Discovery of novel 2,4-diarylaminopyrimidine analogues (DAAPalogues) showing potent inhibitory activities against both wild-type and mutant ALK kinases

Article abstract of DOI:10.1021/jm5005144

We have developed a series of new 2,4-diarylaminopyrimidine analogues (DAAPalogues) bearing a flexible amino acid side chain, different from the majority of the literature reported ALK inhibitors that often possess a structurally constrained arylpiperazine fragment or its equivalents in the solvent-interaction region. Extensive structural elaboration led to compound 15 possessing IC₅₀ values of 2.7 and 15.3 nM, respectively, in the ALK wild-type and gate-keeper mutant L1196M enzymatic assays. This compound not only showed high proliferative inhibition against ALK-addicted cells across different oncogenic forms but also effectively suppressed several ALK secondary mutant cells, including the gate-keeper L1196M and F1174L. Significant antitumor efficacy was achieved in the ALK-driven SUP-M2 xenograft model.

Full text of DOI:10.1021/jm5005144

Article
pubs.acs.org/jmc
Discovery of Novel 2,4-Diarylaminopyrimidine Analogues
(DAAPalogues) Showing Potent Inhibitory Activities against Both
Wild-type and Mutant ALK Kinases
Zilan Song,†,§ Yanhong Yang,‡,§ Zhiqing Liu,† Xia Peng,‡ Junfeng Guo,† Xinying Yang,‡ Kui Wu,†
Jing Ai,*,‡ Jian Ding,‡ Meiyu Geng,*,‡ and Ao Zhang*,†
†CAS Key Laboratory of Receptor Research, Synthetic Organic & Medicinal Chemistry Laboratory, Shanghai Institute of Materia
Medica (SIMM), Chinese Academy of Sciences, Shanghai 201203, China
‡Division of Anti-tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica (SIMM),
Chinese Academy of Sciences, Shanghai 201203, China
S
* Supporting Information
ABSTRACT: We have developed a series of new 2,4-diarylaminopyrimidine analogues (DAAPalogues) bearing a flexible amino
acid side chain, different from the majority of the literature reported ALK inhibitors that often possess a structurally constrained
arylpiperazine fragment or its equivalents in the solvent-interaction region. Extensive structural elaboration led to compound 15
possessing IC50 values of 2.7 and 15.3 nM, respectively, in the ALK wild-type and gate-keeper mutant L1196M enzymatic assays.
This compound not only showed high proliferative inhibition against ALK-addicted cells across different oncogenic forms but
also effectively suppressed several ALK secondary mutant cells, including the gate-keeper L1196M and F1174L. Significant
antitumor efficacy was achieved in the ALK-driven SUP-M2 xenograft model.
FDA in 2011 as the standard treatment for ALK-positive
NSCLCs. Unfortunately, despite the appreciable objective
response rates and impressive progression-free survival times
on crizotinib-treatment,21 resistance to this drug was reported
within a year of starting therapy, which led to the majority of
patients experiencing relapse and tumor regrowth. Although
activation of alternative signaling pathways that bypass ALK
may be one of the resistance mechanisms, analyses of the
resistant patient samples indicated that dozens of ALK gene
secondary mutations22−25 have developed following crizotinib-
treatment, including the gate-keeper mutation L1196M,
F1174L, and others.26,27 Since the gatekeeper mutation
L1196M is believed to cause steric hindrance of crizotinib
binding and is similar to the gatekeeper mutations in the
EGFR-T790M28 and ABL-T315129 kinase domains, a new
INTRODUCTION
■
Anaplastic lymphoma kinase (ALK) is an orphan receptor
tyrosine kinase (RTK), structurally belonging to the insulin
receptor family.1 Implication in brain development and
regulation of specific neurons of the central nervous system
are the general proposed functions of ALK.2−4 In 1994, the
oncogenic nucleophosmin (NPM)−ALK fusion gene was
discovered as an ALK translocation product occurring in 80%
of anaplastic large-cell non-Hodgkin’s lymphoma (ALCL)
cases.5 Subsequently, constitutive activations of ALK due to
chromosomal translocations, amplifications, or point mutations
have been found in other cancers.4 The echinoderm micro-
tubule-associated protein-like 4 (EML4)−ALK fusion gene has
characteristics of “oncogene-addicted tumors” and is implicated
in the largest non-small-cell lung cancer (NSCLC) patient
populations.6−8 The proof-of-concept of the “tumor-addicted
oncogene” EML4−ALK as a novel therapeutic target9−16 has
been validated by Pfizer’s first-generation ALK inhibitor
crizotinib (PF2341066, Xalkori),17−20 which was approved by
Special Issue: New Frontiers in Kinases
Received: April 1, 2014
© XXXX American Chemical Society
A
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Figure 1. Marketed ALK inhibitor 1 and new clinical inhibitors 2−4 (blue color denotes solvent region).
Figure 2. Proposed new DAAPalogues containing an amino acid side chain.
a
Scheme 1. Synthesis of Compounds 10−14
a
Reagents and conditions: (i) for 8a, 8b, 8d, 8e, and 8f, DIPEA, IPA; for 8c, NaH, DMF; (ii) for 10, CSA, IPA, MW, 80 °C; for 11, Pd(OAc)2, X-
Phos, Cs2CO3, 1,4-dioxane, MW, 100 °C; (iii) 1-pyrrolidinecarbonyl chloride, Et3N, CH2Cl2; (iv) (a) Boc-L-Proline, TBTU, DIPEA, DMF; (b)
TFA, CH2Cl2; (v) (a) TFA, CH2Cl2; (b) tert-butylisocyanate, CH2Cl2.
generation30 of ALK inhibitors able to overcome the resistant
mutant L1196M is greatly needed. Currently, a number of
second-generation ALK inhibitors have progressed to clinical
trials including 2,4-diarylaminopyrimidine 2,31,32 triazene 3,33
B
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a
Scheme 2. Synthesis of Compounds 15−27
a
Reagents and conditions: (i) DIPEA, IPA; (ii) CSA, IPA, MW,80 °C.
a
Scheme 3. Synthesis of Compounds 28−48
a
Reagents and conditions: (i) CPA, IPA, MW, 80 °C; (ii) 10% Pd/C, H2, MeOH; (iii) chloroacetyl chloride, Et3N, CH2Cl2; (iv) TBTU, DIPEA,
DMF; (v) K2CO3, Et3N, MeCN.
and tetracyclic 434−36 (Figure 1). All these new ALK inhibitors
showed high potency against both wild-type and mutant ALK
and exerted significant in vivo antitumor efficacy in crizotinib-
resistant ALK-positive NSCLC patients.
selective ALK inhibitor with efficacy against both wild-type
ALK and L1196M mutant. Herein, we present the synthesis
and pharmacological results.
By using a scaffold repurposing strategy,37 we recently
developed38 a series of 2,4-diarylaminopyrimidine analogues
(DAAPalogues) from Cephalon’s tyrosine kinase c-Met
inhibitor 539 (Figure 2). These compounds structurally featured
a 1-methyl/aryl-N3-benzazepine framework and showed high
potency against both c-Met and ALK kinases. As a continuation
of this work,40 we conducted an alternative structural
modification approach by opening the azepanone ring of 5 at
the C3−C4 position, thus leading to a series of new
DAAPalogues bearing a flexible amino acid side chain. These
new compounds differed from most of the reported ALK
inhibitors that often contained a structurally constrained
arylpiperazine fragment or its equivalent in the solvent-
interaction region (as that in Figure 1, blue color). Structural
elaboration indicated that subtle change within the amino acid
component led to significant difference in the ALK potency and
selectivity. Compound 15 was found to be the most potent and
CHEMISTRY
■
As outlined in Scheme 1, 4-amine-2,5-dichloropyrimidines 8a−
f32,38,39 were used as the key intermediates, which were
prepared from 2,4,5-trichloropyrimidine and commercially
available amines 7a−f by following literature procedures.
Substitution of 8a with tert-butyl (2-((3-aminophenyl)-
amino)-2-oxoethyl) carbamate (9a) in the presence of CSA
provided compound 10 in 78% yield.39 Meanwhile, compound
11 was prepared by treating chloride 8b with 9a in the presence
of Pd(OAc)2, X-Phos, and Cs2CO3 in 52% yield.38 Urea
derivative 12 was obtained by treating amine 10 with 1-
pyrrolidinecarbonyl chloride in 48% yield. Condensation of 10
with Boc-L-proline followed by deprotection led to amino
amide 13 in 67% yield.38 Deprotection of compound 11
followed by reacting with 2-isocyanato-2-methylpropane
provided compound 14 in 47% yield.
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a
Table 1. Inhibitory Effects of New Compounds against c-Met and ALK Kinases
a
IC50’s were calculated by Logit method from the results of at least two independent tests with eight concentrations each and expressed as means
SD.
D
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a
Table 2. Inhibitory Effects of New Compounds against c-Met and ALK Kinases
a
IC50’s were calculated by Logit method from the results of at least two independent tests with eight concentrations each and expressed as means
SD.
Meanwhile, dichloropyrimidine 8g was obtained in 76% yield
by treating commercially available 1-(methylsulfonyl)-1,2,3,4-
tetrahydro-quinoxaline with 2,4,5-trichloropyrimidine. Subse-
quent condensation of 9a with pyrimidines 8c−g in the
presence of CSA yielded amino amides 15−19 in 53−82%
yields. Meanwhile, treating 8c with various amino acid
derivatives 20 under a similar condensation conditions
provided DAAPalogues 21−27 in 42−69% yields (Scheme 2).
In addition, N-(3-aminophenyl)-5-chloro-N′-[2-
(isopropylsulfonyl)phenyl]pyrimidine-2,4-diamine (29) was
prepared from commercially available 3-nitroaniline by
following a literature procedure.41 Subsequent condensation38
of 29 with various amino acids provided compounds 31−33,
46, and 47 in 41−68% yields.
Amidation of 29 with 2-chloroacetyl chloride yielded
compound 30. Compounds 34−43 bearing a cyclic amine
functionality were prepared in 47−78% yields by treating
intermediate 30 with corresponding cyclic amine substrates in
the presence of K2CO3 and Et3N.42 Similarly, compounds 44
and 45 were obtained in 48−56% yields by condensation of 30
with corresponding N-Boc amines followed by deprotection
(Scheme 3).
RESULTS AND DISCUSSION
■
Enzymatic Assay. The 2,4-diarylaminopyrimidine (DAAP)
scaffold has long been recognized as a classical kinase inhibitor
motif;43,44 our recent study also confirmed that such
compounds displayed high potency against several kinase
targets, especially c-Met and ALK.38 Therefore, all the new
synthetic compounds were initially assayed for their inhibitory
effects against both c-Met and ALK, and compounds with high
potency and selectivity for ALK over c-Met will be selected for
further profiling. As shown in Table 1, opening the azepanone
ring of Cephalon’s c-Met inhibitor 5 by deletion of the C4−C5
ethylene unit generated urea 12, which retained good inhibitory
effect against c-Met, but the potency at ALK was moderate
(280 nM). Isomerization of 12 to dipeptide 13 led to slightly
enhanced potencies at both c-Met and ALK kinases, with IC50
values of 16 and 80 nM, respectively. N-t-Butyl urea 14 showed
significantly reduced potency at ALK, whereas higher potency
against c-Met was retained. Similar high c-Met potency and
selectivity were observed as well for compound 11 bearing a t-
butoxycarbonyl moiety as the terminal capping group. Quite
interestingly, simple 2-amino-N-ethylacetamide 10 bearing a
naked primary amino group showed high potency at both c-
E
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a
Table 3. Inhibitory Effects of New Compounds against c-Met and ALK Kinases
a
IC50’s were calculated by Logit method from the results of at least two independent tests with eight concentrations each and expressed as means
SD.
