Discovery and Structural Optimization of N5-Substituted 6,7-Dioxo-6,7-dihydropteridines as Potent and Selective Epidermal Growth Factor Receptor (EGFR) Inhibitors against L858R/T790M Resistance Mutation

Article abstract of DOI:10.1021/acs.jmedchem.6b00403

EGFR-targeted inhibitors (gefitinib and erlotinib) provided an effective strategy for the treatment of non-small-cell lung cancer. However, the EGFR T790M secondary mutation has become a leading cause of clinically acquired resistance to these agents. Her

Full text of DOI:10.1021/acs.jmedchem.6b00403

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Article
Discovery and Structural Optimization of N5-Substituted 6,7-dioxo-6,7-
dihydropteridines as Potent and Selective Epidermal Growth Factor
Receptor (EGFR) Inhibitors Against L858R/T790M Resistance Mutation
Yongjia Hao, Xia Wang, Tao Zhang, Deheng Sun, Yi Tong, Yuqiong Xu, Haiyang Chen, Linjiang
Tong, Lili Zhu, Zhenjiang Zhao, Zhuo Chen, Jian Ding, Hua Xie, Yufang Xu, and Honglin Li
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 11 Jul 2016
Downloaded from http://pubs.acs.org on July 11, 2016
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Discovery and Structural Optimization of N5ꢀSubstituted 6,7ꢀdioxoꢀ6,7ꢀ
dihydropteridines as Potent and Selective Epidermal Growth Factor Receptor
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(
EGFR) Inhibitors Against L858R/T790M Resistance Mutation
1,#
1,#
2,#
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Yongjia Hao , Xia Wang , Tao Zhang , Deheng Sun , Yi Tong , Yuqiong Xu , Haiyang
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2,
Chen , Linjiang Tong , Lili Zhu , Zhenjiang Zhao , Zhuo Chen , Jian Ding , Hua Xie *, Yufang
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,
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Xu, * and Honglin Li *
1
Shanghai Key Laboratory of New Drug Design, Shanghai Key Laboratory of Chemical Biology,
State Key Laboratory of Bioreactor Engineering, School of Pharmacy, East China University of
2
Science & Technology, Shanghai, 200237, China; Division of Antiꢀtumor Pharmacology, State
Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, Shanghai, 201203, China.
*To whom correspondence should be addressed. Email: hxie@jding.dhs.org,
yfxu@ecust.edu.cn, hlli@ecust.edu.cn
Please address correspondence and requests for reprints to:
Prof. Honglin Li
School of Pharmacy, East China University of Science and Technology
130 Meilong Road, Shanghai 200237
Phone/Fax: +86ꢀ21ꢀ64250213
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ABSTRACT
EGFRꢀtargeted inhibitors (gefitinib and erlotinib) provided an effective strategy for the treatment
of nonꢀsmall cell lung cancer. However, the EGFR T790M secondary mutation has become a
leading cause of clinically acquired resistance to these agents. Herein, based on the previously
reported irreversible EGFR inhibitor (compound 9), we present a structureꢀbased design
approach, which is rationalized via analyzing its binding model and comparing the differences of
gatekeeper pocket between the T790M mutant and wildꢀtype (WT) EGFR kinases. Guided by
these results, a novel 6,7ꢀdioxoꢀ6,7ꢀdihydropteridine scaffold was discovered and hydrophobic
modifications at N5ꢀpostion were conducted to strengthen nonpolar contacts and improve mutant
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selectivity over EGFR . Finally, the most representative compound 17d was identified. This
work demonstrates the power of structureꢀbased strategy in discovering lead compounds, and
L858R/T790M
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provides molecular insights into the selectivity of EGFR
over EGFR , which may
play an important role in designing new classes of mutantꢀselective EGFR inhibitors.
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INTRODUCTION
Receptor tyrosine kinases (RTKs) play significant roles in cellular signaling pathways and
regulate numerous cellular processes, such as cell proliferation, differentiation, metabolism and
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apoptosis. Moreover, RTKs as key regulators are involved in the development and progression
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,5
of many types of human malignancies. As for the known RTKs, the epidermal growth factor
receptor (EGFR) tyrosine kinase is one of the most important clinically validated targets for antiꢀ
6,7
cancer therapies, especially for nonꢀsmall cell lung cancer (NSCLC) treatment. Up to now,
EGFRꢀtargeted tyrosine kinase inhibitors (TKIs), including firstꢀgeneration EGFR inhibitors
gefitinib (1) and erlotinib (2), have been approved by US Food and Drug Administration (FDA)
for treatment of NSCLC patients with EGFR activating mutations (exon 19 deletion or exon 21
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,9
L858R substitution mutation) in 2003 and 2004, respectively (Figure 1). EGFR L858R point
mutation (substitution of a leucine to an arginine at position 858) in exon 21 is one of the most
common EGFR activating mutations, which appears adjacent to the highly conserved activation
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loop. These two small molecular EGFR inhibitors achieved excellent improvements in overall
response rates (ORR) and median progressionꢀfree survival (PFS) for NSCLC patients harboring
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this activating mutation.
Although the early results of firstꢀgeneration EGFR inhibitors are
impressive, unfortunately, most NSCLC patients with activating mutations initially responded to
the TKIs and invariably developed acquired resistance to EGFR TKIs therapy within months.
The most common mechanism of acquired resistance is the secondary T790M (substitution of
threonine to methionine) gatekeeper point mutation in exon 20 that occurs in cis with an EGFR
activating mutation (e.g., L858R), which accounts for approximately 60% of these acquired
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resistances. In EGFR T790M mutation kinase, the Met790 gatekeeper residue is a more bulky
amino acid than Thr790. Because the volume of the side chain increases, the hydrophobic ATPꢀ
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binding subpocket becomes smaller. Thus, the resulting steric impediment may reduce binding of
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gefitinib and erlotinib.
To overcome the problem of acquired resistance to firstꢀgeneration TKIs, several secondꢀ and
thirdꢀgeneration irreversible EGFR TKIs have been developed. These irreversible EGFR TKIs
were appended with a Michael receptor moiety which can form a covalent bond with the
sulfhydryl group of Cys797 in the ATP binding cleft of EGFR, thereby increasing the potency of
inhibition. For example, afatinib (3), one of the secondꢀgeneration irreversible EGFRꢀTKIs,
which received marketing authorization by FDA in 2013 and showed good potency in vivo in
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xenograft models driven by dualꢀmutant EGFR (EGFR
) (Figure 1).
However,
because of doseꢀlimiting toxicities related to inhibition of wildꢀtype (WT) EGFR, this agent
displayed limited benefit clinically for NSCLC patients who have developed T790M acquired
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resistance.
More recently, thirdꢀgeneration covalent EGFR inhibitors, such as compounds 4
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(
WZ4002), 5 (COꢀ1686, Rociletinib), and 6 (AZD9291, Osimertinib),
etc., have been
L858R/T790M
developed as mutantꢀselective EGFR inhibitors that specifically target EGFR
mutation,
while being less selective for WT EGFR (Figure 1). However, compound 4 did not progress into
clinical development. Compound 5 is currently in phase I/II clinical trials in NSCLC patients
with EGFR T790Mꢀpositive mutation. Recently, compound 6 has been granted accelerated
approval by the FDA to treat advanced EGFR T790M mutationꢀpositive NSCLC patients whose
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disease had progressed following prior treatment with other EGFRꢀblocking therapy. In
addition, researchers in academia have also reported a series of EGFR inhibitors with varying
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degrees of selectivity for dualꢀmutant EGFR over WT EGFR
, such as compounds 7 and 8
developed by Ding’s group on the basis of compound 4, which showed potent enzymatic
L858R/T790M
activities against EGFR
with low nanomolar IC or K values (7, IC = 0.9 nM; 8, K
50 d 50 d
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=
2.6 nM). Notably, their design strategy using conformational constraint to develop thirdꢀ
generation EGFR inhibitors provide inspiration for researchers to design novel selective EGFR
inhibitors to overcome EGFR gatekeeper mutations in NSCLC treatment. Herein, we describe
the design and optimization of 6,7ꢀdioxoꢀ6,7ꢀdihydropteridine derivatives as novel
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L858R/T790M
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EGFR
inhibitors with improved selectivity profiles over EGFR .
Figure 1
CHEMISTRY
As outlined in Scheme 1, compounds 17aꢀe were prepared starting from commercially available
,3ꢀbenzendiamine (10), followed by treatment with diꢀtertꢀbutyl dicarbonate to achieve Bocꢀ
1
protected amine (11). Reacting compound 11 with 2,4ꢀdichloroꢀ5ꢀnitropyrimidine to yield tertꢀ
butyl (3ꢀ((2ꢀchloroꢀ5ꢀnitropyrimidinꢀ4ꢀyl)amino)phenyl)carbamate (12) in a high yield (80%).
