Discovery of a novel class anti-proliferative agents and potential inhibitors of EGFR tyrosine kinases based on 4-anilinotetrahydropyrido[4,3-d]pyrimidine scaffold: Design, synthesis and biological evaluations

Article abstract of DOI:10.1016/j.bmc.2015.05.059

A novel series of 4-arylamino-6/7-substituted-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidines were designed, synthesized and their biological activities as the potential anti-proliferative agents and EGFR kinase inhibitors were evaluated. Both of N-acrylamide fragment in THPPs and 4-aniline groups with substituents played key roles for their significant anti-proliferative activities against four cancer cell lines (HT29, A549, H460 and H1975). Especially inhibitory activity of Gefitinib-resistant H1975 were showed more favorable, which could be observed from compounds 13b, 13c, 13n, 13o, 13p, 13r, 13s, 13u and 24c obviously. By evaluation of inhibiting EGFR and HER2 kinases, seven compounds (13b, 13g, 13n, 13o, 13p, 13r and 13s) showed stronger EGFR potency with IC₅₀ ≤ 18 nM, which could also be understood by preliminary docking study of 13b with EGFR kinase. In view of the primary SAR, bisarylaniline derivatives (13o, 13p, 13r and 13s) showed obvious improvements on HER2 inhibition, which indicated their being potential EGFR/HER2 dual kinase inhibitors.

Full text of DOI:10.1016/j.bmc.2015.05.059

Accepted Manuscript
Discovery of a novel class anti-proliferative agents and potential inhibitors of
EGFR tyrosine kinases based on 4-anilinotetrahydropyrido[4,3-d]pyrimidine
scaffold: design, synthesis and biological evaluations
Yong Zhang, Kai Zhang, Meng Zhao, Lixia Zhang, Mingze Qin, Shuchun Guo,
Yanfang Zhao, Ping Gong
PII:
S0968-0896(15)00494-0
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.05.059
BMC 12356
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
8 April 2015
29 May 2015
30 May 2015
Please cite this article as: Zhang, Y., Zhang, K., Zhao, M., Zhang, L., Qin, M., Guo, S., Zhao, Y., Gong, P., Discovery
of a novel class anti-proliferative agents and potential inhibitors of EGFR tyrosine kinases based on 4-
anilinotetrahydropyrido[4,3-d]pyrimidine scaffold: design, synthesis and biological evaluations, Bioorganic &
Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.05.059
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Discovery of a novel class anti-proliferative agents and potential inhibitors of EGFR tyrosine kinases based on
4-anilinotetrahydropyrido[4,3-d]pyrimidine scaffold: design, synthesis and biological evaluations
Yong Zhang, Kai Zhang, Meng Zhao, Lixia Zhang, Mingze Qin, Shuchun Guo, Yanfang Zhao, Ping Gong*
Key Laboratory of Structure-Based Drug Design and Discovery (Shenyang Pharmaceutical University), Ministry of
Education, 103 Wenhua Road, Shenhe District, Shenyang 110016, PR China
Keywords
tetrahydropyrido[4,3-d]pyrimidine (THPP); anti-proliferative agents; EGFR tyrosine kinase inhibitor.
Abstract
A novel series of 4-arylamino-6/7-substituted-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidines were designed, synthesized
and their biological activities as the potential anti-proliferative agents and EGFR kinase inhibitors were evaluated. Both
of N-acrylamide fragment in THPPs and 4-aniline groups with substituents played key roles for their significant
anti-proliferative activities against four cancer cell lines (HT29, A549, H460 and H1975). Especially inhibitory activity
of Gefitinib-resistant H1975 were showed more favorable, which could be observed from compounds 13b, 13c, 13n, 13o,
13p, 13r, 13s, 13u and 24c obviously. By evaluation of inhibiting EGFR and HER2 kinases, seven compounds (13b, 13g,
13n, 13o, 13p, 13r and 13s) showed stronger EGFR potency with IC50 ≤ 18 nM, which could also be understood by
preliminary docking study of 13b with EGFR kinase. In view of the primary SAR, bisarylaniline derivatives (13o, 13p,
13r and 13s) showed obvious improvements on HER2 inhibition, which indicated their being potential EGFR/HER2
dual kinase inhibitors.
1. Introduction
Non-small cell lung cancer (NSCLC) is one of important causes of cancer mortality globally.1 Most of the NSCLC
patients present with advanced, unresectable or metastatic disease, and a 5-year survival with less than ca. 15%.2
Therefore, novel therapeutic strategies including the design of safe and potent anti-NSCLC drugs are highly desired. One
of the most important strategies for the treatment of NSCLC is to target the epidermal growth factor receptor (EGFR).3,4
which belongs to the erbB family of receptor tyrosine kinases (RTKs) and contains a subfamily of four closely related
receptor tyrosine kinases, EGFR (ErbB-1), erbB2/HER2, erbB3/HER3 and erbB4/HER4.5 As EGFR kinase inhibitors,
4-anilinoquinazoline derivatives have attracted more and more attention since the successful marketing of Gefitinib in
2003 (Figure 1).6 To date, it is remarkable that the other 5 kinase inhibitors with 4-anilinoquinazoline moiety have also
received the approvals from Food and Drug Administration (FDA) to be utilized as anticancer drugs, including EGFR
inhibitors Erlotinib7 and Icotinib,8 EGFR/HER2 dual inhibitors Lapatinib9 and Afatinib,10 VEGFR/EGFR dual inhibitor
Vandetanib.11
(Figure 1 should be listed here.)
More abundant and systematic achievements on the research and development of 4-aminoquinzaline derivatives
triggered people’s interest on its usefulness of being bioisosteres and exploration toward broad non-quinazolines
structures. We focused on the well-known tetrahydropyrido[4,3-d]pyrimidine (THPP) core which have attracted
considerable attention due to their biological and pharmaceutical properties such as anti-proliferative activity (Bcl-2
inhibitor,12 mTOR kinase inhibitor,13,14 PI3K inhibitor,15 P2Y14 receptor antagonist,16 VEGFR-2 inhibitor17 and Erks
inhibitor18) and other biological activities (PDE10 inhibitor,19 DPP-4 inhibitor,20 TASK-3 channel antagonist,21 TGR5
Agonist,22 α1-Adrenoceptor antagonist,23 HIV attachment inhibitor24 etc.), as shown in Figure 2.
(Figure 2 should be listed here.)
*Corresponding author. Tel./fax: +86 24 23986429. E-mail address: gongpinggp@126.com.
1

However, to the best of our knowledge, 4-arylamino-6-substituted-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidines have
never been investigated as EGFR kinase inhibitors. Herein, we disclosed our design and synthesis of this completely new
class of EGFR kinase inhibitors which were also biologically evaluated.
2. Results and Discussion
2.1. Design
The skeleton of 4-arylamino-6-substituted-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine retains the stability of the
quinazoline but also its 6-substitued side chain showed the more flexibility than quinazoline’s on account of fusing
saturated tetrahydropyridine in THPP. We propose such motif can mimic the famous 4-anilinquinazoline and influence
kinase inhibitory activities with better physicochemical properties due to more free orientation of THPP core and
different linker pattern of flexible N-substituted side chains. Based on the structure-activity relationship (SAR) studies of
quinazolines as EGFR inhibitors,25, 26 we installed the 3-chloro-4-fluoro aniline fragment at 4-position as the first choice
because it has been widely utilized in designing many EGFR inhibitors, such as Gefitinib and Afatinib. It has been
established that 4-arylamino fragment can well extend in the hydrophobic pocket in the back of the ATP-binding cleft
and provide the key kinase inhibition. At the same time, many different linker patterns of 6-substituents (series I) were
designed and introduced which pointed at the Phosphate-binding region. Further optimization of 4-arylamino moiety in
the designed compound with the optimal linker pattern led to compound series II as the potential EGFR inhibitors as
evaluated by cancer cell lines and kinases. On the other hand, 4-arylamino-7-substituted-THPP series III were also
designed and evaluated in order to investigate the anticancer activity of outspreading orientation of the side chain due to
different N-position in THPP. (Figure. 3).
(Figure 3 should be listed here.)
In our initial study, structure a1 was designed and aligned with Gefitinib and Erlotinib based on molecule field
theory.27, 28 The result suggested that all three compounds had reasonable overlap as shown in Figure 4, which indicated
the 4-arylamino-6-substituted-THPPs could effectively mimic the 4-anilinquinazoline derivatives.
(Figure 4. should be listed here.)
2.2. Chemistry
The designed 4-arylamino-6-substituted-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidines (series I and II) were synthesized
as shown in Schemes 1 and 2. It was critical to introduce a robust N-protecting group in THPP core to avoid formation of
byproducts occurring at arylamine in subsequent deprotection (especially for arylamines with Cl, Br or NO2 groups).29
Ethyl 1-benzyl-4-oxopiperidine-3-carboxylate (1) was therefore subjected to catalytic hydrogenation to give the
intermediate 2, which was protected again with CbzCl to yield intermediate 3. Cyclization with formamidine acetate
generated 4 efficiently. After chlorination with phosphorus oxychloride to get 5, the optimal base and solvent were
explored and identified to facilitate nucleophilic substitution with different anilines and afford the compounds 6a-q in
satisfactory yields. Deprotection in HBr and acetic acid yielded the key precursors 7a-q cleanly. The target products 8-13
were obtained in various conditions as depicted in Scheme 2. Compound 7a reacted with alkylsulfury chloride to obtain
sulfonamides 8a and 8b, or underwent Michael addition or alkylation to get amines 9a-b, or reacted with diethyl
squarate and subsequently amines to afford compounds 10a-c, or reacted with CDI and amines to prepare ureas 11a-c, or
coupled with chlorocarbonic ester or acyl chloride/aliphatic acid to afford carbamates 12a-d or amides 13a-u and a1,
respectively.
(Scheme 1 should be listed here.)
(Scheme 2 should be listed here.)
On the other hand, in order to investigate the influence of nitrogen atom at different positions in THPP core, the
4-arylamino-7-alkyl substituted-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidine compounds 20-23 and 24 a-c (series III) were
synthesized by adopting the similar methods as for 6-substituted compounds taking ethyl 1-benzyl-3-oxopiperidine-4-
carboxylate as the starting material (Schemes 3 and 4).
2

(Scheme 3 should be listed here.)
(Scheme 4 should be listed here.)
2.3. In vitro anti-proliferative activity
In order to find the best linker pattern at 6-position in THPP, the compounds of series I were initially synthesized and
evaluated by applying the MTT colorimetric assay using Gefitinib as positive control.30,31 The compounds with different
linkers were screened by anti-proliferative activity against HT29 (human colorectal cancer line), A549 (human lung
cancer cell line) and H460 (large cell lung cancer line), as well as Gefitinib-resistant H1975 cancer line (which is a
human lung cancer cell line expressed high levels of EGFR T790M/L858R mutation) as shown in Table 1. The
unsubstituted compound 7a, sulfonamides 8a-b, N-alkylated compounds 9a-b and compounds 10a-b using cyclobutene
dione as linkage showed poor activities in four cancer cell lines, as compared with Gefitinib, although compound 10c in
squarate derivatives displayed comparable activity against three cancer cell lines (IC50 = 20.21 µM for HT29, IC50 =
57.28 µM for A549, IC50 = 24.12 µM for H460), poor activity for H1975 (IC50 = 125.33 µM). With carbonyl as linkage,
the urea 11a-c and carbamates 12a-d also didn’t exhibit more potency. However, urea 11c (IC50 = 12.49 µM for A549)
and carbamate 12a (IC50 = 38.32 µM for H1975) showed comparable anti-proliferative activities against certain cancer
cell lines. Remarkably, amide derivatives 13b with propenamide demonstrated significant activity (IC50 = 42.35 µM for
HT29, IC50 = 22.27 µM for A549, IC50 = 29.73 µM for H460, especially IC50 = 6.32 µM for H1975). Subsequently the
result of comparison of the anti-proliferative activity of propenamide 13b, propynamide 13c and propanamide 13d was
that both Michael acceptors with unsaturated bond displayed better cellar activities, especially against H1975, IC50 of
compound 13c is 6.21 µM.
(Table 1 should be listed here.)
Keeping the N-acrylamide as the optimal fragment in THPP, further optimization and biological evaluations were
focused on the 4-substitued anilines fragment of series II compounds (Table 2). In most cases, the designed compounds
showed comparable or even more significant activities against four cell lines comparing with control compound
Gefitinib, especially inhibitory activity against Gefitinib-resistant H1975. It should be noted that compounds with
electron-withdrawing groups at meta-position of aniline showed stronger activities than those with para- or
ortho-electron-withdrawing groups (13g vs. 13k, 13n vs. 13l). Compound 13m exhibited lower activity than compound
13f against H1975 (IC50 = 34.03 µM vs 12.37 µM) could be perceived that substituted group at the ortho position could
reduce activity such as compound 13h and 13m. The change of substituted group at para position of the compounds
would not sharply decrease the activity. If introducing another aryl moiety at the para-position of 3-halogenaniline group
and with N, O or S as the linker, the anti-proliferative activities could be further improved. The inhibition of compound
13p with Cl (IC50 = 2.79 µM) against H1975 was 15 times more potent than compound 13q (IC50 = 43.42 µM) with F at
meta position of arylamine. In the derivatives with N linker between bisarylamines, urea 13u had more anti-proliferative
effect than amide 13t. There were eight compounds with IC50 ≤ 7µM against H1975 which showed more favorable
activity than the positive control Gefitinib (IC50 = 12.87 µM). From the Table 2 we could see that all the tested
compounds demonstrated good activity against H29, especially compound 13n, 13r, 13s and 13u (IC50 = 7.56 µM, 5.91
µM, 7.48 µM and 5.28 µM respectively). Compound 13p and 13r demonstrated the stronger activity against A549 (IC50
= 9.40 µM and 5.88 µM respectively), and five compounds (13i, 13n, 13r, 13t, 13u) showed potent activity against H460
with IC50 < 7 µM.
(Table 2 should be listed here.)
On the other hand, we also evaluated the anti-proliferative activities of 7-substituted THPPs (series III) in order to
investigate the influence of the side chain orientation resulted from different N positions in THPPs (Table 3). Totally 7
designed compounds showed comparable or poorer activities compared with the corresponding 6-substituted compounds
(20a vs. 7a; 21 vs. 8a; 22 vs. 11a; 23 vs. 12a; 24a vs. 13e; 24b vs. 13b and 24c vs. 13p). The results indicated N-side
chain at 6-position of THPP may have positive effect on the anti-proliferative activities.
3

