Discovery of 9H-purins as potential tubulin polymerization inhibitors: Synthesis, biological evaluation and structure?activity relationships

Article abstract of DOI:10.1016/j.ejmech.2017.07.054

Two series of N-(4-methoxyphenyl)-N-methyl-9H-purin-6-amines (9a-d and 10a-h) and 9-substituted benzyl-6-chloro-9H-purines (11a-h) were designed and synthesized. Their antiproliferative activities against human myelogenous leukemia (K562), human neuroblas

Full text of DOI:10.1016/j.ejmech.2017.07.054

Accepted Manuscript
Discovery of 9H-purins as potential tubulin polymerization inhibitors: Synthesis,
biological evaluation and structure−activity relationships
Zhong-Zhen Zhou, Xiu-Dong Shi, Hong-Fang Feng, Yu-Fang Cheng, Hai-Tao Wang,
Jiang-Ping Xu
PII:
S0223-5234(17)30576-7
10.1016/j.ejmech.2017.07.054
EJMECH 9617
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 February 2017
Revised Date: 19 May 2017
Accepted Date: 23 July 2017
Please cite this article as: Z.-Z. Zhou, X.-D. Shi, H.-F. Feng, Y.-F. Cheng, H.-T. Wang, J.-P. Xu,
Discovery of 9H-purins as potential tubulin polymerization inhibitors: Synthesis, biological evaluation
and structure−activity relationships, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.07.054.
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Graphic Abstract
Discovery of 9H-purins as potential tubulin polymerization inhibitors:
synthesis, biological evaluation and structure−activity relationships
Zhong-Zhen Zhou *, Xiu-Dong Shi, Hong-Fang Feng, Yu-Fang Cheng, Hai-Tao Wang and Jiang-Ping Xu *

ACCEPTED MANUSCRIPT
Discovery of 9H-purins as potential tubulin polymerization inhibitors:
synthesis, biological evaluation and structure−activity relationships
Zhong-Zhen Zhou *, Xiu-Dong Shi, Hong-Fang Feng, Yu-Fang Cheng, Hai-Tao Wang and
Jiang-Ping Xu *
Department of Neuropharmacology and Novel Drug Discovery, School of Pharmaceutical Sciences,
Southern Medical University, Guangzhou 510515, China.
Corresponding author. Tel.: +86 20 61648235; fax: +86 20 61648235; e-mail: zhouzz@smu.edu.cn (Z.-Z.
Zhou), jpx@smu.edu.cn (J.-P. Xu).
Abstract Two series of N-(4-methoxyphenyl)-N-methyl-9H-purin-6-amines (9a-d and 10a-h) and
9-substituted benzyl-6-chloro-9H-purines (11a-h) were designed and synthesized. Their antiproliferative
activities against human myelogenous leukemia (K562), human neuroblastoma (SH-SY5Y) and gastric
cancer (AGS) cell lines were evaluated using the MTT assay. The preliminary results indicated that
compounds 9d and 11e-h displayed low-micromole GI50 values against all tested cell lines. In addition,
compounds 10b and 10d showed wonderful antiproliferative activities towards SH-SY5Y cells with
selectivity of >230-fold over K562 and AGS cells. Among them, compounds 9d, 10b, 10d and 11g with
good antitumor activities exhibited high selectivity for tumor cell lines over immortalized mouse
hippocampal (HT22) cell line. Moreover, compound 9d with sub-micromole GI50 values toward AGS cells
exhibited moderate tubulin polymerization inhibitory activity, and induced apoptosis at G2/M phase arrest
with a dose-dependent manner in the human AGS cells.
Key words: 9H-purins, antiproliferative activity, tubulin-polymerization inhibitors, apoptosis, cell cycle
arrest.
1. Introduction
Cancer with the uncontrolled cell proliferation is a constant threat for human health [1].
Therefore, a continued effort of discovering new anticancer agents remains critically
important. It is well known that nitrogen-containing heterocycles are widely distributed in
nature and essential for the metabolism of all living cells. Among these heterocycles, the
pyrimidine nucleus is an important drug-like scaffold. It is present in many pharmacologically
active compounds, particularly in antitumor agents. Undoubtedly, tubulin is a good target for
its essential in a diverse array of eukaryotic cell functions, and many small molecules have

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been found[2-6]. Among tubulin-polymerization inhibitors, many are fused pyrimidine
derivatives, such as quinazolines (1a-b [7]), cyclopenta[d]pyrimidin-4-amine 2 [8, 9] and
7H-pyrrolo[2,3-d]pyrimidine 3 [8]. 5-methyl-furo[2,3 d]pyrimidine 4 [10] with wonderful
antitumor activity was reported as a potent microtubule depolymerizing agent.
Pyrrolo[2,3-d]pyrimidine 5 [11] bearing the 7-benzyl group displayed good antiproliferative
and antitubulin activities. Pyrazolo[4,3-d]pyrimidine 6 [12] bearing two 4-methoxybenzyl
groups was identified as multi-target compound that targets both tubulin polymerization and
cyclin-dependent kinases 1, 2 and 7.
Fig. 1. Fused pyrimidine derivatives as anticancer agents.
Fig. 2. The structures of target compounds
The replacement of fused pyrimidine nucleus with purine is a promising strategy for the
discovery of novel therapeutic agents, and propitious to increase antitumor molecular
diversity. In addition, purine derivatives have attracted considerable attention for their
important chemo-preventive and chemo-therapeutic effects on cancer [13-19]. For example,
6-chloro-9H-purin-2-amine bearing substituted benzyl moieties (7) [20] was reported to

