Discovery of selective EGFR modulator to inhibit L858R/T790M double mutants bearing a N-9-Diphenyl-9H-purin-2-amine scaffold

Article abstract of DOI:10.1016/j.bmc.2018.02.029

Based upon the modeling binding mode of marketed AZD9291 with T790M, a series of N-9-Diphenyl-9H-purin-2-amine derivatives were designed and synthesized with the purpose to overcome the drug resistance resulted from T790M/L858R double mutations. The most potent compound 23a showed excellent enzyme inhibitory activities and selectivity with nanomolar IC₅₀ values for both the single T790M and double T790M/L858R mutant EGFRs, and was more than 8-fold selective for wild type EGFR. Compound 23a displayed strong antiproliferative activity against the H1975 non-small cell lung cancer (NSCLC) cells bearing T790M/L858R. And it was less potent against A549 (WT EGFR and k-Ras mutation) and HT-29 (non-special gene type) cells, showing a high safety index.

Full text of DOI:10.1016/j.bmc.2018.02.029

Accepted Manuscript
Discoveryof selectiveEGFRmodulatortoinhibitL858R/T790M doublemutants-
bearingaN-9-Diphenyl-9H-purin-2-aminescaffold
Jinxing Hu, Yufei Han, Jingtao Wang, Yue Liu, Yanfang Zhao, Yajing Liu, Ping
Gong
PII:
S0968-0896(18)30008-7
https://doi.org/10.1016/j.bmc.2018.02.029
BMC 14215
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
4 January 2018
15 February 2018
17 February 2018
Please cite this article as: Hu, J., Han, Y., Wang, J., Liu, Y., Zhao, Y., Liu, Y., Ping Gong, Discoveryof
selectiveEGFRmodulatortoinhibitL858R/T790M
doublemutantsbearingaN-9-Diphenyl-9H-purin-2-
aminescaffold, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.02.029
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Discovery of selective EGFR modulator to inhibit L858R/T790M
double mutants bearing a N-9-Diphenyl-9H-purin-2-amine scaffold
Jinxing Hua, Yufei Hana, Jingtao Wanga, Yue Liua, Yanfang Zhaoa, Yajing Liua, *, Ping Gonga, **
aKey Laboratory of Structure-Based Drug Design and Discovery (Shenyang Pharmaceutical
University), Ministry of Education, 103 Wenhua Road, Shenhe District, Shenyang 110016, PR
China
*Corresponding author.
**Corresponding author.
E-mail address: lyjpharm@126.com (Yajing Liu), gongpinggp@126.com (Ping Gong).
Abstract
Based upon the modeling binding mode of marketed AZD9291 with T790M, a series of
N-9-Diphenyl-9H-purin-2-amine derivatives were designed and synthesized with the purpose to
overcome the drug resistance resulted from T790M/L858R double mutations. The most potent
compound 23a showed excellent enzyme inhibitory activities and selectivity with nanomolar IC50
values for both the single T790M and double T790M/L858R mutant EGFRs, and was more than
8-fold selective for wild type EGFR. Compound 23a displayed strong antiproliferative activity
against the H1975 non-small cell lung cancer (NSCLC) cells bearing T790M/L858R. And it was
less potent against A549 (WT EGFR and k-Ras mutation) and HT-29 (non-special gene type) cells,
showing a high safety index.
Keywords: Non-small cell lung cancer; N-9-Diphenyl-9H-purin-2-amine derivatives;
L858R/T790M double mutants
The epidermal growth factor receptor (EGFR) tyrosine kinase played a crucial role in cellular
signal-transduction pathways and was closely related to cell proliferation, survival, adhesion,
migration, and differentiation.[1,2] Thus, EGFR tyrosine kinase has become an attractive
therapeutic target for cancer treatment, especially for nonsmall-cell lung cancer (NSCLC).[3-5]
The first generation of EGFR drugs, gefitinib (1, Fig.1) [6,7] and erlotinib (2, Fig.1) [8]
relied on reversible affinity to achieve potency. More recently, a covalent inhibition strategy has
been used because it delivered distinct pharmacological properties. Irreversible kinase inhibitors
can achieve advantages over reversible compounds by delivering complete and sustained target
engagement in the presence of high intracellular concentrations of ATP in the cells and by
requiring the physical turnover of kinase proteins to restore inhibited signaling pathways.[9-11]
Indeed, second generation, irreversible inhibitors of WT EGFR (afatinib) (3, Fig 1)[12] which
covalently modify EGF Cys797 demonstrate increased cellular potency against T790 mutants
relative to their reversible counterparts. However because these compounds maintained the aniline
moiety that clashed with Met790 side chain, their T790M activity was less than their activity
against the primary activating EGFR mutants. As a result, their clinical efficacy to treat T790M

patients was limited due to dose-limiting toxicity associated with inhibition of WT EGFR.
In response to this unmet need and because irreversible inhibitors demonstrated better
potency against EGFR T790M mutants than reversible inhibitors, an effort was initiated to
discover highly potent irreversible inhibitors of the drug resistant T790M mutants that spare
EGFR WT activity to the greatest extent as a means to minimize mechanism-based toxicities.
Some irreversible inhibitors of EGFR T790M have been pursued, resulting in the identification of
WZ4002 (4, Fig 1) [13], CO-1686 (6, Fig 1) [14] and AZD9291 (5, Fig 1) [15, 16]. In addition,
there are several recent publications discussing the design and SAR for novel third generation
irreversible EGFR inhibitors.[15,17-19] Among them, AZD9291 showed 200-fold selectivity for
T790M/L858R double mutants over WT EGFR [20] and was approved for the treatment of
NSCLC patients with EGFR T790M mutation by FDA in November 2015.
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 [21]. To overcome the drawbacks of AZD9291, in
this manuscript, we described the design, synthesis, in vitro biological evaluation of a series of
novel selective T790M inhibitors.
Fig. 1. Structures of different generation EGFR inhibitors
2. Results and discussion
2.1. 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 (Fig. 2).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 [15]. These interactions were envisioned to be

important for improving the activity and selectivity against mutated EGFR kinase [22].
Fig. 2. Interaction map of AZD9291 with the EGFR T790M mutant and the overview in the binding site.
To our knowledge, the purin moiety have been widely applied as building blocks in drug
design, and they could be found in several therapeutic agents, such as seliciclib and GS-4059
[23,24]. Based upon the binding mode of AZD9291 with T790M active domain, we designed and
synthesized a series of N-9-diphenyl-9H-purin-2-amine analogues (Fig 3) by applying the
conformational constraint strategy, which is an effective method to improve ligand selectivity for
a molecular target. Moreover, additional hydrogen bond donors and acceptors were expected to
contribute to the interaction with EGFR. More importantly, the aminopyrimidines and a Michael
receptor group were expected to contribute to the interaction with EGFR. And a number of side
chain amines (R1) were also modified with the purpose to improve the potency and physical
properties of the inhibitors.
Fig. 3. Design strategy of target compounds.
The novel target scaffold was docked to the EGFR binding pocket (PDB: 3IKA) and overlay
results showed that the conformation of this novel scaffold is well consistent with AZD9291's
binding conformation, which suggested that a N-9-Diphenyl-9H-purin-2-amine Scaffold moiety
could serve as a scaffold from which to build a novel series of EGFR inhibitors (Fig. 4).

Fig.4 Docking structure of target scaffold (red) overlayed with AZD9291 (blue) in the complex of EGFRT790M
mutant. (For interpretation of the references to color in this figure legend, the reader is referred to the web version
of this article.)
2.2. CHEMISTRY
The synthesis of target compounds 21a−t and 22a-c were described in Scheme 1. The
intermediate 8 was synthesized by nitration of commercially available 4-fluoro-2-methoxyaniline
7. Substituted aniline 8 was protected with (Boc)2O generated the intermediate 9, which was
reacted with various aliphatic amines under basic conditions to formed intermediates 10.
Subsequent reduction of the nitro groups of 10 with hydrogen gave amines 11, which were not
purified and used in the next reaction directly. Acylation of 11a-c with acryloyl chloride
conveniently produced compounds 12a-c. The key intermediates 13 was obtained by the
deprotection of 12 in the presence of trifluoroacetic acid.
Anilines regioselectively reacted with the 4-chloro atom of the 2,4-dichloro-5-nitropyrimidine
14 in the presence of N,N-diisopropylethylamine (DIPEA) to produce the key pyrimidine
intermediates 15, which coupled with anilines 13 to obtain pyrimidine template compounds 16a-t.
Subsequent reduction of the nitro groups of 16a-t with stannous chloride gave amines 17a-t in
excellent yield (68−85%). Finally, compounds 17a-t were treated with formic acid or
carbonyldiimidazole to yield the desired 2,9-dianilinopurine derivatives 21a-t or 22a-e.
The general steps for preparing the final compounds 23a-c are outlined in Scheme 2.
Intermediate 20a-c was obtained from intermediate 14 via a three-step reaction using similar
conditions as described for 16 and 17. And the resultant intermediates (20a-c) were treated with
isothiocyanatobenzene to yield the final 2,9-dianilinopurine derivatives 23a-c.

Scheme 1. Reagents and conditions: (a) KNO3/H2SO4, -15°C, 3h; (b)Boc2O, DMAP, DCM, 25°C, 6h; (c)
appropriate alkyl amine, K2CO3, DMF, 65°C, 3-5h; (d) Pd/C, H2, MeOH, 25°C, 12h; (e) acryloyl chloride, DIPEA,
DCM, 25°C, 2h; (f) TFA, DCM, 25°C, 5-7 h; (g) ArNH2, K2CO3, DCM, -5-0°C, 2 h; (h) TFA, 1,4-dioxane/IPA,
.
25°C, 25h; (i) SnCl2 2H2O, MeOH, 65°C,3-4h; (j) formic acid/PPA, 3 h, 125 °C; (k) carbonyldiimidazole, CH2Cl2,
3 h, rt.

