Synthesis and evaluation of 2,9-disubstituted 8-phenylthio/phenylsulfinyl-9H-purine as new EGFR inhibitors

Article abstract of DOI:10.1016/j.bmc.2018.03.025

In present study, we described the synthesis and biological evaluation of a new class of EGFR inhibitors containing 2,9-disubstituted 8-phenylthio/phenylsulfinyl-9H-purine scaffold. Thirty-one compounds were synthesized. Among them, compound C9 displayed the IC₅₀ of 29.4 nM against HCC827 cell line and 1.9 nM against EGFR^L858R. Compound C12 showed moderate inhibitory activity against EGFR^{L858R/T790M/C797S} (IC₅₀ = 114 nM). Western bolt assay suggested that compound C9 significantly inhibited EGFR phosphorylation. In vivo test, compound C9 remarkably exhibited inhibitory effect on tumor growth at 5.0 mg/kg by oral administration in established nude mouse HCC827 xenograft model. These results indicate that the 2,9-disubstituted 8-phenylsulfinyl/phenylsulfinyl-9H-purine derivatives can act as potent EGFR(L858R) inhibitors and effective anticancer agents. Additionally, optimization of compound C12 may result in discovering the fourth-generation EGFR-TKIs.

Full text of DOI:10.1016/j.bmc.2018.03.025

Accepted Manuscript
Synthesis and evaluation of 2,9-disubstituted 8-phenylthio/phenylsulfinyl-9H-
purine as new EGFR Inhibitors
Yuan-Yuan Hei, Ying Shen, Jin Wang, Hao Zhang, Hong-Yi Zhao, Minhang
Xin, Yong-Xiao Cao, Yan Li, San-Qi Zhang
PII:
S0968-0896(18)30220-7
https://doi.org/10.1016/j.bmc.2018.03.025
BMC 14263
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 February 2018
9 March 2018
14 March 2018
Please cite this article as: Hei, Y-Y., Shen, Y., Wang, J., Zhang, H., Zhao, H-Y., Xin, M., Cao, Y-X., Li, Y., Zhang,
S-Q., Synthesis and evaluation of 2,9-disubstituted 8-phenylthio/phenylsulfinyl-9H-purine as new EGFR Inhibitors,
Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.03.025
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Synthesis and evaluation of 2,9-disubstituted
8-phenylthio/phenylsulfinyl-9H-purine as new EGFR Inhibitors
Yuan-Yuan Heia, Ying Shena, Jin Wangb, Hao Zhanga, Hong-Yi Zhaoa, Minhang Xina, Yong-Xiao
Caob, Yan Li,a San-Qi Zhanga*
a
Department of Medicinal Chemistry, School of Pharmacy, Xian Jiaotong University, Xian,
Shaanxi, 710061, PR China
b
Department of Pharmacology, School of Basic Medical Sciences, Xi’an Jiaotong University,
Xi’an, Shaanxi, 710061, PR China
*Corresponding author: San-Qi Zhang, Ph. D., Professor, E-mail address: sqzhang@xjtu.edu.cn (S.
Q. Zhang).
Abstract
In present study, we described the synthesis and biological evaluation of a new
class of EGFR inhibitors containing 2, 9-disubstituted 8-phenylthio/
phenylsulfinyl-9H-purine scaffold. Thirty-one compounds were synthesized. Among
them, compound C9 displayed the IC50 of 29.4 nM against HCC827 cell line and 1.9
nM against EGFRL858R. Compound C12 showed moderate inhibitory activity against
EGFRL858R/T790M/C797S (IC50 = 114 nM). Western bolt assay suggested that compound
C9 significantly inhibited EGFR phosphorylation. In vivo test, compound C9
remarkably exhibited inhibitory effect on tumor growth at 5.0 mg/kg by oral
administration in established nude mouse HCC827 xenograft model. These results
indicate that the 2,9-disubstituted 8-phenylsulfinyl/phenylsulfinyl-9H-purine
derivatives can act as potent EGFR(L858R) inhibitors and effective anticancer agents.
Additionally, optimization of compound C12 may result in discovering the
fourth-generation EGFR-TKIs.
Keywords purine * EGFR-TK inhibitor * drug design * antiproliferative effects *
anticancer agent
1

1. Introduction
The epidermal growth factor receptor (EGFR) belongs to the family of
protein-tyrosine kinase receptor (RTK), which plays a key role in the regulation of
cell growth, differentiation, and survival.1 RTKs have been observed over-expression
and/or constitutive activation in numerous types of human tumor, including colon,
breast, ovarian, head and neck, and non-small cell lung cancers (NSCLC). Among the
known RTKs, EGFR and human epidermal growth factor receptor have been
extensively studied and clinically validated as targets for chemical therapies.2, 3 The
first-generation EGFR tyrosine kinase inhibitor (EGFR-TKI), gefitinib3 and erlotinib4
were approved by FDA for the treatment of NSCLC. For the population of NSCLC
patients whose tumors harbor activating mutations in EGFR, targeted kinase
inhibitors such as gefitinib and erlotinib have proven to be very effective treatments
resulting in tumor regressions and improved progression-free survival.5 However, the
efficacy of small organic molecules inhibitors such as gefitinib, etc. is restricted to a
small subset of patients due to molecular heterogeneity among and within tumors.6, 7
The drug resistance caused by receptor mutation is another issue needed to pay
attention.8-10 Numbers of compounds with different structures were discovered as
EGFR or multi-target inhibitors.11-15 The essential pharmacophore for
4-anilinoquinazolines is the two nitrogen atoms at 1-posotion, 3-position and the
N-aryl at 4-position as well.16 The morpholine moiety in gefitinib is not involved in
any interaction with EGFR and is randomly ordered due to its lower electron density.
Consequently, the modification of the 6-substituted group in 4-anilinoquinazoline
with the group bearing active group provide the inhibitors with improved potency.17-20
Meanwhile, Numbers of compounds with different structures have been developed as
EGFR
or
multi-target
inhibitors.21,22
Salicylanilide
molecule
and
2-aryl-8-hydroxyisoquinolin-1(2H)-one can construct an intramolecular hydrogen
bond and form a pseudo six-membered ring to mimic the pyrimidine ring of
quinazolines. Thus, N-aryl salicylamides and 2-aryl-8-hydroxyisoquinolin-1(2H)-one
were studied as EGFR inhibitors.23,24
2

Figure 1. The structures of EGFR inhibitors
To overcome the resistance, the replacement of 6-substituted group in gefitinib
with acrylamido group produced the second-generation irreversible EGFR inhibitors
including afatinib25 (Figure 1), dacomitinib26 and neratinib.27 Afatinib was
demonstrated activity in preclinical studies against T790M mutations. But the clinical
trial data of afatinib did not demonstrate distinctly improved efficacy.28 The kinase
active site domain is mutated in many NSCLC epithelial tumors and clinical studies
suggest that these mutations aid in tumorigenesis. There are two main types of
mutations. One is activating mutation (e.g., L858R or exon 19 deletion) and the other
is drug resistance mutation (e.g., T790M).29 The latter mutation (T790M) results in an
increased affinity of the kinase toward ATP, rendering these ATP-competitive
inhibitors less effective despite comparable affinities to the different mutant forms of
the protein. The progress of 2-anilinopyrimidine-based small-molecule EGFR-T790M
inhibitors AZD9291 and its analogues has been well summarized.30 However, the
effective treatment of patients that harbor the EGFR-T790M drug resistance mutation
is limited by the emergence of new drug resistances to AZD9291 therapy.31 Therefore,
the development of the fourth-generation EGFR-TKIs has become a new research
hotspot.32,33
Gefitinib, erlotinib and afatinib share the 4-arylaminoquinazoline as their common
scaffold. Compound 4 (Figure 2) with a purine scaffold was discovered to be a
reversible EGFR-TKI, which displayed potent inhibitory active toward EGFR
(L858R).34 However, the high CLogP value (6.40, predicted by ChemBioDraw 14.0)
limited the development of compound 4 as a candidate. In this study, we intend to
replace 8-phenylamino group in compound 4 with 8-phenylthio or phenylsulfinyl
3

moiety to discover new EGFR-TKI and anticancer agents (structures C and D in
Figure 2). Meanwhile, oxygen atom or hydroxyl group is introduced to R1 moiety to
decrease CLogP value. Herein, we report the synthesis and biological activities
evaluation of new compounds.
Figure 2. The design of target compounds
2. Results and discussion
2.1. Chemistry
The synthetic route for the title compound C and D was outlined in Scheme 1.
2,4-Dichloro-5-nitropyrimidine was used as the starting material. The intermediates A
were prepared as reported protocol.34 Catalyzed by Pd/C (5%), the nitro group in
intermediate A was performed hydrogenation-reduction to produce corresponding
amine, which was used in the next step without further purification. The refluxing of
the mixture containing the amine, CS2, KOH, EtOH and H2O afforded intermediates
B. Catalyzed by CuI, the reaction of intermediates B with iodobenzene yielded
products C, 2, 9-disubstituted 8-phenylthio-9H-purine. The oxidation of C with
m-chloroperoxybenzoic acid produced compounds D, 2, 9-disubstituted 8-
phenylsulfinyl-9H-purine.
4

Scheme 1. Synthesis of 2-anilino-8-henylthio/phenylsulfinyl-9H-purine derivatives C and D.
Reagents and conditions: a) CH2Cl2, DIEA, amine, -40°C to room temperature, 60-75%; b) n-butanol,
90 °C, 5 h, 70%-76%; c) H2, 5% Pd/C, MeOH, 50°C, 6 h, 85%-90%; d) CS2, KOH, EtOH:H2O =
10:1, refluxed, 52.6-90.3%; e) PhI, CuI, 1,10-phenanthroline, K2CO3, DMSO, 140°C, 24 h,
55.2%-66.7%; f) m-chloroperoxybenzoic acid, CH2Cl2, 50.2-63.7%.
To elucidate the change of R2 and R3 on activity, compounds C7-C12 and
D7-D12 were synthesized (Scheme 2).
Scheme 2. Synthesis of 2-anilino-8-henylthio/phenylsulfinyl-9H-purine derivatives C and D.
Reagents and conditions: a) aryliodide, CuI, 1,10-phenanthroline, K2CO3, DMSO, 140℃, 24 h,
55.7%-63.6%; b) m-chloroperoxybenzoic acid, CH2Cl2, 49.2-63.2%.
5

