Design, synthesis, and biological evaluation of 2-Oxo-3,4- dihydropyrimido[4,5-d]pyrimidinyl derivatives as new irreversible epidermal growth factor receptor inhibitors with improved pharmacokinetic properties

Article abstract of DOI:10.1021/jm4012388

Structural optimization of a series of 2-oxo-3,4-dihydropyrimido[4,5-d] pyrimidinyl compounds, potential new irreversible EGFR inhibitors, was performed to improve pharmacokinetic properties of the compounds. This led to compound 2v with improved aqueous solubility and good pharmacokinetic properties which at the nanomolar level potently inhibits gefitinib-resistant EGFR ^L858R/T790M kinase and displays strong antiproliferative activity against H1975 nonsmall cell lung cancer cells. The new inhibitor also shows promising antitumor efficacy in a murine EGFR^L858R/T790M-driven H1975 xenograft model without effect on body weight. These studies provide new lead compounds for further development of drugs for treatment of gefitinib-resistant nonsmall cell lung cancer patients.

Full text of DOI:10.1021/jm4012388

Article
pubs.acs.org/jmc
Design, Synthesis, and Biological Evaluation of 2‑Oxo-3,4-
dihydropyrimido[4,5‑d]pyrimidinyl Derivatives as New Irreversible
Epidermal Growth Factor Receptor Inhibitors with Improved
Pharmacokinetic Properties
Shilin Xu,†,§ Tianfeng Xu,†,§ Lianwen Zhang,† Zhang Zhang,† Jinfeng Luo,† Yingxue Liu,† Xiaoyun Lu,†
Zhengchao Tu,† Xiaomei Ren,† and Ke Ding*,†
†Institute of Chemical Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, #190 Kaiyuan
Avenue, Guangzhou 510530, China
§University of Chinese Academy of Sciences, #19 Yuquan Road, Beijing 100049, China
S
* Supporting Information
ABSTRACT: Structural optimization of a series of 2-oxo-3,4-
dihydropyrimido[4,5-d]pyrimidinyl compounds, potential new
irreversible EGFR inhibitors, was performed to improve
pharmacokinetic properties of the compounds. This led to
compound 2v with improved aqueous solubility and good
pharmacokinetic properties which at the nanomolar level
potently inhibits gefitinib-resistant EGFRL858R/T790M kinase and
displays strong antiproliferative activity against H1975 non-
small cell lung cancer cells. The new inhibitor also shows
promising antitumor efficacy in a murine EGFRL858R/T790M
-
driven H1975 xenograft model without effect on body weight.
These studies provide new lead compounds for further development of drugs for treatment of gefitinib-resistant nonsmall cell
lung cancer patients.
With respect to the “on-target” toxicity of the current
irreversible EGFR inhibitors, a strategy that has become
attractive is to develop selective EGFRT790M mutant inhibitors
capable of overcoming the clinically acquired resistance in
NSCLC patients. Recently, WZ4002 was identified to inhibit
selectively the proliferation of Ba/F3 cells stably expressing
EGFRT790M mutation, while displaying relatively low potency
with respect to the wild-type kinase.13 Another selective
inhibitor of the EGFRT790M mutant, CO-1686,14 has just
advanced into phase I clinical trials (NCT01526928).15
We have previously reported a number of novel selective
EGFRT790M mutant inhibitors with different chemical scaf-
folds.16 2-Oxo-3,4-dihydropyrimido[4,5-d]pyrimidine (1) has
been demonstrated to selectively inhibit the proliferation of
H1975 NSCLC cells bearing EGFRT790M with an IC50 value of
INTRODUCTION
■
Epidermal growth factor receptor kinase (EGFR, ErbB1) is a
well-validated target used in anticancer drug discovery.1 Several
EGFR inhibitors, such as gefitinib2 and erlotinib,3 have
achieved significant clinical benefits in the management of
nonsmall cell lung cancer (NSCLC) in patients with activating
mutations in the EGFR kinase domain, including L858R and
delE746-A750).4 However, a secondary threonine790
→
methionine790 mutation (T790M) in the EGFR catalytic
domain is responsible for about 50% of the clinical acquired
drug resistance in NSCLC patients.5
Second-generation irreversible EGFR inhibitors have been
developed to overcome the EGFRT790M mutation-related
resistance. CI-1033,6 HKI-272,7 and PF-00299804,8 for
example, have been advanced into late-stage clinical develop-
ment.9 Most recently, gilotrif (BIBW-2992) was approved by
the US FDA for treatment of late-stage NSCLC patients with
actively mutated EGFR.10 However, almost all of the molecules
lack selectivity between EGFRT790M mutants and the wild-type
kinase, and this leads to relatively low maximum tolerated dose
(MTD) and dosing-limiting toxicity in NSCLC patients.11 Only
limited clinical efficacy was observed for these inhibitors, and
this may be due to the fact that the in vivo concentrations
achieved are insufficient to suppress the functions of
EGFRT790M mutant.11,12
14 nM, although it was significantly less potent on cancer cells
16a
or normal cells with EGFRWT
.
This compound, orally
administered, also showed promising antitumor efficacy in a
H1975 xenograft mouse model. However, its relatively low
aqueous solubility (4.0 μg/mL at pH 6.8) and unsatisfactory
pharmacokinetic properties may limit its further development
(Table 1).17
Received: August 9, 2013
Published: October 14, 2013
© 2013 American Chemical Society
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Table 1. In Vitro EGFR Inhibition and Antiproliferation Data for Compounds 2a−v
a
EGFR kinase activity assays were performed using the FRET-based Z′-Lyte assay according to the manufacturer’s instructions.22 The compounds
b
were incubated with the kinase reaction mixture for 1.5 h before measurement. Reported data are means from two independent experiments. The
antiproliferative activities of the compounds were evaluated using the MTS assay. The data are means from at least four independent experiments.
c
d
H1975 is a gefitinib-resistant human lung cancer cell line (EGFRL858R/T790M). Measured at pH 6.8. Data from ref 16a.
In this paper, we describe further optimization of compound
1 with the aim of improving its aqueous solubility and oral
absorption properties. This has led to the identification of 2v
(Figure 1) which has significantly improved pharmacokinetic
properties. It also displays promising antitumor efficacy in the
H1975 xenograft mouse model and represents a new attractive
lead for further development of compounds that can overcome
EGFRT790M mutation-induced resistance in NSCLC patients.
CHEMISTRY
■
Figure 1. Design of new EGFRT790M inhibitors with improved
pharmacokinetics.
The synthesis of compound 2 is shown in Scheme 1.
Commercially available ethyl 4-chloro-2-(methylthio)-
pyrimidine-5-carboxylate (3) was reacted with the protected
aliphatic amine 4 to produce the adduct 5 in high yields. The
reduction of 5 with lithium aluminum hydride gave alcohol 6,
which was subsequently oxidized with MnO2 to obtain the
aldehyde 7. Reductive amination of aldehyde 7 with diverse
amines yielded the corresponding compound 8. The key
intermediate 10 was prepared by direct cyclization using
triphosgene and subsequent oxidation with m-CPBA. Nucleo-
philic coupling of intermediates 10 with different substituted
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a
Scheme 1. Synthesis of Compounds 2
a
Reagent and conditions: (a) K2CO3 (2 equiv), DMF, 60 °C, 85−97%; (b) LiAlH4 (2 equiv), THF, −40 °C to 0 °C, 40−58%; (c) MnO2, CH2Cl2,
rt, 84−93%; (d) R1NH2, AcOH, NaBH4, MeOH, 0 °C to rt, 72−90%; (e) triphosgene (0.34 equiv), Et3N (3.0 equiv), CH2Cl2, 0 °C to rt, 88−96%;
(f) 3-chloroperbenzoic acid (3.0 equiv), CH2Cl2, rt, 80−92%; (g) RNH2, (1.2 equiv), trifluoroacetic acid (1.2 equiv), 2-BuOH, 110 °C; (h)
trifluoroacetic acid (10 equiv), CH2Cl2, rt, 38−60% (for two steps); (i) acryloyl chloride (1.2 equiv), DIPEA (2 equiv), CH2Cl2, 0 °C to rt, 75−90%.
amines followed by treatment with trifluoroacetic acid afforded
the key intermediates 12, which react with acryloyl chloride to
produce the final products, 2.
are 709 nM and 2.7 μM, respectively (Table 1). Other less
planar linkers such as 4-piperidinyl (2b), (R)-3-piperidinyl
(2c), (S)-3-piperidinyl (2d), (R)-3-pyrrolidinyl (2e), (S)-3-
pyrrolidinyl (2f), and 2-aminoethyl moieties (2g) also
significantly improve the aqueous solubility of the resulting
compounds (Table 1), but these groups have a different impact
on the EGFRL858R/T790M kinase inhibitory activities. For
instance, compound 2b has an aqueous solubility of 9.3 μg/
mL at pH 6.8, slightly lower than that of 2a, but its
EGFRL858R/T790M inhibitory activity is 5 times greater than
that of 2a.
RESULTS AND DISCUSSION
■
Compound 1 was identified as a selective EGFRT790M inhibitor,
which in a FRET-base Z′-Lyte assay potently inhibits
EGFRL858R/T790M kinase activity with an IC50 of 0.7 nM. It
also displays strong antiproliferative effects (IC50 = 14 nM) on
16a
H1975 NSCLC cells harboring EGFRL858R/T790M
.
However,
it has relatively low aqueous solubility (4.0 μg/mL at pH 6.8)
and generally unsatisfactory pharmacokinetic properties in
Sprague−Dawley (SD) rats. Structure feature analysis revealed
that compound 1 is highly planar, and this may greatly
contribute to its low aqueous solubility. Disruption of
molecular planarity is expected to reduce the crystal-stacking
capability of a solid solute in water and becomes an effective
strategy to improve the water solubility of a compound.17a,18
Our previous investigations have demonstrated that the “U-
shaped” configuration of rings A−D (Figure 1), together with
the aniline ring (C) bearing a hydrophilic group and the
acrylamide moiety, is necessary to maintain the EGFR
inhibitory activity.16a The phenyl ring D functions as a linker
suitable for the acrylamide to chelate with Cys797 in EGFR
proteins and provides an ideal position at which to reduce the
molecular planarity, potentially improving the aqueous
solubility (Figure 1).17a,18 Consequently, we first replaced the
phenyl ring D in compound 1 with a less planar cyclohexyl ring.
