Design, Synthesis, and Biological Evaluation of Pyrimido[4,5- d]pyrimidine-2,4(1 H,3 H)-diones as Potent and Selective Epidermal Growth Factor Receptor (EGFR) Inhibitors against L858R/T790M Resistance Mutation

Article abstract of DOI:10.1021/acs.jmedchem.8b00346

First-generation epidermal growth factor receptor (EGFR) inhibitors, gefitinib and erlotinib, have achieved initially marked clinical efficacy for nonsmall cell lung cancer (NSCLC) patients with EGFR activating mutations. However, their clinical benefit was limited by the emergence of acquired resistance mutations. In most cases (approximately 60%), the resistance was caused by the secondary EGFR T790M gatekeeper mutation. Thus, it is still desirable to develop novel third-generation EGFR inhibitors to overcome T790M mutation while sparing wild-type (WT) EGFR. Herein, a series of pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione derivatives were designed and synthesized, among which the most potent compound 20g not only demonstrated significant inhibitory activity and selectivity for EGFR^L858R/T790M and H1975 cells in vitro but also displayed outstanding antitumor efficiency in H1975 xenograft mouse model. The encouraging mutant-selective results at both in vitro and in vivo levels suggested that 20g might be used as a promising lead compound for further structural optimization as potent and selective EGFR^L858R/T790M inhibitors.

Full text of DOI:10.1021/acs.jmedchem.8b00346

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Article
Design, Synthesis, and Biological Evaluation of
Pyrimido[4,5-d]pyrimidine-2,4(1H,3H)-diones as Potent
and Selective Epidermal Growth Factor Receptor (EGFR)
Inhibitors against L858R/T790M Resistance Mutation
Yongjia Hao, Jiankun Lyu, Rong Qu, Yi Tong, Deheng Sun, Fang Feng, Linjiang Tong,
Tingyuan Yang, Zhenjiang Zhao, Lili Zhu, Jian Ding, Yufang Xu, Hua Xie, and Honglin Li
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00346 • Publication Date (Web): 15 Jun 2018
Downloaded from http://pubs.acs.org on June 16, 2018
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Design, Synthesis, and Biological Evaluation of Pyrimido[4,5ꢀd]pyrimidineꢀ
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,4(1H,3H)ꢀdiones as Potent and Selective Epidermal Growth Factor Receptor
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(EGFR) Inhibitors against L858R/T790M Resistance Mutation
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,2,‡
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3,‡
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Yongjia Hao , Jiankun Lyu , Rong Qu , Yi Tong , Deheng Sun , Fang Feng , Linjiang
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Tong , Tingyuan Yang , Zhenjiang Zhao , Lili Zhu , Jian Ding , Yufang Xu *, Hua Xie *,
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Honglin Li *
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Shanghai Key Laboratory of New Drug Design, State Key Laboratory of Bioreactor
Engineering, School of Pharmacy, East China University of Science and Technology, Shanghai
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00237, China.
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School of Pharmacy, Guizhou University of Chinese Medicine, Guiyang 550025, China.
Division of Antiꢀtumor Pharmacology, State Key Laboratory of Drug Research, Shanghai
Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
‡
Authors contributed equally to this work
*
To whom correspondence should be addressed. Email: yfxu@ecust.edu.cn, hxie@jding.dhs.org,
hlli@ecust.edu.cn
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ABSTRACT
Firstꢀgeneration epidermal growth factor receptor (EGFR) inhibitors, gefitinib and erlotinib have
achieved initially marked clinical efficacy for nonꢀsmall cell lung cancer (NSCLC) patients with
EGFR activating mutations. However, their clinical benefit was limited by the emergence of
acquired resistance mutations. In most cases (approximately 60%), the resistance was caused by
the secondary EGFR T790M gatekeeper mutation. Thus, it is still desirable to develop novel
thirdꢀgeneration EGFR inhibitors to overcome T790M mutation while sparing wildꢀtype (WT)
EGFR. Herein, a series of pyrimido[4,5ꢀd]pyrimidineꢀ2,4(1H,3H)ꢀdione derivatives were
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designed and synthesized. Among which, the most potent compound 20g not only demonstrated
L858R/T790M
significant inhibitory activity and selectivity for EGFR
and H1975 cells in vitro, but
also displayed outstanding antitumor efficiency in H1975 xenograft mouse model. The
encouraging mutantꢀselective results at both in vitro and in vivo levels suggested that 20g might
be used as a promising lead compound for further structural optimization as potent and selective
L858R/T790M
EGFR
inhibitors.
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INTRODUCTION
Lung cancer, especially nonꢀsmall cell lung cancer (NSCLC) is one of the most prevalent
malignant tumors, and also a major health problem worldwide, whose fiveꢀyear survival rate is
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less than 15% after diagnosis. It is wellꢀknown that the epidermal growth factor receptor
(EGFR, also known as ErbB1, HER1)ꢀmediated signaling pathways are essential for various
biological processes and cancers development and progression, such as cell proliferation,
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ꢀ4
adhesion, migration, differentiation and survival. Thus, the EGFR tyrosine kinase has been
proven to be an attractive and clinically validated drug target in cancer therapy, in particular for
NSCLC treatment. Firstꢀgeneration reversible and quinazolineꢀbased EGFR tyrosine kinase
inhibitors (TKIs), including gefitinib (1) and erlotinib (2), have received approval from US Food
and Drug Administration (FDA) for the treatment of NSCLC in 2003 and 2004, respectively
5,6
(
Figure 1). Despite the clinical efficacy of the two drugs have been well established for
NSCLC patients with the most common EGFR activating mutations, such as L858R (activating
7
ꢀ9
mutation in exon 21) and del E746−A750 (deletions in exon 19). Unfortunately, a secondary
T790M point mutation (the substitution threonine790 with methionine residue) at the gatekeeper
position of EGFR has caused approximately 60% NSCLC patients who initially responded to the
treatment of firstꢀgeneration EGFR inhibitors to develop acquired resistance after a median of
10
1
0–14 months. Occurrence of the T790M acquired resistance mutation not only seriously
affected the improvement of survival rates, but also impaired the quality of life of NSCLC
patients.
In order to prevent the acquired resistance caused by the T790M mutation, several secondꢀ
1
1ꢀ14
generation irreversible EGFR inhibitors have been developed.
Afatinib (3) is a typical
example, which has been approved by FDA as a new firstꢀline treatment for late stage
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metastatic) NSCLC patients with EGFR exon 19 deletions or exon 21 L858R substitution
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mutations in 2013 (Figure 1). Although afatinib was highly active in vivo in xenograft models
L858R/T790M
driven by EGFR
, it didn’t increase objective response rate in clinical trial of NSCLC
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patients who have been developed resistance to gefitinib or erlotinib on account of the narrow
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therapeutic window towards wildꢀtype (WT) EGFR. Therefore, there is still an unmet clinical
need to develop thirdꢀgeneration EGFR TKIs, namely, mutantꢀselective EGFRꢀTKIs. These
inhibitors have potent selectivity for EGFR T790M while sparing wildꢀtype EGFR, so can
improve the efficacy and reduce the toxicity associated with the inhibition of WT EGFR protein.
1
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WZ4002 (4) with an anilinepyrimidine core was the first such type of mutantꢀselective EGFRꢀ
TKIs, nevertheless, which did not progress into clinical development. Simultaneously, another
two mutantꢀselective small molecule EGFR inhibitors have been publicly reported, that are COꢀ
18,19
20,21
1
686 (5, Rociletinib)
and AZD9291 (6, Osimertinib)
. These two agents demonstrated
good potency and prominent selectivity for EGFR L858R/T790M over WT EGFR (Figure 1).
Furthermore, according to its significant clinical benefits, FDA has accelerated approved
T790M
compound 6 to treat advanced NSCLC patients with EGFR
acquired resistance mutation,
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who have progressed on or after prior treatment with other EGFRꢀtargeted therapy.
Figure 1
Recently, we reported a study of developing a series of mutantꢀselective EGFR inhibitors with
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a C4ꢀalkylꢀ1,4ꢀdihydroꢀ2Hꢀpyrimido[4,5ꢀd][1,3]oxazinꢀ2ꢀones scaffold. By a 3Dꢀcomformer
based scaffold hopping strategy, we designed a new scaffold of pyrimido[4,5ꢀd]pyrimidineꢀ
L858R/T790M
2
,4(1H,3H)ꢀdione as a starting point to develop a series of novel EGFR
selective
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inhibitors. At the end of a 3ꢀround structural optimization, our candidate compound 20g displays
a favorable selectivity against doubleꢀmutant EGFR at both enzymatic and cellular levels.
Compound 20g also shows a good selectivity profile in in vivo study. Our study provides a new
scaffold of mutantꢀselective EGFR inhibitors by an efficient in-silico scheme.
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Figure 2
CHEMISTRY
As shown in Scheme 1, compounds 14aꢀe were synthesized starting from 1,3ꢀbenzendiamine,
followed by treatment with diꢀtertꢀbutyl dicarbonate to give Bocꢀprotected amine (8). Then,
compound (8) was reacted with ethyl 2,4ꢀdichloropyrimidineꢀ5ꢀcarboxylate to yield ethyl 4ꢀ((3ꢀ
(
(tertꢀbutoxycarbonyl)amino)phenyl)amino)ꢀ2ꢀchloropyrimidineꢀ5ꢀcarboxylate (9) in a good
yield. Hydrolysis of the carboxylic ethyl ester group by 1 M NaOH to afford carboxylic acid
10), and the key intermediates (11) were generated with arylamines by replacement of the
(
chloride in the pyrimidine. The amide derivatives (12) were prepared from condensation
carboxylic acid derivatives (11) with methylamine hydrochloride. Direct cyclization with 1,1'ꢀ
carbonyldiimidazole to obtain the ringꢀclosure intermediates (13). Finally, 13 were deprotected
with trifluoroacetic acid in dichloromethane and reacted with acryloyl chloride to produce the
target compounds 14a−e.
