Design, synthesis, SAR discussion, in?vitro and in?vivo evaluation of novel selective EGFR modulator to inhibit L858R/T790M double mutants

Article abstract of DOI:10.1016/j.ejmech.2017.04.036

Based upon the modeling binding mode of marketed AZD9291 with T790M, a series of 5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinoline derivatives were designed and synthesized with the purpose to overcome the drug resistance resulted from T790M/L858R double mutations. The most potent compound 8 showed excellent enzyme inhibitory activities and selectivity with sub nanomolar IC₅₀ values for both the single L858R and double T790M/L858R mutant EGFRs, and was more than 8-fold selective for wild type EGFR. Compound 8 exhibited good microsomes stabilities and pharmacokinetic properties and lower binding affinity to hERG ion channel than AZD9291 and displayed strong antiproliferative activity against the H1975 non-small cell lung cancer (NSCLC) cells bearing T790M/L858R and in?vivo anticancer efficacy in a human NSCLC (H1975) xenograft mouse model.

Full text of DOI:10.1016/j.ejmech.2017.04.036

Accepted Manuscript
Design, synthesis, SAR discussion, in vitro and in vivo evaluation of novel selective
EGFR modulator to inhibit L858R/T790M double mutants
Haoyang Zhang, Wenkui Wu, Chao Feng, Zhaogang Liu, Enhe Bai, Xueyuan Wang,
Meng Lei, Hao Cheng, Huayun Feng, Jingmiao Shi, Jia Wang, Zhao Zhang, Tao Jin,
Shanshan Chen, Shihe Hu, Yongqiang Zhu
PII:
S0223-5234(17)30300-8
10.1016/j.ejmech.2017.04.036
EJMECH 9385
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 January 2017
Revised Date: 11 April 2017
Accepted Date: 13 April 2017
Please cite this article as: H. Zhang, W. Wu, C. Feng, Z. Liu, E. Bai, X. Wang, M. Lei, H. Cheng, H.
Feng, J. Shi, J. Wang, Z. Zhang, T. Jin, S. Chen, S. Hu, Y. Zhu, Design, synthesis, SAR discussion, in
vitro and in vivo evaluation of novel selective EGFR modulator to inhibit L858R/T790M double mutants,
European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.04.036.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
N
O
NH
O
N
N
N
N
N
H
Compound 8
EGFRWT IC50: 21.6 nM
EGFRT790M/L858R IC50: 2.6 nM
T1/2= 4.1 h, F%= 52.4%
hERG IC50: 8.46
Binding of AZD9291 with T790M

ACCEPTED MANUSCRIPT
Design, synthesis, SAR discussion, in vitro and in
vivo evaluation of novel selective EGFR modulator
to inhibit L858R/T790M double mutants
Haoyang Zhang,#,† Wenkui Wu,#,† Chao Feng,‡ Zhaogang Liu,‡ Enhe Bai,§ Xueyuan Wang,†
Meng Lei,§ Hao Cheng,‡ Huayun Feng,§ Jingmiao Shi,‡ Jia Wang,‡ Zhao Zhang,† Tao Jin,†
Shanshan Chen,‡ Shihe Hu,*,‡ Yongqiang Zhu*,†
†College of Life Science, Nanjing Normal University, No. 1 Wenyuan Road, Nanjing 210046, PR
China, ‡Jiangsu Chia Tai Fenghai Pharmaceutical Co. Ltd., No. 9 Weidi Road, Nanjing 210046,
§
PR China and College of Science, Nanjing Forestry University, No. 159 Longpan Road,
Nanjing 210037, PR China
Corresponding Authors
*Prof. Yongqiang Zhu
Phone: 86-25-85891591; Fax: 86-25-85891591; E-mail: zhyqscu@hotmail.com.
*Dr. Shihe Hu
Phone: 86-25-57033246-8280; Fax: 86-25-57033246-8280; E-mail: hsh.2@163.com.
Author Contributions
#These authors contributed equally to the work.
1

ACCEPTED MANUSCRIPT
ABSTRACT: Based upon the modeling binding mode of marketed AZD9291 with T790M, a
series of 5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinoline derivatives were designed and synthesized
with the purpose to overcome the drug resistance resulted from T790M/L858R double mutations.
The most potent compound 8 showed excellent enzyme inhibitory activities and selectivity with
sub nanomolar IC50 values for both the single L858R and double T790M/L858R mutant EGFRs,
and was more than 8-fold selective for wild type EGFR. Compound 8 exhibited good microsomes
stabilities and pharmacokinetic properties and lower binding affinity to hERG ion channel than
AZD9291 and displayed strong antiproliferative activity against the H1975 non-small cell lung
cancer (NSCLC) cells bearing T790M/L858R and in vivo anticancer efficacy in a human NSCLC
(H1975) xenograft mouse model.
KEYWORDS: Non-small cell lung cancer, 5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinoline
derivatives, EGFR modulator, L858R/T790M double mutants
1. Introduction
The ErbB family of receptor tyrosine kinases plays a crucial regulatory role associated with
malignancies, making this protein family an anticancer target [1]. Epidermal growth factor
receptor (EGFR) belongs to the ErbB family and is a key mediator in cellular signaling related
to cell growth, proliferation, survival, and migration [2-4]. Overexpression or mutation of EGFR
is a common feature in many human solid malignancies, especially in non-small cell lung cancer
(NSCLC) [5-7]. Therefore, EGFR represents a valuable target for the design of anticancer
agents [8-11]. The first-generation EGFR inhibitors erlotinib and gefitinib (Fig. 1) have been
used in clinic for the treatment of EGFR-mutated NSCLC (such as L858R and delE746-A750)
[12, 13]. Unfortunately, the clinical efficacies of erlotinib and gefitinib were limited by the
acquired drug resistance, such as that induced by the mutation of the gatekeeper residue
2

