2,4-Diarylamino-pyrimidines as kinase inhibitors co-targeting IGF1R and EGFRL858R/T790M

Article abstract of DOI:10.1016/j.bmcl.2015.07.089

IGF1R amplification was recently implied to be related to the secondary acquired resistance against the 2nd or 3rd generation EGFR inhibitor therapies. We have successfully identified a series of 2,4-diarylamino-pyrimidines as new IGF1R/EGFR^L858R/T79

Full text of DOI:10.1016/j.bmcl.2015.07.089

Accepted Manuscript
2, 4- Diarylamino-pyrimidines as Kinase Inhibitors Co-targeting IGF1R and
EGFRL858R/T790M
Shingpan Chan, Kun Han, Rong Qu, Linjiang Tong, Yingjun Li, Zhang Zhang,
Huimin Cheng, Xiaoyun Lu, Adam Patterson, Jeff Smaill, Xiaomei Ren, Jian
Ding, Hua Xie, Ke Ding
PII:
S0960-894X(15)00808-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.07.089
BMCL 22978
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 May 2015
23 July 2015
27 July 2015
Please cite this article as: Chan, S., Han, K., Qu, R., Tong, L., Li, Y., Zhang, Z., Cheng, H., Lu, X., Patterson, A.,
Smaill, J., Ren, X., Ding, J., Xie, H., Ding, K., 2, 4- Diarylamino-pyrimidines as Kinase Inhibitors Co-targeting
IGF1R and EGFRL858R/T790M, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/
j.bmcl.2015.07.089
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Graphical Abstract
2, 4- Diarylamino-pyrimidines as Kinase
Inhibitors Co-targeting IGF1R and
EGFRL858R/T790M
Leave this area blank for abstract info.
Shingpan Chan, Kun Han, Rong Qu, Linjiang Tong, Yingjun Li, Zhang Zhang, Huimin Cheng, Xiaoyun Lu,
Adam Patterson, Jeff Smaill, Xiaomei Ren, Jian Ding, Hua Xie, and Ke Ding
1

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
2, 4- Diarylamino-pyrimidines as Kinase Inhibitors Co-targeting IGF1R and
EGFRL858R/T790M
Shingpan Chan,a,b Kun Han,c Rong Qu,b,c Linjiang Tong,c Yingjun Li,a,b Zhang Zhang,a,d Huimin Cheng,a
Xiaoyun Lu,a Adam Patterson,e Jeff Smaill,e Xiaomei Ren,a Jian Ding,c Hua Xie,c,* and Ke Dinga,∗
a State Key Laboratory of Respiratory Diseases, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, #190 Kaiyuan Avenue,
Guangzhou 510530, China
b University of Chinese Academy of Sciences, #19 Yuquan Road, Beijing 100049, China
c State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China
d Jinan University, #601 Huangpu Avenue West, Guangzhou 510632, China
e Auckland Cancer Society Research Centre, and Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, #92019 Private Bag, Auckland
1142, New Zealand
ARTICLE INFO
ABSTRACT
Article history:
Received
IGF1R amplification was recently implied to be related to the secondary acquired resistance
against the 2nd or 3rd generation EGFR inhibitor therapies. We have successfully identified a
series of 2, 4-diarylamino-pyrimidines as new IGF1R/EGFRL858R/T790M co-targeting agents. One
of the most promising compounds 8g potently inhibits both kinases with low nanomolar IC50
values, but is significantly less potent in inhibiting the wild type EGFR. The compound also
displays a good kinase selectivity profile against a panel of 468 kinases. Moreover, 8g strongly
suppresses the proliferation of CO-1686-resistant H1975-IGF1R cancer cells, suggesting its
promising potential as a new lead compound for future anticancer drug discovery.
Revised
Accepted
Available online
Keywords:
EGFR
IGF1R
2015 Elsevier Ltd. All rights reserved
.