The resulting compound 2a indeed displays an improved
aqueous solubility of 18.8 μg/mL (pH 6.8), 4 times that of
compound 1. However, the introduction of a cyclohexyl group
causes a loss of about 1000-fold of potency in both
EGFRL858R/T790M suppression and H1975 cell growth inhibition
assays. For suppression of the enzymatic function of
EGFRL858R/T790M and inhibition of the proliferation of H1975
NSCLC cells, the corresponding IC50 values of compound 2a
Further investigation revealed that the chirality of the linker
could affect the EGFRL858R/T790M inhibitory activity of the
compounds. Compound 2c, with a (R)-3-piperidinyl linker has
an IC50 value of 40.3 nM with EGFRL858R/T790M, while the (S)-
3-piperidinyl-linked compound 2d is 5 times less potent. On
the other hand, compound 2f containing a (S)-3-pyrrolidinyl
moiety was about 12 times as potent as the R-enantiomer 2e,
which strongly inhibits EGFRL858R/T790M with an IC50 value of
8.8 nM. Compound 2f also potently suppresses the
proliferation of H1975 NSCLC cells, which possess the
EGFRL858R/T790M-resistant mutant, with an IC50 value of 0.28
μM. The aqueous solubility of compound 2f is 107.1 μg/mL
(pH 6.8), 26 times greater than that of the original compound
1. Interestingly, the replacement of pyrrolidinyl ring with an
ethenyl chain (2g) almost totally abolishes the kinase inhibitory
activity.
In view of its promising enzymatic and cellular potencies and
aqueous solubility, we further investigated the pharmacokinetic
properties of 2f in SD rats (Table 2). The compound displays
encouraging oral bioavailability (F = 24%) and could serve as a
new starting point for further structural optimization. We first
studied the impact of the 3-substituent on the biological activity
of the compound and showed that when the 3-benzyl group in
compound 2f was replaced with methyl (2h), isopropyl (2i),
cyclopentyl (2j), or cyclohexanyl (2k), the EGFRL858R/T790M
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appeared to inhibit EGFR kinase somewhat comparably, and
deletion of both 2′-methoxyl and 4-methylpiperazin-1-yl
moieties, giving 2m, caused a 15-fold potency loss, emphasizing
the relevance of the hydrophilic group to EGFR kinase
inhibition. The compound (2n) lacking a methoxyl group,
however, shows no obvious improvement in its pharmacoki-
netic properties in SD rats. Other modifications, such as
switching the 4′-(4-methylpiperazin-1-yl) group in 2n to the 3′-
position (2o), or replacing the 4′-(4-methylpiperazin-1-yl)
group in 2n with a 4-(N-methylpiperidinyl) (2p) or a 4-(4-
methylpiperazin-1-yl)ethyl moiety (2q), failed to improve the
compounds’ pharmacokinetic properties in SD rats or benefit
the kinase inhibitory activities.
Further investigation revealed that the 3′-methoxylated
compound (2r) and the 2′-methoxylated compound (2l)
have similar EGFRL858R/T790M inhibitory potencies and
pharmacokinetic properties. However, in contrast to our
previous observation that 2′-fluorine substituents in ring C
are detrimental to the kinase inhibitory activity,16a the 3′-
fluorine-substituted compound 2s shows similar potency to that
of 2l. In SD rats, compound 2s displays an improved maximum
concentration (Cmax = 964 μg/L) and lower oral clearance (Cl
= 7.3 L/h/kg) than that of 2l, which might be due to the strong
electron-withdrawing properties of fluorine, decreasing the
electron density in the phenyl ring C.21 However, when the 3′-
F is replaced with 3′-CF3 (2t) or 3′-Cl (2u), the resulting
compounds are 2−3-fold less potent. When a methyl group is
introduced to the 3′-position of phenyl ring C, the resulting
compound 2v not only displays good biological activities (IC50
of EGFRL858R/T790M = 9.2 nM, and IC50 of H1975 = 47 nM) but
also promising pharmacokinetic properties. The compound
displays a reasonable maximum concentration (Cmax = 1040
μg/L), acceptable oral clearance rate (Cl = 10 L/h/kg), and
oral bioavailability (30%) in SD rats at an oral dose of 25 mg/
kg. This might be attributed to the significant improvement of
its aqueous solubility (1265 μg/mL at pH 6.8).
Table 2. Sprague-Dawley Rat PK Parameters for
Compounds 1, 2f, 2l−s, and 2v
a
compd route Cmax (μg/L) AUC(0‑∞) (μg·h/L) Cl (L/h/kg) F (%)
b
1
iv
4369
−
784
1568
−
359
3.2
−
24
44
44
54
20
po
iv
−
2f
14.4
62.6
5.2
po
iv
102
418
2l
1789
613
962
po
iv
2125
2034
4520
857
12.0
2.5
2m
2n
2o
2p
2q
2r
2s
2v
6544
1673
1525
783
po
iv
5.7
5.8
po
iv
2312
746
12.2
6.8
1823
291
po
iv
760
33.9
−
20.1
c
−
359
−
1305
−
po
iv
1413
232
561
9.2
40
20
33
30
po
iv
1126
949
22.9
5.5
3408
420
po
iv
948
28
3263
964
2118
3480
1640
2491
2.4
po
iv
7.3
3975
1040
3.1
po
10.0
a
Compounds were formulated in 2% DMSO, 4% EtOH, 4% castor oil,
and 90% H2O. The pharmacokinetic parameters were determined after
a single oral administration (25 mg/kg, n = 4 rats) and intravenous
b
injection (5 mg/kg, n = 4 rats). Compound 1 was treated as a
mesylate salt and was not detected when administrated by oral dosing.
c
Compound 2p was not administrated by intravenous injection due to
insolubility in the vehicle.
inhibitory activities decrease between 5- and 100-fold,
displaying IC50 values of 525, 864, 58.7, or 42.9 nM,
respectively. When a phenyl group was introduced at the 3-
position, however, the resulting compound 2l showed
inhibitory effect against EGFRL858R/T790M enzyme and the
proliferation of H1975 NSCLC cells comparable to that of 2f,
with IC50 values of 15.2 and 111.0 nM, respectively.
Encouragingly, further studies revealed that compound 2l
displays a 14-fold improvement of aqueous solubility (1535.1
μg/mL at pH 6.8) compared to that of 2f. The compound also
has greater oral bioavailability than that of 2f (F = 44%), higher
maximal plasma concentration (Cmax = 613 μg/L), and lower
clearance rate (Cl = 12 L/h/kg) in SD rats at an oral dose of 25
mg/kg.
The kinase inhibition of 2v was further validated by
investigating its suppressive functions on the phosphorylation
of EGFR and the downstream signaling proteins in NSCLC
H1975 cells harboring EGFRL858R/T790M. As shown in Figure 2,
the treatment of compound 2v causes a dose-dependent
reduction of the phosphorylation of EGFR in H1975 cells. The
protein levels of downstream signals, such as p-AKT and p-
Structural optimization of phenyl ring C, the (N-
methylpiperazinyl)aniline, was also performed to potentially
improve the metabolic stability. It was clear that the 2′-
methoxy-4-(N-methylpiperazinyl)aniline group possesses high
electron density and could be oxidized to a reactive quinone
diimine to give rise to idiosyncratic toxicity and impair the oral
absorption.19 Decreasing the electron density has been proved
to be an efficient approach to increase the metabolic stability of
aromatics and improve the pharmacokinetic properties of a
molecule,20 and we first removed the electron-donating 2′-
methoxyl and 4-methylpiperazin-1-yl moieties in compound 2l.
The resulting compound 2m, at an oral dose of 25 mg/kg,
Figure 2. 2v dose-dependently decreases the phosphorylation of
EGFR, AKT, and ERK. H1975 cells were treated with vehicle
(DMSO) or 3 to 330 nM 2v for 2 h. The cells were lysed, and the
proteins were determined by a Western blot assay. The results are
representative of three independent experiments.
indeed displayed a higher maximum concentration (Cmax
=
1673 μg/L) and a lower oral clearance (Cl = 5.7 L/h/kg) in SD
rats than that of 2l. The investigation also showed that
compound 2n, formed by removal of the 2′-methoxyl group,
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Table 3. Antiproliferative Activities of 2v against a Panel of Cells with Wild-Type EGFR
a
antiproliferation (IC50, μM)
b
b
b
b
c
d
H460
16.0 8.9
95D
4.0 2.4
H1299
5.1 3.4
H358
>20.0
HL-7702
>10.0
HCC827
2v
0.005 0.002
a
The antiproliferative activities of compound 2v were evaluated using the MTS assay. The data are means from at least three independent
b
c
experiments. H460, 95D, H358, and H1299 are human NSCLC cells harboring wild-type EGFR. HL-7702 is a diploid human liver cell line.
d
HCC827 is a human NSCLC cell line with EGFRdel E746‑A750
.
Figure 3. In vivo effect of compound 2v on tumor volume and body weight in xenograft model of H1975. (A) Antitumor efficacy of compound 2v in
a human NSCLC (H1975) xenograft nude mouse model. (B) Body weight of mice during treatment with 2v for 2 weeks. Mice were orally dosed
once daily (qd) for 14 days at 10 mg/kg or 30 mg/kg, and were monitored for signs of morbidity. Tumors and body weight were measured every
other day (*p < 0.05, **p < 0.01).
ERK, are also clearly inhibited by 2v at concentrations of 110
nM and 33 nM, respectively, while the total protein levels of
EGFR, AKT, ERK, and GAPDH remain unchanged. Not
surprisingly, the phosphorylation of EGFR and the downstream
targets are barely affected by gefitinib at the relatively high
concentration of 330 nM. Further investigation revealed that
The antiproliferative effects of 2v against a panel of NSCLC
cells with different EGFR status were also investigated (Table
3). Compound 2v potently inhibits the growth of H1975
NSCLC cells harboring the EGFRL858R/T790M mutation and
HCC827 cells with EGFRdel E746‑A750, at IC50 values of 0.047
and 0.005 μM, respectively. However, it is approximately 85−
4000-fold less potent to other NSCLC cancer cells with
EGFRWT. For example, its IC50 values are 16.0, 4.0, 5.1 and over
20.0 μM against H460, 95D, H1299, and H358 NSCLC cancer
cells, respectively. In addition, 2v also displayed IC50 values
over 200 times greater on normal human liver than that of
H1975, suggesting that its cytotoxic effects are minimal.
Given its strong inhibition of EGFRL858R/T790M kinase and on
NSCLC cell growth, together with its promising pharmacoki-
netic properties, the in vivo antitumor efficacy of 2v was
evaluated using an EGFRL858R/T790M-driven human H1975
xenograft mouse model. Repeated oral administration of 2v
(qd) for 14 days displays dose-dependent inhibition of tumor
growth. The tumor growth inhibition (TGI) values are 35.4%
and 56.8% at dosages of 10 and 30 mg/kg/day, respectively
(Figure 3A). In addition, all doses of 2v are well-tolerated, with
no mortality or significant loss of body weight (<5% relative to
the vehicle-matched controls) observed during treatment
(Figure 3B).
compound 2v also potently inhibited the kinase activities of
L851Q
EGFR T790M, EGFR L858R, and EGFR
mutants with IC50
values of 10.0, 3.0, and 3.0 nM, respectively. But its activity
against EGFRWT is obviously less potent in a Western blot
analysis in A431 human epithelial carcinoma cells with
overexpressed EGFRWT (Supporting Information).
