Design, synthesis and biological evaluations of a series of Pyrido[1,2-a]pyrimidinone derivatives as novel selective FGFR inhibitors

Article abstract of DOI:10.1016/j.ejmech.2021.113499

Aberrant signaling of fibroblast growth factor receptors (FGFRs) has been identified as a driver of tumorigenesis and the development of many solid tumors, making FGFRs a compelling target for anticancer therapy. Herein, we describe the design and synthes

Full text of DOI:10.1016/j.ejmech.2021.113499

European Journal of Medicinal Chemistry 220 (2021) 113499
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Design, synthesis and biological evaluations of a series of Pyrido[1,2-a]
pyrimidinone derivatives as novel selective FGFR inhibitors
,
,
Kai Ran a 1, Jun Zeng a 1, Guoquan Wan a, Xiaojie He a, Zhanzhan Feng a, Wang Xiang a,
Wei Wei a, Xiang Hu a, Ningyu Wang b, Zhihao Liu a **, Luoting Yu a
,
, *
a State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, And Collaborative Innovation
Center for Biotherapy, 17#3rd Section, Ren Min South Road, Chengdu, 610041, China
b School of Life Science and Engineering, Southwest JiaoTong University, Chengdu, Sichuan, 611756, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Aberrant signaling of fibroblast growth factor receptors (FGFRs) has been identified as a driver of
tumorigenesis and the development of many solid tumors, making FGFRs a compelling target for anti-
cancer therapy. Herein, we describe the design and synthesis of pyrido[1,2-a]pyrimidinone derivatives as
potent FGFR inhibitors. Examination of structureeactivity relationships and preliminary assessment
identified 23d as a novel FGFR inhibitor that displayed excellent potency in vitro. Candidate 23d sup-
pressed the phosphorylation of FGFR signaling pathways and induced cell cycle arrest and apoptosis at
low nanomolar concentration. In the kinase inhibition profile, 23d showed excellent kinase selectivity for
the FGFR family. Furthermore, 23d showed higher aqueous solubility than Erdafitinib. Moreover, 23d
exhibited potent antitumor activity (tumor growth inhibition ¼ 106.4%) in FGFR2-amplified SNU-16
gastric cancer xenograft model using a daily oral dose of 30 mg/kg. These results suggest that 23d is a
promising candidate for further drug development.
Received 3 February 2021
Received in revised form
2 April 2021
Accepted 16 April 2021
Available online 26 April 2021
Keywords:
FGFR
pyrido[1,2-a]pyrimidinone
Solubility
Tumor growth inhibition
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
and mutations in the exons of FGFR family members are associated
with tumor cell invasion, metastasis, angiogenesis, tumor recur-
Fibroblast growth factor receptors (FGFRs) are composed of four
highly conserved receptor subtypes (FGFR-1, 2, 3, and 4) and make
up a subfamily of receptor tyrosine kinases [1]. All the FGFR sub-
types consist of an extracellular ligand domain, a single trans-
membrane helix domain, and an intracellular domain with tyrosine
kinase activity [2]. After being activated by extracellular fibroblast
growth factors, FGFR undergoes dimerization and autophosphor-
ylation, resulting in activation of downstream signaling pathways,
rence, and drug resistance [2,7e10]. FGFR genomic aberrations
have been identified in many types of tumors [11e13]. For instance,
FGFR1 amplifications occur in about 20% of squamous non-small
cell lung carcinomas and 10%e15% of breast cancers. FGFR2 trans-
locations occur in 14% of intrahepatic cholangiocarcinomas. FGFR3
mutations have been identified in 60%e80% of non-muscle-
invasive bladder carcinomas, and FGFR3 translocations are also
found in 3% of glioblastomas and about 20% of myelomas. In
addition, FGFR4 amplifications occur in 50% of hepatocellular car-
cinomas. All these gain-of-function alterations result in the over-
activation of downstream growth and proliferation signaling
pathway and promotion of tumorigenesis. Therefore, the FGFR
such as PI3K-Akt, RAS-MEK-ERK, and PLCg pathways. These FGFR
cascades play important roles in cell proliferation, differentiation,
and metastasis [3e6].
Abnormal FGFRs caused by gene amplifications, translocations,
signaling pathway represents
therapeutics.
a promising target for cancer
The early FGFR inhibitors, such as Dovitinib [14], Ponatinib [15],
and Lucitanib [16], usually inhibit a broad range of additional ki-
nases. Although therapies based on inhibiting multiple kinases
have been approved for the treatment of cancers, serious adverse
effects such as VEGFR-2 based dose-limiting toxicities prevent their
extensive clinical application [17]. To overcome these drawbacks,
* Corresponding author. Lab of Medicinal Chemistry, State Key Laboratory of
Biotherapy and Cancer Center, West China Hospital, Sichuan University and
Collaborative Innovation Center for Biotherapy, 17#3rd Section, Ren Min South
Road, Chengdu, 610041, China.
** Corresponding author.
E-mail addresses: liuzhihao@scu.edu.cn (Z. Liu), yuluot@scu.edu.cn (L. Yu).
These authors contribute equally to this work.
1
https://doi.org/10.1016/j.ejmech.2021.113499
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

K. Ran, J. Zeng, G. Wan et al.
European Journal of Medicinal Chemistry 220 (2021) 113499
the second-generation FGFR kinase inhibitors with improved target
selectivity were developed, of which several candidates have
entered clinical trials, including NVP-BGJ398 [18] (1), AZD4547 [19]
(2), LY2874455 [20] (3), CH5183284 [21] (4), Pemigatinib [22] (5),
and Erdafitinib [23] (6) (Fig. 1). The recent approval of two pan-
FGFR inhibitors Erdafitinib and Pemigatinib has further validated
the feasibility of FGFRs inhibitors as therapeutic strategies for FGFR-
related cancers [24,25].
insoluble at pH 7.4) is one of the main factors affecting its phar-
macokinetics [37]. Therefore, measures to increase the aqueous
solubility of compounds would be considered in our following
design and optimization studies.
In 2014, Astex Pharmaceuticals discovered quinazolinone com-
pound 7 as an analogue of Erdafitinib [38], but observed a 40-fold
decrease in FGFR1 potency. In the co-crystal structure of FGFR1 in
complex with Erdafitinib (PDB ID: 5EW8, Fig. 2A) [31], the qui-
noxaline core of Erdafitinib can form a single hydrogen bond with
Ala564 in hinge region, while one of the methoxyl oxygen atoms in
dimethoxyphenyl ring is involved in a hydrogen bond with Asp641.
The methyl pyrazole group could extend towards the solvent
channel and make van der Waals interactions with Lue484. In order
to find out the reason for the decline in FGFR1 activity of 7
compared with Erdafitinib, we conducted a molecular docking
analysis to elucidate the interaction mode of compound 7 with
FGFR1 (PDB ID: 5EW8). As shown in Fig. 2B, compound 7 has a
similar binding mode with Erdafitinib, and maintains the key H-
bonding network observed in the complex of Erdafitinib with
FGFR1. However, the nitrogen bridge in compound 7 tends to force
the methyl pyrazole ring to be perpendicular to the quinazolinone
core, similar to that observed in Idelalisib [39] and BMS-777607
[40]. Such perpendicular rotation of the methyl pyrazole ring
would destroy its interaction with residue Lue484, leading to a
decline in activity.
Despite the advances made in selective FGFR inhibitors, the
clinical efficacy of current FGFR inhibitors is still unsatisfactory
[26e29]. Except for the lack of appropriate biomarkers for FGFR
inhibitor sensitivity, the commonly reported challenge of FGFR
inhibitor efficacy is the development of drug resistance. The
intrinsic or acquired point mutations in the FGFR kinase domain
give the current FGFR inhibitors distinct effects for different mu-
tants. The other barrier limiting the clinical applications of FGFR
inhibitors is that of adverse toxicity, including hyperphosphatemia
and eye disorders, which has led to a narrow therapeutic window.
These undesired side effects are closely related to the target
selectivity and pharmacokinetic properties of current FGFR in-
hibitors [30e33]. Hence, currently medicinal chemists have focus
on developing structural diversification of highly selective and
highly bioactive FGFR inhibitors with potential druggability to
overcome above shortcomings [34,35]. In this study, we designed
and synthesized a series of pyrido[1,2-a]pyrimidinone derivatives
as potent FGFR inhibitors with excellent potency and selectivity.
The compounds displayed significant antitumor efficacy and
improved aqueous solubility compared with approved drug
Erdafitinib.
In view of the structural similarities between 7 and Erdafitinib,
we utilized the scaffold hopping strategy [41] to design the pyrido
[1,2-a]pyrimidinone core, which can form similar hydrogen bond
with FGFR1 and might restore the interaction between adjacent
aryl and residue Lue484 due to more suitable bond angle, to
generate a novel type of FGFR inhibitor 8 (Fig. 2C). Furthermore, a
hydrophilic group was introduced at the Ar or R3 position of 8 in an
effort to improve its solubility.
2. Molecular design
While Erdafitinib, the first FGFR-selective compound approved
by the US Food and Drug Administration, provides encouraging
clinical benefit in cancer patients harboring FGFR genetic alter-
ations, its dose-limiting adverse events are also reported in its in-
struction [24]. Except for hyperphosphatemia caused by the on-
target effect (unwanted inhibition of FGFR1 and FGFR3), other
drug-related adverse events might be related with its unsatisfac-
tory pharmacokinetic profile, such as long half-life (t1/2 ¼ 59 h) and
excessive metabolic stability, which result in its accumulation
in vivo [36]. The poor aqueous solubility of Erdafitinib (practically
3. Chemistry
3.1. Synthesis of compounds 8ae8d
The synthetic route to compounds 8ae8d is shown in Scheme 1.
Under Bredereck reaction conditions, commercially available
methyl 2-(benzyloxy)acetate 9 was converted to enamine 10.
Cyclization of 10 with 2-amino-5-bromopyridine at 90e100 ꢀC
Fig. 1. Reference selective FGFR inhibitors under clinical trials or in the market.
2

K. Ran, J. Zeng, G. Wan et al.
European Journal of Medicinal Chemistry 220 (2021) 113499
Fig. 2. (A) Cocrystal structure of Erdafitinib bound with FGFR1 (PDB ID: 5EW8) [31]; (B) Docking results of 7 in the FGFR1 protein (PDB ID: 5EW8); (C) The design of pyrido[1,2-a]
pyrimidinone derivatives as novel FGFR inhibitors using a scaffold hopping strategy.
Scheme 1. Reagents and conditions: (i) tert-butoxybis(dimethylamino)methane, 90 ꢀCe100 ꢀC; (ii) 2-amino-5-bromopyridine, AcOH, 120e130 ꢀC; (iii) 3,5-dimethoxyaniline,
Pd2(dba)3, BINAP, Cs2CO3, toluene, 100 ꢀC; (iv) 2-isopropylaminoethylchloride hydrochloride, KOH, Bu4NBr, THF, H2O, 50 ꢀC; (v) TFA, 90e100 ꢀC; (vi) (Boc)2O, Et3N, THF, 0 ꢀCert; (vii)
(a) alkyl halide, K2CO3, DMF, 90e120 ꢀC, (b) TFA, DCM, rt.
provided
3-(benzyloxy)-7-bromo-4H-pyrido[1,2-a]pyrimidin-4-
presence of trifluoroacetic acid and subsequent protection with a
Boc group. Finally, the target compounds 8ae8d were obtained
through substitution of 15 with different alkyl halides and subse-
quent Boc deprotection.
one 11. Buchwald-Hartwig cross-coupling of 11 with 3,5-
dimethoxyaniline followed by nucleophilic substitution with 2-
isopropylaminoethylchloride under basic conditions led to 13. In-
termediate 15 was then prepared by debenzylation of 13 in the
3

K. Ran, J. Zeng, G. Wan et al.
European Journal of Medicinal Chemistry 220 (2021) 113499
3.2. Synthesis of compounds 23ae23m
leaving group with various amines furnished target compounds
34ae34c.
Synthesis of compounds 23ae23m is outlined in Scheme 2.
Treatment of 5-bromopyridin-2-amine 17 with 2,2-dimethyl-1,3-
dioxane-4,6-dione in the presence of triethyl orthoformate, fol-
lowed by cyclization in diphenyl oxide at 220 ꢀC led to 7-bromo-
4. Results and discussion
4.1. Structureeactivity relationships
4H-pyrido[1,2-a]pyrimidin-4-one 19. Iodination of 19 at the a-po-
sition of the amide using N-iodosuccinimide gave dihalogenated
intermediate 20. Subsequent Suzuki reaction with different boronic
esters and Buchward coupling with 3,5-dimethoxyaniline afforded
22ae22m. Compounds 23ae23m were prepared by nucleophilic
substitution of 22ae22m with 2-isopropylaminoethylchloride un-
der basic conditions, after which 23b and 23g underwent addi-
tional deprotection in the presence of hydrochloric acid.
All the synthetic compounds were assayed for their biochemical
activity against FGFR1. Potent compounds were further tested for
their antiproliferative effects in FGFR2-amplified SNU-16 cells.
Erdafitinib was used as the positive control. We first focused on
optimizing the R1 group adjacent to the pyrido[1,2-a]pyrimidinone
core by introducing different substituents (see Table 1). Com-
pounds 8ae8d were obtained by introduction of flexible alkyloxy
groups at the R1 position, and all of them showed a complete loss in
FGFR1 inhibitory activity. When R1 was substituted by a partially
saturated ring, the activity of the N-Boc protected tetrahydropyr-
idine ring 23a and corresponding deprotected amine 23b
increased. Inspired by this result, the aromatic heterocycle methyl
pyrazole (23d), dimethyl-substituted pyrazoles (23e and 23f), and
pyrazole (23g) were introduced at the same position. Among these
compounds, 23d and 23g exhibited the most potent enzymatic and
cellular activity with IC50 values below 10 nM. Notably, 23d showed
the highest potency against FGFR1 (IC50 ¼ 0.57 nM) and SNU-16
(IC50 ¼ 3.3 nM). Accordingly, based on the pyrazol-4-yl group, a
series of N-substituents on the pyrazole ring were prepared in the
search for a more effective compound. All the extended N-sub-
stituents (23he23k) displayed good FGFR1 inhibition with IC50
values ranging from 1.0 to 8.0 nM. Of these compounds, 2-
fluoroethyl 23j had the highest antiproliferative effect
(IC50 ¼ 1.9 nM), probably because of its good cell permeability.
Likewise, 23l and 23m featuring bulky tetrahydropyranyl groups
maintained good potency in both enzymatic and cellular activity.
Motivated by good tolerance of N-substituents on the pyrazol-4-
yl group described above, incorporation of a polar group or a basic
center at this position was performed in the following optimization
to improve the solubility (see Table 2). All the obtained compounds
maintained good potency against FGFR1 and SNU-16 cells.
Consistent with our expectation, hydroxyethyl 27a showed about
3.3. Synthesis of compounds 27ae27f and 29ae29f
Scheme 3 presents the synthesis of compounds 27ae27f and
29ae29f. Starting from intermediate 23l, benzyloxycarbonyl (CBZ)
protection of the free amine and subsequent selective tetrahy-
dropyranyl deprotection produced the pyrazole 25. Treatment with
different alkyl halides or alkylene oxides afforded 26ae26f, which
were converted to the target compounds 27ae27f via removal of all
protective groups in the presence of hydrobromic acid. Meanwhile,
the selective Boc deprotection of 26e and 26f in the presence of
trifluoroacetic acid provided 28a and 28b, respectively. Reductive
amination of 28a with different aldehydes followed by CBZ
deprotection gave compounds 29a and 29b. Acylation or meth-
anesulfonylation of 28a and 28b followed by CBZ deprotection
yielded 29ce29f.
3.4. Synthesis of compounds 30a, 30b, 32, and 34ae34c
As shown in Scheme 4, nucleophilic substitution of 22d with
different alkyl halides in the presence of NaH provided 30a and 30b.
Using the same method, treatment of 22d with (2-
bromoethoxy)(tert-butyl)dimethylsilane afforded intermediate 31,
which was then converted to compound 32 via TBDMS (tert-
butyldimethylsilyl) deprotection in the presence of tetrabuty-
lammonium fluoride. Next, methanesulfonylation of alcohol 32
followed by displacement of the resulting methanesulfonyloxy
5-fold improvement in aqueous solubility (147
mg/mL) compared
with Erdafitinib. When two methyl groups were further introduced
Scheme 2. Reagents and conditions: (i) Triethyl orthoformate, 2,2-dimethyl-1,3-dioxane-4,6-dione, EtOH, reflux; (ii) Diphenyl oxide, 220 ꢀC; (iii) NIS, DMF, 80 ꢀC; (iv) Boronic acids
or boronate esters, PdCl2DPPF, K2CO3, dioxane, H2O, 100 ꢀC; (v) 3,5-Dimethoxyaniline, Pd2(dba)3, 2,20-bis(diphenylphosphino)-1,10-dinaphthalene, Cs2CO3, toluene, 100 ꢀC; (vi) (a) 2-
Isopropylaminoethylchloride hydrochloride, KOH, Bu4NBr, THF, H2O, 50 ꢀC; (b) HCl, dioxane, rt.
4

