A structure-guided optimization of pyrido[2,3-d]pyrimidin-7-ones as selective inhibitors of EGFRL858R/T790Mmutant with improved pharmacokinetic properties

Article abstract of DOI:10.1016/j.ejmech.2016.12.006

Structural optimization of pyrido[2,3-d]pyrimidin-7-ones was conducted to yield a series of new selective EGFR^T790Minhibitors with improved pharmacokinetic properties. One of the most promising compound 9s potently suppressed EGFR^L858R/T790Mkinase and inhibited the proliferation of H1975?cells with IC₅₀values of 2.0?nM and 40?nM, respectively. The compound dose-dependently induced reduction of the phosphorylation of EGFR and downstream activation of ERK in NCI[sbnd]H1975?cells. It also exhibited moderate plasma exposure after oral administration and an oral bioavailability value of 16%. Compound 9s may serve as a promising lead compound for further drug discovery overcoming the acquired resistance of non-small cell lung cancer (NSCLC) patients.

Full text of DOI:10.1016/j.ejmech.2016.12.006

Accepted Manuscript
A structure-guided optimization of pyrido[2,3-d]pyrimidin-7-ones as selective inhibitors
L858R/T790M
of EGFR
mutant with improved pharmacokinetic properties
Lei Yu, Minhao Huang, Tianfeng Xu, Linjiang Tong, Xiao-e Yan, Zhang Zhang, Yong
Xu, Caihong Yun, Hua Xie, Ke Ding, Xiaoyun Lu
PII:
S0223-5234(16)31012-1
10.1016/j.ejmech.2016.12.006
EJMECH 9097
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 September 2016
Revised Date: 24 November 2016
Accepted Date: 2 December 2016
Please cite this article as: L. Yu, M. Huang, T. Xu, L. Tong, X.-e Yan, Z. Zhang, Y. Xu, C. Yun, H. Xie,
K. Ding, X. Lu, A structure-guided optimization of pyrido[2,3-d]pyrimidin-7-ones as selective inhibitors of
L858R/T790M
EGFR
mutant with improved pharmacokinetic properties, European Journal of Medicinal
Chemistry (2017), doi: 10.1016/j.ejmech.2016.12.006.
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ACCEPTED MANUSCRIPT
A Structure-Guided Optimization of Pyrido[2,3-d]pyrimidin-7-ones
as Selective Inhibitors of EGFRL858R/T790M Mutant with Improved
Pharmacokinetic Properties
Lei Yua,b, Minhao Huanga,b, Tianfeng Xua,b, Linjiang Tongd, Xiao-e Yane, Zhang Zhanga, Yong Xua,
Caihong Yune, Hua Xied*, Ke Dinga,c*, Xiaoyun Luc*
Structural optimization of pyrido[2,3-d]pyrimidin-7-ones yielded new selective EGFRT790M
inhibitors. Compound 9s exhibited good pharmacokinetic properties with F value of 16%, and
inhibited EGFRL858R/T790M kinase and H1975 cells with IC50 values of 2.0 and 40 nM, respectively.

ACCEPTED MANUSCRIPT
A Structure-Guided Optimization of Pyrido[2,3-d]pyrimidin-7-ones
as Selective Inhibitors of EGFRL858R/T790M Mutant with Improved
Pharmacokinetic Properties
Lei Yua,b, Minhao Huanga,b, Tianfeng Xua,b, Linjiang Tongd, Xiao-e Yane, Zhang Zhanga, Yong Xua,
Caihong Yune, Hua Xied*, Ke Dinga,c*, Xiaoyun Luc*
a
Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, No. 190
Kaiyuan Avenue, Guangzhou 510530, China
b University of Chinese Academy of Sciences, No. 19 Yuquan Road, Beijing 100049, China
c
School of pharmacy, Jinan University, No. 601 Huangpu Avenue West, Guangzhou 510632,
China.
d
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, No. 555 Zu-Chong-Zhi Road, Zhangjiang Hi-Tech Park, Shanghai 201203,
China
e
Peking University Institute of Systems Biomedicine and Department of Biophysics, Peking
University Health Science Center, Beijing 100191, China
Corresponding
authors:
Tel:
+86-20-85221523.
E-mail:
ding_ke@gibh.ac.cn;
luxy2016@jnu.edu.cn.
Abstract
Structural optimization of pyrido[2,3-d]pyrimidin-7-ones was conducted to yield a series of
new selective EGFRT790M inhibitors with improved pharmacokinetic properties. One of the most
promising compound 9s potently suppressed EGFRL858R/T790M kinase and inhibited the
proliferation of H1975 cells with IC50 values of 2.0 nM and 40 nM, respectively. The compound
dose-dependently induced reduction of the phosphorylation of EGFR and downstream activation
of ERK in NCI-H1975 cells. It also exhibited moderate plasma exposure after oral administration
and an oral bioavailability value of 16 %. Compound 9s may serve as a promising lead compound
for further drug discovery overcoming the acquired resistance of non-small cell lung cancer
(NSCLC) patients.
Keyword: EGFRT790M mutant, Pyrido[2,3-d]pyrimidin-7-ones, Pharmacokinetic property, NSCLC,
Irreversible inhibitor

ACCEPTED MANUSCRIPT
1. Introduction
The epidermal growth factor receptor (EGFR, ErbB1, HER1) is a well-validated molecular
target for drug discovery treating non-small-cell lung cancer (NSCLC) patients [1]. Several small
molecule EGFR inhibitors (e.g., the reversible inhibitors gefitinib [2], erlotinib [3] and irreversible
inhibitor afatinib [4] etc.) have achieved significant clinical benefits in NSCLC patients with
active mutations in EGFR tyrosine kinase domain (i.e. L858R mutation in exon 21 and del
E746-A750 mutation in exon 19) [5]. However, acquired resistance against the current inhibitors
becomes a major challenge for clinical management of NSCLC patients [6]. A secondary
threonine790 to methionine790 mutation is a primary mechanism of the resistance accounting for
approximate 50 % resistant NSCLC patients. The 2nd generation irreversible cysteine797-reacting
inhibitors have been developed to potentially overcome the EGFRT790M mutation-mediated
resistance. Nevertheless, most of them suffer from low clinical maximum tolerated dose (MTD) in
resistance patients due to their poor margin over wild-type (WT) EGFR inhibition. To address the
“on-target” toxicity, several third- generation EGFRT790M mutant selective inhibitors [7- 11] (e.g.,
WZ4002 (1) [7], rociletinib (2) [8], osimertinib (AZD9291) (3) [9], olmutinib (4) [10] (Fig. 1))
were developed. These compounds effectively inhibit resistant EGFRT790M mutant while are
obvious less potent to EGFRWT. Significantly, the U.S. FDA recently granted an accelerated
approval for osimertinib to treat EGFRT790M mutated patients whose disease has gotten worse after
treatment of EGFR-blocking therapy [9b].
Figure 1. Chemical structures of newly reported selective irreversible EGFRT790M inhibitors.
Our group had explored great efforts to discover novel EGFRT790M mutant selective inhibitors
(Fig. 1) with distinct chemical scaffolds, such as dihydropyrimido[4,5-d]pyrimidinyl (5 and 6)

ACCEPTED MANUSCRIPT
[12], pyrimidine [4,5-d]pyrimidin-4(1H)-one (7) [13], and pyrido[2,3-d]pyrimidine-7-one
derivatives (8) [14]. The compound 8 displayed highly potent and specific EGFRT790M inhibition
(EGFRL858R/T790M IC50 = 0.8 nM) and strongly suppressed the proliferation of EGFRT790M mutated
H1975 NSCLC cells with an IC50 value of 2.8 nM. However, it possesses unacceptable
pharmacokinetic (PK) parameters such as poor oral bioavailability (F = 10 %), low oral exposure
and rapid clearance which limit the further development. Herein, we describe further structural
optimization of the compound with an aim of improving its PK profiles.
Figure 2. Pharmacokinetic property-based modification of the pyrido[2,3-d]pyrimidine-7-ones as
selective EGFRT790M inhibitors
2. Chemistry
Syntheses of the designed compounds 9a- 9u is outlined in Scheme 1. A directly nucleophilic
coupling of commercially available 5-bromo-2, 4-dichloropyrimidine (10) with N-Boc amine (11)
produced intermediates 12. Compounds 12 were then reacted with trans-but-2-enoic acid under
Heck coupling conditions [14] in the presence of bis(benzonitrile)palladium(II) dichloride,
followed by a treatment with acetic anhydride to produce the key intermediates 13. The
intermediates 13 were coupled with various amines (14) and followed by deprotection to yield
substituted amines 15. Compounds 9a- 9u were finally obtained by a straightforward acryloylation
of 15.

