a number of pharmacotherapy options for T2DM, most of current anti-diabetes drug have shown known adverse effects and loss of their overall efficacy in a long-term glycaemic control^4–6. Thus, there is still a critical need of novel therapeutic targets or approaches for treatment T2DM by good glycaemic control.

G protein-coupled receptor 119 (GPR119) is a member of class A (rhodopsin-type) GPCR family, with highly expressed in pancreatic β-cells and the K and L cells of the gastrointestinal tract^7–9. Activation of GPR119 increases the intracellular cyclic AMP (cAMP) level, which in turn directly stimulate the glucose-dependent insulin secretion and regulate glucagon-like peptide 1 (GLP-1), leading to improve the glucose tolerance in T2DM patients^10–14. In addition, GPR119 agonists showed β-cells function preservation, which is also an important role in current T2DM therapy^15–17. As a result, GPR119 agonists are used for discovery of anti-T2DM agents by lowering the blood glucose level and improving β-cells function. Indeed, numerous synthetic, small molecule GPR119 agonists were revealed by academia and industry to date, and some of which have advanced into clinical trials such as MBX-2982, BMS-903452, LEZ763, ZYG-19^18–30. Despite tremendous endeavours, none of GPR119 agonists were approved to market by FDA up to now.

In our efforts to discover GPR119 agonists, we previously have evaluated some series of pyrimidine derivatives, and some compounds displayed quite good agonistic potency^31,32. Among them, pyrimidopyrimidine compounds 1 and 2 exhibited single digit EC₅₀ values (2.2 nM and 8.1 nM respectively); however, these two agonists did not show the significant glucose-lowering effect in oral glucose tolerance test (oGTT) in mice compared with positive control. Therefore, we sequentially attempted to optimise the core fragment with the aim to enhance the biological activity both in vitro and in vivo. With this purpose, we introduced pyrimido[5,4-b][1,4]oxazine as the core using the strategy of scaffold hopping. We also identified whether introduction of several conformation restricted azabicyclic amines were beneficial to agonistic potency or not (Figure 1). In this paper, we described our optimisation to synthesise and evaluate a series of novel pyrimido[5,4-b][1,4]oxazine derivatives as GPR119 agonists, also including an in vivo efficacy study.

Figure 1. The design of target compounds.

2. Results and discussion

2.1. Chemistry

The intermediates amine or bicyclic amines 3–6 could be purchased or synthesised according to the reported procedures^33–36. Coupling reaction of 4,6-dichloro-5-methoxypyrimidine with 4-amino-3-fluorobenzonitrile in DMF under basic condition afforded compound 7, followed by demethylation using BBr₃ solution in dichloromethane under reflux condition gave hydroxyl compound 8. Cyclisation of 8 with 1-bromo-2-chloroethane and K₂CO₃ in DMF generated key intermediate 9. Buchwald–Hartwig reaction of 9 and amines 3–6 with Pd₂(dba)₃, X-Phos and Cs₂CO₃ under reflux conditions and N₂ atmosphere overnight resulted in target compounds 10–13 (Scheme 1).

Scheme 1. Synthesis of compounds 10–13. Reagents and conditions: (a) 4-amino-3-fluorobenzonitrile, K₂CO₃, DMF, 65 °C, overnight. (b) 1 M BBr₃ in DCM, anhydrous DCM, r. t. – reflux, 2 h. (c) 1-bromo-2-chloroethane, K₂CO₃, DMF, 40 °C, overnight. (d) amines 3–6, Pd₂(dba)₃, X-Phos, Cs₂CO₃, 1,4-Dioxane, reflux, under N₂ overnight.

Removing Boc group of derivative 10 in 3 M HCl ethanol solution obtained amine compound 14 in good yield, which was treated with various chloro-fragments in base conditions to receive desired final compounds 15–20 (Scheme 2).

Scheme 2. Synthesis of compounds 15–20. Reagents and conditions: (a) 3 M HCl in EtOH, r. t., overnight. (b) chloro-fragments, Et₃N, DCM, r. t., overnight or Cs₂CO₃, DMF, r. t., overnight.

The general synthetic procedures of the pyrimidooxazine derivatives 22–25 were synthesised as shown in Scheme 3, following similar methods with compounds 15–20.

Scheme 3. Synthesis of compounds 22–25. Reagents and conditions: (a) 3 M HCl in EtOH, r. t., overnight. (b) chloro-fragments, Et₃N, DCM, r. t., overnight or Cs₂CO₃, DMF, r. t., overnight.

2.2. Biological activity

2.2.1. GPR119 activation

GPR119 agonistic activity of target compounds 10–13, 15–20, and 22–25 were measured using a reporter assay with the human GPR119 receptor stably expressed in CHO K1 cells. A GPR119 agonist GSK-1292263 was chose for the reference. The results expressed the activity as EC₅₀ values and the inherent activity (IA) as percentages (%max) of response which were compared to the reference GSK-1292263 (defined the maximal effect activation).