Scheme 2. Reagents and conditions: (a) appropriate alkyl amine, K2CO3, DCM, -5-0°C, 2h; (b) TFA, 1,4-dioxane
.
/IPA, 25°C, 25h; (c) SnCl2 2H2O, MeOH, reflux, 3-4h; (d) CH2Cl2, DIPEA, DCC, isothiocyanatobenzene, reflux,
12 h.
2.3. Biological evaluation
2.3.1. Inhibitory activity against A549, H1975, and HT-29 cells
NCI-H1975 adenocarcinoma cell lines bearing EGFRL858R,T790M point mutations were used for
potency optimization while A549 epidermoid carcinoma cell lines bearing overexpressed EGFRWT
were used as a counterscreen, allowing an early assessment of selectivity over EGFRWT. In addition,
these compounds were screened in HT-29 colon cancer cells, which expressed a non-special gene
type, to test their toxic effects. The results were expressed as IC50 values and summarized in Table
1. The IC50 values were the average of at least three independent experiments.
As shown in Table 1, All the target compounds showed moderate-to-excellent cytotoxic activity
against H1975 cell line and some exhibited more or similar potent activities against certain cancer
Table 1
Cell cytotoxicity of representative compounds a.
IC50(μM)a
Compd
R1
R2

H1975
HT-29
Selectivity Ratio
A549
A549/H1975
58
-Ph
4-CH3O-Ph
4-CH3-Ph
4-IPE-Ph
4-C2H5-Ph
4-F-Ph
21a
21b
21c
0.58±0.13
3.33±0.04
0.36±0.07
14.52±1.24
1.97±0.18
0.42±0.08
1.86±0.03
1.15±0.04
8.71±0.23
5.82±0.47
12.60±2.34
2.09±0.36
11.2±1.87
3.02±.023
5.19±1.54
2.90±0.21
9.25±0.05
6.31±1.57
4.23±0.13
1.52±0.02
1.23±0.05
2.05±0.67
0.49±0.03
1.17±0.08
18.52±3.19
6.08±1.39
1.42±0.07
0.78±0.04
0.87±0.05
0.11±0.05
0.16±0.07
0.14±0.07
0.074±0.024
0.13±0.08
0.061±0.031
0.085±0.027
0.14±0.06
0.076±0.043
6.72±0.57
1.71±0.35
1.80±0.12
1.61±0.23
0.035±0.012
0.10±0.08
0.33±0.07
1.93±0.42
7.32±0.16
5.10±0.47
29.84±1.69
0.83±0.07
2.14±0.40
10.41±0.21
4.04±0.35
4.31±0.43
0.038±0.014
0.046±0.022
0.23±0.05
0.060±0.022
8.08±0.17
1.74±0.05
9.23±0.02
15.26±3.01
8.18±0,17
11.96±1.22
7.69±0.03
1.46±0.002
9.52±0.18
9.75±1.44
11.33±0.16
16.24±3.34
20.55±2.67
3.64±0.09
1.61±0.04
20.85±3.16
7.69±0.08
20.70±0.15
23.02±1.63
35.14±0.94
4.82±0.07
14.29±0.71
39.30±3.47
1.54±0.16
19.58±0.02
8.70±.034
4.72±0.02
11.44±0.13
0.65±0.06
42
3
196
16
7
21d
21e
21f
4-Cl-Ph
4-Br-Ph
4-I-Ph
30
8
21g
21h
21i
115
<1
7
2-F-Ph
21j
2-Cl-Ph
2-Br-Ph
2,4-(Cl)2-Ph
3-Br-4-F-Ph
3-Cl-4-F-Ph
C5H9
21k
21l
1
7
21m
21n
21o
21p
21q
21r
86
35
9
4-IPE-Ph
4-I-Ph
5
<1
<1
<1
15
1
4-IPE-Ph
4-I-Ph
21s
21t
4-I-Ph
22a
22b
22c
4-IPE-Ph
4-I-Ph
<1
<1
4
4-IPE-Ph
4-I-Ph
22d
22e
CH3
160
31
3
23a
23b
23c
(CH3)2CH2
(CH2)4CH
9
AZD9291
a The biological data are generated from at least three independent experiments.
lines in comparison with AZD9291, which indicated that the replacement of pyrimidine moiety in
AZD9291 with N-9-Diphenyl-9H-purin-2-amine scaffold via cyclization strategy maintained
potent cytotoxicity. In addition, the target compounds suppressed H1975 cells more effectively
than they suppressed A549 cells, which suggests that the observed potent efficacy might be
attributable to selective inhibition of the mutant form of EGFR. The most promising compounds
21n, 23a and 23b exhibited more potent antitumour activity against H1975 cell line than the
positive control AZD9291 with IC50 values from 0.025 μM to 0.046 μM. These compounds are
also evaluated against HT-29 colon cancer cells, which expressed a non-special gene type, to test
their toxic effects. As shown in Table 1 compounds 21n, 23a and 23b were less potent against
A549 (WT EGFR and k-Ras mutation) and HT-29 (non-special gene type) cells, showing a high
safety index. Therefore, compounds 21n, 23a and 23b might be promising candidates to overcome
drug resistance mediated by the EGFR T790 Mutant[25].
Our initial effort was to explore the cytotoxic activity of different substituent compounds on the

phenyl ring in order to understand the structure-activity relationship. Primarily, the introduction of
electron-withdrawing groups exhibited a positive effect on the cytotoxic activityon on H1975 cells,
such as compound 21f (R2=4-fluorophenyl, IC50 =0.061 μM ), 21i (R2 = 4-iodophenyl) and 21n
(R2 =3-bromo-4-fluorophenyl, IC50 =0.035 μM ), which are similar to or better than that of
AZD9291 (IC50 =0.060 μM ). However, compound 21f showed less selectivity between WT and
TL EGFR (the ratio of WT/TL was 7). By contrast, monoelectron-donating groups (EDGs) such as
4-methoxy analogues (21b-21c, 21e) exhibited a negative effect. Exceptively, compound 21d
exhibited a positive effect on the cytotoxic activity (IC50 =0.074 μM ). The greatly influenced in
cytotoxic potency was observed when halogen atom was introduced to the different position of
phenyl ring. For example, shifting the Cl atom from the para position (21g, IC50=0.085 μM against
H1975) to the ortho position (21k, IC50=1.71 μM against H1975) significant decreased inhibition
of H1975 cells, while a slightly reduced the inhibition on A549 and HT-29 cells. Moreover,
incorporation of another halogen atom (21m, R2 = 2, 4-dichloro, IC50 = 1.61 μM against H1975)
showed somewhat a decreased cellular activity compared with 4-chloro analogue (21g: R2 =
4-chloro), which indicated monosubstitution of phenyl is more preferred. Interestingly, Isopropyl
group at R2 position increased the TL activities and selectivity in a certain extent. Compared with
compound 21a, isopropyl substituted compound 21d was merely 1.5-fold potent in activity, but its
selectivity was increased by more than 3 folds. Based on the activities and selectivity, compound
21d and 21i were selected as leads to optimize.
After the optimization of R2 moiety, the modification of amine R1 groups was carried out. As
seen from the data on Table 1, replacing the ethylenediamine group with rigid N-methyl
piperazine group (21q, 21r) or morpholine group (21s, 21t) was lower tolerated and somewhat
decreased the biological activity. For example, compared with 21d (IC50 =0.074 μM ), compound
21q (IC50 =1.93μM ) decreased potency by approximately 26-fold in H1975 cancer, which
demonstrated that the N,N,N’-trimethylethane-1,2-diamine group was essential for high activity.
With regard to compounds 22a-22e, due to the presence of a hydroxyl group on the pyrimidine
core, they exhibited very weak against H1975 activity, with IC50 values of more than 0.23 μM. In
addition, the selectivity between WT and TL EGFR is low (WT / TL <10). Satisfactorily,
2,9-dianilinopurine derivative 23a (IC50 =0.038 μM) exhibited a significant enhancement of
potency against H1975 cells, as well as it showed less potent against A549 (IC50=6.08μM, WT
EGFR and k-Ras mutation) and HT-29 (IC50=8.70μM, non-special gene type) cells, showing a
high safety index. Further studies about 2,9-dianilinopurine derivative are in progress in our
laboratories and will be reported upon in the future.
2.3.2. Inhibitory activity against EGFR kinase
To rationalize their appealing cellular inhibition, the optimized compounds (21d, 21f, 21g, 21i,
21n, 23a and 23b) were evaluated using EGFR kinases with different status including EGFR
T790M/L858R, EGFR T790M, and WT EGFR. The results obtained using a well-established
mobility shift assay are summarized in Table 2, with AZD9291 as the positive controls.
As shown in Table 2, all seven tested compounds exhibited excellent EGFR T790M/L858R
enzymatic potency with IC50 values ranging from 2.1 to 5.1 nM, which were comparable to the
enzymatic activity of AZD9291.
The inhibitory activities of compounds 21n and 23a were particularly more pronounced with
IC50 values less than 3 nM. All the tested compounds showed low-level inhibition of WT EGFR
(IC50, 21.3-42nM) and among them, compounds 23a and 23b were up to 8-fold more selective

against EGFR T790M/L858R than they were against WT EGFR. Although 21n showed high
levels of inhibition of T790M/L858R activity, it was less selective between WT and TL EGFR.
Compound 23a showed the more potent activity with an IC50 value of 2.7 nM, which was
similar to that of the positive control, AZD9291 (IC50 = 2.0 nM), indicating that this compound
deserves further study with regard to its application in the treatment of cancer. So based on the
selectivity and activity data against these three EGFRs, compound 23a were selected to be further
evaluated the drug availability. In addition, it was noteworthy that compounds 21n and 23a
possessed distinct substructures in their skeletons, highlighting a potential for further investigation
of this series.
Table 2
Inhibitory activity against EGFR kinase
Compd
EGFR IC50 (nM)a
T790M/L858R
T790M
ND
5.8
WT
25
TLb/WT selectivity
4.2
3.2
3.9
3.5
2.3
2.7
5.1
2.0
6.0
5.6
7.0
4.9
4.3
8.3
8.0
8.4
21d
21f
18.2
15.6
17
6.4
21g
4.7
21i
ND
3.5
10
21n
23a
22.4
41
ND
3.1
23b
16.7
AZD9291
ND: not determined.
a The biological data are means from at least two replicated experiments.
b TL represents EGFR T790M/L858R.
3. Molecular modeling analysis
To further elucidate the binding mode of compounds, a detail docking analysis was performed.
In our study, the co-crystal structure of WZ4002 with EGFRT790M was selected as the docking
model (PDB ID code: 3IKA ). The docking simulation was conducted using Glide XP
(Schrodinger 2014), since Glide uses a hierarchical series of filters to search for possible locations
of the ligand in the active-site region of the receptor. The shape and properties of the receptor are
represented on a grid by several different sets of fields that provide progressively more accurate
scoring of the ligand poses. The image files were generated using Accelrys DS visualizer 4.0
system. The binding model was exemplified by the interaction of compound 23a with EGFRT790M
.
As shown in Fig.5, compound 23a could tightly contact with EGFRT790M through several
important binding forces, including: (1) The purin core of the molecule formed two hydrogen
bonds with Met793 residue in the kinase hinge; (2) A covalent bond between the acryl amide and
the amino acid Cys797; (3) Furthermore, several vander Waals interactions existed between 23a
and the residues of protein, such as the purin ring with Ala743、Met790, and phenyl ring with
Met766 and ASP855, respectively; (4)The pep interaction is formed between the phenyl ring and
Lys745. All these interactions contribute to the tight binding and greatly enhance the inhibitory
potency of 23a. Compared with AZD9291, van der Waals forces of purine core with Met790 and
the pep interaction of aniline with Lys745 increased the effect with EGFRT790. However, the
aniline of compound 23a lacks the hydrogen bond interaction with EGFRT790M, so that further