Scheme 1
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To compare the influence of activity, we synthesized a series of final compounds with
different substituted alkyl groups at 3ꢀposition of pyrimido[4,5ꢀd]pyrimidineꢀ2,4(1H,3H)ꢀdione
moiety (Scheme 2). Commercially available 2,4ꢀdichloroꢀ5ꢀpyrimidinecarbonyl chloride was
reacted with ammonium hydroxide to give 2,4ꢀdichloropyrimidineꢀ5ꢀcarboxamide (15).
Intermediates (17) were generated with Bocꢀprotected amine and appropriate arylamines by
replacement of the chloride at 4, 2ꢀposition of pyrimidine, respectively. Then, the ringꢀclosure
compounds (18) were obtained based on the same procedure as described in Scheme 1, which
further were alkylated with appropriate alkyl halides to afford the N3ꢀalkylated compounds
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(
19). At last, the final compounds 20a−f were synthesized using the same synthetic methods as
described above. And compounds 20gꢀl were prepared by a method similar to that for compound
0a.
2
Scheme 2
RESULTS AND DISCUSSION
Scaffold Hopping and Binding Mode Analysis. Through a structureꢀbased design strategy
targeting a hydrophobic subꢀpocket induced by a gatekeeper mutant T790M, compound 7
(Figure 2A) was previously identified as a nanomolar irreversible EGFR inhibitor with a doubleꢀ
L858R/T790M
WT
mutant selective profile (IC against EGFR
kinase over EGFR kinase: 4.7 nM vs
50
2
5
474 nM, 101ꢀfold selectivity). After superimposing the docked pose of compound 7 with
another doubleꢀmutantꢀselective EGFR inhibitor 5 (Figure 2B), the binding mode of both
doubleꢀmutant inhibitors share some similar characteristics: 1) The aminopyrimidine moiety of
both inhibitors binds to Met793 residue at the hinge region through two hydrogen bonding
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interactions. 2) The hydrophilic tail on the leftꢀhand side faces toward solventꢀexposure area. 3)
The warhead, metaꢀacrylamide group, forms a covalent bond with Cys797. Besides, there are
two subꢀpockets induced by gatekeeper mutant T790M (Figure 2C). Based on the overlaid
docked poses of compounds 5 and 7, subꢀpocket 1 (S1) is occupied by the fluorine group in the
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C5ꢀCF substituent of compound 5 or the methyl group at N5ꢀposition of compound 7,
3
respectively. In order to further improve the selectivity profile, by occupying both mutantꢀ
induced subꢀpockets (S1 and S2), compound 14a was designed with a novel scaffold:
pyrimido[4,5ꢀd]pyrimidineꢀ2,4(1H,3H)ꢀdione. According to the docked pose of compound 14a
(Figure 2D), the carbonyl group at C4ꢀposition occupies the S1 pocket as well, like compounds 5
3
and 7. And hydrophobic groups with different lengths and volumes at the R position could be
introduced to explore the S2 pocket and to strengthen the nonpolar contacts with the gatekeeper
mutant Met790.
Structureꢀactivity Relationship. 14a inhibited the enzymatic activity against
L858R/T790M
WT
EGFR
and EGFR with IC values of 46 and 180 nM, respectively, and showed
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WT
nearly 4ꢀfold selectivity for EGFR
in comparison with EGFR (Table 1). Its in vitro
antiproliferation activity were tested by using the sulforhodamine B (SRB) colorimetric assay on
H1975 cell lines (expressing EGFRꢀL858R/T790M) and A431 cell lines (harboring overꢀ
WT
expressed EGFR ). However, it exhibited weak antiproliferative effects on both the H1975 and
A431 cell lines (> 10 ꢁM). Thus, we decided to improve the physical properties of 14a by
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exploring the R position. With a N,Nꢀdimethylpiperidinꢀ4ꢀamine substituent at the R position
(
compound 14d), the predicted cell permeability score was improved from 64.1 (14a) to 80.3
14d) (the higher value, the better cell permeability, predicted by Qikprop in Maestro software
(
2
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package ). As we expected, the enzymatic activity of 14d against EGFR kinases and the
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L858R/T790M
WT
selectivity between EGFR
and EGFR remain the same level as 14a (Table 1).
Besides, compound 14d displayed potent inhibitory activity against H1975 cell line (230 nM),
and showed selectivity against the H1975 over the A431 cell lines (approximately 17ꢀfold). As a
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contrast, we also synthesized compound 14e with no modification on R and R . As expected,
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4e lost the selectivity between EGFR
and EGFR , and showed weak
antiproliferative effects on both the H1975 and A431 cell lines (> 9 ꢁM). The results once again
verify the importance of the hydrophilic tail on the leftꢀhand side. Since compound 14d showed
better inhibitory activity at both enzymatic and cellular levels, and acceptable aqueous solubility
(
531 ꢁg/mL), it was selected as a new starting point for the nextꢀround structure modifications.
Table 1
Based on the strategy we described in Figure 2D, probing the S2 pocket is one of the most
important structureꢀbased optimizations to improve the selectivity against double mutants over
wild type. Considering the feasibility of synthesis, hydrophobic groups with varied length and
3
volume were introduced at R position (Table 2). Thus, compounds 20aꢀf were synthesized
3
(
Scheme 2). When varying the substituted group at R position from methyl, ethyl, propyl to
isopropyl (compounds 14d, 20aꢀc), the enzymatic inhibition against doubleꢀmutant EGFR also
boosted correspondingly with the increase of group length and volume. For compounds 20b and
2
0c, their values of IC against doubleꢀmutant EGFR reach the subnanomolar range (0.4 and 0.8
50
nM, respectively, Table 2). Based on the docking simulation (Figure 2E), the hydrophobic
3
groups at R position is able to occupy the T790M mutant induced S2 pocket as well as
strengthen van der Waals (VDWs) contacts with gatekeeper Met790. Compared with the positive
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control compound 5, all three compounds (20aꢀc) showed a doubleꢀmutant preferred selectivity
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(
14ꢀfold vs. 44, 33 and 25ꢀfold), but the longer and bulkier substituents at R position
(compounds 20dꢀf) keep potent inhibitory activity against doubleꢀmutant EGFR, these structural
modifications also increase the inhibition against wildꢀtype EGFR, resulting in complete loss of
doubleꢀmutant selective profile (0.6, 0.7, 2.7ꢀfold, respectively). There are many structures that
show the flexibility of S2 pocket to accommodate a wide range of hydrophobic moieties in both
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EGFR doubleꢀmutant and wildꢀtype structures.
According to our docking simulations,
compound 20f is able to be docked into both doubleꢀmutant and wildꢀtype EGFR structures with
a wide open S2 pocket (Figure 2F and Figure S1). Based on the docked pose of 20f with doubleꢀ
mutant EGFR, we believe that the energy gain from occupying at S2 pocket with bulkier group
compensates the steric strain (1.9 Å: the distance in Figure 2F shows unfavorable VDW
interactions) between the carbonyl group at C4ꢀposition and wild type gatekeeper Thr790. Thus,
3
compounds with longer and bulkier substituents at R position regain inhibitory activity against
wildꢀtype EGFR. At the secondꢀround optimization, compounds 20aꢀc also showed a better
selectivity against H1975 cell lines over A431 cell lines than that of compounds 20dꢀf (12, 11
and 4ꢀfold, respectively). This trend is in consistent with that at the enzymatic level.
At the third round, we moved forward with the most selective one at cellular level (20c) for
further structural optimizations. Previously, some literatures have also reported to modify the
29ꢀ31
leftꢀhand side chain of the compounds to discovery active candidates.
Inspired by that, we
synthesized a set of compounds (compounds 20gꢀl) that possess different leftꢀhand side chains
on the basis of 20c (Table 2). The data revealed that compounds 20h, 20jꢀl displayed varying
degrees selectivity losses in EGFR enzymatic inhibitory activity compared with 20c.
Encouragingly, compounds 20g and 20i demonstrated more potent selectivity for doubleꢀmutant
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EGFR enzyme (263ꢀfold and 34ꢀfold, respectively) and increase of aqueous solubility (262
ꢁ
g/mL and 280 ꢁg/mL, respectively) than 20c. Especially 20g, which showed 263ꢀfold
L858R/T790M
WT
selectivity between the EGFR
and the EGFR , and showed more potent inhibitory
0
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activity and selectivity than the positive control compound 5. In consistent with their enzyme
inhibitory activity, these compounds also showed different results in antiproliferation assays.
Compounds 20gꢀi were roughly equipotent to 20c for the H1975 cell lines, but resulted in
varying degrees drops of selectivity for A431 cells, especially 20h and 20i. And 20jꢀl exhibited
diminished antiproliferation activity for H1975 cells and increased antiproliferation efficiency
for A431 cells as compared to 20c (Table 2).
Table 2
Inhibitory Effects of EGFR and Downstream Signaling Transduction. The selective
inhibition of the representative compound 20g for the phosphorylation of EGFR and the
downstream signaling transduction was further confirmed by using the Western blot analysis
L858R/T790M
(Figure 3). In H1975 cells, 20g inhibited the phosphorylation of EGFR
at the
concentration as low as 1 nM, and potently inhibited EGFR phosphorylation and downstream
molecules AKT phosphorylation at 100 nM, which even showed more potent inhibitory activity
relative to compound 6 (Figure 3A). In A431cells, 20g moderately inhibited the phosphorylation
WT
of EGFR even at 100 nM (Figure 3B). The results clearly showed that compound 20g more
potently inhibited the phosphorylation of EGFR and downstream signaling transduction (pꢀAKT)
L858R/T790M
in cellular level, further suggesting 20g was a potent and selective EGFR
inhibitor.
Figure 3
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Target Duration of Action of Compound 20g in Vitro. In order to get an insight into the
duration of target inhibition in vitro, we performed washout experiment. Compound 20g was
washed out after incubation for 2 h in H1975 cells. Then phosphorylation of target at different
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time points was detected by Western blot. As shown in Figure 4A, inhibition of target
L858R/T790M
EGFR
activation can last for 24 h at least. And inhibition of the signaling protein ERK
activation can also last for more than 12 h. Furthermore, the target duration was similar to that of
control compound 6 (Figure 4B).
