Design, synthesis, and biological evaluation of novel conformationally constrained inhibitors targeting epidermal growth factor receptor threonine 790 → methionine790 mutant

Article abstract of DOI:10.1021/jm201591k

The EGFR^T790M mutant contributes approximately 50% to clinically acquired resistance against gefitinib or erlotinib. However, almost all the single agent clinical trials of the second generation irreversible EGFR inhibitors appear inadequate to overcome the EGFR^T790M-related resistance. We have designed and synthesized a series of 2-oxo-3,4- dihydropyrimido[4,5-d]pyrimidinyl derivatives as novel EGFR inhibitors. The most potent compounds, 2q and 2s, inhibited the enzymatic activities of wild-type and mutated EGFRs, with IC₅₀ values in subnanomolar ranges, including the T790M mutants. The kinase inhibitory efficiencies of the compounds were further validated by Western blot analysis of the activation of EGFR and downstream signaling in cancer cells harboring different mutants of EGFR. The compounds also strongly inhibited the proliferation of H1975 non small cell lung cancer cells bearing EGFR^L858R/T790M, while being significantly less toxic to normal cells. Moreover, 2s displayed promising anticancer efficacy in a human NSCLC (H1975) xenograft nude mouse model.

Full text of DOI:10.1021/jm201591k

Article
pubs.acs.org/jmc
Design, Synthesis, and Biological Evaluation of Novel
Conformationally Constrained Inhibitors Targeting Epidermal
Growth Factor Receptor Threonine790 → Methionine790 Mutant
Shaohua Chang,†,§,# Lianwen Zhang,§,‡,# Shilin Xu,†,§ Jinfeng Luo,† Xiaoyun Lu,† Zhang Zhang,†
Tianfeng Xu,†,§ Yingxue Liu,† Zhengchao Tu,† Yong Xu,† Xiaomei Ren,† Meiyu Geng,⊥ Jian Ding,⊥
Duanqing Pei,† and Ke Ding*,†
†Key Laboratory of Regenerative Biology and Institute of Chemical Biology, Guangzhou Institutes of Biomedicine and Health,
Chinese Academy of Sciences, #190 Kaiyuan Avenue, Guangzhou 510530, China
‡School of Life Science, University of Science and Technology of China, # 96 Jinzhai Road, Hefei 230026, China
§Graduate School of Chinese Academy of Sciences, # 19 Yuquan Road, Beijing 100049, China
⊥State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, # 555 Zu-Chong-Zhi
Road, Zhangjiang Hi-Tech Park, Shanghai 201203, China
S
* Supporting Information
ABSTRACT: The EGFRT790M mutant contributes approx-
imately 50% to clinically acquired resistance against gefitinib or
erlotinib. However, almost all the single agent clinical trials of
the second generation irreversible EGFR inhibitors appear
inadequate to overcome the EGFRT790M-related resistance. We
have designed and synthesized a series of 2-oxo-3,4-
dihydropyrimido[4,5-d]pyrimidinyl derivatives as novel
EGFR inhibitors. The most potent compounds, 2q and 2s,
inhibited the enzymatic activities of wild-type and mutated EGFRs, with IC50 values in subnanomolar ranges, including the
T790M mutants. The kinase inhibitory efficiencies of the compounds were further validated by Western blot analysis of the
activation of EGFR and downstream signaling in cancer cells harboring different mutants of EGFR. The compounds also strongly
inhibited the proliferation of H1975 non small cell lung cancer cells bearing EGFRL858R/T790M, while being significantly less toxic
to normal cells. Moreover, 2s displayed promising anticancer efficacy in a human NSCLC (H1975) xenograft nude mouse
model.
moderately affects the binding of gefinitib and erotinib.
However, this mutation enhances the binding affinity for
ATP with EGFR, which leads to higher concentrations of the
competitive inhibitors being required for the kinase suppres-
sion.10
INTRODUCTION
■
Epidermal growth factor receptor (EGFR) belongs to the ErbB
family of receptor tyrosine kinases which play indispensable
roles in cell proliferation, survival, adhesion, migration, and
differentiation.1 Small molecular EGFR inhibitors, gefinitib and
erlotinib, were approved by US Food and Drug Administration
in 2002 and 2004, respectively, and have achieved significant
clinical benefit for non small cell lung cancer (NSCLC) patients
harboring activating EGFR mutations such as L858R or exon
19 deletions (del E746-A750), etc.2−4 Anti-EGFR antibodies
cetuximab5 and panitumumab6 have also become effective
clinical therapies for different solid tumors associated with
overexpressed EGFR. Despite the clear clinical benefits of
gefinitib and erotinib, their efficacy is eventually diminished
because of acquired point mutations in the kinase domain of
EGFR as well as the upregulation of bypass signaling pathways.7
In order to overcome the T790M mutation related
resistance, a number of irreversible ATP-competitive EGFR
inhibitors11 such as CI-1033,12 BIBW2992,13 HKI-272,14 and
PF00299804,15 etc., have been developed. The irreversible
inhibitors contain a Michael addition receptor moiety which
may form a covalent bind with the conserved cysteine residue
present in the lip of the EGFR ATP binding site (Cys797) to
achieve occupancy greater than that of the reversible
inhibitors.16 The reaction of the Cys797 residue with
irreversible inhibitors also potently prevents its sulfenylation-
enhancing kinase activity.30 However, almost all the single
agent clinical trials of the second generation irreversible EGFR
inhibitors seemed to be disappointing.7b For instance, HKI-272
Particularly, a single T790M point mutation (threonine790
→
methionine790) at the “gatekeeper” position in EGFR accounts
for approximately 50% in clinically acquired resistant patients.8
Different from the fact that T315I-mutated Abl introduces a
steric impediment for imatinib binding,9 EGFRT790M only
Received: November 25, 2011
Published: February 16, 2012
© 2012 American Chemical Society
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(neratinib) has been demonstrated to show little activity against
gefitinib-resistant NSCLC patients harboring EGFR mutations
in a phase II trial, likely due to toxicity-related dose-limitation.17
PF00299804 also failed to show any responses in NSCLC
patients with EGFRT790M mutants.18 In a phase III clinical trial,
BIBW2992 (afatinib) only displayed 7% response rate for
NSCLC patients who progressed after ≥12 weeks of gefinitib
or erlotinib treatment.7b,19 The most promising results came
from a phase Ib trial of a combination of BIBW2992 with
cetuximab, which showed an approximate 40% objective
response rate. However, the progression-free survival and
overall survival results are unpredictable.20 Therefore, the
clinically acquired resistance against first-line EGFR inhibitors
remains one of the major challenging unmet medical needs for
NSCLC treatment. It is highly desirable to identify new
molecules targeting EGFRT790M mutants.11
mate (4) almost quantitatively produced ethyl 4-(3-(tert-
butoxycarbonyl)phenylamino)-2-(methylthio)pyrimidine-5-car-
boxylate (5) as a white solid. The reduction of compound 5
with lithium aluminum hydride yielded tert-butyl 3-(5-
(hydroxymethyl)-2-(methylthio)pyrimidin-4-ylamino)-
phenylcarbamate (6) which was followed by selective oxidation
to produce the aldehyde 7 in a good yield. A variety of diamine
intermediates 8 could be prepared by a reductive amination of
aldehyde 7 with different amines. With diamine compounds 8
in hand, the key intermediates 10 were readily synthesized by
direct cyclization with triphosgene and followed by oxidation.
Compound 10 coupled with different substituted anilines to
produce precursor compounds 11 which were further
deprotected with trifluoroacetic acid and reacted with acryloyl
chloride to obtain the conformation-constrained EGFR
inhibitors 2.
Most recently, Gray et al. reported a novel series of
substituted pyrimidines as irreversible EGFR inhibitors
displaying good selectivity against EGFRT790M mutants over
the wild type kinase.21 One of the most active and selective
inhibitors, WZ4002 (1),21 potently inhibited the proliferation
of the cancer cells or Ba/F3 cells harboring L858R, del E746-
A750, and/or T790M mutated EGFR, while it was obviously
less potent against the growth of wild type EGFR cells.
Furthermore, the compound also displayed promising in vivo
efficacy in gefitinib-resistant NSCLC models, which repre-
sented a new, promising strategy to overcome the acquired
resistance for NSCLC patients. The clinical results of 1 or
related candidates are eagerly awaited. In this paper, we report
the structural design, synthesis, and biological evaluation of
conformation-constrained 2-oxo-3,4-dihydropyrimido[4,5-d]-
pyrimidinyl derivatives as novel EGFR inhibitors which strongly
RESULTS AND DICUSSION
■
Taking 1 as the lead molecule, we designed a series of 2-oxo-
3,4-dihydropyrimido[4,5-d]pyrimidinyl derivatives as new
EGFR inhibitors using a combination of bioisosterism and
conformational constraint strategies. The kinase inhibitory
activities of the designed compounds were evaluated via a well
established FRET-based Z′-Lyte assay22 against different types
of EGFR kinases, and the results are summarized in Table 1.
Under the experimental conditions, compound 1 potently
inhibited EGFRWT, EGFRT790M, EGFRL858R, EGFRL861Q, and
EGFRL858R/T790M with IC50 values of 6.18, 3.77, 5.37, 6.13, and
1.88 nM, respectively, which were similar to previously
reported data.21
We were pleased to find that the conformation-constrained
compound 2a also strongly suppressed the enzymatic activities
of all five types of EGFR kinases with potencies comparable to
that of compound 1 (Table 1). Taking compound 2a as our
new lead compound, we conducted further structure
modification to explore the structure−activity relationships.
The impact of the 1-(4-methylpiperazinyl) group in compound
2a was first inspected by removal or replacement with other
moieties. The results demonstrated that the 1-(4-methylpiper-
azinyl) group was important for the EGFR kinase inhibition.
When the 1-(4-methylpiperazinyl) group was removed (2g) or
replaced with dimethylamino (2b), 1-piperidinyl (2c), or N-
morpholino (2d) moieties, the potencies against EGFR kinases
decreased approximately 5−20 fold. For instance, the IC50
suppressed the enzymatic activities of EGFRWT, EGFRL858R
,
EGFRT790M, EGFRL861Q, and EGFRL858R/T790M. The compounds
also potently inhibited the proliferation of NSCLC cells bearing
EGFRdel E746‑A750 or EGFRL858R/T790M mutants. One of the most
potent inhibitors, 2s, displayed significant in vivo anticancer
efficacy in a H1975 human NSCLC cancer xenograft mouse
model, representing a new, promising lead compound with a
different chemical scaffold for further development of EGFR
inhibitors to overcome EGFRT790M mutation-induced clinical
resistance to gefitinib and erotinib.
values of compound 2a against EGFRWT, EGFRT790M
,
EGFRL858R, EGFRL861Q, and EGFRL858R/T790M were 1.16, 6.56,
1.08, 1.21, and 3.14 nM, while the corresponding values for
compound 2c were decreased to 29.50, 95.71, 10.42, 15.40, and
36.43 nM, respectively. Further investigation also revealed that
the potency loss of compound 2c was partially restored by
introducing a 4-dimethylamino group (2b) at the position of
the original piperidinyl moiety. When the 1-(4-methylpiper-
azinyl) group was replaced with a 1-(4-methyl-1,4-diazepanyl)
group, the resulting compound 2e was almost equally potent to
compound 2a. However, when the substituted aniline in
compound 2a was replaced with a simple methylamino moiety
(2h), the potencies against EGFR kinases were almost
completely abolished.
