Design, synthesis and pharmacological evaluation of N4,N6-disubstituted pyrimidine-4,6-diamine derivatives as potent EGFR inhibitors in non-small cell lung cancer

Article abstract of DOI:10.1016/j.ejmech.2018.08.031

[Figure presented]A novel series of 4, 6-disubstituted pyrimidines derivatives were designed, synthesized, and evaluated as epidermal growth factor receptor (EGFR) inhibitors for non-small cell lung cancer(NSCLC). 4, 6-disubstituted pyrimidines as core structure was utilized to substitute the lead structure AZD3759 of the quinazoline basic skeleton via an approach involving scaffold hopping. It was found that compound Yfq07 exhibited the best inhibitory effect compared with AZD3759 in vitro and in vivo: Yfq07 exhibited a competitive ATP inhibitory effect, multiple target effects, and further featured a stronger activity against H3255, A431, HCC827, PC-9 and H1975 compared to AZD3759. Moreover, a stronger pro-apoptotic effect, inhibition of cell G2/M phase on A431, H3255, HCC827 and H1975 could also be observed. In this study, the ultimate goal was changing the core structure to improve other epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) properties while retaining the overall potency. Yfq07 was further explored as an effective 4, 6-pyrimidine anticancer agent for the treatment of human NSCLC.

Full text of DOI:10.1016/j.ejmech.2018.08.031

Accepted Manuscript
Design, synthesis and pharmacological evaluation of N4,N6-disubstituted
pyrimidine-4,6-diamine derivatives as potent EGFR inhibitors in non-small cell lung
cancer
Yuan Zhang, Handeng Lv, Lu Luo, Yong Xu, Yaqian Pan, Yuewu Wang, Han Lin,
Jianhua Xiong, Ping Guo, Jinsan Zhang, Xiaokun Li, Faqing Ye
PII:
S0223-5234(18)30699-8
10.1016/j.ejmech.2018.08.031
EJMECH 10642
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 May 2018
Revised Date: 11 August 2018
Accepted Date: 11 August 2018
Please cite this article as: Y. Zhang, H. Lv, L. Luo, Y. Xu, Y. Pan, Y. Wang, H. Lin, J. Xiong, P. Guo,
J. Zhang, X. Li, F. Ye, Design, synthesis and pharmacological evaluation of N4,N6-disubstituted
pyrimidine-4,6-diamine derivatives as potent EGFR inhibitors in non-small cell lung cancer, European
Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.08.031.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, Synthesis and pharmacological evaluation of N4,N6-disubstituted
Pyrimidine-4,6-Diamine Derivatives as potent EGFR Inhibitors in
Non-Small Cell Lung Cancer
a,#
a,#
a
a
a
Yuan Zhang , Handeng Lv , Lu Luo , Yong Xu , Yaqian Pan , Yuewu
a
c
b
a
a,
Wang , Han Lin , Jianhua Xiong , Ping Guo , Jinsan Zhang *,
a,
a,
Xiaokun Li *, Faqing Ye *
a
School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, 325035,
China.
b
Wenzhou Peoples Hospital, Wenzhou, Zhejiang, 325000, China
c Department of pharmacy. The first affiliated Hospital of Wenzhou Medical University. Wenzhou,
China
#
These authors contributed equally to this work.
*
Corresponding author:
Faqing Ye, Professor
School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, 325035,
China
Tel: (+86)-577-86689793; Fax: (+86)-577-86689793
E-Mail: yfq664340@163.com

ACCEPTED MANUSCRIPT
ABSTRACT:
A novel series of 4, 6-disubstituted pyrimidines derivatives were designed, synthesized, and
evaluated as epidermal growth factor receptor (EGFR) inhibitors for non-small cell lung
cancer(NSCLC). 4, 6-disubstituted pyrimidines as core structure was utilized to substitute the lead
structure AZD3759 of the quinazoline basic skeleton via an approach involving scaffold hopping.
It was found that compound Yfq07 exhibited the best inhibitory effect compared with AZD3759
in vitro and in vivo: Yfq07 exhibited a competitive ATP inhibitory effect, multiple target effects,
and further featured a stronger activity against H3255, A431, HCC827, PC-9 and H1975
compared to AZD3759. Moreover, a stronger pro-apoptotic effect, inhibition of cell G2 / M phase
on A431, H3255, HCC827 and H1975 could also be observed. In this study, the ultimate goal was
changing the core structure to improve other epidermal growth factor receptor tyrosine kinase
inhibitors (EGFR-TKIs) properties while retaining the overall potency. Yfq07 was further
explored as an effective 4, 6-pyrimidine anticancer agent for the treatment of human NSCLC.
Keywords: EGFR-TKIs; Scaffold hopping; NSCLC
1
. INTRODUCTION
Lung cancer represents a cancer type exhibiting the highest morbidity rate and non–small cell
lung cancer (NSCLC) types, accounting for approximately 90% of all lung cancer cases [1].
NSCLC targeted therapy has now become critically important, and the epidermal growth factor
receptor (EGFR) tyrosine kinase is a particular popular target [2-5].
Presently, small molecule inhibitors, used for EGFR triggered NSCLC, are mainly based on
two types of core structures (Figure 1). One type is based on 2,4-pyrimidines, such as Rociletinib
[6] and Osimertinib [7]. Another type stems from quinazolines compounds, including Gefitinib [8],
Erlotinib [9] and Afatinib [10]. In recent years, a new potent quinazoline EGFR inhibitor,
AZD3759 was discovered and evaluated by Xiaolin Zhang et al. [11]. AZD3759 has demonstrated
an effective inhibitory activity on NSCLC. Moreover, this investigational compound is currently
evaluated in a phase 1 clinical trial in patients suffering from EGFR triggered NSCLC [12-14].