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Scheme 1. Synthesis of compounds 9a- 9u. Reagents and conditions: (i) K2CO3, N,
N-dimethylformamide, 80- 95 %, (ii) a. (E)-but-2-enoic acid, bis(benzonitrile)palladium(II)
dichloride, tri(o-tolyl)phosphine, N, N-diisopropylethylamine, tetrahydrofuran, 70 °C, argon, 16
hrs, b. Acetic anhydride, 90 °C, 24 hrs, 25- 50 %, (iii) a. for 9d, K3PO4,
tris(dibenzylideneacetone)dipalladium(0), 2-(dicyclohexylphosphino)-2',4',6'-triisopropylbiphenyl,
110 °C; for 9a to 9u, 2-butanol, trifluoroacetic acid, 90- 110 °C, 18 hrs, b. dichloromethane,
trifluoroacetic acid, room temperature, 50- 85 % (two steps), (iv) Acrylyl chloride, N,
N-diisopropylethylamine, dichloromethane, 0 °C, 40- 80 %.
3. Results and Discussion
Structural feature analysis of 8 revealed that it adopts a highly planar configuration, which is
believed to contribute to the poor solubility and unacceptable pharmacokinetic parameters. Our
previous studies have demonstrated that the 5-methyl pyrido[2,3-d]pyrimidin-7-one scaffold and
the acrylamide moiety are critical for the selective EGFR kinase inhibition [14]. In order to get
insight understanding on the binding mode of compound 8 with EGFR T790M, a co-crystal structure
of the inhibitor complexed with EGFRT790M was determined with a resolution of 2.8 Å (Fig. 3 and
SI Table 1). It was shown that the inhibitor 8 bound to the ATP binding site of EGFR with a
“U-shaped” configuration. The pyrido[2,3-d]pyrimidine-7-one core formed a bidentate hydrogen
bonding interaction with the “hinge” residue Met793 of the protein (Fig. 3A). The methoxyl
substituent of aniline ring extended towards Leu792 and Pro794 in the hinge region and the
aniline ring formed a hydrophobic interaction with the α-carbon of Gly796. The 5-methyl group
was slipped into the “gatekeeper” residue Met790 which likely contributed to the selectivity of
EGFRT790M over the wild-type EGFR (Fig. 3A) [14].

ACCEPTED MANUSCRIPT
Figure 3. A X-ray crystal structure of compound 8 binding with EGFRT790M (PDB ID: 5GMP). (A)
The EGFRT790M kinase is shown in a blue ribbon representation. Compound 8 is shown in yellow
stick structure. Hydrogen bonds are indicated by black hatched lines to key amino acids. (B) The
X-ray crystal structure with interaction surface is shown.
The X-ray structure analysis also revealed that the left-arm hydrophilic phenylpiperazine
moiety of compound 8 was exposed to a solvent accessible surface (Fig. 3B). It might provide an
ideal position for further structural optimization to improve PK profiles of the molecules without
affecting the selective EGFRT790M suppression. Thus, compounds 9a and 9b which harbored
hydrophilic pyrazole substituents were first designed and evaluated to display strong
EGFRL858R/T790M inhibition with IC50 values of 4.2 and 1.8 nM, respectively (Table 1). Whereas,
both compounds also exhibited significantly less potencies against EGFRWT with IC50 values of
444.7 and 166.2 nM, respectively. Pyridine containing compound 9c and thiazole substituted
molecule 9d were also designed and evaluated to exhibit strong and wild-type sparing
EGFRL858R/T790M inhibition with IC50 values of 1.4 and 2.5 nM, respectively. In addition,
compounds 9c and 9d strongly suppressed the proliferation of EGFRT790M mutated H1975 NSCLC
cells with IC50 values of 64 and 8.0 nM, respectively, while their activities against A431 cells with
wild-type EGFR are significantly less potent. Demethylation of the N-methyl phenylpiperazine
moiety is a possible mechanism contributing to the metabolic liability of compound 8 [15].
Consequently, 3’, 4’- dimethylpiperazine (9e) and 3’, 4’, 5’- trimethylpiperazine (9f) analogues
were synthesized to block the potential in vivo demethylation by increasing steric hindrance. Not
surprisingly, both 9e and 9f exhibited comparable EGFRL858R/T790M inhibitory potencies to that of
lead molecule 8 (Table 1).
Table 1 In vitro EGFR inhibition and anti-proliferation data of compounds 9a- 9f.

ACCEPTED MANUSCRIPT
Kinase inhibitiona
Anti-proliferationb
(IC50, nM)
EGFRL858R/T790M
(IC50, µM)
Compds
R1
R2
EGFRWT
344.3
H1975c
0.042
A431d
1.545
1.604
2e
3e
-
-
4.3
3.4
260.4
0.030
OMe
329.7
444.7
0.8
4.2
0.002
0.289
1.404
4.004
8
9a
166.2
46.8
1.8
1.4
2.5
0.014
0.064
0.008
3.525
2.379
2.698
9b
9c
9d
549.6
OMe
OMe
169.2
97.0
1.2
3.3
0.018
0.004
0.706
1.670
9e
9f
a EGFR activity assays were performed using an ELISA assay according to the reported protocols
[4]. The compounds were incubated with the kinase reaction mixture for 1.0 h before
b
measurement. The data are mean values from 3 independent experiments. The anti-proliferative
activities of the compounds were evaluated using the SRB (sulforhodamine B) assay. The data

ACCEPTED MANUSCRIPT
were means from at least three independent experiments. c H1975 is a human lung cancer cell line
d
e
(EGFRL858R/T790M).
A431 is an epidermoid carcinoma cell line (EGFRWT).
These two
compounds were synthesized in our laboratory [8, 9].
The X-ray structural information further revealed that the right-arm phenyl ring in compound 8
functioned as a linker between the 5-methyl pyrido[2,3-d]pyrimidin-7-one core and an acrylamide
to react with Cys797 residue in EGFR protein (Figure. 3B). Our previous investigations had
demonstrated that a replacement of the phenyl ring with a more flexible aliphatic ring is an
effective strategy to improve aqueous solubility of the molecules [12b]. Thus, we first replaced the
phenyl ring with a flexible 3-methylene-pyrrolidinyl (9g) (Table 2). However, the resulting
compound 9g demonstrated a significant potency loss in both EGFRL858R/T790M kinase (IC50 = 63.8
nM) and H1975 cell growth inhibitory (IC50 = 305 nM) assays. Several other flexible linkers, such
as (S)-3-pyrrolidinyl (9h), (R)-3-pyrrolidinyl (9i) and 4-piperidinyl (9j) were also explored.
Disappointingly, all of these modification caused obvious potency decrease. It was also
noteworthy that the S-enantiomer 9h (IC50 = 91.8 nM) displayed 5 times better EGFRL858R/T790M
inhibitory potency than the corresponding R-enantiomer 9i (IC50 = 436 nM), when a 3-pyrrolidinyl
moiety was utilized as the linker. Nevertheless, both of the compounds were significantly less
L858R/T790M
potent to suppress the proliferation of H1975 cells harboring EGFR
mutant than the
lead molecule 8.
Table 2 In vitro EGFR inhibition and anti-proliferation data of compounds 9g- 9u
Kinase inhibitiona
(IC50, nM)
Anti-proliferationb
(IC50, µM)
Compds
R3
R4
L
EGFRWT
344.3
EGFRL858R/T790M H1975c A431d
2e
3e
-
-
4.3
3.4
0.042
0.030
1.545
1.604
260.4

ACCEPTED MANUSCRIPT
H
H
H
H
329.7
0.8
0.002
0.305
1.404
>10
8
>1000
63.8
9g
H
H
H
H
>1000
>1000
91.8
0.762
5.200
>10.0
>10.0
9h
9i
436.3
H
H
H
H
>1000
36.9
>1000
2.3
2.235
0.081
>10.0
3.622
9j
9k
H
H
H
H
H
H
208.4
472.1
24.0
1.8
3.4
2.4
0.006
0.142
0.120
3.248
1.054
1.443
9l
9m
9n
H
H
>1000
17.7
0.197
0.660
9o
H
H
H
H
26
0.8
0.6
1.8
2.0
1.6
3.3
0.0001
0.007
0.004
0.040
0.012
0.030
0.817
1.428
1.154
1.388
1.712
1.134
9p
9q
9r
9s
9t
33.1
57.0
181.9
66.0
97.0
H
H
H
H
H
Me
Me
Me
9u
a EGFR activity assays were performed using an ELISA assay according to the reported protocols
[4]. The compounds were incubated with the kinase reaction mixture for 1.0 h before
b
measurement. The data are mean values from 3 independent experiments. The anti-proliferative
activities of the compounds were evaluated using the SRB (sulforhodamine B) assay. The data
were means from at least three independent experiments. c H1975 is a human lung cancer cell line
d
e
(EGFRL858R/T790M).
A431 is an epidermoid carcinoma cell line (EGFRWT).
These two

