Design and synthesis of novel pyrimido[5,4-d]pyrimidine derivatives as GPR119 agonist for treatment of type 2 diabetes

Article abstract of DOI:10.1016/j.bmc.2018.06.035

We described the discovery and optimization of a novel series of pyrimidopyrimidine derivatives as G-protein coupled receptor 119 (GPR119) agonists against type 2 diabetes. Most designed compounds displayed significant GPR119 agonistic activities. Optimized analogues 15a and 21e exhibited highly potent agonistic activities with single digit EC₅₀ values (2.2 nM and 8.1 nM, respectively). Therefore, 15a and 21e were evaluated for their oral glucose tolerance test (oGTT) in C57BL/6N mice. Compound 15a reduced the blood glucose area of under curve from 0 to 2 h (AUC_0–2h) to 13.5% at the dose of 15 mg/kg comparing with Metformin reduced 18% of AUC_0–2h at the dose of 300 mg/kg.

Full text of DOI:10.1016/j.bmc.2018.06.035

Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of novel pyrimido[5,4-d]pyrimidine derivatives as
GPR119 agonist for treatment of type 2 diabetes
a
b
a
b,⁎
a
a
a
Yuanying Fang , Jun Xu , Zhifeng Li , Zunhua Yang , Lijuan Xiong , Yi Jin , Qi Wang ,
a
c
d,⁎
Saisai Xie , Wufu Zhu , Sheng Chang
a
National Engineering Research Center for Manufacturing Technology of TCM Solid Preparation, Jiangxi University of Traditional Chinese Medicine, Nanchang 330006,
China
b
c
College of Pharmacy, Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, China
College of Pharmacy, Jiangxi Science & Technology Normal University, Nanchang 330013, China
College of Pharmacy, Jilin Medical University, Jilin 132013, China
d
A R T I C L E I N F O
A B S T R A C T
Keywords:
We described the discovery and optimization of a novel series of pyrimidopyrimidine derivatives as G-protein
coupled receptor 119 (GPR119) agonists against type 2 diabetes. Most designed compounds displayed significant
GPR119 agonistic activities. Optimized analogues 15a and 21e exhibited highly potent agonistic activities with
single digit EC50 values (2.2 nM and 8.1 nM, respectively). Therefore, 15a and 21e were evaluated for their oral
glucose tolerance test (oGTT) in C57BL/6N mice. Compound 15a reduced the blood glucose area of under curve
from 0 to 2 h (AUC0–2h) to 13.5% at the dose of 15 mg/kg comparing with Metformin reduced 18% of AUC0–2h at
the dose of 300 mg/kg.
GPR119 agonist
Type 2 diabetes
Pyrimido[5,4-d]pyrimidine
oGTT
1. Introduction
target for discovery of antidiabetic agents.
After Arena researchers disclosed the first potent and oral small
Type 2 diabetes mellitus (T2DM), a progressive chronic disease, is
molecule GPR119 agonist, in the form of AR231453, a number of
characterized by the inability of the human body to keep the blood
glucose level in the required and balanced range in the fed and/or
pharmaceutical companies and academic institutes devoted to develop
To date, a few compounds as
GPR119 agonists such as MBX-2982, DS-8500a, BMS-903452,
GPR119 agonists against T2DM.1
7–22
1–3
fasting state.
The number of people with T2DM worldwide is more
than 300 million, and the prevalence is predicted to rise to 642 million
LEZ763, ZYG-19 have been pushed into clinical trials. However, none
4
–6
5,23
by 2040.
In clinical therapy for T2DM, multiple antidiabetic agents
of them has been approved by FDA (Fig. 2).
So more and more
such as insulin, metformin, thiazolidiones (TZDs), sulfonylurea deri-
vatives (SUs), glucagon-like peptide 1 (GLP-1) analogues and dipeptidyl
peptidase 4 (DPP4) inhibitors have been used. However, many patients
scaffolds have been discovered and evaluated for their anti-diabetes
potency.
We previously reported a series of 5-nitropyrimidine and quinazo-
line derivatives as GPR119 agonist. Among them, compound 1 con-
taining 5-nitropyrimidine with an endo-azabicyclic amino fragment
7
are still unable to achieve their target glycemic control and all current
8
diabetes drugs have known adverse effects. Therefore, new drugs with
greater safety and efficacy are strongly needed for treatment of dia-
betes.
exhibited
potent
and
full
GPR119
agonistic
activity
(EC50 = 1.5 nM).2
4,25
But nitro-compound always shows the hepato-
GPR119 is a class A type of G Protein coupled receptor, which is
expressed primarily in pancreatic β-cells and the K and L cells of the
toxicity. We found the fused 4,8-disubstitute pyrimidopyrimidine could
be the molecular variation of 5-nitropyrimidine with similar structures
and easily obtainable from synthetic precursor of dipyridamole.
Therefore, we designed to replace the 5-nitropyrimidine with pyr-
imidopyrimidine. Then three anilinos substituted by electron-with-
drawing groups (EWG) were introduced as head moiety connected to
the new scaffold (Fig. 3). We also retained endo- and exo-azabicyclic
fragments as tail moiety to confirm the rationality of scaffold hopping
9
,10
gastrointestinal tract.
Oleoylethanolamide (OEA) and N-oleoyldo-
pamine (OLDA) have been identified and investigated for their biolo-
gical effects as endogenous natural GPR119 agonists (Fig. 1).1
1–13
The
stimulation of GPR119 could increase the cellular cAMP levels, leading
to glucose-dependent insulin secretion from pancreatic β-cells and in-
1
4–16
cretin release.
The dual mechanism makes GPR119 a promising
⁎
Corresponding authors.
E-mail addresses: joshyyy@126.com (Z. Yang), changsheng-pharm@hotmail.com (S. Chang).
https://doi.org/10.1016/j.bmc.2018.06.035
Received 11 June 2018; Received in revised form 25 June 2018; Accepted 26 June 2018
0968-0896/ © 2018 Elsevier Ltd. All rights reserved.
Please cite this article as: Fang, Y., Bioorganic & Medicinal Chemistry (2018), https://doi.org/10.1016/j.bmc.2018.06.035

Y. Fang et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
tetrachloropyrimido[5,4-d]pyrimidine 12 with 2-fluoro-4-(methylthio)
aniline followed by oxidation with 54% yield in two steps. Reaction of
1
1
3 with azabicyclic amines and diisopropylethylamine (DIPEA) yielded
4a–14b, following by dechlorination using 10% palladium hydroxide
(
Pa(OH)
2
) and potassium hydroxide (KOH) under hydrogen to obtain
15a–15b in ca. 10% yield due to occurrence of defluorination. When
triethylamine (TEA) was used instead of KOH, the yield could be im-
proved to some degree.
The synthesis of analogues 18a–18e were shown in Scheme 3, si-
milar to derivatives 15a–15b. Without fluorine on aniline group, de-
chlorination resulted in better yields.
Compounds 21a–21h were synthesized with the similar procedure
with derivatives 15a–15b (Scheme 4). The reaction of intermediate 19
with alcohol fragments 3, 4 and 5 under strong basic condition
(LiHMDS) furnished compounds 20b–20d.
Fig. 1. The structures of OEA and OLDA.