ACCEPTED MANUSCRIPT
(T790M) which was detected in 50% of clinically acquired drug resistant patients [14, 15]. This
mutation decreases the binding of ATP-competitive inhibitors to the kinase and restores ATP
affinity to EGFR [16].
In order to overcome drug resistance induced by T790M, the second-generation irreversible
inhibitors had been developed including afatinib and dacomitinib (Fig. 1). These inhibitors
contained electrophilic Michael-acceptor moieties that could covalently modify the conserved
cysteine residue (Cys797) at the lip of the ATP binding cleft of EGFR [17-19]. However, their
clinical efficacy had been limited by associated skin rash and gastrointestinal toxicity because of
their poor kinase selectivity between EGFR T790M mutants and the wild-type (WT) EGFR [20].
The desire to improve the selectivity of EGFR T790M over the WT EGFR led to the third-
generation of irreversible EGFR inhibitors. Since the discovery of WZ-4002, a number of , -
unsaturated vinyl amide inhibitors entered clinical trials, such as CO-1686, PF-06459988 and
AZD9291 (Fig. 1) [21-24]. Among them, AZD9291 which had an amino pyrimidine scaffold,
showed 200-fold selectivity for T790M/L858R double mutants over WT EGFR [25] and was
approved for the treatment of NSCLC patients with EGFR T790M mutation by FDA in November
2015.
3

ACCEPTED MANUSCRIPT
N
N
N
O
NH
O
O
N
N
NH
N
N
N
O
N
N
N
H
O
Cl
PF-06459988
NH
AZD9291 (Osimertinib)
Fig. 1. Structures of different generation EGFR inhibitors.
AZD9291 had been reported to have a few common adverse events in clinical trials, such as
diarrhea, rash and decreased appetite. And the morbidity of diarrhea, rash and cardiotoxicity
increased with escalating doses of AZD9291 [26]. To overcome the drawbacks of AZD9291, in
this manuscript, we described the design, synthesis, in vitro and in vivo biological evaluation of
a series of novel selective T790M inhibitors.
4

ACCEPTED MANUSCRIPT
2. Results and discussion
2.1. Chemistry
The synthetic routes of target compounds 6-16 and 22-30 were outlined in Schemes 1 and 2,
respectively. For the preparation of compounds 6−12 (Scheme 1), commercially available 2- or
5-substituted pyrimidines 1 were firstly coupled with 1-methyl-1H-indole or 5,6-dihydro-4H-
pyrrolo[3,2,1-ij]quinolone fragments to give intermediates 2 (2a or 2b) in moderate to high
yields (45.0-90.0%) with the catalyst aluminum chloride. And then the 6-chloro groups of 2 were
substituted by 4-fluoro-2-methoxy-5-nitroaniline moiety to give the intermediates 3 with the
yields of 81.5-92.0%. The fluoro groups of 3 were substituted by the N,N,N-trimethylethane-1,2-
diamine to produce intermediates 4 with the yields of 60.0-78.0%. Subsequent reduction of the
nitro groups of 4 with hydrogen gave amines 5, which were not purified and directly used in the
next reaction. After acylation of amines 5 with acryloyl chloride and elimination of
hydrochloride with sodium hydroxide, the desired final molecules 6-12 were obtained with the
yields of 25.0-65.5%.
Scheme 1. Synthetic Route for Target Compounds 6-12a
5

ACCEPTED MANUSCRIPT
aReagents and conditions: (a) 1-methyl-1H-indole (1 equiv) or 5,6-dihydro-4H-pyrrolo [3,2,1-
ij]quinolone (1 equiv), AlCl3 (1 equiv), 1,2-dimethoxyethane (DME), 85 °C, 6 h, 45.0-90.0%; (b)
4-fluoro-2methoxy-5-nitroaniline (1.1 equiv), tosic acid monohydrate (1.1 equiv), 1,4-dioxane,
90 °C, 9 h, 81.5-92.0%; (c) N,N,N'-trimethylethane-1,2-diamine (1.1 equiv), N,N-
diisopropylethyl- amine (DIPEA) (1.5 equiv), N,N-dimethylacetamide (DMAC), 140 °C, 6 h,
60.0-78.0%; (d) 10% Pd/C (0.2 equiv), H2, MeOH, rt, overnight; (e) 3-chloropropanoyl chloride
(1 equiv), NaOH (4 equiv), THF, rt-65 °C, overnight, 25.0-65.5%.
The target compounds 13-16 and 22−30 were prepared from commercially available 2,4-
dichloro pyrimidine 17. Firstly 4-chloro of starting material 17 was substituted by 3-methyl-1H-
indole and 1H-indazole groups to give the key intermediates 18a and 18b with the yields of
86.4% and 80.0%, respectively. For the preparation of intermediate 18c, 4-chloro group of
starting material 17 was substituted by 5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolone with the
catalyst aluminum chloride in high yield (90.1%). Then the 2-chloro groups of compounds 18
were substituted by 4-fluoro-2-methoxy-5-nitroaniline to give the intermediates 19 (yields 84.0-
95.0%). The fluoro groups of 19 were substituted by different amines R to produce intermediates
6

ACCEPTED MANUSCRIPT
20 with moderate to high yields (60.5-80.0%). Subsequent reduction of the nitro groups of 20
with hydrogen gave amines 21, which were not purified and directly used in the next reaction.
After acylation of 21 with acryloyl chloride and elimination of hydrochloride with sodium
hydroxide, the desired compounds 13-16 and 22-30 were obtained with the yields of 24.9-70.0%.
Scheme 2. Synthetic Route for Target Compounds 13-16 and 22-30a
N
N
Ar=
N
N
18b
18a
18c
aReagents and conditions: (a) 3-methyl-1H-indole (1 equiv) or 1H-indazole (1 equiv), K2CO3 (2
equiv), MeCN, reflux, overnight, 80.0-86.4%; 5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolone (1
equiv), AlCl3 (1 equiv), DME, 80 °C, 6 h, 90.1%; (b) 4-fluoro-2-methoxy-5-nitroaniline (1.1
equiv), tosic acid monohydrate (1.1 equiv), 1,4-dioxane, 85 °C, 8 h, 84.0-95.0%; (c) different
substituted amines R (1.1 equiv), DIPEA (1.5 equiv), DMAC, 140 °C, 6 h, 60.5-80.0%; (d) 10%
7

