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 AUC0–2h at the dose of 30 mg/kg, which is stronger than Vildagliptin (23.4% reduction vs. 17.9% reduction).


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][2][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][5][6] . Thus, there is still a critical need of novel therapeutic targets or approaches for treatment T2DM by good glycaemic control.
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 EC 50 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 [5,4b] [1,4]oxazine derivatives as GPR119 agonists, also including an in vivo efficacy study.
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).
The general synthetic procedures of the pyrimidooxazine derivatives 22-25 were synthesised as shown in Scheme 3, following similar methods with compounds 15-20.

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 EC 50 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 (EC 50 ¼ 13 and 200 nM respectively), which contained tropine amine and piperidine amine scaffolds with moderate lipophilicity (ClogP ¼ 4.0 and 3.5 respectively).
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 (EC 50 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 EC 50 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 (EC 50 ¼ 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 EC 50 values than compound 22. Furthermore, compound 15 revealed the strongest inherent activity (%max 146.5%) and best agonistic activity (EC 50 ¼ 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.

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 EC 50 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 EC 50 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.

Chemistry
All starting materials were obtained from commercial suppliers and used without further purification. 1 H-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  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.  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 K 2 CO 3 (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 MgSO 4 , filtered and evaporated. The residue was purified by column chromatography (petroleum ether: EtOAc ¼ 3: 1) to afford the desired product as a claybank solid (0.68 g, 55%). 1

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), Pd 2 (dba) 3 (0.05 mmol), X-Phos (0.05 mmol), and Cs 2 CO 3 (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 MgSO 4 , and evaporated. The residue was purified by column chromatography to give the product.    [16][17][18][19][20] To a solution of compound 14 (0.26 mmol) in DMF (3 ml), chlorofragments (0.34 mmol) and K 2 CO 3 (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 MgSO 4 , filtered and evaporated. The residue was purified by column chromatography to afford the desired product.