F
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Met and ALK, with IC50 values of 6.5 and 32.8 nM,
respectively.
for ALK over c-Met than the nonsubstituted piperazine 36.
High potency was observed on N-acyl substituted piperazine
analogue 39, which showed an IC50 value of 0.7 nM and an
ALK/c-Met selectivity of 145. This compound is superior to
compound 15 in both potency and selectivity for ALK. The N-
containing bicyclic analogues 41−43 generally had moderate
selectivity for ALK over c-Met, but compounds 41 and 43 still
retained high ALK potency, indicating that a remote H-bonding
donor is beneficial for interaction with ALK kinase. High
potency and selectivity against ALK were also obtained from
compounds 44 and 45 bearing an octahydropyrrolo[3,4-
b]pyrrole terminal group. Replacement of the primary amino
group in 15 with a furan (46) or an indole (47) moiety led to
reduction in both potency and selectivity at the ALK kinase.
Cellular Inhibition Study. On the basis of the enzymatic
assays, potent and selective ALK inhibitors were selected for
cellular assay. The antiproliferative effects38 of the compounds
in ALK-addicted lymphoma cell line SUP-M2 harboring
NPM−ALK are listed in Table 4. Meanwhile, tumor cell line
With the moderate ALK inhibitor 10 as our new lead, we
optimized the pyrimidin-2-yl aminobenzamide moiety. It was
found that replacing the N-methylcarbonyl substituent in the
aminobenzamide component of 10 with the isopropylsulfonyl
group as that in clinical ALK inhibitors 2 and 3 significantly
increased ALK potency and repositioned the selectivity from c-
Met to ALK. Compound 15 showed an IC50 value of 2.7 nM
for ALK and was 56-fold more potent against ALK than against
c-Met kinase. Incorporation of the 3-aminobicyclo[2.2.1]hept-
5-ene-2-carboxylic amide fragment, a more lipophilic group
used by Cephalon45 for generation of new ALK inhibitors led
to compound 16, which retained good potency and selectivity
at c-Met, but the potency at ALK was moderate. Compared
with benzamide 10, methanesufonylamine 17 was 3-fold less
potent against the ALK kinase, but much higher potency was
observed when an N-methyl substituent was introduced. N-
Methylmethanesufonylamine 18 showed an IC50 value of 7.7
nM and was 7-fold more potent against ALK than against c-
Met. Tetrahydroquinoxaline 19, formed by connecting the two
N-atoms of the benzene-1,2-diamino component with an
ethylene unit lost potency at both ALK and c-Met. The results
indicated that the free NH connecting to central pyrimidine
core, rather than the NH in the methanesufonylamino moiety,
was critical to the interactions with both kinases.
Since compound 15 displayed high potency and selectivity
for ALK kinase, our further optimization was focused on
exploring the steric and electronic capacities of the amino
acetamide side chain based on the structure 15. As shown in
Table 2, we first tested various amino acids bearing a primary
amino group. Insertion of a methyl group yielded 2-
aminopropanamide 21, which retained high potency against
ALK (1.8 nM), but the selectivity over c-Met was only 4-fold.
More bulky groups such as those in 22−25 led to slightly
increased selectivity, whereas the potency against ALK was
sacrificed. Compound 26 with a one-carbon longer alkyl chain
retained high potency against ALK with an IC50 value of 3.1
nM, which was 14-fold higher than that against c-Met.
However, further enlongating the alkyl chain to 4-amino
butaneamide 27 caused reduction in ALK potency.
Meanwhile, to elucidate the necessity of the primary amino
group for the high ALK potency of compound 15, a large
number of 2-amino acetamido fragments bearing diversified
substitutions on either the amino group or the alkyl chain or
both were investigated. As shown in Table 3, substitution on
the primary amino group by forming an azetidine ring led to
compound 31, which showed an IC50 value of 44 nM against
ALK. Notably, the 3-azetidine regiomer 32 was 2-fold more
potent against ALK than 31 and nearly inactive against c-Met,
thus providing a 50-fold ALK/c-Met selectivity. Extremely high
potency was observed for the N-4-(dimethylamino)butanoyl
substituted azetidine analogue 33, which showed an IC50 value
of 0.8 nM at ALK and was over 1000-fold selective versus c-
Met. Conversion of the primary amino group in 15 to
morpholines 34 and 35 also provided relatively good potency
and selectivity for ALK, but they were much less potent than
either 15 or 33. Interestingly, replacement of the primary
amino group in 15 by a substituted piperazinyl moiety
generated highly potent and selective ALK inhibitors 36−40.
All these compounds displayed IC50 values of less than 10 nM
at ALK, but the selectivity over c-Met differed. The alkyl
substituted piperazine analogues 37 and 38 were more selective
Table 4. Inhibitory Effects of Potent Compounds on Cell
Proliferation
IC50
compd
ALK enzyme IC50 (nM) ALK SUP-M2 (nM) A549 (μM)
15
21
26
33
37
39
43
44
2.7
1.8
3.1
0.8
1.7
0.7
3.0
2.3
3.4
13.7
15.3 3.7
<0.1
1.1 0.4
0.2 0.01
2.3 0.08
3.9 0.2
0.2 0.02
0.2 0.02
0.1 0.02
0.8 0.1
>1
79.0 18.6
365 3.9
0.1 0.04
7.7 2.2
0.2 0.09
5.5 1.4
167 35.9
174 20.8
a
2
crizotinib
>1
a
Compound 2 (LDK378) was reported32 to be more sensitive against
BaF3/NPM−ALK cells with an IC50 value of 26.0 nM.
A549 whose growth was not dependent on ALK was used to
test the potential off-target effects of the selected ALK
inhibitors. In parallel with the enzymatic results, all the selected
compounds displayed high antiproliferative effects in the SUP-
M2 cell lines. Compound 15 bearing a primary amino group
showed an IC50 value of 15.3 nM and 70-fold selectivity over
A549 cells. Compound 21 bearing a methyl group in the side
chain, though equally potent to 15 in the enzymatic assay, was
much more potent (>150-fold) in the cellular assay with an
IC50 value of less than 0.1 nM. Quite disappointingly, this
compound also showed high potency in the A549 cells
indicating that a general cytotoxicity might exist. Although
the exact reason was not clear, the higher cellular potency and
cytotoxicity of 21 might be partially related to the increased
lipophilicity by the methyl group. Compound 26 with a longer
side chain and compound 33 bearing an N-acyl substituted
azetidine moiety displayed moderate potency in the SUP-M2
cells, despite their high enzymatic potencies. Although equally
potent to compound 15 in the ALK enzymatic assay, the
piperazinyl DAAPalogue 37 displayed 150-fold higher cellular
potency with an IC50 value of 0.1 nM. However, this compound
was also highly potent in the A549 cell lines. In comparison to
their high enzymatic potency, N-acyl substituted piperazine
analogue 39 and the octahydropyrrolo[3,4-b]pyrrole 44 were
less potent in the cell and showed higher general cytotoxicity in
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Figure 3. Preliminary PK results.
the A549 cell lines. The bicyclic compound 43 showed 15-fold
higher cellular potency than its enzymatic potency, whereas
significant cytotoxicity was observed as well in A549 cells. All
these new DAAPalogues were more potent than the marketed
drug crizotinib (1) and clinical compound 2 in both ALK
kinase and SUP-M2 cells.
Preliminary Pharmacokinetic Study. From the results
above, compound 15 stood out having high potency against
both ALK enzyme and cells and showed less potential for
cytotoxicity. This compound was further evaluated in our
preliminary pharmacokinetic study37,46 at a dose of 2.5 mg/kg
(i.g., intragastric) in rats. As shown in Figure 3, compound 15
showed a moderate plasma exposure (AUC0‑∞ = 454 ng·h/mL)
and a short half-life. It has a moderate lipophilicity but with
poor aqueous solubility (less than 0.1 mg/mL). However, the
solubility was much improved by forming the corresponding
hydrochloride salt (∼4 mg/mL).
also to show high potency against LTK kinase, a family member
of ALK; otherwise much weaker inhibitory effects were
observed on the remaining 17 tested kinases (IC50 > 1 μM
or 0.1 μM) (Table 5). Therefore, compound 15 was a potent
ALK family inhibitor with against the resistant gatekeeper
mutant.
Antiproliferative Effects against Both ALK-Addicted
and Crizotinib-Resistant Cells. Since activated ALK is
known to trigger cancer cell proliferation, we then set out to
investigate the inhibitory effects of compound 15 on cell
proliferation in a panel of human cancer cell lines harboring
different levels of ALK expression and activation. As shown in
Table 6, compound 15 significantly inhibited cell proliferation
Table 6. Effects of Compound 15 on Cell Proliferation in
a
ALK-Addicted Cell Lines
cell line
IC50 (nM)
Overcoming ALK Resistant Gatekeeper Mutant Study.
To determine the potential of compound 15 in overcoming
crizotinib’s resistance, it was evaluated against the ALK
gatekeeper mutant L1196M.30 An IC50 value of 15.3 nM was
observed indicating that compound 15 was potent against both
wild-type and mutant ALK (Table 5). Meanwhile, compound
15 was further evaluated for the inhibitory activities on a panel
of our in-house47 tyrosine kinases. Compound 15 was found
SUP-M2 (NPM-ALK)
15.3 3.7
15.0 0.3
28.6 2.3
96.8 15.7
41.9 9.7
SU-DHL-1 (NPM-ALK)
NB-1 (ALK Amplification)
H3122 (EML4-ALK variant1)
BaF3/EML4-ALK (V3)
a
The IC50 values are shown as the mean
separate experiments.
SD (nM) from three
Table 5. Kinase Selectivity Profile of Compound 15
of ALK constitutively activated SUP-M2, SU-DHL-1, NB-1,
H3122, and BaF3/EML4−ALK (V3) cells that cover the
frequently occurring oncogenic forms of ALK (ALK
amplification, ALK chromosomal rearrangement), with IC50
values of 15.3, 15.0, 28.6, 96.8, and 41.9 nM, respectively.
These results suggested that compound 15 inhibited ALK-
dependent cell proliferation across different oncogenic forms.