The selectivity profile of 2v against a panel of 456 kinases
(including 395 different wild-type kinases) was also investigated
by using KINOMEscan platform (Ambit Bioscience, San Diego,
CA) at a concentration of 100 nM which was about 70 times of
its Kd value on EGFRL858R/T790M (Kd = 1.4 nM, Supporting
Information). The results clearly revealed that, in addition to
EGFR and the other ERBB family members (ERBB2, ERBB3,
and ERBB4), compound 2v also exhibited strong binding with
Abl1, Blk, Btk, DDR1, Flt3, Kit, Jak3, MEK5, PDGFRβ, RET,
Src, and several other kinases, indicating that this compound
might be used as a lead compound for treatment of many other
types of cancer. However, the potential safety issue of 2v
related to its relative kinase selectivity requires further
investigation.
To validate the irreversible inhibition of compound 2v
against EGFR, an inhibitor washout assay was also performed in
HCC827 NSCLC cells with activating EGFR mutation
(Supporting Information). The results clearly showed that the
EGFR autophosphorylation in HCC827 cells was obviously
recovered soon after the reversible inhibitor was washed out.
However, the EGFR phosphorylation recovery could never be
observed after 2v was removed for 24 h, which strongly
suggested that the compound was an irreversible inhibitor of
EGFR.
SUMMARY
■
We have described structural optimization aimed at improving
the aqueous solubility and pharmacokinetic properties of a
series of irreversible EGFR inhibitors based on a 2-oxo-3,4-
dihydropyrimido[4,5-d]pyrimidinyl analogue. One of the most
promising compounds, 2v, displays an aqueous solubility of
1265 μg/mL, some 300-fold greater than that of the original
lead compound 1 (4.0 μg/mL). Compound 2v potently
inhibits the enzymatic function of EGFRL858R/T790M with an
IC50 value of 9.2 nM and suppresses proliferation of gefitinib-
resistant H1975 human NSCLC cells with an IC50 value of 47
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7a−v. MnO2 (3g, 34.59 mmol) was added to a solution of 6a (4.25 g,
11.53 mmol) in CH2Cl2 (100 mL). The resulting solution was stirred
at room temperature overnight, and then the reaction mixture was
filtered through a pad of Celite. Filtration and removal of the solvent
gave an oily crude product that was subjected to flash column
chromatography (SiO2, petroleum ether/EtOAc stepwise elution, 3:1
nM. The compound also exhibits good pharmacokinetic
properties with oral bioavailability of 30%. Furthermore, it
displayed promising antitumor efficacy in vivo in an
EGFRL858R/T790M-driven human H1975 xenograft mouse
model without obvious toxic signs during the treatment. Our
studies provide a new lead compound with improved
pharmacokinetic properties and acceptable in vivo antitumor
activity for further development of the drug for treatment of
gefitinib-resistant NSCLC. Further investigation on the
compound is in progress and will be reported in due course.
1
to 1:1) to give 7a (3.8 g, yield 90%). H NMR (400 MHz, CDCl3) δ
9.68 (s, 1H), 8.54 (s, 1H), 8.31 (s, 1H), 4.40 (s, 1H), 4.11−4.04 (m,
1H), 3.48 (s, 1H), 2.54 (s, 3H), 2.14−2.09 (m, 4H), 1.45 (s, 9H),
1.41−1.22 (m, 4H). MS (ESI) m/z 367.2 [M + H]+.
tert-Butyl 4-(5-((Benzylamino)methyl)-2-(methylthio)-
pyrimidin-4-ylamino)cyclohexylcarbamate (8a). General proce-
dure for syntheses of 8a−v. Benzylamine (1 g, 9.25 mmol) and AcOH
(0.1 mL) were added at room temperature to a solution of 7a (2.26 g,
6.17 mmol) in MeOH (50 mL). After the amine was converted
completely to the imine, NaBH4 (469 mg, 12.34 mmol) was added to
the reaction mixture at 0 °C. The resulting mixture was concentrated
in vacuo, partitioned between CH2Cl2 and saturated NaHCO3, and
extracted with CH2Cl2. The organic layer was separated, washed with
brine, dried with Na2SO4, filtered, and concentrated in vacuo. The
resulting crude material was subjected to flash column chromatog-
raphy (SiO2, petroleum ether/EtOAc, 3:1) to give 8a (2.46 g, yield
EXPERIMENTAL SECTION
■
General Methods for Chemistry. All reagents and solvents were
used as purchased from commercial sources. Flash chromatography
was performed using 300 mesh silica gel. All reactions were monitored
by TLC, using silica gel plates with fluorescence F254 and UV light
visualization. 1H NMR spectra was recorded on a Bruker AV-400
spectrometer at 400 MHz or a Bruker AV-500 spectrometer at 500
MHz. 13C NMR spectra was recorded on a Bruker AV-500
spectrometer at 125 MHz. Coupling constants (J) are expressed in
hertz (Hz). Chemical shifts (δ) of NMR are reported in parts per
million (ppm) units relative to an internal standard (TMS). Low
resolution ESI-MS were recorded on an Agilent 1200 HPLC-MSD
mass spectrometer and high resolution ESI-MS on an Applied
Biosystems Q-STAR Elite ESI-LC-MS/MS mass spectrometer. Purity
of compounds was determined by reverse-phase high performance
liquid chromatography (HPLC) analysis to be >95%. HPLC
instrument: Dionex Summit HPLC (Column: Diamonsil C18, 5.0
μm, 4.6 × 250 mm (Dikma Technologies); detector: PDA-100
photodiode array; injector: ASI-100 autoinjector; pump: p-680A). A
flow rate of 1.0 mL/min was used with mobile phase of MeOH in H2O
with 0.1% modifier (ammonia, v/v).
1
87%). H NMR (500 MHz, CDCl3) δ 7.71 (s, 1H), 7.36−7.33 (m,
2H), 7.30−7.28 (m, 2H), 7.25 (s, 1H), 4.42 (s, 1H), 3.94−3.92 (m,
1H), 3.70 (s, 2H), 3.65 (s, 2H), 3.47 (s, 1H), 2.48 (s, 3H), 2.12−2.06
(m, 4H), 1.77 (br, 2H), 1.45 (s, 9H), 1.31−1.24 (m, 4H). MS (ESI)
m/z 458.1 [M + H]+.
tert-Butyl 4-(3-Benzyl-7-(methylthio)-2-oxo-3,4-
d i h y d r o p y r i m i d o [ 4 , 5 - d ] p y r i m i d i n - 1 ( 2 H ) - y l ) -
cyclohexylcarbamate (9a). General procedure for syntheses of 9a−
v. N,N-Diisopropylethylamine (2.8 mL, 16.14 mmol) and triphosgene
(0.58 g, 2 mmol) were added to a solution of 8a (2.46 g, 5.38 mmol)
in CH2Cl2 at 0 °C. The mixture was poured into a solution of 10%
aqueous NaHCO3 and extracted with CH2Cl2. The organic layer was
separated, washed with 10% aqueous NaHCO3 and brine, dried with
Na2SO4, filtered, and concentrated in vacuo to give 9a (2.50 g, yield
Ethyl 4-(4-(tert-Butoxycarbonylamino)cyclohexylamino)-2-
(methylthio)pyrimidine-5-carboxylate (5a). General procedure
for syntheses of 5a−v. Compound 4a (6.63 g, 30.94 mmol) and
potassium carbonate (7.1 g, 51.56 mmol) were added to a solution of
3 (6 g, 25.78 mmol) in DMF (50 mL). The resulting solution was
stirred at 60 °C overnight. After cooling to room temperature, ice−
water (500 mL) was added to the reaction mixture. The precipitate
was filtered, and the filter cake was rinsed with additional cool water
and then dried in a vacuum oven to give 5a (9.21 g, yield 87%), which
was used without further purification. 1H NMR (400 MHz, CDCl3) δ
8.62 (s, 1H), 8.22 (d, J = 6.0 Hz, 1H), 4.40 (s, 1H), 4.31 (q, J = 7.2
Hz, 2H), 4.08−3.99 (m, 1H), 3.47 (s, 1H), 2.52 (s, 3H), 2.14−2.07
(m, 4H), 1.45 (s, 9H), 1.41−1.40 (m, 2H), 1.36 (t, J = 7.2 Hz, 3H),
1.31−1.22 (m, 2H). MS (ESI) m/z 411.2 [M + H]+.
tert-Butyl 4-(5-(Hydroxymethyl)-2-(methylthio)pyrimidin-4-
ylamino)cyclohexylcarbamate (6a). General procedure for syn-
theses of 6a−v. A suspension of LiAlH4 (1.7 g, 44.82 mmol) in
anhydrous THF (50 mL) was added dropwise to a solution of 5a (9.2
g, 22.41 mmol) in anhydrous THF (200 mL) under stirring for 0.5 h
at −40 °C. The reaction mixture was stirred until the temperature was
warmed to 0 °C, and then it was treated with 2 mL of H2O to
decompose unreacted LiAlH4. Then, a further 6 mL of H2O was added
to the reaction mixture followed by 15% aqueous NaOH (2 mL). The
resulting mixture was stirred for 10 min at room temperature and
filtered through a pad of Celite. The Celite was washed with THF, and
the washes were combined and then concentrated in vacuo. The
concentrated mixture was poured into H2O and extracted with
CH2Cl2. The organic layer was separated, washed with brine, dried
with Na2SO4, filtered, and concentrated in vacuo. The resultant crude
material was purified by column chromatography (SiO2, CH2Cl2/
MeOH stepwise elution, 40:1 to 20:1) to give 6a (4.25 g, yield 51%).