Figure 4
DMPK Properties of Compound 20g. In the permeability properties assay, compound 20g
−
6
showed modest permeability in the MDCK II cells (Papp, A→B of 1.5 × 10 cm/s, and P ratio of
app
9
.1 at 2 ꢁM). We then examined the in vitro metabolic stability of compound 20g with human
and mouse liver microsomes. At the assay concentration of 0.1 ꢁM, the results demonstrated that
0g exhibited acceptable microsomal stability with halfꢀlife (T ) of 85.4 min (human liver
2
1
/2
microsomes) and 46.1 min (mouse liver microsomes), and MF% of 44.3 and 49.4, respectively
Table S1). In addition, 20g showed weak cytochrome P450 (CYP450) inhibition against five
major human CYP450 enzymes (1A2, 2C9, 2C19, and 2D6: IC > 50 ꢁM; 3A4: IC = 23.9
(
5
0
50
ꢁ
M). We also examined the preliminary in vivo pharmacokinetic properties of compound 20g in
mice following intravenous (IV) and oral administration. As shown in Table 3, 20g demonstrated
a reasonable area under the curve (AUC ) value of 658 ng·h/mL, moderate halfꢀlife (T =1.1 h)
0
ꢀ∞
1/2
and oral bioavailability (F = 35%) during the oral dose of 10 mg/kg. The results indicate that it is
meaningful to further evaluate the preliminary in vivo antitumor efficacy studies of compound
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0g.
Table 3
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In Vivo Antitumor Efficiency Study. On the basis of its outstanding in vitro inhibitory
activity, we selected compound 20g to further evaluate its preliminary in vivo antitumor efficacy
in H1975 and A431 xenograft mouse models (Figure 5). In this study, 4ꢀ6 weeksꢀold mice (n =
6
/group) in treatment group were dosed orally once daily with 20g at 50 mg/kg for 14 days. As
shown in Figure 5A, compared with the vehicle group, 20g significantly inhibited the tumor
growth (TGI = 73%, p<0.001) during the 14 day period in H1975 xenograft mouse mode.
However, 20g exhibited much less potent tumor growth inhibition (TGI = 37%, p<0.05) in A431
xenograft mouse model (Figure 5B). Besides, during the experiment, no significant body weight
loss, mortality and obvious side effects were observed (Figure 5C and 5D), indicating that
compound 20g was wellꢀtolerated at this dose.
Figure 5
CONCLUSIONS
Through a 3D conformer based scaffold hopping protocol based on previously reported mutantꢀ
selective EGFR inhibitors, we designed and synthesized a series of pyrimido[4,5ꢀd]pyrimidineꢀ
L858R/T790M
2
,4(1H,3H)ꢀdione derivatives as novel potent and selective EGFR
inhibitors. For three
compounds (20b, 20c and 20g) in this series, our design strategy to occupy S2 pocket boosts the
inhibitions against doubleꢀmutant EGFR and brings the IC to a subnanomolar level. After three
5
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round SAR explorations, one of the most promising compounds 20g shows a favorable
selectivity at both in vitro and in vivo levels, indicating that compound 20g might be used as a
L858R/T790M
promising drug candidate to overcome EGFR
drugꢀresistance mutation. Further
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evaluation for the candidate compound is still ongoing, and will be reported in due course.
EXPERIMENTAL SECTION
Molecular Modeling. The Xꢀray structures (PDB code: 3IKA and 4G5J) were downloaded
from the Protein Data Bank (PDB, http://www.pdb.org). The protocol of comparative docking
2
3
study was reported in our previous publications : First, compounds 5 and 7 was docked into the
T790M
23
EGFR
(PDB code: 3IKA) as reversible inhibitors using Glide (version 5.5) in extra
precision (XP) mode with default settings; Then, the covalent bond between the Cys797 and the
2
6
electrophilic group was formed by the build panel in Maestro ; Finally, to fix the bond lengths
and eliminate steric clashed, the inhibitorꢀkinase covalent complex was minimized in water
solvent using MacroModel application with a flexible ligand and a restrained protein.
3
2
The scaffold hopping was conducted by an inꢀhouse software: SHAFTS . The query molecule
is the docked pose of compound 7 obtained from the above docking study. The aligned pose of
compound 20c (similarity score is 1.2875) was generated by a fast 3Dꢀsimilarity calculation in
3
3
SHAFTS . Then, formed the covalent bond and minimized complexes with MacroModel
application with the same settings as described above.
For getting docked poses of compound 20f with wildꢀtype and doubleꢀmutant EGFRs. Two
structures (PDB code: 2RGP and 3W2S) were prepared in Maestro and aligned to the structure
with PDB code 3IKA. Compound 20f (similarity score is 1.4038) was aligned to the docked pose
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of compound 7 by SHAFTS. Then manually formed the covalent bond and minimized in
MacroModel.
Reagents and General Methods. All chemical reagents and solvents used in experiments
0
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were purchased from commercial suppliers and used without further purification. Silica gel
(300−400 mesh) used for column chromatography and thinꢀlayer chromatography (TLC) used to
1
monitor progress of the reaction were purchased from Qingdao Haiyang Chemical Co.,Ltd. H
1
3
and C NMR spectra were performed on the Bruker spectrometer (AVꢀ400) at 400 MHz and
1
6
00 MHz, respectively. The lowꢀresolution mass spectra (LCꢀMS) were measured using Agilent
120 LCꢀMS. The highꢀresolution mass spectra (HRMS, Waters LCT Premier XE TOF) were
measured at the Institute of Fine Chemistry of ECUST. Melting points were determined with the
digital melting point apparatus (WRSꢀ1B). The purity of final compounds were analyzed by
HPLC (HewlettꢀPackard 1100), with the purity of all compounds being over than 95%. The
HPLC instrument was equipped with a photodiode array detector using a Zorbax RPꢀ18 column
(5 ꢁm, 4.6 mm×250 mm, reverse phase column). The mobile phases A and B were acetonitrile
and the solution of 10 mM ammonium acetate in purified water (pH = 6.0), respectively.
tertꢀButyl(3ꢀAminophenyl)carbamate (8). To a mixture of 1,3ꢀphenylenediamine (10.800 g,
1
00 mmol), Et N (10.100 g, 100 mmol) in methanol (150 mL) was added Boc O (21.800 g, 100
3 2
o
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 compound 2 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
2
.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,
+
H), 1.51 (s, 9H). LCꢀMS: m/z: 209.1 (M+H) .
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9
Ethyl4ꢀ((3ꢀ((tertꢀbutoxycarbonyl)amino)phenyl)amino)ꢀ2ꢀchloropyrimidineꢀ5ꢀ
carboxylate (9). A mixture of ethylꢀ2,4ꢀdichloropyrimidineꢀ5ꢀcarboxylate (2.210 g, 10 mmol),
compound 8 (2.080 g, 10 mmol) and DIPEA (1.935 g, 15 mmol) in CH CN (40 mL) was heated
3
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0
with reflux for 2 h. After cooling to room temperature, the white solid was collected by filtration
1
and washed with CH CN to give the product 9 (3.371 g, 86%). H NMR (400 MHz, DMSOꢀd ) δ
3
6
1
0.23 (s, 1H), 9.50 (s, 1H), 8.80 (s, 1H), 7.70 (s, 1H), 7.35 (d, J = 7.6 Hz, 1H), 7.29 (t, J = 8.0
Hz, 1H), 7.24 (d, J = 8.4 Hz, 1H), 4.39 (q, J = 7.2 Hz, 2H), 1.49 (s, 9H), 1.36 (t, J = 7.2 Hz, 3H).
+
LCꢀMS: m/z: 393.1 (M+H) .
4
ꢀ((3ꢀ((tertꢀButoxycarbonyl)Amino)phenyl)amino)ꢀ2ꢀchloropyrimidineꢀ5ꢀcarboxylic acid
(
10). A mixture of compound 9 (3.136 g, 8 mmol) in THF and 1 M NaOH (8 mL) was heated at
o
5
0 C for 3 h. After cooling to room temperature, the solution was acidified with 1 M HCl to
give a white solid, which was collected by filtration and washed with THF and water to give the
1
title compound 10 (2.796 g, 96%). H NMR (400 MHz, DMSOꢀd ) δ 10.84 (s, 1H), 9.49 (s, 1H),
6
8.76 (s, 1H), 7.67 (s, 1H), 7.40 (d, J = 8.0 Hz, 1H), 7.28 (t, J = 8.0 Hz, 1H), 7.22 (d, J = 8.4 Hz,
+
1
H), 1.49 (s, 9H). LCꢀMS: m/z: 365.1 (M+H) .
4
ꢀ((3ꢀ((tertꢀButoxycarbonyl)Amino)phenyl)amino)ꢀ2ꢀ(phenylamino)pyrimidineꢀ5ꢀ
carboxylic acid (11). To a solution of compound 10 (2.184 g, 6 mmol) in CH CN (40 mL) was
3
added aniline (0.558 g, 6 mmol), the mixture was heated with reflux for 12 h. After cooling to
room temperature, the residue was collected by filtration and washed with CH CN to give the
3
1
product 11 as a white solid (2.249 g, 89%). H NMR (400 MHz, DMSOꢀd ) δ 10.44 (s, 1H), 9.82
6
(s, 1H), 9.36 (s, 1H), 8.70 (s, 1H), 7.69 (d, J = 8.0 Hz, 2H), 7.63 (s, 1H), 7.43 (s, 1H), 7.26ꢀ7.21
+
(
m, 4H), 6.98 (t, J = 6.8 Hz, 1H), 1.47 (s, 9H). HRMS(ESI) (m/z): (M+H) calcd for C H N O
2
2
24
5
4
4
22.1828, found, 422.1823.
