Design and synthesis of alkyl substituted pyridino[2,3-D]pyrimidine compounds as PI3Kα/mTOR dual inhibitors with improved pharmacokinetic properties and potent in vivo antitumor activity

Article abstract of DOI:10.1016/j.bmc.2018.06.025

Using pyridino[2,3-D]pyrimidine as the core, total 13 pyridino[2,3-D]pyrimidine derivatives with different alkyl substituents at C2 site have been designed and synthesized to search for novel PI3Kα/mTOR dual inhibitors. Most of the target compounds showed potent mTOR inhibition activity with IC₅₀ values ranging from single to double digit nanomole. Five target compounds exhibited pronounced PI3Kα inhibition activity. In vitro cellular assay indicated that most of the target compounds showed excellent antiproliferative activity, especially 3j whose potency against SKOV3 was 8-fold higher than the positive control AZD8055. In vitro metabolic stability study found that 3j had a comparable stability to that of AZD8055. More importantly, 3j showed better antitumor activity and pharmacokinetic properties in vivo as compared with AZD8055.

Full text of DOI:10.1016/j.bmc.2018.06.025

Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of alkyl substituted pyridino[2,3-D]pyrimidine
compounds as PI3Kα/mTOR dual inhibitors with improved pharmacokinetic
properties and potent in vivo antitumor activity
Yinyin Liua, Qinhua Xiaa,⁎, Lei Fangb,⁎
a
Nanjing University of Chinese Medicine, Nanjing 210046, China
Jiangsu Province Hi-Tech Key Laboratory for Bio-medical Research, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, China
b
A R T I C L E I N F O
A B S T R A C T
Keywords:
PI3Kα/mTOR dual inhibitors
Antitumor activity
Using pyridino[2,3-D]pyrimidine as the core, total 13 pyridino[2,3-D]pyrimidine derivatives with different alkyl
substituents at C2 site have been designed and synthesized to search for novel PI3Kα/mTOR dual inhibitors.
Most of the target compounds showed potent mTOR inhibition activity with IC50 values ranging from single to
double digit nanomole. Five target compounds exhibited pronounced PI3Kα inhibition activity. In vitro cellular
assay indicated that most of the target compounds showed excellent antiproliferative activity, especially 3j
whose potency against SKOV3 was 8-fold higher than the positive control AZD8055. In vitro metabolic stability
study found that 3j had a comparable stability to that of AZD8055. More importantly, 3j showed better anti-
tumor activity and pharmacokinetic properties in vivo as compared with AZD8055.
Pyridino[2,3-D]pyrimidine compounds
1. Introduction
However, this success is limited to several rare cancers. In the primary
solid tumor, single dose treatment with rapamycin analogs can achieve
With the deepening insight on cancer genomics and biology, several
key intracellular cancer-related signaling pathways have been dis-
covered. One of the main pathways is the phosphoinositide 3-kinase
only a moderate overall response rate.5 The limited success of these
single-hit agents may be due to the rapid acquired drug resistance and
compensatory mechanisms. In addition, the cross talk and feedback of
downstream members of the PI3K/AKT/mTOR pathway can also at-
tenuate the inhibitory effect on the early components. Thereby, to im-
prove the efficacy, PI3Kα/mTOR dual inhibitors have been developed.
Since such inhibitors could target at two nodal points of the pathway,
the PI3Kα/mTOR dual inhibitors appear to augment efficacy and lower
the likelihood to induce drug resistance.6 Pyridino[2,3-d] pyrimidine
scaffold seems to be a promising template for the development of
PI3Kα/mTOR dual inhibitors. In 2014, Routier’s group disclosed a
series of pyridopyrimidine-based PI3Ka/mTOR dual inhibitors which
showed nanomolar enzymatic and cellular activities on both targets.7
Later, Childers et al. reported another series of pyridopyrimidines with
a phenylurea moiety at the C2 site.8 Besides, AZD8055 (Fig. 1), a well-
known mTORC1/mTORC2 dual inhibitor that containing a pyridino
[2,3-d] pyrimidine core, has also been reported to have inhibitory effect
on PI3Kα, though the potency is mild.9 Referring to the structure-ac-
tivity relationship of pyridino[2,3-d] pyrimidine compounds that
summarized from Pike’s work,9 the structure of the substituent at the
C4 and C7 site of the pyridino[2,3-d] pyrimidine scaffold is conserved.
(PI3K)/protein kinase
B (AKT)/mammalian target of rapamycin
(mTOR) pathway that has become the preferred target for anti-cancer
drug development. The kinase mTOR, existed in two distinct multi-
protein complexes (mTORC1 and mTORC2), has a high degree of active
site similarity with PI3Ks.1,2 AKT attenuates the inhibitory effect of
tuberous sclerosis (TSC) protein on mTORC1 by phosphorylating the
TSC protein, allowing mTORC1 to be activated by GTPase. Activation of
mTOR further achieves a specific gene transcription and translation
through the ribosomal protein kinase p70S6K and transcriptional reg-
ulator protein 4EBP1 etc. Then the conduction process is completed,
which ultimately achieves cell response to extracellular signals.3,4 De-
pending on this extracellular signal, tumor cells regulate their con-
tinued proliferation, invasion, metastasis and anti-apoptotic activity to
maintain their malignant phenotypes.
Due to the central role of mTOR in the vital cellular processes,
mTOR inhibitors have emerged as promising anticancer agents.
Rapamycin analogs, known as the first generation of mTOR selective
inhibitors, have shown to be effective in a series of preclinical models.
Abbreviations: PI3K, phosphoinositide 3-kinase; mTOR, mammalian target of rapamycin; TSC, tuberous sclerosis; DDQ, 2,3-dicyano-5,6-dichlorobenzoquinone; THF, tetrahydrofuran;
DIPEA, N,N-diisopropylethylamine; DMF, N,N-dimethylformamide; MRT, mean retention time
⁎
Corresponding authors.
E-mail addresses: xa-qh@163.com (Q. Xia), lei.fang@seu.edu.cn (L. Fang).
https://doi.org/10.1016/j.bmc.2018.06.025
Received 16 May 2018; Received in revised form 14 June 2018; Accepted 16 June 2018
0968-0896/©2018ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:Liu,Y.,Bioorganic&MedicinalChemistry(2018),https://doi.org/10.1016/j.bmc.2018.06.025

Y. Liu et al.
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the C2 site may allow various structural modifications, leading to the
change of potency, physicochemical and pharmacokinetic properties. A
successful example is AZD8055 which is derived from Ku-0063794 by
replacing the 2,6-dimethyl morpholine with 3-methyl morpholine at
the C2 site. This modification significantly improved the potency of the
enzyme inhibition, aqueous solubility and pharmacokinetic properties.
Thereby, using AZD8055 as the lead compound, we further explored
the structural modifications on the pyridino[2,3-d] pyrimidine scaffold,
paying emphasis on the C2 site. Different from the former studies which
often employed heterocycle as the substituent, our modifications focus
on small alkyl group which connected to C2 site via a CeC bond. In this
work, a series of pyridino[2,3-D]pyrimidine derivatives with different
small alkyl substituents at C2 site have been designed (Fig. 1). We
comprehensively investigated the influence of the modifications on the
PI3Kα/mTOR inhibition activity, antiproliferative activity, metabolic
stability, pharmacokinetic property and the in vivo antitumor activity.
We found a potent PI3Kα/mTOR dual inhibitor (i.e. 3j) with remark-
able antitumor activity both in vitro and in vivo, showing a potential for
further exploration.
Fig. 1. The drug design strategy and the structures of the target compounds
3a–3m.
