Discovery of a potent dual ALK and EGFR T790M inhibitor

Article abstract of DOI:10.1016/j.ejmech.2017.04.079

The mutational activations of anaplastic lymphoma kinase (ALK) and epidermal growth factor receptor (EGFR) are validated oncogenic events and the targets of approved drugs to treat non-small cell lung cancer (NSCLC). Here we report highly potent dual small molecule inhibitors of both ALK and EGFR, particularly the T790M mutant which confers resistance to first generation EGFR inhibitors. Dual ALK/EGFR inhibitors may provide an efficient approach to prevent resistance that arises as a consequence of clinically reported reciprocal activation mechanisms. Our lead compound 7c displayed remarkable inhibitory activities against both ALK and EGFR in enzymatic and cellular assays. We demonstrate that 7c is capable of recapitulating the signaling effects and antiproliferative activity of combined treatment with the approved ALK inhibitor ceritinib and T790M EGFR inhibitor osimertinib against patient-derived non-small cell lung cancer cell line, DFCI032 which harbors both EML4-ALK and activated EGFR.

Full text of DOI:10.1016/j.ejmech.2017.04.079

Accepted Manuscript
Discovery of a potent dual ALK and EGFR T790M inhibitor
Jaebong Jang, Jung Beom Son, Ciric To, Magda Bahcall, So Young Kim, Seock
Yong Kang, Mierzhati Mushajiang, Younho Lee, Pasi A. Jänne, Hwan Geun Choi,
Nathanael S. Gray
PII:
S0223-5234(17)30361-6
10.1016/j.ejmech.2017.04.079
EJMECH 9430
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 March 2017
Revised Date: 27 April 2017
Accepted Date: 30 April 2017
Please cite this article as: J. Jang, J.B. Son, C. To, M. Bahcall, S.Y. Kim, S.Y. Kang, M. Mushajiang,
Y. Lee, P.A. Jänne, H.G. Choi, N.S. Gray, Discovery of a potent dual ALK and EGFR T790M inhibitor,
European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.04.079.
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ACCEPTED MANUSCRIPT
Graphical Abstract

ACCEPTED MANUSCRIPT
Discovery of a Potent Dual ALK and EGFR T790M Inhibitor
Jaebong Janga,c,1, Jung Beom Song,1, Ciric Tob,f, Magda Bahcalld, So Young Kimg, Seock
Yong Kangg, Mierzhati Mushajiangd, Younho Leeg, Pasi A. Jänneb,d,e,h, Hwan Geun
Choig,** and Nathanael S. Graya,c,*
aDepartment of Biological Chemistry & Molecular Pharmacology, bHarvard Medical School,
Boston, Massachusetts 02115, United States
d
e
cDepartment of Cancer Biology, Lowe Center for Thoracic Oncology and Belfer Center for
f
Applied Cancer Science, Department of Medical Oncology, Dana-Farber Cancer Institute,
Boston, Massachusetts 02215, United States
gNew Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu
41061, Republic of Korea
hDepartment of Medicine, Brigham and Women`s Hospital, Boston, Massachusetts 02115,
United States
* Corresponding author. E-mail: nathanael_gray@dfci.harvard.edu
** Corresponding author. E-mail: hgchoi@dgmif.re.kr
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1These authors contributed equally.
ABSTRACT
The mutational activations of anaplastic lymphoma kinase (ALK) and epidermal growth
factor receptor (EGFR) are validated oncogenic events and the targets of approved drugs to treat
non-small cell lung cancer (NSCLC). Here we report highly potent dual small molecule
inhibitors of both ALK and EGFR, particularly the T790M mutant which confers resistance to
first generation EGFR inhibitors. Dual ALK/EGFR inhibitors may provide an efficient approach
to prevent resistance that arises as a consequence of clinically reported reciprocal activation
mechanisms. Our lead compound 7c displayed remarkable inhibitory activities against both ALK
and EGFR in enzymatic and cellular assays. We demonstrate that 7c is capable of recapitulating
the signaling effects and antiproliferative activity of combined treatment with the approved ALK
inhibitor ceritinib and T790M EGFR inhibitor osimertinib against patient-derived non-small cell
lung cancer cell line, DFCI032 which harbors both EML4-ALK and activated EGFR.
Keywords
ALK, EGFR T790M, dual inhibitor, non-small cell lung cancer, rational drug design.
1. Introduction
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Oncogenic mutations and translocations of receptor tyrosine kinases (RTKs) cause the
development and progression of many cancers [1]. Small molecule drugs targeting dysregulated
RTKs have distinct clinical benefits especially for genetically defined patient populations [2].
The anaplastic lymphoma kinase (ALK) and epidermal growth factor receptor (EGFR) have been
well characterized as the leading oncogenic drivers of non-small cell lung cancer (NSCLC) [3].