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tertꢀButyl(3ꢀ((5ꢀ(Methylcarbamoyl)ꢀ2ꢀ(phenylamino)pyrimidinꢀ4ꢀyl)amino)phenyl)carba
mate (12). To a solution of 11 (1.263 g, 3 mmol), DIPEA (0.774 g, 6 mmol) in dry DMF (15
mL) was added HATU (1.368 g, 3.6 mmol). After stirring for 1 h at room temperature,
methylamine hydrochloride (0.306 g, 4.5 mmol) was added and stirred overnight. The reaction
mixture was poured into ice water, the resulting solid was collected by filtration, washed with
0
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9
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1
water and recrystallized from EtOH to give the product 12 (0.612 g, 47%). H NMR (400 MHz,
DMSOꢀd ) δ 11.33 (s, 1H), 9.59 (s, 1H), 9.31 (s, 1H), 8.64 (s, 1H), 8.50 (d, J = 4.8 Hz, 1H), 7.69
6
(d, J = 7.6 Hz, 2H), 7.62 (s, 1H), 7.50ꢀ7.48 (m, 1H), 7.23 (t, J = 7.6 Hz, 2H), 7.19 (d, J = 8.0 Hz,
1
H), 7.15 (d, J = 8.0 Hz, 1H), 6.96 (t, J = 7.2 Hz, 1H), 2.79 (d, J = 4.4 Hz, 3H), 1.47 (s, 9H). LCꢀ
+
MS: m/z: 435.2 (M+H) .
tertꢀButyl(3ꢀ(3ꢀMethylꢀ2,4ꢀdioxoꢀ7ꢀ(phenylamino)ꢀ3,4ꢀdihydropyrimido[4,5ꢀd]pyrimidinꢀ
1
(2H)ꢀyl)phenyl)carbamate (13). To a solution of 12 (0.434 g, 1 mmol), K CO (0.276 g, 2
2 3
mmol) in dry THF (10 mL) was added 1,1'ꢀcarbonyldiimidazole (0.324 g, 2 mmol). The reaction
mixture was heated with reflux overnight. After cooling to room temperature, the reaction
mixture was poured into water, and extracted with CH Cl (3×20 mL). The organic layer was
2
2
washed with saturated aqueous NaCl, dried over anhydrous Na SO and concentrated in vacuo.
2
4
The residue was purified by silica gel chromatography (petroleum ether/ethylacetate = 1:2, v/v)
1
to give the product 13 as a white solid (0.193 g, 42%). H NMR (400 MHz, DMSOꢀd ) δ 9.62 (s,
6
1
H), 8.92 (s, 1H), 7.66 (s, 1H), 7.52 (d, J = 8.0 Hz, 1H), 7.45 (t, J = 8.0 Hz, 1H), 7.36ꢀ7.32 (m,
2
H), 7.03 (d, J = 8.0 Hz, 3H), 6.91 (d, J = 7.2 Hz, 1H), 3.30 (s, 3H), 1.45 (s, 9H). HRMS(ESI)
+
(
m/z): (M+H) calcd for C H N O 461.1937, found, 461.1939.
24
25
6
4
Nꢀ(3ꢀ(3ꢀMethylꢀ2,4ꢀdioxoꢀ7ꢀ(phenylamino)ꢀ3,4ꢀdihydropyrimido[4,5ꢀd]pyrimidinꢀ1(2H)ꢀ
yl)phenyl)acrylamide (14e). To a solution of 13 (0.190 g, 0.41 mmol) in CH Cl (4 mL) was
2
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added trifluoroacetic acid (1 mL). The mixture was stirred for 4 h at room temperature. The
reaction solution was neutralized with saturated aqueous NaHCO , and extracted with CH Cl
2
3
2
(3×15 mL). The organic layer was washed with saturated aqueous NaCl, dried over anhydrous
Na SO and concentrated in vacuo to obtain a white solid (0.136 g, 90%), which was used in the
1
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next reaction without further purification.
To a solution of 1ꢀ(3ꢀaminophenyl)ꢀ3ꢀmethylꢀ7ꢀ(phenylamino)pyrimido[4,5ꢀd] pyrimidineꢀ
2,4(1H,3H)ꢀdione (0.136 g, 0.38 mmol) in NMP (1.5 mL) was added acryloyl chloride (37 ꢁL,
o
0
.46 mmol) dissolved in CH CN (0.5 mL) at 0 C. The reaction solution was stirred overnight at
3
room temperature. The mixture was poured into water, and extracted with CH Cl (3×15 mL).
2
2
The organic layer was washed with saturated aqueous NaCl, dried over anhydrous Na SO and
2
4
concentrated in vacuo. The residue was purified by silica gel chromatography (petroleum
ether/ethylacetate = 1:1.5, v/v) to give the product 14e as a white solid (0.083 g, 53%). mp
o
1
2
90.3ꢀ291.2 C. H NMR (400 MHz, DMSOꢀd ) δ 10.38 (s, 1H), 8.93 (s, 1H), 7.82 (d, J = 3.2
6
Hz, 1H), 7.80 (s, 1H), 7.53 (t, J = 8.0 Hz, 1H), 7.33ꢀ7.29 (m, 2H), 7.15 (d, J = 7.6 Hz, 1H), 7.02ꢀ
.96 (m, 2H), 6.89 (t, J = 6.0 Hz, 1H), 6.44 (dd, J = 17.2 Hz, J = 10.4 Hz, 1H), 6.25 (dd, J = 17.2
6
13
Hz, J = 1.6 Hz, 1H), 5.76 (dd, J = 10.4 Hz, J = 2.0 Hz, 1H), 3.30 (s, 3H). C NMR (100 MHz,
DMSOꢀd ) δ 163.24, 160.11, 158.97, 157.87, 150.86, 139.90, 138.91, 136.19, 131.61, 129.45,
6
+
1
28.11, 127.22, 124.30, 122.53, 120.07, 119.26, 119.07, 27.57. HRMS(ESI) (m/z): (M+H)
calcd for C H N O 415.1519, found, 415.1512. HPLC purity: 97.66%, retention time = 10.68
2
2
19
6
3
min.
The following compounds 14aꢀd were prepared by a method similar to that for the synthesis
of compound 14e.
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Nꢀ(3ꢀ(7ꢀ((4ꢀ(4ꢀAcetylpiperazinꢀ1ꢀyl)ꢀ2ꢀmethoxyphenyl)amino)ꢀ3ꢀmethylꢀ2,4ꢀdioxoꢀ3,4ꢀ
dihydropyrimido[4,5ꢀd]pyrimidinꢀ1(2H)ꢀyl)phenyl)acrylamide (14a). Yellow solid (yield
o
1
49%). mp 228.5ꢀ229.8 C. H NMR (400 MHz, DMSOꢀd ) δ 10.34 (s, 1H), 8.83 (s, 1H), 8.59 (s,
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
H), 7.86 (s, 1H), 7.69 (s, 1H),7.48 (t, J = 8.4 Hz, 1H), 7.18 (d, J = 8.8 Hz, 1H), 7.09 (d, J = 8.0
Hz, 1H), 6.55 (s, 1H), 6.45 (dd, J = 17.2 Hz, J = 10.4 Hz, 1H), 6.26 (dd, J = 17.2 Hz, J = 1.6 Hz,
H), 5.99 (s, 1H), 5.77 (dd, J = 10.0 Hz, J = 1.2 Hz, 1H), 3.76 (s, 3H), 3.58ꢀ3.54 (m, 4H), 3.28
1
13
(
s, 3H), 3.05ꢀ2.99 (m, 4H), 2.05 (s, 3H). C NMR (100 MHz, DMSOꢀd ) δ 168.19, 163.21,
6
1
60.10, 157.83, 139.73, 135.99, 131.67, 129.33, 127.18, 49.15, 48.76, 45.42, 40.60, 27.54, 21.16.
+
HRMS(ESI) (m/z): (M+H) calcd for C H N O 571.2417, found, 571.2417. HPLC purity:
2
9
31
8
5
97.63%, retention time = 9.50 min.
Nꢀ(3ꢀ(7ꢀ((2ꢀMethoxyꢀ4ꢀmorpholinophenyl)amino)ꢀ3ꢀmethylꢀ2,4ꢀdioxoꢀ3,4ꢀ
dihydropyrimido[4,5ꢀd]pyrimidinꢀ1(2H)ꢀyl)phenyl)acrylamide (14b). Yellow solid (yield
o
1
5
1%). mp >300 C. H NMR (400 MHz, DMSOꢀd ) δ 10.36 (s, 1H), 8.80 (s, 1H), 8.44 (s, 1H),
6
8.03 (s, 1H), 7.86 (d, J = 8.0 Hz, 1H), 7.69 (t, J = 2.0 Hz, 1H), 7.52 (t, J = 8.4 Hz, 1H), 7.32 (d, J
8.8 Hz, 1H), 7.09 (d, J = 7.6 Hz, 1H), 6.57 (d, J = 2.0 Hz, 1H), 6.45 (dd, J = 16.8 Hz, J = 10.0
Hz, 1H), 6.27 (dd, J = 17.2 Hz, J = 2.0 Hz, 1H), 5.77 (dd, J = 10.4 Hz, J = 2.0 Hz, 1H), 3.77 (s,
=
1
3
3H), 3.58ꢀ3.54 (m, 4H), 3.06ꢀ2.99 (m, 4H), 2.05 (s, 3H). C NMR (100 MHz, DMSOꢀd ) δ
6
1
1
63.23, 160.09, 158.87, 157.82, 150.86, 149.81, 148.06, 139.74, 135.98, 131.65, 129.34, 127.19,
24.18, 121.08, 120.00, 119.12, 105.61, 99.65, 99.15, 66.03, 55.64, 48.78, 27.54. HRMS(ESI)
+
(
m/z): (M+H) calcd for C H N O 528.1995, found, 528.1993. HPLC purity: 96.86%,
27 28
7
5
retention time = 10.73 min.