Figure 1. Design of conformation-constrained derivatives of 1 as new
EGFR inhibitors.
CHEMISTRY
■
The impact of the R2 methoxy group in 2a was also
investigated. Similar to the previous observation,21 removal of
the R2 methoxy group improved the potencies against all forms
of the EGFR kinases 2- to 3-fold, but the resulting compound
2i could lose the selectivity over JAK3 and the TEC-family
The designed compounds 2 were prepared by using ethyl 4-
chloro-2-(methylthio)pyrimidine-5-carboxylate (3) as the start-
ing material (Scheme 1).29 Briefly, a direct nucleophilic
coupling of compound 3 with tert-butyl 3-aminophenylcarba-
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Scheme 1. Synthesis of the Conformation-Constrained Compounds 2
Reagent and conditions: (a) K2CO3 (2.0 equiv), dimethylformamide (DMF), 60 °C, overnight, 97%; (b) lithium aluminum hydride (LAH, 2.0
equiv), THF, −40 °C to r.t., 57%; (c) MnO2, dichloromethane, r.t., overnight, 85%; (d) R2NH2, AcOH, NaBH4, MeOH, 0 °C to r.t., overnight; (e)
triphosgene (0.34 equiv), Et3N (4.0 equiv), 0 °C to r.t., 2.0 h; (f) 3-chloroperbenzoic acid (m-CPBA, 3.0 equiv), dichloromethane, r.t., 3.0 h; (g)
R1NH2 (1.0 equiv), trifluoroacetic acid (1.0 equiv), 2-BuOH, 110 °C, sealed tube, 18.0 h, 30−60%; (h) trifluoroacetic acide, dichloromethane, r.t.;
(i) acryloyl chloride (1.0 equiv), N,N-diisopropylethylamine (DIPEA, 1.0 equiv), 0 °C to r.t.
kinase.21 When the methoxy group was moved from the 2′-
position to the 3′-position (2j), the inhibitory potency against
EGFRL858R/T790M was barely affected, although the potencies
against EGFRWT, EGFRT790M, and EGFRL858R were improved
2−3 fold. When the 2′-methoxy group in 2a was replaced with a
slightly larger group such as an ethoxy (2k) or isopropoxy (2l)
group, potencies were obviously decreased. Although the 2′-
methyl analogue (2m) displayed potencies similar to that of 2a,
the 2′-fluoro compound (2n) was totally inactive. The potency
loss of 2n might be due to a five-membered ring formation
involving a hydrogen bond between the F atom and the NH
moiety, which could alter the conformation of the molecule.
The results also illustrated that the R3 position in compound
2a might be replaced with a larger hydrophobic group to
improve the potency against EGFR kinase. When the R3 methyl
was replaced with an isopropyl (2o) or cyclopropyl (2p) group,
the inhibitory activities against EGFRWT, EGFRL858R, and
EGFRL861Q were obviously improved, with IC50 values below
1 nM, but the suppressive functions against EGFRT790M and
EGFRL858R/T790M were slightly decreased. The methyl group
could also be replaced with large hydrophobic groups such as 2-
naphthyl (2r), 4-biphenyl (2t), 4-phenoxyphenyl (2u), and 4-
benzoxyphenyl (2v), etc., to maintain the strong inhibition
against EGFR kinase. Interestingly, when the R3 methyl in
compound 2a was replaced with a phenyl (2q) or benzyl (2s)
group, the resulting compounds showed the best potencies. For
instance, compound 2s strongly bound to all the types of EGFR
kinase and inhibited their enzymatic functions with Kd and IC50
values both in subnanomolar ranges (Table 1), which is
obviously more potent than compound 1. However, the
binding affinities with EGFR were significantly decreased over
1000 fold (Kd values about 560−1500 nM) when the α,β-
unsaturated acrylamide moieties were removed (2w and 2x).
Highly consistent with their binding affinity loss, 2w and 2x
were almost totally inactive to inhibit the EGFR enzymatic
activities (IC50 values >1000 nM).
Taking compounds 2q and 2s as examples, we further
profiled the compounds against a panel of 451 kinases using the
Ambit Kinome screening platform to investigate the selectivity
of the new EGFR inhibitors (Supporting Information and
Figure 2). The results suggested that both compounds
displayed excellent selectivity on EGFR, and the specificity
profile of compound 2s was obviously greater than that of 2q.
In addition to EGFR, 2s only exhibited obvious binding to
BLK, BTK, ErbB-2, ErbB4, GAK, and TXK at a concentration
of 100 nM, which was about 100 times its Kd values on EGFR,
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a
Table 1. In Vitro Enzymatic Inhibitory Activities of Compounds 2 against Different Types of EGFR
a
EGFR activity assays were performed using the FRET-based Z′-Lyte assay according to the manufacturer’s instructions. The compounds were
incubated with the kinase reaction mixture for 1.5 h before measurement. The data were means from at least three independent experiments.
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Table 1. continued
bBinding constant values (Kd) were determined from Ambit KINOMEscan. The data were means from two independent experiments. c
Reported
data.21
Figure 2. KINOMEsacn tree spot maps illustrating the selectivity profiles for compounds 2q and 2s versus a panel of 451 kinase targets (including
392 wild-type kinases). The size of the red circle is proportional to the percent of DMSO control, where 0% and 35% of control equals 100% and
65% competition, respectively.
Figure 3. Compounds 2a, 2q, and 2s inhibit the activation of EGFR and downstream signaling in a dose-dependent manner. (A) Compounds 2a,
2q, 2s, and 1 inhibit the activation of EGFR and downstream signaling in H1975 NSCLC cells harboring EGFRL858R/T790M, while gefitinib has no
effects. (B) Compounds 2a, 2q, 2s, 1, and gefitinib inhibit the activation of EGFR and downstream signaling in HCC827 NSCLC cells harboring
EGFRdel E746‑A750. The results are representative of experiments in triplicate. Gfb: gefitinib.
while compound 2q also showed strong binding with BMX,
FRK, JAK3, KITV559D, LCK, MAP3K15, PDGFRβ, TEC, and
TNK2 at the same time.
In order to further validate the activities of these new EGFR
inhibitors, we examined the effects of representative com-
pounds 2a, 2q, and 2s on the activation of EGFR and the
downstream signal in cancer cells harboring a different status of
EGFR kinases, and the results are summarized in Figure 3.
Similar to that of compound 1, inhibitors 2a, 2q, and 2s dose-
dependently inhibited the phosphorylation of EGFR in both
H1975 NSCLC cells bearing EGFRL858R/T790M and HCC827
23
E746‑A750
cells harboring EGFRdel
.
The protein levels of
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Table 2. Antiproliferative Activities of the Conformation-Constrained Inhibitors 2 against Cells Harboring a Different Status of
a
EGFR
IC50 (μM)
compound
HCC827 (Del E746_A750)
H1975 (L858R/T790M)
A431 (WT, overexpression)
A549 (WT, k-Ras mutation)
HL7702 (WT)
1
0.007 0.001
0.018 0.007
0.74 0.08
0.048 0.005
0.047 0.014
0.54 0.08
0.61 0.05
0.15 0.01
0.72 0.02
0.53 0.08
0.17 0.02
20.07 1.65
0.045 0.017
0.024 0.001
0.103 0.002
0.23 0.01
0.20 0.03
8.46 1.26
0.058 0.014
0.054 0.020
0.016 0.006
0.030 0.013
0.014 0.006
0.095 0.008
0.048 0.019
0.033 0.011
>20
1.39 0.28
0.75 0.21
5.62 1.35
5.06 0.66
>10
5.31 0.56
15.94 1.64
>20
2.73 0.23
>10
2a
2b
2c
>10
0.170 0.006
0.13 0.04
12.02 1.21
10.46 2.08
>20
>10
2d
2e
>10
0.073 0.008
0.15 0.05
2.15 0.48
1.39 0.48
6.90 1.57
>10
>10
2f
14.50 1.76
18.02 0.51
>20
>10
2g
0.044 0.01
6.92 3.32
>10
2h
2i
>10
0.014 0.009
0.041 0.018
0.014 0.004
0.027 0.001
0.031 0.015
5.01 0.33
0.55 0.07
0.36 0.17
1.08 0.18
2.11 0.66
0.99 0.30
>10
11.35 0.63
3.33 0.71
10.35 1.59
11.67 2.15
11.92 1.99
11.13 1.87
8.42 2.64
17.90 3.12
2.80 1.35
2.41 0.52
2.66 0.33
2.27 0.11
1.77 0.27
2.47 0.60
11.17 2.17
>30
7.56 1.83
>10
2j
2k
>10
2l
>10
2m
2n
2o
2p
2q
2r
>10
7.18 2.65
>10
0.004 0.001
0.009 0.003
0.003 0.001
0.006 0.002
0.003 0.001
0.018 0.006
0.014 0.003
0.023 0.001
4.89 1.02
0.82 0.13
0.81 0.02
0.11 0.02
0.57 0.02
0.17 0.03
1.21 0.15
1.20 0.24
1.15 0.06
>10
>10
4.02 2.28
3.44 1.31
2.39 0.53
3.69 2.73
>10
2s
2t
2u
2v
2.79 1.35
>10
2w
2x
6.79 2.48
>20
>10
>10
CI-1033
gefitinib
0.001 0.0003
0.008 0.004
0.064 0.003
9.02 1.08
0.15 0.09
2.15 0.15
1.59 0.58
10.21 2.43
2.30 0.35
>10
a
The antiproliferative activities of the compounds were evaluated using the MTS assay. The data were means from at least four independent
experiments.
downstream p-Akt and p-Erk were also obviously decreased,
while the total protein levels of EGFR, Akt, Erk, and GAPDH
remained unchanged. Although gefitinib could potently induce
the inhibition of EGFR activation and downstream signaling in
3−4 times greater than that of 1 against H1975 cancer cell
growth. Further investigation also revealed that the compounds
2q and 2s potently induced G1 phase arrest and apoptosis in
both HCC827 and H1975 NSCLC cells and inhibited the
colony formation of H1975 cancer cells in dose-dependent
manners (Supporting Information). Not surprisingly, the
enzymatically inactive compounds 2n, 2w, and 2x did not
show obvious inhibition on the growth of HCC827 or H1975
cancer cells.