ACCEPTED MANUSCRIPT
Figure 1. Chemical structures of representative examples of the two EGFR inhibitor types
Using molecular docking simulation to mimic the binding mode of inhibitors AZD3759 and
EGFR L858R protein, we found that N-1 of the quinazoline forms an H-bond with back bond NH
of Met-798. H-2 of the quinazoline group was shown to be acidic and could form an oxygen
bridge with water molecules to form H-bonding interactions with Thr-766. However,
N6-methylpiperazine unit was found to be completely exposed to solvent molecules and did not
form interactions with the protein. Thus, besides pyrimidines [15], triazines, purines, and
pyrazines may be selected to replace the core structure of the quinazoline. Interestingly, one report
published by Qiong Zhang et al. [16] showed that 4,6-disubstituted pyrimidines [17] derivatives
exhibited a more potent and selective inhibitory effect in both enzymatic and cellular EGFR
studies compared to other kinase-directed heterocycles including 2,4-pyrimidines [18], triazines,
purines, quinazolines [19], pyrazines [20], etc. In this study, we have used scaffold hopping
approach through changing the core structure and in an effort to improve other structure properties
while retaining the overall potency [21-25]. The ultimate goal was to develop novel compounds
with potentially higher efficacy.
In summary, we selected 4, 6-disubstituted pyrimidines as core structure to synthesize four
series of new 4, 6-diamine pyrimidines derivatives (Figure 2). All compounds were evaluated for
their efficacy as EGFR-TKIs for NSCLC therapy. Our study showed that 4, 6-disubstituted
pyrimidines may indeed be used as new small molecule inhibitors in EGFR triggered NSCLC.

ACCEPTED MANUSCRIPT
Figure 2
.
Schematic representation of the rational design of novel compounds
2
2
. RESULTS AND DISCUSSION
.1 CHEMISTRY
The synthetic routes for the production of the four series of target compounds used in this
study are shown in Scheme 3. The corresponding structures are shown in Tables 1-4.
-pyrimidinamine derivatives, as the key intermediates of the first series of compounds
Yfq01-Yfq24), were prepared from commercially available 4,6-dichloropyrimidine with different
4
(
anilines in the presence of a sufficient amount of potassium iodide. Nucleophilic substitution of
these intermediates with appropriately substituted anilines in the presence of p-toluenesulfonic
acid afforded the target compounds Yfq01-Yfq24. Acyl chlorination of commercially available or
previously synthesized benzoic acids or cinnamic acids with thionyl chloride provided the
corresponding acyl chlorides, which were coupled with 6-chloropyrimidin-4-amine, followed by
nucleophilic substitution with substituted anilines to yield the second (Yfq25-Yfq40) and third
(Yfq41-Yfq61) series of compounds. Furthermore, after esterification of fatty acid chains with
various alcohols, the forth series of compounds were synthesized using a similar procedure the
first series of compounds.

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Scheme 2. Synthesis of target compounds.
Reagents and conditions: (I) KI, EtOH, reflux; (II) EtOH, TsOH, reflux.
Reagents and conditions: (I) SOCl , reflux; (II) 4- chloro- 6- amine pyrimidine, acetone, Et N,
2
3
reflux; (III) substitued amines, dioxane, TsOH, reflux.
Reagents and conditions: (I) SOCl , reflux; (II) 4- chloro-6-aminepyrimidine, acetone, Et N,
2
3
reflux; (III) substitued amines, dioxane, TsOH, reflux.

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Reagents and conditions: (I) Glutaric anhydride or succinic anhydride, dioxane, reflux; (II)
substituted amines, sulfuric acid, KI, acetone, reflux; (III) EtOH; sulfuric acid; reflux.
Scheme 3. Synthetic route for the target compounds of the four series.
Table 1. Chemical structures of target compounds Yfq01-Yfq24.
delE746-A750
IC50 (nM)
974
L858R
EGFR
EGFR
EGFR
3
4
Compd.
R
R
%
inh
IC50 (nM)
%inh
%inh
IC50 (nM)
Yfq01
Yfq02
Yfq03
Yfq04
Yfq05
Yfq06
3’-OMe
3’- OMe
3’- OMe
3’- OMe
3’- OMe
2’,5’- OMe
46.8
16.7
30.7
30.5
48.7
50.2
>10000
--
84.7
48.3
46.2
72.5
90.2
99.3
71.6
1573
--
32.8
48.4
76.6
89.8
96.7
--
--
--
--
>10000
>10000
779
1188
1229
467
1745
1847
619
Yfq07
3’- OMe
96.9
26
97.9
7.7
97.0
18
Yfq08
Yfq09
2’,5’- OMe
2’,5’- OMe
72.5
6.2
336
--
55.9
26.6
208
--
51.6
32.1
230
--
Yfq10
2’,5’- OMe
30.4
--
43.4
--
46.9
--
Yfq11
Yfq12
2’,5’- OMe
2’,5’- OMe
79.0
54.7
288
125
43.3
57.9
>10000
102
72.1
58.2
167
167