ACCEPTED MANUSCRIPT
Pd/C (0.2 equiv), H2, MeOH, rt, overnight; (e) acryloyl chloride (1 equiv), DIPEA (2.0 equiv),
CH2Cl2, -10 °C, 0.5 h, 24.9-70.0%.
2.2. Design strategy of the new compounds
Without the crystal complex of AZD9291 binding to the T790M EGFR kinase domain, we
employed a published T790M structure (PDB code: 3IKA) to model the binding mode with
AZD9291 (Figs. 2 and 3). The docking mode indicated that AZD9291 bound to the outer edge
of the ATP-binding pocket. The pyrimidine core of the molecule formed two hydrogen bonds with
Met793 residue in the kinase hinge: one with the indole group adjacent to the gatekeeper
residue, and the other with the dimethylamine moiety positioned in the solvent channel, while
Cys797 covalently bonded to the acrylamide group [22]. Furthermore, several vander Waals
interactions existed between AZD9291 and the residues of protein, such as the pyrimidine ring
with Ala743, and the indole ring with Val726 and Phe723, respectively (Fig. 3) [27]. These
interactions were envisioned to be important for improving the activity and selectivity against
mutated EGFR kinase [28].
8

ACCEPTED MANUSCRIPT
Fig. 2. Docking pose of AZD9291 in the complex of EGFR T790M mutant.
Fig. 3. Interaction map of AZD9291 with the EGFR T790M mutant and the overview in the
binding site.
Based upon the binding mode of AZD9291 with T790M active domain, we modified the
substitution position of the pyrimidine ring and expanded the indole ring (Fig. 4) to increase the
binding interaction and selectivity of the inhibitors with T790M EGFR kinase, which may reduce
their affinity for the human ether-a-go-go related gene (hERG) channel (cardiotoxicity). And a
number of side chain amines (R) were also modified with the purpose to improve the potency and
physical properties of the inhibitors.
9

ACCEPTED MANUSCRIPT
Fig. 4. Design strategy of the new compounds based on AZD9291.
2.3. Biological evaluation
According to our design strategy (Fig. 4), a series of novel T790M inhibitors with 2- and 4-
amino pyrimidine scaffold were designed and synthesized (Table 1). The kinase inhibitory
activities of the target compounds were evaluated via a well-established FRET-based Z’-Lyte
assay [29] against different types of EGFR kinases (Table 1).
Compared with 2-amino pyrimidine standard AZD9291, most of 4-amino pyrimidine
analogues 12-16 showed greatly decreased activities against wild type (WT) EGFR, single
mutant L858R and double mutant T790M/L858R (TL), but compounds 10 and 11 displayed
strong activities against all of these three EGFRs (all IC50 values were less than 5 nM).
However, these two compounds showed less selectivity between WT and TL EGFR (the ratio of
WT/TL was 1.4 and 1.2, respectively). Screening of 2- and 4-amino substituted pyrimidine cores
indicated that the former could be selected as the template to develop. The biological results
demonstrated that ring expansion of indole moiety dramatically affected the activities and
selectivity for the 2-amino substituted pyrimidine analogues. Introduction of 5,6-dihydro-4H-
10

ACCEPTED MANUSCRIPT
pyrrolo[3,2,1-ij]quinolone group at the indole position led to the same activity and selectivity as
the standard AZD9291. For example, compound 8 inhibited the wild type EGFR, single mutant
L858R and double mutants T790M/L858R (TL) with the IC50 values of 21.6, 6.0 and 2.6 nM,
respectively and the corresponding IC50 values of AZD9291 were 19.7, 6.0 and 2.1 nM,
respectively, which suggested that the spatial position of indole group in AZD9291 close to the
gatekeeper residue would be large enough for the ternary ring structure group such as 5,6-
dihydro-4H-pyrrolo[3,2,1-ij]quinoline moiety. However, the substitution of 1-methyl-1H-indole
(AZD9291) with 3-methyl-1H-indole (compound 6) dramatically decreased the activity and
selectivity (Table 1). Furthermore, the introduction of 1H-indazole (compound 7) moiety led to
complete loss of activities against all of the three EGFR kinases. For the 4-amino compound,
modification of indole position did not change the activity and selectivity in a large extent. For
example, both 1-methyl-1H-indole (compound 10) and 5,6-dihydro-4H-pyrrolo[3,2,1-
ij]quinolone (compound 11) moieties led to the same activities against WT, L858R and TL
kinases (all IC50 values were less than 5 nM) and the similar selectivity. The same results could
be concluded for 1-methyl-1H-indole substituted compound 12 and 5,6-dihydro-4H-
pyrrolo[3,2,1-ij]- quinolone substituted compound 16. Different substitutes at R1 and R2
positions affected the activities and selectivity of both 2- and 4-amino pyrimidine analogues. For
2-amino substituted compounds, the substitution of hydrogen at R2 position (AZD9291) with
methoxy group (compound 9) decreased the TL activity (IC50: 13.0 nM vs 2.0 nM) with 6.5 folds,
but the selectivity was increased with 5.3 folds (WT/TL: 52.6 vs 9.9). For 4-amino substituted
compounds, substitution of the hydrogen at R1 position (compound 10, IC50: 1.6 nM) with fluoro
(compound 13, IC50: 94.8 nM) and methoxy groups (compound 14, IC50: 212.5 nM) dramatically
decreased the TL activities. Different groups at R2 position also had great influence on the
activity and selectivity. Methyl and methoxy groups at R2 position decreased the TL activities
11