(Table 3 should be listed here.)
2.4. In vitro EGFR and HER2 inhibitory activity
On the basis of the anti-proliferative results against four cancer cell lines, totally 7 designed compounds were selected
for further in vitro EGFR and HER2 inhibitory activities studies in comparison with Staurosporine, which had good
inhibitory activities against ERFR and HER2 kinase as a multiple protein kinases inhibitor.34 The results were
summarized in Table 4. All tested compounds exhibited excellent EGFR potency (IC50 ≤ 18 nM). The single arylamine
substituted compounds 13b, 13g and 13n showed low HER2 inhibitory activities but compounds 13o, 13p, 13r and 13s
with another aryl moiety in aniline fragment displayed considerable HER2 inhibitory activities. These biological data
revealed that the single arylamine substituted THPPs might be selective inhibitor of single target of EGFR kinase, and
bisaryl-substituted THPPs might be dual selective inhibitors on the ERFR/HER2 kinase targets.
(Table 4 should be listed here.)
2.5. Molecular docking
Compound 13b was selected for molecular docking study in EGFR crystal structure (PDB ID: 4G5P) for better
understanding the potency of this series of 6-substitued THPPs and further SAR study.35 As shown in Figure 5, the
docking result suggested that the THPP scaffold well fit into the ATP pocket and the N-1 atom had hydrogen bonding
interaction with carbonyl oxygen in Met790 as the quinazoline core.36 At the same time, carbonyl oxygen at the
6-acrylamide tail has hydrogen bonding interaction with NH in residue Cys797. Moreover, the carbon atom of Michael
acceptor was situated 3.73 Å away from the sulfhydroyl group of Cys797, which was suitable for the formation of the
covalent bond.37 The three key interactions might be able to explain the corresponding biological activities of the
designed 4-arylamino-6-substituted-THPPs as irreversible inhibitors of EGFR.
(Figure 5 should be listed here.)
3. Conclusions
A novel series of 4-arylamino-6/7-substituted-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine derivatives were designed,
synthesized and biologically evaluated as potential EGFR tyrosine kinase inhibitor. N-acrylamide fragment in THPPs
and 4-aniline groups with substituents played key roles for the significant anti-proliferative activities against four cancer
cell lines (HT29, A549, H460 and H1975). Especially inhibitory activity of Gefitinib-resistant H1975 were showed more
favorable, which could be observed from compounds 13b, 13c, 13n, 13o, 13p, 13r, 13s, 13u and 24c (IC50 ≤ 8 µM)
obviously. By evaluation of inhibiting EGFR and HER2 kinases, 7 compounds (13b, 13g, 13n, 13o, 13p, 13r and 13s)
showed stronger EGFR potency with IC50 ≤ 18 nM, which could also be mechanistically understood by preliminary
docking study of 13b with EGFR kinase. On the other hand, bisarylaniline derivatives (13o, 13p, 13r and 13s) showed
obvious improvements on HER2 inhibition activities, which indicated their potential being as EGFR/HER2 dual kinase
inhibitors. The further structure scope expanding of these new series and evaluations of their pharmacological profile, as
well as improvement of HER2 inhibition ability are still on-going.
4. Experimental
4.1. Chemistry
Unless otherwise noted, all materials were obtained from commercial suppliers and were used without further
purification. Reactions’ time and purity of the products were monitored by TLC on FLUKA silica gel aluminum cards
(0.2 mm thickness) with fluorescent indicator 254 nm. Column chromatography was run on silica gel (200-300 mesh)
from Qingdao Ocean Chemicals (Qingdao, Shandong, China). All melting points were obtained on a Büchi Melting
Point B-540 apparatus (Büchi Labortechnik, Flawil, Switzerland) and were uncorrected. Mass spectra (MS) were taken
in ESI mode on Agilent 1100 LC–MS (Agilent, Palo Alto, CA, USA).1H NMR and 13C NMR spectra were recorded on
Bruker ARX-400, 400 MHz; or BrukerARX-600, 600 MHz spectrometers (Bruker Bioscience, Billerica, MA, USA) with
TMS as an internal standard. The IR spectra were recorded by means of the KBr pellet technique on a Bruker FTS 135
spectrometer. Elemental analysis was determined on a Carlo-Erba1106Elemental analysis instrument (Carlo Erba, Milan,
4

Italy).(In the mode of measurement C, H, and N, the sample into the combustion tube in pure oxygen atmosphere static
combustion and products by a specific reagent after formation of CO2, H2O, N2 and nitrogen oxides, uniform mixing
under the atmospheric pressure. The thermal conductivity detector is used for determining the content of C, H and N
from mixed gases.).
4.1.1. Ethyl 4-oxopiperidine-3-carboxylate hydrochloride (2)
To compound 1 (160 g, 0.54 mol) in ethanol (1000 mL) was added palladium on carbon (16 g, 10% wt.) and
concentrated aqueous hydrogen chloride (16 mL) and the mixture was hydrogenated under hydrogen (5 atm.) at 40 °C
for 8h. The solution was filtered through a bed of Cellulose microcrystalline and the solvent was removed in vacuo to
give 107 g of compound 2 as a light brown solid without any further purification to be used in the next step. Light brown
solid; Yield: 95 %; MS (ESI) m/z (%): 172.3[M+H]+.
4.1.2. 1-benzyl 3-ethyl 4-oxopiperidine-1,3-dicarboxylate (3)
To a solution of compound 2 (87.7 g, 0.42 mol) and DIPEA (193 g, 1.05 mol) in DCM (600 mL) was added dropwise
slowly CbzCl (86.4 g, 0.50 mol) at 0 °C for an hour. After addition, the reaction solution was allowed to r.t. and stirred
for an hour. The reaction mixture was added water (1000 mL), separated two phase, the organic layer was washed by 1 N
HCl (1000 mL) and brine (1000 mL), then dried (MgSO4), filtered and concentrated to afford 165 g crude product 3.
White gray solid; Yield: 91%; MS (ESI) m/z (%): 304.0[M-H]-.
4.1.3. Benzyl 4-hydroxy-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (4)
To a slurry of sodium methoxide (7.1 g, 131 mmol) in anhydrous ethanol (60 mL) at room temperature was added
formamidine acetate (4.4 g, 24.9 mmol) and compound 3 (10 g, 33.0 mmol) in one portion. After stirring at reflux for 6 h,
the mixture was cooled to r.t.. Water (30 mL) was added, followed by con. HCl to adjust pH = 4. And the mixture was
extracted by DCM (100 mL × 2), the combined organic layer was concentrated and slurred by MTBE (30 mL) to afford
6.2 g compound 4. White solid; Yield: 62%; 1H NMR (DMSO-d6, 400 MHz): δ 8.07 (s, 1H), 7.32-7.38 (m, 5H), 5.11 (s,
2H), 4.22 (br. s., 2H), 3.64 (br. s., 2H), 2.50-2.62 (t, J = 5.7 Hz, 2H); MS (ESI) m/z (%): 285.9[M+H]+.
4.1.4. Benzyl 4-chloro-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (5)
To a solution of compound 4 (12. g, 49.6 mmol) and DIPEA (8.9 g, 58.5 mmol) in CH3CN (80 mL) was added
phosphorus oxychloride (9.12 g, 59.6 mmol) at r.t., and the reaction solution was heated to reflux for an hour, then
cooled to r.t.. The resulting mixture was quenched with saturated aqueous sodium bicarbonate solution (200 mL), the
mixture was then extracted with DCM (100 mL × 2). The combined organic layers were concentrated to give 11 g
compound 5 for next step directly. Brown oil; MS (ESI) m/z (%): 303.7[M+H]+.
4.1.5. General procedure for preparation of benzyl 4-substitutedphenylamino-7,8-dihydropyrido[4,3-d]
pyrimidine-6(5H)-carboxylate (6a-6q)
To a mixture of an appropriate aniline (0.83 mmol) and compound 4 (200 mg, 66 mmol) in THF (5 mL) was added
slowly NaHMDS (92 mg, 1 mmol) in an ice bath. Upon the completion of addition, the reaction mixture was allowed to
room temperature and stirred for 2 h. The mixture was added water (10 mL), extracted with EtOAc (10 mL × 2), washed
with brine (20 mL × 2). The organic phase was separated, dried and evaporated, and the crude product was purified by
chromatography to afford the compounds 6a-6q.
4.1.5.1. Benzyl 4-((3-chloro-4-fluorophenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (6a)
1
White solid; Yield: 79.3%; M.p.: 182-183 °C; H NMR (DMSO-d6, 400 MHz): δ 8.76( s, 1H), 8.44( s, 1H),7.96 (s,
5

1H), 7.66 (s, 1H), 7.34-7.39(m, 6H), 5.17 (s, 2H), 4.54 (br. s., 2H), 3.73 (br. s., 2H), 2.75 (t, J = 5.7 Hz, 2H); MS (ESI)
m/z (%): 413.4[M+H]+.
4.1.5.2. Benzyl 4-((3-(trifluoromethyl) phenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (6b)
White solid; Yield: 74.9%; M.p.: 133-135 °C; MS (ESI) m/z (%): 429.1[M+H]+.
4.1.5.3. Benzyl 4-((3-bromophenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (6c)
White solid; Yield: 80.7%; M.p.: 165-168 °C; MS (ESI) m/z (%): 439.0[M+H]+.
4.1.5.4. Benzyl 4-((2-methyl-5-nitrophenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (6d)
White yellow solid; Yield: 65.4%; M.p.: 149-150 °C; MS (ESI) m/z (%): 419.9[M+H]+.
4.1.5.5. Benzyl 4-((3,4-dimethylphenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (6e)
White solid; Yield: 71.6 %; M.p.: 176-179 °C; MS (ESI) m/z (%): 389.2[M+H]+.
4.1.5.6. Benzyl 4-((3-bromo-5-methoxyphenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (6f)
White solid; Yield: 59.2%; M.p.: 157-159 °C; MS (ESI) m/z (%): 469.3[M+H]+.
4.1.5.7. Benzyl 4-((4-bromophenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (6g)
White solid; Yield: 77.3%; M.p.: 126-127 °C; MS (ESI) m/z (%): 439.2[M+H]+.
4.1.5.8. Benzyl 4-((2,4-dichlorophenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (6h)
White solid; Yield: 69.7%; M.p.: 138-139 °C; MS (ESI) m/z (%): 428.8[M+H]+.
4.1.5.9. Benzyl 4-((2-chloro-5-(trifluoromethyl) phenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-
carboxylate (6i)
White solid; Yield: 68.0%; M.p.: 145-147 °C; MS (ESI) m/z (%): 462.3[M+H]+.
4.1.5.10. Benzyl 4-((3,5-dichlorophenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (6j)
White solid; Yield: 73.2 %; M.p.: 153-155 °C; MS (ESI) m/z (%): 429.0[M+H]+.
4.1.5.11. Benzyl 4-((3-chloro-4-(pyridin-2-ylmethoxy) phenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-
carboxylate (6k)
White solid; Yield: 74.8%; M.p.: 194-196 °C; MS (ESI) m/z (%): 502.1[M+H]+.
4.1.5.12. Benzyl 4-((3-chloro-4-((3-fluorobenzyl) oxy) phenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-
carboxylate (6l)
White solid; Yield: 69.5%; M.p.: 189-191 °C; MS (ESI) m/z (%): 519.0[M+H]+.
4.1.5.13. Benzyl 4-((3-fluoro-4-(pyridin-2-ylmethoxy) phenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-
carboxylate (6m)
White solid; Yield: 63.2%; M.p.: 171-173 °C; MS (ESI) m/z (%): 485.8[M+H]+.
4.1.5.14. Benzyl 4-((3-chloro-4-(3-(trifluoromethyl) phenoxy) phenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidine-
6

