Synthesis and biological evaluation of irreversible EGFR tyrosine kinase inhibitors containing pyrido[3,4-d]pyrimidine scaffold

Article abstract of DOI:10.1016/j.bmc.2018.05.039

In the present study, a new class of compounds containing pyrido[3,4-d]pyrimidine scaffold with an acrylamide moiety was designed as irreversible EGFR-TKIs to overcome acquired EGFR-T790M resistance. The most promising compound 25h inhibited HCC827 and H1975 cells growth with the IC₅₀ values of 0.025 μM and 0.49 μM, respectively. Meanwhile, 25h displayed potent inhibitory activity against the EGFR^L858R (IC₅₀ = 1.7 nM) and EGFR^L858R/T790M (IC₅₀ = 23.3 nM). 25h could suppress EGFR phosphorylation in HCC827 and H1975 cell lines and significantly induce the apoptosis of HCC827 cells. Additionally, compound 25h could remarkably inhibit cancer growth in established HCC827 xenograft mouse model at 50 mg/kg in vivo. These results indicated that the 2,4-disubstituted 6-(5-substituted pyridin-2-amino)pyrido[3,4-d]pyrimidine derivatives can serve as effective EGFR inhibitors and potent anticancer agents.

Full text of DOI:10.1016/j.bmc.2018.05.039

Accepted Manuscript
Synthesis and Biological Evaluation of Irreversible EGFR Tyrosine Kinase In-
hibitors Containing Pyrido[3,4-d]pyrimidine Scaffold
Hao Zhang, Jin Wang, Hong-Yi Zhao, Xue-Yan Yang, Hao Lei, Minhang Xin,
Yong-Xiao Cao, San-Qi Zhang
PII:
S0968-0896(18)30793-4
https://doi.org/10.1016/j.bmc.2018.05.039
BMC 14381
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
24 April 2018
21 May 2018
23 May 2018
Please cite this article as: Zhang, H., Wang, J., Zhao, H-Y., Yang, X-Y., Lei, H., Xin, M., Cao, Y-X., Zhang, S-Q.,
Synthesis and Biological Evaluation of Irreversible EGFR Tyrosine Kinase Inhibitors Containing Pyrido[3,4-
d]pyrimidine Scaffold, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.05.039
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Synthesis and Biological Evaluation of Irreversible EGFR Tyrosine
Kinase Inhibitors Containing Pyrido[3,4-d]pyrimidine Scaffold
Hao Zhang a, Jin Wang b, Hong-Yi Zhao a, Xue-Yan Yanga, Hao Lei a, Minhang Xin a,
Yong-Xiao Cao b, San-Qi Zhang a *
a Department of Medicinal Chemistry, School of Pharmacy, Xian Jiaotong University,
Xian, Shaanxi, 710061, PR China
b
Department of Pharmacology, School of Basic Medical Science, Xian Jiaotong
University, Xian, Shaanxi, 710061, PR China
*Corresponding author: sqzhang@mail.xjtu.edu.cn (S. Q. Zhang).
Abstract
In the present study,
a
new class of compounds containing
pyrido[3,4-d]pyrimidine scaffold with an acrylamide moiety was designed as
irreversible EGFR-TKIs to overcome acquired EGFR-T790M resistance. The most
promising compound 25h inhibited HCC827 and H1975 cells growth with the IC50
values of 0.025 μM and 0.49 μM, respectively. Meanwhile, 25h displayed potent
inhibitory activity against the EGFRL858R (IC50 = 1.7 nM) and EGFRL858R/T790M (IC50 =
23.3 nM). 25h could suppress EGFR phosphorylation in HCC827 and H1975 cell
lines and significantly induce the apoptosis of HCC827 cells. Additionally, compound
25h could remarkably inhibit cancer growth in established HCC827 xenograft mouse
model at 50 mg/kg in vivo. These results indicated that the 2,4-disubstituted
6-(5-substituted pyridin-2-amino)pyrido[3,4-d]pyrimidine derivatives can serve as
effective EGFR inhibitors and potent anticancer agents.
Keywords
pyrido[3,4-d]pyrimidine; EGFR-TKIs; anticancer; irreversible inhibitors; drug design
1

1. Introduction
Receptor tyrosine kinases (RTKs) play an important role in the regulation of cell
growth, survival, and differentiation.1 Constitutive activation and/or over-expression
have been observed in numerous human tumors such as breast, ovarian, colon and
non-small cell lung cancers (NSCLC). Epidermal growth factor receptor (EGFR) is
among the known RTKs and has been extensively studied. As a number of the
families of ErbB, EGFR tyrosine kinase has been validated as one of the most
effective targets for the treatment of NSCLC.2, 3 So far, there are thousands of EGFR
tyrosine kinase inhibitors (EGFR-TKIs) were synthesized and evaluated. For NSCLC
patients harboring activating mutations (e.g., L858R and exon 19 deletion), the
first-generation EGFR-TKIs gefitinib and erlotinib (Fig. 1) showed effective tumor
regressions and improved progression-free survival (PFS).4-6 However, after about one
year of treatment, these anti-tumor effects are counteracted owing to drug-resistance
caused by T790M mutation.7 The transformation of threonine toward bulkier
methionine not only provokes a significant steric repulsion with the
4-aminoquinazoline-based inhibitors but also increases the affinity of EGFR to ATP,
rendering first-generation ATP-competitive inhibitors less effective.8, 9 To overcome
the resistance, the modification of the 6-substituted group in 4-anilinoquinazolines
with the group bearing active group provided the inhibitors with improved
potency.10-13 Likewise, the replacement of 6-substituted group in gefitinib with
acrylamide moiety produced the second-generation irreversible EGFR inhibitors
including afatinib14 (Fig. 1), dacomitinib15 and neratinib16. Afatinib demonstrated high
activity against T790M mutation and approved by FDA in 2013. Nevertheless,
afatinib failed to distinctly improved efficacy in the clinical trial data because of
dose-limiting toxicity (e.g. skin rash and diarrhea) caused by the loss of selectivity
over wild-type EGFR (EGFRWT).17, 18
Fig. 1. The structures of the first- and second-generation EGFR-TKIs
2

To conquer the shortcoming of second-generation EGFR-TKIs, researchers
developed third-generation inhibitors with high potency against T790M resistant
mutation but sparing EGFRWT activity. The strategy of employing an electrophilic
group capable of forming a C-S covalent bond with the conserved Cys797 residue was
still used in drug design and optimization. In the past few years, a family of
2-aminopyrimidine-based compounds, including osimertinib (AZD9291)19-21
rociletinib olmutinib avitinib
(CO-1686)22, (HM61713)23, (AC0010)24,
,
PF-0674777525 and PF-0645998826 have been researched to overcome T790M
mutation-related resistance (Fig. 2). These inhibitors not only effectively target
activated mutations and the T790M resistant mutation but also display better
selectivity over EGFRWT. For its precise biological properties, osimertinib was
approved by FDA in 2015 for the treatment of T790M patients. The progress of
2-aminopyrimidine-based EGFR-T790M inhibitors has been comprehensively
summarized.27-31
Fig. 2. The structures of the third-generation EGFR-TKIs
3

Fig. 3. The structures of new generation EGFR-TKIs with potency to EGFR C797S
Despite great success of third-generation EGFR-TKIs, acquired resistance remains
a significant clinical issue in NSCLC treatment. Resistance mechanisms of the
third-generation EGFR-TKIs have been summarized.27, 30 Among those possible
mechanisms, triple mutation C797S in EGFR accounts for more than 20%. Recently,
compounds 1032, 33, 1134, 35 1236 and 1337 were discovered as a new generation
EGFR-TKIs to combat the C797S-mediated resistance. Lately, we demonstrated that
2,4,6-trisubstitued
pyrido[3,4-d]pyrimidine38
and
2,9-disubstituted
8-phenylthio/phenylsulfinyl-9H-purine39 derivatives could serve as reversible
EGFR-TKIs with potent activities on EGFRL858R/T790M/C797S. Among those novel
compounds, compound 14 (Fig. 4) displayed strong activity against
EGFRL858R/T790M/C797S with IC50 value of 7.2 nM. Compared with the third-generation
EGFR inhibitors, compounds 10-12 and 14 incorporate a 2-naphthyl or
4-fluorophenyl to occupy the back hydrophobic pocket (BHP) formed by gatekeeper
Met790 and the side chain of Lys745, yielding potent activity due to the hydrophobic
interaction.
The innovation of drug structure is crucial in medicinal chemistry research. It is
of great value to develop more active and selective EGFR-T790M small molecule
inhibitors with a novel chemical skeleton. In an effort to acquire more effective
EGFR-T790M inhibitors based on reversible inhibitor 14, (i) we introduced
acrylamide warhead with moderate chemical reactivity as Michael acceptor (marked
in pink) to form the C-S covalent bond with Cys797 residue25, 26; (ii) aminopyrrolidine
and aminopiperidine with higher sp3 character was used as the linker of
pyrido[3,4-d]pyrimidine scaffold and warhead to achieve decreased melting point,
improved solubility and increased activity and selectivity40-43; (iii) in terms of
orientation and geometry, the rigid R1 phenylamino group was introduced to insert
4