The optimal substituent at the C4 site is morpholine or 3S-methyl
morpholine since the morpholine oxygen may form a critical hydrogen
bond with amino acid residues.10 As for the C4 site, 3,4-disubstituted
phenyl group (e.g. 3-hydroxymethyl-4-methoxy phenyl group) seems
the preferred substituent. Changing these substituents to the others
would significantly decrease the activity. In contrast, the substituents at
2. Results and discussion
2.1. Chemistry
The synthetic routes were depicted in Schemes 1 and 2. As shown in
Scheme 1, the desired pyrido[2,3-d]pyrimidine derivatives (3a–3f)
Scheme 1. Synthesis of compounds 3a–3f. Regents and condition: (a) NaCN, DMSO, 140 °C, 2 h; (b) H2O2, DMSO, K2CO3, rt, 12 h; (c) Pd/C, H2, CH3OH, rt, 1 h; (d)
(CH3)3CSi(CH3)2Cl, NEt3, THF, 60 °C, 12 h; (e) NaH, CH2(CO2Et)2, CH3CN, reflux, 3 h; (f) LiBH4, THF, 60 °C, 2 h; (g) CH3SO2CH3, n-BuLi, THF, rt, 12 h; (h) CH3COCl,
CH3OH, 40 °C, 12 h; (i) tert-butyl cyanoacetate, t-BuOK, THF, rt, 12 h; (j) p-toluenesulfonic acid, toluene, reflux, 12 h.
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Scheme 2. Synthesis of compounds 3g–3m. Regents and condition: (a) trimethylsilylacetylene, CuI, Pd(PPh3)4, NEt3, DMF, 100 °C, 12 h; (b) NaOH, CH3OH, rt, 12 h;
(c) NaH, CH2(CO2Et)2, CH3CN, reflux, 3 h; (d) LiCl, DMSO, 150 °C, 2 h; (e) LiOH, THF/H2O, rt, 4 h; (f) SOCl2, CH3OH, then POCl3, CH3CON(CH3)2, DIPEA, 3S-methyl
morpholine, THF, 120 °C, 12 h; (g) LiAlH4, THF, 0 °C, 12 h; (h) (CH3) 3CHO, CuCl2, CH3CH2OH, 75 °C, 12 h; (i) DDQ, CH2Cl2, rt, 1 h; (j) POCl3, 3S-methyl morpholine,
CH2Cl2, rt, 1 h; (h) K2CO3, Pd(PPh3)4, dioxane, 80 °C, 12 h; (i) Pd(PPh3)2Cl2, CuI, DIPEA, DMF, 130 °C, 0.5 h; (j) H2, 10% Pd/C, methanol, rt, 1 h.
were prepared from the key intermediate 2 which was prepared ac-
cording to a literature method.9 Briefly, compound 2 was treated with
NaCN to give 3a, which was further reacted with H2O2 in the presence
of K2CO3 to give 3b. Compound 3c was obtained from the catalytic
hydrogenation of 3a. For the synthesis of 3d, the hydroxyl group of
intermediate 2 was firstly protected by (CH3)3CSi(CH3)2Cl to yield key
intermediate 4. Thereafter compound 5 was prepared via a condensa-
tion reaction of 4 and diethyl malonate. Then 3d was successfully
prepared by reducing 5 with LiBH4. Intermediate 4 was treated with
CH3SO2CH3 and followed the remove of the protection group to offer
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Table 1
IC50 values for enzymatic inhibition of mTOR and PI3Kα.
Compound
IC50 (μM)a
Compound
IC50 (μM)a
mTOR
PI3Kα
mTOR
PI3Kα
AZD8055
BEZ-235
0.008
–
0.001
3.6
0.03
2.6
0.2
0.01
0.5
0.4
3g
3h
3i
3j
3k
3l
0.029
0.095
0.010
0.002
0.072
0.030
0.053
0.008
0.010
0.002
0.001
0.010
0.009
0.015
> 10
> 10
3.0
0.4
1.3
3a
3b
3c
3d
3e
3f
0.032
0.048
0.146
> 0.5
> 0.5
0.088
0.011
0.015
0.030
0.5
0.1
0.2
3.2
> 10
> 10
> 10
> 10
> 10
> 10
3m
0.018
a
The IC50 values are the mean of at least three experiments.
3e. Intermediate 4 was treated with tert-butyl cyanoacetate under basic
condition to give 7 which was refluxed in toluene in the presence of p-
toluenesulfonic acid to give 3f (Scheme 1).
when the hydroxyl group was converted to the ether form (e.g. 3g), the
inhibition activity came back. Among all of the employed substituents
the hydrophobic alkyl groups like methyl, isopropyl and tertiary butyl
groups seemed to be optimal. Moreover, the size of the substituents also
had influence on the activity. It was found that the larger the size is, the
better the activity is. As for the alkyl groups, the order of the activity is
tertiary butyl > methyl > isopropyl. As for the substituent at C4 site,
both morpholine and 3S-methyl morpholine were suitable for the en-
zyme inhibition activity. No obvious difference of the activity was ob-
served between the two groups.
As for the synthesis of target compounds containing 3S-methyl
morpholine group at the C4 site of the core (3g–3m), the synthetic
route was outlined in Scheme 2. With CuI and Pd(PPh3)4 as catalysts,
compound 8 reacted with trimethylsilylacetylene to give 9, which was
treated with NaOH in methanol to yield 3g. Using the similar synthetic
method of 5, compound 11 was prepared from 10 and CH2(CO2Et)2.11
Compound 11 was treated with LiCl and thereafter the protection group
was removed by treating with LiOH to yield 3h. Compound 12, pre-
pared according to the former reported protocol,12 was coupled to 3S-
methyl morpholine to give 13, which was reduced by LiAlH4 to give 3i.
Compound 14 reacted with trimethyl acetaldehyde to give 15 which
was further treated with DDQ to yield 16. Using the similar synthetic
method of 13, 3S-methyl morpholine was coupled to the pyridino[2,3-
d] pyrimidine core, resulting in compound 17. Finally, 3j and 3k were
prepared from 17 and corresponding borate ester via a Suzuki reaction,
respectively. As for 3l and 3m, compound 8 reacted with 18 to give 3l
which was further reduced to offer 3m (Scheme 2).
2.3. Antitumor activity in vitro
The antiproliferative activity of the target compounds was measured
using the CCK-8 assay with AZD8055 as a positive control.13 Since
genetic aberrations of the PI3K pathway frequently occur in various
human cancers, four different human cancer cell lines including U87MG
human glioblastoma cell line, MCF-7 human breast cancer cell line, PC-
3 human prostate cancer cell line, and SKOV-3 human ovarian cancer
cell line, were employed in the assay. The results were shown in
Table 2. Similar to the results of the enzymatic inhibition test, com-
pounds 3h–3j that containing hydrophobic alkyl groups at C2 site
showed better activity than the others. In contrast, compounds 3b–3d
that containing hydrophilic substituents at C2 site showed very weak
antiproliferative effect. The consistence of the enzymatic and cellular
activity suggested that the antiproliferative effect mainly depended on
the enzymatic inhibition of PI3Kα and mTOR. As far as the cancer cell
lines were concerned, U87MG and SKOV-3 were generally more sen-
sitive to the target compounds than the other two. Among all of the
target compounds, 3j exhibited best activity against all of the tested cell
lines. In particular, the inhibition potency of 3j against SKOV-3 cell line
was even 8-fold higher than that of AZD8055, indicating a promising
potential for further exploration.