Then, compound 12 was converted to the intermediate (13) by displacement of 2ꢀchloro in the
pyrimidine with appropriate arylamines in a good yield (69%), subsequent reduction of the nitro
group and reaction with diethyl oxalate to obtain the ringꢀclosure compound (15) in an
acceptable 60% overall yield, which was deprotected with trifluoroacetic acid and reacted with
acryloyl chloride to produce the compound 17a with moderate 41% overall yield of twoꢀstep.
Meanwhile, compound 15 was alkylated according to the reported procedure with appropriate
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alkyl halides to afford the N5ꢀalkylated derivatives (16bꢀe) in moderate 49ꢀ55% yields.
Finally, compounds 16bꢀe were deprotected with trifluoroacetic acid in dichloromethane and
reacted with acryloyl chloride to produce the final target compounds 17b−e with 31ꢀ46% overall
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yields. Compounds 17fꢀj were prepared by a method similar to that for compound 17b in similar
overall yields.
Scheme 1
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RESULTS AND DISCUSSION
Lead Compound and Design Strategy. In our previous studies, compound 9 (Figure 1) was
identified by scaffold hopping as a potent irreversible EGFR kinase inhibitor, which exhibited
competitive inhibitory enzymatic activities with single nanomolar IC values against both wildꢀ
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type and L858R/T790M mutant EGFRs (3.8 nM vs. 1.1 nM). On the basis of molecular
modeling studies, 9 irreversibly binds to the T790M mutant EGFR (PDB 3IKA) by targeting the
Cys797 residue in the ATP binding site via the formation of a covalent bond, and the Nꢀ
methylpiperazine motif on the leftꢀhand side is directed toward the solvent exposed region. The
pteridinꢀ7(8H)ꢀone core forms an expected bidentate hydrogenꢀbonded interaction with the hinge
residue Met793 while the doubleꢀring system contacts the mutant gatekeeper Met790 residue.
Besides, the EGFR L858R mutation adjacent to the activation loop is far from the ligand and
would not affect the binding site (Figure 2A and 2B). The crystal structure of compound 4,
which exhibited similar binding modes but mutantꢀselective activity, encouraged us to do the
T790M
WT
comparative crystal structure analysis for EGFR
mutant over EGFR . As shown in Figure
2B, overlay of the structure of WT EGFR (PDB 4G5J) into T790M mutant EGFR shows that
T790M
selectivity of compound 4 for the gatekeeper EGFR
mutant was obtained through the 5ꢀ
position chlorine substitution that points directly toward the methionine residue at position 790
WT
where it may achieve van der Waals contacts. However, in the EGFR , the bulkier methionine
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residue is replaced by the smaller and more polar threonine residue at position 790, and therefore,
it reduced the hydrophobic interaction between the gatekeeper residue and the chlorine
substituent, leading to lower affinity. Given these differences in the gatekeeper pocket between
the T790M mutant and WT EGFR kinases, we conducted lead optimization studies with
hydrophobic modifications at the 5ꢀpostion moiety to strengthen nonpolar contacts and improve
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selectivity over EGFR . Nevertheless, it is impossible to directly introduce substituents at the
5ꢀposition of compound 9. Therefore, a scaffold hopping of the pteridinꢀ7(8H)ꢀone was
conducted. The pyrazinone part of the pteridinꢀ7(8H)ꢀone was replaced with piperazinediones in
order to add hydrophobic substituents at the 5ꢀposition and thus occupy the lipophilic space
within the gatekeeper pocket (Figure 2C). These efforts resulted in compound 17a. Moreover,
the superposition of compound 4 and compound 9 suggests that little space remains in the
binding site adjacent to the gatekeeper residue, and only small substitutions can be
accommodated. We therefore designed a series of 6,7ꢀdioxoꢀ6,7ꢀdihydropteridine derivatives
WT
with N5ꢀsubstituents as a hydrophobic functional group to improve selectivity over EGFR ,
studied the interactions between EGFR kinases and the inhibitors by molecular modeling to
provide us guidance for the structural optimization, and ultimately synthesized the inhibitors to
verify our hypothesis.
Figure 2
Binding Mode Analysis. The designed inhibitors (17aꢀe) were aligned to the crystal structure
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of compound 4 via inꢀhouse molecular 3D similarity calculation method SHAFTS and using
MacroModel optimization to generate covalent proteinꢀligand complexes. As shown in Figure
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A, the 6,7ꢀdioxoꢀ6,7ꢀdihydropteridine ring extends into the hydrophobic pocket around the
T790M gatekeeper residue, however, the hydrophobic contact between the N5ꢀunsubstituted
doubleꢀring system and Met790 is not as tight as that of the chlorine substituent on the
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pyrimidine ring in compound 4, and would not significantly influence selectivity over EGFR .
A N5ꢀmethyl moiety (17b) was then added as a hydrophobic substitution to target Met790 in
EGFR on the basis of observing the overlay poses of 9 and compound 4 as well as the gatekeeper
pocket differences. As expected, in the case of the T790M mutant EGFR, the 5ꢀsubstituent is in
van der Waals contacts (distance of 3.1 Å) with the methyl group of Met790, while for the WT
EGFR the corresponding distance is much shorter (distance of 1.9 Å), leading to a steric clash
between the 5ꢀsubstituent and the hydroxyl side chain of Thr790 (Figure 3B) which would likely
affect the potency and selectivity.
Based on the above molecular simulations, we increased the bulkiness of the group at Nꢀ5
moiety in inhibitor 17a, such as 5ꢀethyl (17c), 5ꢀisopropyl (17d) and 5ꢀpropyl (17e), to further
explore how large a substituent would be tolerated in terms of potency in the mutant EGFR
WT
kinase and simultaneously benefit selectivity over EGFR . Modeling structure of compound
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7d shows that the 5ꢀisopropyl moiety extends to the methionine gatekeeper pocket and fits the
pocket shape well, resulting in the formation of close hydrophobic interactions with the residues
of Met790, Ala743, Leu844 and Met793 that contribute to the potency of 17d toward the
WT
L858R/T790M mutant (Figure 3C). However, in the model of EGFR /17d, different orientation
of the 5ꢀisopropyl group was adopted. The 5ꢀisopropyl also participates in nonpolar interactions
with the residues of Thr790, Thr854, Ala743 and Leu844 (Figure 3D), which may be responsible
WT
for the similarity in the EGFR potency of 17d compared to compound 17a. As for 17c and
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interactions with the gatekeeper Met790 residue (Figure S1A and S1C), but similar to that of
17d, the orientations of the 5ꢀsubstituents would be influenced to match the gatekeeper Thr790
residue while binding to the active site of the WT EGFR (Figure S1B and S1D), thus possibly
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WT
leading to a decrease of selectivity between EGFR
and EGFR .
Figure 3
Enzyme and Cellular Activities. As a proof, these five compounds have been synthesized (as
shown in Scheme 1) and their IC50 values are shown in Table 1. Compound 17a potently
WT
L858R/T790M
inhibited the enzymatic activity of EGFR and EGFR
mutant with IC values of 2.8
50
and 2.5 nM, respectively, which is similar to those of compound 9 and also showed no selectivity
for L858R/T790M mutant EGFR over WT EGFR. The binding mode shown in Figure 3A also
implied that the hydrophobic contact between the 6,7ꢀdioxoꢀ6,7ꢀdihydropteridine core and the
T790M gatekeeper residue is not as tight as that of the chlorine substituent on the pyrimidine
ring in compound 4. The kinase inhibition of 17a was further validated by investigating its
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suppression on the growth of H1975 cells ectopically expressing EGFR L858R/T790M.
However, it did not account for any significant inhibitory effect on H1975 cells at concentrations
up to 10000 nM. The effects of 17a on A431 cells harboring WT EGFR kinase were also
investigated. The results demonstrated that 17a poorly affected the activation of EGFR in the
corresponding cells although it displayed strong inhibition against EGFR kinases under the in
vitro screening assay, indicating the need for further investigation with respect to
physicochemical properties, such as solubility and cell permeability. The solubility was
measured at pH 7.4 and revealed an acceptable aqueous solubility of 839 ꢁg/mL (Table 2).
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Furthermore, we built simple prediction models of cell permeability described by QPPCaco2
values calculated using Qikprop in Maestro software package. As shown in Table 2, 17a
exhibited poor cell permeability with the QPPCaco2 value of 22.4, which was outside the
recommended ranges (< 25 poor), thus providing a reasonable explanation for the compound’s
loss of cellular efficacy as compared to the inhibitory activity with respect to isolated kinases.
