Design, synthesis and preliminary biological evaluation of purine-2,6-diamine derivatives as cyclin-dependent kinase (CDK) inhibitors

Article abstract of DOI:10.1002/cjoc.201300420

Novel purine-2,6-diamine derivatives were designed and synthesized as cyclin-dependent kinase (CDK) inhibitors. According to the preliminary biological evaluation, most of the compounds show good inhibitory activities in CDK1 enzyme assay and potent antiproliferative activities in some tumor cell lines. Especially, compound 11a (IC₅₀=0.35 μmol/L for CDK1/cyclin B and IC₅₀=0.023 μmol/L for CDK2/cyclin A) possessed better inhibitory effect compared with Roscovitine (IC₅₀=2.54 (mol/L for CDK1/cyclin B and IC₅₀=0.092 μmol/L for CDK2/cyclin A). Copyright

Full text of DOI:10.1002/cjoc.201300420

FULL PAPER
DOI: 10.1002/cjoc.201300420
Design, Synthesis and Preliminary Biological Evaluation of
Purine-2,6-diamine Derivatives as Cyclin-dependent Kinase
(CDK) Inhibitors
Junhua Wang,a Quande Wang,a Liangren Zhang,b and Hao Fang*,a
a Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University,
Jinan, Shandong 250012, China
b State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences,
Peking University, Beijing 100191, China
Novel purine-2,6-diamine derivatives were designed and synthesized as cyclin-dependent kinase (CDK) inhibi-
tors. According to the preliminary biological evaluation, most of the compounds show good inhibitory activities in
CDK1 enzyme assay and potent antiproliferative activities in some tumor cell lines. Especially, compound 11a
(IC50＝0.35 μmol/L for CDK1/cyclin B and IC50＝0.023 μmol/L for CDK2/cyclin A) possessed better inhibitory
effect compared with Roscovitine (IC50 ＝2.54 μmol/L for CDK1/cyclin B and IC50 ＝0.092 μmol/L for
CDK2/cyclin A).
Keywords purine derivatives, CDK inhibitors, antitumor agents, regulation of cell cycle
Introduction
proliferation or to inhibit tumor growth.[3,9] Along this
line, many efforts have been devoted to develop novel
CDK1 inhibitors in recent years.
A variety of genetic and/or epigenetic events in hu-
man cancer are either direct or indirect consequence of
deregulated cell cycle. However, the deregulation of cell
cycle is often related to abnormal of cyclin-dependent
kinases (CDKs) activities which frequently associated
with many types of cancer.[1] CDKs, which consist of a
catalytic CDK subunit and a positive regulatory cyclin
subunit, belong to serine/threonine kinase family. Only
a certain subset of CDKs-cyclins involved in cell cycle
directly, including three interphase CDKs (CDK2,
CDK4 and CDK6), a mitotic CDK (CDK1) and ten cy-
clins.[2] For the subtype CDK1, it can form the
CDK1/cyclin A complex and CDK1/cyclin B complex,
which can control cell cycle to entry into mitosis and
maintenance of the mitotic state.[3] More recently, ge-
netic studies in mice showed that the knockout of the
mitotic kinase CDK1 was lethal and facilitated to the
cell cycle arrest, indicating that CDK1 is essential for
the cell cycle progression and executes all the events for
cell division.[4-7]
During the past decade many small molecule CDK
inhibitors have been developed and identified with
various structures.[3,10,11] Among them, purine heterocy-
cles are one of the most investigated structural skeletons
for CDK inhibitors.[12-17] For example, R-roscovitine
(CYC202, seliciclib), a 2,6,9-trisubstituted purine CDKs
inhibitor, has been studied in phase II clinical trial.[18,19]
However, the moderate inhibitory activities against
CDKs is still a problem for R-roscovitine and limited
this agent in therapeutic application. Therefore, further
structural modification should be carried out on purine
derivatives so as to develop more potent CDK inhibi-
tors.
NH
H3C
HO
N
N
N
Regulation of CDK1 levels has been regarded as a
good way to block cell cycle or induce apoptosis in
higher eukaryotes.[8] Recently, a number of literatures
have illustrated that CDK1 inhibitors could block the
cell cycle progression through mitosis and hold promise
for treating cancer due to their abilities to control cell
N
N
H
CH3
H3C
R-Roscovitine
In our studies, the structural modification was per-
formed based on the structure of Roscovitine. Different
*
E-mail: haofangcn@sdu.edu.cn; Tel.: 0086-0531-88382731; Fax: 0086-0531-88382548
Received May 27, 2013; accepted August 22, 2013; published online September 16, 2013.
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cjoc.2013201300420 or from the author.
Chin. J. Chem. 2013, 31, 1181—1191
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hydroxyalkylamines and amino acids were introduced to
C-2 position and the cyclohexylmethyl was introduced
to N-6 position at the same time. According to litera-
ture,[20] deletion of 9-isopropyl can increase the hydro-
gen bond interaction with the target. Therefore, other
three series of N2,N6-disubstituted 9H-purine-2,6-dia-
mine were designed to increase the CDK1 binding af-
finities. The synthetic routes of all the target compounds
were shown in Schemes 1－4.