6(5H)-carboxylate (6n)
White solid; Yield: 79.6%; M.p.: 192-194 °C; MS (ESI) m/z (%): 555.0[M+H]+.
4.1.5.15. Benzyl 4-((3-chloro-4-((3-fluorobenzyl) thio) phenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidine-
6(5H)-carboxylate (6o)
White solid; Yield: 87.1%; M.p.: 183-184 °C; MS (ESI) m/z (%): 535.2[M+H]+.
4.1.5.16. Benzyl 4-((3-chloro-4-(3-fluorobenzamido) phenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidine-
6(5H)-carboxylate (6p)
White solid; Yield: 48.7 %; M.p.: 187-188 °C; MS (ESI) m/z (%): 532.3[M+H]+.
4.1.5.17. Benzyl 4-((3-chloro-4-(3-(3-fluorophenyl) ureido) phenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidine-
6(5H)-carboxylate (6q)
White solid; Yield: 53.4%; M.p.: 196-198 °C; MS (ESI) m/z (%): 547.1[M+H]+.
4.1.6. General procedure for preparation of N-phenyl-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4- substituted
amine (7a-q)
Compound 6a-q (5 g) was dissolved in acetic acid (10 mL) and HBr (10 mL), the reaction solution was heated and
stirred at 50 °C for 2 h. After finished reaction, the solution was cooled to 0 °C, added water (100 mL), adjusted pH = 10
with 5 N NaOH, extracted with Me-THF (100 mL × 3), washed by brine (100 mL), dried and removed the solvent then
slurried by MTBE (20 mL) to afford the product (7a-q).
4.1.6.1. N-(3-chloro-4-fluorophenyl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-amine (7a)
Beige solid,Yield: 89.4%; M.p.: 170-173°C; 1H NMR (DMSO-d6, 400 MHz): δ 8.46 (s, 2H), 8.04 (dd, J = 6.8, 2.7 Hz,
1H), 7.72 (ddd, J = 9.0, 4.4, 2.7 Hz, 1H), 7.42 (t, J=9.2 Hz, 1H), 3.80 (s, 2H), 3.05 (t, J = 5.8Hz, 2H), 2.69 (t, J = 5.7 Hz,
2H); MS (ESI) m/z (%): 276.5[M+H]+; Anal. calcd. for C15H14ClFN4O (%): C, 56.02; H, 4.34; N, 20.10; Found (%): C,
56.11; H, 4.31; N, 20.20.
4.1.6.2. N-(3-(trifluoromethyl) phenyl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-amine (7b)
1
Beige solid, Yield: 86.2%; M.p.: 129-131 °C; H NMR (DMSO-d6, 400 MHz): δ 8.55 (s, 1H), 8.44 (s, 1H), 8.12 (s,
1H), 8.07 (d, J = 8.2 Hz, 1H), 7.54 (t, J = 7.9 Hz, 1H), 7.36 (d, J = 7.7 Hz, 1H), 3.81 (s, 2H), 3.02 (t, J = 5.7 Hz, 2H),
2.66 (t, J = 5.7 Hz, 2H); MS (ESI) m/z (%): 294.6[M+H]+.
4.1.6.3. N-(3-bromophenyl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-amine (7c)
1
Beige solid, Yield: 76.5%; M.p.: 118-120 °C; H NMR (CD3OD, 400 MHz): δ 8.43 (s, 1H), 7.89-7.99 (m, 1H),
7.57-7.64 (m, 1H), 7.23-7.29 (m, 2H), 3.99 (s, 2H), 3.30 (t, J = 6.0 Hz, 2H), 2.89 (t, J = 6.0 Hz, 2H); MS (ESI) m/z
(%): 303.1[M-H]-.
4.1.6.4. N-(2-methyl-5-nitrophenyl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-amine (7d)
1
Light brown; Yield: 69.4%; M.p.: 84-87 °C; H NMR (CD3OD, 400 MHz): δ 8.27 (s, 1H), 8.21 (d, J = 2.3 Hz, 1H),
8.07 (dd, J = 8.4, 2.3 Hz, 1H), 7.54 (d, J = 8.4 Hz, 1H), 4.03 (s, 2H), 3.31-3.35 (m, 2H), 2.89 (t, J = 5.9 Hz, 2H), 2.34 (s,
3H); MS (ESI) m/z(%): 286.2[M+H]+.
7

4.1.6.5. N-(3,4-dimethylphenyl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-amine (7e)
Beige solid; Yield: 74.6%; M.p.: 184-185 °C; 1H NMR (CD3OD, 400 MHz): δ 8.31 (s, 1H), 7.29 (s, 1H), 7.26 (dd, J =
8.1, 2.1 Hz, 1H), 7.11 (d, J = 8.1 Hz, 1H), 3.98 (s, 2H), 3.31-3.34 (m, 2H), 2.87 (t, J = 6.1 Hz, 2H), 2.27 (s, 3H), 2.26 (s,
3H);MS (ESI) m/z(%): 253.0[M-H]-.
4.1.6.6. N-(3-bromo-5-methoxyphenyl)-5,6,7,8-tetrahydro[4,3-d]pyrimidin-4-amine (7f)
Beige solid; Yield: 67.8%; M.p.: 134-136 °C; MS (ESI) m/z (%): 335.1[M+H]+.
4.1.6.7. N-(4-bromophenyl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-amine (7g)
Beige solid; Yield: 73.2%; M.p.: 110-112 °C; MS (ESI) m/z (%): 305.0[M+H]+.
4.1.6.8. N-(2, 4-dichlorophenyl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-amine (7h)
Beige solid; Yield: 84.6%; M.p.: 135-137 °C; MS (ESI) m/z (%): 295.1[M+H]+.
4.1.6.9. N-(2-chloro-5-(trifluoromethyl) phenyl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-amine (7i)
Beige solid; Yield: 71.0%; M.p.: 121-123 °C; MS (ESI) m/z (%): 329.2[M+H]+.
4.1.6.10. N-(3,5-dichlorophenyl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-amine (7j)
White solid; Yield: 83.4%; M.p.: 146-148 °C; MS (ESI) m/z (%): 295.1[M+H]+.
4.1.6.11. N-(3-chloro-4-(pyridin-2-ylmethoxy) phenyl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-amine (7k)
White solid; Yield: 73.6%; M.p.: 140-141 °C; 1H NMR (DMSO-d6, 400 MHz): δ 8.59 (d, J = 4.3 Hz, 1H), 8.35 (s, 1H),
8.27 (s, 1H), 7.84-7.90 (m, 1H), 7.83 (d, J = 2.2 Hz, 1H), 7.51-7.60 (m, 2H), 7.32-7.39 (m, 1H), 7.19 (d, J = 9.0 Hz, 1H),
5.26 (s, 2H), 3.74 (br. s., 2H), 3.00 (t, J = 5.5 Hz, 2H), 2.59-2.65 (m, 2H). MS (ESI) m/z (%): 366.1[M-H]-.
4.1.6.12. N-(3-chloro-4-((3-fluorobenzyl) oxy) phenyl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-amine (7l)
Beige solid; Yield: 81.4%; M.p.: 181-183 °C; 1H NMR (CD3OD, 400 MHz): δ 8.33 (s, 1H), 7.72 (d, J = 2.6 Hz, 1H),
7.34-7.44 (m, 2H), 7.18-7.30 (m, 2H), 6.97-7.08 (m, 2H), 5.14 (s, 2H), 3.95 (s, 2H), 3.27 (t, J = 5.9 Hz, 2H), 2.85 (t, J =
5.8 Hz, 2H);MS (ESI) m/z (%): 385.1[M+H]+.
4.1.6.13. N-(3-fluoro-4-(pyridin-2-ylmethoxy) phenyl)-5,6,7,8-tetrahydropyrido[4, 3-d]pyrimidin-4-amine (7m)
Beige solid; Yield: 74.8%; M.p.: 152-184 °C; MS (ESI) m/z (%): 351.8[M+H]+.
4.1.6.14. N-(3-chloro-4-(3-(trifluoromethyl) phenoxy) phenyl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-amine
(7n)
Light yellow; Yield: 93.7%; M.p.: 169-170 °C; 1H NMR (CD3OD, 400 MHz): δ 8.42 (s, 1H), 7.96 (d, J = 2.6 Hz, 1H),
7.62 (dd, J = 8.8, 2.6 Hz, 1H), 7.48-7.55 (m, 1H), 7.37 (d, J = 7.7 Hz, 1H), 7.12-7.19 (m, 3H), 3.95 (s, 2H), 3.22-3.27 (m,
2H), 2.80-2.88 (m, 2H);MS (ESI) m/z (%): 421.0[M+H]+.
4.1.6.15. N-(3-chloro-4-((3-fluorobenzyl) thio) phenyl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-amine (7o)
8

Light brown; Yield: 58.1%; M.p.: 186-188 °C; MS (ESI) m/z (%): 398.5[M-H]-.
4.1.6.16. N-(2-chloro-4-((5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-yl) amino) phenyl)-3-fluorobenzamide (7p)
Beige solid; Yield: 65.7%; M.p.: 172-173 °C; 1H NMR (DMSO-d6, 400 MHz): δ 10.14 (s, 1H), 8.54 (s, 1H), 8.46 (s,
1H), 8.01 (d, J = 2.4 Hz, 1H), 7.86 (d, J = 7.8 Hz, 1H), 7.78 (d, J = 9.5 Hz, 1H), 7.72 (dd, J = 8.8, 2.3 Hz, 1H),
7.56-7.65 (m, 1H), 7.42-7.52 (m, 2H), 3.87 (s, 2H), 3.10 (t, J = 5.7 Hz, 2H), 2.71 (t, J = 5.6 Hz, 2H); MS (ESI) m/z (%):
396.7[M-H]-.
4.1.6.17. 1-(2-chloro-4-((5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-yl)amino)phenyl)-3-(3-fluorophenyl)urea(7q)
Beige solid; Yield: 87.2 %; M.p.: 126-128 °C; MS (ESI) m/z (%): 413.2[M+H]+.
4.1.7. General procedure for preparation of N-(3-chloro-4-fluorophenyl)-6-(substituted sulfonyl)-5,6,7,8-
tetrahydropyrido[4,3-d]pyrimidin-4-amine (8a-b)
To a solution of compound 7a (200 mg, 0.72 mmol) and DIPEA (138 mg,1.08 mmol) in DCM (6 mL) was added
dropwise slowly alkyl sulfonyl chloride (0.94 mmol) at °C. After addition, the reaction solution was allowed to r.t., and
stirred for an hour. The reaction solution was washed by Na2CO3 (10 mL) aqueous and brine (10 mL), then dried by
Na2SO4, filtered and concentrated, purified by column to afford compound 8a-b.
4.1.7.1. N-(3-chloro-4-fluorophenyl)-6-(methylsulfonyl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-amine (8a)
White solid; Yield: 85.9%; M.p.: 210-212 °C; 1H NMR (CDCl3, 400 MHz): δ 8.61 (s, 1H), 7.80 (br. s., 1H), 7.41 (d,
J=5.4 Hz, 1H), 7.16 (t, J=8.7 Hz, 1H), 6.44 (br. s., 1H), 4.34 (s, 2H), 3.57-3.75 (m, 2H), 3.05 (br. s., 2H), 2.98 (s, 3H);
MS (ESI) m/z(%): 357.2[M+H]+; Anal. calcd. for C14H14ClFN4O2S (%): C, 47.13; H, 3.95; N, 15.70; Found (%): C,
47.32; H, 4.06; N, 15.77.
4.1.7.2.
(8b)
N-(3-chloro-4-fluorophenyl)-6-(cyclopropylsulfonyl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-amine
White solid; Yield: 96.6 %; M.p.: 203-204 °C; 1H NMR (DMSO-d6, 400 MHz): δ 8.72 (s, 1H), 8.46 (s, 1H), 7.94 (dd,
J = 6.8, 2.6 Hz, 1H), 7.58-7.74 (m, 1H), 7.40 (t, J = 9.1 Hz, 1H), 4.38 (s, 2H), 3.60 (t, J = 5.9 Hz, 2H), 2.87 (t, J = 5.7
Hz, 2H), 2.60-2.72 (m, 1H), 0.87-1.14 (m, 4H); MS (ESI) m/z (%): 382.5[M+H]+; Anal. calcd. for C16H16ClFN4O2S(%):
C, 50.20; H, 4.21; N, 14.63; Found (%): C, 50.28; H, 4.20; N, 14.66.
4.1.8.1. 2-(4-((3-chloro-4-fluorophenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)-1- morpholino
ethanone (9a)
To a solution of compound 7a (200 mg, 0.72 mmol) and K2CO3 (198 mg, 1.44 mmol) in acetone (6 mL) was added
2-chloro-1-morpholinoethanone (176 mg, 1.04 mmol). And the reaction mixture was heated at reflux for 4 h. The
reaction mixture was cooled to r.t., filtered. And the filtrate was removed the solvent, purified by column to afford
1
compound 9a. Light yellow solid; Yield: 74.3%; M.p.: 187–189 °C; H NMR (CDCl3, 400 MHz): δ 8.56 (s, 1H),
7.69-7.77 (m, 1H), 7.32-7.44 (m, 1H), 7.12 (t, J = 8.6 Hz, 1H), 6.48 (br. s., 1H), 3.58-3.73 (m, 10H), 3.49 (br. s., 2H),
2.93 (br. s., 2H); 13C NMR (CDCl3, 101 MHz): δ 168.0, 160.7, 156.5, 155.8, 135.1, 124.0, 121.5, 120.9, 116.5, 110.9,
66.9, 61.0, 50.1, 49.2, 46.3, 42.3, 32.1; MS (ESI) m/z (%): 404.5[M-H]-; Anal. calcd. for C19H21ClFN5O2 (%): C, 56.23;
H, 5.22; N, 17.26;Found (%): C, 56.49; H, 5.21; N, 17.17.
9