2.2. Biological activity
Analogues 15a–b, 18a–18e, 21a–21h were evaluated for their
abilities to activate the human GPR119 receptor in a cell-based cAMP
assay, which were expressed in EC50 and %max values. GSK-1292263
was chosen as the positive control. The EC50 values represent the
concentration of the tested compounds for 50% cAMP stimulation,
while the %max values present the relative response (%) of the tested
compounds compared to the maximal effect of GSK-1292263.
As shown in Table 1, most of pyrimidopyrimidine derivatives de-
signed illustrated the significant GPR119 agonistic activities. Among
them, compounds containing endo-bicyclic moiety exhibited stronger
bioactivities than exo-moiety, which is consistent with our previous
results. Introduction of fluorine to aniline moiety was beneficial to the
agonistic potency. And analogues with bicyclic amines were revealed
better agonistic activities than bicyclic alcohol (21c vs 21e; 21d vs
N
Fig. 2. The structures of typical GPR119 agonists.
in the interaction and to investigate the relationship between the con-
formations and agonistic activity. In this paper, we described the
synthesis and biological evaluation of a series of pyrimidopyrimidine
compounds as GPR119 agonist.
2
1e). As a result, Compounds 18b, 21c and 21g all containing endo-
bicyclic moieties demonstrated the strong agonistic potency
EC50 < 100 nM). Furthermore, 15a bearing endo-azabicyclic amine 6
(
and 2-fluoro-4-methylsulfonylphenylamino moiety displayed the most
potent agonistic activity (87.5% max) with the EC50 value of 2.2 nM,
while compound 21e also containing group 6 and 2-fluoro-4-cyano-
phenylamino group showed the second potent efficacy (83.8% max)
with quite good EC50 value (8.1 nM). Both 15a and 21e were calculated
to have suitable lipophilicity (ClogP: 3.7 and 4.0), which would be a
low risk of pharmacokinetics issues.
Hence, we can summarize the general structure-activity relationship
(SAR) of pyrimidopyrimidine derivatives for GPR119 agonistic activity.
Firstly, 4,8-disubstitute pyrimido[5,4-d]pyrimidine core was discovered
to be a successful surrogate of 5-nitropyrimidine. After installation of
the same aryl amino and bicyclic amino as compound 1 as typical head
and tail, the resulting compound 15a maintains single digit nanomolar
GPR119 agonist potency. Next, in the head moiety of aryl amine, in-
troduction of fluorine in ortho-position will increase the potency
3.1–20-fold when substituted with EWG such as methylsulfonyl or
2
. Results and discussion
2.1. Chemistry
Azabicyclic intermediates 4, 5, 6, 8 and 9 were prepared according
5
,26
to precedent procedures.
Intermediate 7 was synthesized following
the procedures and conditions as shown in Scheme 1. Mesylation of
endo-N-boc-nortropine 4 with mesyl chloride (MsCl) furnished 10 with
good yield (95%), which was treated with sodium azide (NaN
3
) to af-
ford exo-azido 11 in 92% yield. Reduction of compound 11 using 10%
palladium hydroxide (Pa(OH) ) on charcoal under hydrogen generated
exo-azabicyclic amine 7 in 90% yield.
2
7
2
2
8
The synthesis of pyrimidopyrimidine derivatives 15a–15b was
outlined in Scheme 2. Intermediate 13 was generated by coupling of
Fig. 3. The design of GPR119 agonists.
2

Y. Fang et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
Scheme 1. Reagents and conditions: (a) MsCl, TEA,
CH
2
Cl
2
, 0 °C – rt, 5 h; (b) NaN
3
, MeCN, H
2
O, reflux,
overnight; (c) 10% Pd(OH)
overnight.
2
/C, H , MeOH, 40 °C,
2
2 2 2
Scheme 2. Reagents and conditions: (a) i: 2-fluoro-4-(methylthio)aniline, THF, 0 °C – rt, 1 h; ii: mCPBA, CH Cl , 0 °C – rt, overnight; (b) R-NH , DIPEA, THF, rt, 1 h;
2 2 3
(c) 10% Pd(OH) /C, H , KOH or Et N, EtOH, 65 °C, 48 h.
Scheme 3. Reagents and conditions: (a) i: 4-methylthioaniline, THF, 0 °C – rt, 1 h; ii: mCPBA, CH
OH) /C, H , Et N, EtOH, 65 °C, 24 h.
2 2 2
Cl , 0 °C – rt, overnight; (b) R-NH , DIPEA, THF, rt, 1 h; (c) 10% Pd
(
2
2
3
cyano group in the para-position (15a vs 18b; 15b vs 18c). In the tail
moiety of aliphatic ring, it is no doubt that rigid endo conformers of
bicyclic compounds show higher potency and efficacy than exo and
flexible piperidine and serve as full agonists. We also can conclude that
bicyclic amines are superior to ether type and the tropane ring is better
than 2-azabicyclo[2.2.2]octane for the benefit of agonism.
3. Conclusion
In summary, starting from our previous lead compound 1, we re-
placed the 5-nitropyrimidine core with pyrimidopyrimidine to obtain a
series of novel compounds as drug candidates of GPR119 agonist for
treatment of type 2 diabetes. Some derivatives showed good agonistic
activities (EC50 < 100 nM) and potency. SAR studies illustrated that
pyrimidopyrimidine compounds bearing endo-bicyclic moiety were
good for agonistic potency, which is consistent with our former results.
And introduction of fluorine on aniline group could increase the
GPR119 agonistic activity. Derivatives containing bicyclic amine was
revealed better activities than bicyclic ether. As a result, optimized
compounds 15a and 21e displayed more potent agonistic activities with
single digit EC50 values (2.2 nM and 8.1 nM respectively), which were
evaluated for their oGTT with the dose of 5 mg/kg or 15 mg/kg in
C57BL/6N mice. Compound 15a exerted 13.5% reduction in blood
glucose AUC0–2h at the dose of 15 mg/kg comparing with metformin
reduced 18% of AUC0–2h at the dose of 300 mg/kg. Follow up studies
and results will be reported in due course.
According to the promising agonistic potency of compound 15a and
21e in vitro, we conducted the oral glucose tolerance test (oGTT) in
C57BL/6N mice. Single dose of either compound 15a or 21e (5 mg/kg
and 15 mg/kg) were administered to C57BL/6N mice and metformin
was chosen as a positive control with the dose of 300 mg/kg. As was
outlined in Fig. 4, compound 15a and 21e showed blood glucose re-
duction effect in dose-dependent manner. Compound 15a reduced the
area under curve from
0 to 120 min (AUC0–120min) by 6.4%
(
22.10 ± 1.99) and 13.5% (20.44 ± 3.37) at the dosage of 5 mg/kg
and 15 mg/kg, respectively. And 21a reduced the glucose level by 3.5%
and 8.9% respectively. However, metformin exhibited better potency
with the reduction of AUC0–120min by 18.2% (Vehicle: 23.62 ± 3.20,
Met: 19.32 ± 1.76) at the dose of 300 mg/kg.
3

Y. Fang et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
Scheme 4. Reagents and conditions: (a) 2-fluoro-4-cyanoaniline, THF, rt, 1 h; (b) i: R-NH
2
, DIPEA, THF, rt, 1 h; ii: R-OH, LiHMDS, THF, 0 °C – rt, 5 h (c) 10% Pd
2 2 3
(OH) /C, H , EtOH, Et N, 65 °C, 48 h.
Table 1
GPR119 agonist activities of compounds 15a–b, 18a–18e, 21a–21h.
