2-Oxo-3, 4-dihydropyrimido[4, 5-d]pyrimidinyl derivatives as new irreversible pan fibroblast growth factor receptor (FGFR) inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.04.049

A series of 2-oxo-3, 4-dihydropyrimido[4,5-d]-pyrimidinyl derivatives were designed and synthesized as new irreversible inhibitors of the FGFR family. One of the most promising compounds 2l potently inhibited FGFR1/2/3 with IC₅₀ values of 1.06, 0.84 and 5.38?nM, respectively, whereas its potency against FGFR4 was diminished by an order of magnitude. Compound 2l strongly suppresses the proliferation of FGFR1-amplified H520 non-small cell lung cancer cells, FGFR2-amplified SUM52 breast cancer cells and FGFR3-amplified SW780 bladder cancer cells with low nanomolar IC₅₀ values, but was significantly less potent against four FGFR-negative cancer cell lines, with low micromolar IC₅₀ values. Biological investigation also confirmed the irreversible binding of the molecule with the FGFR1-3 target kinases. Compound 2l may serve as a promising new lead for further anticancer drug discovery.

Full text of DOI:10.1016/j.ejmech.2017.04.049

Accepted Manuscript
2-Oxo-3, 4-dihydropyrimido[4, 5-d]pyrimidinyl derivatives as new irreversible pan
fibroblast growth factor receptor (FGFR) inhibitors
Xueqiang Li, Christopher P. Guise, Rana Taghipouran, Yuliana Yosaatmadja, Amir
Ashoorzadeh, Woo-Kyong Paik, Christopher J. Squire, Shuang Jiang, Jinfeng Luo,
Yong Xu, Zheng-Chao Tu, Xiaoyun Lu, Xiaomei Ren, Adam V. Patterson, Jeff B.
Smaill, Ke Ding
PII:
S0223-5234(17)30315-X
10.1016/j.ejmech.2017.04.049
EJMECH 9400
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 March 2017
Revised Date: 18 April 2017
Accepted Date: 20 April 2017
Please cite this article as: X. Li, C.P. Guise, R. Taghipouran, Y. Yosaatmadja, A. Ashoorzadeh, W.-
K. Paik, C.J. Squire, S. Jiang, J. Luo, Y. Xu, Z.-C. Tu, X. Lu, X. Ren, A.V. Patterson, J.B. Smaill, K.
Ding, 2-Oxo-3, 4-dihydropyrimido[4, 5-d]pyrimidinyl derivatives as new irreversible pan fibroblast growth
factor receptor (FGFR) inhibitors, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.04.049.
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ACCEPTED MANUSCRIPT
2-Oxo-3, 4-dihydropyrimido[4, 5-d]pyrimidinyl Derivatives as New
Irreversible Pan Fibroblast Growth Factor Receptor (FGFR)
Inhibitors
Xueqiang Lia,b, Christopher P. Guisec,d, Rana Taghipouranc, Yuliana Yosaatmadjae, Amir
Ashoorzadehc, Woo-Kyong Paike, Christopher J. Squire d,e, Shuang Jianga,b, Jinfeng Luoa, Yong
Xua, Zheng-Chao Tua, Xiaoyun Luf, Xiaomei Renf, Adam V. Pattersonc,d*, Jeff B. Smaillc,d* and Ke
Dinga,f*
Graphical abstract
Structural optimization of 2-oxo-3, 4-dihydropyrimido[4,5-d]-pyrimidinyl derivatives yielded new
selective FGFR inhibitors. Compound 2l covalently inhibited FGFR1-3 kinase with IC50 values of
1.06, 0.84 and 5.38 nM, respectively. Compound 2l significantly suppressed the growth of
FGFR-amplified cancer cell lines and was less potent against FGFR-negative cancer cell lines and
human normal liver HL7702 cells. Western blot analysis revealed that 2l dose-dependently
inhibited the phosphorylation of FGFR in SUM52 cells bearing amplified-FGFR2 at nanomolar
concentrations.

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2-Oxo-3, 4-dihydropyrimido[4, 5-d]pyrimidinyl Derivatives as New
Irreversible Pan Fibroblast Growth Factor Receptor (FGFR)
Inhibitors
Xueqiang Lia,b, Christopher P. Guisec,d, Rana Taghipouranc, Yuliana Yosaatmadjae, Amir
Ashoorzadehc, Woo-Kyong Paike, Christopher J. Squire d,e, Shuang Jianga,b, Jinfeng Luoa, Yong
Xua, Zheng-Chao Tua, Xiaoyun Luf, Xiaomei Renf, Adam V. Pattersonc,d*, Jeff B. Smaillc,d* and Ke
Dinga,f*
a Guangzhou Institutes of Biomedicine and Health, Guangdong Provincial Key Laboratory of
Biocomputing, Chinese Academy of Sciences, No.190 Kaiyuan Avenue, Guangzhou 510530,
China
b University of Chinese Academy of Sciences, No. 19 Yuquan Road, Beijing 100049, China
c
Auckland Cancer Society Research Centre, School of Medical Sciences, The University of
Auckland, Private Bag 92019, Auckland 1142, New Zealand
d Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Private Bag
92019, Auckland 1142, New Zealand
e School of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland 1142,
New Zealand
f School of Pharmacy, Jinan University, No. 601 Huangpu Avenue West, Guangzhou 510632,
China.
* Corresponding authors:
Tel: + 64 9 3737 599 extn 86798. E-mail: j.smaill@auckland.ac.nz;
a.patterson@auckland.ac.nz; Tel: + 8620 85223764. E-mail: dingke@jnu.edu.cn
Abstract
A series of 2-oxo-3, 4-dihydropyrimido[4,5-d]-pyrimidinyl derivatives were designed and
synthesized as new irreversible inhibitors of the FGFR family. One of the most promising
compounds 2l potently inhibited FGFR1/2/3 with IC50 values of 1.06, 0.84 and 5.38 nM,
respectively, whereas its potency against FGFR4 was diminished by an order of magnitude.
Compound 2l strongly suppresses the proliferation of FGFR1-amplified H520 non-small cell lung
cancer cells, FGFR2-amplified SUM52 breast cancer cells and FGFR3-amplified SW780 bladder
cancer cells with low nanomolar IC50 values, but was significantly less potent against four
FGFR-negative cancer cell lines, with low micromolar IC50 values. Biological investigation also
confirmed the irreversible binding of the molecule with the FGFR1-3 target kinases. Compound 2l

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may serve as a promising new lead for further anticancer drug discovery.
Keyword: FGFR, 2-Oxo-3,4-dihydropyrimido[4, 5-d]pyrimidinyl derivatives, Irreversible
inhibitor
1. Introduction
Fibroblast growth factor receptors (FGFRs) belong to a receptor tyrosine kinase superfamily
which consists of four highly conserved functional members (FGFR1-4) [1]. After being activated
by fibroblast growth factor (FGF) ligands, the FGFR family regulate many key biological
processes, such as embryonic development, cell proliferation, differentiation, migration, apoptosis,
angiogenesis and metabolism [2]. FGFR aberrations (i.e., amplification, gene fusion or mutation)
are identified in a variety of human cancers [3]. For instance, it has been reported that FGFR1
amplification is commonly observed in 9-20 % of squamous non-small cell lung cancer [3,4],
10-15 % of breast cancer and 5 % of ovarian cancer. FGFR2 is also amplified in about 10 % of
gastric cancer and 4% of triple-negative breast cancer, and FGFR2 mutation is detected in about
12-14 % of endometrial cancer [3,5]. Activating mutations of FGFR3 are observed in 38-66 % of
non-invasive urothelial carcinoma and its amplification and translocation are frequently found in
bladder cancer [5]. In addition, approximately 50 % of hepatocellular carcinomas exhibit
overexpression of FGFR4 [6]. Abnormal activation of the FGFR family has been associated with
survival and migration of cancer cells, tumor angiogenesis and poor prognosis, and substantial
studies demonstrate that deregulated FGFR signaling is a key driver in the development of
acquired resistance to targeted therapies [7,11]. Thus, the FGFR family members have become
promising molecular targets for new anticancer drug development.
Figure 1. Design of new covalent FGFR inhibitors
Early attempts to identify a selective FGFR inhibitor led to the discovery of PD173074 as a
biologically active prototypic compound [12]. Since then, several selective or nonselective small

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molecular FGFR inhibitors have been advanced into different stages of clinical investigation [13].
While non-selective FGFR inhibitors show efficacy in patients harboring cancers with deregulated
FGFR signaling, severe side effects are also observed due to inhibition of several off target kinases
such as VEGFR, PDGFR, etc. (e.g., BIBF-1120 [14], TKI-258 [15], AZD-2171 [16]) [13,17].
Other selective reversible FGFR inhibitors with a more favorable safety profile have also been
developed into clinical investigation (e.g., BGJ-398 [18], AZD-4547 [19], JNJ-42756493 [20],
CH-5183284 [21]). Preliminary results indicate that clinical benefits are observed in patients with
tumors harboring FGFR alterations [1].
Recently, there is an increasing interest in developing irreversible kinase inhibitors due to
their superior efficacy, functional selectivity and ability of overcoming clinical resistance [22].
The approval of ibrutinib [23], afatinib [24] and osimertinib [25] for the treatment of chronic
lymphocytic leukemia, EGFR-mutant positive non-small cell lung cancer and
EGFR-T790M-positive non-small cell lung cancer, respectively has validated this approach.
FIIN-1 (1), the first reported selective covalent FGFR inhibitor was designed from the
available FGFR1 PD173074 co-crystal structure; it is highly active against FGFR and exhibited
potent antiproliferative activity against various FGFR-dependent tumor cells [26] . Structural
analysis of FIIN-1 revealed that it possessed some key structural elements which form crucial
interactions with the FGFR protein: (1) a heterocyclic moiety (so-called “head region”) forming a
hydrogen bond network with residues in the kinase hinge region; (2)
a
2,
6-dichloro-3,5-dimethoxyphenyl group occupying the hydrophobic pocket behind the gatekeeper
residue; (3) a side chain with an acrylamide moiety targeting an unpaired cysteine on the p-loop of
the FGFR proteins. In 2014, Gray and colleagues reported two further covalent FGFR inhibitor
examples based on this approach, termed FIIN-2 and FIIN-3. They displayed strong
anti-proliferative effects against cells with gatekeeper mutants of FGFR1 or FGFR2 which confer
resistance to BGJ-398 and AZD-4547 [27]. Recently, reports indicate two covalent pan-FGFR
inhibitors (TAS-120 [5] and PRN-1317 [28]) are being evaluated in phase I clinical trial. Clinical
results for these agents are highly anticipated.
We envisioned that the development of new covalent FGFR inhibitors with different
chemical scaffolds may be a valuable strategy to provide more options for the treatment of
FGFR-dependent cancers. Herein, we describe the design, synthesis and optimization of

