Design and synthesis of tetrahydropyridopyrimidine derivatives as dual GPR119 and DPP-4 modulators

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Design and synthesis of tetrahydropyridopyrimidine derivatives as dual GPR119 and DPP-4 modulators Yuanying Fanga,b, Shaokun Zhangb, Wenting Wua, Yanhua Liua, Juan Yangb, Yuyuan Lia, Min Lia, ⁎ ⁎ Huanhuan Donga, Yi Jinb, , Ronghua Liua, Zunhua Yanga, a

College of Pharmacy, Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, China National Engineering Research Center for Manufacturing Technology of TCM Solid Preparation, Jiangxi University of Traditional Chinese Medicine, Nanchang 330006, China

b

ARTICLE INFO

ABSTRACT

Keywords: Tetrahydropyridopyrimidine GPR119 DPP-4 Design Hypoglycemic activity

Based on the approach of merged pharmacophores of GPR119 agonists and DPP-4 inhibitors, a series of tetrahydropyridopyrimidine compounds were designed as dual GPR119 and DPP-4 modulators with hypoglycemic activity. Seven fragments extracted from DPP-4 inhibitors were hybridized with the scaffold of tetrahydropyridopyrimidine. Among them, compound 51 displayed most potent GPR119 agonistic activity (EC50 = 8.7 nM) and good inhibition rate of 74.5% against DPP-4 at 10 μM. Furthermore, the blood glucose AUC0-2h of 51 was reduced to 19.5% in the oral glucose tolerance test (oGTT) at the dose of 30 mg/kg in C57BL/ 6N mice, which was more potent than that of vildagliptin (16.4%) at the same dose. The docking study of compound 51 with DPP-4 indicated GPR119 agonists could inhibit DPP-4 to serve as dual GPR119 and DPP-4 modulators.

1. Introduction Diabetes mellitus is a rapid-growing chronic diseases throughout the world, with a prevalence of approximately 425 million patients in 2017, of which more than 90% are classified as type 2 diabetes mellitus (T2DM) [1,2]. The expenditure on diabetes-related healthcare was up to 727 billion dollars during 2017 [3,4]. In clinical therapy of T2DM, there are a variety of hypoglycemic agents in market [5]. However, most of anti-diabetic drugs cause adverse effects, meanwhile a number of patients are unsuccessful to control the targeted plasma glucose levels [6,7]. Thus, there is a strong need of new orally available antidiabetic drugs with new mechanism of action, improved efficacy and safety [8]. G-protein coupled receptor 119 (GPR119) is expressed predominantly in enteroendocrine L and K cells in the small intestine, also in β-cells of pancreas [9]. Activation of GPR119 simulates intracellular cAMP level, leading to increase glucose-dependent insulin release and glucagon-like peptide-1 (GLP-1) secretion, which improve glucose homeostasis of T2DM patients [9,10]. Hence, GPR119 is an attractive drug target for development of anti-diabetic agents. DPP-4 is a multifunctional membrane-bound serine protease glycoprotein, which can give rise to the rapid inactivation of GLP-1

⁎

[11–13]. As a result, inhibition of DPP-4 has been approved for T2DM treatment because of prolonging the half-life of GLP-1. And DPP-4 inhibitors have been demonstrated to be sensitive to reduce HbA1c and glucose levels, leading to improved glycemic control [14–17]. Since the first DPP-4 inhibitor sitagliptin was launched in 2006, more than ten gliptins had been approved (Fig. 1). In the past decade, GPR119 agonist has received much interest by industrial and academic researchers for discovery of anti-diabetic agents. Unfortunately, none of clinical candidates were lunched successfully in market up to date [18]. But according to reported papers, combination of GPR119 agonist and DPP-4 inhibitor could significantly increase the level of plasma active GLP-1 and accelerate glucose clearance [19–23]. Therefore, based on the complementary and mutual mechanism of action of GPR119 agonist and DPP-4 inhibitor, we proposed a novel strategy of developing fused pyrimidines as dual modulators targeting GPR119 and DPP-4. Comparing with combinations of two or more agents, dual-targeted single molecule modulators showed multiple advantages such as lower toxicity, reduced development costs and avoiding problems with differing pharmacokinetics. We herein report the design and synthesis of dual GPR119 and DPP-4 modulator with tetrahydropyridopyrimidine scaffold.

Corresponding authors. E-mail addresses: [email protected] (Y. Jin), [email protected] (Z. Yang).

https://doi.org/10.1016/j.bioorg.2019.103390 Received 5 September 2019; Received in revised form 16 October 2019; Accepted 21 October 2019 0045-2068/ © 2019 Elsevier Inc. All rights reserved.

Please cite this article as: Yuanying Fang, et al., Bioorganic Chemistry, https://doi.org/10.1016/j.bioorg.2019.103390

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Fig. 1. Representative DPP-4 inhibitors.

2. Results and discussion

available cycloamines, which were reacted with chloroacetyl chloride in good yields (Scheme 1). A2 was obtained by acylation of L-proline amide, followed by dehydration of amide 1. A4 and A5 were synthesized from benzohydrazide via reacting with 3-chloropropionyl chloride or chloroacetyl chloride respectively to afford diacyl hydrazide 2 or 3, followed by cyclization with POCl3 under reflux condition. 4,6-disubstituted tetrahydropyrido[4,3-d]pyrimidine compounds 9–16 were synthesized as shown in Scheme 2. The key intermediate 4 was obtained according to the reported procedures [27–30]. Substitution of 4 with 2-fluoro-4-cyano aniline or 2-fluoro-4-methylsulfonyl aniline resulted in 5 or 6 in 35% and 83% yield. The debenzylation of 5 or 6 afforded intermediates 7 or 8, which was reacted with fragments A1, A3, A4, A5 and A6 respectively in basic conditions to give final compounds 9–16 in 32% − 65% yields. 4,7-disubstituted tetrahydropyrido[3,4-d]pyrimidine compounds 20–23 were synthesized following the similar procedure of 4,6-disubstituted tetrahydropyrido[4,3-d]pyrimidine derivatives (Scheme 3). The starting intermediate 17 was prepared using the reported methods [31–34]. Synthesis of 4,6-disubstituted-2-methyl tetrahydropyrido[4,3-d] pyrimidine compounds 32–44 was outlined in Scheme 4. The starting material benzylamine was reacted with methyl acrylate to afford 24, following by Claisen condensation and cyclization with acetamidine

2.1. Chemistry Although there is pretty big diversity among the structures of most gliptins, they all bind essentially in the same catalytic site of DPP-4. The hydrophobic moieties such as 2-cyanopyrrolindine of vildagliptin, thiazolidine of teneligliptin and trifluorobenzene can occupy the S1 pocket near the catalytic Ser630, while the less hydrophobic moieties such as 3-aminopiperidine of trelagliptin and tetrahydrotriazolopyrazine of sitagliptin will bind S2 pocket [24,25]. These featured fragments can be extracted to serve as pharmacophores and building blocks for the design of DPP-4 inhibitor. We had presented that tetrahydropyridopyrimidine derivatives showed the significant GPR119 agonistic activities [26]. These results inspired us to design dual modulator targeting GPR119 and DPP-4 using pharmacophore combination and scaffold hopping. Our strategy was retaining the fused pyrimidine as core and substituted anilines as tail. Seven various fragments, selected from reported DPP-4 inhibitors was hybridized to the cores and served as head (Fig. 2). We envisaged that synergistic effects of GPR119 agonism and DPP-4 inhibition could be advantageous to blood glucose control. The fragments A1, A3, A6 and A7 were generated from commercial

Fig. 2. Design of dual GPR119 and DPP-4 modulator. 2

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Scheme 1. Reagents and conditions: (a) NaOH, THF, room temperature, 1 h, 85%; (b) K2CO3, DCM, room temperature, 1 h; (c) TFAA, pyridine, DCM, room temperature, 4 h, 72%; (d) TEA, DCM, room temperature, 1 h, 64%; (e) THF, room temperature, 1 h; (f) POCl3, reflux, 3 h, 88% in two steps; (g) THF, room temperature, 1 h; (h) POCl3, reflux, 3 h, 87% in two steps; (i) Na2CO3, dioxane, room temperature, 1 h, 94%; (j) TEA, DCM, room temperature, 1 h, 70%.

hydrochloride to yield 26, which was treated with POCl3 to give chloride 27. Then final compounds 32–44 were obtained using the similar procedure with 4,7-disubstituted tetrahydropyrido[3,4-d]pyrimidine compounds. 4,7-disubstituted-2-methyl tetrahydropyrido[3,4-d]pyrimidine compounds 50–62 were generated using the similar procedures with 4,7-disubstituted tetrahydropyrido[3,4-d]pyrimidine derivatives (Scheme 5). The key intermediate 45 was synthesized via the reported procedures [32,35].

bearing A2, A6 or A7 fragment retained potent GPR119 agonistic activities (EC50 values ranged from 0.030 μM to 0.078 μM) with good efficacy (76.2–98.5%max). Then we selected the compounds with moderate to potent GPR119 agonistic activities (EC50 values less than 10 μM) to test their DPP-4 inhibitory rates at 10 μM concentration. Among them, compound 39 bearing A2 fragment displayed the best inhibitory rate of 83.2% against DPP-4. Compounds containing fragment A5 and A6 showed weak inhibitory activities. Better results were disclosed in the nucleus of 4,7-disubstituted tetrahydropyrido[4,3-d]pyrimidine (Table 2). Compounds with methyl substitution showed similar or slightly stronger potency of GPR119 agonism than non-substitution at C-2 position. The comparison of function between methylsulfonyl and cyano substitution at tail part showed variance. It was clearly found that all compounds bearing A2, A6 or A7 fragment displayed potent GPR119 agonistic activities (EC50 values less than 100 nM) with good efficacy (70.8–102.5%max). Meanwhile, they also showed moderate to good DPP-4 inhibitory activities (58.9–77.9% inhibition at 10 μM). Among them, compound 51 with A2 at head moiety demonstrated the greatest GPR119 agonistic potency (EC50 = 8.7 nM, close to the reference GSK1292263) and good inhibition rate of 74.5% against DPP-4. To predict the physicochemical and drug-like properties, we calculated the logarithm of partition coefficient of all final compounds using BIOVIA Discovery Studio 4.5. Compound 51 was calculated to have suitable lipophilicity (ClogP = 2.1) to lower the risk of pharmacokinetics issues.

2.2. Biological evaluation 2.2.1. GPR119 activation and DPP-4 inhibition GPR119 agonistic activity of target compounds 9–16, 20–23, 32–44, 50–62 were measured using a cell-based cAMP assay. The GPR119 agonist GSK-1292263 was chosen as the reference. The results were expressed as EC50 values representing the concentration at which compounds provoked 50% cAMP stimulation and the inherent activity (IA) as percentages (%max) of response which were compared to the reference GSK-1292263 (defined the maximal effect activation). The in vitro inhibitory activities of selected GPR119 agonists against DPP-4 were also evaluated using vildagliptin as positive control. We firstly investigated the effects of substituents on the GPR119 agonism in the skeleton of 4,6-disubstituted tetrahydropyrido[4,3-d] pyrimidine. As shown in Table 1, methyl substitution at the C-2 (R2) increased the potency of GPR119 agonism (compound 10 vs. 33; 13 vs. 36; 15 vs. 40). Compounds with methylsulfonyl (Ms) substitution at the tail (R4) exhibited slightly potent or similar GPR119 activation with cyano group (compound 10 vs. 15; 33 vs. 40; 35 vs. 42; 36 vs. 43; 37 vs. 44). In case of the head moiety (R6), compound 36, 39, 43 and 44

2.2.2. Oral glucose tolerance test of compound 51 Based on the promising potency towards dual targets in vitro, compound 51 was further evaluated for the oral glucose tolerance test

Scheme 2. Reagents and conditions: (a) 4-amino-3-fluorobenzonitrile, NaH, THF, reflux, overnight, 35% or 2-fluoro-4-(methylsulfonyl)aniline, X-Phos, Pd2(dba)3, Cs2CO3, dioxane, reflux, overnight, 83%; (b) i, 2-chloroethyl chloroformate, 1,2-dichloroethane, reflux, overnight, ii MeOH, reflux, 3 h; (c) A1 or A3 or A4 or A5 or A6, K2CO3, DMF, 70 ℃, overnight. 3

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Scheme 3. Reagents and conditions: (a) 2-fluoro-4-(methylsulfonyl)aniline, X-Phos, Pd2(dba)3, Cs2CO3, dioxane, under N2, reflux, overnight; (b) i, 2-chloroethyl chloroformate, 1,2-dichloroethane, reflux, overnight, ii MeOH, reflux, 3 h; (c) A1 or A2 or A6 or A7, DIPEA, NaI, 65 ℃, overnight.