Compound
hGPR119 activity
EC50 (uM)
ClogPb
Compound
hGPR119 activity
EC50 (uM)
ClogPb
%maxa
%maxa
1
1
1
1
2
2
2
2
5a
8a
8c
8e
1b
1d
1f
0.0022
0.345
1.4
2.1
1.0
> 10
1.7
> 10
87.5
61.3
47.5
40.6
53.4
36.4
58.1
31.7
3.7
2.9
3.5
3.5
3.7
4.2
4.0
4.0
15b
18b
18d
21a
21c
21e
21g
GSK-1292263
0.452
0.044
0.179
0.147
0.036
0.0081
0.049
0.0034
65.3
72.9
71.8
67.2
78.9
83.8
73.3
100
3.7
3.5
3.5
3.5
4.2
4.0
4.0
1h
a
%
max: cAMP stimulation% compared to maximal effect of GSK-1292263.
b
ClogP was calculated using ACD software from Discovery Studio 4.5.
4
. Experimental
was used directly without purification. 1H NMR (300 MHz, CDCl
ppm): 5.05 (m, 1H), 4.29 (brs, 2H), 3.01 (s, 3H), 2.10–1.65 (m, 8H),
1.48 (s, 9H). MS-ESI: [M-H] 304.2.
3
) δ
(
−
4.1. Chemistry
All starting materials were obtained from commercial suppliers and
1
13
4.1.2. tert-butyl (exo)-3-azido-8-azabicyclo[3.2.1]octane-8-carboxylate
used without further purification. H NMR and C NMR spectra were
recorded on a Bruker AVANCE III HD 600 (600 Hz) spectrometer.
Chemical shifts are reported in parts per million (ppm) downfield re-
lative to tetramethylsilane as an internal standard. Peak splitting pat-
terns are abbreviated as s (singlet), br s (broad singlet), d (doublet), t
(
11)
To a solution of compound 10 (1.5 g, 4.9 mmol) in acetonitrile
60 mL) and H O (12 mL) was added NaN (1.3 g, 20 mmol). Then the
(
2
3
reaction was heated to reflux for overnight. The mixture was extracted
with ethyl acetate. The organic phase was washed with brine, dried
(
triplet), dd (doublet of doublet) and m (multiplet). MS spectra were
over MgSO
chromatography (petro ether: EtOAc = 10:1) to give compound 11 as
white solid (1.14 g, 92%). 1H NMR (600 MHz, CDCl
) δ (ppm): 4.34
brs, 2H), 3.21 (m, 1H), 1.83 (m, 4H), 1.67 (m, 4H), 1.49 (s, 9H). MS-
4
, and evaporated. The residue was purified by column
recorded on a Thermo Fisher (LCQ Fleet). HR-MS spectra were recorded
on a AB SCIEX (Triple TOF 5600+). TLC was performed on silica F254
purchased from Branch of Qingdao Haiyang Chemical Co. and detected
by UV light at 254 nm or by charring with sulphuric acid. Column
chromatography was performed on silica gel column (200–300 mesh,
Branch of Qingdao Haiyang Chemical Co.).
3
(
+
ESI: [M + H] 253.1.
4.1.3. tert-butyl (exo)-3-amino-8-azabicyclo[3.2.1]octane-8-carboxylate
4.1.1. tert-butyl
(endo)-3-((methylsulfonyl)oxy)-8-azabicyclo[3.2.1]
(7)
octane-8-carboxylate (10)
To a solution of compounds 11 (1.1 g, 4.4 mmol) in methanol
To a solution of compound 4 (1.2 g, 5.3 mmol) in dry CH
20 mL) was added triethylamine (1.1 g, 10.6 mmol). Then mesyl
chloride (0.91 g, 8 mmol) was added dropwise at 0 °C. The reaction was
stirred at room temperature for 5 h. The reaction was diluted with
2
Cl
2
(15 mL) was added 10% palladium hydroxide on charcoal (110 mg).
The reaction mixture was stirred under hydrogen atmosphere at 40 °C
overnight. The mixture was filtered. The filtrate was evaporated to af-
(
ford the product 7 (0.89 g, 90%) as white crystal without purification.
1
CH
2
Cl
2
, washed with aqueous NH
4
Cl and brine, dried over MgSO
4
, and
H NMR (600 MHz, CDCl ) δ (ppm): 4.32–4.15 (m, 3H), 3.17 (m, 2H),
3
+
evaporated to receive the product 10 (1.5 g, 95%) as white solid, which
1.96 (m, 4H), 1.82 (m, 4H), 1.49 (s, 9H). MS-ESI: [M + H] 227.4.
4

Y. Fang et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
Fig. 4. Single dose of compounds 15a
and 21e on oGTT in C57BL/6N mice.
Compounds 15a and 21e were ad-
ministered to C57BL/6N mice after
16 h starvation, then the oral glucose
tolerance test (3 g/kg) was conducted
after 4 h of the single dose, the blood
glucose level at 0, 15, 30, 60, 90 and
120 min were recorded for area under
curve calculation. The results are pre-
*
sented as the mean ± SE. , p < 0.05
compared to vehicle group (n = 8).
4
.1.4. 2,6,8-trichloro-N-(2-fluoro-4-(methylsulfonyl)phenyl)pyrimido[5,4-
chromatography to give the product.
.1.9. tert-butyl (endo)-3-((8-((2-fluoro-4-(methylsulfonyl)phenyl)amino)
d]pyrimidin-4-amine (13)
A
suspension of tetrachloropyrimido[5,4-d]pyrimidine (0.65 g,
4
2
.4 mmol) in THF (5 mL) and H
2
O (0.6 mL) was added 2-fluoro-4-
pyrimido[5,4-d]pyrimidin-4-yl)amino)-8-azabicyclo[3.2.1]octane-8-
(
methylthio)aniline (0.1 mL, 0.8 mmol) dropwisely at 0 °C and stirred at
ambient temperature for 1 h. The reaction was diluted with CH Cl
10 mL) and added 3-Chloroperoxybenzoic acid (0.36 g, 77%,
.6 mmol) at 0 °C. The reaction mixture was diluted with CH Cl , wa-
shed subsequently with saturated Na solution, NaHCO solution
and brine, dried over MgSO , and evaporated. The residue was purified
carboxylate (15a)
Light yellow solid, 12% yield. 1H NMR (600 MHz, CDCl ) δ (ppm):
2
2
3
(
1
9.36 (d, J = 3.4 Hz, 1H), 9.20 (t, J = 7.8 Hz, 1H), 8.76 (s, 1H), 8.62 (s,
1H), 7.85 (dd, J = 8.8, 1.6 Hz, 1H), 7.79 (dd, J = 10.0, 2.0 Hz, 1H),
7.52 (d, J = 7.6, 1H), 4.54 (m, 1H), 4.39–4.29 (m, 2H), 3.10 (s, 3H),
2.45–2.33 (m, 2H), 2.20 (m, 2H), 2.10–2.04 (m, 2H), 1.93 (m, 2H), 1.52
2
2
2
2
S O
3
3
4
(s, 9H). 13C NMR (150 MHz, CDCl ) δ: 157.8, 156.0, 155.7, 153.6,
by column chromatography (petro ether: EtOAc = 3:1) to give com-
pound 13 as yellow solid (0.18 g, 54% in 2 steps). H NMR (300 MHz,
CDCl
2
3
1
153.4, 152.8, 151.1, 134.6, 132.5, 132.1, 124.5, 121.2, 114.4, 79.6,
3
) δ (ppm): 9.37 (s, 1H), 8.99 (t, J = 8.5 Hz, 1H), 7.95–7.80 (m,
52.8, 52.1, 44.7, 43.5, 35.2, 34.6, 28.5 (×3), 28.3, 27.8. HRMS-ESI (m/
+
+
H), 3.13 (s, 3H). MS-ESI: [M + H] 422.1.
z) calcd for C25H31N O S [M + H] : 544.2142, found 544.2158.