Scheme 3 General synthetic route of the target compounds 11a
－11h
Cl
NH
NH2
N
N
N
N
N
TEA, n-butanol
N
H
Cl
N
N
H
Cl
N
10
9
Scheme 1 General synthetic route of the target compounds 4a
－4h
NH
ArNH2, TFA, n-butanol
N
R
or ArNH2, TFA, TFE
N
H
N
N
H
Cl
NH
11a - 11h
N
NH2
N
N
N
TEA, EtOH
N
F
N
1
Scheme 4 General synthetic route of the target compounds 15a
－15c
N
H
F
N
H
2
O
NH
Br
Cl
Cl
N
N
N
O
N
N
N
N
N
Cl
N
K2CO3, DMSO
F
N
O
K2CO3, DMSO
N
H
H2N
N
5
H2N
3
O
12
NH
N
N
N
NMP, amino acid, DBU, N2
or amino alcohol, DMSO, DIEA, N2
N
R
NH
arylsulfonyl chloride
pyridine
N
NH2
TEA, EtOH
H
N
N
O
4a - 4h
N
H2N
N
O
13
Scheme 2 General synthetic route of the target compounds 8a
－8i
Cl
3 mol/L NaOH
THF
NH
NH
N
NH
N
HBF4, NaNO2
H2O
benzylamine, TEA
N
N
N
N
N
N
N
R
O
n-butanol
N
R
O
N
H
H2N
N
O
N
H
S
H2N
N
S
N
N
5
N
N
O
H
6
O
H
H
O
14a - 14c
15a - 15c
NH
ArNH2, TFA, n-butanol
NH
N
N
N
or ArNH2, TFA, TFE
affinities to CDK1. Only compounds 4e and 4f have the
IC50 value about 5 μmol/L, but still are not good as
Roscovitine (Table 1).
N
N
R
N
N
N
N
H
F
H
H
7
8a - 8i
Literature reported that the derivatives without iso-
propyl substitution at N-9 position would be helpful to
increase the inhibition to CDK. In our studies, these two
series of compounds were synthesized without isopropyl
in N-9 position. The difference between these two series
compounds is the R1 substitution, one is benzyl group
and the other is cyclohexylmethylene fragment. For the
target compounds with benzyl group in R1 position (8a
－8i), the binding affinities to CDK1 did not show sig-
nificantly increase and only two compounds (8e, 8g)
possess the similar inhibition compared with Rosco-
vitine. For the target compounds with cyclohexyl-
methylene substitution in R1 position, introducing ben-
zenesulfonamide fragment in C-2 position (15a－15c)
Results and Discussion
Totally twenty eight purine-2,6-diamine derivatives
were synthesized. The preliminary biological evalua-
tions include their enzyme inhibition against CDK1/
cyclin B, CDK2/cyclin A and antiproliferative activities
against human colon carcinoma (HCT116), human
breast carcinoma (MDA-MB231) and human prostate
carcinoma (PC3) cell lines.
According to the result of CDK1 inhibition, target
compounds 4a－4h with hydroxyalkylamine or amino
acids substitution at C-2 position showed poor binding
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Chin. J. Chem. 2013, 31, 1181—1191

Design, Synthesis and Preliminary Biological Evaluation of Purine-2,6-diamine Derivatives
Table 1 CDK1 inhibitory activity of the target compounds
R1
NH
N
N
R2
N
N
N
H
R3
Compd
R1
R2
R3
Compd
R1
R2
R3
H
−
1
−
1
IC50a/(μmol•L )
IC50a/(μmol•L )
OH
SO2NH2
9.54
2.74
4a
8g
8h
O
S
OH
＞10
＞10
H
H
＞10
＞10
4b
4c
O
OCH3
OH
OH
8i
OH
SO2NH2
＞10
5.13
5.45
11a
11b
11c
H
H
H
0.35
＞10
1.05
4d
4e
4f
OH
OH
F
OH
OH
OH
＞10
＞10
11d
11e
H
H
5.37
4g
4h
O
O
OH
CH3
＞10
Br
OCH3
Br
H
H
H
H
H
H
＞10
＞10
＞10
＞10
3.34
H
H
H
H
H
H
＞10
＞10
1.06
8a
8b
8c
8d
8e
8f
11f
11g
11h
15a
15b
15c
OCH3
CH3
O
S
O
O
S
＞10
＞10
＞10
F
O
O
OH
F
S
NO2
OCF3
O
O
S
＞10
O
(R,S)-Roscovitine
2.54
a IC50 values were the mean values of duplicate experiments.
will significantly lower the binding affinities to CDK1
compared with the corresponding aniline derivatives.