In the past two decades, several wild-type and mutant-directed inhibitors of both ALK and
EGFR have been developed. For example, the first-generation ALK inhibitor, crizotinib [4] and
the first and second generation EGFR inhibitors, gefitinib [5], erlotinib [6] and afatinib [7] have
been developed to treat NSCLC. Despite excellent response rates to these drugs, patients
invariably relapse due to the emergence of drug resistant tumors. The second generation ALK
inhibitors, ceritinib [8], alectinib [9] and brigatinib [10] and the third generation EGFR inhibitor,
osimertinib [11-13] potently overcome the secondary resistant mutations.
Despite extensive exploration of diverse chemotypes as inhibitors of ALK and EGFR,
there have been no reports of small molecules that can act as dual inhibitors of both kinases.
EGFR activation has been demonstrated as one of the compensatory resistance mechanisms of
oncogenic ALK inhibition by small molecule inhibitors such as crizotinib, alectinib and ceritinib
[14-16]. Additionally, with improved ability to perform single-cell sequencing there have been
reports of tumors that harbor oncogenic alleles of both ALK and mutant EGFR concurrently [17-
20].
Here we report the rational design, synthesis and biological evaluation of a dual inhibitor
of ALK and EGFR, especially the T790M resistant mutant, that acts as a reversible ATP-
competitive inhibitor with respect to ALK and as an irreversible, covalent inhibitor of the
T790M mutant of EGFR.
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2. Results and discussion
2.1 Rational drug design
Our study commenced by recognizing a structural similarity that exists between several
of the known ALK inhibitors and EGFR inhibitors. The ALK inhibitors, ceritinib [21], TAE684
[22] and brigatinib [23] share a 2,4-disubstituted pyrimidine scaffold with the third generation
EGFR inhibitors, osimertinib [11-13], WZ4002 [24] and rociletinib [25, 26]. Despite sharing the
2,4-disubstituted pyrimidine core, both ALK and EGFR inhibitors have distinct substituents that
are critical to the recognition of their respective targets. In particular, the T790M EGFR
inhibitors use a meta-acrylamide to enable the formation of a covalent bond with Cys797 located
at the edge of the EGFR ATP binding pocket. The ALK inhibitors exploit an ortho-substituted
isopropylsulfone (TAE684/ceritinib) or a dimethylphosphine oxide (brigatinib) to form a
hydrogen bond with the catalytic Lys1150, which is critical to the binding affinity. Based on this
structural analysis, we decided to combine both the acrylamide and ortho-substituted hydrogen
bond acceptors with the 2,4-disubstituted pyrimidine scaffold with the aim of generating a dual
ALK/EGFR inhibitor. (Figure 1) The ortho-alkoxy group at the C2-aniline substituent is a well-
known functional group which is required to impart kinome-wide selectivity [21, 22]. For
example, the methoxy group of WZ4002 led to improved selectivity for EGFR relative to its
non-substituted analog, WZ3146 [24]. We also selected the methoxy group instead of a bulkier
alkoxy group in our ALK/EGFR dual inhibitor because the isopropoxy substituent of ceritinib
causes a loss of binding affinity for EGFR (Figure 3).
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Figure 1. Rational design of dual ALK and EGFR inhibitor.
2.2 Structure-Activity Relationship
The potencies of newly synthesized compounds were first assessed biochemically using
a Z`-Lyte enzymatic assay for wild type ALK and an ATP-depletion (ADP-Glo) enzymatic assay
for wild type EGFR, EGFR T790M and EGFR T790M/L858R. Compounds were subsequently
evaluated using a proliferation assay employing Ba/F3 cells [27] transformed by either EML4-
ALK, wild type EGFR or mutant EGFRs such as L858R, exon19del, L858R/T70M or
exon19del/T790M. Furthermore, we utilized parental Ba/F3 cells grown in the presence of
interlukin-3 (IL-3) to assess whether the compounds exhibited any general cytotoxicity.
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First, we benchmarked our enzymatic and cellular ALK and EGFR assays using the
ALK inhibitor, ceritinib and the EGFR inhibitors WZ4002 and osimertinib which share the 2,4-
disubstituted pyrimidine core structure (Table 1). As expected, ceritinib exhibited excellent
biochemical potency against wild type ALK with IC50 value of 20 nM and exhibited weak
potency against wild type EGFR with IC50 value of > 2 µM. Interestingly, ceritinib also
displayed potent inhibition of T790M and T790M/L858R EGFR mutants using the ADP-Glo
assays, although less potent than the IC50 values measured for WZ4002 and osimertinib. This
result was reproduced by a Z`-LYTE enzymatic assay (Invitrogen, Table S1). The observed
biochemical potency of ceritinib for T790M EGFR was not observed in the Ba/F3 cellular assay
(Table 2), highlighting the importance of tracking both the biochemical and cellular potencies.
The two EGFR inhibitors displayed excellent potencies against all forms of EGFRs, especially
T790M containing mutants (IC50 = 5 ~ 7 nM), but displayed moderate to low potencies against
wild type ALK (IC50 = 990 nM and 177 nM). These known drugs also showed similar trends in
their cell growth inhibitory activities in the proliferation assays of Ba/F3 cells transformed with
EML4-ALK, wild type EGFR and four different EGFR mutants (Table 2).