NSCLC
dual inhibitor
———
∗ Corresponding author. Tel.: +86-20-32015276 (K.D.). +86-21-50805897 (H.X.); e-mail: ding_ke@gibh.ac.cn (K. Ding). hxie@simm.ac.cn
(H. Xie.).

The first generation epidermal growth factor receptor (EGFR)
inhibitors (e.g. gefitinib and erlotinib) have shown remarkable
benefits for non-small-cell lung cancer (NSCLC) patients
carrying EGFR activating mutations (L858R and exon-19
deletion).1,2 However, an acquired threonine790 →methionine790
(T790M) mutation of EGFR contributes greatly to the clinical
resistance against current drugs.3 The cysteine797 reactive second-
generation inhibitors afatinib, neratinib and dacomitinib were
subsequently developed and demonstrated
a
promising
resistance-overcoming capability in preclinical studies. However,
they display strong, non-selective suppression on wild-type
EGFR which causes unacceptably low Maximum-Tolerated-
Dose (MTD) in resistant patients.4 Recently, several 3rd
generation wild-type sparing EGFRT790M inhibitors, such as CO-
1686 (1),5 AZD9291 (2),6 compound 37 and WZ4002 (4),8 were
discovered. Both 1 and 2 have demonstrated significant clinical
benefit for resistant NSCLC patients and were granted
“Breakthrough Therapy” designations by the US FDA in 2014.9
Figure 1. Chemical structures of selective irreversible EGFRT790M inhibitors,
representative IGF1R inhibitors and the designed EGFRT790M/IGF1R dual
inhibitor 8a.
Most recently, EGFRC797S mutation has been reported to
mediate a secondary acquired resistance against drug 2 in
EGFRT790M mutated NSCLC patients.10 Tyrosine Kinase Inhibitor
dose-escalation investigations also suggested that sustained
activation of Akt could induce insensitivity against drugs 1 and 2
Although the EGFR and IGF1R kinase domains only share
33.4% identity in a sequence alignment analysis (Supplementary
Figure S1), we were pleased to find a reported IGF1R inhibitor 7
harbors an almost identical 2, 4-disubstituted pyrimidinyl
scaffold to that of the well-characterized EGFRT790M inhibitors 1
and 4 (Figure. 1).19 Moreover, a superimposition of co-crystal
structures of EGFRT790M-4 (PDB: 3IKA)8 with IGF1R-7 (PDB:
3QQU)19 revealed that the inhibitors bind to the corresponding
targets with highly similar “U” shaped modes (Figure 2A) where:
1) the conserved 2-aminopyrimidine scaffold (“middle section”)
forms two hydrogen bonds with Met793 or Met1082 in the hinge
region of EGFRT790M or IGF1R, respectively; 2) a hydrophilic
substituted phenyl group (“left section”) captures van der Waals
interaction with a hydrophobic pocket close to solvent accessible
regions; 3) another aromatic moiety occupies a hydrophobic
pocket (“right section”) accessed at approximately 90o to the
middle 2-aminopyrimidine ring. Distinguishingly, an α, β
unsaturated acrylyl group [either in the left (2, 3) or right phenyl
ring (1, 4)] is essential for the EGFRT790M inhibitors to form a
covalent interaction with Cys797 of the protein, whereas a 4-NH
moiety in the pyrimidinyl scaffold is required to form a critical
hydrogen bond with Asp1153 in IGF1R. Based on these
observations, compound 8a was designed as a new potential
IGF1R/EGFRT790M dual inhibitor. A preliminary computational
study suggested that 8a could nicely bind with both proteins in
similar binding poses to that of 4 with EGFRT790M (PDB: 3IKA)
or 7 with IGF1R (PDB: 3QQU), respectively (Figure. 2B & 2C).