At first, we evaluated pyrimidooxazine derivatives 10–13 for the GPR119 agonistic activity and intrinsic activity (Table 1). As a result, good activities were observed with compounds 10 and 12 (EC₅₀ = 13 and 200 nM respectively), which contained tropine amine and piperidine amine scaffolds with moderate lipophilicity (ClogP = 4.0 and 3.5 respectively).

Table 1. GPR119 agonistic activities of compounds 10–13.

Compound	Structure	GPR119 activation		ClogP^b
		EC50 (nM)	%max^a
10		13	83.9	4.0
11		1800	45.9	4.6
12		200	63.5	3.5
13		>10000	22.7	4.5
GSK-1292263		6.6	100

^a %max: cAMP stimulation % compared to maximal effect of GSK1292263.

^b ClogP was calculated using ACD software from Discovery Studio 4.5.

Next, we focussed on the modification of Boc group in head part with various moieties. The biological results were shown in Table 2, all compounds exhibited moderate to potent agonistic activities (EC₅₀ values range from 250 nM to 12 nM). The substitution of 2-pyrimidyl on the nitrogen of tropine ring yielded compounds 16 and 17, which showed the moderate EC₅₀ values (130 nM and 250 nM, respectively). However, the compounds 23 and 24, bearing same substituents on the nitrogen of piperidine ring, demonstrated significant agonistic activities (EC₅₀ = 44 nM and 40 nM) and lower lipophilicity. But the reverse results were observed for carbamate substituted derivatives (15 vs. 22), and compound 15 exhibited 10 times EC₅₀ values than compound 22. Furthermore, compound 15 revealed the strongest inherent activity (%max 146.5%) and best agonistic activity (EC₅₀ = 12 nM) with suitable ClogP value (3.8). Consequently, compounds 15 and 10 were selected and examined the oral glucose tolerance test (oGTT) in vivo as promising GPR119 agonists.

Table 2. GPR119 agonistic activities of compounds 15–20 and 22–25.

Compound	Structure	GPR119 activation		ClogP^b
		EC50 (nM)	%max^a
15		12	146.3	3.8
16		130	60.5	4.6
17		250	71.2	4.3
18		110	100	3.0
19		68	75.6	3.7
20		82	79.6	3.3
22		120	66.1	3.3
23		44	70.7	4.0
24		40	62.7	3.8
25		90	100	2.7
GSK-1292263		6.6	100

^a %max: cAMP stimulation % compared to maximal effect of GSK1292263.

^b ClogP was calculated using ACD software from Discovery Studio 4.5.

2.2.2. oGTT in mice

Based on the good agonistic activity, we conducted oGTT of compounds 10 and 15 with a single dose (15 and 30 mg/kg) in C57BL/6N mice with DPP-4 inhibitor vildagliptin as a positive control. The results were outlined in Figure 2, compounds 10 and 15 both showed blood glucose reduction effect in dose-dependent manner. Vildagliptin reduced the area under curve from 0 to 120 min (AUC_{0–120 min}) by 17.9% (Vehicle: 24.69 ± 3.08, Vildagliptin: 20.26 ± 2.14) at the dose of 30 mg/kg. Meanwhile compounds 10 and 15 reduced AUC_{0–120 min} by 10.7% (22.07 ± 4.28) and 14.5% (21.10 ± 3.92) at the dosage of 15 mg/kg, respectively. However, 10 and 15 showed significant improved efficacy by the reduction of value to 18.8% (20.05 ± 2.27) and 23.4% (18.91 ± 2.58) at the dosage of 30 mg/kg, respectively.

Figure 2. Single dose of compounds 10 and 15 on oGTT in C57BL/6N mice. The results are presented as the mean ± SE. *p < 0.05 compared to vehicle group (n = 8).

3. Conclusion

In summary, we have designed, synthesised and biologically evaluated a series of novel pyrimido[5,4-b][1,4]oxazine derivatives as potent GPR119 agonists. In vitro, half derivatives exhibited strong EC₅₀ values (<100 nM). Among the aliphatic amine moieties of this scaffold, the compound 10 with tropine amine ring displayed much more potent agonistic activity than piperidine amine and other rigid bicyclic amines. In the further optimisation of N-substitution, only isopropyl carbamate of tropine ring 15 improved the EC₅₀ values and showed the greatest inherent activity. Accordingly, compounds 10 and 15 were conducted the oGTT in C57BL/6N mice. Both two agonists demonstrated blood glucose reduction effect in a dose-dependent manner. Furthermore, the optimised compound 15 was exerted improved 23.4% reduction in blood glucose AUC_0–2h at the dose of 30 mg/kg comparing with Vildagliptin (17.9% reduction). Follow-up studies and their results will be reported in due course.