HCC827 cells, it did not show an effect on the EGFRT790M
-
mutated H1975 cells. The effects of 2a, 2q, and 2s on cells
harboring wild type EGFR kinase (EGFR overexpressed
A43124 cells and A54925 cells bearing k-Ras mutation) were
also investigated. The results demonstrated that the com-
pounds only moderately affected the activation of EGFR in the
corresponding cells although they displayed strong inhibition
against EGFR kinase under the in vitro screening assay (Table
1 and Supporting Information).
The antiproliferative effects of the new inhibitors were also
investigated to monitor their potential antitumor activities
(Table 2). Highly consistent with their strong kinase inhibition,
the compounds displayed great antiproliferative effects on
HCC827 and H1975 NSCLC cells harboring EGFRdel E746‑A750
and EGFRL858R/T790M, respectively. Most of the compounds
potently suppressed the growth of HCC827 cancer cells with
IC50 values in the low nanomolar ranges. Compounds 2o, 2p,
2q, 2r, and 2s were equal in potency to that of gefitinib, CI-
1033, and 1 for inhibiting the growth of HCC827 cancer cells,
with IC50 values of 3−9 nM. Moreover, several compounds also
displayed strong antiproliferative effects on gefitinib-resistant
H1975 cells bearing EGFRL858R/T790M. Compounds 2a, 2i, 2j,
2o, 2p, 2r, 2u, and 2v were almost equal in potency to that of
CI-1033 and inhibitor 1, while 2q and 2s displayed potencies
Although the compounds also strongly inhibited the
enzymatic activity of EGFRWT, they were obviously less potent
to suppress the proliferation of A431 human epithelial
carcinoma cells with overexpressed EGFRWT, which might be
due to the complex genomic background and the strong
binding of EGFRWT with ATP.31 For instance, compound 2s
inhibited the enzymatic activities of EGFRWT, EGFRL858R
,
EGFRL861Q, and EGFRL858R/T790M, with IC50 values of 0.29,
0.67, 0.38, 0.72, and 0.93 nM, respectively. It also potently
inhibited the growth of HCC827 and H1975, with IC50 values
of 3 and 14 nM, but its activity on A431 cancer cells was
approximate 10−50 times less potent (IC50 value was about
170 nM). However, an irreversible clinical candidate CI-1033
displayed comparable potency to inhibit the growth of H1975
and A431 cancer cells. Similar to many other reversible and
irreversible EGFR inhibitors, the compounds only showed
moderate inhibitory activities against the growth of A549
NSCLC cells which possess EGFRWT and the k-Ras mutation-
activating bypass MAPK signal pathway.28
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Our data have demonstrated that the new EGFR inhibitors
also strongly inhibited EGFRWT. Because many tissues use wild-
type EGFR for normal cellular processes, the potential for
toxicity from irreversible EGFR inhibitors is a concern.
Therefore, the growth inhibitory activities of the compounds
against HL-7702 normal human liver cells which bear EGFRWT
were also evaluated to monitor the potential toxic effects.26 As
shown in Table 2, only a few very potent EGFR inhibitors
displayed moderate inhibitory activities against the growth of
HL-7702 cells, indicating that the cytotoxic effects of these
compounds were minimal. For instance, the highly potent
inhibitors 2q and 2s inhibited the growth of HL-7702 cells with
IC50 values of 4.02 and 2.39 μM, respectively, which was
approximately 200−1000 fold less potent than their activities
against the HCC827 and H1975 NSCLC cells. The
compounds also displayed moderate or minimal cytotoxicity
on human HLF-1 cells (diploid human lung fibroblasts),27
which further suggested that the compounds might possess a
high safety index (Supporting Information).
the enzymatic activities of different mutants of EGFR as well as
the wild type EGFR kinase. The most potent compounds 2q
and 2s displayed good potencies, with IC50 values in the high
picomolar range against all types of EGFR kinase, including the
clinical resistance-related EGFRT790M mutant. A further kinase
profiling study also suggested that the compounds displayed
remarkable selectivity on EGFR. The kinase inhibitory
efficiencies of the compounds were further validated by
Western blot analysis of the activation of EGFR and the
downstream signaling in cancer cells harboring different
mutants of EGFR. Furthermore, the compounds also strongly
inhibited HCC827 and H1975 non small cell lung cancer cells
E746‑A750
bearing EGFRdel
and EGFRL858R/T790M, respectively,
with potencies better than that of 1 and CI-1033. The
compounds only showed moderate or minimal cytotoxicity to
normal HL-7702 and HLF-1 cells, indicating that they might
possess a high safety index. Moreover, a further in vivo
antitumor efficacy study demonstrated that compound 2s
significantly inhibited the tumor growth and induced tumor
stasis in an EGFRL858R/T790M-driven human NSCLC xenograft
mouse model of H1975 by orally dosing at 30 mg/kg/day. Our
study provides a new lead compound with a different chemical
scaffold for further development of EGFR inhibitors to
overcome EGFRT790M mutation-related clinical resistance to
gefitinib or erotinib. Further extensive pharmacokinetics and
safety evaluation on 2s are in progress and will be reported in
due course.
Given its high potency and remarkable selectivity profile,
compound 2s was further evaluated for in vivo antitumor
efficacy in an EGFRL858R/T790M-driven human NSCLC xenograft
mouse model of H1975 (Figure 4). SCID mice bearing
EXPERIMENTAL SECTION
■
Chemistry. Reagents and solvents were obtained from commercial
suppliers and used without further purification. Flash chromatography
was performed using silica gel (300−400 mesh). All reactions were
monitored by TLC, using silica gel plates with fluorescence F254 and
1
UV light visualization. H and 13C NMR spectra were recorded on a
Bruker AV-400 spectrometer at 400 MHz and Bruker AV-500
spectrometer at 125 MHz, respectively. Coupling constants (J) are
expressed in hertz (Hz). Chemical shifts (δ) of NMR are reported in
parts per million (ppm) units relative to internal control (TMS). The
low or high resolution of ESI-MS was recorded on an Agilent 1200
HPLC-MSD mass spectrometer or Applied Biosystems Q-STAR Elite
ESI-LC-MS/MS mass spectrometer, respectively. The purity of
compounds was determined by reverse-phase high performance liquid
chromatography (HPLC) analysis to be over 95% (>95%). HPLC
instrument: Dionex Summit HPLC (Column: Diamonsil C18, 5.0 μm,
4.6 × 250 mm (Dikma Technologies); detector: PDA-100 photodiode
array; injector: ASI-100 autoinjector; pump: p-680A). Elution: MeOH
in water; flow rate: 1.0 mL/min.
Figure 4. In vivo antitumor effect of compound 2s in a human
NSCLC (H1975) xenograft nude mouse model. The H1975 model
was resistant to gefitinib. Mice were monitored for signs of morbidity
(behavior and body loss), and tumors were measured every other day.
Statistical significance (p < 0.05) for antitumor efficacy, based upon
tumor growth relative to the vehicle controls. Two animals died at day
10 and 12 in the vehicle control group, respectively.
Ethyl 4-(3-(tert-Butoxycarbonylamino)phenylamino)-2-
(methylthio)pyrimidine-5-carboxylate (5). To a solution of com-
pound 4 (20.8 g, 100 mmol) in DMF (300 mL) were added potassium
carbonate (27.6 g, 200 mmol) and compound 3 (23.3 g, 100 mmol).
The reaction was heated to 80 °C and stirred overnight. After being
cooled to room temperature, the reaction mixture was added to ice−
water (1000 mL). The precipitate was filtered, and the filtered cake
was rinsed with additional cool water and dried in a vacuum oven to
give the title compound (38.8 g, 96% yield), which was used without
established H1975 tumor xenografts were dosed orally with 2s
at 10 and 30 mg/kg daily (qd) over a 14-day period. Gefitinib
(50 mg/kg, qd) was used as a reference drug to validate the
resistant models. Both doses of 2s were well tolerated, with no
mortality or significant body weight loss (<5% relative to the
vehicle matched controls) observed during the treatment.
Dosing at 10 mg/kg/day of 2s had little effect on the tumor
growth compared with the control group. However, compound
2s displayed significant in vivo antitumor efficacy (p < 0.05)
and induced tumor stasis at 30 mg/kg/day, indicating that it
might serve as a promising lead compound for further
development of EGFR inhibitors to overcome EGFRT790M
mutation-related clinical resistance to gefitinib or erotinib.
In summary, a series of 2-oxo-3,4-dihydropyrimido[4,5-
d]pyrimidinyl derivatives have been designed and synthesized
as novel EGFR inhibitors. The compounds potently inhibited
1
further purification. H NMR (400 MHz, CDCl3) δ 10.37 (s, 1H),
8.76 (s, 1H), 7.90 (s, 1H), 7.34 (d, J = 8.0 Hz, 1H), 7.24 (t, J = 8.0 Hz,
1H), 7.02 (d, J = 8.0 Hz, 1H), 6.53 (s, 1H), 4.38 (q, J = 7.2, 14.4 Hz,
2H), 2.55 (s, 3H), 1.52 (s, 9H), 1.40 (t, J = 7.2 Hz, 3H).
tert-Butyl 3-(5-(Hydroxymethyl)-2-(methylthio)pyrimidin-4-
ylamino)phenylcarbamate (6). To a solution of compound 5 (20.2
g, 50 mmol) in THF (500 mL) was added 2.0 M lithium aluminum
hydride solution in THF (50 mL, 100 mmol) at −40 °C. The reaction
mixture was stirred until the temperature warmed to room
temperature and was then treated with saturated NH4Cl solution
(75 mL). After the mixture was stirred at room temperature for 30
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Journal of Medicinal Chemistry
Article
min, the solid was filtered off. The filtrate was partitioned between
dichloromethane (500 mL) and water (300 mL). The organic layer
was washed with brine and dried over Na2SO4, the solid was filtered
off, and the filtrate was concentrated under reduced pressure. The
resulting crude product was purified by flash silica gel chromatography
with dichloromethane/methanol (80/1 to 40/1, v/v) to yield the title
product as a white solid (10.33 g, 57%). 1H NMR (400 MHz, CDCl3)
δ 8.02 (s, 1H), 7.86 (s, 1H), 7.80 (s, 1H), 7.36 (dd, J = 1.2, 8.0 Hz,
1H), 7.22 (t, J = 8.0 Hz, 1H), 6.95 (dd, J = 1.2, 8.0 Hz, 1H), 6.52 (s,
1H), 4.61 (s, 2H), 2.52 (s, 3H), 1.52 (s, 9H).
