Design, synthesis, and biological evaluation of novel conformationally constrained inhibitors targeting EGFR

Article abstract of DOI:10.1021/ml4002437

This letter describes the construction of conformationally constrained quinazoline analogues. Structure-activity relationship studies led to the identification of the lead compound 9n. Compound 9n exhibits effective in vitro activity against A431^{WT,overexpression} and H1975 ^{[L858R/T790M]} cancer cell lines but is significantly less effective against EGFR negative cancer cell lines (SW620, A549, and K562). Compound 9n was also assessed for potency in enzymatic assays and in vivo antitumor studies. The results indicated that 9n is a potent kinase inhibitor against both wild-type and T790M mutant EGFR kinase. Meanwhile, an oral administration of 9n at a dose of 200 mg/kg produced a considerable antitumor effect in a A431 xenograft model, as compared to gefitinib. A preliminary pharmacokinetic study of 9n also indicates it has good pharmacokinetic properties, and therefore, it is a good starting point for further development.

Full text of DOI:10.1021/ml4002437

Letter
pubs.acs.org/acsmedchemlett
Design, Synthesis, and Biological Evaluation of Novel
Conformationally Constrained Inhibitors Targeting EGFR
Jianwei Wu,†,⊥ Wenteng Chen,†,⊥ Guangxin Xia,‡ Jing Zhang,‡ Jiaan Shao,† Biqin Tan,§
Chunchun Zhang,‡ Wanwan Yu,† Qinjie Weng,§ Haiyan Liu,‡ Miao Hu,† Hailin Deng,‡ Yu Hao,‡
Jingkang Shen,*,‡,∥ and Yongping Yu*,†
†Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Science, Zhejiang University,
Hangzhou 310058, P. R. China
‡Central Research Institute, Shanghai Pharmaceuticals Holding Co., Ltd., Building 5, No. 898 Ha Lei Road, Shanghai, China
§School of Pharmaceutical Science, Zhejiang University, Hangzhou 310058, P. R. China
∥State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Science, 555 Zu Chong Zhi
Road, Shanghai 201203, China
S
* Supporting Information
ABSTRACT: This letter describes the construction of conformation-
ally constrained quinazoline analogues. Structure−activity relationship
studies led to the identification of the lead compound 9n. Compound
9n exhibits effective in vitro activity against A431WT,overexpression and
H1975[L858R/T790M] cancer cell lines but is significantly less effective
against EGFR negative cancer cell lines (SW620, A549, and K562).
Compound 9n was also assessed for potency in enzymatic assays and in
vivo antitumor studies. The results indicated that 9n is a potent kinase
inhibitor against both wild-type and T790M mutant EGFR kinase.
Meanwhile, an oral administration of 9n at a dose of 200 mg/kg
produced a considerable antitumor effect in a A431 xenograft model, as
compared to gefitinib. A preliminary pharmacokinetic study of 9n also indicates it has good pharmacokinetic properties, and
therefore, it is a good starting point for further development.
KEYWORDS: Anticancer, kinase inhibitor, EGFR, conformationally constrained
he clinical success of a number of kinase-directed drugs
Tindicates that kinase represent therapeutically relevant
targets. The epidermal growth factor receptor (EGFR) belongs
to the ErbB family of receptor tyrosine kinases (RTKs), which
plays an important role in the regulation of cell growth,
differentiation, and survival.1 Because of their multidimensional
role in the progression of cancer, EGFR and its family members
have emerged as attractive targets for anticancer therapy.2
Accordingly, targeting EGFR has been intensely pursued, with
the approval of gefitinib, erlotinib, and lapatinib for use in the
clinic.3−7 As is common with many therapies, challenges with
respect to treatment resistance emerge over time. This situation
is certainly true of EGFR inhibitor therapy. Clinical studies have
demonstrated the occurrence of resistance to gefitinib with the
T790M mutation accounting for 50% of the clinically observed
resistant mutations.