4. Experimental

4.1. Chemistry

All starting materials were obtained from commercial suppliers and used without further purification. ¹H-NMR and 13C-NMR spectra were recorded on a Bruker AVANCE III HD 600 (600 Hz) spectrometer. Chemical shifts are reported in parts per million (ppm) downfield relative to tetramethylsilane as an internal standard. Peak splitting patterns are abbreviated as s (singlet), br s (broad singlet), d (doublet), t (triplet), dd (doublet of doublet), and m (multiplet). MS spectra were recorded on a Thermo Fisher (LCQ Fleet). HR-MS spectra were recorded on an AB SCIEX (Triple TOF 5600+). TLC was performed on silica F254 purchased from Branch of Qingdao Haiyang Chemical Co. (Qingdao, China) and detected by UV light at 254, 365 nm or by charring with sulphuric acid. Column chromatography was performed on silica gel column (200–300 mesh, Branch of Qingdao Haiyang Chemical Co.). Analytical HPLC was performed on a Waters Acquity^® Arc™ with 2998 PDA detector and all final compounds possessed purities of >90% after purification.

4.1.1. 4-((6-Chloro-5-methoxypyrimidin-4-yl)amino)-3-fluorobenzonitrile (7)

To a solution of 4,6-dichloro-5-methoxypyrimidine (1 g, 5.7 mmol) in DMF (20 ml), 4-amino-3-fluorobenzonitrile (0.6 g, 4.4 mmol) and K₂CO₃ (2.4 g, 17 mmol) were added. The reaction was stirrd at 65 °C for overnight. Then the mixture was poured into ice water. The mix solution was extracted with ethyl acetate for two times, washed with brine for two times. The organic layer was dried over MgSO₄, filtered and evaporated. The residue was purified by column chromatography (petroleum ether: EtOAc = 3: 1) to afford the desired product as a claybank solid (0.68 g, 55%). ¹H-NMR (600 MHz, CDCl₃) δ (ppm): 8.94 (t, J = 8.8 Hz, 1H), 8.35 (s, 1H), 7.73 (s, 1H), 7.59 (d, J = 8.8 Hz, 1H), 7.52 (d, J = 10.2 Hz, 1H), 3.10 (s, 3H). MS-ESI: [M + H]⁺: 279.3.

4.1.2. 4-((6-Chloro-5-hydroxypyrimidin-4-yl)amino)-3-fluorobenzonitrile (8)

To a solution of compound 7 (0.5 g, 1.9 mmol) in anhydrous dichloromethane (15 ml), 1 M BBr₃ in dichloromethane solution (5.7 ml, 5.7 mmol) was added at room temperature (r. t.) The reaction was reflux for 2 h. Then the reaction was quenched by water. The mixture was extracted with dichloromethane for two times, washed by brine for two times. The organic layer was dried over MgSO₄, filtered and evaporated. The residue was purified by column chromatography (petroleum ether: EtOAc = 1: 1) to give the desired product as a yellow solid (0.3 g, 72%). ¹H-NMR (600 MHz, CDCl₃) δ (ppm): 8.92 (t, J = 8.4 Hz, 1H), 8.32 (s, 1H), 7.68 (s, 1H), 7.53 (d, J = 8.6 Hz, 1H), 7.46 (d, J = 10.8 Hz, 1H). MS-ESI: [M + H]⁺: 265.1.

4.1.3. 4-(4-Chloro-6,7-dihydro-8H-pyrimido[5,4-b][1,4]oxazin-8-yl)-3-fluorobenzonitrile (9)

To a solution of compound 8 (0.4 g, 1.4 mmol) in DMF (10 ml), 1-bromo-2-chloroethane (0.62 g, 4.3 mmol) and K₂CO₃ (0.6 g, 4.3 mmol) were added. The reaction was stirred at 40 °C for overnight. Then the mixture was poured into ice water. The mix solution was extracted with ethyl acetate for two times, washed with brine for two times. The organic layer was dried over MgSO₄, filtered and evaporated. The residue was purified by column chromatography (petroleum ether: EtOAc = 2: 1) to afford the desired product as a claybank solid (0.68 g, 55%). ¹H-NMR (600 MHz, CDCl₃) δ (ppm): 8.04 (s, 1H), 8.35 (s, 1H), 7.73–7.44 (m, 3H), 4.51 (t, J = 4.4 Hz, 2H), 3.95 (t, J = 4.3 Hz, 2H). MS-ESI: [M + H]⁺: 291.5.

4.1.4. General procedure of compounds 10–13

To the solution of compound 9 (0.22 mmol) and substituted amines (0.22 mmol) in 1,4-dioxane (2 ml), Pd₂(dba)₃ (0.05 mmol), X-Phos (0.05 mmol), and Cs₂CO₃ (0.55 mmol) were added. The reaction was heated to reflux under nitrogen gas for overnight. Then the mixture was diluted with ethyl acetate, washed with brine, dried over MgSO₄, and evaporated. The residue was purified by column chromatography to give the product.