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exhibit excellent antiproliferative activities against various tumor cell lines. Replacing the
skeleton of compounds 2 and 3 with purine ring gave phenylaminopurine 8 [13], which
displayed good antiproliferative activities and induced G2/M phase arrest as reported tubulin
depolymerizing agents.
Inspired by the above considerations, N-(4-methoxyphenyl)-N-methyl-9H-purin-6-amines
(9a-d and 10a-h) and 9-substituted benzyl-6-chloro-9H-purines (11a-h) were designed and
synthesized by replacing 7H-pyrrolo[2,3-d]pyrimidine and furo[2,3 d]pyrimidine ring in
compounds 4 and 5 with purine ring (Fig. 2 and Scheme 1). Their antiproliferative activities
towards three human cancer cell lines (K562, SH-SY5Y and AGS) and one non-tumor cell
line (immortalized mouse hippocampal cell line, HT22) were performed. The apoptosis assay,
cell-cycle analyses and tubulin polymerization assay in vitro of selected compounds have also
been further performed.
2. Results and discussion
2.1 Chemistry
Target compounds 9a-d, 10a-h and 11a-h were synthesized as described in Scheme 1 using
6-chloro-9H-purines 12a-b as starting materials. Compounds 9a-b were obtained directly by
the substitution reaction of 4-methoxy-N-methylaniline with 6-chloro-9H-purines 12a-b.
Compounds 9c-d were synthesized from 9-methyl-6-chloro-9H-purines, which were the
methylation products of compounds 12a-b. The substitution reaction of 6-chloro-9H-purines
12a-b with substituted benzyl bromine gave compounds 11a-h, which could be converted into
compounds 10a-h by C-N cross coupling reaction.
Scheme 1. The synthesis route of target compounds 9a-d, 10a-h and 11a-h.

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2.2 Pharmacology
2.2.1 In vitro antiproliferative activities
Antiproliferative activities of target compounds 9a-d, 10a-h and 11a-h were examined in
three human cancer cell lines, including K562, SH-SY5Y and AGS cell lines, using MTT
method [21]. Fluorouracil (5-Fu) and colchicine were used as a reference cytotoxic compound
in the experiments. The results were expressed as growth inhibitory concentration (GI50)
values which represent the compound concentrations required to produce a 50% growth
inhibition of cell growth after 48 h of incubation compared to untreated controls (Table 1).
As shown in Table 1, the antiproliferative activities (GI50) of target compounds 9a-d, 10a-h
and 11a-h against these cancer cell lines ranged from 0.26 µM to >100 µM. Among these
compounds, compounds 9a-b and 11a-d were found to be inactive toward all tested cells.
However, compounds 9d and 11e-h demonstrated wonderful antiproliferative activities with
low-micromole GI50 values against all tested cancer cell lines. Particularly, compound 9d
showed best antiproliferative activity (GI50 = 0.26 µM) against gastric cancer (AGS) cell lines.
In addition, compounds 10a, 10b, 10d, 10e, 10f and 10h exhibited selective antiproliferative
activities against SH-SY5Y cells. Moreover, compounds 10b and 10d showed wonderful
antiproliferative activities against SH-SY5Y cells with selectivity of >230-fold over K562 and
AGS cells. The GI50 values of compounds 10b and 10d (GI50 = 0.43 µM and 0.26 µM
respectively) were about 28- and 46-fold over that of 5-FU (GI50 = 11.9 µM), respectively. In
addition, compounds 9d, 10b, 10d and 11g with good antitumor activities displayed lower
cytotoxicities toward non-tumor HT22 cell line. More interesting, compound 9d exhibited at
least 23-, 43- and 430-fold higher selectivity for tumor cells over HT22. Compound 10b and
10d showed at least 230- and 88-flod higher selectivity for SH-SY5Y cell over HT22 cell,
respectively.
Careful analysis on SAR of these compounds revealed that the substituents have primary
influences on their antiproliferative activities and selectivity. Firstly, 2-Cl group is one of
primary influences on the antiproliferative activities. For example, N-(4-methoxyphenyl)-N-
methyl-9H-purin-6-amines bearing a Cl atom at 2-position (9c-d and 10e-h) displayed higher
activities against K562 and AGS cells than their corresponding purine derivatives (9a-b,
10a-d). 9-Substituted benzyl-6-chloro-9H-purines bearing a 2-H group (11a-d) displayed