K. Ran, J. Zeng, G. Wan et al.
European Journal of Medicinal Chemistry 220 (2021) 113499
Scheme 3. Reagents and conditions: (i) CbzCl, DIEA, DCM, rt; (ii) 5 N HCl, MeOH, rt; (iii) alkyl halide, Cs2CO3, DMF, 100 ꢀC; (iv) HBr, AcOH, rt; (v) TFA, DCM, rt; (vi) (a) aldehyde,
AcOH, Na(OAc)3BH, 1,2-dichloroethane, rt-60 ꢀC; (b) HBr, AcOH, rt or (c) acyl chloride/sulfuryl chloride, Et3N, DCM, rt; (d) HBr, AcOH, rt.
Scheme 4. Reagents and conditions: (i) alkyl halide, NaH, DMF, 5 ꢀCert; (ii) TBAF, THF, rt; (iii) MsCl, Et3N, CH2Cl2, 0 ꢀC; (iv) amine, CH3CN, 100 ꢀC.
on the hydroxyethyl tail (27b), a 3-fold lower solubility (49.4
m
g/
retrospective determination, the previous highly active compound
mL) was observed. The introduction of a basic center (27c and 27d)
did not increase the solubility. Then we attempted to connect the
pyrazole ring with an unsubstituted piperidine ring. The resulting
compound 27e showed an obvious improvement in solubility
23d showed better solubility (327 mg/mL), and its molecular weight
was less than 500.
We analyzed the co-crystal structure of Erdafitinib/FGFR1
complex and found that the 2-isopropylaminoethyl chain in Erda-
fitinib could extend away from the kinase domain into a solvent
exposed region [34]. Hence, introduction of some polar or hydro-
philic groups at this position of 8 would have the potential to
improve the compound’s solubility while maintaining its activity.
Given that the N-methyl substitution on pyrazole ring provided the
most potent inhibition against FGFR1 and the highest solubility, the
(227 mg/mL) but showed a slight decrease in cellular activity, which
was probably caused by decreased cell permeability. Further
modification of the piperidine ring with methyl (29a) and ethyl
(29b) groups slightly increased the antiproliferative potency, but
also resulted in significantly decreased solubility. To our surprise,
acetylpiperidinyl 29c and (methylsulfonyl)piperidinyl 29d exhibi-
ted both high potency and significantly improved solubility.
Notably, 29d achieved the highest cellular activity (IC50 ¼ 0.67 nM)
scaffold
7-[(3,5-dimethoxyphenyl)amino]-3-(1-methyl-1H-pyr-
azol-4-yl)-4H-pyrido[1,2-a]pyrimidin-4-one was established for
the following investigation of R3 variations. However, all the ob-
tained derivatives (30ae30b, 32 and 34ae34c) showed slightly or
apparently lower potency against FGFR1 or cellular activity
compared with 23d. Overall, with good enzymatic activity, cellular
activity, and aqueous solubility, compound 23d was chosen for
further evaluation.
and aqueous solubility (227 mg/mL). In addition, we used azetidine
instead of piperidine to prepare similar compounds 27f, 29e, and
29f; however, none showed better potency or solubility than
compound 29d. Despite the encouraging profiles for activity and
solubility, the molecular weight of 29d (MW ¼ 610) exceeded 500,
which caused some concern for its pharmacokinetic properties. In a
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K. Ran, J. Zeng, G. Wan et al.
European Journal of Medicinal Chemistry 220 (2021) 113499
Table 1
4.2. Kinase selectivity profile of 23d
Structureeactivity results for chemical modifications at R1.
For a broad assessment of the kinase selectivity, 23d was eval-
uated kinase inhibition profile against a diverse panel of 360 re-
combinant human kinases from Eurofins Discovery using an in vitro
ATP-site competition binding assay at a concentration of 1 mM. As
summarized in Fig. 3 (a list of the tested kinases can be found in
Table S1) [16], kinases were inhibited at a cutoff of 35% of the
dimethyl sulfoxide control. Of these, only 9 hits (FGFR1, FGFR2,
FGFR3, FGFR4, RET, FLT1, FLT4, Lyn, and FMS) including FGFR1-4
had >90% inhibition. Further FGFR1-4 enzymatic assay displayed
potent inhibition with IC50s of 0.57, 2.0, 0.80 and 1.4 nM, respec-
tively. All these data indicated that 23d was a potent and selective
pan-FGFR inhibitor.
Compound
R1
IC50 (nM)
FGFR1a
>100
SNU-16 cellb
N.T.c
8a
8b
>100
>100
N.T.
N.T.
4.3. Molecular docking of 23d
8c
To elucidate the mode of interaction of 23d with FGFR, we
conducted molecular docking analysis using a reported crystal
structure of FGFR1 (PDB ID: 5EW8). As illustrated in Fig. 4A, 23d
showed a binding mode similar to that of Erdafitinib (6) and
maintains the key H-bonding network observed in the complex of
FGFR1 with 6, as shown in Fig. 2A. In terms of the difference be-
tween the two complexes, the pyrazole ring of 23d could undergo
some rotation with respect to the pyrido[1,2-a]pyrimidinone core
(Fig. 4C), while the pyrazole ring of 6 was coplanar with the qui-
noxaline ring [31] (Fig. 4B). Fortunately, the slight rotation of the
pyrazole ring in 23d did not affect its interaction with amino acid
residue Lue484, which resulted in improved enzymatic potency
when compared with the lead 7 (Fig. 2B). Furthermore, this rotation
disrupted the planarity of compound 23d, which may have been a
factor its higher aqueous solubility when compared with 6.
8d
>100
N.T.
23a
3.7
17.7
23b
23c
23d
23e
23f
23g
23h
23i
22
59.9
53.2
3.3
37
0.57
>100
50
N.T.
N.T.
7.3
4.4. Inhibition on FGFR signaling by compound 23d
To investigate the cellular effects of compound 23d in targeting
FGFR signaling pathway, the effects of 23d on the phosphorylation
of FGFR and its major downstream signaling molecules were
analyzed by Western blot in SNU-16 cells (FGFR2-amplification). As
shown in Fig. 5, the phosphorylation of FGFR2 was obviously
inhibited by 23d in a dose-dependent manner. Besides, 23d
significantly inhibited the phosphorylation of FRS2. The phos-
phorylation of AKT and ERK1/2 was also decreased at 5 nM con-
centration, indicating that PI3K-ATK and MAPK signaling were also
significantly repressed.
2.1
8.0
1.0
1.2
2.0
32.4
3.5
23j
1.9
4.5. Inhibition of SNU-16 cell proliferation by compound 23d
23k
6.9
Since FGFRs play a vital role in cell proliferation and survival, we
examined the proliferation inhibitory effect of 23d against FGFR2-
amplified SNU-16 gastric cells using the MTT assay and a cell
counting assay in detail. In the MTT assay, SNU-16 cells were
exposed to graded concentrations of 23d for 24, 48, 72, or 96 h, and
caused marked decreases in viability (Fig. 6A). This showed that
23d inhibited the proliferation of SNU-16 cells in a time-dependent
and concentration-dependent manner. The following cell counting
assay provided a readily visible example of cell proliferation inhi-
bition (Fig. 6B and C). Remarkably, 23d almost completely inhibited
the growth of SNU-16 cells at 10 nM concentration, indicating its
superior anti-proliferative effect in vitro.
23l
1.2
4.7
1.8
1.1
23m
1.5
Erdafitinib
0.19
a
Biochemical assay was performed with ATP at Km concentration, test results
provided by Sundia MediTech Company, Ltd.
b
Tumour cells were treated with compounds for 72 h.
N.T.: Not tested.
c
6

K. Ran, J. Zeng, G. Wan et al.
European Journal of Medicinal Chemistry 220 (2021) 113499
Table 2
Structure, activity and solubility relationships for chemical modifications at R2 and R3.
Compound
R2
R3
IC50 (nM)
FGFR1a
Solubilityc
(mg/mL)
SNU-16 cellb
Erdafitinib
27a
0.19
2.7
1.1
9.1
29.4
147
27b
27c
27d
1.4
1.2
1.2
4.1
5.5
4.8
49.4
<10.0
<10.0
27e
29a
29b
29c
29d
0.65
1.3
15.7
5.9
227
<10.0
<10.0
197
2.4
10.5
1.8
0.82
1.0
0.67
227
27f
29e
29f
1.2
1.6
1.6
87.5
4.4
<10.0
59.1
4.0
<10.0
23d
Me
0.57
3.3
327
30a
30b
Me
Me
>100
N.T.d
44.0
N.T.
N.T.
18
32
Me
Me
1.9
14
23.0
28.7
N.T.
N.T.
34a
34b
34c
Me
Me
85
99.7
12.6
N.T.
N.T.
2.5
a
Biochemical assay was performed with ATP at Km concentration; test results provided by Sundia MediTech Company, Ltd.
Tumour cells were treated with compounds for 72 h.
Solubility at pH 7.4.
b
c
d
N.T.: Not tested.
4.6. Compound 23d induces cell cycle G0/G1 phase arrest and
apoptosis
of 23d-treated SNU-16 cells. The experimental results showed that
23d induced both cell arrest in the G0/G1 phase (Fig. 7A and B) and
cell apoptosis (Fig. 7C and D) when the concentration exceeded
5 nM. The expression of cell cycle-related proteins including CDK2,
CDK4, CDK6, Cyclin D, and Cyclin E were inhibited at a similar
As the inhibition of FGFR activity has the influence on cell cycle
and apoptosis, we conducted flow cytometry assay (FCM) analysis
7

K. Ran, J. Zeng, G. Wan et al.
European Journal of Medicinal Chemistry 220 (2021) 113499
Fig. 3. Kinome-wide selectivity profiling of compound 23d with the Kinase Profile assay by Eurofins Discovery. Measurements were performed at a concentration of 1 mM of the
inhibitor in duplicate. The % control means remaining active kinase percentage. The affinity was defined with respect to a dimethyl sulfoxide (DMSO) control. The TREEspot image
was mapped with the KinMap software tool provided by Cell Signaling Technology, Inc. (www.cellsignal.com). Biochemical IC50s of 23d were determined against FGFR1-4. The
results were provided by Sundia MediTech Company, Ltd., n ¼ 1.
Fig. 4. (A) Docking results of 23d in the FGFR1 protein (PDB ID: 5EW8); (B) Cocrystal structure of 6 bound to FGFR1 (PDB ID: 5EW8) [31]; (C) Rotation of (A).
8

K. Ran, J. Zeng, G. Wan et al.
European Journal of Medicinal Chemistry 220 (2021) 113499
concentration (Fig. 7E), and upregulation of apoptosis-related
proteins, including cleaved caspase-9 and cleaved PARP, was also
observed (Fig. 7F).
4.7. Pharmacokinetic study of compound 23d
Before in vivo efficacy evaluation, the pharmacokinetic profile of
the pan-FGFR inhibitor 23d was tested in Sprague-Dawley rats (iv,
3 mg/kg; oral, 30 mg/kg). Blood samples were collected at 0.083,
0.16, 0.25, 0.5, 1, 2, 4, 6, 10, and 24 h after dosing. The blood drug
level was then determined with liquid chromatographyetandem
mass spectrometry. The pharmacokinetic parameters are listed in
Table 3. In rats, 23d showed slow absorption (Tmax ¼ 4.33 h), with a
peak concentration of 130.34 ng/mL, an AUC value of 948.37 ng h/
mL, and a terminal half-life of 2.65 h following a single oral dose of
30 mg/kg. The oral bioavailability of 23d in rat was 14.94%.
4.8. In vivo antitumor efficacy in human xenografts of 23d
Given that compound 23d showed good kinase selectivity,
excellent activity, and acceptable pharmacokinetic properties, the
efficacy of 23d in vivo was tested in the SNU-16 xenograft model.
23d was orally administered at doses of 15 or 30 mg/kg once daily
for 4 weeks. The results showed that 23d could suppress tumor
growth in a dose-dependent manner, with tumor growth inhibition
of 67.3% and 106.4% at the doses of 15 and 30 mg/kg, respectively
Fig. 5. Compound 23d effectively inhibits the phosphorylation of FGFR2 and the
downstream effectors FRS2, AKT and ERK1/2 in SNU-16 cells. Cells treated with 23d for
24 h at the indicated concentrations were lysed and subjected to Western blot analysis.
Fig. 6. Antiproliferative effect of 23d on SNU-16 cells. (A) SNU-16 cell lines were treated with different concentrations of 23d for 24 h, 48 h, 72 h, and 96 h, respectively; (B) The
statistics of cell counting; (C) Photographs of cell counting at different time after 10 nM treatment of 23d.
9