Nꢀ(3ꢀ(7ꢀ((2ꢀMethoxyꢀ4ꢀthiomorpholinophenyl)amino)ꢀ3ꢀmethylꢀ2,4ꢀdioxoꢀ3,4ꢀ
dihydropyrimido[4,5ꢀd]pyrimidinꢀ1(2H)ꢀyl)phenyl)acrylamide (14c). Yellow solid (yield
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9
o
1
5
2%). mp 246.0ꢀ247.1 C. H NMR (400 MHz, DMSOꢀd ) δ 10.35 (s, 1H), 8.84 (s, 1H), 8.60 (s,
6
1
H), 8.85 (s, 1H), 7.70 (s, 1H), 7.48 (t, J = 8.0 Hz, 1H), 7.17 (d, J = 8.4 Hz, 1H), 7.09 (d, J = 7.6
Hz, 1H), 6.45 (dd, J = 17.2 Hz, J = 10.0 Hz, 1H), 6.27 (dd, J = 16.8 Hz, J = 2.0 Hz, 1H), 5.97ꢀ
1
1
1
1
1
1
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1
1
2
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2
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2
2
2
3
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4
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4
4
4
4
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5
5
5
5
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5
6
0
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2
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5
6
7
8
9
0
1
2
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5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
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7
8
9
0
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2
3
4
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8
9
0
5
3
1
1
5
.95 (m, 1H), 5.77 (dd, J = 10.0 Hz, J = 1.6 Hz, 1H), 3.75 (s, 3H), 3.42ꢀ3.39 (m, 4H), 3.28 (s,
13
H), 2.66ꢀ2.63 (m, 4H). C NMR (100 MHz, DMSOꢀd ) δ 163.24, 160.08, 158.90, 157.81,
6
50.86, 149.99, 139.76, 135.99, 131.63, 129.33, 127.22, 124.19, 121.32, 120.01, 119.10, 106.85,
+
00.43, 99.63, 55.65, 51.46, 27.54, 25.62. HRMS(ESI) (m/z): (M+H) calcd for C H N O S
2
7
28
7
4
46.1923, found, 546.1924. HPLC purity: 95.21%, retention time = 12.49 min.
Nꢀ(3ꢀ(7ꢀ((4ꢀ(4ꢀ(Dimethylamino)piperidinꢀ1ꢀyl)ꢀ2ꢀmethoxyphenyl)amino)ꢀ3ꢀmethylꢀ2,4ꢀ
dioxoꢀ3,4ꢀdihydropyrimido[4,5ꢀd]pyrimidinꢀ1(2H)ꢀyl)phenyl)acrylamide (14d). Yellow solid
o
1
(
yield 47%). mp >300 C. H NMR (400 MHz, DMSOꢀd ) δ 10.36 (s, 1H), 8.83 (s, 1H), 8.54 (s,
6
1
H), 7.88ꢀ7.86 (m, 1H), 7.70 (s, 1H),7.47 (t, J = 8.0 Hz, 1H), 7.16 (d, J = 8.4 Hz, 1H), 7.08 (d, J
7.6 Hz, 1H), 6.50ꢀ6.43 (m, 2H), 6.26 (dd, J = 16.8 Hz, J = 1.6 Hz, 1H), 5.98ꢀ5.96 (m, 1H),
=
5.76 (dd, J = 10.0 Hz, J = 1.2 Hz, 1H), 3.75 (s, 3H), 3.59ꢀ3.58 (m, 2H), 3.28 (s, 3H), 2.60ꢀ2.55
+
(
m, 2H), 2.22 (s, 6H), 1.81 (d, J = 7.6 Hz, 2H), 1.47ꢀ1.40 (m, 2H). HRMS(ESI) (m/z): (M+H)
calcd for C H N O 571.2781, found, 571.2780. HPLC purity: 97.96%, retention time = 11.35
3
0
35
8
4
min.
2,4ꢀDichloropyrimidineꢀ5ꢀcarboxamide (15). To a solution of 2,4ꢀdichloropyrimidineꢀ5ꢀ
carbonyl chloride (4.220 g, 20 mmol) in dichloromethane (15 mL) was added ammonium
o
o
hydroxide (1.400 g, 40 mmol) at 0 C. The reaction mixture was stirred for 2 h at 0 C, and the
mixture was poured into ice water. The resulting solid was collected by filtration, washed with
1
water to give the product 15 as a white solid (3.510 g, 92%). H NMR (400 MHz, DMSOꢀd ) δ
6
8
.91 (s, 1H), 8.20 (s, 1H), 8.08 (s, 1H).
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tertꢀButyl(3ꢀ((5ꢀCarbamoylꢀ2ꢀchloropyrimidinꢀ4ꢀyl)amino)phenyl)carbamate (16).
A
mixture of compound 15 (3.438 g, 18 mmol), DIPEA (3.483 g, 27 mmol), compound 8 (3.744 g,
o
18 mmol) in isopropanol (50 mL) was heated at 40 C for 5 h. After cooling to room
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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6
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0
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2
3
4
5
6
7
8
9
0
temperature, the white solid was collected by filtration and washed with isopropanol to give the
1
product 16 (5.480 g, 84%). H NMR (400 MHz, DMSOꢀd ) δ 11.51 (s, 1H), 9.49 (s, 1H), 8.78 (s,
6
1
7
H), 8.47 (s, 1H), 8.00 (s, 1H), 7.64 (s, 1H), 7.41 (d, J = 8.0 Hz, 1H), 7.27 (t, J = 8.0 Hz, 1H),
+
.20 (d, J = 8.0 Hz, 1H), 1.48 (s, 9H). LCꢀMS: m/z: 364.1 (M+H) .
tertꢀButyl(3ꢀ((5ꢀCarbamoylꢀ2ꢀ((4ꢀ(4ꢀ(dimethylamino)piperidinꢀ1ꢀyl)ꢀ2ꢀmethoxyphenyl)
amino)pyrimidinꢀ4ꢀyl)amino)phenyl)carbamate (17). To a mixture of compound 16 (5.082 g,
4 mmol), trifluoroacetic acid (1.550 mL, 21 mmol) in isopropanol (100 mL) was added 1ꢀ(4ꢀ
1
aminoꢀ3ꢀmethoxyphenyl)ꢀN,Nꢀdimethylpiperidinꢀ4ꢀamine (4.183 g, 16.8 mmol). The reaction
solution was heated with reflux overnight. After cooling to room temperature, the solution was
concentrated with a rotary evaporator. And neutralized with saturated aqueous NaHCO3,
extracted with CH Cl (3×50 mL). The organic layer was washed with saturated aqueous NaCl,
2
2
dried over anhydrous Na SO and concentrated in vacuo. The residue was purified by silica gel
2
4
chromatography (dichloromethane/methanol = 15:1, v/v) to give the product 11 (5.450 g, 67%).
1
H NMR (400 MHz, DMSOꢀd ) δ 11.46 (s, 1H), 9.34 (s, 1H), 8.65 (s, 1H), 8.24 (s, 1H), 8.08 (s,
6
1
H), 7.52 (d, J = 8.8 Hz, 1H), 7.41 (d, J = 7.6 Hz, 1H), 7.37 (s, 1H), 7.12ꢀ7.07 (m, 2H), 6.62 (d, J
1.6 Hz, 1H), 6.45 (d, J = 8.4 Hz, 1H), 3.77 (s, 3H), 3.70 (d, J = 12.0 Hz, 2H), 2.66 (t, J = 11.2
=
Hz, 2H), 2.20 (s, 6H), 1.84 (d, J = 12.0 Hz, 2H), 1.54ꢀ1.49 (m, 2H), 1.47 (s, 9H). LCꢀMS: m/z:
+
5
77.4 (M+H) .
tertꢀButyl(3ꢀ(7ꢀ((4ꢀ(4ꢀ(Dimethylamino)piperidinꢀ1ꢀyl)ꢀ2ꢀmethoxyphenyl)amino)ꢀ2,4ꢀ
dioxoꢀ3,4ꢀdihydropyrimido[4,5ꢀd]pyrimidinꢀ1(2H)ꢀyl)phenyl)carbamate (18). A mixture of
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compound 17 (5.184 g, 9 mmol), K CO (1.863 g, 13.5 mmol), 1,1'ꢀcarbonyldiimidazole (2.916
2
3
g, 18 mmol) in dry THF (40 mL) was heated with reflux overnight. After cooling to room
temperature, the solution was added ice water and extracted with CH Cl (3×30 mL). The
2
2
1
1
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1
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1
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3
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5
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5
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5
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0
1
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7
8
9
0
1
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0
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0
organic layer was washed with saturated aqueous NaCl, dried over anhydrous Na SO and
2
4
concentrated in vacuo. The residue was purified by silica gel chromatography
1
(
dichloromethane/methanol = 12:1, v/v) to give the product 18 (3.289 g, 60%). H NMR (400
MHz, DMSOꢀd ) δ 11.55 (s, 1H), 9.59 (s, 1H), 8.77 (s, 1H), 8.54 (s, 1H), 7.53 (s, 2H), 7.38 (t, J
6
=
8.0 Hz, 1H), 7.17 (d, J = 6.8 Hz, 1H), 6.96 (d, J = 7.2 Hz, 1H), 6.50 (s, 1H), 6.02ꢀ5.99 (m, 1H),
3
.76 (s, 3H), 3.61ꢀ3.59 (m, 2H), 2.62ꢀ2.57 (m, 2H), 2.19 (s, 6H), 1.82 (d, J = 12.0 Hz, 2H), 1.51ꢀ
+
1.48 (m, 2H), 1.46 (s, 9H). LCꢀMS: m/z: 603.3 (M+H) .
tertꢀButyl(3ꢀ(7ꢀ((4ꢀ(4ꢀ(Dimethylamino)piperidinꢀ1ꢀyl)ꢀ2ꢀmethoxyphenyl)amino)ꢀ3ꢀethylꢀ
2
,4ꢀdioxoꢀ3,4ꢀdihydropyrimido[4,5ꢀd]pyrimidinꢀ1(2H)ꢀyl)phenyl)carbamate (19). To a
mixture of compound 18 (0.602 g, 1 mmol), Cs CO (0.489 g, 1.5 mmol) in DMF (5 mL) was
2
3
added iodoethane (95 ꢁL, 1.2 mmol). The mixture was stirred at room temperature overnight.
The solution was added ice water and extracted with CH Cl (3×25 mL). The organic layer was
2
2
washed with saturated aqueous NaCl, dried over anhydrous Na SO and concentrated in vacuo.