ACCEPTED MANUSCRIPT
compounds were synthesized in our laboratory [8, 9].
Given the fact that a flexible linker is detrimental to EGFRL858R/T790M inhibition, a different
optimization strategy is needed to improve pharmacokinetic properties of the molecules. X-ray
structure analysis suggested that the right-arm phenyl ring in 8 was accommodated in a partially
opened hydrophobic pocket formed by Leu718, Ser719, V726 and Leu844 residues (Fig. 3B). The
2”- or 3”- positions of the phenyl group might be used to introduce an extra substituent to improve
PK parameters by disrupting the molecular planarity and symmetry [16] without obviously
affecting EGFRL858R/T790M inhibitory potency. Based on this hypothesis, 2”- methyl (9k) and 3”-
methyl (9l) derivatives were first designed and synthesized. The compounds indeed exhibited
strong EGFRL858R/T790M inhibitory activities with IC50 values of 2.3 and 1.8 nM, respectively,
which is similar to that of 8. Further analysis also revealed that 3”- methyl compound (9l)
displayed obviously greater target selectivity than the corresponding 2”- methyl analogue (9k)
with a factor of 115- fold (Table 2). Thus, 3”- position of the phenyl group might be an optimal
position for introducing substituents to maintain high EGFRL858R/T790M potency and selectivity.
Further investigation suggested that 3”- position was well tolerated to several other hydrophobic
substituents, such as ethyl (9m), isopropyl (9n), tert-butyl (9o), fluoro (9p), chloro (9q), bromo
(9r) and trifluoromethyl (9s) moieties to keep the strong EGFRL858R/T790M inhibition. Compounds
9m, 9n, 9p, 9q, and 9r exhibited comparable EGFRL858R/T790M inhibitory potencies to the lead
molecule 8. These compounds also displayed strong anti-proliferative activities against H1975
NSCLC cells with EGFRL858R/T790M mutation. However, the significantly decreased target
specificity of these molecules (comparing with 8) might limit their potential for further
development. It was also noteworthy that the introduction of a tert-butyl group (9o) in 3”- position
caused a 20-fold potency loss with IC50 value of 17.7 nM (Table 2). This might be due to the steric
conflict between bulky group and the hydrophobic residues (Leu718 and Ser719) in the protein.
Encouragingly, when a trifluoromethyl group was introduced to the 3”- position, the resulting
compound 9s exhibited comparable EGFRL858R/T790M inhibitory potency to that of 8 with an IC50 of
2.0 nM, and also strong anti-proliferative activity with an IC50 of 40 nM. This molecule also
displayed an approximate 90-fold EGFRL858R/T790M selectivity over EGFRWT (Table 2). The 3’,
4’-dimethyl (9t) and 3’, 4’, 5’-trimethyl (9u) derivatives based on 9s also demonstrated strong
EGFRL858R/T790M inhibition and promising target specificity.

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Pharmacokinetic parameters of the most potent and selective molecules (i.e. 9d, 9e, 9f, 9s, 9t
and 9u) were further determined in male Sprage-Dawley rats (Table 3). It was shown that
compound 9d displayed poor bioavailability (F = 0.6 %, Table 3), suggesting the left-arm
heterocyclic replacements didn’t meet our initial goal for PK parameter improvement. Compounds
9e and 9f displayed improved PK properties with bioavailability values of 24.3 % and 13.3 %,
respectively. The maximum plasma concentration (Cmax) and oral exposure (AUC) of compound
9e were 154.7 µg/ L and 526.4 µg/ L*h, respectively, while the corresponding values of compound
9f were 64.43 µg/ L and 612.72 µg/ L*h after a single oral administration at 25 mg/kg (Table 3).
Compound 9s also exhibited improved pharmacokinetics parameters with an oral bioavailability
of 16 %, a Cmax value of 160.7 µg/ L and an AUC value of 670.9 µg/ L*h after an oral dosing of 25
mg/ kg. In addition, compound 9s demonstrated relatively lower clearance rate (CLz = 6.0 L/ h/Kg)
than that of compound 8. These data collectively suggested that an introduction of trifluoromethyl
group at 3’’- position of phenyl ring might benefit the PK profile improvement. However, there is
no much advantage achieved by combination of the structural elements from 9s and 9e or 9f with
that from 9s. The resulting compounds 9t and 9u displayed significantly lower Cmax and AUC
values after oral administration at same dosages (Table 3).
Table 3. Sprague-Dawley rat PK parameters by intravenous and oral administrationa
Compds route AUC(0→∞) (µg/L*h)
Cmax(µg/L)
1366.3
83.1
Tmax(h)
1.563
0.25
1.667
4.00
2
CLz(L/h/Kg)
11.4
F(%)
10.8
iv
po
iv
450.2
243.2
887.9
26.6
8b
1910
9dc
9eb
9fd
5.7
0.6
24.3
13.33
po
iv
10.6
432.7
526.4
622.21
612.72
831.2
670.9
398.48
1334.2
154.7
772.27
64.43
2455
11.8
8.12
po
iv
po
iv
9sb
9td
6.1
16.1
5.58
po
iv
160.7
259.17
2.25

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po
iv
111.22
409.65
89.66
21.36
592.08
17.45
1.67
9ud
12.29
4.38
po
6
a The pharmacokinetic parameters were determined after a single oral administration (25 mg/ kg, n
b
= 3 rats) and intravenous injection (5 mg/ kg, n = 3 rats). Compounds formulated in 2 %
dimethyl sulfoxide, 4 % ethanol, 4 % castor oil, and 90 % H2O. They were treated as a mesylate
salt. c Compounds formulated in 5 % ethanol, 3 % tween-80 and 92 % H2O and were treated as a
d
mesylate salt. Compounds were dissolved in 5 % dimethyl sulfoxide, 40 % PEG400 and saline.
They were treated as a mesylate salt.
The kinase inhibition of the compound 9s was further validated by investigating its suppressive
functions on the activation of EGFR and the downstream signaling proteins in H1975 NSCLC
cells harboring EGFRL858R/T790M mutant. As shown in Figure 4, compound 9s caused a
dose-dependent reduction of the phosphorylation of EGFR and downstream ERK in H1975 cells.
However, it was significantly less potent in A431 cells harboring EGFRWT. Thus, compound 9s
displayed selective suppression of phos-EGFR in H1975 cells, which was consistent with our
previous investigation [14].
Figure. 4 Compound 9s potently and selectively inhibits the activation of EGFR signals in H1975
NSCLC cells (A), while less potent in A431 cancer cells (B). Cells were treated with or without
compound 9s and 3 for 4 hrs at indicated concentration, respectively. Cells were then stimulated
by 100 ng/ mL EGFR for 10 min and harvested for Western blot analysis.
4. Conclusion
Based on structural feature analysis of EGFRT790M complex with compound 8, structural
optimization of pyrido[2,3-d]pyrimidin-7-ones were conducted with an aim to improve the
pharmacokinetics profiles of the new irreversible wild-type sparing EGFRL858R/T790M inhibitors.

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One of the most promising compound 9s exhibited moderate pharmacokinetic properties with an
oral bioavailability value of 16 %, strong EGFRL858R/T790M inhibitory potency with an IC50 of 2.0
nM and promising anti-proliferative activity against gefitinib-resistant H1975 human NSCLC cells
with an IC50 of 40 nM. The further structural optimization and in vivo antitumor efficacy of this
compound in different models are undergoing and the results will be disclosed in due course.
5. Experimental Section
5.1 General methods for chemistry
All reagents and solvents used as were purchased from commercial sources without further
purification. Flash chromatography was performed using silica gel (200- 300 mesh). All reactions
were monitored by TLC, using silica gel plates with fluorescence F254 and UV light visualization.
1H NMR and 13C NMR spectra were recorded on a Brucker AV- 400 spectrometer at 400 MHz
and Brucker AV- 500 spectrometer at 125 MHz using deuterated solvents as internal standard.
Coupling constants (J) are expressed in hertz (Hz). Chemical shifts (δ) are given in parts per
million (ppm). High resolution ESI- MS were recorded on an Applied Biosystems Q-STAR Elite
ESI- LC- MS/ MS mass spectrometer. The purity of compounds was determined with
reverse-phase HPLC analysis to be over 95 % (see supporting information). HPLC instrument:
Dionex Summit HPLC (Column: Diamonsil C18, 5.0 µm, 4.6 × 250 mm (Agilent Technologies);
detector: PDA- 100 photodiode array; injector: ASI- 100 autoinjector; pump: p-680A). A flow rate
of 1.0 mL/ min was used with mobile phase of MeOH in H2O with 0.1 % modifier (ammonia v/v).
Tert-butyl (3-((5-bromo-2-chloropyrimidin-4-yl)amino)phenyl)carbamate (12a)
General procedure for the synthesis of 12b- 12u. To a solution of 5-bromo-2,
4-dichloropyrimidine (22.79 g, 100 mmol) and tert-butyl (3-aminophenyl)carbamate (20.83 g, 100
mmol) in DMF (100 mL), K2CO3 (27.64 g, 200 mmol) was added. The suspension was stirred
overnight. 200 mL of ice water was added to the reaction mixture and then extracted with
dichloromethane (DCM), washed with brine and dried over Na2SO4. After filtration, evaporation,
the condensation was purified with a silica gel column to yield compound 12a (39.4 g, 99 %). 1H
NMR (400 MHz, DMSO-d6), δ 9.43 (s, 1 H), 9.28 (s, 1 H), 8.44 (s, 1 H), 7.66 (s, 1 H), 7.25 (d 1 H,
J = 2.8 Hz), 7.24 (s, 1 H), 7.13 (dd, 1 H, J = 2.4, 4.0 Hz), 1.48 (s, 9 H).
Tert-butyl (3-(2-chloro-5-methyl-7-oxopyrido[2,3-d]pyrimidin-8(7H)-yl)-phenyl)carbamate
(13a)