ACCEPTED MANUSCRIPT
and increased the selectivity in a certain extent. Compared with compound 10, methoxy
substituted compound 16 was 6-fold less potent in activity, but its selectivity was increased by
more than 40 folds, while the methyl substituted compound 15 was 37-fold less potent in activity,
but the selectivity was increased in only 2 folds. Based on the activities and selectivity,
compound 8 was selected as a lead to optimize.
Table 1. Enzymatic Inhibitory Activities of Compounds 6-16 and AZD9291a
IC50 (nM)a
Selectivity
(WT/TL)
Compd.
R1
R2
Ar
X
Y
WTb
L858R TLc
N
-
H
C
N
714.5 185.6 287.4
2.5
6
-
-
H
H
C
N
NAd
21.6
>600
6.0
>600
2.6
-
7
8
C
N
8.3
12

ACCEPTED MANUSCRIPT
-
OMe
C
N
685.0 154.6 13.0
52.6
1.4
9
H
H
H
N
C
2.3
3.1
2.2
1.2
1.6
2.6
10
11
H
N
N
C
C
1.2
H
OMe
543.0 181.1 22.0
24.7
12
13
14
15
16
F
H
N
C
C
C
C
32.1
9.9
94.8
212.5
60.6
0.34
1.0
OMe
H
N
N
N
222.5 55.5
H
CH3
OMe
192.0 46.5
3.2
H
636.0 123.4 10.9
58.3
13

ACCEPTED MANUSCRIPT
-
H
C
N
19.7
6.1
2.0
9.9
AZD9291
aEnzymatic inhibitory-activity assays were performed using the FRET-based Z
′-Lyte assay. All
c
experiments were repeated at least three times. bWT, wild type EGFR. TL, T790M/L858R
double mutants. dNA, not active.
After the optimization of ring expansion and pyrimidine moiety, the modification of amine R
groups was carried out. Different amine moieties were screened and the results were showed in
Table 2. Firstly, the terminal N atom (compound 8) was changed to its isostere O atom
(compound 22) to investigate the activity and selectivity by application of the bioisosterism
method. The results indicated that the activities of compound 22 against three kinds of kinases
were dramatically decreased although the selectivity was 3-fold higher than that of compound 8.
Then the effect of the amidation of terminal tertiary N was evaluated. Amides 23 and 24 were
inactive in the inhibition of WT kinase and only showed micromole level against L858R and TL
kinases, which may be due to the loss of hydrogen donor after amidation of terminal N atom.
Then the effect of different substituents of N atom linked to the core was investigated. After
cyclization with four membered ring (compound 25), the activities against L858R and TL kinases
nearly decreased 2 folds compared with compound 8. When the distance of two nitrogen atoms
of R group was increased, the activities against three kinases were all decreased. For example,
compounds 26 and 27 lost the potencies against all forms of the EGFR kinases compared with
compound 8. Compound 28, which was cyclized with five membered ring, remained the potencies
against EGFR kinases, but the selectivity slightly reduced. When the amine moiety R was
replaced with piperazine moiety (compound 29), all the inhibitory activities reduced significantly
and compound 30 was inactive in the inhibition of all forms of the EGFR kinases after amidation
14

ACCEPTED MANUSCRIPT
of piperazine ring. The optimized results illustrated that the R position might be N,N,N -
trimethylethane-1,2-diamine group to maintain the stronger inhibition against EGFR kinases
and the better selectivity.
Table 2. Enzymatic Inhibitory Activities of 22-30 and AZD9291a
IC50 (nM)a
Selectivity
Compd.
R
WTb
21.6
L858R
5.3
TLc
2.9
(TL/WT)
7.4
N
8
N
N
OH
838.5
192.3
498.0
34.8
24.1
>3.6
22
23
>1000
278.2
>1000
63.9
385.5
13.6
188.2
4.9
>5.3
13.0
24
25
N
N
15

ACCEPTED MANUSCRIPT
202.1
251.1
11.6
14.5
6.7
30.2
21.3
26
27
11.8
8.8
4.0
1.9
4.6
28
29
877.4
64.3
55.5
15.8
NAd
13.8
427.7
4.2
1230.0
1.9
-
30
7.2
AZD9291
aEnzymatic inhibitory-activity assays were performed using the FRET-based Z
′-Lyte assay. All
b
c
experiments were repeated at least three times. WT, wild type EGFR. TL, T790M/L858R
double mutants. dNA, not active.
The antiproliferative effects of the new potential inhibitors with the IC50 values of enzymatic
inhibition less than 10 nM were further investigated to evaluate their potential antitumor
activities in cancer cell lines (Table 3). The results displayed that compounds 10, 11 and 28 were
active against A431, HCC827 and H1975 NSCLC cells harboring wild-type EGFR, EGFR
delE746-A750 and EGFR L858R/T790M, respectively. But these compounds showed less
selectivity between wild-type and mutant cell lines. Compounds 8, 9, 12, 16, 25 and 26 were
nearly inactive in the inhibition of wild-type A431 cell and much active against mutant cell lines
16