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3
’,4’,5’-O
Me
Yfq13
79.6
901
10.9
>10000
16.2
>10000
Yfq14
Yfq15
Yfq16
Yfq17
Yfq18
Yfq19
Yfq20
Yfq21
2’,5’- OMe
3’- Cl
19.6
65.5
-2.9
6.8
--
170
--
44.3
44.2
-12.1
-12.0
0.7
--
43.9
43.8
-4.5
-8.3
12.8
97.5
35.4
8.5
--
>10000
>10000
3’- Cl
--
--
--
--
3’- Cl
--
3’- Cl
9.5
--
--
--
4’- Cl
89.8
12.3
-4.9
324
--
97.5
14.2
12.6
229
--
236
--
4’- Cl
4’- Cl
--
--
--
Yfq22
Yfq23
Yfq24
4’- Cl
4’- F
34.6
25.3
-0.4
--
--
--
40.9
33.0
-14.4
--
--
--
45.5
41.6
-1.5
--
--
--
4
’-ethyl
formate
Note: "--" indicates IC greater than 30000 nM
5
0
Table 2. Chemical structures of target compounds Yfq25-Yfq40.
delE746-A750
L858R
EGFR
EGFR
EGFR
5
6
Compd.
R
R
%inh
IC50 (nM)
%inh
IC50 (nM)
%inh
IC50 (nM)
Yfq25
Yfq26
H
H
18.6
96.0
--
47.8
--
39.6
--
81
98.0
26
97.9
60

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Yfq27
Yfq28
Yfq29
Yfq30
Yfq31
Yfq32
H
H
12.8
-1.2
-2.9
11.2
10.1
2.7
>10000
61.2
-7.2
-17.6
-3.3
3.8
754
--
73.2
-0.2
-4.2
2.4
1209
--
--
--
--
--
--
H
--
--
H
--
--
H
2.1
--
4’- F
-8.8
-0.8
--
Yfq33
4’- F
14.0
--
32.5
37.0
--
Yfq34
Yfq35
Yfq36
4’- F
4’- F
4’- F
-4.2
34.4
-8.1
--
>10000
--
-15.7
79.7
-5.2
-3.7
82.7
-0.5
--
1608
--
1409
--
Yfq37
Yfq38
Yfq39
Yfq40
4’- F
4’-F
-3.3
83.3
-1.2
1.7
--
131
--
-6.4
99.0
-7.3
--
63
--
-1.0
97.7
-5.5
--
96
--
4’- F
4’- F
--
-11.3
--
-10.3
--
Note: "--" indicates IC greater than 30000 nM
5
0
Table 3. Chemical structures of target compounds Yfq41-Yfq61.
delE746-A750
L858R
EGFR
EGFR
EGFR
7
8
Compd.
R
R
%inh
IC50 (nM)
%inh
IC50 (nM)
%inh
IC50 (nM)

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Yfq41
H
-0.3
--
13.0
--
-2.1
--
Yfq42
Yfq43
H
H
-0.4
1.9
--
--
28.2
24.3
--
--
-4.0
-6.5
--
--
Yfq44
H
-4.0
--
-5.2
--
-3.5
--
Yfq45
Yfq46
Yfq47
Yfq48
H
H
-5.7
-11.1
7.3
24.3
40.5
42.7
47.7
--
--
--
--
-8.7
5.8
7.7
2.2
--
--
--
--
--
--
--
4’-F
4’-F
-5.1
Yfq49
Yfq50
Yfq51
4’-F
-7.6
2.7
2.1
--
--
--
-12.5
-12.7
-12.2
--
--
--
-9.0
-11.8
-9.2
--
--
--
2’-Br-4’-F
2’-Br-4’-F
Yfq52
Yfq53
2’-Br-4’-F
4’- F
-7.7
2.9
--
--
-12.9
-9.0
--
--
-12.2
-10.3
--
--
Yfq54
Yfq55
4’-OMe
4’- OMe
22.7
-6.9
--
--
36.6
0.8
--
--
0.1
--
--
-9.0
Yfq56
Yfq57
Yfq58
Yfq59
4’- OMe
4’- OMe
4’- F
-2.9
4.1
--
--
--
--
-13.3
17.0
47.3
-4.5
--
--
--
--
-8.4
-2.8
11.3
-9.8
--
--
--
--
7.1
2’-Br-4’-F
-1.6

ACCEPTED MANUSCRIPT
Yfq60
Yfq61
4’-OMe
4’-OMe
7.6
--
--
-7.9
8.9
--
--
-7.2
--
--
-1.8
26.1
Note: "--" indicates IC greater than 30000 nM
5
0
Table 4. Chemical structures of target compounds Yfq62-Yfq95.
delE746-A750
IC50 (nM)
792
L858R
EGFR
EGFR
%inh
EGFR
9
10
Compd.
n
R
R
%
inh
IC50 (nM)
%inh
84.0
IC (nM)
50
Yfq62
Yfq63
Yfq64
Yfq65
Yfq66
2
2
2
2
2
H
H
H
H
H
34.7
-17.8
11.7
35.2
36.5
>10000
90.8
-6.3
825
--
--
--
--
--
--
--
--
--
-4.5
-4.4
39.3
47.9
-11.9
28.6
35.6
--
--
--
Yfq67
2
H
26.3
--
1.7
--
34.6
--
Yfq68
Yfq69
Yfq70
2
3
3
H
H
H
10.2
88.3
56.0
--
25.4
96.5
69.7
--
38.9
96.0
79.1
--
>10000
>10000
105
944
183
1322
Yfq71
Yfq72
Yfq73
3
3
3
H
H
H
73.1
39.4
91.3
27
--
98.9
39.6
7
--
98.4
43.6
21
--
48
100.4
21
100.6
69

ACCEPTED MANUSCRIPT
Yfq74
3
H
68.4
68
60.6
>10000
56.7
>10000
Yfq75
Yfq76
3
3
H
H
96.4
90.5
37
70
79.5
-5.2
12
83.9
-1.5
31
>10000
>10000
Yfq77
Yfq78
Yfq79
Yfq80
Yfq81
Yfq82
3
3
2
2
1
2
H
68.3
41.4
65.9
46.8
43.6
39.4
53
95.8
-10.2
82.3
8.8
11
--
99.5
-4.5
82.1
-8.7
-4.4
-9.5
27
--
H
--
ethyl
ethyl
ethyl
ethyl
>10000
933
--
1345
--
--
--
--
5.9
--
--
-7.2
--
--
Yfq83
Yfq84
Yfq85
2
2
2
ethyl
ethyl
ethyl
41.0
24.2
45.0
--
--
--
-1.1
5.9
--
--
--
-6.2
-5.6
-8.9
--
--
--
-6.5
Yfq86
Yfq87
Yfq88
2
2
2
ethyl
ethyl
ethyl
6.0
3.6
--
--
-4.9
3.7
--
--
-4.9
-4.0
10.6
--
--
85.7
27
45.6
>10000
>10000
Yfq89
Yfq90
3
3
ethyl
ethyl
-1.4
7.2
--
--
-19.0
-4.9
--
--
-10.9
-7.3
--
--