ACCEPTED MANUSCRIPT
General procedure for the synthesis of 13b- 13u. Compound 12a (10.3 g, 25.76 mmol) and
trans-crotonic acid (11.08 g, 128.8 mmol) were mixed in dry THF (50 mL) and DIPEA (44.8 mL)
under argon. The slurry was stirred, evacuated and refilled with argon before
bis(benzonitrile)palladium(II) dichloride (0.494 g, 1.29 mmol) and tri-o-tolylphosphine (0.392 g,
1.29 mmol) were added. The mixture was then heated and stirred at 70 °C for 16 hrs and then 6
mL of Ac2O was added. The reaction mixture was heated and stirred at 90 °C for an additional 24
hrs. The solvent was filtered and then removed under reduced pressure and the residue was diluted
with DCM. The organic layer was separated and washed with 1N HCl (3 ×) and brine, dried over
anhydrous Na2SO4, concentrated and purified by silica gel chromatography to afford compound
13a (2.235 g, 25 %). 1H NMR (500 MHz, DMSO-d6), δ 9.56 (s, 1 H), 9.09 (s, 1 H), 7.46-7.38 (m,
3 H), 6.88 (d, 1 H, J = 7.5 Hz) 6.71 (d, 1 H, J = 1.0 Hz), 2.49 (s, 3 H), 1.46 (s, 9 H).
8-(3-Aminophenyl)-2-((1-(2-(dimethylamino)ethyl)-1H-pyrazol-4-yl)amino)-5-methyl-pyrido[
2,3-d]pyrimidin-7(8H)-one (15a)
General procedure for the synthesis of 15b- 15u. To a solution of compound 13a (0.41 g, 1.06
mmol) in 2-butanol (5 mL), 1-(2-(dimethylamino)ethyl)-1H-pyrazol-4-amine (0.196 g, 1.27 mmol)
and trifluoroacetic acid (94 µL) were added in a sealed tube. The reaction was heated at 95 °C for
18 hrs. The reaction mixture was then allowed to cool to room temperature. The mixture was
transferred to a round-bottom flask and then the solvent was removed under reduced pressure. The
residue was dissolved in DCM (2.0 mL) and TFA (2.0 mL), and the resulting mixture was stirred
for 5 hrs at room temperature. The solvent was removed under reduced pressure, and the residue
was neutralized with saturated NaHCO3 aqueous solution. The water layer was extracted with
DCM. The organic layer was combined and washed with brine, dried over Na2SO4, filtered,
concentrated, and purified by silica gel chromatography to afford 15a as a yellow solid (0.264 g,
65 % for two steps). 1H NMR (500 MHz, DMSO-d6), δ 10.00 (s, 1 H), 8.78 (s, 1 H), 7.25-7.22 (m,
1 H), 7.18 (s, 1 H), 6.95 (s, 1 H), 6.73 (d, 1 H, J = 8.0 Hz), 6.44 (s, 1 H), 6.40 (d, 1 H, J = 8.0 Hz),
6.26 (s, 1 H), 5.34 (s, 2 H), 3.89 (t, 2 H, J = 6.5 Hz), 2.43-2.40 (m, 5 H), 2.16 (s, 6 H).
N-(3-(2-((1-(2-(Dimethylamino)ethyl)-1H-pyrazol-4-yl)amino)-5-methyl-7-oxopyrido[2,3-d]p
yrimidin-8(7H)-yl)phenyl)acrylamide (9a)
General procedure for the synthesis of 9a- 9u. Acryloyl chloride (110 µL) was added dropwise
to a mixture of 15a (264 mg, 0.65 mmol) and DIPEA (0.23 mL) in dry DCM (20 mL) at 0 °C. The

ACCEPTED MANUSCRIPT
reaction mixture was stirred at 0 °C for 30 min and concentrated under reduced pressure. The
resulting crude product was purified by silica gel chromatography to afford 9a as a yellow solid
1
(149 mg, 50 %). H NMR (500 MHz, DMSO-d6) δ 10.40 (s, 1 H), 10.06 (s, 1 H), 8.81 (s, 1 H),
7.86 (d, 1 H, J = 8 Hz), 7.65 (s, 1 H), 7.59 (t, 1 H, J = 8.0 Hz), 7.13 (s, 1 H), 7.05 (d, 1 H, J = 7.6
Hz), 6.77 (s, 1 H), 6.42 (dd, 1 H, J = 10.1, 10.1 Hz), 6.30-6.23 (m, 2 H), 5.76 (d, 1 H, J = 10.2 Hz),
3.83-3.76 (m, 2 H), 2.46 (s, 3 H), 2.43-2.40 (m, 2 H), 2.09 (s, 6 H). 13C NMR (125 MHz,
DMSO-d6), δ 163.3, 162.2, 157.6, 156.6, 156.5, 147.1, 140.2, 137.6, 131.6, 129.7, 129.1, 127.2,
124.1, 122.2, 119.6, 119.0, 118.9, 116.0, 105.3, 58.7, 49.5, 44.9, 17.0. HRMS (ESI) for
C24H26N8O2 [M+H]+, Calcd: 459.22515, Found: 459.22604, RT= 0.41 min. HPLC analysis:
MeOH-H2O (75: 25), RT= 4.08 min, 98.6 % purity.
N-(3-(5-Methyl-2-((1-(1-methylpiperidin-4-yl)-1H-pyrazol-4-yl)amino)-7-oxopyrido[2,3-d]py
rimidin-8(7H)-yl)phenyl)acrylamide (9b)
1H NMR (400 MHz, DMSO-d6), δ 10.38 (s, 1 H), 10.04 (s, 1 H), 8.81 (s, 1 H), 7.92 (d, 1 H, J =
8.2 Hz), 7.61 (s, 1 H), 7.58 (t, 1 H, J = 8.0 Hz), 7.18 (s, 1 H), 7.03 (d, 1 H, J = 8.5 Hz), 6.86 (s, 1
H), 6.43 (dd, 1 H, J = 10.1, 10.1 Hz), 6.29 (d, 1 H, J = 0.9 Hz), 6.25 (dd, 1 H, J = 1.9, 2.0 Hz),
5.77 (dd, 1 H, J = 1.9, 2.0 Hz), 3.60-3.53 (m, 1 H), 2.84-2.76 (m, 2 H), 2.46 (s, 3 H), 2.22 (s, 3 H),
2.06-1.99 (m, 2 H), 1.80-1.70 (m, 2 H), 1.63-1.55 (m, 2 H). 13C NMR (125 MHz, DMSO-d6), δ
164.2, 163.1, 158.6, 157.5, 147.9, 141.0, 138.5, 132.5, 130.6, 129.8, 128.1, 125.1, 122.8, 120.6,
120.0, 118.7, 116.9, 106.2, 59.0, 55.0, 54.9, 46.6, 32.8, 17.9. HRMS (ESI) for C26H28N8O2
[M+H]+, Calcd: 485.2408, Found: 485.24059, RT= 0.41 min. HPLC analysis: MeOH-H2O (75:
25), RT= 5.68 min, 99.4 % purity.
N-(3-(5-methyl-2-((6-(4-methylpiperazin-1-yl)pyridin-3-yl)amino)-7-oxo-pyrido[2,3-d]pyrimi
din-8(7H)-yl)phenyl)acrylamide (9c)
1H NMR (500 MHz, DMSO-d6), δ 8.65 (s, 1 H), 8.49 (s, 1 H), 7.82 (br, 2 H), 7.45 (s, 1 H), 7.39 (t,
1 H, J = 7.8 Hz), 7.29 (s, 1 H), 7.25 (s, 1 H), 6.88 (d, 1 H, J = 7.7 Hz), 6.39 (s, 1 H), 6.33 (d, 1 H,
J = 16.8 Hz), 6.23 (br, 1 H), 6.12 (dd, 1 H, J = 10.2, 10.2 Hz), 5.64 (d, 1 H, J = 10.2 Hz), 3.43 (s,
4 H), 2.49-2.48 (m, 7 H), 2.33 (s, 3 H). 13C NMR (125 MHz, DMSO-d6), δ 163.2, 162.1, 158.5,
156.6, 156.2, 154.8, 146.9, 139.9, 138.3, 137.0, 131.7, 129.5, 128.4, 127.5, 127.1, 123.9, 119.7,
118.9, 116.4, 105.9, 105.7, 54.3, 45.8, 45.1, 16.9. HRMS (ESI) for C27H28N8O2 [M+H]+, Calcd:
497.2408, Found: 497.24036, RT= 0.3 min. HPLC analysis: MeOH-H2O (80: 20), RT= 5.63 min,