4.1.5. tert-Butyl (endo)-3-((8-(4-cyano-2-fluorophenyl)-7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazin-4-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylate (10)

Yellowish solid, 52% yield. ¹H-NMR (600 MHz, CDCl₃) δ (ppm): 7.93 (s, 1H), 7.54–7.50 (m, 1H), 7.47 (m, 2H), 5.38 (d, J = 7.0 Hz, 1H), 4.46–4.37 (m, 2H), 4.33 (m, 2H), 4.22 (s, 1H), 3.89 (s, 2H), 2.39 (s, 1H), 2.30–2.20 (m, 1H), 2.12 (t, J = 7.4 Hz, 2H), 2.07–1.92 (m, 2H), 1.84 (d, J = 27.1 Hz, 2H), 1.49 (s, 9H). 13C-NMR (150 MHz, CDCl₃) δ (ppm): 156.4 (d, J = 250.5 Hz), 153.4, 151.3, 149.8, 143.9, 135.4 (d, J = 10.5 Hz), 128.5 (d, J = 3.5 Hz), 128.4 (d, J = 2.6 Hz), 121.8, 120.6 (d, J = 23.7 Hz), 117.6 (d, J = 2.6 Hz), 109.4 (d, J = 9.2 Hz), 79.5, 64.5, 53.1, 52.2, 48.2 (d, J = 3.9 Hz), 43.1, 35.8, 35.3, 28.5 (x3), 28.3, 27.9. HRMS-TOF (m/z) calcd for C₂₅H₂₉FN₆O₃ [M + H]⁺: 481.2358, found 481.2436.

4.1.6. tert-Butyl (exo)-3-(((8-(4-cyano-2-fluorophenyl)-7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazin-4-yl)amino)methyl)-8-azabicyclo[3.2.1]octane-8-carboxylate (11)

Yellowish solid, 45% yield. ¹H-NMR (600 MHz, CDCl₃) δ (ppm): 7.92 (s, 1H), 7.55–7.51 (m, 1H), 7.50–7.44 (m, 2H), 5.00 (t, J = 6.1 Hz, 1H), 4.39–4.35 (m, 2H), 4.30 (s, 1H), 4.20 (s, 1H), 3.95–3.81 (m, 2H), 3.37 (d, J = 23.7 Hz, 2H), 2.29–2.14 (m, 1H), 1.97 (s, 2H), 1.72–1.63 (m, 4H), 1.65 (s, 2H), 1.49 (s, 9H). HRMS-TOF (m/z) calcd for C₂₆H₃₁FN₆O₃ [M + H]⁺: 495.2514, found 495.2592.

4.1.7. tert-Butyl 4-((8-(4-cyano-2-fluorophenyl)-7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazin-4-yl)amino)piperidine-1-carboxylate (12)

Yellowish solid, 76% yield. ¹H-NMR (600 MHz, CDCl₃) δ (ppm): 7.93 (s, 1H), 7.56–7.51 (m, 1H), 7.48 (m, 2H), 4.83 (d, J = 8.1 Hz, 1H), 4.38 (t, J = 4.3 Hz, 2H), 4.17–4.11 (m, 3H), 3.89–3.87 (t, J = 3.7, 2H), 2.98 (m, 2H), 2.07–2.05 (m, 2H), 1.49 (s, 9H), 1.43–1.40 (m, 2H). 13C-NMR (150 MHz, CDCl₃) δ (ppm): 156.4 (d, J = 250.7 Hz), 154.8, 151.2, 149.7, 144.2, 135.4 (d, J = 10.5 Hz), 128.5 (d, J = 3.6 Hz), 128.4 (d, J = 2.6 Hz), 121.7, 120.6 (d, J = 23.7 Hz), 117.6 (d, J = 2.7 Hz), 109.5 (d, J = 9.0 Hz), 79.7, 64.3, 48.2 (d, J = 3.8 Hz), 47.6, 43.0, 32.5 (x2), 28.5 (x3). HRMS-TOF (m/z) calcd for C₂₃H₂₇FN₆O₃ [M + H]⁺: 455.2201, found 455.2275.

4.1.8. tert-Butyl 3-((8-(4-cyano-2-fluorophenyl)-7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazin-4-yl)amino)-9-azabicyclo[3.3.1]nonane-9-carboxylate (13)

Yellowish solid, 52% yield. ¹H-NMR (600 MHz, CDCl₃) δ (ppm): 7.91 (s, 1H), 7.52 (t, J = 7.8 Hz, 1H), 7.50–7.43 (m, 2H), 4.71 (d, J = 8.3 Hz, 1H), 4.60 (t, J = 9.0 Hz, 1H), 4.46 (d, J = 12.3 Hz, 1H), 4.36 (dt, J = 6.2, 4.8 Hz, 2H), 4.03 (m, 1H), 3.93–3.81 (m, 2H), 2.52 (m, 2H), 2.09–1.94 (m, 2H), 1.85 (m, 1H), 1.77–1.74 (m, 1H), 1.72–1.69 (m, 1H), 1.66-.1.61 (m, 1H), 1.58–1.57 (m, 1H), 1.50 (s, 9H), 1.43–1.41 (m, 2H). HRMS-TOF (m/z) calcd for C₂₆H₃₁FN₆O₃ [M + H]⁺: 495.2514, found 495.2600.