To boost the structural diversity of the title compounds, 8-benzylthio/
benzylsulfinyl-9H-purine was synthesized. In the presence of K2CO3, the nucleophilic
substitution of B with benzyl bromide yielded 8-benzylthio-9H-purine E, which was
treated with m-chloroperoxybenzoic acid to produce compounds F.
Scheme 3. Synthesis of 2,8,9-trisubstitued purine derivatives E and F. Reagents and conditions: a)
benzyl bromide or 3-chloromethylpyridine hydrochloride, acetone, K2CO3, 42.4%-72.9%; b)
m-chloroperoxybenzoic acid, CH2Cl2, 46.3-68.2%.
1
All the newly synthesized title compounds were characterized by H-NMR,
13C-NMR and MS.
2.2. The antiproliferative effects in vitro
The antiproliferative effects of synthesized compounds were evaluated against
human lung carcinoma cell line HCC827 (Del E746-A750) by applying the 3-[4,
5-dimethylthiazol-2-yl]-2, 5-diphenyl-2H-tetrazolium bromide (MTT) colorimetric
assay. The EGFR-TKI gefitinib and AZD9291 were used as the positive controls. The
results of compounds C are summarized in Table 1. Firstly, to explore the effect of
substituent at the N-9 position of purine scaffold, we synthesized compounds C1-C6,
which contained substituents of different sizes at the N-9 purine position. Compounds
C1, C2 and C3 displayed moderate activities against HCC827 with the IC50 values of
289.9-659.9 nM. Increasing the substituent volume, the antiproliferative effects of
compounds C4, C5 and C6 significantly improved. Compound C5 (IC50 = 42.6 nM)
exhibited equipotent antiproliferative effect with AZD9291 (IC50 = 43.1 nM) and
slightly increased in activity over compound C4 (IC50 = 51.2 nM). Compound C6
(IC50 = 47.9 nM), attached a hydrophilic group to the substitute group of N-9 position
of the purine scaffold, did not improve its antiproliferative effect. These results
6

suggested that
Table 1. Antiproliferative effects of compounds C against HCC827 cell lines (
, n = 3)
x  s
Compds
R1
R2
R3
H
IC50 (nM)
289.9±33.6
304.5±33.5
659.9±42.1
51.2±10.3
42.6±10.7
47.9±9.3
C1
H
H
C2
C3
H
C4
H
C5
H
C6
H
44.3±0.9
C7
H
498.8±32.4
29.4±4.4
C8
H
C9
H
139.8±23.9
51.4±4.2
C10
C11
C12
3-F
4-F
47.8±0.7
33.3±4.2
43.1±1.1
gefitinib
AZD9291
Next, compounds C7-C10 were synthesized to explore the influence of the
substituent at position of R2 on the antiproliferative effect. Compound C7 (IC50 = 44.3
nM) with 2-dimethylaminoethoxy at R2 position displayed the same potency
7

compared with C5 (IC50 = 42.6 nM). The replacement of N-methylpiperazinyl in C5
with N-(2-(dimethylamino)ethyl)-N-methylamino (C8) or 1-methylpiperidin-4-yloxy
group (C10) resulted in a decrease in cell-based effect. However, compound C9 with
4-N, N-dimethylaminopiperidin-1-yl at position of R2 showed the most potent
antiproliferative effect against HCC827 (IC50 = 29.4 nM), which was comparable to
the positive controls gefitinib and AZD9291. When fluorine atom was presented at
the meta-position (C11, IC50 = 51.4 nM) and para-position (C12, IC50 = 47.8 nM) of
the phenyl ring of the 8-phenylthio, the antiproliferative effect of the two compounds
was not changed distinctly against HCC827.
Table 2. Antiproliferative effects of compounds D against HCC827 cell lines (
, n = 3)
x  s
R1
R2
R3
H
IC50 (nM)
Compds
214.5±25.5
1627.8±208.4
838.3±26.3
64.7±18.1
D1
H
H
H
H
H
H
H
H
D2
D3
D4
D5
D6
D7
D8
D9
410.6±20.1
268.3±80.7
50.3±8.8
1329.3±56.8
1287.6±61.0
8

H
852.7±17.2
358.7±30.7
553.1±62.2
D10
D11
D12
3-F
4-F
33.3±4.2
43.1±1.1
gefitinib
AZD9291
The antiproliferative effects of 2,9-disubstituted 8-phenylsulfinyl-9H-purine,
compounds D were listed in table 2. Compounds D4 and D7 showed potent
antiproliferative effects on HCC827 with IC50 values of 64.7 nM and 50.3 nM,
respectively. However, the rest compounds D displayed moderate antiproliferative
effects in the HCC827 cellular assay with IC50 values of 214.5-1627.8 nM. Compared
with compounds C with a phenylthio at 8-position of the purine scaffold, compounds
D with a phenylsulfinyl at 8-position of the purine scaffold were less effective against
HCC827.
To further study structure-activity relationship of compounds, we changed the
8-phenylthio/phenylsulfinyl to 8-benzylthio/benzylsulfinyl to produce compound E
and F. The antiproliferative effects of compounds E1-E4 and F1-F3 against HCC827
are summarized in Table 3. Compared with compounds C and positive controls, these
compounds showed a significant drop in the cell-based activity, indicating that
benzylthio/benzylsulfinyl at the C-8 position of purine scaffold was not suitable.
Table 3. Antiproliferative effects of 8-benzylthio/ benzylsulfinyl-9H-purine E and F against
HCC827 cell lines ( x  s , n = 3)
9

Compds
R
X
IC50 (nM)
N
8832.6±243.5
E1
N
N
517.3±112.1
499.6±20.5
106.5±9.9
E2
E3
E4
F1
F2
F3
CH
N
2353.3±532.4
>10000
N
CH
433.1±11.1
33.3±4.2
43.1±1.1
gefitinib
AZD9291
In order to investigate the cell-based selectivity, compounds with potent
antiproliferative effects were selected to measure their antiproliferative effects against
human lung carcinoma cell lines H1975 and A549 cell lines. The results were
summarized in Table 4. The data in Table 4 indicated that compounds C4 and C6
showed moderate antiproliferative effects against A549 cell line. The other tested
compounds exhibited weak antiproliferative effects against H1975 and A549 cell lines.
In addition, the most active compound C9 displayed 340-fold selectivity against
HCC827 cell lines over A549 cell line. Overall, these data suggested that tested
compounds were significantly more active against HCC827 cell lines compared to the
H1975 and A549 cell lines.
Table 4. Antiproliferative effects and cell-based selectivity of compounds against HCC827,
H1975 and A549 cell lines ((IC50, nM), x  s , n = 3)
H1975/
HCC827
109
A549/
HCC827
5
Compds
HCC827
H1975
A549
51.2±10.3
42.6±10.7
42.6±9.3
44.3±0.9
5580±190
2260±120
1320±220
9610±360
C4
C5
C6
C7
237±10
2945±123
426±16
53
69
31
10
>10000
217
>226
10

29.4±4.4
51.4±4.2
47.8±0.71
64.7±18.1
50.3±8.8
33.3±4.2
43.1±1.1
4990±147
2770±280
6260±680
5110±980
>10000
>10000
3362±820
4850±313
>10000
170
54
>340
65
C9
C11
131
79
101
>154
>198
-
C12
D4
>10000
>198
-
D7
>10000
1260
gefitinib
AZD9291
47.2±5.8
486 ± 170
-
-
2.3. EGFR enzymatic activity assay
Then, compounds C4-C7, C9, C11, C12, D4 and D7 exhibited nanomolar
antiproliferative effects and were selected to evaluate their inhibitory activity against
EGFR to elucidate the mechanism of antiproliferative effects. AZD9291 was used as
the positive drug. The data are listed in Table 5.
Table 5. Enzymatic activity of C4-C7, C9, C11, C12, D4 and D7 (IC50, nM, n = 2)
EGFR
(WT)
nd
EGFR
(L858R)
4.2
EGFR
EGFR
Compds
(L858R/T790M) (L858R/T790M/C797S)
C4
C5
nd
nd
nd
892
529
556
331
805
114
nd
nd
2.0
C6
nd
2.4
nd
C7
nd
1.7
nd
C9
1.6
nd
1.9
104
nd
C11
C12
D4
5.8
2.5
nd
1.2
189
nd
4.2
D7
nd
2.6
nd
nd
AZD9291
1.4
2.1
1.8
120
“nd”: not determined.
The data in Table 5 indicated that all the tested compounds displayed potent
inhibitory activity against EGFR (L858R), weak activity toward EGFR
(L858R/T790M) and EGFR (L858R/T790M/C797S). At the same time, compounds
C9, C12 and AZD9291 exhibited potent inhibitory activity against EGFR (WT) at the
11

same test conditions. It should be noticed that compound C7 and C12 displayed the
IC50 of 556 nM and 114 nM against EGFR triple mutant (C797S), respectively. The
data suggested that a compound (C12) with 8-(4-fluorophenylthio) moiety may
improve its activity on EGFR triple mutant. This hints that optimization of compound
C12 may result in discovering potent EGFR (L858R/T790M/C797S) inhibitors.
So far, thirty-one compounds were synthesized and their antiproliferative effects
were evaluated. Nine compounds were determined their inhibitory activities against
EGFR. The property comparison of compounds C7, C9, 4 and AZD9291 was listed in
Table 6. The data in Table 6 indicated that compounds C7 and C9 displayed close
antiproliferative effects against HCC827 and enzyme inhibitory on EGFR (L858R)
compared with compound 4 and AZD9291. However, compounds C7 and C9,
presented in our study, showed higher growth inhibition ratio on HCC827 cell than
compound 4 and AZD9291 at 1 M. In the meantime, predicted CLogP value was less
than 5.
Table 6. Property comparison of compounds C7, C9, 4, and AZD9291
c
Emax
IC50 (nM),
IC50(nM)
compds
MWa
CLogPb
EGFR(L858R)
(%, 1 M)
(HCC827)
491
530
469
500
4.89
4.41
6.40
4.60
44.4±0.9
29.4±4.4
35.9±4.2
25.4±1.5
78
81
70
73
1.7
1.9
nd
C7
C9
4
AZD9291
2.1
“nd”: not determined. a: molecule weight (MW) was calculated from ChemBioDraw 14.0. b:
CLogP was predicted by ChemBioDraw 14.0. c: growth inhibition rate on HCC827 cell at 1 M.
2.4. Western blot assay of inhibition of EGFR autophosphorylation
To further verify the mechanism of the antiproliferative activity of compound C9,
Western blot assay was employed to evaluate the inhibition of EGFR
autophosphorylation of compound C9 in HCC827 cell line (Figure 3). The results
suggested that compound C9 can block EGFR phosphorylation in a dose-dependent
manner in the HCC827 cell line.
12