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Introduction of a methyl group at the 5ꢀposition led to a compound (17b) which was roughly
L858R/T790M
equipotent to compound 17a in the kinase inhibitory activities against EGFR
(IC =
50
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4
.7 nM) and also approximately 101ꢀfold selective over the EGFR (IC = 474 nM). These
50
T790M
results were consistent with the modeling structure of EGFR
in complex with compound
17b (Figure 3B), showing that the 5ꢀmethyl moiety was directed toward the gatekeeper region of
WT
the ATPꢀbinding site, and was detrimental to the kinase inhibition of EGFR due to the steric
clashes, thus leading to a 170ꢀfold potency loss compared with the parent compound 17a (474
nM vs. 2.8 nM). Furthermore, 17b displayed a decreased solubility of 392 ꢁg/mL and an
increased QPPCaco2 value of 47.4, with the suppressive potency against H1975 cell lines
increasing to 1772 nM, but still suffering a significantly drop in potency from enzyme to cell,
which might be due to the poor octanol/water partition coefficient log P (Table 2).
As expected, the introduction of bulkier substitutions at the 5ꢀposition resulted in further
L858R/T790M
enhancement of inhibitory activities against EGFR
as observed for the 5ꢀisopropyl
(17d) and 5ꢀpropyl (17e) derivatives. Both of these compounds strongly inhibited
L858R/T790M
WT
EGFR
with IC values below 1 nM. However, their EGFR inhibitory potency (2.0
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and 2.2 nM, respectively) were also improved relative to compound 17b, resulting in a slight
L858R/T790M
WT
decrease of selectivity between EGFR
and EGFR . As shown in the predicted
binding mode (Figure 3C and 3D), the 5ꢀisopropyl group adopted different orientations in
binding with the T790M mutant and WT EGFR kinases, and could form nonpolar interactions
with both Thr790 and Met790, which may account for the increased EGFR potency and
decreased selectivity. Additionally, 5ꢀethyl derivative (17c) displayed an IC value of 1.2 nM on
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L858R/T790M
WT
EGFR
, but its IC value on EGFR (IC = 81 nM) was 6 times greater than that of
50 50
17b, further highlighting that larger groups than methyl at the 5ꢀposition might strengthen the
hydrophobic interactions with the threonine gatekeeper, and thus showing decreased selectivity
over WT EGFR. The effects of all three compounds were further investigated on the activation
of H1975 and A431 cells. It was shown that compounds 17c and 17e exhibited moderate potency
in suppressing the growth of H1975 cells with IC values of 126 and 119 nM, respectively,
5
0
whereas their corresponding values for A431 cells were about 18ꢀ70ꢀfold greater. Besides, the
most enzymatically potent compound 17d also exhibited potent growthꢀinhibitory activities in
the H1975 cells with an IC value of 18 nM, which was comparable to that of compound 5.
5
0
More significantly, the corresponding antiproliferative activity on A431 cells was about 7773
nM and was less potent by over 432 fold than that against H1975 cells, suggesting that 17d may
serve as an excellent selective inhibitor for EGFR mutations. The pK , especially the aqueous
a
solubility of compounds 17c, 17d, and 17e were much lower than that of compound 17a, along
with their higher QPPCaco2 values, leading to improved cellular antiproliferative activities.
Besides, in agreement with the findings that the mutant EGFRs exhibit substantially increased
3
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autoꢀphosphorylating activity relative to WT EGFR , 17d suffered a 3887ꢀfold drop in potency
of WT EGFR from enzyme to cell, yet a relatively less drop for L858R/T790M mutant EGFR
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(
60ꢀfold), indicating the enhanced catalytic activity and increased sensitivity of EGFR mutants
WT
compared to EGFR .
On the basis of compound 17a, we introduced alkyl groups, such as methyl and ethyl at the Nꢀ
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moiety to improve selectivity over EGFR kinase, the resulting compounds 17b and 17c
L858R/T790M
WT
indeed showed 101ꢀ and 67ꢀfold selectivity between EGFR
and EGFR . These
results confirmed our initial hypothesis that hydrophobic optimization at 5ꢀposition to strengthen
nonpolar contacts is extremely important to inhibitors of L858R/T790M EGFR mutants with
selectivity over WT EGFR. Furthermore, substituting groups at the 5ꢀposition are actually sizeꢀ
constrained, namely introduction of bulkier substitutions, such as isopropyl (17d) and propyl
WT
(17e) at the 5ꢀposition resulted slight decrease of selectivity over EGFR kinase, suggesting that
WT
bulkier substituents may improve the inhibitory activities against EGFR
through the
hydrophobic interactions within the gatekeeper pocket, and therefore resulting in a decrease of
L858R/T790M
WT
selectivity between EGFR
and EGFR .
Modifications to the LeftꢀHand Side Chain. Considering compound 17d containing a 5ꢀ
isopropyl moiety showed remarkable enzymatic and cellular inhibitory activities against
L858R/T790M mutant EGFR, we selected it as a new lead compound for further structural
optimization. Previous investigations have demonstrated that the 2ꢀposition side chain bearing
hydrophobic interactions with the backbone of the hinge region has a significant impact on the
2
7,33,36
inhibitory activity.
We synthesized a set of compounds with different leftꢀhand side chains
while maintaining the 5ꢀisopropyl substitution on the dihydropteridine ring. The resulting IC50
values are listed in Table 1. Replacements of Nꢀmethylpiperazine moiety with N,N,N’ꢀ
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trimethylethylenediamine (compound 17f), N,Nꢀdimethylethanolamine (compound 17g) and 1ꢀ
methylꢀ4ꢀ(piperidinꢀ4ꢀyl)piperazine (compound 17h), produced compounds that showed
improved pK and aqueous solubility relative to 17d (Table 2), while these compounds
a
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demonstrated diminished potency in the enzyme and proliferation assays compared to 17d. Next,
we kept Nꢀmethylpiperazine motif constant, removed the methoxyl at 2ꢀposition of benzene and
introduced methyl or methoxy groups at 3ꢀposition and produced compounds 17i and 17j. Their
pK and aqueous solubility were also greater than 17d, which was in accordance with the
a
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previous study. However, these two compounds also exhibited a slight drop in enzymatic and
cellular assays in comparison to 17d (Table 1). These molecules displayed modest efficiency but
less selectivity in inhibiting the growth of H1975 cancer cells than 17d, indicating that the initial
leftꢀhand side chain was most favourable for mutant EGFR potency and selectivity.
Table 2
The Reactivity to Glutathione of 17d. In order to preliminarily explore its reactivity to other
thiols sources, such as glutathione, 17d was selected as representative compound to perform the
experiment. According to the reported literature, the experiment was performed with compound
o
17d (1 mM), glutathione (10 mM) in PBS (0.1 M, pH 7.4) at 37 C in the presence of
3
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dimethylacetamide (10%). However, we did not detect the glutathione adduct using highꢀ
resolution mass spectra (HRMS) even though the reaction has been mixed for 24 h. Furthermore,
considering the intracellular concentrations of glutathione are quite low, the reactivity of 17d to
glutathione might be lower.
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Kinase Selectivity Profile of 17d. As compound 17d demonstrated promising enzymatic
potencies, we next evaluated its kinase selectivity profile against a diverse panel of 468 kinases
at a concentration of 1000 nM by using the profiling platform of DiscoveRx KINOMEscan
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(
Figure 4). The results demonstrated that 17d only showed similar strong binding interaction
(percent of control value <1%) against ASK2, CAMK1, ERBB2, ERBB4, GAK, and TXK
kinases excluding EGFR at the screening concentration of 1000 nM, suggesting that the
compound has excellent selectivity on EGFR.
Figure 4
Inhibitory Effects of 17d on EGFR Activation and Downstream Signaling Transduction
in Cancer Cells. In order to test the inhibition on the phosphorylation of EGFR and the
downstream signaling transduction, we conducted the Western blot analysis of the representative
compound 17d in H1975 and A431 cells, respectively (Figure 5). The results showed that, 17d
L858R/T790M
potently inhibited the phosphorylation of EGFR
and ERK in H1975 cells at the
concentration of 100 nM, which showed similar inhibitory activity to compound 6 (Figure 5A).
WT
Meanwhile, 17d exhibited moderate inhibition on the phosphorylation of EGFR even at 1000
nM in A431 cells (Figure 5B). The results clearly demonstrated that 17d was at least 10ꢀfold
L858R/T790M
WT
more potent against EGFR
7d was a potent and selective EGFR
than EGFR at the cellular level, further indicating that
L858R/T790M
1
inhibitor.
