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

Article abstract of DOI:10.1016/j.bioorg.2019.103390

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 AUC_0-2h 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.

Full text of DOI:10.1016/j.bioorg.2019.103390

BioorganicChemistryxxx(xxxx)xxxx
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
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
b National Engineering Research Center for Manufacturing Technology of TCM Solid Preparation, Jiangxi University of Traditional Chinese Medicine, Nanchang 330006,
China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Based on the approach of merged pharmacophores of GPR119 agonists and DPP-4 inhibitors, a series of tetra-
hydropyridopyrimidine 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 tetra-
hydropyridopyrimidine. 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.
Tetrahydropyridopyrimidine
GPR119
DPP-4
Design
Hypoglycemic activity
1. Introduction
[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 in-
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 le-
vels [6,7]. Thus, there is a strong need of new orally available anti-
diabetic drugs with new mechanism of action, improved efficacy and
safety [8].
hibitors 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 suc-
cessfully 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 pro-
posed a novel strategy of developing fused pyrimidines as dual mod-
ulators 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.
G-protein coupled receptor 119 (GPR119) is expressed pre-
dominantly 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 gly-
coprotein, which can give rise to the rapid inactivation of GLP-1
⁎ Corresponding authors.
E-mail addresses: yinghua616909@hotmail.com (Y. Jin), joshyyy@126.com (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/©2019ElsevierInc.Allrightsreserved.
Pleasecitethisarticleas:YuanyingFang,etal.,BioorganicChemistry,https://doi.org/10.1016/j.bioorg.2019.103390

Y. Fang, et al.
BioorganicChemistryxxx(xxxx)xxxx
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.
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
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]. Substitu-
tion 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.
such
as
3-aminopiperidine
of
trelagliptin
and
tetra-
hydrotriazolopyrazine 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.
4,7-disubstituted tetrahydropyrido[3,4-d]pyrimidine compounds
20–23 were synthesized following the similar procedure of 4,6-dis-
ubstituted 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
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 si-
milar procedure with 4,7-disubstituted tetrahydropyrido[3,4-d]pyr-
imidine compounds.
bearing A2, A6 or A7 fragment retained potent GPR119 agonistic ac-
tivities (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 frag-
ment A5 and A6 showed weak inhibitory activities.
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].
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 ac-
tivities (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.
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.
To predict the physicochemical and drug-like properties, we calcu-
lated 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 pharmacoki-
netics issues.
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
activity of the GPR119 agonist compound 51.
3. Conclusion
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.
In summary, a series of tetrahydropyridopyrimidine derivatives
were designed and synthesized for the discovery of dual ligands tar-
geting GPR119 and DPP-4. Seven fragments of DPP-4 inhibitor phar-
macophores A1–A7 were connected to the head of tetra-
hydropyridopyrimidine 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 ac-
tivity (EC50 = 8.7 nM) and good inhibition rate of 74.5% against DPP-4
at 10 μM. Moreover, compound 51 also demonstrated stronger hy-
poglycemic 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.
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
(n = 8).
SE. *, p < 0.05 compared to vehicle group
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 2-
cyanopyrrolidine 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]pyr-
imidine moiety interacted with the residues Glu205 and Arg125 in S2
pocket. Furthermore, the tail fragment of 2-fluoro-4-cyanoaniline in-
teracted 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