Figure 3. Inhibition of EGFR autophosphorylation of compound C9 in HCC827 cell line
2.5. Anticancer effects in vivo
Finally, we explored whether potent compound could inhibit tumor growth in
established mouse xenografts models. Compound C9 displayed potent inhibitory
activity against EGFR (L858R) and antiproliferative effect on HCC827. Then C9 was
selected to evaluate its anticancer effect in vivo. A study using mice bearing HCC827
xenografts was performed. Compound C9 was dosed orally at 1 mg/kg or 5 mg/kg
once a day for 20 days. The results were shown in Figure 4.
vehicle
1 mg/kg
5 mg/kg
Figure 4. The anticancer effect of compound C9 on HCC827 xenografts model (n = 6 in each
group). (A) The photograph of tumors in each group after C9 and vehicle treatment. (B) Tumor
growth curves during treatment with C9 and vehicle. **p < 0.01 vs vehicle. (C) Body weight of
13

mice during treatment with C9 and vehicle. (D) Comparison of the final tumor weights in each
group after the 20-day treatment of C9. Numbers in columns indicate the mean tumor weight in
each group. *p < 0.05, **p < 0.01 vs vehicle.
The results indicated that compound C9 can significantly inhibit tumor growth at
a dosage of 5 mg/kg. Meanwhile, mouse body weights of tested animals were
increased during treatment.
2.6. Molecular docking studies
To further explain the EGFR inhibitory activity of compounds C7, D7 and C9,
we performed a docking analysis utilizing the C-DOCKER program within Discovery
Studio 2.5 software packages. The binding mode of compounds C7, D7 and C9
within the active site of the T790M mutant predicted by molecular docking were
depicted in Figure 5. From the docking results, we observed that compounds C7, D7
and C9 were favorably located into the receptor pocket and had three same binding
characteristics: (1) the purine ring was located in the ATP-binging pocket and
sandwiched between the N- and C-lobes of the kinase. (2) The hydrogen atom of
aniline at the C-2 position of purine scaffold and the 1-nitrogen atom from purine core
formed a hydrogen bond with Met793, respectively. (3) The 2-aniline branch
extended the solvent and the phenyl from 8-phenylthio moiety or 8-phenylsulfinyl
was directed into the hydrophobic pocket of the ATP-binding domain. The sp3
hybridization of sulfur atom in 8-phenylthio moiety or 8-phenylsulfinyl may be
conducive to this combination. It was noted that the 8-phenylsulfinyl in compound D7
could form two extra hydrogen bonds with Lys745 and Asp855, respectively.
However, compound D9 did not produce a suitable docking result. These could
explain why compound D7 displayed more potent inhibitory activity than other
compounds D in enzyme and cell-based evaluation.
14

Figure 5. The docking mode of Compounds C7, D7 and C9 with the T790M EGFR mutant
(PDB code: 2JIU). (A) The docking pose of compound C7 with EGFR. (B) The docking pose of
compound D7 with EGFR. (C) The docking pose of compound C9 with EGFR. Compounds C7,
D7 and C9 were shown as sticks. Hydrogen bonds within 3.0 Å were shown as yellow dashed
lines.
3. Conclusions
In summary, thirty-one 2, 9-disubstituted 8-phenylthio/phenylsulfinyl
/benzylthio-9H-purine derivatives were synthesized. Some compounds displayed
potent antiproliferative effects against HCC827 cancer cell line and compound C9
exhibited potent inhibitory activities against the EGFRL858R (IC50 = 1.9 nM).
Compound C12 showed moderate inhibitory activity against EGFRL858R/T790M/C797S
(IC50 = 114 nM). Western bolt assay suggested that compound C9 significantly
inhibited EGFR phosphorylation. Additionally, compound C9 remarkably exhibited
inhibitory effect on tumor growth at 5.0 mg/kg by oral administration in established
nude mouse HCC827 xenograft model. These results indicate that 2,9-disubstituted
8-phenylsulfinyl-9H-purine derivatives can act as potent EGFR(L858R) inhibitors and
effective anticancer agents. Additionally, optimization of compound C12 may result
in discovering the fourth-generation EGFR-TKIs.
4. Experimental protocols
4.1. Chemistry and chemical methods
Unless specified otherwise, all starting materials, reagents and solvents were
commercially available. All the reactions were monitored by thin-layer
15

chromatography on silica gel plates (GF-254) and visualized with UV light. All the
melting points were determined on a Shanghai micro melting-point apparatus (model:
SGW® X-4B) and thermometer was uncorrected. NMR spectra were recorded on a
400 Bruker NMR spectrometer with tetramethylsilane (TMS) as an internal reference.
All chemical shifts were reported in parts per million (ppm). Mass measurements
were performed using electrospray ionization (positive mode) on a quadrupole
time-of-flight (QTOF) mass spectrometer (Skyray Instrument).
4.1.1. General procedure for the synthesis of intermediates B1-B10.
The reaction of 2, 4-dichloro-5-nitropyrimidine with isopropylamine produced
intermediate 2-chloro-N-isopropyl-5-nitropyrimidin-4-amine. 4-Fluoronitrobenzene
reacted with 1-methylpiperazine in DMSO yielded the intermediate
1-methyl-4-(4-nitrophenyl)piperazine in the presence of K2CO3. The catalytic
hydrogenation of 1-methyl-4-(4-nitrophenyl)piperazine with palladium on carbon
(Pd/C, 5%) quantificationally provided the desired 4-(4-methylpiperazin-1-yl) aniline.
Refluxing of the 2-chloro-N-isopropyl-5-nitropyrimidin-4-amine with 4-(4-methyl
piperazin-1-yl)aniline in n-butanol yielded N4-isopropyl-N2-(4-(4-methylpiperazin-
1-yl)phenyl)-5-nitropyrimidine-2,4-diamine, which was reduced to intermediate A1
with a good yield by catalytic hydrogenation using Pd/C as a catalyst. Intermediates A
were prepared as these steps and used for the next step without further purification.
These processes were carried out as reported.34
The mixture of A (0.88 mmol), CS2 (85 µL, 0.88 mmol) and KOH (51 mg, 0.88
mmol) in EtOH (30 mL) and H2O (3 mL) was refluxed for 4 h, evaporated under
reduced pressure. The resulting crude product was purified by silica gel column
chromatography using DCM/MeOH = 20:1 (V/V) as eluent to afford the compound B
as a solid.
4.1.1.1. 9-Isopropyl-2-((4-(4-methylpiperazin-1-yl)phenyl)amino)-9H-purine-8-thiol
(B1). Off-white solid; Yield 66.7%; mp 239.9-241.1 °C; 1H NMR (400 MHz, CDCl3)
δ 8.13 (s, 1H, Ar-H), 7.51 (d, J = 8.9 Hz, 2H, Ar-H), 7.13 (s, 1H, NH), 6.98 (d, J = 8.9
Hz, 2H, Ar-H), 5.38 (dt, J = 13.9, 6.9 Hz, 1H, CH), 3.26 – 3.21 (m, 4H, CH2×2),
16

2.69 – 2.64 (m, 4H, CH2×2), 2.41 (s, 3H, CH3), 1.68 (d, J = 6.9 Hz, 6H, CH3×2).
13C NMR (100 MHz, DMSO-d6) δ 169.1, 156.3, 152.0, 146.4, 137.6, 133.4, 120.2
(2C), 117.1, 116.3(2C), 55.1 (2C), 49.3(2C), 47.8, 46.2, 19.8 (2C). MS (ESI, m/z):
384.6 [M + H]+.
4.1.1.2. 9-Cyclopropyl-2-((4-(4-methylpiperazin-1-yl)phenyl)amino)-9H-purine-8-thi-
1
ol (B2). Off-white solid; Yield 63.8%; mp >250 °C; H NMR (400 MHz, CDCl3) δ
8.12 (s, 1H, Ar-H), 7.53 (d, J = 8.9 Hz, 2H, Ar-H), 7.02 (s, 1H, NH), 6.98 (d, J = 9.0
Hz, 2H, Ar-H), 3.28 (s, 1H, CH), 3.24 – 3.20 (m, 4H, CH2×2), 2.64 (s, 4H, CH2×2),
13
2.40 (s, 3H, CH3), 1.43 (s, 2H, CH×2), 1.26 (d, J = 6.5 Hz, 2H, CH×2). C NMR
(100 MHz, DMSO-d6) δ 171.4, 156.5, 153.0, 146.3, 137.2, 133.5, 120.1 (2C), 117.0,
116.4 (2C), 55.1 (2C), 49.3 (2C), 46.1, 26.1, 6.7 (2C). MS (ESI, m/z): 382.6 [M +
H]+.
4.1.1.3.
9-(Cyclopropylmethyl)-2-((4-(4-methylpiperazin-1-yl)phenyl)amino)-9H-
1
purine-8-thiol (B3). Gray solid; Yield 52.6%; mp >250 °C; H NMR (400 MHz,
DMSO-d6) δ 9.35 (s, 1H, NH), 8.19 (s, 1H, Ar-H), 7.59 (d, J = 9.0 Hz, 2H, Ar-H),
6.88 (d, J = 9.1 Hz, 2H, Ar-H), 4.02 (d, J = 7.2 Hz, 2H, CH2), 3.10 – 3.04 (m, 4H,
CH2×2), 2.49 – 2.45 (m, 4H, CH2×2), 2.23 (s, 3H, CH3), 1.39 (dd, J = 13.7, 6.2 Hz,
1H, CH), 0.51 (dd, J = 8.1, 6.6 Hz, 4H, CH2×2). 13C NMR (100 MHz, DMSO-d6) δ
169.7, 156.9, 152.5, 146.4, 137.4, 133.4, 120.2 (2C), 117.3, 116.3 (2C), 55.2 (2C),
49.3 (2C), 46.7, 46.2, 10.3, 4.1 (2C). MS (ESI, m/z): 395.2 [M + H]+.
4.1.1.4. 9-Cyclopentyl-2-((4-(4-methylpiperazin-1-yl)phenyl)amino)-9H-purine-8-thi-
1
ol (B4). Off-white solid; Yield 70.9%; mp >250 °C; H NMR (400 MHz, CDCl3) δ
8.12 (s, 1H, Ar-H), 7.50 (d, J = 8.9 Hz, 2H, Ar-H), 7.36 (s, 1H, NH), 6.95 (d, J = 8.9
Hz, 2H, Ar-H), 5.54 – 5.40 (m, 1H, CH), 3.28 – 3.23 (m, 4H, CH2×2), 2.79 – 2.73 (m,
4H, CH2×2), 2.47 – 2.39 (m, 5H, CH3, CH×2), 2.05 (d, J = 6.3 Hz, 4H, CH×4),
13
1.77 – 1.69 (m, 2H, CH×2). C NMR (100 MHz, DMSO-d6) δ 169.7, 156.3, 151.7,
146.5, 137.7, 133.2, 120.4 (2C), 117.2, 116.3 (2C), 55.7, 55.1 (2C), 49.3 (2C), 46.1,
28.7 (2C), 25.1 (2C). MS (ESI, m/z): 410.9 [M + H]+.
4.1.1.5. 2-((4-(4-Methylpiperazin-1-yl)phenyl)amino)-9-(tetrahydro-2H-pyran-4-yl)-
9H-purine-8-thiol (B5). Off-white solid; Yield 90.3%; mp >250 °C; 1H NMR (400
17