According to the data in Table 1, most of the target
compounds with aniline substitution in C-2 position
(11a－11h) exhibit similar or even better inhibition
compared with Roscovitine. The substituent in the ani-
line also has the important impact on the inhibitory ac-
tivities. For example, compounds 11e, 11f and 11g with
methyl, methoxy and bromo group substituted respec-
tively showed very poor inhibition. But the compounds
Chin. J. Chem. 2013, 31, 1181—1191
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11a (IC50＝0.35 μmol/L) and 11c (IC50＝1.05 μmol/L)
with sulfonamide and hydroxyl substitution exhibit
more potent activities to CDK1/cyclin B than Rosco-
vitine (IC50＝2.54 μmol/L).
more potent binding affinity (Table 3). It also indicated
that the growth of the tumor cell lines (HCT116,
MDA-MB231 and PC3) could be significantly inhibited
by the active compounds 11a, 11c and 11h. Moreover,
these three compounds possessed better growth inhibi-
tion towards the HCT116 cell than that of MDA-MB231
and PC3.
Then MTT assay was used to investigate the antipro-
liferative activities of the target compounds. The pre-
liminary evaluation was performed using HCT116 cell
line (Table 2) and the results suggest that compounds
15a－15c and most of the compounds 4a－4h show
poor antiproliferative activities. Only compounds 4a, 4e
and 4f exhibit better inhibition compared with Rosco-
vitine. Most of the compounds 8a－8i and all the com-
pounds of 11a－11h have better antiproliferative activi-
ties than Roscovitine. Especially compounds 11a, 11c
and 11h not only showed strong inhibition to HCT116
tumor cell line, but also exhibit best binding affinities to
CDK1. Therefore, these three compounds were chosen
to perform further biological evaluation.
To check the effect of the active compounds 11a,
11c and 11h against the other subtype of CDKs, the
three compounds were tested the inhibitory activity
against CDK2/cyclin A. The results suggest that the
compounds also show good inhibition to CDK2/cyclin
A compared with Roscovitine, especially 11a exhibits
Conclusions
In summary, we report the synthesis and biological
evaluation of new purine-2,6-diamine derivatives as
potential antitumor agents. The active compounds 11a,
11c and 11h not only showed significant inhibitory ac-
tivities against CDK1/cyclin B enzyme, CDK2/cyclin A
enzyme, but also had obvious inhibition to HCT116,
MDA-MB231 and PC3 tumor cell lines. These results
suggest that these series of purine-2,6-diamine deriva-
tives could be used as lead compounds to investigate
new potent CDK inhibitors.
Experimental
Chemistry: general procedures
All the materials used were commercially available
Table 2 Antiproliferative activities of the target compounds to HCT116
IC50a,b/(μmol•L−1)
Compd
5.38±1.8 8g
Compd
IC50a,b/(μmol•L−1)
19.7±4.6
12.2±3.2
27.9±6.6
7.79±2.4
9.58±3.0
4.99±2.3
8.06±2.7
15.0±3.8
4.95±1.2
6.37±1.7
6.96±1.6
＞100
4a
4b
4c
4d
4e
4f
4g
4h
8a
8b
8c
8d
8e
8f
23.2±4.1
24.6±8.1
16.4±8.7
8.44±2.7
7.74±2.4
＞100
8h
8i
11a
11b
11c
11d
11e
11f
11g
11h
15a
15b
15c
＞100
6.37±2.2
12.0±4.6
45.9±34.3
9.94±2.3
8.60±3.2
8.96±2.9
15.7±1.7
＞100
＞100
(R,S)-Roscovitine
a IC50 values were the mean values of three repeated experiments. b IC50 values represent the concentration which results in 50% decrease
in cell growth after 48 h incubation.
Table 3 Biological activities of compounds 11a, 11c and 11h against CDKs (Cyclin-dependent kinases): CDK1/cyclin B, CDK2/cyclin
A and against the tumor cell lines HCT116, PC-3, MDA-MB-231
−
1
−
1
Enzyme activities IC50/(μmol•L )
Anti-proliferative IC50a,b/(μmol•L )
Compd
CDK1/Cyclin B
CDK2/Cyclin A
HCT116
MDA-MB231
21.3±6.4
27.4±5.6
23.6±6.8
49.4±9.0
PC3
0.35
1.05
1.06
2.54
0.023
0.19
11a
7.79±2.4
4.99±2.3
6.96±1.6
15.7±1.7
13.3±2.7
13.4±2.1
14.6±2.8
42.8±4.5
11c
0.35
11h
(R,S)-Roscovitine
0.092
a IC50 values were the mean values of three repeated experiments. b IC50 values represent the concentration which results in 50% decrease
in cell growth after 48 h incubation.