1H NMR (400 MHz, CDCl3) δ 7.63 (s, 1H), 5.86 (d, J = 7.2 Hz, 1H),
1
96%), which was used without further purification. H NMR (500
MHz, CDCl3) δ 7.94 (s, 1H), 7.36−7.33 (m, 2H), 7.32−7.28 (m, 3H),
4.73−4.68 (m, 1H), 4.63 (s, 2H), 4.40 (s, 1H), 4.12 (s, 2H), 3.52 (s,
1H), 2.55 (s, 3H), 2.14 (d, J = 8.8 Hz, 2H), 1.76 (d, J = 8.8 Hz, 2H),
1.45 (s, 9H), 1.31−1.22 (m, 4H). MS (ESI) m/z 484.2 [M + H]+.
tert-Butyl 4-(3-Benzyl-7-(methylsulfonyl)-2-oxo-3,4-
d i h y d r o p y r i m i d o [ 4 , 5 - d ] p y r i m i d i n - 1 ( 2 H ) - y l ) -
cyclohexylcarbamate (10a). General procedure for syntheses of
10a−v. m-CPBA (75%) (2.4 g, 10.34 mmol) was added to a solution
of 9a (2.5 g, 5.17 mmol) in anhydrous CH2Cl2 (50 mL) at 0 °C under
argon. The reaction mixture was allowed to warm to room
temperature and then stirred for 4 h. The solution was diluted with
CH2Cl2 and then treated with 50% Na2S2O3/NaHCO3 solution. The
organic layer was separated, washed with brine, dried with Na2SO4,
filtered, and concentrated in vacuo. The resulting crude material was
subjected to flash column chromatography (SiO2, CH2Cl2/MeOH
1
stepwise elution, 40:1 to 20:1) to give 10a (2.4 g, yield 90%). H
NMR (500 MHz, CDCl3) δ 8.26 (s, 1H), 7.39−7.33 (m, 3H), 7.32−
7.29 (m, 2H), 4.77−4.71 (m, 1H), 4.66 (s, 2H), 4.40 (s, 1H), 4.27 (s,
2H), 3.53 (s, 1H), 3.31 (s, 3H), 2.71−2.64 (m, 2H), 2.16 (d, J = 8.4
Hz, 2H), 1.78 (d, J = 9.2 Hz, 2H), 1.60 (s, 9H), 1.34−1.27 (m, 2H).
1-(4-Aminocyclohexyl)-3-benzyl-7-(2-methoxy-4-(4-methyl-
piperazin-1-yl)phenylamino)-3,4-dihydropyrimido[4,5-d]-
pyrimidin-2(1H)-one (12a). General procedure for syntheses of
12a−v. 2-Methoxy-4-(4-methylpiperazin-1-yl)aniline (464 mg, 2.10
mmol) was added to a solution of 10a (0.9 g, 1.75 mmol), in 2-butyl
alcohol (5 mL), followed by trifluoroacetic acid (157 μL, 2.10 mmol).
The reaction mixture was stirred for 18 h at 110 °C in a sealed tube.
The reaction mixture was cooled to room temperature and
concentrated in vacuo to give crude product 11a which was used in
the next step without characterization. Then trifluoroacetic acid (0.5
mL) was added to a solution of 11a in CH2Cl2 (5 mL), and the
resulting mixture was stirred at room temperature for 4 h. The reaction
mixture was diluted with CH2Cl2, basified with saturated aqueous
4.46 (s, 2H), 4.45 (s, 1H), 4.00−3.93 (m, 1H), 3.41 (s, 1H), 2.83 (s,
1H), 2.47 (s, 3H), 2.13−2.03 (m, 4H), 1.43 (s, 9H), 1.35−1.26 (m,
4H). MS (ESI) m/z 369.2 [M + H]+.
tert-Butyl 4-(5-Formyl-2-(methylthio)pyrimidin-4-ylamino)-
cyclohexylcarbamate (7a). General procedure for syntheses of
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NaHCO3 to pH = 9, and extracted with CH2Cl2. The organic layer was
separated, washed with 10% aqueous NaHCO3 and brine, dried with
Na2SO4, filtered, and concentrated in vacuo. The resultant crude
material was purified by column chromatography (SiO2, CH2Cl2/
MeOH/NH4OH, 40:1:0.4) to give 12a (485 mg, yield 50% for two
(S)-1-(1-Acryloylpiperidin-3-yl)-3-benzyl-7-(2-methoxy-4-(4-
methylpiperazin-1-yl)phenylamino)-3,4-dihydropyrimido[4,5-
d]pyrimidin-2(1H)-one (2d). [α]2D0 −124.090 (c 0.137, CHCl3). H
1
NMR (400 MHz, CDCl3, a mixture of rotamers) δ 8.00 (d, J = 8.8 Hz,
1H), 7.89 (s, 0.6H), 7.86 (s, 0.4H), 7.37−7.29 (m, 6H), 6.67−6.60 (m,
1H), 6.53−6.47 (m, 2H), 6.30 (t, J = 16.4 Hz, 1H), 5.72−5.61 (m,
1H), 4.83−4.77 (m, 1H), 4.72−4.55 (m, 3H), 4.18−4.15 (m, 0.5H),
4.11 (s, 2H) 4.00−3.97 (m, 1H), 3.87 (s, 3H), 3.83−3.77 (m, 0.5H),
3.18−3.16 (m, 4H), 3.10−2.67(m, 2H), 2.62−2.60 (m, 4H), 2.37 (s,
3H), 1.98−1.86 (m, 2H), 1.63−1.60 (m, 1H). 13C NMR (125 MHz,
CDCl3) δ 165.6, 165.1, 159.3, 157.3, 153.7, 153.5, 149.8, 149.6, 147.7,
147.6, 136.2, 136.1, 128.8, 128.0, 127.8, 127.7, 127.3, 121.9, 121.5,
121.1, 120.7, 108.0, 102.2, 102.0, 100.6, 100.3, 55.6, 55.1, 52.5, 51.3,
50.1, 49.9, 48.2, 46.1, 44.4, 43.7, 42.3, 27.5, 27.3, 26.5, 25.4. HRMS
(ESI) for C33H40N8O3 [M + H]+, calcd: 597.3296, found: 597.3291.
HPLC analysis: MeOH−H2O (78:22), 12.63 min, 98.5% purity.
(R)-1-(1-Acryloylpyrrolidin-3-yl)-3-benzyl-7-(2-methoxy-4-
(4-methylpiperazin-1-yl)phenylamino)-3,4-dihydropyrimido-
[4,5-d]pyrimidin-2(1H)-one (2e). [α]2D0 +1.075 (c 0.186, CHCl3).
1H NMR (400 MHz, CDCl3, a mixture of rotamers) δ 7.95 (d, J = 8.4
1
steps). H NMR (500 MHz, CDCl3) δ 8.07−8.06 (m, 1H), 7.82 (s,
1H), 7.34−7.32 (m, 2H), 7.29−7.26 (m, 4H), 6.55−6.54 (m, 2H),
4.73−4.66 (m, 1H), 4.61 (s, 2H), 4.06 (s, 2H), 3.86 (s, 3H), 3.18 (t, J
= 4.0 Hz, 4H), 2.81−2.76 (m, 1H), 2.64−2.57 (m, 6H), 2.35 (s, 3H),
1.98 (d, J = 9.2 Hz, 2H), 1.75 (d, J = 8.8 Hz, 2H), 1.32−1.23 (m, 2H).
MS (ESI) m/z 557.3 [M + H]+.
N-(4-(3-Benzyl-7-(2-methoxy-4-(4-methylpiperazin-1-yl)-
phenylamino)-2-oxo-3,4-dihydropyrimido[4,5-d]pyrimidin-
1(2H)-yl)cyclohexyl)acrylamide (2a). General procedure for
syntheses of 2a−v. N,N-Diisopropylethylamine (300 μL, 1.742
mmol) and acryloyl chloride (85 μL, 1.045 mmol) were added to a
solution of 12a (485 mg, 0.871 mmol) in anhydrous CH2Cl2 (10 mL)
with stirring at 0 °C. The reaction was allowed to warm to room
temperature, and then, after stirring for 4 h, the reaction mixture was
poured into a solution of 10% aqueous NaHCO3 and extracted with
CH2Cl2. The organic layer was separated, washed with 10% aqueous
NaHCO3 and brine, dried with Na2SO4, filtered, and concentrated in
vacuo. The resultant crude material was purified by column
chromatography (SiO2, CH2Cl2/MeOH/NH4OH, 40:1:0.4) to give
Hz, 1H), 7.90 (s, 0.6H), 7.89 (s, 0.4H), 7.38−7.23 (m, 6H), 6.58−6.38
(m, 4H), 5.72−5.55 (m, 2H), 4.72−4.56 (m, 2H), 4.31−4.27 (m,
0.6H), 4.13 (s, 1.2H), 4.12 (s, 0.8H), 4.10−3.89 (m, 1.7H), 3.87 (s,
1.8H), 3.86 (s, 1.2H), 3.85−3.80 (m, 0.7H), 3.67−3.51 (m, 1H),
3.20−3.15 (m, 4H), 2.99−2.85 (m, 1H), 2.62−2.59 (m, 4H), 2.37 (s,
3H), 2.28−2.08 (m, 1H). 13C NMR (125 MHz, CDCl3) δ 164.6,
164.3, 159.2, 157.1, 157.0, 153.7, 153.6, 153.5, 149.7, 149.5, 147.7,
136.1, 136.0, 128.8, 128.3, 128.1, 127.9, 127.8, 127.4, 127.3, 121.6,
121.3, 120.7, 120.3, 107.9, 107.8, 102.2, 100.4, 100.1, 55.6, 55.1, 52.4,
51.6, 51.4, 50.8, 50.0, 49.7, 46.9, 46.7, 46.1, 45.7, 44.4, 43.8, 28.2, 25.8.
HRMS (ESI) for C32H38N8O3 [M + H]+, calcd: 583.3140, found:
583.3138. HPLC analysis: MeOH−H2O (78:22), 11.28 min, 98.5%
purity.
1
2a (425 mg, yield 80%). H NMR (500 MHz, CDCl3) δ 8.07 (d, J =
8.5 Hz, 1H), 7.84 (s, 1H), 7.36−7.29 (m, 5H), 7.24 (s, 1H), 6.70 (d, J
= 8.5 Hz, 1H), 6.29 (d, J = 17.0 Hz, 1H), 6.08 (dd, J = 10.0, 17.0 Hz,
1H), 5.64 (d, J = 10.0 Hz, 1H), 5.42 (d, J = 7.5 Hz, 1H), 4.71 (t, J =
12.0 Hz, 1H), 4.63 (s, 2H), 4.08 (s, 2H), 4.04−3.97 (m, 1H), 3.89 (s,
3H), 3.21 (s, 4H), 2.78 (q, J = 12.0 Hz, 2H), 2.61 (s, 4H), 2.37 (s,
3H), 2.16 (d, J = 12.0 Hz, 2H), 1.78 (s, 2H), 1.34 (q, J = 12.0 Hz, 2H).
13C NMR (125 MHz, CDCl3) δ 164.6, 159.4, 157.7, 154.2, 153.1,
149.7, 147.6, 136.4, 131.2, 128.7, 128.1, 127.7, 126.1, 121.8, 120.9,
108.3, 102.5, 100.4, 55.6, 55.2, 54.2, 51.5, 49.9, 48.0, 46.0, 43.8, 33.0,
27.6. HRMS (ESI) for C34H42N8O3 [M + H]+, calcd: 611.3453, found:
611.3440. HPLC analysis: MeOH−H2O (85:15), 7.68 min, 99.58%
purity.