7
4
4
.1.5. General procedure for 4,8-diamino-2,6-dichloropyrimido[5,4-d]
4.1.10. tert-butyl (exo)-3-((8-((2-fluoro-4-(methylsulfonyl)phenyl)amino)
pyrimido[5,4-d]pyrimidin-4-yl)amino)-8-azabicyclo[3.2.1]octane-8-
carboxylate (15b)
pyrimidines (14a–14b)
To the solution of compound 13 (0.11 g, 0.25 mmol) and bicyclic
amine (0.38 mmol) in THF (3 mL) was added DIPEA (0.38 mmol). After
stirred at R.T. for 1 h, the reaction mixture was diluted with EtOAc,
washed with brine, dried over MgSO
purified by column chromatography to give the product.
Light yellow solid, 10% yield. 1H NMR (600 MHz, CDCl ) δ (ppm):
3
9.38 (d, J = 3.2 Hz, 1H), 9.19 (t, J = 8.0 Hz, 1H), 8.76 (s, 1H), 8.61 (s,
1H), 7.85 (dd, J = 8.6, 1.4 Hz, 1H), 7.79 (dd, J = 10.0, 2.0 Hz, 1H),
6.76 (d, J = 8.5, 1H), 4.81–4.74 (m, 1H), 4.40–4.31 (m, 2H), 3.11 (s,
4
, and evaporated. The residue was
1
3
3H), 2.10 (m, 2H), 1.91 (m, 2H), 1.83–1.65 (m, 2H), 1.53 (s, 9H).
C
4
.1.6. tert-butyl (endo)-3-((2,6-dichloro-8-((2-fluoro-4-(methylsulfonyl)
NMR (150 MHz, CDCl ) δ: 158.2, 156.1, 155.8, 153.4, 153.2, 152.8,
3
phenyl)amino)pyrimido[5,4-d]pyrimidin-4-yl)amino)-8-azabicyclo[3.2.1]
151.1, 134.6, 132.3, 132.1, 124.5, 121.2, 114.3, 79.6, 53.3, 52.7, 44.7,
octane-8-carboxylate (14a)
42.9, 37.7, 36.9, 29.7, 28.6 (×3), 27.8. HRMS-ESI (m/z) calcd for
Yellow solid, 32% yield. 1H NMR (300 MHz, CDCl
) δ (ppm): 9.12
d, J = 3.2 Hz, 1H), 8.95 (t, J = 8.1 Hz, 1H), 7.80 (m, 2H), 7.45 (d,
J = 7.7 Hz, 1H), 4.50–4.20 (m, 3H), 3.10 (s, 3H), 2.50–2.10 (m, 4H),
.00–1.80 (m, 4H), 1.50 (s, 9H). HRMS-ESI (m/z) calcd for
25 31 7 4
C H N O
S [M + H]+: 544.2142, found 544.2167.
3
(
4.1.11. 2,6,8-trichloro-N-(4-(methylsulfonyl)phenyl)pyrimido[5,4-d]
pyrimidin-4-amine (16)
2
+
C
25
H
28Cl
2
7
FN O
4
S [M + Na] : 634.1182, found 634.1234.
Follow the similar synthetic procedure of compound 13. Yellow
1
solid, 65% yield in two steps. H NMR (600 MHz, CDCl
3
) δ (ppm): 9.12
+
4.1.7. tert-butyl
(exo)-3-((2,6-dichloro-8-((2-fluoro-4-(methylsulfonyl)
(s, 1H), 8.20–8.00 (m, 4H), 3.10 (s, 3H). MS-ESI: [M + H] 404.1.
phenyl)amino)pyrimido[5,4-d]pyrimidin-4-yl)amino)-8-azabicyclo[3.2.1]
octane-8-carboxylate (14b)
4.1.12. tert-butyl 4-((2,6-dichloro-8-((4-(methylsulfonyl)phenyl)amino)
pyrimido[5,4-d]pyrimidin-4-yl)amino)piperidine-1-carboxylate (17a)
Follow the similar synthetic procedure of compounds 14a–14b.
Yellow solid, 38% yield. 1H NMR (600 MHz, CDCl
) δ (ppm): 9.17
d, J = 3.2 Hz, 1H), 8.98 (t, J = 8.6 Hz, 1H), 7.88 (dd, J = 8.6, 1.5 Hz,
H), 7.81 (dd, J = 9.8, 2.0 Hz, 1H), 6.78 (d, J = 8.4 Hz, 1H), 4.74–4.69
m, 1H), 4.33–4.26 (m, 2H), 3.12 (s, 3H), 2.08 (m, 4H), 1.88 (m, 2H),
.70 (m, 2H), 1.53 (s, 9H). HRMS-ESI (m/z) calcd for C25 28Cl FN
3
(
1
(
1
Yellow solid, 45% yield. 1H NMR (600 MHz, CDCl ) δ (ppm): 8.89 (s,
3
1H), 8.05 (m, 2H), 7.97 (m, 2H), 6.88 (d, J = 7.9 Hz, 1H), 4.31 (m, 1H),
H
2
7
O
4
S
4.18 (m, 2H), 3.04 (s, 3H), 2.93 (m, 2H), 2.05 (m, 2H), 1.59 (m, 2H),
+
+
[
M + Na] : 634.1182, found 634.1243.
1.44 (s, 9H). HRMS-ESI (m/z) calcd for C23H29Cl N O S [M + Na]
:
2
7 4
590.1120, found 590.1186.
4.1.8. General procedure for 4,8-diaminopyrimido[5,4-d]pyrimidines
(
15a–15b)
To the solution of 2,6-dichloropyrimido[5,4-d]pyrimidines
4.1.13. tert-butyl (endo)-3-((2,6-dichloro-8-((4-(methylsulfonyl)phenyl)
amino)pyrimido[5,4-d]pyrimidin-4-yl)amino)-8-azabicyclo[3.2.1]octane-
8-carboxylate (17b)
(
(
0.1 mmol) in EtOH (3 mL) was added Pd(OH)
0.3 mmol). The reaction was connected with the H
2
(15 mg) and KOH
balloon and stirred
2
Follow the similar synthetic procedure of compounds 14a–14b.
at 65 °C for 48 h. The mixture was cooled down, diluted with CH
and filterer over Celite. The filtrate was washed with brine, dried over
MgSO and evaporated. The residue was purified by column
2
Cl
2
,
Yellow solid, 38% yield. 1H NMR (600 MHz, CDCl
1H), 8.15–7.95 (m, 4H), 7.45 (d, J = 7.7 Hz, 1H), 4.55–4.20 (m, 3H),
3.08 (s, 3H), 2.50–2.10 (m, 4H), 2.00–1.80 (m, 4H), 1.50 (s, 9H).
3
) δ (ppm): 8.89 (s,
4
,
5

Y. Fang et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
2.10 (m, 4H), 1.90 (m, 2H), 1.79–1.67 (m, 2H), 1.53 (s, 9H). C NMR
S [M + Na]+: 616.1277,
13
HRMS-ESI (m/z) calcd for C25
H29Cl
2
N
7
O
4
found 616.1330.