In order to overcome the T790M mutation related drug
resistance, a variety of irreversible inhibitors were developed.
These compounds contain a Michael acceptor moiety designed
to form a covalent bond with the conserved cysteine residue
(Cys797) at the lip of the EGFR ATP binding site.8,9 So far,
canertinib,10,11 neratinib,12,13 dacomitinib,14 and afatinib15 have
been developed as second generation irreversible inhibitors with
limited clinical efficacy. These drugs demonstrate the utility of
quinazoline derivatives as an attractive scaffold for the develop-
ment of EGFR inhibitors. To date, many studies have been
targeted at finding new structures based on quinazolines that are
potent EGFR inhibitors.16−18 All of these inhibitors share a
common feature: upon the binding to EGFR, a hydrogen bond
forms between the N1 atom of the compound and the backbone
NH of Met769 in the hinge region.
To investigate the potential use of a novel scaffold as an EGFR
ATP binding site inhibitor, a series of conformationally
constrained quinazoline derivatives were synthesized (consisting
of 34 structurally characterized compounds with purity >98%).
(For general synthetic procedures refer to Schemes 1 and 2 and
Supporting Information.) From this set of new compounds, a
specific analogue was identified that inhibits the growth of both
gefitinib-resistant (H1975[T790M/L858R]) and gefitinib-sensitive
(A431WT,overexpression) cell lines but is not effective against EGFR-
negative (A549, K562, and SW620) cells.
Received: June 27, 2013
Accepted: August 6, 2013
© XXXX American Chemical Society
A
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ACS Medicinal Chemistry Letters
Letter
that possessed approximately equal IC50 values against
A431WT,expression and H1975[L858R/T790M] as the reference
inhibitors (Figure 1 and Tables S2 and S3, Supporting
Information).
Scheme 1. Synthesis of Compounds 7a−e and 8a−b
Scheme 2. Synthesis of Compounds 9a−9p
The screening result (Table S1, Supporting Information) of
the synthesized analogues 7a−7o and 8a−8b did not show
obvious inhibition on the proliferation of A431 human epithelial
carcinoma cells with overexpressed EGFRWT, but surprisingly,
some of the synthesized analogues suppressed the enzymatic
activity of EGFRWT, which might be due to the complex genomic
background and the strong binding of EGFRWT with ATP.19 For
instance, 7b inhibited the enzymatic activities of EGFRWT with
an IC50 value of 1.29 nM, but its activity on A431 cancer cells was
approximately 20 000 times less potent (IC50 value was more
than 30 μM). The results indicated that a further modification of
the scaffold might prove to be an effective strategy for identifying
more potent inhibitors. The impact of the R3 group was first
studied by replacement with other acrylamide groups. Interest-
ingly, the inhibitory potency against A431WT,overexpression and wild-
type EGFR was significantly increased by over 20-fold (Table S2,
Supporting Information). For instance, when the hydrogen on
the R3 substitution was replaced with acrylamide, the resulting
compounds 9a−9e are almost 5 times more potent than 7a−7e.
However, when the substituted acrylamide was replaced with a
slightly hydrophobic moiety (9f−9h), the potencies against
A431WT,overexpression and wild-type EGFR kinase were almost
completely abolished. Further investigation also revealed that the
potency loss of 9f−9h was partially restored by introducing a N-
morpholino moiety (9i−9l) at the position of the original phenyl
moiety. The result suggests that the substituted acrylamide at the
R3 position can be replaced with a more hydrophilic group in
order to improve the potency against the A431 and wild-type
EGFR kinases. This hypothesis was verified by the in vitro
screening result of 9m−9p. The inhibitory activities against
A431WT,overexpression and EGFRWT were improved, with IC50 values
almost equal to the reference irreversible inhibitor, canertinib.