BHP for lipophilic interactions. Herein, we describe the synthesis and biological
activities evaluation of pyrido[3,4-d]pyrimidine derivatives bearing flexible linker and
acrylamide warhead to target EGFR and overcome T790M resistance.
Fig. 4. Design of novel irreversible EGFR-TKIs containing pyrido[3,4-d]pyrimidine scaffold.
Het, heterocyclic scaffold; BHP, back hydrophobic pocket; HR, hydrophobic region; SER,
solvent exposed region; RP, ribose pocket; PBS, phosphate binding site.
2. Results and discussion
2.1. Chemistry
The synthetic route for compounds 20a-d was outlined in Scheme 1.
Commercially available 5-amino-2-chloroisonicotinic acid was used as the starting
material. The heating of the starting material with formamidine acetate produced
intermediate 15 in a solvent-free condition. Intermediate 15 was converted into
4,6-dichloropyrido[3,4-d]pyrimidine (16) by the reaction with thionyl chloride.
Depending on distinct difference in reactivity, 4-chloride in intermediate 16 was
substituted by pyrrolidine at room temperature to yield intermediate 17. The Boc
protecting group was removed by TFA, and the free amino group reacted with
acryloyl chloride to obtain intermediate 18. With the aid of Buchwald-Hartwig
amination, treatment of intermediate 18 with substituted aniline or 5-substituted
pyridin-2-amine yielded title compounds 20a-d.
5

Scheme 1. Synthetic route of compounds 20a-d. Reagents and conditions: (i) formamidine acetate,
160 °C, 0.5 h, 90.4%; (ii) SOCl2, DMF(cat.), reflux, 4 h; (iii) amine, DIPEA, THF, rt, 2 h, 65.1%,
over two steps; (iv) TFA, DCM, rt, 1.5 h; (v); acryloyl chloride, TEA, DCM, 0 °C–rt, 2 h, 79.8%,
over two steps; (vi) Pd(OAc)2, X-phos, Cs2CO3, 1,4-dioxane, reflux, 4 h, 56.4%–83.6%.
The synthetic route for compounds 25a-h was depicted in Scheme 2.
2,4,6-Trichloropyrido[3,4-d]pyrimidine (21) was prepared from 5-amino-2-
chloroisonicotinic acid as we reported.38 Depending on distinct difference in reactivity,
three chlorine atoms at the 4,2,6-position in 21 were successively replaced by amines.
Intermediates 22a-h were obtained by stirring the mixture of intermediate 21, DIPEA
and different amines in THF. The Boc group in 22a-h was removed by TFA, and the
free amino group reacted with acryloyl chloride to obtain intermediates 23a-h.
Intermediates 24a-h were produced by refluxing intermediates 23a-h and substituted
aniline in 2-butanol. With the aid of Buchwald-Hartwig amination, treatment of
intermediates 24a-h with 5-substituted pyridin-2-amine yielded title compounds
25a-h.
To
boost
the
structural
diversity
of
the
title
compounds,
29a-g
6-(acrylamidopyridin-2-amino)-4-(pyrrolidin-1-yl)pyrido[3,4-d]pyrimidines
(Scheme 3) were synthesized with similar approach.
6

Scheme 2. Synthetic route of compounds 25a-h. Reagents and conditions: (i) amines, DIPEA,
THF, rt, 1–2 h, 72.0%–100%; (ii) TFA, DCM, 0.5 h–2 h; (iii) acryloyl chloride, DIPEA, DCM,
0 °C–rt, 2 h–4 h, 24.9%–79.2%, over two steps; (iv) anilines, p-TsOH, 2-butanol, reflux, 2 h–4 h,
64.4%–100%; (v) Pd(OAc)2, X-phos, Cs2CO3, 1,4-dioxane, reflux, 2 h–4 h, 23.7%–67.7%.
Scheme 3. Synthesis route of compounds 29a-g. Reagents and conditions: (i) amines, DIPEA,
THF, rt, 0.5 h, 90.9%; (ii) for 27a and 27b, anilines, p-TsOH, 2-butanol, reflux, 2 h, 91.8–99.2%;
for 27c-g, alcohols, NaH, THF, rt, 2 h, 98.0%–100%; (v) Pd(OAc)2, X-phos, Cs2CO3, 1,4-dioxane,
reflux, 2 h–4 h, 17.6%–41.9%.
2.2. Biological evaluations
2.2.1. The antiproliferative activity in vitro
By employing the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-2H-tetrazolium bromide
(MTT) assay, we first evaluate the antiproliferative activities of synthesized
compounds against human epithelial carcinoma cell lines including A549 (EGFRWT),
HCC827 (harboring activated mutant EGFRDel E746-A750) and H1975 (drug-resistant,
carrying EGFRL858R/T790M). The third-generation EGFR inhibitor osimertinib was used
7

as the positive drug. The results of compounds 20a-d and 25a-h were listed in Table
1.
Table 1. Antiproliferative effects of compounds 20a-d and 25a-h (Mean ± SD, n = 3)
IC50 (μM)
Compds
linker
R3
HCC827
>10a
H1975
A549
ND
NDb
20a
>10
ND
ND
20b
>10
ND
>10
ND
ND
20c
20d
25a
3.52 ± 0.16
0.020 ± 0.003
1.44 ± 0.06
1.19 ± 0.33
0.069 ± 0.013
0.85 ± 0.16
0.64 ± 0.13
25b
0.054 ± 0.023
0.073 ± 0.005
1.75 ± 0.42
1.40 ± 0.02
0.81 ± 0.02
0.81 ± 0.26
25c
25d
0.17 ± 0.01
0.27 ± 0.02
0.44 ± 0.08
0.86 ± 0.25
1.40 ± 0.24
0.65 ± 0.10
1.67 ± 0.40
0.81 ± 0.05
25e
25f
25g
0.16 ± 0.02
8

0.025 ± 0.005
0.027 ± 0.01
0.49 ± 0.08
0.99 ± 0.04
25h
0.019 ± 0.008 0.53 ± 0.09
osimertinib
a inhibition rate < 50% at 10 μM; b ND, not determined.
To explore the effect of substituent at the 6-position of pyrido[3,4-d]pyrimidine
scaffold, we first synthesized compounds 20a-c. Disappointingly, no significant
antiproliferative activities against HCC827 cells were observed in compounds 20a-c.
We suspected that it is difficult for 20a-c to maintain pharmacophoric conformation
owing to the steric repulsion between the hydrogen atom at 5-position of
pyrido[3,4-d]pyrimidine and the hydrogen atom at 2-position of aniline. To overcome
the problem of steric clash, pyridin-2-yl was used to replace phenyl and compound
20d were obtained. In contrast with compounds 20a-c, compound 20d displayed
improved antiproliferative activity against HCC827 cells with IC50 value of 3.52 M.
The results indicated that the replacement of the phenyl with pyridin-2-yl was proved
to be effective in the promotion of antiproliferative activity. This finding is consistent
with our previous study of reversible EGFR-TKIs.38 Subsequently, to further enhance
the activity, compounds 25a-h with a phenylamino group at 2-position of scaffold
were synthesized. Compound 25a displayed potent activity against HCC827 with the
IC50 value of 0.020 M, which is more than 170-fold higher than compound 20d.
Obviously, 2-phenylamino moiety is very important to improve antiproliferative
activity. Compounds 25a and 25b with a 3-(acrylamido)pyrrolidin-1-yl group attached
to the 4-position of pyrido[3,4-d]pyrimidine scaffold exhibited potent antiproliferative
effects on HCC827 cells. While in compounds 25e and 25f with a
1-acrylamidopyrrolidin-3-yl-amino group attached to the 4-position of
pyrido[3,4-d]pyrimidine scaffold, only moderate antiproliferative effects were
observed. In these cases, (R)-configuration of chiral carbon is favorable for
antiproliferative effect. Compounds 25c and 25d with a 3-(acrylamido)piperidin-1-yl
group attached to the 4-position of pyrido[3,4-d]pyrimidine scaffold exhibited potent
antiproliferative effects on HCC827 cells. Compounds 25h with
(S)-1-(acrylamido)piperidin-3-yl group attached to the 4-position
a
of
pyrido[3,4-d]pyrimidine scaffold exhibited potent antiproliferative effects on HCC827
cells (IC50 = 0.025 M ) and mild antiproliferative effects on H1975 cells (IC50 = 0.49
M ). Moreover, compounds 25a-h showed more potent activities against HCC827
9

than the other cells.
To extend the diversity of compound structures and further study
structure-activity relationship (SAR), we moved the acrylamide moiety from
4-postion to 6-postion of pyrido[3,4-d]pyrimidine scaffold to produce compounds
29a-g. However, compared with compounds 25a-h and positive control, these
compounds showed a significant drop in the cell-based activity (Table 2). Only
compounds 29c and 29f displayed moderate activity against HCC827 cells with IC50
value of 2.74 μM and 8.70 μM, respectively. We noticed that compounds 29a-g
showed poorer solubility than compounds 25a-h due to the lack of
4-methylpiperazin-1-yl. And not only that, the polar 4-methylpiperazin lacking in
compounds 29a-g can stretch into solvent exposed region (SER), which hinted that
the polar group may be helpful for activity. The results indicated that 6-acrylamido
pyridine-2-ylamino at the 6-position of pyrido[3,4-d]pyrimidine scaffold was not
suitable.
Table 2. Antiproliferative effects of compounds 29a-g (Mean ± SD, n = 3)
IC50 (μM)
Compds
HCC 827
> 10a
29a
> 10
2.74 ± 0.58
> 10
29b
29c
29d
> 10
29e
8.70 ± 2.10
> 10
29f
29g
0 027 ± 0.01
osimertinib
a inhibition rate < 50% at 10 μM.
2.2.2. EGFR kinase activities assay
Compounds 25a-h, the active inhibitors against HCC827 cells, were selected to
determine their kinase inhibitory activity against EGFR to verify the mechanism of
10