structural modifications will be focused on the hydrogen bond donor / acceptor substituents in the
aniline.
Fig. 5. The EGFRT790M enzyme (PDB code: 3IKA) active site in complex with compound 23a. Compound 23a was shown in colored
sticks (yellow:carbon atom, blue: nitrogen atom, red: oxygen atom). H-bonding interactions between the 23a and EGFRT790M were
indicated with dotted lines in green. pep interactions were shown with dotted lines in pink. (For interpretation of the references to color in
this figure legend, the reader is referred to the web version of this article.)
In addition, we wanted the new compound to be “druglike” with respect to their predicted
physicochemical properties. One of our success criteria was that the resulting compounds should
be lead-like with respect to their physicochemical properties. As a metric to demonstrate
lead-likeness, we chose to evaluate the lipophilic ligand efficiency (LIPE)26-29 of our most potent
analogues and compare them to AZD9291. It was gratifying to note that 23a displayed very good
predicted druglike characteristics (LIPE 4.44), comparing favorably to AZD9291, the results are
shown in Table 3. Although the predicted water solubility was equivalent to that of AZD9291
(solubility at pH 7.4 in water: <0.01 mg/mL)30, further analysis showed 23a to have a favorable
topological polar surface area (TPSA)31 value of 109 Å2.

Table 3
Calculated physicochemical parameters of 23a and AZD9291
cLogPa
4.13
cLogDa
3.01
LIPEb
4.44
Compound
23a
pIC50
8.57
8.70
MWt
515.6
499.6
4.49
2.65
4.21
AZD9291
aCalculated using instant JChem. bLIPE = pIC50 − cLogP.
4. 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
structureactivity relationship (SAR) was discussed in detail and the results demonstrated that
substitution with bearing a N,9-Diphenyl-9H-purin-2-amine scaffold was considered as a novel
strategy for maintaining EGFR T790M inhibitory activity and selectivity. Among the most
promising inhibitors, compound 23a potently inhibited the enzymatic function of EGFR
T790M/L858R with an IC50 value of 2.7 nM and NSCLC H1975 cell with an IC50 value of 38 nM.
It was also less potent against A549 (WT EGFR and k-Ras mutation) and HT-29 (non-special
gene type) cells, showing a high safety index. Subsequent satisfactory predictions were obtained
by predicting the physicochemical properties of compound 23a. Further studies on structural
optimization and biological activities about these derivatives are still underway in our laboratory
and will be reported in the future.
5. Experimental section
5.1. Chemistry
Reagents and solvents were obtained from commercial sources and used without further purification.
The purity of the synthesized compounds was measured by high performance liquid chromatography
(HPLC, Agilent, USA). Flash chromatography was performed using silica gel (200-300 mesh). All
reactions were monitored by TLC on silica gel plates. Melting points were deter determined on Büchi
1
Melting Point B-540 apparatus (Büchi Labortechnik, Flawil, Switzerland). H NMR and 13C NMR
spectra were recorded on Bruker ARX-400, 400MHz or Bruker ARX-600, 600 MHz spectrometers
(Bruker Bioscience Billerica, MA, USA) with TMS as an internal standard. Mass spectra (MS) were
measured in ESI mode on an Aglient 1100 LC-MS (Agilent, Palo Alto, CA, USA). Elemental analysis
was determined on a Carlo-Erba 1106 Elemental analysis instrument (Carlo Erba, Milan, Italy). The IR
spectra were recorded by means of the KBr pellet technique on a Bruker FTS 135 spectrometer.
5.2 Preparation of 4-Fluoro-2-methoxy-5-nitro-phenylamine (8)
4-Fluoro-2-methoxyaniline(12.0g, 85.0 mmol) was added to cooled concentrated H2SO4 (80 mL).
The mixture was stirred at 0-10°C for 15-30 min. KNO3 (8.6g, 85.0 mmol) was added to the mixture.
The resulting mixture was stirred at 0-5°C for 1-2 h and then poured into ice/water. The mixture was
neutralised with concentrated K2CO3. The resulting solid was filtered off and dried under vacuum. The
1
corresponding amine was used in the next step without further purification. H NMR (400 MHz,
DMSO) δ 7.35 (d, J = 7.8 Hz, 1H), 7.04 (d, J = 13.4 Hz, 1H), 5.28 (s, 1H), 3.91 (s, 1H).

5.3. Preparation of tert-butyl (4-fluoro-2-methoxy-5-nitrophenyl)carbamate (9)
A solution of 8 (8.0g, 43.0 mmol) in CH2Cl2 (100 mL) was cooled to 0-5°C in an ice/water bath.
Boc2O (9.4g, 43.0 mmol) in DCM (20 mL) was added to the mixture slowly. The progress of the
reaction was monitored by TLC. After completion of the reaction, the reaction mixture was
concentrated to dryness under reduced pressure. The crude product was purified by flash silica
chromatography, with gradient 10−40% EtOAc in hexane to yield intermediate (9) (12.0 g, 42.1 mmol,
98%) as a yellow solid. m.p. 88-90 °C (EtOAc /hexane); 1H NMR (400 MHz, CDCl3) δ 8.91 (br, 1H),
6.99 (s, 1H), 6.72 (d, J=12.0, 1H), 4.0 (s, 3H), 1.567 (s, 9H); 13C NMR (100 MHz, CDCl3) δ 153.5,
152.6, 152.5,152.2, 150.9, 124.7, 124.7, 114.7, 100.0, 99.7, 56.8, 28.3, 7.9; IR 3432, 2985, 1723, 1536,
1485, 1158. MS calcd for C12H15FN2O5[M-H]— 285.0887; found: 285.0865
5.4 General procedure for preparation of compounds (10a-c)
Appropriate alkyl amine (2.7 g, 27.0 mmol) was added to a solution of intermediate (9) (6.3g, 22.0
mmol) and DIPEA (N,N-Diisopropylethylamine) (3.82 mL, 22.0 mmol) in DMF (100 mL). The
mixture was heated to 60 °C and stirred at this temperature for 2h. Water (200 mL) was added to the
reaction mixture, which was then extracted with DCM (50 mL*3). The combined extracts were dried
over anhydrous magnesium sulfate. The solvent was removed under vacuum to afford compounds
10a-c, respectively, which were used without further purification.
5.4.1. tert-butyl (4-((2-(dimethylamino)ethyl)(methyl)amino)- 2-methoxy-5 -nitrophenyl)
carbamate(10a)
1H NMR (400MHz，DMSO-d6) δ 8.12 (m, 2H), 6.74 (s, 1H), 3.90 (s, 3H), 3.21 (m, 2H), 2.81 (s, 3H),
2.44 (m, 2H), 2.14 (s, 6H), 1.45 (s, 9H); 13C NMR (100MHz，DMSO-d6) δ 153.5, 144.9, 132.1, 120.0,
102.4, 79.8, 56.8, 56.7, 53.1, 45.9, 40.8, 40.6, 40.4, 28.5, 27.9; IR (KBr) cm-1: 3441, 2979, 2359, 1708,
1546, 1162; ESI-MS m/z: 369.2 [M+H]+.
5.4.2. tert-butyl (2-methoxy-4-(4-methylpiperazin-1-yl)-5-nitrophenyl)carbamate(10b)
Yellow solid. Yield: 78%. ESI-MS m/z: 367.2 [M +H]+.
5.4.3. tert-butyl (2-methoxy-4-morpholino-5-nitrophenyl)carbamate(10c)
Yellow solid. Yield: 75%. ESI-MS m/z: 354.3[M +H]+.
5.5. General procedure for preparation of compounds (11a-c)
To a solution of 10 (22.6 mmol) in ethanol (150 mL) was added 10% Pd/C (0.5 g). The reaction
mixture was stirred at room temperature under an atmosphere of H2 for 9 h. After completion of the
reaction, the resulting mixture was filtered through Celite, and the filtered catalyst was washed with
ethanol. The filtrate was concentrated under high vacuum to afford compound 11a-c, respectively.
5.5.1 tert-butyl (5-amino-4-((2-(dimethylamino)ethyl)(methyl)amino)- 2-methoxyphenyl)
carbamate(11a ).
Brown liquid. Yield: 78%. 1H NMR (DMSO-d6) δ 8.12 (m, 2H), 6.74 (s, 1H), 3.90 (s, 3H), 3.21 (m,
2H), 2.81 (s, 3H), 2.44 (m, 2H), 2.14 (s, 6H), 1.45 (s, 9H). ESI-MS m/z: 369.2 [M+H]+.
5.5.2. tert-butyl (5-amino-2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)carbamate(11b)