K. Ran, J. Zeng, G. Wan et al.
European Journal of Medicinal Chemistry 220 (2021) 113499
Fig. 7. (A) FCM analysis for cell cycle effects of 23d on SNU-16 cells. Cell were treated with increasing concentration of 23d for 48 h, harvested, fixed and stained with propidium
iodide; (B) Data analysis for cell cycle FCM analysis (*, p < 0.05); (C) FCM analysis for apoptosis effects of 23d. SNU-16 cells were treated with 23d for 72 h with increasing
concentration, followed by staining with 7-AAD/Annexin V; (D) Data analysis for cell apoptosis FCM analysis (**, p < 0.01, ***, p < 0.001); (E) Immunoblotting analysis of cell cycle
related protein expression after 48 h of treatment of 23d; (F) Immunoblotting analysis of apoptosis related protein expression after 72 h of treatment of 23d. Each experiment was
repeated three times.
Table 3
Pharmacokinetic profiles of 23d in rats.
Does (mg/kg)
Cmax (ng/mL)
Tmax (h)
AUC0-t (ng$h/mL)
MRT (h)
T1/2 (h)
F%
3 mg/kg iv
30 mg/kg po
466.20 96.14
130.34 76.51
0.08 0.00
4.33 4.93
634.76 118.47
948.37 370.10
1.38 0.15
5.29 2.41
1.39 0.25
2.65 0.89
14.94%
(Fig. 8A). The final average tumor weight in the treated group was
much lower than that of the vehicle-treated group (Fig. 8C). In
addition, no significant change of body weight (Fig. 8B) or blood
biochemical parameters (Fig. 8D and E) were observed after 23d
treatment. Therefore, 23d can be considered an effective and safe
FGFR inhibitor candidate that is suitable for further drug
development.
novel selective FGFR inhibitors. Systematic exploration and opti-
mization of several substitutions on the pyrido[1,2-a]pyrimidinone
scaffold led to the identification of optimal compound 23d. The
incorporation of pyrido[1,2-a]pyrimidinone in 23d afforded the
pyrazole ring slight rotation while retaining its interaction with
amino acid residue Lue484 of FGFR1. This led to increased potency
against FGFR1 when compared with the lead 7. The small rotation
of pyrazole ring may also be helpful to improve the aqueous solu-
bility of 23d compared with Erdafitinib by disrupting its molecular
planarity. Full kinase spectrum screening indicated that 23d
possessed relatively high selectivity against the FGFR family.
Western blot assay also confirmed that the antitumor activities
5. Conclusion
This article describes the design, synthesis, and biological
evaluation of a series of pyrido[1,2-a]pyrimidinone derivatives as
10

K. Ran, J. Zeng, G. Wan et al.
European Journal of Medicinal Chemistry 220 (2021) 113499
Fig. 8. In-vivo efficacy of 23d in SNU-16 xenograft model. NOD/SCID mice bearing SNU-16 (n ¼ 5) were treated with vehicle control, Erdafitinib (30 mg/kg) and 23d (15 mg/kg and
30 mg/kg) once per day for 4 weeks. (A) Tumor volumes were measured three times per week (***, p < 0.001, two-way ANOVA); (B) Tumor weight after 4 weeks of treatment (**,
p < 0.01, ***, p < 0.001, t-test); (C) Body weight change in SNU-16 xenograft during treatment; (D) CBC analysis after treatment (Unit for WBC, RBC, PLT, HGB is 109/L, 1012/L, 1011/L,
10 g/L, respectively); (E) Blood biochemical analysis after treatment.
were indeed on-target. Furthermore, compound 23d stimulated
tumor cell cycle arrest in the G0/G1 phase and induced apoptosis at
low nanomolar concentration. After oral administration of com-
pound 23d at a daily dose of 30 mg/kg, almost complete tumor
stasis was observed in nude mice bearing SNU-16 human gastric
cancer xenograft after 4 weeks of initial administration. These re-
sults highlight the status of 23d as a promising FGFR inhibitor for
further drug development.
d
8.96 (s, 1H), 8.55 (s, 1H), 8.21 (s, 1H), 7.77 (d, J ¼ 9.2 Hz, 1H), 7.29
(dd, J ¼ 9.2, 2.7 Hz, 1H), 7.16 (d, J ¼ 2.6 Hz, 1H), 6.48 (d, J ¼ 2.2 Hz,
2H), 6.42 (t, J ¼ 2.2 Hz, 1H), 3.94 (s, 3H), 3.89 (t, J ¼ 7.0 Hz, 2H), 3.75
(s, 6H), 2.81 (t, J ¼ 7.0 Hz, 2H), 2.75-2.67 (m, 1H), 0.96 (d, J ¼ 6.2 Hz,
6H). 13C NMR (101 MHz, DMSO‑d6):
d 161.90, 149.57, 148.48, 147.53,
143.98, 140.09, 138.27, 135.96, 131.18, 129.49, 121.80, 120.87, 109.58,
104.35, 97.52, 55.77, 52.86, 48.53, 44.34, 39.32, 23.38. HRMS:
calculated for C25H30N6O2 [(M þ H)þ], 447.2504; found 447.2507.
6. Experimental section
6.1.2. 6-((3,5-dimethoxyphenyl)(2-(isopropylamino)ethyl)amino)-
3-(1-methyl-1H-pyrazol-4-yl)quinazolin-4(3H)-one (7)
6.1. Chemistry
7 was synthesized using the procedure described in the patent
application WO2014/174307A1 [38]. 1H NMR (400 MHz, DMSO‑d6):
Unless otherwise noted, all materials were obtained from
commercial suppliers and used without further purification. The 1H
and 13C NMR spectra were recorded on a Bruker Avance 400
spectrometer at 25 ꢀC using DMSO‑d6 or CDCl3 as the solvent.
d
8.27 (s, 1H), 8.21 (s, 1H), 7.82 (s,1H), 7.61 (d, J ¼ 2.8 Hz,1H), 7.59 (d,
J ¼ 9.0 Hz, 1H), 7.44 (dd, J ¼ 9.0, 2.8 Hz, 1H), 6.33 (d, J ¼ 2.0 Hz, 2H),
6.29 (t, J ¼ 2.0 Hz, 1H), 3.91 (s, 3H), 3.83 (t, J ¼ 7.0 Hz, 2H), 3.71 (s,
6H), 2.76 (t, J ¼ 7.0 Hz, 2H), 2.73-2.67 (m, 1H), 0.95 (d, J ¼ 6.2 Hz,
Chemical shifts (
d
ppm) are reported in ppm relative to Me4Si (in-
6H). 13C NMR (101 MHz, DMSO‑d6):
d 161.82, 159.43, 148.91, 147.36,
ternal standard), coupling constants (J Hz) are reported in hertz,
and peak multiplicity are reported as s (singlet), d (doublet), t
(triplet), q (quartet), m (multiplet), or brs (broad singlet). High
resolution mass analysis is performed on a Waters Q-TOF Premier
mass spectrometer with electron spray ionization (ESI). Thin layer
chromatography (TLC) was performed on 0.20 mm silica gel F-254
plates (Qingdao Haiyang Chemical, China). Visualization of TLC was
accomplished with UV light and/or aqueous potassium perman-
ganate or I2 in silica gel. Column chromatography was performed
using silica gel 60 of 300e400 mesh (Qingdao Haiyang Chemical,
China).
144.37, 141.02, 134.55, 128.77, 127.34, 126.36, 122.82, 120.22, 112.80,
102.23, 95.99, 55.68, 52.59, 48.51, 44.17, 39.99, 23.23. HRMS:
calculated for C25H30N6O3 [(M þ H)þ], 463.2453; found 463.2456.
6.1.3. 7-((3,5-dimethoxyphenyl)(2-(isopropylamino)ethyl)amino)-
3-(2-methoxyethoxy)-4H-pyrido[1,2-a]pyrimidin-4-one (8a)
Step 1: 3-(benzyloxy)-7-bromo-4H-pyrido[1,2-a]pyrimidin-4-
one (11). A mixture of methyl 2-(benzyloxy)acetate (9, 7.0 g,
39 mmol) and Bredereck’s reagent (8.2 g, 47 mmol) was stirred
at 90e100 ꢀC for 12 h. TLC detected that the reaction was
completed. The mixture was concentrated in vacuo. Then the
resulting solid was dissolved in AcOH (90 mL), 5-bromopyridin-
2-amine (6.8 g, 39 mmol) was added. The reaction mixture was
stirred at 120e130 ꢀC for 16 h. Upon completion of the reaction,
AcOH was removed under vacuum. The residue was dissolved in
6.1.1. N1-(3,5-dimethoxyphenyl)-N2-isopropyl-N1-(3-(1-methyl-
1H-pyrazol-4-yl)quinoxalin-6-yl)ethane-1,2-diamine (6)
6 was synthesized using the procedure described in the patent
application WO2011/135376A1 [42]. 1H NMR (400 MHz, DMSO‑d6):
11