ACCEPTED MANUSCRIPT
and showed high selectivity. The SAR analysis of these compounds indicated that after
cyclization with four membered ring (compounds 25 and 26) of amine R groups, the selectivity
against HCC827 and H1975 cancer cells nearly remained compared with compound 8, but the
activities decreased slightly. Interestingly, introduction of a methoxy group in the 6-position of
the 2-aminopyrimidine ring (compound 9) and the 2-position of the 4-aminopyrimidine rings
(compounds 12 and 16) led to a significant loss in potencies against the cancer cells. So based
on the selectivity and activity data against the mutant cancer cell lines, compound 8 were
selected to be further evaluated the drug availability.
Table 3. Cell Viabilities of Representative Compoundsa
NSCLC cell lines IC50 (nM)
Selectivity
(TL/WT)
Compd. A431 (WTb) HCC827 (del E746-A750)
H1975 (TLc)
102.6
319.9
33.8
869.8
109.2
1166.0
16.2
8.5
8
>10000
128.4
>31.3
3.8
9
10
11
12
16
25
26
28
121.7
24.3
37.8
3.2
>10000
>10000
1123.0
1468.0
514.2
1207.0
925.0
98.2
563.0
210.2
143.3
166.2
99.9
>17.8
>47.6
7.8
289.7
71.7
8.8
5.1
17

ACCEPTED MANUSCRIPT
615.6
61.6
67.0
9.2
AZD9291
a
b
c
All experiments were repeated at least three times. WT, wild type EGFR. TL, T790M/L858R
double mutant.
The metabolic stability of the promising compound 8 was determined with various species of
liver microsomes, such as human, mouse, rat, dog and monkey liver microsomes (Data were
shown in Table 4). And the marketed AZD9291 was selected as the standard. The half-life (T1/2)
and intrinsic clearance (CLint) parameters were used to evaluate their metabolic stabilities. It
could give a good indication of the in vivo hepatic clearance when the overall clearance
mechanism was metabolic and when oxidative metabolism dominates (i.e., CLmetabolic
≫ CLrenal +
CLbiliary + CLother) [30, 31]. Table 4 revealed that compound 8 displayed good metabolic
stabilities. The half-life of 8 in human liver microsome were more than 180 minutes, which
showed that it almost was not metabolized by human liver microsome and the control AZD9291
showed similar result. For other species, mouse liver microsome metabolized compound 8 in
11.9 minutes, which was more rapidly than other microsomes. Furthermore, compound 8 was
metabolized in shorter time than AZD9291 in mouse, rat and monkey liver microsomes except
dog. So this compound could be metabolized more rapidly than AZD9291 after binding with
target and showed less toxicity than AZD9291, which was displayed during our in vivo
experiments.
Table 4. Stabilities of Compounds 8 and AZD9291 in Liver Microsomes of Various Species
8
AZD9291
Liver microsomes
T1/2
CLint
T1/2
CLint
18

ACCEPTED MANUSCRIPT
(min) (mL/min/kg) (min)
(mL/min/kg)
Human
Mouse
Rat
>180
11.9
46.2
31.5
28.9
NDa
456.8
54.0
63.4
64.8
NDa
23.9
62.4
21.0
63.0
NDa
228.4
40.0
95.0
29.7
Dog
Monkey
aND: not detected.
Furthermore, data of the metabolic stabilities in human and rat hepatocytes for compound 8
and AZD9291 showed the similar results as that of microsome stabilities (Data were shown in
Table 5). Compound 8 was metabolized more rapidly than AZD9291 in both human and rat
hepatocytes. And the metabolic degradation of compound 8 in human hepatocytes was slower
than rat one, which was consistent with the results of liver microsomes stabilities.
Table 5. Stabilities of Compounds 8 and AZD9291 in Hepatocyte
8
AZD9291
CLint
Hepatocyte
T1/2
CLint
(ꢀL/min/106 cell)
T1/2
(min)
(min)
(ꢀL/min/106 cell)
19

ACCEPTED MANUSCRIPT
Human
Rat
138.6
33.0
5.0
>180
57.8
NDa
12.0
21.0
aND: not detected.
With these encouraging in vitro data, candidate 8 was further investigated by profiling po and
iv pharmacokinetics in male Sprague-Dawley (SD) rats. The results were illustrated in Table 6.
After oral administration in male SD rats, compound 8 showed longer half life time than
AZD9291 (T1/2: 4.1 vs 3.8 h), partially because of its lower systemic clearance (CL: 5169.7 vs
6600.0 (mL/h)/kg). Furthermore, good exposure was observed for compound 8 (AUC0-inf: 1021.0
h·ng/mL). It also showed a good oral bioavailability (52.4%), approximately equal to AZD9291
in our test (66.5%).
Table 6. In Vivo Pharmacokinetic Parameters for Compounds 8 and AZD9291a
Parameters
Dose (mg/kg)
T1/2(h)
8
AZD9291
10.0 (po)
4.1
1.0 (iv)
4.5
10.0 (po)
1.0 (iv)
4.5
3.8
Tmax(h)
3.3
-
2.7
-
Cmax(ng/mL)
AUC0-t(h·ng/mL)
AUC0-inf(h·ng/mL)
CL((mL/h)/kg)
MRT(h)
111.0
994.7
1021.0
-
-
111.0
937.3
954.3
-
-
189.7
194.0
5169.7
4.7
141.0
152.7
6600.0
4.8
5.7
5.7
20

ACCEPTED MANUSCRIPT
F(%)
52.4
-
66.5
-
aPharmacokinetic parameters following administration in male Sprague−Dawley rats.
Because of its potent activity in inhibiting EGFR T790M/L858R kinases and NSCLC cell
growth and good pharmacokinetic properties, compound 8 was further evaluated for in vivo
antitumor efficacy using an EGFR L858R/T790M-driven human NSCLC xenograft mouse model
of H1975 (Table 7 and Fig.5). SCID mice bearing established H1975 tumor xenografts were
dosed orally with compound 8 at 5, 10 and 20 mg/kg daily over an 18-day period. The results
showed that the tumor volume inhibition was dose-dependent (Fig.5). Dosing at 5 mg/kg/day, the
tumor volume was reduced in a certain extent compared with the control PBS and treatment with
10 mg/kg/day of compound 8 nearly inhibited tumor growth. When the dose was increased to 20
mg/kg/day, compound 8 displayed significant in vivo antitumor efficacy and induced tumor
stasis. The tumor growth values (T/C%) were 56.4%, 25.7% and 12.1% at three different doses,
respectively. And the tumor growth inhibition values (TGI%) were 46.8%, 83.5% and 96.3%,
respectively (Table 7). In addition, compound 8 was well-tolerated and no mortality or
significant loss of body weight was observed during treatment at all doses.
21