Figure 5
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Metabolic Stability and In Vivo Pharmacokinetics Properties. We then examined the in
vitro metabolic stability of compound 17d with mouse liver microsomes. The results showed that
1
7d exhibited acceptable microsomal stability with a halfꢀlife (T ) of 51 min at the
1/2
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concentration of 1 ꢁM. Additionally, the preliminary in vivo pharmacokinetic properties of
compound 17d in mice following intravenous (IV) and oral administration were also investigated.
The PK profiles are shown in the Figure 6, and the analysis of PK parameters is summarized in
Table 3. Following an oral dose of 10 mg/kg, 17d showed a reasonable area under the curve
(AUC ) value of 680 ng·h/mL, moderate T of 1.1 h, and acceptable oral bioavailability (F =
0
ꢀ∞
1/2
48%). These results suggest that 17d would be a suitable candidate for further in vivo antitumor
efficacy studies.
Figure 6
Table 3
In Vivo Antitumor Efficacy Study. The most potent inhibitor, 17d, was further evaluated for
its in vivo antitumor efficacy in xenograft mouse models of H1975 and A431 (Figure 7). As
mentioned above, afatinib is an irreversible secondꢀgeneration EGFR inhibitor, which potently
3
9
inhibits the EGFR, HER2 and HER4 tyrosine kinases. According to the reported data, oral
administration of compound 5 (100 mg/kg once daily or 50 mg/kg twice daily) could induce
tumor regression in H1975 xenograft model, while afatinib (20 mg/kg/d) showed more
2
2
significant antiꢀtumor activity in A431 xenograft model relative to compound 5. Therefore,
compound 5 and afatinib were used as reference molecules in the H1975 and A431 xenograft
models, and oral doses of these two agents were selected as 50 mg/kg/d and 25 mg/kg/d,
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respectively. In H1975 (EGFRꢀL858R/T790M) xenograft mouse model, mice (n = 3/group) were
dosed with 17d at 50 mg/kg (PO, once daily) for 14 consecutive days. As illustrated in Figure
7
A, compared with the vehicle group, 17d significantly inhibited the tumor growth at this dose
TGI: 52%, P < 0.05), which was comparable with the reference compound 5. In A431 xenograft
mouse model (Figure 7B), mice (n = 6/group) were also dosed orally with 17d at 50 mg/kg/d for
4 days. 17d exhibited a moderate tumor growth inhibition (TGI: 50%, P < 0.05) compared with
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the afatinib group (TGI: 94%, P < 0.05). During the course of the experiment, 17d did not cause
significant body weight loss of mice and no mortality was observed (Figure S2A and S2B),
indicating that it was tolerated well at this dose.
The effects of compound 17d on phosphorylation of target and expression of Kiꢀ67, a tumor
proliferation marker, were evaluated by immunohistochemistry of H1975 tumor tissues after the
14 days of treatment. The expression of pꢀEGFR and Kiꢀ67 was significantly decreased by 17d
(Figures 7C and D). In this model, 17d significantly inhibited tumor cell proliferation (Kiꢀ67)
and pꢀEGFR expression compared with the vehicle groups (Figure 7C and D). Briefly, the
encouraging in vivo antitumor selectivity of compound 17d demonstrated that it might be used as
a promising lead compound for further development as a selective EGFR inhibitor to overcome
L858R/T790M
EGFR
resistance mutation.
Figure 7
CONCLUSIONS
Starting with a previously reported irreversible EGFR inhibitor, we developed 6,7ꢀdioxoꢀ6,7ꢀ
L858R/T790M
dihydropteridineꢀbased selective and cellꢀactive inhibitors of EGFR
mutants. Analysis
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T790M
WT
of the crystal structure of EGFR
and EGFR suggested that introduction of substituents at
the Nꢀ5 position of the dihydropteridine scaffold would lead to an increase in both
L858R/T790M
EGFR
activity and selectivity because these can provide suitable vectors toward the
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WT
gatekeeper Met790 pocket. While in the EGFR kinase, the smaller and more polar threonine
residue would reduce the hydrophobic interaction between the gatekeeper residue and the 5ꢀ
subsitutent, leading to lower affinity. As expected, introduction of hydrophobic groups, such as
L858R/T790M
an isopropyl (17d) at the 5ꢀposition, led to an increase in EGFR
inhibitory activity,
which agrees well with our design hypothesis. Importantly, 17d displayed strong
antiproliferation activities on H1975 cancer cells, while its effect on the A431 cancer cells was
significantly less potent. A further in vivo antitumor efficacy evaluation on 17d suggested that it
significantly inhibited the tumor growth in H1975 NSCLC xenograft mouse model by PO dosing
at 50 mg/kg/day for 14 days. The encouraging selectivity and in vivo antitumor effects of
compound 17d supported that it might be used as a promising lead compound for further
development as a selective EGFR inhibitor to overcome the EGFR resistance mutations.
EXPERIMENTAL SECTION
1. Molecular Modeling and Physiochemical Properties Calculation. Using the reported
bioactive conformation of compound 4 (with Michael addition receptor deleted) in the EGFR
T790M crystal structure (PDB 3IKA) as the query, SHAFTS was utilized to generate the
alignment poses of the novel designed inhibitors (17aꢀe). SHAFTS is a fast 3D similarity method
in which the 3D similarity calculation is driven by the hybrid information of molecular shape and
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chemotype features. The details of the algorithm and application are provided elsewhere and can
4
0
be accessed at the ChemMapper server.
The designed compounds were first docked into the EGFR proteins as a reversible inhibitor
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using Glide (version 5.5) , and then the covalent bond between the sulfur atom on the Cys797
thiol and the terminal carbon atom on the acrylamide group was formed along with any bond
order changes that occur upon formation of the covalent bond. Consequently, the covalent
complexes are minimized in vacuum to restore normal bond lengths and resolve steric clashes
using MacroModel module in Maestro with the OPLS_2005 force field as well as with flexible
ligand and rigid protein. Finally, the physicochemical properties of compounds 17aꢀj were
predicted using QikProp in Maestro software package.
2. Reagents and General Methods. All chemical reagents and solvents were purchased from
commercial sources (Energy Chemical, Titan chemical and Accela ChemBio), and used without
further purification. Compound 5 and afatinib were purchased from Yingrui Chemical
Technology and Adamasꢀbeta, respectively. Column chromatography was performed using
300−400 mesh silica gel purchased from Qingdao Haiyang Chemical Co.,Ltd. Thinꢀlayer
1
13
chromatography (TLC) was used to monitor progress of the reaction. H and C NMR spectra
were recorded on a Bruker AVꢀ400 spectrometer at 400 MHz and 100 MHz, respectively.
Chemical shifts (δ) are expressed in parts per million (ppm) units using TMS as the internal
standard. The 2D HMBC NMR spectra and highꢀresolution mass spectra (HRMS, Waters LCT
Premier XE TOF) were measured at the Institute of Fine Chemistry of ECUST. The lowꢀ
resolution mass spectra were measured with Agilent 6120 LCꢀMS. Melting points were
determined using a WRSꢀ1B digital melting point apparatus. The purity of final compounds was
determined by high performance liquid chromatography (HPLC) analysis, with the purity of all
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compounds being higher than 95% (>95%). HPLC instrument: HewlettꢀPackard 1100 HPLC
equipped with a photodiode array detector using a Zorbax RPꢀ18, 5 ꢁm, 4.6 mm×250 mm
column (reverse phase). The mobile phase A was acetonitrile and mobile phase B was 10 mM
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NH OAc in water (pH 6.0). A gradient of 10−100% A over 20 min was run at a flow rate of 1.0
4
mL/min.
tertꢀButyl (3ꢀAminophenyl)carbamate (11). To a mixture of 1,3ꢀphenylenediamine (10.800
g, 100 mmol), Et N (10.100 g, 100 mmol) in methanol (150 mL) was added (Boc) O (21.800 g,
3
2
o
1
00 mmol) at 0 C. The reaction solution was stirred for 24 h at room temperature. Then, the
solution was concentrated with a rotary evaporator. The crude product was purified by silica gel
chromatography (petroleum ether/ethylacetate = 4:1, v/v) to obtain the title product as white
1
powder (13.310 g, 64%). H NMR (400 MHz, CDCl ) δ 7.03 (t, J = 8.0 Hz, 1H), 6.96 (s, 1H),
3
6.55 (dd, J = 8.0 Hz, J = 1.2 Hz, 1H), 6.43 (s, 1H), 6.36 (dd, J = 8.0 Hz, J = 1.6 Hz, 1H), 3.54 (s,
+
2
H), 1.51 (s, 9H). LCꢀMS: m/z: 209.1 (M+H) .
tertꢀButyl (3ꢀ((2ꢀChloroꢀ5ꢀnitropyrimidinꢀ4ꢀyl)amino)phenyl)carbamate (12). To a
mixture of compound 11 (1.940 g, 10 mmol), Na CO (1.590 g, 15 mmol) in DMF (20 mL) was
2
3
added dropwise to a solution of tertꢀbutyl(3ꢀaminophenyl) carbamate (2.080 g, 10 mmol) in
o
DMF (20 mL) at ꢀ70 C. After stirring for 1 h, the reaction mixture was added to ice water (300
mL). The precipitate was filtered and dried in a vacuum oven to give the title product as a yellow
1
solid (2.918 g, 80%), which was used without further purification. H NMR (400 MHz, CDCl ):
3
δ 10.19 (s, 1H), 9.20 (s, 1H), 7.84 (s, 1H), 7.36 (d, J = 5.2 Hz, 2H), 7.21ꢀ7.18 (m, 1H), 6.63 (s,
+
1
H), 1.56 (s, 9H). LCꢀMS: m/z: 366.1 (M+H) .