MHz, CDCl3) δ 8.15 (s, 1H, Ar-H), 7.53 (d, J = 8.7 Hz, 2H, Ar-H), 7.18 (s, 1H, NH),
6.98 (d, J = 8.7 Hz, 2H, Ar-H), 5.27 (t, J = 12.3 Hz, 1H, CH), 4.18 (d, J = 7.1 Hz, 2H,
CH×2), 3.62 (t, J = 11.7 Hz, 2H, CH×2), 3.25 (s, 4H, CH2×2), 3.04 – 2.89 (m, 2H,
CH×2), 2.71 (s, 4H, CH2×2), 2.44 (s, 3H, CH3), 1.81 (d, J = 10.1 Hz, 2H, CH×2).
13C NMR (100 MHz, DMSO-d6) δ 169.3, 156.4, 152.1, 146.3, 142.3, 137.8, 132.9,
120.2 (2C), 116.3 (2C), 67.1 (2C), 55.2 (2C), 52.7, 49.4 (2C), 46.2, 29.3(2C). MS
(ESI, m/z): 426.6 [M + H]+.
4.1.1.6. (1S, 4S)-4-(8-Mercapto-2-((4-(4-methylpiperazin-1-yl)phenyl)amino)-9H-pur-
in-9-yl)cyclohexan-1-ol (B6). Yellow solid; Yield 73.6%; mp 235.7-239.2 °C; 1H
NMR (400 MHz, CDCl3) δ 8.13 (s, 1H, Ar-H), 7.50 (d, J = 8.9 Hz, 2H, Ar-H), 7.11 (s,
1H, NH), 6.97 (d, J = 9.0 Hz, 2H, Ar-H), 5.02 (t, J = 12.4 Hz, 1H, CH), 3.80 (dd, J =
20.6, 9.7 Hz, 1H, CH), 3.25 – 3.22 (m, 4H, CH2×2), 2.73 – 2.69 (m, 4H, CH2×2),
2.44 (s, 3H, CH3), 2.18 (s, 2H, CH×2), 1.89 (d, J = 11.5 Hz, 2H, CH×2), 1.56 (d, J
= 13.6 Hz, 2H, CH×2), 1.29 (d, J = 13.2 Hz, 2H, CH×2). 13C NMR (100 MHz,
DMSO-d6) δ 169.2, 156.2, 151.9, 146.4, 137.8, 133.4, 120.2 (2C), 117.0, 116.3 (2C),
68.7, 55.2 (2C), 54.6, 49.4 (2C), 46.2, 35.1 (2C), 27.1 (2C). MS (ESI, m/z): 440.6 [M
+ H]+.
4.1.1.7. 2-((4-((2-(Dimethylamino)ethyl)(methyl)amino)phenyl)amino)-9-(tetrahydro-
2H-pyran-4-yl)-9H-purine-8-thiol (B7). Off-white solid; Yield 63.6%; mp 184.2-
187.7℃; 1H NMR (400 MHz, CDCl3) δ 7.89 (s, 1H, Ar-H), 7.34 (d, J = 8.9 Hz, 2H,
Ar-H), 7.04 (d, J = 15.1 Hz, 1H, NH), 6.64 (d, J = 9.0 Hz, 2H, Ar-H), 5.33 – 5.24 (m,
1H, CH), 4.17 (dd, J = 11.4, 4.2 Hz, 2H, CH2), 3.64 – 3.52 (m, 4H, CH2, CH×2),
3.01 – 2.93 (m, 5H, CH3, CH×2), 2.69 (t, J = 6.6 Hz, 2H, CH×2), 2.46 (s, 6H, CH3
×2), 1.82 (d, J = 9.6 Hz, 2H, CH×2). 13C NMR (100 MHz, DMSO-d6) δ 169.1,
156.5, 152.2, 144.9, 137.6, 130.6, 121.2 (2C), 117.1, 112.7 (2C), 67.2 (2C), 55.9, 52.7,
50.8, 45.9 (2C), 38.9, 29.3 (2C). MS (ESI, m/z): 428.6 [M + H]+.
4.1.1.8. 2-((4-(4-(Dimethylamino)piperidin-1-yl)phenyl)amino)-9-(tetrahydro-2H-pyr-
1
an-4-yl) -9H-purine-8-thiol (B8). Off-white solid; Yield 55.7%; mp >250 °C; H
NMR (400 MHz, DMSO-d6) δ 9.35 (s, 1H, NH), 8.19 (s, 1H, Ar-H), 7.59 (d, J = 9.0
Hz, 2H, Ar-H), 6.89 (d, J = 9.1 Hz, 2H, Ar-H), 5.12 (s, 1H, CH), 4.37 (s, 1H, CH),
18

4.03 (d, J = 7.0 Hz, 2H, CH×2), 3.64 (d, J = 12.1 Hz, 2H, CH×2), 3.45 (d, J = 6.7
Hz, 2H, CH×2), 2.85 – 2.73 (m, 2H, CH×2), 2.62 (d, J = 11.7 Hz, 2H, CH×2), 2.43
(s, 6H, CH3×2), 1.92 (d, J = 12.4 Hz, 2H, CH×2), 1.67 (d, J = 9.3 Hz, 2H, CH×2),
1.58 (d, J = 8.5 Hz, 2H, CH×2). 13C NMR (100 MHz, DMSO-d6) δ 156.1, 153.8,
148.9, 147.9, 147.2, 132.5, 131.7 (2C), 130.8, 129.6 (2C), 67.5 (2C), 62.1, 54.2, 50.1
(2C), 41.7 (2C), 30.4 (2C), 28.4 (2C). MS (ESI, m/z): 454.6 [M + H]+.
4.1.1.9. 2-((4-((1-Methylpiperidin-4-yl)oxy)phenyl)amino)-9-(tetrahydro-2H-pyran-4
1
-yl)-9H-purine-8-thiol (B9). Off-white solid; Yield 55.7%; mp 247.6-249.8 °C; H
NMR (400 MHz, CDCl3) δ 8.18 (s, 1H, Ar-H), 7.54 (d, J = 8.9 Hz, 2H, Ar-H), 7.20 (s,
1H, NH), 6.95 (d, J = 9.0 Hz, 2H, Ar-H), 5.27 (ddd, J = 12.3, 7.9, 4.2 Hz, 1H, CH),
4.35 (s, 1H, CH), 4.18 (dd, J = 11.5, 4.3 Hz, 2H, CH×2), 3.62 (t, J = 11.5 Hz, 2H,
CH×2), 2.93 (qd, J = 12.5, 4.7 Hz, 2H, CH×2), 2.82 (s, 2H, CH×2), 2.48 (s, 2H,
CH×2), 2.41 (s, 3H, CH3), 2.09 (dd, J = 10.3, 6.4 Hz, 2H, CH×2), 1.93 (s, 2H, CH
×2), 1.81 (d, J = 9.7 Hz, 2H, CH×2). 13C NMR (100 MHz, DMSO-d6) δ 169.4,
156.1, 153.8, 152.2, 137.6, 134.4, 120.6 (2C), 117.4, 114.8 (2C), 67.1 (2C), 66.3, 58.2
(2C), 52.7, 45.9 (3C), 29.3 (2C). MS (ESI, m/z): 441.6 [M + H]+.
4.1.1.10. 2-((4-(2-(Dimethylamino)ethoxy)phenyl)amino)-9-(tetrahydro-2H-pyran-4-
1
yl)-9H-purine-8-thiol (B10). Gray solid; Yield 56.2%; mp 228.6-229.6 °C; H NMR
(400 MHz, CDCl3) δ 8.06 (s, 1H, Ar-H), 7.46 (d, J = 9.0 Hz, 2H, Ar-H), 7.16 (s, 1H,
NH), 6.83 (d, J = 9.0 Hz, 2H, Ar-H), 5.34 – 5.22 (m, 1H, CH), 4.19 – 4.13 (m, 4H,
CH2, CH×2), 3.63 (d, J = 11.7 Hz, 2H, CH2), 2.97 – 2.86 (m, 4H, CH×4), 2.50 (s,
13
6H, CH3×2), 1.80 (d, J = 9.4 Hz, 2H, CH×2). C NMR (100 MHz, DMSO-d6) δ
169.5, 156.1, 152.2, 152.0, 137.6, 134.6, 120.7 (2C), 117.7, 116.6 (2C), 74.5, 72.5,
67.1 (2C), 52.7 (2C), 46.1, 30.9 (2C), 29.3 (2C). MS (ESI, m/z): 415.6 [M + H]+.
4.1.2. General procedure for the synthesis of compounds C1-C12.
The mixture of B (0.52 mmol), iodobenzene (80 µL, 0.78 mmol), CuI (8.2 mg,
0.026 mmol), 1,10-phenanthroline (16.3 mg, 0.052 mmol), K2CO3 (160.4 mg, 1.04
mmol) and DMSO (5 mL) was stirred at 140 °C for 24 h under argon atmosphere,
cooled to room temperature. To the mixture was added 20 mL of DCM. The organic
phase was washed three times with water, and dried over Na2SO4, filtered,
19