(150 MHz, CDCl
32.3, 131.8, 129.0 (×2), 120.1 (×2), 79.8, 53.5, 52.8, 45.0, 43.0,
37.8, 37.1, 28.7 (×3), 28.6, 28.0. HRMS-ESI (m/z) calcd for
S [M + H]+: 526.2236, found 526.2262.
3
) δ: 158.4, 156.4, 155.6, 153.8, 153.4, 143.1, 134.9,
1
4.1.14. tert-butyl
(exo)-3-((2,6-dichloro-8-((4-(methylsulfonyl)phenyl)
amino)pyrimido[5,4-d]pyrimidin-4-yl)amino)-8-azabicyclo[3.2.1]octane-
25 31 7 4
C H N O
8-carboxylate (17c)
Follow the similar synthetic procedure of compounds 14a–14b.
1
4.1.20. tert-butyl (endo)-5-((8-((4-(methylsulfonyl)phenyl)amino)pyrim-
ido[5,4-d]pyrimidin-4-yl)amino)-2-azabicyclo[2.2.2]octane-2-carboxylate
Yellow solid, 32% yield. H NMR (600 MHz, CDCl
3
) δ (ppm): 8.94 (s,
1
(
2
H), 8.11 (m, 2H), 8.03 (m, 2H), 6.79 (d, J = 8.4 Hz, 1H), 4.73–4.67
m, 1H), 4.40 (s, 1H), 4.31 (s, 1H), 3.10 (s, 3H), 2.09 (m, 4H), 1.88 (m,
H), 1.70 (m, 2H), 1.53 (s, 9H). HRMS-ESI (m/z) calcd for
(
18d)
Follow the similar synthetic procedure of compounds 15a–15b.
1
+
Light yellow solid, 34% yield. H NMR (600 MHz, CDCl
3
) δ (ppm): 9.12
25 2 7 4
C H29Cl N O S [M + Na] : 616.1277, found 616.1318.
(
d, J = 6.4 Hz, 1H), 8.75 (s, 1H), 8.59 (s, 1H), 8.18 (m, 2H), 8.01 (m,
2
3
2
H), 7.05 (m, 1H), 4.52 (m, 1H), 4.23 (m, 0.6H), 4.08 (m, 0.4H),
4
.1.15. tert-butyl (endo)-5-((2,6-dichloro-8-((4-(methylsulfonyl)phenyl)
.63–3.59 (m, 1H), 3.49–3.45 (m, 1H), 3.10 (s, 3H), 2.65–2.61 (m, 1H),
amino)pyrimido[5,4-d]pyrimidin-4-yl)amino)-2-azabicyclo[2.2.2]octane-
1
3
.37–2.32 (m, 1H), 2.09–1.76 (m, 5H), 1.52 (s, 9H).
) δ: 158.6, 156.3, 155.4, 154.8, 153.7, 142.9, 134.8,
132.2, 131.5, 128.9 (×2), 120.0 (×2), 79.4, 48.0, 47.4, 42.8, 35.5,
0.1, 29.6, 28.6 (×3), 26.7, 18.2. HRMS-ESI (m/z) calcd for
S [M + H]+: 526.2236, found 526.2245.
C NMR
2-carboxylate (17d)
(
150 MHz, CDCl
3
Follow the similar synthetic procedure of compounds 14a–14b.
1
Yellow solid, 40% yield. H NMR (600 MHz, CDCl
3
) δ (ppm): 8.94 (s,
3
1
4
3
H), 8.11 (m, 2H), 8.03 (m, 2H), 7.03 (d, J = 7.6 Hz, 1H), 4.55 (m, 1H),
25 31 7 4
C H N O
.24 (m, 0.6H), 4.09 (m, 0.4H), 3.60 (m, 1H), 3.45 (m, 1H), 3.10 (s,
H), 2.61 (m, 1H), 2.33 (m, 1H), 1.90–1.83 (m, 5H), 1.53 (s, 9H).
+
HRMS-ESI (m/z) calcd for C25
found 616.1325.
H29Cl
2
N
7
O
4
S [M + Na] : 616.1277,
4.1.21. tert-butyl (exo)-5-((8-((4-(methylsulfonyl)phenyl)amino)pyrimido
[
(
5,4-d]pyrimidin-4-yl)amino)-2-azabicyclo[2.2.2]octane-2-carboxylate
18e)
4.1.16. tert-butyl
(exo)-5-((2,6-dichloro-8-((4-(methylsulfonyl)phenyl)
Follow the similar synthetic procedure of compounds 15a–15b.
amino)pyrimido[5,4-d]pyrimidin-4-yl)amino)-2-azabicyclo[2.2.2]octane-
1
Light yellow solid, 38% yield. H NMR (600 MHz, CDCl
3
) δ (ppm): 9.10
2-carboxylate (17e)
(
d, J = 10.1 Hz, 1H), 8.75 (s, 1H), 8.60 (s, 1H), 8.18 (m, 2H), 8.01 (m,
Follow the similar synthetic procedure of compounds 14a–14b.
2
0
2
H), 7.06 (dd, J = 7.7 Hz, 1H), 4.47 (m, 1H), 4.25 (m, 0.6H), 4.11 (m,
1
Yellow solid, 36% yield. H NMR (600 MHz, CDCl
1
4
3
) δ (ppm): 8.95 (s,
.4H), 3.62 (m, 1H), 3.56–3.47 (m, 1H), 3.10 (s, 3H), 2.41 (m, 1H),
.32 (m, 1H), 1.98–1.83 (m, 4H), 1.68 (m, 1H), 1.52 (s, 9H). C NMR
H), 8.12 (m, 2H), 8.05 (m, 2H), 7.06 (d, J = 7.9 Hz, 1H), 4.55 (m, 1H),
.24 (m, 0.6H), 4.10 (m, 0.4H), 3.59 (m, 1H), 3.43–3.33 (m, 1H), 3.11
13
(
1
3
3
150 MHz, CDCl ) δ: 158.6, 156.3, 155.4, 154.7, 153.8, 142.9, 134.8,
(
s, 3H), 2.40 (m, 1H), 2.34 (m, 1H), 1.84–1.79 (m, 5H), 1.51 (s, 9H).
32.3, 131.3, 128.9 (×2), 119.9 (×2), 79.5, 47.0, 44.8, 42.9, 36.1,
+
2 7 4
HRMS-ESI (m/z) calcd for C25H29Cl N O S [M + Na] : 616.1277,
found 616.1314.
0.3, 29.7, 28.6 (×3), 25.5, 22.9. HRMS-ESI (m/z) calcd for
S [M + H]+: 526.2236, found 526.2213.
C
25
H
31
N
7
O
4
4
.1.17. tert-butyl 4-((8-((4-(methylsulfonyl)phenyl)amino)pyrimido[5,4-
4
.1.22. 3-fluoro-4-((2,6,8-trichloropyrimido[5,4-d]pyrimidin-4-yl)amino)
d]pyrimidin-4-yl)amino)piperidine-1-carboxylate (18a)
benzonitrile (19)
Follow the similar synthetic procedure of compounds 15a–15b.