Brown liquid. Yield: 69%. ESI-MS m/z: 337.2 [M+H]+.
5.5.3. tert-butyl (5-amino-2-methoxy-4-morpholinophenyl)carbamate(11c)
Brown liquid. Yield: 75%. 1H NMR (400 MHz, DMSO-d6) δ 7.60 (s, 1H), 7.10 (s, 1H), 6.62 (s, 1H),
4.47 (s, 1H), 3.76 – 3.70 (m, 4H), 3.69 (s, 3H), 2.80 – 2.71 (m, 4H), 1.44 (s, 9H). ESI-MS m/z: 324.2
[M+H]+.
5.6. General procedure for preparation of compounds (12a-c)
To a solution of 11 (2.0 g, 7.9 mmol) and DIPEA (1.38 mL, 7.9 mmol) in CH2Cl2 (50 mL) at 0 °C,
acryloyl chloride (0.64 mL, 7.9 mmol) was added dropwise. The mixture was stirred for 1 h, then
extracted with water (2-50 mL). The organic layerwas dried over anhydrous Na2SO4, and evaporated to
give the crude residue. Compounds (12a-c) was obtained as a pale-yellow solid after purification by
silica gel column chromatography ((CH2Cl2:MeOH, 30:1 to 15:1 v/v).
5.6.1. tert-butyl 5-acrylamido-4-((2-(dimethylamino)ethyl)(methyl)amino) -2-methoxyphenyl)
carbamate(12a)
Brown solid. Yield: 54%. m.p. 92-94°C. ESI-MS m/z: 393.5[M+ H]+.
5.6.2. tert-butyl (5-acrylamido-2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)carbamate (12b).
Brown solid. Yield: 57%. ESI-MS m/z: 393.5[M+ H]+.
5.6.3. tert-butyl (5-acrylamido-2-methoxy-4-morpholinophenyl)carbamate(12c)
Brown solid. Yield: 65%. 1H NMR (400 MHz, DMSO-d6) δ 9.06 (s, 1H), 8.17 (s, 1H), 7.88 (s, 1H),
6.80 (s, 1H), 6.63 (dd, J = 16.8, 10.6 Hz, 1H), 6.21 (d, J = 17.1 Hz, 1H), 5.72 (d, J = 10.1 Hz, 1H), 3.80
(s, 3H), 3.77 (s, 4H), 2.80 (s, 4H), 1.44 (s, 9H).ESI-MS m/z: 378.2[M+ H]+.
5.7. General procedure for preparation of compounds (13a-c)
TFA (20 mmol) was added to a solution of 12 (2 mmol) in CH2Cl2 (50 mL) at room temperature.
The reaction mixture was stirred for 2-7 h, and was monitored by TLC. The mixture was extracted with
water (2*100 mL), the combined aqueous layer was neutralized with aqueous K2CO3 to pH 7-8. The
precipitate was filtered and dried to generate the compounds 13a-c, respectively.
5.7.1. N-(5-amino-2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxyphenyl)acrylamide (13a)
1
Gray solid. Yield: 75.2%. H NMR (400 MHz, DMSO-d6) δ 9.94 (s, 1H), 7.59 (s, 1H), 6.78 (s, 1H),
6.45 (s, 1H), 6.19 (dd, J = 16.9, 1.9 Hz, 1H), 5.69 (dd, J = 10.1, 1.9 Hz, 1H), 4.64 (s, 2H), 3.74 (s, 3H),
2.86 (s, 2H), 2.57 (s, 3H), 2.29 (s, 8H); 13C NMR (100 MHz, DMSO-d6) δ 163.3, 143.6, 133. 9, 132.4,
132.3, 129.9, 125.9, 107.1, 105.2, 57.2, 55.8, 45.15, 44.2; IR 3452, 3342, 2935, 2823, 1670, 1526, 1207;
ESI-MS m/z: 293.2[M+ H]+
5.7.2. N-(5-amino-4-methoxy-2-(4-methylpiperazin-1-yl)phenyl)acrylamide(13b)
Gray solid. Yield: 73.1%. ESI-MS m/z: 291.2[M+ H]+
5.7.3. N-(5-amino-4-methoxy-2-morpholinophenyl)acrylamide(13c)
Gray solid. Yield: 67.3%. ESI-MS m/z: 278.1[M+ H]+

5.8. General procedure for preparation of compounds (16a-t)
A solution of 13 (3.0 g, 12.4 mmol) and 15 (2.2 g, 12.4 mmol) in 1,4-dioxane/IPA (20 mL) added
10% TFA (300 mg), and the resulting suspension was stirred at 25°C under an atmospheric pressure of
N2 for 12 h. The resulting precipitate was collected by filtration, rinsing with EtOH, to give compounds
16a-t as its hydrochloride salt.
5.8.1. N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((5-nitro-4-(phenylamino)pyrimidin
-2-yl)amino)phenyl)acrylamide(16a)
Yellow solid; Yield: 58.3%; 1H NMR (400 MHz, DMSO-d6 ) δ 10.12 (s, 1H), 8.75 (s, 1H), 7.75 (dd,
J = 15.1, 3.1 Hz, 2H), 7.33 (t, J = 15.0 Hz, 2H), 7.12 (s, 1H), 7.05 (t, J = 3.0 Hz, 1H), 6.43 (s, 1H), 6.28
(dd, J = 34.1, 19.2 Hz, 2H), 6.05 (dd, J = 20.0, 4.6 Hz, 1H), 5.69 (dd, J = 33.3, 4.6 Hz, 1H), 3.86 (s,
3H), 3.57 – 3.47 (m, 2H), 2.75 (s, 3H), 2.50 (t, J = 10.4 Hz, 2H), 2.21 (s, 6H). MS (ESI) m/z(%): 492.4
[M+H]+, 514.4 [M+Na]+.
5.8.2. N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((4-((4-methoxyphenyl)amino)-5-nit
ropyrimidin-2-yl)amino)phenyl)acrylamide(16b)
Yellow solid; Yield: 55.6%; MS (ESI) m/z(%): 537.2 [M+H]+, 559.2 [M+Na]+.
5.8.3. N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((5-nitro-4-(p-tolylamino)pyrimidin
-2-yl)amino)phenyl)acrylamide(16c)
Yellow solid; Yield: 50.1%; MS (ESI) m/z(%): 521.3 [M+H]+, 543.3 [M+Na]+.
5.8.4. N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-5-((4-((4-isopropylphenyl)amino)-5-nitropyrimid
in-2-yl)amino)-4-methoxyphenyl)acrylamide(16d)
Yellow solid; Yield: 43.7%; MS (ESI) m/z(%): 549.3 [M+H]+, 556.2 [M+Na]+.
5.8.5. N-(5-((4-((4-ethylphenyl)amino)-5-nitropyrimidin-2-yl)amino)-4-methoxy-2-morpholinophenyl)
acrylamide(16e)
Yellow solid; Yield: 45.5%; MS (ESI) m/z(%): 520.2 [M+H]+, 571.3 [M+Na]+
5.8.6. N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-5-((4-((4-fluorophenyl)amino)-5-nitropyrimidin-
2-yl)amino)-4-methoxyphenyl)acrylamide(16f)
Yellow solid; Yield: 67.5%; MS (ESI) m/z(%): 525.2 [M+H]+, 547.2 [M+Na]+
5.8.7. N-(5-((4-((4-chlorophenyl)amino)-5-nitropyrimidin-2-yl)amino)-2-((2-(dimethylamino)ethy
l)(methyl)amino)-4-methoxyphenyl)acrylamide(16g)
Light yellow solid; Yield: 72.1%; 1H NMR (400 MHz, DMSO-d6) δ 10.30 (s, 1H), 9.07 (s, 1H), 8.19 (s,
1H), 7.74 (s, 1H), 7.19 (s, 2H), 7.00 (s, 2H), 6.22 (dd, J = 26.4, 9.0 Hz, 2H), 5.74 – 5.67 (m, 1H), 3.76
(s, 3H), 2.96 (s, 2H), 2.69 – 2.64 (m, 3H), 2.31 (s, 8H). MS (ESI) m/z(%): 541.2 [M+H]+, 563.2
[M+Na]+
5.8.8. N-(5-((4-((4-bromophenyl)amino)-5-nitropyrimidin-2-yl)amino)-2-((2-(dimethylamino)ethy
l)(methyl)amino)-4-methoxyphenyl)acrylamide(16h)

Yellow solid; Yield: 69.5%; MS (ESI) m/z(%): 585.1 [M+H]+, 607.1 [M+Na]+
5.8.9. N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-5-((4-((4-iodophenyl)amino)-5-nitropyrimidin-2-
yl)amino)-4-methoxyphenyl)acrylamide(16i)
1
Yellow solid; Yield: 73.5%; H NMR (400 MHz, DMSO-d6) δ 10.38 (s, 1H), 9.04 (s, 1H), 8.16 (s,
1H), 7.71 (s, 2H), 7.53 (s, 2H), 7.23 (s, 1H), 7.07 (s, 1H), 6.82 (s, 1H), 6.52 (s, 1H), 6.24 (s, 1H), 5.74
(s, 1H), 3.78 (s, 3H), 2.93 (s, 2H), 2.70 (s, 3H), 2.24 (d, J = 36.8 Hz, 8H). MS (ESI) m/z(%): 633.1
[M+H]+, 655.1 [M+Na]+
5.8.10. N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-5-((4-((2-fluorophenyl)amino)-5-nitropyrimidin
-2-yl)amino)-4-methoxyphenyl)acrylamide(16j)
Yellow solid; Yield: 73.5%; MS (ESI) m/z(%): 525.2 [M+H]+, 563.2 [M+K]+
5.8.11. N-(5-((4-((2-chlorophenyl)amino)-5-nitropyrimidin-2-yl)amino)-2-((2-(dimethylamino)eth
yl)(methyl)amino)-4-methoxyphenyl)acrylamide(16k)
Yellow solid; Yield: 75.5%; MS (ESI) m/z(%): 541.2[M+H]+, 563.2 [M+Na]+
5.8.12. N-(5-((4-((2-bromophenyl)amino)-5-nitropyrimidin-2-yl)amino)-2-((2-(dimethylamino)eth
yl)(methyl)amino)-4-methoxyphenyl)acrylamide(16l)
Yellow solid; Yield: 78.5%; 1H NMR (400 MHz, DMSO-d6) δ 10.03 (s, 1H), 9.11 (s, 1H), 8.14 (d, J
= 33.3 Hz, 2H), 7.60 (s, 1H), 7.01 (s, 2H), 6.73 (s, 1H), 6.21 (dd, J = 18.7, 13.5 Hz, 1H), 5.73 (dd, J =
12.8, 9.0 Hz, 1H), 3.79 (d, J = 19.8 Hz, 3H), 3.04 (s, 2H), 2.69 (s, 3H), 2.38 (s, 8H); MS (ESI) m/z(%):
585.1[M+H]+, 607.1 [M+Na]+
5.8.13. N-(5-((4-((2,4-dichlorophenyl)amino)-5-nitropyrimidin-2-yl)amino)-2-((2-(dimethylamino)ethy
l)(methyl)amino)-4-methoxyphenyl)acrylamide(16m)
Yellow solid; Yield: 62.5%; MS (ESI) m/z(%): 575.2[M+H]+, 597.2 [M+Na]+
5.8.14. N-(5-((4-((4-bromo-3-fluorophenyl)amino)-5-nitropyrimidin-2-yl)amino)-2-((2-(dimethylamino)
ethyl)(methyl)amino)-4-methoxyphenyl)acrylamide(16n)
Yellow solid; Yield: 67.5%; 1H NMR (400 MHz, DMSO-d6) δ 10.09 (s, 1H), 9.04 (s, 1H), 8.18 (dd,
J = 6.3, 2.5 Hz, 1H), 8.01 (s, 1H), 7.69 – 7.66 (m, 1H), 7.39 (s, 1H), 7.27 (s, 1H), 6.96 (d, J = 1.9 Hz,
1H), 6.21 (d, J = 16.4 Hz, 1H), 5.71 (dd, J = 9.6, 1.0 Hz, 1H), 3.80 (s, 3H), 3.25 (s, 2H), 3.12 (s, 3H),
2.76 (d, J = 4.8 Hz, 6H), 2.60 (s, 2H); MS (ESI) m/z(%): 603.1[M+H]+, 625.1 [M+Na]+
5.8.15. N-(5-((4-((3-chloro-4-fluorophenyl)amino)-5-nitropyrimidin-2-yl)amino)-2-((2-(dimethylamino)
ethyl)(methyl)amino)-4-methoxyphenyl)acrylamide(16o)
Yellow solid; Yield: 56.3%; MS (ESI) m/z(%): 559.2[M+H]+, 582.2 [M+Na]+
5.8.16. N-(5-((4-(cyclopentylamino)-5-nitropyrimidin-2-yl)amino)-2-((2-(dimethylamino)ethyl)(methyl)
amino)-4-methoxyphenyl)acrylamide(16p)
Yellow solid; Yield: 58.6%; MS (ESI) m/z(%): 499.3[M+H]+, 521.3[M+Na]+