We further profiled the synthesized analogues 9a−9p against
NSCLC cell line H1975. The H1975 cell line bears the drug
resistant mutation in EGFR[L858R/T790M] and is a typical EGFR-
driven cell lines. Several compounds (8c−8d and 9n−9p) also
displayed strong antiproliferative effects on gefitinib-resistant
H1975 cells, with the IC50 value equal to or more potent than
that of canertinib (Table S2, Supporting Information). Three
closely related analogues, 9n−9p, were identified from the screen
Figure 1. Compounds 9n−9p are novel EGFR inhibitors, suppress the
growth of EGFRWT/EGFRL858R/T790M containing cancer cell lines, and
inhibit the enzymatic activities of EGFRWT/EGFRT790M kinase. (a)
Chemical structures of 9n−9p. IC50 values for A431 (top left), H1975
(top, right), EGFRWT (bottom, left), and EGFRT790M (bottom, right).
Growth inhibitory was assessed using the MTT.
To better understand how our conformationally constrained
compounds interact with the ATP-binding cleft of EGFR and
how this relates to their potency for EGFRT790M, we determined a
putative binding mode of one representative compound 9n
within the active binding pocket of the EGFRT790M (Figure 2).
The morpholine fused scaffold is located in the ATP-binding
pocket. Two highly conserved hydrogen bonds are formed
between the quinazoline core and the hinge region. The
molecule is oriented such that 6-substituted acrylamide branch
extends toward the solvent, and the 4-aniline moiety is directed
into the backpocket of EGFR kinase. The docking result is shown
to form a covalent bond with Cys797 as expected, with a
measured distance of 2.54 Å. The prediction is consistent with
observed result from Western blot studies (Figure 3). In
addition, a salt bridge was formed between the carboxyl group of
Asp800 and the nitrogen atom of the dimethylamino group.
Furthermore, the protonated dimethylamino group might
facilitate the nucleophilic attack of the sulfhydryl group of
Cys797.20 The prediction is also consistent with our observations
that a hydrophobic substituent (e.g., phenyl) led to a decrease in
the potency in comparison with the dimethylamino group.
Compound 9n and canertinib inhibit the autophosphorylation
of EGFR in no wash (left) and wash out (right), while gefitinib
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ACS Medicinal Chemistry Letters
Letter
low potential for cardiac toxicity (Table S4, Supporting
Information).
Furthermore, 9n and 9p have a good pharmacokinetic in vitro
ADME profiles: the inhibition of cytochrome P450 was assessed
in recombinant human cytochrome P450 isoforms (1A2, 2C9,
2D6, and 3A4), and the IC50 values were higher than 10 μM,
suggesting a low potential for 9n and 9p to be involved in any
drug−drug interactions. The intrinsic clearance found in liver
microsomes predicts good to excellent in vivo clearance (Table
S5, Supporting Information).
A pharmacokinetic study of 9n and 9p was performed in
BALB/c mice. As shown in Table 2, 9n demonstrated desirable
a
Table 2. Pharmacokinetic Parameters for 9n
AUC(0−24 h) (μM·
t1/2
(min)
tmax
(min)
Cmax
(μM)
route
min)
F%
i.v.
25.4
133
762
548
2
0.29
0.36
p.o.
45
26.1
Figure 2. Putative binding mode of 9n within the active pocket of the
a
The pharmacokinetic parameters are obtained after a single i.v. (3
mg/kg) or oral (10 mg/kg) administration. The data are obtained
from 12 mice in each treatment group.
EGFRT790M (PDB code: 4I24).
results with a half-life (9.1 h) and oral bioavailability (26.1%),
while 9p displayed an unsatisfactory pharmacokinetic prolife (F
= 6.42%) (Table S6, Supporting Information).
We further determined the effectiveness of 9n in vivo by using
a nude mouse xenograft model harboring EGFRWT,expression. We
chose 9n for the in vivo studies because, in vitro, it is effective
against EGFR expressing cancer cell lines and has a good ADME
prolife. Gefitinib (200 mg/kg) was used as a reference drug to
validate the models. In the A431 tumor model, the mice were
treated via oral gavage once daily with either 9n or gefitinib once
the tumor had grown to a volume of 50−150 mm2. Oral
administration of 9n and gefitinib at 200 mg/kg/day inhibited
tumor growth at 66.8% and 35.9%, respectively. No mortality or
significant weight loss was observed during the treatment (Figure
4).