We next investigated which combination of functional groups are critical to achieving
potent inhibition of ALK and EGFR, including the aforementioned ortho-hydrogen bond
acceptor and the meta-acrylamide functional groups. We first synthesized and evaluated 7a
containing the ortho-isopropylsulfone at C4-aniline substituent and the meta-acrylamide at C2-
aniline substituent of the pyrimidine core. 7a displayed moderate enzyme inhibitory activities
against wild type ALK, T790M EGFR and L858R/T790M EGFR with IC50 values of 103 nM, 26
nM and 86 nM respectively and it showed weak potency against wild type EGFR (Table 1).
Consistent with the biochemical activities, 7a showed moderate antiproliferative potencies
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against mutant EGFR transformed Ba/F3 cells [EC50 = 78 nM (L858R), 195 nM (exon19del),
183 nM (T790M/L858R), 158 nM (exon19del/T790M)] while exhibiting weak proliferative
inhibition of wild type EGFR (EC50 = 827 nM) as well as no meaningful inhibition against
parental Ba/F3 cells (EC50
≥ 5000 nM). However, contrary to the biochemical assay, compound
7a displayed poor antiproliferative activity against EML4-ALK transformed Ba/F3 cells which
encouraged us to explore further the structure-activity relationships.
Previously, the C5-substituents of pyrimidine-based kinase inhibitors have been
demonstrated as a key functional group for improving potency and kinome selectivity via the
interaction with the gatekeeper residue [23, 28, 29]. Ceritinib, TAE684, brigatinib, and WZ4002
all possess a chlorine substituent at C5-pyrimidine which is required to achieve potent inhibition
of ALK or EGFR. For WZ4002 an interaction of the C5-chlorine with the thioether of M790 was
observed in the crystal structure and was critical to achieving selectivity for T790M over wild
type EGFR [24, 30]. Similar to the known ALK and EGFR inhibitors, introduction of diverse
halogens such as fluorine, chlorine and iodine at C5 of the pyrimidine core led to remarkable
increases in inhibitory activities against both ALK and EGFRs (Tables 1 and 2). Especially
compound 7c containing a chlorine substituent, exhibited more than 300-fold improved anti-
proliferative activity against EML4-ALK transformed Ba/F3 cells. Moreover, inhibition of wild
type EGFR and mutant EGFRs in enzymatic and cellular assays were improved by 15 ~ 30-fold,
with maintenance of selectivity between wild type EGFR and mutant EGFRs. The fluorine
derivative (7b) was roughly equipotent and had similar selectivity to 7c but the iodine derivative
(7d) showed slightly lower potencies against both ALK and EGFR while retaining selectivity for
wild type and mutant EGFRs.
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We next directed our attention to evaluate the combination of various ortho-hydrogen
bond acceptors such as dimethylsulfonamide and dimethylphosphine oxide at C4-aniline with
C5-halogen substituents (Table 1 and 2, 7e - l). The dimethylphosphine oxide analogs (7e - 7h)
were slightly less potent against ALK than the isopropylsulfone analogs (7a - 7d) but, equipotent
against wild type EGFR and mutant EGFRs in enzymatic assays and retained the potencies
against mutant EGFR transformed Ba/F3 cells. However, these analogs totally lost their growth
inhibitory activity against EML4-ALK transformed Ba/F3 cells. In contrast to the
dimethylphosphine oxide, introduction of the dimethylsulfonamide group led to increases in
inhibitory activities of both ALK and EGFRs in enzymatic and cellular assays (7a and 7i) and
when combined with the halogen substituents at C5 of the pyrimidine as in 7j - 7l, biochemical
and cellular potencies were similar to those of the isopropylsulfone analogs (7b - 7d). However,
these analogs showed a slight cytotoxicity against parental Ba/F3 cells (EC50 = 2.3 ~ 4.7 µM)
which led us to choose compound 7c as a lead compound.
ALK (IC50, nM)
EGFR (IC50, nM)
Compd. No.
Ceritinib
R1
R2
wt
20
wt
2156
120
114
T790M
T790M/L858R
28
5
19
4
WZ4002
990
177
Osimertinib
5
7
7a
7b
7c
7d
H
F
103
17
2480
46
26
2
86
2
Cl
I
18
151
865
2
4
91
9
33
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7e
7f
H
F
695
123
40
149
106
65
5
2
2
2
35
5
7g
7h
Cl
I
12
8
41
54
7i
7j
H
F
31
15
17
38
307
172
204
244
4
2
2
3
12
3
7k
7l
Cl
I
4
5
Table 1. Enzymatic Inhibitory Activities against ALK and EGFR.