K. Ran, J. Zeng, G. Wan et al.
European Journal of Medicinal Chemistry 220 (2021) 113499
ethyl acetate (40 mL). The undissolved solid was collected by
filtration, washed with a small amount of ethyl acetate and dried
in vacuum to give 11 as off-white solid (8.4 g, 65%). 1H NMR
was dissolved in DCM (3 mL), TFA (0.5 mL) was added. The
resulting solution was stirred at room temperature overnight.
After completion (monitored by TLC), the reaction solution was
concentrated under reduced pressure. The residue was purified
on TLC-preparative plates to afford the desired product 8a as
(400 MHz, DMSO‑d6):
d
8.93 (d, J ¼ 2.0 Hz, 1H), 8.25 (s, 1H), 7.83
(dd, J ¼ 9.5, 2.0 Hz, 1H), 7.56 (d, J ¼ 9.5 Hz, 1H), 7.47 (d, J ¼ 7.2 Hz,
2H), 7.40 (t, J ¼ 7.2 Hz, 2H), 7.38-7.34 (m,1H), 5.22 (s, 2H). ESI-MS
m/z 331.2 [MþH]þ.
light yellow oil (31 mg, 80%). 1H NMR (400 MHz, CDCl3):
d 8.60
(s, 1H), 8.15 (s, 1H), 7.42 (d, J ¼ 9.7 Hz, 1H), 7.37 (dd, J ¼ 9.7,
2.0 Hz, 1H), 6.27 (s, 3H), 4.39-4.29 (m, 2H), 3.90 (t, J ¼ 6.6 Hz,
2H), 3.82-3.72 (m, 8H), 3.46 (s, 3H), 2.95 (t, J ¼ 6.6 Hz, 2H), 2.89-
2.82 (m, 1H), 1.08 (d, J ¼ 6.2 Hz, 6H). HRMS: calculated for
Step 2: 3-(benzyloxy)-7-((3,5-dimethoxyphenyl)amino)-4H-
pyrido[1,2-a]pyrimidin-4-one (12). A mixture of 11 (1.67 g,
5 mmol), 3,5-dimethoxyaniline (0.92 g, 6 mmol), Pd2(dba)3
(0.23 g, 0.25 mmol), ( )-BINAP (0.16 g, 0.25 mmol) and cesium
carbonate (3.26 g, 10 mmol) in 1,4-dioxane (30 mL) wꢀas
degassed with N2 for 10 min. The reaction was heated to 95 C
overnight under N2, and then cooled to room temperature. The
mixture was filtrated through a Celite, and the filter cake was
washed with DCM/MeOH (10/1). After concentration of the
filtrate, the residue was purified by column chromatography to
give 12 as yellow solid (1.05 g, 52%). 1H NMR (400 MHz,
C
24H32N4O5 [(M þ H)þ], 457.2447; found 457.2451.
6.1.4. 7-((3,5-dimethoxyphenyl)(2-(isopropylamino)ethyl)amino)-
3-(2-(dimethylamino)ethoxy)-4H-pyrido[1,2-a]pyrimidin-4-one
(8b)
8b was obtained according to the similar procedure of preparing
8a using N,N-dimethylamino chloroethane hydrochloride instead
of 1-bromo-2-methoxyethane. Light yellow solid, yield 79%. 1H
DMSO‑d6):
d
8.72-8.61 (m, 2H), 8.15 (s, 1H), 7.63 (d, J ¼ 2.2 Hz,
1H), 7.61 (s, 1H), 7.47 (d, J ¼ 7.0 Hz, 2H), 7.39 (t, J ¼ 7.0 Hz, 2H),
7.36-7.30 (m, 1H), 6.33 (d, J ¼ 2.0 Hz, 2H), 6.16 (t, J ¼ 2.0 Hz, 1H),
5.20 (s, 2H), 3.74 (s, 6H). ESI-MS m/z 403.4 [MþH]þ.
NMR (400 MHz, CDCl3):
d
8.56 (d, J ¼ 2.4 Hz, 1H), 8.09 (s, 1H), 7.41
(d, J ¼ 9.6 Hz, 1H), 7.36 (dd, J ¼ 9.6, 2.4 Hz, 1H), 6.28 (s, 3H), 4.25 (t,
J ¼ 5.6 Hz, 2H), 3.92 (t, J ¼ 6.8 Hz, 2H), 3.76 (s, 6H), 2.96 (t, J ¼ 6.8 Hz,
2H), 2.90-2.85 (m, 1H), 2.82 (t, J ¼ 5.6 Hz, 2H), 2.38 (s, 6H), 1.09 (d,
Step
3:
3-(benzyloxy)-7-((3,5-dimethoxyphenyl)(2-(iso-
propylamino)ethyl)amino)-4H-pyrido[1,2-a]pyrimidin-4-one
(13). To a stirred mixture of 2-methyltetrahydrofuran (12 mL)
and KOH (0.336 g, 6 mmol) was added water (0.4 mL). Then 12
(1.6 g, 4 mmol)) and tetrabutylammonium bromide (0.322 g,
1 mmol) were added and the mixture was heated at 50 ꢀC for 1 h
while stirring. Then N-(2-chloroethyl)-2-propanamine hydro-
chloride (1.14 g, 7.2 mmol) was added in one portion and the
mixture was stirred for 18 h at 50 ꢀC. When the conversion was
complete, water was added. The mixture was extracted with
ethyl acetate twice. The combined organic layer was washed
with brine, dried over anhydrous sodium sulfate, and concen-
trated in vacuum. The residue was purified via silica gel chro-
matography to afford 13 (1.39 g, 71%). 1H NMR (400 MHz,
J ¼ 6.3 Hz, 6H). 13C NMR (101 MHz, CDCl3):
d 161.84, 147.57, 143.25,
138.24, 138.13, 137.72, 130.75, 125.91, 111.25, 102.44, 96.74, 68.16,
58.20, 55.47, 52.48, 49.08, 45.59, 45.55 43.45, 29.70, 22.43. HRMS:
calculated for C25H35N5O4 [(M þ H)þ], 470.2763; found 470.2766.
6.1.5. 7-((3,5-dimethoxyphenyl)(2-(isopropylamino)ethyl)amino)-
3-(2-morpholinoethoxy)-4H-pyrido[1,2-a]pyrimidin-4-one (8c)
8c was obtained according to the similar procedure of preparing
8a using 4-(2-chloroethyl)morpholine hydrochloride instead of 1-
bromo-2-methoxyethane. Light yellow solid, yield 70%. 1H NMR
(400 MHz, CDCl3):
d
8.54 (d, J ¼ 2.0 Hz, 1H), 8.08 (s, 1H), 7.41 (d,
J ¼ 9.6 Hz, 1H), 7.36 (dd, J ¼ 9.6, 2.0 Hz, 1H), 6.28 (s, 3H), 4.27 (t,
J ¼ 5.5 Hz, 2H), 3.96 (t, J ¼ 6.9 Hz, 2H), 3.77-3.73 (m, 10H), 3.00 (t,
J ¼ 6.9 Hz, 2H), 2.97-2.90 (m, 1H), 2.85 (t, J ¼ 5.5 Hz, 2H), 2.65-2.57
DMSO‑d6):
d 8.52-8.47 (m, 1H), 8.15 (s, 1H), 7.56-7.50 (m, 2H),
7.47 (d, J ¼ 7.2 Hz, 2H), 7.40 (t, J ¼ 7.2 Hz, 2H), 7.34 (t, J ¼ 7.2 Hz,
1H), 6.33 (d, J ¼ 2.0 Hz, 2H), 6.28 (t, J ¼ 2.0 Hz, 1H), 5.20 (s, 2H),
3.80 (t, J ¼ 6.7 Hz, 2H), 3.71 (s, 6H), 2.76 (t, J ¼ 6.7 Hz, 2H), 2.74-
2.66 (m, 1H), 0.96 (d, J ¼ 6.2 Hz, 6H). ESI-MS m/z 488.6 [MþH]þ.
Step 4: tert-butyl (2-((3,5-dimethoxyphenyl)(3-hydroxy-4-oxo-
4H-pyrido[1,2-a]pyrimidin-7-yl)amino)ethyl)(isopropyl)carba-
mate (15). A mixture of 13 (1.3 g, 2.2 mmol) and trifluoroacetic
acid (TFA, 5 mL) was stirred at 90e100 ꢀC for 3 h. Upon
completion of the reaction, the mixture was concentrated in
vacuum. The residue was dissolved in tetrahydrofuran (13 mL).
(Boc)2O (0.5 g, 2.3 mmol) and Et3N (6.4 mL, 46 mmol) were
added, and the reaction mixture was stirred at room tempera-
ture overnight. TLC detected that the reaction was completed.
The mixture was concentrated under vacuum, the residue was
purified by column chromatography to afford 15 as light yellow
(m, 4H), 1.12 (d, J ¼ 6.3 Hz, 6H). 13C NMR (101 MHz, CDCl3):
d 161.87,
153.47, 147.37, 143.24, 138.18, 138.16, 137.84, 130.69, 125.95, 111.10,
102.51, 96.83, 67.57, 66.80, 57.78, 55.49, 55.47, 53.86, 51.93 (2H),
49.30, 43.13, 22.00. HRMS: calculated for C27H37N5O5 [(M þ H)þ],
512.2869; found 512.2867.
6.1.6. 7-((3,5-dimethoxyphenyl)(2-(isopropylamino)ethyl)amino)-
3-((tetrahydro-2H-pyran-4-yl)oxy)-4H-pyrido[1,2-a]pyrimidin-4-
one (8d)
8d was obtained according to the similar procedure of preparing
8a using 4-bromotetrahydro-2H-pyran instead of 1-bromo-2-
methoxyethane. Light yellow solid, yield 62%. 1H NMR (400 MHz,
CDCl3):
d
8.59 (d, J ¼ 2.0 Hz, 1H), 8.13 (s, 1H), 7.45 (d, J ¼ 9.6 Hz, 1H),
7.40 (dd, J ¼ 9.6, 2.0 Hz, 1H), 6.29 (s, 3H), 4.75-4.64 (m, 1H), 4.06-
4.00 (m, 2H), 3.88 (t, J ¼ 6.7 Hz, 2H), 3.77 (s, 6H), 3.57-3.49 (m, 2H),
2.94 (t, J ¼ 6.7 Hz, 2H), 2.86-2.78 (m, 1H), 2.06-1.19 (m, 2H), 1.88-
1.79 (m, 2H), 1.06 (d, J ¼ 6.2 Hz, 6H). HRMS: calculated for
solid (0.56 g, 51%). 1H NMR (400 MHz, CDCl3):
d 8.60-8.43 (m,
1H), 8.20 (s, 1H), 7.57-7.40 (m, 2H), 6.45-6.18 (m, 3H), 4.00-3.83
(m, 3H), 3.77 (s, 6H), 3.39 (brs, 2H),1.44 (s, 9H),1.15 (d, J ¼ 6.4 Hz,
6H). ESI-MS m/z 498.6 [MþH]þ.
C
26H34N4O5 [(M þ H)þ], 483.2603; found 483.2605.
Step 5: 7-((3,5-dimethoxyphenyl)(2-(isopropylamino)ethyl)
amino)-3-(2-methoxyethoxy)-4H-pyrido[1,2-a]pyrimidin-4-
one (8a). A mixture of 22 (50 mg, 0.1 mmol), 1-bromo-2-
6.1.7. tert-butyl 4-(7-((3,5-dimethoxyphenyl)(2-(isopropylamino)
ethyl)amino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-3,6-
dihydropyridine-1(2H)-carboxylate (23a)
methoxyethane (12
mL, 0.12 mmol) and K2CO3 (42 mg,
0.3 mmol) in DMF (4 mL) was stirred at 90e100 ꢀC for 2 h. Upon
completion of the reaction, the reaction mixture was quenched
with water and extracted with ethyl acetate twice. The com-
bined extracts were washed with brine, dried over anhydrous
Na2SO4 and concentrated under vacuum. The resulting residue
Step
1:
(E)-5-(((5-bromopyridin-2-yl)imino)methyl)-2,2-
mixture of triethyl
dimethyl-1,3-dioxane-4,6-dione (18).
A
orthoformate (8.5 g, 0.08 mol) and 2,2-dimethyl-1,3-dioxane-
4,6-dione (11.5 g, 0.08 mol) was heated at 60 ꢀC for 2 h. Then a
solution of 5-bromopyridin-2-amine (17, 13.8 g, 0.08 mol) in
12

K. Ran, J. Zeng, G. Wan et al.
European Journal of Medicinal Chemistry 220 (2021) 113499
EtOH (80 mL) was added slowly, and the resulting reaction
mixture was stirred at 60 ꢀC for additional 2 h. Upon completion
of the reaction, the mixture was cooled to room temperature.
The precipitate was filtered, and the filter cake was washed with
a small amount of EtOH and dried in a vacuum oven to afford 18
0.24 mmol) was added water (0.1 mL). Then intermediate 22a
(77 mg, 0.16 mmol)) and tetrabutylammonium bromide (13 mg,
0.04 mmol) were added and the mixture was heated at 50 ꢀC for
1 h while stirring. Then N-(2-chloroethyl)-2-propanamine hy-
drochloride (46 mg, 0.29 mmol) was added in one portion and
the mixture was stirred for 18 h at 50 ꢀC. When the conversion
was complete, water was added. The mixture was extracted
with ethyl acetate twice. The combined organic layer was
washed with brine, dried over anhydrous sodium sulfate, and
concentrated in vacuum. The residue was purified via silica gel
chromatography to afford 23a as yellow solid (55 mg, 61%). 1H
as white solid (16.6g, 64%). 1H NMR (400 MHz, CDCl3):
d 11.39-
11.24 (m, 1 H), 9.38-9.28 (m, 1 H), 8.47 (d, J ¼ 2.4 Hz, 1H), 7.86
(dd, J ¼ 8.5, 2.4 Hz, 1H), 6.95 (d, J ¼ 8.5 Hz, 1H), 1.76 (s, 6H). ESI-
MS m/z 327.1 [MþH]þ.
Step 2: 7-bromo-4H-pyrido[1,2-a]pyrimidin-4-one (19). Ph2O
was heated to 220 ꢀC, then 18 (16.6 g, 0.051 mol) was slowly
added into the solution. The mixture was stirred at 220 ꢀC for
30 min. TLC detected that the reaction was completed. The
mixture was cooled and purified by column chromatography to
NMR (400 MHz, CDCl3): d 8.72 (s, 1H), 8.24 (s, 1H), 7.53-7.44 (m,
2H), 6.55 (s, 1H), 6.30 (s, 3H), 4.13 (brs, 2H), 3.88 (t, J ¼ 6.6 Hz,
2H), 3.77 (s, 6H), 3.67 (d, J ¼ 5.8 Hz, 2H), 2.93 (t, J ¼ 6.6 Hz, 2H),
2.84-2.76 (m, 1H), 2.62 (brs, 2H), 1.50 (s, 9H), 1.05 (d, J ¼ 6.2 Hz,
afford 19 (11.5 g, 100%). 1H NMR (400 MHz, DMSO‑d6):
d 9.03 (d,
J ¼ 2.2 Hz, 1H), 8.32 (d, J ¼ 6.4 Hz, 1H), 8.08 (dd, J ¼ 9.4, 2.2 Hz,
1H), 7.65 (d, J ¼ 9.4 Hz, 1H), 6.46 (d, J ¼ 6.4 Hz, 1H). ESI-MS m/z
225.0 [MþH]þ.
6H). 13C NMR (101 MHz, CDCl3):
d 161.87, 155.77, 154.82, 149.64,
147.60, 146.41, 138.96, 132.21, 130.66, 125.89, 124.53, 115.89,
111.85, 102.65, 96.82, 79.56, 55.41, 55.39, 53.18, 48.80, 43.81,
29.66, 28.48, 27.38, 26.87, 22.96. HRMS: calculated for
Step 3: 7-bromo-3-iodo-4H-pyrido[1,2-a]pyrimidin-4-one (20).
To a stirred solution of 19 (7.4 g, 0.033 mol) in DMF (50 mL) wꢀas
added NIS (10.4 g, 0.046 mol), the mixture was stirred at 80 C
for 5 h. Upon completion of the reaction, the reaction mixture
was cooled to room temperature, and then added with water
(50 mL) under stirring. The precipitate was collected by filtra-
tion, washed with water dried to a constant weight to afford 20
C
31H41N5O5 [(M þ H)þ], 564.3182; found 564.3179.
6.1.8. 7-((3,5-dimethoxyphenyl)(2-(isopropylamino)ethyl)amino)-
3-(1,2,3,6-tetrahydropyridin-4-yl)-4H-pyrido[1,2-a]pyrimidin-4-
one (23b)
Compound 23a (85 mg, 0.15 mmol) was dissolved in a solution
of HCl/MeOH (4 mL, 4 M in methanol) and the mixture was
continually stirred overnight at room temperature. The mixture
was concentrated and the residue was purified by silica gel chro-
matography (dichloromethane/methanol, v/v, 15:1) to give the title
product 23b as yellow solid (38 mg, 55%). 1H NMR (400 MHz,
(12.1 g,104%). 1H NMR (400 MHz, DMSO‑d6):
d
9.02 (d, J ¼ 2.0 Hz,
1H), 8.75 (s, 1H), 8.13 (dd, J ¼ 9.4, 2.0 Hz, 1H), 7.67 (d, J ¼ 9.4 Hz,
1H). ESI-MS m/z 350.9 [MþH]þ.
Step 4: tert-butyl 4-(7-bromo-4-oxo-4H-pyrido[1,2-a]pyr-
imidin-3-yl)-3,6-dihydropyridine-1(2H)-carboxylate (21a). 20
(14.1 g, 40 mmol) and tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate
(12.4 g, 40 mmol), Na2CO3 (8.5 g, 6 mmol) were dissolved in
dioxane (100 mL) and H2O (25 mL). The suspension was
degassed under nitrogen bubbling for 10 min before
Pd(dppf)2Cl2 (2.9 g, 0.15 mmol) was added. The reaction mixture
was heated to 100 ꢀC for 5 h and then diluted with ethyl acetate.
The solution was washed with water and brine successively,
dried over anhydrous sodium sulfate, and concentrated in vac-
uum. The residue was purified via silica gel chromatography to
CDCl3):
d
8.72 (d, J ¼ 1.6 Hz,1H), 8.25 (s,1H), 7.51-7.44 (m, 2H), 6.64-
6.59 (m, 1H), 6.29 (s, 3H), 3.87 (t, J ¼ 6.7 Hz, 2H), 3.77 (s, 6H), 3.63-
3.58 (m, 2H), 3.15 (t, J ¼ 5.6 Hz, 2H), 2.93 (t, J ¼ 6.7 Hz, 2H), 2.83-
2.75 (m,1H), 2.57 (s, 2H), 1.04 (d, J ¼ 6.2 Hz, 6H). 13C NMR (101 MHz,
CDCl3):
d 161.87, 155.89, 149.62, 147.73, 146.41, 138.82, 132.23,
130.74, 127.06, 125.91, 116.50, 112.11, 102.52, 96.72, 55.44, 55.42,
53.24, 48.81, 45.42, 43.88, 43.08, 29.68, 27.56, 23.01. HRMS: calcu-
lated for C26H33N5O3 [(M þ H)þ], 464.2657; found 464.2655.
afford 21a (10.7 g, 66%). 1H NMR (400 MHz, CDCl3):
d 9.23 (d,
6.1.9. 7-((3,5-dimethoxyphenyl)(2-(isopropylamino)ethyl)amino)-
3-(pyridin-3-yl)-4H-pyrido[1,2-a]pyrimidin-4-one (23c)
J ¼ 2.0 Hz, 1H), 8.31 (s, 1H), 7.72 (dd, J ¼ 9.4, 2.0 Hz, 1H), 7.53 (d,
J ¼ 9.4 Hz, 1H), 6.61 (s, 1H), 4.18-4.11 (m, 2H), 3.66 (t, J ¼ 5.5 Hz,
2H), 2.63-2.54 (m, 2H), 1.50 (s, 9H). ESI-MS m/z 406.1 [MþH]þ.
Step 5: tert-butyl 4-(7-((3,5-dimethoxyphenyl)amino)-4-oxo-
4H-pyrido[1,2-a]pyrimidin-3-yl)-3,6-dihydropyridine-1(2H)-
carboxylate (22a). A mixture of 21a (10.7 g, 26.3 mmol), 3,5-
dimethoxyaniline (4.8 g, 31.6 mmol), Pd2(dba)3 (2.4 g,
2.63 mmol), ( )-BINAP (2.5 g, 3.95 mmol) and cesium carbonate
(12.9 g, 39.5 mmol) in anhydrous toluene (120 mL) was
degassed with N2 for 10 min. The reaction was heated to 100 ꢀC
overnight under N2, and then cooled to room temperature. The
mixture was filtrated through a Celite, and the filter cake was
washed with DCM/MeOH (10/1). After concentration of the
filtrate, the residue was purified by column chromatography to
23c was obtained according to the similar procedure of pre-
paring 23a using pyridin-3-ylboronic acid instead of tert-butyl 4-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-
dihydropyridine-1(2H)-carboxylate. Yellow solid, yield 42%. 1H
NMR (400 MHz, CDCl3):
d
8.98 (d, J ¼ 1.8 Hz, 1H), 8.78 (d, J ¼ 2.0 Hz,
1H), 8.58 (dd, J ¼ 4.8, 1.8 Hz, 1H), 8.48 (s, 1H), 8.26-8.21 (m, 1H), 7.58
(dd, J ¼ 9.6, 2.4 Hz, 1H), 7.54 (d, J ¼ 9.6 Hz, 1H), 7.41-7.36 (m, 1H),
6.33 (s, 3H), 3.92 (t, J ¼ 6.7 Hz, 2H), 3.78 (s, 6H), 2.96 (t, J ¼ 6.7 Hz,
2H), 2.88-2.80 (m, 1H), 1.07 (d, J ¼ 6.3 Hz, 6H). HRMS: calculated for
C
26H29N5O3 [(M þ H)þ], 460.2344; found 460.2340.
6.1.10. 7-((3,5-dimethoxyphenyl)(2-(isopropylamino)ethyl)amino)-
3-(1-methyl-1H-pyrazol-4-yl)-4H-pyrido[1,2-a]pyrimidin-4-one
(23d)
give 22a (7.1 g, 56%). 1H NMR (400 MHz, CDCl3):
d 8.89 (d,
J ¼ 2.2 Hz, 1H), 8.25 (s, 1H), 7.65 (dd, J ¼ 9.6, 2.2 Hz, 1H), 7.59 (d,
J ¼ 9.6 Hz, 1H), 6.55 (s, 1H), 6.25 (d, J ¼ 2.0 Hz, 2H), 6.17 (t,
J ¼ 2.0 Hz, 1H), 6.10 (s, 1H), 4.15-4.09 (m, 2H), 3.77 (s, 6H), 3.65
(d, J ¼ 5.0 Hz, 2H), 2.67-2.56 (m, 2H), 1.49 (s, 9H). ESI-MS m/z
479.2 [MþH]þ.
23d was obtained according to the similar procedure of pre-
paring
23a
using
1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)-1H-pyrazole instead of tert-butyl 4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-
carboxylate. Yellow solid, yield 80%. 1H NMR (400 MHz, DMSO‑d6):
Step
6:
tert-butyl
4-(7-((3,5-dimethoxyphenyl)(2-(iso-
d
8.72 (s, 1H), 8.66 (d, J ¼ 2.2 Hz, 1H), 8.37 (s, 1H), 8.12 (s, 1H), 7.66
propylamino)ethyl)amino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-
3-yl)-3,6-dihydropyridine-1(2H)-carboxylate (23a). To a stirred
mixture of 2-methyltetrahydrofuran (2 mL) and KOH (14 mg,
(dd, J ¼ 9.6, 2.2 Hz, 1H), 7.61 (d, J ¼ 9.6 Hz, 1H), 6.38 (d, J ¼ 2.0 Hz,
2H), 6.33-6.29 (m, 1H), 3.90 (s, 3H), 3.82 (t, J ¼ 6.7 Hz, 2H), 3.72 (s,
6H), 2.78 (t, J ¼ 6.7 Hz, 2H), 2.75-2.67 (m, 1H), 0.96 (d, J ¼ 6.2 Hz,
13