(S)-1-(1-Acryloylpyrrolidin-3-yl)-3-benzyl-7-(2-methoxy-4-
(4-methylpiperazin-1-yl)phenylamino)-3,4-dihydropyrimido-
[4,5-d]pyrimidin-2(1H)-one (2f). [α]2D0 −1.105 (c 0.181, CHCl3).
1H NMR (400 MHz, CDCl3, a mixture of rotamers) δ 7.95 (d, J = 8.8
Hz, 1H), 7.90 (s, 0.6H), 7.89 (s, 0.4H), 7.38−7.23 (m, 6H), 6.58−6.44
(m, 3H), 6.40−6.38 (m, 1H), 5.72−5.55 (m, 2H), 4.72−4.56 (m, 2H),
4.31−4.26 (m, 0.6H), 4.13 (s, 1.2H), 4.12 (s, 0.8H), 4.10−3.89 (m,
1.7H), 3.87 (s, 1.8H), 3.86 (s, 1.2H), 3.85−3.80 (m, 0.7H), 3.67−3.51
(m, 1H), 3.20−3.15 (m, 4H), 2.99−2.85 (m, 1H), 2.62−2.58 (m, 4H),
2.37 (s, 3H), 2.28−2.08 (m, 1H). 13C NMR (125 MHz, CDCl3) δ
164.6, 164.3, 159.2, 159.1, 157.1, 157.0, 153.7, 153.6, 153.5, 149.7,
149.5, 147.7, 136.1, 136.0, 128.8, 128.3, 128.1, 128.0, 127.8, 127.3,
121.5, 121.3, 120.7, 120.3, 107.8, 107.7, 102.2, 100.4, 100.1, 55.6, 55.1,
52.4, 51.5, 51.4, 50.7, 49.9, 49.7, 46.9, 46.7, 46.1, 45.7, 44.4, 43.7, 28.2,
25.8. HRMS (ESI) for C32H38N8O3 [M + H]+, calcd: 583.3140, found:
583.3135. HPLC analysis: MeOH−H2O (78:22), 11.31 min, 97.8%
purity.
1-(1-Acryloylpiperidin-4-yl)-3-benzyl-7-(2-methoxy-4-(4-
methylpiperazin-1-yl)phenylamino)-3,4-dihydropyrimido[4,5-
1
d]pyrimidin-2(1H)-one (2b). H NMR (400 MHz, CDCl3) δ 8.08
(d, J = 8.8 Hz, 1H), 7.87 (s, 1H), 7.37−7.33 (m, 3H), 7.31−7.28 (m,
3H), 6.63 (dd, J = 10.8, 16.8 Hz, 1H), 6.54 (d, J = 2.4 Hz, 1H), 6.49
(dd, J = 2.4, 8.8 Hz, 1H), 6.29 (dd, J = 2.0, 16.8 Hz, 1H), 5.69 (dd, J =
2.0, 10.4 Hz, 1H), 5.01−4.95 (m, 1H), 4.89−4.87 (m, 1H), 4.62 (s,
2H), 4.16−4.12 (m, 1H), 4.10 (s, 2H), 3.88 (s, 3H), 3.17 (t, J = 4.8
Hz, 4H), 3.13−3.10 (m, 1H), 2.86−2.71 (m, 3H), 2.59 (t, J = 4.8 Hz,
4H), 2.35 (s, 3H), 1.85−1.79 (m, 2H). 13C NMR (125 MHz, CDCl3)
δ 165.5, 159.1, 157.4, 153.9, 153.4, 149.2, 147.4, 136.3, 128.8, 128.1,
127.8, 127.4, 121.8, 119.8, 107.9, 102.2, 100.3, 55.6, 55.2, 53.0, 51.4,
49.9, 46.2, 46.1, 43.8, 42.5, 29.3, 28.5. HRMS (ESI) for C33H40N8O3
[M + H]+, calcd: 597.3296, found: 597.3297. HPLC analysis: MeOH−
H2O (85:15), 7.96 min, 97.18% purity.
N-(2-(3-Benzyl-7-(2-methoxy-4-(4-methylpiperazin-1-yl)-
phenylamino)-2-oxo-3,4-dihydropyrimido[4,5-d]pyrimidin-
1
1(2H)-yl)ethyl)acrylamide (2g). H NMR (500 MHz, CDCl3) 8.09
(d, J = 8.5 Hz, 1H), 7.90 (s, 1H), 7.38−7.28 (m, 7H), 7.02 (s, 1H),
6.59−6.56 (m, 2H), 6.21−6.17 (m, 2H), 5.98 (dd, J = 10.5, 17.0 Hz,
1H), 5.56 (d, J = 10.5 Hz, 1H), 4.68 (s, 2H), 4.37−4.35 (m, 2H), 4.24
(s, 2H), 3.90 (s, 3H), 3.72−3.71 (m, 2H), 3.27 (s, 4H), 2.75 (s, 4H),
2.47 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 165.8, 159.1, 156.3,
154.5, 153.6, 149.3, 147.4, 135.8, 131.1, 128.8, 128.0, 127.8, 125.5,
121.7, 120.2, 108.1, 100.9, 100.4, 55.6, 55.0, 51.6, 49.9, 46.0, 44.0, 40.3.
HRMS (ESI) for C30H36N8O3 [M + H]+, calcd: 557.2983, found:
557.2987. HPLC analysis: MeOH−H2O (82:18), 7.59 min, 97.01%
purity.
(S)-1-(1-Acryloylpyrrolidin-3-yl)-7-(2-methoxy-4-(4-methyl-
piperazin-1-yl)phenylamino)-3-methyl-3,4-dihydropyrimido-
[4,5-d]pyrimidin-2(1H)-one (2h). [α]2D0 −4.000 (c 0.100, CHCl3).
1H NMR (400 MHz, CDCl3, a mixture of rotamers) δ 7.96 (s, 0.6H),
7.95 (s, 0.4H), 7.93 (d, J = 8.8 Hz, 1H), 7.31 (s, 0.6H), 7.22 (s, 0.4H),
6.55−6.42 (m, 3H), 6.38−6.35 (m, 1H), 5.71−5.51 (m, 2H), 4.28−
4.21 (m, 0.6H), 4.24 (s, 2H), 4.04−3.91 (m, 1.7H), 3.86 (s, 2H), 3.85
(s, 1H), 3.78−3.74 (m, 0.7H), 3.64−3.48 (m, 1H), 3.18−3.14 (m,
(R)-1-(1-Acryloylpiperidin-3-yl)-3-benzyl-7-(2-methoxy-4-(4-
methylpiperazin-1-yl)phenylamino)-3,4-dihydropyrimido[4,5-
d]pyrimidin-2(1H)-one (2c). [α]2D0 +123.337 (c 0.154, CHCl3). H
1
NMR (400 MHz, CDCl3, a mixture of rotamers) δ 7.99 (d, J = 8.4 Hz,
1H), 7.89 (s, 0.6H), 7.86 (s, 0.4H), 7.37−7.25 (m, 6H), 6.67−6.60 (m,
1H), 6.53−6.46 (m, 2H), 6.30 (t, J = 16.0 Hz, 1H), 5.72−5.61 (m,
1H), 4.82−4.76 (m, 1H), 4.72−4.64 (m, 2H), 4.62−4.55 (m, 1H),
4.15−4.13 (m, 0.4H), 4.11 (s, 2H), 4.00−3.97 (m, 1H), 3.87 (s, 3H),
3.82−3.77 (m, 0.6H), 3.18−3.14 (m, 4H), 3.09−2.63 (m, 2H), 2.60−
2.58 (m, 4H), 2.35 (s, 3H), 1.98−1.86 (m, 2H), 1.63−1.55 (m, 1H).
13C NMR (125 MHz, CDCl3) δ 165.6, 165.1, 159.4, 157.3, 153.8,
153.5, 149.8, 149.6, 147.7, 147.6, 136.2, 136.1, 128.8, 128.0, 127.8,
127.7, 127.3, 121.9, 121.5, 121.1, 120.6, 108.0, 102.1, 102.0, 100.6,
100.3, 55.6, 55.1, 52.5, 51.3, 50.1, 49.9, 48.2, 46.1, 44.4, 43.8, 43.7,
42.3, 27.5, 27.3, 26.5, 25.4. HRMS (ESI) for C33H40N8O3 [M + H]+,
calcd: 597.3296, found: 597.3292. HPLC analysis: MeOH−H2O
(78:22), 12.63 min, 97.39% purity.
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4H), 3.03 (s, 1.8H), 3.02 (s, 1.2H), 2.93−2.79 (m, 1H), 2.60−2.58 (m,
4H), 2.35 (s, 3H), 2.22−2.02 (m, 1H). 13C NMR (125 MHz, CDCl3)
δ 164.6, 164.3, 159.2, 157.2, 157.1, 153.7, 153.6, 153.4, 149.8, 149.5,
147.6, 128.8, 128.3, 127.3, 121.6, 121.4, 120.7, 120.3, 107.9, 107.8,
102.1, 100.4, 100.2, 55.6, 55.1, 50.5, 49.9, 49.7, 46.8, 46.6, 46.4, 46.1,
46.0, 45.7, 44.4, 35.7, 35.6, 28.2, 25.8. HRMS (ESI) for C26H34N8O3
[M + H]+, calcd: 507.2827, found: 507.2822. HPLC analysis: MeOH−
H2O (78:22), 6.66 min, 97.09% purity.
(S)-1-(1-Acryloylpyrrolidin-3-yl)-3-isopropyl-7-(2-methoxy-
4-(4-methylpiperazin-1-yl)phenylamino)-3,4-dihydropyrimido-
[4,5-d]pyrimidin-2(1H)-one (2i). [α]2D0 +9.589 (c 0.146, CHCl3). 1H
NMR (400 MHz, CDCl3, a mixture of rotamers) δ 8.00 (s, 0.6H), 7.99
(s, 0.4H), 7.98−7.94 (m, 1H), 7.32 (s, 0.7H), 7.23 (s, 0.3H), 6.57−
6.36 (m, 4H), 5.71−5.61 (m, 1H), 5.55−5.43 (m, 1H), 4.75−4.67 (m,
1H), 4.28−4.23 (m, 0.6H), 4.10 (s, 1.2H), 4.09 (s, 0.8H), 4.05−3.89
(m, 1.7H), 3.87 (s, 1.8H), 3.86 (s, 1.2H), 3.83−3.78 (m, 0.7H), 3.65−
3.49 (m, 1H), 3.21−3.16 (m, 4H), 2.97−2.83 (m, 1H), 2.65−2.61 (m,
4H), 2.39 (s, 1.8H), 2.38 (s, 1.2H), 2.26−2.05 (m, 1H), 1.22−1.18 (m,
6H). 13C NMR (125 MHz, CDCl3) δ 164.6, 159.2, 157.4, 157.3, 153.4,
153.3, 153.2, 149.7, 149.4, 147.6, 128.8, 128.3, 127.3, 121.7, 121.5,
120.6, 120.3, 107.9, 107.8, 102.8, 102.7, 100.4, 100.2, 55.6, 55.1, 52.5,
50.9, 49.9, 49.7, 47.0, 46.7, 46.1, 46.0, 45.9, 45.7, 44.4, 37.3, 28.2, 25.7,
19.2. HRMS (ESI) for C28H38N8O3 [M + H]+, calcd: 535.3140, found:
535.3138. HPLC analysis: MeOH−H2O (78:22), 8.34 min, 98.42%
purity.