EGFR (EC50, nM)
EML4-ALK
Parental
T790M/L T790M/
Compd. No. R1
Ceritinib
R2
(EC50, nM)
26
wt
> 3000
989
L858R
3000
26
Del
2500
68
858R
2500
31
Del
2660
27
(EC50, nM)
> 5000
WZ4002
6258
> 5000
Osimertinib
1748
66
6
18
20
16
> 5000
7a
7b
7c
H
F
2182
20
827
25
78
2
195
5
183
5
158
5
> 5000
> 5000
> 5000
Cl
7
32
4
6
6
5
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7d
7e
7f
I
H
F
87
> 5000
2506
961
240
988
178
68
19
236
68
47
680
157
44
22
830
162
45
20
400
74
> 5000
> 5000
> 5000
> 5000
> 5000
7g
7h
Cl
I
21
30
2357
96
23
84
31
32
7i
7j
H
F
217
39
8
93
31
19
8
21
6
22
6
19
5
> 5000
4661
7k
Cl
69
4
6
6
5
2904
7l
I
21
262
19
24
19
17
2270
Table 2. Antiproliferation Activities against EML4-ALK Ba/F3 and EGFR Ba/F3 Cells.
2.3 Characterization of compound 7c
We next performed detailed characterization of 7c including a molecular modeling study,
kinome selectivity profile, effects on ALK and EGFR-dependent signaling pathways,
antiproliferative activity on patient-derived NSCLC and pharmacokinetic profile in mouse.
To better rationalize the structural-basis for the observed dual inhibition of ALK and
EGFR, we performed a molecular modeling based on the co-crystal structures of ALK and
ceritinib (PDB 4MKC), wild type EGFR and osimertinib (PDB 4ZAU) and T790M EGFR and
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WZ4002 (PDB 3IKA) (Figure 2). 7c is predicted to bind to the ATP binding site similarly to
ceritinib and WZ4002 making identical hydrogen bonds to the hinge residues Met1199 and
Met793. We carefully investigated the binding modes of two distinct functional groups such as
the meta-acrylamide in ALK and the ortho-isopropylsulfone in EGFR. The residue
corresponding to the Cys797 in EGFR, which forms the covalent bond with the meta-acrylamide
(Table S2), is the Asp1203 in ALK. We did not find an additional interaction between the meta-
acrylamide of 7c and the Asp1203, but it is well accommodated by this residue at the edge of the
ATP binding pocket. On the other hand, ALK and EGFR have the same residues around the
region of the binding pocket occupied by the ortho-isopropylsulfone, except for the DFG-1
residue (Gly1269 in ALK and Thr854 in EGFR), which creates similar binding pockets. As a
result, the ortho-isopropylsulfone is positioned in proximity to the catalytic Lys745 in wild type
EGFR and T790M mutant EGFR, which forms an additional hydrogen bond as observed in the
ceritinib-ALK co-crystal structure.
(A)
(B)
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(C)
Figure 2. Molecular docking models of compound 7c bound to (A) wild type ALK, (B) wild
type EGFR and (C) T790M EGFR. Hydrogen bonds are indicated by dashed lines.
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We assessed the kinase selectivity of 7c using the KINOMEscan technology [DiscoverX]
[31, 32] against a diverse panel of 468 kinases at a concentration of 1 µM and compared it to the
profiles obtained for both TAE684 (panel of 353 kinases) and ceritinib (Figure 3). Hydrogen
bond acceptors such as sulfone, sulfonamide and phosphine oxide, are well known functional
groups which interact with the conserved catalytic lysine. Installing these functional groups at
ortho-position of C4-anilinopyrimidine leads to poor kinome selectivity. However, ceritinib,
which has a bulky ortho-isopropoxy substituent at C2-anilinopyrimidine, achieved good kinase
selectivity [S-score(1) = 0.02] even though it contains the ortho-isopropylsulfone at the C4-
anilinopyrimidine, compared to TAE684 which has the ortho-methoxy group [S-score(1) = 0.37].
However, ceritinib lost affinity for EGFR proteins in KINOMEscan which encouraged us to
retain the ortho-methoxy substituent of 2-anilinopyrimidine. Fortunately, 7c displayed superior
overall kinome selectivity [S-score(1) = 0.07] relative to TAE684, and as expected it still bound
to both wild type and mutants ALK and EGFR. The profiling revealed a number of potential off-
targets such as ErbB2, ErbB4, BTK and BLK, which have a cysteine residue at the same position
as the Cys797 of EGFR [24], and several non-receptor tyrosine kinases (Table S3).
(A)
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(B)
14

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(C)
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Figure 3. Kinome-wide selectivity profiles of (A) TAE684, (B) ceritinib and (C) compound 7c at
a 1 µM concentration. Wild type and mutants ALK and EGFR are indicated by blue spots.