Y. Fang, et al.
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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, 1,2-
dichloroethane, reflux, overnight, ii MeOH, reflux, 3 h; (c) A1–A7, K2CO3, DMF, 70 ℃, overnight.
Table 1
Table 2
GPR119 agonistic and DPP-4 inhibitory activities of 4,6-disubstituted tetra-
GPR119 agonistic and DPP-4 inhibitory activities of 4,7-disubstituted tetra-
hydropyrido[4,3-d]pyrimidine compounds.
hydropyrido[4,3-d]pyrimidine compounds.
ClogPb
Compd. R2
R4
R7 GPR119 activation
DPP-4 inhibition
ClogPb
Compd. R2
R4
R6 GPR119 activation
DPP-4 inhibition
EC50 (μM)
%maxa
%inhibition (10 μM)
EC50 (μM)
> 10
%maxa
%inhibition (10 μM)
9
H
H
H
H
H
H
H
H
CN A1
CN A3
CN A4
CN A5
> 10
> 10
> 10
> 10
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
–
–
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
–
20
21
22
23
50
51
52
53
54
55
56
57
58
59
60
61
62
H
H
H
H
Ms A1
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
–
–
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
–
10
11
12
13
14
15
16
32
33
34
35
36
37
38
39
40
41
42
43
44
–
Ms A2 0.090
Ms A6 0.021
Ms A7 0.085
75.8
67.1
77.9
–
–
–
Me CN A1
> 10
CN A6 0.45
33.5
–
Me CN A2 0.0087
74.5
–
Ms A1
Ms A3 7.9
Ms A4 > 10
> 10
> 10
Me CN A3
Me CN A4
Me CN A5
> 10
> 10
> 10
26.7
–
–
Me CN A1
–
–
Me CN A3 8.2
30.0
–
Me CN A6 0.012
Me CN A7 0.057
64.7
18.5
–
Me CN A4
> 10
Me Ms A1
> 10
Me CN A5 7.7
Me CN A6 0.036
Me CN A7 0.13
54.3
44.7
28.8
–
Me Ms A2 0.042
72.5
–
Me Ms A4
> 10
Me Ms A5 6.8
Me Ms A6 0.024
Me Ms A7 0.068
11.3
70.4
58.9
–
Me Ms A1
> 10
Me Ms A2 0.054
83.2
52.4
–
Me Ms A3 7.2
Me Ms A4
> 10
GSK1292263
Vildagliptin
0.0066
–
99.6
–
Me Ms A5 8.3
Me Ms A6 0.030
Me Ms A7 0.078
19.5
65.1
5.7
–
a
b
%max: cAMP stimulation % compared to maximal effect of GSK1292263.
GSK1292263
Vildagliptin
0.0066
–
ClogP was calculated using ACD software from Discovery Studio 4.5.
99.6
–
4.1.2. (S)-1-(2-chloroacetyl)pyrrolidine-2-carbonitrile (A2)
a
b
%max: cAMP stimulation % compared to maximal effect of GSK1292263.
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 pro-
duct 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.
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 atmo-
sphere. 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 com-
bined, washed with brine (10 mL), dried over anhydrous MgSO4, fil-
tered 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

Y. Fang, et al.
BioorganicChemistryxxx(xxxx)xxxx
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 atmo-
sphere. The reaction mixture was warmed to ambient temperature and
stirred for 1 h. The mixture was diluted with CH2Cl2 (30 mL) and wa-
shed with brine (10 mL × 2). The organic layer was dried over anhy-
drous 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
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).
1H 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.
(600 MHz, DMSO)
δ (ppm): 4.61 (d, J = 16.9 Hz, 2H), 4.09 (d,
4.1.5. 2-(Chloromethyl)-5-phenyl-1,3,4-oxadiazole (A5)
J = 14.1 Hz, 2H), 3.89 (t, J = 6.4 Hz, 1H), 3.86 (t, J = 6.2 Hz, 1H),
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.
3.15 (t, J = 6.2 Hz, 1H), 3.05 (t, J = 6.4 Hz, 1H). MS-ESI: [M + H]+
:
165.2.
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 re-
fluxed 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

Y. Fang, et al.
BioorganicChemistryxxx(xxxx)xxxx
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)
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 atmo-
sphere. The reaction mixture was warmed to ambient temperature and
stirred for 1 h. The mixture was diluted with CH2Cl2 (30 mL) and wa-
shed with brine (10 mL × 2). The organic layer was dried over anhy-
drous 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.
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
4.1.12. 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)-5,6,7,8-
tetrahydropyrido[4,3-d]pyrimidin-4-amine (6)
4.1.14. 3-fluoro-4-((6-(2-oxo-2-(thiazolidin-3-yl)ethyl)-5,6,7,8-
To the solution of 6-benzyl-4-chloro-5,6,7,8-tetrahydropyrido[4,3-
d]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 over-
night. 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.
tetrahydropyrido[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)-5,6,7,8-
tetrahydropyrido[4,3-d]pyrimidin-4-yl)amino)benzonitrile (11)
4.1.10. 3-fluoro-4-((5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-yl)
amino)benzonitrile (7)
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,
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 (pet-
roleum 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,
32.1, 24.2. HRMS-TOF (m/z) calcd for
C
24H20FN7O [M + H]+
:
442.1792, found 442.1781. HPLC purity, 96.6%.
4.1.16. 3-fluoro-4-((6-((5-phenyl-1,3,4-oxadiazol-2-yl)methyl)-5,6,7,8-
tetrahydropyrido[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