1
A
suspension of tetrachloropyrimido[5,4-d]pyrimidine (0.65 g,
Light yellow solid, 45% yield. H NMR (600 MHz, CDCl
3
) δ (ppm): 9.11
2
0
.4 mmol) in THF (5 mL) was added 2-fluoro-4-cyanoaniline (0.11 g,
.8 mmol) at 0 °C and stirred at ambient temperature for 1 h. The re-
(
s, 1H), 8.70 (d, J = 10.9 Hz, 1H), 8.56 (d, J = 10.1, 1H), 4.31 (m, 1H),
4
1
1
1
.12 (m, 2H), 3.11–3.00 (m, 2H), 3.07 (s, 3H), 2.12–2.04 (m, 2H),
1
3
action diluted with EtOAc, washed with brine, dried over MgSO , and
evaporated. The residue was purified by column chromatography
.57–1.53 (m, 2H), 1.48 (s, 9H). C NMR (150 MHz, CDCl
3
) δ: 158.2,
4
56.2, 155.3, 154.7, 153.7, 142.9, 134.8, 132.2, 131.6, 128.8 (×2),
19.9 (×2), 79.9, 48.2, 45.8, 44.8, 31.6, 30.0, 28.4 (×3). HRMS-ESI
(
petro ether: EtOAc = 5:1) to give compound 19 as yellow solid (0.22 g,
1
+
74% yield). H NMR (600 MHz, CDCl ) δ (ppm): 9.37 (s, 1H), 8.94 (t,
J = 8.2 Hz, 1H), 7.66 (d, J = 8.6 Hz, 1H), 7.58 (dd, J = 10.4, 1.7 Hz,
1
(
m/z) calcd for C23
H
29
N
7
O
4
S [M + H] : 522.1899, found 522.1955.
3
H). MS-ESI: [M + H]+ 469.2.
4
.1.18. tert-butyl (endo)-3-((8-((4-(methylsulfonyl)phenyl)amino)pyrim-
ido[5,4-d]pyrimidin-4-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylate
18b)
Follow the similar synthetic procedure of compounds 15a–15b.
(
4
.1.23. tert-butyl
4-((2,6-dichloro-8-((4-cyano-2-fluorophenyl)amino)
pyrimido[5,4-d]pyrimidin-4-yl)amino)piperidine-1-carboxylate (20a)
Follow the similar synthetic procedure of compounds 14a–14b.
1
Light yellow solid, 33% yield. H NMR (600 MHz, CDCl
s, 1H), 8.74 (s, 1H), 8.59 (s, 1H), 8.18 (m, 2H), 8.01 (m, 2H), 7.52 (d,
J = 7.6 Hz, 1H), 4.53 (m, 1H), 4.39–4.27 (m, 2H), 3.10 (s, 3H),
3
) δ (ppm): 9.10
(
Yellow solid, 41% yield. 1H NMR (600 MHz, CDCl
3
) δ (ppm): 9.10 (s,
H), 8.85 (t, J = 8.2 Hz, 1H), 7.55 (d, J = 8.9 Hz, 1H), 7.46 (d,
J = 10.4 Hz, 1H), 6.85 (d, J = 7.9 Hz, 1H), 4.26 (m, 1H), 4.13 (m, 2H),
.93 (m, 2H), 2.02 (m, 4H), 1.44 (s, 9H). HRMS-ESI (m/z) calcd for
[M + Na]+: 555.1203, found 555.1251.
23Cl FN
1
2
9
1
4
.35–2.20 (m, 2H), 2.18 (m, 2H), 2.05 (m, 2H), 1.94 (m, 2H), 1.51 (s,
1
3
H). C NMR (150 MHz, CDCl ) δ: 157.9, 156.2, 155.4, 153.4, 143.0,
3
2
C
34.8, 132.4, 131.4, 128.9 (×2), 119.9 (×2), 79.6, 52.9, 52.1, 44.8,
23
H
2
8 2
O
3.5, 35.2, 34.8, 28.5 (×3), 28.3, 27.7. HRMS-ESI (m/z) calcd for
+
C
25
H
31
N
7
O
4
S [M + H] : 526.2236, found 526.2226.
4.1.24. General procedure for 4,8-diamino-2,6-dichloropyrimido[5,4-d]
4.1.19. tert-butyl (exo)-3-((8-((4-(methylsulfonyl)phenyl)amino)pyrimido
pyrimidines (20b-20d)
[
(
5,4-d]pyrimidin-4-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylate
18c)
To the solution of compound 19 (1 mmol) and intermediates 3, 4 or
5 (2.5 mmol) in anhydrous THF (10 mL) was added LiHMDS (3 mmol)
dropwisely at 0 °C. After stirred at rt for 12 h, the reaction mixture was
diluted with EtOAc, washed with brine, dried over MgSO , and eva-
4
porated. The residue was purified by column chromatography to give
Follow the similar synthetic procedure of compounds 15a–15b.
1
Light yellow solid, 40% yield. H NMR (600 MHz, CDCl
3
) δ (ppm): 9.10
(
s, 1H), 8.73 (s, 1H), 8.59 (s, 1H), 8.17 (m, 2H), 8.00 (m, 2H), 6.76 (d,
J = 8.5 Hz, 1H), 4.80–4.73 (m, 1H), 4.40–4.31 (m, 2H), 3.10 (s, 3H),
the product.
6

Y. Fang et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
4
.1.25. tert-butyl
4-((2,6-dichloro-8-((4-cyano-2-fluorophenyl)amino)
J = 10.3 Hz, 1H), 7.06 (d, J = 7.2 Hz, 1H), 4.46 (m, 1H), 4.26 (m,
0.6H), 4.11 (m, 0.4H), 3.57 (m, 1H), 3.38 (m, 1H), 2.39 (m, 1H), 2.31
(m, 1H), 1.98–1.83 (m, 4H), 1.67 (m, 1H), 1.52 (s, 9H). HRMS-ESI (m/
pyrimido[5,4-d]pyrimidin-4-yl)oxy)piperidine-1-carboxylate (20b)
1
Yellow solid, 31% yield. H NMR (600 MHz, CDCl
3
) δ (ppm): 9.25
2 8 2
z) calcd for C25H25Cl FN O
[M + Na]+: 581.1360, found 581.1423.
(
d, J = 3.4 Hz, 1H), 8.95 (t, J = 8.2 Hz, 1H), 7.63 (d, J = 8.6 Hz, 1H),
7
2
C
.54 (dd, J = 10.4, 1.7 Hz, 1H), 5.59 (m, 1H), 3.98 (s, 2H), 3.28 (m,
H), 2.13 (m, 2H), 1.98 (m, 2H), 1.50 (s, 9H). HRMS-ESI (m/z) calcd for
4.1.32. tert-butyl 4-((8-((4-cyano-2-fluorophenyl)amino)pyrimido[5,4-d]
pyrimidin-4-yl)amino)piperidine-1-carboxylate (21a)
+
23
H
22Cl
2
7
FN O
3
[M + Na] : 556.1043, found 555.1007.
Follow the similar synthetic procedure of compounds 15a–15b.