We investigated the cell growth inhibitory activity of 7c against patient-derived lung
cancer cell line, DFCI032, which harbors wild type EML4-ALK together with activated EGFR
[33]. We also tested DFCI076 cell line, which harbors both activated EGFR and an L1152R
mutation in the ALK kinase domain, which renders cells resistant to most ALK inhibitors [14, 34]
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(Figure 4A and 4B). As expected, ceritinib or osimertinib alone were not able to efficiently
inhibit the proliferation of DFCI032 (EC50 = 4.44 µM and 4.73 µM, respectively). In contrast,
our lead compound 7c exhibited remarkable inhibitory effect against DFCI032 cells with an EC50
value of 0.17 µM, which is 25-fold more potent than ceritinib or osimertinib alone and
equipotent to a 1:1 combination treatment of ceritinib and osimertinib (EC50 = 0.19 µM). On the
other hand, the 1:1 combination of ceritinib and osimertinib was not able to efficiently inhibit the
proliferation of DFCI076 due to the loss of ALK inhibition. 7c was also less effective against
DFCI076, but was 5-fold more potent than the 1:1 combination of ceritinib and osimertinib,
which was presumably due to stronger ALK inhibition than ceritinib. This result strongly
supports the ability of 7c to inhibit both ALK and EGFR in a patient-derived cellular context, in
good agreement with the engineered Ba/F3 cellular assays.
To assess the capability of 7c to downregulate ALK and EGFR signaling pathways
simultaneously in a cellular context, we performed Western blotting for phosphorylated ALK
and EGFR as well as downstream signaling mediators such as Akt and ERK in DFCI032 cells
(Figure 4C). Ceritinib and osimertinib suppressed phosphorylation of their primary target
respectively but were not able to sufficiently reduce phosphorylation of Akt and ERK. On the
other hand, the 1:1 combination of ceritinib and osimertinib effectively inhibited phosphorylation
of both ALK and EGFR as well as Akt and ERK. Consistent with the cell growth inhibitory
activity, 7c alone also showed dual inhibition of ALK and EGFR phosphorylation which led to
better suppression of both PI3K/Akt and MAPK/ERK pathways than the 1:1 combination of
ceritinib and osimertinib.
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(A)
(B)
Patient-
derived
cell line
EML4- Crizotinib
Resistance
mechanism
EC50 (µM)
ALK
sensitivity
Ceritinib Osimertinib
7c
Ceritinib/Osimertinib
(1:1)
variant
DFCI032
DFCI076
1
3
Resistant
Resistant
WT ALK TKD; EGFR
activation
4.44
3.49
4.73
7.93
0.17
0.82
0.19
ALK L1152R mutation;
EGFR activation
3.21
(C)
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Figure 4. Profiles of compound 7c in patient-derived NSCLC. (A) Dose-dependent cell growth
curves of ceritinib (orange), osimertinib (cyan), compound 7c (red) and the 1:1 combination of
ceritinib and osimertinib (black) against DFCI032 and DCI076. (B) Cellular contexts of
DFCI032 and DFCI076 and EC50 values. (C) Effects on phosphorylation of ALK, EGFR, Akt
and ERK in DFCI032 cells.
We profiled the in vitro metabolic stability of newly synthesized compounds using
human, dog, rat and mouse liver microsomes and we evaluated their ability to inhibit cytochrome
P450 (CYP) in vitro (Table 3). Generally, the dimethylphosphine oxide analogs showed superior
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microsomal stability and lower CYP enzyme inhibition than the corresponding isopropylsulfone
analogs and the dimethylsulfonamide analogs which displayed 30 - 60 % inhibition of CYP 3A4
and approximately 20 % inhibition of other CYP enzymes. Our lead compound 7c was not stable
in liver microsomes of all species whereas corresponding dimethylphosphine oxide analog 7g
displayed good microsomal stability. This result was not surprising because the
dimethylphosphine oxide analogs showed lower clog P values than the isopropylsulfone analogs
and dimethylsulfonamide analogs.
Cytochrome P450 inhibition
(% of control)
Liver microsomal stability
(% remaining after 30 min)
Compd.
No.
R1
H
R2
1A2 2C9 2C19 2D6
3A4
Human Dog Rat
Mouse
cLog Pa
3.9
7a
7b
7c
80
91
89
90
94
83
87
93
86
67
95
90
40
5
48
27
17
22
11
11
8
F
51
26
16
15
12
4.1
Cl
44
4.7
7d
7e
7f
I
H
F
81
84
88
90
86
82
87
47
89
14
70
84
61
65
19
73
87
77
93
14
78
87
76
81
10
76
80
66
57
5.1
2.0
2.2
2.8
3.2
>100
>100 >100 >100 >100 >100
7g
7h
Cl
I
93
91
>100
>100
98
99
85
80
91
88
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7i
7j
H
F
82
>100
87
88
88
59
85
85
91
75
67
79
>100
88
56
71
39
54
10
9
50
33
31
35
6
7
5
3.9
4.1
4.6
5.0
10
23
12
7k
Cl
11
13
12
7l
I
79
93
13
aCalculated log P using ChemDraw Professional version 16.0.0.82.
Table 3. Metabolic Stability in Liver Microsome and Cytochrome P450 Enzyme Inhibition of
Compound 7a-l.
7c displayed reasonable mouse pharmacokinetic properties with a moderate T1/2 of 1.25
hour and AUClast value of 1551 ng·h/mL following an oral dose of 10 mg/Kg (Table 4A).