Meanwhile, antiproliferative effects of compound 15 in a
number of crizotinib-resistant cell lines were tested as well
(Table 7). Notably, compound 15 showed an equally high
potency against both native EML4−ALK and EML4−ALK
L1196M-mediated NIH/3T3 cell proliferation with IC50 values
of 142 and 149 nM, respectively. Moreover, compound 15
% or IC50
a
kinase
ALK
15
1 (crizotinib)
2.7 0.8 nM
15.3 0.1 nM
93% @ 0.1 μM
32.2% @ 0.1 μM
>1 μM
13.7 1.6 nM
122 20.4 nM
ALK-L1196M
LTK
RON
AXL
Tyto3
>1 μM
Mer
>1 μM
PDGFR-α
PDGFR-β
EGFR
EGFRT790M‑L858R
FGFR1
c-Src
>1 μM
>1 μM
>1 μM
significantly inhibited the proliferation of Kelly cells (IC50
=
35% @ 0.1 μM
50% @ 0.1 μM
>1 μM
Table 7. Antiproliferative Effects of Compound 15 in
a
Crizotinib-Resistant Cells
RET
43% @ 0.1 μM
>1 μM
KDR
IC50 (nM)
Flt-1
>1 μM
cell line
15
crizotinib
c-Kit
>1 μM
NIH/3T3/EML4−ALK (V1)
NIH/3T3/EML4−ALK (V1)-L1196M
Kelly (ALK F1174L)
142 26.6
149 7.8
28.1 6.9
31.3 2.3
95.4 0.3
Abl
>1 μM
606 51.5
>1000
ErbB4
12% @ 0.1 μM
a
LAN-5 (ALK R1275Q)
>1000
IGF1R, insulin-like growth factor 1 receptor; PDGFR, platelet
derived growth factor receptor; EGFR, epidermal growth factor
receptor; KDR, kinase insert domain receptor; FGFR1, fibroblast
growth factor receptor 1; Flt-1: Fms-like tyrosine kinase.
a
The IC50 values are shown as the mean
separate experiments.
SD (nM) from three
H
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28.1 nM), which harbor crizotinib-induced resistant mutant
F1174L. In addition, compound 15 significantly inhibited the
proliferation of cell line LAN-5 (IC50 = 31.3 nM) harboring
another hot spot-activating mutation (R1275Q) in neuro-
blastoma and a mutant much less sensitive to crizotinib (Table
7).23 These data further confirmed that compound 15 was
capable of blocking the ALK resistant mutants.
In Vivo Antitumor Efficacy Study. Since compound 15
possessed high ALK enzymatic and cellular potencies and was
also effective in blocking the ALK resistant mutants, we
proceeded to evaluate its antitumor efficacy in vivo. The SUP-
M2 xenograft model,47,48 which was specifically driven by
constitutively active ALK, was used (Figure 4). Compound 15
EXPERIMENTAL SECTION
■
Chemistry. 1H NMR spectral data were recorded in CDCl3,
CDCl3/CD3OD, or DMSO-d6 on Varian Mercury 300 or 400 NMR
spectrometer, and 13C NMR was recorded in CDCl3 on Varian
Mercury 400 or 500 NMR spectrometer. Low-resolution mass spectra
(MS) and high-resolution mass spectra (HRMS) were recorded at
anionizing voltage of 70 eV on a Finnigan/MAT95 spectrometer.
Column chromatography was carried out on silica gel (200−300
mesh). All reactions were monitored using thin layer chromatography
(TLC) on silica gel plates. Compounds 8a−f32,38,39 were prepared
according to the literature procedures.
General Procedure for Synthesis of Compounds 10, 15−19, 21−
27, and 28. A microwave tube (10 mL) was charged with 4-amine-2,5-
dichloropyrimidines (8a−g) (0.2 mmol), an appropriate aniline (9a or
20) bearing an amino acid side chain (0.28 mmol), (1S)-(+)-10-
camphorsulfonic acid (CSA, 0.4 mmol), and isopropanol (3 mL). The
mixture was heated to 80 °C under microwave irradiation (80 W) for 1
h. The solution was concentrated under vacuum, washed with
saturated NaHCO3 and brine, dried over Na2SO4, and purified by
chromatography (CHCl3/MeOH = 10:1) to afford corresponding
target compound.
2-((2-((3-(2-Aminoacetamido)phenyl)amino)-5-chloropyrimidin-
1
4-yl)amino)-N-methylbenzamide (10). White solid (78%). H NMR
(300 MHz, DMSO) δ 11.68 (s, 1H), 9.47 (d, J = 3.4 Hz, 1H), 8.85−
8.68 (m, 2H), 8.25−8.17 (m, 1H), 7.85 (s, 1H), 7.79−7.68 (m, 1H),
7.49−7.33 (m, 2H), 7.31−7.06 (m, 3H), 3.28 (s, 2H), 3.25 (d, J = 3.1
Hz, 2H), 2.79 (t, J = 3.8 Hz, 3H). 13C NMR (126 MHz, DMSO) δ
171.6, 168.9, 157.7, 154.9, 154.4, 140.5, 139.3, 138.8, 131.5, 128.5,
127.9, 121.8, 121.2, 120.4, 115.0, 113.0, 110.8, 105.2, 45.3, 26.3. MS
(ESI) m/z 426 [M + H]+. HRMS: calcd for C22H21ClN7O2 [M + H]+,
426.1445; found 426.1446.
Figure 4. Antitumor activity of compound 15 in SUP-M2 xenograft
model. Mice bearing SUP-M2 were orally administered compound 15
in the HCl salt form with 0.5% CMC-Na (sodium carboxymethyl
cellulose) or crizotinib in water once daily for 14 days after the tumor
volume reached 80 to 100 mm3 at the indicated doses. The results
were expressed as the mean standard error (drug-treated group, n =
5; vehicle group, n = 10) **p < 0.01 vs vehicle group on final day,
using Student’s t test.
2-Amino-N-(3-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)-
pyrimidin-2-yl)amino)phenyl)acetamide (15). Off-white solid (82%).
1H NMR (300 MHz, DMSO) δ 10.13 (s, 1H), 9.57 (s, 1H), 8.65 (d, J
= 7.7 Hz, 1H), 8.26 (s, 1H), 7.91−7.76 (m, 4H), 7.68 (t, J = 7.5 Hz,
1H), 7.39−7.06 (m, 5H), 6.19 (s, 1H), 3.65 (s, 1H), 3.23 (s, 2H), 1.15
(d, J = 6.7 Hz, 6H).13C NMR (126 MHz, CD3OD) δ 164.2, 158.1,
151.8, 141.9, 138.8, 136.2, 135.6, 134.7, 131.3, 129.4, 128.1, 126.4,
126.1, 119.5, 117.4, 114.9, 106.2, 55.5, 40.8, 13.9. MS (ESI) m/z 475
[M + H] +. The free base was converted to its HCl salt. Anal. Calcd for
C21H23N6O3S.2HCl.0. 5H2O: C, 45.29, H, 4.71, N, 15.09; found C:
45.17, H: 4.96, N: 15.08.
(1S,2S,3R,4R)-3-((2-((3-(2-Aminoacetamido)phenyl)amino)-5-
chloropyrimidin-4-yl)amino)-N-methylbicyclo[2.2.1]hept-5-ene-2-
carboxamide (16). Off-white solid (78%). 1H NMR (400 MHz,
DMSO) δ 9.32 (s, 1H), 8.31 (d, J = 4.6 Hz, 1H), 7.96 (d, J = 7.4 Hz,
2H), 7.68 (d, J = 7.7 Hz, 1H), 7.51−7.43 (m, 1H), 7.18 (d, J = 5.9 Hz,
2H), 6.31 (dd, J = 5.6, 2.8 Hz, 1H), 6.22 (dd, J = 5.4, 3.0 Hz, 1H), 4.17
(t, J = 7.9 Hz, 1H), 2.83 (s, 1H), 2.76 (s, 1H), 2.62 (d, J = 4.5 Hz,
3H), 2.53 (s, 1H), 2.12 (d, J = 8.6 Hz, 1H), 1.39 (d, J = 8.6 Hz, 1H).
13C NMR (101 MHz, DMSO) δ 174.4, 172.1, 158.5, 156.8, 153.3,
was administered orally as its hydrochloride salt with 0.5%
CMC-Na (sodium carboxymethyl cellulose) at doses of 50 or
30 mg/kg once daily for 14 consecutive days. Compared with
the vehicle treatment, compound 15 significantly inhibited
tumor growth at both doses with tumor growth inhibitory rates
of >89% (p < 0.01) and with no significant body weight loss.
CONCLUSION
■
141.5, 139.1, 137.1, 128.9, 114.5, 112.8, 110.5, 104.1, 52.4, 48.1, 47.3,
45.8, 44.8, 44.2, 26.1. MS (ESI) m/z 442 [M + H]+. HRMS: calcd for
C21H25ClN7O2 [M + H], 442.1758; found 442.1754.
In summary, we have developed a series of new DAAPalogues
bearing a flexible amino acid side chain. These new compounds
differed from most of the literature reported ALK inhibitors,
which often contain a structurally constrained arylpiperazine
fragment or its equivalents in the solvent-interaction region.
Structural elaboration indicated that subtle change within the
amino acid fragment led to significant difference in both the
ALK potency and selectivity. Compound 15 was identified as a
potent and selective ALK inhibitor possessing IC50 values of 2.7
and 15.3 nM against the ALK wild-type and L1196 mutant
enzymes, respectively. It showed high antiproliferative effects
toward a number of ALK-dependent cells across different
oncogenic forms and was potent as well against several ALK
resistant mutant cells, including the gate-keeper L1196M and
L1174M. Significant antitumor efficacy was achieved for
compound 15 in the ALK-driven SUP-M2 xenograft model.
2-Amino-N-(3-((5-chloro-4-((2-(methylsulfonamido)phenyl)-
amino)pyrimidin-2-yl)amino)phenyl)acetamide (17). Off-white solid
(62%). 1H NMR (400 MHz, CDCl3/MeOD) δ 7.98 (s, 1H), 7.90 (d, J
= 7.9 Hz, 1H), 7.55 (s, 1H), 7.33 (d, J = 7.7 Hz, 1H), 7.29−7.25 (m,
1H), 7.18 (d, J = 8.2 Hz, 3H), 7.11 (d, J = 8.0 Hz, 1H), 3.35 (s, 2H),
2.90 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 171.3, 157.5, 156.2,
154.2, 139.8, 137.7, 134.3, 129.1, 128.9, 127.6, 126.7, 125.8, 125.6,
115.6, 113.9, 110.8, 105.5, 44.6, 38.9. MS (ESI) m/z 462 [M + H]+.
HRMS: calcd for C19H20ClN7O3S [M + H], 462.1115; found
462.1123.