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inactivities for all tested cells, while 9-substituted benzyl-2,6-dichloro-9H-purines bearing a
2-Cl group (11e-h) shown wonderful antiproliferative activities against all tested cells.
Table 1 In vitro evaluation of compounds 9a-d, 10a-h and 11a-h.
OMe
OMe
OMe
OMe
Me
N
Cl
Me
N
Me
N
Me
N
Cl
N
N
N
N
N
N
N
N
N
N
N
N
N
R2
N
N
N
N
R2
N
N
N
Cl
(OMe)n
N
Cl
H
N
H
N
Cl
N
H
(OMe)n
(OMe)n
(OMe)n
9a-b
9c-d
10a-d
1ea-h
11a-d
11e-h
antiproliferative activity (GI50, µM) a
CC50 (uM)b
HT22
tubulin
Compounds
polymerization
inhibition (%)c
NTd
R2 or
K562
>100
SH-SY5Y
AGS
H
>100
>100
>100
>100
NT
NT
9a
9b
methyl >100
> 100
methyl 2.3±0.7
4-methoxybenzyl >100
NT
H
>100
30±1.5
NT
NT
9c
3.4±0.6
27±2.3
0.43±0.05
>100
0.26±0.05
>100
>100
NT
41±1.5
NT
9d
10a
10b
10c
3,4-dimethoxybenzyl >100
3,5-dimethoxybenzyl 42±1.2
3,4,5-trimethoxybenzyl >100
4-methoxybenzyl >100
> 100
>100
NT
18±0.6
NT
>100
0.26±0.07
31±1.9
7.3±1.0
>100
>100
23±3
NT
20±0.5
NT
10d
10e
>100
3,4-dimethoxybenzyl 27±2.1
3,5-dimethoxybenzyl 42±2.7
3,4,5-trimethoxybenzyl 41±2.3
56±2.5
69±3.9
85±4.1
29±1.3
>100
NT
25±0.8
NT
10f
NT
10g
10h
11ae
11b
11c
3.0±0.5
> 50
NT
12±0.6
NT
4-methoxybenzyl
3,4-dimethoxybenzyl
3,5-dimethoxybenzyl
3,4,5-trimethoxybenzyl
> 50
> 50
>100
> 50
NT
>100
NT
NT
>100
>100
NT
NT
>100
> 50
NT
NT
11d
11ef
11f
4-methoxybenzyl 36±2.1
3,4-dimethoxybenzyl 7.0±1.3
3,5-dimethoxybenzyl 4.6±1.1
3,4,5-trimethoxybenzyl 4.3±0.6
19±1.1
7.7±1.6
5.5±1.1
1.7±0.7
9.6±0.9
12±1.2
19±1.5
11±1.2
3.8±0.5
9.4±0.8
25±1.0
0.04±0.1
NT
25±1.1
22±0.6
13±0.8
13±1.1
NT
NT
>100
NT
11g
11h
5-Fu
Colchicine
NT
0.02±0.1 0.04±0.1
NT
77±3
a
50% Growth inhibition and data are expressed as means ± SDs (standard deviations) from at least two independent
experiments.
b CC50 values represent the 50% cytotoxic concentration after 48h, the mean values of three repeated experiments.
c The tested concentration of compounds is 10 µM, and tubulin concentration is 5 mg/mL. Data are expressed as means ± SDs
(standard deviations) from three independent experiments.
d NT stands for “Not Tested”.
e The structure of compound 9a has been reported[22]
f The structure of compound 9e has been reported [23, 24]
Secondly, the substituents exert significant influences on their antiproliferative activities
against SH-SY5Y cells. For example, the replacement of 9-H or 9-Me group of compound

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9a-d with substituted benzyl group lead to compounds 10a, 10b, 10d, 10e, 10f and 10h with
selective antiproliferative activities towards SH-SY5Y cells. However, compounds 10c and
10g bearing 3,5-dimethoxyphenyl group are inactive for SH-SY5Y cells. In addition,
compounds 10a-d bearing a 2-H group exhibited higher activities against SH-SY5Y cells than
corresponding purine derivatives 10e-h bearing a 2-Cl group. The aforementioned phenomena
illustrate the position of methoxy moiety on the benzyl group and 2-substituents (R1) have
impacts on their activities against SH-SY5Y cells.
Base on the obtained antiproliferative activities, in vitro tubulin polymerization inhibition
test was performed on the nine selected compounds with good activities at 10 µM, and
colchicine was used as the reference (Table 1). Interestingly, their tubulin polymerization
inhibitory activities are related to their antiproliferative activities. For example, compound 9d
also exhibited the most potent antitubulin polymerization inhibition activity (41% at 10 µM,
Figure S1 and Table 1).
2.2.2. Effects on cell cycle distribution and apoptosis assay
Many studies have confirmed that the tubulin polymerization inhibitors can arrest cells in
the G2/M phase of the cell cycle, and induce their apoptosis[6, 25-27]. The excellent
inhibitory activity of 9d promoted us to further investigate its cellular mechanisms by flow
cytometry analysis. As shown in Fig. 3, AGS cells treated with compound 9d were arrested at
the G2/M phase in a dose-dependent manner, and induced an increase in the percentage of
cells at G2/M. The percentages of cells at G2/M phase were increased to 64.39% at high
concentration from 9.18% for the blank control group, which is accompanied by a concurrent
reduction in the percentage of cells at G0/G1 and S phase.
To investigate whether compound 9d induces apoptosis, the apoptotic effect of compound
9d was also evaluated by an annexin V FITC/PI (AV/PI) dual staining assay using DMSO as
the control. The AGS cells were treated with compound 9d at different concentration to
examine the apoptotic effect. Annexin V-FITC/propidium iodide analysis revealed that
compound 9d induced apoptosis of AGS cells in a concentration-dependent manner. As
shown in Fig. 4, compound 9d induced 33.9% apoptosis in the cancer cells at higher
concentration.

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Fig. 3. Cell cycle distribution of AGS cells cell lines after treatment with compound 9d at different
concentration. It determined by flow cytometry analysis after 24 h co-culture using DNA intercalating dye,
propidium iodide (PI). DMSO was used as blank control, and colchicine was used as positive control.
Fig. 4. Annexin V-FITC/propidium iodide analyses on apoptosis of AGS cells 24 h after co-culture with
compound 9d. The four quadrants identified as: live, early apoptotic; late apoptotic; Dead. DMSO was used
as control.
2.2.3. Molecular docking
To support the discussion of the previous biological results, compound 9d was chosen to
dock in the tubulin at the colchicine site (PDB: 4O2B, 2.30 Å[28], Fig. 5). As shown in Fig. 5,
the docked conformation of compound 9d (magenta) exhibits similar conformation with
colchicine (white) with some apparent variances in the colchicine site. The 9H-purin scaffold
of compound 9d overlaps the trimethoxybenzene ring of colchicine, hydrophobically interacts