Y. Fang, et al.
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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)-7,8-
dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)acetyl)piperidin-3-yl)carbamate
(13)
4.1.23. General procedure for compounds 20–23
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
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 eva-
porated. The residue was purified by column chromatography to afford
the desired product.
4.1.24. 2-(4-((2-fluoro-4-(methylsulfonyl)phenyl)amino)-5,8-
dihydropyrido[3,4-d]pyrimidin-7(6H)-yl)-1-(3-(trifluoromethyl)-5,6-
dihydro-[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
(x3), 22.6. HRMS-TOF (m/z) calcd for
C
26H32FN7O3 [M + H]+
:
510.2629, found 510.2619. HPLC purity, 95.2%.
4.1.18. 2-(4-((2-fluoro-4-(methylsulfonyl)phenyl)amino)-7,8-
dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)-1-(3-(trifluoromethyl)-5,6-
dihydro-[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%.
C
22H22F4N8O3S [M + H]+: 555.1550, found 555.1534. HPLC purity,
92.6%.
4.1.25. (S)-1-(2-(4-((2-fluoro-4-(methylsulfonyl)phenyl)amino)-5,8-
dihydropyrido[3,4-d]pyrimidin-7(6H)-yl)acetyl)pyrrolidine-2-carbonitrile
(21)
4.1.19. 2-(4-((2-fluoro-4-(methylsulfonyl)phenyl)amino)-7,8-
dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)-1-(thiazolidin-3-yl)ethan-1-one
(15)
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),
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%.
2.16–1.96 (m, 2H). HRMS-TOF (m/z) calcd for
C21H23FN6O3S
[M + H]+: 459.1615, found 459.1613. HPLC purity, 94.9%.
4.1.26. 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),
4.1.20. N-(2-fluoro-4-(methylsulfonyl)phenyl)-6-(2-(5-phenyl-1,3,4-
oxadiazol-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,
1.39 (s, 9H). HRMS-TOF (m/z) calcd for C26H35FN6O5S [M + H]+
:
563.2452, found 563.2444. HPLC purity, 95.3%.
J = 7.4 Hz, 2H), 3.24 (t, J = 7.6 Hz, 2H), 3.09 (s, 3H), 2.99 (s, 4H). 13
C
4.1.27. Tert-butyl
4-(2-(4-((2-fluoro-4-(methylsulfonyl)phenyl)amino)-
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. HRMS-
5,8-dihydropyrido[3,4-d]pyrimidin-7(6H)-yl)acetyl)piperazine-1-
carboxylate (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),
TOF (m/z) calcd for
C
24H23FN6O3S [M + H]+
: 495.1615, found
495.1598. HPLC purity, 95.6%.
4.1.21. 7-benzyl-N-(2-fluoro-4-(methylsulfonyl)phenyl)-5,6,7,8-
tetrahydropyrido[3,4-d]pyrimidin-4-amine (18)
1.48 (s, 9H). HRMS-TOF (m/z) calcd for C25H33FN6O5S [M + H]+
:
549.2295, found 549.2282. HPLC purity, 95.0%.
The title intermediate was generated from 17 using similar proce-
dure 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

Y. Fang, et al.
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overnight under nitrogen atmosphere. The reaction mixture was eva-
porated 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-5,6,7,8-
tetrahydropyrido[4,3-d]pyrimidin-4-amine (31)
Follow the similar procedure of 7–8. Light yellow solid, 72% yield.
1H 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)-5,6-
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 (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)-5,6,7,8-
tetrahydropyrido[4,3-d]pyrimidin-4-yl)amino)benzonitrile (33)
4.1.30. 6-Benzyl-2-methyl-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-ol
(26)
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%.
To the solution of methyl 1-benzyl-4-oxopiperidine-3-carboxylate
(2.5 g, 10.1 mmol) in MeOH (15 mL) was added acetamidine hydro-
chloride (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 fil-
tered 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,
4.1.31. 6-Benzyl-4-chloro-2-methyl-5,6,7,8-tetrahydropyrido[4,3-d]
pyrimidine (27)
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,
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.
32.0, 25.8, 24.2. HRMS-TOF (m/z) calcd for C25H22FN7O [M + H]+
:
456.1948, found 456.1933. HPLC purity, 95.3%.
4.1.32. 4-((6-benzyl-2-methyl-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-
4.1.39. 3-fluoro-4-((2-methyl-6-((5-phenyl-1,3,4-oxadiazol-2-yl)methyl)-
4-yl)amino)-3-fluorobenzonitrile (28)
5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-yl)amino)benzonitrile (35)
Follow the similar procedure of 6. Light yellow solid, 62% yield. 1
H
Follow the similar procedure of 9–16. Yellow solid, 51% yield. m.p.
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.
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). 13
C
4.1.33. 6-benzyl-N-(2-fluoro-4-(methylsulfonyl)phenyl)-2-methyl-5,6,7,8-
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. HRMS-
tetrahydropyrido[4,3-d]pyrimidin-4-amine (29)
Follow the similar procedure of 6. Light yellow solid, 72% yield. 1
H
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.
TOF (m/z) calcd for
C
24H20FN7O [M + H]+
: 442.1792, found
442.1793. HPLC purity, 97.0%.
4.1.40. Tert-butyl
(R)-(1-(2-(4-((4-cyano-2-fluorophenyl)amino)-2-
methyl-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)acetyl)piperidin-3-yl)
carbamate (36)
4.1.34. 3-fluoro-4-((2-methyl-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-
4-yl)amino)benzonitrile (30)
Follow the similar procedure of 9–16. Yellow solid, 66% yield. m.p.
Follow the similar procedure of 7–8. Light yellow solid, 58% yield.
1H 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.
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),
9