in H1975 NSCLC cells.5,11
A similar independent study
demonstrated that amplification of Insulin-like Growth Factor 1
Receptor (IGF1R) was involved in over-activation of Akt. A
selective IGF1R inhibitor BMS536924 (5) successfully restored
the drug sensitivity to 4 in the WZ4002-Resistant (WZR)
NSCLC cells, and a combination of 4 with 5 completely
prevented the emergence of drug-resistant clones in the
experimental system.12
IGF1R is another tyrosine kinase whose overexpression is
closely correlated to malignant transformation and acquired
resistance of various human cancers.13 Although most IGF1R
based therapies displayed relatively disappointing clinical
outcomes with metabolic side effects (e.g. hyperglycemia) when
utilized as
a
single agent in non-selected patients,14,15
combinations of IGF1R inhibitors with other drugs were recently
demonstrated synergistic therapeutic benefits in metastatic
pancreatic cancer and lung cancer patients.16,17 There results
collectively imply that co-targeting EGFRT790M and IGF1R may
be a tractable strategy to overcome potential resistance against
the 2nd or 3rd generation EGFR inhibitors. Encouragingly, several
AZD9291 derivatives were recently reported to strongly inhibit
both EGFRT790M and IGF1R and display promising in vivo
antitumor efficacy.6,18
Herein, we report the design and synthesis of 2, 4-
diarylamino-pyrimidines as new IGF1R and EGFRT790M dual
inhibitors with capability to suppress the proliferation of CO-
1686-resistant H1975-IGF1R cancer cells.
Figure 2. (A) Superimposition of EGFRT790M-drug 4 (green, PDB: 3IKA)
with IGF1R-7 (blue, PDB: 3QQU). (B) Predicted binding model of 8a with
IGF1R (PDB: 3QQU). (C) Predicted binding model of 8a with EGFRT790M by
using Schrodinger software (PDB: 3IKA).
Compound 8a and the derivatives were readily prepared by
following procedures outlined in Scheme 1. Briefly,
a
regioselective nucleophilic substitution of 2, 4-dichloro
pyrimidines (9) with commercially available chemicals 10
provided intermediates 11 or 14. Compounds 11 or 14 were then
reacted with appropriately substituted nitroanilines to produce 12
or 15 either by a p-toluenesulfonic acid (p-TSA) catalyzed SNAr
reaction, or under Buchwald-Hartwig coupling conditions (for
2

compounds where X = O). Subsequent iron-mediated nitro
reduction of 13 or 16 then yielded key intermediates 13 or 16.
Compounds 13 or 16 were coupled with (E)-4-
(dimethylamino)but-2-enoic acid to produce the final compounds
8a-8x.
Kinase Inhibition
NO.
X
R1
R2
R3
(IC50, µM)
EGFR
(WT)
1.33
EGFR
(DM)
0.150
IGF-
1R
8a
8b
8c
8d
8e
8f
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NMe
O
Cl
Cl
a
b
c
d
e
f
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
0.154
4.56
1.30
0.191
0.066
0.041
0.038
0.018
0.038
0.044
0.031
0.032
0.047
0.293
0.159
0.056
0.032
0.023
0.073
0.102
0.023
0.031
0.024
0.056
0.029
0.026
0.154
0.031
0.052
0.017
0.015
0.013
0.037
0.038
0.481
>1
Cl
Cl
0.976
0.400
0.260
0.848
0.615
0.295
1.56
Cl
Cl
8g
8h
8i
Cl
g
h
i
Cl
Cl
8j
Cl
j
Scheme 1. Syntheses of compounds 8a-8x. Reagents and conditions:
(a) Na2CO3, EtOH, rt or n-BuLi, THF, -40 oC, 68-90%; (b) p-TSA, 2-
pentanol, reflux or Pd(OAc)2, Xantphos, Cs2CO3, 1,4-dioxane, reflux, 83-
95%; (c) Fe, NH4Cl, EtOH, water, 60 oC, 72-90%; (d) (E)-4-
(dimethylamino)but-2-enoic acid, 1-[Bis(dimethylamino) methylene]-1H-
1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU), N,N-
diisopropylethylamine (DIEA), THF, rt, 60-88%.