K. Ran, J. Zeng, G. Wan et al.
European Journal of Medicinal Chemistry 220 (2021) 113499
6H). 13C NMR (101 MHz, DMSO‑d6):
d
161.86, 154.50, 148.47, 148.10,
preparing 23a using 1-propyl-4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)-1H-pyrazole instead of tert-butyl 4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-
carboxylate. Yellow solid, yield 42%. 1H NMR (400 MHz, CDCl3):
145.77, 139.17, 136.91, 132.54, 129.57, 126.58, 115.69, 113.27, 108.43,
101.86, 96.29, 55.75, 52.94, 48.52, 44.03, 39.05, 23.33. HRMS
calculated for C25H30N6O3 [(M þ H)þ], 463.2453; found, 463.2454.
d
8.73 (d, J ¼ 2.0 Hz, 1H), 8.62 (s, 1H), 8.32 (s, 1H), 8.00 (s, 1H), 7.50
6.1.11. 7-((3,5-dimethoxyphenyl)(2-(isopropylamino)ethyl)amino)-
3-(1,5-dimethyl-1H-pyrazol-4-yl)-4H-pyrido[1,2-a]pyrimidin-4-
one (23e)
23e was obtained according to the similar procedure of pre-
paring 23a using 1,5-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)-1H-pyrazole instead of tert-butyl 4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-
carboxylate. Yellow solid, yield 21%. 1H NMR (400 MHz, CDCl3):
(d, J ¼ 9.6 Hz, 1H), 7.46 (dd, J ¼ 9.6, 2.0 Hz, 1H), 6.33-6.27 (m, 3H),
4.14 (t, J ¼ 7.0 Hz, 2H), 3.95 (t, J ¼ 6.7 Hz, 2H), 3.76 (s, 6H), 2.99 (t,
J ¼ 6.7 Hz, 2H), 2.93-2.85 (m, 1H), 1.98-1.90 (m, 2H), 1.10 (d,
J ¼ 6.4 Hz, 6H), 0.96 (t, J ¼ 7.4 Hz, 3H). HRMS: calculated for
C
27H34N6O3 [(M þ H)þ], 491.2766; found 491.2767.
6.1.16. 7-((3,5-dimethoxyphenyl)(2-(isopropylamino)ethyl)amino)-
3-(1-(2-fluoroethyl)-1H-pyrazol-4-yl)-4H-pyrido[1,2-a]pyrimidin-
4-one (23j)
d
8.71 (s, 1H), 8.49 (s, 1H), 8.03 (s, 1H), 7.51-7.42 (m, 2H), 6.34-6.26
(m, 3H), 3.96 (t, J ¼ 6.6 Hz, 2H), 3.86 (s, 3H), 3.75 (s, 6H), 3.03-2.90
23j was obtained according to the similar procedure of prepar-
ing 23a using 1-(2-fluoroethyl)-4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)-1H-pyrazole instead of tert-butyl 4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-
carboxylate. Yellow solid, yield 37%. 1H NMR (400 MHz, CDCl3):
(m, 3H), 2.08 (s, 3H), 1.15 (d, J ¼ 6.2 Hz, 6H). HRMS: calculated for
C
26H32N6O3 [(M þ H)þ], 477.2610; found 477.2611.
6.1.12. 7-((3,5-dimethoxyphenyl)(2-(isopropylamino)ethyl)amino)-
3-(1,3-dimethyl-1H-pyrazol-4-yl)-4H-pyrido[1,2-a]pyrimidin-4-
one (23f)
23f was obtained according to the similar procedure of pre-
paring 23a using 1,3-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)-1H-pyrazole instead of tert-butyl 4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-
carboxylate. Yellow solid, yield 54%. 1H NMR (400 MHz, CDCl3):
d
8.74 (d, J ¼ 2.0 Hz, 1H), 8.62 (s, 1H), 8.41 (s, 1H), 8.07 (s, 1H), 7.53-
7.45 (m, 2H), 6.34-6.28 (m, 3H), 4.88 (t, J ¼ 4.8 Hz, 1H), 4.76 (t,
J ¼ 4.8 Hz, 1H), 4.52 (t, J ¼ 4.8 Hz, 1H), 4.45 (t, J ¼ 4.8 Hz, 1H), 3.90 (t,
J ¼ 6.7 Hz, 2H), 3.77 (s, 6H), 2.95 (t, J ¼ 6.7 Hz, 2H), 2.85-2.78 (m,
1H), 1.06 (d, J ¼ 6.2 Hz, 6H). HRMS: calculated for C26H31FN6O3
[(M þ H)þ], 495.2516; found 495.2516.
d
8.71 (s, 1H), 8.47 (s, 1H), 8.05 (s, 1H), 7.48 (s, 2H), 6.32-6.25 (m,
6.1.17. 7-((3,5-dimethoxyphenyl)(2-(isopropylamino)ethyl)amino)-
3-(1-(2-methoxyethyl)-1H-pyrazol-4-yl)-4H-pyrido[1,2-a]
pyrimidin-4-one (23k)
23k was obtained according to the similar procedure of pre-
paring 23a using 1-(2-methoxyethyl)-4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)-1H-pyrazole instead of tert-butyl 4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-
carboxylate. Yellow solid, yield 39%. 1H NMR (400 MHz, CDCl3):
3H), 3.98 (t, J ¼ 6.6 Hz, 2H), 3.89 (s, 3H), 3.76 (s, 6H), 3.04-2.91 (m,
3H), 2.44 (s, 3H), 1.12 (d, J ¼ 6.2 Hz, 6H). 13C NMR (101 MHz, CDCl3):
d
161.91, 155.96, 149.67, 147.35,146.19,145.79, 138.77, 132.50, 131.79,
126.08, 112.55, 111.96, 110.19, 102.57, 96.86, 55.48, 55.44, 52.03,
49.29, 43.19, 38.71, 22.05, 14.16. HRMS: calculated for C26H32N6O3
[(M þ H)þ], 477.2610; found 477.2610.
6.1.13. 7-((3,5-dimethoxyphenyl)(2-(isopropylamino)ethyl)amino)-
3-(1H-pyrazol-4-yl)-4H-pyrido[1,2-a]pyrimidin-4-one (23g)
d
8.75 (d, J ¼ 2.0 Hz, 1H), 8.61 (s, 1H), 8.37 (s, 1H), 8.05 (s, 1H), 7.53-
7.43 (m, 2H), 6.35-6.25 (m, 3H), 4.36 (t, J ¼ 5.4 Hz, 2H), 3.93-3.88 (m,
2H), 3.81 (t, J ¼ 5.4 Hz, 2H), 3.77 (s, 6H), 3.36 (s, 3H), 2.96 (t,
J ¼ 6.7 Hz, 2H), 2.87-2.79 (m, 1H), 1.06 (d, J ¼ 6.2 Hz, 6H). HRMS:
calculated for C27H34N6O4 [(M þ H)þ], 507.2716; found 507.2713.
Compound 23l (56 mg, 0.11 mmol) was dissolved in hydro-
chloric acid solution (4 M in methanol, 3 mL) and the mixture was
continually stirred overnight at room temperature. The mixture
was concentrated and the residue was purified by silica gel chro-
matography (dichloromethane/methanol, v/v, 15:1) to give the title
product 23g as yellow solid (35 mg, 71%). 1H NMR (400 MHz,
6.1.18. 7-((3,5-dimethoxyphenyl)(2-(isopropylamino)ethyl)amino)-
3-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)-4H-pyrido[1,2-
a]pyrimidin-4-one (23l)
CDCl3): d 8.73 (s, 1H), 8.58 (s, 1H), 8.47 (s, 1H),8.06 (s, 1H), 7.49-7.35
(m, 2H), 6.39-6.26 (m, 3H), 3.98 (t, J ¼ 6.7 Hz, 2H), 3.79 (s, 6H), 3.06-
23l was obtained according to the similar procedure of prepar-
2.90 (m, 3H), 1.19 (d, J ¼ 6.2 Hz, 6H). HRMS: calculated for
ing
tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole instead of tert-
butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-
dihydropyridine-1(2H)-carboxylate. Yellow solid, yield 55%. 1H
NMR (400 MHz, DMSO‑d6):
8.78 (s, 1H), 8.65 (d, J ¼ 2.4 Hz, 1H),
23a
using
1-(tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-
C
24H28N6O3 [(M þ H)þ], 449.2297; found 449.2302.
6.1.14. 7-((3,5-dimethoxyphenyl)(2-(isopropylamino)ethyl)amino)-
3-(1-ethyl-1H-pyrazol-4-yl)-4H-pyrido[1,2-a]pyrimidin-4-one
(23h)
d
8.54 (s, 1H), 8.20 (s, 1H), 7.68 (dd, J ¼ 9.6, 2.4 Hz, 1H), 7.63 (d,
J ¼ 9.6 Hz, 1H), 6.39 (d, J ¼ 2.0 Hz, 2H), 6.35-6.28 (m, 1H), 5.49-5.43
(m, 1H), 3.98-3.91 (m, 1H), 3.83 (t, J ¼ 6.6 Hz, 2H), 3.71 (s, 6H), 3.70-
3.62 (m, 1H), 2.79 (t, J ¼ 6.6 Hz, 2H), 2.75-2.66 (m, 1H), 2.15-2.05 (m,
1H), 1.98-1.92 (m, 1H), 1.74-1.67 (m, 1H), 1.60-1.53 (m, 1H), 0.96 (d,
J ¼ 6.2 Hz, 6H), 0.88-0.83 (m, 2H). HRMS: calculated for C29H36N6O4
[(M þ H)þ], 533.2872; found 533.2875.
23h was obtained according to the similar procedure of pre-
paring
23a
using
1-ethyl-4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)-1H-pyrazole instead of tert-butyl 4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-
carboxylate. Yellow solid, yield 53%. 1H NMR (400 MHz, DMSO‑d6):
d
8.78 (s, 1H), 8.69 (s, 1H), 8.39 (s, 1H), 8.22 (s, 1H), 7.76 (dd, J ¼ 9.6,
2.2 Hz, 1H), 7.70 (d, J ¼ 9.6 Hz, 1H), 6.42-6.33 (m, 3H), 3.98-3.89 (m,
5H), 3.81 (t, J ¼ 6.7 Hz, 2H), 3.74 (s, 6H), 2.74 (t, J ¼ 6.7 Hz, 2H), 2.75-
2.67 (m, 1H), 1.32-1.28 (m, 3H), 1.02 (d, J ¼ 6.2 Hz, 6H). HRMS:
calculated for C26H32N6O3 [(M þ H)þ], 477.2610; found 477.2611.
6.1.19. 7-((3,5-dimethoxyphenyl)(2-(isopropylamino)ethyl)amino)-
3-(1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl)-4H-pyrido[1,2-
a]pyrimidin-4-one (23m)
23m was obtained according to the similar procedure of pre-
6.1.15. 7-((3,5-dimethoxyphenyl)(2-(isopropylamino)ethyl)amino)-
3-(1-propyl-1H-pyrazol-4-yl)-4H-pyrido[1,2-a]pyrimidin-4-one
(23i)
paring
tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole instead of tert-
butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-
dihydropyridine-1(2H)-carboxylate. Yellow solid, yield 42%. 1H
23a
using
1-(tetrahydro-2H-pyran-4-yl)-4-(4,4,5,5-
23i was obtained according to the similar procedure of
14