2-Amino-N-(3-((5-chloro-4-((2-(N-methylmethylsulfonamido)-
phenyl)amino)pyrimidin-2-yl)amino)phenyl)acetamide (18). Off-
1
white solid (72%). H NMR (400 MHz, DMSO) δ 10.44 (s, 1H),
9.55 (s, 1H), 8.41 (s, 2H), 8.22 (s, 1H), 7.83 (s, 1H), 7.64 (d, J = 7.9
Hz, 1H), 7.44−7.38 (m, 2H), 7.23 (dd, J = 19.7, 8.5 Hz, 3H), 3.68 (s,
2H), 3.21 (s, 3H), 3.12 (s, 3H). 13C NMR (101 MHz, CDCl3) δ
I
dx.doi.org/10.1021/jm5005144 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
+
166.4, 158.2, 155.3, 154.7, 141.1, 138.8, 137.0, 132.9, 129.1, 127.4,
124.8, 123.4, 115.8, 113.5, 111.2, 105.1, 42.2, 39.3, 35.5. MS (ESI) m/z
476 [M + H] +. HRMS: calcd for C20H22ClN7O3S [M + H], 476.1272;
found 476.1268.
(ESI) m/z 555[M + H] . HRMS: calcd for C25H28ClN8O3S [M +
H],: 555.1688; found 555.1688.
3-Amino-N-(3-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)-
pyrimidin-2-yl)amino)phenyl)propanamide (26). Off-white solid
(45%). H NMR (400 MHz, CDCl3) δ 10.02 (s, 1H), 9.62 (s, 1H),
1
2-Amino-N-(3-((5-chloro-4-(4-(methylsulfonyl)-3,4-dihydroqui-
noxalin-1(2H)-yl)pyrimidin-2-yl)amino)phenyl)acetamide (19). Off-
8.62 (d, J = 7.8 Hz, 1H), 8.16 (s, 1H), 7.89 (dd, J = 8.0, 1.5 Hz, 1H),
7.79 (s, 1H), 7.63−7.55 (m, 2H), 7.41 (d, J = 8.0 Hz, 1H), 7.23 (ddd, J
= 8.0, 3.2, 1.8 Hz, 2H), 7.16 (d, J = 8.3 Hz, 1H), 3.25 (dt, J = 13.7, 6.9
Hz, 1H), 3.14−3.04 (m, 2H), 2.51−2.44 (m, 2H), 1.89 (s, 2H), 1.32
(d, J = 6.9 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 170.6, 157.7,
154.8, 154.6, 139.3, 138.5, 137.9, 134.0, 130.7, 128.7, 123.9, 123.0,
122.6, 115.0, 113.7, 110.9, 105.9, 55.1, 38.3, 37.4, 14.8. MS (ESI) m/z
489 [M + H]+. HRMS: calcd for C22H25ClN6O3S [M + H], 489.1476;
found 489.1476.
1
white solid (53%). H NMR (400 MHz, CDCl3/MeOD) δ 8.13 (s,
1H), 8.09 (s, 1H), 7.67 (dd, J = 7.7, 1.9 Hz, 1H), 7.22 (dd, J = 12.5,
5.0 Hz, 2H), 7.11−7.02 (m, 3H), 6.78 (dd, J = 7.6, 1.8 Hz, 1H), 4.16
(t, J = 6.1 Hz, 2H), 3.99 (t, J = 6.2 Hz, 2H), 3.40 (s, 2H), 2.85 (s, 3H).
13C NMR (101 MHz, CDCl3) δ171.1, 158.2, 158.0, 157.9, 139.7,
138.1, 135.4, 129.2, 128.9, 125.6, 125.2, 124.1, 120.5, 115.0, 113.7,
110.3, 109.7, 48.0, 47.2, 44.6, 37.3. MS (ESI) m/z 488 [M + H]+.
HRMS: calcd for C21H22ClN7O3S [M + H], 488.1272; found
488.1280.
4-Amino-N-(3-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)-
pyrimidin-2-yl)amino)phenyl)butanamide (27). White solid (48%).
1H NMR (400 MHz, CDCl3) δ 9.06 (s, 1H), 8.60 (d, J = 8.3 Hz, 1H),
(S)-2-Amino-N-(3-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)-
amino)pyrimidin-2-yl)amino)phenyl)propanamide (21). Off-white
1
solid (69%). H NMR (400 MHz, CDCl3) δ 9.62 (s, 1H), 9.49 (s,
8.11 (s, 1H), 7.87 (dd, J = 8.0, 1.4 Hz, 1H), 7.73 (s, 1H), 7.67 (s, 1H),
7.56 (t, J = 7.2 Hz, 1H), 7.43 (d, J = 7.3 Hz, 1H), 7.24−7.12 (m, 3H),
3.66 (s, 2H), 3.27−3.19 (m, 1H), 2.89 (t, J = 6.5 Hz, 2H), 2.49 (t, J =
6.9 Hz, 2H), 1.95−1.88 (m, 2H), 1.30 (d, J = 6.9 Hz, 6H). 13C NMR
(101 MHz, CDCl3) δ 171.0, 157.1, 154.7, 154.5, 139.3, 138.4, 137.8,
134.0, 130.7, 128.6, 123.8, 122.9, 122.6, 115.3, 113.7, 110.9, 105.9,
55.2, 40.0, 34.3, 25.9, 14.8. MS (ESI) m/z 503 [M + H]+. HRMS:
calcd for C23H27ClN6O3S [M + H], 503.1632; found 503.1629.
Synthesis of N-(2-((3-((5-Chloro-4-((2-(methylcarbamoyl)phenyl)-
amino)pyrimidin-2-yl)amino)phenyl)amino)-2-oxoethyl)-
pyrrolidine-1-carboxamide (12). A solution of pyrrolidine-1-carbonyl
chloride (27 mg, 0.2 mmol) in CH2Cl2 was added to the solution of
compound 10 (42 mg, 0.1 mmol) and Et3N (40 mg, 0.4 mmol) in
CH2Cl2 at 0 °C. The mixture was stirred for 5 h, poured into water,
and then extracted with CH2Cl2. The organic phase was dried over
Na2SO4, concentrated in vacuo, and purified by chromatography to
afford the corresponding product 12 as a white solid in 48% yield. 1H
NMR (300 MHz, CDCl3+CD3OD) δ 8.74 (d, J = 8.4 Hz, 1H), 8.08 (s,
1H), 7.68 (s, 1H), 7.54 (s, 1H), 7.44 (d, J = 7.5 Hz, 1H), 7.25 (m,
2H), 7.13 (m, 2H), 6.95 (t, J = 7.3 Hz, 1H), 5.57 (s, 1H), 3.81 (s, 2H),
3.25 (s, 4H), 2.86 (d, J = 3.9 Hz, 3H), 1.81 (s, 4H). 13C NMR (126
MHz, DMSO) δ 169.1, 168.9, 157.7, 156.5, 154.9, 154.4, 140.4, 139.3,
139.0, 131.6, 128.4, 127.9, 121.8, 121.2, 120.4, 115.0, 113.2, 111.1,
105.2, 45.3, 44.1, 26.3, 25.0. MS (ESI) m/z 545 [M + Na]+. HRMS:
calcd for C25H27ClN8O3 [M + Na], 545.1792; found 545.1799.
Synthesis of Pyrimidin-2-yl(amino)-N-(2-((3-(5-chloro-4-(2-
(methylcarbamoyl)phenyl)amino)phenyl)amino)-2-oxoethyl)-
pyrrolidine-2-carboxamide (13). TBTU (128 mg, 0.4 mmol) and
DIPEA (103 mg, 0.8 mmol) were added successively to a solution
Boc-L-proline (43 mg, 0.3 mmol) and compound 10 (85 mg, 0.2
mmol) in DMF (3 mL). The reaction was stirred overnight at rt and
then diluted with water (8 mL). After filtration, the filter cake was
dried to provide tert-butyl 2-((2-((3-((5-chloro-4-((2-
(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)-
amino)-2-oxoethyl)carbamoyl)pyrrolidine-1-carboxylate as a crude
product. The crude compound was then dissolved in a mixed solvent
system (CH2Cl2/TFA = 1:1, 5 mL), and the mixture was stirred at rt
for 2 h. After removal of the solvent, the residue was taken up in water
(5 mL), alkalified with NaHCO3 to pH > 7, and then extracted with
CHCl3.The combined organic layer was washed with brine (10 mL),
dried, filtered, and then evaporated. The residue was subjected to
column chromatography on silica gel (CHCl3/MeOH, 20/1) to give
1H), 8.61 (d, J = 8.2 Hz, 1H), 8.18 (s, 1H), 7.94−7.84 (m, 2H), 7.64−
7.56 (m, 1H), 7.47−7.39 (m, 2H), 7.24 (ddd, J = 8.1, 7.5, 3.0 Hz, 3H),
3.63 (q, J = 7.0 Hz, 1H), 3.31−3.21 (m, 1H), 1.95 (s, 2H), 1.43 (d, J =
7.0 Hz, 3H), 1.32 (d, J = 6.9 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ
173.7, 157.5, 155.2, 155.1, 139.8, 138.3, 134.5, 131.1, 129.2, 124.3,
123.4, 123.0, 115.5, 113.7, 110.8, 106.4, 55.5, 51.1, 21.5, 15.3. MS
(ESI) m/z 489 [M + H]+. HRMS: calcd for C22H26ClN6O3S [M +
H]+, 489.1476; found 489.1487.
(S)-2-Amino-N-(3-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)-
amino)pyrimidin-2-yl)amino)phenyl)-3-methylbutanamide (22).
Off-white solid (65%). 1H NMR (400 MHz, DMSO) δ 9.59 (s,
1H), 8.72 (d, J = 8.1 Hz, 1H), 8.30 (s, 1H), 7.84 (dd, J = 7.8, 1.5 Hz,
2H), 7.72 (t, J = 7.9 Hz, 1H), 7.37 (dd, J = 14.3, 7.2 Hz, 2H), 7.27 (d,
J = 8.2 Hz, 1H), 7.18 (t, J = 8.0 Hz, 1H), 3.50−3.43 (m, 1H), 3.11 (d,
J = 5.3 Hz, 1H), 1.94 (dd, J = 12.5, 6.6 Hz, 1H), 1.17 (d, J = 6.8 Hz,
6H), 0.92 (d, J = 6.8 Hz, 3H), 0.84 (d, J = 6.8 Hz, 3H). 13C NMR (101
MHz, DMSO) δ 174.1, 158.1, 155.6, 140.7, 139.4, 138.5, 135.4, 131.3,
129.0, 124.3, 123.9, 115.5, 113.8, 111.4, 105.4, 61.0, 55.3, 32.1, 20.1,
17.6, 15.3. MS (ESI) m/z 517 [M + H]+. HRMS: calcd for
C24H30ClN6O3S [M + H], 517.1789; found 517.1790.
1-Amino-N-(3-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)-
pyrimidin-2-yl)amino)phenyl)cyclopropanecarboxamide (23). Off-
white solid (62%). 1H NMR (400 MHz, DMSO) δ 9.95 (s, 1H), 9.59
(s, 1H), 9.53 (s, 1H), 8.68 (d, J = 8.1 Hz, 1H), 8.31 (s, 1H), 7.90 (s,
1H), 7.84 (dd, J = 8.0, 1.4 Hz, 1H), 7.72 (t, J = 7.8 Hz, 1H), 7.40−7.29
(m, 2H), 7.26 (d, J = 8.2 Hz, 1H), 7.18 (t, J = 8.0 Hz, 1H), 3.50−3.41
(m, 1H), 1.39 (s, 2H), 1.17 (d, J = 6.8 Hz, 6H), 1.16−1.13 (m, 2H),
0.89 (dd, J = 6.9, 3.6 Hz, 2H). 13C NMR (126 MHz, DMSO) δ173.6,
157.6, 155.2, 154.7, 140.2, 138.7, 137.9, 134.9, 130.9, 128.5, 124.0,
123.5, 115.0, 113.1, 110.7, 104.8, 54.8, 36.0, 26.3, 18.1, 14.8. MS (ESI)
m/z 501 [M + H]+. HRMS: calcd for C23H26ClN6O3S [M + H],
501.1476; found 501.1479.