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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%.
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
C
25H25FN6O3S [M + H]+: 509.1771, found 509.1762. HPLC purity,
92.3%.
4.1.46. N-(2-fluoro-4-(methylsulfonyl)phenyl)-2-methyl-6-((5-phenyl-
1,3,4-oxadiazol-2-yl)methyl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-
amine (42)
4.1.41. Tert-butyl 4-(2-(4-((4-cyano-2-fluorophenyl)amino)-2-methyl-7,8-
dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)acetyl)piperazine-1-carboxylate
(37)
Follow the similar procedure of 9–16. Yellow solid, 53% yield. m.p.
Follow the similar procedure of 9–16. Yellow solid, 73% yield. m.p.
189.3–190.6 °C. 1H NMR (600 MHz, CDCl3)
δ (ppm): 8.90 (t,
213.8–215.6 °C. 1H NMR (600 MHz, CDCl3)
δ
(ppm): 8.86 (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%.
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
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)
3), 25.8. HRMS-TOF (m/z) calcd for
C
26H32FN7O3 [M + H]+
:
510.2629, found 510.2626. HPLC purity, 96.3%.
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,
4.1.42. 2-(4-((2-fluoro-4-(methylsulfonyl)phenyl)amino)-2-methyl-7,8-
dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)-1-(3-(trifluoromethyl)-5,6-
dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)ethan-1-one (38)
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). HRMS-
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).
13C 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%.
TOF (m/z) calcd for
C
27H37FN6O5S [M + H]+
: 577.2608, found
577.2593. HPLC purity, 96.6%.
4.1.48. Tert-butyl 4-(2-(4-((2-fluoro-4-(methylsulfonyl)phenyl)amino)-2-
methyl-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)acetyl)piperazine-1-
carboxylate (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)-2-
methyl-5,8-dihydropyrido[3,4-d]pyrimidin-7(6H)-yl)acetyl)pyrrolidine-2-
carbonitrile (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),
4.1.49. 4-((7-benzyl-2-methyl-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-
4-yl)amino)-3-fluorobenzonitrile (46)
2.06–1.99 (m, 2H). HRMS-TOF (m/z) calcd for
C
22H25FN6O3S
Follow the similar procedure of 6. Light yellow solid, 71% yield. 1
H
[M + H]+: 473.1771, found 473.1758. HPLC purity, 98.1%.
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-7,8-
dihydropyrido[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.
4.1.50. 7-benzyl-N-(2-fluoro-4-(methylsulfonyl)phenyl)-2-methyl-5,6,7,8-
221.5–223.7 °C. 1H NMR (600 MHz, CDCl3)
δ
(ppm): 8.92 (t,
tetrahydropyrido[3,4-d]pyrimidin-4-amine (47)
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%.
Follow the similar procedure of 6. Light yellow solid, 54% yield. 1
H
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-phenyl-
1,3,4-oxadiazol-2-yl)ethyl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-
amine (41)
4.1.51. 3-fluoro-4-((2-methyl-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-
4-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,
Follow the similar procedure of 9–16. Yellow solid, 65% yield. m.p.
10