5.8.17. N-(5-((4-((4-isopropylphenyl)amino)-5-nitropyrimidin-2-yl)amino)-4-methoxy-2-(4-methylpipe
razin-1-yl)phenyl)acrylamide(16q)
Yellow solid; Yield: 77.6%; MS (ESI) m/z(%): 547.3[M+H]+, 569.3[M+Na]+
5.8.18. N-(5-((4-((4-iodophenyl)amino)-5-nitropyrimidin-2-yl)amino)-4-methoxy-2-(4-methylpiperazin
-1-yl)phenyl)acrylamide(16r)
Yellow solid; Yield: 73.4%; MS (ESI) m/z(%): 631.1[M+H]+, 653.1[M+Na]+
5.8.19. N-(5-((4-((4-isopropylphenyl)amino)-5-nitropyrimidin-2-yl)amino)-4-methoxy-2-morpholinoph
enyl)acrylamide(16s)
Yellow solid; Yield: 54.5%; MS (ESI) m/z(%): 534.2[M+H]+, 556.2[M+Na]+
5.8.20. N-(5-((4-((4-iodophenyl)amino)-5-nitropyrimidin-2-yl)amino)-4-methoxy-2-morpholinophenyl)
acrylamide(16t)
Yellow solid; Yield: 59.5%; 1H NMR (600 MHz, DMSO-d6) δ 10.21 (s, 1H), 9.10 (d, J = 45.0 Hz,
2H), 7.93 (s, 1H), 7.38 (d, J = 70.3 Hz, 3H), 6.93 (s, 1H), 6.76 – 6.60 (m, 1H), 6.20 (d, J = 16.9 Hz,
1H), 5.73 (d, J = 10.0 Hz, 1H), 3.85 (s, 3H), 3.75 (s, 4H), 2.92 (s, 4H); MS (ESI) m/z(%):618.1[M+H]+,
640.1[M+Na]+
5.9. General procedure for preparation of compounds (21a-t)
Compound 16 was dissolved in a mixture of EtOH and water ((v/v) 3.6/1). Iron powder and NH4Cl
were added to it, and the mixture was then stirred in reflux for 8 h, cooled to room temperature, and
filtered through a pad of Celite. The filtrate was concentrated in vacuo. The residue was extracted with
EtOAc, and the organic extract was washed with saturated NaHCO3, water, and brine and dried over
anhydrous MgSO4 . It was then filtered and concentrated in vacuo to generate the compounds 17. A
solution of 17 (3 mmol), frmic acid (1.5 equiv) and PPA (1.5 equiv) in a mixture of DMF (10 mL) was
stirred at 125°C for 4h. The volatiles were evaporated in vacuo, and the residue was purified by column
chromatography (CH2Cl2/MeOH 15:1) to generate the compounds 21a-t, respectively.
5.9.1. N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((9-phenyl-9H-purin-2-yl)amino)ph
enyl)acrylamide (21a)
Yield: 75.6%, M.p.:134.2-135.1oC ; H NMR (400 MHz, DMSO-d6) δ 10.13 (s, 1H), 8.90 (d, J = 6.7
1
Hz, 2H), 8.69 (s, 1H), 8.22 (s, 1H), 7.96 (d, J = 7.8 Hz, 2H), 7.51 (t, J = 7.8 Hz, 2H), 7.38 (t, J = 7.4 Hz,
1H), 6.99 (s, 1H), 6.52 (s, 1H), 6.28 (d, J = 16.4 Hz, 1H), 5.80 (d, J = 10.1 Hz, 1H), 3.84 (s, 3H), 2.90
(s, 2H), 2.68 (s, 3H), 2.38 (s, 2H), 2.26 (s, 6H); MS (ESI) m/z(%): 487.2 [M+H]+; Anal. calcd. for
C26H30N8O2 (%): C, 64.18; H, 6.21; N, 23.03; Found (%): C, 64.22; H, 6.17; N, 23.07.
5.9.2. N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((9-(4-methoxyphenyl)-9H-purin-2-
yl)amino)phenyl)acrylamide(21b)
Yield: 65.5%, M.p.:143.7-144.5oC; H NMR (400 MHz, CDCl3) δ 9.57 (s, 1H), 9.32 (s, 1H), 8.80 (s,
1
1H), 8.17 (s, 1H), 7.70 (d, J = 8.8 Hz, 2H), 7.04 (d, J = 8.9 Hz, 2H), 6.66 (s, 1H), 6.52 (dd, J = 16.8,
1.6 Hz, 1H), 5.80 (dd, J = 10.1, 1.6 Hz, 1H), 3.90 (s, 3H), 3.82 (s, 3H), 3.32 (s, 2H), 3.17 (s, 2H), 2.81
(s, 6H), 2.73 (s, 3H). MS (ESI) m/z(%): 517.3 [M+H]+; Anal. calcd. For C27H32N8O3 (%): C, 62.77; H,
6.24; N, 21.69;. Found (%): C, 62.80; H, 6.27; N, 21.65.

5.9.3. N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((9-(p-tolyl)-9H-purin-2-yl)amino)p
henyl)acrylamide(21c)
Yield: 78.6%, M.p.: 162.2-163.1oC; H NMR (400 MHz, DMSO-d6) δ 10.14 (s, 1H), 8.89 (d, J = 4.7
1
Hz, 2H), 8.65 (s, 1H), 8.17 (s, 1H), 7.83 (d, J = 8.3 Hz, 2H), 7.31 (d, J = 8.1 Hz, 2H), 6.98 (s, 1H), 6.50
(dd, J = 17.0, 10.1 Hz, 1H), 6.29 (dd, J = 16.9, 1.6 Hz, 1H), 5.82 (dd, J = 10.1, 1.6 Hz, 1H), 3.85 (s,
3H), 2.89 (s, 2H), 2.69 (s, 3H), 2.32 (s, 5H), 2.24 (s, 6H). MS (ESI) m/z(%): 517.3 [M+H]+; Anal.
calcd. For C27H32N8O2 (%): C, 64.78; H, 6.44; N, 22.38; Found (%): C, 64.85; H, 6.58; N, 23.29.
5.9.4. N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-5-((9-(4-isopropylphenyl)-9H-purin-2-yl)amino)-
4-methoxyphenyl)acrylamide(21d)
Yield: 63.2%, M.p.: 114.2-115.3oC; 1H NMR (400 MHz, DMSO-d6) δ 10.22 (s, 1H), 8.97 (s, 1H), 8.90
(s, 1H), 8.64 (s, 1H), 8.14 (s, 1H), 7.87 (d, J = 8.3 Hz, 2H), 7.37 (d, J = 8.2 Hz, 2H), 7.00 (s, 1H), 6.47
(dd, J = 16.7, 10.3 Hz, 1H), 6.33 (d, J = 16.8 Hz, 1H), 5.83 (d, J = 10.1 Hz, 1H), 3.84 (s, 3H), 2.88 –
2.82 (m, 2H), 2.70 (s, 3H), 2.29 – 2.24 (m, 2H), 2.21 (s, 6H). 13C NMR (100 MHz, DMSO-d6) δ 167.38,
154.45, 153.03, 150.50, 150.40, 149.68, 146.35, 136.06, 134.99, 130.02, 128.24, 127.95, 125.00,
124.33, 123.54, 122.63, 115.33, 107.54, 57.31, 56.83, 52.23, 45.57, 39.57, 33.96, 23.38. MS (ESI)
m/z(%): 529.2 [M+H]+; Anal. calcd. For C29H36N8O2 (%): C, 65.89; H, 6.86; N, 21.20; Found (%): C,
65.80; H, 6.78; N, 21.29.
5.9.5. N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-5-((9-(4-ethylphenyl)-9H-purin-2-yl)amino)-4-m
ethoxyphenyl)acrylamide(21e)
Yield: 71.2%, M.p.: 152.9-153.7oC; H NMR (400 MHz, DMSO-d6) δ 10.19 (s, 1H), 8.91 (d, J = 9.6
1
Hz, 2H), 8.65 (s, 1H), 8.17 (s, 1H), 7.86 (d, J = 8.3 Hz, 2H), 7.34 (d, J = 8.3 Hz, 2H), 6.99 (s, 1H), 6.48
(dd, J = 16.8, 10.1 Hz, 1H), 6.31 (dd, J = 17.0, 1.8 Hz, 1H), 5.82 (dd, J = 10.1, 1.8 Hz, 1H), 3.84 (s,
3H), 2.87 (t, J = 5.5 Hz, 2H), 2.68 (d, J = 12.7 Hz, 3H), 2.61 (dd, J = 15.4, 7.8 Hz, 2H), 2.32 (d, J = 5.3
Hz, 2H), 2.23 (s, 6H), 1.18 (t, J = 7.6 Hz, 3H).MS (ESI) m/z(%): 515.2 [M+H]+; Anal. calcd. For
C28H34N8O2 (%): C, 65.35; H, 6.66; N, 21.77; Found (%): C, 65.42; H, 6.75; N, 21.60.
5.9.6. N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-5-((9-(4-fluorophenyl)-9H-purin-2-yl)amino)-4-
methoxyphenyl)acrylamide(21f)
Yield: 62.6%, M.p.:186.6-187.5oC; 1H NMR (400 MHz, DMSO-d6) δ 10.12 (s, 1H), 8.90 (d, J = 6.1 Hz,
2H), 8.66 (s, 1H), 8.20 (s, 1H), 8.00 (s, 2H), 7.34 (t, J = 8.5 Hz, 2H), 6.99 (s, 1H), 6.49 (d, J = 9.8 Hz,
1H), 6.29 (dd, J = 17.0, 0.9 Hz, 1H), 5.88 – 5.75 (m, 1H), 3.85 (s, 3H), 2.90 (s, 2H), 2.69 (s, 3H), 2.37
(s, 2H), 2.29 (d, J = 25.9 Hz, 6H). MS (ESI) m/z(%): 505.2 [M+H]+; Anal. calcd. For C26H29FN8O2 (%):
C, 61.89; H, 5.79; N, 22.21. Found (%): C, 62.83; H, 5.84; N, 22.25.
5.9.7. N-(5-((9-(4-chlorophenyl)-9H-purin-2-yl)amino)-2-((2-(dimethylamino)ethyl)(methyl)amino)-4-
methoxyphenyl)acrylamide(21g)
Yield: 52.5%, M.p.:125.3-126.2oC; 1H NMR (400 MHz, DMSO-d6) δ 10.18 (s, 1H), 8.92 (d, J = 8.4 Hz,
2H), 8.71 (s, 1H), 8.23 (s, 1H), 8.04 (d, J = 8.7 Hz, 2H), 7.56 (d, J = 8.7 Hz, 2H), 7.00 (s, 1H), 6.49 (dd,
J = 17.0, 9.9 Hz, 1H), 6.30 (dd, J = 16.9, 1.7 Hz, 1H), 5.83 (dd, J = 10.3, 1.4 Hz, 1H), 3.85 (s, 3H),
2.88 (t, J = 5.6 Hz, 2H), 2.70 (s, 3H), 2.33 (d, J = 5.2 Hz, 2H), 2.23 (s, 6H). MS (ESI) m/z(%): 521.3