2.2. Enzymatic inhibition of PI3Kα and mTOR
All of the target compounds were screened for the enzymatic in-
hibition activity against PI3Kα and mTOR using Lance Ultra assay and
Kinase-Glo Plus luminescent assay, respectively. BEZ-235 (a potent
PI3Kα inhibitor) and AZD8055 were used as the positive controls. The
results were shown in Table 1. Similar to the lead compound AZD8055,
most of the target compounds, except 3c, 3d and 3e, exhibited potent
inhibitory activity towards mTOR with IC50 values ranging from single
to double digit nM. In particular, the potency of compound 3j was 4-
fold higher than that of AZD8055. When PI3Kα was regarded, five
target compounds showed positive inhibitory effect on PI3Kα with IC50
values at micromole level. The potency of the active target compounds
was lower than the positive control BEZ-235, but superior to the lead
compound AZD8055. Notably, 3j again showed the best inhibition ac-
tivity with an IC50 value of 0.4 μM, a tenth of that of AZD8055. Inter-
estingly, the enzymatic inhibition potency of compound 3k, which had
a similar structure to 3j but possessed a 3-(methylcarbamoyl)phenyl
group (a moiety from mTOR inhibitor AZD2014) at C7 site instead of
the 3-hydroxymethyl-4-methoxy phenyl group of 3j, significantly de-
creased as compared with 3j, indicating 3-hydroxymethyl-4-methoxy
phenyl group was optimal for C7 site. This result is consistent with the
Pike’s findings.9
2.4. Western blot analysis
To further confirm the intracellular PI3Kα and mTOR inhibitory
activity of 3j, we performed western blot analysis to investigate the
activity of the two major kinases, Akt and mTORC1, after the treatment
of U87MG cells with 3j. The kinase activity was determined by mon-
itoring the phosphorylation level of AKT and its downstream target S6
in U87MG cells. As shown from Fig. 2, 3j could completely inhibit the
phosphorylation of AKT at the dose of 200 and 20 nM. Even at the dose
of 2 nM, a significant inhibition was also observed. As for S6, a dose-
dependent inhibition of the phosphorylation of S6 was also found after
the treatment of 3j, though the potency was relatively lower than that
to AKT. These results are in accordance with the results of enzymatic
assays, which confirms the intracellular inhibition activity of 3j against
PI3K pathway.
By analyzing the structure-activity relationship, it can be found that
introduction of a sulfonyl group to the C2 site of the pyridino[2,3-d]
pyrimidine core (e.g. 3e) significantly decreased the inhibition activity
towards both PI3Kα and mTOR. Meanwhile, the hydrophilic sub-
stituents at C2 site seemed also unfavorable for the inhibition activity.
For example, when 1,3-propanediol was connected to the core resulting
in 3d, no inhibition activity was observed in the assay. Interestingly,
4

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Table 2
activity with an inhibition rate of 28%. In addition, no body weight loss
was observed after the administration of the tested compound (data not
shown), indicating a good safety 3j possessed. The results clearly in-
dicated that 3j could effectively inhibit tumor growth in vivo in a dose-
dependent manner, and its potency was higher than AZD8055. Thus, on
the basis of its potent in vivo efficacy, 3j was selected as a candidate for
further pharmacokinetic investigation.
Antiproliferative activities of the target compounds.
Compound
IC50 (nM)
U87MG
SEMa
MCF-7
PC-3
SKOV-3
AZD8055
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
21.2
7.9
27.0
8.5
36.7
6.8
7.9
17.0
5.1
12.0
7.0
55.0
9.0
25.0
12.0
920.0
821.0
> 1000
> 1000
> 1000
90.3
25.8
32.8
> 1000
> 1000
> 1000
> 1000
> 1000
185.7
72.0
77.3
54.0
6.0
> 1000
> 1000
> 1000
> 1000
> 1000
66.6
163.1
66.0
155.0
23.0
660.0
640.5
> 1000
> 1000
> 1000
54.5
52.4
49.1
44.7
2.7. Pharmacokinetic investigation
In order to investigate whether the structural modification has an
effect on the pharmacokinetic properties or not, compounds 3j was
selected for single dose studies on the pharmacokinetics property.
Beagle dogs were treated with 3j at a dose of 1 mg/kg by gavage, and
after 24 h the pharmacokinetic parameters of the half life (tl/2), max-
imum plasma concentration (Cmax), time to reach Cmax (Tmax), mean
residence time (MRT) and area under the plasma concentration–time
curve for 0–24 h after dosing (AUC0−24h) were determined (Table 4).
Compared with AZD8055, 3j demonstrated a general higher plasma
concentration (288 ng/mL vs 228 ng/mL), longer half-life (5.4 h vs
4.5 h) and mean residence time (4.3 h vs 3.1 h), suggesting that the
designed pyridino[2,3-D]pyrimidine derivative showed a better per-
formance than AZD8055 in pharmacokinetic.
2.7
5.2
4.2
89.2
91.9
33.8
7.2
8.0
2.9
7.6
2.3
2.5
2.2
2.0
6.5
8.7
7.7
4.3
3.2
0.7
7.0
1.1
32.5
85.5
94.2
4.0
12.1
14.0
31.0
225
443
110
212
476.0
7.0
8.1
15.0
58.6
125
118.5
5.0
11.0
9.8
3m
13.6
a
Data is the mean value of triplicate measurements. The cell viability was
measured after 48 h drug exposure.
3j
AZD8055
Vehicle 0.2
2
20
200 200 20
2
0.2 nM
3. Conclusion
pAKTS473
pAKTT308
A series of pyridino[2,3-D]pyrimidine derivatives with different
alkyl substituents at C2 site have been designed and synthesized to
search for novel PI3Kα/mTOR dual inhibitors. In the enzyme assay,
most of the target compounds showed potent mTOR inhibition activity
with IC50 values ranging from single to double digit nM. Five target
compounds exhibited pronounced PI3Kα inhibition activity.
Particularly, compound 3j showed most potent inhibitory activity to-
wards both kinases among all of the target compounds. In vitro cellular
assay indicated that most of the target compounds showed excellent
antiproliferative activity, especially 3j whose potency against SKOV3
was 8-fold higher than the positive control AZD8055. The consistence
of the enzymatic and cellular results suggested that PI3Kα/mTOR
probably were the targets of the designed compounds. Indeed, western
blot analysis further confirmed the intracellular inhibition effect of 3j
on the phosphorylation level of AKT and its downstream target S6. In
vitro metabolic stability study found that 3j had a better stability than
that of AZD8055. More importantly, 3j showed better antitumor ac-
tivity and pharmacokinetic properties in vivo as compared with
AZD8055. All of the findings suggested a promising potential of 3j for
further exploration.
AKT
pS6
S6
Fig. 2. Effects of 3j and AZD8055 on the phosphorylation level of AKT and its
downstream target S6 in U87MG cells after 24 h exposure.
2.5. Stability in human liver microsomes
The metabolic stability of compound 3j and AZD8055 was ex-
amined in vitro using human liver microsomes. The results were sum-
marized in Table 3. It was found that 3j showed excellent stability in
human liver microsomes with t1/2 value of 30.9 min, much longer than
that of AZD8055 (t1/2 = 21.0 min). This results indicated that structural
modification by replacing the 3-methyl morpholine moiety with small
alkyl group could improve the stability, which conformed to our pre-
conceived ideas. The clearance rate of 3j and AZD8055 was at the same
level.
4. Experiment section
4.1. Chemistry
4.1.1. Materials and instruments
All chemicals were obtained from commercial purchase and sol-
vents were purified and dried by standard procedures. Flash chroma-
tography (FC): silica gel (SiO2; 40 mm, 200–300 mesh). Melting points
were uncorrected and were determined using a capillary apparatus
(RDCSY-I). 1H NMR and 13C NMR Spectra: Bruker AVANCE-400 Digital
NMR Spectrometer, ESI-MS: Thermo Finnigan LCQ advantage MAX.
Elemental analysis was performed on a Vario EL III apparatus.
2.6. In vivo antitumor activity
In view of the good results of the enzymatic and cellular tests in
vitro, compounds 3j (3, 10 and 30 mg/kg) was selected for further in
vivo efficacy studies in U87MG tumor xenograft mouse model (Fig. 3).