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2-oxo-3,4-dihydropyrimido[4,5-d]pyrimidinyl derivatives with a particular focus on varying the
phenyl acrylamide linker chemistry of FIIN-1, while introducing the dimethylaminocrotonamide
Michael acceptor common to several clinical stage covalent kinase inhibitors [24,29], providing a
potent, novel series of covalent FGFR inhibitors (Fig. 1).
2. Chemistry
Synthesis of the designed compounds 2 is outlined in Scheme 1. Briefly, commercially
available ethyl 4-chloro-2-(methylthio)-pyrimidine-5-carboxylate (3) was reacted with the
protected aliphatic amine 4 (a-f) to yield the adduct 5 (a-f), which was reduced to the alcohol 6
(a-f) by treatment with lithium aluminum hydride. Oxidation of the alcohol 6 (a-f) by MnO2
afforded the aldehyde 7 (a-f). The aniline intermediate 8 (a-f) was readily prepared by reductive
amination of 7 (a-f) with 3, 5-dimethoxyaniline. Cyclization of the intermediate 8 (a-f) using
triphosgene produced the heterocyclic intermediate 9 (a-f). Chlorination of compound 9 (a-f) with
SO2Cl2 yielded compound 10 (a-f), which was oxidized with m-CPBA to give the key
methylsulfonyl intermediate 11 (a-f). Nucleophilic substitution of intermediate 11 (a-f) with
different amines followed by Boc-deprotection employing trifluoroacetic acid afforded the amine
12 (a-v), which was coupled with trans-4-dimethylaminocrotonic acid hydrochloride to afford the
final product 2 (a-v).
Scheme 1. Synthesis of compounds 2a-2v a.

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R1
L
R1
L
OMe
Cl
12a
12d
12f
NH
NH
N
N
N
N
OMe
Cl
R1
N
N
L
O
H
NH
NH
12b
12c
NH
NH
NH
N
N
12a-12f
12e
N
a Reagent and conditions: (a) K2CO3, DMF, 60 °C, 85-92 %, (b) LiAlH4, THF, −40 °C to 0 °C,
40-55 %, (c) MnO2, CH2Cl2, rt, overnight, 81-90 %, (d) 3,5-Dimethoxyaniline, CH3COOH,
NaBH4, CH3OH, 0 °C to rt, 67-82 %, (e) triphosgene, ethyl di-isopropylamine, CH2Cl2, 0 °C to
rt ,75-86 %, (f) SO2Cl2, CH3CN, 0 °C; DIPEA，Di-tert-butyl dicarbonate, CH2Cl2, 64-72 % (two
steps), (g) m-CPBA, CH2Cl2, rt, 80-90 %, (h) R1NH2, 1,4-dioxane, 110 °C, for 2a to 2k; TFA,
CH2Cl2, rt, 65-75 %, (two steps), (i) R1NH2, TFA, 1,4-dioxane, 110 °C, for 2l to 2v; TFA, CH2Cl2,
rt, 72-86 %, (two steps), (j) trans-4-dimethylaminocrotonic acid hydrochloride, HATU, DIPEA,
CH3CN, rt, 70-85 %.
3. Results and discussion
The FGFR1 kinase inhibitory potencies of the designed compounds were first determined by
using FRET-base Z'-Lyte biochemical kinase assay. FIIN-1 was utilized as a positive control to
validate the screening conditions. FIIN-1 displayed an IC50 value of 3.65 nM against FGFR1,
which is similar to the reported data [26]. To our delight, the first alternate linker investigated in
compound 2a, an (S)-pyrrolidin-3-yl moiety, showed potent inhibition of FGFR1 with an IC50
value of 2.33 nM (Table 1). Taking 2a as our new lead, further modifications to the group linking
the
dimethylaminocrotonamide
Michael
acceptor
and
the
hinge
binding
2-oxo-3,4-dihydropyrimido[4,5-d]pyrimidine scaffold were made to explore the structure-activity
relationships. First, the importance of the chirality of the linker was assessed. A molecule with a
(R)-pyrrolidin-3-yl linker (2b) was synthesized. It was shown that compound 2b displayed a

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4-fold decreased in FGFR1 inhibitory potency with an IC50 value of 10.7 nM. Further
investigation also suggested that several hetero cyclic or linear linkers such as (S)-piperidin-3-yl
(2c), (R)-piperidin-3-yl (2d), piperidin-4-yl (2e) and 2-aminoethyl (2f) were well tolerated
displaying strong FGFR1 inhibitory activities. The piperidin-4-yl linker in particular (compound
2e) displayed the strongest FGFR1 inhibition with an IC50 value of 0.73 nM. It was also
noteworthy that the chirality of the 5-membered ring system pyrrolidin-3-yl linker had an
influence on the FGFR1 inhibitory potency (compound 2a and 2b), while in the 6-membered
example the (S)-piperidin-3-yl compound 2c and the (R)-piperidin-3-yl inhibitor 2d exhibited
similar FGFR1 inhibitory potencies with IC50 values of 3.08 and 3.54 nM, respectively. In view of
its excellent potency and convenient lack of chirality, compound 2e represented a new starting
point for further optimization.
We next turned our attention to the 7-position of the 2-oxo-3,4-dihydropyrimido [4,5-d]
pyrimidine scaffold (R1 group) exploring the effect of introducing a range of aliphatic and
aromatic substituents. As shown in Table 1, a secondary amine (R1-NH) at the R1 position is
critical for maintaining potency. Introduction of a tertiary amine at this position resulted in over
270-fold loss of FGFR1 inhibitory potency (2e vs 2g). This result strongly implied that a hydrogen
bond was being formed between the secondary amine H-bond donor (NH) and the hinge region of
FGFR1, in an identical manner to that observed in the reported FGFR1/PD173074 co-crystal
structure [30]. This was later confirmed by solving an inhibitor-bound crystal structure of
compound 2l with FGFR1 (as discussed below). Molecular docking of 2e into the FGFR1
co-structure suggested that a relatively large hydrophobic group can be accommodated at the R1
position of the molecule (Supplementary Material, Figure S1). Thus, a series of new inhibitors
with a variety of R1 substituents were designed and synthesized by fixing the piperidin-4-yl group
as the optimal linker between the acrylamide warhead and the 2-oxo-3, 4-dihydropyrimido [4,
5-d]-pyrimidinyl scaffold (Table 1). It was shown that changes in the R1 position were indeed well
tolerated with a variety of branched alkyl, aliphatic, aliphatic ethers or aromatic cyclic
hydrophobic groups displaying strong FGFR1 inhibition with IC50 values ranging from 1.06 to
1.32 nM (2h-2l). For instance, the R1-iso-propyl compound 2h displayed an IC50 value of 1.07 nM
against FGFR1, while the R1-phenyl molecule 2l exhibited almost identical potency. Further
investigation also suggested that the phenyl group in 2l could be mono- or poly-substituted while

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maintaining the strong FGFR1 suppressing potency. Particularly, when a para-methoxyl
substitution was introduced, the resulting compound 2v exhibited remarkable FGFR1 inhibitory
potency with an IC50 value of 0.59 nM.
Table 1. In Vitro FGFR1 Kinase Inhibitory Activities of Inhibitors 2a-2v.
aFGFR1
Compds Formula
L
R1
IC50(nM)
2a
2b
I
I
2.33
10.7
2c
2d
2e
2f
I
I
3.08
3.54
0.73
1.77
200
I
I
2g
II
2h
2i
II
II
II
II
II
1.07
1.23
1.06
1.32
1.06
2j
2k
2l
2m
2n
2o
II
II
II
II
II
1.18
1.16
1.21
1.81
4.59
2p
2q

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2r
II
2.28
2s
2t
II
II
II
1.97
0.99
0.91
2u
2v
II
0.59
3.65
FIIN-1
aFGFR1 activity were performed using the FRET-based Z-Lyte assay. The data are means from at
least three independent experiments and the variations are less than 20%. FIIN-1 was obtained
from a commercial source.
In view of their excellent FGFR1 inhibitory potencies, compounds 2e, 2h, 2i, 2j, 2k, 2l, 2m,
2n 2o, 2t, 2u and 2v were selected to further evaluate their inhibitory potencies against the other
FGFR family members, e.g., FGFR2, FGFR3 and FGFR4, and the results are summarized in Table
2. In general, all of the selected compounds exhibited strong pan inhibition against FGFR1,
FGFR2 and FGFR3 with low nanomolar potencies. However, they were significantly less potent
with respect to inhibition of FGFR4. For instance, compound 2v strongly inhibited the enzymatic
activities of FGFR1/2/3 with IC50 values of 0.59, 1.21 and 5.09 nM, respectively. However,
potency for inhibition of FGFR4 was diminished, with an IC50 value of 269 nM .
The antiproliferative activity of the compounds was also evaluated against a panel of
FGFR-driven cancer cell lines. PD173074 and FIIN-1 were chosen as positive controls, while the
kinase assay inactive molecule 2g was utilized as a negative reference. Highly consistent with
their strong FGFR1/2/3 kinase suppressing potency, all of the selected compounds demonstrated
strong inhibitory effects against the proliferation of FGFR1-amplified H520 non-small cell lung
cancer cells, FGFR2-amplified SUM52 breast cancer cells and FGFR3-amplified SW780 bladder
cancer cells with low nM IC50 values. For instance, compound 2l potently inhibited the growth of
H520, SUM52 and SW780 cells with IC50 values of 4.14, 0.65 and 9.73 nM, respectively. This
antiproliferative activity was 5-30 fold greater than that observed for the positive control FIIN-1.
Importantly, the cell growth inhibitory activity of compound 2l was also assessed against
FGFR-negative cancer cells where it was demonstrated to be significantly less potent. For instance,
the compound demonstrated IC50 values of 1.26, 2.75, 1.40 and 2.48 µM against the