8k
8l
Cl
f
0.094
10.0
Cl
f
0.297
0.347
0.099
0.151
0.021
0.103
0.182
0.193
0.041
0.170
0.198
0.117
0.163
8m
8n
8o
8p
8q
8r
8s
S
Cl
f
10.0
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
H
f
0.054
0.131
0.254
0.225
0.225
0.173
2.07
F
f
Br
Me
OMe
CN
CF3
Cl
f
Kinase inhibitory activity of the compounds (IC50) was
evaluated by using well-established ELISA assays (Table 1).20
Inhibitor 1 and NVP-AEW541 (6) were included as positive
controls to validate the screening conditions for EGFR and
IGF1R, respectively. Under the experimental conditions,
inhibitor 1 potently inhibits EGFRL858R/T790M with an IC50 value of
0.005 µM, whereas its potencies against EGFRWT and IGF1R are
significantly lower.5 Compound 6 also displayed comparable
IGF1R inhibitory activity to the reported results.21
f
f
f
8t
f
8u
8v
8w
8x
f
4.80
Cl
f
F
1.40
Cl
f
Me
2.22
Cl
f
OEt
1.46
0.185
0.30b
0.005
0.022b
1.55
>0.1b
0.029
0.14b
Consistent with our computational prediction, 8a indeed co-
targeted the EGFRL858R/T790M and IGF1R enzymes with almost
identical IC50 values of 0.15 µM, while being about 10-fold less
potent against EGFRWT. Further investigation revealed that
chloride substitution at different positions of the R2-phenyl ring
1
6
-
-
aThe kinase inhibitory activity assays were performed using enzyme-
linked immunosorbent assay (ELISA).19 The data shown is the mean from at
least three independent experiments and the variations are <20%. b Reported
data. 5,21
achieved
a diverse impact on the dual inhibition of
EGFRL858R/T790M/IGF1R. For instance, ortho-chloro derivative 8b
displayed similar EGFRL858R/T790M and IGF1R enzyme inhibitory
activities to that of 8a. However, the meta- or para- substituted
compounds 8c or 8d showed 2- to 5-fold improved potency
against both kinases. It was also shown that 3, 4-dichloro
substitution (8e) maintained strong EGFRL858R/T790M inhibition,
while the potency against IGF1R was further improved by 2 folds
(compared with 8c). Inspired by the above observations, the β-
naphthylamino compound 8f was designed and synthesized to
potently inhibit EGFRL858R/T790M and IGF1R with IC50 values of
0.018 µM and 0.015 µM, respectively. This compound also
spared EGFRWT with a ratio of 14-fold [IC50 (EGFRWT) / IC50
(EGFRL858R/T790M)]. The other bicyclic compounds 8g, 8h and 8i
displayed comparable potencies and selectivity to that of 8f.
However, the α-naphthyl analogue 8j was significantly less
potent. It has been suggested that the “NH” group at the 4-
position of 8a is critical for forming a hydrogen bond with
IGF1R (Figure. 2B). Indeed, when the 4- NH was methylated or
was replaced with an oxygen or sulfur atom, the resulting
compounds (8k, 8l and 8m, respectively) displayed obviously
less IGF1R inhibitory potencies.
It was also determined that lipophilic substitution at the 5-
position of 8f was well tolerated with respect to inhibition of
EGFRL858R/T790M. Compounds 8n-8q, 8s and 8t, which contain H,
F, Br, Me, CN, or CF3 at the 5- position, respectively, displayed
comparable IC50 values against EGFRL858R T790M
to that of 8f.