concentrated in vacuum. The residue was purified by column chromatography on
silica gel using DCM/MeOH = 10:1 (V/V) as the eluent to afford the compound C as
a solid.
4.1.2.1. 9-Isopropyl-N-(4-(4-methylpiperazin-1-yl)phenyl)-8-(phenylthio)-9H-purin-2
–amine (C1). Off-white solid; Yield 66.7%; mp 190.1-191.2 °C; 1H NMR (400 MHz,
CDCl3) δ 8.68 (s, 1H, Ar-H), 7.56 (t, J = 6.1 Hz, 2H, Ar-H), 7.48 (dt, J = 8.6, 2.3 Hz,
2H, Ar-H), 7.43 – 7.34 (m, 3H, Ar-H), 7.34 – 7.30 (m, 1H, NH), 6.97 (d, J = 9.0 Hz,
2H, Ar-H), 4.98 – 4.82 (m, 1H, CH), 3.24 – 3.19 (m, 4H, CH2×2), 2.66 – 2.61 (m,
13
4H, CH2×2), 2.39 (s, 3H, CH3), 1.63 (d, J = 6.9 Hz, 6H, CH3×2). C NMR (100
MHz, CDCl3) δ 155.9, 153.8, 148.6, 146.8, 132.8, 131.7 (2C), 130.8, 129.6 (2C),
129.1, 128.5, 120.3 (2C), 116.9 (2C), 55.2 (2C), 49.9 (2C), 49.3, 46.1, 20.7 (2C). MS
(ESI, m/z): 415.6 [M + H]+.
4.1.2.2.
9-Cyclopropyl-N-(4-(4-methylpiperazin-1-yl)phenyl)-8-(phenylthio)-9H-
purin-2-amine (C2). Off-white solid; Yield 69.7%; mp 210.4-213.2 °C; 1H NMR (400
MHz, CDCl3) δ 8.58 (s, 1H, Ar-H), 7.62 (s, 4H, Ar-H), 7.44 (s, 3H, Ar-H), 7.25 (s, 1H,
NH), 6.97 (d, J = 7.2 Hz, 2H, Ar-H), 3.23 (s, 4H, CH2×2), 3.05 (s, 1H, CH), 2.67 (s,
4H, CH2×2), 2.43 (d, J = 17.4 Hz, 3H, CH3), 1.36 (s, 2H, CH2), 1.24 (s, 2H, CH2).
13C NMR (100 MHz, CDCl3) δ 156.1, 155.3, 152.7, 147.5, 146.7, 133.4 (2C), 133.0,
129.6 (2C), 129.1, 128.9, 128.5, 120.2 (2C), 117.2 (2C), 55.1 (2C), 49.9 (2C), 46.0,
24.4, 7.2 (2C). MS (ESI, m/z): 458.2 [M + H]+.
4.1.2.3. 9-(Cyclopropylmethyl)-N-(4-(4-methylpiperazin-1-yl)phenyl)-8-(phenylthio)-
1
9H-purin-2-amine (C3). Off-white solid; Yield 58.3%; mp 172.7-173.6 °C; H NMR
(400 MHz, CDCl3) δ 8.68 (s, 1H, Ar-H), 7.57 (d, J = 8.5 Hz, 2H, Ar-H), 7.53 (d, J =
6.8 Hz, 2H, Ar-H), 7.39 (d, J = 7.1 Hz, 3H, Ar-H), 6.97 (d, J = 8.5 Hz, 2H, Ar-H),
4.06 (d, J = 6.9 Hz, 2H, CH2), 3.22 (s, 4H, CH2×2), 2.65 (s, 4H, CH2×2), 2.40 (s,
3H, CH3), 1.33 (s, 1H, CH), 0.58 – 0.49 (m, 4H, CH2×2). 13C NMR (100 MHz,
CDCl3) δ 156.7, 154.3, 148.6, 148.3, 146.9, 132.7, 131.9 (2C), 130.2, 129.6 (2C),
128.8, 128.6, 120.5 (2C), 117.1 (2C), 55.2 (2C), 49.8 (2C), 47.7, 46.1, 11.0, 4.3 (2C).
MS (ESI, m/z): 472.6 [M + H]+.
4.1.2.4. 9-Cyclopentyl-N-(4-(4-methylpiperazin-1-yl)phenyl)-8-(phenylthio)-9H-purin
20

-2-amine (C4). Off-white solid; Yield 57.6%; mp 179.4-181.0 °C; 1H NMR (400 MHz,
CDCl3) δ 8.68 (s, 1H, Ar-H), 7.55 (d, J = 9.0 Hz, 2H, Ar-H), 7.50 – 7.47 (m, 1H,
Ar-H), 7.46 (d, J = 1.4 Hz, 1H, Ar-H), 7.41 – 7.34 (m, 3H, Ar-H), 7.17 (s, 1H, NH),
6.95 (d, J = 9.0 Hz, 2H, Ar-H), 4.96 (t, J = 8.7 Hz, 1H, CH), 3.26 – 3.19 (m, 4H, CH2
×2), 2.68 – 2.61 (m, 4H, CH2×2), 2.50 – 2.42 (m, 2H, CH2), 2.40 (s, 3H, CH3), 2.05
(dd, J = 7.1, 5.4 Hz, 2H, CH2), 1.94 – 1.85 (m, 2H. CH2), 1.68 (dd, J = 11.3, 5.6 Hz,
13
2H, CH2). C NMR (100 MHz, CDCl3) δ 155.9, 148.9, 148.7, 146.8, 132.9, 131.9,
131.6 (2C), 130.9, 129.7, 129.6 (2C), 128.4, 120.4 (2C), 117.1 (2C), 57.5, 55.1 (2C),
49.8 (2C), 45.9, 30.1 (2C), 25.1 (2C). MS (ESI, m/z): 486.6 [M + H]+.
4.1.2.5. N-(4-(4-Methylpiperazin-1-yl)phenyl)-8-(phenylthio)-9-(tetrahydro-2H-pyran
1
-4-yl)-9H-purin-2-amine (C5). Off-white solid; Yield 56.7%; mp 235.6-236.0 °C; H
NMR (400 MHz, CDCl3) δ 8.70 (s, 1H, Ar-H), 7.58 (d, J = 8.9 Hz, 2H, Ar-H), 7.50
(dd, J = 7.9, 1.6 Hz, 2H, Ar-H), 7.44 – 7.31 (m, 3H, Ar-H), 7.29 (s, 1H, NH), 6.98 (d,
J = 9.0 Hz, 2H, Ar-H), 4.72 – 4.62 (m, 1H, CH), 4.12 (dd, J = 11.5, 4.2 Hz, 2H, CH×
2), 3.44 (t, J = 11.4 Hz, 2H, CH×2), 3.27 – 3.14 (m, 4H, CH2×2), 2.93 (qd, J = 12.5,
4.5 Hz, 2H, CH×2), 2.69 – 2.55 (m, 4H, CH2×2), 2.38 (s, 3H, CH3), 1.55 (dd, J =
12.4, 2.5 Hz, 2H, CH×2). 13C NMR (100 MHz, CDCl3) δ 156.0, 153.8, 148.9, 147.9,
146.9, 132.6, 131.8 (2C), 130.8, 129.7 (2C), 128.8, 128.7, 120.2 (2C), 117.0 (2C),
67.5 (2C), 55.2 (2C), 54.2, 49.8 (2C), 46.1, 30.4 (2C). MS (ESI, m/z): 502.6 [M +
H]+.
4.1.2.6. (1S, 4S)-4-(2-((4-(4-methylpiperazin-1-yl)phenyl)amino)-8-(phenylthio)-9H-
purin-9-yl)cyclohexan-1-ol (C6). Off-white solid; Yield 55.2%; mp 203.9-207.1 °C;
1H NMR (400 MHz, CDCl3) δ 8.68 (s, 1H, Ar-H), 7.55 (d, J = 6.8 Hz, 2H, Ar-H), 7.49
(s, 2H, Ar-H), 7.37 (s, 4H, Ar-H, NH), 6.96 (d, J = 6.6 Hz, 2H, Ar-H), 4.46 (s, 1H,
CH), 3.80 (s, 1H, CH), 3.21 (s, 4H, CH2×2), 2.63 (s, 4H, CH2×2), 2.39 (s, 3H, CH3),
2.14 (s, 4H, CH2×2), 1.67 (d, J = 9.5 Hz, 2H, CH×2), 1.40 (d, J = 10.9 Hz, 2H, CH
13
×2). C NMR (100 MHz, CDCl3) δ 155.9, 153.6, 148.8, 148.2, 146.9, 132.7, 131.9
(2C), 130.7, 129.6 (2C), 128.7, 128.9, 120.4 (2C), 116.9 (2C), 69.6, 56.0, 55.2 (2C),
49.9 (2C), 46.1, 34.8 (2C), 27.9 (2C). MS (ESI, m/z): 516.1 [M + H]+.
4.1.2.7. N-(4-(2-(Dimethylamino)ethoxy)phenyl)-8-(phenylthio)-9-(tetrahydro-2H-py-
21

ran-4-yl)-9H-purin-2-amine (C7). Off-white solid; Yield 47.3%; mp 172.7-173.5 °C;
1H NMR (400 MHz, CDCl3) δ 8.70 (s, 1H, Ar-H), 7.57 (t, J = 6.1 Hz, 2H, Ar-H), 7.50
(dd, J = 7.9, 1.6 Hz, 2H, Ar-H), 7.42 – 7.32 (m, 4H, Ar-H, NH), 6.95 (d, J = 8.9 Hz,
2H, Ar-H), 4.66 (ddd, J = 12.1, 7.9, 4.2 Hz, 1H, CH), 4.14 – 4.07 (m, 4H, CH×4),
3.44 (t, J = 11.6 Hz, 2H, CH2), 2.92 (qd, J = 12.5, 4.5 Hz, 2H, CH×2), 2.75 (t, J = 5.7
Hz, 2H, CH2), 2.37 (s, 6H, CH3×2), 1.56 (dd, J = 12.4, 2.4 Hz, 2H, CH×2). 13C
NMR (100 MHz, CDCl3) δ 156.1, 154.4, 153.8, 148.8, 148.1, 133.2, 131.8 (2C),
130.7, 129.7 (2C), 128.9, 128.7, 120.7 (2C), 115.0 (2C), 67.5 (2C), 66.4, 58.4, 54.2,
45.9 (2C), 30.4 (2C). MS (ESI, m/z): 491.1 [M + H]+.
4.1.2.8. N1-(2-(Dimethylamino)ethyl)-N1-methyl-N4-(8-(phenylthio)-9-(tetrahydro-2H
-pyran-4-yl)-9H-purin-2-yl)benzene-1,4-diamine (C8). Off-white solid; Yield 55.3%;
1
mp 164.4-165.6 °C; H NMR (400 MHz, CDCl3) δ 8.69 (s, 1H, Ar-H), 7.52 (s, 1H,
Ar-H), 7.50 (d, J = 2.3 Hz, 2H, Ar-H), 7.49 – 7.47 (m, 1H, Ar-H), 7.41 – 7.34 (m, 3H,
Ar-H), 6.77 (d, J = 9.0 Hz, 2H, Ar-H), 4.71 – 4.62 (m, 1H, CH), 4.11 (dd, J = 11.5, 4.2
Hz, 2H, CH×2), 3.48 – 3.45 (m, 2H, CH2), 3.42 (d, J = 11.7 Hz, 2H, CH×2), 2.96 (s,
3H, CH3), 2.95 – 2.86 (m, 2H, ), 2.55 – 2.47 (m, 2H, CH2), 2.31 (s, 6H, CH3×2), 1.55
(dd, J = 12.4, 2.5 Hz, 2H, CH×2). 13C NMR (100 MHz, CDCl3) δ 156.5, 153.95,
148.95, 147.6, 145.6, 131.6 (2C), 130.9, 129.7 (2C), 129.6, 128.7, 128.6, 121.3 (2C),
113.0 (2C), 67.5 (2C), 55.9, 54.2, 51.6, 45.9 (2C), 38.9, 30.5 (2C). MS (ESI, m/z):
504.2 [M + H]+.
4.1.2.9. N-(4-(4-(Dimethylamino)piperidin-1-yl)phenyl)-8-(phenylthio)-9-(tetrahydro-
2H-pyran-4-yl)-9H-purin-2-amine (C9). Off-white solid; Yield 51.4%; mp
242.3-244.8 °C; 1H NMR (400 MHz, CDCl3) δ 8.70 (s, 1H, Ar-H), 7.57 (d, J = 9.0 Hz,
2H, Ar-H), 7.49 (dd, J = 7.9, 1.6 Hz, 2H, Ar-H), 7.38 (tdd, J = 7.0, 5.0, 1.9 Hz, 3H,
Ar-H), 7.31 (s, 1H, NH), 6.98 (d, J = 9.0 Hz, 2H, Ar-H), 4.66 (dd, J = 10.1, 6.0 Hz,
1H, CH), 4.12 (dd, J = 11.5, 4.2 Hz, 2H, CH×2), 3.68 (d, J = 12.3 Hz, 2H, CH×2),
3.44 (t, J = 11.4 Hz, 2H, CH×2), 2.93 (td, J = 12.4, 7.9 Hz, 2H, CH×2), 2.70 (td, J =
12.1, 1.9 Hz, 2H, CH×2), 2.35 (s, 6H, CH3×2), 2.28 (dd, J = 9.2, 5.7 Hz, 1H, CH),
1.95 (d, J = 12.4 Hz, 2H, CH×2), 1.69 (tt, J = 12.0, 6.1 Hz, 2H, CH×2), 1.55 (dd, J
22