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FGFR-negative non-small cell lung cancer cell lines PC-9, H661, H460 and H1299, respectively.
Compound 2l also exhibited less cytotoxic effect against human normal liver HL7702 cells with
an IC50 value of 1.89 µM. This data offers reassurance that the observed antiproliferative activity
in FGFR-amplified cell lines is dependent on-target inhibition and not off-target effects resulting
from the electrophilic crotonamide moiety present in the compound. Compounds 2k and 2o also
exhibited obviously better antiproliferative potency than that of FIIN-1. Not surprisingly, the
kinase inhibition inactive compound 2g displayed significantly less cell growth inhibitory potency
against H520, SUM52 and SW780 cancer cells with IC50 values of 2240, 224 and 570 nM,
respectively, again supporting the non-toxic nature of the crotonamide common to compounds 2l
and 2g.
Table 2. Inhibitory Activities of Selected Compounds against FGFR1-4 isolated enzymes and
FGFR-amplified cancer cell lines
In vitro Kinase inhibitiona
Cell growth inhibitionb
(IC50 nM)
(IC50 nM)
Compds
FGFR1 FGFR2 FGFR3 FGFR4 H520 SUM52 SW780
2e
0.73
1.07
1.23
1.06
1.32
1.06
1.18
1.16
1.21
0.99
0.91
0.28
200
1.62
2.61
3.63
1.39
2.39
0.84
1.58
1.65
1.77
1.45
2.73
1.21
414
7.59
12.9
20.5
11.1
16.3
5.38
7.15
7.12
7.04
5.07
11.6
5.09
452
296
79.2
180
166
68.7
47.8
186
44.2
78.1
383
269
56.2
4.91
70.0
13.6
7.82
4.14
19.3
5.3
2.0
0.65
4.0
5.2
12.3
32.0
14.5
3.34
9.73
5.4
2h
2i
2j
0.84
0.57
0.65
1.0
2k
2l
2m
2n
1.1
15.1
9.0
2o
6.5
1.1
2t
8.69
39.8
3.0
0.77
3.3
16.1
27.5
0.3
2u
2v
0.8
2g
>1µM >1µM 2420
281
224
12.3
2.9
570
84.3
277
PD173074
FIIN-1
3.65
2.29
5.99
261
121
aFGFR1-4 activity assays were performed using FRET-based Z'-Lyte assay. bThe anti-proliferative
activities of the compounds were evaluated using the SRB assay. The reported data are means
from three independent experiments and the variations are < 20%. H520 is an FGFR1-amplified
non-small cell lung cancer cell line. SUM52 is an FGFR2-amplified breast cancer cell line.
SW780 is an FGFR3-amplified bladder cancer cell line. PD173074 and FIIN-1 were obtained
from commercial sources.

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As an early available representative example of the class, compound 2t was screened for
biochemical inhibition of a panel of 456 kinases (DiscoveRx KINOMEscan) at a set concentration
of 1 µM. This determined that compound 2t displays excellent selectivity for the FGFR family
with a S(35) selectivity score of 0.082 indicating that a concentration of 1µM provides a Percent
Control < 35 for only 8.2% of the non-mutant kinases studied (33/403) (Fig. 2). Outside of the
FGFR family potent inhibition was also observed for BLK, CSF1R and MAP3K2.
Figure 2. TreeSpotTM analysis of compound 2t following screening against 456 kinases.
Taking compound 2l as an example of the minimum preferred pharmacophore, the potent
FGFR inhibition of the compound was further validated by testing its ability to inhibit FGF
ligand-mediated activation of FGFR2 and its down-stream signaling partner ERK1/2 in SUM52
cells. As shown in Figure 3, the compound significantly inhibited the phosphorylation of FGFR
and ERK1/2 in SUM52 cells at nanomolar concentrations. It was noteworthy that a 30 minute
exposure to a concentration of 30 nM of compound 2l was sufficient to totally block the activation
of FGFR2 and ERK1/2, indicating the impressive inhibitory potency of the compound against the
FGFR2 target.
Figure 3. Dose-dependent inhibition of phosphorylation of FGFR and ERK1/2 in SUM52 cells
following exposure of cells to compound 2l at the stated concentration for 30 minutes prior to

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FGF-stimulation, cell lysis and Western blot assay relative to α-tubulin as a protein loading
control. The results are representative of two independent experiments.
The new inhibitors were rationally designed as irreversible inhibitors by targeting the
cysteine residue at the p-loop region of the FGFR proteins [26]. In order to validate the
irreversible nature of binding for compound 2l with FGFR1-3, a comparative “drug washout”
Western blot assay was also performed in the FGFR-amplified cancer cell lines. Here,
maintenance of signal inhibition following extensive cell washing is indicative of irreversible
receptor binding. PD173074 and FIIN-1 were used as respective reversible and irreversible control
compounds. Exposure of the SUM52 and H520 cell lines to 100 nM of the test compounds for 30
minutes, followed by cell lysis and Western blot, resulted in complete inhibition of
phosphorylation of ERK1/2, consistent with complete inhibition of phosphorylation of the primary
FGFR1/2 targets at this test concentration. This was confirmed by Western blot for phospho-FGFR
in SUM52 cells (Fig 4; no drug washout). It was notable that only compound 2l completely
inhibited phospho-ERK1/2 in FGFR3-amplified SW780 cells at 100 nM, with FIIN-1 and
PD173074 appearing less effective against FGFR3 at this concentration. Exposure of each cell line
to 100 nM of test compounds for 30 minutes, followed by extensive drug washout prior to cell
lysis and Western blot, demonstrated restoration of phospho-FGFR and phospho-ERK1/2
signaling for the reversible control compound PD173074 as expected. For FIIN-1, inhibition of
phosphorylation of FGFR and ERK1/2 was conserved in SUM52 cells despite drug washout, as
was inhibition of phospho-ERK1/2 in H520 cells. Surprisingly, restoration of phospho-ERK1/2
signaling was observed in SW780 cells following drug washout (Fig 4; drug washout).
Collectively these results are consistent with FIIN-1 acting as an irreversible inhibitor of FGFR1
(H520 cells) and FGFR2 (SUM52), and a reversible inhibitor of FGFR3 (SW780) under the test
conditions. Pleasingly, compound 2l demonstrated continued inhibition of phosphorylation of
ERK1/2 in all three cell lines (in addition to inhibition of phospho-FGFR in SUM52 cells)
following drug washout (Fig 4; drug washout), indicating compound 2l is a potent irreversible
inhibitor of FGFR1-3 in cells.

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Figure 4. Western blot for inhibition of phosphorylation of FGFR and ERK1/2 in SUM52, H520
and SW780 cells following exposure of cells to 100 nM of FIIN-1 (control irreversible inhibitor),
PD173074 (control reversible inhibitor) and compound 2l for 30 minutes, with and without drug
washout prior to FGF-stimulation and cell lysis. PD173074 and FIIN-1 were obtained from
commercial sources.
The reversible binding mode of compound 2l to the active site of FGFR1 has been
determined by protein X-ray crystallography [31] (Fig. 5). Compound 21 binds as expected in the
active site with the 2,6-dichloro-3,5-dimethoxyphenyl group buried in a hydrophobic pocket
forming a single hydrogen bond from the 3-methoxy substituent to Asp641 of the protein. The
central heterocyclic system forms the anticipated hydrogen bond donor acceptor interaction to
Ala564 in the hinge region of the protein backbone. The acrylamide side chain is presented into
solvent accessible space and is available for nucleophilic attack by the target cysteine in the p-loop
of the protein. In the current protein construct the target cysteine has been mutated to an alanine,
as described previously [30], facilitating appropriate crystal formation. This prevents us trapping
an irreversible binding mode for compound 2l in the present structure. However, this structure
does allow us to model the likely location of the target cysteine side chain [32] . To our surprise,
the site of the modeled cysteine sulfur atom is ~12 Å distant from the reactive acrylamide site.

ACCEPTED MANUSCRIPT
Covalent binding to this cysteine, to provide the irreversible inhibition observed in cells, would
require the dynamic movement of the acrylamide side chain of compound 21 and/or the “closure”
of the p-loop to bring the reactive sites into close proximity. Efforts towards producing an
irreversible binding mode by X-ray crystallography using a wild type protein sequence are
ongoing.
Figure 5. The reversible binding mode of compound 21 determined by X-ray crystallography
(PDB ID: 5UR1). A section of the acrylamide side chain was not modeled in the crystal structure
and is shown in the foreground in white; the covalent bond-forming Michael acceptor carbon of
the acrylamide is indicated by an asterisk. The p-loop target cysteine 488 is located ~12 Å away
from the acrylamide functionality. Hydrogen bonds between compound 21 and the protein are
illustrated (right hand side) and the hydrophobic pocket that encloses the
2,6-dichloro-3,5-dimethoxyphenyl group is indicated by a bold, dashed green line.
4. Conclusion
In summary, a series of 2-oxo-3, 4-dihydropyrimido[4,5-d]pyrimidinyl derivatives were
designed and synthesized as highly potent covalent FGFR1-3 inhibitors. One of the most
promising compounds, 2l, potently inhibited FGFR1/2/3 with IC50 values of 1.06, 0.84 and 5.38
nM, respectively. However, its potency against FGFR4 was diminished with an IC50 value of 68.7
nM. Compound 2l strongly suppresses the proliferation of FGFR1-amplified H520 non-small cell
lung cancer cells, FGFR2-amplified SUM52 breast cancer cells and FGFR3-amplified SW780
bladder cancer cells with IC50 values of 4.14, 0.65 and 9.73 nM, respectively, but is significantly
less potent for FGFR-negative cancer cells and human normal liver cells. Biological investigation
also confirmed the irreversible binding of the molecule with the target FGFR1-3 kinases. Further
preclinical investigation of the compound is ongoing and will be disclosed in due course.
5. Experimental Section