2-Amino-N-(3-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)-
pyrimidin-2-yl)amino)phenyl)-2-methylpropanamide (24). White
1
solid (65%). H NMR (400 MHz, CDCl3) δ 9.88 (s, 1H), 9.63 (s,
1H), 8.62 (d, J = 8.3 Hz, 1H), 8.20 (s, 1H), 7.93−7.87 (m, 2H), 7.61
(t, J = 7.2 Hz, 1H), 7.41 (d, J = 7.6 Hz, 1H), 7.34 (s, 1H), 7.24 (t, J =
5.1 Hz, 2H), 3.26 (dt, J = 13.8, 6.8 Hz, 1H), 1.45 (s, 6H), 1.33 (d, J =
6.9 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ175.7, 157.6, 155.3,
155.2, 139.8, 138.7, 138.3, 134.5, 131.1, 129.2, 124.4, 123.4, 123.0,
115.4, 113.7, 110.7, 106.5, 55.6, 55.3, 29.2, 15.3. MS (ESI) m/z 503
[M + H]+. HRMS: calcd for C23H27ClN6O3S [M + H], 503.1632;
found 503.1642.
1
compound 13 as a white solid in 67% yield. H NMR (400 MHz,
DMSO) δ 11.71 (s, 1H), 9.98 (s, 1H), 9.50 (s, 1H), 8.80 (dd, J = 18.6,
6.3 Hz, 2H), 8.30 (t, J = 5.5 Hz, 1H), 8.22 (s, 1H), 7.83 (s, 1H), 7.76
(d, J = 7.6 Hz, 1H), 7.44 (dd, J = 19.7, 7.3 Hz, 2H), 7.21 (d, J = 7.6
Hz, 2H), 7.13 (t, J = 7.5 Hz, 1H), 3.92 (d, J = 5.8 Hz, 2H), 3.63 (dd, J
= 8.8, 5.4 Hz, 1H), 2.89 (dd, J = 11.6, 5.0 Hz, 1H), 2.81 (d, J = 4.3 Hz,
3H), 2.04−1.92 (m, 2H), 1.73 (dd, J = 12.5, 5.7 Hz, 1H), 1.63 (dd, J =
6.7, 3.7 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 174.4, 168.9, 167.4,
157.7, 154.9, 154.4, 140.5, 139.3, 138.8, 131.5, 128.5, 127.8, 121.8,
121.2, 120.4, 115.1, 113.1, 111.0, 105.2, 60.0, 46.6, 42.3, 30.2, 26.2,
(S)-2-Amino-N-(3-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)-
amino)pyrimidin-2-yl)amino)phenyl)-3-(1H-imidazol-4-yl)-
propanamide (25). Off-white solid (42%). 1H NMR (300 MHz,
CDCl3) δ 9.62 (s, 2H), 8.58 (d, J = 8.2 Hz, 1H), 8.15 (s, 1H), 7.92−
7.77 (m, 2H), 7.56 (s, 2H), 7.38 (d, J = 7.3 Hz, 1H), 7.20 (s, 3H), 6.87
(s, 1H), 3.74 (s, 1H), 3.49 (s, 1 H) 3.25 (m, 2H), 3.07 (m, 2H), 1.31
(d, J = 6.7 Hz, 6H). 13C NMR (126 MHz, CDCl3) δ 172.9, 157.5,
155.2, 155.0, 139.9, 138.3, 138.2, 135.2,134.5, 131.2, 129.2, 124.2,
123.3, 123.1, 115.8, 113.9, 110.0, 106.5, 55.6, 55.5, 29.7, 15.3. MS
J
dx.doi.org/10.1021/jm5005144 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
25.6. MS (ESI) m/z 523 [M + H]+. HRMS: calcd for C25H28ClN8O3
[M + H], 523.1973; found 523.1969.
= 8.3, 3.9 Hz, 1H), 3.27 (dt, J = 13.6, 6.8 Hz, 1H), 2.73 (d, J = 8.0 Hz,
1H), 2.51−2.40 (m, 1H), 1.34 (d, J = 6.4 Hz, 6H). 13C NMR (101
MHz, CDCl3) δ 172.0, 157.5, 155.3, 155.1, 139.8, 138.3, 138.2, 134.5,
131.2, 129.3, 124.5, 123.4, 123.1, 115.6, 113.8, 110.6, 106.6, 59.5, 55.6,
43.4, 26.5, 15.3, 15.3. MS (ESI) m/z 501 [M + H]+. HRMS: calcd for
C23H25ClN6O3S [M + H]+, 501.1476; found 501.1476.
Synthesis of tert-Butyl (2-((3-((5-Chloro-4-((2-fluoro-6-
(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)-
amino)-2-oxoethyl)carbamate (11). A microwave tube was charged
with 8b (66 mg, 0.22 mmol), 9a (53 mg, 0.2 mmol), Pd (OAc)2 (7
mg, 0.03 mmol), X-Phos (35 mg, 0.06 mmol), Cs2CO3 (160 mg, 0.5
mmol), and 1,4-dioxane (3 mL) under N2 atmosphere. The mixture
was heated to 100 °C under 80 W for 1.5 h and then concentrated in
vacuum. The residue was taken up in water and CHCl3. The organic
phase was washed with brine, dried over Na2SO4, and concentrated in
vacuo, and the residue was purified by chromatography to afford
compound 11 as an off-white solid in 52% yield. 1H NMR (300 MHz,
DMSO) δ 9.73 (s, 1H), 9.29 (s, 2H), 8.52 (d, J = 4.6 Hz, 1H), 8.15 (s,
1H), 7.53−7.42 (m, 3H), 7.38 (dd, J = 7.3, 4.5 Hz, 1H), 7.29 (d, J =
8.3 Hz, 1H), 7.11 (d, J = 8.1 Hz, 1H), 7.06−6.93 (m, 2H), 3.68 (d, J =
5.9 Hz, 2H), 3.33 (s, 1H), 2.71 (d, J = 4.5 Hz, 3H), 1.37 (s, 9H). 13C
NMR (126 MHz, DMSO) δ 168.4, 167.6, 158.3, 158.2, 156.5, 156.4,
156.3, 154.9, 140.9, 139.2, 132.2, 128.7, 126.9, 126.8, 125.8, 125.7,
124.1, 118.7, 118.6, 114.5, 113.3, 110.6, 105.0, 78.4, 44.1, 28.6, 26.6.
MS (ESI) m/z 566 [M + Na]+. HRMS: calcd for C25H27ClN7O4F [M
+ Na], 566.1695; found 566.1696.
Synthesis of tert-Butyl 2-((2-((3-(2-(3-(tert-Butyl)ureido)-
acetamido)phenyl)amino)-5-chloropyrimidin-4-yl)amino)-3-fluoro-
N-methylbenzamide (14). Amine 15 (54 mg, 0.1 mmol) was
dissolved in a mixed solvent system (CH2Cl2/TFA = 1:1, 5 mL)
and stirred at rt for 2 h. After removal of the solvent, the residue was
taken up in water (5 mL), alkalified with NaHCO3 to pH > 7, and
then extracted with CHCl3. The combined organic layer was washed
with brine, dried, filtered, and then evaporated. The residue was
dissolved in CH2Cl2 (5 mL), and 2-isocyanato-2-methylpropane (30
mg, 0.3 mmol) was added. The mixture was stirred for 24 h at rt and
extracted with water and CH2Cl2. The combined organic layer was
washed with brine, dried, filtered, and then evaporated. The residue
was purified by chromatography to afford compound 14 as an off-
white solid in 47% yield. 1H NMR (300 MHz, DMSO) δ 9.77 (s, 1H),
9.29 (d, J = 4.9 Hz, 2H), 8.52 (d, J = 4.6 Hz, 1H), 8.15 (s, 1H), 7.56−
7.41 (m, 3H), 7.37 (dd, J = 7.4, 4.5 Hz, 1H), 7.28 (d, J = 8.2 Hz, 1H),
7.11 (d, J = 8.2 Hz, 1H), 6.97 (t, J = 8.1 Hz, 1H), 6.04 (s, 1H), 5.93 (t,
J = 5.4 Hz, 1H), 3.76 (d, J = 5.4 Hz, 2H), 2.71 (d, J = 4.5 Hz, 3H),
1.20 (s, 9H). 13C NMR (126 MHz, DMSO) δ 169.3, 167.6, 158.3,
157.7, 156.4, 154.9, 140.9, 139.1, 132.2, 128.7, 126.9, 125.8, 125.7,
124.1, 118.7, 114.6, 113.4, 110.8, 105.0, 49.5, 43.5, 29.7, 26.6. MS
(ESI) m/z 543 [M + H]+. HRMS: calcd for C25H29ClN8O3F [M + H]
543.1957; found 543.2045.
N-(3-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)-
pyrimidin-2-yl)amino)phenyl)azetidine-3-carboxamide (32). White
1
solid (63%). H NMR (400 MHz, CDCl3+MeOD) δ 8.58 (s, 1H),
8.06 (d, J = 3.9 Hz, 1H), 7.83 (s, 1H), 7.66 (s, 1H), 7.55 (s, 1H), 7.42
(s, 1H), 7.20 (s, 3H), 3.75 (s, 1H), 3.62 (d, J = 6.3 Hz, 1H), 3.21 (s,
1H), 3.10−2.92 (m, 2H), 2.84 (s, 1H), 1.26 (d, J = 4.0 Hz, 6H). 13C
NMR (126 MHz, CDCl3) δ 170.2, 157.5, 155.2, 154.8, 139.6, 138.4,
138.1, 134.6, 131.1, 129.1, 124.0, 123.4, 123.2, 116.3, 114.4, 111.6,
106.1, 55.6, 50.4, 43.1, 41.2, 15.1. MS (ESI) m/z 501[M + H]+.
HRMS: calcd for C23H26ClN6O3S [M + H], 501.1471; found
501.1470.