Y. Fang, et al.
BioorganicChemistryxxx(xxxx)xxxx
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),
4.1.52. N-(2-fluoro-4-(methylsulfonyl)phenyl)-2-methyl-5,6,7,8-
tetrahydropyrido[3,4-d]pyrimidin-4-amine (49)
2.62 (s, 3H). HRMS-TOF (m/z) calcd for C24H20FN7O [M + H]+
:
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). MS-
ESI: [M + H]+: 284.3.
442.1792, found 442.1785. HPLC purity, 99.0%.
4.1.58. Tert-butyl
(R)-(1-(2-(4-((4-cyano-2-fluorophenyl)amino)-2-
methyl-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,
4.1.53. 3-fluoro-4-((2-methyl-7-(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[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%.
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%.
4.1.59. Tert-butyl 4-(2-(4-((4-cyano-2-fluorophenyl)amino)-2-methyl-5,8-
dihydropyrido[3,4-d]pyrimidin-7(6H)-yl)acetyl)piperazine-1-carboxylate
(56)
4.1.54. (S)-1-(2-(4-((4-cyano-2-fluorophenyl)amino)-2-methyl-5,8-
dihydropyrido[3,4-d]pyrimidin-7(6H)-yl)acetyl)pyrrolidine-2-carbonitrile
(51)
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,
Follow the similar procedure of 9–16. Yellow solid, 47% yield. m.p.
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,
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
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
C
26H32FN7O3 [M + H]+: 510.2629, found 510.2616. HPLC purity,
96.8%.
C
22H22FN7O [M + H]+: 420.1948, found 420.1947. HPLC purity,
95.9%.
4.1.60. 2-(4-((2-fluoro-4-(methylsulfonyl)phenyl)amino)-2-methyl-5,8-
dihydropyrido[3,4-d]pyrimidin-7(6H)-yl)-1-(3-(trifluoromethyl)-5,6-
dihydro-[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.
4.1.55. 3-fluoro-4-((2-methyl-7-(2-oxo-2-(thiazolidin-3-yl)ethyl)-5,6,7,8-
tetrahydropyrido[3,4-d]pyrimidin-4-yl)amino)benzonitrile (52)
188.7–190.9 °C. 1H NMR (600 MHz, CDCl3)
δ (ppm): 9.01 (t,
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 = 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
J = 12.9 Hz, 3H). HRMS-TOF (m/z) calcd for C20H21FN6OS [M + H]+
:
(150 MHz, CDCl3)
δ (ppm): 168.2 (d, J = 41.8 Hz), 164.8 (d,
413.1560, found 413.1552. HPLC purity, 93.3%.
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%.
4.1.56. 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,
4.1.61. (S)-1-(2-(4-((2-fluoro-4-(methylsulfonyl)phenyl)amino)-2-
methyl-5,8-dihydropyrido[3,4-d]pyrimidin-7(6H)-yl)acetyl)pyrrolidine-2-
carbonitrile (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
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%.
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,
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
25.8, 25.3, 22.1. HRMS-TOF (m/z) calcd for C22H25FN6O3S [M + H]+
:
473.1771, found 473.1762. HPLC purity, 94.0%.
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 accu-
mulation 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-sus-
pended 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™-anti-
cAMP 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 con-
centration.
4.1.62. N-(2-fluoro-4-(methylsulfonyl)phenyl)-2-methyl-7-(2-(5-phenyl-
1,3,4-oxadiazol-2-yl)ethyl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-
amine (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
C
25H25FN6O3S [M + H]+: 509.1771, found 509.1760. HPLC purity,
92.4%.
4.1.63. N-(2-fluoro-4-(methylsulfonyl)phenyl)-2-methyl-7-((5-phenyl-
1,3,4-oxadiazol-2-yl)methyl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-
amine (60)
4.3. DPP-4 inhibition assay
DPP-4 inhibition assay was performed by using Gly-Pro-PNA as
substrate, which could be cleaved by the enzyme to release yellow p-
nitroaniline (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 in-
cubation at 37 °C, then the percentage of inhibition was calculated as:
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%.
[(ODenzyme+substrate
−
ODsubstrate
)
−
(ODenzyme+substrate+compd
−
ODsubstrate+compd)]/(OD
− ODsubstrate) × 100%.
enzyme+substrate
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)
For the acute single dose study, vehicle (0.5% carbox-
ymethylcellulose 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).
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,
Declaration of Competing Interest
The authors declared that there is no conflict of interest.
Acknowledgements
21.8, 21.2. HRMS-TOF (m/z) calcd for C27H37FN6O5S [M + H]+
:
577.2608, found 577.2591. HPLC purity, 95.2%.
4.1.65. Tert-butyl 4-(2-(4-((2-fluoro-4-(methylsulfonyl)phenyl)amino)-2-
methyl-5,8-dihydropyrido[3,4-d]pyrimidin-7(6H)-yl)acetyl)piperazine-1-
carboxylate (62)
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).
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),
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