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tertꢀButyl (3ꢀ((2ꢀ((2ꢀMethoxyꢀ4ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)phenyl)amino)ꢀ5ꢀnitropyrimidinꢀ
ꢀyl)amino)phenyl)carbamate (13). To a mixture of compound 12 (2.555 g, 7 mmol), DIPEA
4
(
1.806 g, 14 mmol) in THF (30 mL) was added 2ꢀmethoxyꢀ4ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)aniline
1.547 g, 7 mmol), and the reaction solution was stirred at room temperature overnight. The
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(
resulting precipitate was filtered and washed with dichloromethane to give the title product as a
1
yellow solid (2.655 g, 69%), which was used without further purification. H NMR (400 MHz,
DMSOꢀd ): δ 10.24 (s, 1H), 9.42 (s, 1H), 9.19 (s, 1H), 9.03 (s, 1H), 7.54 (s, 1H), 7.39 (d, J = 8.4
6
Hz, 1H), 7.22 (t, J = 8.8 Hz, 2H), 7.09 (t, J = 8.0 Hz, 1H), 6.62ꢀ6.60 (m, 1H), 6.34 (d, J = 7.6 Hz,
1H), 3.76 (s, 3H), 3.15 (t, J = 4.4 Hz, 4H), 2.47 (t, J = 4.4 Hz, 4H), 2.24 (s, 3H), 1.47 (s, 9H).
+
LCꢀMS: m/z: 551.4 (M+H) .
tertꢀButyl (3ꢀ((5ꢀAminoꢀ2ꢀ((2ꢀmethoxyꢀ4ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)phenyl)amino)pyrimid
inꢀ4ꢀyl)amino)phenyl)carbamate (14). A solution of compound 13 (2.600 g, 4.7 mmol) in
methanol (80 mL) was treated with 10% Pd/C (10 wt %) and hydrogenated for 10 h. The catalyst
was filtered off through Celite, and the filtrate was concentrated under reduced pressure. The
resulting crude product was recrystallized from ethanol to give the title compound 14 (2.110 g,
1
8
7
1
6%). H NMR (400 MHz, DMSOꢀd ): δ 9.32 (s, 1H), 8.17 (s, 1H), 7.99 (d, J = 8.8 Hz, 1H),
6
.96 (s, 1H), 7.59 (s, 1H), 7.32 (d, J = 8.0 Hz, 1H), 7.16 (t, J = 8.0 Hz, 1H), 7.06 (d, J = 8.0 Hz,
H), 6.98 (s, 1H), 6.60 (d, J = 2.4 Hz, 1H), 6.38 (dd, J = 8.8 Hz, J = 2.4 Hz, 1H), 4.39 (s, 2H),
3.82 (s, 3H), 3.07 (t, J = 4.4 Hz, 4H), 2.48 (t, J = 4.4 Hz, 4H), 2.25 (s, 3H), 1.48 (s, 9H). LCꢀMS:
+
m/z: 521.3 (M+H) .
tertꢀButyl (3ꢀ(2ꢀ((2ꢀMethoxyꢀ4ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)phenyl)amino)ꢀ6,7ꢀdioxoꢀ6,7ꢀdihy
dropteridinꢀ8(5H)ꢀyl)phenyl)carbamate (15). To a mixture of compound 14 (2.100 g, 4
mmol), Et N (0.808 g, 8 mmol) in ethanol (25 mL) was added diethyl oxalate (1.168 g, 8 mmol).
3
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The reaction mixture was stirred at reflux for 30 h. After being cooled to room temperature, the
resulting precipitate was filtered and washed with ethanol to give the title product as a yellow
1
solid (1.606 g, 70%). H NMR (400 MHz, DMSOꢀd ): δ 9.64 (s, 1H), 8.14 (s, 1H), 7.59 (s, 1H),
6
10
11
12
13
14
15
16
17
18
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20
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54
55
56
57
58
59
60
7
.57 (s, 1H), 7.55 (d, J = 8.0 Hz, 1H), 7.43 (t, J = 8.0 Hz, 1H), 7.26 (d, J = 8.8 Hz, 1H), 6.95 (d,
J = 7.6 Hz, 1H), 6.55 (d, J = 2.4 Hz, 1H), 6.06 (d, J = 8.0 Hz, 1H), 3.77 (s, 3H), 3.03 (t, J = 4.4
+
Hz, 4H), 2.44 (t, J = 4.4 Hz, 4H), 2.22 (s, 3H), 1.45 (s, 9H). HRMS(ESI) (m/z): (M+H) calcd
for C H N O 575.2730, found, 575.2725.
2
9
35
8
5
Nꢀ(3ꢀ(2ꢀ((2ꢀMethoxyꢀ4ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)phenyl)amino)ꢀ6,7ꢀdioxoꢀ6,7ꢀdihydropte
ridinꢀ8(5H)ꢀyl)phenyl)acrylamide (17a). To a solution of compound 15 (0.574 g, 1 mmol) in
CH Cl (5 mL) was added trifluoroacetic acid (1 mL). The mixture was stirred for 5 h at room
2
2
temperature. The reaction solution was neutralized with saturated aqueous NaHCO , and
3
extracted with CH Cl (3×15 mL). The organic layer was washed with saturated aqueous NaCl,
2
2
dried over anhydrous Na SO and concentrated in vacuo to obtain a yellow solid (0.355 g, 75%),
2
4
which was used in the next reaction without further purification.
To a solution of 8ꢀ(3ꢀaminophenyl)ꢀ2ꢀ((2ꢀmethoxyꢀ4ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl) phenyl)amino)ꢀ
5,8ꢀdihydropteridineꢀ6,7ꢀdione (0.340 g, 0.7 mmol) in NMP (1.5 mL) was added acryloyl
o
chloride (74 ꢁL, 0.9 mmol) dissolved in CH CN (1.0 mL) at 0 C. The reaction solution was
3
stirred overnight at room temperature. The mixture was poured into saturated aqueous NaHCO3,
and extracted with CH Cl (3×20 mL). The organic layer was washed with saturated aqueous
2
2
NaCl, dried over anhydrous Na SO and concentrated in vacuo. The residue was purified by
2
4
silica gel chromatography (dichloromethane/methanol = 15:1, v/v) to give the product 17a as a
o
1
yellow solid (0.200 g, 54%). mp >300 C. H NMR (400 MHz, DMSOꢀd ) δ 10.69 (s, 1H), 8.22
6
(s, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.76 (s, 1H), 7.64 (s, 1H), 7.52 (t, J = 8.0 Hz, 1H), 7.28 (d, J =
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.8 Hz, 1H), 7.08 (d, J = 8.0 Hz, 1H), 6.59ꢀ6.52 (m, 2H), 6.26 (dd, J = 16.8 Hz, J = 1.2 Hz, 1H),
.07 (d, J = 8.4 Hz, 1H), 5.77 (dd, J = 10.0 Hz, J = 1.2 Hz, 1H), 3.78 (s, 3H), 3.31ꢀ3.29 (m, 4H),
1
3
.20ꢀ3.18 (m, 4H), 2.74 (s, 3H). C NMR (100 MHz, DMSOꢀd ) δ 163.36, 156.77, 154.48,
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
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0
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8
9
0
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8
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53.10, 148.88, 146.75, 145.19, 143.85, 139.99, 135.80, 131.78, 129.41, 127.08, 123.52, 121.72,
119.47, 119.36, 113.10, 106.82, 100.31, 55.74, 52.37, 46.39, 42.33. HPLC purity: 97.63%,
+
retention time = 9.50 min. HRMS(ESI) (m/z): (M+H) calcd for C H N O 529.2312, found,
2
7
29
8
4
529.2312.