= 12.4, 2.5 Hz, 2H, CH×2). 13C NMR (100 MHz, CDCl3) δ 156.1, 153.8, 148.9,
147.9, 147.2, 132.5, 131.7 (2C), 130.8, 129.6 (2C), 128.7, 120.2 (2C), 117.5 (2C),
67.5 (2C), 62.1, 54.2, 50.1 (2C), 41.7 (2C), 30.4 (2C), 28.4 (2C). MS (ESI, m/z):
530.2 [M + H]+.
4.1.2.10. N-(4-((1-Methylpiperidin-4-yl)oxy)phenyl)-8-(phenylthio)-9-(tetrahydro-2H
-pyran-4-yl)-9H-purin-2-amine (C10). Off-white solid; Yield 59.6%; mp
177.9-179.2 °C; 1H NMR (400 MHz, CDCl3) δ 8.70 (s, 1H, Ar-H), 7.58 (s, 1H, Ar-H),
7.56 (d, J = 3.4 Hz, 1H, Ar-H), 7.51 (d, J = 1.9 Hz, 1H, Ar-H), 7.49 (d, J = 1.4 Hz, 1H,
Ar-H), 7.43 – 7.34 (m, 3H, Ar-H), 7.32 (s, 1H, NH), 6.97 – 6.92 (m, 2H, Ar-H), 4.67
(ddd, J = 12.1, 7.9, 4.2 Hz, 1H, CH×2), 4.35 – 4.24 (m, 1H, CH×2), 4.12 (dd, J =
11.5, 4.2 Hz, 2H, CH×2), 3.44 (t, J = 11.4 Hz, 2H, CH×2), 2.92 (qd, J = 12.5, 4.6
Hz, 2H, CH×2), 2.72 (s, 2H, CH×2), 2.33 (s, 3H, CH3), 2.29 (s, 2H, CH×2), 2.07 –
1.97 (m, 2H, CH×2), 1.91 – 1.81 (m, 2H, CH×2), 1.56 (dd, J = 12.4, 2.5 Hz, 2H,
CH×2). 13C NMR (100 MHz, CDCl3) δ 156.0, 153.8, 152.9, 148.8, 148.1, 133.4,
131.8 (2C), 130.6, 129.7 (2C), 128.9, 128.7, 120.6 (2C), 116.9 (2C), 67.5 (2C), 54.2
(2C), 52.7, 46.2 (2C), 30.9 (2C), 30.4 (2C). MS (ESI, m/z): 517.1 [M + H]+.
4.1.2.11. N-(4-(2-(Dimethylamino)ethoxy)phenyl)-8-((3-fluorophenyl)thio)-9-(tetrah-
ydro-2H-pyran-4-yl)-9H-purin-2-amine (C11). Off-white solid; Yield 55.2%; mp
146.2-150.3 °C; 1H NMR (400 MHz, CDCl3) δ 8.74 (s, 1H, Ar-H), 7.58 (d, J = 8.8 Hz,
2H, Ar-H), 7.36 (td, J = 8.0, 5.9 Hz, 1H, Ar-H), 7.29 – 7.18 (m, 3H, NH, Ar-H), 7.12
– 7.02 (m, 1H, Ar-H), 6.96 (d, J = 8.9 Hz, 2H, Ar-H), 4.74 – 4.60 (m, 1H, CH), 4.13
(dd, J = 12.1, 6.7 Hz, 4H, CH2, CH×2), 3.47 (t, J = 12.0 Hz, 2H, CH×2), 3.02 –
2.88 (m, 2H, CH×2), 2.81 (t, J = 5.5 Hz, 2H, CH2), 2.41 (s, 6H, CH3×2), 1.62 (d, J
13
= 12.6 Hz, 2H, CH×2). C NMR (100 MHz, CDCl3) δ 162.6 (d, JC-F = 250), 156.2,
154.4, 153.7, 149.2, 146.7, 133.1, 130.9, 126.8, 120.7 (2C), 118.3, 118.1, 115.8, 115.6,
114.9 (2C), 67.5 (2C), 66.2, 58.3, 54.2, 45.8 (2C), 30.5 (2C). MS (ESI, m/z): 509.2
[M + H]+.
4.1.2.12.
N-(4-(2-(Dimethylamino)ethoxy)phenyl)-8-((4-fluorophenyl)thio)-9-
(tetrahy-dro-2H-pyran-4-yl)-9H-purin-2-amine (C12). Off-white solid; Yield 47.2%;
mp 142.7-147.3 °C; 1H NMR (400 MHz, CDCl3) δ 8.66 (s, 1H, Ar-H), 7.62 – 7.53 (m,
23

4H, Ar-H), 7.18 (s, 1H, NH), 7.13 (td, J = 8.5, 1.5 Hz, 2H, Ar-H), 7.00 – 6.92 (m, 2H,
Ar-H), 4.71 – 4.57 (m, 1H, CH), 4.16 (dd, J = 11.6, 3.9 Hz, 2H, CH×2), 4.10 (t, J =
5.7 Hz, 2H, CH2), 3.51 (t, J = 12.0 Hz, 2H, CH×2), 2.95 (qd, J = 12.4, 4.2 Hz, 2H,
CH×2), 2.77 (t, J = 5.6 Hz, 2H, CH2), 2.38 (t, J = 3.8 Hz, 6H, CH3×2), 1.66 (d, J =
12.2 Hz, 2H, CH×2). 13C NMR (100 MHz, CDCl3) δ 163.3 (d, JC-F = 265 Hz), 155.9,
154.5, 153.9, 148.8, 148.4, 135.0, 134.9, 133.1, 128.9, 124.7, 120.6 (2C), 117.1, 116.9,
115.0 (2C), 67.5 (2C), 66.3, 58.4, 54.1, 45.9 (2C), 30.5 (2C). MS (ESI, m/z): 509.2
[M + H]+.
4.1.3. General procedure for the synthesis of compounds E1-E4.
The mixture of B (0.52 mmol), 3-chloromethylpyridine hydrochloride (87 mg,
0.52 mmol), K2CO3 (180 mg, 1.30 mmol) and acetone (10 mL) was refluxed for 6 h.
The mixture was concentrated in vacuum. The residue was purified by column
chromatography on silica gel using DCM/MeOH = 10:1 as the eluent to afford the
compound E as an off-white solid.
4.1.3.1. 9-Cyclopropyl-N-(4-(4-methylpiperazin-1-yl)phenyl)-8-((pyridin-3-ylmethyl)
1
thio)-9H-purin-2-amine (E1). Off-white solid; Yield 42.4%; mp 164.6-165.0 °C; H
NMR (400 MHz, CDCl3) δ 8.75 (d, J = 1.7 Hz, 1H, Ar-H), 8.59 (s, 1H, Ar-H), 8.55
(dd, J = 4.7, 1.3 Hz, 1H, Ar-H), 7.84 (d, J = 7.9 Hz, 1H, Ar-H), 7.60 (d, J = 8.9 Hz,
2H, Ar-H), 7.29 (d, J = 5.5 Hz, 1H, Ar-H), 7.20 (s, 1H, NH), 6.97 (d, J = 9.0 Hz, 2H,
Ar-H), 4.57 (s, 2H, CH2), 3.24 – 3.19 (m, 4H, CH2×2), 3.07 – 2.98 (m, 1H, CH),
2.67 – 2.62 (m, 4H, CH2×2), 2.40 (s, 3H, CH3), 1.25 (t, J = 6.5 Hz, 2H, CH2), 1.23 –
1.18 (m, 2H, CH2). 13C NMR (100 MHz, CDCl3) δ 155.9, 155.7, 153.7, 150.4, 149.0,
146.7, 146.1, 136.7, 133.1, 132.7, 128.6, 123.5, 120.2 (2C), 117.2 (2C), 55.1 (2C),
49.9 (2C), 46.0, 32.8, 23.7, 6.9 (2C). MS (ESI, m/z): 473.5 [M + H]+.
4.1.3.2. 9-Cyclopentyl-N-(4-(4-methylpiperazin-1-yl)phenyl)-8-((pyridin-3-ylmethyl)
1
thio)-9H-purin-2-amine (E2). Off-white solid; Yield 72.9%; mp 178.4-179.5 °C; H
NMR (400 MHz, CDCl3) δ 8.72 (d, J = 2.0 Hz, 1H, Ar-H), 8.61 (s, 1H, Ar-H), 8.54
(dd, J = 4.8, 1.5 Hz, 1H, Ar-H), 7.81 (dt, J = 7.8, 1.8 Hz, 1H, Ar-H), 7.54 (d, J = 8.9
Hz, 2H, Ar-H), 7.28 – 7.25 (m, 1H, Ar-H), 7.10 (s, 1H, NH), 6.96 (d, J = 9.0 Hz, 2H,
24