2
4
The residue was purified by silica gel chromatography (dichloromethane/methanol = 13:1, v/v)
1
to give the title compound 19 (0.378 g, 60%). H NMR (400 MHz, DMSOꢀd ) δ 9.60 (s, 1H),
6
8
7
3
1
.83 (s, 1H), 8.58 (s, 1H), 7.57 (s, 1H), 7.53ꢀ7.51 (m, 1H), 7.39 (t, J = 7.6 Hz, 1H), 7.18 (d, J =
.6 Hz, 1H), 6.97 (d, J = 7.2 Hz, 1H), 6.51 (s, 1H), 6.03ꢀ6.01 (m, 1H), 3.93 (q, J = 6.4 Hz, 2H),
.76 (s, 3H), 3.63ꢀ3.60 (m, 2H), 2.63ꢀ2.58 (m, 2H), 2.24 (s, 6H), 1.83 (d, J = 12.0 Hz, 2H), 1.52ꢀ
+
.47 (m, 2H), 1.46 (s, 9H), 1.17 (t, J = 6.8 Hz, 3H). LCꢀMS: m/z: 631.3 (M+H) .
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Nꢀ(3ꢀ(7ꢀ((4ꢀ(4ꢀ(Dimethylamino)piperidinꢀ1ꢀyl)ꢀ2ꢀmethoxyphenyl)amino)ꢀ3ꢀethylꢀ2,4ꢀ
dioxoꢀ3,4ꢀdihydropyrimido[4,5ꢀd]pyrimidinꢀ1(2H)ꢀyl)phenyl)acrylamide (20a). To
a
solution of compound 19 (0.348 g, 0.55 mmol) in CH Cl (5 mL) was added trifluoroacetic acid
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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8
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2
3
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7
8
9
0
1
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3
4
5
6
7
8
9
0
(1.5 mL). The mixture was stirred for 5 h at room temperature. The reaction solution was
neutralized with saturated aqueous NaHCO , and extracted with CH Cl (3×30 mL). The organic
3
2
2
layer was washed with saturated aqueous NaCl, dried over anhydrous Na SO and concentrated
2
4
in vacuo to obtain a yellow solid (0.241 g, 82%), which was used in the next reaction without
further purification.
To
a
solution
of
1ꢀ(3ꢀaminophenyl)ꢀ7ꢀ((4ꢀ(4ꢀ(dimethylamino)piperidinꢀ1ꢀyl)ꢀ2ꢀ
methoxyphenyl)amino)ꢀ3ꢀethylpyrimido[4,5ꢀd]pyrimidineꢀ2,4(1H,3H)ꢀdione (0.220 g, 0.4
mmol) in NMP (1.5 mL) was added acryloyl chloride (42 ꢁL, 0.5 mmol) dissolved in CH CN
3
o
(
0.5 mL) at 0 C. The reaction solution was stirred overnight at room temperature. The mixture
was poured into water, and extracted with CH Cl (3×20 mL). The organic layer was washed
2
2
with saturated aqueous NaCl, dried over anhydrous Na SO and concentrated in vacuo. The
2
4
residue was purified by silica gel chromatography (dichloromethane/methanol = 12:1, v/v) to
o
1
give the title product 20a as a yellow solid (0.116 g, 49%). mp 212.1ꢀ213.6 C. H NMR (400
MHz, DMSOꢀd ) δ 10.64 (s, 1H), 10.56 (s, 1H), 8.84 (s, 1H), 8.62 (s, 1H), 7.92ꢀ7.90 (m, 1H),
6
7
.71 (s, 1H),7.48 (t, J = 8.0 Hz, 1H), 7.17 (d, J = 6.8 Hz, 1H), 7.10 (d, J = 7.2 Hz, 1H), 6.55ꢀ6.49
(m, 2H), 6.28 (dd, J = 16.8 Hz, J = 1.6 Hz, 1H), 6.00 (s, 1H), 5.78 (dd, J = 11.2 Hz, J = 1.2 Hz,
1
H), 3.94 (q, J = 6.4 Hz, 2H), 3.76 (s, 3H), 3.72ꢀ3.68 (m, 2H), 2.73 (s, 6H), 2.60 (t, J = 8.0 Hz,
1
H), 2.63ꢀ2.58 (m, 2H), 2.09 (d, J = 11.2 Hz, 2H), 1.72ꢀ1.68 (m, 2H), 1.18 (t, J = 6.4 Hz, 3H).
+
HRMS(ESI) (m/z): (M+H) calcd for C H N O 585.2938, found, 585.2928. HPLC purity:
3
1
37
8
4
9
6.26%, retention time = 9.95 min.
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The following compounds 20bꢀl were prepared by a synthesis route similar to that for compound
2
0a.
Nꢀ(3ꢀ(7ꢀ((4ꢀ(4ꢀ(Dimethylamino)piperidinꢀ1ꢀyl)ꢀ2ꢀmethoxyphenyl)amino)ꢀ2,4ꢀdioxoꢀ3ꢀ
propylꢀ3,4ꢀdihydropyrimido[4,5ꢀd]pyrimidinꢀ1(2H)ꢀyl)phenyl)acrylamide (20b). Yellow
1
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1
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2
2
2
2
2
2
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
o
1
solid (yield 39%). mp >300 C. H NMR (400 MHz, DMSOꢀd ) δ 10.54 (s, 1H), 8.84 (s, 1H),
6
8
.63 (s, 1H), 7.92 (s, 1H), 7.69 (s, 1H), 7.49 (t, J = 8.0 Hz, 1H), 7.19 (d, J = 6.8 Hz, 1H), 7.10 (d,
J = 7.8 Hz, 1H), 6.54ꢀ6.48 (m, 2H), 6.28 (dd, J = 16.8 Hz, J = 1.2 Hz, 1H), 6.00 (s, 1H), 5.78
dd, J = 10.4 Hz, J = 1.6 Hz, 1H), 3.86 (t, J = 6.8 Hz, 2H), 3.76 (s, 3H), 3.72ꢀ3.69 (m, 2H), 3.26ꢀ
(
3
1
5
.21 (m, 1H), 2.72 (s, 6H), 2.62ꢀ2.57 (m, 2H), 2.07 (d, J = 11.2 Hz, 2H), 1.70ꢀ1.67 (m, 2H),
+
.65ꢀ1.60 (m, 2H), 0.90 (t, J = 7.2 Hz, 3H). HRMS(ESI) (m/z): (M+H) calcd for C H N O
32 39
8
4
99.3094, found, 599.3099. HPLC purity: 97.85%, retention time = 10.96 min.
Nꢀ(3ꢀ(7ꢀ((4ꢀ(4ꢀ(Dimethylamino)piperidinꢀ1ꢀyl)ꢀ2ꢀmethoxyphenyl)amino)ꢀ3ꢀisopropylꢀ2,4ꢀ
dioxoꢀ3,4ꢀdihydropyrimido[4,5ꢀd]pyrimidinꢀ1(2H)ꢀyl)phenyl)acrylamide (20c). Yellow solid
o
1
(yield 41%). mp >300 C. H NMR (400 MHz, DMSOꢀd ) δ 10.55 (s, 1H), 8.83 (s, 1H), 8.60 (s,
6
1
H), 7.91 (s, 1H), 7.69 (s, 1H), 7.48 (t, J = 8.0 Hz, 1H), 7.19 (d, J = 5.6 Hz, 1H), 7.10 (d, J = 7.6
Hz, 1H), 6.55ꢀ6.48 (m, 2H), 6.28 (dd, J = 17.2 Hz, J = 1.6 Hz, 1H), 6.00 (s, 1H), 5.78 (dd, J =
0.0 Hz, J = 1.6 Hz, 1H), 5.17ꢀ5.10 (m, 1H), 3.76 (s, 3H), 3.72ꢀ3.68 (m, 2H), 3.26ꢀ3.20 (m, 1H),
1
2.72 (s, 6H), 2.63ꢀ2.59 (m, 2H), 2.07 (d, J = 10.8 Hz, 2H), 1.70ꢀ1.67 (m, 2H), 1.44 (d, J = 6.8
13
Hz, 6H). C NMR (100 MHz, DMSOꢀd ) δ 163.29, 160.19, 157.83, 139.83, 135.83, 135.95,
6
1
31.81, 129.22, 127.00, 124.26, 120.04, 119.06, 106.58, 99.91, 62.21, 55.67, 47.77, 45.00, 25.55,
+
19.25. HRMS(ESI) (m/z): (M+H) calcd for C H N O 599.3094, found, 599.3079. HPLC
3
2
39
8
4
purity: 98.38%, retention time = 11.12 min.
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Nꢀ(3ꢀ(3ꢀBenzylꢀ7ꢀ((4ꢀ(4ꢀ(dimethylamino)piperidinꢀ1ꢀyl)ꢀ2ꢀmethoxyphenyl)amino)ꢀ2,4ꢀ
dioxoꢀ3,4ꢀdihydropyrimido[4,5ꢀd]pyrimidinꢀ1(2H)ꢀyl)phenyl)acrylamide (20d). Yellow solid
o
1
(yield 39%). mp 285.4ꢀ286.6 C. H NMR (400 MHz, DMSOꢀd ) δ 10.54 (s, 1H), 8.87 (s, 1H),
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8
.69 (s, 1H), 7.91 (s, 1H), 7.69 (s, 1H), 7.48 (t, J = 8.0 Hz, 1H), 7.38 (d, J = 7.2 Hz, 2H), 7.33 (t,
J = 7.2 Hz, 2H), 7.26 (t, J = 7.2 Hz, 1H), 7.19 (d, J = 8.0 Hz, 1H), 7.11 (d, J = 6.8 Hz, 1H), 6.54ꢀ
.47 (m, 2H), 6.27 (dd, J = 16.8 Hz, J = 1.6 Hz, 1H), 6.01ꢀ5.99 (m, 1H), 5.77 (dd, J = 10.0 Hz, J
6
=
1.6 Hz, 1H), 5.10 (s, 2H), 3.76 (s, 3H), 3.72ꢀ3.69 (m, 2H), 3.27ꢀ3.21 (m, 1H), 2.73 (s, 6H),
13
2
.62ꢀ2.57 (m, 2H), 2.08 (d, J = 11.2 Hz, 2H), 1.71ꢀ1.68 (m, 2H). C NMR (100 MHz, DMSOꢀ
d6) δ 163.31, 159.94, 139.88, 136.98, 135.84, 131.82, 129.27, 128.30, 127.63, 127.14, 127.01,
+
62.24, 59.74, 55.68, 47.72, 43.83, 25.47, 20.74. HRMS(ESI) (m/z): (M+H) calcd for
C H N O 647.3094, found, 647.3088. HPLC purity: 95.30%, retention time = 12.72 min.