4.1.8.2. 3-(4-((3-chloro-4-fluorophenyl) amino)-7,8-dihydropyrid[4,3-d]pyrimidin-6(5H)-yl)propanamide (9b)
The solution of compound 7a (200 mg, 0.72 mmol) and acrylamide (64 mg, 0.90 mmol) in ethanol (6 mL) was heated
at reflux for 8 h. The reaction mixture was cooled to r.t., and removed the solvent, purified by column to afford
compound 9b. White solid; Yield: 96.2%; M.p.: 177-179 °C; 1H NMR (DMSO-d6, 400 MHz): δ 8.50 (br. s., 1H), 8.41 (s,
1H), 7.96 (dd, J = 6.8, 2.7 Hz, 1H), 7.66 (ddd, J = 9.0, 4.3, 2.7 Hz, 1H), 7.39 (s, 1H), 7.34-7.38 (m, 1H), 6.80 (br. s., 1H),
+
3.48 (s, 2H), 2.79 (t, J = 7.2 Hz, 2H), 2.73 (s, 4H), 2.38 (t, J = 7.3 Hz, 2H); MS (ESI) m/z (%):349.8[M+H] ; Anal.
calcd. for C16H17ClFN5O (%): C, 54.94; H, 4.90; N, 20.02; Found (%): C, 55.13; H, 4.92; N, 20.08.
4.1.9.1. 3-(4-((3-chloro-4-fluorophenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)-4-ethoxycyclobut-
3-ene-1,2-dione (10a)
To a solution of compound 7a (200 mg, 0.72 mmol) and TEA (146 mg, 1.44 mmol) in ethanol (6 mL) was added
diethyl squarate (246 mg, 1.44 mmol) and heated at reflux for 4 h. The reaction solution was cooled to r.t., and removed
1
the solvent, purified by column to afford compound 10a. Light yellow; Yield: 72.6%; M.p.: 211-213°C; H NMR
(DMSO-d6, 400 MHz): δ 8.71-8.94 (m, 1H), 8.47 (s, 1H), 7.91 (br. s., 1H), 7.61 (br. s., 1H), 7.40 (t, J = 9.1 Hz, 1H),
4.52-5.05 (m, 4H), 3.72-4.21 (m, 2H), 2.92 (br. s., 2H), 1.41 (t, J = 6.5 Hz, 3H); MS (ESI) m/z (%): 401.3[M-H] -; Anal.
calcd. for C19H16ClFN4O3(%): C, 56.65; H, 4.00; N, 13.91; Found (%): C, 56.69; H, 4.11; N, 13.86.
4.1.9.2. 3-(4-((3-chloro-4-fluorophenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)-4-(dimethylamino)
cyclobut-3-ene-1,2-dione (10b)
To the solution of compound 7a (200 mg, 0.72 mmol) and TEA (146 mg, 1.44 mmol) in ethanol (6 mL) was added
diethyl squarate (246 mg, 1.44 mmol) and heated at reflux for 4 h. After added diethylamine (1.8 ml, 3.6 mmol), the
reaction was continued to stir at the same temperature for 4 h, removed the solvent, purified by column to afford the
1
compound 10b. White solid; Yield: 77.9 %; M.p.: 266-267 °C; H NMR (DMSO-d6, 400 MHz): δ 8.68 (s, 1H), 8.46 (s,
1H), 7.90 (dd, J = 6.7, 2.4 Hz, 1H), 7.55-7.70 (m, 1H), 7.40 (t, J = 9.1 Hz, 1H), 4.69 (s, 2H), 4.60-4.76 (m, 2H), 3.93 (t,
-
J = 5.6 Hz, 2H), 3.23 (s, 6H), 2.89 (t, J = 5.4 Hz, 2H); MS (ESI) m/z (%): 400.2[M-H] ; Anal. calcd. for
C19H17ClFN5O2 (%): C, 56.79; H, 4.26; N, 17.43; Found (%): C, 56.83; H, 4.28; N, 17.33.
4.1.9.3. 3-(4-((3-chloro-4-fluorophenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)-4-((2-(dimethylamino)
ethyl) amino) cyclobut-3-ene-1,2-dione (10c)
Preparation of compound 10c is followed the procedure for compound 10b. White solid; Yield: 81.4%; M.p.: 224-226
1
°C; H NMR (DMSO-d6, 400 MHz): δ 8.83 (br. s., 1H), 8.45 (s, 1H), 8.20 (br. s., 1H), 8.02 (d, J = 6.0 Hz, 1H),
7.63-7.78 (m, 1H), 7.37 (t, J = 9.0 Hz, 1H), 4.77 (br. s., 2H), 3.99 (br. s., 2H), 3.78 (d, J = 5.4 Hz, 2H), 2.85 (t, J = 5.4
Hz, 2H), 2.70 (br. s., 2H), 2.36 (br. s., 6H) ; MS (ESI) m/z (%): 443.2 [M-H] -; Anal. calcd. for C21H22ClFN6O2 (%): C,
56.69; H, 4.98; N, 18.89; Found (%): C, 56.75; H, 4.92; N, 18.92.
4.1.10.1.
4-((3-chloro-4-fluorophenyl)
amino)-N,
N-dimethyl-7,8-dihydropyrid[4,3-d]pyrimidine-6(5H)-
carboxamide (11a)
The solution of compound 7a (200 mg, 0.72 mmol) and CDI (154 mg, 0.94 mmol) in DMF (2 mL) was heated at 80
°C for 4 h. After added diethylamine (1.8 ml,3.6 mmol), the reaction was continued to stir at the same temperature for 4
h, cooled to r.t., added water (10 mL), extracted by Me-THF (10 mL × 3), washed by brine (10 mL), dried by Na2SO4,
10

1
removed the solvent, purified by column to afford the compound 11a. White solid; Yield: 78.4%; M.p.: 201-203 °C; H
NMR (CDCl3, 400 MHz): δ 8.54 (s, 1H), 7.67-7.78 (m, 1H), 7.34-7.45 (m, 1H), 7.11 (t, J = 8.7 Hz, 1H), 7.05 (br. s., 1H),
-
4.27 (s, 2H), 3.44-3.54 (m, 2H), 2.95 (m, 2H), 2.91 (s, 6H); MS (ESI) m/z (%): 348.4[M-H] ; Anal. calcd. for
C16H17ClFN5O (%): C, 54.94; H, 4.90; N, 20.02; Found (%): C, 54.91; H, 4.87; N, 20.25.
4.1.10.2. (4-((3-chloro-4-fluorophenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)(4-methylpiperazin-
1-yl) methanone (11b)
Preparation of compound 11b is followed the procedure for compound 11a. White solid; Yield: 72.5 %; M.p.: 213-214
°C; 1H NMR (DMSO-d6, 400 MHz): δ 8.71 (s, 1H), 8.42 (s, 1H), 7.94 (dd, J = 6.8, 2.6 Hz, 1H), 7.65 (ddd, J = 9.0, 4.3,
2.7 Hz, 1H), 7.38 (t, J = 9.1 Hz, 1H), 4.26 (s, 2H), 3.46 (t, J = 5.6 Hz, 2H), 3.28 (br. s., 4H), 2.79 (t, J = 5.4 Hz, 2H),
2.45 (br. s., 4H), 2.27 (s, 3H); MS (ESI) m/z (%): 403.5[M-H] -; Anal. calcd. for C19H22ClFN6O (%): C, 56.36; H, 5.48;
N, 20.76; Found (%): C, 56.46; H, 5.52; N, 20.80.
4.1.10.3. 4-((3-chloro-4-fluorophenyl) amino)-N-(3-(dimethylamino) propyl)-7,8-dihydropyrid[4,3-d]pyrimidine-
6(5H)-carboxamide (11c)
Preparation of compound 11c is followed the procedure for compound 10a. White solid; Yield: 72.8%; M.p.: 182-185
°C; 1H NMR (DMSO-d6, 400 MHz): δ 8.77 (s, 1H), 8.45 (s, 1H), 8.08 (dd, J = 6.8, 2.6 Hz, 1H), 7.75-7.81 (m, 1H), 7.37
(t, J = 9.1 Hz, 1H), 6.98 (t, J = 5.3 Hz, 1H), 4.51 (s, 2H), 3.64 (t, J = 5.6 Hz, 2H), 3.10-3.17 (m, 2H), 2.66-2.75 (m, 4H),
2.46 (s, 6H), 1.65-1.78 (m, 2H); MS (ESI) m/z (%): 407.3[M+H]+; Anal. calcd. for C19H24ClFN6O (%): C, 56.09; H, 5.95;
N, 20.65; Found (%): C, 56.24; H, 5.92; N, 20.71.
4.1.11.1. Methyl 4-((3-chloro-4-fluorophenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (12a)
To a solution of compound 7a (200 mg, 0.72 mmol) and DIPEA (138 mg, 1.08 mmol) in DCM (6 mL) was added
dropwise slowly methyl carbonochloridate (82 mg, 0.86 mmol) at 0 °C. After addition, the reaction solution was allowed
to r.t., and stirred for an hour. The reaction solution was washed by Na2CO3 (10 mL) aqueous solution and brine (10 mL),
then dried by Na2SO4, filtered and concentrated, purified by column to afford compound 12a. Gray solid; Yield: 83.2%;
M.p.: 183-185 °C; 1H NMR (CDCl3, 400 MHz): δ 8.58 (br. s., 1H), 7.80 (br. s., 1H), 7.41 (d, J = 8.3 Hz, 1H), 7.06-7.17
(m, 1H), 6.82 (br. s., 1H), 4.53 (br. s., 2H), 3.77(s, 3H), 3.72-3.89 (m, 2H), 2.91 (br. s., 2H); MS (ESI) m/z(%):
335.2[M-H]-; Anal. calcd. for C15H14ClFN4O2 (%): C, 53.50; H, 4.19; N, 16.64; Found (%): C, 53.71; H, 4.44; N, 16.61.
4.1.11.2. Ethyl 4-((3-chloro-4-fluorophenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidine-6(5H)-carboxylate (12b)
Preparation of compound 12b is followed the procedure for compound 12a. White solid; Yield: 93.2%; M.p.: 182-184
°C; 1H NMR (CDCl3, 400 MHz): δ 8.58 (s, 1H), 7.79 (br. s., 1H), 7.42 (d, J = 8.2 Hz, 1H), 7.15 (t, J = 8.7 Hz, 1H), 4.55
(br. s., 2H), 4.15-4.26 (m, 2H), 3.82 (t, J = 5.7 Hz, 2H), 2.96 (t, J = 5.7 Hz, 2H), 1.27-1.36 (m, 3H); MS (ESI) m/z(%):
348.7[M-H] -; Anal. calcd. for C16H16ClFN4O2(%): C, 54.78; H, 4.60; N, 15.97; Found (%): C, 54.87; H, 4.63; N, 16.04.
4.1.11.3. 2-(4-(2-hydroxyethyl) piperazin-1-yl) ethyl 4-((3-chloro-4-fluorophenyl) amino)-7,8-dihydropyrido[4,3-d]
pyrimidine-6(5H)-carboxylate (12c)
Preparation of compound 12c is followed the procedure for compound 12a.White solid; Yield: 59.1 %; M.p.: 174-176
1
°C; H NMR (CD3OD, 400 MHz): δ 8.41 (s, 1H), 7.86 (d, J = 4.4 Hz, 1H), 7.56 (br. s., 1H), 7.22 (t, J = 8.9 Hz, 1H),
4.48-4.67 (m, 2H), 4.32 (t, J = 5.6 Hz, 2H), 3.83 (br. s., 2H), 3.68 (br. s., 2H), 2.85 (t, J = 5.8 Hz, 2H), 2.73 (t, J = 5.6
11

Hz, 2H), 2.29-2.70 (m, 9H); MS (ESI) m/z (%): 476.6[M-H] -; Anal. calcd. for C22H28ClFN6O3(%): C, 55.17; H, 5.89; N,
17.55; Found (%): C, 55.22; H, 5.90; N, 17.53.
4.1.11.4. 2-(dimethylamino) ethyl 4-((3-chloro-4-fluorophenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidine-
6(5H)-carboxylate (12d)
Preparation of compound 12d is followed the procedure for compound 12a. White solid; Yield: 65.3%; M.p.:179-180
1
°C; H NMR (CD3OD, 400 MHz): δ 8.40 (s, 1H), 7.86 (d, J = 2.9 Hz, 1H), 7.54 (br. s., 1H), 7.22 (t, J = 8.9 Hz, 1H),
4.51-4.64 (m, 2H), 4.31 (t, J = 5.5 Hz, 2H), 3.81 (d, J = 2.9 Hz, 2H), 2.85 (t, J = 5.8 Hz, 2H), 2.73 (br. s., 2H), 2.37 (br.
s., 6H); MS (ESI) m/z (%):391.6[M-H]-; Anal. calcd. for C18H21ClFN5O2 (%): C, 54.89; H, 5.37; N, 17.78; Found (%): C,
54.96; H, 5.35; N, 17.79.
4.1.12.1. 1-(4-((3-chloro-4-fluorophenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl) ethanone (13a)
To a solution of compound 7a (200 mg, 0.72 mmol) and DIPEA (138 mg, 1.08 mmol) in DCM (6 mL) was added
dropwise slowly methyl acetyl chloride(67.5 mg, 0.86 mmol) at 0 °C. After addition, the reaction solution was allowed to
r.t., and stirred for an hour. The reaction solution was washed by Na2CO3 (10 mL) aqueous solution and brine (10 mL),
then dried by Na2SO4, filtered and concentrated, purified by column to afford compound 13a. White solid; Yield: 91.7%;
1
M.p.: 159-160 °C; H NMR (CDCl3, 400 MHz): δ 8.54 (s, 1H), 7.94 (br. s., 1H), 7.74 (dd, J = 6.5, 2.5 Hz, 1H),
7.39-7.52 (m, 1H), 7.13 (t, J = 8.7 Hz, 1H), 4.74 (s, 2H), 3.79 (t, J = 5.9 Hz, 2H), 2.98 (t, J = 5.6 Hz, 2H), 2.17 (s, 3H);
MS (ESI) m/z (%): 318.9[M-H]-; Anal. calcd. for C15H14ClFN4O (%): C, 56.17; H, 4.40; N, 17.47; Found (%): C, 56.31;
H, 4.38; N, 17.52.
4.1.12.2. 1-(4-((3-chloro-4-fluorophenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl) prop-2-en-1-one
(13b)
Preparation of compound 13b is followed the procedure for compound 13a. White solid; Yield: 81.2%; M.p.: 166-168
°C; 1H NMR (CDCl3, 400 MHz): δ 8.58 (s, 1H), 7.79 (dd, J = 6.4, 2.3 Hz, 1H), 7.44 (dt, J = 8.8, 3.4 Hz, 1H), 7.26 (br. s.,
1H), 7.12 (t, J = 8.7 Hz, 1H), 6.69 (dd, J = 16.9, 10.6 Hz, 1H), 6.28 (d, J = 16.9 Hz, 1H), 5.80 (dd, J = 10.6, 1.5 Hz, 1H),
4.77 (s, 2H), 3.91 (t, J = 5.7 Hz, 2H), 2.98 (t, J = 5.6 Hz, 2H); 13C NMR (DMSO-d6, 101 MHz): δ 165.2, 160.9, 156.6,
155.5, 151.9, 137.2, 128.7, 128.3, 123.8, 122.7, 119.2, 116.8, 111.6, 42.3, 38.3, 31.9; MS (ESI) m/z (%): 330.7[M-H] -;
IR (KBr) cm-1: 3482.5, 3324.6, 3218.8, 3121.5, 3010.2, 1645.5, 1615.1, 1497.6, 1423.5, 1349.3, 1264.1, 1213.6, 1133.8,
1056.5, 962.3, 923.4, 879.4, 787.8, 692.9, 563.6, 543.7, 515.1, 501.8; Anal. calcd. for C16H14ClFN4O (%): C, 57.75; H,
4.24; N, 16.84; Found (%): C, 57.81; H, 4.27; N, 16.77.
4.1.12.3. 1-(4-((3-chloro-4-fluorophenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl) prop-2-yn-1-one
(13c)
To a mixture of compound 7a (200 mg, 0.72 mmol) and propargylic acid (75 mg, 1.08 mmol) in CH3CN (6 mL) was
added EDCI.HCl (208 mg, 1.08 mmol) and HOBT.H2O (166 mg, 1.08 mmol), then added dropwise DIPEA (186 mg,
2.88 mmol) at r.t.. After addition, the reaction solution was heated at 60 °C for 6 hour. The reaction solution was added
water (10 mL), extracted with EtOAc (10 mL × 3), washed by brine (10 mL), then dried by Na2SO4, filtered and
1
concentrated, purified by column to afford compound 13c.White solid; Yield: 58.4%; M.p.: 171-174 °C; H NMR
(DMSO-d6, 400 MHz): δ 8.80 (br. s., 1H), 8.31-8.55 (m, 1H), 7.96 (dd, J = 6.8, 2.6 Hz, 1H), 7.56-7.73 (m, 1H),
12