ACCEPTED MANUSCRIPT
Yfq91
Yfq92
2
2
3.6
--
--
24.7
1.6
--
--
0.1
--
--
-2.4
-10.4
Yfq93
Yfq94
2
2
-7.3
8.8
--
--
-0.2
-3.0
--
--
-13.0
-4.9
--
--
Yfq95
2
-2.5
--
-9.8
--
-5.0
--
Note: "--" indicates IC greater than 30000 nM
5
0
2
.2. Biological evaluation
2
.2.1 Kinase Inhibitory Activity and Kinase Selectivity Profile Compounds Yfq01-95 were
screened using a mobility shift assay against EGFR (wt), EGFR (L858R), EGFR (delE746-A750),
FGFR1 and KDR, AZD3759 as positive control and an ATP concentration at Km. As shown in
Supplementary Table S1 and Table 1-4, all compounds suppressed EGFR (wt), EGFR (L858R),
and EGFR (delE746-A750) more selectively than FGFR1 and KDR. As shown in Table 1-4,
Yfq07 and Yfq71 suppressed EGFR (wt) (IC = 26 nM), (IC = 27 nM) respectively and EGFR
5
0
50
(delE746-A750) (Yfq07 IC = 18 nM), (Yfq71 IC = 21 nM) more effectively than AZD3759
50 50
(
IC = 30 nM, IC = 41 nM). As for EGFR (L858R), the inhibitory activity of Yfq07 (IC = 7.7
5
0
50
50
nM) and Yfq71 (IC = 7 nM) were close to AZD3759 (IC = 5.5 nM).
5
0
50
In order to further verify the effects of the drug targets, AURA, EGFR, FGFR1, FGFR2,
FGFR4, FLT3, HER2, HER4, IKKβ, JAK1, JAK2, MET, MAP4K2, MAP4K4, PAK2, PDGFRb,
RET, SRC, TYK2, TRK-A, BRAF, FLT1 (VEGFR1), FLT4 (VEGFR3), IGF1R, PDGFRa, AKT1
and cKIT 27 as different statuses of RTKs were selected. As expected, all compounds exhibited a
more potent inhibitory activity to EGFR than other kinase species (Table 5.).
Table 5. In vitro percent inhibitory activity of partial compounds against 27 kinase species.
%inhibiton
RTK.
Yfq07
26.9
Yfq26
21.7
Yfq71
43.0
Yfq73
Yfq75
8.0
Yfq77
9.2
PDGFRa
PDGFRb
EGFR
4.7
30.4
29.4
74.1
24.2
93.2
10.3
86.5
10.0
84
86.2
99.0
96.5

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HER2
HER4
FGFR1
FGFR2
FGFR4
RET
-4.2
11.3
4.8
55.8
71.4
-1.6
8.3
57.3
88.6
13.5
16.5
8.8
4.9
27.1
5.6
-1.9
21.7
24.0
21.4
21.6
7.7
-1.5
8.3
5.6
9.8
7.2
5.9
4.8
1.8
-0.6
10.4
4.4
4.0
16.3
7.2
2.9
16.8
4.0
8.5
MET
3.3
12.0
16.6
17.7
18.8
10.1
6.8
9.2
SRC
33.2
0.7
27.7
-4.2
5.6
83.7
12.7
9.6
11.4
4.2
11.7
0.2
JAK2
TRK-A
IGF1R
AKT1
FLT3
6.8
8.4
6.0
6.3
5.3
13.2
0.0
9.2
4.0
-2.1
59.1
-1.4
0.0
-3.4
6.9
4.7
-3.0
16.4
-2.4
2.5
35.5
-1.0
-0.6
13.3
9.2
11.5
-3.1
-1.4
5.9
10.5
-0.7
-4.4
13.3
10.5
13.5
11.3
13.3
18.9
11.8
29.4
19.8
JAK1
-0.8
-7.9
26.0
-3.2
44.8
16.6
3.5
TYK2
CKIT
0.4
-4.4
3.3
MAP4K2
MAP4K4
BRAF
IKKB
PAK2
FLT1
5.4
5.5
53.5
47.5
-2.1
4.7
78.2
79.3
1.1
56.3
12.7
1.2
21.6
16.7
-3.2
9.4
13.4
2.3
16.9
12.3
29.4
8.7
15.7
2.9
18.1
47.3
52.3
-9.5
9.5
FLT4
21.6
2.6
28.2
17.3
AURa
49.0
Note: Compound concentrations were 1µΜ
2
.2.2 Cellular Activities Compounds Yfq01 to Yfq95 were screened against H3255, A431,
HCC827, H1975, PC-9 and BEAS-2B, AZD3759 as positive control using an MTT assay. As
shown in Table 6, compound Yfq07 inhibits A431 (IC50 = 0.74±0.43 µM) and H1975 (IC50
.89±0.03 µM) more effectively than AZD3759 (IC50 = 3.50±0.30 µM, IC50 = 24.16±0.35 µM).
=
0
Although compound Yfq07 exhibited a reduced inhibitory activity against H3255 and HCC827
cells compared to AZD3759, an overall suitable inhibitory effect could be observed. Compound
Yfq07 featured an IC50 = 21.43±0.15 µM towards Beas-2B meaning that Yfq07 exhibited a low
toxicity towards normal lung bronchial cells.