36
39
8
4
Nꢀ(3ꢀ(7ꢀ((4ꢀ(4ꢀ(Dimethylamino)piperidinꢀ1ꢀyl)ꢀ2ꢀmethoxyphenyl)amino)ꢀ2,4ꢀdioxoꢀ3ꢀ
phenethylꢀ3,4ꢀdihydropyrimido[4,5ꢀd]pyrimidinꢀ1(2H)ꢀyl)phenyl)acrylamide (20e). Yellow
o
1
solid (yield 40%). mp 196.5ꢀ197.7 C. H NMR (400 MHz, DMSOꢀd ) δ 10.58 (s, 1H), 8.85 (s,
6
1
H), 8.67 (s, 1H), 7.91 (s, 1H), 7.72 (s, 1H), 7.49 (t, J = 8.0 Hz, 1H), 7.32 (t, J = 7.6 Hz, 2H),
.26ꢀ7.23 (m, 3H), 7.19 (d, J = 12.8 Hz, 1H), 7.07 (d, J = 6.8 Hz, 1H), 6.56ꢀ6.49 (m, 2H), 6.28
7
(dd, J = 17.2 Hz, J = 1.6 Hz, 1H), 6.01ꢀ5.99 (m, 1H), 5.78 (dd, J = 10.4 Hz, J = 1.6 Hz, 1H),
4
2
.10ꢀ4.08 (m, 2H), 3.77 (s, 3H), 3.72ꢀ3.69 (m, 2H), 3.24ꢀ3.19 (m, 1H), 2.90 (t, J = 7.6 Hz, 2H),
13
.71 (s, 6H), 2.62ꢀ2.57 (m, 2H), 2.07 (d, J = 11.2 Hz, 2H), 1.70ꢀ1.68 (m, 2H). C NMR (100
MHz, DMSOꢀd ) δ 163.33, 159.68, 139.90, 138.51, 135.82, 131.83, 129.26, 128.59, 128.50,
6
+
1
27.02, 126.39, 62.23, 55.70, 47.74, 41.92, 33.28, 25.52. HRMS(ESI) (m/z): (M+H) calcd for
C H N O 661.3251, found, 661.3251. HPLC purity: 99.09%, retention time = 13.71 min.
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Nꢀ(3ꢀ(7ꢀ((4ꢀ(4ꢀ(Dimethylamino)piperidinꢀ1ꢀyl)ꢀ2ꢀmethoxyphenyl)amino)ꢀ2,4ꢀdioxoꢀ3ꢀ(3ꢀ
phenylpropyl)ꢀ3,4ꢀdihydropyrimido[4,5ꢀd]pyrimidinꢀ1(2H)ꢀyl)phenyl)acrylamide
(20f).
o
1
Yellow solid (yield 38%). mp 252.1ꢀ253.8 C. H NMR (400 MHz, DMSOꢀd ) δ 10.72 (s, 1H),
6
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8
7
1
.81 (s, 1H), 8.60 (s, 1H), 7.92 (s, 1H), 7.78 (s, 1H), 7.48 (t, J = 8.0 Hz, 1H), 7.28ꢀ7.19 (m, 5H),
.14 (t, J = 7.2 Hz, 1H), 7.07 (d, J = 7.2 Hz, 1H), 6.61ꢀ6.52 (m, 2H), 6.29 (dd, J = 17.2 Hz, J =
.6 Hz, 1H), 6.02 (s, 1H), 5.78 (dd, J = 10.0 Hz, J = 1.6 Hz, 1H), 3.94 (t, J = 7.2 Hz, 2H), 3.76
(
s, 3H), 3.22ꢀ3.17 (m, 1H), 2.69 (s, 6H), 2.68ꢀ2.63 (m, 4H), 2.08 (d, J = 10.4 Hz, 2H), 1.94 (t, J =
13
6
1
.8 Hz, 2H). C NMR (100 MHz, DMSOꢀd ) δ 163.33, 159.89, 157.82, 141.32, 139.88, 135.89,
6
31.84, 129.23, 128.21, 128.12, 126.99, 125.68, 62.20, 47.76, 40.52, 32.51, 28.57, 25.57.
+
HRMS(ESI) (m/z): (M+H) calcd for C H N O 675.3407, found, 675.3403. HPLC purity:
3
8
43
8
4
9
5.85%, retention time = 14.42 min.
Nꢀ(3ꢀ(3ꢀIsopropylꢀ7ꢀ((2ꢀmethoxyꢀ4ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)phenyl)amino)ꢀ2,4ꢀdioxoꢀ3,4ꢀ
dihydropyrimido[4,5ꢀd]pyrimidinꢀ1(2H)ꢀyl)phenyl)acrylamide (20g). Yellow solid (yield
o
1
39%). mp >300 C. H NMR (400 MHz, DMSOꢀd ) δ 10.64 (s, 1H), 8.83 (s, 1H), 8.63 (s, 1H),
6
7
.85 (d, J = 7.2 Hz, 1H), 7.73 (s, 1H), 7.47 (t, J = 8.0 Hz, 1H), 7.20 (d, J = 8.4 Hz, 1H), 7.09 (d,
J = 7.6 Hz, 1H), 6.56ꢀ6.51 (m, 2H), 6.27 (dd, J = 17.2 Hz, J = 1.6 Hz, 1H), 6.03ꢀ6.01 (m, 1H),
5.77 (dd, J = 10.4 Hz, J = 1.2 Hz, 1H), 5.17ꢀ5.10 (m, 1H), 3.76 (s, 3H), 3.29 (t, J = 4.0 Hz, 4H),
1
3
3
.20 (t, J = 4.0 Hz, 4H), 2.75 (s, 3H), 1.44 (d, J = 6.4 Hz, 6H). C NMR (100 MHz, DMSOꢀd6)
δ 163.33, 160.20, 157.83, 139.79, 135.94, 131.79, 129.21, 127.05, 120.09, 100.00, 55.74, 52.25,
+
4
5
6.05, 45.02, 42.29, 19.24. HRMS(ESI) (m/z): (M+H) calcd for C H N O 571.2781, found,
30 35
8
4
71.2775. HPLC purity: 98.71%, retention time = 11.25 min.
Nꢀ(3ꢀ(3ꢀIsopropylꢀ7ꢀ((3ꢀmethoxyꢀ4ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)phenyl)amino)ꢀ2,4ꢀdioxoꢀ3,4ꢀ
dihydropyrimido[4,5ꢀd]pyrimidinꢀ1(2H)ꢀyl)phenyl)acrylamide (20h). Yellow solid (yield
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4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
o
1
4
7%). mp 221.3ꢀ222.5 C. H NMR (400 MHz, DMSOꢀd ) δ 10.51 (s, 1H), 10.18 (s, 1H), 8.88
6
(
s, 1H), 7.89 (s, 1H), 7.72 (s, 1H), 7.50 (t, J = 8.0 Hz, 1H), 7.13 (d, J = 8.0 Hz, 1H), 6.91 (s, 1H),
6.49 (dd, J = 16.8 Hz, J = 10.0 Hz, 1H), 6.43ꢀ6.41 (m, 1H), 6.27 (dd, J = 16.8 Hz, J = 1.6 Hz,
0
1
2
3
4
5
6
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
1
4
5
H), 5.77 (dd, J = 10.0 Hz, J = 1.6 Hz, 1H), 5.18ꢀ5.11 (m, 1H), 3.56 (s, 3H), 3.04ꢀ3.01 (m, 8H),
13
.63 (s, 3H), 1.44 (d, J = 6.8 Hz, 6H). C NMR (100 MHz, DMSOꢀd ) δ 163.29, 160.17, 151.55,
6
50.38, 139.87, 136.09, 134.47, 131.73, 129.33, 127.13, 124.33, 120.10, 119.23, 55.29, 53.27,
+
7.95, 45.06, 43.21, 19.23. HRMS(ESI) (m/z): (M+H) calcd for C H N O 571.2781, found,
3
0
35
8
4
71.2773. HPLC purity: 97.35%, retention time = 10.11 min.
Nꢀ(3ꢀ(3ꢀIsopropylꢀ7ꢀ((3ꢀmethylꢀ4ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)phenyl)amino)ꢀ2,4ꢀdioxoꢀ3,4ꢀ
dihydropyrimido[4,5ꢀd]pyrimidinꢀ1(2H)ꢀyl)phenyl)acrylamide (20i). Yellow solid (yield
o
1
4
9%). mp 295.5ꢀ296.9 C. H NMR (400 MHz, DMSOꢀd ) δ 10.54 (s, 1H), 10.22 (s, 1H), 8.88
6
(
s, 1H), 7.90 (d, J = 7.2 Hz, 1H), 7.74 (s, 1H), 7.51 (t, J = 8.0 Hz, 1H), 7.18 (s, 1H), 7.13 (d, J =
.0 Hz, 1H), 7.08 (d, J = 6.4 Hz, 1H), 6.69 (d, J = 7.6 Hz, 1H), 6.50 (dd, J = 17.2 Hz, J = 10.4
Hz, 1H), 6.26 (dd, J = 17.2 Hz, J = 1.6 Hz, 1H), 5.76 (dd, J = 10.4 Hz, J = 1.6 Hz, 1H), 5.18ꢀ
.11 (m, 1H), 3.12 (t, J = 4.0 Hz, 4H), 2.92 (t, J = 4.0 Hz, 4H), 2.70 (s, 3H), 1.97 (s, 3H), 1.44 (d,
8
5
1
3
J = 7.2 Hz, 6H). C NMR (100 MHz, DMSOꢀd ) δ 163.29, 160.18, 159.05, 157.91, 150.40,
6
139.92, 136.16, 134.60, 132.03, 131.74, 129.37, 127.10, 124.26, 121.38, 119.95, 119.35, 118.62,
+
1
17.58, 53.35, 48.94, 45.04, 42.84, 19.23, 17.44. HRMS(ESI) (m/z): (M+H) calcd for
C H N O 555.2832, found, 555.2831. HPLC purity: 99.05%, retention time = 10.93 min.