/
However, their potency against IGF1R varied widely. The 5-Br
analogue 8p showed an almost identical IC50 value against
IGF1R to that of 8f, but the other compounds were obviously less
potent. Interestingly, the 5-methoxy compound 8r was
significantly less active than 8f against both kinases. Our studies
further established that the R3-methoxy group of 8f could be
replaced by H (8u), F (8v), Me (8w), or EtO- (8x) without
affecting the EGFRT790M inhibition, but in each case the potency
against IGF1R was decreased.
The anti-proliferative activity of the compounds was also
investigated against H1975 NSCLC cells, harboring
EGFRL858R/T790M mutation, and A431 cells, expressing EGFR wild
type. It was shown that 8g possessed the strongest cell growth
inhibitory activity with an IC50 value of 0.13 µM, which was
almost equal potent to drug 1 (Supplementary Table S1).
Table 1. In Vitro Kinase Inhibitory Activity of Compounds
8a-8xa
3

The strong and selective dual inhibition of 8g against
EGFRL858R/T790M/IGF1R was further confirmed by determining its
binding affinities to the target proteins. It was shown that 8g
effectively bound with EGFRL858R/T790M and IGF1R with Kd
values of 15.0 and 18.0 nM, respectively (Supplementary Table
S2). However, its binding affinity with EGFRWT was about 60-
fold less potent. A profiling investigation against 468 kinases
revealed that 8g possesses a good to moderate kinase selectivity
profile with an S(10) score21 of 0.05 at a concentration of 1.0 µM
(Supplementary Table S3). In addition to the primary targets, 8g
also binds a number of other cancer related kinases including
ALK (anaplastic lymphoma kinase), Btk (Bruton’s tyrosine
kinase); ERBB2 (erb-b2 receptor tyrosine kinase 2); FAK (focal
adhesion kinase); FER (fer (fps/fes related) tyrosine kinase);
LTK(leukocyte receptor tyrosine kinase); TLT3 (triggering
receptor expressed on myeloid cells-like 3) etc. (Supplementary
Table S3), indicating its potential to serve as a lead molecule for
other cancer therapeutic development. However, the potential
safety issues related to its relative kinase selectivity profile
requires further investigation.
inhibition against other kinases. In addition, drug 1 was
about 10-fold less potent at inhibiting the proliferation of
H1975-IGF1R cells than parental H1975 cells, whereas, 8g was
equally potent at inhibiting the growth of both cell lines
with IC50 values of 0.15 and 0.13 µM, respectively
(Supplementary Figure S3b).
In summary, a series of 2, 4-diarylamino-pyrimidines
were designed
and
synthesized
as new dual
IGF1R/EGFRL858R/T790M co-targeted agents. A representative
compound 8g potently inhibited both of the kinases with
low nanomolar IC50 values, but was significantly less potent
at inhibiting wild type EGFR. This compound also displayed
a good to moderate kinase selectivity profile against a panel
of 468 kinases. Moreover, 8g strongly suppresses the
proliferation of CO-1686-resistant H1975-IGF1R cancer cells,
suggesting its promising potential as a new lead compound
for drug discovery overcoming acquired resistance to the 2nd
and 3rd generation EGFR inhibitors.
Acknowledgments
Compound 8g dose-dependently suppressed the
phosphorylation of EGFR and decreased the levels of p-Akt
and p-Erk1/2 in EGFRL858R/T790M mutated H1975 cells with a
similar potency to that of drug 1 (Supplementary Figure S2a).
It also inhibited the activation of IGF1R and downstream
targets in IGF1R amplified HT-29 cancer cells (Supplementary
Figure S2b). These results further supported the
EGFRL858R/T790M/IGF1R dual inhibition of 8g.
We thank financial support from Ministry of Science and
Technology of China (2014DFG32100), the National Natural
Science Foundation (81425021, 881173080 and 81321092),
National High Technology Research and Development (863) for
Young Scientists program (2015AA020906), and the Health
Research Council of New Zealand (13/1020).
Supplementary data
Supplementary data associated with this article can be found,
in the online version.
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