tert-Butyl 3-(5-Formyl-2-(methylthio)pyrimidin-4-ylamino)-
phenylcarbamate (7). To a solution of compound 6 (10.0 g, 27.6
mmol) in dichloromethane (300 mL) was added activated manganese-
(IV) oxide (24.0 g, 276 mmol) at room temperature, and the mixture
was stirred overnight, the solid was filtered off, and the filtrate was
concentrated under reduced pressure. The resulting crude product was
purified by flash silica gel chromatography with petroleum ether/ethyl
acetate (5/1 to 3/1, v/v) to yield the title product as a white solid
(8.36 g, 84%). 1H NMR (400 MHz, CDCl3) δ 10.61 (s, 1H), 9.77 (s,
1H), 8.43 (s, 1H), 7.98 (s, 1H), 7.36 (dd, J = 0.8, 8.0 Hz, 1H), 7.25−
7.29 (m, 1H), 7.03 (dd, J = 1.2, 8.0 Hz, 1H), 6.51 (s, 1H), 2.59 (s,
3H), 1.53 (s, 9H).
tert-Butyl 3-(7-(2-Methoxy-4-(4-methylpiperazin-1-yl)-
phenylamino)-3-methyl-2-oxo-3,4-dihydropyrimido[4,5-d]-
pyrimidin-1(2H)-yl)phenylcarbamate (11a). To a solution of
compound 10a (260 mg, 0.6 mmol) in butan-2-ol (2 mL) were
added 2-methoxy-4-(4-methylpiperazin-1-yl)aniline (133 mg, 0.6
mmol) and trifluoroacetic acid (48 μL, 0.6 mmol). The reaction
mixture was stirred for 18 h at 110 °C in a sealed tube. The reaction
mixture was cooled to room temperature and partitioned between
dichloromethane (30 mL) and saturated NaHCO3 (10 mL), and the
water layer was extracted with dichloromethane (10 mL × 2). The
combined organic layer was washed with brine and dried with Na2SO4,
the solid was filtered off, and the filtrate was concentrated under
reduced pressure. The resulting crude product was purified by silica gel
chromatography with dichloromethane/methanol (40/1 to 20/1, v/v)
1
to give the title product as a yellow solid (151 mg, 44%). H NMR
(400 MHz, CDCl3) δ 7.99 (s, 1H), 7.48 (d, J = 7.5 Hz, 1H), 7.44 (s,
1H), 7.41 (t, J = 8.0 Hz, 1H), 7.35 (s, 1H), 6.97 (d, J = 7.5 Hz, 1H),
6.63 (s, 1H), 6.42 (d, J = 2.0 Hz, 1H), 6.16 (d, J = 6.4 Hz, 1H), 4.42
(s, 2H), 3.80 (s, 3H), 3.22 (m, 4H), 3.08 (s, 3H), 2.79 (m, 4H), 1.47
(s, 9H). LCMS (ESI): m/z 575.3 [M + H]+.
1-(3-Aminophenyl)-7-(2-methoxy-4-(4-methylpiperazin-1-yl)-
phenylamino)-3-methyl-3,4-dihydropyrimido[4,5-d]pyrimidin-
2(1H)-one (2w). To a solution of compound 11a (143 mg, 0.25
mmol) in dichloromethane (1 mL) was added trifluoroacetic acid (0.2
mL, 2.7 mmol). The reaction mixture was stirred for 1 h at room
temperature. The reaction mixture was cooled to room temperature
and partitioned between dichloromethane (10 mL) and saturated
NaHCO3 (2 mL), and the water layer was extracted with
dichloromethane (5 mL × 2). The combined organic layer was
washed with brine and dried with Na2SO4, the solid was filtered off,
and the filtrate was concentrated under reduced pressure. The
resulting crude product was purified by silica gel chromatography with
dichloromethane/methanol (40/1 to 20/1, v/v) to give the title
product as a yellow solid (100 mg, 84%). 1H NMR (400 MHz,
CDCl3) δ 7.99 (s, 1H), 7.61 (s, 1H), 7.46 (s, 1H), 7.28 (t, J = 8.0 Hz,
2H), 6.78 (dd, J = 2.0, 8.0 Hz, 1H), 6.67 (d, J = 7.6 Hz, 1H), 6.61 (t, J
= 2.0 Hz, 1H), 6.45 (d, J = 2.4 Hz, 1H), 6.21 (d, J = 7.6 Hz, 1H), 4.42
(s, 2H), 3.81 (s, 3H), 3.13 (t, J = 4.8 Hz, 4H), 3.09 (s, 3H), 2.64 (t, J =
4.8 Hz, 4H), 2.29 (s, 3H). LCMS (ESI): m/z 475.2 [M + H]+.
N-(3-(7-(2-Methoxy-4-(4-methylpiperazin-1-yl)phenylamino)-3-
methyl-2-oxo-3,4-dihydropyrimido[4,5-d]pyrimidin-1(2H)-yl)-
phenyl)acrylamide (2a). To a solution of compound 2x (95 mg, 0.20
mmol) in dichloromethane (2 mL) were added diisopropyethylamine
(40 μL, 0.20 mmol) and acryloyl chloride (16 μL, 0.20 mmol). The
reaction mixture was stirred for 1 h at room temperature. The resulting
crude product was purified by silica gel chromatography with
dichloromethane/methanol (40/1 to 20/1, v/v) to give the title
product as a yellow solid (90 mg, 85%). 1H NMR (400 MHz, DMSO-
d6) δ 10.31 (s, 1H), 8.11 (s, 1H), 7.81 (d, J = 7.6 Hz, 1H), 7.56 (s,
1H), 7.48 (s, 1H), 7.43 (t, J = 8.0 Hz, 1H), 7.27 (d, J = 8.4 Hz, 1H),
6.95 (d, J = 8.0 Hz, 1H), 6.51 (s, 1H), 6.44 (dd, J = 10.0, 16.8 Hz, 1H),
6.25 (d, J = 16.8 Hz, 1H), 6.02 (d, J = 8.0 Hz, 1H), 5.76 (d, J = 10.0
Hz, 1H), 4.46 (s, 2H), 3.76 (s, 3H), 2.99 (m, 4H), 2.96 (s, 3H), 2.50
(m, 4H), 2.22 (s, 3H). 13C NMR (125 MHz, DMSO-d6) δ 163.12,
158.45, 157.23, 154.03, 152.54, 146.70, 139.52, 137.49, 131.76, 128.92,
126.92, 124.92, 120.79, 120.72, 118.24, 106.41, 101.15, 99.75, 55.61,
54.59, 48.72, 45.82, 45.70, 34.99. HRMS (ESI): exact mass calcd for
C28H32N8O3 [M + H]+, 529.2670, found 529.2675.
tert-Butyl 3-(5-((Methylamino)methyl)-2-(methylthio)pyrimidin-
4-ylamino)phenylcarbamate (8a). To a solution of compound 7
(7.21 g, 20.0 mmol) in methanol (200 mL) were added sodium acetate
(8.2 g, 100 mmol) and methanaminium chloride (6.75 g, 100 mmol)
at room temperature, and the mixture was stirred for 1 h. NaBH4 (1.51
g, 40.0 mmol) was added. The reaction mixture was partitioned
between dichloromethane (200 mL) and saturated NaHCO3 (100
mL), and then the water layer was extracted with dichloromethane
(100 mL × 2). The combined organic layer was washed with brine and
dried over Na2SO4, the solid was filtered off, and the filtrate was
concentrated under reduced pressure. The resulting crude product was
purified by silica gel chromatography with dichloromethane/methanol
(80/1 to 40/1, v/v) to give the title product as a white solid (5.25 g,
1
71%). H NMR (400 MHz, CDCl3) δ 10.12 (s, 1H), 7.89 (s, 1H),
7.78 (s, 1H), 7.35 (d, J = 8.8 Hz, 1H), 7.21 (t, J = 8.0 Hz, 1H), 6.97
(dd, J = 1.2, 8.0 Hz, 1H), 6.50 (s, 1H), 3.74 (s, 2H), 3.55 (s, 3H), 2.44
(s, 3H), 1.52 (s, 9H).
tert-Butyl 3-(3-mMethyl-7-(methylthio)-2-oxo-3,4-
dihydropyrimido[4,5-d]pyrimidin-1(2H)-yl)phenylcarbamate (9a).
To a solution of compound 8a (5.20 g, 13.8 mmol) in THF (140
mL) were added DIEA (8 mL, 55.2 mmol) and 0.2 M triphosgene (25
mL, 5.05 mmol) at 0 °C, and the mixture was stirred at room
temperature for 1 h and then partitioned between dichloromethane
(200 mL) and saturated NaHCO3 (100 mL) solution. The organic
layer was washed with brine and dried over Na2SO4, the solid was
filtered off, and the filtrate was concentrated under reduced pressure.
The resulting crude product was purified by silica gel chromatography
with dichloromethane/methanol (60/1 to 30/1, v/v) to give the title
product as a white solid (4.71 g, 85%). 1H NMR (400 MHz, CDCl3) δ
8.10 (s, 1H), 7.44 (s, 1H), 7.34 (t, J = 8.0 Hz, 1H), 7.23 (d, J = 7.2 Hz,
1H), 6.90 (d, J = 8.0 Hz, 1H), 6.55 (s, 1H), 4.46 (s, 2H), 3.08 (s, 3H),
2.14 (s, 3H), 1.50 (s, 9H).
tert-Butyl 3-(3-Methyl-7-(methylsulfonyl)-2-oxo-3,4-
dihydropyrimido[4,5-d]pyrimidin-1(2H)-yl)phenylcarbamate (10a).
To a solution of compound 9a (4.0 g, 10.0 mmol) in dichloromethane
(100 mL) was added 85% m-chloroperbenzoic acid (6.1 g, 30.0 mmol)
in batches at 0 °C. The reaction mixture was stirred for 3 h at room
temperature. The reaction mixture was partitioned between dichloro-
methane (200 mL) and saturated NaHCO3 (100 mL). The organic
layer was washed with brine and dried over Na2SO4, the solid was
filtered off, and the filtrate was concentrated under reduced pressure.
The resulting white solid (3.90 g, 90% yield) was recrystallized from a
minimum amount of ethyl acetate, which was used for the next step
The following compounds (2b−x) were prepared from compound
10 and the corresponding aniline by a method similar to that for 2a.