1
4
.1.26. tert-butyl
(endo)-3-((2,6-dichloro-8-((4-cyano-2-fluorophenyl)
Light yellow solid, 18% yield. H NMR (600 MHz, CDCl
3
) δ (ppm): 9.36
amino)pyrimido[5,4-d]pyrimidin-4-yl)oxy)-8-azabicyclo[3.2.1]octane-8-
carboxylate (20c)
(d, J = 3.1 Hz, 1H), 9.13 (t, J = 8.3 Hz, 1H), 8.75 (s, 1H), 8.62 (s, 1H),
7.58 (d, J = 8.6 Hz, 1H), 7.50 (dd, J = 10.6, 1.8 Hz, 1H), 6.91 (d,
J = 8.1 Hz, 1H), 4.36–4.32 (m, 1H), 4.17 (m, 2H), 3.02 (m, 2H), 2.13
Yellow solid, 26% yield. 1H NMR (600 MHz, CDCl
3
) δ (ppm): 9.22
(
d, J = 3.7 Hz, 1H), 8.95 (t, J = 8.3 Hz, 1H), 7.63 (d, J = 8.7 Hz, 1H),
8 2
(m, 2H), 1.50 (s, 9H). HRMS-ESI (m/z) calcd for C23H25FN O
7
.55 (dd, J = 10.4, 1.6 Hz, 1H), 5.74 (m, 1H), 4.34–4.26 (m, 2H),
[M + H]+: 465.2163, found 465.2231.
2
.35–2.27 (m, 4H), 2.11–2.05 (m, 4H), 1.52 (s, 9H). HRMS-ESI (m/z)
+
calcd for C25
H24Cl
2
FN
7
O
3
[M + Na] : 582.1199, found 582.1249.
4.1.33. tert-butyl 4-((8-((4-cyano-2-fluorophenyl)amino)pyrimido[5,4-d]
pyrimidin-4-yl)oxy)piperidine-1-carboxylate (21b)
4
.1.27. tert-butyl
(exo)-3-((2,6-dichloro-8-((4-cyano-2-fluorophenyl)
Follow the similar synthetic procedure of compounds 15a–15b.
1
amino)pyrimido[5,4-d]pyrimidin-4-yl)oxy)-8-azabicyclo[3.2.1]octane-8-
carboxylate (20d)
Light yellow solid, 15% yield. H NMR (600 MHz, CDCl
3
) δ (ppm): 9.45
(d, J = 3.6 Hz, 1H), 9.15 (t, J = 8.3 Hz, 1H), 8.97 (s, 1H), 8.83 (s, 1H),
7.59 (d, J = 8.6 Hz, 1H), 7.50 (dd, J = 10.5, 1.8 Hz, 1H), 5.62–5.58 (m,
1H), 4.03 (s, 2H), 3.25–3.21 (m, 2H), 2.17 (m, 2H), 1.99–1.93 (m, 2H),
Yellow solid, 24% yield. 1H NMR (600 MHz, CDCl
) δ (ppm): 9.25
d, J = 3.7 Hz, 1H), 8.94 (t, J = 8.2 Hz, 1H), 7.63 (d, J = 8.5 Hz, 1H),
.55 (dd, J = 10.4, 1.5 Hz, 1H), 5.83 (m, 1H), 4.25–4.22 (m, 2H), 2.26
m, 2H), 2.10–2.07 (m, 4H), 1.85–1.83 (m, 2H), 1.52 (s, 9H). HRMS-ESI
3
(
7
(
(
1.50 (s, 9H). 13C NMR (150 MHz, CDCl
154.3, 152.7, 151.0, 135.1, 134.1, 131.3, 129.5, 121.6, 118.5, 106.6,
) δ: 165.3, 156.3, 155.5, 154.8,
3
+
m/z) calcd for
C
25
H24Cl
2
FN
7
O
3
[M + Na]
:
582.1199, found
79.9, 74.7, 41.6, 38.7, 30.4, 29.7, 28.4 (×3). HRMS-ESI (m/z) calcd for
23 7 3
C H24FN O
[M + H]+: 466.2003, found 466.2055.
5
82.1267.
4
.1.28. tert-butyl
(endo)-3-((2,6-dichloro-8-((4-cyano-2-fluorophenyl)
4.1.34. tert-butyl (endo)-3-((8-((4-cyano-2-fluorophenyl)amino)pyrimido
[5,4-d]pyrimidin-4-yl)oxy)-8-azabicyclo[3.2.1]octane-8-carboxylate
(21c)
amino)pyrimido[5,4-d]pyrimidin-4-yl)amino)-8-azabicyclo[3.2.1]octane-
8-carboxylate (20e)
Follow the similar synthetic procedure of compounds 14a–14b.
Follow the similar synthetic procedure of compounds 15a–15b.
1
1
Yellow solid, 37% yield. H NMR (600 MHz, CDCl
3
) δ (ppm): 9.13 (s,
Light yellow solid, 14% yield. H NMR (600 MHz, CDCl
3
) δ (ppm): 9.45
1
H), 8.92 (t, J = 8.3 Hz, 1H), 7.61 (d, J = 8.6 Hz, 1H), 7.52 (dd,
J = 10.4, 1.7 Hz, 1H), 7.45 (d, J = 7.7 Hz, 1H), 4.51 (m, 1H), 4.32 (m,
H), 2.45–2.34 (m, 2H), 2.22–2.18 (m, 2H), 2.02–1.96 (m, 2H), 1.87
m, 2H), 1.52 (s, 9H). HRMS-ESI (m/z) calcd for C25 25Cl FN
(d, J = 3.5 Hz, 1H), 9.16 (t, J = 8.3 Hz, 1H), 8.96 (s, 1H), 8.82 (s, 1H),
7.59 (dd, J = 10.5, 1.7 Hz, 1H), 7.51 (dd, J = 10.5, 1.7, 1H), 5.75 (m,
1H), 4.30 (m, 2H), 2.33 (m, 4H), 2.11 (m, 4H), 1.52 (s, 9H). HRMS-ESI
2
(
[
2
8
O
2
(m/z) calcd for C25
7 3
H26FN O
[M + H]+: 492.2159, found 492.2225.
H
+
M + Na] : 581.1360, found 581.1420.
4.1.35. tert-butyl (exo)-3-((8-((4-cyano-2-fluorophenyl)amino)pyrimido
4.1.29. tert-butyl
(exo)-3-((2,6-dichloro-8-((4-cyano-2-fluorophenyl)
[5,4-d]pyrimidin-4-yl)oxy)-8-azabicyclo[3.2.1]octane-8-carboxylate
amino)pyrimido[5,4-d]pyrimidin-4-yl)amino)-8-azabicyclo[3.2.1]octane-
(21d)
8
-carboxylate (20f)
Follow the similar synthetic procedure of compounds 14a–14b.
Follow the similar synthetic procedure of compounds 15a–15b.
1
Light yellow solid, 15% yield. H NMR (600 MHz, CDCl
3
) δ (ppm): 9.46
1
Yellow solid, 32% yield. H NMR (600 MHz, CDCl
3
) δ (ppm): 9.14 (s,
(d, J = 3.4 Hz, 1H), 9.15 (t, J = 8.2 Hz, 1H), 8.97 (s, 1H), 8.83 (s, 1H),
7.59 (d, J = 8.8 Hz, 1H), 7.51 (dd, J = 10.6, 1.7 Hz, 1H), 5.93 (m, 1H),
4.45–4.35 (m, 2H), 2.29 (m, 2H), 2.17 (m, 2H), 2.09–2.06 (m, 4H), 1.48
1
H), 8.91 (t, J = 8.2 Hz, 1H), 7.60 (d, J = 8.6 Hz, 1H), 7.52 (dd,
J = 10.4, 1.7 Hz, 1H), 6.78 (d, J = 8.4 Hz, 1H), 4.71 (m, 1H), 4.39–4.32
m, 2H), 2.08 (m, 4H), 1.88 (m, 2H), 1.74 (m, 2H), 1.53 (s, 9H). HRMS-
7 3
(s, 9H). HRMS-ESI (m/z) calcd for C25H26FN O
[M + H]+: 492.2159,
(
+
ESI (m/z) calcd for C25
H
25Cl
2
FN
8
O
2
[M + Na] : 581.1360, found
found 492.2201.