(oGTT) with a single dose (15 and 30 mg/kg) in C57BL/6N mice. The DPP-4 inhibitor vildagliptin was selected as positive control. Compound 51 was observed to reduce the area under the curve of blood glucose from 0 to 120 min (AUC0-120min) by 12.3% (22.18 ± 3.99) and 19.5% (20.35 ± 3.73) at the dosage of 15 mg/kg and 30 mg/kg, respectively. Vildagliptin reduced AUC0–120min by 16.4% (Vehicle: 25.28 ± 3.48, Vildagliptin: 21.13 ± 3.84) at the dose of 30 mg/kg. Compound 51 exhibited greater improvement of glucose tolerance capacity than the positive control vildagliptin (Fig. 3). After 16 h starvation, compounds 51 was administered to the mice, then the oral glucose tolerance test (3 g/kg) was conducted after 4 h of the single dose, the blood glucose concentration at 0, 15, 30, 60, 90 and 120 min were recorded for area under curve calculation. The results are presented as the mean ± SE. *, p < 0.05 compared to vehicle group (n = 8).

activity of the GPR119 agonist compound 51. 3. Conclusion In summary, a series of tetrahydropyridopyrimidine derivatives were designed and synthesized for the discovery of dual ligands targeting GPR119 and DPP-4. Seven fragments of DPP-4 inhibitor pharmacophores A1–A7 were connected to the head of tetrahydropyridopyrimidine skeleton. Compounds bearing A2, A6 or A7 fragment retained potent GPR119 agonistic activities (EC50 < 100 nM) with good receptor efficacy. Among them, compound 51 containing A2 at head moiety was identified to displayed most potent agonistic activity (EC50 = 8.7 nM) and good inhibition rate of 74.5% against DPP-4 at 10 μM. Moreover, compound 51 also demonstrated stronger hypoglycemic effect in the oral glucose tolerance test in C57BL/6N mice than vildagliptin at the same dose. Further study on GPR119 and DPP-4 dual modulator is in progress.

2.3. Molecular docking Since the 3D structure of GPR119 was not reported yet, we input the structure of the dual modulator compound 51 to dock with DPP-4 (PDB ID: 6B1E) to understand the binding mode of DPP-4 and 51 using Discovery Studio 4.5 software (Fig. 4). Similar with vildagliptin, the 2cyanopyrrolidine moiety of 51 occupied the S1 hydrophobic pocket, and the nitrile group formed hydrogen bonds with Try666 and Tyr547. The binding modes also indicated the tetrahydropyrido[4,3-d]pyrimidine moiety interacted with the residues Glu205 and Arg125 in S2 pocket. Furthermore, the tail fragment of 2-fluoro-4-cyanoaniline interacted with Phe357 in S2′ extensive subsite within the target enzyme, and the cyano group formed hydrogen bond with Arg669. The docking model indicated that the head and core moieties of compound 51 could fit the desired S1 and S2 pockets of DPP-4 respectively, and the tail fragment of anilino group provided additional binding affinity with S2′ extensive subsite, which might partially explain the DPP-4 inhibitory

4. Experimental 4.1. Chemistry All starting materials were commercially available and used without further purification. NMR spectra were recorded on a Bruker AVANCE III HD 600 (600 Hz) spectrometer. Chemical shifts were reported in parts per million (ppm) downfield relative to tetramethylsilane as an internal standard. Proton coupling patterns were 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 a AB SCIEX (Triple TOF 5600 + ). Analytical HPLC was performed on a Waters Acquity® ArcTM with 2998 PDA detector. Melting points were measured on a SGW X-4B melting point apparatus (INESA, Shanghai, China) and were

Scheme 4. Reagents and conditions: (a) methyl acrylate, MeOH, 60 ℃, 12 h; (b) EtONa, toluene, 100 ℃, 3 h; (c) acetamidine hydrochloride, MeONa, 40 ℃, overnight; (d) POCl3, 110 ℃, 5 h; (e) substituted amines, X-Phos, Pd2(dba)3, Cs2CO3, dioxane, under N2, overnight, reflux; (f) i, 2-chloroethyl chloroformate, 1,2-dichloroethane, reflux, overnight, ii MeOH, reflux, 3 h; (g) A1–A7, K2CO3, DMF, 70 ℃, overnight. 4

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Scheme 5. Reagents and conditions: (a) substituted amines, X-Phos, Pd2(dba)3, Cs2CO3, 1,4-Dioxane, overnight, reflux; (b) i, 2-chloroethyl chloroformate, 1,2dichloroethane, reflux, overnight, ii MeOH, reflux, 3 h; (c) A1–A7, K2CO3, DMF, 70 ℃, overnight. Table 1 GPR119 agonistic and DPP-4 inhibitory activities of 4,6-disubstituted tetrahydropyrido[4,3-d]pyrimidine compounds.

Compd.

R2

9 H 10 H 11 H 12 H 13 H 14 H 15 H 16 H 32 Me 33 Me 34 Me 35 Me 36 Me 37 Me 38 Me 39 Me 40 Me 41 Me 42 Me 43 Me 44 Me GSK1292263 Vildagliptin a b

R4

CN CN CN CN CN Ms Ms Ms CN CN CN CN CN CN Ms Ms Ms Ms Ms Ms Ms

R6

A1 A3 A4 A5 A6 A1 A3 A4 A1 A3 A4 A5 A6 A7 A1 A2 A3 A4 A5 A6 A7

GPR119 activation

DPP-4 inhibition

EC50 (μM)

%maxa

%inhibition (10 μM)

> 10 > 10 > 10 > 10 0.45 > 10 7.9 > 10 > 10 8.2 > 10 7.7 0.036 0.13 > 10 0.054 7.2 > 10 8.3 0.030 0.078 0.0066 –

29.4 32.1 37.5 27.7 91.0 33.6 45.2 38.9 30.4 49.7 38.2 50.7 95.5 90.6 35.7 76.2 54.5 32.7 59.1 98.5 90.6 100.0 –

– – – – 33.5 – 26.7 – – 30.0 – 54.3 44.7 28.8 – 83.2 52.4 – 19.5 65.1 5.7 – 99.6

Table 2 GPR119 agonistic and DPP-4 inhibitory activities of 4,7-disubstituted tetrahydropyrido[4,3-d]pyrimidine compounds.

ClogPb

Compd.

R2

20 H 21 H 22 H 23 H 50 Me 51 Me 52 Me 53 Me 54 Me 55 Me 56 Me 57 Me 58 Me 59 Me 60 Me 61 Me 62 Me GSK1292263 Vildagliptin

2.3 2.2 3.8 3.6 3.0 1.9 1.8 3.5 2.3 2.2 3.8 3.5 3.0 2.5 1.9 1.7 1.8 3.4 3.2 2.6 2.2 – –

a b

R4

Ms Ms Ms Ms CN CN CN CN CN CN CN Ms Ms Ms Ms Ms Ms

R7

A1 A2 A6 A7 A1 A2 A3 A4 A5 A6 A7 A1 A2 A4 A5 A6 A7

GPR119 activation

DPP-4 inhibition

EC50 (μM)

%maxa

%inhibition (10 μM)

> 10 0.090 0.021 0.085 > 10 0.0087 > 10 > 10 > 10 0.012 0.057 > 10 0.042 > 10 6.8 0.024 0.068 0.0066 –

34.3 70.8 95.0 89.4 38.2 85.4 49.2 31.7 40.8 95.7 88.0 40.1 74.5 42.3 66.3 102.5 97.5 100.0 –

– 75.8 67.1 77.9 – 74.5 – – – 64.7 18.5 – 72.5 – 11.3 70.4 58.9 – 99.6

ClogPb

2.0 1.8 2.8 2.3 2.3 2.1 2.2 3.9 3.6 3.1 2.6 2.0 1.8 3.5 3.3 2.7 2.2 – –

%max: cAMP stimulation % compared to maximal effect of GSK1292263. ClogP was calculated using ACD software from Discovery Studio 4.5.

4.1.2. (S)-1-(2-chloroacetyl)pyrrolidine-2-carbonitrile (A2) To a mixture of L-proline amide (5.0 g, 43.8 mmol) and K2CO3 (12.1 g, 87.6 mmol) in CH2Cl2 (50 mL) was added chloroacetyl chloride (7.3 mL, 87.6 mmol) dropwise at 0 °C. The reaction mixture was warmed to ambient temperature and stirred for 1 h. The mixture was diluted with CH2Cl2 (100 mL) and washed with brine (30 mL × 2). The organic layer was dried over anhydrous MgSO4, filtered and evaporated to give the crude product as a yellow solid (7.9 g). To the solution of amide and pyridine (10.0 mL, 123.9 mmol) in CH2Cl2 (75 mL) was added trifluoroacetic acid anhydride (8.7 mL, 61.9 mmol) dropwise at 0 °C under nitrogen atmosphere. The reaction mixture was warmed to ambient temperature and stirred for 4 h and quenched with ice water (60 mL). The organic layer was washed with 15% HCl (50 mL). and dried over anhydrous MgSO4, filtered and evaporated. The crude product was purified by silica gel column chromatography (petroleum ether: EtOAc = 2: 1) to afford the desired product as a yellow oil (5.4 g, 72% in two steps). 1H NMR (600 MHz, DMSO) δ (ppm): 4.78 (dd, J = 8.0, 2.6 Hz, 1H), 4.08 (d, J = 3.7 Hz, 2H), 3.79–3.71 (m, 2H), 3.69–3.59 (m, 2H), 2.40–2.29 (m, 2H), 2.24 (m, 2H). MS-ESI: [M + H]+: 173.1.

%max: cAMP stimulation % compared to maximal effect of GSK1292263. ClogP was calculated using ACD software from Discovery Studio 4.5.

uncorrected. 4.1.1. 2-Chloro-1-(3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a] pyrazin-7(8H)-yl)ethan-1-one (A1) A mixture of 3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo [4,3-a]pyrazine hydrochloride (1.0 g, 4.4 mmol) in EtOAc (10 mL) was treated with aqueous NaOH solution (1.0 mL, 30% w/w) to give a free base. To the solution of the base in THF (10 mL) was added chloroacetyl chloride (4.0 mL, 48.3 mmol) dropwise at 0 °C under nitrogen atmosphere. The reaction mixture was warmed to ambient temperature and stirred for 1 h. The mixture was neutralized with aqueous 25% NaOH and extracted with EtOAc (20 mL × 2). The organic layers were combined, washed with brine (10 mL), dried over anhydrous MgSO4, filtered and evaporated. The crude product was purified by silica gel column chromatography (petroleum ether: EtOAc = 1: 1) to afford the desired product as a yellow solid (1.0 g, 85%). 1H NMR (600 MHz, DMSO) δ (ppm): 5.07 (s, 2H), 4.35 (s, 1H), 4.22 (m, 3H), 4.15 (s, 1H), 4.07 (s, 1H). MS-ESI: [M + H]+: 269.1.

4.1.3. 2-Chloro-1-(thiazolidin-3-yl)ethan-1-one (A3) To a solution of thiazolidine (1.0 g, 11.2 mmol) and triethyl amine 5

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Fig. 3. Single dose of compounds 51 on oGTT in C57BL/6N mice.