Furthermore, 2 hours after an intravenous (2 mg/Kg) or an oral (10 mg/Kg) dose, 7c showed
higher lung exposure (2.36 µM and 4.64 µM) than plasma exposure (0.10 µM and 0.15 µM)
which lasted for 8 hours (0.11 µM and 0.74 µM versus 0.00 µM and 0.01 µM). Despite low
bioavailability (24 %), the preferential accumulation of 7c in lung tissue over plasma would be
advantageous to use in studying the effects of dual ALK/EGFR inhibition in murine models of
lung cancer (Table 4B).
(A)
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ID route
dose
matrix
T1/2
Tmax
Cmax
AUClast
Cl_obs
Vss_obs
F
(mg/Kg)
(hr)
(hr) (ng/mL)
(hr·ng/mL)
(mL/min/Kg)
(L/Kg)
(%)
7c
IV
2
plasma
plasma
1.25
882
1287
1551
28.99
2.66
PO
10
1.17
520
114.54
24
(B)
ID
7c
matrix
plasma
route
dose (mg/Kg)
time (hr)
conc. (µM)
0.10
IV
PO
IV
2
10
2
2
2
8
8
2
2
8
8
0.15
0.00
PO
IV
10
2
0.01
7c
lung
2.36
PO
IV
10
2
4.64
0.11
PO
10
0.74
Table 4. (A) Pharmacokinetic Properties and (B) In vivo Lung Tissue Availability of Compound
7c.
2.4 Chemistry
The synthesis, which is described in scheme 1, followed well established synthetic routes
for similar compounds [13, 21, 23]. All variations were made at the first nucleophilic aromatic
substitution (SNAr) reaction step using commercially available 2,4-dichloropyrimidine cores and
three different anilines. Then, we performed a four-step reaction sequence to synthesize the
isopropylsulfone and dimethylsulfoamide analogs (7a - d and 7i - l) including two SNAr
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reactions, nitro-reduction and acrylamide formation to install the C2-aniline substituent. The
dimethylphosphine oxide analogs (7e - h) were also made via similar route except for a
Buchwald coupling which was employed to install the 4-fluoro-2-methoxy-5-nitroaniline at C2-
position of pyrimidine.
(i) 2-1 and 2-3 : NaH, DMF/DMSO (9:1), 0 °C; 2-2 : K2CO3, DMF, 70 °C; (ii) 3a - d and 3i - l :
4-fluoro-2-methoxy-5-nitroaniline, TFA, 2-BuOH, 100 °C; 3e - h : 4-fluoro-2-methoxy-5-
nitroaniline, Pd2dba3, Xphos, K2CO3, 2-BuOH; (iii) N1,N1,N2-trimethylethane-1,2-diamine, 1,4-
dioxane, 80 °C; (iv) 5a - d and 5i - l : SnCl2·2H2O, HCl, EtOAc; 5e - h : Pd/C, H2 gas, MeOH;
(v) acryloyl chloride, sat. NaHCO3/THF (1:1).
23

ACCEPTED MANUSCRIPT
Scheme 1. Synthetic Routes for Compound 7a-l.
3. Conclusion
Based on our previous effort developing covalent T790M EGFR inhibitor, WZ4002, we
have rationally designed and synthesized the potent dual inhibitor of ALK and EGFR,
particularly the T790M resistant mutant, by combining the crucial functional groups of known
ALK inhibitors ceritinib and brigatinib, and EGFR inhibitors WZ4002 and osimertinib, in the
2,4-diarylaminopyrimidine scaffold. This effort resulted in the identification of 7c as a potent
dual inhibitor of ALK and EGFRs, including T790M and L858R/T790M mutants, in
biochemical assays with low nanomolar IC50 values, which was well correlated with cell growth
inhibitory activities against EML4-ALK, wild type EGFR, L858R, exon19del, L858R/T790M or
exon19del/T790M EGFR transformed Ba/F3 cells. Moreover, compound 7c recapitulated the
cell growth inhibitory activity observed upon combinatorial treatment with both ceritinib and
osimertinib against the patient-derived NSCLC cell line, DFCI032 which harbors both EML4-
ALK and activated EGFR, and inhibited the cellular phosphorylation of ALK and EGFR, as well
as their downstream signaling proteins Akt and ERK. These results suggest that 7c is a promising
lead compound to develop advanced ALK and EGFR dual inhibitors. Further optimization of this
scaffold to improve metabolic stability and kinome-wide selectivity is currently underway.
4. Experimental protocols
24

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4.1 Chemistry
Starting materials, reagents and solvents were purchased from commercial suppliers and
were used without further purification unless otherwise noted. All reactions were monitored
using a Waters Acquity UPLC/MS system (Waters PDA eλ Detector, QDa Detector, Sample
manager – FL, Binary Solvent Manager) using Acquity UPLC® BEH C18 column (2.1 x 50
mm, 1.7 µm particle size): solvent gradient = 90 % A at 0 min, 1 % A at 1.6 min; solvent A = 0.1
% formic acid in Water; solvent B = 0.1 % formic acid in Acetonitrile; flow rate : 0.6 mL/min.