581.1411.
4.1.36. tert-butyl (endo)-3-((8-((4-cyano-2-fluorophenyl)amino)pyrimido
4.1.30. tert-butyl
(endo)-5-((2,6-dichloro-8-((4-cyano-2-fluorophenyl)
[5,4-d]pyrimidin-4-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylate
amino)pyrimido[5,4-d]pyrimidin-4-yl)amino)-2-azabicyclo[2.2.2]octane-
(21e)
2
-carboxylate (20g)
Follow the similar synthetic procedure of compounds 14a–14b.
Follow the similar synthetic procedure of compounds 15a–15b.
1
Light yellow solid, 11% yield. H NMR (600 MHz, CDCl
3
) δ (ppm): 9.32
1
Yellow solid, 42% yield. H NMR (600 MHz, CDCl
3
) δ (ppm): 9.16 (s,
(s, 1H), 9.11 (t, J = 8.2 Hz, 1H), 8.74 (s, 1H), 8.60 (s, 1H), 7.56 (d,
J = 8.5 Hz, 1H), 7.51–7.47 (m, 2H), 4.52 (m, 1H), 4.38–4.28 (m, 2H),
2.44–2.33 (m, 2H), 2.19 (m, 2H), 2.04 (m, 2H), 1.92 (m, 2H), 1.51 (s,
1
H), 8.92 (t, J = 8.3 Hz, 1H), 7.61 (d, J = 8.7 Hz, 1H), 7.53 (dd,
J = 10.4, 1.7 Hz, 1H), 7.03 (d, J = 7.4 Hz, 1H), 4.51 (m, 1H), 4.24 (m,
.6H), 4.08 (m, 0.4H), 3.60 (m, 1H), 3.46 (m, 1H), 2.64 (m, 1H), 2.31
m, 1H), 1.98–1.70 (m, 4H), 1.53 (m, 1H), 1.51 (s, 9H). HRMS-ESI (m/
0
(
9H). 13C NMR (150 MHz, CDCl
152.6, 150.1, 132.4, 131.7, 131.6, 129.4, 121.3, 118.4, 106.0, 79.6,
3
) δ: 157.8, 156.0, 155.7, 153.6, 153.4,
+
z) calcd for C25
H
25Cl
2
FN
8
O
2
[M + Na] : 581.1360, found 581.1435.
52.9, 52.1, 43.5, 35.2, 34.6, 28.5 (×3), 28.4, 27.9. HRMS-ESI (m/z)
8 2
calcd for C25H27FN O
[M + H]+: 491.2319, found 491.2347.
4.1.31. tert-butyl
(exo)-5-((2,6-dichloro-8-((4-cyano-2-fluorophenyl)
amino)pyrimido[5,4-d]pyrimidin-4-yl)amino)-2-azabicyclo[2.2.2]octane-
4.1.37. tert-butyl (exo)-3-((8-((4-cyano-2-fluorophenyl)amino)pyrimido
[5,4-d]pyrimidin-4-yl)amino)-8-azabicyclo[3.2.1]octane-8-carboxylate
(21f)
2-carboxylate (20h)
Follow the similar synthetic procedure of compounds 14a–14b.
1
Yellow solid, 35% yield. H NMR (600 MHz, CDCl
1
3
) δ (ppm): 9.15 (s,
Follow the similar synthetic procedure of compounds 15a–15b.
1
H), 8.93 (t, J = 8.3 Hz, 1H), 7.61 (d, J = 8.5 Hz, 1H), 7.48 (d,
Light yellow solid, 10% yield. H NMR (600 MHz, CDCl
3
) δ (ppm): 9.35
7

Y. Fang et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
(
7
d, J = 3.4 Hz, 1H), 9.13 (t, J = 8.3 Hz, 1H), 8.75 (s, 1H), 8.61 (s, 1H),
.57 (d, J = 8.8 Hz, 1H), 7.49 (dd, J = 10.6, 1.8 Hz, 1H), 6.74 (d,
J = 8.5 Hz, 1H), 4.80–4.74 (m, 1H), 4.41–4.31 (m, 2H), 2.10 (m, 4H),
.91 (m, 2H), 1.79 (m, 2H), 1.53 (s, 9H). HRMS-ESI (m/z) calcd for
for 1 h before being read on Envision (Perkin Elmer). The assay was
performed for three replicates for each concentration.
1
C
4.3. oGTT in C57BL/6N mice
+
25
H
27FN
8 2
O [M + H] : 491.2319, found 491.2361.
For the acute single dose study, vehicle (0.5% carbox-
ymethylcellulose sodium, 10 mL/kg), compounds 15a (5 and 15 mg/
kg), 21e (5 and 15 mg/kg) and Metformin (300 mg/kg) were adminis-
tered to C57BL/6N mice after 16-hrsstarvation, 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).
4
.1.38. tert-butyl (endo)-5-((8-((4-cyano-2-fluorophenyl)amino)pyrimido
[
(
5,4-d]pyrimidin-4-yl)amino)-2-azabicyclo[2.2.2]octane-2-carboxylate
21g)
Follow the similar synthetic procedure of compounds 15a–15b.
1
Light yellow solid, 14% yield. H NMR (600 MHz, CDCl
3
) δ (ppm): 9.34
(
s, 1H), 9.11 (t, J = 8.3 Hz, 1H), 8.75 (s, 1H), 8.60 (d, J = 3.6 Hz, 1H),
7
1
.56 (d, J = 8.8 Hz, 1H), 7.48 (d, J = 10.5, 1H), 7.04 (d, J = 7.3 Hz,
H), 4.51 (m, 1H), 4.22 (m, 0.6H), 4.07 (m, 0.4H), 3.60 (m,. 1H), 3.46
Acknowledgments
(
1
(
1
3
m, 1H), 2.67–2.59 (m, 1H), 2.36–2.31 (m, 1H), 1.99–1.93 (m, 2H),
13
.84–1.76 (m, 2H), 1.70–1.65 (m, 1H), 1.50 (s, 9H).
) δ: 158.5, 156.1, 155.7, 154.7, 153.4, 152.6, 150.9,
32.3, 131.9, 131.5, 129.4, 121.3, 118.4, 106.1, 79.4, 48.0, 47.8, 43.5,
8 2
5.6, 30.0, 28.6 (×3), 26.7. HRMS-ESI (m/z) calcd for C25 27FN O
C NMR
This work was supported by National Natural Science Foundation of
China (81460526), Jiangxi Provincial Department of Science and
Technology (20171BAB205103), Hongcheng Haiou Plan of Nanchang
City (Zunhua Yang, 2017) and Health and Family Planning Commission
of Jiangxi Province (2016A013, 20185521 and 20185523).
150 MHz, CDCl
3
H
+
[
M + H] : 491.2319, found 491.2355.
4.1.39. tert-butyl (exo)-5-((8-((4-cyano-2-fluorophenyl)amino)pyrimido
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[
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Light yellow solid, 15% yield. H NMR (600 MHz, CDCl
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8