antiproliferative activities. Osimertinib was used as the positive control. The data
were listed in Table 3. We can see that the selected compounds 25a-h displayed very
potent activities against activating mutation EGFRL858R with nanomolar range
inhibitory activity (IC50 values from 1.7 nM to 14.2 nM). Meanwhile, compounds 25a,
25b and 25h also showed strong inhibitory activity on EGFRL858R/T790M with the IC50
values of 36.6, 26.5 and 23.3 nM, respectively. Moreover, weak activities against
triple mutation EGFRL858R/T790M/C797S were observed in 25b (IC50 = 285.0 nM) and
25h (IC50 = 582.2 nM). Compared to reversible inhibitor 14 we have previously
descried, the irreversible compounds 25a-h showed weak inhibitory activity against
EGFRL858R/T790M/C797S in spite of introduction of acrylamide. Indeed, the acrylamide
moiety is not very important for the activity against EGFRL858R/T790M/C797S but crucial
for the T790M inhibition. The results of kinase activities assay also showed that
compounds 25a-h and osimertinib exhibited potent inhibitory activity against
EGFRWT at the same test conditions. It can be seen that the kinase inhibitory activities
of tested compounds are consistent with the effects on two selected cell lines HCC827
and H1975 harboring different EGFR types.
Table 3. Enzymatic activities of compounds against different types of EGFR (n = 2)
IC50 (nM)
L858R/T790M L858R/T790M/C797S
Compds
WT
2.7
L858R
4.7
36.6
26.5
NDa
285.0
> 500b
> 500
> 500
> 500
> 500
582.2
109.0
25a
25b
1.5
3.2
5.4
6.5
163.7
361.1
106.4
238.1
130.2
23.3
25c
12.0
3.3
14.2
4.6
25d
25e
9.9
10.8
4.5
25f
4.7
25g
1.9
1.7
25h
0.9
1.2
2.4
osimertinib
< 0.5c
2d
< 0.5c
1d
< 0.5c
< 1d
77d
a ND, not determined; b Inhibition rate < 50% at 500 nM; c The data reported by Gunther33; d The
data reported by Engel31.
2.2.3. Western blotting assay
To further verify the mechanism of the antiproliferative activity of compound
25h, Western blotting assay was employed to characterize inhibition of p-EGFR in
HCC827 and H1975 cell lines. As depicted in Fig. 5, compound 25h showed
11

significant suppression of p-EGFR in HCC827 at all tested concentrations range (Fig.
5A) while showed different suppression of p-EGFR in H1975 in a dose-dependent
manner (Fig. 5B). In addition, the inhibition of phosphorylation of EGFR in HCC827
and H1975 is consistent with antiproliferative activity in MTT assay.
Fig. 5. Effects of compound 25h on EGFR signaling in HCC827 (A) and H1975 (B) cells. Cells
were treated with the indicated concentrations for 8 h.
2.2.4. Cell apoptosis assay
To characterize whether compound 25h can induce cell apoptosis, the
annexinV/propidium iodide (PI) biparametric cytofluorimetric assay was performed
on HCC827 cells. As illustrated in Fig. 6, the percentages of apoptotic cells for
osimertinib and 25h at 3 μM were 76.38% and 75.84%, respectively, which indicated
that compound 25h could significantly induce the apoptosis of HCC827 cells.
Fig. 6. The apoptosis effect of compound 25h on HCC827 cells. (A) Without drug or DMSO treat
(control) and after treated with 0.03% DMSO, 3 μM of AZD (osimertinib) and 25h for 24 h. The
upper left quadrant Q1 represented necrotic cells, the upper right quadrant Q2 represented for late
apoptotic cells, the low left quadrant Q3 represented live cells and the low right quadrant Q4
represented for early apoptotic cells. Results are mean of three independent experiments. (B) The
apoptosis rate was the sum of Q2+Q4. Data are the means ± SD of values from three independent
12

experiments (ns, not significant vs control; *** p < 0.001 vs 0.03% DMSO).
2.2.5. Anticancer effect in vivo
Finally, the most promising inhibitor 25h, with potent inhibitory activity against
EGFRL858R and antiproliferative effect on HCC827, was evaluated for its antitumor
efficacy in HCC827 xenograft mouse model in vivo. According to reported data of
related third-generation EGFR-TKIs43-46, oral administration doses of 25h were
selected 10 mg/kg and 50 mg/kg. In the HCC827 xenograft model, mice (n =
5-6/group) were oral dosed once daily for 20 consecutive days. As depicted in Fig. 7A,
25h significantly inhibited the tumor growth at the dose of 10 mg/kg (TGI = 60.6%)
and 50 mg/kg (TGI = 86.5%) compared with the vehicle group. In addition, during the
course of the experiment, compound 25h did not cause significant body weight loss
and no mortality of mice was observed (Fig. 7C), suggesting that 25h was tolerated
well with low toxicity at those doses.
Fig. 7. In vivo antitumor efficacy of 25h in HCC827 xenograft mouse model at 10 mg/kg and 50
mg/kg. (A) Tumor volumes. (B) Tumor weights. (C) Body weight of mice. (D) Images of the
tumor collected from the mice on d21. All data represent mean ± SEM. (* p < 0.05, ** p < 0.01 vs
vehicle).
13

2.2.6. Molecular docking study
In order to explore the binding mode of our compounds, Sybyl-X 2.0 software
was used to dock 20d, 25a and 25h into ATP binding site of EGFRT790M (PDB code
2JIU).8 The results were depicted in Fig. 8. As the foremost pharmacophore, the
nitrogen atom at 7-position of pyrido[3,4-d] pyrimidine and the hydrogen atom from
aminopyridine at 6-position formed two vital hydrogen bonds with Met793 in the
hinge region. Hydrogen atom in aniline at 2-position of pyridopyrimidine scaffold
formed a hydrogen bond with gatekeeper residue Met790. The aminopyrrolidine or
aminopiperidine moiety at 4-position occupied the ATP ribose pocket and generated
Van der Waals interaction with Phe856 and Asp855 from the Asp-Phe-Gly (DFG)
motif. In potent inhibitors 25a and 25h, aniline at 2-position of
pyrido[3,4-d]pyrimidine inserted into the BHP (marked in red circle) (Fig. 8E/8F and
Fig. 8H/I) to form hydrophobic interactions. However, owing to the lack of aniline to
occupy the BHP (Fig. 8B/C), compound 20d only displayed weak activity on
HCC827 cells. Those findings implied that the hydrophobic interaction formed by
BHP with inhibitor was closely linked to inhibitory activity. In addition, the distance
(2.7 Å) of Cys797 toward acrylamide moiety in compound 25h (Fig. 8H) indicated
the potential possibility of forming covalent bond, which hinted 25h could serve as
irreversible EGFR inhibitor. Obviously, the docking results further verified our design
strategy.
14

Fig. 8. (A) Structure of 20d, (B) docking result of 20d with EGFRT790M and (C) gray surface
representation of 20d docked into ATP-binding site. (D) Structure of 25a, (E) docking result of
25a with EGFRT790M and (F) gray surface representation of 25a docked into ATP-binding site. (G)
Structure of 25h, (H) docking result of 25h with EGFRT790M and (I) gray surface representation of
25h docked into ATP-binding site. Hydrogen bonds are indicated as yellow dashed lines.
inhibitors (yellow) and key protein residues (green) are highlighted in stick form. Clear hydrogen
bond interactions with Met793, Met 790 and Gly 796 are observed. Red arrow indicates the
distance (2.7 Å) of Cys797 and acrylamide moiety in 25h. Red circles represent the back
hydrophobic pocket (BHP). Some parts of the protein are omitted for clarity.
3. Conclusions
In summary, we demonstrated that the 2,4-disubstituted 6-(5-substituted
15

pyridin-2-amino)pyrido[3,4-d]pyrimidine derivatives can serve as effective EGFR
inhibitors and potent anticancer agents. The most promising compound 25h inhibited
HCC827 and H1975 cells growth with the IC50 values of 0.025 μM and 0.49 μM,
respectively. Meanwhile, 25h displayed potent inhibitory activity against the
EGFRL858R (IC50 = 1.7 nM) and EGFRL858R/T790M (IC50 = 23.3 nM). Further
experiments indicated that 25h could suppress EGFR phosphorylation in HCC827 and
H1975 cell lines and significantly induce the apoptosis of HCC827 cells. Additionally,
compound 25h at 50 mg/kg could remarkably inhibit cancer growth in established
HCC827 nude mouse xenograft model in vivo.
4. Experimental protocols
4.1. Chemistry and chemical methods
Unless specified otherwise, all the starting materials, reagents and solvents were
commercially available. All reactions were monitored by thin-layer chromatography
(TLC) on silica gel plates (GF-254) and visualized with UV light (254 nm and 365
nm). All melting points were determined on a Shanghai micro melting-point
instrument (SGW® X-4B) and the thermometer was uncorrected. NMR spectra were
recorded on a 400 Bruker NMR spectrometer with tetramethylsilane (TMS) as an
internal reference. All chemical shifts are reported in parts per million (ppm). The
following abbreviations were used to describe peak splitting patterns when
appropriate: s (singlet), d (doublet), t (triplet), m (multiplet), br (broad signal), dd
(doublet of doublets). Coupling constants (J) are expressed in hertz unit (Hz). Mass
spectrum (MS) was obtained by ESI-MS (Skyray instrument LC-MS 1000). High
resolution mass spectrum (HRMS) was obtained via using electrospray ionization
(positive mode) on an Ultra performance liquid chromatography-Quadrupole-time of
flight Mass Spectrometer (WATERS I-Class VION IMS Q-TOF). Polarimeter
(IP-digi300EDU, Shanghai InsMark Instrument Technology Co., Ltd) was used to
obtain specific rotations when a sufficient sample was available. Specific rotations
20
based on the equation [α] = (100⋅α)/(l⋅c) was reported as [α]D (c 2.0, MeOH, where
the concentration c is in g/l00 mL and the path length l is in decimeters)..
4.1.1. 6-Chloropyrido[3,4-d]pyrimidin-4-ol (15)
The mixture of 5-amino-2-chloroisonicotinic acid (5.00 g, 29.07 mmol) and
formamidine acetate (6.00 g, 58.14 mmol) was stirred at 160 °C for 0.5 h, cooled,
16