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5.1 Chemistry
All reagents and solvents were obtained from commercial sources. Flash chromatography was
performed using silica gel (300 mesh). All reactions were monitored by TLC, using silica gel
plates with fluorescence F254 and UV light visualization. 1H NMR was recorded on a Brucker AV-
13
400 spectrometer at 400 MHz or a Brucker AV-500 spectrometer at 125 MHz. C NMR spectra
was recorded on a Brucker AV-500 spectrometer at 125 MHz. Coupling constants (J) are
expressed in hertz (Hz). Chemical shifts (δ) are reported in parts per million (ppm). The high
resolution of ESI-MS was recorded on an Applied Biosystems Q-STAR Elite ESI- LC- MS/MS
mass spectrometer. The purity of compounds was determined to be over 95% using reverse-phase
HPLC analysis. HPLC instrument: Dionex Summit HPLC (Column: Diamonsil C18, 5.0 mm,
4.6×250 mm (Agilent Technologies); detector: PDA-100 photodiode array; injector: ASI-100
autoinjector; pump: p-680A). The flow rate is 1.0 mL/min and mobile phase is MeOH in H2O with
0.1% modifier (ammonia v/v).
Ethyl
4-((1-(tert-butoxycarbonyl)piperidin-4-yl)amino)-2-(methylthio)pyrimidine
-5-
carboxylate (5e)
General procedure for the synthesis of 5a-5f. To a solution of 3 (21 g, 90.3 mmol) and 4e (21.7
g, 108.3 mmol) in DMF (300 mL) was added K2CO3 (25 g, 180.9 mmol). The reaction was heated
to 60 °C overnight. After the mixture was cooled to room temperature, it was added to ice-water.
The precipitate was filtered, and the filtered cake was washed with cool water and dried in a
vacuum oven to afford 5e (31.1 g, 86.9 %). 1H NMR (400 MHz, CDCl3), δ 8.63 (s, 1H), 8.50 (d, J
= 7.2 Hz, 1H), 4.31 (q, J = 7.2 Hz, 2H), 4.27-4.21 (m, 1H), 4.00-3.97 (m, 2H), 3.02 (t, J = 11.4 Hz,
2H), 2.51 (s, 3H), 2.03-1.99 (m, 2H), 1.54-1.49 (m, 2H), 1.46 (s, 9H), 1.36 (t, J = 7.2 Hz, 3H). MS
(ESI) m/z 397.1 [M+H]+.
Tert-butyl
4-((5-(hydroxymethyl)-2-(methylthio)pyrimidin-4-yl)amino)piperidine-1-
carboxylate (6e)
General procedure for the synthesis of 6a- 6f. A suspension of LiAlH4 (4.41 g, 116.0 mmol) in dry
THF (70 mL) was added to a stirred solution of 5e (23 g, 58.0 mmol) in dry THF (160 mL) at
−40 °C under argon. The mixture was warmed to 0 °C. After the reaction was completed, 4.4 mL
of H2O was added dropwise. After that, 15% aqueous NaOH (4.4 mL) was added, followed by a
further 13.2 mL of water. The resulting mixture was stirred at room temperature for 20 min and

ACCEPTED MANUSCRIPT
filtered through a pad of Celite. The Celite was washed with CH2Cl2, and the washes were
combined and then dried in vacuo. CH2Cl2 was added to the residue and washed with saturated
aqueous NaHCO3.The organic layer was separated, washed with brine, dried with Na2SO4 and
concentrated in vacuo. The resulting crude product was purified by column chromatography
giving 6e (11.72 g, 57 %). 1H NMR (400 MHz, CDCl3), δ 7.63 (s, 1H), 5.95 (d, J = 7.2 Hz, 1H),
4.48 (s, 2H), 4.21-4.14 (m, 1H), 3.99-3.96 (m, 2H), 2.95 (t, J = 11.4 Hz, 2H), 2.47 (s, 3H),
2.03-1.99 (m, 2H), 1.45 (s, 9H), 1.43-1.37 (m, 2H). MS (ESI) m/z 355.1 [M+H]+.
Tert-butyl 4-((5-formyl-2-(methylthio)pyrimidin-4-yl)amino)piperidine-1-carboxylate (7e)
General procedure for the synthesis of 7a- 7f. Compound 6e (11.72 g, 33.1mmol) was dissolved
in CH2Cl2 (250 mL) and MnO2 (17.3 g, 198 mmol) was added in three portions. The mixture was
stirred at room temperature overnight. The mixture was filtered through a pad of Celite. The
filtrate was concentrated and the resulting crude product was purified by column chromatography
1
to afford 7e (11 g, 94 %). H NMR (400 MHz, CDCl3), δ 9.70 (s, 1H), 8.59 (d, J = 6.8 Hz, 1H),
8.31 (s, 1H), 4.34-4.25 (m, 1H), 4.03-4.01 (m, 1H), 3.01 (t, J = 11.4 Hz, 2H), 2.54 (s, 3H),
2.04-2.00 (m, 2H), 1.54-1.50 (m, 2H), 1.47 (s, 9H). MS (ESI) m/z 353.0 [M+H]+.
Tert-butyl
4-((5-(((3,5-dimethoxyphenyl)amino)methyl)-2-(methylthio)pyrimidin-4-yl)
amino)piperidine-1-carboxylate (8e)
General procedure for the synthesis of 8a-8f. To a solution of compound 7e (10.8 g, 30.6 mmol)
and 3,5-dimethoxyaniline (7.04 g, 46.0 mmol) in anhydrous CH3OH (150 mL) were added
CH3COOH (0.76 mL). The mixture was stirred at room temperature overnight. After the
completion of the reaction, NaBH4 (3.5 g, 92 mmol) was added to the reaction mixture at 0 °C in
tree portions. The resulting mixture was concentrated, partitioned between CH2Cl2 and saturated
NaHCO3 and extracted with CH2Cl2. The organic phase was separated and concentrated. The
1
crude compound was purified by column chromatography to afford 8e (12.7 g, 85 %). H NMR
(400 MHz, CDCl3), δ 7.85 (s, 1H), 5.97(s, 1H), 5.89 (m, 2H), 4.18-4.16 (m, 1H), 4.06 (s, 2H),
3.93 (m, 2H), 3.75 (s, 6H), 2.97-2.91 (m, 2H), 2.50 (s, 3H), 1.99-1.95 (m, 2H), 1.45 (s, 9H),
1.40-1.35 (m, 2H). MS (ESI) m/z 490.2 [M+H]+.
Tert-butyl 4-(3-(3,5-dimethoxyphenyl)-7-(methylthio)-2-oxo-3,4-dihydropyrimido [4,5-d]
pyrimidin-1(2H)-yl)piperidine-1-carboxylate (9e)
General procedure for the synthesis of 9a-9f. Compound 8e (9.10 g, 18.6 mmol) was dissolved

ACCEPTED MANUSCRIPT
in CH2Cl2 (110 mL) and DPIEA (9.62 mL, 55.3 mmol) was added dropwise a solution of
triphosgene (1.84 g, 6.2 mmol) in anhydrous CH2Cl2 (10 mL) at 0 °C. The mixture was warmed to
room temperature. After the reaction was over, aqueous saturated NaHCO3 was poured into the
mixture. The organic layer was separated, washed with brine, dried with Na2SO4, filtered, and
concentrated in vacuo. The crude product was subjected to column chromatography to give 9e (8.6
g, 89.6 %). 1H NMR (400 MHz, CDCl3), δ 8.09 (s, 1H), 6.45-6.44 (m, 2H), 6.39 (m, 1H), 4.83 (s,
1H), 4.61 (s, 2H), 4.32-4.28 (m, 2H), 3.79 (s, 6H), 2.81-2.72 (m, 4H), 2.54 (s, 3H), 1.99-1.95 (m,
2H), 1.46 (s, 9H). MS (ESI) m/z 516.2 [M+H]+.
Tert-butyl
4-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-7-(methylthio)-2-oxo-3,4-
dihydropyrimido[4,5-d]pyrimidin-1(2H)-yl)piperidine-1-carboxylate (10e)
General procedure for the synthesis of 10a-10f. To a solution of compound 9e (4.85 g, 9.41
mmol) in anhydrous CH3CN (60 mL) was added SO2Cl2 (1.52 mL, 18.82 mmol) dropwise. The
mixture was stirred in ice-water bath for 30 min and quenched by addition of saturated aqueous
NaHCO3. The mixture was basified with saturated aqueous NaHCO3 and extracted with CH2Cl2.
The organic layer was separated, dried over Na2SO4, and concentrated in vacuo. The resulting
crude product was dissolved in CH2Cl2 (60 mL). DIPEA (1.5 mL, 9.44 mmol) and Di-tert-butyl
dicarbonate (4 mL, 9.44 mmol) were added to the mixture. The mixture was stirred at room
temperature for 4 h. After removal of CH2Cl2, the crude product was purified by column
chromatography to afford 10e (3.96 g, 72 %). 1H NMR (400 MHz, CDCl3), δ 8.09 (s, 1H), 6.59 (s,
1H), 4.88 (m, 1H), 4.53 (s, 2H), 4.31-4.20 (m, 2H), 3.93 (s, 6H), 2.74-2.71 (m, 4H), 2.54 (s, 3H),
1.75 (m, 2H), 1.45 (s, 9H). MS (ESI) m/z 585.1 [M+H]+.
Tert-butyl
4-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-7-(methylsulfonyl)-2-oxo-3,4-
dihydropyrimido[4,5-d]pyrimidin-1(2H)-yl)piperidine-1-carboxylate (11e)
General procedure for the synthesis of 11a-11f. To a solution of compound 10e (3.3 g, 5.7
mmol) in CH2Cl2 (50 mL) was added m-CPBA (2.78 g, 12.0 mmol). The mixture was stirred at
room temperature for 5 h. The solution was diluted with CH2Cl2 and washed with saturated
aqueous NaHCO3 three times. The organic layer was separated, washed with brine, dried with
Na2SO4 and concentrated in vacuo. The resulting material was subjected to column
chromatography affording 11e (2.8 g, 81 %). 1H NMR (500 MHz, CDCl3), δ 8.40 (s, 1H), 6.61 (s,
1H), 4.93-4.87 (m, 1H), 4.68 (s, 2H), 4.32-4.20 (m, 2H), 3.94 (s, 6H), 2.81 (m, 2H), 2.70-2.64 (m,

ACCEPTED MANUSCRIPT
2H), 1.77-1.74 (m, 2H), 1.60-1.55 (m, 2H), 1.45 (s, 9H). MS (ESI) m/z 617.1 [M+H]+.
(E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-(1-(4-(dimethylamino)but-2-enoyl)piperidin-4-yl
)-7-(isopropylamino)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (2h)
General procedure for the synthesis of 2a-2k. To a solution of 11e (0.9 g, 1.46 mmol) in
1,4-dioxance (10 mL) was added isopropylamine (0.37 ml, 4.38 mmol). The mixture was stirred at
110 °C in a sealed tube for 5 h. The mixture was cooled to room temperature and concentrated in
vacuo. The crude product was dissolved in CH2Cl2 (10 mL), then TFA (0.5 mL) was added. The
resulting mixture was stirred at room temperature for 4 h. The mixture was basified with saturated
aqueous NaHCO3. The organic layer was separated, washed with brine, dried with Na2SO4,
filtered and concentrated. The resulting material was used without any further purification.
Trans-4-dimethylaminocrotonic acid hydrochloride (0.21 g, 1.3 mmol) and HATU (0.57 g, 1.5
mmol) were added to a flask charged with the rude compound, followed by adding CH3CN (10
mL) and DIPEA (0.49 mL, 3.0 mmol). The resulting mixture was stirred at room temperature
overnight. The reaction mixture was diluted with CH2Cl2, basified with saturated aqueous
NaHCO3 and extracted with CH2Cl2. The organic layer was separated, washed with brine, dried
with Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by column
chromatography to give compound 2h (0.42 g, 69 %). 1H NMR (500 MHz, DMSO-d6), δ 7.96 (s,
1H), 6.97 (s, 2H), 6.63-6.59 (m, 2H), 4.83 (s, 1H), 4.62-4.46 (m, 1H), 4.37 (s, 2H), 4.15 (d, J =
13.6 Hz, 1H), 3.95 (s, 6H), 3.90-3.68 (m, 1H), 3.10-3.05 (m, 1H), 3.01 (d, J = 4.4 Hz, 2H),
2.61-2.59 (m, 3H), 2.13 (s, 6H), 1.71-1.68 (m, 2H), 1.11 (d, J = 6.4 Hz, 6H). 13C NMR (125 MHz,
DMSO-d6), δ 163.78, 160.57, 156.60, 154.68, 154.12, 151.46, 141.68, 137.99, 122.29, 113.15,
98.11, 59.91, 56.84, 45.00, 44.79, 42.21, 41.78, 29.18, 28.05, 22.35. HRMS (ESI) for
C28H37Cl2N7O4 [M+H]+, calcd: 606.2357, found: 606.2353. HPLC analysis: MeOH−H2O (85: 15),
5.91 min, 97.16 % purity.
3-(2,6-dichloro-3,5-dimethoxyphenyl)-7-(phenylamino)-1-(piperidin-4-yl)-3,4-dihydropyrimi
do[4,5-d]pyrimidin-2(1H)-one (12l)
General procedure for the synthesis of 12l-12v. Aniline (0.3 ml, 2.92 mmol) was added to a
solution of 11e (0.9 g, 1.46 mmol) in 1,4-dioxance (10 mL), followed by adding TFA (0.12 mL,
1.61 mmol).The reaction mixture was stirred at 110 °C for 18 h in a sealed tube. The mixture was
cooled to room temperature and concentrated in vacuo. The crude product was dissolved in