N-(3-(7-(4-(Dimethylamino)-2-methoxyphenylamino)-3-methyl-
2-oxo-3,4-dihydropyrimido[4,5-d]pyrimidin-1(2H)-yl)phenyl)-
1
acrylamide (2b). H NMR (400 MHz, DMSO-d6) δ 10.28 (s, 1H),
8.09 (s, 1H), 7.79 (d, J = 8.8 Hz, 1H), 7.57 (s, 1H), 7.42 (t, J = 8.0 Hz,
2H), 7.22 (d, J = 8.8 Hz, 1H), 6.95 (d, J = 8.0 Hz, 1H), 6.43 (dd, J =
10.0, 16.8 Hz, 1H), 6.30 (d, J = 2.4 Hz, 1H), 6.25 (dd, J = 2.0, 16.8 Hz,
1H), 5.87 (d, J = 7.6 Hz, 1H), 5.75 (dd, J = 2.0, 10.0 Hz, 1H), 4.45 (s,
2H), 3.76 (s, 3H), 2.96 (s, 3H), 2.78 (s, 6H). 13C NMR (125 MHz,
DMSO-d6) δ163.12, 158.42, 157.20, 153.98, 152.58, 146.82, 139.51,
137.51, 131.76, 128.85, 126.91, 124.93, 120.70, 118.59, 118.16, 103.81,
1
without further purification. H NMR (400 MHz, CDCl3) δ 8.45 (s,
1H), 7.58 (s, 1H), 7.35 (t, J = 8.0 Hz, 1H), 7.15 (dd, J = 1.2, 8.0 Hz,
1H), 6.89 (dd, J = 1.2, 8.0 Hz, 1H), 6.67 (s, 1H), 4.62 (s, 2H), 3.11 (s,
3H), 2.98 (s, 3H), 1.48 (s, 9H).
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100.82, 96.77, 55.48, 45.81, 40.58, 34.98. HRMS (ESI): exact mass
calcd for C25H27N7O3 [M + H]+, 474.2248, found 474.2252.
N-(3-(7-(2-Methoxy-4-(piperidin-1-yl)phenylamino)-3-methyl-2-
oxo-3,4-dihydropyrimido[4,5-d]pyrimidin-1(2H)-yl)phenyl)-
(d, J = 10.4 Hz, 1H), 4.37 (s, 2H), 2.93 (s, 3H), 2.59 (s, 3H). 13C
NMR (125 MHz, DMSO-d6) δ 163.11, 161.56, 157.11, 153.74, 152.93,
139.14, 137.44,131.75, 128.50, 126.92, 125.14, 120.87, 118.23, 45.68,
35.01. HRMS (ESI): exact mass calcd for C17H18N6O2 [M + H]+,
339.1564, found 339.1566.
1
acrylamide (2c). H NMR (400 MHz, DMSO-d6) δ 10.30 (s, 1H),
8.11 (s, 1H), 7.82 (d, J = 8.0 Hz, 1H), 7.56 (s, 1H), 7.47 (s, 1H), 7.43
(t, J = 8.0 Hz, 1H), 7.25 (d, J = 8.0 Hz, 1H), 6.95 (d, J = 8.0 Hz, 1H),
6.49 (d, J = 2.2 Hz, 1H), 6.43 (dd, J = 8.0, 16.0 Hz, 1H), 6.25 (dd, J =
1.6, 8.0 Hz, 1H), 6.02 (d, J = 7.4 Hz, 1H), 5.76 (dd, J = 1.6, 8.0 Hz,
1H), 4.46 (s, 2H), 3.75 (s, 3H), 2.96 (m, 7H), 1.59 (m, 4H), 1.49−
1.504 (m, 2H). 13C NMR (125 MHz, DMSO-d6) δ163.57, 158.70,
157.68, 154.48, 153.00, 148.08, 139.99, 137.95, 132.20, 129.37, 127.36,
125.37, 121.15, 118.63, 107.50, 101.56, 100.87, 56.02, 50.90, 46.27,
35.44, 25.82, 24.30. HRMS (ESI): exact mass calcd for C28H31N7O3
[M + H]+, 514.2561, found 514.2565.
N-(3-(3-Methyl-7-(4-(4-methylpiperazin-1-yl)phenylamino)-2-
oxo-3,4-dihydropyrimido[4,5-d]pyrimidin-1(2H)-yl)phenyl)-
1
acrylamide (2i). H NMR (400 MHz, DMSO-d6) δ 10.31 (s, 1H),
9.17 (s, 1H), 8.13 (s, 1H), 7.84 (d, J = 8.0 Hz, 1H), 7.56 (s, 1H), 7.44
(t, J = 8.0 Hz, 1H), 7.12 (d, J = 7.2 Hz, 2H), 6.96 (d, J = 7.6 Hz, 1H),
6.53 (d, J = 8.0 Hz, 2H), 6.43 (dd, J = 9.6, 16.4 Hz, 1H), 6.25 (d, J =
16.4 Hz, 1H), 5.76 (d, J = 10.0 Hz, 1H), 4.46 (s, 2H), 2.97 (s, 3H),
2.92 (s, 4H), 2.41 (s, 4H), 2.20 (s, 3H). 13C NMR (125 MHz, DMSO-
d6) δ 163.15, 158.41, 157.12, 154.03, 152.63, 145.53, 139.58, 137.68,
132.69, 131.75, 129.00, 126.99, 124.99, 120.79, 119.07, 118.22, 115.44,
100.58, 54.64, 48.83, 45.77, 35.01. HRMS (ESI): exact mass calcd for
C27H30N8O22 [M + H]+, 499.2564, found 499.2562.
N-(3-(7-(2-Methoxy-4-morpholinophenylamino)-3-methyl-2-
oxo-3,4-dihydropyrimido[4,5-d]pyrimidin-1(2H)-yl)phenyl)-
1
acrylamide (2d). H NMR (400 MHz, DMSO-d6) δ 10.30 (s, 1H),
N-(3-(7-(3-Methoxy-4-(4-methylpiperazin-1-yl)phenylamino)-3-
8.12 (s, 1H), 7.81 (d, J = 6.8 Hz, 1H), 7.56 (s, 1H), 7.50 (s, 1H), 7.43
(t, J = 8.0 Hz, 1H), 7.28 (d, J = 10.0 Hz, 1H), 6.96 (d, J = 10.0 Hz,
1H), 6.52 (d, J = 2.0 Hz, 1H), 6.43 (dd, J = 10.0, 16.8 Hz, 1H), 6.22−
6.27 (m, 1H), 6.02 (d, J = 8.0 Hz, 1H), 5.74−5.77 (m, 1H), 4.46 (s,
2H), 3.76 (s, 3H), 3.71 (t, J = 4.4 Hz, 4H), 2.96 (m, 7H). 13C NMR
(125 MHz, DMSO-d6) δ 163.58, 158.68, 157.68, 154.49, 152.98,
147.23, 139.96, 137.95, 132.20, 129.38, 127.42, 125.39, 121.56, 121.19,
118.71, 106.61, 101.65, 100.02, 66.53, 56.09, 49.66, 46.27, 35.44.
HRMS (ESI): exact mass calcd for C27H29N7O4 [M + H]+, 516.2354,
found 516.2358.
N-(3-(7-(2-Methoxy-4-(4-methyl-1,4-diazepan-1-yl)-
phenylamino)-3-methyl-2-oxo-3,4-dihydropyrimido[4,5-d]-
pyrimidin-1(2H)-yl)phenyl)acrylamide (5e). 1H NMR (400 MHz,
DMSO-d6) δ 10.30 (s, 1H), 8.19 (s, 1H), 7.85 (s, 1H), 7.69−7.70 (m,
2H), 7.51 (t, J = 8.0 Hz, 1H), 7.45 (t, J = 8.0 Hz, 1H), 6.96−6.99 (m,
2H), 6.40−6.46 (m, 2H), 6.24 (dd, J = 1.6, 16.8 Hz, 1H), 5.75 (dd, J =
1.6, 10.0 Hz, 1H), 4.50 (s, 2H), 3.33 (m, 7H), 2.98 (s, 3H), 2.74 (t, J =
4.4 Hz, 4H), 2.49 (m, 2H), 2.24 (s, 3H). 13C NMR (125 MHz,
DMSO-d6) δ 163.11, 158.51, 157.21, 154.01, 152.63, 149.63, 139.54,
137.55, 131.77, 128.88, 126.93, 124.94, 120.69, 118.14, 117.40, 102.43,
100.64, 95.24, 57.14, 56.31, 55.45, 48.25, 47.90, 46.01, 45.84, 35.00,
26.93. HRMS (ESI): exact mass calcd for C29H34N8O3 [M + H]+,
543.2827, found 543.2832.
methyl-2-oxo-3,4-dihydropyrimido[4,5-d]pyrimidin-1(2H)-yl)-
1
phenyl)acrylamide (2j). H NMR (400 MHz, DMSO-d6) δ 10.30 (s,
1H), 9.13 (s, 1H), 8.15 (s, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.58 (s, 1H),
7.42 (d, J = 8.0 Hz, 1H), 6.91−6.97 (m, 2H), 6.86 (s, 1H), 6.39−6.46
(m, 2H), 6.24 (d, J = 16.8 Hz, 1H), 5.75 (d, J = 10.0 Hz, 1H), 4.46 (s,
2H), 3.58 (s, 3H), 2.96 (s, 3H), 2.79 (s, 4H), 2.39 (s, 4H), 2.19 (s,
3H). 13C NMR (125 MHz, DMSO-d6) δ 163.11, 158.46, 157.14,
153.92, 152.64, 151.68, 139.55, 137.55, 135.51, 135.36, 131.74, 128.89,
126.93, 124.95, 120.74, 118.20, 117.34, 110.55, 103.72, 101.06, 55.20,
54.95, 50.18, 45.86, 45.82, 35.01. HRMS (ESI): exact mass calcd for
C28H32N8O3 [M + H]+, 529.2670, found 529.2667.