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Table 6. Anti-proliferative activities of partial Yfq01-95 against cells using a different status of
EGFR.
Compound
IC ±SD(nM)a
5
0
b
c
d
e
f
g
H3255
A431
HCC827
H1975
PC-9
BEAS-2B
Yfq01
--
15.20±0.32
4.04±0.18
--
1.66±0.21
17.60±0.18
Yfq07
Yfq08
Yfq11
Yfq19
Yfq26
Yfq27
Yfq35
Yfq38
Yfq62
Yfq69
Yfq71
Yfq77
Yfq88
AZD3759
0.86±0.09
--
0.74±0.43
--
1.14±0.17
1.72±0.16
0.89±0.03
--
0.45±0.07
0.64±0.09
--
21.43±0.
17.96±0.10
18.10±0.30
18.05±0.22
18.52±0.20
17.43±0.31
18.56±0.16
20.95±0.29
18.98±0.23
17.91±0.30
18.02±0.21
17.90±0.20
21.07±0.12
18.66±0.28
--
--
24.29±0.53
--
--
--
--
--
1.18±0.10
0.66±0.08
2.09±0.12
0.82±0.08
0.87±0.04
0.79±0.06
0.94±0.09
2.00±0.10
0.56±0.04
--
8.88±0.27
9.17±0.45
--
9.46±0.25
6.54±0.15
--
5.35±0.16
7.00±0.13
--
11.40±0.26
7.07±0.21
17.57±0.32
1.97±0.06
2.73±0.11
1.85±0.09
--
1.87±0.36
4.37±0.12
2.94±0.22
--
1.13±0.10
1.90±0.13
1.19±0.12
--
1.96±0.09
3.13±0.14
2.22±0.13
4.02±0.16
1.56±0.09
--
5.69±0.13
--
0.78±0.07
0.49±0.02
3.50±0.30
1.16±0.08
--
0.09±0.02
0.03±0.01
24.16±0.35
0.05±0.02
a
Note: "--" indicates IC greater than 30000 nM. The inhibitory effects of individual compounds
5
0
on the proliferation of cancer cell lines were determined by an MTT assay. The data are
b
represented as means ± SD from at least three independent experiments. H3255 is a human lung
c
cancer cell line (EGFR L858R). A431 is a human epithelial carcinoma cell line (overexpressed
d
e
WT EGFR). HCC827 is a human lung cancer cell line (EGFR del E746_A750). H1975 is a
f
human lung cancer cell line (EGFR L858R/T790M). PC-9 is a human lung cancer cell line
g
(
EGFR del E746_A750). BEAS-2B is a human bronchial epithelial cell line

ACCEPTED MANUSCRIPT
2
.2.3 Structure–activity relationship analysis Four series of 95 novel N4, N6-disubstituted
pyrimidine-4, 6-diamine derivatives were evaluated using kinases and EGFR triggered NSCLC.
Most of these species exhibited EGFR inhibitory activity, indicating that N4, N6-disubstituted
pyrimidine-4, 6-diamine as the core structure could effectively inhibit EGFR and EGFR triggered
NSCLC. The first series of these compounds, namely compound Yfq07, was found to be the most
prominent active structure, with the methoxy groups on the benzene ring substituted by
4
,6-diamine, respectively. Moreover, the substitution of the methoxy group on the benzene ring
was more prominent than chlorine substitution, and the methoxy groups in 3-position on the
benzene ring exhibited an improved EGFR inhibitory activity. Different from the first series of
compounds, the second series bearing a phenylamide group attached on the N4-position of the
N4,N6-disubstituted pyrimidine-4,6-diamine, featured reduced overall activities compared to the
first series. Among them, compound Yfq26 also featured a methoxyphenyl group, and surprisingly,
it still exhibited an excellent inhibitory activity. As for the third series, upon increasing the length
of the branch in N4-position by introducing a Michael acceptor molecule, we found that the
overall inhibitory activities of the third series were not enhanced. Presumably, the Michael
acceptor molecule was not positioned in its right place. Moreover, the fourth series of compounds
without aniline substitution and selected straight chain, further increased the length of the side
chain and retained a Michael acceptor molecule. However, compounds of the fourth series of
compounds exhibited a reasonable inhibitory activity on EGFR kinase but did not exhibit the same
inhibitory activities in cellular assays of Yfq73, Yfq75 etc. Furthermore, compounds featuring a
branched 3-carbon chain proved to be exhibit a more potent inhibitory activity than others. Taken
in concert, via structure–activity relationship analysis of the four series, compounds of the first
series with a relatively simple structure could be further modified to obtain compounds with
improved characteristics.
2
.2.4 Inhibitory Effects of Yfq07 on EGFR Mediated Signaling Pathways in Cancer Cells In
order to investigate the inhibitory effect on the phosphorylation of EGFR and the downstream
signaling transduction, Western blot analysis of the representative compound Yfq07 in H3255,
HCC827, A431 and H1975 cells was used ( Figure 6.). A dosage of 30 ng/ml of EGF was used for
the following set of experiments. The results indicated that Yfq07 significantly inhibited EGFR
auto-phosphorylation at Y1068, including the downstream AKT and ERK1/2 phosphorylation, in a
dose-dependent manner.