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Design, Synthesis and Preliminary Biological Evaluation of Purine-2,6-diamine Derivatives
General synthesis of target compounds 4d－4f
and used without further purification. Solvents were
distilled prior to use and flash chromatography was
performed using silica gel (60 Å, 200－300 mesh). All
reactions were monitored by thin-layer chromatography
on 0.25 mm silica gel plates (GF-254) and visualized
with UV light, or iodine vapour. Melting points were
determined on an electrothermal melting point apparatus
and thermometer is uncorrected. Proton nuclear mag-
netic resonance (1H NMR) spectra were recorded on a
Brucker Avance-300, Brucker Avance-400 or Varian
Inova-600 spectrometers using trimethylsilane as an
internal standard. High-resolution mass spectral (HRMS)
data were reported as m/z (relative intensity). All tested
compounds are＞95% pure by HPLC analysis, per-
formed on an Agilent 1100 HPLC instrument using a
Luna 5μ C18 (2) column (150 mm×4.60 mm), eluted
with different ratios of acetonitrile or methanol/water
(containing 0.1% formic acid) over 15 min, with detec-
tion at 259 nm and a flow rate of 1.0 mL/min.
A mixture of 3 (0.3 g, 1 mmol), L-Leucinol/L-
Valinol/L-Alaninol (6.7 mmol) and N,N-diisopropyl
ethylamine (1.7 mL, 10 mmol) in 5 mL DMSO was
stirred at 160 ℃. After cooling, H2O was added and the
solution was extracted with ethyl acetate. The combined
organic layer was washed with brine, dried over MgSO4,
filtered and evaporated. The residue was purified by
column chromatography and then conversed to the cor-
responding HCl salt with saturated HCl/EtOAc.
General synthesis of target compounds 4g－4h
3 (0.5 g, 1.7 mmol) was dissolved in 10 mL
N-methyl-2-pyrrolidinone, L-Ser/L-Thr/L-Ala (34 mmol)
and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 5.3 mL,
34 mmol) were then added successively and the mixture
was stirred under N2 at 160 ℃. After cooling, the mix-
ture was diluted with 10% critic acid and CH2Cl2. The
organic phase was separated and washed with brine,
then dried over MgSO4 and purified by column chro-
matography to afford corresponding compounds.
N-(Cyclohexylmethyl)-2-fluoro-9H-purine-6-amine
(2)
2-(6-(Cyclohexyl-methylamino)-9-isopropyl-9H-pu-
Cyclohexymethanamine (4.5 mL, 34.9 mmol) and
triethylamine (TEA, 5.9 mL, 43.6 mmol) were added to
a stirred solution of 6-chloro-2-fluoro purine (1, 5.0 g,
29 mmol) in 30 mL ethanol successively. The mixture
was refluxed for 5 h. Then the solution was allowed to
cool to room temperature and solvent was concentrated
in vaccum. The residue was purified by column chro-
matography to afford 2 as white solid (4.8 g, 66%). m.p.
rine-2-ylamino)butan-1-ol (4a)
Yield 32%. m.p. 116－118 ℃; 1H NMR (600 MHz,
CDCl3) δ: 0.97－1.01 (m, 2H), 1.03 (t, J＝7.8 Hz, 3H),
1.13－1.26 (m, 3H), 1.53 (d, J＝6 Hz, 6H), 1.56－1.60
(m, 2H), 1.61－1.67 (m, 2H), 1.71－1.73 (m, 2H),
1.80－1.82 (m, 2H), 3.38 (s, 2H), 3.63 (dd, J＝10.8 Hz
and 7.2 Hz, 1H), 3.82－3.89 (m, 2H), 4.57－4.61 (m,
1H), 4.88 (s, 1H), 5.45－5.67 (m, 2H), 7.49 (s, 1H);
HRMS calcd for C19H32N6O 361.2716, found 361.2711.
Anal. RP-HPLC tR ＝ 5.8 min [methanol ∶ water
(containing 0.1% formic acid) ＝ 60 ∶ 40; purity ＝
96.8%].
1
164－168 ℃; H NMR (300 MHz, DMSO-d6) δ: 0.88
－0.99 (m, 2H), 1.14－1.16 (m, 3H), 1.61－1.72 (m,
6H), 3.25 (s, 2H), 8.06 (s, 1H), 8.17 (s, 1H), 12.98 (s,
1H); ESI-MS m/z: 250.4 [M＋1]＋.