K. Ran, J. Zeng, G. Wan et al.
European Journal of Medicinal Chemistry 220 (2021) 113499
NMR (400 MHz, CDCl3):
d
8.73 (d, J ¼ 2.0 Hz, 1H), 8.63 (s, 1H), 8.40
6.1.21. 7-((3,5-dimethoxyphenyl)(2-(isopropylamino)ethyl)amino)-
3-(1-(2-hydroxy-2-methylpropyl)-1H-pyrazol-4-yl)-4H-pyrido[1,2-
a]pyrimidin-4-one (27b)
(s,1H), 8.02 (s,1H), 7.54-7.45 (m, 2H), 6.31 (s, 3H), 4.46-4.36 (m,1H),
4.17-4.10 (m, 2H), 3.92 (t, J ¼ 6.7 Hz, 2H), 3.77 (s, 6H), 3.60-3.52 (m,
2H), 2.96 (t, J ¼ 6.7 Hz, 2H), 2.88-2.81 (m, 1H), 2.19 -2.12 (m, 4H),
1.07 (d, J ¼ 6.2 Hz, 6H). HRMS: calculated for C29H36N6O4
[(M þ H)þ], 533.2872; found 533.2874.
27b was obtained according to the similar procedure of pre-
paring 27a using 2,2-dimethyloxirane instead of (2-
bromoethoxy)(tert-butyl)dimethylsilane. Yellow solid, yield 28%.
1H NMR (400 MHz, CDCl3):
d 8.74 (s, 1H), 8.62 (s, 1H), 8.37 (s, 1H),
8.06 (s, 1H), 7.54-7.46 (m, 2H), 6.31 (s, 3H), 4.13 (s, 2H), 3.90 (t,
J ¼ 6.6 Hz, 2H), 3.77 (s, 7H), 2.95 (t, J ¼ 6.6 Hz, 2H), 2.86-2.78 (m,
1H), 1.25 (s, 6H), 1.06 (d, J ¼ 6.2 Hz, 6H). HRMS: calculated for
6.1.20. 7-((3,5-dimethoxyphenyl)(2-(isopropylamino)ethyl)amino)-
3-(1-(2-hydroxyethyl)-1H-pyrazol-4-yl)-4H-pyrido[1,2-a]
pyrimidin-4-one (27a)
C
28H36N6O4 [(M þ H)þ], 521.2872; found 521.2873.
Step 1: benzyl (2-((3,5-dimethoxyphenyl)(4-oxo-3-(1-(tetrahy-
dro-2H-pyran-2-yl)-1H-pyrazol-4-yl)-4H-pyrido[1,2-a]pyr-
6.1.22. 7-((3,5-dimethoxyphenyl)(2-(isopropylamino)ethyl)amino)-
3-(1-(2-(dimethylamino)ethyl)-1H-pyrazol-4-yl)-4H-pyrido[1,2-a]
pyrimidin-4-one (27c)
imidin-7-yl)amino)ethyl)(isopropyl)carbamate (24). To
a
mixture of 23l (1.76 g, 3.3 mmol) and DIEA (2.7 mL, 16.5 mmol)
in DCM (20 mL) was added benzyl chloroformate (1.9 mL,
13.2 mmol) dropwise at 0 ꢀC. The mixture was stirred at room
temperature for 2 h. Upon completion of the reaction, the re-
action mixture was quenched with water and extracted with
DCM twice. The combined extracts were washed with brine,
dried over anhydrous Na2SO4 and concentrated under vacuum.
The residue was purified via silica gel chromatography to give 24
27c was obtained according to the similar procedure of pre-
paring 27a using N,N-dimethylamino chloroethane hydrochloride
instead of (2-bromoethoxy)(tert-butyl)dimethylsilane. Yellow solid,
yield 33%. 1H NMR (400 MHz, CDCl3):
d
8.74 (d, J ¼ 2.2 Hz, 1H), 8.61
(s, 1H), 8.37 (s, 1H), 8.01 (s, 1H), 7.51 (d, J ¼ 9.6 Hz, 1H), 7.46 (dd,
J ¼ 9.6, 2.2 Hz, 1H), 6.31 (s, 3H), 4.30 (t, J ¼ 6.8 Hz, 2H), 3.90 (t,
J ¼ 6.7 Hz, 2H), 3.77 (s, 6H), 2.95 (t, J ¼ 6.7 Hz, 2H), 2.86-2.78 (m,
3H), 2.30 (s, 6H), 1.06 (d, J ¼ 6.2 Hz, 6H). 13C NMR (101 MHz, CDCl3):
as yellow solid (1.5 g, 68%). 1H NMR (400 MHz, CDCl3):
d
8.90-
d 161.92, 155.16, 148.07, 147.58, 145.55, 139.09, 136.51, 131.47, 129.17,
8.72 (m, 1H), 8.61 (s, 1H), 8.58-8.46 (m, 1H), 8.08 (s, 1H), 7.68-
7.49 (m, 1H), 7.46-7.27 (m, 5H), 6.43-6.13 (m, 3H), 5.43 (dd,
J ¼ 9.6, 2.2 Hz, 1H), 5.31-5.18 (m, 2H), 4.43-4.25 (m, 1H), 4.14-
4.04 (m, 1H), 4.04-3.84 (m, 2H), 3.82-3.63 (m, 7H), 3.54-3.36 (m,
2H), 2.26-2.15 (m, 1H), 2.15-1.99 (m, 2H), 1.77-1.55 (m, 3H), 1.13
(d, J ¼ 6.2 Hz, 6H). ESI-MS m/z 666.8 [MþH]þ.
126.09, 115.39, 111.59, 109.22, 102.83, 97.00, 59.13, 55.49, 55.47,
53.07, 50.42, 48.90, 45.61, 45.58, 43.83, 22.89. HRMS: calculated for
C
28H37N7O3 [(M þ H)þ], 520.3032; found 520.3033.
6.1.23. 7-((3,5-dimethoxyphenyl)(2-(isopropylamino)ethyl)amino)-
3-(1-(2-morpholinoethyl)-1H-pyrazol-4-yl)-4H-pyrido[1,2-a]
pyrimidin-4-one (27d)
27d was obtained according to the similar procedure of pre-
paring 27a using 4-(2-chloroethyl)morpholine hydrochloride
instead of (2-bromoethoxy)(tert-butyl)dimethylsilane. Yellow solid,
Step 2: benzyl (2-((3,5-dimethoxyphenyl)(4-oxo-3-(1H-pyr-
azol-4-yl)-4H-pyrido[1,2-a]pyrimidin-7-yl)amino)ethyl)(-
isopropyl)carbamate (25). A mixture of 24 (1.5 g, 2.2 mmol) and
HCl/MeOH (4 M, 5.5 mL, 22 mmol) was stirred at room tem-
perature overnight. Upon completion of the reaction, the reac-
tion mixture was quenched with saturated aqueous NaHCO3,
then extracted with DCM twice. The combined extracts were
washed with brine, dried over anhydrous Na2SO4 and concen-
trated under vacuum to afford the crude product 25 as yellow
solid (1.4 g, 109%), which was used in next step without further
purification. ESI-MS m/z 582.7 [MþH]þ.
yield 41%. 1H NMR (400 MHz, CDCl3):
d
8.74 (d, J ¼ 2.0 Hz, 1H), 8.61
(s, 1H), 8.38 (s, 1H), 8.01 (s, 1H), 7.51 (d, J ¼ 9.6 Hz, 1H), 7.47 (dd,
J ¼ 9.6, 2.0 Hz, 1H), 6.31 (s, 3H), 4.31 (t, J ¼ 6.7 Hz, 2H), 3.92 (t,
J ¼ 6.6 Hz, 2H), 3.77 (s, 6H), 3.73-3.69 (m, 4H), 2.96 (t, J ¼ 6.7 Hz,
2H), 2.89-2.81 (m, 3H), 2.53-2.48 (m, 4H),1.07 (d, J ¼ 6.2 Hz, 6H). 13
C
NMR (101 MHz, CDCl3):
d 161.93, 155.14, 148.05, 147.51, 145.57,
139.11, 136.46, 131.49, 129.26, 126.10, 115.43, 111.61, 109.15, 102.84,
97.00, 66.93, 58.26, 55.49, 55.46, 53.68, 52.92, 49.79, 48.97, 43.72,
22.76. HRMS: calculated for C30H39N7O4 [(M þ H)þ], 562.3138;
found 562.3138.
Step 3: 7-((3,5-dimethoxyphenyl)(2-(isopropylamino)ethyl)
amino)-3-(1-(2-hydroxyethyl)-1H-pyrazol-4-yl)-4H-pyrido[1,2-
a]pyrimidin-4-one (27a). A mixture of 25 (87 mg, 0.15 mmol),
(2-bromoethoxy)(tert-butyl)dimethylsilane (36 mg, 0.15 mmol)
and Cs2CO3 (98 mg, 0.3 mmol) in DMF (5 mL) was stirred at 50 ꢀC
overnight. After completion (monitored by TLC), the reaction
mixture was quenched with water, then extracted with ethyl
acetate twice. The combined extracts were washed with water
and brine successively, dried over anhydrous Na2SO4 and
concentrated under vacuum to give 26a. The residue was dis-
solved in AcOH (2 mL), HBr (48%, 0.5 mL) was added. The
mixture was stirred at room temperature overnight. After
completion (monitored by TLC), the solvent was removed under
reduced pressure. The residue was basified with saturated
aqueous NaHCO3, then extracted with DCM twice. The com-
bined extracts were washed with brine, dried over anhydrous
Na2SO4 and concentrated. The residue was purified on TLC-
preparative plates to afford the desired product 27a as yellow
6.1.24. 7-((3,5-dimethoxyphenyl)(2-(isopropylamino)ethyl)amino)-
3-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)-4H-pyrido[1,2-a]pyrimidin-
4-one (27e)
27e was obtained according to the similar procedure of pre-
paring 27a using tert-butyl 4-iodopiperidine-1-carboxylate instead
of (2-bromoethoxy)(tert-butyl)dimethylsilane. Yellow solid, yield
25%. 1H NMR (400 MHz, CDCl3):
d
8.75 (d, J ¼ 2.0 Hz, 1H), 8.62 (s,
1H), 8.39 (s, 1H), 8.02 (s, 1H), 7.51 (d, J ¼ 9.6 Hz,1H), 7.47 (dd, J ¼ 9.6,
2.0 Hz, 1H), 6.38-6.25 (m, 3H), 4.34-4.25 (m, 1H), 3.90 (t, J ¼ 6.6 Hz,
2H), 3.77 (s, 6H), 3.35-3.25 (m, 2H), 2.95 (t, J ¼ 6.6 Hz, 2H), 2.86-2.74
(m, 3H), 2.27-2.18 (m, 2H), 2.01-1.97 (m, 2H),1.06 (d, J ¼ 6.2 Hz, 6H).
HRMS: calculated for C29H37N7O3 [(M þ H)þ], 532.3032; found
532.3035.
solid (15 mg, 20%). 1H NMR (400 MHz, CDCl3):
d
8.71 (s, 1H), 8.56
6.1.25. 3-(1-(azetidin-3-yl)-1H-pyrazol-4-yl)-7-((3,5-
dimethoxyphenyl)(2-(isopropylamino)ethyl)amino)-4H-pyrido[1,2-
a]pyrimidin-4-one (27f)
27f was obtained according to the similar procedure of pre-
paring 27a using tert-butyl 3-iodoazetidine-1-carboxylate instead
of (2-bromoethoxy)(tert-butyl)dimethylsilane. Yellow solid, yield
(s, 1H), 8.35 (s, 1H), 8.02 (s, 1H), 7.54-7.45 (m, 2H), 6.37-6.26 (m,
3H), 4.35-4.28 (m, 2H), 4.10-4.02 (m, 2H), 3.91 (t, J ¼ 6.5 Hz, 2H),
3.77 (s, 6H), 2.96 (t, J ¼ 6.5 Hz, 2H), 2.89-2.81 (m, 1H), 1.08 (d,
J ¼ 6.2 Hz, 6H). HRMS: calculated for C26H32N6O4 [(M þ H)þ],
493.2559; found 493.2558.
15