AZD8055 (10 mg/kg) was used as positive control. It was found that 3j
exhibited significant in vivo efficacy. At a high dose of 30 mg/kg, the
tumor inhibition rate of 3j reached 83%. At a middle dose of 10 mg/kg,
3j also exhibited a good tumor inhibiting activity with an inhibition
rate of 64%, higher than that of equal dose of AZD8055 (59%). Even at
a low dose of 3 mg/kg, 3j still showed a moderate tumor inhibiting
4.1.2. 7-(3-(Hydroxymethyl)-4-methoxyphenyl)-4-morpholinopyrido[2,3-
d]pyrimidine-2-carbonitrile (3a)
Compound 2 (50 mg, 0.13 mmol) and NaCN (7 mg, 0.14 mmol)
were dissolved in 2 mL of DMSO. The obtained solution was heated to
140 °C and kept stirring for 2 h. After cooling to room temperature,
20 mL of water was added and the solution was extracted with AcOEt
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4.5
4
B
Vehicle
3.5
3
Vehicle
AZD8055 (10 mg/kg)
3j (3 mg/kg)
3j (3 mg/kg)
2.5
2
3j (10 mg/kg)
3j (30 mg/kg)
3j (10 mg/kg)
3j (30 mg/kg)
1.5
1
0.5
0
AZD8055
(10 mg/kg)
0
3
6
9
12
Days after the drug administration
Fig. 3. (A) Relative volumes of U87MG tumors after the treatment of different dose of 3j and AZD8055 in xenograft mouse models (n = 5). (B) Images of the tumors
after treatments with different formulations.
4.1.4. (5-(2-(Aminomethyl)-4-morpholinopyrido[2,3-d]pyrimidin-7-yl)-2-
methoxyphenyl)methanol (3c)
Table 3
Metabolic stability of 3j and AZD805 in human liver microsomes.
Compound 2 (6 mg, 0.02 mmol) was dissolved in 2 mL of methanol.
To the obtained solution ammonia solution (28%, 0.1 mL) and Pd/C
(10%, 3 mg) were added and H2 gas was charged. The mixture was
stirred for 1 h at room temperature. The mixture was then filtrated and
the filtrate was concentrated in vacuo and the crude product was pur-
ified by column chromatography (CH2Cl2:EtOAc = 5:1) to give com-
pound 3c as yellow solid. Yield 83%; m.p. 105–107 °C. 1HNMR
(400 MHz, CD3OD): δ 8.32 (s, 1H), 7.99–7.97 (m, 2H), 7.56 (d, 1H),
7.04 (d, 1H), 5.39 (s, 2H), 4.85 (s, 1H), 4.72 (s, 2H), 4.04–3.93 (m, 4H),
3.89 (s, 3H), 3.77–3.72 (m, 4H), 2.28–2.26 (m, 2H); 13CNMR (100 MHz,
CD3OD): δ 188.3, 180.2, 158.3, 156.8, 150.8, 145.7, 140.5, 136.7,
134.6, 132.8, 122.7, 114.9, 100.4, 66.0, 62.5, 55.3, 48.1, 47.2; ESI-MS:
382.2 [M+H]+; Element Analysis for C20H23N5O3 (%) C: 62.98%, H:
6.08%, N: 18.36% Found C: 62.86%, H: 6.20%, N: 18.44%.
Compound
T1/2 (min)
CLF (μL/min/mg)
AZD8055
3j
21.0
30.9
28.6
28.1
Table 4
Pharmacokinetic profile of 3j and AZD8055.a
Compd.
Dosea
(mg/kg)
Cmax (ng/mL) AUC (ng/mL * h) t1/2 (h)
MRT (h)
AZD8055
3j
1
1
228
288
39.2
44.5
787
857
108.5
98.4
4.5
5.4
0.7 3.1
0.5 4.3
0.5
0.4
a
Dosed as a solution of stroke-physiological saline solution with 5% DMSO.
(20 mL × 2). The organic phase was successively washed by water
(40 mL × 3) and saturated salt solution (40 mL × 3), and then dried
over anhydrous Na2SO4, filtrated, and purified by column chromato-
graphy (CH2Cl2:EtOAc = 5:1) to give compound 3a as yellow solid.
Yield 21%; m.p. 98–99 °C. 1HNMR (400 MHz, CD3OD): δ 8.57 (d, 1H),
8.33 (s, 1H), 8.25 (d, 1H), 8.13 (d, 1H), 7.19 (d, 1H), 5.37 (s, 1H), 4.75
(s, 2H), 4.13–4.09 (m, 4H), 3.98 (s, 3H), 3.89–3.88 (m, 4H); 13CNMR
(100 MHz, CD3OD): δ 174.8, 158.3, 156.8, 153.8, 151.6, 144.6, 141.2,
136.7, 134.6, 132.2, 126.8, 117.6, 114.4, 99.9, 66.5, 62.5, 55.3, 47.2;
ESI-MS: 378.0 [M+H]+; Element Analysis for C20H19N5O3 (%) C:
63.65%, H: 5.07%, N: 18.56% Found C: 63.56%, H: 5.01%, N: 18.53%.
4.1.5. 4-(7-(3-(((tert-Butyldimethylsilyl)oxy)methyl)-4-methoxyphenyl)-
2-chloropyrido[2,3-d]pyrimidin-4-yl)morpholine (4)
Compound 2 (300 mg, 0.78 mmol), (CH3)3CSi(CH3)2Cl (140 mg,
0.93 mmol) and NEt3 (0.2 mL, 1.55 mmol) were dissolved in 10 mL of
THF. The solution was stirred for 12 h at 60 °C. The mixture was then
filtrated and the filtrate was successively washed by saturated Na2CO3
solution (40 mL × 3) and saturated salt solution (40 mL × 3), and then
the organic phase was dried over anhydrous Na2SO4, filtrated, and
concentrated in vacuo to give the yellow crude product which was used
for the next reaction without purification.
4.1.6. Diethyl 2-(7-(3-(((tert-butyldimethylsilyl)oxy)methyl)-4-
methoxyphenyl)-4-morpholinopyrido[2,3-d]pyrimidin-2-yl)malonate (5)
NaH (28.80 mg, 1.20 mmol) and CH2(CO2Et)2 (192 mg, 1.20 mmol)
were dissolved in 10 mL of acetonitrile. The mixture was stirred for
0.5 h and then compound 4 (300 mg, 0.60 mmol) was added. The ob-
tained mixture was refluxed for 3 h. After cooling to room temperature,
the reaction mixture was poured into 30 mL of ice-water. The mixture
was extracted with AcOEt (40 mL × 3). The organic phase was washed
by saturated salt solution (50 mL × 2), and then dried over anhydrous
Na2SO4, filtrated, concentrated in vacuo and purified by column chro-
matography (CH2Cl2:EtOAc = 5:1) to give compound 5 as brown solid.
4.1.3. 7-(3-(Hydroxymethyl)-4-methoxyphenyl)-4-morpholinopyrido[2,3-
d]pyrimidine-2-carboxamide (3b)
Compound 3a (130 mg, 0.33 mmol), 30% H2O2 aqueous solution
(112 mg, 0.99 mmol) and K2CO3 (92 mg, 0.66 mmol) were added into
5 mL of DMSO and the obtained mixture was stirred for 12 h at room
temperature. Then 20 mL of water was added and the solution was
extracted with AcOEt (20 mL × 2). The organic phase was successively
washed by water (40 mL × 3) and saturated salt solution (40 mL × 3),
and then dried over anhydrous Na2SO4, filtrated, and purified by
column chromatography (CH2Cl2:EtOAc = 5:1) to give compound 3b as
yellow solid. Yield 16%; m.p. 112–114 °C. 1HNMR (400 MHz, CD3OD):
δ 8.32 (s, 1H), 7.92–7.90 (m, 2H), 7.56 (d, 1H), 7.04 (d, 1H), 5.39 (s,
1H), 4.86 (s, 2H), 4.00 (s, 3H), 3.90–3.85 (m, 8H); 13CNMR (100 MHz,
CD3OD): δ 186.6, 168.1, 166.9, 156.8, 156.0, 149.4, 144.4, 143.4,
136.7, 134.6, 132.2, 126.6, 114.4, 104.1, 66.5, 62.8, 55.5, 47.8; ESI-
MS: 396.2 [M+H]+; Element Analysis for C20H21N5O4 (%) C: 60.75%,
H: 5.35%, N: 17.71% Found C: 60.66%, H: 5.30%, N: 17.59%.