(2.2 mL, 15.7 mmol) in CH2Cl2 (10 mL) was added chloroacetyl chloride (1.3 mL, 15.7 mmol) dropwise at 0 °C under nitrogen atmosphere. The reaction mixture was warmed to ambient temperature and stirred for 1 h. The mixture was diluted with CH2Cl2 (30 mL) and washed with brine (10 mL × 2). The organic layer was dried over anhydrous MgSO4, filtered and evaporated. The crude product was purified by silica gel column chromatography (petroleum ether: EtOAc = 5: 1) to afford the desired product as a yellow oil (1.2 g, 64%). 1H NMR (600 MHz, DMSO) δ (ppm): 4.61 (d, J = 16.9 Hz, 2H), 4.09 (d, J = 14.1 Hz, 2H), 3.89 (t, J = 6.4 Hz, 1H), 3.86 (t, J = 6.2 Hz, 1H), 3.15 (t, J = 6.2 Hz, 1H), 3.05 (t, J = 6.4 Hz, 1H). MS-ESI: [M + H]+: 165.2.

combined, washed with brine (10 mL × 2), dried over anhydrous MgSO4, filtered and evaporated. The crude product was purified by silica gel column chromatography (petroleum ether: EtOAc = 2: 1) to afford the desired product as a yellow solid (2.7 g, 88% in two steps). 1 H NMR (600 MHz, CDCl3) δ (ppm): 8.10–8.05 (m, 1H), 7.60–7.52 (m, 2H), 3.99 (t, J = 6.9 Hz, 1H), 3.45 (t, J = 6.9 Hz, 1H). MS-ESI: [M + H]+: 209.1. 4.1.5. 2-(Chloromethyl)-5-phenyl-1,3,4-oxadiazole (A5) The intermediate was prepared in 87% yield in two steps by using chloroacetyl chloride as acylating agent instead of 3-chloropropanoyl chloride following the similar procedure with A4. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.10 (d, J = 7.4 Hz, 2H), 7.60 (t, J = 7.3 Hz, 1H), 7.55 (t, J = 7.4 Hz, 2H), 4.81 (s, 2H). MS-ESI: [M + H]+: 195.4.

4.1.4. 2-(2-Chloroethyl)-5-phenyl-1,3,4-oxadiazole (A4) To a solution of benzohydrazide (2.0 g, 14.7 mmol) in THF (15 mL) was added 3-chloropropanoyl chloride (1.6 mL, 16.7 mmol) dropwise at 0 °C under nitrogen atmosphere. The reaction mixture was warmed to ambient temperature and stirred for 1 h. The precipitate was collected by filtration and dried to give the diacyl hydrazide as a white solid (3 g). The intermediate solid was dissolved in POCl3 (4 mL) and refluxed for 3 h. The mixture was poured slowly into ice water (30 mL) and extracted with EtOAc (20 mL × 2). The organic layers were

4.1.6. Tert-butyl (R)-(1-(2-chloroacetyl)piperidin-3-yl)carbamate (A6) To a solution of R-3-(Boc-amino)piperidine (1.0 g, 5.0 mmol) in dioxane (10 mL) was added 10% Na2CO3 (5 mL). The mixture was stirred for 20 min, then cooled in an ice water bath, and chloroacetyl chloride (0.8 mL, 9.7 mmol) was added dropwise at 0 °C. The reaction mixture was basified with 10% Na2CO3 to pH 9, warmed to ambient temperature and stirred for 1 h. The precipitate was collected by

Fig. 4. Docking study of compounds 51with DPP-4 (PDB ID: 6B1E). 6

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filtration and dried to give the desired product as a white solid (1.3 g, 94%). 1H NMR (600 MHz, CDCl3) δ (ppm): 4.60 (s, 1H), 4.13 (m, 2H), 4.10 (s, 1H), 3.70 (m, 2H), 3.50 (s, 1H), 3.37 (s, 1H), 1.89 (m, 2H), 1.71 (m, 2H), 1.62 (s, 9H). MS-ESI: [M + H]+: 277.5.

J = 10.9, 1.8 Hz, 1H), 6.59 (d, J = 4.1 Hz, 1H), 3.96 (s, 2H), 3.25 (t, J = 5.8 Hz, 2H), 2.90 (t, J = 5.8 Hz, 2H). MS-ESI: [M + H]+: 270.5. 4.1.11. N-(2-Fluoro-4-(methylsulfonyl)phenyl)-5,6,7,8-tetrahydropyrido [4,3-d]pyrimidin-4-amine (8) The title intermediate was generated from benzyl intermediate 6 using similar procedure of 7. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.93 (t, J = 8.2 Hz, 1H), 8.70 (s, 1H), 7.78 (dd, J = 8.7, 1.3 Hz, 1H), 7.72 (dd, J = 10.4, 2.0 Hz, 1H), 6.62 (d, J = 4.4 Hz, 1H), 3.97 (s, 2H), 3.25 (t, J = 5.8 Hz, 2H), 3.08 (s, 3H), 2.90 (t, J = 5.8 Hz, 2H). MS-ESI: [M + H]+: 323.1

4.1.7. Tert-butyl 4-(2-chloroacetyl)piperazine-1-carboxylate (A7) To a solution of 1-Boc-piperazine (1.0 g, 5.4 mmol) and triethyl amine (1.1 mL, 7.6 mmol) in CH2Cl2 (10 mL) was added chloroacetyl chloride (0.6 mL, 7.3 mmol) dropwise at 0 °C under nitrogen atmosphere. The reaction mixture was warmed to ambient temperature and stirred for 1 h. The mixture was diluted with CH2Cl2 (30 mL) and washed with brine (10 mL × 2). The organic layer was dried over anhydrous MgSO4, filtered and evaporated. The crude product was purified by silica gel column chromatography (petroleum ether: EtOAc = 5: 1) to afford the desired product as a yellow solid (1.0 g, 70%). 1H NMR (600 MHz, CDCl3) δ (ppm): 4.10 (s, 2H), 3.62 (s, 2H), 3.53 (s, 4H), 3.47 (s, 2H), 1.50 (s, 9H). MS-ESI: [M + H]+: 263.3.

4.1.12. General procedure for compounds 9–13 To a solution of intermediate 7 (100 mg, 0.37 mmol) in DMF (4 mL) was added K2CO3 (71 mg, 0.52 mmol) and fragment chloride (0.52 mmol). The reaction mixture was stirred at 70 °C overnight. The mixture was cooled down and extracted with EtOAc. The organic layer was dried over anhydrous MgSO4, filtered and evaporated. The crude product was purified by silica gel column chromatography.

4.1.8. 4-((6-benzyl-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-yl) amino)-3-fluorobenzonitrile (5) To a solution of 6-benzyl-4-chloro-5,6,7,8-tetrahydropyrido[4,3-d] pyrimidine (1.5 g, 5.8 mmol) and 4-amino-3-fluorobenzonitrile (0.86 g, 6.3 mmol) in THF (20 mL) was added NaH (0.46 g, 60%, 11.6 mmol) portionwise at 0 °C under nitrogen atmosphere. The reaction mixture was heated to 65 °C and stirred overnight. The mixture was diluted with EtOAc (50 mL) and washed with brine (10 mL × 2). The organic layer was dried over anhydrous MgSO4, filtered and evaporated. The crude product was purified by silica gel column chromatography (petroleum ether: EtOAc = 1: 1) to afford the desired product as a yellow solid (0.7 g, 35%). 1H NMR (600 MHz, CDCl3) δ (ppm): 8.91 (t, J = 8.4 Hz, 1H), 8.73 (s, 1H), 7.59 (d, J = 8.8 Hz, 1H), 7.49–7.21 (m, 6H), 6.62 (d, J = 4.2 Hz, 1H), 3.96 (s, 2H), 3.78 (s, 2H), 3.25 (t, J = 5.8 Hz, 2H), 2.90 (t, J = 5.8 Hz, 2H). MS-ESI: [M + H]+: 360.3.

4.1.13. 3-fluoro-4-((6-(2-oxo-2-(3-(trifluoromethyl)-5,6-dihydro-[1,2,4] triazolo[4,3-a]pyrazin-7(8H)-yl)ethyl)-5,6,7,8-tetrahydropyrido[4,3-d] pyrimidin-4-yl)amino)benzonitrile (9) Yellow solid, 75% yield. m.p. 192.2–194.1 °C. 1H NMR (600 MHz, DMSO-d6) δ (ppm): 8.73 (s, 1H), 8.39 (d, J = 4.6 Hz, 1H), 7.88 (dd, J = 10.5, 1.7 Hz, 1H), 7.75 – 7.66 (m, 2H), 5.13–4.92 (m, 2H), 4.25–4.16 (m, 2H), 4.11–3.98 (m, 2H), 3.65 (s, 2H), 3.62 (s, 2H), 2.87 (m, 2H), 2.83–2.70 (m, 2H). 13C NMR (150 MHz, DMSO-d6) δ (ppm): 169.1, 161.8, 156.6 (d, J = 3.1 Hz), 156.1, 155.4 (d, J = 4.1 Hz), 154.5, 151.4 (d, J = 4.4 Hz), 133.1 (d, J = 11.0 Hz), 129.5, 127.0, 120.2 (d, J = 24.0 Hz), 118.9 (q, J = 268.1 Hz), 118.5, 113.2, 107.4 (d, J = 9.5 Hz), 60.2, 49.6, 49.2, 44.3, 41.6, 39.0, 32.1. HRMS-TOF (m/z) calcd for C22H19F4N9O [M + H]+: 502.1727, found 502.1714. HPLC purity, 95.3%.

4.1.9. 6-benzyl-N-(2-fluoro-4-(methylsulfonyl)phenyl)-5,6,7,8tetrahydropyrido[4,3-d]pyrimidin-4-amine (6) To the solution of 6-benzyl-4-chloro-5,6,7,8-tetrahydropyrido[4,3d]pyrimidine (1.5 g, 5.8 mmol) and 2-fluoro-4-(methylsulfonyl)aniline (1.3 g, 6.9 mmol) in 1,4-dioxane (30 mL) was added Pd2(dba)3 (1.1 g, 1.2 mmol), X-Phos (0.57 g, 1.2 mmol) and Cs2CO3 (0.81 g, 2.5 mmol) under nitrogen atmosphere. The reaction was heated to reflux overnight. After cooled down to room temperature the mixture was diluted with EtOAc (60 mL) and washed with brine. 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 yellow solid (2.0 g, 83%). 1H NMR (600 MHz, CDCl3) δ (ppm): 8.96 (t, J = 8.2 Hz, 1H), 8.72 (s, 1H), 7.78 (m, 1H), 7.72 (m, 1H), 7.35–7.21 (m, 5H), 6.71 (d, J = 4.5 Hz, 1H), 3.97 (s, 2H), 3.77 (s, 2H), 3.27 (t, J = 5.9 Hz, 2H), 3.10 (s, 3H), 2.92 (t, J = 5.9 Hz, 2H). MS-ESI: [M + H]+: 399.3.

4.1.14. 3-fluoro-4-((6-(2-oxo-2-(thiazolidin-3-yl)ethyl)-5,6,7,8tetrahydropyrido[4,3-d]pyrimidin-4-yl)amino)benzonitrile (10) Yellow solid, 65% yield. m.p. 215.4–216.9 °C. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.81 (t, J = 8.3 Hz, 1H), 8.69 (s, 1H), 7.51 (d, J = 8.6 Hz, 1H), 7.49–7.38 (m, 1H), 6.59 (s, 1H), 4.65 (d, J = 9.6 Hz, 2H), 3.92 (t, J = 6.4 Hz, 1H), 3.87 (t, J = 6.2 Hz, 1H), 3.76 (d, J = 11.5 Hz, 2H), 3.56 (d, J = 15.1 Hz, 2H), 3.14 (t, J = 6.2 Hz, 1H), 3.09–2.97 (m, 5H). 13C NMR (150 MHz, CDCl3) δ (ppm):167.3 (d, J = 37.4 Hz), 162.3, 155.6, 155.4, 151.4 (d, J = 243.6 Hz), 132.2 (d, J = 8.8 Hz), 129.3 (d, J = 3.6 Hz), 122.0, 118.3 (d, J = 22.9 Hz), 118.0 (d, J = 2.8 Hz), 112.6 (d, J = 9.5 Hz), 105.4 (d, J = 9.3 Hz), 61.0, 60.9, 49.6, 48.8, 48.6, 48.1, 31.5. HRMS-TOF (m/z) calcd for C19H19FN6OS [M + H]+: 399.1403, found 399.1404. HPLC purity, 92.6%. 4.1.15. 3-fluoro-4-((6-(2-(5-phenyl-1,3,4-oxadiazol-2-yl)ethyl)-5,6,7,8tetrahydropyrido[4,3-d]pyrimidin-4-yl)amino)benzonitrile (11) Yellow solid, 54% yield. m.p. 215.0–217.2 °C. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.81 (t, J = 8.1 Hz, 1H), 8.68 (s, 1H), 8.10–7.98 (m, 2H), 7.62–7.49 (m, 4H), 7.44 (dd, J = 10.8, 1.8 Hz, 1H), 6.67 (s, 1H), 3.70 (s, 2H), 3.31 (d, J = 6.5 Hz, 2H), 3.26 (d, J = 6.0 Hz, 2H), 3.00 (s, 4H). 13C NMR (150 MHz, CDCl3) δ (ppm): 165.1, 165.0, 162.6, 155.6, 155.4, 151.4 (d, J = 246.3 Hz), 132.3 (d, J = 10.4 Hz), 131.7, 129.4 (d, J = 3.6 Hz), 129.1 (x 2), 126.8 (x 2), 123.9, 121.7, 118.2 (d, J = 22.8 Hz), 118.0, 112.7, 105.4 (d, J = 8.8 Hz), 54.2, 49.2, 49.0, 32.1, 24.2. HRMS-TOF (m/z) calcd for C24H20FN7O [M + H]+: 442.1792, found 442.1781. HPLC purity, 96.6%.