tertꢀButyl (3ꢀ(5ꢀEthylꢀ2ꢀ((2ꢀmethoxyꢀ4ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)phenyl)amino)ꢀ6,7ꢀdioxoꢀ
,7ꢀdihydropteridinꢀ8(5H)ꢀyl)phenyl)carbamate (16c). To a mixture of compound 15 (0.861
6
g, 1.5 mmol), Cs CO (0.587 g, 1.8 mmol) in DMF (8 mL) was added iodoethane (155 ꢁL, 1.9
2
3
mmol). The mixture was stirred at room temperature overnight. The solution was added ice water
and extracted with CH Cl (3×30 mL). The organic layer was washed with saturated aqueous
2
2
NaCl, dried over anhydrous Na SO and concentrated in vacuo. The residue was purified by
2
4
silica gel chromatography (dichloromethane/methanol = 15:1, v/v) to give the title product 16c as
1
a yellow solid (0.477 g, 53%). H NMR (500 MHz, DMSOꢀd ) δ 9.68 (s, 1H), 8.54 (s, 1H), 7.69
6
(s, 1H), 7.65 (s, 1H), 7.60 (d, J = 6.4 Hz, 1H), 7.49 (t, J = 6.4 Hz, 1H), 7.30 (d, J = 6.8 Hz, 1H),
7
.00 (d, J = 6.4 Hz, 1H), 6.61 (s, 1H), 6.12 (d, J = 6.0 Hz, 1H), 4.18 (q, J = 5.2 Hz, 2H), 3.82 (s,
3H), 3.19 (t, J = 4.4 Hz, 4H), 2.88 (t, J = 4.4 Hz, 4H), 2.54 (s, 3H), 1.49 (s, 9H), 1.30 (t, J = 5.6
+
Hz, 3H). HRMS(ESI) (m/z): (M+H) calcd for C H N O 603.3043, found, 603.3047. The
3
1
39
8
5
obtained 2D HMBC spectrum was provided in the Supporting Information.
Nꢀ(3ꢀ(2ꢀ((2ꢀMethoxyꢀ4ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)phenyl)amino)ꢀ5ꢀethylꢀ6,7ꢀdioxoꢀ6,7ꢀdihy
dropteridinꢀ8(5H)ꢀyl)phenyl)acrylamide (17c). To a solution of compound 16c (0.421 g, 0.7
mmol) in CH Cl (5 mL) was added trifluoroacetic acid (1 mL). The mixture was stirred for 5 h
2
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at room temperature. The reaction solution was neutralized with saturated aqueous NaHCO , and
3
extracted with CH Cl (3×15 mL). The organic layer was washed with saturated aqueous NaCl,
2
2
dried over anhydrous Na SO and concentrated in vacuo to obtain a yellow solid (0.299 g, 85%),
2
4
10
11
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13
14
15
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17
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which was used in the next reaction without further purification.
To a solution of 8ꢀ(3ꢀaminophenyl)ꢀ5ꢀethylꢀ2ꢀ((2ꢀmethoxyꢀ4ꢀ(4ꢀmethylpiperazin ꢀ1ꢀ
yl)phenyl)amino)ꢀ5,8ꢀdihydropteridineꢀ6,7ꢀdione (0.299 g, 0.6 mmol) in NMP (1.5 mL) was
o
added acryloyl chloride (65 ꢁL, 0.8 mmol) dissolved in CH CN (1.0 mL) at 0 C. The reaction
3
solution was stirred overnight at room temperature. The mixture was poured into saturated
aqueous NaHCO , and extracted with CH Cl (3×20 mL). The organic layer was washed with
3
2
2
saturated aqueous NaCl, dried over anhydrous Na SO and concentrated in vacuo. The residue
2
4
was purified by silica gel chromatography (dichloromethane/methanol = 15:1, v/v) to give the
o
1
product 17c as a yellow solid (0.178 g, 53%). mp >300 C. H NMR (400 MHz, DMSOꢀd ) δ
6
1
0.42 (s, 1H), 8.57 (s, 1H), 7.92 (s, 1H), 7.89 (s, 1H), 7.70 (s, 1H), 7.53 (t, J = 8.0 Hz, 1H), 7.32
(d, J = 8.8 Hz, 1H), 7.10 (d, J = 7.6 Hz, 1H), 6.53 (d, J = 2.4 Hz, 1H), 6.45 (dd, J = 17.2 Hz, J =
0.4 Hz, 1H ), 6.26 (dd, J = 16.8 Hz, J = 1.6 Hz, 1H), 6.07ꢀ6.05 (m, 1H), 5.77 (dd, J = 10.0 Hz, J
1.6 Hz, 1H), 4.42 (q, J = 6.8 Hz, 2H), 3.77 (s, 3H), 3.02 (t, J = 4.4 Hz, 4H), 2.45 (t, J = 4.4 Hz,
1
=
1
3
4
H), 2.23 (s, 3H), 1.40 (t, J = 7.2 Hz, 3H). C NMR (100 MHz, DMSOꢀd ) δ 163.26, 155.10,
6
152.45, 151.89, 149.55, 139.88, 135.39, 131.63, 129.52, 127.20, 123.59, 120.27, 119.41, 117.32,
106.24, 99.71, 63.02, 55.62, 54.53, 48.56, 45.64, 14.06. HPLC purity: 98.90%, retention time =
+
1
0.74 min. HRMS(ESI) (m/z): (M+H) calcd for C H N O 557.2625, found, 557.2615.
29
33
8
4
The following compounds 17b, 17dꢀj were prepared by a method similar to that for compound
7c.
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Nꢀ(3ꢀ(2ꢀ((2ꢀMethoxyꢀ4ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)phenyl)amino)ꢀ5ꢀmethylꢀ6,7ꢀdioxoꢀ6,7ꢀ
o
dihydropteridinꢀ8(5H)ꢀyl)phenyl)acrylamide (17b). Yellow solid (yield 43%). mp >300 C.
1
H NMR (400 MHz, DMSOꢀd ) δ 10.46 (s, 1H), 8.48 (s, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.69 (s,
6
0
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2
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1
H), 7.66 (s, 1H), 7.53 (t, J = 8.0 Hz, 1H), 7.22 (d, J = 8.8 Hz, 1H), 7.07 (d, J = 8.0 Hz, 1H),
6.53 (d, J = 1.6 Hz, 1H), 6.46 (dd, J = 16.8 Hz, J = 10.0 Hz, 1H ), 6.26 (dd, J = 17.2 Hz, J = 1.6
Hz, 1H ), 6.02 (d, J = 8.4 Hz, 1H), 5.77 (dd, J = 10.0 Hz, J = 1.2 Hz, 1H ), 3.77 (s, 3H), 3.55ꢀ
1
3
3
.53 (m, 4H), 3.06ꢀ3.02 (m, 4H), 2.57 (s, 3H), 2.32 (s, 3H). C NMR (100 MHz, DMSOꢀd ) δ
6
163.24, 155.48, 154.76, 153.13, 146.80, 143.85, 139.96, 135.82, 131.67, 129.56, 127.14, 123.50,
1
20.84, 119.36, 119.26, 114.92, 106.37, 99.84, 55.63, 54.34, 48.43, 45.31, 29.03. HPLC purity:
+
9
5.77%, retention time = 10.81 min. HRMS(ESI) (m/z): (M+H) calcd for C H N O
28 31
8
4
543.2468, found, 543.2470.