ACCEPTED MANUSCRIPT
CH2Cl2 (10 mL), then TFA (0.5 mL) was added. The resulting mixture was stirred at room
temperature for 4 h. The mixture was basified with saturated aqueous NaHCO3. The organic layer
was separated, washed with brine and concentrated in vacuo. The resultant crude material was
1
purified by column chromatography furnishing the tittle compound 12l (0.62 g, 80 %). H NMR
(400 MHz, CDCl3), δ 9.56 (s, 1H), 8.16 (s, 1H), 7.75-7.73 (m, 2H), 7.30-7.26 (m, 2H), 6.98 (s,
1H), 6.96-6.94 (m, 1H), 4.71-4.66 (m, 1H), 4.48 (s, 2H), 3.96 (s, 6H), 3.06-3.04 (m, 2H),
2.56-2.42 (m, 4H), 1.62-1.59 (m, 2H). MS (ESI) m/z 530.1 [M+H]+.
(E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-(1-(4-(dimethylamino)but-2-enoyl)piperidin-4-yl
)-7-(phenylamino)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (2l)
General procedure for the synthesis of 2l-2v. To a solution of 12l (0.49 g, 0.9 mmol), trans-4-
dimethyl- aminocrotonic acid hydrochloride (0.2 g, 1.2 mmol) and HATU (0.5 g, 1.4 mmol) in
CH3CN (12 mL) was added DIPEA (0.46 mL, 2.75 mmol). The resulting mixture was stirred at
room temperature overnight. The reaction mixture was diluted with CH2Cl2, basified with
saturated aqueous NaHCO3. The organic layer was separated, washed with brine and concentrated.
The crude material was purified by column chromatography to give compound 2l (0.47 g, 79.4 %).
1H NMR (500 MHz, DMSO-d6), δ 9.61 (s, 1H), 8.19 (s, 1H), 7.69 (d, J = 8.0 Hz, 2H), 7.45-7.22
(m, 2H), 6.98 (s, 1H), 6.94 (t, J = 7.3 Hz, 1H), 6.73-6.46 (m, 2H), 4.87 (m, 1H), 4.61 (d, J = 12.6
Hz, 1H), 4.48 (s, 2H), 4.19 (d, J = 13.9 Hz, 1H), 3.95 (s, 6H), 3.08 (t, J = 13.4 Hz, 1H), 3.01 (d, J
= 4.2 Hz, 2H), 2.63 (t, J = 12.9 Hz, 1H), 2.47-2.35 (m, 2H), 2.13 (s, 6H), 1.77 (d, J = 11.9 Hz, 2H).
13C NMR (125 MHz, DMSO-d6), δ 163.97, 158.92, 156.82, 154.71, 154.10, 151.03, 141.92,
140.17, 137.76, 128.46, 122.35, 121.60, 113.17, 102.74, 98.20, 59.93, 56.87, 52.54, 45.31, 45.03,
44.80, 41.78, 29.07, 28.08. HRMS (ESI) for C31H35Cl2N7O4 [M+H] +, calcd: 640.2200, found:
640.2204. HPLC analysis: MeOH−H2O (85: 15), 6.36 min, 97.59 % purity.
(S, E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-(1-(4-(dimethylamino)but-2-enoyl) pyrrolidin
-3-yl)-7-(methylamino)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (2a)
[α] 2D0 -14.81 (c 0.405, CH2Cl2). 1H NMR (400 MHz, CDCl3), δ 7.91 (d, J = 7.0 Hz, 1H), 6.88 (dt, J
= 15.2, 6.1 Hz, 1H), 6.58 (d, J = 5.5 Hz, 1H), 6.27 (ddt, J = 23.0, 15.2, 1.6 Hz, 1H), 5.61 (q, J =
8.5 Hz, 1H), 5.15 (q, J = 5.0 Hz, 0.6H), 5.06 (d, J = 5.4 Hz, 0.4H), 4.45 (dd, J = 8.6, 0.8 Hz, 2H),
4.16 (s, 0.6H), 3.99 (ddd, J = 12.7, 8.1, 5.0 Hz, 1.4H), 3.94-3.90 (m, 6.3H), 3.86 (t, J = 9.4 Hz,

ACCEPTED MANUSCRIPT
0.7H), 3.57 (ddt, J = 46.0, 12.3, 8.1 Hz, 1H), 3.06 (td, J = 6.6, 1.6 Hz, 2H), 2.95 (dd, J = 5.1, 1.5
Hz, 3H), 2.77 (d, J = 8.3 Hz, 1H), 2.31 (dtd, J = 13.0, 8.7, 4.7 Hz, 0.3H), 2.23 (d, J = 5.8 Hz, 6H),
2.17 (ddd, J = 11.8, 8.3, 3.4 Hz, 0.7H). 13C NMR (100 MHz, CDCl3), δ 164.79, 164.41, 162.21,
162.11, 157.34, 157.07, 155.23, 155.15, 154.00, 153.87, 151.79, 151.60, 142.07, 141.91, 138.21,
138.17, 123.69, 123.19, 114.92, 114.80, 114.74, 97.47, 60.82, 56.84, 56.81, 52.34, 50.65, 47.67,
46.02, 45.57, 45.47, 44.78, 29.00, 28.57, 26.60. HRMS (ESI) for C25H31Cl2N7O4 [M+H]+, calcd:
564.1887, found: 564.1884. HPLC analysis: MeOH−H2O (75: 25), 7.27 min, 96.04 % purity.
(R, E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-(1-(4-(dimethylamino)but-2-enoyl) pyrrolidin
-3-yl)-7-(methylamino)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (2b)
[α] 2D0 10.000 (c 0.22, CH2Cl2). 1H NMR (500 MHz, DMSO-d6), δ 8.11-7.95 (m, 1H), 7.11 (s, 1H),
7.07-6.94 (m, 1H), 6.60 (dtd, J = 15.3, 6.1, 3.0 Hz, 1H), 6.36 (dd, J = 25.9, 15.2 Hz, 1H), 5.47 (s,
1H), 4.42 (d, J = 2.6 Hz, 2H), 3.95 (s, 6H), 3.91-3.79 (m, 1H), 3.73 (s, 2H), 3.57 (d, J = 8.9 Hz,
1H), 3.00 (dd, J = 9.8, 6.0 Hz, 2H), 2.76 (d, J = 5.5 Hz, 3H), 2.58 (s, 1H), 2.22 (d, J = 8.8 Hz, 1H),
2.13 (d, J = 8.6 Hz, 6H). 13C NMR (125 MHz, DMSO-d6), δ 163.20, 163.10, 161.74, 154.70,
151.15, 141.23, 141.19, 137.73, 123.74, 123.15, 113.12, 98.22, 59.85, 59.75, 56.87, 55.99, 54.86,
51.65, 50.17, 47.04, 45.15, 45.01, 44.98, 44.78, 44.09, 39.02, 28.21, 27.74, 26.12, 18.51. HRMS
(ESI) for C25H31Cl2N7O4 [M+H]+, calcd: 564.1887, found: 564.1887. HPLC analysis: MeOH−
H2O (75: 25), 7.28 min, 96.74 % purity.
(S, E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-(1-(4-(dimethylamino)but-2-enoyl) piperidin-
3-yl)-7-(methylamino)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (2c)
[α] 2D0 -10.000 (c 0.20, CH2Cl2). 1H NMR (500 MHz, DMSO-d6), δ 8.00 (s, 1H), 7.10 (s, 1H), 6.98
(s, 1H), 6.59 (d, J = 6.0 Hz, 2H), 4.58 (d, J = 30.9 Hz, 1H), 4.41 (s, 3H), 4.07-4.01(m, 2H), 3.95 (s,
6H), 3.15-2.89 (m, 3H), 2.79 (s, 3H), 2.64 (s, 1H), 2.12 (d, J = 17.1 Hz, 6H), 1.80 (s, 2H), 1.42 (s,
1H). 13C NMR (125 MHz, DMSO-d6), δ 164.12, 161.83, 156.55, 154.69, 154.19, 151.28, 141.89,
137.90, 122.43, 113.20, 98.17, 59.91, 56.86, 55.99, 54.86, 47.39, 45.01, 44.83, 41.80, 27.87, 24.94,
18.51. HRMS (ESI) for C26H33Cl2N7O4 [M+H]+, calcd: 578.2044, found: 578.2039. HPLC
analysis: MeOH−H2O (85: 15), 5.02 min, 97.35 % purity.
(R, E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-(1-(4-(dimethylamino)but-2-enoyl) piperidin
-3-yl)-7-(methylamino)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (2d)