ACCEPTED MANUSCRIPT
99.1 % purity.
N-(3-(5-Methyl-2-((5-methyl-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridin-2-yl)amino)-7-oxopyri
do[2,3-d]pyrimidin-8(7H)-yl)phenyl)acrylamide (9d)
1H NMR (400 MHz, DMSO-d6), δ 11.74 (s, 1 H), 10.33 (s, 1 H), 8.95 (s, 1 H), 7.82 (d, 1 H, J =
8.3 Hz), 7.64 (s, 1 H), 7.51 (t, 1 H, J = 8.0 Hz), 7.01 (d, 1 H, J = 8.6 Hz), 6.44 (dd, 2 H, J = 10.2,
11.6 Hz), 6.25 (dd, 1 H, J = 1.7, 1.8 Hz), 5.76 (dd, 1 H, J = 1.7, 1.8 Hz), 3.23 (s, 2 H), 2.60 (d, 2 H,
J = 5.26 Hz), 2.52 (d, 2 H, J = 6.58 Hz), 2.32 (s, 3 H). 13C NMR (125 MHz, DMSO-d6), δ 163.18,
162.03, 156.88, 156.01, 155.69, 146.81, 142.44, 140.58, 137.19, 131.69, 129.97, 127.05, 124.3,
120.2, 119.0, 118.7, 117.8, 107.2, 51.9, 50.9, 44.7, 32.9, 31.5, 29.4, 28.9, 25.9, 17.0. HRMS (ESI)
for C30H33N7O3 [M+H]+, Calcd: 474.17067, Found: 474.17037, RT= 0.42 min. HPLC analysis:
MeOH-H2O (85: 15), RT= 5.12 min, 96.7 % purity.
N-(3-(2-((4-(3,4-Dimethylpiperazin-1-yl)-2-methoxyphenyl)amino)-5-methyl-7-oxopyrido-[2,
3-d]pyrimidin-8(7H)-yl)phenyl)acrylamide (9e)
1H NMR (400 MHz, DMSO-d6), δ 10.34 (s, 1 H), 8.79 (s, 1 H), 8.10 (s, 1 H), 7.91 (s, 1 H), 7.53 (s,
1 H), 7.49 (d, 1 H, J = 7.9 Hz), 7.26 (s, 1 H, J = 8.3 Hz), 6.96 (d, 1 H, J = 7.4 Hz), 6.50 (s, 1 H),
6.46-6.39 (m, 1 H), 6.32 (s, 1 H), 6.25 (d 1 H, J = 16.6 Hz), 5.97 (s, 1 H), 5.76 (d, 1 H, J = 9.8 Hz),
3.77 (s, 3 H), 3.39-3.39 (m, 2 H), 2.78 (d, 1 H, J = 10.6 Hz), 2.62 (s, 1 H), 2.53 (s, 1 H), 2.45 (s, 3
H), 2.25 (s, 2 H), 2.20 (s, 3 H), 2.09-2.08 (m, 1 H). 13C NMR (125 MHz, DMSO-d6), δ 164.1,
163.0, 157.5, 157.2, 147.8, 140.9, 138.0, 132.7, 130.4, 128.0, 124.9, 120.6, 119.6, 117.4, 107.0,
100.4, 58.1, 56.6, 55.9, 49.5, 43.1, 17.9, 17.8. HRMS (ESI) for C30H33N7O3 [M+H]+, Calcd:
540.27176, Found: 540.27057, RT= 0.41 min. HPLC analysis: MeOH-H2O (85: 15), RT= 5.12
min, 96.8 % purity.
N-(3-(2-((2-Methoxy-4-(3,4,5-trimethylpiperazin-1-yl)phenyl)amino)-5-methyl-7-oxopyrido[2
,3-d]pyrimidin-8(7H)-yl)phenyl)acrylamide（9f）
1H NMR (500 MHz, DMSO-d6), δ 10.36 (s, 1 H), 8.79 (s, 1 H), 8.12 (s, 1 H), 7.96 (s, 1 H), 7.50 (t,
2 H, J = 7.7 Hz), 7.26 (d, 1 H, J = 8.8 Hz), 6.96 (d, 1 H, J = 7.9 Hz), 6.50 (s, 1 H), 6.46- 6.40 (m,
1 H), 6.32 (s, 1 H), 6.26 (d, 1 H, J = 17.0 Hz), 5.96 (s, 0.7 H), 5.77 (s, 1 H, J = 10.4 Hz), 3.77 (s, 3
H), 2.45 (s, 3 H), 2.25 (s, 4 H), 2.18 (s, 3 H), 1.08 (d, 6 H, J = 4.9 Hz). 13C NMR (125 MHz,
DMSO-d6), δ 164.2, 163.1, 157.5, 157.2, 147.9, 140.9, 138.0, 132.7, 130.5, 128.1, 124.9, 120.6,
119.7, 117.4, 107.1, 106.9, 100.3, 58.2, 56.7, 56.5, 38.4, 18.8, 17.9. HRMS (ESI) for C27H33N7O3

ACCEPTED MANUSCRIPT
[M+H]+, Calcd: 554.28741, Found: 554.28756, RT= 0.41 min. HPLC analysis: MeOH-H2O (80:
20), RT= 11.5 min, 99.9 % purity.
8-((1-Acryloylpyrrolidin-3-yl)methyl)-2-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amin
o)-5-methylpyrido[2,3-d]pyrimidin-7(8H)-one (9g)
1H NMR (400 MHz, CDCl3) δ 8.62 (d, 1 H, J = 3.76 Hz), 8.16 (dd, 1 H, J = 8.44, 8.72 Hz), 7.68
(d, 1 H, J = 25.4 Hz), 6.58-6.51 (m, 2 H), 6.40-6.31 (m, 2 H), 6.27 (s, 1 H), 5.65-5.58 (m, 1 H),
4.54-4.35 (m, 2 H), 3.90 (s, 3 H), 3.78-3.58 (m, 2 H), 3.50-3.40 (m, 2 H), 3.19-3.18 (m, 4 H),
2.97-2.85 (m, 1 H), 2.60 (t, 4 H, J = 9.8 Hz), 2.40 (s, 3 H), 2.36 (s, 3 H), 2.06-1.85 (m, 2 H). 13C
NMR (125 MHz, CDCl3), δ 164.4, 163.4, 159.2, 159.0, 155.8, 155.6, 149.9, 149.5, 148.3, 148.1,
145.3, 128.6, 127.2, 120.9, 120.8, 120.6, 117.2, 107.6, 107.1, 100.2, 55.7, 55.1, 50.3, 49.7, 49.3,
46.0, 45.8, 45.3, 42.4, 42.2, 38.8, 36.0, 29.6, 29.4, 28.5, 17.2. HRMS (ESI) for C28H35N7O3
[M+H]+, Calcd: 518.28741, Found: 518.28699, RT= 0.42 min. HPLC analysis: MeOH-H2O (80:
20), RT= 6.81 min, 98.0 % purity.
(S)-8-(1-Acryloylpyrrolidin-3-yl)-2-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-5-
methylpyrido[2,3-d]pyrimidin-7(8H)-one (9h)
25
1
[α] D 25.862 (c 0.015, CH3OH). H NMR (400 MHz, DMSO-d6), δ 8.73 (s, 1 H), 8.69 (s, 1 H),
7.33 (s, 1 H), 6.55-6.39 (m, 2 H), 6.18-6.12 (m, 2 H), 5.88 (s, 1 H), 5.66 (dd, 1 H, J = 11.7, 10.7
Hz), 3.82 (t, 0.7 H, J = 9.8 Hz), 3.75 (s, 3 H), 3.67 (s, 0.8 H), 3.59-3.51 (m, 2 H), 3.14-3.12 (m, 5
H), 2.80-2.68 (m, 1 H), 2.55 (s, 4 H), 2.35 (s, 3 H), 2.27 (d, 3 H, J = 7.4 Hz), 1.90 (d, 1 H, J = 42.9
Hz). 13C NMR (125 MHz, DMSO-d6), δ 163.1, 163.0, 162.5, 162.4, 159.8, 159.7, 156.9, 156.8,
155.2, 153.1, 152.8, 149.5, 149.4, 146.2, 146.1, 129.5, 128.9, 126.6, 125.3, 125.2, 118.8, 118.6,
115.8, 106.4, 106.3, 99.5, 55.4, 55.2, 54.4, 49.7, 48.3, 48.1, 47.8, 45.5, 45.4, 45.1, 44.7, 43.9, 26.4,
24.5, 16.6. HRMS (ESI) for C27H33N7O3 [M+H]+, Calcd: 504.27176, Found: 504.27127, RT= 0.42
min. HPLC analysis: MeOH-H2O (80: 20), RT= 6.87 min, 95.9 % purity.
(R)-8-(1-Acryloylpyrrolidin-3-yl)-2-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-5-
methylpyrido[2,3-d]pyrimidin-7(8H)-one (9i)
[α] 2D5 -39.216 (c -0.020, CH3OH). H NMR (400 MHz, CDCl3), δ 8.76 (d, 2 H, J = 17.1 Hz), 7.35
1
(d, 1H, J = 23.1 Hz), 6.59 (s, 1 H), 6.48-6.43 (m, 2 H), 6.16 (dd, 2 H, J = 5.8, 9.8 Hz), 5.87 (s, 1
H), 5.69 (dd, 1 H, J = 10.1, 10.5 Hz), 4.07 (t, 1 H, J = 9.3 Hz), 3.82 (t, 1 H, J = 11.0 Hz), 3.76 (d,