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with β/Ala250, β/Asp251, β/Lys254 and β/Lys255. 9-Methyl group of compound 9d
hydrophobically interacts hydrophobic interactions with β/Cys241and β/Leu242, while the
2-chloro group interacts with β/Ala317, β/Thr353 and β/Ala354. The N-methyl group of
compound 9d simulates bridging role of the cycloheptane ring of colchicine, and forms
hydrophobic interactions with β/Lys254, β/Leu255 and β/Asn258 of tubulin. The
4-meothoxyphenyl
ring
of
compound
9d
occupies
the
place
of
the
cyclohepta-2,4,6-trien-1-one of colchicine with some variances, and forms hydrophobic
interactions with β/Asn258, β/Met259, β/Val351, β/Lys352 and α/Val181 of tubulin.
Fig. 5. Potential configuration of compound 9d in the colchicine site of tubulin (PDB: 4O2B[28]). The C
atoms in compound 9d are colored in magenta, while the C atoms in colchicine is colored in white. The
total docking score of compound 9d is 6.689, while the colchicine is 8.722.
3. Conclusion
In summary, seventeen 9H-purins were designed and synthesized in this paper. Their
preliminary antiproliferative activities against K562, SH-SY5Y and AGS cell lines indicated
that their activities varied along with the substituents. Among them, compounds 9d and 11e-h
showed higher antiproliferative activities against all tested cells than 5-Fu. Moreover,
2-chloro-9-methyl-N-(4-methoxyphenyl)-N-methyl-9H-purin-6-amine (9d) exhibited good
tubulin polymerization inhibitory activity, and induce apoptosis at G2/M phase arrest in a
dose-dependent manner in the human AGS cells. Meanwhile, 9-substituted
benzyl-N-(4-methoxyphenyl)-N-methyl-9H-purin-6-amines 10a-b, 10d, 10e-f and 10h
displayed selective antiproliferative activities against SH-SY5Y cells. Compounds 10b
and10d showed the wonderful antiproliferative activities against SH-SY5Y cells with
sub-micromole GI50 values. In addition, compounds 9d, 10b, 10d and 11g showed very lower

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cytotoxicities toward non-tumor HT22 cell line. Considering their attractive antitumor
activities and selectivity, further efforts for novel 9H-purins analogues with higher anticancer
activities are presently going on in our laboratories.
4. Experimental Section
4.1 Chemistry
1
13
ESI spectra were measured on a Waters UPLC/Quattro Premier XE mass spectrometer. H and C
NMR spectra were recorded in CDCl3 or DMSO-d6 using a Varian Mercury 400 spectrometer and
TMS as an internal reference. Element analyses were carried out on a Vario ELIII CHNSO elemental
analyzer. Analyses indicated by the symbols of the elements or functions were within ±0.4% of the
theoretical values. All the chemicals were of analytical grade and used without further purification.
4.1.2 General procedures of compounds 9a-b
6-chloro-9-H-purines (0.5 mmol), isopropanol (3 mL) and 4-methoxy-N-methylaniline (82 mg, 0.6
mmol) was added into a round flask (5 mL) with a stir bar. The resulting mixture was stirred at 80 oC
until the starting material disappeared on TLC. Then the solvent was removed under reduced pressure
to afford crude product, which was purified by chromatography on silica gel (petroleum ether/acetic
ether = 1: 1, v/v) to get target compound.
4.1.2.1 N-(4-methoxyphenyl)-N-methyl-9H-purin-6-amine 9a: White solid. Yield: 22%. 1H NMR (400
MHz, DMSO-d6) δ 3.78 (s, 3H), 3.79 (s, 3H), 6.95 (d, J = 8.6 Hz, 2H), 7.24 (d, J = 8.6 Hz, 2H), 8.04
(s, 1H), 8.18 (s, 1H), 12.98 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 40.6, 55.6, 114.5, 119.6, 128.5,
138.9, 139.1, 151.9, 152.0, 154.5, 157.7. ESI-MS (m/z): 271.14 ([M+Na]+). And Anal. Calcd. for
C13H13N5O: C, 61.17; H, 5.13; N, 27.43; Found: C, 61.54; H, 5.36; N, 27.09.
1
4.1.2.2 2-chloro-N-(4-methoxyphenyl)-N-methyl-9H-purin-6-amine 9b: White solid. Yield: 36%. H
NMR (400 MHz, DMSO-d6) δ 3.73 (s, 3H), 3.80 (s, 3H), 6.97 (d, J = 8 Hz, 2H), 7.27(d, J = 8 Hz, 2H),
8.03 (s, 1H), 13.12 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 40.8, 55.6, 114.6, 118.7, 128.4, 138.2,
139.5, 152.5, 153.152, 154.8, 158.1. ESI-MS (m/z): 290.70 ([M+H]+). And Anal. Calcd. for
C13H12ClN5O: C, 53.89; H, 4.17; N, 24.17; Found: C, 53.71; H, 4.52; N, 24.39.
4.1.3 General procedures of compounds 9c-d
To a solution of 9H-purines (1.5 mmol) and sodium hydride (0.12 g, 60%) in anhydrous DMF (3
mL) was added iodomethane (0.42g, 3 mmol). The resulting mixture was stirred at room temperature