N-(3-(7-(2-Ethoxy-4-(4-methylpiperazin-1-yl)phenylamino)-3-
methyl-2-oxo-3,4-dihydropyrimido[4,5-d]pyrimidin-1(2H)-yl)-
phenyl)acrylamide (2k). 1H NMR (400 MHz, CDCl3) δ 8.28 (s, 1H),
7.99 (s, 1H), 7.93 (d, J = 7.6 Hz, 1H), 7.48 (s, 1H), 7.40 (t, J = 8.0 Hz,
2H), 7.28 (s, 1H), 6.96 (d, J = 8.0 Hz, 1H), 6.38 (s, 1H), 6.34 (d, J =
16.4 Hz, 1H), 6.18 (dd, J = 10.0, 16.8 Hz, 1H), 6.10 (d, J = 8.4 Hz,
1H), 5.65 (d, J = 10.4 Hz, 1H), 4.42 (s, 2H), 4.00 (q, J = 6.8, 13.6 Hz,
2H), 3.13 (s, 4H), 3. 06 (s, 3H), 2.70 (s, 4H), 2.44 (s, 3H), 1.39 (t, J =
6.8 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 163.39, 158.58, 157.56,
153.72, 147.44, 146.03, 139.35, 137.15, 131.54, 129.63, 127.12, 125.19,
122.57, 121.09, 119.70, 118.94, 108.52, 101.04, 100.11, 64.19, 54.78,
49.44, 46.96, 45.49, 35.62, 14.91. HRMS (ESI): exact mass calcd for
C29H34N8O3 [M + H]+, 543.2827, found 543.2834.
N-(3-(7-(4-(4-(Dimethylamino)piperidin-1-yl)-2-methoxyphenyla-
mino)-3-methyl-2-oxo-3,4-dihydropyrimido[4,5-d]pyrimidin-1(2H)-
N-(3-(7-(2-Isopropoxy-4-(4-methylpiperazin-1-yl)phenylamino)-
1
yl)phenyl)acrylamide (5f). H NMR (400 MHz, DMSO-d6) δ 10.34
3-methyl-2-oxo-3,4-dihydropyrimido[4,5-d]pyrimidin-1(2H)-yl)-
1
(s, 1H), 8.11 (s, 1H), 7.84 (d, J = 7.6 Hz, 1H), 7.55 (s, 1H), 7.48 (s,
1H), 7.42 (t, J = 8.0 Hz, 1H), 7.25 (d, J = 8.4 Hz, 1H), 6.95 (d, J = 7.2
Hz, 1H), 6.51 (s, 1H), 6.45 (dd, J = 10.0, 17.2 Hz, 1H), 6.25 (d, J =
16.8 Hz, 1H), 6.04 (s, 1H), 5.76 (d, J = 9.6 Hz, 1H), 4.46 (s, 2H), 3.75
(s, 3H), 3.56 (d, J = 12.4 Hz, 2H), 3.35 (s, 3H), 2.87 (m, 1H), 2.49−
2.56 (m, 2H), 2.34 (s, 6H), 1.86 (d, J = 10.4 Hz, 2H), 1.46−1.55 (m,
2H). 13C NMR (125 MHz, DMSO-d6) δ163.12, 158.24, 157.23,
154.04, 152.54, 146.93, 139.54, 137.50, 131.77, 128.91, 126.90, 124.92,
120.71, 120.65, 118.20, 106.98, 101.12, 100.32, 61.37, 55.59, 48.87,
45.83, 41.45, 34.99, 27.85. HRMS (ESI): exact mass calcd for
C30H36N8O3 [M + H]+, 557.2983, found 557.2989.
phenyl)acrylamide (2l). H NMR (400 MHz, DMSO-d6) δ 8.40 (s,
1H), 7.96 (s, 1H), 7.91 (d, J = 8.0 Hz, 1H), 7.49 (s, 1H), 7.37 (t, J =
8.0 Hz, 2H), 7.25 (s, 1H), 6.94 (d, J = 7.6 Hz, 1H), 6.41 (d, J = 2.0 Hz,
1H), 6.32 (d, J = 12.8 Hz, 1H), 6.14−6.18 (m, 1H), 6.08−6.11 (m,
1H), 5.62 (d, J = 10.0 Hz, 1H), 4.47−4.53 (m, 1H), 4.39 (s, 2H), 3.05
(t, J = 4.8 Hz, 4H), 3.03 (s, 3H), 2.56 (t, J = 4.6 Hz, 4H), 2.34 (s, 3H),
1.30 (d, J = 6.0 Hz, 6H). 13C NMR (125 MHz, DMSO-d6) δ 163.39,
158.50, 157.46, 153.70, 146.39, 139.54, 136.95, 131.65, 129.61, 126.83,
124.95, 123.15, 121.02, 119.75, 118.92, 108.52, 102.48, 99.86, 71.26,
55.05, 49.80, 46.94, 45.99, 35.61, 22.19. HRMS (ESI): exact mass calcd
for C30H36N8O3 [M + H]+, 557.2987, found 557.2990.
N-(3-(3-Methyl-2-oxo-7-(phenylamino)-3,4-dihydropyrimido[4,5-
N-(3-(3-Methyl-7-(2-methyl-4-(4-methylpiperazin-1-yl)-
1
d]pyrimidin-1(2H)-yl)phenyl)acrylamide (2g). H NMR (400 MHz,
phenylamino)-2-oxo-3,4-dihydropyrimido[4,5-d]pyrimidin-1(2H)-
1
DMSO-d6) δ 10.30 (s, 1H), 9.41 (s, 1H), 8.18 (s, 1H), 7.43 (d, J = 8.0
Hz, 1H), 7.66 (s, 1H), 7.45 (t, J = 8.0 Hz, 1H), 7.27 (d, J = 8.0 Hz,
2H), 6.99 (d, J = 8.0 Hz, 1H), 6.93 (t, J = 7.6 Hz, 2H), 6.75 (t, J = 7.2
Hz, 1H), 6.42 (dd, J = 10.4, 16.8 Hz, 1H), 6.24(d, J = 16.8 Hz, 1H),
5.75 (d, J = 10.4 Hz, 1H), 4.48 (s, 2H), 2.97 (s, 3H). 13C NMR (125
MHz, DMSO-d6) δ163.14, 158.32, 157.17, 154.05, 152.52, 140.33,
139.59, 137.66, 131.70, 128.94, 127.93, 127.00, 125.06, 120.75, 120.67,
118.26, 118.05, 101.39, 45.87, 34.99. HRMS (ESI): exact mass calcd
for C22H20N6O2 [M + H]+, 401.1721, found 401.1735.
yl)phenyl)acrylamide (2m). H NMR (400 MHz, CDCl3) δ 8.35 (s,
1H), 7.97 (s, 1H), 7.62 (d, J = 7.6 Hz, 1H), 7.45 (s, 1H), 7.27−7.33
(m, 2H), 6.91 (d, J = 8.0 Hz, 1H), 6.64 (s, 1H), 6.56 (s, 1H), 6.49 (d, J
= 8.0 Hz, 1H), 6.33 (d, J = 16.8 Hz, 1H), 6.17 (dd, J = 10.0, 16.8 Hz,
1H), 5.64 (d, J = 10.4 Hz, 1H), 4.44 (s, 2H), 3.14 (m, 4H), 3.10 (s,
3H), 2.65 (m, 4H), 2.41 (s, 3H), 2.14 (s, 3H). 13C NMR (125 MHz,
CDCl3) δ 163.47, 159.65, 157.61, 153.65, 147.23, 139.42, 136.82,
131.65, 129.92, 129.27, 126.80, 125.04, 121.15, 119.65, 118.22, 114.43,
100.52, 54.90, 49.17, 46.87, 45.69, 35.67, 18.38. HRMS (ESI): exact
mass calcd for C28H32N8O2 [M + H]+, 513.2721, found 513.2719.
N-(3-(7-(2-Fluoro-4-(4-methylpiperazin-1-yl)phenylamino)-3-
methyl-2-oxo-3,4-dihydropyrimido[4,5-d]pyrimidin-1(2H)-yl)-
phenyl)acrylamide (2n). 1H NMR (400 MHz, DMSO-d6) δ 10.30 (s,
1H), 8.19 (s, 1H), 7.86 (s, 1H), 7.69−7.70 (m, 2H), 7.51 (t, J = 8.0
N-(3-(3-Methyl-7-(methylamino)-2-oxo-3,4-dihydropyrimido[4,5-
1
d]pyrimidin-1(2H)-yl)phenyl)acrylamide (2h). H NMR (400 MHz,
DMSO-d6) δ 10.23 (s, 1H), 8.01 (s, 1H), 7.61 (d, J = 8.0 Hz, 1H),
7.55 (s, 1H), 7.36 (t, J = 8.0 Hz, 1H), 6.91 (d, J = 7.6 Hz, 1H), 6.71 (s,
1H), 6.43 (dd, J = 10.0, 16.8 Hz, 1H), 6.25 (d, J = 16.8 Hz, 1H), 5.76
2719
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Article
Hz, 1H), 7.45 (t, J = 8.0 Hz, 1H), 6.96−6.99 (m, 2H), 6.40−6.47 (m,
2H), 6.24 (dd, J = 2.0, 17.2 Hz, 1H), 5.75 (dd, J = 2.0, 10.0 Hz, 1H),
4.50 (s, 2H), 2.98 (s, 3H), 2.74 (t, J = 4.4 Hz, 4H), 2.50 (m, 4H), 2.24
(s, 3H). 13C NMR (125 MHz, DMSO-d6) δ 163.63, 158.28, 158.10,
157.76, 156.37, 154.66, 152.80, 142.47, 142.41, 140.00, 137.93, 132.14,
130.90, 129.41, 127.48, 125.45, 121.17, 118.80, 110.27, 110.10, 108.35,
108.17, 102.59, 55.42, 51.48, 46.28, 45.98, 35.45. HRMS (ESI): exact
mass calcd for C27H29FN8O2 [M + H]+, 517.2470, found 517.2473.
N-(3-(3-Isopropyl-7-(2-methoxy-4-(4-methylpiperazin-1-yl)-
phenylamino)-2-oxo-3,4-dihydropyrimido[4,5-d]pyrimidin-1(2H)-
5.76 (dd, J = 2.0, 10.0 Hz, 1H), 4.64 (s, 2H), 4.40 (s, 2H), 3.75 (s,
3H), 2.99 (t, J = 4.8 Hz, 4H), 2.42 (t, J = 4.8 Hz, 4H), 2.21 (s, 3H).
13C NMR (125 MHz, DMSO-d6) δ 163.14, 158.28, 157.08, 154.24,
152.61, 146.81, 139.51, 137.49, 136.68, 131.75, 128.96, 128.61, 127.75,
127.40, 126.94, 125.00, 120.77, 120.73, 118.35, 106.41, 101.00, 99.74,
55.61, 54.63, 50.52, 48.75, 45.75, 43.73. HRMS (ESI): exact mass calcd
for C34H36N8O3 [M + H]+, 605.2983, found 605.2984.