ACCEPTED MANUSCRIPT
[α] 2D0 5.405 (c 0.37, CH2Cl2). 1H NMR (500 MHz, DMSO-d6), δ 8.00 (s, 1H), 7.09 (s, 1H), 6.98 (s,
1H), 6.74-6.46 (m, 2H), 4.77-4.24 (m, 4.5H), 4.23-4.00 (m, 1.5H), 3.95 (s, 6H), 3.14-2.91 (m, 3H),
2.96-2.72 (m, 3H), 2.64 (s, 1H), 2.14 (d, J = 16.6 Hz, 6H), 1.79 (d, J = 13.3 Hz, 2H), 1.41 (d, J =
12.5 Hz, 1H). 13C NMR (125 MHz, DMSO-d6), δ 164.09, 161.79, 156.50, 154.67, 151.25, 141.53,
137.87, 122.63, 113.17, 99.46, 98.18, 59.79, 56.85, 55.97, 54.83, 44.88, 44.81, 39.09, 39.02, 27.85,
18.49. HRMS (ESI) for C26H33Cl2N7O4 [M+H]+, calcd: 578.2044, found: 578.2038. HPLC
analysis: MeOH−H2O (90: 10), 5.28 min, 95.92 % purity.
(E)-N-(3-(3-(2,6-dichloro-3,5-dimethoxyphenyl)-7-(methylamino)-2-oxo-3,4-
dihydropyrimido[4,5-d]pyrimidin-1(2H)-yl)propyl)-4-(dimethylamino)but-2-enamide (2f)
1H NMR (500 MHz, DMSO-d6), δ 7.97 (br, 2H), 7.03 (br, 1H), 6.99 (s, 1H), 6.52 (dt, J = 15.4, 6.1
Hz, 1H), 5.99 (dt, J = 15.5, 1.6 Hz, 1H), 4.44 (s, 2H), 3.99-3.96 (m, 2H) , 3.95 (s, 6H), 3.18-3.13
(m, 2H), 2.96 (dd, J = 6.2, 1.5 Hz, 2H), 2.79 (d, J = 4.7 Hz, 3H), 2.12 (s, 6H), 1.75 (t, J = 7.3 Hz,
2H). 13C NMR (125 MHz, DMSO-d6), δ 164.41, 162.00, 155.63, 154.71, 154.00, 150.91, 138.97,
137.78, 125.99, 113.19, 98.22, 59.69, 56.83, 55.97, 54.82, 44.98, 44.78, 36.32, 28.15, 27.76, 18.45.
HRMS (ESI) for C24H31Cl2N7O4 [M+H]+, calcd: 552.1887, found: 552.1888. HPLC analysis:
MeOH−H2O (80: 20), 5.30 min, 95.44 % purity.
(E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-(1-(4-(dimethylamino)but-2-enoyl) piperidin -4
-yl)-7-(methylamino)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (2e)
1H NMR (400 MHz, CDCl3), δ 7.97 (s, 1H), 7.36 (d, J = 1.2 Hz, 1H), 6.89 (dt, J = 15.0, 6.1 Hz,
1H), 6.68 (s, 1H), 6.63-6.40 (m, 1H), 5.40 (d, J = 1.2 Hz, 1H), 5.17 (d, J = 5.1 Hz, 1H), 5.04 (d, J
= 11.9 Hz, 1H), 4.88 (d, J = 12.8 Hz, 1H), 4.54 (d, J = 7.6 Hz, 2H), 4.20 (d, J = 13.0 Hz, 1H), 4.02
(s, 6H), 3.57 (d, J = 1.3 Hz, 1H), 3.34-3.12 (m, 3H), 3.05 (d, J = 4.9 Hz, 3H), 2.92 (d, J = 12.6 Hz,
2H), 2.78 (t, J = 12.9 Hz, 1H), 2.35 (d, J = 1.3 Hz, 6H). 13C NMR (125 MHz, DMSO-d6), δ
164.02, 161.78, 156.64, 154.68, 151.37, 141.71, 137.99, 122.36, 113.17, 98.12, 59.92, 56.85,
56.00, 51.95, 45.26, 44.99, 44.79, 27.84, 18.52. HRMS (ESI) for C26H33Cl2N7O4 [M+H]+, calcd:
578.2044, found: 578.2029. HPLC analysis: MeOH−H2O (80: 20), 5.83 min, 97.04 % purity.
(E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-7-(dimethylamino)-1-(1-(4-(dimethylamino)but-2-e
noyl)piperidin-4-yl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (2g)

ACCEPTED MANUSCRIPT
1H NMR (500 MHz, DMSO-d6), δ 8.04 (s, 1H), 6.97 (s, 1H), 6.81-6.44 (m, 2H), 5.06-4.72 (m,
1H), 4.52 (d, J = 12.4 Hz, 1H), 4.40 (s, 2H), 4.14 (d, J = 13.8 Hz, 1H), 3.95 (s, 6H), 3.14-3.04 (m,
9H), 2.63 (m, 3H), 2.15 (s, 6H), 1.68 (d, J = 17.0 Hz, 2H). 13C NMR (125 MHz, DMSO-d6), δ
164.01, 160.78, 156.36, 154.65, 153.75, 151.41, 141.41, 137.98, 122.48, 113.16, 99.76, 98.15,
59.81, 56.83, 54.82, 52.10, 44.88, 44.71, 36.64, 18.48. HRMS (ESI) for C27H35Cl2N7O4 [M+H]+,
calcd: 592.2200, found: 592.2203. HPLC analysis: MeOH−H2O (85: 15), 6.45min, 98.43 % purity.
(E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-(1-(4-(dimethylamino)but-2-enoyl) piperidin-4-
yl)-7-((tetrahydrofuran-3-yl)amino)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (2i)
1H NMR (500 MHz, DMSO-d6), δ 8.01 (s, 1H), 7.43 (brs, 1H), 6.98 (s, 1H), 6.84-6.40 (m, 2H),
4.85-4.83(m, 1H), 4.56 (d, J = 12.5 Hz, 1H), 4.40 (s, 2H), 4.29-4.12 (m, 2H), 3.96 (s, 6H), 3.82 (t,
J = 7.4 Hz, 2H), 3.68 (s, 1H), 3.64-3.49 (m, 1H), 3.10 (m, 1H) , 3.03 (d, J = 4.6 Hz, 2H),
2.72-2.53 (m, 3H), 2.14 (s, 6H), 2.08 (s, 1H), 1.86 (s, 1H), 1.72-1.69(m, 2H). 13C NMR (125 MHz,
DMSO-d6), δ 163.87, 160.88, 165.61, 151.33, 141.78, 137.93, 122.31, 113.15, 98.14, 72.55, 66.46,
59.91, 56.85, 54.87, 51.72, 44.99, 44.77, 41.75, 32.11, 29.18, 28.06. HRMS (ESI) for
C29H37Cl2N7O5 [M+H]+, calcd: 634.2306, found: 634.2304. HPLC analysis: MeOH−H2O (80: 20),
5.23 min, 95.76 % purity.
(E)-7-(cyclohexylamino)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-(1-(4-(dimethylamino)but
-2-enoyl)piperidin-4-yl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (2j)
1H NMR (500 MHz, DMSO-d6), δ 7.96 (s, 1H), 7.06 (brs, 1H), 6.97 (s, 1H), 6.64-6.56(m, 2H),
4.79 (s, 1H), 4.59 (d, J = 13.2 Hz, 1H), 4.37 (s, 2H), 4.17 (d, J = 13.4 Hz, 1H), 3.95 (s, 6H), 3.54
(brs, 1H), 3.09-3.03 (m, 1H), 3.01 (d, J = 4.6 Hz, 2H), 2.63-2.58 (m, 2H), 2.13 (s, 6H), 1.84 (brs,
2H), 1.69 (brs, 4H), 1.52 (brs, 1H), 1.23 (brs, 4H), 1.14-1.09 (m, 1H), 1.07-1.04 (m, 1H). 13C
NMR (125 MHz, DMSO-d6), δ 160.58, 154.66, 141.80, 137.99, 122.26, 113.16, 98.13, 59.92,
56.83, 55.97, 54.83, 44.98, 44.79, 32.43, 25.35, 24.71, 18.49. HRMS (ESI) for C31H41Cl2N7O4
[M+H]+, calcd: 646.2670, found: 646.2666. HPLC analysis: MeOH−H2O (85: 15), 7.79 min,
97.70 % purity.
(E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-(1-(4-(dimethylamino)but-2-enoyl) piperidin -4
-yl)-7-((tetrahydro-2H-pyran-4-yl)amino)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one
(2k)

ACCEPTED MANUSCRIPT
1H NMR (500 MHz, DMSO-d6), δ 7.98 (s, 1H), 7.19 (s, 1H), 6.97 (s, 1H), 6.75-6.39 (m, 2H), 4.80
(s, 1H), 4.58 (s, 1H), 4.38 (s, 2H), 4.17 (d, J = 13.5 Hz, 1H), 3.95 (s, 6H), 3.90-3.68 (m, 3H),
3.43-3.40 (m, 3H), 3.21-2.97 (m, 3H), 2.64-2.56 (m, 2H), 2.13 (s, 6H), 1.77-1.70 (m, 4H),
1.64-1.37 (m, 2H). 13C NMR (125 MHz, DMSO-d6), δ 160.55, 154.68, 151.49, 142.04, 137.96,
122.27, 113.15, 98.13, 66.02, 59.92, 56.85, 47.27, 45.01, 41.69, 32.49. HRMS (ESI) for
C30H39Cl2N7O5 [M+H]+, calcd: 648.2462, found: 648.2463. HPLC analysis: MeOH−H2O (85: 15),
4.58 min, 98.30 % purity.
(E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-(1-(4-(dimethylamino)but-2-enoyl)piperidin-4-yl
)-7-((3-methoxyphenyl)amino)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (2m)
1H NMR (500 MHz, DMSO-d6), δ 9.59 (s, 1H), 8.19 (s, 1H), 7.35 (s, 1H), 7.32 (dd, J = 8.0, 1.9
Hz, 1H), 7.19 (t, J = 8.1 Hz, 1H), 6.98 (s, 1H), 6.74-6.57 (m, 2H), 6.55 (dd, J = 8.2, 2.5 Hz, 1H),
4.90 (ddt, J = 11.7, 7.8, 4.0 Hz, 1H), 4.59 (d, J = 13.0 Hz, 1H), 4.48 (s, 2H), 4.19 (d, J = 13.6 Hz,
1H), 3.95 (s, 6H), 3.73 (s, 3H), 3.10 (t, J = 13.4 Hz, 1H), 3.01 (d, J = 4.8 Hz, 2H), 2.66 (t, J = 12.8
Hz, 1H), 2.42 (q, J = 13.4 Hz, 2H), 2.13 (s, 6H), 1.78 (d, J = 12.0 Hz, 2H). 13C NMR (125 MHz,
DMSO-d6), δ 163.98, 159.52, 158.85, 156.78, 154.69, 154.02, 150.88, 141.78, 141.32, 137.72,
129.17, 122.34, 113.18, 111.60, 106.28, 105.59, 102.73, 98.22, 59.89, 56.85, 54.97, 54.82, 52.42,
44.98, 44.77. HRMS (ESI) for C32H37Cl2N7O5 [M+H]+, calcd: 670.2306, found: 670.2281. HPLC
analysis: MeOH−H2O (85: 15), 6.77 min, 95.36 % purity.
(E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-(1-(4-(dimethylamino)but-2-enoyl) piperidin -4-
yl)-7-(m-tolylamino)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (2n)
1H NMR (500 MHz, DMSO-d6), δ 9.52 (s, 1H), 8.18 (s, 1H), 7.66-7.50 (m, 1H), 7.46 (s, 1H), 7.17
(t, J = 7.8 Hz, 1H), 6.98 (s, 1H), 6.77 (d, J = 7.5 Hz, 1H), 6.70-6.50 (m, 2H), 4.92-4.85 (m, 1H),
4.59 (d, J = 12.8 Hz, 1H), 4.47 (s, 2H), 4.18 (d, J = 13.5 Hz, 1H), 3.95 (s, 6H), 3.10-3.07 (m, 1H),
3.05 (d, J = 5.2 Hz, 2H), 2.63-2.60 (m, 1H), 2.47-2.39 (m, 2H), 2.29 (s, 3H), 2.16 (s, 6H), 1.78 (d,
J = 11.9 Hz, 2H). 13C NMR (125 MHz, DMSO-d6), δ 163.91, 158.97, 156.80, 154.70, 154.08,
150.95, 141.36, 140.07, 137.74, 137.48, 128.27, 122.64, 122.40, 119.58, 116.40, 113.17, 102.55,
98.22, 59.76, 56.86, 55.97, 54.83, 52.37, 44.85, 44.77, 21.34, 18.49. HRMS (ESI) for
C32H37Cl2N7O4 [M+H]+, calcd: 654.2357, found: 654.2342. HPLC analysis: MeOH−H2O (80: 20),
12.44 min, 96.11 % purity.