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p-EGFR/EGFR
p-AKT/AKT
p-ERK/ERK
H3255
2
1
1
0
0
.0
.5
.0
.5
.0
*
*
*
**
NS
NS
*
**
#
#
#
NS
##
NS
#
#
#
#
NS
#
##
#
#
###
##
##
#
µM
Yfq07
EGF
AZD3759
Yfq07
AZD3759
Yfq07
EGF
AZD3759
EGF

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p-EGFR/EGFR
p-AKT/AKT
p-ERK/ERK
HCC827
1
1
0
0
.5
.0
.5
.0
*
*
*
*
***
NS
#
NS
##
#
#
#
#
NS
NS
##
#
#
#
#
##
#
#
#
#
##
µM
Yfq07
EGF
AZD3759
Yfq07
EGF
AZD3759
Yfq07
EGF
AZD3759

ACCEPTED MANUSCRIPT
p-EGFR/EGFR
p-AKT/AKT
p-ERK/ERK
H1975
1
1
0
0
.5
.0
.5
.0
***
**
*
NS
NS
** NS
NS
NS
NS
NS
#
NS
NS
#
#
**
#
## ##
NS
#
#
#
#
##
µM
Yfq07
EGF
AZD3759
Yfq07
EGF
AZD3759
Yfq07
EGF
AZD3759
Figure 3. Inhibitory effect of Yfq07 on EGFR and its downstream ERK1/2 and AKT
phosphorylation levels in H3255, A431, HCC827 and H1975 cell lines ;* P < 0.05, ** p < 0.01,
*
** p <0.001 compared with the blank control group; # P < 0.05, ## p < 0.01, ### p <0.001
compared with the positive drug AZD3759.
2
.2.5 Compound Yfq07 Induces Cellular Apoptosis and Cell Cycle Arrest in G2/M Phase in
NSCLC We examined the pro-apoptotic effects of compound Yfq07 using an Annexin
V/propidium iodide (PI) assay. Non-treated cells were used as a negative control and AZD3759
was used as a positive control. As shown in Figure 4, the cell lines H3255, A431, HCC827 and
H1975 were treated for 48 h. We found that a concentration of 500 nM exhibited a significant
effect on the overall apoptosis rate. Compound Yfq07 was found to promote apoptosis in a
dose-dependent manner. These results revealed that Yfq07 was the most effective apoptotic
inducer, exhibiting an apoptotic potential higher than AZD3759.
We used flow cytometry to determine the anti-mitogenic effects of Yfq07 in H3255, A431,
HCC827 and H1975 cell lines. As shown in Figure 5, compound Yfq07 was found to block the
cell cycle in a dose-dependent manner. Furthermore, it was found that compound Yfq07 blocked
the cells in the G2/M phase, while AZD3759 demonstrated no significant effect. Compound
Yfq07 in particular blocked cells in the G2/M phase in H1975, which would be the main reason
for inhibiting the proliferation of H1975 cells. Therefore, these results revealed that Yfq07 may
represent a potential cell cycle inhibitor blocking G2/M phase.

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H3255
A431
6
4
2
0
0
0
0
HCC827
H1975
*
**
*
**
#
#
#
**
##
#
##
***
***
**
***
*
***
*
***
*
*
***
***
**
**
*
**
Yfq07 AZD3759
Yfq07 AZD3759
Yfq07 AZD3759
Yfq07 AZD3759

ACCEPTED MANUSCRIPT
Figure 4. Effects of Yfq07 on apoptosis in H3255, A431, HCC827 and H1975 cell lines; * P
<
0.05, ** p < 0.01, *** p < 0.001 compared with the blank control group; # P < 0.05, ## p < 0.01;
#
## p < 0.001 compared with the positive drug AZD3759.

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H3255
A549
HCC827
60
H1975
#
##
###
#
###
*
**
***
**
***
*
40
*
NS
**
*
*
**
NS
NS
NS
NS
NS
20
0
Yfq07 AZD3759
Yfq07 AZD3759
Yfq07 AZD3759
Yfq07 AZD3759
Figure 5. Effects of compound Yfq07 on cell cycle arrest in A431, H3255, HCC827 and
H1975 cell lines; * P < 0.05, ** p < 0.01, *** p < 0.001 compared with the blank control group.
2
.2.6 Molecular Docking and Molecular Dynamics Simulation To kinetically validate that drug
binding could block ATP from binding to this site, we carried out ATP competition experiments by
measuring the inhibitory capacity of Yfq07 and Yfq071 at increasing ATP concentrations. As
shown at Figures 6A and 6B, we found that compounds Yfq07 and Yfq071 were dependent on the
ATP concentration. Hence, both compounds were confirmed to be ATP-competitive inhibitors.
Through docking results of the active compound Yfq07 and EGFR L858R (Figure 6C), it
could be concluded that the 2,5-dimethoxyphenyl group introduced to the nitrogen atom in
4
-position of the modified Yfq07 pyrimidine nucleus penetrated into the ATP binding cleft of the
receptor protein and one of the methoxy groups formed a hydrogen bond with Asp-855. Moreover,
-methoxyaniline in 6-position of the pyrimidine moiety formed one H-bond with the Met-793
3
residue. In addition, the binding pattern of Yfq07 with the receptor differed from the lead
compound AZD3759, suggesting that the modified 4,6-pyrimidine diamines may exhibit an
improved molecular flexibility.