Ar-H), 4.71 – 4.62 (m, 1H, CH), 4.58 (s, 2H, CH2), 3.23 – 3.18 (m, 4H, CH2×2),
2.67 – 2.62 (m, 4H, CH2×2), 2.44 – 2.38 (m, 5H, CH3, CH2), 2.04 (dd, J = 15.6, 9.4
Hz, 4H, CH2×2), 1.70 (d, J = 5.3 Hz, 2H, CH2). 13C NMR (100 MHz, CDCl3) δ
155.6, 154.4, 151.1, 150.3, 149.0, 146.7, 146.4, 136.6, 132.7, 129.3, 123.5, 120.4 (2C),
117.1 (2C), 56.9, 55.2 (2C), 49.9 (2C), 45.9, 33.7, 30.1 (2C), 24.9 (2C). MS (ESI,
m/z): 501.7 [M + H]+.
4.1.3.3.
N-(4-(4-Methylpiperazin-1-yl)phenyl)-8-((pyridin-3-ylmethyl)thio)-9-
(tetrahydro-2H-pyran-4-yl)-9H-purin-2-amine (E3). Off-white solid; Yield 64.5%; mp
193.4-196.1 °C; 1H NMR (400 MHz, CDCl3) δ 8.70 (s, 1H, Ar-H), 8.62 (s, 1H, Ar-H),
8.57 – 8.50 (m, 1H, Ar-H), 7.83 – 7.75 (m, 1H, Ar-H), 7.56 (d, J = 9.0 Hz, 2H, Ar-H),
7.25 (dd, J = 7.7, 4.9 Hz, 1H, Ar-H), 6.96 (d, J = 8.9 Hz, 2H, Ar-H), 4.57 (s, 2H, CH2),
4.41 – 4.30 (m, 1H, CH), 4.19 – 4.09 (m, 2H, CH×2), 3.49 (t, J = 11.5 Hz, 2H, CH×
2), 3.26 – 3.13 (m, 4H, CH2×2), 2.86 (qd, J = 12.4, 4.5 Hz, 2H, CH×2), 2.65 – 2.56
(m, 4H, CH2×2), 2.36 (s, 3H, CH3), 1.71 (d, J = 12.5 Hz, 2H, CH×2). 13C NMR
(100 MHz, CDCl3) δ 155.7, 154.5, 150.3, 150.1, 149.1, 146.8, 146.6, 136.6, 132.9,
132.5, 128.9, 123.5, 120.2 (2C), 117.0 (2C), 67.4 (2C), 55.2 (2C), 53.5, 49.9 (2C),
46.1, 33.8, 30.5 (2C). MS (ESI, m/z): 517.2 [M + H]+.
4.1.3.4. 8-(Benzylthio)-N-(4-(4-methylpiperazin-1-yl)phenyl)-9-(tetrahydro-2H-pyran
1
-4-yl)-9H-purin-2-amine (E4). Off-white solid; Yield 56.3%; mp 179.0-182.1 °C; H
NMR (400 MHz, CDCl3) δ 8.65 (s, 1H, Ar-H), 7.58 (d, J = 8.9 Hz, 2H, Ar-H), 7.43 (d,
J = 6.8 Hz, 2H, Ar-H), 7.40 – 7.29 (m, 3H, Ar-H), 7.27 (s, 1H, NH), 6.98 (d, J = 8.9
Hz, 2H, Ar-H), 4.59 (s, 2H, CH2), 4.47 – 4.33 (m, 1H, CH), 4.14 (dd, J = 11.6, 4.1 Hz,
2H, CH×2), 3.49 (t, J = 11.7 Hz, 2H, CH×2), 3.30 – 3.18 (m, 4H, CH2×2), 2.89 (qd,
J = 12.5, 4.6 Hz, 2H, CH×2), 2.72 – 2.62 (m, 4H, CH2×2), 2.41 (s, 3H, CH3), 1.70
(dd, J = 12.5, 2.5 Hz, 2H, CH×2). 13C NMR (100 MHz, CDCl3) δ 155.6, 154.5,
151.1, 146.6, 146.5, 136.1, 133.1, 129.1 (2C), 128.8 (3C), 127.9, 120.1 (2C), 117.2
(2C), 67.5 (2C), 55.1 (2C), 53.5, 49.8 (2C), 46.1, 37.2, 30.4 (2C). MS (ESI, m/z):
516.2 [M + H]+.
4.1.4. General procedure for the synthesis of compounds D1-D12 and F1-F3.
A solution of 3-chloroperbenzoic acid (45.3 mg, 0.22 mmol) in DCM (10 mL)
25

was added dropwise to a mixture of C or E (0.22 mmol) in DCM (10 mL) at -15 °C.
The reaction mixture was stirred at room temperature for 30 min and concentrated
under reduced pressure. The resulting crude product was purified by silica gel
chromatography using DCM/MeOH = 5:1 (V/V) as the eluent to afford D or F as a
yellow solid.
4.1.4.1.
9-Isopropyl-N-(4-(4-methylpiperazin-1-yl)phenyl)-8-(phenylsulfinyl)-9H-
1
purin-2-amine (D1). Yellow solid; Yield 55.6%; mp 176.6-177.7 °C; H NMR (400
MHz, CDCl3) δ 8.67 (s, 1H, Ar-H), 7.60 (d, J = 9.0 Hz, 3H, Ar-H), 7.48 (d, J = 6.4 Hz,
2H, Ar-H), 7.37 (q, J = 5.8 Hz, 3H, Ar-H, NH), 6.97 (t, J = 7.6 Hz, 2H, Ar-H), 4.88
(dt, J = 13.6, 6.8 Hz, 1H, CH), 3.76 (t, J = 11.0 Hz, 2H, CH×2), 3.52 (t, J = 9.8 Hz,
2H, CH×2), 3.37 (d, J = 3.5 Hz, 4H, CH2×2), 3.33 (s, 3H, CH3), 1.62 (d, J = 6.8 Hz,
6H, CH3×2). 13C NMR (100 MHz, CDCl3) δ 155.8, 153.8, 148.5, 148.3, 145.2, 134.1,
131.8 (2C), 130.6, 129.6 (2C), 129.1, 128.6, 120.2 (2C), 117.9 (2C), 65.8 (2C), 60.5,
49.3, 45.2 (2C), 20.7 (2C). MS (ESI, m/z): 476.2 [M + H]+.
4.1.4.2. 9-Cyclopropyl-N-(4-(4-methylpiperazin-1-yl)phenyl)-8-(phenylsulfinyl)-9H-
1
purin-2-amine (D2). Yellow solid; Yield 60.3%; mp 140.7-143.1 °C; H NMR (400
MHz, CDCl3) δ 8.56 (s, 1H, Ar-H), 7.69 – 7.57 (m, 4H, Ar-H), 7.43 (d, J = 3.6 Hz, 3H,
Ar-H), 6.98 (d, J = 8.3 Hz, 2H, Ar-H), 3.75 (t, J = 10.9 Hz, 2H, CH×2), 3.54 (s, 2H,
CH×2), 3.39 (d, J = 32.6 Hz, 7H, CH2×2, CH3), 3.04 (d, J = 3.5 Hz, 1H, CH), 1.31
13
– 1.19 (m, 4H, CH2×2). C NMR (100 MHz, CDCl3) δ 155.9, 155.2, 152.9, 147.4,
145.2, 140.1, 134.2, 133.6 (2C), 129.6 (2C), 129.2, 128.8, 120.1 (2C), 118.0 (2C),
65.8, 60.3, 45.3 (2C), 24.4 (2C), 7.2 (2C). MS (ESI, m/z): 474.1 [M + H]+.
4.1.4.3. 9-(Cyclopropylmethyl)-N-(4-(4-methylpiperazin-1-yl)phenyl)-8-(phenylsulfi-
nyl)-9H-purin-2-amine (D3). White solid; Yield 58.8%; mp 136.1-140.5 °C; 1H NMR
(400 MHz, CDCl3) δ 8.66 (s, 1H, Ar-H), 7.60 (d, J = 7.5 Hz, 2H, Ar-H), 7.53 (d, J =
5.6 Hz, 2H, Ar-H), 7.38 (s, 4H, Ar-H, NH), 6.97 (d, J = 7.1 Hz, 2H, Ar-H), 4.05 (d, J
= 5.9 Hz, 2H, CH2), 3.46 (d, J = 41.9 Hz, 11H, CH2×4, CH3), 1.32 (s, 1H, CH), 0.53
13
(d, J = 11.9 Hz, 4H, CH2×2). C NMR (100 MHz, CDCl3) δ 156.4, 154.3, 149.0,
148.1, 145.1, 134.1, 132.0 (2C), 129.9, 129.6 (2C), 128.8, 128.7, 120.4 (2C), 117.9
(2C), 65.5, 60.0, 47.7, 45.1 (2C), 11.0 (2C), 4.3 (2C). MS (ESI, m/z): 488.2 [M + H]+.
26

4.1.4.4. 9-Cyclopentyl-N-(4-(4-methylpiperazin-1-yl)phenyl)-8-(phenylsulfinyl)-9H-
1
purin-2-amine (D4). White solid; Yield 54.3%; mp 192.0-194.4 °C; H NMR (400
MHz, CDCl3) δ 8.68 (s, 1H, Ar-H), 7.59 (d, J = 8.9 Hz, 2H, Ar-H), 7.48 (dd, J = 7.9,
1.7 Hz, 2H, Ar-H), 7.41 – 7.35 (m, 3H, Ar-H), 7.28 (s, 1H, NH), 6.98 (d, J = 9.0 Hz,
2H, Ar-H), 4.96 (p, J = 8.7 Hz, 1H. CH), 3.80 (dd, J = 17.5, 7.0 Hz, 2H, CH2), 3.54
(td, J = 11.5, 3.3 Hz, 2H, CH2), 3.41 – 3.34 (m, 7H, CH3, CH2×2), 2.44 (td, J = 16.5,
8.2 Hz, 2H, CH2), 2.04 (dd, J = 13.8, 7.8 Hz, 2H, CH2), 1.97 – 1.86 (m, 2H, CH2),
1.76 – 1.64 (m, 2H, CH2). 13C NMR (100 MHz, CDCl3) δ 155.7, 153.5, 149.2, 148.6,
145.2, 134.1, 131.7 (2C), 130.7, 129.6 (2C), 129.3, 128.6, 120.3 (2C), 117.9 (2C),
65.7 (2C), 60.1, 57.5, 45.2 (2C), 30.1 (2C), 25.1 (2C). MS (ESI, m/z): 502.5 [M +
H]+.
4.1.4.5. N-(4-(4-Methylpiperazin-1-yl)phenyl)-8-(phenylsulfinyl)-9-(tetrahydro-2H-
pyran-4-yl)-9H-purin-2-amine (D5). White solid; Yield 63.7%; mp 170.9-171.6 °C;
1H NMR (400 MHz, CDCl3) δ 8.69 (s, 1H, Ar-H), 7.62 (d, J = 8.5 Hz, 2H, Ar-H), 7.55
(s, 1H, Ar-H), 7.49 (d, J = 6.5 Hz, 2H, Ar-H), 7.37 (d, J = 6.9 Hz, 3H, Ar-H, NH),
6.99 (d, J = 8.5 Hz, 2H, Ar-H), 4.64 (d, J = 11.8 Hz, 1H, CH), 4.11 (d, J = 8.1 Hz, 2H,
CH×2), 3.74 (t, J = 11.1 Hz, 2H, CH×2), 3.57 – 3.37 (m, 8H, CH2×4), 3.34 (s, 3H,
CH3), 2.91 (dt, J = 12.0, 8.4 Hz, 2H, CH×2), 1.55 (d, J = 11.0 Hz, 2H, CH×2). 13C
NMR (100 MHz, CDCl3) δ 155.9, 153.8, 148.8, 148.2, 145.2, 133.9, 131.8 (2C),
130.6, 129.7 (2C), 128.9, 128.7, 120.2 (2C), 117.9 (2C), 67.5 (2C), 65.8, 60.4, 54.2
(2C), 45.1 (2C), 30.4 (2C). MS (ESI, m/z): 518.6 [M + H]+.
4.1.4.6. (1S, 4S)-4-(2-((4-(4-Methylpiperazin-1-yl)phenyl)amino)-8-(phenylsulfinyl)-
1
9H-purin-9-yl)cyclohexan-1-ol (D6). Brown oil; Yield 58.2%; H NMR (400 MHz,
DMSO-d6) δ 9.55 (s, 1H, NH), 8.72 (s, 1H, Ar-H), 7.69 (d, J = 8.8 Hz, 2H, Ar-H),
7.53 – 7.40 (m, 5H, Ar-H), 6.98 (d, J = 8.9 Hz, 2H, Ar-H), 4.38 (t, J = 12.0 Hz, 1H,
CH), 3.72 – 3.61 (m, 4H, CH2×2), 3.52 (d, J = 11.7 Hz, 4H, CH2×2), 3.32 (s, 4H,
CH, CH3), 2.55 (s, 2H, CH×2), 1.93 (d, J = 10.2 Hz, 2H, CH×2), 1.58 (d, J = 10.8
Hz, 2H, CH×2), 1.26 (s, 2H, CH×2). 13C NMR (100 MHz, DMSO-d6) δ 156.1,
153.4, 149.1, 147.2, 144.9, 134.2, 131.9 (2C), 131.1, 130.2 (2C), 129.1, 128.5, 120.1
(2C), 116.9 (2C), 68.5, 64.3 (2C), 58.4, 56.1, 44.3 (2C), 34.9 (2C), 28.2 (2C). MS
27