water (100 mL) was added. The mixture was refluxed under vigorous stirring for 20
min, cooled again. The precipitate formed was collected, washed with water and dried
to give 4.74 g intermediate 15 as off-white power. Yield 90.4%. mp > 320 °C. MS m/z
(ESI) 182.4 [M+H]+.
4.1.2. Tert-butyl (R)-(1-(6-chloropyrido[3,4-d]pyrimidin-4-yl)pyrrolidin-3-yl)
carbamate (17)
The mixture of intermediate 15 (2.00 g, 11.01 mmol), SOCl2 (20 mL) and DMF
(5 drops) was refluxed for 4 h. The remaining SOCl2 was distilled off. The residue
was cooled, suspended with water, extracted with ethyl acetate (30 mL×3). The
organic layer was separated, washed with saturated NaCl solution, dried over
anhydrous Na2SO4 and concentrated to give 4,6-dichloropyrido[3,4-d]pyrimidine
(intermediate 16) as light yellow solid. Intermediate 16 was dissolved in isopropanol
(25mL), DIPEA (3 mL, 16.52 mmol) and tert-butyl (R)-pyrrolidin-3-ylcarbamate
(2.05 g, 11.01 mmol) was added. The mixture was stirred at room temperature for 2 h.
The solvent was removed under reduced pressure. The residue was suspended with
water and extracted with ethyl acetate (25 mL×3). The organic phase was washed with
saturated NaCl solution, dried over anhydrous Na2SO4 and concentrated. The crude
product was suspended in cooled ethanol (15 mL) and filtered to give intermediate 17
as light yellow power. Yield 2.50 g, 65.1%, over two steps. mp 89.5–91.4 °C. 1H
NMR (400 MHz, CDCl3) δ 9.09 (s, 1H, Ar-H), 8.65 (s, 1H, Ar-H), 7.90 (s, 1H, Ar-H),
4.95–4.65 (m, 1H, NH), 4.53–4.30 (m, 1H, CH), 4.24–3.95 (m, 3H, NCH2), 3.90–3.75
(m, 1H, NCH2), 2.40–2.30 (m, 1H, CH2), 2.18–2.08 (m, 1H, CH2), 1.46 (s, 9H,
C(CH3)3). MS m/z (ESI) 350.7 [M+H]+.
4.1.3. (R)-N-(1-(6-chloropyrido[3,4-d]pyrimidin-4-yl)pyrrolidin-3-yl)acrylamide
(18)
To the solution of intermediate 17 (200 mg, 0.59 mmol) in DCM (8 mL), TFA (2
mL) was added, and the mixture was stirred at room temperature for 1.5 h. The
solvent was removed under reduced pressure to get crude amine which was used for
the next step without furfure purification. To the mixture of the amine and DIPEA (0.3
mL, 1.77 mmol) in DCM (15 mL), acryloyl chloride (60 μL, 0.71 mmol) diluted with
DCM (5 mL) was added dropwise. The mixture was stirred at room temperature and
for 2 h, quenched with aqueous NaHCO3 solution and extracted with DCM (30
17

mL×2). The organic layer was washed with saturated NaCl solution, dried over
anhydrous Na2SO4, and concentrated under reduced pressure to give intermediate 18
1
as white solid. Yield 150 mg, 79.8%, over two steps. mp 232.8–235.3 °C. H NMR
(400 MHz, DMSO-d6) δ 8.95 (s, 1H, Ar-H), 8.60 (s, 1H, Ar-H), 8.44 (d, J = 6.4 Hz,
1H, NH), 8.09 (s, 1H), 6.25–6.09 (m, 2H, CH=CH2), 5.62 (dd, J = 9.6, 2.7 Hz, 1H,
CH=CH2), 4.53–4.44 (m, 1H, CH), 4.26–3.76 (m, 4H, NCH2), 2.30–2.18 (m, 1H,
CH2), 2.08–1.95 (m, 1H, CH2). MS m/z (ESI) 320.7 [M+H]+.
4.1.4. Synthesis of intermediates 19a-d
2-Methoxy-4-(2-methoxyethoxy)aniline (19a), 4-(2-(dimethylamino)ethoxy)-2-
methoxyaniline (19b) and 2-methoxy-4-(4-methylpiperazin-1-yl)aniline (19c) were
synthesized from 4-fluoro-2-methoxy-1-nitrobenzene.47 5-Bromo-2-nitropyridine was
used as starting material to prepare 5-(4-methylpiperazin-1-yl)pyridine-2-amine
(19d).48
4.1.5. 2,4,6-Trichloropyrido[3,4-d]pyrimidine (21)
Intermediate 21 was prepared from 5-amino-2-chloroisonicotinic acid as we
previously reported.38
4.1.6. General procedure for the synthesis of intermediates 22a-h
To a mixture of intermediate 21 (1 mmol) and DIPEA (1.5 mmol) in THF, amine
(1.1 mmol) was added under stirring. The mixture was stirred for about 0.5 h–2 h at
room temperature. The solvent was removed under reduced pressure and the residue
was suspended in water. The solid was collected by filtration, washed with water and
dried to obtain intermediates 22a-h.
4.1.6.1. tert-butyl
(R)-(1-(2,6-dichloropyrido[3,4-d]pyrimidin-4-yl)pyrrolidin-3-yl)
carbamate (22a)
Light yellow solid. Yield 78.7%. mp 95.6–97.8 °C. 1H NMR (400 MHz, CDCl3)
δ 8.98 (s, 1H, Ar-H), 7.89 (s, 1H, Ar-H), 4.76 (s, 1H, NH), 4.51–4.36 (m, 1H, NCH2),
4.25–4.01 (m, 3H, NCH2), 3.90–3.81 (m, 1H, CH), 2.41–2.07 (m, 2H, CH2), 1.45 (s,
9H, (CH3)3). MS m/z (ESI) 384.2 [M+H]+.
18

4.1.6.2. tert-butyl
(S)-(1-(2,6-dichloropyrido[3,4-d]pyrimidin-4-yl)pyrrolidin-3-yl)
carbamate (22b)
1
Light yellow solid. Yield 100.0%. mp 101.3–103.5 °C. H NMR (400 MHz,
CDCl3) δ 8.97 (s, 1H, Ar-H), 7.89 (s, 1H, Ar-H), 4.76 (s, 1H, NH), 4.52–4.36 (m, 1H,
NCH2), 4.26–3.98 (m, 3H, NCH2), 3.92–3.79 (m, 1H, CH), 2.43–2.29 (m, 1H, CH2),
2.21–1.95 (m, 1H, CH2), 1.45 (s, 9H, (CH3)3). MS m/z (ESI) 384.2 [M+H]+.
4.1.6.3. tert-butyl
(R)-(1-(2,6-dichloropyrido[3,4-d]pyrimidin-4-yl)piperidin-3-yl)
carbamate (22c)
1
Light yellow solid. Yield 89.3%. mp 209.3–212.6 °C. H NMR (400 MHz,
CDCl3) δ 9.03 (s, 1H, Ar-H), 8.05 (s, 1H, Ar-H), 4.95–4.63 (m, 1H, NH), 4.38–4.17
(m, 2H, NCH2), 3.98–3.75 (m, 1H, CH), 3.57–3.41 (m, 2H, NCH2), 2.17–2.05 (m, 1H,
CH2), 2.00–1.94 (m, 1H, CH2), 1.85–1.77 (m, 1H, CH2), 1.74–1.63 (m, 1H, CH2),
1.43 (s, 9H, (CH3)3). MS m/z (ESI) 398.0 [M+H]+.
4.1.6.4. tert-butyl
(S)-(1-(2,6-dichloropyrido[3,4-d]pyrimidin-4-yl)piperidin-3-yl)
carbamate (22d)
1
Light yellow solid. Yield 84.3%. mp 204.9–206.2 °C. H NMR (400 MHz,
CDCl3) δ 9.05 (s, 1H, Ar-H), 8.06 (s, 1H, Ar-H), 4.80 (s, 1H, NH), 4.31–4.27 (m, 2H,
NCH2), 3.89 (s, 1H, CH), 3.47–3.43 (m, 2H, NCH2), 2.04–2.00 (m, 2H, CH2),
1.79–1.74 (m, 2H, CH2), 1.43 (s, 9H, (CH3)3). MS m/z (ESI) 398.1 [M+H]+.
4.1.6.5. (R)-1-(3-((2,6-dichloropyrido[3,4-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)
prop-2-en-1-one (22e)
1
Light yellow solid. Yield 72.0%. H NMR (400 MHz, CDCl3) δ 8.97 (s, 1H,
Ar-H), 8.12 (s, 1H, Ar-H), 7.61 (br, 1H, NH), 5.06–4.88 (m, 1H, CH), 3.77–3.48 (m,
1H, NCH2), 2.38–2.12 (m, 2H, CH2) 1.51 (s, 9H, C(CH3)3). MS m/z (ESI) 384.6
[M+H]+.
4.1.6.6. (S)-1-(3-((2,6-dichloropyrido[3,4-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)
prop-2-en-1-one (22f)
1
Light yellow solid. Yield 78.9%. H NMR (400 MHz, CDCl3) δ 8.94 (s, 1H,
Ar-H), 8.18 (s, 1H, Ar-H), 7.84 (s, 1H, NH), 5.05–4.80 (m, 1H, CH), 3.75–3.48 (m,
19