ACCEPTED MANUSCRIPT
C
Figure 6. Yfq07 and Yfq071 inhibit EGFR L858R through a mechanism that proved to be
ATP-concentration-dependent. Selective ATP-competitive kinase assay of compounds (A) Yfq07,
(B) Yfq071. (C) Molecular docking diagram and two-dimensional diagram highlighting the
binding modes of Yfq07 and EGFRL858R.
2
.2.7 Pharmacokinetic Evaluation and In Vivo Antitumor Efficacy Study We evaluated the PK
properties of compound Yfq07 in rats following oral administration (Table 8 and Figure 7). Upon
oral administration of compound Yfq07, a Cmax value of 1056.37 ± 394.23 ng/mL was obtained.
After oral administration, a half-life of 4.17 ± 2.06 hours was obtained.
Furthermore, compound Yfq07 was evaluated for antitumor efficacy evaluation in vivo using
a PC-9 cell xenograft mouse model. Over a 23-day period, compound Yfq07 (15 mg/kg and 30
mg/kg) was administered intraperitoneally. Moreover, the positive control compound AZD3759
(30 mg/kg) was administered intraperitoneally [14]. As shown in Figure 8, we found that
compound Yfq07 exhibited a significant antitumor efficacy in vivo. Furthermore, the mice
exhibited no body weight loss throughout the experiment. The immunoblotting assays performed
on the tumor tissue confirmed that the treatment with compound Yfq07 effectively inhibited
phosphorylation of EGFR, AKT and ERK1/2 (Figure 9). Immunohistochemical analysis
confirmed these results (Figure 10).

ACCEPTED MANUSCRIPT
Table 8. The pharmacokinetic parameters of compound Yfq07 in rat plasma after oral
administration at a dosage of 30 mg/kg, n=6.
Parameters
t (h)
Mean ± SD
4.17 ± 2.06
1056.37 ± 394.23
6.66 ± 2.74
5700.32
1
/2
Cmax (ng/mL)
MRT0→t (h)
AUC0→t (ng/mL•h)
AUC0→∞ (ng/mL•h)
5822.04
V (L/kg)
d
0.03
t , elimination half-life; Cmax, peak concentration; MRT(0–t), mean residence time (from time
1/2
point 0 to time t); AUC (0–t), area under the curve from time zero to the last sampling time point,
AUC (0–∞), area under the curve from time zero to the infinity; V , volume of distribution.
d
1
1
1
1
600
400
200
000
8
6
4
2
00
00
00
00
0
0
5
10
Tim e (h)
15
20
25
Figure 7. Concentration versus time curves of compound Yfq07 in rats after a single dose (30
mg/kg) via oral administration.

ACCEPTED MANUSCRIPT
Figure 8. Effects of Yfq07 and AZD3759 on the growth of nude mice; (A) Representative
tumor-bearing mice after various treatments. (B) Tumor weights. (C) Tumor volumes. (D) Tumor
weight statistics. * P < 0.05, ** p < 0.01, *** p < 0.001 compared with the blank control group.

ACCEPTED MANUSCRIPT
1
0
p-EGFR/EGFR
p-AKT/AKT
p-ERK/ERK
NS
8
6
4
2
0
**
***
**
*
*
**
*
**
*
**
***
**
*
Figure 9. Effects of Yfq07 on phosphorylation of EGFR and downstream AKT as well as
ERK1/2 in tumor tissue; * P < 0.05, ** p < 0.01, *** p < 0.001 compared with the blank control
group.

ACCEPTED MANUSCRIPT
Figure 10. Subcutaneous PC-9 xenografts, treated with compound Yfq07 and the positive-control
AZD3759. The statuses of p-EGFR, p-AKT and cleaved-caspase 3 were evaluated by
immunohistochemistry.
3
. CONCLUSIONS
In this study, we reported the synthesis of a series of N4, N6-disubstituted pyrimindine-4,
6
-diamines and evaluation of their biological activities. Most of the compounds exhibited
inhibitory activities towards non-small cell lung cancer cells, similar to AZD3759. Using a
mobility shift assay and Latha screen assay to screen the compounds demonstrated that an
inhibitory effect was only observed on EGFR kinase in vitro. The anti-proliferative activity of the
test compounds in non-small cell lung cancer cells including different types of EGFR mutation
L858R
del E746_A750
cells (H3255 (EGFR
), A431 (overexpressed WT EGFR), HCC827 (EGFR
),
L858R/T790M
del E746_A750
H1975 (EGFR
) and PC-9 (EGFR
)) was evaluated using an MTT assay. In
doing so, similar effects as using AZD3759 could be observed. While compound Yfq07
significantly increased apoptosis and blockage in the G2/M phase in above mentioned NSCLC
cell lines. Furthermore, compound Yfq07 demonstrated the ability to down-regulate expression of

ACCEPTED MANUSCRIPT
EGFR, AKT and ERK1/2 phosphorylation. Moreover, compound Yfq07 significantly inhibited a
PC-9-driven xenograft mouse model. Overall, these results showed that compound Yfq07 exhibits
advantages over similar compounds, particularly with respects to its anti-EGFR kinase activity,
anti-proliferative effects against non-small cell lung cancer cell lines, induction of apoptosis and
blocking of cell cycles. The preliminary biological activity screenings of our novel series of N4,
N6-disubstituted pyrimindine-4,6-diamine derivatives demonstrate that these compounds may
potentially be useful for treatment of non-small cell lung cancer.
4
. EXPERIMENTAL SECTION
4
4
.1 Chemistry
.1.1 General Melting points were determined using a SGWX-4 microscopic melting point meter
1
13
and are reported in uncorrected form. H and C NMR spectra were recorded on a Bruker 600
MHz NMR spectrometer, using CDCl or DMSO-d as solvents. Chemical shifts are expressed in
3
6
ppm with TMS as internal reference. J values are provided in hertz. Mass spectra were recorded
on a Waters Xevo TQ-S Micro mass spectrometer. High resolution mass spectrometric data were
obtained on a Thermo Fisher Scientific LTQ FTICR-MS instrument. IR spectra were recorded on
a Varian 660 IR spectrometer. Reactions were monitored by thin layer chromatography (TLC) on
glass plates coated with silica gel GF-254. The purity of the all biologically tested compounds was
≥
95%. Column chromatography was performed with 200-300 mesh silica gel.
4
.1.2 General Procedure for the Synthesis of All Compounds in the First Series To a round
bottom flask, 4, 6-dichloropyrimidine 1.00 g was added along with ethanol 30 ml and KI 5 mg.
The substituted aniline was then added dropwise and the resulting mixture was stirred at 80 °C for
2
h. The resulting crude product was filtered off, recrystallized and used in the next reaction step
without further purification. A mixture of the above crude product, another substituted amine,
p-methylbenzene sulfonicacid in ethanol was stirred at 80 °C for 6 h. The mixture was cooled to
room temperature and the organic layer was washed with saturated aqueous NaCl, dried over
anhydrous Na SO , and concentrated in vacuo. The residue was purifed by silica gel
2
4
chromatography to provide the desired product.
4
.1.3 General Procedure for the Synthesis of All Compounds in the Second and the Third
Series To a round-bottomed flask was added 4-chloro-6-aminopyrimidine (500 mg), benzoyl
chloride, acetone 20 ml, and triethylamine 100 µL. The resulting solution was stirred at 0 °C for 8
h. Then, the solution was concentrated using a rotary evaporator and upon addition of saturated
aqueous Na CO , precipitation occurred. The mixture was stirred for 3 h and then washed with
2
3
1
0ml dichloromethane and ethyl acetate and filtered. The resulting crude product was
recrystallized from a mixture of dichloromethane and ethyl acetate three times to obtain the