ACCEPTED MANUSCRIPT
Fig. 5. In vivo activity of 8 in the human NSCLC (H1975) xenograft nude mouse model.
Table 7. In Vivo Antitumor Effect of 8 in Human H1975 Xenograft Nude Mouse Model
TWe Mean
TVa Mean
(mm3)
RTV
SFd
Group
RTVb Mean
T/Cc (%)
TGIf (%)
（
Day18, g）
Control (PBSg)
8 (5 mg/kg)
948.05
527.78
239.33
113.52
6.77
3.82
1.74
0.82
1.88
1
56.4%
25.7%
12.1%
**
**
**
46.8%
83.5%
96.3%
8 (10 mg/kg)
8 (20 mg/kg)
0.31
0.07
aTV: tumor volume; bRTV: relative tumor volume, the individual RTV was calculated as follows:
RTV = Vt/V0, where Vt is the volume on each day of measurement, and V0 is the volume on the
c
d
day of initial treatment; T/C(%)=RTVtreatment/RTVcontrol ×100%; SF: statistical significance
e
f
(*p<0.05, **p<0.01); TW: tumor weight, TGI: tumor growth inhibition, TGI(%)= (TWcontrol
-
TWtreatment ) /TWcontrol
×
100%; gPBS: phosphate buffer saline.
The human ether-a-go-go related gene (hERG) ion channel inhibition, which may result in a
concomitant risk of sudden death, must be avoided during drug development [32]. The inhibition
of the hERG channel by compounds 8 and AZD9291 was evaluated in HEK293 cells using an in
vitro Ion Works Quattro study [33]. Fig. 6 highlighted the observation that compound 8 showed
14.8-fold lower affinities for the hERG ion channel than AZD9291. This result suggested that
compound 8 may have the less probability to induce QT interval prolongation than AZD9291 for
the patients.
22

ACCEPTED MANUSCRIPT
Fig. 6. hERG current inhibition of compounds 8 and AZD9291.
3. Conclusion
A novel series of selective covalent EGFR T790M inhibitors were designed and synthesized
based on the modeling binding mode of marketed drug AZD9291 with EGFR T790M mutant.
The structure-activity relationship (SAR) was discussed in detail and the results demonstrated
that substitution with 5,6-dihydro-4H-pyrrolo-[3,2,1-ij] quinolone moiety at the para-position of
2-aminopyrimidine was considered as a novel strategy for maintaining EGFR T790M inhibitory
activity and selectivity. Among the most promising inhibitors, compound 8 potently inhibited the
enzymatic function of EGFR T790M/L858R with an IC50 value of 2.6 nM and NSCLC H1975 cell
with an IC50 value of 102.6 nM. In vitro and in vivo metabolic stability studies also suggested
that compound 8 also exhibited good pharmacokinetic properties with oral bioavailability of
52.4%. Moreover, an in vivo antitumor efficacy study demonstrated that compound 8
significantly inhibited tumor growth in an EGFR T790M/L858R-driven human NSCLC xenograft
mouse model by po dosing at 20 mg/kg/day. The lower binding affinities of compound 8 to the
hERG ion channel than AZD9291 suggested that this candidate may be less cardiotoxic than
23

ACCEPTED MANUSCRIPT
AZD9291. Therefore, the in vitro and in vivo data indicated that compound 8 had very desirable
profiles to be selected as a clinical candidate.
4. Experimental section
4.1. Chemistry
General methods for chemistry. Unless otherwise specified, reagents were purchased
from commercial suppliers and used without further purification. Absolutely anhydrous solvents
(CH2Cl2, THF, DMF, etc.) were purchased from Aldrich packaged under nitrogen in Sure/Seal
bottles. Yields refer to chromatography unless otherwise stated. All reactions were monitored by
thin-layer chromatography carried out on silica gel aluminum sheets (60F-254) and spots were
visualized with UV light or iodine. All reactions involving air or moisture-sensitive reagents
1
were performed under an argon atmosphere. H NMR and 13C NMR spectra were recorded at
room temperature on BRUKER Avance 400 spectrometers. Chemical shifts were reported in
parts per million (ppm, units), and tetramethylsilane (TMS) was used as an internal reference.
Coupling constants (J) were expressed in Hertz. Mass spectra were obtained using Agilent LC-
MS (1956B) instruments in electrospray positive and negative ionization modes. High-resolution
mass spectra (HRMS) were recorded on a ZAB-HS instrument using an electrospray source
(ESI). All the final products had purity of 95%. The purity of the final products was determined
by UPLC (Thermo) on a Syncronis C18 column (50 mm
×2.1 mm, 1.7 µm) with 0.04% TFA/ACN
(gradient eluted program: 0-7 min 90/10 v/v; 7.1-10 min 0/100 v/v; 10.1-13 min 90/10 v/v) at 0.4
mL/min flow rate and 254 nm detector wavelength.
4.1.1 1-(2-chloropyrimidin-4-yl)-3-methyl-1H-indole (18a)
General procedure for the synthesis of compounds 18a and 18b. To a solution of 2,4-
dichloropyrimidine (12.49 g, 83.63 mmol) in acetonitrile (160 mL) was added 3-methyl-1H-
indole (10.00 g, 76.23 mmol) and K2CO3 (21.07 g, 152.47 mmol) and the mixture stirred at reflux
24