N-(3-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)-
pyrimidin-2-yl)amino)phenyl)-2-(furan-2-yl)acetamide (46). White
solid (78%). 1H NMR (400 MHz, DMSO) δ 10.13 (s, 1H), 9.57 (d, J
= 16.3 Hz, 2H), 8.69 (d, J = 7.9 Hz, 1H), 8.30 (s, 1H), 7.90−7.80 (m,
2H), 7.69 (s, 1H), 7.57 (s, 1H), 7.36 (t, J = 6.0 Hz, 2H), 7.19 (d, J =
7.8 Hz, 2H), 6.40 (s, 1H), 6.26 (d, J = 3.0 Hz, 1H), 3.72 (s, 2H),
3.50−3.40 (m, 1H), 1.17 (d, J = 6.8 Hz, 6H). 13C NMR (101 MHz,
CDCl3) δ 166.9, 158.0, 155.5, 155.1, 149.9, 142.4, 139.7, 138.3, 135.2,
131.4, 128.9, 124.3, 124.1, 123.8, 115.7, 113.7, 111.4, 110.9, 108.0,
105.3, 55.3, 36.5, 15.3. MS (ESI) m/z 526 [M + H]+. HRMS: calcd for
C25H24ClN5O4S [M +H], 526.1316; found 526.1315.
N-(3-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)-
pyrimidin-2-yl)amino)phenyl)-2-(1H-indol-2-yl)acetamide (47).
1
White solid (72%). H NMR (300 MHz, CDCl3+MeOD) δ 8.48 (d,
J = 8.4 Hz, 1H), 8.02 (s, 1H), 7.78 (dd, J = 7.9, 1.5 Hz, 1H), 7.49 (dd,
J = 16.2, 9.8 Hz, 3H), 7.33 (dd, J = 12.5, 4.6 Hz, 2H), 7.15−7.02 (m,
5H), 6.93 (d, J = 8.0 Hz, 1H), 3.78 (s, 2H), 3.15 (dd, J = 13.7, 6.8 Hz,
1H), 1.22 (d, J = 6.9 Hz, 6H). 13C NMR (126 MHz, CDCl3) δ 170.6,
157.4, 155.1, 154.7, 139.5, 138.0, 136.4, 134.5, 131.0, 129.0, 126.7,
124.1, 123.4, 123.2, 122.3, 119.8, 118.3, 116.3, 114.5, 111.7, 111.6,
107.6, 55.6, 34.3, 15.2. MS (ESI) m/z 573 [M − H]. HRMS: calcd for
(C29H26N6O3ClS), 573.1470; found 573.1480.
Synthesis of N-(3-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)-
amino)pyrimidin-2-yl)amino)phenyl)-1-(4-(dimethylamino)-
butanoyl)azetidine-3-carboxamide (33). TBTU (128 mg, 0.4 mmol)
and DIPEA (103 mg, 0.8 mmol) were added successively to a solution
(E)-4-(dimethylamino)but-2-enoic acid (39 mg, 0.3 mmol) and
compound 29 (100 mg, 0.2 mmol) in DMF (3 mL). The reaction
was stirred overnight at rt and then diluted with water (8 mL). After
filtration, the filter cake was dried. The residue was dissolved in MeOH
(10 mL), and 10% Pd/C (15%) was added. The mixture was stirred
for 2 h under H2 atmosphere and then filtered. The filtrate was
concentrated in vacuum, and the residue was subjected to column
chromatography on silica gel (CHCl3/MeOH, 20/1) to give
Synthesis of 2-Chloro-N-(3-((5-chloro-4-((2-(isopropylsulfonyl)-
phenyl)amino)pyrimidin-2-yl)amino)phenyl)acetamide (30). To a
crude compound 29 (417 mg, 1 mmol) and Et3N (404 mg, 4 mmol)
in CH2Cl2 (20 mL) was added dropwise a solution of 2-chloroacetyl
chloride (224 mg, 2 mmol) in CH2Cl2 (5 mL). The mixture was
stirred at rt for 1 h. After being quenched with aqueous sodium
bicarbonate, the mixture was extracted with ethyl acetate. The organic
phase was washed with brine, dried over Na2SO4, filtered, and
concentrated. The residue was used directly without further
purification.
1
compound 33 (white solid, 65%). H NMR (300 MHz, CDCl3) δ
9.61 (s, 1H), 8.59 (d, J = 12.7 Hz, 2H), 8.12 (s, 1H), 7.86 (d, J = 7.2
Hz, 1H), 7.75 (s, 1H), 7.58 (d, J = 18.1 Hz, 2H), 7.41 (s, 1H), 7.20 (s,
3H), 4.42 (d, J = 5.3 Hz, 1H), 4.18 (d, J = 28.9 Hz, 3H), 3.45 (s, 1H),
3.31−3.12 (m, 1H), 2.30 (d, J = 6.6 Hz, 2H), 2.21 (s, 6H), 2.11 (d, J =
6.7 Hz, 2H), 1.76 (d, J = 6.2 Hz, 2H), 1.29 (s, 6H). 13C NMR (126
MHz, CDCl3) δ 173.2, 169.9, 157.5, 155.2, 155.0, 139.9, 138.4, 134.5,
131.2, 129.2, 124.1, 123.3, 123.1, 116.1, 114.3, 111.4, 106.5, 58.7, 55.6,
52.2, 50.8, 45.1, 33.7, 28.7, 22.3, 15.3. MS (ESI) m/z 612 [M − H].
HRMS: calcd for C29H35N4O7ClS [M − H], 612.2154; found
612.2160.
General Procedure for Synthesis of Compounds 34−37 and 40−
44. An appropriate amine (0.2 mmol), Et3N (1 mmol), and K2CO3
(1.2 mmol) were added successively to a solution of compound 30
(0.1 mmol) in MeCN (10 mL). The reaction was heated to reflux
overnight. After removal of the solvent, the residue was diluted with
water and extracted with CHCl3.The combined organic layers were
washed with brine, dried, filtered, and then evaporated. The residue
General Procedure for Synthesis of Compounds 31, 32, 46, and
47. TBTU (64 mg, 0.2 mmol) and DIPEA (52 mg, 0.4 mmol) were
added successively to a solution of an appropriate acid (0.15 mmol)
and compound 30 (42 mg, 0.1 mmol) in DMF (2 mL). The reaction
was stirred overnight at rt, diluted with water (8 mL), and extracted
with CHCl3. The combined organic layers were washed with brine (10
mL) and dried over Na2SO4. After filtration, the solvents were
evaporated. The residue was subjected to column chromatography on
silica gel (CHCl3/MeOH, 20/1) to give the expected compound.
N-(3-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)-
pyrimidin-2-yl)amino)phenyl)azetidine-2-carboxamide (31). White
1
solid (68%). H NMR (400 MHz, CDCl3) δ 9.64 (s, 1H), 9.58 (s,
1H), 8.62 (d, J = 8.5 Hz, 1H), 8.19 (s, 1H), 7.92 (dd, J = 8.0, 1.6 Hz,
2H), 7.64 (t, J = 7.1 Hz, 1H), 7.44 (dd, J = 7.6, 3.5 Hz, 1H), 7.28−7.18
(m, 2H), 4.46 (t, J = 8.5 Hz, 1H), 3.86 (q, J = 8.5 Hz, 1H), 3.37 (td, J
K
dx.doi.org/10.1021/jm5005144 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
was subjected to column chromatography on silica gel (CHCl3/
MeOH, 20/1) to give the expected compound.
2.99−2.91 (m, 2H), 2.68 (d, J = 9.6 Hz, 1H), 2.26 (s, 1H), 1.82 (d, J =
9.6 Hz, 1H), 1.66 (d, J = 9.8 Hz, 1H), 1.32 (d, J = 6.9 Hz, 6H). 13C
NMR (126 MHz, CDCl3) δ 169.0, 157.5, 155.1, 155.0, 139.8, 138.2,
138.0, 134.4, 131.1, 129.1, 124.2, 123.3, 122.9, 115.7, 113.7, 110.9,
106.4, 63.3, 62.6, 59.6, 56.9, 55.5, 49.7, 36.2, 15.2. MS (ESI) m/z 556
[M + H]+. HRMS: calcd for C26H30ClN7O3S [M + Na], 578.1717;
found 578.1719.
2-(8-Oxa-3-azabicyclo[3.2.1]octan-3-yl)-N-(3-((5-chloro-4-((2-
(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)-
acetamide (42). Off-white solid (52%). 1H NMR (300 MHz, CDCl3)
δ 9.64 (s, 1H), 9.14 (s, 1H), 8.60 (d, J = 8.4 Hz, 1H), 8.20−8.11 (m,
1H), 7.89 (dd, J = 5.7, 1.9 Hz, 2H), 7.59 (dd, J = 10.8, 4.9 Hz, 1H),
7.47 (d, J = 7.9 Hz, 1H), 7.29 (d, J = 2.6 Hz, 1H), 7.22 (d, J = 2.6 Hz,
1H), 7.08 (d, J = 7.9 Hz, 1H), 4.35 (s, 2H), 3.29−3.18 (m, 1H), 3.06
(d, J = 2.2 Hz, 2H), 2.63 (s, 4H), 2.01 (s, 4H), 1.30 (d, J = 6.8 Hz,
6H). 13C NMR (101 MHz, CDCl3) δ 167.7, 157.0, 154.8, 154.6, 139.6,
137.9, 137.6, 134.0, 130.8, 129.0, 123.9, 122.9, 122.6, 115.2, 112.8,
110.0, 106.2, 73.9, 60.9, 58.2, 55.2, 28.1, 14.9. MS (ESI) m/z 571 [M
+H]+. HRMS: calcd for C27H31ClN6O4S [M + Na], 593.1714; found
593.1713.
N-(3-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)-
pyrimidin-2-yl)amino)phenyl)-2-(3-hydroxy-8-azabicyclo[3.2.1]-
octan-8-yl)acetamide (43). Off-white solid (41%). 1H NMR (300
MHz, CDCl3) δ 9.64 (s, 2H), 8.61 (d, J = 8.4 Hz, 1H), 8.15 (s, 1H),
7.89 (d, J = 7.9 Hz, 1H), 7.82 (s, 1H), 7.58 (t, J = 7.7 Hz, 1H), 7.48 (d,
J = 8.1 Hz, 1H), 7.33 (s, 1H), 7.21 (d, J = 8.1 Hz, 1H), 7.12 (d, J = 7.7
Hz, 1H), 4.09 (s, 1H), 3.19 (s, 3H), 3.10 (s, 2H), 2.20 (d, J = 7.5 Hz,
2H), 2.13 (s, 1H), 2.09 (s, 1H), 2.04 (s, 1H), 1.96 (s, 2H), 1.79 (d, J =
14.6 Hz, 2H), 1.31 (d, J = 6.8 Hz, 6H). 13C NMR (126 MHz, CDCl3)
δ 168.6, 157.1, 154.8, 154.5, 139.4, 137.9, 137.7, 134.0, 130.7, 128.8,
123.9, 122.9, 122.6, 115.4, 113.4, 110.5, 106.1, 63.5, 59.5, 55.2, 26.0,
14.8. MS (ESI) m/z 583 [M − H]. HRMS: calcd for C28H32ClN6O4S
[M − H], 583.1889; found 583.1897.