4.1.10. 3-fluoro-4-((5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-yl) amino)benzonitrile (7) To a solution of intermediate 5 (0.7 g, 1.9 mmol) in dichloroethane (10 mL) was added 1-chloroethyl chloroformate (0.3 mL, 2.8 mmol) dropwise at 0 °C under nitrogen atmosphere. The reaction mixture was heated to reflux and stirred overnight. The mixture was evaporated to dryness and the residue was dissolved in MeOH (5 mL) and refluxed for 2 h. The solvent was removed and the residue was partitioned between EtOAc and water. The aqueous layer was basified with saturated NaHCO3 to pH 9 and extracted with CH2Cl2 (30 mL × 2). The organic layer was dried over anhydrous MgSO4, filtered and evaporated. The crude product was purified by silica gel column chromatography (petroleum ether: EtOAc = 1: 1) to afford the desired product as a yellow solid (0.4 g, 76%). 1H NMR (600 MHz, CDCl3) δ (ppm): 8.89 (t, J = 8.4 Hz, 1H), 8.70 (s, 1H), 7.52 (d, J = 8.7 Hz, 1H), 7.44 (dd,

4.1.16. 3-fluoro-4-((6-((5-phenyl-1,3,4-oxadiazol-2-yl)methyl)-5,6,7,8tetrahydropyrido[4,3-d]pyrimidin-4-yl)amino)benzonitrile (12) Yellow solid, 59% yield. m.p. 178.6–180.2 °C. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.75 (t, J = 8.3 Hz, 1H), 8.69 (s, 1H), 8.09 (dd, 7

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J = 8.3, 1.3 Hz, 2H), 7.63–7.47 (m, 4H), 7.44 (dd, J = 10.7, 1.8 Hz, 1H), 6.65 (s, 1H), 4.25 (s, 2H), 3.80 (s, 2H), 3.11 (t, J = 5.7 Hz, 2H), 3.06 (d, J = 5.3 Hz, 2H). 13C NMR (150 MHz, CDCl3) δ (ppm): 165.7, 162.7, 162.2, 155.7, 155.4, 151.6 (d, J = 240.0 Hz), 132.2, 132.1, 129.3 (d, J = 3.5 Hz), 129.2 (x 2), 127.0 (x 2), 123.6, 122.0, 118.3 (d, J = 22.8 Hz), 118.0, 112.3, 105.6 (d, J = 9.4 Hz), 51.5, 49.2, 48.3, 32.2. HPLC purity, 95.2%.

J = 4.1 Hz, 1H), 3.80 (s, 2H), 3.70 (s, 2H), 3.09 (s, 3H), 2.93 (s, 2H), 2.75 (s, 2H). MS-ESI: [M + H]+: 413.3. 4.1.22. N-(2-fluoro-4-(methylsulfonyl)phenyl)-5,6,7,8-tetrahydropyrido [3,4-d]pyrimidin-4-amine (19) Follow the similar procedure of 7–8. Light yellow solid, 57%. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.99 (s, 1H), 8.69 (s, 1H), 7.80 (d, J = 8.1 Hz, 1H), 7.74 (d, J = 10.0 Hz, 1H), 6.90 (s, 1H), 4.04 (s, 2H), 3.30 (s, 2H), 3.09 (s, 3H), 2.66 (s, 2H). MS-ESI: [M + H]+: 323.1.

4.1.17. Tert-butyl (R)-(1-(2-(4-((4-cyano-2-fluorophenyl)amino)-7,8dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)acetyl)piperidin-3-yl)carbamate (13) Yellow solid, 62% yield. m.p. 226.2–227.8 °C. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.80 (t, J = 8.3 Hz, 1H), 8.68 (s, 1H), 7.51 (dt, J = 8.5, 1.4 Hz, 1H), 7.43 (dd, J = 10.8, 1.9 Hz, 1H), 6.66 (d, J = 4.3 Hz, 1H), 4.76 (d, J = 6.2 Hz, 1H), 3.86 (d, J = 13.3 Hz, 1H), 3.80–3.49 (m, 8H), 3.45 (m, 1H), 3.31 (dd, J = 13.1, 7.1 Hz, 1H), 3.12–2.93 (m, 4H), 1.93 (m, 1H), 1.74 (m, 1H), 1.40 (s, 9H). 13C NMR (150 MHz, CDCl3) δ (ppm): 168.2, 162.4, 155.5, 155.4, 155.2, 151.6 (d, J = 244.1 Hz), 132.3, 129.3, 122.1, 118.3 (d, J = 22.5 Hz), 118.0, 112.9, 105.4 (d, J = 9.4 Hz), 59.7, 50.4, 49.5, 48.9, 47.3, 42.3, 32.1, 29.8, 28.4, 28.3 (x3), 22.6. HRMS-TOF (m/z) calcd for C26H32FN7O3 [M + H]+: 510.2629, found 510.2619. HPLC purity, 95.2%.

4.1.23. General procedure for compounds 20–23 To a solution of 19 (1 mmol) and fragment A (1.2 mmol) in THF was add DIPEA (1.5 mmol) and NaI (0.1 mmol). The reaction was stirred at 65 °C for overnight. The mixture was extracted with EtOAc and washed with brine. The organic layer was dried over MgSO4, filtered and evaporated. The residue was purified by column chromatography to afford the desired product. 4.1.24. 2-(4-((2-fluoro-4-(methylsulfonyl)phenyl)amino)-5,8dihydropyrido[3,4-d]pyrimidin-7(6H)-yl)-1-(3-(trifluoromethyl)-5,6dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)ethan-1-one (20) Yellow solid, 55% yield. m.p. 173.2–175.4 °C. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.96 (t, J = 8.0 Hz, 1H), 8.69 (s, 1H), 7.80 (d, J = 8.5 Hz, 1H), 7.74 (d, J = 10.2 Hz, 1H), 6.87 (s, 1H), 5.26 – 5.02 (m, 2H), 4.21 (t, J = 5.4 Hz, 2H), 4.14 (t, J = 5.3 Hz, 2H), 3.71 (d, J = 53.6 Hz, 2H), 3.55 (d, J = 46.1 Hz, 2H), 3.09 (s, 3H), 3.01 (t, J = 5.5 Hz, 2H), 2.75 (t, J = 5.6 Hz, 2H). HRMS-TOF (m/z) calcd for C22H22F4N8O3S [M + H]+: 555.1550, found 555.1534. HPLC purity, 92.6%.

4.1.18. 2-(4-((2-fluoro-4-(methylsulfonyl)phenyl)amino)-7,8dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)-1-(3-(trifluoromethyl)-5,6dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)ethan-1-one (14) Yellow solid, 65% yield. m.p. 194.5–196.8 °C. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.83 (t, J = 8.3 Hz, 1H), 8.71 (s, 1H), 7.78 (d, J = 8.5 Hz, 1H), 7.73 (d, J = 10.1 Hz, 1H), 6.60 (s, 1H), 5.12 (m, 2H), 4.23 (t, J = 5.4 Hz, 2H), 4.16 (m, 2H), 3.71 (m, 2H), 3.66 (s, 2H), 3.09 (s, 3H), 3.01 (s, 4H). HRMS-TOF (m/z) calcd for C22H22F4N8O3S [M + H]+: 555.1550, found 555.1552. HPLC purity, 93.8%.

4.1.25. (S)-1-(2-(4-((2-fluoro-4-(methylsulfonyl)phenyl)amino)-5,8dihydropyrido[3,4-d]pyrimidin-7(6H)-yl)acetyl)pyrrolidine-2-carbonitrile (21) Yellow solid, 45% yield. m.p. 206.7–208.5 °C. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.96 (t, J = 8.1 Hz, 1H), 8.69 (s, 1H), 7.79 (d, J = 8.8 Hz, 1H), 7.74 (d, J = 10.3 Hz, 1H), 6.90 (s, 1H), 4.81 (d, J = 6.3 Hz, 1H), 3.83 (s, 1H), 3.77 (m, 1H), 3.75 – 3.53 (m, 2H), 3.48 (m, 2H), 3.09 (s, 4H), 2.77 (t, J = 5.6 Hz, 2H), 2.45–2.15 (m, 4H), 2.16–1.96 (m, 2H). HRMS-TOF (m/z) calcd for C21H23FN6O3S [M + H]+: 459.1615, found 459.1613. HPLC purity, 94.9%.

4.1.19. 2-(4-((2-fluoro-4-(methylsulfonyl)phenyl)amino)-7,8dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)-1-(thiazolidin-3-yl)ethan-1-one (15) Yellow solid, 71% yield. m.p. 211.6–212.8 °C. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.85 (t, J = 8.1 Hz, 1H), 8.70 (s, 1H), 7.78 (dd, J = 8.7, 1.6 Hz, 1H), 7.73 (dd, J = 10.3, 2.0 Hz, 1H), 6.61 (s, 1H), 4.65 (d, J = 9.2 Hz, 2H), 3.92 (t, J = 6.4 Hz, 1H), 3.87 (t, J = 6.2 Hz, 1H), 3.77 (d, J = 11.7 Hz, 2H), 3.57 (d, J = 15.0 Hz, 2H), 3.14 (t, J = 6.2 Hz, 1H), 3.09 (s, 3H), 3.04 (dt, J = 7.6, 5.2 Hz, 5H). HRMS-TOF (m/z) calcd for C19H22FN5O3S2 [M + H]+: 452.1226, found 452.1216. HPLC purity, 95.2%.

4.1.26. Tert-butyl (R)-(1-(2-(4-((2-fluoro-4-(methylsulfonyl)phenyl) amino)-5,8-dihydropyrido[3,4-d]pyrimidin-7(6H)-yl)acetyl)piperidin-3-yl) carbamate (22) Yellow solid, 51% yield. m.p. 230.7–232.6 °C. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.98 (d, J = 7.9 Hz, 1H), 8.69 (s, 1H), 7.79 (d, J = 8.5 Hz, 1H), 7.74 (d, J = 10.3 Hz, 1H), 6.89 (s, 1H), 3.79 (m, 3H), 4.79 (s, 1H), 3.64 (m, 2H), 3.47 (m, 3H), 3.32 (m, 1H), 3.09 (s, 3H), 3.04 (m, 2H), 2.77 (m, 2H), 2.03 (m, 1H), 1.90 (s, 1H), 1.47 (s, 2H), 1.39 (s, 9H). HRMS-TOF (m/z) calcd for C26H35FN6O5S [M + H]+: 563.2452, found 563.2444. HPLC purity, 95.3%.

4.1.20. N-(2-fluoro-4-(methylsulfonyl)phenyl)-6-(2-(5-phenyl-1,3,4oxadiazol-2-yl)ethyl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-amine (16) Yellow solid, 62% yield. m.p. 178.4–180.3 °C. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.89 (t, J = 8.2 Hz, 1H), 8.70 (s, 1H), 8.16–7.99 (m, 2H), 7.79 (dd, J = 8.8, 2.1 Hz, 1H), 7.74 (dd, J = 10.3, 2.1 Hz, 1H), 7.61–7.48 (m, 3H), 6.61 (d, J = 4.7 Hz, 1H), 3.68 (s, 2H), 3.30 (t, J = 7.4 Hz, 2H), 3.24 (t, J = 7.6 Hz, 2H), 3.09 (s, 3H), 2.99 (s, 4H). 13C NMR (150 MHz, CDCl3) δ (ppm): 165.1, 165.0, 162.6, 155.6, 155.4, 151.6 (d, J = 245.9 Hz), 133.9 (d, J = 6.0 Hz), 132.7 (d, J = 9.1 Hz), 131.8, 129.1 (x 2), 126.8 (x 2), 124.5 (d, J = 3.4 Hz), 123.9, 121.7, 114.2 (d, J = 22.5 Hz), 112.7, 54.2, 49.2, 49.0, 44.7, 32.1, 24.2. HRMSTOF (m/z) calcd for C24H23FN6O3S [M + H]+: 495.1615, found 495.1598. HPLC purity, 95.6%.