7.13-7.43 (m, 1H), 4.53-4.86 (m, 2H), 4.60 (s, 1H), 3.71-4.14 (m, 2H), 2.65-2.91 (m, 2H); MS (ESI) m/z (%):
329.2[M-H] -; Anal. calcd. for C16H12ClFN4O (%): C, 58.10; H, 3.66; N, 16.94; Found (%): C, 58.27; H, 3.65; N, 16.89.
4.1.12.4. 1-(4-((3-chloro-4-fluorophenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl) propan-1-one (13d)
Preparation of compound 13d is followed the procedure for compound 13c. White solid; Yield: 93.7%; M.p.: 163-166
°C; 1H NMR (DMSO-d6, 400 MHz): δ 8.73 (br. s., 1H), 8.43 (s, 1H), 7.80-8.05 (m, 1H), 7.57-7.75 (m, 1H), 7.27-7.49 (m,
1H), 4.43-4.67 (m, 2H), 3.63-3.85 (m, 2H), 2.61-2.87 (m, 2H), 2.35-2.57 (m, 3H), 1.05 (t, J = 7.3 Hz, 3H); MS (ESI)
-
m/z (%): 333.3[M-H] ; Anal. calcd. for C16H16ClFN4O (%): C, 57.40; H, 4.82; N, 16.74; Found (%): C, 57.55; H, 4.79;
N, 16.81.
4.1.12.5. 1-(4-((3-chloro-4-fluorophenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)-2-(dimethylamino)
ethanone (13e)
Preparation of compound 13e is followed the procedure for compound 13c.White solid; Yield: 84.7%; M.p.: 165-168
1
°C; H NMR (CD3OD, 400 MHz): δ 8.34-8.40 (m, 1H), 7.78-7.90 (m, 1H), 7.44-7.60 (m, 1H), 7.08-7.22 (m, 1H),
4.56-4.66 (m, 2H), 3.87 (t, J = 5.5 Hz, 2H), 3.34-3.45 (m, 2H), 2.74-2.92 (m, 2H), 2.34 (s, 6H); MS (ESI) m/z (%):
362.4[M-H] -; Anal. calcd. for C17H19ClFN5O(%): C, 56.12; H, 5.26; N, 19.25; Found (%): C, 56.33; H, 5.30; N, 19.41.
4.1.12.6. 1-(4-((3-chloro-4-fluorophenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)-4-(dimethylamino)
butan-1-one (a1)
Preparation of compound a1 is followed the procedure for compound 13e. White solid; Yield: 89.2%; M.p.: 165-167
°C; 1H NMR (DMSO-d6, 400 MHz): δ 9.09-9.48 (m, 1H), 8.59 (s, 1H), 7.91 (dd, J = 6.8, 2.6 Hz, 1H), 7.65 (ddd, J = 9.0,
4.3, 2.7 Hz, 1H), 7.40 (t, J = 9.0 Hz, 1H), 4.62 (s, 2H), 3.77-3.81 (m, 2H), 3.07-3.16 (m, 2H), 2.81 (s, 6H), 2.61 (t, J =
-
7.0 Hz, 2H), 2.51 (dt, J = 3.8, 1.8 Hz, 2H), 1.81-2.01 (m, 2H); MS (ESI) m/z (%): 390.5 [M-H] ; Anal. calcd. for
C19H23ClFN5O(%): C, 58.23; H, 5.92; N, 17.87; Found (%): C, 58.41; H, 5.99; N, 17,64.
4.1.12.7. 1-(4-((3-(trifluoromethyl) phenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl) prop-2-en-1-one
(13f)
Preparation of compound 13f is followed the procedure for compound 13a. White solid; Yield: 82.0%; M.p.: 167-169
°C; 1H NMR (CDCl3, 400 MHz): δ 8.62 (s, 1H), 7.96 (br. s., 1H), 7.84 (d, J = 8.1 Hz, 1H), 7.48 (t, J = 7.9 Hz, 1H), 7.38
(d, J = 7.6 Hz, 1H), 7.21 (br. s., 1H), 6.70 (dd, J = 16.8, 10.6 Hz, 1H), 6.32 (d, J = 16.6 Hz, 1H), 5.81 (dd, J = 10.6, 1.3
Hz, 1H), 4.80 (s, 2H), 3.92 (t, J = 5.6 Hz, 2H), 3.00 (t, J = 5.6 Hz, 2H); MS (ESI) m/z (%): 347.1[M-H]-; Anal. calcd. for
C17H15F3N4O (%): C, 58.62; H, 4.34; N, 16.08; Found (%): C, 58.67; H, 4.29; N, 16.13.
4.1.12.8. 1-(4-((3-bromophenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl) prop-2-en-1-one (13g)
Preparation of compound 13g is followed the procedure for compound 13a. White solid; Yield: 92.4 %; M.p.: 154-155
1
°C; H NMR (DMSO-d6, 400 MHz): δ 8.61-8.93 (m, 1H), 8.51 (s, 1H), 7.96-8.18 (m, 1H), 7.79 (d, J = 8.1 Hz, 1H),
7.22-7.47 (m, 2H), 7.00 (dt, J = 16.4, 10.9 Hz, 1H), 6.09-6.39 (m, 1H), 5.63-5.94 (m, 1H), 4.63-4.86 (m, 2H), 3.80-4.01
(m, 2H), 2.70-2.91 (m, 2H); 13C NMR (DMSO-d6, 101 MHz): δ 165.1, 160.7, 156.1,155.5 141.8, 130.7, 128.7, 128.3,
125.8, 124.4, 121.5, 121.0, 110.1, 42.3, 37.9, 32.5; MS (ESI) m/z (%): 357.4[M-H]-; IR (KBr) cm-1: 3424.6, 3286.8,
3185.2, 3106.1, 2999.6, 2923.2, 2807.1, 1645.5, 1617.9, 1575.1, 1511.3, 1475.6, 1428.0, 1346.0, 1242.8, 1228.9, 1139.3,
1072.1, 921.6, 864.8, 782.0, 683.4, 561.9, 475.9; Anal. calcd. for C16H15BrN4O(%): C, 53.50; H, 4.21; N, 15.60; Found
(%): C, 53.57; H, 4.27; N, 15.53.
13

4.1.12.9. 1-(4-((2-methyl-5-nitrophenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl) prop-2-en-1-one
(13h)
Preparation of compound 13h is followed the procedure for compound 13a. Light brown solid; Yield: 81.7%; M.p.:
193-196 °C; 1H NMR (CDCl3, 400 MHz): δ 8.49 (s, 1H), 8.40 (br. s., 1H), 7.93-8.05 (m, 1H), 7.66 (br. s., 1H), 7.41 (d, J
= 8.3 Hz, 1H), 7.33 (br. s., 1H), 6.50-6.64 (m, 1H), 6.02 (d, J = 16.8 Hz, 1H), 5.60 (d, J = 10.6 Hz, 1H), 4.86 (br. s., 2H),
3.90 (br. s., 2H), 2.96 (t, J = 5.0 Hz, 2H), 2.34 (s, 3H); MS (ESI) m/z (%):338.4[M-H]-; Anal. calcd. for C17H17N5O3 (%):
C, 60.17; H, 5.05; N, 20.64; Found (%): C, 60.15; H, 5.14; N, 20.59.
4.1.12.10. 1-(4-((3, 4-dimethylphenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl) prop-2-en-1-one (13i)
Preparation of compound 13i is followed the procedure for compound 13a. Light yellow solid; Yield: 87.0%; M.p.:
1
145-147 °C; H NMR (CDCl3, 400 MHz): δ 8.53 (s, 1H), 7.22-7.27 (m, 2H), 7.11 (d, J = 7.9 Hz, 1H), 6.83 (br. s., 1H),
6.67 (dd, J = 16.8, 10.6 Hz, 1H), 6.32 (d, J = 16.6 Hz, 1H), 5.78 (dd, J = 10.6, 1.1 Hz, 1H), 4.69 (s, 2H), 3.87 (t, J = 5.6
Hz, 2H), 2.92 (t, J = 5.6 Hz, 2H), 2.26 (s, 3H), 2.24 (s, 3H); MS (ESI) m/z(%): 307.4[M-H]-; Anal. calcd. for C18H20N4O
(%): C, 70.11; H, 6.54; N, 18.17; Found (%): C, 70.23; H, 6.49; N, 18.21.
4.1.12.11. 1-(4-((3-bromo-5-methoxyphenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl) prop-2-en-1-one
(13j)
Preparation of compound 13j is followed the procedure for compound 13a. White solid; Yield: 77.3%; M.p.: 169-161
°C; 1H NMR (CDCl3, 400 MHz): δ 8.82 (br. s., 1H), 8.67 (s, 1H), 7.16 (dd, J = 8.6, 2.1 Hz, 1H), 7.05 (br. s., 1H), 6.79 (d,
J = 8.7 Hz, 1H), 6.70 (dd, J = 16.8, 10.6 Hz, 1H), 6.41 (dd, J = 16.8, 1.3 Hz, 1H), 5.83 (dd, J = 10.6, 1.3 Hz, 1H), 4.69
(br. s., 2H), 3.94 (s, 3H), 3.90 (br. s., 2H), 2.97 (t, J = 5.3 Hz, 2H); MS (ESI) m/z (%): 387.2[M-H]-; Anal. calcd. for
C17H17BrN4O2 (%): C, 52.46; H, 4.40; N, 14.39; Found (%): C, 52.41; H, 4.39; N, 14.48.
4.1.12.12. 1-(4-((4-bromophenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl) prop-2-en-1-one (13k)
Preparation of compound 13k is followed the procedure for compound 13a. White solid; Yield: 90.1 %; M.p.:155-158
1
°C; H NMR (CDCl3, 400 MHz): δ 8.54 (br. s., 1H), 8.07 (br. s., 1H), 7.36-7.61 (m, 4H), 6.65 (dd, J = 16.1, 10.8 Hz,
1H), 6.20 (d, J = 16.6 Hz, 1H), 5.77 (d, J = 10.5 Hz, 1H), 4.82 (br. s., 2H), 3.88 (br. s., 2H), 3.02 (br. s., 2H); MS (ESI)
m/z(%):357.2[M-H]-; Anal. calcd. for C16H15BrN4O(%): C, 53.50; H, 4.21; N, 15.60; Found (%): C, 53.47; H, 4.23; N,
15.67.
4.1.12.13. 1-(4-((2, 4-dichlorophenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl) prop-2-en-1-one (13l)
Preparation of compound 13l is followed the procedure for compound 13a. White solid; Yield: 87.5 %; M.p.:181-183
°C; 1H NMR (CDCl3, 400 MHz): δ 8.63 (s, 1H), 8.42 (d, J = 8.4 Hz, 1H), 7.46 (d, J = 2.3 Hz, 1H), 7.32 (dd, J = 8.9, 2.1
Hz, 1H), 6.84 (br. s., 1H), 6.70 (dd, J = 16.8, 10.5 Hz, 1H), 6.40 (d, J = 16.8 Hz, 1H), 5.83 (d, J = 10.5 Hz, 1H), 4.75 (br.
s., 2H), 3.93 (br. s., 2H), 3.00 (t, J = 5.1 Hz, 2H); MS (ESI) m/z (%): 347.1[M-H]-; Anal. calcd. for C16H14Cl2N4O (%): C,
55.03; H, 4.04; N, 16.04; Found (%): C, 55.19; H, 4.07; N, 16.11.
4.1.12.14. 1-(4-((2-chloro-5-(trifluoromethyl) phenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)
prop-2-en-1-one (13m)
Preparation of compound 13m is followed the procedure for compound 13a. White solid; Yield: 91.3%; M.p.: 185-187
14