[M+H]+; Anal. calcd. For C26H29ClN8O2 (%): , C, 59.94; H, 5.61; N, 21.51; Found (%): C, 59.98; H,
5.57; N, 21.56
5.9.8. N-(5-((9-(4-bromophenyl)-9H-purin-2-yl)amino)-2-((2-(dimethylamino)ethyl)(methyl)amino)-4-
methoxyphenyl)acrylamide(21h)
Yield: 52.6%, M.p.:186.9-187.8oC; 1H NMR (400 MHz, DMSO-d6) δ 10.19 (s, 1H), 8.92 (d, J = 4.0 Hz,
2H), 8.71 (s, 1H), 8.23 (s, 1H), 7.98 (d, J = 8.7 Hz, 2H), 7.70 (d, J = 8.6 Hz, 2H), 7.01 (s, 1H), 6.47 (dd,
J = 16.9, 10.0 Hz, 1H), 6.30 (d, J = 16.5 Hz, 1H), 5.83 (d, J = 10.8 Hz, 1H), 3.85 (s, 3H), 2.87 (s, 2H),
2.70 (s, 3H), 2.29 (s, 2H), 2.22 (s, 6H). MS (ESI) m/z(%): 565.2 [M+H]+, 567.2 [M+H]; Anal. calcd.
For C26H29BrN8O2 (%): , C, 55.23; H, 5.17; N, 19.82; Found (%): C, 55.27; H, 5.21; N, 19.86.
5.9.9. N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-5-((9-(4-iodophenyl)-9H-purin-2-yl)amino)-4-me
thoxyphenyl)acrylamide(21i)
Yield: 62.4%, M.p.:154.1-155.2oC; 1H NMR (400 MHz, DMSO-d6) δ 10.22 (s, 1H), 8.92 (d, J = 10.0
Hz, 2H), 8.70 (s, 1H), 8.22 (s, 1H), 7.84 (s, 4H), 7.13 – 6.86 (m, 1H), 6.70 – 6.03 (m, 2H), 5.84 (dd, J =
8.1, 1.6 Hz, 1H), 3.85 (s, 3H), 2.86 (s, 2H), 2.71 (s, 3H), 2.24 (d, J = 21.9 Hz, 8H). MS (ESI) m/z(%):
613.4 [M+H]+; Anal. calcd. For C26H29IN8O2 (%): C, 50.99; H, 4.77; N, 18.30; Found (%): C, 50.87; H,
4.70; N, 18.18.
5.9.10. N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-5-((9-(2-fluorophenyl)-9H-purin-2-yl)amino)-4-
methoxyphenyl)acrylamide(21j)
Yield: 66.2%, M.p.:154.1-155.2oC; 1H NMR (400 MHz, DMSO-d6) δ 9.99 (s, 1H), 9.51 (s, 1H), 8.84 (s,
1H), 8.24 (s, 2H), 7.45 (d, J = 61.5 Hz, 2H), 6.95 (s, 2H), 6.44 (s, 1H), 6.22 (d, J = 17.2 Hz, 1H), 5.79 –
5.66 (m, 1H), 3.83 (s, 3H), 2.89 (s, 2H), 2.66 (s, 3H), 2.25 (s, 8H). MS (ESI) m/z(%): 505.6 [M+H]+;
Anal. calcd. For C26H29FN8O2 (%): C, 61.89; H, 5.79;N, 22.21; Found (%): C, 61.81; H, 5.70; N, 22.15.
5.9.11. N-(5-((9-(2-chlorophenyl)-9H-purin-2-yl)amino)-2-((2-(dimethylamino)ethyl)(methyl)amino)-4
-methoxyphenyl)acrylamide(21k)
Yield: 71.2%, M.p.:164.1-165.2oC; 1H NMR (400 MHz, DMSO-d6) δ 9.99 (s, 1H), 9.51 (s, 1H), 8.84 (s,
1H), 8.24 (s, 2H), 7.45 (d, J = 61.5 Hz, 2H), 6.95 (s, 2H), 6.44 (s, 1H), 6.22 (dd, J = 16.9, 1.2 Hz, 1H),
5.79 – 5.66 (m, 1H), 3.83 (s, 3H), 2.89 (s, 2H), 2.66 (s, 3H), 2.25 (s, 8H). MS (ESI) m/z(%): 521.2
[M+H]+; Anal. calcd. For C26H29ClN8O2(%): C, 59.94; H, 5.61; N, 21.51; Found (%): C, 59.82; H, 5.69;
N, 21.71.
5.9.12. N-(5-((9-(2-bromophenyl)-9H-purin-2-yl)amino)-2-((2-(dimethylamino)ethyl)(methyl)amino)-4
-methoxyphenyl)acrylamide(21l)
Yield: 65.2%, M.p.:123.9-124.6oC; 1H NMR (400 MHz, DMSO-d6) δ 10.01 (s, 1H), 8.86 (s, 1H), 8.65
(s, 1H), 8.41 (s, 1H), 8.20 (s, 1H), 7.88 (d, J = 7.8 Hz, 1H), 7.73 (d, J = 7.5 Hz, 1H), 7.56 (t, J = 7.5 Hz,
1H), 7.48 (t, J = 7.1 Hz, 1H), 6.93 (s, 1H), 6.41 (dd, J = 15.8, 9.7 Hz, 1H), 6.24 (d, J = 16.7 Hz, 1H),
5.76 (d, J = 9.9 Hz, 1H), 3.78 (s, 3H), 2.86 (s, 2H), 2.66 (s, 3H), 2.32 (s, 2H), 2.22 (s, 6H). MS (ESI)
m/z(%): 565.2 [M+H]+, 567.2 [M+H]; Anal. calcd. For C26H29BrN8O2(%): C, 55.23; H, 5.17; N, 19.82;
Found (%): C, 55.34; H, 5.25; N, 19.73.