N-(Cyclohexylmethyl)-2-fluoro-9-isopropyl-9H-
purine-6-amine (3)
2-(6-(Cyclohexyl-methylamino)-9-isopropyl-9H-
purine-2-ylamino)ethanol (4b)
To a solution of 2 (4.80 g, 19.3 mmol) in DMSO (15
mL) were added 2-bromopropane (4.5 mL, 48.1 mmol)
and K2CO3 (8.0 g, 57.8 mmol). The mixture stayed at 80
℃ overnight. After addition of H2O, the mixture was
extracted with ethyl acetate, and the combined organic
layers were washed with brine, dried over MgSO4, con-
centrated and chromatographed on silica gel to give 3
1
Yield 32%. m.p. 95－98 ℃; H NMR (600 MHz,
CDCl3) δ: 0.94－1.03 (m, 2H), 1.18－1.27 (m, 3H) 1.53
(d, J＝6.8 Hz, 6H), 1.55－1.61 (m, 1H), 1.62－1.64 (m,
1H), 1.69－1.72 (m, 2H), 1.78－1.81 (m, 2H), 3.38 (s,
2H), 3.55－3.58 (m, 2H), 3.83 (t, J＝4.4 Hz, 2H), 4.57
－4.64 (m, 1H), 5.02 (br s, 1H), 5.37 (t, J＝5.4 Hz, 1H),
5.87 (s, 1H), 7.50 (s, 1H); HRMS calcd for C17H28N6O
333.2401, found 333.2418. Anal. RP-HPLC tR＝7.8 min
[methanol∶water (containing 0.1% formic acid)＝50∶
50; purity＝99.4%].
1
(4.0 g, 72%) as a white solid. m.p. 112－115 ℃; H
NMR (400 MHz, DMSO-d6) δ: 0.92－0.96 (m, 2H),
1.14－1.16 (m, 3H), 1.49 (d, J＝6.8 Hz, 6H), 1.64－
1.67 (m, 6H), 3.26 (br s, 2H), 4.59－4.65 (m, 1H), 8.20
(s, 1H), 8.29 (s, 1H); ESI-MS m/z: 292.4 [M＋1]＋.
2-(6-(Cyclohexylmethylamino)-9-isopropyl-9H-
purine-2-ylamino)propane-1,3-diol (4c)
General synthesis of target compounds 4a－4c
Yield 41%. m.p. 166－168 ℃; 1H NMR (600 MHz,
CDCl3) δ: 0.93－0.99 (m, 2H), 1.09－1.27 (m, 3H),
1.50 (d, J＝7.2 Hz, 6H), 1.53－1.60 (m, 1H), 1.63－
1.65 (m, 1H), 1.69－1.71 (m, 2H), 1.77－1.79 (m, 2H),
3.32 (s, 2H), 3.82－3.90 (m, 4H), 4.08 (t, J＝3 Hz, 1H),
4.56 (hept., J＝7.2 Hz, 1H), 5.12 (s, 2H), 5.70 (d, J＝
5.4 Hz, 1H), 5.96 (s, 1H), 7.50 (s, 1H); HRMS calcd for
A mixture of 3 (0.3 g, 1 mmol) and different amino
alcohol (6.5 mmol) in 5 mL DMSO was stirred under
N2 at 160 ℃ till the reaction was completed. Then the
mixture was cooled, water was added and extracted with
ethyl acetate. The combined organic layer was washed
with brine, dried over MgSO4 and then purified by
column chromatography to afford the target compounds.
Chin. J. Chem. 2013, 31, 1181—1191
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C18H30N6O2: 363.2508, found 363.2536. Anal. RP-
HPLC tR＝5.2 min [methanol∶water (containing 0.1%
formic acid)＝50∶50; purity＝99.7%].
1.85 (m, 2H), 3.38 (s, 2H), 4.43－4.48 (m, 1H), 4.61－
4.68 (m, 1H), 7.82 (s, 1H); HRMS calcd for C18H28N6O2
361.2352, found 361.2354. Anal. RP-HPLC tR＝9.1 min
[acetonitrile∶water (containing 0.1% formic acid)＝
28∶72; purity＝98.4%].
2-(6-(Cyclohexylmethylamino)-9-isopropyl-9H-
purine-2-ylamino)-4-methylpentane-1-ol (4d)
N6-Benzyl-9H-purine-2,6-diamine (6)
Yield 18%. m.p. 145－149 ℃; 1H NMR (400 MHz,
CD3OD) δ: 1.02 (t, J＝6.4 Hz, 6H), 1.07－1.15 (m, 2H),
1.25－1.36 (m, 3H), 1.51－1.59 (m, 2H), 1.62－1.64
(m, 6H), 1.73－1.81 (m, 4H), 1.89－1.92 (m, 2H), 3.43
－3.47 (m, 2H), 3.64－3.72 (m, 2H), 4.24－4.27 (m,
1H), 4.74－4.80 (m, 1H), 8.51 (s, 1H); HRMS calcd for
C21H36N6O 389.3029, found 389.3007. Anal. RP-HPLC
tR ＝ 14.3 min [methanol ∶ water (containing 0.1%
formic acid)＝60∶40; purity＝98.3%].
To a stirred solution of 2-amino-6-chloro purine (5,
10.0 g, 59.0 mmol) in n-butanol (150 mL) at room
temperature, benzylamine (9.7 mL, 88.5 mmol) and
TEA (20 mL, 147.5 mmol) were added successively.