ACCEPTED MANUSCRIPT
(E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-(1-(4-(dimethylamino)but-2-enoyl) piperidin -
4-yl)-7-((3-fluorophenyl)amino)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (2o)
1H NMR (500 MHz, DMSO-d6), δ 9.85 (s, 1H), 8.23 (s, 1H), 7.73 (d, J = 12.4 Hz, 1H), 7.51-7.39
(m, 1H), 7.31 (q, J = 8.0 Hz, 1H), 6.99 (s, 1H), 6.83-6.70 (m, 1H), 6.67-6.48 (m, 2H), 4.92-4.87(m,
1H), 4.61 (d, J = 12.7 Hz, 1H), 4.50 (s, 2H), 4.20 (d, J = 14.1 Hz, 1H), 3.95 (s, 6H), 3.11-3.07 (m,
1H), 3.01 (d, J = 4.6 Hz, 2H), 2.68-2.62 (m, 1H), 2.46-2.37 (m, 2H), 2.13 (s, 6H), 1.81-1.79 (m,
2H). 13C NMR (125 MHz, DMSO-d6), δ 163.99, 163.22, 161.31, 158.60, 156.69, 154.70, 154.07,
150.81, 142.17, 142.08, 141.88, 137.66, 129.97, 129.90, 122.29, 114.67, 113.15, 107.75, 107.58,
105.49, 105.28, 103.35, 98.24, 59.90, 56.86, 52.58, 44.99, 44.75, 29.07, 28.06. HRMS (ESI) for
C31H34Cl2FN7O4 [M+H]+, calcd: 658.2106, found: 658.2108. HPLC analysis: MeOH−H2O (85:
15), 7.42 min, 95.97 % purity.
(E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-7-((3,5-difluorophenyl)amino)-1-(1-(4-(dimethyla
mino)but-2-enoyl)piperidin-4-yl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (2p)
1H NMR (500 MHz, DMSO-d6), δ 10.05 (s, 1H), 8.26 (s, 1H), 7.59-7.42 (m, 2H), 6.99 (s, 1H),
6.75 (tt, J = 9.2, 2.4 Hz,1H), 6.66-6.50 (m, 2H), 4.88 (tt, J = 11.9, 4.0 Hz, 1H), 4.59 (d, J = 12.9
Hz, 1H), 4.51 (s, 2H), 4.20 (d, J = 13.5 Hz, 1H), 3.95 (s, 6H), 3.16-3.03 (m, 1H), 3.01 (d, J = 5.1
Hz, 2H), 2.66 (d, J = 13.6 Hz, 1H), 2.46-2.31 (m, 2H), 2.13 (s, 6H), 1.82 (d, J = 12.0 Hz, 2H). 13C
NMR (125 MHz, DMSO-d6), δ 164.02, 163.50, 163.37, 161.58, 161.45, 158.35, 156.98, 154.71,
154.05, 150.66, 142.97, 141.84, 137.58, 122.28, 113.15, 103.98, 101.41, 101.17, 98.27, 96.16,
59.88, 56.87, 55.97, 54.82, 52.66, 44.98, 44.73, 18.49. HRMS (ESI) for C31H33Cl2F2N7O4,
[M+H]+, calcd: 676.2012, found: 676.2017. HPLC analysis: MeOH−H2O (85: 15), 9.43 min,
98.01 % purity.
(E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-7-((3,4-difluorophenyl)amino)-1-(1-(4-(dimethyla
mino)but-2-enoyl)piperidin-4-yl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (2q)
1H NMR (500 MHz, DMSO-d6), δ 9.84 (s, 1H), 8.22 (s, 1H), 7.89-7.86 (m, 1H), 7.43-7.33 (m,
2H), 6.99 (s, 1H), 6.64-6.56 (m, 2H), 4.88-4.84 (m, 1H), 4.61-4.59 (m, 1H), 4.49 (s, 2H),
4.20-4.18 (m, 1H), 3.95(s, 6H), 3.12-3.06 (m, 1H), 3.02-3.01 (m, 2H), 2.67-2.62 (m, 1H),
2.45-2.36 (m, 2H), 2.13 (s, 6H), 1.79-1.78 (m, 2H). 13C NMR (125 MHz, DMSO-d6), δ 163.97,
158.53, 156.92, 154.71, 154.08, 150.84, 141.87, 137.66, 137.45, 137.38, 122.31, 117.16, 117.02,
115.08, 113.15, 107.77, 107.59, 103.41, 98.24, 59.89, 56.87, 52.58, 44.98, 44.75, 29.09, 28.05.

ACCEPTED MANUSCRIPT
HRMS (ESI) for C31H33Cl2F2N7O4 [M+H]+, calcd: 676.2012, found: 676. 2025. HPLC analysis:
MeOH−H2O (85: 15), 7.80 min, 98.38 % purity.
(E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-(1-(4-(dimethylamino)but-2-enoyl)
piperidin-4-yl) -7-((4-fluoro-3-methoxyphenyl)amino)-3,4-dihydropyrimido[4,5-d] pyrimidin
-2(1H)-one (2r)
1H NMR (500 MHz, DMSO-d6), δ 9.52 (s, 1H), 8.18 (s, 1H), 7.41 (ddd, J = 8.9, 3.9, 2.6 Hz, 1H),
7.34 (d, J = 7.8 Hz, 1H), 7.12 (dd, J = 11.5, 8.9 Hz, 1H), 6.98 (s, 1H), 6.79-6.46 (m, 2H),
5.00-4.77 (m, 1H), 4.59 (d, J = 12.9 Hz, 1H), 4.47 (d, J = 2.0 Hz, 2H), 4.18 (d, J = 13.5 Hz, 1H),
3.95 (s, 6H), 3.81 (s, 3H), 3.09-3.04(m, 1H), 3.01 (d, J = 4.2 Hz, 2H), 2.64-2.59 (m, 1H),
2.47-2.37 (m, 2H), 2.13 (s, 6H), 1.76 (d, J = 11.9 Hz, 2H). 13C NMR (125 MHz, DMSO-d6), δ
163.94, 158.85, 156.85, 154.69, 153.95, 150.96, 147.88, 146.76, 146.67, 145.98, 141.88, 137.72,
136.91, 122.29, 115.39, 115.24, 113.15, 111.08, 111.04, 106.01, 102.89, 98.22, 59.90, 56.86,
56.02, 52.38, 44.98, 44.74. HRMS (ESI) for C32H36Cl2FN7O5 [M+H]+, calcd: 688.2212, found:
688.2216. HPLC analysis: MeOH−H2O (85: 15), 6.05 min, 95.08 % purity.
(E)-7-((3-chloro-4-fluorophenyl)amino)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-(1-(4-(dimet
hylamino)but-2-enoyl)piperidin-4-yl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (2s)
1H NMR (500 MHz, DMSO-d6), δ 9.80 (s, 1H), 8.22 (s, 1H), 7.99-7.97 (m, 1H), 7.60-7.58 (m,
1H), 7.34 (t, J = 9.1 Hz, 1H), 6.99 (s, 1H), 6.63-6.56 (m, 2H), 4.89-4.83 (m, 1H), 4.58 (d, J = 12.8
Hz, 1H), 4.49 (s, 2H), 4.18 (d, J = 13.6 Hz, 1H), 3.95 (s, 6H), 3.14-3.09 (m, 1H), 3.03-3.00 (m,
2H), 2.69-2.65 (m, 1H), 2.45-2.37 (m, 2H), 2.12 (s, 6H), 1.79 (s, 2H).13C NMR (125 MHz,
DMSO-d6), δ 163.93, 158.52, 156.89, 154.69, 154.10, 153.07, 151.15, 150.80, 141.87, 137.64,
137.54, 122.25, 119.99, 119.19, 119.14, 118.18, 118.73, 116.66, 116.49, 113.13, 103.33, 98.25,
59.88, 56.86, 52.49, 44.97, 44.72, 29.01, 28.00. HRMS (ESI) for C31H33Cl3FN7O4 [M+H]+, calcd:
692.1716, found: 692.1717. HPLC analysis: MeOH−H2O (85: 15), 9.43 min, 98.38 % purity.
(E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-(1-(4-(dimethylamino)but-2-enoyl)piperidin-4-yl
)-7-((4-fluorophenyl)amino)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (2t)
1H NMR (500 MHz, DMSO-d6), δ 9.62 (s, 1H), 8.18 (s, 1H), 7.84-7.57 (m, 2H), 7.13 (t, J = 8.9
Hz, 2H), 6.98 (s, 1H), 6.61 (m, 2H), 4.84 (td, J = 11.7, 5.8 Hz, 1H), 4.60 (d, J = 12.8 Hz, 1H),
4.47 (s, 2H), 4.18 (d, J = 13.6 Hz, 1H), 3.95 (s, 6H), 3.09 (dd, J = 16.9, 10.8 Hz, 1H), 3.04-2.97
(m, 1H), 2.64 (t, J = 12.8 Hz, 1H), 2.46-2.29 (m, 2H), 2.13 (s, 6H), 1.76 (d, J = 12.0 Hz, 1H). 13C