°C; 1H NMR (CDCl3, 400 MHz): δ 8.95 (br. s., 1H), 8.70 (s, 1H), 7.56 (d, J = 8.3 Hz, 1H), 7.32 (d, J = 8.2 Hz, 1H), 7.03
(br. s., 1H), 6.70 (dd, J = 16.8, 10.6 Hz, 1H), 6.40 (dd, J = 16.8, 1.2 Hz, 1H), 5.83 (d, J = 10.5 Hz, 1H), 4.78 (br. s., 2H),
3.93 (br. s., 2H), 3.01 (t, J = 5.4 Hz, 2H); MS (ESI) m/z (%): 381.4[M-H]-; Anal. calcd. for C17H14ClF3N4O (%): C,
53.34; H, 3.69; N, 14.64; Found (%): C, 53.41; H, 3.71; N, 14.58.
4.1.12.14. 1-(4-((3, 5-dichlorophenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl) prop-2-en-1-one (13n)
Preparation of compound 13n is followed the procedure for compound 13a. White solid; Yield: 79.3 %; M.p.:
1
177-179 °C; H NMR (CD3OD, 400 MHz): δ 8.50 (br. s., 1H), 7.78 (s, 2H), 7.14 (s, 1H), 6.92 (dd, J = 16.7, 10.7 Hz,
1H), 6.31 (d, J = 16.8 Hz, 1H), 5.85 (d, J = 10.8 Hz, 1H), 4.73 (br. s., 2H), 3.98 (t, J = 5.9 Hz, 2H), 2.83-2.99 (m, 2H);
MS (ESI) m/z(%): 347.2[M-H]-; Anal. calcd. for C16H14Cl2N4O (%): C, 55.03; H, 4.04; N, 16.04; Found (%): C, 55.23; H,
4.07; N, 16.11.
4.1.12.15. 1-(4-((3-chloro-4-(pyridin-2-ylmethoxy) phenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)
prop-2-en-1-one (13o)
Preparation of compound 13o is followed the procedure for compound 13a. Light gray solid; Yield: 90.5%; M.p.:
178-180 °C; 1H NMR (DMSO-d6, 400 MHz): δ 8.62 (d, J = 4.4 Hz, 1H), 8.47-8.77 (m, 1H), 8.43 (br. s., 1H), 7.76-7.99
(m, 2H), 7.61 (d, J = 7.6 Hz, 2H), 7.33-7.43 (m, 1H), 7.16-7.32 (m, 1H), 6.82-7.08 (m, 1H), 6.11-6.35 (m, 1H),
5.63-5.88 (m, 1H), 5.30 (s, 2H), 4.55-4.81 (m, 2H), 3.74-4.02 (m, 2H), 2.64-2.94 (m, 2H); 13C NMR (DMSO-d6,
101MHz): δ 165.4, 160.6, 160.5, 156.9, 155.6, 149.6, 137.5, 134.0, 128.9, 128.3, 124.5, 123.5, 121.8, 121.2, 114.5, 111.2,
110.6, 71.6, 42.3, 38.5, 32.5, 31.2; MS (ESI) m/z (%): 420.3[M-H]-; IR (KBr) cm-1: 3491.9, 3159.5, 3094.0, 2923.4,
2852.9, 1643.3, 1592.0, 1501.5, 1453.7, 1423.1, 1348.2, 1302.3, 1270.5, 1220.1, 1133.9, 1097.0, 1065.8, 1037.5, 977.9,
923.5, 864.0, 805.1, 747.7, 664.3, 623.7, 575.0, 500.0; Anal. calcd. for C22H20ClN5O2(%): C, 62.63; H, 4.78; N, 16.60;
Found (%): C, 62.71; H, 4.83; N, 16.70.
4.1.12.16. 1-(4-((3-chloro-4-((3-fluorobenzyl) oxy) phenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)
prop-2-en-1-one (13p)
Preparation of compound 13p is followed the procedure for compound 13a. White solid; Yield: 77.1%; M.p.: 195-197
°C; 1H NMR (CDCl3, 400 MHz): δ 8.53 (s, 1H), 7.65 (d, J = 2.2 Hz, 1H), 7.42 (s, 1H), 7.32-7.40 (m, 2H), 7.18-7.25 (m,
2H), 6.98-7.06 (m, 1H), 6.91 (d, J = 8.9 Hz, 1H), 6.65 (dd, J = 16.8, 10.6 Hz, 1H), 6.23 (d, J = 16.8 Hz, 1H), 5.75 (dd, J
= 10.7, 1.2 Hz, 1H), 5.13 (s, 2H), 4.77 (s, 2H), 3.88 (t, J = 5.6 Hz, 2H), 2.94 (t, J = 5.4 Hz, 2H); 13C NMR (CD3OD, 101
MHz): δ 166.0, 163.9, 161.4, 159.1, 156.7, 155.7, 150.5, 138.8, 132.2, 129.8, 128.7, 126.7, 124.8, 123.0, 122.0, 114.5,
113.9, 113.6, 109.5, 70.1, 42.4, 39.2, 30.6; MS (ESI) m/z (%): 437.3[M-H]-; IR (KBr) cm-1: 3422.7, 3321.7, 2923.5,
2954.3, 1643.6, 1605.0, 1498.4, 1447.9, 1423.2, 1384.3, 1269.8, 1251.3, 1218.1, 1136.0, 1060.0, 926.0, 859.5, 804.8,
780.3, 682.0, 573.4, 521.8; Anal. calcd. for C23H20ClFN4O2(%): C, 62.94; H, 4.59; N, 12.77; Found (%): C, 62.98; H,
4.64; N, 12.69.
4.1.12.17. 1-(4-((3-fluoro-4-(pyridin-2-ylmethoxy) phenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)
prop-2-en-1-one (13q)
Preparation of compound 13q is followed the procedure for compound 13a. White solid; Yield: 83.3%; M.p.: 187-189
°C; 1H NMR (CDCl3, 400 MHz): δ 8.60 (d, J = 4.3 Hz, 1H), 8.55 (s, 1H), 7.75 (t, J = 7.2 Hz, 1H), 7.50-7.63 (m, 2H),
7.25 (d, J = 6.8 Hz, 1H), 7.16 (d, J = 8.7 Hz, 1H), 6.99 (t, J = 8.9 Hz, 1H), 6.66 (dd, J = 16.5, 10.8 Hz, 1H), 6.27 (d, J =
16.8 Hz, 1H), 5.77 (d, J = 11.1 Hz, 1H), 5.27 (s, 2H), 4.73 (br. s., 2H), 3.89 (br. s., 2H), 2.99 (br. s., 2H); 13C NMR
(CD3OD, 101 MHz): δ 165.9, 159.1, 156.5, 155.6, 153.2, 150.7, 148.8, 142.8, 136.6, 132.0, 128.7, 126.6, 122.5, 121.0,
117.7, 115.1, 111.3, 109.5, 71.8, 42.3, 39.1, 31.7; MS (ESI) m/z (%): 406.1[M+H]+; IR (KBr) cm-1: 3425.3, 3312.9,
15

3113.4, 2922.9, 1645.9, 1645.9, 1615.5, 1574.9, 1522.5, 1449.1, 1433.7, 1300.7, 1253.3, 1216.4, 1129.1, 1094.9, 1052.7,
982.9, 906.7, 867.5, 807.7, 761.0, 660.6, 559.8, 470.3; Anal. calcd. for C22H20FN5O2 (%): C, 65.17; H, 4.97; N, 17.27;
Found (%): C, 65.21; H, 4.54; N, 17.23.
4.1.12.18. 1-(4-((3-chloro-4-(3-(trifluoromethyl) phenoxy) phenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidin-
6(5H)-yl) prop-2-en-1-one (13r)
Preparation of compound 13r is followed the procedure for compound 13a. White solid; Yield: 76.8 %; M.p.: 247-248
°C; 1H NMR (CDCl3, 400 MHz): δ 8.57 (s, 1H), 8.25 (s, 1H), 7.87 (d, J = 2.3 Hz, 1H), 7.59 (dd, J = 8.8, 2.4 Hz, 1H),
7.37-7.43 (m, 1H), 7.31 (d, J = 7.7 Hz, 1H), 7.16 (s, 1H), 7.09 (d, J = 8.1 Hz, 1H), 7.01 (d, J = 8.8 Hz, 1H), 6.67 (dd, J
= 16.9, 10.8 Hz, 1H), 6.19 (d, J = 16.9 Hz, 1H), 5.75 (d, J = 11.5 Hz, 1H), 4.88 (s, 2H), 3.89 (t, J = 5.6 Hz, 2H),
2.92-3.00 (m, 2H); 13C NMR (CDCl3, 101 MHz): δ 166.6, 159.8, 158.0, 157.1, 156.0, 147.1, 136.8, 132.3, 130.5, 129.3,
127.4, 126.6, 124.6, 122.3, 122.1, 120.3, 119.7, 114.0, 110.8, 43.0, 40.1, 32.3; MS (ESI) m/z (%): 473.0[M-H]-; Anal.
calcd. for C23H18ClF3N4O2(%): C, 58.17; H, 3.82; N, 11.80; Found (%): C, 58.21; H, 3.78; N, 11.74.
4.1.12.19. 1-(4-((3-chloro-4-((3-fluorobenzyl) thio) phenyl) amino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)
prop-2-en-1-one (13s)
Preparation of compound 13s is followed the procedure for compound 13a. Light yellow solid; Yield: 75.7%; M.p.:
1
223-226 °C; H NMR (CD3OD, 400 MHz): δ 8.44 (br. s., 1H), 7.84 (br. s., 1H), 7.47 (d, J = 8.6 Hz, 1H), 7.19-7.32 (m,
3H), 7.10 (d, J = 7.7 Hz, 1H), 7.04 (d, J = 9.9 Hz, 1H), 6.81-6.97 (m, 2H), 6.29 (d, J = 16.8 Hz, 1H), 5.83 (d, J = 10.6
Hz, 1H), 4.66 (br. s., 2H), 4.09-4.17 (m, 2H), 3.94 (t, J = 5.9 Hz, 2H), 2.79-2.96 (m, 2H); 13C NMR (CD3OD, 101 MHz):
δ 167.8, 165.1, 162.5, 158.9, 155.2, 141.3, 139.6, 132.6, 131.1, 130.6, 129.9, 129.4, 128.8, 125.8, 124.3, 122.0, 116.5,
114.9, 112.3, 43.2, 39.9, 38.2, 31.9; MS (ESI) m/z (%): 455.2[M+H]+; Anal. calcd. for C20H18ClFN4S (%): C, 59.92; H,
4.53; N, 13.98; Found (%): C, 60.03; H, 4.50; N, 13.92.
4.1.12.20.
N-(4-((6-acryloyl-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-yl)
amino)-2-chlorophenyl)-3-fluoro
benzamide (13t)
Preparation of compound 13t is followed the procedure for compound 13a. White solid; Yield: 93.1 %; M.p.: 242-243
°C; 1H NMR (DMSO-d6, 400 MHz): δ 10.18 (s, 1H), 8.61-9.02 (m, 1H), 8.51 (br. s., 1H), 7.97-8.09 (m, 1H), 7.89 (d, J =
7.8 Hz, 1H), 7.82 (d, J = 9.5 Hz, 1H), 7.76 (d, J = 8.6 Hz, 1H), 7.63 (td, J = 7.9, 6.1 Hz, 1H), 7.43-7.57 (m, 2H),
6.85-7.12 (m, 1H), 6.11-6.36 (m, 1H), 5.57-5.94 (m, 1H), 4.55-4.81 (m, 2H), 3.57-4.00 (m, 2H), 2.71-2.92 (m, 2H); 13
C
NMR (DMSO-d6, 101 MHz): δ 165.4, 164.6, 163.6, 161.2, 160.9, 156.6, 155.5, 139.4, 136.7, 131.1, 129.9, 129.0, 128.6,
124.3, 122.6, 121.1, 119.1, 115.0, 114.8, 111.8, 42.2, 38.1, 32.5; MS (ESI) m/z (%): 450.2[M-H]-; Anal. calcd. for
C23H19ClFN5O2(%): C, 61.13; H, 4.24; N, 15.50; Found (%): C, 61.17; H, 4.23; N, 15.60.
4.1.12.21. 1-(4-((6-acryloyl-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-yl) amino)-2-chlorophenyl)-3-(3-fluoro
phenyl) urea (13u)
Preparation of compound 13u is followed the procedure for compound 13a. White solid; Yield: 84.5 %; M.p.: 274-276
1
°C; H NMR (CD3OD, 400 MHz): δ 8.45 (br. s., 1H), 8.09 (d, J = 8.9 Hz, 1H), 7.81-7.88 (m, 1H), 7.43-7.53 (m, 2H),
7.28 (td, J = 8.1, 6.7 Hz, 1H), 7.10 (dd, J = 8.2, 1.2 Hz, 1H), 6.87-7.01 (m, 1H), 6.75 (td, J = 8.3, 2.1 Hz, 1H), 6.32 (d, J
= 16.5 Hz, 1H), 5.85 (d, J = 10.6 Hz, 1H), 4.71 (br. s., 2H), 3.98 (t, J = 5.8 Hz, 2H), 2.82-2.97 (m, 2H); MS (ESI) m/z
16