The reaction mixture was refluxed at 130 ℃ for 6 h
and then cooled to room temperature. The solvent was
removed under low pressure and the residue was
washed with n-propanol to afford 6 as a white solid
(13.9 g, 98%) after drying under vacuum, m.p. 234－
238 ℃. 1H NMR (600 MHz, DMSO-d6) δ: 4.64 (s, 2H),
5.75 (s, 2H), 7.20 (t, J＝7.2 Hz, 2H), 7.28 (t, J＝7.8 Hz,
2H), 7.34 (d, J＝7.2 Hz, 2H), 7.68 (s, 1H), 12.15 (s, 1H);
HRMS calcd for C12H12N6 241.1201, found 241.1218.
2-(6-(Cyclohexylmethylamino)-9-isopropyl-9H-
purine-2-ylamino)-3-methylbutan-1-ol (4e)
1
Yield 18%. m.p. 75－79 ℃; H NMR (400 MHz,
CD3OD) δ: 1.03－1.07 (m, 6H), 1.11－1.13 (m, 2H),
1.24－1.35 (m, 3H), 1.59 (dd, J＝6.5 Hz and 2.8 Hz,
6H), 1.72－1.81 (m, 4H), 1.86－1.89 (m, 2H), 2.07 (t,
J＝5.7 Hz, 1H), 3.41 (s, 2H), 3.75 (d, J＝4.8 Hz, 2H),
4.02 (s, 1H), 4.67－4.73 (m, 1H), 8.04 (s, 1H); HRMS
calcd for C20H34N6O 375.2872, found 375.2884. Anal.
RP-HPLC tR＝8.0 min [methanol∶water (containing
0.1% formic acid)＝60∶40; purity＝97.2%].
N-Benzyl-2-fluoro-9H-purin-6-amine (7)
An aqueous solution of sodium nitrite (1.4 g, 200
mmol) was added with stirring to a solution of 6 (2.4 g,
10 mmol) in 48% fluoroboric acid (30 mL) at –15 ℃.
After the reaction mixture was stirred at –15 ℃ for 30
min, solution was neutralized to pH＝7. The resulting
precipitate was collected and extracted with ethyl ace-
tate. The organic layer was concentrated in vaccum and
then purified by chromatography to yield 7 (0.83 g,
34%). m.p. 225－227 ℃; 1H NMR (600 MHz, DMSO-
d6) δ: 4.64 (d, J＝6 Hz, 2H), 7.24 (d, J＝6 Hz, 1H), 7.31
－7.36 (m, 4H), 8.11 (s, 1H), 8.80 (s, 1H), 13.05 (s, 1H);
HRMS calcd for C12H10FN5 244.0998, found 244.0997.
2-(6-(Cyclohexylmethylamino)-9-isopropyl-9H-
purine-2-ylamino)propane-1-ol (4f)
Yield 71%. m.p. 184－188 ℃; 1H NMR (400 MHz,
CD3OD) δ: 1.09－1.17 (m, 2H), 1.24－1.30 (m, 3H),
1.34 (d, J＝6.6 Hz, 3H), 1.67 (d, J＝6.7 Hz, 6H), 1.72
－1.74 (m, 2H), 1.79－1.82 (m, 2H), 1.92－1.95 (m,
2H), 3.52 (d, J＝6.8 Hz, 2H), 3.63－3.74 (m, 2H), 4.20
－4.24 (m, 1H), 4.84－4.90 (m, 1H), 9.00 (s, 1H);
HRMS calcd for C18H30N6O 347.2559, found 347.2556.
Anal. RP-HPLC tR ＝ 7.1 min [acetonitrile ∶ water
(containing 0.1% formic acid) ＝ 28 ∶ 72; purity ＝
99.1%].
General synthesis of N6-benzyl-N2-(substituted
phenyl)-9H-purine-2,6-diamine (8a－8f)
A mixture of 7 (0.25 g, 1 mmol), substituted aniline
(7 mmol) and trifluoroacetic acid (TFA, 0.1 mL, 1.3
mmol) in n-butanol was heated to reflux at 130 ℃
overnight. The reaction mixture was cooled and the pre-
cipitate was collected, then purified by column chroma-
tography to give the corresponding compounds.
2-(6-(Cyclohexylmethylamino)-9-isopropyl-9H-
purine-2-ylamino)-3-hydroxypropanoic acid (4g)
1
Yield 17%. m.p. 158－160 ℃; H NMR (300 MHz,
General synthesis of N6-benzyl-N2-(substituted
phenyl)-9H-purine-2,6-diamine (8g, 8h)
CD3OD) δ: 0.98－1.12 (m, 2H), 1.29－1.40 (m, 3H),
1.58 (d, J＝6.6 Hz, 6H), 1.65－1.73 (m, 2H), 1.78－
1.89 (m, 4H), 3.38 (s, 2H), 4.03 (d, J＝4.2 Hz, 2H),
4.61 (t, J＝7.2 Hz, 1H), 4.66－4.72 (m, 1H), 7.86 (s,
1H); HRMS calcd for C18H28N6O3 377.2301, found
377.2319. Anal. RP-HPLC tR＝6.5 min [acetonitrile∶
water (containing 0.1% formic acid)＝25∶75; purity＝
96.7%].