N-(3-(3-(Biphenyl-4-yl)-7-(2-methoxy-4-(4-methylpiperazin-1-yl)-
phenylamino)-2-oxo-3,4-dihydropyrimido[4,5-d]pyrimidin-1(2H)-
1
yl)phenyl)acrylamide (2t). H NMR (400 MHz, DMSO-d6) δ 10.33
1
yl)phenyl)acrylamide (2o). H NMR (400 MHz, DMSO-d6) δ 10.32
(s, 1H), 8.19 (s, 1H), 7.83 (d, J = 7.6 Hz, 1H), 7.69−7.74 (m, 5H),
7.46−7.58 (m, 6H), 7.37 (t, J = 7.2 Hz, 1H), 7.32 (d, J = 8.8 Hz, 1H),
7.09 (d, J = 7.6 Hz, 1H), 6.52 (s, 1H), 6.45 (dd, J = 10.0, 16.4 Hz, 1H),
6.26 (d, J = 16.8 Hz, 1H), 6.06 (d, J = 6.8 Hz, 1H), 5.77 (d, J = 10.0
Hz, 1H), 4.92 (s, 2H), 3.77 (s, 3H), 3.01 (m, 4H), 2.43 (m, 4H), 2.22
(s, 3H). 13C NMR (125 MHz, DMSO-d6) δ 163.14, 158.44, 157.17,
154.09, 152.15, 146.93, 141.69, 139.54, 139.43, 137.78, 137.27, 131.74,
128.97, 128.91, 127.41, 126.94, 126.88, 126.58, 125.83, 124.98, 120.80,
120.71, 118.46, 106.43, 101.81, 99.76, 55.61, 54.60, 48.71, 46.60,
45.70. HRMS (ESI): exact mass calcd for C39H38N8O3 [M + H]+,
667.3140, found 667.3146.
(s, 1H), 8.14 (s, 1H), 7.80 (d, J = 6.8 Hz, 1H), 7.57 (s, 1H), 7.48 (s,
1H), 7.42 (t, J = 8.0 Hz, 1H), 7.29 (d, J = 7.6 Hz, 1H), 6.97 (d, J = 8.0
Hz, 1H), 6.52 (s, 1H), 6.43 (dd, J = 10.0, 17.2 Hz, 1H), 6.25 (d, J =
16.8 Hz, 1H), 6.04 (d, J = 9.2 Hz, 1H), 5.76 (d, J = 10.4 Hz, 1H),
4.52−4.55 (m, 1H), 4.37 (s, 2H), 3.76 (s, 3H), 3.04 (m, 4H), 2.57 (m,
4H), 2.32 (s, 3H), 1.19 (d, J = 6.8 Hz, 6H). 13C NMR (125 MHz,
DMSO-d6) δ163.11, 158.31, 157.12, 154.03, 152.13, 146.79, 139.44,
137.58, 131.75, 128.85, 126.91, 125.01, 120.80, 118.22, 106.43, 101.45,
99.75, 55.59, 54.63, 48.77, 45.74, 45.65, 37.28, 18.74. HRMS (ESI):
exact mass calcd for C30H36N8O3 [M + H]+, 557.2983, found
557.2985.
N-(3-(7-(2-Methoxy-4-(4-methylpiperazin-1-yl)phenylamino)-2-
oxo-3-(4-phenoxyphenyl)-3,4-dihydropyrimido[4,5-d]pyrimidin-
N-(3-(3-Cyclopropyl-7-(2-methoxy-4-(4-methylpiperazin-1-yl)-
1
1(2H)-yl)phenyl)acrylamide (2u). H NMR (400 MHz, DMSO-d6) δ
phenylamino)-2-oxo-3,4-dihydropyrimido[4,5-d]pyrimidin-1(2H)-
1
yl)phenyl)acrylamide (2p). H NMR (400 MHz, DMSO-d6) δ 10.31
10.32 (s, 1H), 8.17 (s, 1H), 7.81 (d, J = 8.4 Hz, 1H), 7.67 (s, 1H), 7.56
(s, 1H), 7.40−7.47 (m, 5H), 7.30 (d, J = 8.8 Hz, 1H), 7.16 (t, J = 7.6
Hz, 1H), 7.04−7.07 (m, 5H), 6.52 (d, J = 2.4 Hz, 1H), 6.44 (dd, J =
10.0, 16.8 Hz, 1H), 6.25 (dd, J = 2.0, 16.8 Hz, 1H), 6.04 (s, 1H), 5.76
(dd, J = 2.0, 10.0 Hz, 1H), 4.86 (s, 2H), 3.77 (s, 3H), 3.00 (t, J = 4.4
Hz, 4H), 2.43 (t, J = 4.4 Hz, 4H), 2.22 (s, 3H). 13C NMR (125 MHz,
DMSO-d6) δ 163.13, 158.40, 157.19, 156.49, 154.71, 154.07, 152.20,
146.90, 139.51, 137.69, 137.27, 131.74, 130.07, 128.95, 127.46, 126.94,
124.96, 123.61, 120.77, 120.70, 118.73, 118.61, 118.41, 106.41, 101.77,
99.75, 55.61, 54.62, 48.73, 47.01, 45.73. HRMS (ESI): exact mass calcd
for C39H38N8O4 [M + H]+, 683.3089, found 683.3095.
(s, 1H), 8.12 (s, 1H), 7.81 (d, J = 7.6 Hz, 1H), 7.55 (s, 1H), 7.48 (s,
1H), 7.43 (t, J = 8.0 Hz, 1H), 7.27 (d, J = 7.6 Hz, 1H), 6.96 (d, J = 7.6
Hz, 1H), 6.50 (s, 1H), 6.43 (dd, J = 10.0, 17.2 Hz, 1H), 6.25 (d, J =
16.8 Hz, 1H), 6.03 (s, 1H), 5.76 (d, J = 10.0 Hz, 1H), 4.43 (s, 2H),
3.75 (s, 3H), 3.00 (m, 4H), 2.67 (m, 1H), 2.46 (m, 4H), 2.24 (s, 3H),
0.76 (d, J = 5.6 Hz, 2H), 0.68 (m, 2H). 13C NMR (125 MHz, DMSO-
d6) δ 163.11, 158.27, 157.11, 154.01, 153.89, 146.78, 139.49, 137.42,
131.74, 128.91, 126.92, 124.91, 120.77, 120.73, 118.28, 106.41, 101.87,
99.74, 55.59, 54.62, 48.76, 45.74, 44.50, 30.04, 6.86. HRMS (ESI):
exact mass calcd for C30H34N8O3 [M + H]+, 555.2827, found
555.2821.
N-(3-(7-(2-Methoxy-4-(4-methylpiperazin-1-yl)phenylamino)-2-
oxo-3-phenyl-3,4-dihydropyrimido[4,5-d]pyrimidin-1(2H)-yl)-
phenyl)acrylamide (2q). 1H NMR (400 MHz, DMSO-d6) δ 10.32 (s,
1H), 8.17 (s, 1H), 7.82 (d, J = 8.0 Hz, 1H), 7.68 (s, 1H), 7.56 (s, 1H),
7.41−7.47 (m, 5H), 7.26−7.32 (m, 2H), 7.07 (d, J = 7.6 Hz, 1H), 6.52
(d, J = 2.0 Hz, 1H), 6.44 (dd, J = 10.0, 16.8 Hz, 1H), 6.26 (dd, J = 1.6,
16.8 Hz, 1H), 6.05 (d, J = 7.2 Hz, 1H), 5.76 (d, J = 11.6 Hz, 1H), 4.87
(s, 2H), 3.77 (s, 3H), 3.01 (m, 4H), 2.43 (m, 4H), 2.22 (s, 3H). 13C
NMR (125 MHz, DMSO-d6) δ 163.14, 158.43, 157.20, 154.08, 152.17,
146.90, 142.39, 139.53, 137.28, 131.75, 128.97, 128.73, 126.96, 126.16,
125.60, 124.98, 120.79, 120.70, 118.44, 106.42, 101.82, 99.76, 55.61,
54.63, 48.75, 46.74, 45.75. HRMS (ESI): exact mass calcd for
C33H34N8O3 [M + H]+, 591.2827, found 591.2823.
N-(3-(3-(4-(Benzyloxy)phenyl)-7-(2-methoxy-4-(4-methylpipera-
zin-1-yl)phenylamino)-2-oxo-3,4-dihydropyrimido[4,5-d]pyrimidin-
1
1(2H)-yl)phenyl)acrylamide (2v). H NMR (400 MHz, DMSO-d6) δ
10.31 (s, 1H), 8.15 (s, 1H), 7.81 (d, J = 8.4 Hz, 1H), 7.66 (s, 1H), 7.54
(s, 1H), 7.29−7.47 (m, 8H), 7.04−7.07 (m, 3H), 6.52 (d, J = 2.0 Hz,
1H), 6.44 (dd, J = 10.0, 16.8 Hz, 1H), 6.25 (dd, J = 2.0, 16.8 Hz, 1H),
6.05 (d, J = 8.0 Hz, 1H), 5.76 (dd, J = 2.0, 10.0 Hz, 1H), 5.13 (s, 2H),
4.81 (s, 2H), 3.77 (s, 3H), 3.00 (t, J = 4.4 Hz, 4H), 2.42 (t, J = 4.4 Hz,
4H), 2.22(s, 3H). 13C NMR (125 MHz, DMSO-d6) δ 163.13, 158.37,
157.23, 156.52, 154.03, 152.21, 146.84, 139.51, 137.33, 136.97, 135.44,
131.74, 128.93, 128.39, 127.79, 127.63, 127.13, 126.93, 124.97, 120.78,
120.72, 118.37, 114.89, 106.42, 101.77, 99.75, 69.36, 55.61, 54.63,
48.75, 47.24, 45.75. HRMS (ESI): exact mass calcd for C40H40N8O4
[M + H]+, 697.3245, found 697.3252.
N-(3-(7-(2-Methoxy-4-(4-methylpiperazin-1-yl)phenylamino)-3-
N-(3-(7-(2-Methoxy-4-(4-methylpiperazin-1-yl)phenylamino)-3-
methyl-2-oxo-3,4-dihydropyrimido[4,5-d]pyrimidin-1(2H)-yl)-
(naphthalen-1-yl)-2-oxo-3,4-dihydropyrimido[4,5-d]pyrimidin-
1
1
phenyl)propionamide (2x). H NMR (400 MHz, DMSO-d6) δ 9.99
1(2H)-yl)phenyl)acrylamide (2r). H NMR (400 MHz, DMSO-d6) δ
(s, 1H), 8.10 (s, 1H), 7.69 (d, J = 8.4 Hz, 1H), 7.50 (s, 1H), 7.46 (s,
1H), 7.39 (t, J = 8.0 Hz, 1H), 7.26 (d, J = 8.4 Hz, 1H), 6.90 (d, J = 8.0
Hz, 1H), 6.51 (d, J = 2.4 Hz, 1H), 6.03 (d, J = 7.6 Hz, 1H), 4.45 (s,
2H), 3.76 (s, 3H), 3.01 (t, J = 4.4 Hz, 4H), 2.96 (s, 3H), 2.43 (t, J =
4.4 Hz, 4H), 2.30 (q, J = 7.2, 14.8 Hz, 2H), 2.22 (s, 3H), 1.06 (t, J =
7.2 Hz, 3H). 13C NMR (125 MHz, DMSO-d6) δ171.97, 158.24,
157.26, 153.99, 152.54, 146.78, 139.89, 137.41, 128.76, 124.32, 120.79,
120.31, 117.87, 106.44, 101.13, 99.74, 55.61, 54.66, 48.79, 45.81,
45.75, 39.00, 29.55, 9.60. HRMS (ESI): exact mass calcd for
C28H34N8O3 [M + H]+, 531.2827, found 531.2819.