(S)-1-(1-Acryloylpyrrolidin-3-yl)-3-cyclopentyl-7-(2-me-
thoxy-4-(4-methylpiperazin-1-yl)phenylamino)-3,4-
dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (2j). [α]2D0 +6.667 (c
0.120, CHCl3). 1H NMR (500 MHz, CDCl3, a mixture of rotamers) δ
8.00 (s, 0.7H), 7.98 (s, 0.3H), 7.97−7.94 (m, 1H), 7.32 (s, 0.6H), 7.24
(s, 0.4H), 6.56−6.33 (m, 4H), 5.71−5.61 (m, 1H), 5.52−5.43 (m,
1H), 4.85−4.79 (m, 1H), 4.27−4.20 (m, 0.6H), 4.11 (s, 1.2H), 4.10 (s,
0.8H), 4.06−3.89 (m, 1.7H), 3.82−3.77 (m, 0.7H), 3.87 (s, 3H),
3.65−3.48 (m, 1H), 3.25−3.22 (m, 4H), 2.95−2.83 (m, 1H), 2.72−
2.69 (m, 4H), 2.45 (s, 1.8H), 2.43 (s, 1.2H), 2.26−2.06 (m, 1H),
1.91−1.85 (m, 2H), 1.76−1.72 (m, 2H), 1.66−1.55 (m, 4H). 13C
NMR (125 MHz, CDCl3) δ 164.5, 164.2, 159.2, 159.1, 157.4, 157.3,
153.7, 153.5, 153.2, 153.0, 149.6, 149.3, 147.5, 147.4, 128.8, 128.3,
127.2, 121.7, 121.4, 120.5, 120.2, 107.8, 107.7, 102.8, 100.3, 100.1,
56.0, 55.8, 55.5, 55.0, 52.5, 50.9, 49.8, 49.6, 46.9, 46.6, 46.0, 45.9, 45.6,
44.3, 38.6, 28.2, 27.9, 27.8, 25.7, 24.1, 24.0. HRMS (ESI) for
C30H40N8O3 [M + H]+, calcd: 561.3296, found: 561.3290. HPLC
analysis: MeOH−H2O (85:15), 7.21 min, 99.51% purity.
157.2, 153.5, 153.4, 153.3, 153.2, 149.7, 149.5, 147.7, 142.0, 141.9,
129.2, 129.1, 128.8, 128.3, 127.3, 126.8, 126.7, 125.3, 125.2, 121.5,
121.3, 120.7, 120.3, 107.9, 107.8, 102.8, 100.4, 100.1, 55.6, 55.1, 52.7,
51.0, 49.9, 49.7, 47.2, 47.1, 47.0, 46.7, 46.1, 45.7, 44.4, 28.4, 25.8.
HRMS (ESI) for C31H36N8O3 [M + H]+, calcd: 569.2983, found:
569.2980. HPLC analysis: MeOH−H2O (78:22), 8.79 min, 96.98%
purity.
(S)-1-(1-Acryloylpyrrolidin-3-yl)-3-phenyl-7-(phenylamino)-
3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (2m). [α]D20
1
−2.564 (c 0.078, CHCl3). H NMR (500 MHz, CDCl3, a mixture of
rotamers) δ 8.05 (s, 0.6H), 8.03 (s, 0.4H), 7.57−7.51 (m, 2H), 7.48−
7.41 (m, 3H), 7.34−7.28 (m, 5H), 7.08−7.05 (m, 1H), 6.52−6.38 (m,
2H), 5.72−5.63 (m, 1H), 5.61−5.49 (m, 1H), 4.70−4.62 (m, 2H),
4.22−4.18 (m, 0.6H), 4.09−3.99 (m, 1.4H), 3.90−3.84 (m, 1H),
3.63−3.58 (m, 0.4H), 3.54−3.48 (m, 0.6H), 2.86−2.75 (m, 1H),
2.32−2.17 (m, 1H). 13C NMR (125 MHz, CDCl3) δ 165.1, 164.4,
159.1, 157.5, 157.1, 153.3, 153.2, 153.0, 141.9, 139.0, 138.9, 129.2,
128.9, 128.8, 128.1, 127.6, 127.5, 126.9, 126.8, 125.3, 125.2, 123.2,
123.1, 120.2, 119.7, 103.9, 52.9, 51.2, 47.7, 47.1, 47.0, 45.7, 44.7, 28.4,
26.3. HRMS (ESI) for C25H24N6O2 [M + H]+, calcd: 441.2034, found:
441.2030. HPLC analysis: MeOH−H2O (85:15), 4.73 min, 99.24%
purity.
(S)-1-(1-Acryloylpyrrolidin-3-yl)-7-(4-(4-methylpiperazin-1-
yl)phenylamino)-3-phenyl-3,4-dihydropyrimido[4,5-d]-
pyrimidin-2(1H)-one (2n). [α]2D0 +1.770 (c 0.113, CHCl3). 1H NMR
(500 MHz, CDCl3, a mixture of rotamers) δ 8.01 (s, 0.6H), 8.00 (s,
0.4H), 7.44−7.36 (m, 4H), 7.32−7.27 (m, 3H), 7.09 (s, 0.6H), 6.92−
6.88 (m, 2.4H), 6.52−6.33 (m, 2H), 5.70−5.63 (m, 1H), 5.57−5.47
(m, 1H), 4.67−4.58 (m, 2H), 4.23−4.20 (m, 0.6H), 4.09−3.98 (m,
1.4H), 3.87−3.83 (m, 1H), 3.62−3.57 (m, 0.4H), 3.52−3.46 (m,
0.6H), 3.24−3.20 (m, 4H), 2.85−2.77 (m, 1H), 2.69−2.65 (m, 4H),
2.42 (s, 1.8H), 2.41 (s, 1.2H), 2.29−2.14 (m, 1H). 13C NMR (125
MHz, CDCl3) δ 164.7, 164.2, 159.6, 159.5, 157.3, 157.0, 153.4, 153.3,
147.5, 141.9, 131.3, 129.1, 128.7, 128.2, 127.4, 126.7, 126.6, 125.2,
125.1, 122.1, 121.6, 116.5, 102.8, 55.0, 54.9, 52.7, 51.0, 49.4, 49.2, 47.2,
46.9, 46.0, 45.9, 45.6, 44.5, 28.2, 26.0. HRMS (ESI) for C30H34N8O2
[M + H]+, calcd: 539.2878, found: 539.2872. HPLC analysis: MeOH−
H2O (81:19), 5.74 min, 98.71% purity.
(S)-1-(1-Acryloylpyrrolidin-3-yl)-7-(3-(4-methylpiperazin-1-
yl)phenylamino)-3-phenyl-3,4-dihydropyrimido[4,5-d]-
pyrimidin-2(1H)-one (2o). [α]2D0 −5.825 (c 0.103, CHCl3). 1H
NMR (500 MHz, CDCl3, a mixture of rotamers) δ 8.05 (s, 0.6H), 8.04
(s, 0.4H), 7.44−7.40 (m, 2H), 7.32−7.25 (m, 4H), 7.23−7.04 (m,
3H), 6.65−6.63 (m, 1H), 6.52−6.34 (m, 2H), 5.71−5.53 (m, 2H),
4.70−4.60 (m, 2H), 4.19 −4.16 (m, 0.6H), 4.12−4.03 (m, 1.3H),
3.94−3.84 (m, 1.1H), 3.65−3.60 (m, 0.4H), 3.53−3.47 (m, 0.6H),
3.30−3.29 (m, 4H), 2.83−2.76 (m, 1H), 2.75−2.65 (m, 4H), 2.45 (s,
1.2H), 2.43 (s, 1.8H), 2.34−2.18 (m, 1H). 13C NMR (125 MHz,
CDCl3) δ 165.0, 164.2, 159.3, 159.2, 157.4, 157.0, 153.4, 153.3, 153.2,
152.9, 151.9, 141.9, 140.0, 139.9, 129.5, 129.4, 129.2, 128.8, 128.2,
127.5, 127.4, 126.8, 126.7, 125.2, 111.6, 111.2, 110.9, 107.8, 107.4,
103.4, 54.9, 54.9, 52.8, 51.0, 48.8, 47.9, 47.2, 47.0, 45.9, 45.7, 44.7,
28.6, 26.4. HRMS (ESI) for C30H34N8O2 [M + H]+, calcd: 539.2878,
found: 539.2872. HPLC analysis: MeOH−H2O (75:25), 9.27 min,
99.87% purity.
(S)-1-(1-Acryloylpyrrolidin-3-yl)-7-(4-(1-methylpiperidin-4-
yl)phenylamino)-3-phenyl-3,4-dihydropyrimido[4,5-d]-
pyrimidin-2(1H)-one (2p). [α]2D0 −28.000 (c 0.100, CHCl3). 1H
NMR (400 MHz, CDCl3, a mixture of rotamers) δ 8.04 (s, 0.6H), 8.03
(s, 0.4H), 7.50−7.40 (m, 4H), 7.32−7.29 (m, 3H), 7.21−7.18 (m,
2.6H), 7.02 (s, 0.4H), 6.55−5.37 (m, 2H), 5.72−5.63 (m, 1H), 5.61−
5.51 (m, 1H), 4.69−4.60 (m, 2H), 4.23−4.19 (m, 0.6H), 4.11−3.99
(m, 1.4H), 3.95−3.85 (m, 1H), 3.66−3.60 (m, 0.4H), 3.54−3.46 (m,
0.6H), 3.14−3.10 (m, 2H), 2.89−2.74 (m, 1H), 2.55−2.50 (m, 1H),
2.45 (s, 1.7H), 2.43 (s, 1.3H), 2.33−2.29 (m, 1H), 2.26−2.17 (m, 2H),
2.02−1.92 (m, 4H). 13C NMR (125 MHz, CDCl3) δ 165.0, 164.3,
159.3, 159.2, 157.4, 157.0, 153.4, 153.3, 153.1, 141.9, 141.1, 141.0,
137.0, 129.2, 128.8, 128.2, 127.6, 127.2, 126.8, 126.7, 125.3, 125.2,
120.2, 120.0, 103.5, 103.4, 56.3, 56.2, 52.8, 51.2, 47.6, 47.1, 47.0, 46.3,
45.7, 44.6, 41.3, 41.2, 33.4, 33.3, 28.4, 26.2. HRMS (ESI) for
(S)-1-(1-Acryloylpyrrolidin-3-yl)-3-cyclohexyl-7-(2-methoxy-
4-(4-methylpiperazin-1-yl)phenylamino)-3,4-dihydropyrimido-
[4,5-d]pyrimidin-2(1H)-one (2k). [α]2D0 +5.085 (c 0.118, CHCl3).