4.1.9. 4-(4-(((endo)-8-Azabicyclo[3.2.1]octan-3-yl)amino)-6,7-dihydro-8H-pyrimido[5,4-b][1,4]oxazin-8-yl)-3-fluorobenzonitrile (14)

To a solution of compound 10 (1 mmol) in 3 M HCl/EtOH (40 ml) was stirred at r. t. for overnight. Then the mixture was filtered to obtain the product 14, which was used for next step without purification. MS-ESI: [M + H]⁺: 381.3.

4.1.10. Isopropyl (endo)-3-((8-(4-cyano-2-fluorophenyl)-7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazin-4-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylate (15)

To a solution of compound 14 (0.1 g, 0.26 mmol) in dichloromethane (3 ml), isopropyl carbonochloridate (30 μL, 0.34 mmol) and Et₃N (73 μL, 1 mmol) were added. The reaction was stirred at r.t. for overnight. Then mixture was diluted with dichloromethane, washed with brine for two times. The organic layer was dried over MgSO₄, filtered and evaporated. The residue was purified by column chromatography (petroleum ether: EtOAc = 1: 1) to afford the desired product as a light yellow solid (70 mg, 58%). ¹H-NMR (600 MHz, CDCl₃) δ (ppm): 7.93 (s, 1H), 7.54–7.51 (m, 1H), 7.48 (m, 2H), 5.38 (d, J = 6.9 Hz, 1H), 4.98 (dt, J = 12.5, 6.2 Hz, 1H), 4.40–4.37 (m, 3H), 4.34 (m, 2H), 3.89 (m, 2H), 2.37 (m, 1H), 2.24 (m, 1H), 2.13 (m, 2H), 2.02 (m, 2H), 1.87 (m, 2H), 1.28 (s, 3H), 1.27 (s, 3H). 13C-NMR (150 MHz, CDCl₃) δ (ppm): 156.4 (d, J = 250.5 Hz), 153.6, 151.3, 149.8, 143.9, 135.4 (d, J = 10.5 Hz), 128.5 (d, J = 3.6 Hz), 128.4 (d, J = 2.5 Hz), 121.8, 120.6 (d, J = 23.7 Hz), 117.6 (d, J = 2.4 Hz), 109.4 (d, J = 9.2 Hz), 68.1, 64.5, 53.1, 52.6, 48.2 (d, J = 4.4 Hz), 43.1, 35.9, 35.4, 28.3, 27.9, 22.3 (x2). HRMS-TOF (m/z) calcd for C₂₄H₂₇FN₆O₃ [M + H]⁺: 467.2201, found 467.2282.

4.1.11. General procedure of compounds 16–20

To a solution of compound 14 (0.26 mmol) in DMF (3 ml), chloro-fragments (0.34 mmol) and K₂CO₃ (1 mmol) were added. The reaction was stirred at r. t. for overnight. Then the mixture was poured into ice water. The mix solution was extracted with ethyl acetate for two times, washed with brine for 2 times. The organic layer was dried over MgSO₄, filtered and evaporated. The residue was purified by column chromatography to afford the desired product.

4.1.12. 4-(4-(((endo)-8-(5-Ethylpyrimidin-2-yl)-8-azabicyclo[3.2.1]octan-3-yl)amino)-6,7-dihydro-8H-pyrimido[5,4-b][1,4]oxazin-8-yl)-3-fluorobenzonitrile (16)

Yellowish solid, 47% yield. ¹H-NMR (600 MHz, CDCl₃) δ (ppm): 8.22 (s, 2H), 7.92 (s, 1H), 7.55–7.51 (m, 1H), 7.47 (m, 2H), 5.52 (d, J = 7.2 Hz, 1H), 4.78 (s, 2H), 4.43–4.37 (m, 2H), 4.30 (q, J = 6.6 Hz, 1H), 3.92–3.85 (m, 2H), 2.50 (q, J = 7.6 Hz, 2H), 2.38 (m, 2H), 2.25–2.18 (m, 2H), 2.13 (m, 2H), 1.89 (d, J = 14.2 Hz, 2H), 1.23 (t, J = 7.6 Hz, 3H). 13C-NMR (150 MHz, CDCl₃) δ (ppm): 159.1, 157.5 (x2), 156.4 (d, J = 250.7 Hz), 151.4, 149.9, 143.8, 135.5 (d, J = 10.4 Hz), 128.5 (d, J = 3.5 Hz), 128.4 (d, J = 2.6 Hz), 124.6, 121.8, 120.6 (d, J = 23.7 Hz), 117.6 (d, J = 2.7 Hz), 109.4 (d, J = 9.2 Hz), 64.5, 52.6 (x2), 48.3 (d, J = 3.9 Hz), 43.5, 34.7 (x2), 28.1 (x2), 22.8, 15.6. HRMS-TOF (m/z) calcd for C₂₆H₂₇FN₈O [M + H]⁺: 487.2365, found 487.2462.