(%):465.2[M-H]-; Anal. calcd. for C23H20ClFN6O2(%): C, 59.17; H, 4.32; N, 18.00; Found (%): C, 59.28; H, 4.29; N,
18.15.
4.1.13. Ethyl 3-oxopiperidine-4-carboxylate hydrochloride (15)
Preparation of compound 15 is followed the procedure for compound 2. Light brown solid; Yield: 91.4%; MS (ESI)
m/z (%): 172.2[M+H]+.
4.1.14. 1-benzyl 4-ethyl 3-oxopiperidine-1, 4-dicarboxylate (16)
1
Preparation of compound 16 is followed the procedure for compound 3. White solid; Yield: 87.3 %; H NMR
(METHANOL-d4, 400 MHz): δ 7.20-7.48 (m, 6H), 5.13 (s, 2H), 4.21 (q, J = 7.1 Hz, 2H), 4.05 (br. s., 2H), 3.52 (br. s.,
2H), 2.28 (br. s., 2H), 1.28 (t, J = 7.1 Hz, 3H); MS (ESI) m/z (%): 304.0[M-H]-.
4.1.15. Benzyl 4-hydroxy-5,6-dihydropyrido[3,4-d]pyrimidine-7(8H)-carboxylate (17)
1
Preparation of compound 17 is followed the procedure for compound 4. White solid; Yield: 91.6%; H NMR
(DMSO-d6, 400 MHz): δ 12.39 (br. s., 1H), 8.06 (s, 1H), 7.28-7.45 (m, 5H), 5.12 (s, 2H), 4.31 (br. s., 2H), 3.60 (br. s.,
2H), 2.43 (t, J = 5.7 Hz, 2H); MS (ESI) m/z (%): 286.1[M+H]+.
4.1.16. Benzyl 4-chloro-5,6-dihydropyrido[3,4-d]pyrimidine-7(8H)-carboxylate (18)
Preparation of compound 18 is followed the procedure for compound 5. Brown oil; Yield: 96%; MS (ESI) m/z (%):
303.7[M+H]+.
4.1.17.1. Benzyl 4-(3-chloro-4-fluorophenylamino)-5,6-dihydropyrido[3,4-d]pyrimidine-7(8H)-carboxylate (19a)
Preparation of compound 19a is followed the procedure for compound 6a. White solid; Yield: 73.6 %; M.p.: 184-185
°C; MS (ESI) m/z (%): 413.1[M+H]+.
4.1.17.2. Benzyl 4-((3-chloro-4-((3-fluorobenzyl) oxy) phenyl) amino)-5,6-dihydropyrido[3,4-d]pyrimidine-
7(8H)-carboxylate (19b)
Preparation of compound 19b is followed the procedure for compound 6a. White solid; Yield: 77.1 %; M.p.: 196-197
°C; MS (ESI) m/z (%): 519.2[M+H]+.
4.1.18.1. N-(3-chloro-4-fluorophenyl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-amine (20a)
Preparation of compound 20a is followed the procedure for compound 7a. Gray solid; Yield: 78.3 %; M.p.: 177-179
1
°C; H NMR (CD3OD, 400 MHz): δ 8.36 (s, 1H), 7.88 (dd, J = 6.7, 2.6 Hz, 1H), 7.55 (ddd, J = 8.9, 4.0, 2.7 Hz, 1H),
7.20 (t, J = 8.9 Hz, 1H), 3.84 (s, 2H), 3.17 (t, J = 5.9 Hz, 2H), 2.62 (t, J = 5.7 Hz, 2H); MS (ESI) m/z (%): 278.6[M+H]+;
Anal. calcd. for C13H12ClFN4(%): C, 56.02; H, 4.34; N, 20.10; Found (%): C, 56.18; H, 4.33; N, 20.16.
4.1.18.2. N-(3-chloro-4-((3-fluorobenzyl) oxy) phenyl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-amine (20b)
Preparation of compound 20b is followed the procedure for compound 7a. White solid; Yield: 69.6%; M.p.: 180-181
°C; MS (ESI) m/z (%): 385.1[M+H]+.
4.1.19. N-(3-chloro-4-fluorophenyl)-7-(methylsulfonyl)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4-amine (21)
17

Preparation of compound 21 is followed the procedure for compound 8a. White solid; Yield: 79.6 %; M.p.: 223-225
°C; 1H NMR (CD3OD, 400 MHz): δ 8.42 (s, 1H), 7.89 (dd, J = 6.7, 2.6 Hz, 1H), 7.50-7.62 (m, 1H), 7.22 (t, J = 9.0 Hz,
1H), 4.33 (s, 2H), 3.64 (t, J = 5.9 Hz, 2H), 2.98 (s, 3H), 2.80 (t, J = 5.7 Hz, 2H); MS (ESI) m/z (%): 356.5[M+H]+; Anal.
calcd. for C14H14ClFN4O2S (%): C, 47.13; H, 3.95; N, 15.70; Found (%): C, 47.38; H, 3.84; N, 15.90.
4.1.20.
4-((3-chloro-4-fluorophenyl)
amino)-N,
N-dimethyl-5,6-dihydropyrido[3,4-d]pyrimidine-7(8H)-
carboxamide (22)
Preparation of compound 22 is followed the procedure for compound 11a. White solid; Yield: 67.9 %; M.p.:188-190
1
°C; H NMR (CDCl3, 400 MHz): δ 8.52 (s, 1H), 7.76 (dd, J = 6.5, 2.6 Hz, 1H), 7.55-7.71 (m, 1H), 7.45-7.53 (m, 1H),
7.28 (s, 1H), 7.13 (t, J = 8.7 Hz, 1H), 4.45 (s, 2H), 3.61 (t, J = 5.6 Hz, 2H), 2.89 (s, 6H), 2.82 (t, J = 5.4 Hz, 2H); MS
(ESI) m/z (%): 348.3[M-H]-; Anal. calcd. for C16H17ClFN5O (%): C, 54.94; H, 4.90; N, 20.02; Found (%): C, 55.18; H,
4.85; N, 20.13.
4.1.21. Methyl 4-((3-chloro-4-fluorophenyl) amino)-5,6-dihydropyrido[3,4-d]pyrimidine-7(8H)-carboxylate (23)
Preparation of compound 23 is followed the procedure for compound 13a. White solid; Yield: 86.5%; M.p.:185-187
°C; 1H NMR (CDCl3, 400 MHz): δ 8.53 (s, 1H), 7.80 (d, J = 4.8 Hz, 1H), 7.40-7.57 (m, 1H), 7.14 (t, J = 8.7 Hz, 1H),
4.66 (s, 2H), 3.84 (br. s., 2H), 3.76 (s, 3H), 2.77 (br. s., 2H); MS (ESI) m/z (%): 335.4[M-H]-; Anal. calcd. for
C15H14ClFN4O2(%): C, 53.50; H, 4.19; N, 16.64; Found (%): C, 53.64; H, 4.25; N, 16.65.
4.1.22.1. 1-(4-((3-chloro-4-fluorophenyl) amino)-5,6-dihydropyrido[3,4-d]pyrimidin-7(8H)-yl)-2-(dimethylamino)
ethanone (24a)
Preparation of compound 24a is followed the procedure for compound 13c. White solid; Yield: 79.8%; M.p.: 176-178
°C; 1H NMR (DMSO-d6, 400 MHz): δ 8.76 (br. s., 1H), 8.46 (s, 1H), 7.99-8.08 (m, 1H), 7.63-7.75 (m, 1H), 7.38 (t, J =
9.1 Hz, 1H), 4.69 (s, 1H), 4.48 (s, 1H), 3.85 (t, J = 5.6 Hz, 1H), 3.74-3.79 (m, 1H), 3.11-3.21 (m, 2H), 2.58-2.80 (m, 2H),
2.12-2.24 (m, 6H); MS (ESI) m/z (%): 364.2[M+H]+; Anal. calcd. for C17H19ClFN5O (%): C, 56.12; H, 5.26; N, 19.25;
Found (%): C, 56.29; H, 5.37; N, 19.41.
4.1.23.1. 1-(4-((3-chloro-4-fluorophenyl) amino)-5,6-dihydropyrido[3,4-d]pyrimidin-7(8H)-yl) prop-2-en-1-one
(24b)
Preparation of compound 24b is followed the procedure for compound 13a. White solid; Yield: 83.2%; M.p.: 177-179
°C; 1H NMR (DMSO-d6, 400 MHz): δ 8.69 (d, J = 8.2 Hz, 1H), 8.46 (s, 1H), 7.99 (d, J = 4.2 Hz, 1H), 7.60-7.72 (m, 1H),
7.38 (t, J = 9.1 Hz, 1H), 6.79-7.05 (m, 1H), 6.09-6.23 (m, 1H), 5.64-5.83 (m, 1H), 4.52-4.70 (m, 2H), 3.77-3.99 (m, 2H),
2.69 (d, J = 18.2 Hz, 2H); MS (ESI) m/z (%): 332.5[M+H]+; Anal. calcd. for C16H14ClFN4O (%): C, 57.75; H, 4.24; N,
16.84; Found (%): C, 57.81; H, 4.23; N, 16.89.
4.1.23.2. 1-(4-((3-chloro-4-((3-fluorobenzyl) oxy) phenyl) amino)-5,6-dihydropyrido[3,4-d]pyrimidin-7(8H)-yl)
prop-2-en-1-one (24c)
Preparation of compound 24c is followed the procedure for compound 13a. White solid; Yield: 76.0%; M.p.: 194-196
°C; 1H NMR (CD3OD, 400 MHz): δ 8.34 (s, 1H), 7.71 (br. s., 1H), 7.35-7.46 (m, 2H), 7.28 (d, J = 7.7 Hz, 1H), 7.23 (d,
J = 9.9 Hz, 1H), 7.09 (d, J = 8.9 Hz, 1H), 7.04 (td, J = 8.6, 2.4 Hz, 1H), 6.74-6.94 (m, 1H), 6.27 (d, J = 16.5 Hz, 1H),
5.81 (d, J = 10.8 Hz, 1H), 5.18 (s, 2H), 4.68 (s, 2H), 3.97 (t, J = 5.9 Hz, 2H), 2.70 (m, 2H); MS (ESI) m/z(%):
439.1[M+H]+; Anal. calcd. for C23H20ClFN4O2 (%): C, 62.94; H, 4.59; N, 12.77; Found (%): C, 62.87; H, 4.57; N, 12.90.
18

4.2 Cytotoxicity assay
The anti-proliferative activities of compounds 7a-24d were evaluated against HT29, A549, H460 and H1975 cell lines
by the standard MTT assay in vitro, with Gefitinib as the positive control.38 The cancer cell lines were cultured in
minimum essential medium (MEM) supplement with 10% fetal bovine serum (FBS). Approximate 4 × 103 cells,
suspended in MEM medium, were plated into each well of a 96-well plate and incubated in 5% CO2 at 37 ºC for 24 h.
The tested compounds at the indicated final concentrations were added to the culture medium and incubated for 72 h.
Fresh MTT was added to each well at the terminal concentration of 5 µg/mL, and incubated with cells at 37 ºC for 4 h.
The formazan crystals in each well were dissolved in 100 µL DMSO, and the absorbency at 492 nm (for absorbance of
MTT formazan) and 630 nm (for the reference wavelength) was measured with an ELISA reader. All of the compounds
were tested three times in each of the cell lines. The results, expressed as IC50 (inhibitory concentration 50%), were the
averages of three determinations and calculated by the Bacus Laboratories Incorporated Slide Scanner (Bliss) software.
4.3 In vitro enzymatic activity assay
The EGFR and HER2 kinase assays of tested compounds were performed by homogeneous time-resolved
fluorescence (HTRF) assay as previously reported protocol.39 Briefly, 20 µg/mL poly (Glu, Tyr) 4:1 (Sigma) was
preloaded as a substrate in 384-well plates. Then 50 µL of 10 mM ATP (Invitrogen) solution diluted in kinase reaction
buffer (50 mM HEPES, Ph 7.0, 1 mM DTT, 1 mM MgCl2, 1 mM MnCl2, 0.1% NaN3) was added to each well. Various
concentrations of compounds were diluted in 10 µL of 1% DMSO (v/v), with blank DMSO solution as the negative
control. The kinase reaction was initiated by the addition of purified tyrosine kinase proteins diluted in 39 µL of kinase
reaction buffer solution. Reactions were incubated for 30 min at 25 ºC and stopped by the addition of 5 µL
Streptavidin-XL665 and 5 µL Tk Antibody Cryptate working solution to all of wells. The plate was read by Envision
(Perkinelmer) at 320 nm and 615 nm. IC50 values were calculated from the inhibition curves.
4.4 Molecular docking
The crystal structures of the proteins complex were retrieved from the RCSB Protein Data Bank
(http://www.rcsb.org/pdb/home/home.do). The three-dimensional structures of Afatinib with EGFR kinase was selected
as the docking model (PDB ID: 4G5P). The docking simulation was conducted with Glide XP (Schrödinger 2014), since
Glide uses ahierarchical series of filters to search for possible locations of the ligand in the active-site region of the
receptor. The shape and properties of the receptor were represented on a grid by several different sets of fields that
provide progressively more accurate scoring of the ligand poses. The image files were generated by AccelrysDS
visualizer 4.0 system. The binding model was exemplified by the interaction of compound 13b with EGFR.
Acknowledgments
The work was supported by Program for Innovative Research Team of the Ministry of Education of the People’s
Republic of China and Program for Liaoning Innovative Research Team in University (IRT1073).
References and notes
1. Bruce, E. J.; Daniel, C. I.; Mary, J. M.; Paul, A. B.; Alan, Z.; Robert, W. M.; Anita, J.; Martin, H. C.; Eli, G.; John, D. M. J. Med. Chem.
1986, 80, 1103-10.
2. Douglas, A. A.; Michael, P. K.; Bruno, D.;Steven, L. K.; Scott, R. B. S.; Marie, D. B.; Mark, D. I.; Robert, M. S. Cancer Immunol.
19