4H, NCH2), 2.39–2.11 (m, 2H, CH2), 1.52 (s, 9H, C(CH3)3). MS m/z (ESI) 384.5
[M+H]+.
4.1.6.7. (R)-1-(3-((2,6-dichloropyrido[3,4-d]pyrimidin-4-yl)amino)piperidin-1-yl)
prop-2-en-1-one (22g)
1
Light yellow solid. Yield 89.5%. mp 110.5–112.5 °C. H NMR (400 MHz,
CDCl3) δ 8.96 (s, 1H, Ar-H), 7.58 (s, 1H, Ar-H), 4.47–4.29 (m, 1H, NCH2), 4.25–2.97
(m, 4H, NCH2+CH), 2.46–1.45 (m, 13H, CH2+(CH3)3). MS m/z (ESI) 398.6 [M+H]+.
4.1.6.8. (S)-1-(3-((2,6-dichloropyrido[3,4-d]pyrimidin-4-yl)amino)piperidin-1-yl)
prop-2-en-1-one (22h)
1
Light yellow solid. Yield 75.0%. mp 113.4–115.7 °C. H NMR (400 MHz,
CDCl3) δ 8.96 (s, 1H, Ar-H), 7.58 (s, 1H, Ar-H), 4.46–4.27 (m, 1H, NCH2), 4.24–2.99
(m, 4H, NCH2+CH), 2.45–1.43 (m, 13H, CH2+(CH3)3). MS m/z (ESI) 398.6 [M+H]+.
4.1.7. Synthesis of intermediates 23a-h
Intermediates 23a-h was synthesized from intermediates 22a-h with similar
process to that of Intermediate 18.
4.1.7.1. (R)-N-(1-(2,6-dichloropyrido[3,4-d]pyrimidin-4-yl)pyrrolidin-3-yl)
acrylamide (23a)
1
Light yellow solid. Yield 46.0%. mp 206.5–208.7 °C. H NMR (400 MHz,
CDCl3) δ 8.89 (s, 1H, Ar-H), 7.83 (s, 1H, Ar-H), 6.62 (d, J = 5.2 Hz, 1H, NH), 6.37
(dd, J = 16.9, 1.0 Hz, 1H, CH=CH2), 6.18 (dd, J = 17.0, 10.3 Hz, 1H, CH=CH2), 5.70
(dd, J = 10.3, 1.1 Hz, 1H, CH=CH2), 4.89–4.68 (m, 1H, CH), 4.34–4.13 (m, 1H,
NCH2), 4.12–3.92 (m, 3H, NCH2), 2.45–2.34 (m, 1H, CH2), 2.34–2.17 (m, 1H, CH2).
MS m/z (ESI) 338.5 [M+H]+.
4.1.7.2. (S)-N-(1-(2,6-dichloropyrido[3,4-d]pyrimidin-4-yl)pyrrolidin-3-yl)
acrylamide (23b)
1
Light yellow solid. Yield 24.9%. mp 186.5–188.7 °C. H NMR (400 MHz,
CDCl3) δ 8.89 (s, 1H, Ar-H), 7.83 (s, 1H, Ar-H), 6.68 (s, 1H, NH), 6.37 (dd, J = 17.0,
1.3 Hz, 1H, CH=CH2), 6.18 (dd, J = 17.0, 10.3 Hz, 1H, CH=CH2), 5.70 (dd, J = 10.3,
1.3 Hz, 1H, CH=CH2), 4.80–4.75 (m, 1H, CH), 4.25–4.19 (m, 1H, NCH2),4.05–3,98
20

(m, 3H, NCH2), 2.44–2.33 (m, 1H, CH2), 2.32–2.16 (m, 1H, CH2). MS m/z (ESI)
338.4 [M+H]+.
4.1.7.3. (R)-N-(1-(2,6-dichloropyrido[3,4-d]pyrimidin-4-yl)piperidin-3-yl)acrylamide
(23c)
1
White solid. Yield 66.0%. mp 109.9–112.0 °C. H NMR (400 MHz, CDCl3) δ
9.03 (s, 1H, Ar-H), 7.91 (s, 1H, Ar-H), 6.41 (d, J = 5.2 Hz, 1H, NH), 6.33 (dd, J =
17.0, 1.1 Hz, 1H, CH=CH2), 6.12 (dd, J = 17.0, 10.3 Hz, 1H, CH=CH2), 5.70 (dd, J =
10.3, 1.1 Hz, 1H, CH=CH2), 4.29–4.21 (m, 1H, CH), 4.15–4.06 (m, 2H, NCH2),
3.81–3.71 (m, 2H, NCH2), 2.09–2.03 (m, 1H, CH2), 2.00–1.87 (m, 2H, CH2),
1.85–1.77 (m, 1H, CH2). MS m/z (ESI) 351.9 [M+H]+.
4.1.7.4. (S)-N-(1-(2,6-dichloropyrido[3,4-d]pyrimidin-4-yl)piperidin-3-yl)acrylamide
(23d)
1
White solid. Yield 79.2%. mp 113.0–114.8 °C. H NMR (400 MHz, CDCl3) δ
9.04 (s, 1H, Ar-H), 7.91 (s, 1H, Ar-H), 6.45 (s, 1H, NH), 6.33 (dd, J = 17.0, 1.2 Hz,
1H, CH=CH2), 6.13 (dd, J = 17.0, 10.3 Hz, 1H, CH=CH2), 5.69 (dd, J = 10.3, 1.2 Hz,
1H, CH=CH2), 4.29–4.25 (m, 1H, CH), 4.13–4.09 (m, 2H, NCH2), 3.84–3.78 (m, 2H,
NCH2), 2.08–2.00 (m, 1H, CH2), 1.97–1.90 (m, 2H, CH2), 1.88–1.80 (m, 1H, CH2).
MS m/z (ESI) 352.0 [M+H]+.
4.1.7.5. (R)-1-(3-((2,6-dichloropyrido[3,4-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)
prop-2-en-1-one (23e)
White solid. Yield 59.8%. 1H NMR (400 MHz, DMSO-d6) δ 9.06 (t, J = 5.3 Hz,
1H, NH), 8.89 (d, J = 2.1 Hz, 1H, Ar-H), 8.50 (d, J = 5.7 Hz, 1H, Ar-H), 6.73–6.52 (m,
1H, CH=CH2), 6.26–6.10 (m, 1H, CH=CH2), 5.79–5.65 (m, 1H, CH=CH2), 4.86–4.62
(m, 1H, NH2), 3.99–3.57 (m, 4H, NCH2+CH), 2.33–2.01 (m, 2H, CH2). mp
282.6–284.7 °C. MS m/z (ESI) 338.5 [M+H]+.
4.1.7.6. (S)-1-(3-((2,6-dichloropyrido[3,4-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)
prop-2-en-1-one (23f)
White solid. Yield 74.4%. mp 278.6–280.3 °C. 1H NMR (400 MHz, DMSO-d6) δ
9.08 (t, J = 5.6 Hz, 1H, NH), 8.90 (d, J = 2.0 Hz, 1H, Ar-H), 8.52 (d, J = 5.3 Hz, 1H,
Ar-H), 6.75–6.52 (m, 1H, CH=CH2), 6.29–6.08 (m, 1H, CH=CH2), 5.75–5.65 (m, 1H,
21

CH=CH2), 4.87–4.64 (m, 1H, NCH2), 4.00–3.54 (m, 4H, NCH2+CH), 2.36–2.00 (m,
2H, CH2). MS m/z (ESI) 338.5 [M+H]+.
4.1.7.7. N-((3R)-3-((2,6-dichloropyrido[3,4-d]pyrimidin-4-yl)amino)cyclohexyl)
acrylamide (23g)
1
White solid. Yield 63.3%. H NMR (400 MHz, DMSO-d6) δ 9.01–8.74 (m, 2H,
NH+Ar-H), 8.48 (d, J = 17.3 Hz, 1H, Ar-H), 6.89–6.77 (m, 1H, CH=CH2), 6.17–6.06
(m, 1H, CH=CH2), 5.73–5.63 (m, 1H, CH=CH2), 4.59–4.13 (m, 1H, NCH2),
4.07–3.87 (m, 1H, NCH2), 3.14–3.01 (m, 1H, NCH2), 2.92–2.70 (m, 1H, CH),
2.13–2.01 (m, 1H, CH2), 1.92–1.47 (m, 3H, CH2). MS m/z (ESI) 352.0[M+H]+.
4.1.7.8. N-((3R)-3-((2,6-dichloropyrido[3,4-d]pyrimidin-4-yl)amino)cyclohexyl)
acrylamide (23h)
1
White solid. Yield 68.8%. H NMR (400 MHz, DMSO-d6) δ 9.04–8.78 (m, 2H,
NH+Ar-H), 8.48 (d, J = 16.4 Hz, 1H, Ar-H), 6.91–6.79 (m, 1H, CH=CH2), 6.16–6.06
(m, 1H, CH=CH2), 5.74–5.63 (m, 1H, CH=CH2), 4.58–4.09 (m, 2H, NCH2),
4.07–3.98 (m, 1H, NCH2), 3.15–2.98 (m, 1H, NCH2), 2.94–2.70 (m, 1H, CH),
2.21–1.99 (m, 1H, CH2), 1.99–1.61 (m, 2H, CH2), 1.62–1.43 (m, 1H, CH2). mp
126.5–128.3 °C. MS m/z (ESI) 352.0 [M+H]+.
4.1.8. General procedure for the synthesis of intermediates 24a-h
To a mixture of intermediates 23a-h (1 mmol) and p-toluenesulfonic acid (1.2
mmol) in 2-butyl alcohol (2-BuOH), aniline (1.1 mmol) was added. The mixture was
refluxed for 1 h–2 h under nitrogen atmosphere. The solvent was removed under
reduced pressure. The residue was suspended in NaHCO3 solution and stirred at room
temperature for 0.5 h. The yellow precipitate was collected and washed with water to
give intermediates 24a-h.
4.1.8.1. (R)-N-(1-(6-chloro-2-(phenylamino)pyrido[3,4-d]pyrimidin-4-yl)pyrrolidin
-3-yl)acrylamide (24a)
Light yellow solid. Yield 100%. mp 120.2–122.8 °C. 1H NMR (400 MHz, CDCl3)
δ 8.60 (s, 1H, Ar-H), 7.61 (d, J = 7.8 Hz, 2H, Ar-H), 7.58 (s, 1H, Ar-H), 7.31 (t, J =
7.9 Hz, 2H, Ar-H), 7.10 (s, 1H, NH), 7.04 (t, J = 7.4 Hz, 1H, Ar-H), 6.75 (s, NH), 6.40
(dd, J = 17.0, 1.4 Hz, 1H, CH=CH2), 6.22 (dd, J = 16.9, 10.2 Hz, 1H, CH=CH2), 5.71
22