K. Ran, J. Zeng, G. Wan et al.
European Journal of Medicinal Chemistry 220 (2021) 113499
30%. 1H NMR (400 MHz, CDCl3):
d
8.72 (s, 1H), 8.62 (s, 1H), 8.43 (s,
0.6 mmol) in DCM (5 mL) was added acetyl chloride (32 mL,
1H), 8.09 (s, 1H), 7.51-7.44 (m, 2H), 6.35-6.25 (m, 3H), 5.00-4.91 (m,
1H), 4.48-4.32 (m, 2H), 3.95-3.89 (m, 2H), 3.80-3.72 (m, 8H), 3.06-
2.91 (m, 3H), 1.11 (d, J ¼ 6.2 Hz, 6H). HRMS: calculated for
0.45 mmol) dropwise at 0 ꢀC. Then the reaction mixture was stirred
at room temperature for 1 h. Following this time the reaction
mixture was diluted water and then extracted with DCM twice. The
combined extracts were washed with 0.5 N HCl (aq), dried over
anhydrous Na2SO4, and concentrated under reduced pressure. The
residue was uptaken in AcOH (3 mL) and HBr (48% in water, 0.5 mL)
was added. The mixture was stirred at room temperature over-
night. After completion (monitored by TLC), the solvent was
removed under reduced pressure. The residue was basified with
ammonia (7 M in methanol) and purified by column chromatog-
raphy to give the intermediate 29c as yellow solid (41 mg, 48%). 1H
C
27H33N7O3 [(M þ H)þ], 504.2719; found 504.2723.
6.1.26. 7-((3,5-dimethoxyphenyl)(2-(isopropylamino)ethyl)amino)-
3-(1-(1-methylpiperidin-4-yl)-1H-pyrazol-4-yl)-4H-pyrido[1,2-a]
pyrimidin-4-one (29a)
Step 1: benzyl (2-((3,5-dimethoxyphenyl)(4-oxo-3-(1-(piper-
idin-4-yl)-1H-pyrazol-4-yl)-4H-pyrido[1,2-a]pyrimidin-7-yl)
amino)ethyl)(isopropyl)carbamate (28a). To a solution of tert-
NMR (400 MHz, CDCl3):
d
8.72 (d, J ¼ 2.0 Hz, 1H), 8.61 (s, 1H), 8.38
butyl
4-(4-(7-((2-(((benzyloxy)carbonyl)(isopropyl)amino)
(s, 1H), 8.01 (s, 1H), 7.51 (d, J ¼ 9.6 Hz, 1H), 7.47 (dd, J ¼ 9.6, 2.0 Hz,
1H), 6.31 (s, 3H), 4.77-4.71 (m, 1H), 4.46- 4.36 (m, 1H), 4.01-3.89 (m,
3H), 3.77 (s, 6H), 3.31-3.22 (m, 1H), 2.97 (t, J ¼ 6.6 Hz, 2H), 2.89
-2.75 (m, 2H), 2.29-2.18 (m, 2H), 2.15 (s, 3H), 2.08-1.98 (m, 2H), 1.08
ethyl)(3,5-dimethoxyphenyl)amino)-4-oxo-4H-pyrido[1,2-a]
pyrimidin-3-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate
(26e, 291 mg, 0.38 mmol) in DCM (5 mL) was added TFA (1 mL).
The mixture was stirred at room temperature for 2 h, and then
concentrated under vacuum. The residue was basified with
ammonia (7 M in methanol) and purified by column chroma-
tography to give the intermediate 28a as yellow solid (220 mg,
87%). ESI-MS m/z 665.8 [MþH]þ.
(d, J ¼ 6.2 Hz, 6H). 13C NMR (101 MHz, CDCl3):
d 169.01, 161.93,
155.11, 148.06, 147.40, 145.54, 139.18, 136.26, 131.44, 126.61, 126.10,
115.40, 111.40, 108.94, 102.92, 97.07, 59.06, 55.49, 55.45, 52.77,
49.00, 45.31, 43.63, 40.51, 32.79, 31.99, 22.66, 21.44. HRMS: calcu-
lated for C31H39N7O4 [(M þ H)þ], 574.3138; found 574.3140.
Step 2: 7-((3,5-dimethoxyphenyl)(2-(isopropylamino)ethyl)
amino)-3-(1-(1-methylpiperidin-4-yl)-1H-pyrazol-4-yl)-4H-
pyrido[1,2-a]pyrimidin-4-one (29a). To a solution of 28a (99 mg,
0.15 mmol) in acetonitrile (5 mL) was added formaldehyde so-
6.1.29. 7-((3,5-dimethoxyphenyl)(2-(isopropylamino)ethyl)amino)-
3-(1-(1-(methylsulfonyl)piperidin-4-yl)-1H-pyrazol-4-yl)-4H-
pyrido[1,2-a]pyrimidin-4-one (29d)
lution (37 wt % in water, 61 mg, 0.75 mmol), acetic acid (20
m
L)
29d was obtained according to the similar procedure of pre-
paring 29c using methanesulfonyl chloride instead of acetyl chlo-
and sodium triacetoxyborohydride (96 mg, 0.45 mmol). The
reaction mixture was stirred for 16 h at room temperature.
Following this time the reaction mixture was diluted water and
saturated aqueous sodium hydrogen carbonate solution. The
reaction mixture was extracted with ethyl acetate. The solvent
was removed under reduced pressure. The residue was uptaken
in AcOH (3 mL) and HBr (48%, 0.5 mL) was added. The mixture
was stirred at room temperature overnight. After completion
(monitored by TLC), the solvent was removed under reduced
pressure. The residue was basified with ammonia (7 M in
methanol) and purified by column chromatography to give the
intermediate 29a as yellow solid (8 mg, 10%). 1H NMR (400 MHz,
ride. Yellow solid, yield 45%. 1H NMR (400 MHz, CDCl3):
d 8.72 (s,
1H), 8.61 (s, 1H), 8.39 (s, 1H), 8.02 (s, 1H), 7.54-7.45 (m, 2H), 6.35-
6.25 (m, 3H), 4.37-4.28 (m, 1H), 3.94-3.90 (m, 3H), 3.77 (s, 6H),3.02-
2.96 (m, 4H), 2.86 (s, 3H), 2.33-2.29 (m, 2H), 2.28-2.18 (m, 4H), 1.09
(d, J ¼ 6.2 Hz, 6H). 13C NMR (101 MHz, CDCl3):
d 161.96, 155.14,
148.13, 147.42, 145.61, 139.35, 136.35, 131.50, 126.59, 126.16, 115.61,
111.52, 108.88, 102.92 (2H), 97.09, 58.19, 55.51, 55.47, 52.76, 49.07,
44.88, 43.64, 35.69, 31.83, 22.63. HRMS: calculated for C30H39N7O5S
[(M þ H)þ], 610.2807; found 610.2811.
6.1.30. 3-(1-(1-acetylazetidin-3-yl)-1H-pyrazol-4-yl)-7-((3,5-
dimethoxyphenyl)(2-(isopropylamino)ethyl)amino)-4H-pyrido[1,2-
a]pyrimidin-4-one (29e)
CDCl3): d 8.76-8.70 (m, 1H), 8.61 (s, 1H), 8.38 (s, 1H), 8.01 (s, 1H),
7.53-7.45 (m, 2H), 6.31 (s, 3H), 4.23-4.17 (m, 1H), 3.94 (t,
J ¼ 6.8 Hz, 2H), 3.77 (s, 6H), 3.68-3.59 (m, 2H), 3.08-3.01 (m, 2H),
2.97 (t, J ¼ 6.8 Hz, 2H), 2.90-2.83 (m, 1H), 2.37 (s, 3H), 2.25-2.21
(m, 2H), 2.02-1.95 (m, 2H), 1.09 (d, J ¼ 6.2 Hz, 6H). HRMS:
29e was obtained according to the similar procedure of pre-
paring 29c using tert-butyl 3-iodoazetidine-1-carboxylate instead
of tert-butyl 4-iodopiperidine-1-carboxylate. Yellow solid, yield
calculated for
546.3192.
C30H39N7O3 [(M
þ
H)þ], 546.3188; found
39%. 1H NMR (400 MHz, CDCl3):
d 8.69 (s, 1H), 8.60 (s, 1H), 8.44 (s,
1H), 8.07 (s, 1H), 7.49 (t, J ¼ 9.6 Hz, 2H), 6.35-6.27 (m, 3H), 5.21-5.12
(m,1H), 4.67-4.55 (m, 2H), 4.54-4.42 (m, 2H), 4.01 (t, J ¼ 6.6 Hz, 2H),
3.77 (s, 6H), 3.06-2.93 (m, 3H), 1.96 (s, 3H), 1.15 (d, J ¼ 6.3 Hz, 6H).
6.1.27. 7-((3,5-dimethoxyphenyl)(2-(isopropylamino)ethyl)amino)-
3-(1-(1-ethylpiperidin-4-yl)-1H-pyrazol-4-yl)-4H-pyrido[1,2-a]
pyrimidin-4-one (29b)
13C NMR (101 MHz, CDCl3):
d 170.78, 162.00, 155.11, 148.27, 147.08,
145.68, 139.26, 137.64, 131.53, 128.45, 126.21, 116.23, 111.42, 108.45,
103.00, 97.29, 57.47, 55.54, 55.51, 55.27, 49.50, 49.44, 43.19, 29.70,
29b was obtained according to the similar procedure of pre-
paring 29a using acetaldehyde instead of formaldehyde. Yellow
22.00, 19.05. HRMS: calculated for
546.2825; found 546.2827.
C
29H35N7O4 [(M
þ
H)þ],
solid, yield 17%. 1H NMR (400 MHz, CDCl3):
d 8.74-8.69 (m, 1H), 8.60
(s,1H), 8.38 (s,1H), 8.01 (s,1H), 7.53-7.46 (m, 2H), 6.37-6.27 (m, 3H),
4.31-4.23 (m,1H), 4.00 (t, J ¼ 6.8 Hz, 2H), 3.77 (m, 6H), 3.67-3.60 (m,
2H), 3.23-3.16 (m, 2H), 3.01 (t, J ¼ 6.8 Hz, 2H), 2.97-2.91 (m, 1H),
2.63-2.59 (m, 2H), 2.33-2.28 (m, 2H), 2.05-1.95 (m, 2H), 1.21-1.17 (s,
3H), 1.14 (d, J ¼ 6.2 Hz, 6H). HRMS: calculated for C31H41N7O3
[(M þ H)þ], 560.3345; found 560.3345.
6.1.31. 7-((3,5-dimethoxyphenyl)(2-(isopropylamino)ethyl)amino)-
3-(1-(1-(methylsulfonyl)azetidin-3-yl)-1H-pyrazol-4-yl)-4H-pyrido
[1,2-a]pyrimidin-4-one (29f)
29f was obtained according to the similar procedure of pre-
paring 29d using tert-butyl 3-iodoazetidine-1-carboxylate instead
of tert-butyl 4-iodopiperidine-1-carboxylate. Yellow solid, yield
55%. 1H NMR (400 MHz, CDCl3):
d 8.56 (s, 1H), 8.41 (s, 1H), 8.29 (s,
6.1.28. 3-(1-(1-acetylpiperidin-4-yl)-1H-pyrazol-4-yl)-7-((3,5-
dimethoxyphenyl)(2-(isopropylamino)ethyl)amino)-4H-pyrido[1,2-
a]pyrimidin-4-one (29c)
1H), 7.95 (s, 1H), 7.55-7.45 (m, 2H), 6.39-6.32 (m, 3H), 5.09-5.02 (m,
1H), 4.38-4.26 (m, 2H), 4.80-3.71 (m, 8H), 3.45-3.38 (m, 2H), 3.22-
3.15 (m, 2H), 2.98-2.91 (m, 4H), 1.18 (d, J ¼ 6.2 Hz, 6H). 13C NMR
To a mixture of 28a (100 mg, 0.15 mmol) and Et3N (84
m
L,
(101 MHz, CDCl3): d 162.16, 154.59, 148.38, 145.29, 140.78, 139.50,
16