4.1.27. Tert-butyl 4-(2-(4-((2-fluoro-4-(methylsulfonyl)phenyl)amino)5,8-dihydropyrido[3,4-d]pyrimidin-7(6H)-yl)acetyl)piperazine-1carboxylate (23) Yellow solid, 62% yield. m.p. 193.1–194.9 °C. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.97 (t, J = 8.1 Hz, 1H), 8.69 (s, 1H), 7.79 (d, J = 8.7 Hz, 1H), 7.73 (d, J = 10.3 Hz, 1H), 6.89 (d, J = 3.7 Hz, 1H), 3.76 (s, 2H), 3.63 (s, 2H), 3.58 (s, 2H), 3.49 (d, J = 13.8 Hz, 2H), 3.44 (s, 4H), 3.09 (s, 3H), 3.01 (t, J = 5.5 Hz, 2H), 2.74 (t, J = 5.3 Hz, 2H), 1.48 (s, 9H). HRMS-TOF (m/z) calcd for C25H33FN6O5S [M + H]+: 549.2295, found 549.2282. HPLC purity, 95.0%.

4.1.21. 7-benzyl-N-(2-fluoro-4-(methylsulfonyl)phenyl)-5,6,7,8tetrahydropyrido[3,4-d]pyrimidin-4-amine (18) The title intermediate was generated from 17 using similar procedure of 6. Light yellow solid, 64%. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.97 (t, J = 8.2 Hz, 1H), 8.67 (s, 1H), 7.79 (d, J = 8.7 Hz, 1H), 7.73 (dd, J = 10.3, 1.9 Hz, 1H), 7.39 (m, 4H), 7.32 (m, 1H), 6.87 (d,

4.1.28. Dimethyl 3,3′-(benzylazanediyl)dipropionate (24) The solution of benzyl amine (10.0 mL, 91.5 mmol) and methyl acrylate (18.0 mL, 198.6 mmol) in MeOH (20 mL) was heated at 60 °C 8

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overnight under nitrogen atmosphere. The reaction mixture was evaporated to afford the desired product as a slightly yellow oil (24.9 g, 97%), which was used directly in next step without purification. 1H NMR (600 MHz, CDCl3) δ (ppm): 7.33– 7.25 (m, 5H), 3.67 (s, 6H), 3.61 (s, 2H), 2.83 (t, J = 7.1 Hz, 4H), 2.50 (t, J = 7.1 Hz, 4H). MS-ESI: [M + H]+: 280.3.

4.1.35. N-(2-fluoro-4-(methylsulfonyl)phenyl)-2-methyl-5,6,7,8tetrahydropyrido[4,3-d]pyrimidin-4-amine (31) Follow the similar procedure of 7–8. Light yellow solid, 72% yield. 1 H NMR (400 MHz, CDCl3) δ (ppm): 9.00 (t, J = 8.2 Hz, 1H), 7.78 (d, J = 8.7 Hz, 1H), 7.71 (d, J = 10.4 Hz, 1H), 6.59 (d, J = 3.9 Hz, 1H), 3.93 (s, 2H), 3.23 (t, J = 5.8 Hz, 2H), 3.08 (s, 3H), 2.85 (t, J = 5.6 Hz, 2H), 2.63 (s, 3H). MS-ESI: [M + H]+: 337.1.

4.1.29. Methyl 1-benzyl-4-oxopiperidine-3-carboxylate (25) To a mixture of sodium ethoxide (1.95 g, 28.6 mmol) in toluene (50 mL) was added diester (4.0 g, 14.3 mmol) dropwise at 0 °C. The reaction was stirred at 100 °C for 3 h. Then the reaction was quenched with H2O (9 mL) and acetic acid (1.6 mL, 28.0 mmol). The mixture was stirred at room temperature for 1 h and extracted with EtOAc (50 mL). The organic layer was washed with brine (20 mL), dried over MgSO4, filtered and evaporated to give the product (2.5 g, 70% yield) without further purification. 1H NMR (600 MHz, CDCl3) δ (ppm): 7.38–7.35 (m, 5H), 3.75 (s, 3H), 3.67 (m, 3H), 3.21 (t, J = 1.5 Hz, 2H), 2.65 (t, J = 5.9 Hz, 2H), 2.43 (ddd, J = 5.9, 4.3, 1.6 Hz, 2H). MS-ESI: [M + H]+: 248.5.

4.1.36. 3-fluoro-4-((2-methyl-6-(2-oxo-2-(3-(trifluoromethyl)-5,6dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)ethyl)-5,6,7,8tetrahydropyrido[4,3-d]pyrimidin-4-yl)amino)benzonitrile (32) Follow the similar procedure of 9–16. Yellow solid, 73% yield. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.88 (t, J = 7.9 Hz, 1H), 7.52 (d, J = 8.5 Hz, 1H), 7.44 (d, J = 10.9 Hz, 1H), 6.52 (s, 1H), 5.13 (m, 2H), 4.22 (s, 2H), 4.15 (d, J = 10.3 Hz, 2H), 3.65 (m, 4H), 2.97 (m, 4H), 2.64 (s, 3H). HRMS-TOF (m/z) calcd for C23H21F4N9O [M + H]+: 516.1883, found 516.1878. HPLC purity, 91.5%. 4.1.37. 3-fluoro-4-((2-methyl-6-(2-oxo-2-(thiazolidin-3-yl)ethyl)-5,6,7,8tetrahydropyrido[4,3-d]pyrimidin-4-yl)amino)benzonitrile (33) Follow the similar procedure of 9–16. Yellow solid, 56% yield. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.83 (t, J = 8.1 Hz, 1H), 8.69 (s, 1H), 7.77 (d, J = 8.6 Hz, 1H), 7.73 (dd, J = 10.3, 2.0 Hz, 1H), 6.63 (s, 1H), 4.65 (d, J = 7.4 Hz, 2H), 3.91 (t, J = 6.4 Hz, 1H), 3.87 (t, J = 6.2 Hz, 1H), 3.76 (d, J = 11.5 Hz, 2H), 3.56 (d, J = 15.0 Hz, 2H), 3.13 (t, J = 6.2 Hz, 1H), 3.09 (s, 3H), 3.07–2.99 (m, 5H). HRMS-TOF (m/z) calcd for C20H21FN6OS [M + H]+: 413.1560, found 413.1558. HPLC purity, 90.2%.

4.1.30. 6-Benzyl-2-methyl-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-ol (26) To the solution of methyl 1-benzyl-4-oxopiperidine-3-carboxylate (2.5 g, 10.1 mmol) in MeOH (15 mL) was added acetamidine hydrochloride (1.2 g, 12.7 mmol) and sodium methoxide (1.4 g, 25.9 mmol) at 0 °C under nitrogen atmosphere. The reaction was stirred at 40 °C for overnight. After the reaction completed, the solution was removed under reduced pressure. The residual yellow\solid was dissolved in water (2 mL) and neutralized with acetic acid. The precipitate was filtered to afford a white solid (1.3 g, 50% yield). 1H NMR (600 MHz, CDCl3) δ (ppm): 12.44 (brs, 2H), 7.44–7.29 (m, 5H), 3.75 (s, 2H), 3.50 (s, 2H), 2.85–2.64 (m, 4H), 2.44 (s, 3H). MS-ESI: [M + H]+: 256.5.

4.1.38. 3-fluoro-4-((2-methyl-6-(2-(5-phenyl-1,3,4-oxadiazol-2-yl)ethyl)5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-yl)amino)benzonitrile (34) Follow the similar procedure of 9–16. Yellow solid, 64% yield. m.p. 222.4–224.4 °C. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.90 (t, J = 8.4 Hz, 1H), 8.05 (d, J = 7.7 Hz, 2H), 7.62–7.48 (m, 4H), 7.43 (d, J = 10.9 Hz, 1H), 6.57 (d, J = 3.6 Hz, 1H), 3.63 (s, 2H), 3.29 (t, J = 7.0 Hz, 2H), 3.22 (t, J = 7.0 Hz, 2H), 2.95 (dd, J = 8.7, 4.1 Hz, 4H), 2.62 (s, 3H). 13C NMR (150 MHz, CDCl3) δ (ppm): 165.1, 165.0, 164.9, 162.5, 155.4, 151.2 (d, J = 243.3 Hz), 132.5 (d, J = 8.7 Hz), 131.7, 129.4 (d, J = 3.4 Hz), 129.1 (x 2), 126.8 (x 2), 123.9, 121.5, 118.1, 118.0 (d, J = 22.8 Hz), 109.4, 104.9 (d, J = 9.4 Hz), 54.3, 49.1, 49.0, 32.0, 25.8, 24.2. HRMS-TOF (m/z) calcd for C25H22FN7O [M + H]+: 456.1948, found 456.1933. HPLC purity, 95.3%.

4.1.31. 6-Benzyl-4-chloro-2-methyl-5,6,7,8-tetrahydropyrido[4,3-d] pyrimidine (27) Intermediate 26 (1.3 g, 5.1 mmol) was dissolved in POCl3 (3 mL) at room temperature, then the reaction mixture was heated to reflux for 5 h. The mixture was cooled down, poured into ice water and adjusted to pH 7 with saturated NaHCO3. The precipitate was filtered to afford the product as a yellow solid (1.0 g, 71%). 1H NMR (600 MHz, CDCl3) δ (ppm): 7.30–7.17 (m, 5H), 3.73 (s, 2H), 3.65 (s, 2H), 2.86–2.71 (m, 4H), 2.40 (s, 3H). MS-ESI: [M + H]+: 274.3.

4.1.39. 3-fluoro-4-((2-methyl-6-((5-phenyl-1,3,4-oxadiazol-2-yl)methyl)5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-yl)amino)benzonitrile (35) Follow the similar procedure of 9–16. Yellow solid, 51% yield. m.p. 183.9–186.1 °C. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.86 (t, J = 8.4 Hz, 1H), 8.09 (d, J = 7.1 Hz, 2H), 7.56 (m, 4H), 7.43 (dd, J = 10.8, 1.8 Hz, 1H), 6.56 (d, J = 4.3 Hz, 1H), 4.24 (s, 2H), 3.76 (s, 2H), 3.09 (t, J = 5.8 Hz, 2H), 3.00 (t, J = 5.7 Hz, 2H), 2.63 (s, 3H). 13C NMR (150 MHz, CDCl3) δ (ppm): 165.6, 165.1, 162.7, 162.0, 155.5, 151.2 (d, J = 243.6 Hz), 132.4 (d, J = 8.6 Hz), 132.1, 129.3 (d, J = 3.3 Hz), 129.1 (x 2), 127.0 (x 2), 123.5, 121.7, 118.2 (d, J = 22.8 Hz), 118.1, 109.1, 105.1, 51.5, 49.3, 48.3, 32.0, 25.8. HRMSTOF (m/z) calcd for C24H20FN7O [M + H]+: 442.1792, found 442.1793. HPLC purity, 97.0%.

4.1.32. 4-((6-benzyl-2-methyl-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin4-yl)amino)-3-fluorobenzonitrile (28) Follow the similar procedure of 6. Light yellow solid, 62% yield. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.89 (d, J = 6.7 Hz, 1H), 7.55 (s, 1H), 7.49–7.39 (m, 5H), 6.77 (s, 1H), 6.52 (s, 1H), 3.82 (s, 1H), 3.53 (s, 2H), 2.76 (s, 2H), 2.63 (s, 3H), 2.08 (s, 2H). MS-ESI: [M + H]+: 374.5. 4.1.33. 6-benzyl-N-(2-fluoro-4-(methylsulfonyl)phenyl)-2-methyl-5,6,7,8tetrahydropyrido[4,3-d]pyrimidin-4-amine (29) Follow the similar procedure of 6. Light yellow solid, 72% yield. 1H NMR (600 MHz, CDCl3) δ (ppm): 9.00–8.92 (m, 1H), 7.78 (dd, J = 8.7, 1.4 Hz, 1H), 7.71 (dd, J = 10.4, 2.0 Hz, 1H), 7.43–7.37 (m, 4H), 7.34 (dd, J = 9.4, 4.4 Hz, 1H), 6.53 (d, J = 4.5 Hz, 1H), 3.82 (s, 2H), 3.54 (s, 2H), 3.08 (s, 3H), 2.94 (t, J = 5.6 Hz, 2H), 2.87 (t, J = 5.7 Hz, 2H), 2.64 (s, 3H). MS-ESI: [M + H]+: 427.3.