(dd, J = 10.2, 1.4 Hz, 1H, CH=CH2), 4.79–4.67 (m, 1H, CH), 4.09–4.01 (m, 1H,
NCH2), 3.99–3.87 (m, 2H, NCH2), 3.84–3.77 (m, 1H, NCH2), 2.34–2.19 (m, 2H,
CH2). MS m/z (ESI) 395.1 [M+H]+.
4.1.8.2. (S)-N-(1-(6-chloro-2-(phenylamino)pyrido[3,4-d]pyrimidin-4-yl)pyrrolidin
-3-yl)acrylamide (24b)
1
Light yellow solid. Yield 100%. mp 125.0–127.5 °C. H NMR (400 MHz, CDCl3) δ
8.59 (s, 1H, Ar-H), 7.60 (d, J = 7.9 Hz, 2H, Ar-H), 7.56 (d, J = 12.1 Hz, 1H, Ar-H),
7.30 (dd, J = 11.3, 4.5 Hz, 2H, Ar-H), 7.11 (s, 1H, NH), 7.03 (t, J = 7.4 Hz, 1H, Ar-H),
6.88 (s, 1H, NH), 6.40 (dd, J = 17.0, 1.4 Hz, 1H, CH=CH2), 6.23 (dd, J = 16.8, 10.2
Hz, 1H, CH=CH2), 5.71 (dd, J = 10.2 Hz, 1.4 Hz, 1H, CH=CH2), 4.76–4.71 (m, 1H,
CH), 4.06–3.78 (m, 4H, NCH2), 2.35–2.20 (m, 2H, CH2). MS m/z (ESI) 395.1
[M+H]+.
4.1.8.3. (R)-N-(1-(6-chloro-2-(phenylamino)pyrido[3,4-d]pyrimidin-4-yl)piperidin
-3-yl)acrylamide (24c)
1
Light yellow solid. Yield 76.1%. mp 203.3–205.5 °C. H NMR (400 MHz,
CDCl3) δ 8.82 (s, 1H, Ar-H), 7.69 (d, J = 7.6 Hz, 2H, Ar-H), 7.63 (s, 1H, Ar-H), 7.35
(t, J = 7.4 Hz, 2H, Ar-H), 7.07 (t, J = 7.3 Hz, 1H, Ar-H), 6.37 (s, 1H, NH), 6.28 (dd, J
= 17.0, 1.1 Hz, 1H, CH=CH2), 5.99 (d, J = 17.0, 10.3 Hz, 1H, CH=CH2), 5.62 (dd, J =
10.3, 1.1 Hz, 1H, CH=CH2), 4.34–4.24 (m, 1H, CH), 3.97–3.90 (m, 1H, NCH2),
3.86–3.77 (m, 1H, NCH2), 3.75–3.64 (m, 2H, NCH2), 2.06–1.98 (m, 1H, CH2),
1.94–1.86 (m, 2H, CH2), 1.83–1.77 (m, 1H, CH2). MS m/z (ESI) 409.1 [M+H]+.
4.1.8.4. (S)-N-(1-(6-chloro-2-(phenylamino)pyrido[3,4-d]pyrimidin-4-yl)piperidin
-3-yl)acrylamide (24d)
1
Light yellow solid. Yield 64.4%. mp 197.2–199.3 °C. H NMR (400 MHz,
CDCl3) δ 8.82 (s, 1H, Ar-H), 7.69 (d, J = 7.9 Hz, 2H, Ar-H), 7.65 (s, 1H, Ar-H), 7.35
(t, J = 7.9 Hz, 2H, Ar-H), 7.07 (t, J = 7.4 Hz, 1H, Ar-H), 6.34 (s, 1H, NH), 6.28 (dd, J
= 16.9, 1.0 Hz, 1H, CH=CH2), 5.99 (dd, J = 17.0, 10.3 Hz, 1H, CH=CH2), 5.62 (d, J =
11.2 Hz, 1H, CH=CH2), 4.32–4.26 (m, 1H, CH), 3.99–3.65 (m, 4H, NCH2), 2.07–1.75
(m, 4H, CH2). MS m/z (ESI) 409.1 [M+H]+.
23

4.1.8.5. (R)-1-(3-((6-chloro-2-(phenylamino)pyrido[3,4-d]pyrimidin-4-yl)amino)
pyrrolidin-1-yl)prop-2-en-1-one (24e)
1
Light yellow solid. Yield 88.6%. mp 151.5–154.0 °C. H NMR (400 MHz,
DMSO-d6) δ 9.44 (s, 1H, NH), 8.62 (d, J = 2.8 Hz, 1H, Ar-H), 8.50–8.30 (m, 1H, NH),
8.29 (d, J = 7.4 Hz, 1H, Ar-H), 7.90 (d, J = 7.9 Hz, 2H, Ar-H), 7.30 (t, J = 7.9 Hz, 2H,
Ar-H), 6.98–6.90 (m, 1H, Ar-H), 6.68–6.52 (m, 1H, CH=CH2), 6.30–6.05 (m, 1H,
CH=CH2), 5.80–5.55 (m, 1H, CH=CH2), 4.87–4.65 (m, 1H, CH), 3.90–3.44 (m, 4H,
NCH2), 2.35–2.05 (m, 2H, CH2). MS m/z (ESI) 395.6 [M+H]+.
4.1.8.6. (S)-1-(3-((6-chloro-2-(phenylamino)pyrido[3,4-d]pyrimidin-4-yl)amino)
pyrrolidin-1-yl)prop-2-en-1-one (24f)
1
Light yellow solid. Yield 100%. mp 128.5–130.3 °C. H NMR (400 MHz,
DMSO-d6) δ 9.46 (s, 1H, NH), 8.63 (d, J = 2.8 Hz, 1H, Ar-H), 8.51–8.34 (m, 1H, NH),
8.29 (d, J = 7.4 Hz, 1H, Ar-H), 7.91 (d, J = 7.9 Hz, 2H, Ar-H), 7.30 (t, J = 7.9 Hz, 2H,
Ar-H), 6.99–6.92 (m, 1H, Ar-H), 6.70–6.54 (m, 1H, CH=CH2), 6.28–6.07 (m, 1H,
CH=CH2), 5.78–5.57 (m, 1H, CH=CH2), 4.89–4.67 (m, 1H, CH), 3.91–3.45 (m, 4H,
NCH2), 2.36–2.07 (m, 2H, CH2). MS m/z (ESI) 395.6 [M+H]+.
4.1.8.7. N-((3R)-3-((6-chloro-2-(phenylamino)pyrido[3,4-d]pyrimidin-4-yl)amino)
cyclohexyl)acrylamide (24g)
1
Light yellow solid. Yield 96.9%. mp 117.1–119.2 °C. H NMR (400 MHz,
DMSO-d6) δ 9.42 (s, 1H, NH), 8.62 (s, 1H, Ar-H), 8.29–8.15 (m, 2H, Ar-H+NH), 7.86
(d, J = 7.6 Hz, 2H, Ar-H), 7.26 (t, J = 6.3 Hz, 2H, Ar-H), 7.03–6.97 (m, 1H, Ar-H),
6.57–6.51 (m, 1H, CH=CH2), 6.25–5.94 (m, 1H, CH=CH2), 5.81–5.46 (m, 1H,
CH=CH2), 4.57–4.01 (m, 3H, NCH2), 3.32–2.75 (m, 2H, NCH2+NH), 2.14–2.03 (m,
1H, CH2), 1.92–1.43 (m, 3H, CH2). MS m/z (ESI) 409.0 [M+H]+.
4.1.8.8. N-((3S)-3-((6-chloro-2-(phenylamino)pyrido[3,4-d]pyrimidin-4-yl)amino)
cyclohexyl)acrylamide (24h)
1
Light yellow solid. Yield 86.7%. mp 118.0–120.3 °C. H NMR (400 MHz,
DMSO-d6) δ 9.40 (s, 1H, NH), 8.62 (s, 1H, Ar-H), 8.27–8.12 (m, 2H, Ar-H+NH), 7.86
(d, J = 7.6 Hz, 2H, Ar-H), 7.26 (t, J = 6.3 Hz, 2H, Ar-H), 7.01–6.95 (m, 1H, Ar-H),
6.59–6.51 (m, 1H, CH=CH2), 6.27–5.97 (m, 1H, CH=CH2), 5.80–5.45 (m, 1H,
24