A mixture of 7 (0.25 g, 1 mmol), substituted aninli-
nes (3 mmol), TFA (0.74 mL, 10 mmol), and 2,2,2-tri-
fluoroethanol (10 mL) was refluxed at 85 ℃ for 8 h.
The solvent was concentrated in vaccum after cooling.
The residue was purified by column chromatography on
silica gel to afford target compounds.
2-(6-(Cyclohexylmethylamino)-9-isopropyl-9H-
N6-Benzyl-N2-(4-bromophenyl)-9H-purine-2,6-dia
purine-2-ylamino) propanoic acid (4h)
mine (8a)
1
Yield 28%. m.p. 84－90 ℃; H NMR (400 MHz,
Yield 23%. m.p. 264－268 ℃; 1H NMR (600 MHz,
DMSO-d6) δ: 4.74 (t, J＝5.4 Hz, 2H), 7.21 (t, J＝7.2 Hz,
1H), 7.31 (t, J＝7.8 Hz, 4H), 7.38 (d, J＝7.8 Hz, 2H),
7.71 (d, J＝4.8 Hz, 2H), 7.84 (s, 1H), 8.12 (s, 1H), 9.02
CD3OD) δ: 0.98－1.07 (m, 2H), 1.19－1.33 (m, 3H),
1.51 (d, J＝7.2 Hz, 3H), 1.56 (dd, J＝6.8 and 1.6 Hz,
6H), 1.68－1.70 (m, 2H), 1.74－1.77 (m, 2H), 1.82－
1186
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© 2013 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2013, 31, 1181—1191

Design, Synthesis and Preliminary Biological Evaluation of Purine-2,6-diamine Derivatives
(s, 1H), 12.49 (s, 1H); HRMS calcd for C18H15BrN6
395.0620, found 395.0614. Anal. RP-HPLC tR＝12.7
min [acetonitrile∶water (containing 0.1% formic acid)
＝35∶65; purity＝99.5%].
N6-benzyl-N2-(4-methoxyphenyl)-9H-purine-2,6-
diamine (8b)
Yield 32%. m.p. 229－232 ℃; 1H NMR (600 MHz,
DMSO-d6) δ: 3.69 (s, 3H), 4.69 (s, 2H), 6.77 (d, J＝8.4
Hz, 2H), 7.20 (t, J＝7.8 Hz, 1H), 7.30 (t, J＝7.8 Hz,
2H), 7.37 (d, J＝7.8 Hz, 2H), 7.61 (d, J＝8.4 Hz, 2H),
7.78 (s, 1H), 7.96 (s, 1H), 8.60 (s, 1H), 12.37 (s, 1H);
HRMS calcd for C19H18N6O 347.1620, found 347.1610.
Anal. RP-HPLC tR ＝ 13.3 min [methanol ∶ water
(containing 0.1% formic acid) ＝ 50 ∶ 50; purity ＝
98.9%].
min [methanol∶water (containing 0.1% formic acid)＝
50∶50; purity＝100%].
4-(6-(Benzylamino)-9H-purine-2-ylamino)benzene-
sulfonamide (8g)
1
Yield 8%. m.p. 215－218 ℃; H NMR (600 MHz,
DMSO-d6) δ: 4.72 (s, 2H), 7.10 (s, 2H), 7.22 (s, 1H),
7.32 (s, 2H), 7.39 (d, J＝6 Hz, 2H), 7.60 (d, J＝7.2 Hz,
2H), 7.86 (s, 2H), 7.90 (s, 1H), 8.19 (s, 1H), 9.32 (s, 1H),
12.55 (s, 1H); HRMS calcd for C18H17N7O2S 396.1242,
found 396.1238. Anal. RP-HPLC tR ＝ 5.6 min
[acetonitrile∶water (containing 0.1% formic acid)＝
25∶75; purity＝98.6%].
N6-Benzyl-N2-(4-(methylsulfonyl)phenyl)-9H-purine-
2,6-diamine (8h)
1
Yield 4%. m.p. 241－244 ℃; H NMR (600 MHz,
N6-Benzyl-N2-(p-tolyl)-9H-purine-2,6-diamine (8c)
DMSO-d6) δ: 3.14 (s, 3H), 4.78 (s, 2H), 7.25 (s, 1H),
7.35 (s, 2H), 7.42 (d, J＝5.4 Hz, 2H), 7.73 (d, J＝7.8
Hz, 2H), 7.94 (s, 2H), 8.52 (s, 1H), 8.74 (s, 1H), 9.81 (s,
1H), 13.27 (s, 1H); HRMS calcd for C18H17N7O2S
395.1290, found 395.1305. Anal. RP-HPLC tR＝8.0 min
[acetonitrile∶water (containing 0.1% formic acid)＝
28∶72; purity＝100%].