4.1.40. Tert-butyl (R)-(1-(2-(4-((4-cyano-2-fluorophenyl)amino)-2methyl-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)acetyl)piperidin-3-yl) carbamate (36) Follow the similar procedure of 9–16. Yellow solid, 66% yield. m.p. 224.3–226.2 °C. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.88 (t, J = 8.4 Hz, 1H), 7.50 (d, J = 8.7 Hz, 1H), 7.42 (dd, J = 10.9, 1.8 Hz, 1H), 6.55 (s, 1H), 4.64 (d, J = 6.6 Hz, 2H), 3.91 (t, J = 6.4 Hz, 1H), 3.86 (t, J = 6.2 Hz, 1H), 3.77–3.73 (m, 1H), 3.73–3.67 (m, 2H), 3.54 (d, J = 15.0 Hz, 2H), 3.12 (t, J = 6.2 Hz, 1H), 3.04 (d, J = 6.4 Hz, 1H),

4.1.34. 3-fluoro-4-((2-methyl-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin4-yl)amino)benzonitrile (30) Follow the similar procedure of 7–8. Light yellow solid, 58% yield. 1 H NMR (600 MHz, CDCl3) δ (ppm): 8.95 (t, J = 8.4 Hz, 1H), 7.52 (d, J = 8.7 Hz, 1H), 7.43 (dd, J = 11.0, 1.7 Hz, 1H), 6.58 (d, J = 4.1 Hz, 1H), 3.94 (s, 2H), 3.25 (t, J = 5.9 Hz, 2H), 2.87 (t, J = 5.8 Hz, 2H), 2.64 (s, 3H). MS-ESI: [M + H]+: 284.5. 9

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208.3–210.6 °C. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.96 (t, J = 8.2 Hz, 1H), 8.10–7.98 (m, 2H), 7.79 (dd, J = 8.7, 1.6 Hz, 1H), 7.73 (dd, J = 10.4, 2.0 Hz, 1H), 7.59–7.49 (m, 3H), 6.59 (d, J = 4.0 Hz, 1H), 3.65 (s, 2H), 3.30 (t, J = 6.9 Hz, 2H), 3.22 (t, J = 7.0 Hz, 2H), 3.09 (s, 3H), 3.01–2.92 (m, 4H), 2.63 (s, 3H). HRMS-TOF (m/z) calcd for C25H25FN6O3S [M + H]+: 509.1771, found 509.1762. HPLC purity, 92.3%.

3.03–2.98 (m, 2H), 2.96 (m, 2H), 2.63 (s, 3H), 1.35 (s, 1H), 1.30 (s, 2H), 1.29–1.22 (m, 10H). 13C NMR (150 MHz, CDCl3) δ (ppm): 167.4, 167.2, 164.9 (d, J = 3.5 Hz), 162.2, 155.4, 151.4 (d, J = 243.6 Hz), 132.5 (d, J = 8.8 Hz), 129.4 (d, J = 3.4 Hz), 121.6, 118.1 (d, J = 22.3 Hz), 118.0, 109.4 (d, J = 8.9 Hz), 104.9 (d, J = 9.3 Hz), 61.0, 58.5, 49.7, 48.7, 48.7, 48.6, 48.2, 32.1, 31.5, 29.7 (x 3), 25.8, 22.7. HRMS-TOF (m/z) calcd for C27H34FN7O3 [M + H]+: 524.2785, found 524.2775. HPLC purity, 92.1%.

4.1.46. N-(2-fluoro-4-(methylsulfonyl)phenyl)-2-methyl-6-((5-phenyl1,3,4-oxadiazol-2-yl)methyl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4amine (42) Follow the similar procedure of 9–16. Yellow solid, 53% yield. m.p. 189.3–190.6 °C. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.90 (t, J = 8.1 Hz, 1H), 8.09 (d, J = 7.6 Hz, 2H), 7.78 (d, J = 8.7 Hz, 1H), 7.72 (d, J = 10.3 Hz, 1H), 7.56 (m, 3H), 6.60 (s, 1H), 4.24 (s, 2H), 3.78 (s, 2H), 3.10 (s, 2H), 3.09 (s, 3H), 3.01 (t, J = 5.4 Hz, 2H), 2.63 (s, 3H). HRMS-TOF (m/z) calcd for C24H23FN6O3S [M + H]+: 495.1615, found 495.1606. HPLC purity, 97.5%.

4.1.41. Tert-butyl 4-(2-(4-((4-cyano-2-fluorophenyl)amino)-2-methyl-7,8dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)acetyl)piperazine-1-carboxylate (37) Follow the similar procedure of 9–16. Yellow solid, 73% yield. m.p. 213.8–215.6 °C. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.86 (t, J = 8.3 Hz, 1H), 7.50 (dd, J = 8.6, 1.9 Hz, 1H), 7.41 (dd, J = 10.8, 1.9 Hz, 1H), 6.55 (d, J = 4.6 Hz, 1H), 3.64 (m, 4H), 3.59–3.52 (m, 4H), 3.44 (m, 4H), 2.95 (d, J = 4.7 Hz, 2H), 2.94 (d, J = 4.7 Hz, 2H), 2.62 (s, 3H), 1.47 (s, 9H). 13C NMR (150 MHz, CDCl3) δ (ppm): 167.7, 164.9, 162.2, 155.4, 154.5, 151.4 (d, J = 243.5 Hz), 132.5 (d, J = 8.8 Hz), 129.3 (d, J = 3.3 Hz), 121.7, 118.2, 118.1, 109.3, 105.0 (d, J = 9.1 Hz), 80.4, 60.4, 49.5, 48.9, 45.4 (x2), 41.7 (x2), 32.1, 28.4 (x 3), 25.8. HRMS-TOF (m/z) calcd for C26H32FN7O3 [M + H]+: 510.2629, found 510.2626. HPLC purity, 96.3%.

4.1.47. Tert-butyl (R)-(1-(2-(4-((2-fluoro-4-(methylsulfonyl)phenyl) amino)-2-methyl-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)acetyl) piperidin-3-yl)carbamate (43) Follow the similar procedure of 9–16. Yellow solid, 57% yield. m.p. 229.7–232.0 °C. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.92 (t, J = 8.1 Hz, 1H), 7.81–7.75 (m, 1H), 7.72 (dd, J = 10.3, 2.0 Hz, 1H), 6.68 (s, 1H), 4.81 (d, J = 5.4 Hz, 1H), 3.83 (d, J = 12.5 Hz, 1H), 3.75–3.59 (m, 4H), 3.53 (m, 3H), 3.34 (m, 1H), 3.09 (s, 3H), 3.03 – 2.94 (m, 4H), 2.63 (s, 3H), 1.91 (s, 2H), 1.61 (m, 2H), 1.39 (s, 9H). HRMSTOF (m/z) calcd for C27H37FN6O5S [M + H]+: 577.2608, found 577.2593. HPLC purity, 96.6%.

4.1.42. 2-(4-((2-fluoro-4-(methylsulfonyl)phenyl)amino)-2-methyl-7,8dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)-1-(3-(trifluoromethyl)-5,6dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)ethan-1-one (38) Follow the similar procedure of 9–16. Yellow solid, 47% yield. m.p. 215.4–217.1 °C. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.96–8.90 (m, 1H), 7.78 (d, J = 8.4 Hz, 1H), 7.71 (dd, J = 10.2, 2.2 Hz, 1H), 6.57 (s, 1H), 5.12 (m, 2H), 4.22 (m, 2H), 4.15 (m, 2H), 3.75–3.58 (m, 4H), 3.09 (s, 3H), 2.98 (d, J = 5.3 Hz, 2H), 2.95 (d, J = 5.4 Hz, 2H), 2.64 (s, 3H). 13 C NMR (150 MHz, CDCl3) δ (ppm): 168.3 (d, J = 64.5 Hz), 165.2 (d, J = 21.5 Hz), 161.9 (d, J = 35.2 Hz), 155.5 (d, J = 8.2 Hz), 151.6 (d, J = 244.9 Hz), 150.2, 149.7, 133.7, 132.9 (d, J = 8.9 Hz), 124.4, 121.7 (d, J = 10.7 Hz), 118.2 (d, J = 269.6 Hz), 114.2 (d, J = 22.7 Hz), 108.8 (d, J = 26.4 Hz), 61.1, 60.6, 49.7, 48.9, 44.8, 43.2, 42.9, 38.5, 31.9. HRMS-TOF (m/z) calcd for C23H24F4N8O3S [M + H]+: 569.1706, found 569.1696. HPLC purity, 95.8%.

4.1.48. Tert-butyl 4-(2-(4-((2-fluoro-4-(methylsulfonyl)phenyl)amino)-2methyl-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)acetyl)piperazine-1carboxylate (44) Follow the similar procedure of 9–16. Yellow solid, 58% yield. m.p. 185.1–187.1 °C. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.92 (t, J = 8.2 Hz, 1H), 7.85–7.76 (m, 1H), 7.72 (dd, J = 10.3, 2.1 Hz, 1H), 6.59 (d, J = 4.7 Hz, 1H), 3.65 (m, 4H), 3.56 (m, 4H), 3.46 (m, 4H), 3.09 (s, 3H), 2.98 (d, J = 4.5 Hz, 2H), 2.96 (d, J = 4.6 Hz, 2H), 2.63 (s, 3H), 1.48 (s, 9H). 13C NMR (150 MHz, CDCl3) δ (ppm): 167.7, 165.0, 162.2, 155.5, 154.5, 151.5 (d, J = 245.8 Hz), 133.6, 132.9 (d, J = 8.7 Hz), 124.4 (d, J = 3.4 Hz), 121.6, 114.1 (d, J = 22.6 Hz), 109.4, 80.5, 60.4, 49.5, 48.9, 45.5 (x2), 44.8, 41.7 (x2), 32.2, 28.4 (x 3), 25.8. HRMS-TOF (m/z) calcd for C26H35FN6O5S [M + H]+: 563.2452, found 563.2449. HPLC purity, 93.7%.

4.1.43. (S)-1-(2-(4-((2-fluoro-4-(methylsulfonyl)phenyl)amino)-2methyl-5,8-dihydropyrido[3,4-d]pyrimidin-7(6H)-yl)acetyl)pyrrolidine-2carbonitrile (39) Follow the similar procedure of 9–16. Yellow solid, 39% yield. m.p. 219.6–221.2 °C. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.90 (dd, J = 14.9, 6.9 Hz, 2H), 7.78 (dd, J = 8.7, 2.0 Hz, 2H), 7.75–7.68 (m, 2H), 6.66 (s, 1H), 4.84–4.76 (m, 1H), 3.82–3.71 (m, 2H), 3.61–3.45 (m, 2H), 3.09 (s, 5H), 3.07–2.93 (m, 4H), 2.63 (s, 3H), 2.39–2.15 (m, 4H), 2.06–1.99 (m, 2H). HRMS-TOF (m/z) calcd for C22H25FN6O3S [M + H]+: 473.1771, found 473.1758. HPLC purity, 98.1%.

4.1.49. 4-((7-benzyl-2-methyl-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin4-yl)amino)-3-fluorobenzonitrile (46) Follow the similar procedure of 6. Light yellow solid, 71% yield. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.99 (t, J = 8.4 Hz, 1H), 7.52 (d, J = 8.7 Hz, 1H), 7.42 (dd, J = 11.0, 1.8 Hz, 1H), 7.40–7.29 (m, 5H), 6.81 (d, J = 4.7 Hz, 1H), 3.75 (s, 2H), 3.61 (s, 2H), 2.89 (t, J = 5.8 Hz, 2H), 2.68 (t, J = 5.7 Hz, 2H), 2.61 (s, 3H). MS-ESI: [M + H]+: 374.5.

4.1.44. 2-(4-((2-fluoro-4-(methylsulfonyl)phenyl)amino)-2-methyl-7,8dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)-1-(thiazolidin-3-yl)ethan-1-one (40) Follow the similar procedure of 9–16. Yellow solid, 38% yield. m.p. 221.5–223.7 °C. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.92 (t, J = 8.1 Hz, 1H), 7.78 (dd, J = 8.6, 1.5 Hz, 1H), 7.72 (dd, J = 10.4, 2.1 Hz, 1H), 6.61 (s, 1H), 4.70–4.58 (m, 2H), 3.89 (m, 2H), 3.75–3.69 (m, 2H), 3.55 (m, 2H), 3.13 (t, J = 6.2 Hz, 1H), 3.09 (s, 3H), 3.08–2.95 (m, 5H), 2.63 (s, 3H). HRMS-TOF (m/z) calcd for C20H24FN5O3S2 [M + H]+: 466.1383, found 466.1375. HPLC purity, 97.6%.