(ESI, m/z): 532.1 [M + H]+.
4.1.4.7.
N-(4-(2-(Dimethylamino)ethoxy)phenyl)-8-(phenylsulfinyl)-9-(tetrahydro-
2H-pyran-4-yl)-9H-purin-2-amine (D7). White solid; Yield 59.4%; mp
159.5-161.6 °C; 1H NMR (400 MHz, CDCl3) δ 8.70 (s, 1H, Ar-H), 7.61 (d, J = 8.8 Hz,
2H, Ar-H), 7.50 (d, J = 6.3 Hz, 2H, Ar-H), 7.39 (d, J = 7.0 Hz, 3H, Ar-H), 7.26 (s, 1H,
NH), 6.95 (d, J = 8.7 Hz, 2H, Ar-H), 4.67 (t, J = 12.0 Hz, 1H, CH), 4.59 (s, 2H, CH2),
4.12 (dd, J = 11.3, 3.5 Hz, 2H, CH×2), 3.78 (s, 2H, CH×2), 3.51 – 3.26 (m, 8H,
CH2, CH3×2), 2.92 (tt, J = 12.1, 6.2 Hz, 2H, CH×2), 1.57 (d, J = 12.2 Hz, 2H, CH
×2). 13C NMR (100 MHz, DMSO-d6) δ 156.2, 153.7, 153.0, 148.9, 134.9, 132.2 (2C),
130.9, 130.2 (3C), 129.1, 128.6, 120.6 (2C), 114.9 (2C), 68.9, 66.9 (2C), 62.8, 59.6,
54.0 (2C), 30.5 (2C). MS (ESI, m/z): 507.1 [M + H]+.
4.1.4.8. N1-(2-(Dimethylamino)ethyl)-N1-methyl-N4-(8-(phenylsulfinyl)-9-(tetrahydro
-2H-pyran-4-yl)-9H-purin-2-yl)benzene-1,4-diamine (D8). Yellow solid; Yield 61.2%;
mp 144.8-146.4 °C; 1H NMR (400 MHz, CDCl3) δ 8.67 (s, 1H, Ar-H), 7.54 (d, J = 8.9
Hz, 2H, Ar-H), 7.48 (dd, J = 7.8, 1.5 Hz, 2H, Ar-H), 7.41 – 7.34 (m, 4H, Ar-H), 6.84
(d, J = 9.0 Hz, 2H, Ar-H), 4.65 (tt, J = 11.9, 4.0 Hz, 1H, CH), 4.09 (dd, J = 11.6, 4.0
Hz, 2H, CH×2), 3.97 (t, J = 6.7 Hz, 2H, CH2), 3.47 – 3.44 (m, 2H, CH2), 3.41 (d, J =
12.4 Hz, 2H), 3.27 (s, 6H, CH3×2), 2.99 (s, 3H, CH3), 2.91 (dd, J = 12.5, 4.4 Hz, 2H,
13
CH×2), 1.58 – 1.50 (m, 2H, CH×2). C NMR (100 MHz, CDCl3) δ 156.3, 153.9,
148.9, 147.8, 144.8, 131.7 (2C), 130.8, 130.7, 129.6 (2C), 128.8, 128.7, 121.4 (2C),
113.9 (2C), 67.5 (2C), 66.6, 59.6 (2C), 54.1, 47.7, 39.6, 30.4 (2C). MS (ESI, m/z):
520.2 [M + H]+.
4.1.4.9.
N-(4-(4-(Dimethylamino)piperidin-1-yl)phenyl)-8-(phenylsulfinyl)-9-
(tetrahydro-2H-pyran-4-yl)-9H-purin-2-amine (D9). Yellow solid; Yield 49.2%; mp
151.6-152.8 °C; 1H NMR (400 MHz, CDCl3) δ 8.67 (s, 1H, Ar-H), 7.56 (d, J = 8.5 Hz,
2H, Ar-H), 7.48 (d, J = 6.2 Hz, 2H, Ar-H), 7.42 (s, 1H, NH), 7.36 (d, J = 7.1 Hz, 3H,
Ar-H), 6.93 (d, J = 8.8 Hz, 2H, Ar-H), 4.64 (t, J = 11.8 Hz, 1H, CH), 4.13 – 4.07 (m,
2H, CH×2), 3.75 (d, J = 10.9 Hz, 2H, CH×2), 3.42 (t, J = 11.7 Hz, 3H, CH×2, CH),
3.26 (s, 6H, CH3×2), 2.96 – 2.82 (m, 2H, CH×2), 2.75 (t, J = 11.4 Hz, 2H, CH×2),
28

2.38 (d, J = 8.4 Hz, 2H, CH×2), 1.89 (d, J = 9.6 Hz, 2H, CH×2), 1.53 (d, J = 10.3
13
Hz, 2H, CH×2). C NMR (100 MHz, CDCl3) δ 155.9, 153.8, 148.8, 148.1, 145.9,
133.3, 131.8 (2C), 130.6, 129.7 (2C), 128.9, 128.7, 120.20 (2C), 117.8 (2C), 76.38,
67.5 (2C), 55.1 (2C), 54.2, 49.7 (2C), 30.4 (2C), 26.6 (2C). MS (ESI, m/z): 546.2 [M
+ H]+.
4.1.4.10.
N-(4-((1-Methylpiperidin-4-yl)oxy)phenyl)-8-(phenylsulfinyl)-9-
(tetrahydro-2H-pyran-4-yl)-9H-purin-2-amine (D10). Yellow solid; Yield 63.2%; mp
1
83.1-85.1 °C; H NMR (400 MHz, CDCl3) δ 8.70 (s, 1H, Ar-H), 7.61 (d, J = 8.9 Hz,
2H, Ar-H), 7.54 – 7.44 (m, 2H, Ar-H), 7.40 (dt, J = 11.6, 4.2 Hz, 3H, Ar-H), 7.28 (s,
1H, NH), 6.94 (d, J = 8.9 Hz, 2H, Ar-H), 4.74 – 4.57 (m, 2H, OCH, NCH), 4.12 (dd, J
= 11.4, 4.1 Hz, 2H, CH×2), 3.60 (t, J = 11.2 Hz, 2H, CH×2), 3.45 (t, J = 11.6 Hz,
2H, CH×2), 3.32 (s, 3H, CH3), 3.24 (d, J = 11.1 Hz, 2H, CH×2), 2.91 (dd, J = 12.4,
4.4 Hz, 2H, CH×2), 2.72 (t, J = 13.4 Hz, 2H, CH×2), 1.99 (d, J = 14.7 Hz, 2H, CH
13
×2), 1.57 (d, J = 9.8 Hz, 2H, CH×2). C NMR (100 MHz, CDCl3) δ 155.8, 153.8,
151.9, 148.8, 148.5, 133.9, 131.9 (2C), 130.4, 129.7 (2C), 129.1, 128.9, 120.6 (2C),
116.7 (2C), 67.5 (2C), 67.4, 61.5 (2C), 60.9, 54.2, 30.4 (2C), 24.9 (2C). MS (ESI,
m/z): 533.3 [M + H]+.
4.1.4.11. N-(4-(2-(Dimethylamino)ethoxy)phenyl)-8-((3-fluorophenyl)sulfinyl)-9-(tet-
rahydro-2H-pyran-4-yl)-9H-purin-2-amine (D11). White solid; Yield 63.9%; mp
166.8-167.4 °C; 1H NMR (400 MHz, CDCl3) δ 8.71 (s, 1H, Ar-H), 7.61 (d, J = 8.5 Hz,
2H, Ar-H), 7.51 (s, 1H, NH), 7.35 (dd, J = 14.0, 7.8 Hz, 1H, Ar-H), 7.21 (dd, J = 17.9,
8.3 Hz, 2H, Ar-H), 7.05 (t, J = 8.3 Hz, 1H, Ar-H), 6.94 (d, J = 8.5 Hz, 2H, Ar-H), 4.65
(t, J = 12.0 Hz, 1H, CH), 4.58 (s, 2H, CH2), 4.13 (d, J = 8.1 Hz, 2H, CH×2), 3.70 (s,
2H, CH2), 3.46 (t, J = 11.9 Hz, 2H, CH×2), 3.34 (s, 6H, CH3×2), 3.00 – 2.89 (m, 2H,
CH×2), 1.60 (d, J = 10.7 Hz, 2H, CH×2). 13C NMR (100 MHz, DMSO-d6) δ 162.6
(d, JC-F = 250), 156.3, 153.6, 153.1, 149.3, 134.8, 131.9, 128.6, 127.6, 120.7 (2C),
118.3, 118.1, 116.0, 115.8, 114.9 (2C), 68.8, 66.9, 62.9 (2C), 60.1 (2C), 54.1, 30.57
(2C). MS (ESI, m/z): 525.2 [M + H]+.
4.1.4.12. N-(4-(2-(Dimethylamino)ethoxy)phenyl)-8-((4-fluorophenyl)sulfinyl)-9-(tet-
rahydro-2H-pyran-4-yl)-9H-purin-2-amine (D12). White solid; Yield 58.6%; mp
29