4.1.13. 4-(4-(((endo)-8-(5-Chloropyrimidin-2-yl)-8-azabicyclo[3.2.1]octan-3-yl)amino)-6,7-dihydro-8H-pyrimido[5,4-b][1,4]oxazin-8-yl)-3-fluorobenzonitrile (17)

Yellowish solid, 51% yield. ¹H-NMR (600 MHz, CDCl₃) δ (ppm): 8.27 (s, 2H), 7.92 (s, 1H), 7.53 (t, J = 7.8 Hz, 1H), 7.51–7.44 (m, 2H), 5.48 (d, J = 7.1 Hz, 1H), 4.87–4.67 (m, 2H), 4.49–4.38 (m, 2H), 4.28 (q, J = 6.6 Hz, 1H), 3.95–3.83 (m, 2H), 2.34 (m, 2H), 2.26–2.19 (m, 2H), 2.14 (m, 2H), 1.96–1.88 (m, 2H). 13C-NMR (150 MHz, CDCl₃) δ (ppm): 158.0, 156.4 (d, J = 250.6 Hz), 156.3 (x2), 151.3, 149.9, 143.9, 135.4 (d, J = 10.5 Hz), 128.5 (d, J = 3.6 Hz), 128.4 (d, J = 2.5 Hz), 121.8, 120.6 (d, J = 23.7 Hz), 118.1, 117.6 (d, J = 2.6 Hz), 109.4 (d, J = 9.2 Hz), 64.5, 52.8 (x2), 48.2 (d, J = 3.9 Hz), 43.4, 34.6 (x2), 28.1 (x2). HRMS-TOF (m/z) calcd for C₂₄H₂₂ClFN₈O [M + H]⁺: 493.1662, found 493.1751.

4.1.14. 3-Fluoro-4-(4-(((endo)-8-(2-oxo-2-(3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)ethyl)-8-azabicyclo[3.2.1]octan-3-yl)amino)-6,7-dihydro-8H-pyrimido[5,4-b][1,4]oxazin-8-yl)benzonitrile (18)

Yellowish solid, 44% yield. ¹H-NMR (600 MHz, CDCl₃) δ (ppm): 7.91 (s, 1H), 7.53–7.50 (m, 1H), 7.49–7.43 (m, 2H), 5.30 (m, 2H), 5.05 (s, 1H), 4.38 (m, 2H), 4.33–4.16 (m, 4H), 4.11 (t, J = 5.6 Hz, 1H), 3.87 (t, J = 4.3 Hz, 2H), 3.36 (d, J = 3.8 Hz, 2H), 3.26 (m, 2H), 2.29–2.08 (m, 4H), 1.97 (m, 2H), 1.82 (m, 2H). HRMS-TOF (m/z) calcd for C₂₈H₂₈FN₁₀O₂ [M + H]⁺: 613.2406, found 613.2508.

4.1.15. tert-Butyl ((R)-1-(2-((endo)-3-((8-(4-cyano-2-fluorophenyl)-7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazin-4-yl)amino)-8-azabicyclo[3.2.1]octan-8-yl)acetyl)piperidin-3-yl)carbamate (19)

Yellowish solid, 50% yield. ¹H-NMR (600 MHz, CDCl₃) δ (ppm): 7.92 (s, 1H), 7.52 (m, 1H), 7.47 (m, 2H), 5.47–5.32 (m, 2H), 4.44–4.35 (m, 2H), 4.30 (m, 1H), 3.88 (m, 2H), 3.74–3.67 (m, 3H), 3.61–3.15 (m, 6H), 2.49–2.30 (m, 2H), 2.23 (m, 2H), 1.97 (m, 2H), 1.93–1.74 (m, 6H), 1.48 (s, 9H). HRMS-TOF (m/z) calcd for C₃₂H₄₁FN₈O₄ [M + H]⁺: 621.3308, found 621.3405.

4.1.16. tert-Butyl 4-(2-((endo)-3-((8-(4-cyano-2-fluorophenyl)-7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazin-4-yl)amino)-8-azabicyclo[3.2.1]octan-8-yl)acetyl)piperazine-1-carboxylate (20)

Yellowish solid, 68% yield. ¹H-NMR (600 MHz, CDCl₃) δ (ppm): 7.92 (s, 1H), 7.55–7.50 (m, 1H), 7.47 (m, 2H), 5.34 (d, J = 7.1 Hz, 1H), 4.44–4.35 (m, 2H), 4.26 (q, J = 6.7 Hz, 1H), 3.91–3.84 (m, 2H), 3.72–3.67 (m, 2H), 3.60 (s, 2H), 3.53–3.47 (m, 2H), 3.43 (m, 2H), 3.32 (m, 2H), 3.28 (m, 2H), 2.29 (m, 2H), 2.21–2.13 (m, 2H), 1.96 (m, 2H), 1.83 (d, J = 14.3 Hz, 2H), 1.50 (s, 9H). 13C-NMR (150 MHz, CDCl₃) δ (ppm): 168.8, 156.4 (d, J = 250.6 Hz), 154.6, 151.3, 149.8, 143.8, 135.5 (d, J = 10.5 Hz), 128.5 (d, J = 3.5 Hz), 128.4 (d, J = 2.5 Hz), 121.8, 120.6 (d, J = 23.7 Hz), 117.6 (d, J = 2.5 Hz), 109.4 (d, J = 9.1 Hz), 80.3, 70.6, 64.4, 58.8, 56.0 (x2), 48.2 (d, J = 3.8 Hz), 45.8, 43.5 (x2), 42.4, 41.7, 36.9 (x2), 28.4 (x3), 26.2. HRMS-TOF (m/z) calcd for C₃₁H₃₉FN₈O₄ [M + H]⁺: 607.3151, found 607.3239.