5.9.13. N-(5-((9-(2,4-dichlorophenyl)-9H-purin-2-yl)amino)-2-((2-(dimethylamino)ethyl)(methyl)amin
o)-4-methoxyphenyl)acrylamide(21m)
Yield: 47.5%, M.p.:182.9-183.8oC; 1H NMR (400 MHz, DMSO-d6) δ 10.01 (s, 1H), 8.88 (s, 1H), 8.77
(s, 1H), 8.42 (s, 1H), 8.15 (s, 1H), 7.91 (d, J = 2.1 Hz, 1H), 7.80 (d, J = 8.6 Hz, 1H), 7.58 (dd, J = 8.6,
2.1 Hz, 1H), 6.95 (s, 1H), 6.37 (dd, J = 16.7, 10.0 Hz, 1H), 6.26 (dd, J = 16.7, 1.7 Hz, 1H), 5.77 (dd, J
= 9.6, 2.0 Hz, 1H), 3.80 (s, 3H), 2.83 (t, J = 5.8 Hz, 2H), 2.67 (s, 3H), 2.25 (t, J = 5.7 Hz, 2H), 2.18 (s,
6H). 13C NMR (100 MHz, DMSO) δ 162.86, 157.51, 153.31, 150.27, 147.07, 143.82, 138.86, 134.95,
132.69, 132.00, 131.58, 131.05, 130.40, 128.89, 127.80, 127.49, 126.58, 125.23, 105.59, 56.93, 56.46,
55.38, 49.06, 45.23, 42.95. MS (ESI) m/z(%): 555.5 [M+H]+; Anal. calcd. For C26H28Cl2N8O2 (%): C,
56.22; H, 5.08; N, 20.17; Found (%): C, 56.12; H, 5.16; N, 20.26.
5.9.14. N-(5-((9-(3-bromo-4-fluorophenyl)-9H-purin-2-yl)amino)-2-((2-(dimethylamino)ethyl)(methyl)
amino)-4-methoxyphenyl)acrylamide(21n)
Yield: 57.5%, M.p.:114.9-115.8oC; 1H NMR (400 MHz, DMSO-d6) δ 10.02 (s, 1H), 8.89 (s, 1H), 8.80
(s, 1H), 8.68 (s, 1H), 8.36 – 8.26 (m, 2H), 8.13 – 8.05 (m, 1H), 7.48 (t, J = 8.8 Hz, 1H), 7.00 (s, 1H),
6.43 (s, 1H), 6.21 (dd, J = 16.8, 1.6 Hz, 1H), 5.76 (dd, J = 10.6, 1.5 Hz, 1H), 3.84 (s, 3H), 2.89 (s, 2H),
2.69 (s, 3H), 2.28 (d, J = 33.4 Hz, 8H).MS (ESI) m/z(%): 582.9 [M+H]+，584.9 [M+H]+; Anal. calcd.
For C26H28BrFN8O2 (%): C, 53.52; H, 4.84; N, 19.21; Found (%): C, 53.42; H, 4.90; N, 19.26.
5.9.15. N-(5-((9-(3-chloro-4-fluorophenyl)-9H-purin-2-yl)amino)-2-((2-(dimethylamino)ethyl)(methyl)
amino)-4-methoxyphenyl)acrylamide(21o)
Yield: 47.5%, M.p.:181.3-182.3oC; 1H NMR (400 MHz, DMSO-d6) δ 9.99 (s, 1H), 8.89 (s, 1H), 8.71 (s,
2H), 8.35 (s, 1H), 8.24 (dd, J = 6.3, 2.6 Hz, 2H), 8.09 – 8.01 (m, 2H), 7.87 (d, J = 8.0 Hz, 1H), 7.54 (t,
J = 9.0 Hz, 1H), 6.97 (s, 1H), 6.20 (dd, J = 16.7, 1.1 Hz, 1H), 5.73 (dd, J = 10.2, 1.2 Hz, 1H), 3.85 (s,
3H), 3.21 (s, 2H), 3.18 – 3.11 (m, 3H), 2.62 (d, J = 11.7 Hz, 8H).MS (ESI) m/z(%): 539.5 [M+H]+;
Anal. calcd. For C26H28ClFN8O2 (%): C, 57.94; H, 5.24; N, 20.79; Found (%): C, 57.82; H, 5.32; N,
20.68.
5.9.16. N-(5-((9-cyclopentyl-9H-purin-2-yl)amino)-2-((2-(dimethylamino)ethyl)(methyl)amino)-4-meth
oxyphenyl)acrylamide(21p)
Yield: 78.5%, M.p.:123.9-124.8oC; 1H NMR (400 MHz, DMSO-d6) δ 10.11 (s, 1H), 9.45 (s, 1H), 8.77
(s, 1H), 8.35 (s, 1H), 7.94 (s, 1H), 7.00 (s, 1H), 6.44 (dd, J = 16.7, 9.7 Hz, 1H), 6.23 (dd, J = 17.0, 1.6
Hz, 1H), 5.74 (dd, J = 10.3, 0.9 Hz, 1H), 3.88 (s, 3H), 2.92 – 2.83 (m, 2H), 2.69 (s, 3H), 2.38 – 2.29 (m,
2H), 2.23 (s, 6H). MS (ESI) m/z(%): 479.2 [M+H]+; Anal. calcd. For C25H34N8O2 (%): C, 62.74; H,
7.16; N, 23.41; Found (%): C, 62.65; H, 7.23; N, 23.55.
5.9.17. N-(5-((9-(4-isopropylphenyl)-9H-purin-2-yl)amino)-4-methoxy-2-(4-methylpiperazin-1-yl)phen
yl)acrylamide(21q)
Yield: 57.7%, M.p.:215.5-216.4oC; 1H NMR (400 MHz, DMSO-d6) δ 9.04 (s, 1H), 8.89 (s, 1H), 8.68 (s,
1H), 8.65 (s, 1H), 8.11 (s, 1H), 7.85 (d, J = 8.4 Hz, 2H), 7.40 (d, J = 8.4 Hz, 2H), 6.85 (s, 1H), 6.70 (dd,
J = 16.9, 10.2 Hz, 1H), 6.31 (dd, J = 17.0, 1.5 Hz, 1H), 5.80 (dd, J = 10.3, 1.3 Hz, 1H), 3.87 (s, 3H),
2.93 (dt, J = 13.7, 6.8 Hz, 1H), 2.84 (t, J = 4.4 Hz, 4H), 2.50 (s, 4H), 2.25 (s, 3H), 1.23 (d, J = 6.9 Hz,
6H). MS (ESI) m/z(%): 527.6 [M+H]+; Anal. calcd. For C29H34N8O2(%): C, 66.14; H, 6.51; N, 21.28;
Found (%): C, 66.23; H, 6.45; N, 21.36.

5.9.18. N-(5-((9-(4-iodophenyl)-9H-purin-2-yl)amino)-4-methoxy-2-(4-methylpiperazin-1-yl)phenyl)ac
rylamide(21r)
Yield: 55.4%, M.p.:161.5-162.4oC; H NMR (400 MHz, DMSO-d6) δ 9.10 (s, 1H), 8.91 (s, 1H), 8.68
1
(d, J = 24.0 Hz, 2H), 8.17 (s, 1H), 7.90 (d, J = 7.4 Hz, 2H), 7.82 (d, J = 7.0 Hz, 2H), 6.85 (s, 1H), 6.69
(dd, J = 16.9, 9.3 Hz, 1H), 6.31 (d, J = 17.2 Hz, 1H), 5.83 (d, J = 9.5 Hz, 1H), 3.88 (s, 3H), 2.85 (s, 4H),
2.49 – 2.46 (m, 3H), 2.25 (s, 4H). MS (ESI) m/z(%): 611.4 [M+H]+; Anal. calcd. For C26H27IN8O2(%):
C, 51.16; H, 4.46; N, 18.36; Found (%): C, 51.23; H, 4.40; N, 18.46.
5.9.19. N-(5-((9-(4-isopropylphenyl)-9H-purin-2-yl)amino)-4-methoxy-2-morpholinophenyl)acrylamid
e(21s)
Yield: 65.5%, M.p.:190.4-191.3oC; H NMR (400 MHz, DMSO-d6) δ 9.42 (s, 1H), 8.87 (s, 1H), 8.63
1
(d, J = 6.3 Hz, 1H), 8.13 (s, 1H), 7.85 (d, J = 8.4 Hz, 2H), 7.43 (d, J = 8.5 Hz, 2H), 6.88 (s, 1H), 6.79
(dd,J=16.5,1.7Hz, 1H), 5.75 (dd, J = 12.3, 1.5 Hz, 1H), 3.84 (s, 3H), 3.78 (d, J = 2.9 Hz, 4H), 2.88 –
2.77 (m, 4H), 2.29 (d, J = 3.8 Hz, 1H), 1.26 (d, J = 6.9 Hz, 6H). MS (ESI) m/z(%): 514.2 [M+H]+;
Anal. calcd. For C28H31N7O3(%): C, 65.48; H, 6.08; N, 19.09; Found (%): C, 65.34; H, 6.20; N, 18.86.
5.9.20. N-(5-((9-(4-iodophenyl)-9H-purin-2-yl)amino)-4-methoxy-2-morpholinophenyl)acrylamide(21t)
Yield: 54.9%, M.p.:201.1-202.3oC; 1H NMR (400 MHz, DMSO) δ 9.42 (s, 1H), 8.89 (s, 1H), 8.69 (s,
1H), 8.65 (s, 1H), 8.17 (s, 1H), 7.93 (d, J = 8.7 Hz, 2H), 7.81 (d, J = 8.8 Hz, 2H), 6.88 (s, 1H), 6.52 (dd,
J=17.2, 1.5Hz, 1H), 5.76 (dd, J=10.6, 1.5Hz, 1H), 3.86 (s, 3H), 3.82 – 3.75 (m, 4H), 2.89 – 2.78 (m,
4H). MS (ESI) m/z(%): 598.1 [M+H]+; Anal. calcd. For C25H24IN7O3(%): C, 50.26; H, 4.05; N, 16.41;
Found (%): C, 50.34; H, 4.20; N, 16.56.
5.10. General procedure for preparation of compounds (22a-e)
To a solution of 17 (0.32 mmol, 1.0 eq.) in 3 mL of CH2Cl2 was added 115 mg (0.71 mmol, 2.2 eq.)
of carbonyl diimidazole. The reaction mixture was stirred at room temperature for 16 h. The mixture
was diluted with 20 mL of chloroform and 10 mL of sat. NaHCO3 (aq) and the layers separated. the
organic extract was washed with saturated NaHCO3, water, and brine and dried over anhydrous MgSO4.
It was then filtered and concentrated in vacuo to generate the compounds 22a-e, respectively.
5.10.1. N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-5-((9-(4-iodophenyl)-8-oxo-8,9-dihydro-7H-pur
in-2-yl)amino)-4-methoxyphenyl)acrylamide(22a)
1H NMR (400 MHz, CDCl3) δ 9.96 (s, 1H), 9.04 (s, 1H), 7.91 (s, 1H), 7.78 (d, J = 8.5 Hz, 2H), 7.52 (d,
J = 8.5 Hz, 2H), 7.33 (s, 1H), 6.67 (s, 1H), 6.51 (d, J = 16.7 Hz, 1H), 5.83 (d, J = 11.1 Hz, 1H), 3.77 (s,
3H), 3.09 (s, 2H), 2.69 (s, 3H), 2.65 – 2.33 (m, 8H). MS (ESI) m/z(%): 629.1[M+H]+; Anal. calcd. For
C26H29IN8O3 (%): C, 49.69; H, 4.65; N, 17.83; Found (%): C, 49.55; H, 4.73; N, 17.92.
5.10.2. N-(5-((9-(4-isopropylphenyl)-8-oxo-8,9-dihydro-7H-purin-2-yl)amino)-4-methoxy-2-(4-methyl
piperazin-1-yl)phenyl)acrylamide(22b)
Yield: 53.7%, M.p.:186.4-187.3oC; 1H NMR (400 MHz, DMSO-d6) δ 10.20 (s, 1H), 8.97 (s, 1H), 8.50
(s, 1H), 8.01 (s, 1H), 7.66 (s, 1H), 7.55 (d, J = 8.3 Hz, 2H), 7.35 (d, J = 8.4 Hz, 2H), 6.80 (s, 1H), 6.64
(dd, J = 17.1, 10.0 Hz, 1H), 6.34 – 6.18 (m, 1H), 5.76 (d, J = 10.1 Hz, 1H), 3.82 (s, 3H), 2.97 – 2.89 (m,
1H), 2.80 (d, J = 4.0 Hz, 4H), 2.50 – 2.49 (m, 4H), 2.24 (s, 3H), 1.23 (d, J = 6.9 Hz, 6H). MS (ESI)