ACCEPTED MANUSCRIPT
NMR (125 MHz, DMSO-d6), δ 163.92, 158.85, 158.18, 156.83, 156.28, 154.69, 154.07, 151.02,
141.88, 137.74, 136.48, 122.29, 120.83, 120.76, 115.02, 114.85, 113.17, 102.78, 98.21, 59.90,
56.85, 55.97, 54.83, 52.52, 44.97, 44.76, 18.49. HRMS (ESI) for C31H34Cl2FN7O4 [M+H]+, calcd:
658.2106, found: 658.2088. HPLC analysis: MeOH−H2O (85: 15), 6.55 min, 98.47 % purity.
(E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-(1-(4-(dimethylamino)but-2-enoyl)piperidin-4-yl
)-7-((2-methoxyphenyl)amino)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (2u)
1H NMR (500 MHz, DMSO-d6), δ 8.15 (d, J = 5.4 Hz, 2H), 8.02-7.87 (m, 1H), 7.08-6.96 (m, 3H) ,
6.93 (td, J = 7.5, 7.0, 2.0 Hz, 1H), 6.75-6.53 (m, 2H), 4.87-4.69 (m, 1H), 4.56 (d, J = 12.8 Hz, 1H),
4.47 (s, 2H), 4.13 (d, J = 13.6 Hz, 1H), 3.95 (s, 6H), 3.84 (s, 3H), 3.19 (d, J = 5.9 Hz, 2H), 3.03 (d,
J = 13.6 Hz, 1H), 2.60 (t, J = 13.2 Hz, 1H), 2.39 (d, J = 12.3 Hz, 2H), 2.26 (s, 6H), 1.72 (d, J =
11.8 Hz, 2H).13C NMR (125 MHz, DMSO-d6), δ 163.71, 159.01, 156.85, 154.68, 154.06, 150.99,
149.69, 137.74, 128.18, 123.76, 123.29, 121.15, 120.17, 113.15, 110.89, 102.85, 98.21, 59.36,
56.85, 55.97, 55.64, 54.82, 52.41, 44.75, 44.41, 28.94, 27.93, 18.48. HRMS (ESI) for
C32H37Cl2N7O5 [M+H]+, calcd: 670.2310, found: 670.2308. HPLC analysis: MeOH−H2O (85: 15),
7.83 min, 97.47 % purity.
(E)-3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-(1-(4-(dimethylamino)but-2-enoyl)
piperidin
-4-yl) -7-((4-methoxyphenyl)amino)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (2v)
1H NMR (500 MHz, DMSO-d6), δ 9.42 (s, 1H), 8.15 (s, 1H), 7.75-7.47 (m, 2H), 6.99 (s, 1H), 6.93
-6.79 (m, 2H), 6.81-6.40 (m, 2H), 5.07-4.71 (m, 1H), 4.61 (d, J = 12.6 Hz, 1H), 4.46 (s, 2H), 4.19
(d, J = 13.5 Hz, 1H), 3.96 (s, 6H), 3.72 (s, 3H), 3.21-2.93 (m, 3H), 2.72-2.59 (m, 1H), 2.46-2.42
(m, 2H), 2.16 (s, 6H), 1.76 (d, J = 11.9 Hz, 2H). 13C NMR (125 MHz, DMSO-d6), δ 162.87,
159.11, 156.74, 154.69, 154.39, 154.15, 151.08, 137.78, 133.24, 131.89, 129.07, 120.96, 113.69,
113.20, 113.30, 102.07, 98.19, 57.21, 56.87, 55.16, 52.24, 45.39, 44.79, 42.19, 29.00, 27.99.
HRMS (ESI) for C32H37Cl2N7O5 [M+H]+, calcd: 670.2306, found: 670.2312. HPLC analysis:
MeOH−H2O (80: 20), 9.04 min, 99.37 % purity.
5.2 In Vitro Kinase Enzyme Assay
FGFR1-4 and the Z'-Lyte Kinase Assay Kit were purchased from Invitrogen. The concentration
gradients from 5.1 × 10−11 to 1.0 × 10−6 mol/L were used for all the test compounds. The
experiments were performed according to the instructions of the manufacturer.
5.3 Cell proliferation assays

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5.3.1 FGFR-positive and FGFR-negative cancer cell Proliferation Assay.
Cell lines were cultured in RPMI 1640 supplemented with 10% FBS (v/v) (H520, SUM52 and
SW780) or α-MEM supplemented with 5% FBS (v/v) (PC-9, H661, H460 and H1299) in a
humidified incubator (37 °C, 5% CO2). Cells were harvested when the monolayer achieved 80%
density and counted using a Z2 Coulter® Particle Count and Size Analyzer. Cells were maintained
in culture for <3 months cumulative passage from authenticated stocks.
Cells were seeded into 96-well plates in a total volume of 150 µl/well at densities of 3000
(H661), 2000 (H520, SUM52 and SW780), 1000 (PC9) or 400 (H460 and H1299) cells/well; and
incubated overnight at 37 ˚C to allow attachment. All drugs were prepared as stocks in DMSO and
stored at -80 °C. Working solutions of each drug were made in RPMI + 10% FBS or α-MEM +5%
FBS at 3 times the required final concentration for the top well. A 3-fold serial dilution was
performed along the plate. Following drug addition, cells were incubated in the presence of drug
(37 °C, 5% CO2) for a period of 5 days (with the exception of H520 cells which were incubated
for 7 days). The plates were removed from the incubator and fixed by adding 50 µL of cold 40%
(w/v) TCA solution per well and incubating for 1 hour at 4 °C. The plates were subsequently
washed (3X) with running tap water before being stained with 50 µL of 0.4% (w/v) SRB and
incubated in the dark 30 (min). Excess stain was removed by rinsing the plates (4X) in water
containing 1% (v/v) acetic acid. The stain was solubilized with 100 µL of 10 mM unbuffered Tris
(pH 10.5) per well and the plates were left on a shaker in the dark at RT for 1 hour. The
absorbance values were determined for each well by an EL808 Absorbance Microplate Reader
(490 nm; reference filter 450 nm). The IC50 values were determined by the KC4 microplate data
analysis software (KC4TM, Bio-Tek).
5.3.2 Human normal liver HL7702 cells Proliferation Assay.
HL7702 cells in the logarithmic phase were plated in 96-well culture dishes (~3000 cells/ well).
24 hours later, cells were treated with the corresponding compounds 2l or vehicle control at the
indicated concentration for 72 hours. CCK-8 was added into the 96- well plates (10 µl / well) and
incubated with the cells for 3 hours. OD450 and OD650 were determined by a micro-plate reader.
Absorbance rate (A) for each well was calculated as OD450- OD650. The cell viability rate for
each well was calculated as V% =(As-Ac)/(Ab-Ac)×100% and the data were further analyzed
using Graphpad Prism5 (Graphpad Software ,Inc.) As, Absorbance rate of the test compound well;

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Ac, absorbance rate of the well without either cell or test compound; Ab, absorbance rate of the
well with cell and vehicle control.
5.4 Western blot analysis and washout assay
Cells were seeded on the day prior to drug treatment (1 million cells/well; 6-well plate). For
SW780 and SUM52, cells were seeded in 3 ml RPMI 1640 + 10 % FBS and incubated for 6 hr to
allow adherence. Media was then aspirated, cells washed with PBS and 3 ml of fresh, serum-free
RPMI 1640 was added to serum-starve the cells overnight (18 hours) (all subsequent steps used
serum-free media). Serum starvation was not performed in experiments using H520 cells; instead
cells were seeded in RPMI 1640 + 10 % FBS and incubated overnight. Drug exposure - no
washout: Drug stocks were prepared in media using 1 mM or 10 mM DMSO stock solutions.
Media was aspirated from each well and replaced with 2 ml of drug-free media for control wells
or 2 ml of diluted drug for treated wells. Cells were exposed to drug for 30 min. Recombinant
FGF (50 ng/well) was added to each well 15 minutes prior to the end of the drug exposure and
protein was extracted as below. Drug exposure - with washout: Cells were exposed to drug for 30
min as above. Media was aspirated and each well was washed with 5 ml drug-free media. The
wash media was aspirated and replaced with 2 ml drug-free media and cells incubated for a further
30 minutes, after which the wash protocol was repeated. Cells were incubated for a further 90
minutes in drug-free media. Recombinant FGF (50 ng/well) was added to each well 15 minutes
prior to the end of the drug-free incubation and protein was extracted as below. Protein extraction
and Western blotting: Cell lysates were prepared in 250 µL of RIPA lysis buffer containing
protease inhibitor (1:100). After incubation on ice (30 min) and centrifugation (13,000 rpm, 5 min,
4 °C) the supernatants containing the proteins were stored (-20 °C) until required. Bicinchoninic
acid (BCA) assay was used to determine the protein concentration of individual cell lysate
samples. Western blots were prepared using the following primary antibodies: p-p44/42 MAPK
(pERK1/2) (Thr202/Tyr204) rabbit monoclonal (Cell Signalling Technology #9101); pFGFR
mouse monoclonal (Cell signalling Technology #3476S); and α-Tubulin mouse monoclonal
(Sigma-Aldrich #T6074).
Acknowledgements
The authors appreciate the financial support from National Natural Science Foundation of
China (81425021 and 81673285), Chinese National Programs for Key Research and Development

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(2016YFB0201701)，the Health Research Council of New Zealand (13/196 and 13/1020),
International collaborative funds from Guangdong Province (2016A050502035), Guangdong
Province (2013A022100038, 2015A030312014 and 2015A030306042, 2016A050502041),
Guangzhou City (201508030036), Jinan University , Guangzhou Branch of the Supercomputing
Center of Chinese Academy of Sciences.
Abbreviations
Ala, alanine; FGFR, Fibroblast growth factor receptor; BLK, B-lymphoid tyrosine kinase; CSF1R,
Colony stimulating factor 1 receptor; ERK, extracellular regulated protein kinases; DIPEA,
ethyldiisopropylamine; THF, tetrahydrofuran; TFA, trifluoroacetic acid; DMF, N,
N-dimethylformamide; HATU, 1-[Bis(dimethylamino)methylene] -1H-1,2,3- triazolo[4,5-b]
pyridinium3-oxid hexafluorophosphate; m-CPBA, 3-chloroperbenzoic acid; TLC, thin-layer
chromatography; UV, ultraviolet; ppm, parts per million; SRB, Sulforhodamine B.
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