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until the starting material disappeared on TLC. Then the reaction solution was poured into water (20
mL), and extracted three times with EtOAc (15 mL × 3). The combined organic layer was dried over
anhydrous Na2SO4 and concentrated under reduced pressure to yield crude 9-methyl-9H-purine, which
was used in next step without further purification.
And then the synthesis of compounds 9c-d was performed as previous procedures of compounds
9a-b using obtained 9-methyl-9H-purine as reactant.
1
4.1.3.1 N-(4-methoxyphenyl)-N,9-dimethyl-9H-purin-6-amine 9c: White solid. Yield: 23%. H NMR
(400 MHz, CDCl3) δ 3.80 (s, 3H), 3.85 (s, 6H), 6.97 (d, J = 8 Hz, 2H), 7.25 (d, J = 8 Hz, 2H), 7.66 (s,
13
1H), 8.41 (s, 1H). C NMR (100 MHz, CDCl3) δ 29.6, 40.7, 55.3, 114.5, 120.3, 127.9, 138.7, 139.6,
151.4, 152.4, 154.8, 158.0. ESI-MS (m/z): 270.50 ([M+H]+). And Anal. Calcd. for C14H15N5O: C,
62.44; H, 5.61; N, 26.01; Found: C, 62.32; H, 5.28; N, 25.98.
4.1.3.2 2-chloro-N-(4-methoxyphenyl)-N,9-dimethyl-9H-purin-6-amine 9d: White solid. Yield: 53%.
1H NMR (400 MHz, CDCl3) δ 3.72 (s, 3H), 3.73 (s, 3H), 3.85 (s, 3H), 6.95 (d, J = 8 Hz, 2H), 7.22 (d,
J = 8 Hz, 2H), 7.54 (s, 1H). 13C NMR (100 MHz, CDCl3) δ 29.9, 40.7, 55.4, 114.4, 119.3, 127.9,
137.9, 140.0, 152.6, 153.7, 155.0, 158.4. ESI-MS (m/z): 305.02 ([M+H]+). And Anal. Calcd. for
C14H14ClN5O2: C, 55.36; H, 4.65; N, 23.06; Found: C, 55.49; H, 4.34; N, 22.85.
4.1.2 General procedures of compounds 10a-h and 11a-h.
To a solution of 9H-purines (1.5 mmol) and N,N-diisopropylethylamine (DIPEA, 3 mmol) in DMF
(4 mL) was added substituted benzyl bromide (1.5 mmol). The resulting mixture was stirred at room
temperature until the starting material disappeared on TLC. Then the reaction solution was poured into
water (40 mL), and extracted three times with EtOAc (15 mL × 3). The combined organic layer was
dried over anhydrous Na2SO4 and concentrated under reduced pressure to yield crude product.
Purification was accomplished by chromatography on a silica gel column, eluted with petroleum
ether-acetic ether (1/l, v/v), to give compounds 11. And then the synthesis of compounds 10a-h was
performed as previous procedures of compounds 9a-b using obtained compounds 11a-h as reactants.
4.1.3.1 9-(4-methoxybenzyl)-N-(4-methoxyphenyl)-N-methyl-9H-purin-6-amine 10a: White solid.
Yield: 59%. 1H NMR (400 MHz, CDCl3) δ 3.76 (s, 6H), 4.22 (s, 3H), 5.32 (s, 2H), 6.42 (s, 1H), 6.46
(s, 2H), 8.10 (s, 1H). 13C NMR (100 MHz, CDCl3) δ 41.2, 46.8, 55.3, 55.4, 114.4, 114.6, 120.2, 127.5,
127.9, 138.2, 139.3, 150.7, 151.6, 158.4, 159.7. ESI-MS (m/z): 376.50 ([M+H]+), 398.50 ([M+Na]+).
And Anal. Calcd. For C21H21N5O2: C, 67.18; H, 5.64; N, 18.65; Found: C, 66.92; H, 5.71; N,18.42.
4.1.3.2 9-(3,4-dimethoxybenzyl)-N-(4-methoxyphenyl)-N-methyl-9H-purin-6-amine 10b: White solid.

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1
Yield: 30%. H NMR (400 MHz, CDCl3) δ 3.82 (s, 3H), 3.84 (s, 6H), 3.85 (s, 3H), 5.26 (s, 2H),
6.79-6.87 (m, 3H), 6.96 (d, J = 8 Hz, 2H), 7.24 (d, J = 8 Hz, 2H), 7.64 (s, 1H), 8.42 (s, 1H). 13C NMR
(100 MHz, CDCl3) δ 40.7, 47.0, 55.4, 56.0, 111.3, 114.5, 120.5, 128.0, 128.2, 138.7, 138.9, 149.1, 149.
4, 151.1, 152.5, 154.9, 158.1. ESI-MS (m/z): 406.85 ([M+H]+), 429.13 ([M+Na]+). And Anal. Calcd.
for C22H23N5O3: C, 65.17; H, 5.72; N, 17.27; Found: C, 64.98; H, 5.464; N,17.13.
4.1.3.3 9-(3,5-dimethoxybenzyl)-N-(4-methoxyphenyl)-N-methyl-9H-purin-6-amine 10c: White solid.
Yield: 14%. 1H NMR (400 MHz, CDCl3) δ 3.74 (s, 6H), 3.84 (s, 6H), 5.26 (s, 2H), 6.38 (s, 1H), 6.40
(s, 2H), 6.96 (d, J = 8 Hz, 2H), 7.25 (d, J = 8 Hz, 2H), 7.65 (s, 1H), 8.42 (s, 1H). 13C NMR (100 MHz,
CDCl3) δ 40.8, 47.1, 55.4, 55.4, 99.9, 105.8, 114.5, 120.3, 128.0, 138.0, 138.6, 139.1, 151.1, 152.5,
154.9, 158.1, 161.3. ESI-MS (m/z): 406.87 ([M+H]+), 428.62 ([M+Na]+). And Anal. Calcd. for
C22H23N5O3: C, 65.17; H, 5.72; N, 17.27; Found: C, 65.34; H, 5.41; N,17.57.
4.1.3.4 9-(3,4,5-trimethoxybenzyl)-N-(4-methoxyphenyl)-N-methyl-9H-purin-6-amine 10d: White solid.
Yield: 29%. 1H NMR (400 MHz, CDCl3) δ 3.81 (s, 9H), 3.84 (s, 6H), 5.25 (s, 2H), 6.54 (s, 2H), 6.97
13
(d, J = 8 Hz, 2H), 7.25 (d, J = 8 Hz, 2H), 7.66 (s, 1H), 8.43 (s, 1H). C NMR (100 MHz, CDCl3) δ
40.8, 47.4, 55.4, 56.2, 60.8, 105.2, 114.5, 120.3, 128.0, 131.3, 138.0, 138.6, 139.0, 151.0, 152.5, 153.6,
154.9, 158.2. ESI-MS (m/z): 436.54 ([M+H]+), 458.56 ([M+Na]+). And Anal. Calcd. for C23H25N5O4:
C, 64.44; H, 5.79; N, 16.08; Found: C, 64.71; H, 5.46; N,15.98.
4.1.3.5 2-chloro-9-(4-methoxybenzyl)-N-(4-methoxyphenyl)-N-methyl-9H-purin-6-amine 10e: White
solid. Yield: 82%. 1H NMR (400 MHz, CDCl3) δ 3.70 (s, 3H), 3.78 (s, 3H), 3.83 (s, 3H), 5.19 (s, 2H),
6.84 (d, J = 8 Hz, 2H), 6.94 (d, J = 8 Hz, 2H), 7.20-7.22 (m, 4H), 7.47 (s, 1H). 13C NMR (100 MHz,
CDCl3) δ 29.7, 40.6, 46.7, 55.3, 55.4, 114.37, 114.41, 119.3, 127.3, 128.0, 129.6, 137.9, 139.2, 152.2,
153.8, 155.0, 158.4, 159.7. ESI-MS (m/z): 410.30 ([M+H]+). And Anal. Calcd. for C21H20ClN5O2: C,
61.54; H, 4.92; N, 17.09; Found: C, 61.35; H, 4.73; N, 16.89.
4.1.3.6 2-chloro-9-(3,4-dimethoxybenzyl)-N-(4-methoxyphenyl)-N-methyl-9H-purin-6-amine 10f:
White solid. Yield: 52%. 1H NMR (400 MHz, CDCl3) δ 3.70 (s, 3H), 3.83 (s, 6H), 3.85 (s, 3H), 5.18 (s,
2H), 6.81 (q, J = 8 Hz, 2H), 6.90 (s, 1H), 6.94 (d, J = 8 Hz, 2H), 7.22 (d, J = 8 Hz, 2H), 7.50 (s, 1H).
13C NMR (100 MHz, CDCl3) δ 40.6, 47.2, 55.4, 55.9, 111.2, 111.6, 114.4, 119.3, 120.7, 127.6, 127.9,
137.8, 139.2, 149.2, 149.3, 152.2, 153.7, 155.0, 158.4. ESI-MS (m/z): 440.4 ([M+H]+), 462.4
([M+Na]+). And Anal. Calcd. for C22H22ClN5O3: C, 60.07; H, 5.04; N, 15.92; Found: C, 59.70; H, 4.86;
N,15.67.
4.1.3.7 2-chloro-9-(3,5-dimethoxybenzyl)-N-(4-methoxyphenyl)-N-methyl-9H-purin-6-amine 10g:

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White solid. Yield: 72%. 1H NMR (400 MHz, CDCl3) δ 3.71 (s, 3H), 3.75 (s, 6H), 3.85(s, 3H), 5.20 (s,
2H), 6.38 (s, 1H), 6.41 (s, 2H), 6.96 (d, J = 8 Hz, 2H), 7.22 (d, J = 8 Hz, 2H) 7.70 (s, 1H). 13C NMR
(100 MHz, CDCl3) δ 40.7, 47.8, 55.4, 55.5, 100.2, 106.2, 114.7, 127.9, 137.0, 139.5, 161.3. ESI-MS
(m/z): 440.6 ([M+H]+), 462.5 ([M+Na]+). And Anal. Calcd. for C22H22ClN5O3: C, 60.07; H, 5.04; N,
15.92; Found: C, 60.34; H, 4.89; N,15.87.
4.1.3.8 2-chloro-9-(3,4,5-trimethoxybenzyl)-N-(4-methoxyphenyl)-N-methyl-9H-purin-6-amine 10h:
White solid. Yield: 55%. 1H NMR (400 MHz, CDCl3) δ 3.71 (s, 3H), 3.82 (s, 9H), 3.84(s, 3H), 5.19 (s,
2H), 6.58 (s, 2H), 6.96 (d, J = 8 Hz, 2H), 7.22 (d, J = 8 Hz, 2H), 7.69 (s, 1H). 13C NMR (100 MHz,
CDCl3) δ 40.70, 47.87, 55.4, 56.2, 60.8, 105.7, 114.6, 127.9, 130.5, 137.5, 138.2, 139.3, 152.0, 153.6,
154.0, 154.8, 158.7. ESI-MS(m/z): 470.5 ([M+H]+), 492.5 ([M+Na]+). And Anal. Calcd. for
C23H24ClN5O4: C, 58.79; H, 5.15; N, 14.90; Found: C, 58.64; H, 5.01; N,14.78.
1
4.1.3.9 6-chloro-9-(4-methoxybenzyl)-9H-purine 11a[22]: White solid. Yield: 33%. H NMR (400
MHz, CDCl3) δ 3.79 (s, 3H), 5.38 (s, 2H), 6.89 (d, J = 8 Hz, 2H), 7.28 (d, J = 8 Hz, 2H), 8.08 (s, 1H),
8.77 (s, 1H). 13C NMR (100 MHz, CDCl3) δ 47.4, 55.3, 114.5, 126.4, 128.5, 129.5, 144.9, 150.9, 151.7,
152.0, 159.9. ESI-MS (m/z): 275.16 ([M+H]+). ESI-MS (m/z): 275.16 ([M+H]+). And Anal. Calcd. for
C13H11ClN4O: C, 56.84; H, 4.04; N, 20.40; Found: C, 56.51; H, 3.90; N, 20.35.
1
4.1.3.10 6-chloro-9-(3,4-dimethoxybenzyl)-9H-purine 11b: White solid. Yield: 36%. H NMR (400
MHz, CDCl3) δ 3.84 (s, 3H), 3.88 (s, 3H), 5.39 (s, 2H), 6.84-6.92 (m, 3H), 8.08 (s, 1H), 8.79 (s, 1H).
13C NMR (100 MHz, CDCl3) δ 47.9, 56.0, 56.0, 111.3, 111.5, 120.8, 126.8, 131.6, 144.9, 149.6, 151.2,
151.8, 152.1. ESI-MS (m/z): 305.92 ([M+H]+). And Anal. Calcd. for C14H13ClN4O2: C, 55.18; H, 4.30;
N, 18.39; Found: C, 55.40; H, 4.11; N, 18.50;
1
4.1.3.11 6-chloro-9-(3,5,-dimethoxybenzyl)-9H-purine 11c: White solid. Yield: 34%. H NMR (400
MHz, CDCl3) δ 3.76 (s, 6H), 5.37 (s, 2H), 6.43 (s, 3H), 8.11 (s, 1H), 8.79 (s, 1H). 13C NMR (100 MHz,
CDCl3) δ 48.0, 55.4, 100.25, 106.1, 131.4, 136.6, 145.0, 151.2, 151.8, 152.3, 161.5. ESI-MS (m/z):
305.6 ([M+H]+). And Anal. Calcd. for C14H13ClN4O2: C, 55.18; H, 4.30; N, 18.39; Found: C, 55.42; H,
4.29; N, 18.17.
1
4.1.3.12 6-chloro-9-(3,4,5-trimethoxybenzyl)-9H-purine 11d: White solid. Yield: 30%. H NMR (400
13
MHz, CDCl3) δ 3.82 (s, 6H), 3.83 (s, 3H), 5.37 (s, 2H), 6.52 (s, 2H), 8.12 (s, 1H), 8.81 (s, 1H). C