ACCEPTED MANUSCRIPT
overnight. The reaction was cooled to room temperature, water (100 mL) was added, the mixture
was extracted with dichloromethane (DCM) (3 × 100 mL), dried with Na2SO4, filtered and
concentrated to give a tan solid (16.05 g, 86.4 % yield): 1H NMR(400 MHz, DMSO-d6) 8.65 (d,
J = 5.8 Hz, 1H), 8.56 (d, J = 8.3 Hz, 1H), 7.95 (s, 1H), 7.79 (d, J = 5.9 Hz, 1H), 7.59 (d, J = 7.6
Hz, 1H), 7.38 (t, J = 7.7 Hz, 1H), 7.29 (t, J = 7.4 Hz, 1H), 2.29 (s, 3H). MS (ESI) m/z 244.2
[M+H]+.
4.1.2 N-(4-fluoro-2-methoxy-5-nitrophenyl)-4-(3-methyl-1H-indol-1-yl)pyrimidin-2-amine (19)
1-(2-chloropyrimidin-4-yl)-3-methyl-1H-indole (18a) (20.00 g, 82.07 mmol), 4-fluoro-2-
methoxy-5-nitroaniline (16.80 g, 90.28 mmol) and 4-methyl-benzenesulfonic acid monohydrate
(17.17 g, 90.28 mmol) were heated at 85 °C in 1,4-dioxane (300 mL) for 8 h. The reaction was
cooled to room temperature and diluted with water (200 mL) and 40% aq NaOH added until pH
= 9. The solid was collected by filtration and washed with EtOH (50 mL) to afford the yellow
solid (30.00 g, 92.9 % yield): 1H NMR (400 MHz, DMSO-d6) 8.86 (s, 1H), 8.77 (d, J = 8.2 Hz,
1H), 8.55-8.44 (m, 2H), 7.95 (s, 1H), 7.57 (d, J = 7.2 Hz, 1H), 7.41 (d, J = 13.3 Hz, 1H), 7.34-
7.20 (m, 3H), 3.99 (s, 3H), 2.30 (s, 3H). MS (ESI) m/z 394.2 [M+H]+.
4.1.3
N′-(2-(dimethylamino)ethyl)-5-methoxy-N′-methyl-N-(4-(3-methyl-1H-indol-1-
yl)pyrimidin -2-yl)-2-nitrobenzene-1,4-diamine (20)
N,N,N'-trimethylethane-1,2-diamine (2.86 g, 27.96 mmol) was added to a suspension of N-(4-
fluoro-2-methoxy-5-nitrophenyl)-4-(3-methyl-1H-indol-1-yl)pyrimidin-2-amine (19) (10.00 g,
25.42 mmol), DIPEA (4.91 g, 38.13 mmol) and N,N-dimethyl acetamide (DMAC) (30 mL) in
sealed tube. The mixture was heated at 140 °C for 6 h. After being cooled to room temperature,
the reaction mixture was added to ice water (60 mL). The precipitate was filtered, and the
filtered cake was rinsed with additional cooled MeOH (20 mL) and dried in a vacuum oven to
give a red solid (9.60 g, 79.4% yield), which was used without further purification.
25

ACCEPTED MANUSCRIPT
4.1.4
N1-(2-(dimethylamino)ethyl)-5-methoxy-N1-methyl-N4-(4-(3-methyl-1H-indol-1-yl)
pyrimidin-2-yl) benzene-1,2,4-triamine (21)
N -(2-(dimethylamino)ethyl)-5-methoxy-N -methyl-N-(4-(3-methyl-1H-indol-1-yl) pyrimidin-2-
yl)-2-nitrobenzene-1,4-diamine (20) (5.00 g, 10.52 mmol) and Pd/C (10% by weight) (230 mg,
2.10 mmol) were suspended in a mixture of MeOH (100 mL) and stirred under a hydrogen
atmosphere for 20 h. The reaction mixture was filtered and the filtrate was concentrated in
vacuo. The resulting residue was purified by silica gel chromatography (DCM: MeOH:
NH3·H2O=20:1:0.1) to afford the title compound (3.50 g, 74.7% yield) : 1H NMR (400 MHz,
DMSO-d6) 8.54 (s, 1H), 8.39 (s, 1H), 8.34 (d, J = 5.7 Hz, 1H), 7.84 (s, 1H), 7.55-7.49 (m, 1H),
7.24-7.11 (m, 3H), 6.99 (d, J = 5.7 Hz, 1H), 6.79 (s, 1H), 3.70 (s, 3H), 2.93 (t, J = 6.6 Hz, 2H),
2.67 (s, 3H), 2.39 (t, J = 6.6 Hz, 2H), 2.28 (s, 3H), 2.17 (s, 6H). MS (ESI) m/z 446.2 [M+H]+.
4.1.5 N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((4-(3-methyl-1H-indol-1-
yl)pyrimidin-2-yl)amino)phenyl)acrylamide (6)
Acryloyl chloride (710 mg, 7.85 mmol) in DCM (5 mL) was added dropwise to a stirred
solution of N1-(2-(dimethylamino)ethyl)-5-methoxy-N1-methyl-N4-(4-(3-methyl-1H-indol-1-
yl)pyrimidi n-2-yl)benzene-1,2,4-triamine (21) (3.50 g, 7.85 mmol) and DIPEA (2.03 g, 15.70
mmol) in DCM (30 mL) at -10 °C. The resulting solution was maintained at -10 °C for 1 h and
then washed with brine, dried over Na2SO4, and concentrated. The resulting residue was purified
by silica gel chromatography (DCM: MeOH: NH3·H2O=40:1:0.1) to afford N-(2-((2-
(dimethylamino)ethyl)
(methyl)amino)-4-methoxy-5-((4-(3-methyl-1H-indol-1-yl)pyrimidin-2-
1
yl)amino)phenyl)acryl-amide (6) (1.50 g, 38.2% yield) as a light yellow solid. H NMR (400
MHz, DMSO-d6) 10.14 (s, 1H), 8.63 (s, 1H), 8.54 (s, 1H), 8.37 (s, 1H), 8.35 (s, 1H), 7.99 (s,
1H), 7.53 (d, J = 7.5 Hz, 1H), 7.20-7.07 (m, 2H), 7.06 (d, J = 5.3 Hz, 2H), 6.41 (dd, J = 16.9,
26