Yield 64%; m.p. 85–87 °C. ESI-MS: 382.2 [M+H]+
.
4.1.7. 2-(7-(3-(Hydroxymethyl)-4-methoxyphenyl)-4-morpholinopyrido
[2,3-d]pyrimidin-2-yl)propane-1,3-diol (3d)
Compound 5 (120 mg, 0.20 mmol) and LiBH4 (17 mg, 0.80 mmol)
were added into 10 mL of THF. The mixture was stirred for 2 h at 60 °C.
After cooling to room temperature, the reaction solution was con-
centrated in vacuo and purified by column chromatography
(CH2Cl2:EtOAc = 5:1) to give compound 3d as yellow solid. Yield 59%;
6

Y. Liu et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
4.1.12. (S)-(2-Methoxy-5-(4-(3-methylmorpholino)-2-((trimethylsilyl)
ethynyl)pyrido[2,3-d]pyrimidin-7-yl)phenyl)methanol (9)
m.p. 89–90 °C. 1HNMR (400 MHz, CD3OD): δ 8.33 (s, 1H), 8.01–7.96
(m, 2H), 7.51 (d, 1H), 7.10 (d, 1H), 5.02 (s, 1H), 4.97 (s, 2H), 4.78 (s,
2H), 3.87–3.85 (m, 7H), 3.71–3.55 (m, 8H), 2.97–2.96 (m, 1H);
13CNMR (100 MHz, CD3OD): δ 174.1, 172.1, 156.8, 153.2, 147.4,
145.5, 140.7, 136.7, 134.0, 132.0, 122.7, 114.0, 92.4, 66.5, 62.5, 58.8,
Compound 8 (150 mg, 0.37 mmol), trimethylsilylacetylene (74 mg,
0.75 mmol), CuI (8 mg, 0.037 mmol), Pd(PPh3)4 (15 mg, 0.037 mmol)
and NEt3 (114 mg, 1.10 mmol) were dissolved in 5 mL of DMF. The
solution was stirred for 12 h at 100 °C. The solution was then con-
centrated in vacuo and purified by column chromatography
(CH2Cl2:EtOAc = 5:1) to give compound 9 as yellow solid. Yield 80%;
55.1, 47.2, 45.9; ESI-MS: 427.1 [M+H]+
;
Element Analysis for
22H26N4O5 (%) C: 61.96%, H: 6.15%, N: 13.14% Found C: 62.05%, H:
6.21%, N: 13.01%.
C
m.p. 110–112 °C. ESI-MS: 463.2 [M+H]+
.
4.1.8. 4-(7-(3-(((tert-Butyldimethylsilyl)oxy)methyl)-4-methoxyphenyl)-
2-((methylsulfonyl)methyl)pyrido [2,3-d]pyrimidin-4-yl)morpholine (6)
To a solution of CH3SO2CH3 (282 mg, 3 mmol) in 5 mL of THF was
added n-BuLi dropwise, and the obtained solution was stirred for 0.5 h.
Then compound 4 (150 mg, 0.30 mmol) was added and the reaction
solution was stirred for 12 h at room temperature. Thereafter, the so-
lution was concentrated in vacuo and purified by column chromato-
graphy (CH2Cl2:EtOAc = 5:1) to give compound 6 as yellow solid. Yield
4.1.13. (S)-(5-(2-(2,2-Dimethoxyethyl)-4-(3-methylmorpholino)pyrido
[2,3-d]pyrimidin-7-yl)-2-methoxy-phenyl)methanol (3g)
Compound 9 (100 mg, 0.21 mmol) and NaOH (17 mg, 0.42 mmol)
were dissolved in 5 mL of methanol. The solution was stirred for 12 h at
room temperature. The solvent was then removed and the residue was
added into 50 mL of AcOEt. The solution was washed by saturated salt
solution (15 mL × 3), and then dried over anhydrous Na2SO4, filtrated,
concentrated in vacuo and purified by column chromatography
(CH2Cl2:EtOAc = 5:1) to give compound 3g as yellow solid. Yield 25%;
m.p. 86–88 °C. 1HNMR (400 MHz, DMSO‑d6): δ 6.91 (d, 1H), 6.81 (s,
1H), 6.71 (d, 1H), 6.48 (d, 1H), 5.62 (d, 1H), 3.59–3.56 (m, 1H), 3.18
(s, 3H), 3.17 (m, 1H), 2.77–2.73 (m, 1H), 2.48–2.46 (m, 1H), 2.32 (s,
3H), 2.28–2.17 (m, 4H), 1.85 (s, 6H), 1.77 (m, 2H), 1.65 (m, 2H);
13CNMR (100 MHz, DMSO‑d6): δ 173.0, 163.5, 156.8, 153.0, 152.1,
145.5, 142.0, 136.5, 134.6, 132.2, 127.9, 114.0, 105.4, 95.8, 72.1,
39%; m.p. 80–82 °C. ESI-MS: 599.2 [M+H]+
.
4.1.9. (2-Methoxy-5-(2-((methylsulfonyl)methyl)-4-morpholinopyrido
[2,3-d]pyrimidin-7-yl)phenyl) methanol (3e)
Compound
6 (10 mg, 0.02 mmol) and acetyl chloride (5 mg,
0.05 mmol) were dissolved in 10 mL of methanol. The solution was
stirred for 12 h at 40 °C. After cooling to room temperature, the reaction
solution was concentrated in vacuo and purified by column chroma-
tography (CH2Cl2:EtOAc = 5:1) to give compound 3e as yellow solid.
Yield 63%; m.p. 95–97 °C. 1HNMR (400 MHz, CD3OD): δ 8.55 (s, 1H),
8.45 (d, 1H), 8.15 (d, 1H), 7.67 (d, 1H), 7.05 (d, 1H), 5.39 (s, 1H), 4.86
(s, 2H), 4.83 (s, 2H), 4.14–4.10 (m, 4H), 3.97 (s, 3H), 3.88–3.83 (m,
4H), 3.15 (s, 3H); 13CNMR (100 MHz, CD3OD): δ 178.4, 167.1, 157.4,
156.8, 154.0, 143.7, 141.4, 136.7, 134.5, 132.2, 125.8, 114.4, 98.1,
68.0, 66.5, 62.5, 53.0, 47.2, 42.3; ESI-MS: 445.1 [M+H]+; Element
Analysis for C21H24N4O5S (%) C: 56.74%, H: 5.44%, N: 12.60% Found
C: 57.00%, H: 5.51%, N: 12.78%.
68.3, 62.5, 55.3, 54.9, 48.9, 47.1, 42.4, 13.2; ESI-MS: 455.2 [M+H]+
Element Analysis for C24H30N4O5 (%) C: 63.42%, H: 6.65%, N: 12.33%
Found C: 63.20%, H: 6.45%, N: 12.39%.
;
4.1.14. (S)-2-(7-(4-Methoxy-3-((pivaloyloxy)methyl)phenyl)-4-(3-
methylmorpholino)pyrido[2,3-d] pyrimidin-2-yl)malonate diethyl (11)
NaH (133 mg, 3.27 mmol) was dissolved in 35 mL of THF.