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NMR (100 MHz, CDCl3) δ 48.3, 56.2, 60.8, 105.4, 129.7, 131.1, 138.5, 144.9, 151.1, 151.7, 152.2,
153.8. ESI-MS (m/z): 335.76 ([M+H]+). And Anal. Calcd. for C15H15ClN4O3: C, 53.82; H, 4.52; N,
16.74; Found: C, 53.51; H, 4.42; N, 16.89.
1
4.1.3.13 2,6-dichloro-9-(4-methoxybenzyl)-9H-purine 11e [23, 24]: White solid. Yield: 41%. H NMR
(400 MHz, CDCl3) δ 3.81 (s, 3H), 5.34 (s, 2H), 6.91 (d, J = 8.4 Hz, 2H), 7.28 (d, J = 10.4 Hz, 2H),
8.02 (s, 1H). 13C NMR (100 MHz, CDCl3) δ 47.71, 55.4, 114.7, 125.9, 129.8, 130.3, 130.7, 145.5,
151.8, 153.1, 160.2. ESI-MS (m/z): 309.91 ([M+H]+). And Anal. Calcd. for C13H10Cl2N4O: C, 50.51; H,
3.26; N, 18.12; Found: C, 50.21; H, 3.20; N, 18.20;
4.1.3.14 2,6-dichloro-9-(3,4-dimethoxybenzyl)-9H-purine 11f: White solid. Yield: 52%. 1H NMR (400
MHz, CDCl3) δ 3.86 (s, 3H), 3.88 (s, 3H), 5.33 (s, 2H), 6.86 (d, J = 8 Hz, 1H), 6.90-6.92 (m, 2H), 8.04
(s, 1H). 13C NMR (100 MHz, CDCl3) δ 48.2, 56.0, 56.0, 111.5, 121.0, 126.2, 130.8, 130.9, 145.9,
149.7, 151.9, 153.1. ESI-MS (m/z): 340.14 ([M+H]+). And Anal. Calcd. for C14H12Cl2N4O2: C, 49.58;
H, 3.57; N, 16.52; Found: C, 49.61; H, 3.55; N, 16.45;
1
4.1.3.15 2,6-dichloro-9-(3,5-dimethoxybenzyl)-9H-purine 11g: White solid. Yield: 35%. H NMR
(400 MHz, CDCl3) δ 3.77 (s, 6H), 5.32 (s, 2H), 6.43 (s, 3H), 8.06 (s, 1H). 13C NMR (100 MHz, CDCl3)
δ 48.1, 55.5, 100.4, 106.3, 130.7, 136.0, 145.6, 151.9, 153.2, 153.2, 161.5. ESI-MS (m/z): 339.88
([M+H]+). And Anal. Calcd. for C14H12Cl2N4O2: C, 49.58; H, 3.57; N, 16.52; Found: C, 49.31; H, 3.49;
N, 16.61;
1
4.1.3.16 2,6-dichloro-9-(3,4,5-trimethoxybenzyl)-9H-purine 11h: White solid. Yield: 34%. H NMR
(400 MHz, CDCl3) δ 3.84 (s, 9H), 5.32 (s, 2H), 6.59 (s, 2H), 8.09 (s, 1H). 13C NMR (100 MHz, CDCl3)
δ 48.6, 56.3, 60.9, 105.7, 129.3, 130.8, 138.7, 145.5, 152.0, 153.1, 153.1, 153.9. ESI-MS(m/z): 361.90
([M+H]+). And Anal. Calcd. for C15H14Cl2N4O3: C, 48.80; H, 3.82; N, 15.18; Found: C, 48.57; H, 3.75;
N, 15.07;
4.2 Pharmacology
4.2.1. Compounds
Target compounds were dissolved in DMSO and stored as stock solutions (100 mM) at -20°C. For
experimental use, all the compounds were prepared from stock solutions, diluted with growth medium