Synthesis of N-(3-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)-
amino)pyrimidin-2-yl)amino)phenyl)-2-(4-(oxetan-3-yl)piperazin-1-
yl)acetamide (38). To a solution of 37 (54 mg, 0.1 mmol) in MeOH
(5 mL), oxetan-3-one (14 mg, 0.2 mmol) was added. The mixture was
stirred for 1 h, NaHB(CN)3 (25 mg, 0.4 mmol) was added, and the
reaction was stirred overnight. The reaction was quenched with
saturated NH4Cl. After the solvent was evaporated, the residue was
diluted with water and then extracted with CHCl3. The combined
organic layers were washed with brine, dried, filtered, and then
evaporated. The crude product was subjected to column chromatog-
raphy on silica gel (CHCl3/MeOH, 20/1) to give compound 38
(white solid, 64%). 1H NMR (400 MHz, CDCl3) δ 9.66 (s, 1H), 9.07
(s, 1H), 8.62 (d, J = 8.5 Hz, 1H), 8.19 (s, 1H), 7.92 (dd, J = 7.9, 1.4
Hz, 1H), 7.87 (s, 1H), 7.63 (t, J = 7.9 Hz, 1H), 7.52 (d, J = 8.2 Hz,
1H), 7.25 (d, J = 7.6 Hz, 1H), 7.16−7.05 (m, 2H), 4.67 (dt, J = 24.3,
6.3 Hz, 4H), 3.60−3.51 (m, 1H), 3.27 (dt, J = 13.5, 6.8 Hz, 1H), 3.17
(s, 2H), 2.70 (s, 4H), 2.43 (s, 4H), 1.34 (d, J = 6.9 Hz, 6H). 13C NMR
(126 MHz, CDCl3) δ 168.2, 157.4, 155.3, 155.1, 139.9, 138.3, 138.0,
134.5, 131.2, 129.4, 124.4, 123.4, 123.1, 115.7, 113.7, 110.8, 106.7,
75.4, 61.9, 59.0, 55.6, 53.0, 49.7, 15.3. MS (ESI) m/z 600 [M +H]+.
HRMS: calcd for C28H34ClN7O4S [M + Na], 622.1979; found
622.1980.
N-(3-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)-
pyrimidin-2-yl)amino)phenyl)-2-morpholinoacetamide (34). White
1
solid (61%). H NMR (400 MHz, CDCl3) δ 9.66 (s, 1H), 9.07 (s,
1H), 8.62 (d, J = 8.4 Hz, 1H), 8.19 (s, 1H), 7.92 (dd, J = 8.0, 1.6 Hz,
1H), 7.87 (t, J = 1.9 Hz, 1H), 7.65−7.58 (m, 1H), 7.50 (dd, J = 8.1, 1.5
Hz, 1H), 7.29 (d, J = 8.8 Hz, 1H), 7.28−7.22 (m, 2H), 7.12 (dd, J =
8.0, 1.3 Hz, 1H), 3.82−3.73 (m, 4H), 3.31−3.21 (m, 1H), 3.15 (s,
2H), 2.67−2.60 (m, 4H), 1.33 (d, J = 6.9 Hz, 6H). 13C NMR (101
MHz, CDCl3) δ 167.9, 157.4, 155.2, 155.0, 139.9, 138.3, 137.9, 134.4,
131.2, 129.3, 124.4, 123.3, 123.0, 115.8, 113.7, 110.8, 106.7, 66.9, 62.4,
55.6, 53.7, 15.3. MS (ESI) m/z 545[M + H]+. HRMS: calcd for
C25H30ClN6O4S [M + H], 545.1738; found 545.1732.
N-(3-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)-
pyrimidin-2-yl)amino)phenyl)-2-((2S,6R)-2,6-dimethylmorpholino)-
1
acetamide (35). White solid (65%). H NMR (400 MHz, CDCl3) δ
9.67 (s, 1H), 9.08 (s, 1H), 8.63 (d, J = 8.4 Hz, 1H), 8.20 (s, 1H),
7.94−7.85 (m, 2H), 7.62 (t, J = 7.9 Hz, 1H), 7.50 (dd, J = 7.9, 1.7 Hz,
1H), 7.29−7.23 (m, 2H), 7.12 (dd, J = 8.0, 1.2 Hz, 1H), 3.72 (ddd, J =
10.0, 6.2, 2.0 Hz, 2H), 3.30−3.22 (m, 1H), 3.12 (s, 2H), 2.76 (d, J =
10.4 Hz, 2H), 2.08−2.01 (m, 2H), 1.33 (d, J = 6.9 Hz, 6H), 1.20 (d, J
= 6.3 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 168.1, 157.5, 155.3,
155.1, 139.9, 138.3, 138.0, 134.5, 131.2, 129.3, 124.4, 123.3, 123.1,
115.7, 113.6, 110.8, 106.7, 71.9, 62.0, 59.4, 55.6, 18.9, 15.3. MS (ESI)
m/z: 573 [M + H]+. HRMS: calcd for C27H33ClN6O4S [M + H],
573.2051; found 573.2052.
N-(3-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)-
pyrimidin-2-yl)amino)phenyl)-2-(piperazin-1-yl)acetamide (36).
1
White solid (53%). H NMR (400 MHz, CDCl3) δ 9.65 (s, 1H),
9.16 (s, 1H), 8.62 (d, J = 8.4 Hz, 1H), 8.19 (s, 1H), 7.91 (dd, J = 7.9,
1.5 Hz, 1H), 7.86 (s, 1H), 7.61 (s, 1H), 7.50 (d, J = 8.1 Hz, 1H), 7.30
(s, 1H), 7.24 (d, J = 7.4 Hz, 1H), 7.12 (d, J = 7.9 Hz, 1H), 3.26 (dt, J =
13.8, 6.9 Hz, 1H), 3.12 (s, 2H), 2.99−2.92 (m, 4H), 2.60 (d, J = 4.3
Hz, 4H), 1.33 (d, J = 6.9 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ
168.4, 157.4, 155.2, 155.1, 139.9, 138.3, 138.0, 134.4, 131.2, 129.3,
124.4, 123.4, 123.0, 115.7, 113.7, 110.8, 106.6, 62.6, 55.6, 54.6, 46.2,
15.3. MS (ESI) m/z 544 [M + H]+. HRMS: calcd for C25H30ClN7O3S
[M + H], 544.1898; found 544.1889.
N-(3-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)-
pyrimidin-2-yl)amino)phenyl)-2-(4-methylpiperazin-1-yl)acetamide
1
(37). Off-white solid (73%). H NMR (400 MHz, CDCl3) δ 9.65 (s,
1H), 9.12 (s, 1H), 8.62 (d, J = 8.2 Hz, 1H), 8.19 (s, 1H), 7.91 (dd, J =
8.0, 1.6 Hz, 1H), 7.86 (t, J = 1.9 Hz, 1H), 7.63−7.56 (m, 1H), 7.50
(dd, J = 7.9, 1.9 Hz, 1H), 7.40 (s, 1H), 7.30−7.27 (m, 1H), 7.23 (dd, J
= 10.1, 2.9 Hz, 1H), 7.13−7.09 (m, 1H), 3.26 (dt, J = 13.7, 6.8 Hz,
1H), 3.14 (s, 2H), 2.66 (s, 4H), 2.51 (s, 3H), 2.34 (s, 3H), 2.26 (s,
1H), 1.33 (d, J = 6.9 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 168.3,
157.5, 155.2, 155.0, 139.9, 138.3, 138.0, 134.4, 131.1, 129.2, 124.3,
123.4, 123.0, 115.8, 113.7, 110.9, 106.5, 61.8, 55.6, 55.1, 53.3, 45.9,
15.3. MS (ESI) m/z 558 [M + H]+. HRMS: calcd for C26H33ClN7O3S
[M + H], 558.2054; found 558.2056.
N-(3-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)-
pyrimidin-2-yl)amino)phenyl)-2-(4-(2-hydroxyacetyl)piperazin-1-
1
yl)acetamide (40). White solid (55%). H NMR (300 MHz, CDCl3/
MeOD) δ 8.54 (d, J = 8.4 Hz, 1H), 8.05 (s, 1H), 7.80 (dd, J = 8.0, 1.5
Hz, 1H), 7.66 (s, 1H), 7.51 (t, J = 7.9 Hz, 1H), 7.40 (d, J = 8.2 Hz,
1H), 7.17 (td, J = 7.9, 4.9 Hz, 2H), 7.07 (d, J = 7.2 Hz, 1H), 4.10 (s,
2H), 3.64 (s, 2H), 3.30 (d, J = 5.1 Hz, 2H), 3.17 (dt, J = 13.7, 6.9 Hz,
1H), 3.10 (s, 2H), 2.58−2.52 (m, 4H), 1.23 (d, J = 6.9 Hz, 6H). 13C
NMR (101 MHz, CDCl3) δ 170.2, 167.9, 157.4, 155.2, 154.8, 139.9,
138.1, 137.5, 134.5, 129.2, 123.1, 116.4, 114.0, 111.3, 106.2, 61.7, 59.6,
55.6, 52.9, 52.6, 43.4, 42.0, 15.1. MS (ESI) m/z 602 [M + H]+. HRMS:
calcd for C27H32ClN7O5S [M + H], 602.1952; found 602.1945.
2-(2,5-Diazabicyclo[2.2.1]heptan-2-yl)-N-(3-((5-chloro-4-((2-
(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)-
acetamide (41). Off-white solid (47%). 1H NMR (400 MHz, CDCl3)
δ 9.64 (s, 1H), 9.20 (s, 1H), 8.62 (d, J = 8.4 Hz, 1H), 8.18 (s, 1H),
7.92−7.84 (m, 2H), 7.64−7.55 (m, 2H), 7.50−7.43 (m, 1H), 7.27−
7.20 (m, 2H), 7.12 (d, J = 8.9 Hz, 1H), 3.65 (s, 1H), 3.40 (d, J = 5.9
Hz, 1H), 3.35 (s, 1H), 3.31−3.21 (m, 2H), 3.11 (d, J = 10.4 Hz, 1H),
Synthesis of (E)-N-(3-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)-
amino)pyrimidin-2-yl)amino)phenyl)-2-(4-(4-(dimethylamino)but-
2-enoyl)piperazin-1-yl)acetamide (39). This compound was prepared
from (E)-4-(dimethylamino)but-2-enoic acid (39 mg, 0.3 mmol) and
compound 37 as an off-white solid in 78% yield by following a similar
1
procedure as that for preparation of compound 33. H NMR (400
MHz, CDCl3) δ 9.66 (s, 1H), 8.99 (s, 1H), 8.62 (d, J = 8.4 Hz, 1H),
8.19 (s, 1H), 7.95−7.85 (m, 2H), 7.62 (t, J = 7.9 Hz, 1H), 7.50 (dd, J
= 7.9, 1.7 Hz, 1H), 7.26 (dd, J = 10.0, 5.2 Hz, 2H), 7.12 (dd, J = 7.9,
1.2 Hz, 1H), 6.89 (dt, J = 15.2, 5.9 Hz, 1H), 6.45 (d, J = 15.2 Hz, 1H),
3.71 (d, J = 40.5 Hz, 4H), 3.26 (dt, J = 13.7, 6.9 Hz, 1H), 3.18 (s, 2H),
3.11 (dd, J = 5.9, 1.4 Hz, 2H), 2.67−2.60 (m, 4H), 2.29 (s, 6H), 1.33
(d, J = 6.9 Hz, 6H). 13C NMR (126 MHz, CDCl3) δ 167.6, 165.2,
157.4, 155.3, 155.1, 143.1, 140.0, 138.3, 137.9, 134.4, 131.2, 129.4,
124.4, 123.3, 123.1, 121.5, 115.8, 113.7, 110.8, 106.8, 61.9, 60.6, 55.6,
L
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45.4, 15.3. MS (ESI) m/z 655 [M + H]+. HRMS: calcd for
C31H40ClN8O4S [M + H]+, 655.2852; found 655.2853.