4.1.50. 7-benzyl-N-(2-fluoro-4-(methylsulfonyl)phenyl)-2-methyl-5,6,7,8tetrahydropyrido[3,4-d]pyrimidin-4-amine (47) Follow the similar procedure of 6. Light yellow solid, 54% yield. 1H NMR (400 MHz, CDCl3) δ (ppm): 9.03 (s, 1H), 7.78 (d, J = 8.7 Hz, 1H), 7.71 (d, J = 10.5 Hz, 1H), 7.35 (m, 5H), 6.84 (s, 1H), 3.74 (s, 2H), 3.61 (s, 2H), 3.07 (s, 3H), 2.89 (t, J = 5.7 Hz, 2H), 2.68 (s, 2H), 2.60 (s, 3H). MS-ESI: [M + H]+: 427.3.

4.1.45. N-(2-fluoro-4-(methylsulfonyl)phenyl)-2-methyl-6-(2-(5-phenyl1,3,4-oxadiazol-2-yl)ethyl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4amine (41) Follow the similar procedure of 9–16. Yellow solid, 65% yield. m.p.

4.1.51. 3-fluoro-4-((2-methyl-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin4-yl)amino)benzonitrile (48) Follow the similar procedure of 7–8. Yellow solid, 49% yield. 1H NMR (600 MHz, CDCl3) δ (ppm): 9.01 (t, J = 8.4 Hz, 1H), 7.52 (d, 10

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J = 8.7 Hz, 1H), 7.43 (dd, J = 11.0, 1.8 Hz, 1H), 6.85 (d, J = 4.6 Hz, 1H), 3.99 (s, 2H), 3.28 (t, J = 5.8 Hz, 2H), 2.63 (s, 3H), 2.60 (t, J = 5.7 Hz, 2H), 1.27 (s, 1H). MS-ESI: [M + H]+: 284.3.

4.1.57. 3-fluoro-4-((2-methyl-7-((5-phenyl-1,3,4-oxadiazol-2-yl)methyl)5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yl)amino)benzonitrile (54) Follow the similar procedure of 9–16. Yellow solid, 71% yield. m.p. 192.9–194.6 °C. 1H NMR (400 MHz, DMSO) δ (ppm): 8.97 (t, J = 8.4 Hz, 1H), 8.08 (d, J = 7.3 Hz, 2H), 7.64–7.35 (m, 5H), 6.81 (s, 1H), 4.16 (s, 2H), 3.84 (s, 2H), 3.08 (d, J = 5.0 Hz, 2H), 2.73 (s, 2H), 2.62 (s, 3H). HRMS-TOF (m/z) calcd for C24H20FN7O [M + H]+: 442.1792, found 442.1785. HPLC purity, 99.0%.

4.1.52. N-(2-fluoro-4-(methylsulfonyl)phenyl)-2-methyl-5,6,7,8tetrahydropyrido[3,4-d]pyrimidin-4-amine (49) Follow the similar procedure of 7–8. Yellow solid, 42% yield. 1H NMR (400 MHz, CDCl3) δ (ppm): 9.05 (t, J = 8.3 Hz, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.72 (d, J = 10.5 Hz, 1H), 6.86 (s, 1H), 3.98 (s, 2H), 3.27 (s, 2H), 3.08 (s, 3H), 2.62 (s, 3H), 2.61 (s, 2H), 1.27 (s, 1H). MSESI: [M + H]+: 284.3.

4.1.58. Tert-butyl (R)-(1-(2-(4-((4-cyano-2-fluorophenyl)amino)-2methyl-5,8-dihydropyrido[3,4-d]pyrimidin-7(6H)-yl)acetyl)piperidin-3-yl) carbamate (55) Follow the similar procedure of 9–16. Yellow solid, 62% yield. m.p. 209.3–211.1 °C. 1H NMR (600 MHz, CDCl3) δ (ppm): 9.07–8.83 (m, 1H), 7.51 (d, J = 8.5 Hz, 1H), 7.42 (d, J = 10.9 Hz, 1H), 6.85 (s, 1H), 4.86 (d, J = 5.5 Hz, 1H), 3.83–3.57 (m, 4H), 3.52–3.37 (m, 2H), 3.33 (m, 1H), 2.98 (dd, J = 15.9, 10.7 Hz, 2H), 2.80–2.66 (m, 2H), 2.61 (s, 3H), 1.86 (s, 4H), 1.66–1.52 (m, 2H), 1.42 (s, 9H). 13C NMR (150 MHz, CDCl3) δ (ppm): 168.0, 164.5, 161.7, 156.9, 155.1, 151.2 (d, J = 243.5 Hz), 132.7, 129.5, 121.1, 118.2, 118.0 (d, J = 22.8 Hz), 109.2 (d, J = 28.1 Hz), 104.6 (d, J = 9.4 Hz), 79.6, 59.8, 59.4, 57.4, 57.1, 50.5, 49.1, 47.0, 42.3, 28.3 (x 3), 25.7, 21.8. HRMS-TOF (m/z) calcd for C27H34FN7O3 [M + H]+: 524.2785, found 524.2783. HPLC purity, 96.7%.

4.1.53. 3-fluoro-4-((2-methyl-7-(2-oxo-2-(3-(trifluoromethyl)-5,6dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)ethyl)-5,6,7,8tetrahydropyrido[3,4-d]pyrimidin-4-yl)amino)benzonitrile (50) Follow the similar procedure of 9–16. Yellow solid, 57% yield. m.p. 212.2–214.1 °C. 1H NMR (600 MHz, DMSO‑d6) δ (ppm): 8.50 (d, J = 8.9 Hz, 1H), 7.96 (dt, J = 10.8, 5.4 Hz, 1H), 7.92–7.86 (m, 1H), 7.72–7.62 (m, 1H), 5.11–4.91 (m, 2H), 4.24–4.16 (m, 2H), 4.09–3.97 (m, 2H), 3.57–3.54 (m, 4H), 2.85 (d, J = 5.9 Hz, 2H), 2.76–2.59 (m, 2H), 2.32 (s, 3H). 13C NMR (150 MHz, DMSO‑d6) δ (ppm): 169.0, 163.6, 161.5 (d, J = 5.6 Hz), 158.1, 155.5, 153.8 (d, J = 6.8 Hz), 151.5, 133.3 (d, J = 10.8 Hz), 129.5 (d, J = 3.3 Hz), 119.9 (d, J = 23.7 Hz), 118.9 (q, J = 268.4 Hz), 118.5, 109.7 (d, J = 9.0 Hz), 106.6 (d, J = 9.7 Hz), 59.7, 56.8, 49.3, 44.3, 41.6, 39.0, 25.8, 22.7. HPLC purity, 95.5%.

4.1.59. Tert-butyl 4-(2-(4-((4-cyano-2-fluorophenyl)amino)-2-methyl-5,8dihydropyrido[3,4-d]pyrimidin-7(6H)-yl)acetyl)piperazine-1-carboxylate (56) Follow the similar procedure of 9–16. Yellow solid, 71% yield. m.p. 196.6–197.9 °C. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.98 (t, J = 8.4 Hz, 1H), 7.52 (dt, J = 8.8, 1.3 Hz, 1H), 7.42 (dd, J = 10.9, 1.9 Hz, 1H), 6.83 (d, J = 4.9 Hz, 1H), 3.69 (s, 2H), 3.62 (t, J = 5.5 Hz, 2H), 3.59–3.57 (m, 2H), 3.45–3.42 (m, 6H), 2.97 (t, J = 5.8 Hz, 2H), 2.68 (t, J = 5.9 Hz, 2H), 2.62 (s, 3H), 1.47 (s, 9H). 13C NMR (150 MHz, CDCl3) δ (ppm): 167.6, 164.7, 161.6, 156.9, 154.6, 151.2 (d, J = 243.4 Hz), 132.6 (d, J = 8.5 Hz), 129.5 (d, J = 3.4 Hz), 121.2, 118.1, 118.0, 109.0, 104.7 (d, J = 9.2 Hz), 80.4, 60.1, 57.3, 49.0, 45.5 (x2), 41.7 (x2), 28.4 (x 3), 25.8, 22.1. HRMS-TOF (m/z) calcd for C26H32FN7O3 [M + H]+: 510.2629, found 510.2616. HPLC purity, 96.8%.

4.1.54. (S)-1-(2-(4-((4-cyano-2-fluorophenyl)amino)-2-methyl-5,8dihydropyrido[3,4-d]pyrimidin-7(6H)-yl)acetyl)pyrrolidine-2-carbonitrile (51) Follow the similar procedure of 9–16. Yellow solid, 47% yield. m.p. 208.4–209.8 °C. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.98 (t, J = 8.4 Hz, 1H), 7.52 (d, J = 8.7 Hz, 1H), 7.43 (dd, J = 10.9, 1.8 Hz, 1H), 6.84 (d, J = 4.7 Hz, 1H), 4.80 (dd, J = 8.0, 2.5 Hz, 1H), 3.75 (s, 2H), 3.69–3.53 (m, 2H), 3.52–3.39 (m, 2H), 3.04 (dd, J = 6.6, 5.3 Hz, 2H), 2.71 (t, J = 5.7 Hz, 2H), 2.62 (s, 3H), 2.40–2.13 (m, 4H). 13C NMR (150 MHz, CDCl3) δ (ppm): 168.2, 164.5, 161.6, 156.9 (d, J = 4.4 Hz), 151.2 (d, J = 243.5 Hz), 132.7, (d, J = 8.5 Hz), 129.5 (d, J = 3.2 Hz), 121.2, 118.3, 118.1, 118.0, 109.1, 104.7 (d, J = 9.1 Hz), 59.6, 57.3, 49.1, 46.6, 32.5, 25.8, 25.3, 22.1. HRMS-TOF (m/z) calcd for C22H22FN7O [M + H]+: 420.1948, found 420.1947. HPLC purity, 95.9%.

4.1.60. 2-(4-((2-fluoro-4-(methylsulfonyl)phenyl)amino)-2-methyl-5,8dihydropyrido[3,4-d]pyrimidin-7(6H)-yl)-1-(3-(trifluoromethyl)-5,6dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)ethan-1-one (57) Follow the similar procedure of 9–16. Yellow solid, 64% yield. m.p. 188.7–190.9 °C. 1H NMR (600 MHz, CDCl3) δ (ppm): 9.01 (t, J = 8.2 Hz, 1H), 7.80–7.74 (m, 1H), 7.70 (dd, J = 10.3, 2.1 Hz, 1H), 6.85 (t, J = 5.3 Hz, 1H), 5.19–5.06 (m, 2H), 4.19 (d, J = 5.5 Hz, 2H), 4.13 (d, J = 5.8 Hz, 2H), 3.69 (m, 2H), 3.56 (s, 2H), 3.08 (s, 3H), 2.98 (t, J = 5.8 Hz, 2H), 2.70 (t, J = 5.9 Hz, 2H), 2.62 (s, 3H). 13C NMR (150 MHz, CDCl3) δ (ppm): 168.2 (d, J = 41.8 Hz), 164.8 (d, J = 26.0 Hz), 160.9 (d, J = 12.7 Hz), 157.0, 151.4 (d, J = 246.1 Hz), 150.3, 149.9, 133.2 (d, J = 68.9 Hz), 129.9 (d, J = 5.3 Hz), 124.5 (d, J = 3.3 Hz), 121.3, 118.2 (q, J = 268.8 Hz), 114.0 (d, J = 22.4 Hz), 108.8, 60.5, 56.9, 49.5, 44.7, 43.2, 42.8, 38.5, 25.7, 22.0. HRMS-TOF (m/z) calcd for C23H24F4N8O3S [M + H]+: 569.1706, found 569.1692. HPLC purity, 94.7%.