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and used immediately.
4.2.2. Statistical Analysis.
Statistical analysis was performed and figures were produced using Prism 5 for Windows
(Graph-Pad Software, Inc., San Diego, CA, USA). Data were expressed as mean ± standard Deviation
(SD). All data were analyzed using one-way analysis of variance (ANOVA) followed by Bonferroni’s
Multiple Comparison tests for post hoc analyses between the groups, results were considered
statistically significant when p < 0.05.
4.2.3 Cell culture
SH-SY5Y human neuroblastoma, K562 human myelogenous leukemia and AGS gastric cancer cell
lines were cultured in RPMI 1640, supplemented with 4% fetal bovine serum and 100 U/mL penicillin
and 100 µg/mL streptomycin (all from Invitrogen) at 37°C in a humidified atmosphere of 5% CO2 and
95% air.
4.2.4. In vitro anticancer activity
The in vitro cytotoxic activities of tested compounds was determined by MTT assay [21, 29, 30].
Briefly, cells in the log-phase of growth were harvested by trypsinization, seeded in 96 well plates
(Nunc, Denmark) for 24 h. Then, the cells were treated with various concentrations of the compounds
for 48 h. Fluorouracil (5-Fu) and DMSO were used as positive and negative controls, respectively. The
final concentration of DMSO was less than 1%. After 48 h, the culture medium was removed, and
cells were incubated with 200 mL of MTT solution (0.5 mg/mL) for 4 h. Then, the supernatant was
removed and the formazan crystals were dissolved using DMSO. The stained formazan product was
determined spectrophotometrically at 570 nm in GENios Pro microplate reader (TECAN).
4.2.5. Annexin V-FITC apoptosis assay
Further evaluation of the pro-apoptotic effect of compound 9d on AGS cells was analyzed with the
Annexin V/7-AAD assays using Muse® Annexin V and Dead Cell Assay Kit from Millipore. Annexin
V, which is membrane permeable, labels all cells containing a nucleus. The second component of the
kit is 7-AAD, which stains the membrane of the cells that have been compromised and are dying or
dead. This synergistic functioning of the two components differentiates between cell populations based

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on their health. The assay was performed according to manufacturer’s protocol. Briefly, AGS cells
were grown in 6-well culture plates (3 × 105 cells/well) and treated with compounds for 24 h.
Following the treatment, cells were pelleted and then resuspended in 100 mL of complete growing
media, followed by adding 100 mL of Muse Annexin V and Dead Cell Reagent to each tube. The tubes
were mixed thoroughly and then incubated at room temperature for 20 min without light exposure.
After incubation, the tubes were read individually using Muse TM Cell Analyzer. Default gate settings
were used to distinguish the cells in the live, early-apoptotic, late-apoptotic and dead categories.
4.2.6. Cell cycle analysis
The SH-SY5Y cells were treated with different concentrations of compound 9d for 24 h. After
treatment, the cells were washed twice with ice-cold PBS, collected by centrifugation, and fixed in
ice-cold 70% (v/v) ethanol, washed with PBS, re-suspended with 0.1 mg/mL RNase, stained with 40
mg/mL PI, and analyzed by flow cytometry using FACScalibur (Becton Dickinson). The cell cycle
distributions were analyzed using Cell- Quest software (Becton Dickinson).
4.2.7. In vitro tubulin polymerization assay
According to method described by Shelanski et al. [31], pig brain microtubule protein was isolated
by employing three cycles of temperature-dependent assembly/disassembly. Homogeneous tubulin
was prepared from microtubule protein by phosphocellulose (P11) chromatography as has been
described previously [32]. The purified proteins were stored in aliquots at -70oC. Microtubule
polymerization of tubulin protein was monitored at 37oC in solutions containing compounds (10 µM)
in PEM buffer (100 mM PIPES, 1 mM MgCl2, and 1 mM EGTA), 1 mM GTP, and 5% glycerol by
using light scattering at 340 nm with a SPECTRA MAX 190 (MD) spectrophotometer. And
colchicine was chosen as standard inhibitor of tubulin polymerization, while DMSO was used as
negative control, and tubulin concentration is 5 mg/mL. The percent inhibition values for selected
compounds were compared to the value of colchicine and measured the same day under the same
conditions.
4.3. Molecular docking

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Surflex-Dock program in Sybyl 7.3 was employed to dock compound 9d in colchicine binding site
of αβ-tubulin (PDB: 4O2B, 2.30 Å [28]). And an empirical scoring function and a patented search
engine [33, 34] was adopted. Docking was initiated by removing all ligands and water followed by
addition of random hydrogen atoms. The following procedures are performed according to our
reported procedures [35-37]. Ligand was docked into the corresponding protein-binding site guided by
protomol, which is an idealized representation of a ligand that makes every potential interaction with
the binding site. To validate docking reliability, the co-crystalized ligand (colchicine) was removed
from the active site and docked back into the known binding pocket. The root means square deviation
(RMSD) between the predicted conformation and the actual conformation from the crystal structure of
ligand was 1.96 Å, which was smaller than the resolution of X-ray crystallography 2.30 Å [28]. Thus,
the parameters set for this Surflex-dock simulation successfully reproduced the ligand-binding motif in
the X-ray structure, and were extrapolated to predict the binding conformations of the synthesized
inhibitors.
Acknowledgments
This work was financially supported by Foundation for Guangdong Distinguished Young Teachers in
Higher Education of China, and Science and Technology Program of Guangdong Province of China
(No. 2016A020217008) awarded to Z.Z.Z, and the National Science and Technology Major Projects of
China for “Major New Drug Innovation and Development” (No. 2012ZX09J1211003C),
NSFC-Guangdong Joint Fund (No. U1032006), and the National Natural Science Foundation of China
(No. 81373384) awarded to J. P. X.
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ACCEPTED MANUSCRIPT
Research Highlights
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Two series of 9H-purin-6-amines and 6-dichloro-9H-purines were designed and synthesized.
Compounds 9d and 11e-h exhibited low-micromole GI50 values against all test cell lines.
Compound 9d induced apoptosis at G2/M phase arrest in the human AGS cells.
Compounds 10b and 10d showed selective antiproliferative activities towards SH-SY5Y cells.
Compounds 10b and 10d displayed sub-micromole GI50 values against SH-SY5Y cells.