CH=CH2), 4.60–4.00 (m, 3H, NCH2), 3.30–2.79 (m, 2H, NCH2+NH), 2.15–2.02 (m,
1H, CH2), 1.94–1.42 (m, 3H, CH2). MS m/z (ESI) 409.0 [M+H]+.
4.1.9. 2,6-Dichloro-4-(pyrrolidin-1-yl)pyrido[3,4-d]pyrimidine (26)
Intermediate 26 was synthesized from intermediate 21 and tetrahydropyrrol with
similar process to that of intermediates 22a-h. Light yellow solid. Yield 91.0%. mp
246.8–249.5 °C. 1H NMR (400 Mz, CDCl3) δ 8.98 (s, 1H, Ar-H), 7.94 (s, 1H, Ar-H),
3.98 (s, 4H, NCH2), 2.13 (s, 4H, CH2). MS m/z (ESI) 233.7 [M+H]+.
4.1.10. Synthesis of intermediates 27a and 27b
Intermediates 27a and 27b was synthesized from intermediate 26 with similar
operations to that of intermediates 24a-h.
4.1.10.1. 6-Chloro-N-phenyl-4-(pyrrolidin-1-yl)pyrido[3,4-d]pyrimidin-2-amine
(27a)
1
Light yellow solid. Yield 99.2%. mp 270.8–272.8 °C. H NMR (400 MHz,
CDCl3) δ 8.75 (s, 1H, Ar-H), 7.80 (s, 1H, Ar-H), 7.70 (d, J = 7.7 Hz, 2H, Ar-H), 7.34
(t, J = 8.0 Hz, 2H, Ar-H), 7.16 (s, 1H, NH), 7.04 (t, J = 7.4 Hz, 1H, Ar-H), 3.91 (s, 4H,
NCH2), 2.09 (t, J = 6.5 Hz, 4H, CH2). MS m/z (ESI) 326.0 [M+H]+.
4.1.10.2. 6-Chloro-N-(3-fluorophenyl)-4-(pyrrolidin-1-yl)pyrido[3,4-d]pyrimidin-2-
amine (27b)
1
Light yellow solid. Yield 91.8%. mp 232.7–234.8 °C. H NMR (400 MHz,
CDCl3) δ 8.76 (s, 1H, Ar-H), 7.86–7.81 (m, 1H, Ar-H), 7.81 (s, 1H, Ar-H), 7.28–7.17
(m, 4H, Ar-H), 6.76–6.67 (m, 1H, Ar-H), 3.96–3.88 (s, 4H, NCH2), 2.13–2.08 (m, 4H,
CH2). MS m/z (ESI) 344.5 [M+H]+.
4.1.11. Synthesis of intermediates 27c-g
Sodium hydride (NaH, 60%, 3 mmol) was added to a solution of alcohol (1.1
mmol) in dried THF. The mixture was stirred at room temperature for 0.5 h. Then
intermediate 26 (1 mmol) was added and the mixture was stirred at room temperature
for another 2 h, concentrated under reduced pressure. The residue was suspended in
water. The precipitate was collected, washed with water and dried to give
intermediates 27c-g.
25

4.1.11.1. 6-Chloro-4-(pyrrolidin-1-yl)-2-((tetrahydro-2H-pyran-4-yl)oxy)pyrido[3,4-
d]pyrimidine (27c)
Light yellow solid. Yield 100%. mp 130.5–132.9 °C. 1H NMR (400 MHz, CDCl3)
δ 8.80 (s, 1H, Ar-H), 7.87 (s, 1H, Ar-H), 5.36–5.23 (m, 1H, CH), 4.08–4.00 (m, 2H,
OCH2), 3.98–3.89 (m, 4H, NCH2), 3.64–3.56 (m, 2H, OCH2), 2.16–2.11 (m, 2H, CH2),
2.10–2.06 (m, 4H, CH2), 1.96–1.87 (m, 2H, CH2). MS m/z (ESI) 335.0 [M+H]+.
4.1.11.2. 2-(Benzyloxy)-6-chloro-4-(pyrrolidin-1-yl)pyrido[3,4-d]pyrimidine (27d)
1
Light yellow solid. Yield 99.0%. mp 161.8–163.5 °C. H NMR (400 MHz,
CDCl3) δ 8.83 (s, 1H, ArH), 7.87 (s, 1H, ArH), 7.50 (d, J = 7.2 Hz, 2H, ArH),
7.37–7.27 (m, 3H, ArH), 5.48 (s, 2H, OCH2), 3.96–3.89 (m, 4H, NCH2), 2.11–2.05 (m,
4H, CH2). MS m/z (ESI) 341.1 [M+H]+.
4.1.11.3. 6-Chloro-2-((4-fluorobenzyl)oxy)-4-(pyrrolidin-1-yl)pyrido[3,4-d]
pyrimidine (27e)
1
Light yellow solid. Yield 90.0%. mp 212.0–215.2 °C. H NMR (400 MHz,
CDCl3) δ 8.83 (s, 1H, Ar-H), 7.87 (s, 1H, Ar-H), 7.48 (dd, J = 8.5, 5.5 Hz, 2H, Ar-H),
7.02 (dd, J = 12.1, 5.3 Hz, 2H, Ar-H), 5.44 (s, 2H, OCH2), 3.99–3.85 (m, 4H, NCH2),
2.14–2.01 (m, 4H, CH2). MS m/z (ESI) 359.1 [M+H]+.
4.1.11.4. 6-Chloro-4-(pyrrolidin-1-yl)-2-((tetrahydro-2H-pyran-4-yl)methoxy)pyrido
[3,4-d]pyrimidine (27f)
1
Light yellow solid. Yield 98.0%. mp 250.0–251.7 °C. H NMR (400 MHz,
CDCl3) δ 8.82 (s, 1H, Ar-H), 7.87 (s, 1H, Ar-H), 4.26 (d, J = 6.9 Hz, 2H, OCH2CH),
4.03–3.97 (m, 2H, OCH2), 3.98–3.86 (m, 4H, NCH2), 3.49–3.37 (m, 2H, OCH2),
2.21–2.12 (m, 1H, OCH2CH), 2.12–2.02 (m, 4H, CH2), 1.83–1.76 (m, 2H, CH2),
1.50–1.39 (m, 2H, CH2). MS m/z (ESI) 349.2 [M+H]+.
4.1.11.5. 6-Chloro-2-(cyclohexylmethoxy)-4-(pyrrolidin-1-yl)pyrido[3,4-d]
pyrimidine (27g)
1
Light yellow solid. Yield 98.0%. mp 146.9–149.6 °C. H NMR (400 MHz,
CDCl3) δ 8.82 (s, 1H, Ar-H), 7.86 (s, 1H, Ar-H), 4.21 (d, J = 6.3 Hz, 2H, OCH2),
3.97–3.89 (m, 4H, NCH2), 2.13–2.04 (m, 4H, NCH2), 1.91–1.86 (m, 2H, CH2),
1.80–1.71 (m, 2H, CH2), 1.70–1.65 (m, 1H, CH), 1.29–1.22 (m, 4H, CH2), 1.09–1.00
26

(m, 2H, CH2). MS m/z (ESI) 347.1 [M+H]+.
4.1.12. Synthesis of intermediate 28
2,6-Diaminopyridine was used to prepare N-(6-aminopyridin-2-yl)acrylamide
(28).49
4.1.13. General procedure for the synthesis of title compounds 20a-d, 25a-h and
29a-g
The mixture of intermediate 18 (or 24a-h or 27a-g, 0.50 mmol), intermediate 19
(or 28, 0.55 mmol), Pd(OAc)2 (0.05 mmol), X-phos (or Xantphos, 0.15 mmol),
Cs2CO3 (1.25 mmol) and 1,4-dioxane (or 1,2-dimethoxyethane, DME) were refluxed
under argon atmosphere for 1 h–4 h. The solvent was removed under reduced pressure
and the residue was purified by silica gel column chromatography to produce title
compounds 20a-d, 25a-h and 29a-g.
4.1.13.1. (R)-N-(1-(6-((2-methoxy-4-(2-methoxyethoxy)phenyl)amino)pyrido[3,4-d]
pyrimidin-4-yl)pyrrolidin-3-yl)acrylamide (20a)
1
Light yellow solid. Yield 46.3%. mp 167.5–169.4 °C. H NMR (400 MHz,
DMSO-d6) δ 8.71 (s, 1H, Ar-H), 8.54 (d, J = 6.5 Hz, 1H, NH), 8.30 (s, 1H, NH), 8.28
(s, 1H, Ar-H), 7.74 (d, J = 8.7 Hz, 1H, Ar-H), 7.41 (s, 1H, Ar-H), 6.66 (d, J = 2.0 Hz,
1H, Ar-H), 6.51 (dd, J = 8.7, 2.0 Hz, 1H, CH=CH2), 6.30–6.09 (m, 1H,
Ar-H+CH=CH2), 5.68–5.54 (m, 1H, CH=CH2), 4.52–4.43 (m, J = 3.9 Hz, 1H, NCH2),
4.10–4.07 (m, 2H, OCH2), 3.99–3.87 (m, 2H, NCH2), 3.83 (s, 3H, Ar-OCH3),
3.78–3.72 (m, 1H, NCH2), 3.69–3.64 (m, 2H, OCH2), 3.63–3.54 (m, 1H, NCH2), 3.33
13
(s, 3H, OCH3), 2.25–1.96 (m, 2H, CH2). C NMR (100 MHz, DMSO-d6) δ 165.0,
157.8, 155.1, 153.8, 152.1, 151.9, 149.8, 139.2, 131.9, 126.0, 123.6, 123.0, 122.5,
105.1, 100.0, 99.7, 71.0, 67.5, 58.6, 56.1, 55.8, 53.8, 48.8, 42.0.MS m/z (ESI)
465.9[M+H]+.
4.1.13.2. (R)-N-(1-(6-((4-(2-(dimethylamino)ethoxy)-2-methoxyphenyl)amino)
pyrido[3,4-d]pyrimidin-4-yl)pyrrolidin-3-yl)acrylamide (20b)
1
Light yellow solid. Yield 35.7%. mp 236.9–238.4 °C. H NMR (400 MHz,
CDCl3) δ 8.94 (s, 1H, Ar-H), 8.47 (s, 1H, Ar-H), 7.55 (d, J = 8.6 Hz, 1H, Ar-H),
7.41–7.28 (m, 3H, Ar-H+CH=CH2), 6.71 (s, 1H, Ar-H), 6.63 (d, J = 2.5 Hz, 1H,
27