CH2(CO2Et)2 (525 mg, 3.27 mmol) was added into the reaction solution
dropwise. The obtained solution was stirred for 0.5 h at room tem-
perature. Thereafter a solution of compound 10 (700 mg, 1.49 mmol) in
20 mL THF was added dropwise. The reaction solution was then re-
fluxed for 12 h. The solvent was then removed and the residue was
added into 50 mL of AcOEt. The solution was successively washed by
water (15 mL × 2) and saturated salt solution (15 mL × 3), and then
dried over anhydrous Na2SO4, filtrated, concentrated in vacuo to give
compound 11 as yellow solid which was used for the next reaction
without purification.
4.1.10. tert-Butyl 2-(7-(3-(((tert-butyldimethylsilyl)oxy)methyl)-4-
methoxyphenyl)-4-morpholinopyrido [2,3-d]pyrimidin-2-yl)-2-
cyanoacetate (7)
t-BuOK (40 mg, 0.35 mmol) and tert-butyl cyanoacetate (140 mg,
1.00 mmol) were dissolved in 5 mL of THF. The mixture was stirred for
0.5 h. Then compound 4 was added and the solution was stirred for
12 h. The solvent was then removed and the residue was added into
30 mL of water. The mixture was extracted with AcOEt (40 mL × 3).
The organic phase was then successively washed by saturated Na2CO3
solution (40 mL × 3) and saturated salt solution (40 mL × 3), and then
dried over anhydrous Na2SO4, filtrated, and concentrated in vacuo to
give yellow product which was used for the next reaction without
purification.
4.1.15. (S)-(2-Methoxy-5-(2-methyl-4-(3-methylmorpholino)pyrido[2,3-
d]pyrimidin-7-yl)phenyl)methanol (3h)
Compound 11 (860 mg, 1.41 mmol) and LiCl (60 mg, 1.41 mmol)
were dissolved in 15 mL of DMSO. The obtained solution was stirred for
2 h at 150 °C. After cooling to room temperature, 30 mL water and
30 mL AcOEt were added into the reaction solution. The organic phase
was dried over anhydrous Na2SO4, filtrated, concentrated in vacuo to
give pale yellow oil which was used for the next reaction without
purification.
The obtained oil and LiOH (28 mg, 0.65 mmol) were dissolved in a
mixture of 5 mL THF and 1 mL water. The solution was stirred for 4 h at
room temperature. The solution was then concentrated in vacuo and
purified by column chromatography (CH2Cl2:EtOAc = 5:1) to give
compound 3h as pale yellow solid. Yield 58%; m.p. 52–55 °C. 1HNMR
(400 MHz, DMSO‑d6): δ 8.29 (d, 1H), 8.26 (s, 1H), 8.17 (d, 1H), 7.77 (d,
1H), 7.03 (d, 1H), 4.79 (s, 2H), 4.56–4.55 (m, 1H), 4.09–4.06 (m, 1H),
4.01–3.99 (m, 1H), 3.96 (s, 3H), 3.86–3.83 (m, 1H), 3.76–3.73 (m, 3H),
2.71 (s, 3H), 1.52 (d, 3H); 13CNMR (100 MHz, DMSO‑d6): δ 175.4,
174.2, 156.8, 152.8, 152.0, 145.5, 139.9, 136.7, 134.6, 132.0, 127.5,
114.0, 97.5, 72.1, 68.0, 63.3, 55.3, 48.9, 47.1, 24.6, 13.2; ESI-MS:
455.2 [M+H]+; Element Analysis for C21H24N4O3 (%) C: 66.30%, H:
6.36%, N: 14.73% Found C: 66.20%, H: 6.46%, N: 14.66%.
4.1.11. 2-(7-(3-(Hydroxymethyl)-4-methoxyphenyl)-4-morpholinopyrido
[2,3-d]pyrimidin-2-yl)acetonitrile (3f)
Compound
7 (100 mg, 0.16 mmol) and p-toluenesulfonic acid
(10 mg, 0.03 mmol) were dissolved in 10 mL of toluene. The solution
was firstly stirred for 12 h at room temperature and then further stirred
for 3 h at 65 °C. The solvent was then removed and the residue was
added into 50 mL of AcOEt. The solution was washed by saturated
NH4Cl solution (15 mL × 3), and then dried over anhydrous Na2SO4,
filtrated, concentrated in vacuo and purified by column chromato-
graphy (CH2Cl2:EtOAc = 5:1) to give compound 3f as yellow solid.
Yield 23%; m.p. 116–118 °C. 1HNMR (400 MHz, CDCl3): δ 8.48 (s, 1H),
8.26 (d, 1H), 8.02 (d, 2H), 6.97 (d, 1H), 4.81 (s, 2H), 4.45 (s, 2H),
4.15–4.06 (m, 4H), 3.97 (s, 3H), 3.89 (m, 4H); 13CNMR (100 MHz,
CDCl3): δ 175.2, 157.1, 156.8, 154.5, 150.4, 145.5, 142.0, 136.7, 134.6,
132.2, 127.7, 120.8, 114.4, 99.1, 66.5, 62.5, 55.3, 47.2, 27.0; ESI-MS:
392.1 [M+H]+; Element Analysis for C21H21N5O3 (%) C: 64.44%, H:
5.41%, N: 17.89% Found C: 64.19%, H: 5.21%, N: 17.78%.
7

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Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
4.1.16. Methyl 5-(2-isopropyl-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-
7-yl)-2-methoxybenzoate (13)
4.1.21. (S)-(5-(2-(tert-Butyl)-4-(3-methylmorpholino)pyrido[2,3-d]
pyrimidin-7-yl)-2-methoxyphenyl) methanol (3j)
Compound 12 (200 mg, 0.60 mmol) and SOCl2 (1 mL) were dis-
solved in 5 mL of methanol. The solution was stirred and refluxed for
12 h. The solvent was then removed and the residue was added into a
mixture of 5 mL of CH2Cl2 and 5 mL of water. The organic phase was
washed by saturated salt solution (15 mL × 3), and then dried over
anhydrous Na2SO4, filtrated, concentrated in vacuo to give yellow solid
which was used for the next reaction without purification.
The obtained yellow solid was added into 3 mL of POCl3 and the
solution was refluxed for 6 h. The solution was concentrated in vacuo.
The residue was dissolved in a solution of 5 mL CH3CON(CH3)2, 3S-
methyl morpholine (100 mg, 1 mmol) and 1 mL DIPEA. The solution
was stirred for 12 h at 120 °C. Then 20 mL of water was added into the
reaction solution which was further extracted with EtOAc (25 mL × 2).
The organic phase was washed by saturated salt solution (15 mL × 3),
and then dried over anhydrous Na2SO4, filtrated, concentrated in vacuo
to give crude product which was used for the next reaction without
purification.
Compound 17 (60 mg, 0.19 mmol), phenylborate ester (59 mg,
0.22 mmol), Pd(PPh3)4 (6 mg, 10%) and K2CO3 (78 mg, 0.56 mmol)
were added into a mixture of dioxane/H2O (V/V = 4:1). The reaction
solution was stirred for 12 h at 80 °C. Thereafter the solution was
concentrated in vacuo. The residue was added into a solution of 3S-
methyl morpholine (100 mg, 1 mmol) in 5 mL CH2Cl2. And the obtained
solution was stirred for 1 h at room temperature. The solution was then
concentrated in vacuo and purified by column chromatography
(CH2Cl2:CH3OH = 20:1) to give compound 3j as pale yellow solid.