4.1.17. 3-Fluoro-4-(4-(piperidin-4-ylamino)-6,7-dihydro-8H-pyrimido[5,4-b][1,4]oxazin-8-yl)benzonitrile (21)

Follow the similar procedure of 14. Yellow solid, 72% yield. MS-ESI: [M + H]⁺: 355.7.

4.1.18. Isopropyl 4-((8-(4-cyano-2-fluorophenyl)-7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazin-4-yl)amino)piperidine-1-carboxylate (22)

Follow the similar procedure of 15. Yellowish solid, 70% yield. ¹H-NMR (600 MHz, CDCl₃) δ (ppm): 7.93 (s, 1H), 7.56–7.51 (m, 1H), 7.48 (m, 2H), 4.95 (dt, J = 12.5, 6.2 Hz, 1H), 4.83 (d, J = 8.1 Hz, 1H), 4.43–4.33 (m, 2H), 4.27–4.08 (m, 3H), 3.93–3.85 (m, 2H), 3.00 (t, J = 12.0 Hz, 2H), 2.13–2.04 (m, 2H), 1.50–1.37 (m, 2H), 1.28 (s, 3H), 1.27 (s, 3H). 13C-NMR (150 MHz, CDCl₃) δ (ppm): 156.4 (d, J = 250.6 Hz), 155.2, 151.2, 149.8, 144.2, 135.4 (d, J = 10.4 Hz), 128.5 (d, J = 3.8 Hz), 128.4 (d, J = 2.6 Hz), 121.7, 120.6 (d, J = 23.7 Hz), 117.6 (d, J = 2.7 Hz), 109.5 (d, J = 9.2 Hz), 68.7, 64.3, 48.2 (d, J = 3.9 Hz), 47.6, 42.8 (x2), 32.5 (x2), 22.3 (x2). HRMS-TOF (m/z) calcd for C₂₂H₂₅FN₆O₃ [M + H]⁺: 441.2045, found 441.2144.

4.1.19. 4-(4-((1-(5-Ethylpyrimidin-2-yl)piperidin-4-yl)amino)-6,7-dihydro-8H-pyrimido[5,4-b][1,4]oxazin-8-yl)-3-fluorobenzonitrile (23)

Follow the similar procedure of 16–20. Yellowish solid, 54% yield. ¹H-NMR (600 MHz, CDCl₃) δ (ppm): 8.20 (s, 2H), 7.95 (s, 1H), 7.56–7.51 (m, 1H), 7.48 (m, 2H), 4.87 (d, J = 8.2 Hz, 1H), 4.68 (m, 2H), 4.42–4.32 (m, 2H), 4.32–4.17 (m, 1H), 3.92–3.84 (m, 2H), 3.24–3.10 (m, 2H), 2.49 (q, J = 7.6 Hz, 2H), 2.21–2.12 (m, 2H), 1.50 (m, 2H), 1.21 (t, J = 7.6 Hz, 3H). 13C-NMR (150 MHz, CDCl₃) δ (ppm): 160.8, 157.2 (x2), 156.4 (d, J = 250.9 Hz), 151.4, 149.8, 144.1, 135.4 (d, J = 10.5 Hz), 128.5 (d, J = 3.7 Hz), 128.4 (d, J = 2.6 Hz), 124.5, 121.7, 120.6 (d, J = 23.8 Hz), 117.7 (d, J = 2.6 Hz), 109.4 (d, J = 9.0 Hz), 64.3, 48.2 (d, J = 3.9 Hz), 48.0, 43.1 (x2), 32.5 (x2), 22.7, 15.7. HRMS-TOF (m/z) calcd for C₂₄H₂₅FN₈O [M + H]⁺: 461.2208, found 461.2318.