ACCEPTED MANUSCRIPT
3 H, J = 6.5 Hz), 3.64 (s, 1 H), 3.55 (d, 2 H, J = 10.7 Hz), 3.41 (m, 3 H), 3.13 (m, 4 H), 2.79-2.68
(m, 5 H), 2.36 (s, 3H), 1.91 (dd, 1 H, J = 7.2, 4.1 Hz). 13C NMR (125 MHz, CDCl3)), δ 163.2,
163.1, 162.4, 162.3, 159.7, 159.6, 156.8, 156.7, 155.2, 152.9, 152.8, 147.8, 146.0, 145.9, 129.4,
129.0, 126.6, 126.4, 125.2, 124.9, 120.0, 119.7, 116.1, 115.9, 107.0, 106.4, 100.4, 100.3, 55.6,
55.5, 53.2, 52.0, 51.9, 49.7, 48.5, 45.7, 45.4, 44.8, 43.9, 41.9, 41.8, 41.4, 26.6, 24.6, 17.8, 16.6,
16.5, 11.8. HRMS (ESI) for C27H33N7O3 [M+H]+, Calcd: 504.27176, Found: 504.26978, RT= 0.31
min. HPLC analysis: MeOH-H2O (80: 20), RT= 5.70 min, 99.6 % purity.
8-(1-Acryloylpiperidin-4-yl)-2-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-5-meth
ylpyrido[2,3-d]pyrimidin-7(8H)-one (9j)
1H NMR (400 MHz, CDCl3), δ 8.60 (s, 1 H), 8.13 (d, 1 H, J = 8.2 Hz), 7.65 (s, 1 H), 6.64 (dd, 1 H,
J = 10.6, 10.5 Hz), 6.54-6.51 (m, 2 H), 6.32-6.22 (m, 2 H), 5.70 (d, 1 H, J = 11.1 Hz), 5.65 (m, 1
H), 4.91 (d, 1 H, J = 10.8 Hz), 4.16 (d, 1 H, J = 12.4 Hz), 3.90 (s, 3 H), 3.20 (t, 4 H, J = 4.5 Hz),
3.02 (s, 2 H), 2.78 (t, 1 H, J = 12.5 Hz), 2.60 (t, 4 H, J = 4.6 Hz). 2.37 (s, 6 H), 1.76-1.68 (m, 3 H).
13C NMR (125 MHz, DMSO-d6), δ 164.2, 162.6, 159.3, 156.5, 155.2, 151.8, 148.9, 145.5, 128.5,
126.7, 123.6, 119.1, 116.7, 106.6, 106.3, 99.7, 55.5, 54.5, 50.9, 48.3, 45.6, 16.4. HRMS (ESI) for
C28H35N7O3 [M+H]+, Calcd: 518.28741, Found: 518.28524, RT= 0.32 min. HPLC analysis:
MeOH-H2O (80: 20), RT= 8.33 min, 99.3 % purity.
N-(5-(2-((2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-5-methyl-7-oxopyrido[2,3-d]p
yrimidin-8(7H)-yl)-2-methylphenyl)acrylamide (9k)
1H NMR (400 MHz, DMSO-d6), δ 9.56 (s, 1 H), 8.77 (s, 1 H), 8.08 (s, 1 H), 7.50 (s, 1 H), 7.39 (d,
1 H, J = 8 Hz), 7.25 (d, 1 H, J = 8.7 Hz), 6.98 (d, 1 H, J = 7.5 Hz), 6.61-6.55 (m, 1 H), 6.53 (s, 1
H), 6.29 (s, 1 H), 6.21 (d, 1 H, J = 16.6 Hz), 6.01 (s, 1 H), 5.73 (d, 1 H, J = 9.7 Hz), 3.77 (s, 3 H),
3.05 (s, 4 H), 2.44 (s, 7 H), 2.36 (s, 3 H), 2.22 (s, 3 H). 13C NMR (125 MHz, DMSO-d6), δ 163.1,
162.2, 156.4, 156.3, 146.7, 136.9, 134.4, 131.6, 130.8, 130.6, 126.7, 125.6, 123.8, 119.7, 116.4,
106.3, 106.1, 99.6, 55.6, 54.6, 48.6, 45.7, 17.8, 16.9. HRMS (ESI) for C30H33N7O3 [M+H]+, Calcd:
540.27176, Found: 540.27135, RT= 0.31 min. HPLC analysis: MeOH-H2O (85: 15), RT= 5.63
min, 97.3 % purity.
N-(3-(2-((2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-5-methyl-7-oxopyrido[2,3-d]p
yrimidin-8(7H)-yl)-5-methylphenyl)acrylamide (9l)
1H NMR (400 MHz, DMSO-d6) δ 10.25 (s, 1 H), 8.79 (s, 1 H), 8.09 (s, 1 H), 7.72 (s, 1 H), 7.36 (s,

ACCEPTED MANUSCRIPT
1 H), 7.30 (d, 1 H, J = 8.9 Hz), 6.80 (s, 1 H), 6.53 (d, 1 H, J = 2.0 Hz), 6.43 (dd, 1 H, J = 10.0,
10.1 Hz), 6.31 (s, 1 H), 6.24 (dd, 1 H, J = 1.6, 1.6 Hz), 6.02 (br, 1 H), 5.75 (d, 1 H, J = 11.7 Hz),
3.78 (s, 3 H), 3.03 (s, 4 H), 2.45-2.44 (m, 7 H), 2.34 (s, 3 H), 2.22 (s, 3 H). 13C NMR (125 MHz,
DMSO-d6), δ 163.1, 162.1, 156.4, 156.2, 146.8, 139.7, 138.8, 136.8, 131.7, 126.9, 124.5, 119.8,
119.2, 116.9, 116.5, 106.1, 99.6, 55.7, 54.5, 48.5, 45.7, 21.1, 16.9. HRMS (ESI) for C30H33N7O3
[M+H]+, Calcd: 540.27176, Found: 540.27197, RT= 0.11 min. HPLC analysis: MeOH-H2O (80:
20), RT= 10.27 min, 99.7 % purity.
N-(3-Ethyl-5-(2-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-5-methyl-7-oxopyrido
[2,3-d]pyrimidin-8(7H)-yl)phenyl)acrylamide (9m)
1H NMR (500 MHz, DMSO-d6) δ 10.27 (s, 1 H), 8.79 (s, 1 H), 8.08 (s, 1 H), 7.75 (s, 1 H), 7.38
(s ,1 H), 7.26 (d, 1 H, J = 8.9 Hz), 6.83 (s, 1 H), 6.52 (d, 1 H, J = 2.4 Hz), 6.42 (dd, 1 H, J = 10.1,
10.2 Hz), 6.31 (s, 1 H), 6.24 (dd, 1 H, J = 1.9, 2.0 Hz), 5.97 (br, 1 H), 5.75 (dd, 1 H, J = 1.8, 1.9
Hz), 3.77 (s, 3 H), 3.02 (s, 4 H), 2.65 (q, 2 H, J = 7.5 Hz), 2.45 (s, 3 H), 2.44-2.42 (m, 4 H), 2.22
(s, 3 H), 1.19 (t, 3 H, J = 7.5 Hz). 13C NMR (125 MHz, DMSO-d6), δ 163.1, 162.1, 156.4, 156.3,
146.7, 145.2, 139.8, 136.9, 131.7, 126.9, 123.3, 119.8, 117.9, 117.1, 116.5, 106.1, 106.1, 99.6,
55.6, 54.5, 48.6, 45.7, 28.0, 16.9, 15.1. HRMS (ESI) for C31H35N7O3 [M+H]+, Calcd: 554.28741,
Found: 554.28706, RT= 0.11 min. HPLC analysis: MeOH-H2O (80: 20), RT= 12.41 min, 96.9 %
purity.
N-(3-Isopropyl-5-(2-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-5-methyl-7-oxo-p
yrido[2,3-d]pyrimidin-8(7H)-yl)phenyl)acrylamide (9n)
1H NMR (400 MHz, DMSO-d6), δ 10.27 (s, 1 H), 8.79 (s, 1 H), 8.06 (s, 1 H), 7.78 (s, 1 H), 7.38 (s,
1 H),7.22 (d, 1 H, J = 8.9 Hz), 6.84 (s, 1 H), 6.51 (d, 1 H, J = 2.3 Hz), 6.45-6.37 (m, 1 H), 6.31 (s,
1 H), 6.23 (dd, 1 H, J = 1.9, 2.0 Hz), 5.59 (s, 0.7 H), 5.74 (dd, 1 H, J = 1.9, 2 Hz), 3.77 (s, 3 H),
3.01 (s, 4 H), 2.95-2.88 (m, 1 H), 2.44 (s, 4 H), 2.42 (d, 3 H, J = 4.8 Hz), 2.22 (s, 3 H), 1.20 (dd, 6
H, J = 4, 4 Hz). 13C NMR (125 MHz, DMSO-d6), δ 163.1, 162.0, 156.4, 156.3, 149.9, 146.7,
139.8, 137.0, 131.7, 126.8, 121.9, 119.9, 117.1, 116.5, 106.1, 106.0, 99.6, 55.6, 54.5, 48.5, 45.6,
33.3, 23.7, 23.6, 16.9. HRMS (ESI) for C32H37N7O3 [M+H]+, Calcd: 568.30306, Found:
568.30285, RT= 0.35 min. HPLC analysis: MeOH-H2O (85: 15), RT= 7.07 min, 98.4 % purity.
N-(3-(Tert-butyl)-5-(2-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-5-methyl-7-oxo
pyrido[2,3-d]pyrimidin-8(7H)-yl)phenyl)acrylamide (9o)