30
35
8
3
Nꢀ(3ꢀ(7ꢀ((4ꢀ((2ꢀ(Dimethylamino)ethyl)(methyl)amino)ꢀ2ꢀmethoxyphenyl)amino)ꢀ3ꢀ
isopropylꢀ2,4ꢀdioxoꢀ3,4ꢀdihydropyrimido[4,5ꢀd]pyrimidinꢀ1(2H)ꢀyl)phenyl)acrylamide (20j).
o
1
Yellow solid (yield 37%). mp 150.7ꢀ152.4 C. H NMR (400 MHz, CDCl ) δ 8.94 (s, 1H), 8.45
3
(
s, 1H), 7.95 (s, 1H), 7.83 (s, 1H), 7.68 (d, J = 8.0 Hz, 1H), 7.46 (t, J = 8.0 Hz, 1H), 7.35 (d, J =
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.8 Hz, 1H), 7.04 (d, J = 7.6 Hz, 1H), 6.38 (dd, J = 16.8 Hz, J = 1.6 Hz, 1H), 6.27ꢀ6.17 (m, 2H),
.83 (dd, J = 9.6 Hz, J = 1.6 Hz, 1H), 5.69 (d, J = 10.4 Hz, 1H), 5.36ꢀ5.29 (m, 1H), 3.81 (s, 3H),
5
3.39 (t, J = 7.6 Hz, 2H), 2.88 (s, 3H), 2.45 (t, J = 7.6 Hz, 2H), 2.32 (s, 6H), 1.55 (d, J = 6.8 Hz,
+
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
H). HRMS(ESI) (m/z): (M+H) calcd for C H N O 573.2938, found, 573.2932. HPLC
3
0
37
8
4
purity: 95.07%, retention time = 10.56 min.
Nꢀ(3ꢀ(7ꢀ((4ꢀ(2ꢀ(Dimethylamino)ethoxy)ꢀ2ꢀmethoxyphenyl)amino)ꢀ3ꢀisopropylꢀ2,4ꢀdioxoꢀ
3,4ꢀdihydropyrimido[4,5ꢀd]pyrimidinꢀ1(2H)ꢀyl)phenyl)acrylamide (20k). Yellow solid (yield
o
1
4
1
7
1
3
1%). mp 195.4ꢀ196.7 C. H NMR (400 MHz, DMSOꢀd ) δ 10.39 (s, 1H), 8.84 (s, 1H), 8.64 (s,
6
H), 7.80 (d, J = 4.4 Hz, 1H), 7.71 (s, 1H), 7.48 (t, J = 8.0 Hz, 1H), 7.23 (d, J = 5.6 Hz, 1H),
.10 (d, J = 7.6 Hz, 1H), 6.52ꢀ6.42 (m, 2H), 6.27 (dd, J = 17.2 Hz, J = 1.6 Hz, 1H), 6.01ꢀ5.99 (m,
H), 5.77 (dd, J = 10.0 Hz, J = 1.2 Hz, 1H), 5.15ꢀ5.11 (m, 1H), 3.95 (t, J = 5.2 Hz, 2H), 3.75 (s,
13
H), 2.60 (t, J = 5.2 Hz, 2H), 2.22 (s, 6H), 1.44 (d, J = 6.8 Hz, 6H). C NMR (100 MHz,
DMSOꢀd ) δ 163.19, 160.20, 159.16, 157.83, 155.23, 150.33, 139.68, 136.00, 131.65, 129.27,
6
127.16, 124.32, 121.37, 120.08, 119.10, 103.84, 100.07, 98.76, 65.73, 57.64, 55.73, 45.47, 44.99,
+
1
9.23. HRMS(ESI) (m/z): (M+H) calcd for C H N O 560.2621, found, 560.2625. HPLC
2
9
34
7
5
purity: 98.57%, retention time = 8.78 min.
Nꢀ(3ꢀ(3ꢀIsopropylꢀ7ꢀ((2ꢀmethoxyꢀ4ꢀ(4ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)piperidinꢀ1ꢀyl)phenyl)
amino)ꢀ2,4ꢀdioxoꢀ3,4ꢀdihydropyrimido[4,5ꢀd]pyrimidinꢀ1(2H)ꢀyl)phenyl)acrylamide (20l).
o
1
Yellow solid (yield 43%). mp >300 C. H NMR (400 MHz, DMSOꢀd ) δ 10.57 (s, 1H), 8.82 (s,
6
1
H), 8.57 (s, 1H), 7.91 (s, 1H), 7.70 (s, 1H), 7.47 (t, J = 8.0 Hz, 1H), 7.18 (d, J = 6.8 Hz, 1H),
7.09 (d, J = 7.6 Hz, 1H), 6.56ꢀ6.49 (m, 2H), 6.27 (dd, J = 16.8 Hz, J = 1.6 Hz, 1H), 5.99ꢀ5.97 (m,
1
H), 5.77 (dd, J = 10.0 Hz, J = 1.6 Hz, 1H), 5.17ꢀ5.10 (m, 1H), 3.76 (s, 3H), 3.64ꢀ3.61 (m, 2H),
.96ꢀ2.75 (m, 6H), 2.60ꢀ2.56 (m, 6H), 1.87ꢀ1.85 (m, 1H), 1.54ꢀ1.50 (m, 2H), 1.44 (d, J = 6.8 Hz,
2
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H). C NMR (100 MHz, DMSOꢀd ) δ 163.31, 160.19, 159.07, 157.81, 150.34, 139.83, 135.92,
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31.79, 129.22, 126.99, 119.99, 99.79, 60.77, 55.61, 52.86, 48.21, 46.52, 45.02, 43.23, 27.01,
+
19.24. HRMS(ESI) (m/z): (M+H) calcd for C H N O 654.3516, found, 654.3519. HPLC
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purity: 99.38%, retention time = 11.76 min.
In Vitro Enzymatic Inhibition and Cell Antiproliferation Activity Assay. The enzymatic
inhibition and cell antiproliferation activity assays were evaluated by using the wellꢀestablished
ELISAꢀbased assay and sulforhodamine B (SRB) colorimetric assay, respectively. The detailed
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experiments were performed according to the published literature, and the detailed
characterizations are provided in the Supporting Information.
Western Blot Analysis. H1975 and A431 cells were collected and suspended in lysis buffer
(TrisꢀHCl, pH 6.8, 100 mmol/L; DTT, 200 mmol/L; 0.2% bromophenol blue; 4% SDS; 20%
glycerol). Equivalent amounts of proteins were loaded and separated by SDSꢀPAGE (8%), then
transfered to nitrocellulose membranes. Western blot analysis was subsequently performed with
standard procedures. The antibodies were used for immune detection of proteins, including pꢀ
EGFR (Y1068; #3777S), EGFR (#4267S), pꢀAKT (Ser473), AKT, and GAPDH (Cell Signaling
Technologies, Cambridge, MA).
Human and Mouse Liver Microsomal Stability Assays. The incubation is performed as
follows: microsomes in 0.1 M Tris/HCl buffer of pH 7.4 (0.33 mg/mL microsomal protein), coꢀ
factor MgCl (5 mM), tested compound (final concentration of 0.1 µM, coꢀsolvent (0.01%
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DMSO) and 0.005% Bovin serum albumin) and NADPH (1 mM) at 37 C for 60 min. The
reaction can be started by the addition of liver microsomes, or the tested compound or NADPH.
Aliquots were sampled at 0, 7, 17, 30 and 60 min incubation and enzymatic reaction is stopped
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Page 29 of 52
Journal of Medicinal Chemistry
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by protein precipitation in methanol. After centrifugation, samples are then analysed by LCꢀ
MS/MS (Waters UPLC IꢀClass system; Xevo TQꢀS mass spectrometer).
Measurements of Aqueous Solubility, CYP Inhibition Assays, and Measurement of
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Permeability. Determination of the aqueous solubility was performed according to the published
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literature. The CYP inhibition assays and measurement of permeability of compound 20g were
conducted by WuXi AppTec, and the detailed characterizations are provided in the Supporting
Information.
In Vivo Pharmacokinetics Study in Mice. The in vivo pharmacokinetic study in mice was
conducted by Shanghai Medicilon, Inc. Briefly, the dosing solution (1 mg/mL) was prepared by
dissolving the appropriate amount of the compound in 0.5% CMCꢀNa aqueous solution for oral
administration, and the 0.2 mg/mL dosing solution was prepared by dissolving the appropriate
amount of the compound in mixed solvent (0.15 mL DMSO+1.200 mL PEG400+1.650 mL
Saline) for intravenous administration. Male ICR mice were separately administered to some
groups (three mice /group) for oral (10 mg/kg) or intravenous (1 mg/kg) administration. At time
points 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 h after dosing, the blood sample was collected from
o
each animal and stored in 0−4 C, then separated by centrifugation (8000 r/min for 6 min). All
samples were analyzed using LC−MS/MS (SIMADZU LC system; Applied Biosystems mass
spectrometer), and the acquired data were analyzed by using the WinNonlin (v5.2).
In Vivo Antitumor Efficiency Study. BALB/c mice (4ꢀ6 weeksꢀold) were purchased from
Shanghai Lab. Animal Research Center (Shanghai, China). Briefly, H1975 cell lines and A431
cell lines were cultured in RPMIꢀ1640 (GE Healthcare) supplemented with 10% (v/v) fetal
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bovine serum (Gibico). Then a total volume of 0.1 mL /mouse (approximately 2×10 cells) were
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