Reaction products were purified by flash column chromatography using CombiFlash®Rf with
Teledyne Isco RediSep®Rf High Performance Gold or Silicycle SiliaSepTM High Performance
columns (4 g, 12 g, 24 g, 40 g, or 80 g) and Waters HPLC system using SunFireTM Prep C18
column (19 x 100 mm, 5 µm particle size): solvent gradient = 80 % A at 0 min, 10 % A at 25
min; solvent A = 0.035 % TFA in Water; solvent B = 0.035 % TFA in MeOH; flow rate : 25
1
mL/min. H NMR spectra were recorded on 400 MHz and 500 MHz Bruker Avance III
spectrometers and 13C NMR spectrum was recorded on 125 MHz Bruker Avance III
spectrometer. The mass spectrometry data were measured in positive electrospray ionization
(ESI) mode on LCMS-2020 system (Shimadzu, Tokyo, Japan). Chemical shifts are reported
1
relative to methanol (
δ
= 3.30), chloroform (
δ
= 7.24) or dimethyl sulfoxide (
δ
= 2.50) for H
13
NMR and C NMR. Data are reported as (br = broad, s = singlet, d = doublet, t = triplet, q =
quartet, m = multiplet).
4.1.1 Representative procedure for the synthesis of compounds 7a - 7d and 7i - 7l
25

ACCEPTED MANUSCRIPT
2,5-Dichloro-N-(2-(isopropylsulfonyl)phenyl)pyrimidin-4-amine (3c). To a solution of 2-
(isopropylsulfonyl)aniline (3.0 g, 15.1 mmol) in DMF (80 mL) was added sodium hydride (1.2 g,
30.1 mmol, 60 % in mineral oil) at 0 °C. After stirring for 30 min, 2,4,5-trichloropyrimidine was
added to the reaction mixture followed by warming the mixture to room temperature. After
stirring for 2 h, the reaction mixture was quenched with ice and diluted with excess water. The
precipitate was filtered and the solid was dried by blowing nitrogen gas to obtain 3c as an off-
1
white solid (3.75 g, 72 %). H NMR (500 MHz, DMSO-d6)
δ 9.81 (s, 1H), 8.56 (s, 1H), 8.33 -
8.31 (m, 1H), 7.89 (dd, J = 7.9, 1.5 Hz, 1H), 7.88 - 7.84 (m, 1H), 7.48 (td, J = 7.6, 1.2 Hz, 1H),
3.58 - 3.48 (m, 1H), 1.16 (d, J = 6.7, 6H); LC/MS (ESI) m/z 346.18 [M+H]+.
5-Chloro-N2-(4-fluoro-2-methoxy-5-nitrophenyl)-N4-(2-(isopropylsulfonyl)phenyl)pyrimidine-
2,4-diamine (4c). To a suspension of 2,5-dichloro-N-(2-(isopropylsulfonyl)phenyl)pyrimidin-4-
amine (3c) (1.0 g, 2.89 mmol) and 4-fluoro-2-methoxy-5-nitroaniline (0.565 g, 3.03 mmol) in 2-
butanol (10 mL) was added trifluoroacetic acid (1.1 mL, 14.5 mmol). Then, the mixture was
stirred at 80 °C for 6 h followed by cooling to room temperature. The resulting mixture was
concentrated under reduced pressure and the residue was purified by flash column
chromatography on silica gel (0 to 10 % MeOH in DCM) to give 4c as a brown solid (1.23 g, 86
1
%). H NMR (500 MHz, DMSO-d6)
δ 9.53 (s, 1H), 8.72 (s, 1H), 8.54 (d, J = 8.2 Hz, 1H), 8.44
(d, J = 7.3 Hz, 1H), 8.33 (s, 1H), 7.82 (dd, J = 7.9, 1.5 Hz, 1H), 7.62 - 7.55 (m, 1H), 7.37 - 7.31
(m, 2H), 3.95 (s, 3H), 3.50 - 3.40 (m, 1H), 1.16 (d, J = 6.7, 6H); LC/MS (ESI) m/z 496.36
[M+H]+.
26

ACCEPTED MANUSCRIPT
5-Chloro-N2-(4-((2-(dimethylamino)ethyl)(methyl)amino)-2-methoxy-5-nitrophenyl)-N4-(2-
(isopropylsulfonyl)phenyl)pyrimidine-2,4-diamine (5c). To a solution of 5-chloro-N2-(4-fluoro-2-
methoxy-5-nitrophenyl)-N4-(2-(isopropylsulfonyl)phenyl)pyrimidine-2,4-diamine (4c) (0.800 g,
1.61 mmol) in dioxane (5 mL) was added N1,N1,N2-trimethylethane-1,2-diamine (0.640 mL, 4.84
mmol). The reaction mixture was stirred at 100 °C for 2 h. After completion of the reaction, the
resulting mixture was concentrated under reduced pressure and purified by flash column
chromatography on silica gel (0 to 20 % MeOH in DCM) to give 5c as a sticky liquid (838 mg,
1
90 %) H NMR (500 MHz, DMSO-d6)
δ 9.53 (s, 1H), 8.55 (s, 1H), 8.49 (br, 1H), 8.27 (s, 1H),
8.17 (br, 1H), 7.81 (dd, J = 7.9, 1.5 Hz, 1H), 7.60 - 7.51 (m, 1H), 7.34 - 7.29 (m, 1H), 6.84 (s,
1H), 3.90 (s, 3H), 3.49 - 3.40 (m, 1H), 3.40 - 3.24 (m, 2H), 2.88 (br, 2H), 2.83 (s, 1H), 2.47 (br,
6H), 1.16 (d, J = 6.7, 6H); LC/MS (ESI) m/z 578.59 [M+H]+.