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1H NMR (400 MHz, DMSO-d6) δ 10.34 (s, 1 H), 8.86 (s, 1 H), 8.11 (s, 1 H), 7.98 (s, 1 H), 7.51 (s,
1 H), 7.26 (d, 1 H, J = 8.7 Hz), 7.04 (s, 1 H), 6.59 (s, 1 H), 6.49 (dd, 1 H, J = 10.0, 10.1 Hz), 6.38
(s, 1 H), 6.31 (d, 1 H, J = 16.8 Hz), 6.01 (br, 1 H), 5.82 (d, 1 H, J = 10.1 Hz), 3.85 (s, 3 H), 3.08 (s,
4 H), 2.52-2.50 (m, 7 H), 2.29 (s, 3 H), 1.34 (s, 9 H). 13C NMR (125 MHz, DMSO-d6), δ 163.1,
162.1, 158.1, 156.4,156.3, 152.4, 146.7, 139.7, 136.9, 131.9, 131.8, 126.8, 125.4, 120.8, 119.9,
116.8, 116.6, 115.8, 106.2, 106.1, 99.6, 55.7, 54.4, 48.6, 45.7, 34.6, 31.0, 16.9. HRMS (ESI) for
C33H39N7O3 [M+H]+, Calcd: 582.31871 Found: 582.31817, RT= 0.1 min. HPLC analysis:
MeOH-H2O (80: 20), RT= 18.03 min, 94.8 % purity.
N-(3-Fluoro-5-(2-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-5-methyl-7-oxopyrid
o[2,3-d]pyrimidin-8(7H)-yl)phenyl)acrylamide (9p)
1H NMR (400 MHz, CDCl3) δ 8.65 (s, 1 H), 8.53 (s, 1 H), 8.02 (s, 1 H), 7.84 (s, 1 H), 7.41 (s, 1
H), 6.84 (s, 1 H), 6.66 (d, 1 H, J = 7.4 Hz), 6.41 (d, 1 H, J = 8.8 Hz), 6.34 (d, 2 H, J = 5.5 Hz),
6.14- 6.08 (m, 2 H), 5.67 (d, 1 H, J = 10.3 Hz), 3.12 (s, 3 H), 2.57 (s, 4 H), 2.46 (s, 3 H), 2.35 (s, 3
H). 13C NMR (125 MHz, DMSO-d6), δ 163.4, 163.18, 162.2 (d, 1 C, J = 239.5 Hz), 156.5, 156.1,
146.9, 140.8 (d, 1 C, J = 12.6 Hz), 138.3 (d, 1 C, J = 12.5 Hz), 131.3, 127.7, 119.5, 116.3, 115.9,
111.3 (d, 1 C, J = 23.5 Hz), 106.0, 105.8, 105.6, 99.6, 55.6, 54.5, 48.4, 45.6, 16.9. HRMS (ESI)
for C29H30FN7O3 [M+H]+, Calcd: 544.24669 Found: 544.2458, RT= 0.41 min. HPLC analysis:
MeOH-H2O (75: 25), RT= 11.16 min, 98.9 % purity.
N-(3-Chloro-5-(2-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-5-methyl-7-oxopyri
do[2,3-d]pyrimidin-8(7H)-yl)phenyl)acrylamide (9q)
1H NMR (500 MHz, DMSO-d6) δ 10.51 (s, 1 H), 8.80 (s, 1 H), 8.22 (s, 1 H), 8.07 (s, 1 H), 7.42 (s,
1 H), 7.27 (d, 1 H, J = 8.8 Hz), 7.17 (s, 1 H), 6.52 (s, 1 H), 6.41 (dd, 1 H, J = 10.0, 10.1 Hz),
6.31-6.27 (m, 2 H), 6.05 (br, 1 H), 5.81 (d, 1 H, J = 11.1 Hz), 3.77 (s, 3 H), 3.03 (s, 4 H), 2.44 (s, 3
H), 2.43 (s, 4 H), 2.22 (s, 3 H). 13C NMR (125 MHz, DMSO-d6), δ 163.5, 161.8, 156.5, 156.1,
147.0, 140.8, 138.2, 133.3, 131.3, 127.7, 124.0, 119.5, 118.6, 118.3, 116.3, 106.1, 105.9, 99.6,
55.6, 54.5, 48.5, 45.7, 16.9. HRMS (ESI) for C29H30ClN7O3 [M+H]+, Calcd: 560.21714, Found:
560.21667, RT= 0.1 min. HPLC analysis: MeOH-H2O (80: 20), RT= 11.89 min, 98.7 % purity.
N-(3-Bromo-5-(2-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-5-methyl-7-oxopyri
do[2,3-d]pyrimidin-8(7H)-yl)phenyl)acrylamide (9r)
1H NMR (400 MHz, DMSO-d6) δ 10.48 (s, 1 H), 8.80 (s, 1 H), 8.21 (s, 2 H), 7.46 (s, 1 H),

ACCEPTED MANUSCRIPT
7.29-7.26 (m, 2 H), 6.53 (d, 1 H, J = 2.1 Hz), 6.40 (dd, 1 H, J = 9.9, 10 Hz), 6.31-6.26 (m, 2 H),
6.09 (br, 1 H), 5.81 (dd, 1 H, J = 1.7, 1.9 Hz), 3.77 (s, 3 H), 3.04 (s, 4 H), 2.45-2.43 (m, 7 H), 2.22
(s, 3 H). 13C NMR (125 MHz, DMSO-d6), δ 163.5, 161.9, 156.5, 156.1, 147.0, 140.9, 138.4, 131.3,
127.7, 126.7, 121.4, 121.1, 119.6, 118.9, 116.3, 106.1, 105.9, 99.7, 69.7, 55.6, 54.5, 48.5, 45.7,
16.9. HRMS (ESI) for C29H30BrN7O3 [M+H]+, Calcd: 604.16663, Found: 604.16643, RT= 0.1
min. HPLC analysis: MeOH-H2O (80: 20), RT= 12.32 min, 99.5 % purity.
N-(3-(2-((2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-5-methyl-7-oxopyrido[2,3-d]p
yrimidin-8(7H)-yl)-5-(trifluoromethyl)phenyl)acrylamide (9s)
1H NMR (400 MHz, DMSO-d6), δ 10.65 (s, 1 H), 8.81 (s, 1 H), 8.33 (s, 1 H), 8.23 (s, 1 H), 7.74 (s,
1 H), 7.47 (s, 1 H), 7.13 (d, 1 H, J = 8.4 Hz), 6.51 (s, 1 H), 6.42 (dd, 1 H, J = 10.2, 9.9 Hz), 6.33 (s,
2 H), 5.93 (s, 0.6 H), 5.83 (d, 1 H, J = 9.9 Hz), 3.75 (s, 3 H), 3.00 (s, 4 H), 2.46 (s, 3 H), 2.42 (s, 4
H), 2.21 (s, 3 H). 13C NMR (125 MHz, DMSO-d6), δ 164.1, 162.4, 157.0, 156.6, 147.6, 141.1,
138.6, 131.7, 130.7 (q, 1 C, J = 31.0 Hz), 128.5, 124.2 (q, 1 C, J = 271.0 Hz), 123.9, 121.3, 119.9,
116.7, 115.5, 106.6, 106.2, 100.1, 60.2, 56.1, 55.3, 54.9, 48.9, 46.1, 17.4. HRMS (ESI) for
C30H30F3N7O3 [M+H]+, Calcd: 594.2435, Found: 594.24518, RT= 0.31 min. HPLC analysis:
MeOH-H2O (85: 15), RT= 6.39 min, 95.3 % purity.
N-(3-(2-((4-(3,4-Dimethylpiperazin-1-yl)-2-methoxyphenyl)amino)-5-methyl-7-oxopyrido[2,3
-d]pyrimidin-8(7H)-yl)-5-(trifluoromethyl)phenyl)acrylamide (9t)
1H NMR (400 MHz, CDCl3), δ 9.02 (s, 1 H), 8.66 (s, 1 H), 8.31 (s, 1 H), 7.75 (s, 1 H), 7.53 (s, 1
H), 7.21 (s, 2 H), 6.36 (s, 2 H), 6.33 (s, 1 H), 6.23-6.18 (m, 1 H), 6.05 (s, 1 H), 5.67 (d, 1 H, J =
8.2 Hz), 3.81 (s, 3 H), 3.37 (s, 2 H), 3.13 (s, 2 H), 2.78 (s, 1 H), 2.69 (s, 2 H), 2.54 (s, 3 H), 2.48 (s,
3 H), 1.32 (s, 3 H). 13C NMR (125 MHz, DMSO-d6), δ 163.7, 161.9, 156.5, 156.1, 147.1, 140.8,
138.1, 131.3, 130.2 (q, 1 C, J = 32 Hz), 129.8, 127.8, 127.0, 123.5, 123.8 (q, 1 C, J = 271 Hz),
120.7, 120.5, 119.9, 116.3, 115.1, 106.2, 105.9, 99.9, 59.7, 57.6, 55.7, 54.0, 53.7, 53.2, 47.1, 16.9.
HRMS (ESI) for C31H32F3N7O3 [M+H]+, Calcd: 608.25915, Found: 608.25938, RT= 0.41 min.
HPLC analysis: MeOH-H2O (85: 15), RT= 6.63 min, 98.4 % purity.
N-(3-(2-((2-Methoxy-4-(3,4,5-trimethylpiperazin-1-yl)phenyl)amino)-5-methyl-7-oxopyrido[2
,3-d]pyrimidin-8(7H)-yl)-5-(trifluoromethyl)phenyl)acrylamide (9u)
1H NMR (400 MHz, DMSO-d6), δ 10.66 (s, 1 H), 8.81 (s, 1 H), 8.38 (s, 1 H), 8.23 (s, 1 H), 7.70 (s,
1 H), 7.46 (s, 1 H), 7.14 (d, 1 H, J = 8.8 Hz), 6.49 (s, 1 H), 6.45-6.39 (m, 1 H), 6.33 (s, 2 H), 5.85