m/z(%): 543.6 [M+H]+; Anal. calcd. For C29H34N8O3(%): C, 64.19; H, 6.32; N, 20.65; Found (%): C,
64.23; H, 6.45; N, 20.54.
5.10.3. N-(5-((9-(4-iodophenyl)-8-oxo-8,9-dihydro-7H-purin-2-yl)amino)-4-methoxy-2-(4-methylpiper
azin-1-yl)phenyl)acrylamide(22c)
Yield: 52.1%, M.p.:256.4-257.4oC; 1H NMR (400 MHz, DMSO-d6) δ 9.02 (s, 1H), 8.51 (s, 1H), 8.00 (s,
1H), 7.83 (d, J = 8.5 Hz, 2H), 7.56 (d, J = 8.5 Hz, 2H), 6.80 (s, 1H), 6.65 (dd, J = 16.9, 10.3 Hz, 1H),
6.32 – 6.20 (m, 1H), 5.83 – 5.73 (m, 1H), 3.84 (s, 3H), 2.81 (s, 4H), 2.50 – 2.47 (m, 4H), 2.24 (s, 3H).
MS (ESI) m/z(%): 627.5 [M+H]+; Anal. calcd. For C26H27IN8O3(%): C, 49.85; H, 4.34; N, 17.89;
Found (%): C, 49.76; H, 4.25; N, 17.96.
5.10.4. N-(5-((9-(4-isopropylphenyl)-8-oxo-8,9-dihydro-7H-purin-2-yl)amino)-4-methoxy-2-morpholin
ophenyl)acrylamide(22d)
Yield: 66.5%, M.p.:136.4-137.3oC; 1H NMR (400 MHz, DMSO-d6) δ 9.78 (s, 1H), 8.45 (s, 1H), 8.27 (s,
1H), 7.53 (d, J = 15.0 Hz, 2H), 7.12 (s, 1H), 7.06 (d, J = 14.9 Hz, 2H), 6.43 (s, 1H), 6.29 (dd, J = 33.5,
19.6 Hz, 1H), 6.05 (dd, J = 33.5, 4.6 Hz, 1H), 5.69 (dd, J = 19.6, 4.6 Hz, 1H), 4.71 (s, 1H), 3.86 (s, 3H),
3.72 (t, J = 8.9 Hz, 4H), 3.18 (t, J = 9.0 Hz, 4H), 2.87 (dt, J = 25.4, 12.7 Hz, 1H), 1.20 (d, J = 12.8 Hz,
6H).13C NMR (100 MHz, DMSO-d6) δ 163.41, 155.31, 152.76, 151.16, 148.15, 146.82, 141.03, 139.27,
134.98, 132.90, 130.94, 127.13, 126.37, 125.29, 118.83, 116.01, 115.64, 103.86, 66.83, 56.47, 52.38,
33.68, 24.33. MS (ESI) m/z(%): 530.2 [M+H]+; Anal. calcd. For C28H31N7O4(%): C, 63.50; H, 5.90; N,
18.51; Found (%): C,63.68; H, 5.83; N, 18.76.
5.10.5. N-(5-((9-(4-iodophenyl)-8-oxo-8,9-dihydro-7H-purin-2-yl)amino)-4-methoxy-2-morpholinophe
nyl)acrylamide(22e)
Yield: 59.5%, M.p.:168.4-169.3oC; 1H NMR (400 MHz, DMSO-d6) δ 9.10 (s, 1H), 8.54 (s, 1H), 8.04 (s,
1H), 7.84 (d, J = 8.6 Hz, 2H), 7.71 (s, 1H), 7.53 (d, J = 8.7 Hz, 2H), 6.84 (s, 1H), 6.69 (dd, J = 16.8,
10.2 Hz, 1H), 6.27 (dd, J = 17.1, 1.6 Hz, 1H), 5.79 (dd, J = 10.4, 1.1 Hz, 1H), 3.84 (s, 3H), 3.76 (d, J =
4.3 Hz, 4H), 2.87 – 2.75 (m, 4H). MS (ESI) m/z(%): 614.1 [M+H]+; Anal. calcd. For C25H24IN7O4(%):
C, 48.95; H, 3.94; N, 15.98; Found (%): C,48.78; H, 3.83; N, 15.76.
5.11. General procedure for preparation of compounds (23a-c)
A suspension of compound 20a-c (2.82 mmol), EDCI (0.6 g, 3.11 mmol), DIEA (0.5 mL, 3.78 mmol),
and phenyl isothiocyanate (0.4 mL, 3.78 mmol) in CH2Cl2 (20 mL) was refluxed for 12 h until TLC
showed that the reaction was complete. The reaction mixture was evaporated under reduced pressure,
and the residue was chromatographed over silica gel eluting with CH2Cl2/MeOH (70:1) to give a brown
solid, which was further washed with diethyl ether to afford pure product 23a-c.
5.11.1. N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((9-methyl-8-(phenylamino)-9H-p
urin-2-yl)amino)phenyl)acrylamide(23a)
Yield: 55.8%, M.p.:135.2-2136.1oC; 1H NMR (400 MHz, DMSO-d6) δ 10.09 (s, 1H), 9.17 (s, 1H), 8.36
(s, 1H), 7.90 (d, J = 7.9 Hz, 2H), 7.65 (s, 1H), 7.35 (t, J = 7.8 Hz, 2H), 7.01 (d, J = 9.4 Hz, 2H), 6.52 –
6.40 (m, 1H), 6.25 (dd, J = 16.7, 1.8 Hz, 1H), 5.75 (dd, J = 10.1, 1.6 Hz, 1H), 3.89 (s, 3H), 3.80 (s, 3H),
2.91 (s, 2H), 2.68 (s, 3H), 2.29 (d, J = 11.6 Hz, 8H). MS (ESI) m/z(%): 516.6 [M+H]+; Anal. calcd. For
C27H33N9O2 (%): C, 62.89; H, 6.45; N, 24.45; Found (%): C, 62.78; H, 6.55; N, 24.36.

5.11.2. N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-5-((9-isopropyl-8-(phenylamino)-9H-purin-2-yl)
amino)-4-methoxyphenyl)acrylamide(23b)
Yield: 74.7%, M.p.:141.1-142.2oC; 1H NMR (400 MHz, DMSO-d6) δ 10.03 (s, 1H), 9.00 (d, J = 13.1
Hz, 2H), 8.35 (s, 1H), 7.82 (d, J = 7.8 Hz, 2H), 7.62 (s, 1H), 7.33 (t, J = 7.9 Hz, 2H), 7.02 – 6.93 (m,
2H), 6.45 (dd, J = 17.4, 10.4 Hz, 1H), 6.24 (dd, J = 16.9, 2.0 Hz, 1H), 5.73 (dd, J = 10.1, 1.8 Hz, 1H),
4.89 (dt, J = 13.5, 6.7 Hz, 1H), 4.26 (t, J = 5.2 Hz, 1H), 3.88 (s, 3H), 2.91 (s, 2H), 2.68 (s, 3H), 2.25 (s,
6H), 1.63 (d, J = 6.7 Hz, 6H). MS (ESI) m/z(%): 544.6 [M+H]+; Anal. calcd. For C29H37N9O2 (%): C,
64.07; H, 6.86; N, 23.19; Found (%): C, 64.23; H, 6.82; N, 23.25.
5.11.3. N-(5-((9-cyclopentyl-8-(phenylamino)-9H-purin-2-yl)amino)-2-((2-(dimethylamino)ethyl)(meth
yl)amino)-4-methoxyphenyl)acrylamide(23c)
Yield: 65.5%, M.p.:143.5-144.4oC; 1H NMR (400 MHz, DMSO-d6) δ 10.13 (s, 1H), 9.17 (s, 1H), 8.71
(s, 1H), 8.36 (s, 1H), 7.85 (d, J = 7.8 Hz, 2H), 7.66 (s, 1H), 7.32 (t, J = 7.9 Hz, 2H), 7.01 – 6.93 (m,
2H), 6.23 (dd, J = 16.8, 0.9 Hz, 1H), 5.76 – 5.67 (m, 1H), 4.99 (dd, J = 17.4, 8.8 Hz, 1H), 3.86 (s, 3H),
3.14 (s, 2H), 2.65 (s, 3H), 2.46 – 2.13 (m, 8H), 2.02 – 1.79 (m, 6H), 1.57 – 1.50 (m, 2H). MS (ESI)
m/z(%): 570.6 [M+H]+; Anal. calcd. For C31H39N9O2 (%): C, 65.36; H, 6.90; N, 22.13; Found (%): C,
65.46; H, 6.82; N, 22.25.
6. Pharmacology
6.1. In vitro enzymatic activity assay
The experiments were carried out by a well-established mobility shift assay, and EGFR kinases
(EGFR T790M/L858R, EGFR T790M and WT EGFR) were purchased from Invitrogen. The
kinase base buffer was consist of 50 mM HEPES (pH 7.5), 0.0015% Brij35 and 2 mM DTT, while
the stop buffer contained a mixture of 100 mM HEPES (pH 7.5), 0.015% Brij-35, 0.2% Coating
Reagent and 50 mM EDTA. Initially, the tested compounds were diluted to 50-fold of the final
desired highest concentration in reaction by 100% DMSO. Subsequently, 30 mL of the solution
was transferred to 60 mL of 100% DMSO in the next well and so forth for a total of 5
concentrations. No compound and no enzyme controls were prepared by adding 100 mL DMSO to
two empty wells in the same 96-well plate. Then, 10 mL of compound was transferred to a new
96-well plate, which was marked as the intermediate plate. Additional 90 mL of kinase buffer was
added to each well of intermediate plate. The mixture in intermediate plate was shaked for 10 min.
The assay plate was prepared after transferring 5 mL of each well from the 96-well intermediate
plate to a 384-well plate in duplicates. The prepared enzyme solution (appropriate EGFR in kinase
base buffer) was added to the assay plate, which was then incubated at room temperature for 10
min, followed by the addition 10 mL of prepared peptide solution (FAM-labeled peptide and ATP
in kinase base buffer). The mixture was incubated at 28℃ for another 1 h, then 25 mL of stop
buffer was added. The conversion data was copied from Caliper program, and the values were
converted to inhibition values. Percent inhibition ¼ (max -conversion)/ (max -min) ×100.
6.2. MTT assay
The target compounds were screened in A549, H1975 and HT-29 cells by a standard MTT assay.
The cancer cell lines were cultured in minimum essential medium (MEM) supplement with 10%
fetal bovine serum (FBS). Approximate 4×103 cells, suspended in MEM medium, were plated
onto each well of a 96-well plate and incubated in 5% CO2 at 37℃for 24 h. The tested compounds

were added to the culture medium and the cell cultures were continued for 72 h. Fresh MTT was
added to each well at a terminal concentration of 5 mg/mL, and incubated with cells at 37℃for 4 h.
The formazan crystals were dissolved in 100 mL DMSO each well, and the absorbency at 492 nm
(for absorbance of MTT formazan) and 630 nm (for the reference wavelength) was measured with
an ELISA reader. All of the compounds were tested three times in each of the cell lines. The
results expressed as IC50 (inhibitory concentration 50%) were the averages of at least three
determinations and calculated by using the Bacus Laboratories Incorporated Slide Scanner (Bliss)
software.
Conflict of interest
The authors have declared no conflict of interest.
Acknowledgements
We thank the Progarm for Innovation Research Team of the Ministry of Education and Program
for Liaoning Innovative Research Team in University (IR1073) for financial support.
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Graphical Abstract