CH=CH2), 6.54 (dd, J = 8.6, 2.5 Hz, 1H, CH=CH2), 4.43–4.29 (m, 2H, OCH2), 3.88 (s,
3H, OCH3), 3.19–3.09 (m, 2H, NCH2), 2.68 (s, 6H, N(CH3)2). MS m/z (ESI) 479.0
[M+H]+.
4.1.13.3. (R)-N-(1-(6-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)pyrido
[3,4-d]pyrimidin-4-yl)pyrrolidin-3-yl)acrylamide (20c)
1
Light yellow solid. Yield 38.6%. mp 297.9–300.1 °C. H NMR (400 MHz,
DMSO-d6) δ 8.73 (s, 1H, NH), 8.59–8.43 (m, 2H, Ar-H+NH), 8.40 (s, 1H, Ar-H), 7.79
(d, J = 8.7 Hz, 1H, Ar-H), 7.54 (s, 1H, Ar-H), 6.73 (d, J = 2.2 Hz, 1H, Ar-H), 6.55 (dd,
J = 8.8, 2.2 Hz, 1H, Ar-H), 6.33–5.99 (m, 2H, CH=CH2), 5.64 (dd, J = 9.8, 2.4 Hz,
1H, CH=CH2), 4.56–4.46 (m, 1H, NCH2), 4.30–3.70 (m, 10H, OCH3+NCH2),
3.60–3.47 (m, 2H, NCH2), 3.26–3.13 (m, 2H, NCH2), 2.99–2.92 (m, 1H, CH), 2.88 (s,
3H), 2.30–2.18 (m, 1H, CH2), 2.10–1.96 (m, 1H, CH2). 13C NMR (100 MHz,
DMSO-d6) δ 165.1, 158.0, 154.5, 151.4, 150.3, 146.5, 146.4, 131.8, 126.2, 123.1,
122.5, 122.1, 108.0, 101.5, 101.1, 63.4, 56.4, 56.1, 52.7 (C×2), 49.6, 46.8 (C×2), 42.5,
41.9. MS m/z (ESI) 490.0 [M+H]+.
4.1.13.4. (R)-N-(1-(6-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)pyrido[3,4-d]
pyrimidin-4-yl)pyrrolidin-3-yl)acrylamide (20d)
1
Light yellow solid. Yield 35.6%. mp 292.5–297.5 °C. H NMR (400 MHz,
DMSO-d6) δ 9.78 (s, 1H, NH), 9.12 (s, 1H, Ar-H), 8.81 (s, 1H, Ar-H), 8.57 (d, J = 6.4
Hz, 1H, Ar-H), 8.37 (s, 1H, Ar-H), 7.98 (d, J = 2.8 Hz, 1H, Ar-H), 7.47 (dd, J = 9.1,
2.9 Hz, 1H, Ar-H), 7.27 (d, J = 9.0 Hz, 1H, Ar-H), 6.27 (dd, J = 17.1, 10.0 Hz, 1H,
CH=CH2), 6.14 (dd, J = 17.1, 2.3 Hz, 1H, CH=CH2), 5.64 (dd, J = 10.0, 2.3 Hz, 1H,
CH=CH2), 4.60–4.49 (m, 1H, CH), 4.41–4.17 (m, 1H, NCH2), 4.14–3.94 (m, 2H,
NCH2), 3.93–3.84 (m, 1H, NCH2), 3.32–3.17 (m, 4H, NCH2), 3.15–2.94 (m, 4H,
NCH2), 2.62 (s, 3H, NCH3), 2.32–2.19 (m, 1H, CH2), 2.11–1.96 (m, 1H, CH2). MS
m/z (ESI) 460.4 [M+H]+.
4.1.13.5. (R)-N-(1-(6-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)-2-
(phenylamino)pyrido[3,4-d]pyrimidin-4-yl)pyrrolidin-3-yl)acrylamide (25a)
1
Light yellow solid. Yield 62.4%. mp 243.5–247.0 °C. H NMR (400 MHz,
DMSO-d6) δ 9.47 (s, 1H, NH), 9.05 (s, 1H, Ar-H), 9.02 (s, 1H, Ar-H), 8.58 (s, 1H,
NH), 8.55 (d, J = 6.7 Hz, 1H, Ar-H), 7.91 (m, 3H, Ar-H), 7.42 (dd, J = 9.1, 2.9 Hz, 1H,
28

Ar-H), 7.24 (m, 3H, NH+Ar-H), 6.88 (t, J = 7.3 Hz, 1H, Ar-H), 6.27 (dd, J = 17.1,
10.0 Hz, 1H, CH=CH2), 6.14 (dd, J = 17.1, 2.3 Hz, 1H, CH=CH2), 5.64 (dd, J = 10.0,
2.3 Hz, 1H, CH=CH2), 4.62–4.48 (m, 1H, CH), 4.37–4.18 (m, 1H, NCH2), 4.17–3.96
(m, 2H, NCH2), 3.94–3.85 (m, 1H, NCH2), 3.19–3.07 (m, 4H, NCH2), 2.79–2.62 (m,
4H, NCH2), 2.39 (s, 3H, NCH3), 2.31–2.22 (m, 1H, CH2), 2.14–2.00 (m, 1H, CH2).
13C NMR (100 MHz, DMSO-d6) δ 165.1, 159.2, 155.8, 149.0, 148.2, 147.5, 142.7,
142.0, 141.0, 134.7, 131.9, 128.8 (Ar-C×2), 127.7, 126.0, 120.7, 118.9, 118.8
(Ar-C×2), 112.2, 102.9, 56.0, 54.7 (C×2), 49.2 (C×2), 48.9, 45.8. HRMS m/z (ESI):
calcd for C29H37FN9O [M+H]+ 551.29898, found 551.29895.
4.1.13.6. (S)-N-(1-(6-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)-2-
(phenylamino)pyrido[3,4-d]pyrimidin-4-yl)pyrrolidin-3-yl)acrylamide (25b)
1
Light yellow solid. Yield 54.0%. mp 246.5–250.5 °C. H NMR (400 MHz,
DMSO-d6) δ 9.46 (s, 1H, NH), 9.04 (s, 1H, Ar-H), 9.02 (s, 1H, Ar-H), 8.58 (s, 1H,
NH), 8.54 (d, J = 6.7 Hz, 1H, Ar-H), 7.92 (s, 1H, NH), 7.90 (s, 1H, Ar-H), 7.41 (dd, J
= 9.0, 2.8 Hz, 1H, Ar-H), 7.26 (t, J = 7.8 Hz, 2H, Ar-H), 7.21 (d, J = 9.0 Hz, 1H,
Ar-H), 6.88 (t, J = 7.3 Hz, 1H, Ar-H), 6.26 (dd, J = 17.1, 10.0 Hz, 1H, CH=CH2), 6.15
(dd, J = 17.1, 2.3 Hz, 1H, CH=CH2), 5.64 (dd, J = 10.0, 2.2 Hz, 1H, CH=CH2),
4.64–4.45 (m, 1H, CH), 4.42–4.18 (m, 1H, NCH2), 4.19–3.95 (m, 2H, NCH2),
3.95–3.80 (m, 1H, NCH2), 3.15–3.01 (m, 4H, NCH2), 2.67–2.53 (s, 4H, NCH2), 2.32
13
(s, 3H, NCH3), 2.30–2.23 (m, 1H, CH2), 2.15–1.97 (m, 1H, CH2). C NMR (100
MHz, DMSO-d6) δ 165.1, 159.2, 155.8, 149.0, 148.2, 147.5, 142.7, 142.0, 141.0,
134.7, 131.9, 128.8 (Ar-C×2), 127.7, 126.0, 120.8, 118.9, 118.8 (Ar-C×2), 112.2,
102.7, 56.0, 54.7 (C×2), 49.2 (C×2), 48.9, 45.8. HRMS m/z (ESI): calcd for
C29H37FN9O [M+H]+ 551.29898, found 551.29895.
4.1.13.7. (R)-N-(1-(6-((5-(4-methylpiperazin-1-yl)pyridin-2-yl)amino)-2-
(phenylamino)pyrido[3,4-d]pyrimidin-4-yl)piperidin-3-yl)acrylamide (25c)
1
Light yellow solid. Yield 40.4%. mp 194.8–196.7 °C. H NMR (400 MHz,
DMSO-d6) δ 9.51 (s, 1H, NH), 9.22 (s, 1H, NH), 8.67 (s, 1H, Ar-H), 8.63 (s, 1H,
Ar-H), 8.27 (d, J = 7.6 Hz, 1H, CONH), 7.92 (d, J = 7.9 Hz, 2H, Ar-H), 7.88 (d, J =
2.9 Hz, 1H, Ar-H), 7.40 (dd, J = 9.1, 3.0 Hz, 1H, Ar-H), 7.27 (t, J = 7.9 Hz, 2H, Ar-H),
7.15 (d, J = 9.1 Hz, 1H, Ar-H), 6.91 (t, J = 7.3 Hz, 1H, Ar-H), 6.26 (dd, J = 17.1, 10.1
Hz, 1H, CH=CH2), 6.11 (dd, J = 17.1, 2.2 Hz, 1H, CH=CH2), 5.61 (dd, J = 10.1, 2.2
29