1H NMR (400 MHz, CDCl3, a mixture of rotamers) δ 8.02−7.95 (m,
2H), 7.32 (s, 0.6H), 7.25 (s, 0.4H), 6.55−6.47 (m, 2H), 6.42−6.33 (m,
2H), 5.72−5.61 (m, 1H), 5.54−5.45 (m, 1H), 4.30−4.27 (m, 1H),
4.25−4.22 (m, 0.6H), 4.13 (s, 2H), 4.02−3.94 (m, 1.7H), 3.87 (s, 3H),
3.83−3.77 (m, 0.7H), 3.67−3.49 (m, 1H), 3.25−3.22 (m, 4H), 2.96−
2.81 (m, 1H), 2.73−2.70 (m, 4H), 2.46 (s, 1.6H), 2.44 (s, 1.4H),
2.24−2.05 (m, 1H), 1.86−1.68 (m, 5H), 1.52−1.36 (m, 4H), 1.15−
1.07 (m, 1H). 13C NMR (125 MHz, CDCl3) δ 164.6, 164.2, 159.2,
157.4, 157.3, 153.5, 153.3, 153.2, 153.1, 149.6, 149.3, 147.4, 128.8,
128.3, 127.3, 121.8, 121.6, 120.5, 120.1, 107.9, 102.9, 100.4, 100.1,
55.6, 55.1, 55.0, 54.3, 54.1, 52.5, 50.9, 49.9, 49.6, 47.0, 46.7, 46.0, 45.9,
45.7, 44.4, 38.6, 29.7, 28.3, 25.8, 25.6, 25.5. HRMS (ESI) for
C31H42N8O3 [M + H]+, calcd: 575.3453, found: 575.3446. HPLC
analysis: MeOH−H2O (82:18), 10.51 min, 98.94% purity.
(S)-1-(1-Acryloylpyrrolidin-3-yl)-7-(2-methoxy-4-(4-methyl-
piperazin-1-yl)phenylamino)-3-phenyl-3,4-dihydropyrimido-
[4,5-d]pyrimidin-2(1H)-one (2l). [α]2D0 −8.421 (c 0.095, CHCl3). 1H
NMR (400 MHz, CDCl3, a mixture of rotamers) δ 8.02−7.99 (m,
2H), 7.44−7.39 (m, 3H), 7.32−7.28 (m, 3H), 6.54−6.37 (m, 4H),
5.70−5.61 (m, 1H), 5.59−5.51 (m, 1H), 4.68−4.58 (m, 2H), 4.31−
4.27 (m, 0.8H), 4.07−3.91 (m, 1.5H), 3.89 (s, 1.7H), 3.88 (s, 1.3H),
3.86−3.80 (m, 0.7H), 3.66−3.52 (m, 1H), 3.22−3.18 (m, 4H), 2.98−
2.82 (m, 1H), 2.65−2.61 (m, 4H), 2.39 (s, 3H), 2.33−2.12 (m, 1H).
13C NMR (125 MHz, CDCl3) δ 164.6, 164.2, 159.4, 159.3, 157.3,
8810
dx.doi.org/10.1021/jm4012388 | J. Med. Chem. 2013, 56, 8803−8813

Journal of Medicinal Chemistry
Article
C31H35N7O2 [M + H]+, calcd: 538.2925, found: 538.2921. HPLC
analysis: MeOH−H2O (85:15), 10.26 min, 99.75% purity.
7.28 (m, 5H), 7.07−7.02 (m, 1H), 6.53−6.33 (m, 2H), 5.71−5.50 (m,
2H), 4.77−4.62 (m, 2H), 4.18−3.89 (m, 3H), 3.68−3.63 (m, 0.4H),
3.54−3.48 (m, 0.6H), 3.18 (s, 4H), 2.86−2.82 (m, 4H), 2.75−2.71 (m,
1H), 2.53 (s, 3H), 2.38−2.22 (m, 1H). 13C NMR (125 MHz, CDCl3)
δ 165.0, 164.3, 159.0, 158.9, 157.5, 157.1, 153.3, 153.1, 152.9, 144.4,
141.8, 135.1, 129.1, 128.9, 128.8, 128.1, 127.5, 126.8, 126.7, 125.2,
122.1, 121.9, 120.5, 120.4, 119.0, 118.8, 103.7, 55.2, 52.9, 51.3, 51.2,
51.1, 47.9, 47.0, 46.9, 45.9, 45.7, 44.7, 28.5, 26.5. HRMS (ESI) for
C30H33ClN8O2 [M + H]+, calcd: 573.2488, found: 573.2483. HPLC
analysis: MeOH−H2O (85:15), 7.52 min, 98.38% purity.
(S)-1-(1-Acryloylpyrrolidin-3-yl)-7-(4-(2-(4-methylpiperazin-
1-yl)ethyl)phenylamino)-3-phenyl-3,4-dihydropyrimido[4,5-d]-
pyrimidin-2(1H)-one (2q). [α]2D0 −5.455 (c 0.110, CHCl3). 1H NMR
(400 MHz, CDCl3, a mixture of rotamers) δ 8.04 (s, 0.6H), 8.02 (s,
0.4H), 7.48−7.40 (m, 4H), 7.32−7.27 (m, 3H), 7.23 (s, 0.6H), 7.15
(d, J = 8.0 Hz, 2H), 7.03 (s, 0.4H), 6.54−6.37 (m, 2H), 5.72−5.62 (m,
1H), 5.60−5.49 (m, 1H), 4.69−4.59 (m, 2H), 4.22−4.18 (m, 0.6H),
4.11−3.99 (m, 1.4H), 3.93−3.85 (m, 1H), 3.65−3.59 (m, 0.4H),
3.54−3.47 (m, 0.6H), 2.88−2.83 (m, 1H), 2.81−2.77 (m, 2H), 2.73−
2.66 (m, 2H), 2.64−2.47 (m, 8H), 2.35 (s, 3H), 2.31−2.16 (m, 1H).
13C NMR (125 MHz, CDCl3) δ 164.9, 164.3, 159.3, 159.2, 157.4,
(S)-1-(1-Acryloylpyrrolidin-3-yl)-7-(3-methyl-4-(4-methylpi-
perazin-1-yl)phenylamino)-3-phenyl-3,4-dihydropyrimido[4,5-
d]pyrimidin-2(1H)-one (2v). [α]2D0 −42.478 (c 0.113, CHCl3). H
1
157.0, 153.4, 153.3, 153.1, 141.9, 137.1, 137.0, 135.1, 135.0, 129.1,
129.0, 128.7, 128.1, 127.5, 126.8, 126.7, 125.2, 120.2, 120.0, 103.4,
60.3, 55.0, 53.0, 52.8, 51.1, 47.6, 47.1, 47.0, 45.9, 45.7, 44.6, 32.8, 32.7,
28.4, 26.3. HRMS (ESI) for C32H38N8O2 [M + H]+, calcd: 567.3191,
found: 567.3185. HPLC analysis: MeOH−H2O (85:15), 6.29 min,
99.55% purity.
(S)-1-(1-Acryloylpyrrolidin-3-yl)-7-(3-methoxy-4-(4-methyl-
piperazin-1-yl)phenylamino)-3-phenyl-3,4-dihydropyrimido-
[4,5-d]pyrimidin-2(1H)-one (2r). [α]2D0 −25.263 (c 0.095, CHCl3).
1H NMR (500 MHz, CDCl3, a mixture of rotamers) δ 8.04 (s, 0.7H),
NMR (400 MHz, CDCl3, a mixture of rotamers) δ 8.03 (s, 0.6H), 8.02
(s, 0.4H), 7.42−7.40 (m, 3H), 7.35−7.29 (m, 4H), 7.14−6.96 (m,
2H), 6.54−6.33 (m, 2H), 5.73−5.51 (m, 2H), 4.68−4.59 (m, 2H),
4.19−3.85 (m, 3H), 3.67−3.61 (m, 0.4H), 3.52−3.47 (m, 0.6H), 3.13
(s, 4H), 2.96 (s, 4H), 2.87−2.77 (m, 1H), 2.62 (s, 3H), 2.28 (s, 3H),
2.24−2.19 (m, 1H). 13C NMR (125 MHz, CDCl3) δ 164.9, 164.3,
159.4, 159.3, 157.5, 157.1, 153.5, 153.4, 153.3, 153.0, 146.8, 141.9,
134.5, 133.4, 129.2, 128.8, 128.2, 127.6, 126.8, 126.7, 125.3, 125.2,
123.0, 119.7, 118.5, 118.4, 103.2, 55.4, 52.8, 51.4, 51.3, 51.2, 47.7, 47.2,
47.0, 45.7, 44.7, 28.5, 26.4, 17.9. HRMS (ESI) for C31H36N8O2 [M +
H]+, calcd: 553.3034, found: 553.3032. HPLC analysis: MeOH−H2O
(85:15), 7.09 min, 99.95% purity.
Cell Lines and Reagents. Cell lines H1975 (NSCLC,
EGFRL858R/T790M) were obtained from ATCC. The cells were
maintained at 37 °C in 5% CO2 incubator in RPMI 1460 (Gibco,
Invitrogen) containing 10% fetal bovine serum (Gibco, Invitrogen).
H460 (NSCLC, EGFRWT), 95D (NSCLC, EGFRWT), H358 (NSCLC,
EGFRWT), H1299 (NSCLC, EGFRWT), and HL-7702 (diploid human
liver cell, EGFRWT) were gifts from Prof. Duanqing Pei. The EGFR
gene of every cell line was sequenced before use. Gefitinib was
synthesized in our chemistry laboratory.
In Vitro Kinase Enzyme Assay. EGFR (L858R/T790M) mutant
and the Z′-Lyte Kinase Assay Kit were purchased from Invitrogen. The
concentration gradients from 5.1 × 10−11 to 1.0 × 10−6 mol/L were
used for all the test compounds. The experiments were performed
according to the instructions of the manufacturer.