ACCEPTED MANUSCRIPT
desired intermediate. Then, to a solution of the above intermediate (1.00 mmol), the substituted
amines (2.5 mmol) and benzoic acid hydrate (2.00 g) were added. The mixture was stirred at
8
0 °C for 10 h. After reaction completion, the solution was concentrated on a rotary evaporator,
washed with the saturated aqueous Na CO , and concentrated in vacuo. The residue was purified
2
3
by silica gel chromatography to provide the desired product.
4
.1.4 General Procedure for the Synthesis of All Compounds in the Fourth Series A mixture
of 4-chloro-6-aminopyrimidine (2.00 g), glutaric anhydride (3.50 g) or succinic anhydride (3.10 g)
in dioxane (80 ml) was refluxed for 20 h. The solution was concentrated on a rotary evaporator,
washed with the saturated aqueous Na CO , and concentrated in vacuo. To the filtrate, 5%
2
3
hydrochloric acid was added and the pH was adjusted to 2-3. The forming precipitate was filtered
off and dried to obtain the desired intermediate. To a round-bottomed flask was added
intermediate I, the substituted amines, sulfuric acid 60 µL, and KI 200 mg in acetone (35 ml). The
mixture was refluxed for 20 h. The resulting crude product was filtered off and recrystallized to
obtain intermediate II. To a solution of intermediate II in ethanol was added the substitute and a
small amount of concentrated sulfuric acid. The reaction was refluxed for 5 h. Upon reaction
completion, the solution was concentrated using a rotary evaporator, washed with the saturated
aqueous Na CO , and concentrated in vacuo. The residue was purified by silica gel
2
3
chromatography to provide the desired product.
4
.1.5 N4-(3-Chlorophenyl)-N6-(3-methoxyphenyl)pyrimidine-4,6-diamine (Yfq01) Yield: 87.5%;
1
mp: 183-185 °C; H NMR (600 MHz, DMSO-d ), δ 9.71 (s, 1H, NH), 9.21(s, 1H, H-2), 8.33 (s,
6
1
H, H-5), 7.88-7.89 (m, 1H, N4-5’-PhH), 7.43-7.55 (m, 1H, N6-5’-PhH), 7.29-7.30 (m, 1H,
N6-4’-PhH), 7.21-7.22 (m, 2H, N4-5’,6’-PhH), 7.12 (s, 1H, N4-2’-PhH), 6.97-6.99 (m, 1H,
1
3
N4-4’-PhH), 6.57 (s, 1H, N6-2’-PhH), 6.21 (s, 1H, -NH), 3.74 (s, 3H, OCH3). C NMR (150
MHz, DMSO-d ) δ 160.5, 160.2, 159.7, 157.6, 142.2, 141.5, 133.0, 130.2, 129.5, 120.7, 118.5,
6
-
1
17.5, 112.3, 107.2, 105.7, 87.3, 55.0. HRMS (ESI) m/z: [M-H] calc = 325.0856; found 325.0850
for C H N ClO.
17
15
4
4
.1.6 N4-(4-Chlorophenyl)-N6-(3-methoxyphenyl)pyrimidine-4,6-diamine (Yfq02) Yield: 86.8%;
1
mp: 227-228 °C; ESI-MS [M+H]+: 326; H NMR (600 MHz, DMSO-d ), δ 9.30 (s, 1H, NH), 9.17
6
(s, 1H, H-2), 8.29 (s, 1H, H-5), 7.63(d, 2H, J = 9.0 Hz, N4-2’, 6’-PhH), 7.33 (d, 2H, J = 9.0 Hz,
N4-3’,5’-PhH), 7.20-7.21 (m, 2H, N6-5’-PhH, N6-2’-PhH), 7.02 (d, 1H, J = 9.0 Hz, N6-4’-PhH),
1
3
6
.54 (d, 1H, J = 9.0 Hz, N6-6’-PhH), 6.18 (s, 1H, -NH), 3.74 (s, 3H, -OCH3). C NMR (150
MHz, DMSO-d ) δ 160.5, 160.3, 159.7, 157.6, 141.5, 139.6, 129.5, 126.5, 125.0, 120.9, 112.2,
6
1
07.2, 105.8, 89.0, 54.9.
4
.1.7 N4-(3-Bromophenyl)-N6-(3-methoxyphenyl)pyrimidine-4,6-diamine (Yfq03) Yield: 82.8 %;