Immun. 2000, 49, 63-70.
3. Sharma, S. V.; Bell, D. W.; Settleman, J.; Haber, D. A. Nat. Rev. Cancer. 2007, 7, 169-181.
4. Janku, F.; Stewart, D. J.; Kurzrock, R. Nat. Rev. Clin. Oncol. 2011, 8, 384-385.
5. Burgess, A. W.; Cho, H. S.; Eigenbrot, C.; Ferguson, K. M.; Garrett, T. P.; Leahy, D. J.; Lemmon, M. A.; Sliwkowski, M. X.; Ward, C. W.;
Yokoyama, S. Mol. Cell. 2003, 12, 541-552.
6. Barker, A. J.; Gibson, K. H.; Grundy, W.; Godfrey, A. A.; Barlow, J. J.; Healy, M. P.; Woodburn, J. R.; Ashton, S. E.; Curry, B. J.; Scarlett,
L.; Henthorn, L.; Richards, L. Med. Chem. Lett. 2001, 11, 1911-1914.
7. Sorbera, L. A.; Castañer, J.; Silvestre, J. S.; Bayes, M. Drugs Future 2002, 27, 923.
8. Tan, F.; Shen, X.; Wang, D.; Xie, G.; Zhang, X.; Ding, L.; Hu, Y.; He, W.; Wang, Y.; Wang, Y. Lung Cancer. 2012, 76, 177.
9. Wood, E. R.; Truesdale, A. T.; McDonald, O. B.; Yuan, D.; Hassell, A.; Dickerson, S. H.; Ellis, B.; Pennisi, C.; Horne, E.; Lackey, K.;
Alligood, K. J.; Rusnak, D. W.; Gilmer, T. M.; Shewchuk, L. Cancer Res. 2004, 64, 6652-6659.
10. Eskens, F. A. L. M.; Mom, C. H.; Planting, A. S. T.; Gietema, J. A.; Amelsberg, A.; Huisman, H.; Doorn, L.; Burger, H.; Stopfer, P.;
Verweij, J.; Vries, E. G. Br. J. Cancer. 2008, 98, 80-85.
11. Hennequin, L. F.; Stokes, E.S.; Thomas, A. P.; Johnstone, C.; Ple, P. A.; Ogilvie, D. J.; Dukes, M.; Wedge, S. R.; Kendrew, J.; Curwen, J.
O. J. Med. Chem. 2002,45, 1300.
12. Barry, T.; Karen, M. M.; Naeem, Y.; Lawrence, P.; Michael, D.; Carol, J.; Walter, M.; Lucian, D.; Simon, P.; Michael, M.; Francois, L.;
Madelene, H.; Rajesh, K.; Clayton, S.; John, S.; Kymberly, L.; Catherine, F.; Xie, X.; Raviraj, K.; Kara, H.; Dale, P.; Michael, V. ACS
Med Chem Lett. 2013, 4, 186−190.
13. Michael, F. T.; Koehler, P. B.; Elizabeth, B.; Krista, K. B.; Chen, Y. H.; Gauri, D.; Ding, X.; Jennifer, E.; Kevin, L.; Leslie, L.; Liu, L. C.;
Ly, C.; Shiva, M.; Jim, N.; Jason, O.; Daniel, F.; Ortwine, D. S.; Steve, S.; Lan, T.; Tom, T.; Wu, J. S.; Pei, Z. H.; Joseph, P. L.; Orally, B.
J. Med. Chem. 2012, 55, 10958-10971.
14. Anthony, A. E.; Daniel, G. S.; Elizabeth, B.; Yung-Hsiang, C.; Gauri, D.; Xiao, D.; Antonio, G; Jennifer, A. E.; Lori, S. F.; Michael, F. T.;
Lichuan, L.; Shiva, M.; Daniel, F. O. Zhonghua, P.; Steve, S.; Frederic, S.; Lan, T.; Tom, T.; Joseph, P. L. J. Med. Chem. 2013, 56,
−3101
3090
.
15. Cooke, N. G.; Fernandes, G. S.; Paulo, A.; Furet, P.; Hebach, C.; Hoegenauer, K.; Hollingworth, G.; Kalis, C.; Lewis, I.; Smith, A. B.;
Soldermann, N. From PCT Int. Appl. 2013, WO 2013088404.
16. Guay, D.; Beaulieu, C.; Belley, M.; Crane, S. N.; DeLuca, J.; Gareau, Y.; Hamel, M.; Henault, M.; Hyjazie, H.; Kargman, S. Bioorg. Med.
Chem. Lett. 2011, 21, 2832-2835.
17. Artman, G. D.; Elliott, J. M.; Ji, N.; Liu, D. l.; Ma, F.; Mainolfi, N.; Meredith, E.; Miranda, K.; Powers, J. J.; Rao, C. From PCT. Int. Appl.
2010, WO 2010066684.
18. James, F. B.; John, J. G.; Jason, D. M.; Peter, M.; Mark, C.; Tian, H.; Rustam, G.; Allen, T.; Christopher, S. S.; Michael, B. W.; Gabrielle,
K.; Robert, K.; Michael, B.; Matthew, M.; Chen, H.; Brian, D.; Danette, A. D.; Stephen, E. G.; Patricia, P.; Sheerin, S. L.; Wang, W.;
Kristina, W.; Yin, J.; John, M.; Jacob, B. S. Bioorg. Med. Chem. Lett. 2014, 24, 2635–2639.
19. Izzat, T. R.; Michael, J. B.; Christine, F.; Joy, F.; Nicole, H.; Sarah, H.; Monika, K.; Somang, H. K.; Bennett, M.; Georgia, M.; John, J. R.;
John, D. S.; Sujata, S.; Sean, S.; Jason, U.; Yan, Y. W.; Paul, J. C.; Christopher, D. C. Bioorg. Med. Chem. Lett. 2012, 22, 5903-5908.
20. Lim, K. S.; Cho, J.; Kim, B. H. et al. Brit. J. Clin. Pharmaco. 2009, 68, 883-890.
21. Craig, A. C.; Luo, Y. F.; Cui, M. X; Wang, J. B; Richard, S.; Dong, J. C.; Hu, B.; Michael A. L.; Vincent, P. S.; Richard, L. K.; Yun, G.;
Wang, Y.; Steven, V. F.; Jacquelyn, B.; Scott, M. D.; Duane, R. R.; Pamela, L. T.; Anthony, L. G.; Peter, T. M.; John, J. R.
ChemMedChem. 2012, 7, 123-133.
22. Piotrowski, D. W.; Futatsugi, K.; Warmus, J. S.; Orr, S. T. M.; Freeman-Cook, K. D.; Londregan, A. T.; Wei, L. Q.; Jennings, S. M.; Herr,
M.; Coffey, S. B. Med. Chem. Lett.. 2013, 4, 63-68.
23. Terrence, J. C.; Michael, M.; Keshab, S. Org. Process Res. & Dev. 2005, 9, 80-87.
24. Regueiro-Ren, A.; Swidorski, J.; Liu, Z.; Wang, T.; Zhang, Z; Hamann, L. G.; Meanwel, N. A.; Carini, D. J.; Li, W. From PCT. Int. Appl.
2009, WO 2009158396.
25. Tsou, H. R.; Mamuya, N.; Johnson, B. D.; Reich, M. F.; Gruber, B. C.; Ye, F.; Nilakantan, R.; Shen, R.; Discafani, C.; DeBlanc, R.; Davis,
R.; Koehn, F. E.; Greenberger, L. M.; Wang, Y. F.; Wissner, A. J. Med. Chem. 2001, 44, 2719-2734.
20

26. Rewcastle, G. W.; Denny, W. A.; Bridges, A. J.; Zhou, H.; Cody, D. R.; McMichael, A.; Fry, D. W. J. Med. Chem. 1995, 38, 3482-3487.
27. Totrov, M. Chem. Biol. Drug Des. 2008, 71, 15-27.
28. Cheeseright, T.; Mackey, M.; Rose, S.; Vinter, A. J. Chem. Inf. Model. 2006, 46, 665-76.
29. Treder, A. P.; Andruszkiewicz, R.; Zgoda, W.; Ford, C.; Hudson, A. L. Bioorg. Med. Chem. Lett. 2009, 19, 1009-1011.
30. Han, C.; Wan, L. D.; Ji, H. B.; Ding, K.; Huang, Z. J.; Lai, Y. S.; Peng, S. X.; Zhang, Y. H.. Eur. J. Med. Chem. 2014, 77, 75-83.
31. Mao, Y. J.; Zhu, W. X.; Kong, X. G.; Wang, Z.; Xie, H.; Ding, J.; Nicholas, K. T.; Shen, J. K.; Shen, J. S. Bioorg. Med. Chem. 2013, 21,
3090-3104.
32. Hwei-Ru, T.; Elsebe, G. O. K.; William, A. H.; Marvin, F. R. M.; Brawner, F.; Bernard, D. J.; Ronald, S. M.; Ramaswamy, N.; Carolyn,
D.; Jonathan, G.; Sridhar, K. R.; Ru, S.; Shi, X. Q.; Wang, Y. F.; Janis, U.; Allan, W. J. Med. Chem. 2005, 48, 1107-1131.
33. Siavosh, M.; Andreas, S.; Matthias, W.; Emerich, E.; Herwig, P.; Thomas, C.; Thomas, B.; Thomas, M.; Thomas, B. J. Med. Chem. 2010,
53, 8546–8555.
34. Karaman, M. W.; Herrgard, S.; Treiber, D. K.; Gallant, P.; Atteridge, C. E.; Campbell, B. T.; Chan, K. W.; Ciceri, P.; Davis, M. I.; Edeen,
P. T.; Faraoni, R.; Floyd, M.; Hunt, J. P.; Lockhart, D. J.; Milanov, Z. V.; Morrison, M. J.; Pallares, G.; Patel, H. K.; Pritchard, S.;
Wodicka, L. M.; Zarrinkar, P. P. Nat. Biotechnol. 2008, 26, 127-132.
35. Solca, F.; Dahl, G.; Zoephel, A.; Bader, G.; Sanderson, M.; Klein, C.; Kraemer, O.; Himmelsbach, F.; Haaksma, E.; Adolf, G. R. J.
Pharmacol. Exp. Ther. 2012,343, 342-350.
36. Zhou, W.; Liu, X. F.; Tu, Z. C.; Zhang, L. W.; Ku, X.; Bai, F.; Zhao, Z. J.; Xu, Y. F.; Ding, K.; Li, H. L. J. Med. Chem. 2013, 56,
7821-7837.
37. Allan, W.; Philip, R. H.; Ramaswamy, N.; Lee, M. G.; Ye, F.; Timothy, A. R.; Frank, L. Bioorg. Med. Chem. Lett. 2004, 14, 1411-1416.
38. Tiedt, R.; Degenkolbe, E.; Furet, P.; Appleton, B. A.; Wagner, S.; Schoepfer, J.; Buck, E.; Ruddy, D. A.; Monahan, J. E.; Jones, M. D.;
Blank, J.; Haasen, D.; Drueckes, P.; Wartmann, M.; McCarthy, C.; Sellers, W. R.; Hofmann, F. Cancer Res. 2011, 71, 5255.
39. Li, H. Q.; Li, D. D.; Lu, X.; Xu, Y. Y.; Zhu, H. L. Bioorg. Med. Chem. 2012, 20, 317-323.
Legends
Figure 1 Representative EGFR inhibitors with 4-arylamino-quinazoline.
Figure 2 Representative THPP derivatives with biological and pharmaceutical properties.
Figure 3 Design strategy, screening and optimization of 4-arylamino-6-substituted-THPPs.
Figure 4 The structure of compound a1 and overlay of compound a1 with Gefitinib and Erlotinib. Carbon atom in compound a1 was
shown in purple, cyan for Gefitinib, and grey for Erlotinib. Nitrogen was colored in blue, oxygen in red, chlorine in green.
Scheme 1 Reagents and conditions: (a) H2, Pd/C, 5 atm., HCl, ethanol, 40 °C, 8 h; (b) CbzCl, DIPEA, DCM, r.t., 2 h; (c) formamidine
acetate, ethanol, NaOCH3, 6 h, reflux; (d) POCl3, DIPEA, CH3CN, reflux, 6 h; (e) NH2ArR1, NaHMDS, THF, 0 °C, 2 h; (f) HBr, acetic acid,
50 °C, 2 h.
Scheme 2 Reagents and conditions: (a) ClO2SR2, DIPEA, DCM, 0 °C, 2h; (b) ClCH2R2, DIPEA, DCM, 0 °C, 1 h or CH2CHR2, ethanol, 60
°C, 8 h; (c) i) diethyl squarate, ethanol, 60 °C, 4 h; ii) NR1R2, ethanol, 60 °C, 4 h; (d) i) CDI, ethanol, reflux, 4 h; ii) NR1R2, ethanol, reflux,
12h; (e) ClOCOR2, DIPEA, CH3CN, 0 °C, 1 h;(f) ClOCR2, DIPEA, DCM, 0 °C, 1 h or HO2CR2 EDCI, HOBT, DIPEA, CH3CN, 60 °C, 6 h.
Scheme 3 Reagents and conditions: (a) H2, Pd/C, 5 atm., ethanol, 40 °C, 8 h; (b) CbzCl, DIPEA, DCM, r.t., 2 h; (c) formamidine acetate,
ethanol, NaOCH3, 6 h, reflux; (d) POCl3, DIPEA, DCE, reflux, 6 h; (e) NH2ArR1, NaHMDS, THF, 0 °C, 2 h; (f) HBr, acetic acid, 50 °C, 2 h.
Scheme 4 Reagents and conditions: (a) ClO2SCH3, DIPEA, DCM, 0 °C, 2 h; (b) CDI, ethanol, reflux, 4 h; ii) N(CH3)2, ethanol, reflux, 12 h;
(c) ClOCOCH3, DIPEA, CH3CN, 0 °C, 1 h; (d) ClOCR2, DIPEA, DCM, 0 °C, 1 h or HO2CR2, EDCI, HOBT, DIPEA, CH3CN, 60 °C, 6 h.
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Table 1
Structures and cytotoxicities of compounds 7a-13e against HT29, A549, H460 and H1975 cancer cell lines in vitro.
Table 2
Structures and cytotoxicities of compounds 13f-u against HT29, A549, H460 and H1975 cancer cell lines in vitro.
Table 3
Structures and cytotoxicities of compounds 20-24c against HT29, A549, H460 and H1975 cancer cell lines in vitro.
Table 4
EGFR and HER2 kinase activity of selected compounds 13b, 13g, 13n, 13o, 13p, 13r and 13s in vitro.
Figure 5 Proposed binding model for compound 13b bound to the kinase domain of EGFR. The described hydrogen bonds and key distance
to Cys-797 are shown.
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