4.1.55. 3-fluoro-4-((2-methyl-7-(2-oxo-2-(thiazolidin-3-yl)ethyl)-5,6,7,8tetrahydropyrido[3,4-d]pyrimidin-4-yl)amino)benzonitrile (52) Follow the similar procedure of 9–16. Yellow solid, 66% yield. m.p. 228.3–230.9 °C. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.99–8.91 (m, 1H), 7.50 (d, J = 8.7 Hz, 1H), 7.44–7.35 (m, 1H), 6.81 (d, J = 4.5 Hz, 1H), 4.73–4.53 (m, 2H), 3.94–3.78 (m, 2H), 3.78–3.68 (m, 2H), 3.51–3.36 (m, 2H), 3.02 (m, 4H), 2.72–2.65 (m, 2H), 2.61 (d, J = 12.9 Hz, 3H). HRMS-TOF (m/z) calcd for C20H21FN6OS [M + H]+: 413.1560, found 413.1552. HPLC purity, 93.3%. 4.1.56. 3-fluoro-4-((2-methyl-7-(2-(5-phenyl-1,3,4-oxadiazol-2-yl)ethyl)5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-yl)amino)benzonitrile (53) Follow the similar procedure of 9–16. Yellow solid, 63% yield. m.p. 185.8–187.3 °C. 1H NMR (600 MHz, CDCl3) δ (ppm): 8.97 (t, J = 8.4 Hz, 1H), 8.10–7.99 (m, 2H), 7.59–7.48 (m, 4H), 7.42 (dd, J = 10.9, 1.8 Hz, 1H), 6.81 (d, J = 4.7 Hz, 1H), 3.72 (s, 2H), 3.25 (t, J = 7.2 Hz, 2H), 3.14 (t, J = 7.2 Hz, 2H), 2.98 (t, J = 5.8 Hz, 2H), 2.67 (t, J = 5.6 Hz, 2H), 2.62 (s, 3H). 13C NMR (150 MHz, CDCl3) δ (ppm): 165.1, 164.9, 164.6, 161.7, 156.9, 151.2 (d, J = 243.3 Hz), 132.7 (d, J = 8.6 Hz), 131.7, 129.5 (d, J = 3.4 Hz), 129.1 (x 2), 126.8 (x 2), 123.9, 121.2, 118.1, 118.0, 109.2, 104.7 (d, J = 9.1 Hz), 57.1, 53.9, 49.3, 25.8, 24.0, 22.2. HRMS-TOF (m/z) calcd for C25H22FN7O [M + H]+: 456.1948, found 456.1936. HPLC purity, 95.4%.

4.1.61. (S)-1-(2-(4-((2-fluoro-4-(methylsulfonyl)phenyl)amino)-2methyl-5,8-dihydropyrido[3,4-d]pyrimidin-7(6H)-yl)acetyl)pyrrolidine-2carbonitrile (58) Follow the similar procedure of 9–16. Yellow solid, 39% yield. m.p. 215.3–217.4 °C. 1H NMR (600 MHz, CDCl3) δ (ppm): 9.01 (t, J = 8.1 Hz, 1H), 7.77 (d, J = 8.8 Hz, 1H), 7.70 (dd, J = 10.3, 2.0 Hz, 1H), 6.87 (d, J = 4.8 Hz, 1H), 4.79 (t, J = 4.6 Hz, 1H), 3.75 (s, 2H), 3.70 (s, 1H), 3.60–3.52 (m, 1H), 3.50–3.37 (m, 2H), 3.08 (s, 3H), 3.03 11

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(t, J = 6.0 Hz, 2H), 2.71 (t, J = 5.9 Hz, 2H), 2.61 (s, 3H), 2.40–2.21 (m, 2H), 2.20–2.13 (m, 1H), 1.95 (s, 1H). 13C NMR (150 MHz, CDCl3) δ (ppm): 168.2, 164.6, 161.6, 157.0, 151.4 (d, J = 248.5 Hz), 133.3 (d, J = 6.2 Hz), 133.1 (d, J = 8.9 Hz), 124.5 (d, J = 3.3 Hz), 121.3, 118.3, 114.0 (d, J = 22.5 Hz), 109.2, 59.6, 57.2, 49.2, 46.6, 46.2, 44.7, 32.5, 25.8, 25.3, 22.1. HRMS-TOF (m/z) calcd for C22H25FN6O3S [M + H]+: 473.1771, found 473.1762. HPLC purity, 94.0%.

44.8, 41.7 (x2), 32.0, 28.4 (x3), 25.8. HRMS-TOF (m/z) calcd for C26H35FN6O5S [M + H]+: 563.2452, found 563.2441. HPLC purity, 93.8%. 4.2. Human GPR119 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 uM) and incubated for 1 h. Cell lysis buffers (4X Eu-cAMP tracer solution and 4X ULight™-anticAMP solution) were added to each well, and the plates were incubated at room temperature for 1 h before being read on Envision (Perkin Elmer). The assay was performed for three replicates for each concentration.

4.1.62. N-(2-fluoro-4-(methylsulfonyl)phenyl)-2-methyl-7-(2-(5-phenyl1,3,4-oxadiazol-2-yl)ethyl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4amine (59) Follow the similar procedure of 9–16. Yellow solid, 59% yield. m.p. 194.7–197.2 °C. 1H NMR (600 MHz, CDCl3) δ (ppm): 9.12–8.97 (m, 1H), 8.05 (dt, J = 6.8, 1.5 Hz, 2H), 7.84–7.77 (m, 1H), 7.72 (dd, J = 10.4, 2.1 Hz, 1H), 7.60–7.45 (m, 3H), 6.83 (d, J = 4.9 Hz, 1H), 3.73 (s, 2H), 3.25 (t, J = 7.2 Hz, 2H), 3.15 (t, J = 7.2 Hz, 2H), 3.08 (s, 3H), 2.99 (t, J = 5.8 Hz, 2H), 2.68 (t, J = 5.8 Hz, 2H), 2.63 (s, 3H). 13C NMR (150 MHz, CDCl3) δ (ppm): 165.2, 164.9, 164.6, 161.8, 157.0, 151.4 (d, J = 245.8 Hz), 133.3 (d, J = 6.0 Hz), 133.1 (d, J = 8.6 Hz), 129.1 (x 2), 126.8 (x 2), 124.5, 123.9, 121.2, 114.0 (d, J = 22.5 Hz), 109.3, 57.2, 53.9, 49.3, 44.8, 25.8, 24.1, 22.3. HRMS-TOF (m/z) calcd for C25H25FN6O3S [M + H]+: 509.1771, found 509.1760. HPLC purity, 92.4%.

4.3. DPP-4 inhibition assay

4.1.63. N-(2-fluoro-4-(methylsulfonyl)phenyl)-2-methyl-7-((5-phenyl1,3,4-oxadiazol-2-yl)methyl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4amine (60) Follow the similar procedure of 9–16. Yellow solid, 58% yield. m.p. 190.8–192.2 °C. 1H NMR (600 MHz, CDCl3) δ (ppm): 9.02 (t, J = 8.3 Hz, 1H), 8.08–8.00 (m, 2H), 7.79 (d, J = 8.8 Hz, 1H), 7.76–7.67 (m, 1H), 7.55 (m, 3H), 6.85 (d, J = 4.8 Hz, 1H), 4.16 (s, 2H), 3.84 (s, 2H), 3.08 (m, 5H), 2.74 (t, J = 6.0 Hz, 2H), 2.62 (s, 3H). 13C NMR (150 MHz, CDCl3) δ (ppm): 165.6, 164.7, 162.9, 161.4, 157.0, 151.4 (d, J = 245.7 Hz), 133.4 (d, J = 6.1 Hz), 133.0 (d, J = 8.7 Hz), 132.0, 129.1 (x 2), 127.0 (x 2), 124.5 (d, J = 3.4 Hz), 123.6, 121.2, 114.0 (d, J = 22.6 Hz), 108.9, 56.8, 51.1, 48.9, 44.8, 25.8, 22.1. HRMS-TOF (m/ z) calcd for C24H23FN6O3S [M + H]+: 495.1615, found 495.1598. HPLC purity, 96.0%.

DPP-4 inhibition assay was performed by using Gly-Pro-PNA as substrate, which could be cleaved by the enzyme to release yellow pnitroaniline (PNA). The absorbance of characteristic peak at 405 nm was detected to represent the enzymatic activity. A test well contained 10 μL of compound solution (100 μM), 10 μL of DPP-4 enzyme (4.5 mU/ mL), 10 μL of Gly-Pro-PNA (0.05 mM) and 70 μL of Tris–HCl buffer (pH 8.2) in a 96-well plate. Each well was filled with buffer to the volume of 100 μL. The absorbance (OD) at 405 nm was detected after 1 h of incubation at 37 °C, then the percentage of inhibition was calculated as: [(ODenzyme+substrate − ODsubstrate) − (ODenzyme+substrate+compd − ODsubstrate+compd)]/(OD enzyme+substrate − ODsubstrate) × 100%. 4.4. oGTT in C57BL/6N mice

4.1.64. Tert-butyl (R)-(1-(2-(4-((2-fluoro-4-(methylsulfonyl)phenyl) amino)-2-methyl-5,8-dihydropyrido[3,4-d]pyrimidin-7(6H)-yl)acetyl) piperidin-3-yl)carbamate (61) Follow the similar procedure of 9–16. Yellow solid, 55% yield. m.p. 235.4–237.2 °C. 1H NMR (600 MHz, CDCl3) δ (ppm): 9.05 (m, 1H), 7.79 (dd, J = 8.7, 2.2 Hz, 1H), 7.72 (dt, J = 10.4, 2.4 Hz, 1H), 6.87 (s, 1H), 4.85 (d, J = 6.6 Hz, 1H), 3.84–3.76 (m, 1H), 3.75–3.29 (m, 8H), 3.09 (s, 3H), 3.00 (m, 2H), 2.72 (m, 2H), 2.63 (s, 3H), 2.08–1.83 (m, 2H), 1.67–1.53 (m, 2H), 1.38 (s, 9H). 13C NMR (150 MHz, CDCl3) δ (ppm): 167.4, 163.8, 161.2, 156.3, 154.5, 150.7 (d, J = 246.0 Hz), 132.6, 129.3, 123.9, 120.4, 113.4 (d, J = 22.3 Hz), 108.6 (d, J = 28.1 Hz), 79.0, 58.8, 56.4, 49.9, 48.5, 46.4, 44.1, 41.7, 27.8, 27.7 (x 3), 25.2, 21.8, 21.2. HRMS-TOF (m/z) calcd for C27H37FN6O5S [M + H]+: 577.2608, found 577.2591. HPLC purity, 95.2%.

For the acute single dose study, vehicle (0.5% carboxymethylcellulose sodium, 10 mL/kg), compound 51 (15 and 30 mg/kg) and vildagliptin (30 mg/kg) were administered to C57BL/6N mice after 16-hrs 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 (AUC0-2h). Declaration of Competing Interest The authors declared that there is no conflict of interest. Acknowledgements 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).

4.1.65. Tert-butyl 4-(2-(4-((2-fluoro-4-(methylsulfonyl)phenyl)amino)-2methyl-5,8-dihydropyrido[3,4-d]pyrimidin-7(6H)-yl)acetyl)piperazine-1carboxylate (62) Follow the similar procedure of 9–16. Yellow solid, 63% yield. m.p. 183.2–185.7 °C. 1H NMR (600 MHz, CDCl3) δ (ppm): 9.05 (t, J = 8.2 Hz, 1H), 7.85–7.78 (m, 1H), 7.73 (dd, J = 10.4, 2.1 Hz, 1H), 6.85 (d, J = 5.0 Hz, 1H), 3.70 (s, 2H), 3.63 (s, 2H), 3.59 (m, 2H), 3.46 (s, 2H), 3.44 (m, 4H), 3.09 (s, 3H), 2.98 (t, J = 5.8 Hz, 2H), 2.70 (t, J = 5.9 Hz, 2H), 2.64 (s, 3H), 1.48 (s, 9H). 13C NMR (150 MHz, CDCl3) δ (ppm): 167.6, 164.7, 161.6, 156.9, 154.6, 151.4 (d, J = 246.3 Hz), 133.2 (d, J = 32.4 Hz), 129.9 (d, J = 5.0 Hz), 124.5 (d, J = 3.3 Hz), 121.1, 114.0 (d, J = 22.5 Hz), 109.0, 80.4, 60.1, 57.3, 49.1, 45.5 (x2),

References [1] Y. Fang, J. Xu, Z. Li, Z. Yang, L. Xiong, Y. Jin, Q. Wang, S. Xie, W. Zhu, S. Chang, Design and synthesis of novel pyrimido[5,4-d]pyrimidine derivatives as GPR119 agonist for treatment of type 2 diabetes, Bioorg. Med. Chem. 26 (2018) 4080–4087. [2] J. Lan, Y. Zhao, F. Dong, Z. Yan, W. Zheng, J. Fan, G. Sun, Meta-analysis of the

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