K. Ran, J. Zeng, G. Wan et al.
European Journal of Medicinal Chemistry 220 (2021) 113499
methanesulfonate (33). To a mixture of 32 (2.5 g, 5.9 mmol) and
Et3N (2.1 mL, 14.75 mmol) in DCM (30 mL) was added meth-
anesulfonyl chloride (0.917 mL, 11.8 mmol) dropwise at 0 ꢀC
under argon. The reaction mixture was stirred at room tem-
perature for 2 h. After completion (monitored by TLC), the re-
action mixture was quenched with water, then extracted with
DCM twice. The combined extracts were washed with water and
brine successively, dried over anhydrous Na2SO4 and concen-
trated under vacuum. The residue was purified by column
chromatography to give 33 (2.2 g, 75%). ESI-MS m/z 499.5
[MþH]þ.
136.07, 131.29, 126.29, 126.14, 117.10, 111.57, 107.70, 103.45, 100.00,
55.78, 55.72, 51.11, 47.96, 47.94, 45.17, 41.63, 41.39, 40.39, 22.72.
HRMS: calculated for C28H35N7O5S [(M þ H)þ], 582.2494; found
546.2494.
6.1.32. 7-((cyclopropylmethyl)(3,5-dimethoxyphenyl)amino)-3-(1-
methyl-1H-pyrazol-4-yl)-4H-pyrido[1,2-a]pyrimidin-4-one (30a)
To a solution of 22d (60 mg, 0.16 mmol) in dry DMF (3 mL) at
0 ꢀC was added NaH (13 mg, 0.32 mmol) slowly. After being stirred
at 0 ꢀC for 10 min, (bromomethyl)cyclopropane (32
mL, 0.32 mmol)
was added, and the mixture was stirred at room temperature
overnight. TLC detected that the reaction was completed. The
mixture was quenched with water, then extracted with ethyl ace-
tate twice. The combined extracts were washed with brine, dried
over anhydrous Na2SO4 and concentrated in vacuum. The residue
was purified by column chromatography to afford 30a as light
Step 2: 7-((2-(cyclopropylamino)ethyl)(3,5-dimethoxyphenyl)
amino)-3-(1-methyl-1H-pyrazol-4-yl)-4H-pyrido[1,2-a]pyr-
imidin-4-one (34a). A mixture of 33 (40 mg, 0.08 mmol) and
cyclopropylamine (91 mg, 1.6 mmol) in acetonitrile (6 mL) was
heated at 100 ꢀC overnight. After completion (monitored by
TLC), the reaction mixture was concentrated under vacuum, and
the residue was purified on TLC-preparative plates to afford the
desired product 34a as light yellow solid (9 mg, 18%). 1H NMR
yellow solid (39 mg, 57%). 1H NMR (400 MHz, CDCl3):
d 8.55 (d,
J ¼ 2.6 Hz, 1H), 8.42 (s, 1H), 8.19 (s, 1H), 7.82 (s, 1H), 7.29 (d,
J ¼ 9.6 Hz, 1H), 7.20 (dd, J ¼ 9.6, 2.6 Hz, 1H), 6.18-6.04 (m, 3H), 3.96
(s, 3H), 3.62 (s, 6H), 3.41 (d, J ¼ 6.6 Hz, 2H), 0.69-0.60 (m, 1H), 0.38-
0.32 (m, 2H), 0.10-0.00 (m, 2H). HRMS: calculated for C24H25N5O3
[(M þ H)þ], 432.2031; found 432.2033.
(400 MHz, DMSO‑d6):
d
8.72 (s, 1H), 8.64 (d, J ¼ 2.0 Hz, 1H), 8.36
(s, 1H), 8.11 (s, 1H), 7.66 (dd, J ¼ 9.6, 2.0 Hz, 1H), 7.59 (d,
J ¼ 9.6 Hz, 1H), 6.39-6.32 (m, 3H), 3.94 (s, 3H), 3.81 (t, J ¼ 6.8 Hz,
2H), 3.77 (s, 6H), 2.71 (t, J ¼ 6.8 Hz, 2H), 2.33-2.26 (m, 1H), 0.89-
0.81 (m, 2H), 0.13-0.02 (m, 2H). HRMS: calculated for
6.1.33. 7-((3,5-dimethoxyphenyl)(prop-2-yn-1-yl)amino)-3-(1-
methyl-1H-pyrazol-4-yl)-4H-pyrido[1,2-a]pyrimidin-4-one (30b)
30b was obtained according to the similar procedure of pre-
paring 30a using 3-bromoprop-1-yne instead of (bromomethyl)
cyclopropane. Yellow solid, yield 77%. 1H NMR (400 MHz, CDCl3):
C
25H28N6O3 [(M þ H)þ], 461.2297; found 461.2298.
6.1.36. 7-((3,5-dimethoxyphenyl)(2-(oxetan-3-ylamino)ethyl)
amino)-3-(1-methyl-1H-pyrazol-4-yl)-4H-pyrido[1,2-a]pyrimidin-
4-one (34b)
d
8.88 (d, J ¼ 2.6 Hz, 1H), 8.62 (s, 1H), 8.32 (s, 1H), 8.00 (s, 1H), 7.53
(d, J ¼ 9.6 Hz, 1H), 7.43 (dd, J ¼ 9.6, 2.6 Hz, 1H), 6.34 (s, 3H), 4.48 (d,
J ¼ 2.2 Hz, 2H), 3.97 (s, 3H), 3.77 (s, 6H), 2.37 (d, J ¼ 2.2 Hz, 1H). 13
C
34b was obtained according to the similar procedure of pre-
paring 34a using oxetan-3-amine instead of cyclopropylamine.
NMR (101 MHz, CDCl3):
d 161.83, 155.19, 148.05, 147.31, 145.87,
Yellow solid, yield 33%. 1H NMR (400 MHz, DMSO‑d6):
d 8.73 (s, 1H),
138.04, 136.44, 131.39, 129.77, 126.12, 115.50, 112.56, 109.44, 102.97,
97.57, 77.71, 74.19, 55.47, 42.79, 39.03. HRMS: calculated for
8.64 (s, 1H), 8.38 (s, 1H), 8.12 (s, 1H), 7.68-7.63 (m, 1H), 7.61 (d,
J ¼ 9.6 Hz, 1H), 6.37 (d, J ¼ 1.6 Hz, 2H), 6.33-6.28 (s, 1H), 4.62 (t,
J ¼ 6.5 Hz, 2H), 4.31 (t, J ¼ 6.0 Hz, 2H), 3.97-3.86 (m, 4H), 3.81 (t,
J ¼ 6.6 Hz, 2H), 3.72 (s, 6H), 2.73 (t, J ¼ 6.6 Hz, 2H). HRMS: calculated
for C25H28N6O4 [(M þ H)þ], 477.2246; found 477.2243.
C
23H21N5O3 [(M þ H)þ], 416.1718; found 416.1722.
6.1.34. 7-((3,5-dimethoxyphenyl)(2-hydroxyethyl)amino)-3-(1-
methyl-1H-pyrazol-4-yl)-4H-pyrido[1,2-a]pyrimidin-4-one (32)
To a solution of 22d (4.0 g, 10.6 mmol) in DMF (60 mL) was
added sodium hydride (60% dispersion in mineral oil, 933 mg,
23.32 mmol) slowly at 0 ꢀC under argon. After being stirred at this
temperature for 30 min, (2-bromoethoxy)(tert-butyl)dimethylsi-
lane (4.6 mL, 21.2 mmol) was added, and then the reaction mixture
was stirred at room temperature overnight. Following this time the
reaction mixture was diluted water and then extracted with ethyl
acetate twice. The combined extracts were washed with water and
brine, dried over anhydrous Na2SO4, and concentrated under
reduced pressure. The residue was dissolved in tetrahydrofuran
(20 mL) and tetrabutylammonium fluoride (1 M in tetrahydrofuran,
21.2 mL, 21.2 mmol) was added. The reaction mixture was stirred at
room temperature overnight. Following this time the reaction
mixture was concentrated under vacuum and the resulting residue
was purified by column chromatography to give the title compound
6.1.37. 7-((3,5-dimethoxyphenyl)(2-((4-hydroxycyclohexyl)amino)
ethyl)amino)-3-(1-methyl-1H-pyrazol-4-yl)-4H-pyrido[1,2-a]
pyrimidin-4-one (34c)
34c was obtained according to the similar procedure of pre-
paring 34a using 4-aminocyclohexan-1-ol instead of cyclopropyl-
amine. Yellow solid, yield 41%. 1H NMR (400 MHz, DMSO‑d6):
d 8.73
(s, 1H), 8.65 (d, J ¼ 2.4 Hz, 1H), 8.37 (s, 1H), 8.12 (s, 1H), 7.65 (dd,
J ¼ 9.6, 2.4 Hz, 1H), 7.61 (d, J ¼ 9.6 Hz, 1H), 6.37 (d, J ¼ 2.0 Hz, 2H),
6.30 (t, J ¼ 2.0 Hz 1H), 4.42 (d, J ¼ 4.1 Hz, 1H), 3.89 (s, 3H), 3.81 (t,
J ¼ 6.6 Hz, 2H), 3.72 (s, 6H), 2.80 (t, J ¼ 6.6 Hz, 2H), 2.34-2.28 (m,
1H), 1.86-1.66 (m, 5H), 1.14-1.06 (m, 2H), 1.04 -0.96 (m, 2H). HRMS:
calculated for C28H34N6O4 [(M þ H)þ], 519.2716; found 519.2723.
6.2. Solubility assay
32 as yellow solid (2.5g, 56%). 1H NMR (400 MHz, DMSO‑d6):
d 8.77-
8.71 (m, 2H), 8.38 (s, 1H), 8.12 (s, 1H), 7.68 (dd, J ¼ 9.6, 2.6 Hz, 1H),
7.61 (d, J ¼ 9.6 Hz, 1H), 4.96 (t, J ¼ 5.0 Hz, 1H), 3.93-3.83 (m, 5H),
3.71 (s, 6H), 3.68-3.61 (m, 2H). HRMS: calculated for C22H23N5O4
[(M þ H)þ], 422.1824; found 422.1821.
A sufficient amount of sample was added to the PBS buffer (pH
7.4). After 30 min of sonication, the solution was shaken at 37 ꢀC
water bath for 24 h to form a supersaturated solution and then
filtered by Whatman Unifilter. Concentration of the filtrate was
analyzed by HPLC based on the calibration curve of each sample.
6.1.35. 7-((2-(cyclopropylamino)ethyl)(3,5-dimethoxyphenyl)
amino)-3-(1-methyl-1H-pyrazol-4-yl)-4H-pyrido[1,2-a]pyrimidin-
4-one (34a)
6.3. Kinase inhibition assay
Including FGFR1 (Carna, Cat. No. 08e133, Lot. No.12CBS-0123K),
FGFR2 (Carna, Cat. No. 08e134, Lot. No. 13CBS-0735H), FGFR3
(Carna, Cat. No. 08e135, Lot. No. 12CBS-0744 M), FGFR4 (Carna, Cat.
Step 1: 2-((3,5-dimethoxyphenyl)(3-(1-methyl-1H-pyrazol-4-
yl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-7-yl)amino)ethyl
17

K. Ran, J. Zeng, G. Wan et al.
European Journal of Medicinal Chemistry 220 (2021) 113499
No. 08e136, Lot. No. 12CBS-0076L) and VEGFR2 (Carna, Cat. No.
08e191, Lot. No. 13CBS-0442H) were tested. The concentration
gradients from 1.5 ꢁ 10ꢂ11 to 1.0 ꢁ 10ꢂ7 mol/L were used for all the
test compounds. The experiments were conducted by Sundia
MediTech Company, Ltd. (China).
7-AAD/Annexin-V-PE staining kit (BD Pharmingen, 559763) ac-
cording to the manufacturer’s protocol. The data were analyzed by
NovoExpress 1.4.0.
6.9. Immunoblotting analysis
The activity of 23d, at a concentration of 1
mM using an ATP
concentration of 10 M, was screened against a panel of 360 human
protein kinases by Eurofins using the Eurofins Kinase Profiler
Selectivity Testing Service.
m
Cells were treated with test compound at indicated time. Then
harvested, followed by lysed in 1 ꢁ RIPA buffer (Millipore, 20e188),
which contained protease inhibitor cocktail (Bimake, B14001) and
phosphatase inhibitor cocktail (Bimake, B15001), for 30 min and
equalized before loading. The sample were separated on SDS-PAGE
gel and transferred onto nitrocellulose filter membranes (Millipore,
HATF00010). Then the membranes were incubated with relevant
primary antibody and corresponding secondary antibody. Specific
protein bands were detected via chemiluminescence detection. The
antibodies used in this article were purchased from Cell Signaling
Technology (FGFR2, p-FGFR2, AKT, p-AKT, p-FRS2, ERK1/2, p-ERK1/
2, caspase 3, cleaved-caspase3), Wanleibio (FRS2) and ABclonal
Technology (CDK2, CDK4, CDK6, Cyclin D, Cyclin E, Cleaved-PARP,
6.4. Molecular docking
The 3D structure of FGFR1 was downloaded from the PDB
(http://www.rcsb.org/, PDB ID: 5EW8). The protein was prepared
with discovery studio 3.1. Molecule 7 and 23d were built with
ChemBio3D and optimized at molecular mechanical level. Then 7
and 23d were docked to the binding site of JNJ42756493 by
employing a protein-ligand docking program GOLD2.5, respec-
tively. Scoring function GOLDSCORE was used for exhaustive
searching, solid body optimizing, and interaction scoring. The final
results for molecular docking were visualized by using PyMol
program.
PARP, b-actin).
6.10. In vivo PK assay
6.5. Cell culture and regents
All animal experiments have been approved by the Institutional
Animal Care and Treatment Committee of Sichuan University in
China. The pharmacokinetic profiles were determined in SD rats.
Compound 23d (12.5% ethanol, 12.5% Kolliphor EL and 75% saline)
was subjected to PK studies in SD rats. The test compound was
administered via IV at 3 mg/kg and PO at 30 mg/kg. After admin-
istration, blood samples were collected. The blood samples were
centrifuged to obtain the plasma fraction. The plasma samples were
deproteinized with methanol containing an internal standard. After
centrifugation, the supernatant was diluted with methanol and
centrifuged again. The compound concentrations of the target
compound in the supernatant were measured by LCꢂMS/MS.
All cell lines were purchased from National Collection of
Authenticated Cell Cultures. Human Gastric Cancer Cell line SNU-16
was cultured in Rosewell Park Memorial Institute (RPMI) 1640
Media (BasalMedia, L210KJ) supplemented with 10% fetal bovine
serum (FBS) (GEMINI, 900-108)and Penicillin-Streptomycin
(Corning, 30-002-CI) under humidified conditions with 5% of CO2,
37 ꢀC.
6.6. Cell proliferation assay
SNU-16 cells were seeded in 96-well plates with different cell
density, 0.5 ꢁ 104 per well for 96 h, 1 ꢁ 104 per well for 72 h,
1.5 ꢁ 104 per well for 48 h, 2 ꢁ 104 per well for 24 h. Different doses
of test compounds were treated as soon as the cell seeded in 96-
well plates. After indicated times, cell viability was determined by
using MTT (CSNpharm, CSN12440) assay. The data was analyzed by
using GraphPad Prism 8 software.
6.11. Xenograft mouse studies
Female NOD/SCID mice (6-week-old, weight 18e20 g) in this
study were obtained from Beijing HJF bioscience CO. LTD, Beijing,
China and maintained in a specific-pathogen-free (SPF) condition
facility. A total of 1.0 ꢁ 107 SNU-16 Cells in PBS mixed with BD
Matrigel (1:1) were inoculated subcutaneously on the right flank of
each mouse. When the mean tumor volume had reached approx-
imately 200 mm3, mice were randomly divided into four groups
(five mice each): i. vehicle group; ii. 15 mg/kg 23d group; iii. 30 mg/
kg 23d group; iv. 30 mg/kg erdafitinib group. The test compound
was dissolve in ethanol, then mixed with Kolliphor EL, and filled
with saline to calculated volume. The formula was 12.5% ethanol,
12.5% Kolliphor EL and 75% saline. Compounds were administrated
once per day by oral gavage for 25 days. The tumor volume was
measured 3 times per week by using a caliper and calculated ac-
cording to the following formula: V ¼ 0.5 ꢁ length ꢁ width2. Mice
were monitored for 25 days. Data are expressed as mean SEM. At
the end of study, mice were euthanized, then tumors, blood and
organs were extracted. The complete blood count (CBC) and blood
biochemical analysis was completed by WestChina-Frontier Phar-
maTech (WCFP), Chengdu, China. The TGI values were calculated
with the following formula: TGI ¼ [1-(TN-T0)/(CNeC0)] ꢁ 100%, TN
and T0 represent average tumor volume before treatment and that
of n days after treatment in indicated group. CN and C0 represent
average tumor volume before treatment and that of n days after
treatment in indicated group, in this study, n is 25th day.
6.7. Cell counting assay
SNU-16 cells were seeded in 6-well plates with 1 ꢁ 105 per well.
The test compounds were treated as soon as the cell seeded in 6-
well plates. After indicated time, the imaged were captured under
an inverted microscope. Then resuspended the cells in the captured
well, and took counts of the number of living cells by using trypan
blue (Biosharp, BL627A) rejection method.
6.8. Cell cycle and apoptosis analysis
The cell cycle and apoptosis analysis were both conducted on
flow cytometry (FCM). Cells were seeded in 6-well plates with
1 ꢁ 106 per well. The test compounds were administrated as soon
as the cell seeded into plates. For cell cycle analysis, the test com-
pounds were treated with 48 h. After the treatment, cells were
harvested and washed twice, then fixed by 70% of ethanol. After
fixation, washed twice and stained with Cell Cycle detection Kit
(KeyGEN, KGA512) according to the manufacturer’s protocol. For
apoptosis analysis, the test compounds were treated with 72 h, and
harvested after administration. Then washed twice and stained by
18

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Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgments
We are grateful for Shuhui Xu of State Key Laboratory of Bio-
therapy (Sichuan University) for NMR measurements and Professor
Lijuan Chen’s team of State Key Laboratory of Biotherapy (Sichuan
University) for HRMS measurements. This work was supported by
National S&T Major Special Project on Major New Drug Innovations
(2018ZX09201018), National Natural Science Foundation of China
(81903441), Sichuan University Postdoctoral Science Research
Foundation (2020SCU12020), and Post-Doctor Research Project,
West China Hospital, Sichuan University, China (2018HXBH009).
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Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113499.
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