Cell Proliferation Assay. Cells were seeded in 96-well tissue
culture plates. On the next day, cells were exposed to various
concentrations of compounds and further cultured for 72 h. Cell
proliferation was then determined using sulforhodamine B (SRB,
Sigma) or thiazolyl blue tetrazolium bromide (MTT, Sigma) assay.
IC50 values were calculated by concentration−response curve fitting
using the four-parameter method.
General Procedure for Synthesis of Compounds 44 and 45. An
appropriate amine (0.2 mmol), Et3N (1 mmol), and K2CO3 (1.2
mmol) were added successively to a solution of compound 30 (0.1
mmol) in MeCN (10 mL). The reaction was heated to reflux
overnight. After removal of the solvent, the residue was diluted with
water and extracted with CHCl3. The combined organic layers were
washed with brine, dried, filtered, and then evaporated. The residue
was then dissolved in a mixed solvent system (CH2Cl2/TFA = 1:1, 5
mL) and the mixture was stirred at rt for 2 h. After removal of the
solvent, the residue was taken up in water (5 mL), alkalified with
NaHCO3 to pH > 7, and then extracted with CHCl3.The combined
organic layer was washed with brine, dried, filtered, and then
evaporated. The residue was subjected to column chromatography
on silica gel (CHCl3/MeOH, 20/1) to give the corresponding
product.
N-(3-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)-
pyrimidin-2-yl)amino)phenyl)-2-(hexahydropyrrolo[3,4-b]pyrrol-
1(2H)-yl)acetamide (45). Off-white solid (48%). 1H NMR (400 MHz,
CDCl3) δ 9.64 (s, 1H), 9.26 (s, 1H), 8.62 (d, J = 8.3 Hz, 1H), 8.18 (s,
1H), 7.91 (dd, J = 8.0, 1.5 Hz, 1H), 7.83 (s, 1H), 7.62 (dd, J = 11.4,
4.3 Hz, 1H), 7.52 (d, J = 7.9 Hz, 1H), 7.27−7.17 (m, 4H), 3.48 (d, J =
16.7 Hz, 1H), 3.23 (ddd, J = 11.0, 10.5, 4.7 Hz, 3H), 3.14−3.05 (m,
1H), 2.98 (t, J = 7.8 Hz, 1H), 2.87 (d, J = 6.3 Hz, 2H), 2.75 (s, 1H),
2.62 (dd, J = 11.5, 4.7 Hz, 1H), 2.43−2.35 (m, 1H), 2.14 (d, J = 13.7
Hz, 1H), 1.62−1.53 (m, 1H), 1.33 (d, J = 6.9 Hz, 6H). 13C NMR (126
MHz, CDCl3) δ 169.4, 157.5, 155.3, 155.1, 139.7, 138.3, 134.5, 131.2,
129.2, 124.3, 123.4, 123.1, 115.8, 114.3, 111.3, 106.6, 70.3, 58.8, 55.7,
55.6, 54.0, 52.4, 42.6, 32.4, 15.3. MS (ESI) m/z 570 [M + H]+. HRMS:
calcd for C27H33ClN7O3S [M + H], 570.2054; found 570.2052.
N-(3-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)-
pyrimidin-2-yl)amino)phenyl)-2-(hexahydropyrrolo[3,4-c]pyrrol-
2(1H)-yl)acetamide (44). Off-white solid (56%). 1H NMR (400 MHz,
CDCl3) δ 9.65 (s, 1H), 9.34 (s, 1H), 8.63 (d, J = 8.5 Hz, 1H), 8.16 (s,
1H), 7.91 (d, J = 6.4 Hz, 1H), 7.78 (s, 1H), 7.62 (t, J = 7.9 Hz, 1H),
7.54 (d, J = 8.1 Hz, 1H), 7.40 (s, 1H), 7.33 (d, J = 8.4 Hz, 1H), 7.25 (t,
J = 7.8 Hz, 2H), 3.31−3.24 (m, 3H), 3.22 (s, 2H), 3.01 (d, J = 11.2
Hz, 2H), 2.87 (s, 2H), 2.80 (d, J = 9.6 Hz, 2H), 2.61 (d, J = 6.2 Hz,
2H), 1.33 (d, J = 6.8 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 168.6,
157.5, 155.3, 155.0, 139.8, 138.4, 138.3, 134.6, 131.2, 129.2, 124.3,
123.5, 123.1, 115.8, 114.3, 111.3, 106.5, 60.4, 58.9, 55.6, 53.4, 42.4,
15.3. MS (ESI) m/z 570 [M + H]+. HRMS: calcd for C27H32ClN7O3S
[M + H], 570.2054; found 570.2051.
In Vivo Antitumor Activity Assay. Female nude mice (4−6
weeks) were housed five mice per cage in a specific pathogen-free
room with a 12 h light/dark schedule at 25 1 °C and were feed an
autoclaved chow diet and water ad libitum. All animal experiments
were performed according to institutional ethinical guidelines of
animal care. The cells at a density of (5−10) × 106 in 200 μL were first
implanted sc into the right flank of each nude mouse and then allowed
to grow to 700−800 mm3, defined as a well-developed tumor. After
that, the well-developed tumors were cut into 1 mm3 fragments and
transplanted sc into the right flank of nude mice using a trocar. When
the tumor volume reached 80−100 mm3, the mice were randomly
assigned into control and treatment groups (n = 5 in treated group, n
= 10 in vehicle group). Control groups were given vehicle alone, and
treatment groups received compound 15 hydrochloride salt with
indicated doses via oral administration (with 0.5% CMC-Na (sodium
carboxymethyl cellulose)) or crizotinib hydrochloride salt with water
via oral administration once daily for 2 weeks. The sizes of the tumors
were measured twice per week using a microcaliper. The tumor
volume (TV) was calculated as TV = (length × width2)/2. Relative
tumor volume (RTV) = TVDay‑N/TVDay‑0 × 100%. Data was shown on
indicated days as the median RTV SE. Percent (%) inhibition values
were measured on the final day of study for drug-treated compared
with vehicle-treated mice and are calculated as 100 × (1 − [(treated
final day − treated day 1)/(control final day − control day 1)]).
Significant differences between the treated versus control groups were
determined using Student’s t test.
ASSOCIATED CONTENT
■
S
* Supporting Information
1
Copies of the H and 13C spectra of all new compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Authors
ELISA Kinase Assay. Kinase activity was evaluated according to
the following procedure. Briefly, in enzyme-linked-immunosorbent
assay (ELISA), 20 μg/mL poly(Glu, Tyr)4:1 (Sigma) was precoated as
a substrate in 96-well plates. ATP solution (50 μL of 10 μM) diluted in
kinase reaction buffer (50 mM HEPES, pH 7.4, 50 mM MgCl2, 0.5
mM MnCl2, 0.2 mM Na3VO4, 1 mM DTT) was added to each well.
One microliter of various concentrations of tested compounds diluted
in 1% DMSO (v/v) (Sigma) was added to each reaction well. DMSO
(1% v/v) was used as the negative control. The kinase reaction was
initiated after the addition of purified tyrosine kinase proteins diluted
in 49 μL of kinase reaction buffer solution. After incubation for 60 min
at 37 °C, the plate was washed three times with phosphate buffered
saline (PBS) containing 0.1% Tween 20 (T-PBS). Anti-phosphotyr-
osine (PY99) antibody (100 μL, 1:500 diluted in 5 mg/mL BSA T-
PBS) was then added. After 30 min incubation at 37 °C, the plate was
washed three times. Horseradish peroxidase-conjugated goat anti-
mouse IgG (100 μL, 1:2000 diluted in 5 mg/mL BSA T-PBS) was
added. The plate was then incubated at 37 °C for 30 min and washed 3
times. A solution (100 μL) containing 0.03% H2O2 and 2 mg/mL o-
phenylenediamine in 0.1 mol/L citrate buffer, pH 5.5, was added. The
reaction was terminated by the addition of 50 μL of 2 mol/L H2SO4 as
color changed, and the plate was read using a multiwell
spectrophotometer (SpectraMAX 190, Molecular Devices) at 490
nm. The inhibition rate (%) was calculated using the following
equation: [1 − A490/(A490 control)] × 100%. IC50 values were
calculated from the inhibition curves from two separate experiments.
*For Jing Ai: e-mail jai@simm.ac.cn; fax 86-21-50806072; tel
86-21-50806072.
*For Meiyu Geng: e-mail mygeng@simm.ac.cn; fax 86-21-
50806072; tel 86-21-50806072.
*For Ao Zhang: e-mail aozhang@simm.ac.cn; tel 86-21-
50806035; fax 86-21-50806035.
Author Contributions
§Z. Song and Y. Yang contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by grants from Chinese NSF (Nos.
81125021, 81373277, 81102461, and 91229205) and National
Science & Technology Major Project on ‘Key New Drug
Creation and Manufacturing Program’, China (Grants
2012ZX09103-101-035, 2012ZX09301001-007). Supporting
grants from the “Interdisciplinary Cooperation Team” Program
for Science and Technology Innovation (CAS), the National
Program on Key Basic Research Project of China (Grant
2012CB910704), the Natural Science Foundation of China for
Innovation Research Group (Grant 81321092), and the State
M
dx.doi.org/10.1021/jm5005144 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
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Key Laboratory of Drug Research (Grant SIMM1302KF-08),
Shanghai Institute of Materia Medica, were also appreciated.
ABBREVIATIONS USED
■
ALK, anaplastic lymphoma kinase; RTK, receptor tyrosine
kinase; ALCL, anaplastic large-cell lymphoma; NSCLC, non-
small-cell lung cancer; EML4, echinoderm microtubule-
associated protein-like 4; NPM, nucleophosmin; c-Met,
mesenchymal epithelial transition growth factor; DAAPalogues,
2,4-diarylaminopyrimidine analogues; crizotinib, 3-[(1R)-1-
(2,6-dichloro-3-fluorophenyl)ethoxy]-5-(1-piperidin-4-ylpyra-
zol-4-yl)pyridine-2-amine; CSA, (1S)-(+)-10-camphorsulfonic
acid; X-Phos, 2-(dicyclohexylphosphino)-2′,4′,6′-triisopropyl
biphenyl; IGF1R, insulin-like growth factor 1 receptor;
PDGFR, platelet derived growth factor receptor; EGFR,
epidermal growth factor receptor; KDR, kinase insert domain
receptor; FGFR1, fibroblast growth factor receptor 1; Flt-1,
fms-like tyrosine kinase
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