Nꢀ(3ꢀ(2ꢀ((2ꢀMethoxyꢀ4ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)phenyl)amino)ꢀ5ꢀisopropylꢀ6,7ꢀdioxoꢀ6,7ꢀ
o
1
dihydropteridinꢀ8(5H)ꢀyl)phenyl)acrylamide (17d). Yellow solid (yield 49%). mp >300 C. H
NMR (400 MHz, DMSOꢀd ) δ 10.63 (s, 1H), 8.57 (s, 1H), 7.96 (s, 1H), 7.90 (d, J = 8.4 Hz, 1H),
6
7
.75 (s, 1H), 7.53 (t, J = 8.0 Hz, 1H), 7.36 (d, J = 8.8 Hz, 1H), 7.11 (d, J = 7.6 Hz, 1H), 6.60 (d,
J = 2.0 Hz, 1H), 6.54 (dd, J = 16.8 Hz, J = 10.0 Hz, 1H), 6.27 (dd, J = 16.8 Hz, J = 1.6 Hz, 1H),
6
.12ꢀ6.10 (m, 1H), 5.77 (dd, J = 10.0 Hz, J = 1.6 Hz, 1H), 5.38ꢀ5.30 (m, 1H), 3.79 (s, 3H), 3.31ꢀ
13
3
.29 (m, 4H), 3.26ꢀ3.24 (m, 4H), 2.79 (s, 3H), 1.40 (d, J = 6.4 Hz, 6H). C NMR (100 MHz,
DMSOꢀd ) δ 163.35, 155.00, 152.01, 151.87, 149.40, 139.96, 135.40, 131.74, 129.45, 127.11,
6
1
23.56, 120.95, 119.45, 117.43, 106.61, 100.13, 69.98, 55.71, 52.99, 46.98, 43.30, 21.49. HPLC
+
purity: 97.32%, retention time = 11.84 min. HRMS(ESI) (m/z): (M+H) calcd for C H N O
3
0
35
8
4
571.2781, found, 571.2780.
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Nꢀ(3ꢀ(2ꢀ((2ꢀMethoxyꢀ4ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)phenyl)amino)ꢀ6,7ꢀdioxoꢀ5ꢀpropylꢀ6,7ꢀ
dihydropteridinꢀ8(5H)ꢀyl)phenyl)acrylamide (17e). Yellow solid (yield 47%). mp 298.1ꢀ299.6
o
1
C. H NMR (400 MHz, DMSOꢀd ) δ 10.70 (s, 1H), 8.58 (s, 1H), 7.97 (s, 1H), 7.91 (d, J = 8.0
6
10
11
12
13
14
15
16
17
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Hz, 1H), 7.77 (s, 1H), 7.53 (t, J = 8.0 Hz, 1H), 7.35 (d, J = 8.8 Hz, 1H), 7.11 (d, J = 8.0 Hz, 1H),
6.59ꢀ6.52 (m, 2H), 6.26 (dd, J = 16.8 Hz, J = 1.2 Hz, 1H), 6.11ꢀ6.09 (m, 1H), 5.77 (dd, J = 10.0
Hz, J = 1.6 Hz, 1H), 4.32 (t, J = 6.8 Hz, 2H), 3.78 (s, 3H), 3.31ꢀ3.29 (m, 4H), 3.16ꢀ3.14 (m, 4H),
2.72 (s, 3H), 1.85ꢀ1.77 (m, 2H), 1.01 (t, J = 7.2 Hz, 3H). HPLC purity: 97.20%, retention time =
+
12.17 min. HRMS(ESI) (m/z): (M+H) calcd for C H N O 571.2781, found, 571.2780.
30
35
8
4
Nꢀ(3ꢀ(2ꢀ((4ꢀ((2ꢀ(Dimethylamino)ethyl)(methyl)amino)ꢀ2ꢀmethoxyphenyl)amino)ꢀ5ꢀisopro
pylꢀ6,7ꢀdioxoꢀ6,7ꢀdihydropteridinꢀ8(5H)ꢀyl)phenyl)acrylamide (17f). Redꢀorange solid (yield
o
1
44%). mp >300 C. H NMR (400 MHz, CDCl ) δ 8.58 (s, 1H), 8.42 (s, 1H), 7.65ꢀ7.63 (m, 2H),
3
7.40 (t, J = 8.0 Hz, 1H), 6.94 (d, J = 7.6 Hz, 1H), 6.32 (dd, J = 16.8 Hz, J = 1.2 Hz, 1H), 6.22 (d,
J = 2.4 Hz, 1H), 6.11 (dd, J = 16.8 Hz, J = 10.0 Hz, 1H), 5.90ꢀ5.85 (m, 1H), 5.62 (dd, J = 10.0
Hz, J = 1.2 Hz, 1H), 5.51ꢀ5.45 (m, 1H), 3.79 (s, 3H), 3.53 (t, J = 7.2 Hz, 2H), 2.87 (s, 3H), 2.42
13
(
t, J = 7.6 Hz, 2H), 2.29 (s, 6H), 1.50 (d, J = 6.0 Hz, 6H). C NMR (100 MHz, CDCl ) δ 163.56,
3
1
1
56.66, 156.01, 153.36, 151.60, 149.14, 145.62, 140.35, 134.86, 131.69, 129.93, 127.03, 123.24,
20.37, 119.22, 118.61, 117.41, 104.11, 96.16, 72.22, 55.95, 55.53, 51.53, 49.93, 38.97, 21.69.
+
HPLC purity: 95.07%, retention time = 10.56 min. HRMS(ESI) (m/z): (M+H) calcd for
C H N O 573.2938, found, 573.2939.
30
37
8
4
Nꢀ(3ꢀ(2ꢀ((4ꢀ(2ꢀ(Dimethylamino)ethoxy)ꢀ2ꢀmethoxyphenyl)amino)ꢀ5ꢀisopropylꢀ6,7ꢀdioxoꢀ
,7ꢀdihydropteridinꢀ8(5H)ꢀyl)phenyl)acrylamide (17g). Yellow solid (yield 46%). mp 207.7ꢀ
6
2
8
o
1
09.1 C. H NMR (400 MHz, DMSOꢀd ) δ 10.64 (s, 1H), 8.57 (s, 1H), 8.01 (s, 1H), 7.87 (d, J =
6
.4 Hz, 1H), 7.78 (s, 1H), 7.53 (t, J = 8.0 Hz, 1H), 7.39 (d, J = 7.2 Hz, 1H), 7.11 (d, J = 8.0 Hz,
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H), 6.60 (d, J = 2.4 Hz, 1H), 6.54 (dd, J = 17.2 Hz, J = 10.4 Hz, 1H), 6.26 (dd, J = 17.2 Hz, J =
.0 Hz, 1H), 6.16ꢀ6.14 (m, 1H), 5.76 (dd, J = 10.0 Hz, J = 1.6 Hz, 1H), 5.37ꢀ5.31 (m, 1H), 4.21
2
(t, J = 4.8 Hz, 2H), 3.78 (s, 3H), 3.26 (t, J = 4.8 Hz, 2H), 2.69 (s, 6H), 1.40 (d, J = 6.0 Hz, 6H).
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8
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0
1
2
3
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0
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2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
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0
13
C NMR (100 MHz, DMSOꢀd ) δ 163.37, 155.02, 152.00, 151.92, 149.39, 140.01, 135.39,
6
131.78, 129.40, 127.02, 123.55, 121.97, 119.46, 117.53, 104.48, 99.21, 70.00, 63.39, 55.82,
+
43.37, 21.48. HPLC purity: 95.28%, retention time = 11.59 min. HRMS(ESI) (m/z): (M+H)
calcd for C H N O 560.2621, found, 560.2625.
2
9
34
7
5
Nꢀ(3ꢀ(5ꢀIsopropylꢀ2ꢀ((2ꢀmethoxyꢀ4ꢀ(4ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)piperidinꢀ1ꢀyl)phenyl)ami
no)ꢀ6,7ꢀdioxoꢀ6,7ꢀdihydropteridinꢀ8(5H)ꢀyl)phenyl)acrylamide (17h). Yellow solid (yield
o
1
49%). mp >300 C. H NMR (400 MHz, DMSOꢀd ) δ 10.58 (s, 1H), 8.56 (s, 1H), 7.94 (d, J = 8.0
6
Hz, 1H), 7.91 (s, 1H), 7.72 (s, 1H), 7.53 (t, J = 8.0 Hz, 1H), 7.32 (d, J = 8.8 Hz, 1H), 7.11 (d, J =
.4 Hz, 1H), 6.55ꢀ6.48 (m, 2H), 6.27 (dd, J = 17.2 Hz, J = 2.0 Hz, 1H), 6.08ꢀ6.06 (m, 1H), 5.78
dd, J = 10.0 Hz, J = 1.6 Hz, 1H), 5.36ꢀ5.30 (m, 1H), 3.77 (s, 3H), 3.62ꢀ3.60 (m, 2H), 3.01ꢀ2.92
8
(
(m, 6H), 2.69 (s, 3H), 2.56 (t, J = 11.6 Hz, 4H), 2.42ꢀ2.39 (m, 2H), 1.82ꢀ1.81 (m, 2H), 1.54ꢀ1.49
13
(m, 1H), 1.39 (d, J = 6.4 Hz, 6H). C NMR (100 MHz, DMSOꢀd ) δ 163.31, 155.01, 152.02,
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151.82, 149.40, 139.98, 135.40, 131.76, 129.46, 127.07, 123.55, 120.27, 119.39, 119.35, 117.35,
106.84, 100.27, 69.95, 55.63, 48.52, 21.49. HPLC purity: 99.03%, retention time = 12.34 min.