Cell Proliferation and Growth Inhibition Assay. Cell
proliferation was assessed by MTS assay. Cells were exposed to
treatment for 72 h, and the number of cells used per experiment for
each cell line was adjusted to obtain an absorbance of 0.5 to 1.2 at 570
nm. Compounds were tested at six concentrations (0.1 nM to 10 μM),
with each concentration duplicated six times. All experiments were
repeated at least four times. The data were calculated using GraphPad
Prism version 4.0. Dose−response curves were fitted using a nonlinear
regression model with a sigmoidal dose−response.
Antibodies and Western Blot Assay. H1975 cells were plated in
6-cm dishes. After 24 h of growth, the cells were treated with the
compounds at the indicated concentrations for 2 h. Cell lysates were
collected with 1 × lysis buffer (CST) and were briefly sonicated.
Western blot analyses were conducted after separation by SDS/PAGE
electrophoresis and transfer to PVDF membranes. Membranes were
blocked in 5% bovine serum albumin/TBST and probed with the
indicated antibodies, followed by a peroxidase-conjugated antimouse
or rabbit secondary antibody (Thermo). Antiphospho Akt (Ser 173),
antitotal Akt, anti-EGFR, antiphospho specific EGFR (pY1068),
antitotal Erk1/2, and antiphospho Erk1/2 (pT202/pY204) antibodies
were obtained from Cell Signaling Technology Inc.
Determination of Pharmacokinetic Parameters in Rats. Male
Sprague−Dawley rats (180−220 g) were dosed with the test
compounds intravenously (iv) at 5 mg/kg and by oral gavage (po)
at 25 mg/kg. The compound was dissolved in a vehicle of 2% DMSO,
4% EtOH, 4% castor oil, and 90% H2O. Blood samples (0.3 mL) were
then obtained via orbital sinus puncture at 2 min, 10 min, 30 min, 1 h,
2 h, 3 h, 4 h, 6 h, 8 h, 12 h, 21 h, 24 h, 30 h, 36 h, 48 h, and 72 h time
points and collected into heparinized tubes. Blood samples were
centrifuged for cell removal, and the plasma supernatant was then
transferred to a clean vial and subsequently stored in 4 °C prior to
8.02 (s, 0.3H), 7.44−7.40 (m, 2H), 7.32−7.28 (m, 3H), 7.22−6.89 (m,
4H), 6.53−6.35 (m, 2H), 5.72−5.51 (m, 2H), 4.69−4.60 (m, 2H),
4.18−4.15 (m, 0.6H), 4.10−3.99 (m, 1.4H), 3.94−3.89 (m, 1H), 3.87
(s, 1.8H), 3.86 (s, 1.2H), 3.64−3.59 (m, 0.4H), 3.51−3.46 (m, 0.6H),
3.19 (s, 4H), 2.87−2.77 (m, 4H), 2.75−2.69 (m, 1H), 2.50 (s, 3H),
2.32−2.18 (m, 1H). 13C NMR (125 MHz, CDCl3) δ 165.0, 164.3,
159.3, 159.2, 157.4, 157.0, 153.4, 153.3, 153.0, 152.5, 141.9, 137.0,
136.8, 134.7, 134.5, 129.2, 128.7, 128.1, 127.6, 127.5, 126.8, 126.7,
125.2, 118.4, 112.6, 112.1, 104.8, 104.3, 103.3, 55.5, 55.4, 55.2, 52.7,
51.0, 50.6, 50.5, 48.0, 47.1, 47.0, 45.9, 45.7, 44.7, 28.5, 26.5. HRMS
(ESI) for C31H36N8O3 [M + H]+, calcd: 569.2983, found: 569.2975.
HPLC analysis: MeOH−H2O (75:25), 8.83 min, 99.39% purity.
(S)-1-(1-Acryloylpyrrolidin-3-yl)-7-(3-fluoro-4-(4-methylpi-
perazin-1-yl)phenylamino)-3-phenyl-3,4-dihydropyrimido[4,5-
d]pyrimidin-2(1H)-one (2s). [α]2D0 −1.538 (c 0.130, CHCl3). 1H
NMR (500 MHz, CDCl3, a mixture of rotamers) δ 8.04 (s, 0.6H), 8.03
(s, 0.4H), 7.56−7.49 (m, 1H), 7.44−7.40 (m, 2H), 7.32−7.28 (m,
4H), 7.16−7.08 (m, 1H), 6.93−6.89 (m, 1H), 6.53−6.33 (m, 2H),
5.71−5.48 (m, 2H), 4.69−4.61 (m, 2H), 4.18−4.08 (m, 1.5H), 4.01−
3.87 (m, 1.5H), 3.66−3.61 (m, 0.4H), 3.53−3.47 (m, 0.6H), 3.18−
3.17 (m, 4H), 2.88−2.82 (m, 1H), 2.78−2.67 (m, 4H), 2.47 (s, 1.8H),
2.46 (s, 1.2H), 2.36−2.21 (m, 1H). 13C NMR (125 MHz, CDCl3) δ
165.1, 164.4, 158.9, 157.5, 157.0, 156.4, 154.5, 153.3, 153.0, 141.9,
135.2, 134.4, 129.2, 128.8, 128.3, 127.6, 127.5, 126.9, 126.8, 125.2,
119.2, 115.5, 115.4, 108.7, 108.6, 108.4, 103.7, 55.0, 52.9, 51.3, 50.5,
50.4, 48.1, 47.0, 45.9, 45.7, 44.8, 28.5, 26.6. HRMS (ESI) for
C30H33FN8O2 [M + H]+, calcd: 557.2783, found: 557.2775. HPLC
analysis: MeOH−H2O (75:25), 10.74 min, 99.9% purity.
(S)-1-(1-Acryloylpyrrolidin-3-yl)-7-(4-(4-methylpiperazin-1-
yl)-3-(trifluoromethyl)phenylamino)-3-phenyl-3,4-
dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (2t). [α]2D0 −36.190
(c 0.105, CHCl3).1H NMR (400 MHz, CDCl3, a mixture of rotamers)
δ 8.08−7.99 (m, 2H), 7.74−7.60 (m, 2H), 7.46−7.42 (m, 3H), 7.32−
7.29 (m, 3H), 6.56−6.37 (m, 2H), 5.75−5.52 (m, 2H), 4.79−4.64 (m,
2H), 4.20−3.88 (m, 3H), 3.69−3.62 (m, 1H), 3.52−3.44 (m, 4H),
3.07 (s, 4H), 2.84 (s, 3H), 2.68−2.60 (m, 1H), 2.36−2.26 (m, 1H).
13C NMR (125 MHz, CDCl3) δ 165.2, 164.4, 158.8, 157.6, 157.2,
153.4, 153.1, 152.8, 146.6, 141.8, 136.5, 136.4, 129.3, 129.2, 128.8,
128.2, 128.0, 127.7, 127.6, 126.9, 125.3, 125.0, 124.9, 123.4, 122.7,
118.1, 118.0, 104.29, 55.3, 53.0, 52.8, 51.4, 48.5, 47.2, 47.0, 45.7, 44.9,
28.6, 26.9. HRMS (ESI) for C31H33F3N8O2 [M + H]+, calcd:
607.2751, found: 607.2750. HPLC analysis: MeOH−H2O (85:15),
8.56 min, 99.62% purity.
(S)-1-(1-Acryloylpyrrolidin-3-yl)-7-(3-chloro-4-(4-methylpi-
perazin-1-yl)phenylamino)-3-phenyl-3,4-dihydropyrimido[4,5-
1
d]pyrimidin-2(1H)-one (2u). [α]2D0 −23.704 (c 0.135, CHCl3). H
NMR (500 MHz, CDCl3, a mixture of rotamers) δ 8.05 (s, 0.6H), 8.04
(s, 0.4H), 7.87 (s, 0.6H), 7.79 (s, 0.4H), 7.44−7.40 (m, 2H), 7.36−
8811
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analysis. Test sample concentrations were determined by LC/MS
using propranolol as an internal standard. Pharmacokinetic parameters
were calculated using DAS 2.0 software.
factor receptors β; RET, rearranged during transfection kinase;
Src, Rous sarcoma oncogene cellular homologue
Mouse Tumor Xenograft Efficacy Study. The efficacy study was
conducted in accordance with the guidelines for the Care and Use of
Laboratory Animals in Guangzhou Institute of Biomedicine and
Health (GIBH, CAS). Six week old SCID mice were inoculated
subcutaneously with H1975 NSCLC cells (2 × 106/mouse) in the
right flank. Upon attaining an average tumor volume of 200−300 mm3
(10−12 days post implantation), animals were randomized into
treatment groups (n = 5 per group). Each group was dosed orally for
14 days with either vehicle only or with compound 2v at 10 or 30 mg/
kg daily (qd). Final concentration of vehicle was 1% DMSO, 5%
EtOH, 15% castor oil, and 79% H2O. The doses were 0.1 mL/20 g of
the animal body weight. The length (L) and width (W) of the tumor
mass were measured, and the tumor volume (TV) was calculated as:
TV = W2(L/2). Tumor growth inhibition (TGI) was calculated using
the following formula: TGI = [1 − (T − T0)/(C − C0)] × 100, where
T and T0 are the mean tumor volumes on a specific experimental day
and on the first day of treatment, respectively, for the experimental
groups, and likewise where C and C0 are the mean tumor volumes for
the control group.
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ASSOCIATED CONTENT
■
S
* Supporting Information
1H NMR, 13C NMR, and purity determination for compounds
2a−v, and biological assay details and results. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: +86-20-32015276. Fax: +86-20-32015299. E-mail: ding_
ke@gibh.ac.cn.
Notes
■
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ACKNOWLEDGMENTS
■
We thank National Basic Research Program of China (no.
2010CB529706, 2009CB940904), National Natural Science
Foundation (no. 21072192), Key Project on Innovative Drug
of Guangdong Province (no. 2011A080501013), Strategic
Collaborative project of Guangdong Province and Chinese
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ABBREVIATIONS USED
■
T, threonine; M, methionine; L, leucine; R, arginine; E,
glutamic acid; A, alanine; EGFR, epidermal growth factor
receptor; NSCLC, nonsmall cell lung cancer; MTD, maximum
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C
max, maximum concentration; Cl, clearance rate; AUC, area
under curve; WT, wild-type; m-CPBA, 3-chloroperbenzoic acid;
AKT, protein kinase B; ERK, extracellular signal-regulated
kinases; TGI, tumor growth inhibition; ABL1, abelson murine
leukemia viral oncogene homologue 1; Blk, B lymphocyte
kinase; Btk, Bruton’s tyrosine kinase; DDR1, discoidin domain
receptor 1; Flt3, FMS-like tyrosine kinase 3; Kit, mast/stem cell
growth factor receptor; Jak3, Janus kinase 3; MEK5, mitogen-
activated protein kinase 5; PDGFRβ, platelet-derived growth
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