N4-(5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)-N1-(2-
(dimethylamino)ethyl)-5-methoxy-N1-methylbenzene-1,2,4-triamine (6c). To a suspension of 5-
chloro-N2-(4-((2-(dimethylamino)ethyl)(methyl)amino)-2-methoxy-5-nitrophenyl)-N4-(2-
(isopropylsulfonyl)phenyl)pyrimidine-2,4-diamine (5c) (0.274 g, 0.473 mmol) and tin (II)
chloride dihydrate (1.00 g, 4.74 mmol) in ethyl acetate (7 mL) was added conc. HCl (1 mL).
Then, the resulting mixture was stirred at 50 °C for 5 h and quenched with NH4OH (pH 5),
Na2CO3 solid (pH 7) and the resulting suspension was filtered through Celite. The filtrate was
1
concentrated under reduced pressure to afford 6c as a brown solid (217 mg, 92 %). H NMR
(500 MHz, DMSO-d6)
δ 9.58 (s, 1H), 8.67 (d, J = 8.6 Hz, 1H), 8.32 (s, 1H), 8.20 (s, 1H), 7.80
(dd, J = 7.9, 1.5 Hz, 1H), 7.69 - 7.63 (m, 1H), 7.33 - 7.27 (m, 1H), 7.08 (bs, 1H), 6.81 (s, 1H),
27

ACCEPTED MANUSCRIPT
3.70 (s, 3H), 3.49 - 3.39 (m, 1H), 3.27 (br t, J = 6.0 Hz, 2H), 3.19 (br t, J = 5.8 Hz, 2H), 2.80 (s,
6H), 2.55 (s, 3H), 1.17 (d, J = 6.7 Hz, 6H); LC/MS (ESI) m/z 548.58 [M+H]+.
N-(5-((5-Chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-2-((2-
(dimethylamino)ethyl)(methyl)amino)-4-methoxyphenyl)acrylamide (7c). To a solution of N4-(5-
chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)-N1-(2-(dimethylamino)ethyl)-5-
methoxy-N1-methylbenzene-1,2,4-triamine (6c) (120 mg, 0.219 mmol) in THF (2 mL) and
aq.NaHCO3 (2 mL) was added dropwise acryloyl chloride (0.027 mL, 0.329 mmol) at 0 °C.
After stirring at 0 °C for 10 min, the reaction mixture was diluted with excess DCM and washed
with brine. The organic layer was dried over Na2SO4, filtered and concentrated under reduced
pressure. The residue was purified by preparative HPLC to give 7c as an off-white solid (52 mg,
53 %). 1H NMR (500 MHz, DMSO-d6)
δ 10.02 (br s, 1H), 9.52 (s, 1H), 8.57 - 8.46 (m, 2H), 8.39
(s, 1H), 8.22 (s, 1H), 7.76 (dd, J = 7.9, 1.5 Hz, 1H), 7.47 (t, J = 7.0 Hz, 1H), 7.23 (t, J = 7.5 Hz,
1H), 7.00 (s, 1H), 6.48 - 6.30 (m, 1H), 6.15 (dd, J = 16.8, 1.8 Hz, 1H), 5.72 (dd, J = 10.2, 1.8 Hz,
1H), 3.76 (s, 3H), 3.47 - 3.38 (m, 1H), 2.90 (br s, 2H), 2.71 (s, 3H), 2.36 (br s, 2H), 2.24 (br s,
6H), 1.16 (d, J = 6.7 Hz, 6H); 13C NMR (125 MHz, DMSO-d6)
δ 162.5, 158.5, 155.4, 154.5,
148.8, 140.1, 138.0, 134.5, 132.1, 130.8, 126.9, 126.2, 123.8, 123.4, 122.9, 122.9, 117.8, 105.4,
104.1, 56.7, 55.7, 54.9, 54.9, 45.1, 45.1, 42.2, 14.9, 14.9; LC/MS (ESI) m/z 602.69 [M+H]+;
HRMS (ESI) calcd for C28H37ClN7O4S [M+H]+ 602.2311; found 602.2304.
4.1.1.1
N-(5-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-2-((2-
7c-R). 7c-R was
(dimethylamino)ethyl)(methyl)amino)-4-methoxyphenyl)propionamide
(
synthesized using the analog method that used to synthesize 7c. To a solution of N4-(5-chloro-4-
28