10.34 (s, 1H), 8.21 (s, 1H), 7.93−7.97 (m, 4H), 7.83 (d, J = 8.4 Hz,
1H), 7.72 (s, 1H), 7.63 (dd, J = 2.0, 8.4 Hz, 1H), 7.59 (s, 1H), 7.50−
7.57 (m, 2H), 7.47 (t, J = 8.0 Hz, 1H), 7.33 (d, J = 8.8 Hz, 1H), 7.11
(d, J = 7.6 Hz, 1H), 6.53 (d, J = 2.0 Hz, 1H), 6.45 (dd, J = 10.0, 16.8
Hz, 1H), 6.26 (dd, J = 1.6, 16.8 Hz, 1H), 6.06 (d, J = 8.0 Hz, 1H), 5.77
(dd, J = 1.6, 10.0 Hz, 1H), 5.01 (s, 2H), 3.78 (s, 3H), 3.01 (m, 4H),
2.44 (m, 4H), 2.22 (s, 3H). 13C NMR (125 MHz, DMSO-d6) δ
163.15, 158.48, 157.18, 154.11, 152.35, 140.09, 139.54, 137.25, 133.06,
131.75, 131.09, 128.98, 127.95, 127.52, 127.46, 126.94, 126.38, 125.89,
124.99, 122.26, 120.81, 120.71, 118.49, 106.44, 101.91, 99.78, 55.62,
54.57, 48.68, 46.74, 45.65. HRMS (ESI): exact mass calcd for
C37H36N8O3 [M + H]+, 641.2983, found 641.2985.
Cell Lines and Reagents. H1975 (NSCLC, EGFRL858R/T790M),
HCC827 (NSCLC, EGFR del E746‑A750), A431 (epidermoid carcinoma,
EGFR overexpression), and A549 (NSCLC, EGFR wild type) cells
were obtained from ATCC. The cells were maintained at 37 °C in a
5% CO2 incubator in RPMI 1640 (Gibco, Invitrogen) containing 10%
fetal bovine serum (Gibco, Invitrogen). HLF-1(diploid human lung
fibroblasts) and HL-7702 (diploid human liver cell) were gifts from
Prof. Duanqing Pei. The HLF-1 was maintained in Ham’s F12K
medium (Gibco, Invitrogen) with 15% FBS. The HL-7702 was
N-(3-(3-Benzyl-7-(2-methoxy-4-(4-methylpiperazin-1-yl)-
phenylamino)-2-oxo-3,4-dihydropyrimido[4,5-d]pyrimidin-1(2H)-
1
yl)phenyl)acrylamide (2s). H NMR (400 MHz, DMSO-d6) δ 10.30
(s, 1H), 8.08 (s, 1H), 7.84 (d, J = 8.0 Hz, 1H), 7.60 (s, 1H), 7.49 (s,
1H), 7.45 (t, J = 8.0 Hz, 1H), 7.33−7.41 (m, 4H), 7.26−7.32 (m, 1H),
7.02 (d, J = 7.6 Hz, 1H), 6.50 (d, J = 2.4 Hz, 1H), 6.43 (dd, J = 10.0,
16.4 Hz, 1H), 6.25 (dd, J = 2.0, 16.8 Hz, 1H), 6.03 (d, J = 6.8 Hz, 1H),
2720
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Journal of Medicinal Chemistry
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cultured in RPMI 1640 containing 10% FBS. The EGFR gene of every
cell line was sequenced before use. Gefitinib was synthesized in our
chemistry laboratory.
In Vitro Enzymatic Activity Assay. Wild type and different
EGFR mutants (T790M, L858R, L861Q, L858R/T790M) and the Z′-
Lyte Kinase Assay Kit were purchased from Invitrogen. Ten
concentration gradients from 5.1 × 10−11 to 1.0 × 10−6 mol/L were
set for all the tested compounds. The experiments were performed
according to the instructions of the manufacturer.
(10−12 days post implantation), animals were randomized into
treatment groups (n = 6 mice/group). Each group was dosed orally for
14 days with either vehicle only or with compound 2s at 10 or 30 mg/
kg or gefitinib at 50 mg/kg daily (qd). The doses were in a volume of
0.1 mL/20 g of the animal body weight. Tumor volumes were
measured every other day using vernier calipers, and volumes were
calculated using the following formula: tumor volume (mm3) = W2(L/
2), where W = width and L = length in mm.
Kinase Profiling Study and Kd Determination. The kinase
profiling study and Kd determination were conducted using the Ambit
Kinome screening platform (www.kinomescan.com). Kinases were
produced displayed on T7 phage or by expression in HEK-293 cells
and tagged with DNA. Binding reactions were performed at r.t. for 1 h,
and the fraction of kinase not bound to test compound was
determined by capture with an immobilized affinity ligand and
quantitation by quantitative PCR. Each kinase was tested individually
against each compound. Kd values were determined using 11 serial 3-
fold dilutions and presented as mean values from experiments
performed in duplicate.
Cell Proliferation and Growth Inhibition Assay. Cell
proliferation was assessed by MTS assay. The cells were exposed to
treatment for 72 h, and the number of cells used per experiment for
each cell line was adjusted to obtain an absorbance of 1.3 to 2.2 at 490
nm. Six concentrations (0.1 nM to 10 μM) were set for the
compounds. At least six parallels of every concentration were used. All
experiments were repeated at least four times. The data were
calculated using GraphPad Prism version 4.0. The IC50 were fitted
using a nonlinear regression model with a sigmoidal dose−response.
Colony Formation Assay. The H1975 cells were plated in 6-cm
dishes with densities of 500 cells/dish. After 24-h growth, the cells
were treated with compounds 2q and 2s under concentrations ranging
from 0.1 to 10 nM. The medium with compounds were changed every
3 days. After 9-day incubation, plates were fixed with 4% form-
aldehyde, and 0.2% crystal violet was used for staining.
Antibodies and Western Blotting. Cells were plated in 6-cm
dishes. After 24-h growth, the cells were treated with the compounds
under the indicated concentrations for 24 h. Cell lysates were collected
with 1× lysis buffer (CST) and were briefly sonicated. Western blot
analyses were conducted after separation by SDS/PAGE electro-
phoresis and transfer to PVDF membranes. Membranes were blocked
in 5% bovine serum albumin/TBST and probed with the indicated
antibodies, followed by a peroxidase-conjugated antimouse or rabbit
secondary antibody. Blots were developed by enhanced chemilumi-
nescence (Thermo). Anti-phospho Akt (Ser 173), anti-total Akt, anti-
EGFR, anti-phospho specific EGFR (pY1068), anti-total ERK1/2, and
anti-phospho ERK1/2 (pT202/pY204) antibodies were obtained from
Cell Signaling Technology.
Flow Cytometry Assay. For the apoptosis assay, cells were plated
in six-well plates overnight and were treated with the compounds
under indicated concentrations for 48 h. Trypsin (0.25%) was used,
and a single cell suspension was prepared according to the instructions
of the manufacturer. Apoptosis was assessed using a PE-Annexin V Kit
(BD Pharmingen). The samples were detected with a FACS Calibur
flow cytometer (Becton Dickinson)
ASSOCIATED CONTENT
* Supporting Information
■
S
1H NMR spectrum and purity determination for compounds
2a−x, apoptosis, cell cycle arrest, and colony formation
inhibition induced by compounds 2q and 2s, Western blot
analysis on A549 and A431 cells, growth inhibition on HLF-1
cells. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: +86-20-32015276. Fax: +86-20-32015299. E-mail: ding_
ke@gibh.ac.cn.
■
Author Contributions
#These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank National Basic Research Program of China (#
2010CB529706, 2009CB940904), National Natural Science
Foundation (# 21072192), Key Project on Innovative Drug of
Guangdong Province (# 2011A080501013), Strategic Collab-
orative Project of Guangdong Province and Chinese Academy
of Sciences, Key Project on Innovative Drug of Guangzhou
City (grant # 2009Z1-E911, 2010J-E551), and the 100-talent
program of Chinese Academy of Sciences (CAS) for their
financial support.
ABBREVIATIONS USED
■
T, threonine; M, methionine; I, isoleucine; L, leucine; R,
arginine; E, glutamic acid; A, alanine; Q, glutamine; EGFR,
epidermal growth factor receptor; WT, wild-type; NSCLC, non
small cell lung cancer; ATP, adenosine-triphosphate; LAH,
lithium aluminum hydride; m-CPBA, 3-chloroperbenzoic acid;
DIPEA, N,N-diisopropylethylamine; JAK3, Janus kinase 3; Kd,
binding constant; BLK, B lymphocyte kinase; BTK, Bruton’s
tyrosine kinase; GAK, cyclin G associated kinase; FRK, Fyn-
related kinase; LCK, lymphocyte-specific protein tyrosine
kinase; MAPK, mitogen-activated protein kinase signaling
pathway
The cell cycle analysis was performed by using Cycletest Plus DNA
Reagent Kit (BD Pharmingen). Cells seeded in six-well plates were
serum-starved for 24 h to synchronize cells in the G0−G1 phase of the
cell cycle. Then the cells were treated with 2q and 2s under the
indicated concentrations for 24 h. The cells were lightly trypsinized,
and samples were prepared according to the instructions of the
manufacturer. Samples were analyzed on a FACS Calibur flow
cytometer (Becton Dickinson), and data were analyzed using the
Modfit software package.
Mouse Tumor Xenograft Efficacy Study. The efficacy study was
conducted in accordance with the guidelines for the Care and Use of
Laboratory Animals in Guangzhou Institute of Biomedicine and
Health (GIBH, CAS). Six week old SCID mice were inoculated
subcutaneously with H1975 NSCLC cells (2 × 106/mouse) in the
right flank. Upon reaching an average tumor volume of 380−420 mm3
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