ACCEPTED MANUSCRIPT
10.1 Hz, 1H), 6.22 (dd, J = 16.9, 2.0 Hz, 1H), 5.74 (dd, J = 10.1, 1.9 Hz, 1H), 3.79 (s, 3H), 2.91
13
(t, J = 5.6 Hz, 2H), 2.75 (s, 3H), 2.34 (t, J = 5.6 Hz, 2H), 2.29 (s, 3H), 2.22 (s, 6H). C NMR
(100 MHz, DMSO-d6) 162.39, 160.70, 159.25, 158.05, 148.67, 139.74, 134.79, 132.15, 131.18,
127.35, 126.02, 124.10, 123.28, 122.90, 121.54, 118.66, 117.31, 115.95, 115.67, 105.59, 98.79,
56.86, 55.74, 45.19, 42.22, 9.28. MS (ESI) m/z 500.3 [M+H]+. HRMS calcd for C28H34N7O2
[M+H]+ 500.2769, found 500.2761.
4.1.6 1-(2-chloropyrimidin-4-yl)-5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolone (18c)
A suspension of 2,4-dichloropyrimidine (11.37 g, 76.33 mmol) and aluminum chloride (10.18
g, 76.33 mmol) in DME (100 mL) was stirred at ambient temperature for 20 min. To this was
added 2,3-dihydro-1H-pyrrolo[3,2,1-ij]quinoline (10.00 g, 63.61 mmol) and the mixture was
heated to 80 °C for 6 h. The cooled reaction mixture was added to stirring water (100 mL) and
the mixture was stirred for 2 h, filtered and the solid was washed with EtOH (40 mL). The crude
product was dried in a vacuum oven to give a red solid (15.46 g, 90.1% yield): 1H NMR (400
MHz, DMSO-d6) 8.50 (d, J = 5.5 Hz, 1H), 8.45 (s, 1H), 8.09 (d, J = 7.9 Hz, 1H), 7.80 (d, J =
5.5 Hz, 1H), 7.21-7.11 (m, 1H), 7.01 (d, J = 7.1 Hz, 1H), 4.33-4.18 (m, 2H), 2.96 (t, J = 5.9 Hz,
2H), 2.20-2.11 (m, 2H). MS (ESI) m/z 270.2 [M+H]+.
4.1.7 3-(6-chloropyrimidin-4-yl)-1-methyl-1H-indole (2a)
General procedure for the synthesis of 2a and 2b. A suspension of 4,6-dichloro-pyrimidine
(5.70 g, 38.16 mmol) and aluminum chloride (5.10 g, 38.16 mmol) in DME (60 mL) was stirred
at ambient temperature for 10 min. To this was added 1-methyl-1H-indole (5.00 g, 38.16 mmol)
and the mixture was stirred at reflux overnight. The cooled reaction mixture was added to
stirring water (100 mL) and the mixture was stirred for 1 h, filtered and the solid was washed
with EtOH (20 mL). The crude product was dried in a vacuum oven to give a red solid (4.30 g,
27

ACCEPTED MANUSCRIPT
46.2% yield): 1H NMR (400 MHz, DMSO-d6) 8.92 (s, 1H), 8.58-8.44 (m, 2H), 7.98 (s, 1H), 7.56
(d, J = 8.0 Hz, 1H), 7.33-7.23 (m, 2H), 3.88 (s, 3H). MS (ESI) m/z 244.2 [M+H]+.
4.2. Biological assay
4.2.1 Kinase inhibition assay.
IC50 determinations for EGFR and its mutants (Invitrogen) were performed with the HTRF
(Homogenous Time-Resolved Fluorescence) KinEASE-TK assay from Cisbio according to the
manufacturer’s instructions. A typical enzyme reaction contained 0.04 ng/ L wild-type EGFR or
0.02 ng/ L EGFR (T790M/L858R), 0.5 M TK-substrate-biotin, 10 M ATP, 1 mM DTT, 1 mM
MnCl2 and 5 mM MgCl2. Compounds were screened at serial diluted concentration in the
presence of 1% DMSO with a 15 min pre-incubation of kinase and compounds. All reactions
were started by the addition of ATP and TK-substrate-biotin, incubated at room temperature for
60 min and quenched with the stop buffer containing 62.5 nM Strep-XL665 and TK Ab-Cryptate.
The plates were incubated for 1 h before being read on ClARIOstar Microplate Reader (BMG
LABTECH) using standard HTRF settings. And IC50 values were determined using the GraphPad
Prism 5.0 software. Each reaction was performed in duplicate, and at least three independent
determinations of each IC50 were made.
4.2.2 Cellular phosphorylation assay.
A431 (epidermoid carcinoma, EGFR wild type), HCC827 (NSCLC, EGFR delE746-A750)
and NCI-H1975 (NSCLC, EGFR L858R/T790M) cells were obtained from the Cell Bank of the
Chinese Academy of Sciences (Shanghai, China). A431 cell was maintained in EMEM medium
supplemented with 10% FBS (Gibco). HCC827 and NCI-H1975 cells were separately
maintained in RPMI-1640 medium supplemented with 10% FBS (Gibco). Assays to measure
cellular phosphorylation of endogenous p-EGFR in cell lysates were carried out according to
the protocol described in the Pan phospho-EGFR Cellular Assay Kit (Cisbio, no. 64HR1PEG).
28