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(s, 0.7 H), 5.83 (d, 1 H, J = 10.4 Hz), 3.76 (s, 3 H), 2.46 (s, 3 H), 2.28 (t, 3 H, J = 10.7 Hz), 2.24 (s,
3 H), 2.18 (s, 3 H), 1.07 (d, 6 H, J = 5.8 Hz). 13C NMR (125 MHz, DMSO-d6), δ 163.6, 161.9,
156.7, 156.4, 156.1, 147.1, 140.6, 138.1, 131.2, 130.3 (q, 1 C, J = 32.0 Hz), 127.9, 123.7 (q, 1 C, J
= 271.0 Hz), 123.6, 120.8, 119.3, 116.3, 115.0, 106.1, 105.5, 99.4, 56.9, 55.8, 55.7, 55.6, 37.5,
17.8, 16.9. HRMS (ESI) for C32H34F3N7O3 [M+H]+, Calcd: 622.2748, Found: 622.27507, RT=
0.41 min. HPLC analysis: MeOH-H2O (85: 15), RT= 10.36 min, 96.3 % purity.
5.2 Cell lines and reagents
Cell lines H1975 (NSCLC, EGFRL858R/T790M) and A431 (NSCLC, EGFRWT) were obtained from
American Type Culture Collection (ATCC, Rockville, MD). The cells were maintained at 37 °C in
5 % CO2 incubator in RPMI 1640 (Gibco, Invitrogen) containing 10 % fetal bovine serum (Gibco,
Invitrogen) and 1 % penicillin-streptomycin liquid (Gibco, Invitrogen). The EGFR gene of every
cell line was sequenced before use. Rociletinib and osimertinib were synthesized in our chemistry
laboratory.
5.3 Enzyme-linked immunosorbent assay (ELISA) kinase assay
Poly (Glu, Tyr) 4:1 (Sigma, St. Louis, MO) (20 µg/ mL) was precoated in 96-well ELISA plates
as the substrate. The active kinases were incubated with indicated compounds in 1× reaction
buffer (50 mmol/ L HEPES pH 7.4, 20 mmol/ L MgCl2, 0.1 mmol/ L MnCl2, 0.2 mmol/ L Na3VO4,
1 mmol/ L DTT) containing 5 µmol/ L ATP at 37 °C for 1 h. After incubation, the wells was
washed with PBS, and then incubated with anti-phosphotyrosine (PY99) antibody (Santa Cruz,
CA) and horseradish peroxidase (HRP)-conjugated secondary antibody in sequence. The wells
were visualized using o-phenylenediamine (OPD) and read using a multi-well spectrophotometer
(VERSAmax™, Molecular Devices, Sunnyvale, CA, USA) at 492 nm.
5.4 Cell proliferation assays.
NCI-H1975 and A431 cancer cells were maintained in strict accordance with the instruction and
established procedures. The cell proliferation assay was evaluated using SRB (Sulforhodamine B)
assay treated for 72 hrs with different concentrations of compounds. The data was calculated using
Graph Pad Prism version 4.0. The IC50 were fitted using a non-linear regression model with a
sigmodial dose-response.
5.5 Western blot analysis.
Western blotting was conducted as previously reported. In brief, cells were seeded to six-well

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plates and incubated overnight, and then were treated with or without different concentrations of
compounds for 4 hrs and then stimulated with or without EGF (100 ng/ mL) for 10 min. Cell
samples were then lysed in 1× SDS lysis buffer. Proteins were resolved by SDS-PAGE and
transferred onto polyvinylidene difluoride membranes (Millipore), which were blocked with
nonfat milk and hybridized with specific primary antibodies. The bands were visualized using an
enhanced chemiluminescence reagent (GE Healthcare) after hybridization with a HRP-conjugated
secondary antibody.
5.6 Determination of pharmacokinetic parameters in rats.
Male Sprague-Dawley rats (180-300 g) were dosed with the test compounds intravenously (iv)
at 5 mg/ kg and by oral gavage (po) at 25 mg/ kg. The compound was dissolved in 5 % DMSO,
40 % PEG400 and Saline. Blood samples (0.2 mL) were then obtained via orbital sinus puncture
at 0 min, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 16 h, 24 h, time points and collected
into heparinized tubes. Blood samples were centrifuged for cell removal, and the plasma was then
transferred to a clean vial and subsequently stored at -80 °C prior to analysis. Test sample
concentrations were determined by LC-MS/ MS. Pharmacokinetic parameters were calculated
using WinNonlin software.
Acknowledgements
We thank National High Technology Research and Development (863) for Young Scientists
program (2015AA020906), Guangdong Province (2015A030306042, 2014TQ01R341 and
2015A030312014), Guangzhou City (201508030036 and 201506010086) for their financial
support.
Abbreviations
T, threonine; M, methionine; NSCLC, non-small-cell lung cancer; EGFR, epidermal growth factor
receptor; L, leucine; R, arginine; E, glutamic acid; A, alanine; MTD, maximum tolerated dose; PK,
pharmacokinetics; F, oral bioavailability; DMF, N, N-dimethylformamide; DIPEA,
ethyldiisopropylamine; THF, tetrahydrofuran; Ac2O, acetic anhydride; TFA, trifluoroacetic acid;
DCM, dichloromethane; Cmax, maximum serum concentration; AUC, area under curve; CLz,
clearance rate; iv, intravenous administration; po, oral gavage; Erk, extracellular regulated protein
kinases; TLC, thin-layer chromatography; UV, ultraviolet; ppm, parts per million; SRB,
Sulforhodamine B; ELISA, enzyme-linked immunosorbent assay.

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Captions
Figure 1. Chemical structures of newly reported selective irreversible EGFRT790M inhibitors.
Figure 2. Pharmacokinetic property-based modification of the pyrido[2,3-d]pyrimidine-7-ones as
selective EGFRT790M inhibitors
Figure 3. A X-ray crystal structure of compound 8 binding with EGFRT790M (PDB ID: 5GMP). (A)
The EGFRT790M kinase is shown in a blue ribbon representation. Compound 8 is shown in yellow
stick structure. Hydrogen bonds are indicated by black hatched lines to key amino acids. (B) The
X-ray crystal structure with interaction surface is shown.
Figure. 4 Compound 9s potently and selectively inhibits the activation of EGFR signals in H1975
NSCLC cells (A), while less potent in A431 cancer cells (B). Cells were treated with or without
compound 9s and 3 for 4 hrs at indicated concentration, respectively. Cells were then stimulated
by 100 ng/ mL EGFR for 10 min and harvested for Western blot analysis.
Table 1 In vitro EGFR inhibition and anti-proliferation data of compounds 9a- 9f.
Table 2 In vitro EGFR inhibition and anti-proliferation data of compounds 9g- 9u
Table 3. Sprague-Dawley rat PK parameters by intravenous and oral administration
Scheme 1. Synthesis of compounds 9a- 9u.

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Optimization of pyrido[2,3-d]pyrimidin-7-ones with improved pharmacokinetic properties.
Compound 9s potently suppressed EGFRL858R/T790M kinase and H1975 cells.
Compound 9s exhibited moderate plasma exposure and an oral bioavailability value of 16 %.