Yield 65%; m.p. 73–75 °C. 1HNMR (400 MHz, DMSO‑d6): δ 8.27–8.25
(m, 1H), 8.23–8.22 (m, 1H), 8.18–8.16 (m, 1H), 7.77–7.75 (m, 1H),
7.03–7.01 (m, 1H), 4.80 (s, 2H), 4.47–4.45 (m, 1H) 4.06–4.98 (m, 2H),
3.96 (s, 3H), 3.84–3.69 (m, 4H), 1.66–1.64 (m, 3H), 1.49 (s, 9H);
13CNMR (100 MHz, DMSO‑d6): δ 189.9, 174.7, 157.4, 152.5, 151.5,
145.5, 138.6, 136.7, 134.0, 132.5, 127.5, 114.4, 97.6, 72.1, 68.5, 62.0,
55.3, 48.9, 47.1, 29.4, 26.1, 13.2; ESI-MS: 423.2 [M+H]+; Element
Analysis for C24H30N4O3 (%) C: 68.22%, H: 7.16%, N: 13.26% Found C:
68.28%, H: 7.25%, N: 13.39%.
4.1.17. (S)-(5-(2-Isopropyl-4-(3-methylmorpholino)pyrido[2,3-d]
pyrimidin-7-yl)-2-methoxyphenyl) methanol (3i)
4.1.22. (S)-3-(2-(tert-Butyl)-4-(3-methylmorpholino)pyrido[2,3-d]
pyrimidin-7-yl)-N-methylbenzamide (3k)
Compound 13 (40 mg, 0.09 mmol) was dissolved in 5 mL of THF.
The solution was cooled to 0 °C, and then LiAlH4 (5 mg, 0.11 mmol) was
added. The solution was stirred for 12 h at room temperature, then
concentrated in vacuo and purified by column chromatography
(CH2Cl2:EtOAc = 5:1) to give compound 3i as yellow solid. Yield 21%;
m.p. 81–83 °C. 1HNMR (400 MHz, DMSO‑d6): δ 8.30–8.25 (m, 2H), 8.17
(d, 1H), 7.78 (d, 1H), 7.03 (d, 1H), 4.79 (s, 2H), 4.52–4.50 (m, 1H),
4.10–4.02 (m, 1H), 3.96 (s, 3H), 3.78–3.73 (m, 2H), 3.44–3.38 (m, 2H),
2.91–2.89 (m, 2H), 1.69 (s, 6H), 1.53–1.51 (m, 3H); 13CNMR (100 MHz,
DMSO‑d6): δ 179.4, 174.1 156.8, 152.4, 150.7, 145.7, 139.5, 136.7,
134.6, 132.2, 127.9, 114.0, 99.7, 72.1, 68.3, 62.5, 55.3, 48.9, 47.1,
Yield 65%; m.p. 73–75 °C. 1HNMR (400 MHz, CDCl3): δ 8.80 (s, 1H),
8.27–8.22 (m, 2H), 8.03–8.01 (m, 1H), 7.86–7.84 (m, 1H), 7.59–7.55
(m, 1H), 6.55 (s, 1H), 4.57–4.56 (m, 1H), 4.30–4.20 (m, 1H), 4.05–4.01
(m, 1H), 3.85–3.77 (m, 4H), 3.09–3.08 (m, 3H), 1.60–1.56 (m, 3H),
1.51 (s, 9H); 13CNMR (100 MHz, CDCl3): δ 192.0, 174.7, 168.0, 154.3,
152.6, 143.0, 137.4, 135.8, 133.0, 132.0, 131.3, 129.7, 128.9, 97.0,
72.5, 68.3, 48.9, 47.5, 29.5, 26.1, 26.1, 13.1; ESI-MS: 420.4 [M+H]+
Element Analysis for C24H29N5O2 (%) C: 68.71%, H: 6.97%, N: 16.69%
Found C: 68.52%, H: 6.88%, N: 16.45%.
;
35.0, 20.8, 13.2; ESI-MS: 409.2 [M+H]+
23H28N4O3 (%) C: 67.63%, H: 6.91%, N: 13.72% Found C: 67.48%, H:
6.85%, N: 13.79%.
;
Element Analysis for
4.1.23. (S)-(5-(2-(3,6-Dihydro-2H-pyran-4-yl)-4-(3-methylmorpholino)
pyrido[2,3-d]pyrimidin-7-yl)-2-methoxyphenyl)methanol (3l)
C
Compound 8 (250 mg, 0.67 mmol), 18 (300 mg, 0.80 mmol), Pd
(PPh3)2Cl2 (47 mg, 0.067 mmol), CuI (15 mg, 0.07 mmol) and DIPEA
(0.23 mL, 1.34 mmol) were added into 5 mL DMF. The reaction solution
was stirred for 0.5 h at 130 °C. Thereafter 25 mL water was added into
the solution. The obtained solution was extracted with AcOEt
(30 mL × 2), and the organic phase was washed by saturated salt so-
lution (20 mL × 2), and then dried over anhydrous Na2SO4, filtrated,
concentrated in vacuo and purified by column chromatography
(CH2Cl2:CH3OH = 20:1) to give compound 3l as yellow solid. Yield
29%; m.p. 78–80 °C. 1HNMR (400 MHz, DMSO‑d6): δ 8.27–8.26 (m,
2H), 8.22–8.20 (d, 1H), 7.80 (d, 1H), 7.07–7.05 (d, 1H), 5.96–5.94 (m,
1H), 4.86 (s, 2H), 4.56–4.54 (m, 1H), 4.48–4.46 (m, 2H), 4.06–4.04 (m,
2H), 4.00 (s, 3H), 3.98–3.96 (m, 1H), 3.83–3.80 (m, 3H), 3.78–3.76 (m,
1H), 2.92–2.90 (m, 2H), 2.35 (s, 2H), 1.52 (d, 2H); 13CNMR (100 MHz,
DMSO‑d6): δ 174.2, 170.0, 156.8, 156.0, 151.6, 144.4, 143.1, 137.9,
136.7, 134.6, 132.2, 126.6, 124.0, 114.4, 101.8, 72.1, 68.3, 67.7, 67.1,
62.5, 55.3, 48.9, 47.1, 23.8, 13.2; ESI-MS: 449.2 [M+H]+; Element
Analysis for C25H28N4O4 (%) C: 66.95%, H: 6.29%, N: 12.49% Found C:
66.90%, H: 6.18%, N: 12.39%.
4.1.18. 2-(tert-Butyl)-7-chloro-2,3-dihydropyrido[2,3-d]pyrimidin-4(1H)-
one (15)
Compound 14 (500 mg, 2.90 mmol), (CH3)3CHO (754 mg,
8.80 mmol) and CuCl2 (1.20 g, 8.80 mmol) were added into 5 mL of
ethanol. The obtained mixture was stirred at 75 °C for 12 h. Then the
mixture was filtrated and the filtrate was concentrated in vacuo and
purified by column chromatography (CH2Cl2:CH3OH = 20:1) to give
compound 15 as white solid. Yield 29%; m.p. 121–123 °C. ESI-MS:
240.2 [M+H]+
.
4.1.19. 2-(tert-Butyl)-7-chloropyrido[2,3-d]pyrimidin-4(3H)-one (16)
Compound 15 (110 mg, 0.46 mmol) and DDQ (210 mg, 0.92 mmol)
were dissolved in 5 mL of CH2Cl2. The obtained solution was stirred at
room temperature for 1 h. Then 5 mL of water was added and the so-
lution was extracted with CH2Cl2 (10 mL × 3), and the organic phase
was washed by saturated NaHCO3 solution (10 mL × 2), and then dried
over anhydrous Na2SO4, filtrated, concentrated in vacuo and purified
by column chromatography (CH2Cl2:CH3OH = 20:1) to give compound
16 as gray solid. Yield 75%; m.p. 133–134 °C. ESI-MS: 238.2 [M+H]+
.
4.1.24. (S)-(2-Methoxy-5-(4-(3-methylmorpholino)-2-(tetrahydro-2H-
pyran-4-yl)pyrido[2,3-d]pyrimidin- 7-yl)phenyl)methanol (3m)
4.1.20. (S)-4-(2-(tert-Butyl)-7-chloropyrido[2,3-d]pyrimidin-4-yl)-3-
methylmorpholine (17)
Compound 3l (25 mg, 0.05 mmol) and 10% Pd/C (10 mg) were
added into 20 mL of methanol. After charging with H2 gas, the reaction
solution was stirred for 3 h at room temperature. Thereafter the reac-
tion mixture was filtrated and the filtrate was concentrated in vacuo
and purified by column chromatography (CH2Cl2:CH3OH = 20:1) to
give compound 3m as pale yellow solid. Yield 79%; m.p. 92–95 °C.