4.1.20. 4-(4-((1-(5-Chloropyrimidin-2-yl)piperidin-4-yl)amino)-6,7-dihydro-8H-pyrimido[5,4-b][1,4]oxazin-8-yl)-3-fluorobenzonitrile (24)

Follow the similar procedure of 16–20. Yellowish solid, 58% yield. ¹H-NMR (600 MHz, CDCl₃) δ (ppm): 8.25 (s, 2H), 7.95 (s, 1H), 7.58–7.51 (m, 1H), 7.48 (m, 2H), 4.86 (d, J = 8.1 Hz, 1H), 4.74–4.61 (m, 2H), 4.47–4.33 (m, 2H), 4.28 (m, 1H), 3.96–3.79 (m, 2H), 3.27–3.11 (m, 2H), 2.21–2.12 (m, 2H), 1.48 (m, 2H). 13C-NMR (150 MHz, CDCl₃) δ (ppm): 159.8, 156.4 (d, J = 250.9 Hz), 155.9 (x2), 151.3, 149.8, 144.2, 135.4 (d, J = 10.4 Hz), 128.5 (d, J = 3.6 Hz), 128.4 (d, J = 2.5 Hz), 121.7, 120.6 (d, J = 23.7 Hz), 118.0, 117.6, 109.4 (d, J = 9.2 Hz), 64.3, 48.2 (d, J = 3.8 Hz), 47.8, 43.2 (x2), 32.8 (x2). HRMS-TOF (m/z) calcd for C₂₂H₂₀ClFN₈O [M + H]⁺: 467.1505, found 467.1613.

4.1.21. tert-Butyl 4-(2-(4-((8-(4-cyano-2-fluorophenyl)-7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazin-4-yl)amino)piperidin-1-yl)acetyl)piperazine-1-carboxylate (25)

Follow the similar procedure of 16–20. Yellowish solid, 63% yield. ¹H-NMR (600 MHz, CDCl₃) δ (ppm): 7.91 (s, 1H), 7.54–7.49 (m, 1H), 7.46 (m, 2H), 4.84 (d, J = 8.1 Hz, 1H), 4.50–4.30 (m, 2H), 4.01 (m, 1H), 3.94–3.81 (m, 2H), 3.59 (s, 4H), 3.51–3.45 (m, 2H), 3.42 (s, 2H), 3.22 (s, 2H), 2.89 (m, 2H), 2.32 (m, 2H), 2.08 (m, 2H), 1.65–1.53 (m, 2H), 1.49 (s, 9H). 13C-NMR (150 MHz, CDCl₃) δ (ppm): 168.4, 156.4 (d, J = 250.6 Hz), 154.7, 151.4, 149.8, 144.0, 135.5 (d, J = 10.5 Hz), 128.5 (d, J = 3.6 Hz), 128.4 (d, J = 2.5 Hz), 121.7, 120.6 (d, J = 23.7 Hz), 117.6 (d, J = 2.6 Hz), 109.4 (d, J = 9.1 Hz), 80.3, 64.3, 61.4, 52.8, 50.8, 48.2 (d, J = 3.8 Hz), 47.0, 45.5, 43.6 (x2), 41.7, 32.6 (x2), 28.4 (x3). HRMS-TOF (m/z) calcd for C₂₉H₃₇FN₈O₄ [M + H]⁺: 581.2995, found 581.3136.

4.2. hGPR119 agonistic activity

CHO K1 cells stably transfected with human GPR119 were grown at 37 °C, 95%O₂ and 5% CO₂ in 75 cm flasks containing DMEM/F12 (1:1) media with added 10% FBS (Gibco^®), Geneticin (Gibco^®) and grown until 90% confluent. Cells were then washed (PBS), lifted with cell dissociation solution (Invtrogen^®), counted and used for cAMP accumulation assays and/or passaging (1:10). Following the manufacturer’s instructions for the LANCE^® Ultra cAMP assay (Perkin Elmer), cell transfected with hGPR119 were centrifuged (1000 rpm, 5 min), re-suspended in cAMP assay buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES) and seeded at 5000 cells/well in optiplate-384 (Perkin Elmer). Cells were treated with compounds or reference GSK1292263 over a range of concentrations (10 μM-0.6 μM) and incubated for 1 h. Cell lysis buffers (4X Eu-cAMP tracer solution and 4X ULight™-anti-cAMP solution) were added to each well, and the plates were incubated at r. t. for 1 h before being read on Envision (Perkin Elmer). The assay was performed for three replicates for each concentration.

4.3. oGTT in C57BL/6N mice

For the acute single dose study, vehicle (0.5% carboxymethylcellulose sodium, 10 ml/kg), compound 10, compound 15 (15 and 30 mg/kg) and vildagliptin (30 mg/kg) were administered to C57BL/6N mice after 16-h starvation, then the oral glucose tolerance test (3 g/kg) was conducted after 4 h of the single dose, the blood glucose level at 0, 15, 30, 60, 90, and 120 min were recorded for area under curve calculation (AUC_0–2h). AUC_0–2h(mmol/L) = (BG₀+BG₁₅)×0.25/2 + (BG₁₅+BG₃₀)×0.25/2 + (BG₃₀+BG₆₀)×0.5/2 + (BG₆₀+BG₉₀)×0.5/2 + (BG₉₀+BG₁₂₀)×0.5/2.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was financially supported by National Natural Science Foundation of China [81460526], Jiangxi Provincial Department of Science and Technology [20171BAB205103, 20181BAB215043, 20192ACB21012], Education Department of Jiangxi Province on Science and Technology Project Foundation [GJJ180667] and Traditional Chinese pharmacology discipline construction of Jiangxi University of Traditional Chinese Medicine [JXSYLXK-ZHYAO013].