+
HRMS(ESI) (m/z): (M+H) calcd for C H N O 654.3516, found, 654.3512.
3
5
44
9
4
Nꢀ(3ꢀ(2ꢀ((3ꢀMethylꢀ4ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)phenyl)amino)ꢀ5ꢀisopropylꢀ6,7ꢀdioxoꢀ6,7ꢀ
o
1
dihydropteridinꢀ8(5H)ꢀyl)phenyl)acrylamide (17i). Yellow solid (yield 54%). mp >300 C. H
NMR (400 MHz, DMSOꢀd ) δ 10.54 (s, 1H), 9.61 (s, 1H), 8.61 (s, 1H), 7.91 (d, J = 8.0 Hz, 1H),
6
7
.76 (s, 1H), 7.55 (t, J = 8.0 Hz, 1H), 7.20 (s, 1H), 7.13 (d, J = 8.0 Hz, 1H), 6.69 (d, J = 8.8 Hz,
H), 6.50 (dd, J = 17.2 Hz, J = 10.4 Hz, 1H), 6.26 (dd, J = 17.2 Hz, J = 2.0 Hz, 1H), 5.76 (dd, J
1
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=
10.0 Hz, J = 1.6 Hz, 1H), 5.37ꢀ5.31 (m, 1H), 2.94ꢀ2.91 (m, 4H), 2.85ꢀ2.82 (m, 4H), 2.58 (s,
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3
3
1
1
H), 1.99 (s, 3H), 1.40 (d, J = 6.0 Hz, 6H). C NMR (100 MHz, DMSOꢀd ) δ 163.29, 156.39,
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55.06, 152.12, 151.81, 149.31, 144.40, 140.07, 135.68, 135.60, 131.91, 131.71, 129.56, 127.14,
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23.54, 119.51, 119.28, 118.60, 117.30, 116.66, 69.94, 53.87, 49.66, 43.61, 21.50, 17.42. HPLC
+
purity: 99.38%, retention time = 11.50 min. HRMS(ESI) (m/z): (M+H) calcd for C H N O
3
0
35
8
3
555.2832, found, 555.2833.
Nꢀ(3ꢀ(2ꢀ((3ꢀMethoxyꢀ4ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)phenyl)amino)ꢀ5ꢀisopropylꢀ6,7ꢀdioxoꢀ6,7ꢀ
o
1
dihydropteridinꢀ8(5H)ꢀyl)phenyl)acrylamide (17j). Yellow solid (yield 43%). mp >300 C. H
NMR (400 MHz, DMSOꢀd ) δ 10.59 (s, 1H), 9.57 (s, 1H), 8.62 (s, 1H), 7.90 (d, J = 8.4 Hz, 1H),
6
7
.75 (s, 1H), 7.54 (t, J = 8.0 Hz, 1H), 7.12 (d, J = 7.6 Hz, 1H), 7.04ꢀ6.99 (m, 2H), 6.55ꢀ6.49 (m,
2H), 6.27 (dd, J = 17.2 Hz, J = 2.0 Hz, 1H), 5.77 (dd, J = 10.0 Hz, J = 1.6 Hz, 1H), 5.37ꢀ5.31 (m,
1
3
1
H), 3.57 (s, 3H), 3.19ꢀ3.07 (m, 8H), 2.74 (s, 3H), 1.40 (d, J = 6.0 Hz, 6H). C NMR (100 MHz,
DMSOꢀd ) δ 163.35, 156.39, 155.00, 152.10, 151.85, 151.66, 149.33, 140.07, 135.99, 135.49,
6
133.96, 131.75, 129.50, 127.15, 123.59, 119.49, 119.43, 117.69, 117.43, 110.44, 69.97, 55.24,
5
2.85, 47.49, 42.47, 21.49. HPLC purity: 98.62%, retention time = 10.41 min. HRMS(ESI)
+
(
m/z): (M+H) calcd for C H N O 571.2781, found, 571.2782.
30 35
8
4
WT
3. In Vitro Enzymatic Activity Assay. Kinases domain of wildꢀtype EGFR (EGFR ), dualꢀ
L858R/T790M
mutant EGFR (EGFR
) were expressed using the BacꢀtoꢀBac™ baculovirusexpression
system (Invitrogen, Carlsbad, CA, USA) and purified in NiꢀNTA columns (QIAGEN Inc.,
Valencia, CA, USA). The kinase activity was evaluated with enzymeꢀlinked immunosorbent
assay (ELISA). The detailed characterization are provided in the Supporting Information.
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. Cell Proliferation Inhibition Assay. H1975 and A431 cells were obtained from the
American Type Culture Collection. Cell lines were cultured according to the suppliers'
instructions. Cells were seeded in 96ꢀwell plates (3,000 cells per well) and grown overnight. The
cells were treated with various concentrations of the compounds for 72 h, and the cells were then
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fixed with 10% preꢀcooled trichloroacetic acid (TCA) for 2 h at 4 C and stained for 15 min at
room temperature with 100 ꢁL of 4 mg/mL sulforhodamine B (SRB, Sigma) solution in 1%
acetic acid. After washing the plates for three times, the SRB solution was dissolved in 150 ꢁL of
10 mmol/L Tris base for 5 min and measured at 515 nm using a multiwell spectrophotometer
(
VERSAmax, Molecular Devices). The inhibition rate for cell proliferation was calculated as [1–
(
A515 treated/A515 control)]×100%. The IC value was obtained using the Logit method.
50
5. Kinase Selectivity Profile. The kinase selectivity profile was performed by using the
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DiscoveRx KINOMEscan platform (http://www.kinomescan.com/). The compound was
screened at a concentration of 1000 nM against a panel of 468 kinases. The results were defined
as a percentage of signal between the negative (DMSO, 100% control) and the positive (control
compound, 0% control) control, where the “% control” was calculated as follows: (%) = [(test
compound signal ꢀ positive control signal)/(negative control signal ꢀ positive control signal)
×
100].
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. Western Blot Analysis. Cells were collected and suspended in lysis buffer (100 mmol/L
TrisꢀHCl, pH6.8, 200 mmol/L DTT, 4% SDS, 0.2% bromophenol blue, 20% glycerol).
Equivalent amounts of proteins were loaded and separated by 8% SDSꢀPAGE followed by
transfer to nitrocellulose membranes. Western blot analysis was subsequently performed using
standard procedures. Antibodies used for immune detection of proteins were pꢀEGFR (Y1068;
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#
3777S), EGFR (#4267S), pꢀERK (T202/Y204; #4370L), ERK (#4695S), and Tubulin (#2128L;
Cell Signaling).
7. Mouse Liver Microsomal Stability Assay. This assay was performed by Shanghai
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Medicilon, Inc. Briefly, 1 ꢁM compound was incubated with mouse liver microsomes (0.5
o
mg/mL) and NADPH (1 mg/mL) at 37 C. Then samples were collected at time points 0 min, 10
min, 15 min, 30 min, 45 min, and 60 min and were terminated by the addition of cold methanol.
Samples were separated by centrifugation (15000 r/min for 5 min) and analyzed by using LCꢀ
MS/MS (Waters UPLC system; Applied Biosystems mass spectrometer).
8. In Vivo Pharmacokinetics Study in Mice. The pharmacokinetic parameters of the
compound in mice were conducted by Shanghai Medicilon, Inc. A 0.2 mg/mL dosing solution
was prepared by dissolving 0.6 mg of the compound in mixed solvent (0.15 mL DMSO+1.200
mL PEG400+1.650 mL Saline) for intravenous administration, and the 1 mg/mL dosing solution
was prepared by dissolving 6.0 mg of the compound in 0.5% CMCꢀNa aqueous solution for oral
administration. Male ICR mice were separately administered to a group of three mice per time
point for intravenous (1 mg/kg) or oral administration (10 mg/kg). At time points 0.083 h, 0.25 h,
0.5 h, 1 h, 2 h, 4 h, 8 h, and 24 h after dosing, a blood sample was collected from each mouse and
separated by centrifugation (8000 r/min for 6 min). Then the samples were analyzed by
LC−MS/MS (SIMADZU LC system; Applied Biosystems mass spectrometer), and the acquired
data were analyzed by using the WinNonlin (v5.2).
9. In Vivo Efficacy for Mouse Tumor Xenografts. 4ꢀ6 weeksꢀold BALB/c mice were
purchased from the Shanghai Laboratory Animal Research Center (Shanghai, China). All the
procedures related to animal handling, care and treatment in this article were performed in
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