In summary, a series of conformationally constrained
quinazoline derivatives have been designed and synthesized
and shown utility as EGFR inhibitors. The most potent
compounds 9n and 9p strongly inhibited the enzymatic activities
of wild-type EGFR kinase as well as clinical resistant EGFRT790M
mutant kinase. The kinase inhibitory efficiency of the
compounds were further validated by Western Blot analysis for
the activation of EGFR. Further in vitro assay demonstrated that
9n and 9p are effective against H1975 nonsmall cell lung cancer
cells bearing EGFR[L858R/T790M], with potencies better than
gefitinib. Compounds 9n and 9p showed minimal cytotoxicity to
K562 and SW620. A hERG assay demonstrated their unlikely
cardiac toxicity, indicating that these analogues might possess a
high safety index. An in vivo antitumor assay demonstrates that
an oral once daily dose of 9n at 200 mg/kg produces considerable
tumor inhibition in the A431 xenograft model, as compared to
gefitinib. The pharmacokinetic studies indicate that 9n possesses
good pharmacokinetic properties. In conclusion, our studies
identified a new lead compound containing a novel scaffold
ideally suited for the development of therapeutically relevant
EGFR inhibitor effective against EGFRT790M mutations. Further
detailed studies of the mechanisms of action for 9n are currently
underway.
Figure 3. Inhibition of EGFR autophosphorylation in A431 by Western
blot assay. G, gefitinib; C, canertinib.
has no effects in the wash out group. The result is the evidence of
irreversible binding mode, suggesting 9n is acting the same as
irreversible inhibitor canertinib.
The in vitro data shown in Figure 1 demonstrates that the new
synthesized analogues 9n−9p strongly inhibit the EGFRWT
kinase. Because many tissues use wild-type EGFR for normal
cellular processes, the potential for toxicity from irreversible
EGFR inhibitors is a concern. Therefore, the growth inhibitory of
the compounds against various cancer cell lines was evaluated to
control the potential toxicity. As shown in Table 1, the potent
Table 1. In Vitro Growth Inhibitory Activities against Various
Cancer Cell Lines
9n
9p
gefitinib
(μM)
cancer type
cell line
characteristic
(μM) (μM)
epidemal
NSCLC
NSCLC
colon
A431
EGFRWT,overexpression
EGFRL858R/T790M
EGFRWT,κ‑Ras mutation
EGFR negative
2.2
0.9
4.0
H1975
A549
4.9
3.61
>10
6.2
10.48
>10
>10
25.4
>10
SW620
K562
28.20
>10
malignant
myeloid
EGFR negative
>10
EGFR inhibitor 9n did not display obvious inhibition on the
growth of A549, SW620, and K562 cancer cells, all of which have
low levels of EGFR expression. The results indicate that the
cytotoxic effects of 9n are minimal. Meanwhile, in the human
ether-a-go-go-related gene (hERG) potassium channel patch
clamp assay,21 9n has an IC50 >10 μM, which indicates it has a
C
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ACS Medicinal Chemistry Letters
Letter
Funding
This research was supported by the National Natural Science
Foundation of China (No. 81072516 and No. 81273356),
Natural Science Foundation of Zhejiang Province (No.
Z2110655), Program for Zhejiang Leading Team of S&T
Innovation, and the Osteoporosis and Breast Cancer Research
Center, USA.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful to the financial support and biological activity test
from Shanghai Pharmaceuticals Holding Co., Ltd. We also
appreciated Marc A. Giulianotti (Torrey Pines Institute for
Molecular Studies, USA) for critical reading of the manuscript.
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Details of the synthesis and characterization of all compounds
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experiments, some biological data, and computational methods
used for molecular docking This material is available free of
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