1HNMR (400 MHz, DMSO‑d6): δ 8.29 (d, 1H), 8.25 (d, 1H), 8.18 (d,
1H), 7.78 (d, 1H), 7.04 (d, 1H), 4.80 (s, 2H), 4.56–4.54 (m, 1H), 4.17
(d, 2H), 3.99 (d, 1H), 3.96 (s, 3H), 3.82–3.80 (m, 1H), 3.75 (d, 3H),
Compound 16 (80 mg, 0.33 mmol) was added into 2 mL of POCl3
and the solution was refluxed for 0.5 h. The reaction solution was then
concentrated in vacuo. The residue was added into a solution of 3S-
methyl morpholine (100 mg, 1 mmol) in 5 mL CH2Cl2 and the obtained
solution was stirred for 1 h at room temperature. The solvent was then
removed in vacuo to give crude product which was used for the next
reaction without purification.
8

Y. Liu et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
3.58–3.56 (m, 2H), 3.22 (s, 1H), 2.14–2.12 (m, 3H), 2.02–2.00 (m, 2H),
1.56 (d, 3H); 13CNMR (100 MHz, DMSO‑d6): δ 173.5, 173.2, 156.8,
152.8, 148.1, 145.5, 139.9, 136.7, 134.6, 132.2, 127.6, 114.4, 102.8,
72.1, 70.2, 68.3, 62.5, 55.3, 48.9, 47.1, 38.7, 26.9, 13.2; ESI-MS: 451.2
[M+H]+; Element Analysis for C25H30N4O4 (%) C: 66.65%, H: 6.71%,
N: 12.44% Found C: 66.54%, H: 6.59%, N: 12.31%.
biotransformations were conducted using 1 mg/mL of HLMs in 200 μL
of reaction buffer containing 0.1 M Tris–HCl (pH 7.4) and the respective
test compound with a final volume of 50 μM. The reaction mixture was
preincubated at 37 °C for 5 min, and then, the reaction was commenced
by adding 50 μL of NADPH Regeneration System. The reaction was
ended after 120 min by the addition of cold methanol (200 μL). The
mixture was next centrifuged at 14,000 rpm for 15 min, and HPLC
analysis was performed.
4.2. Biological studies
4.2.1. Enzymatic inhibition
4.2.5. In vivo efficacy study
All of the compounds are tested for their activities against mTOR
using Lance Ultra Assay. The kinase reaction is done in 384-well black
plate by adding 2.5 mL of different concentration of compound solu-
tion, 2.5 mL mTOR (final 10 nM, control wells were added 2.5 mL ki-
nase buffer) solution and 5 mL substrate solution of ULight-4E-BP1
peptide substrate (final 50 nM) and ATP (final 13 mM) in kinase buffer
to each well in order. The assay plate was incubated at room tem-
perature for 1 h. Then 10 mL detection solution buffer was added to
each well of the assay plate to stop the reaction. The assay plate was
read on a plate reader Envision program for luminescence (Ex320/
Em665).
Total 25 BALB/c small nude mice (divided into five groups), female
for 6–7 weeks and body weight 16–20 g, are purchased from Shanghai
SLAC Laboratory Animal CO. LTD; feed the mice under grade SPF.
U87MG cells (5 × 106) were inoculated subcutaneously into the BALB/
c nude mice; when the tumor grows up to 100–150 mm3, allocate the
mice in groups randomly (D0). Respectively dose each group of mice by
intragastric administration with indicated dosage of the tested com-
pounds every day for 10 days; weigh the weight of mice twice every
3 days and record the data. Gross tumor volume (V): V = 1/2ab2. In
which, a means length; b means width.
For the Kinase-Glo® Luminescent Kinase Assay, the compound,
PI3Kα enzyme, the PIP2 (Life) substrate, and ATP (25 μM) were diluted
in kinase buffer to the indicated concentrations. The assay plate was
covered and incubated at room temperature for 1 h. Then, the Kinase-
Glo reagent was added to the PI3Kα plate to stop the reaction and
shaken for 15 min, followed by the addition of kinase detection reagent,
shaken for 1 min, and equilibrated for 60 min. The data were collected
on FlexStation and presented in Excel. The curves were fitted by
Graphpad Prism 5.0.
4.2.6. Pharmacokinetic study
Three male Beagle dogs (9–10 months old, body weight 9–10 kg)
were administrated with tested compounds at a daily dose of 1 mg/kg
by oral gavage, respectively. Blood collection was performed at time
points of 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10 and 24 h following
administration. Blood samples (∼1 mL) from Beagle dogs were col-
lected on ice until centrifugation was performed with in 1 h (1000 ×g
and 4 °C for 15 min). All plasma samples were stored at −20 °C before
analysis. The pharmacokinetic analyses were performed using
WinNonlin version 5.2.1 (Pharsight Corporation, Mountain View, CA,
USA). The pharmacokinetic parameters included half-life (t1/2), area
under the plasma concentration versus time curve within the 24-h
dosing intervals (AUC0–24) were determined by standard model in-
4.2.2. Antitumor test in vitro
The cells were plated in 96-well culture plates at density of 5000
cells per well and incubated for 24 h at 37 °C in a water-atmosphere (5%
CO2). The compounds with desired concentration were obtained by
dissolving in DMSO and diluting with culture medium (DMSO final
concentration < 0.4%). Then the diluted solution of compounds was
treated with the cells for 48 h at 37 °C in a 5% CO2 incubator. After that,
10 µL of a freshly diluted CCK-8 solution (5 mg/mL in PBS) were added
to each well and the plates were incubated for 1 h. The cell survival was
evaluated by measuring the absorbance at 540 nm. IC50 values were
determined from the chart of cell viability (%) against compound
concentration (nM). All experiments were carried out in triplicate.
dependent methods based on the plasma concentration-time data. Cmax
MRT, and Tmax were determined directly from the plasma concentra-
tion-time data.
,
Acknowledgments
The work is supported by the Fundamental Research Funds for the
Central Universities. Dr. Fang is thankful to the support of Natural
Science Foundation of Jiangsu Province – China (No. BK20151402) and
the Priority Academic Program Development of Jiangsu Higher
Education Institutions. The authors also thank Dr. A. Lv and Jiangsu
Hansoh Pharmaceutical Corporation for the valuable help on the
chemistry and pharmacology work.
4.2.3. Western blot analysis
U87MG cells were treated with 3j and AZD8055 at the indicated
concentrations for 24 h at 37 °C, then the cells were harvested, washed
in ice-cold PBS, and lysed with RIPA buffer, protease inhibitors, phos-
phatase cocktails A and B, and PMSF (1 mM). Protein concentration was
determined by the BCA Protein Assay Kit (beyotime#p0012s). The
samples were subjected to SDS–PAGE and then transferred onto PVDF
membranes. The membranes were incubated overnight at 4 °C with the
primary antibody in 5% BSA/TBST buffer with gentle shaking, then
washed with 1 × TBS/T 3 times, followed by incubation for 1 h with a
1/5000 dilution of secondary HRP antibody in 5% nonfat milk/TBST.
Primary antibodies used were anti-AKT, anti-pAKTser473,
antipAKTthr308, anti-S6RP, and anti-pS6RP Ser235/236, and the target
blots were detected with chemiluminescence system.
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4.2.4. Stability in human liver microsomes
Commercial, pooled, human (adult male and female) liver micro-
somes (HLMs) were purchased from Sigma-Aldrich. The
9