Yield 48%. m.p. 277－279 ℃; 1H NMR (600 MHz,
DMSO-d6) δ: 2.21 (s, 3H), 4.70 (s, 2H), 6.97 (d, J＝8.4
Hz, 2H), 7.20 (t, J＝7.2 Hz, 1H), 7.30 (t, J＝7.2 Hz,
2H), 7.38 (d, J＝7.2 Hz, 2H), 7.61 (d, J＝6.6 Hz, 2H),
7.79 (s, 1H), 8.01 (s, 1H), 8.69 (s, 1H), 12.40 (s, 1H);
HRMS calcd for C19H18N6 331.1671, found 331.1668.
Anal. RP-HPLC tR ＝ 16.9 min [methanol ∶ water
(containing 0.1% formic acid) ＝ 50 ∶ 50; purity ＝
95.1%].
N6-Benzyl-N2-(3-methoxyphenyl)-9H-purine-2,6-
diamine (8i)
The mixture of compound 7 (0.25 g, 1 mmol),
3-methoxyaniline (0.86 g, 7 mmol), TFA (0.1 mL, 1.3
mmol) and n-butanol (7 mL) was refluxed at 130 ℃
for 6 h. The reaction mixture was cooled to room tem-
perature and diluted with water. The solution was neu-
tralized to pH＝7 with NaHCO3 and extracted with
ethyl acetate. The combined organic layer was dried
with MgSO4, concentrated under reduced pressure, and
purified by column chromatography to obtain 8i. Yield
42%. m.p. 236 － 238 ℃ ; 1H NMR (600 MHz,
DMSO-d6) δ: 3.69 (s, 3H), 4.72 (s, 2H), 7.06 (t, J＝7.8
Hz, 1H), 7.20 (t, J＝7.8 Hz, 1H), 7.29－7.32 (m, 3H),
7.39 (d, J＝7.8 Hz, 2H), 7.57 (t, J＝8.4 Hz, 2H), 7.82 (s,
1H), 8.04 (s, 1H), 8.81 (s, 1H), 12.45 (s, 1H); HRMS
calcd for C19H18N6O 347.1620, found 347.1614. Anal.
RP-HPLC tR＝7.0 min [acetonitrile∶water (containing
0.1% formic acid)＝28∶72; purity＝100%].
N6-Benzyl-N2-phenyl-9H-purine-2,6-diamine (8d)
Yield 83%. m.p. 261－262 ℃; 1H NMR (600 MHz,
DMSO-d6) δ: 4.74 (s, 2H), 6.88 (t, J＝7.2 Hz, 1H), 7.20
(t, J＝7.8 Hz, 2H), 7.25 (d, J＝7.2 Hz, 1H), 7.33 (t, J＝
7.2 Hz, 2H), 7.39 (d, J＝7.8 Hz, 2H), 7.70 (d, J＝7.2 Hz,
2H), 8.11 (s, 1H), 8.32 (s, 1H), 9.10 (s, 1H), 12.81 (s,
1H); HRMS calcd for C18H16N6 317.1514, found
317.1521. Anal. RP-HPLC tR＝12.1 min [methanol∶
water (containing 0.1% formic acid)＝50∶50; purity＝
99.8%].
N6-Benzyl-N2-(4-hydroxyphenyl)-9H-purine-2,6-
diamine (8e)
Yield 41%. m.p. 258－260 ℃; 1H NMR (600 MHz,
DMSO-d6) δ: 4.71 (t, J＝6.6 Hz, 2H), 6.60 (d, J＝8.4
Hz, 2H,), 7.20 (t, J＝7.8 Hz, 1H), 7.29 (t, J＝7.2 Hz,
2H), 7.37 (d, J＝7.2 Hz, 2H), 7.46 (t, J＝7.8 Hz, 2H),
7.75 (s, 1H), 7.94 (s, 1H), 8.45 (s, 1H), 8.83 (s, 1H),
12.32 (s, 1H); HRMS calcd for C18H16N6O 333.1464,
found 333.1462. Anal. RP-HPLC tR ＝ 6.4 min
[acetonitrile∶water (containing 0.1% formic acid)＝
20∶80; purity＝97.6%].
2-Chloro-N-(cyclohexylmethyl)-9H-purine-6-amine
(10)
To a stirred solution of 2,6-dichloropurine (9, 5.0 g,
26.5 mmol) in n-butanol (30 mL) at room temperature,
aminomethylcyclohexane (4.2 mL, 31.7 mmol) and
TEA (5.4 mL, 40 mmol) were added successively. The
reacting mixture was refluxed at 130 ℃ for 5 h. Then
the solution was allowed to cool to room temperature.
The precipitate was collected and washed with
n-propanol to afford 10 (6.4 g, 91%) after drying. m.p.
N6-Benzyl-N2-(4-fluorophenyl)-9H-purine-2,6-
diamine (8f)
Yield 21%. m.p. 247－250 ℃; 1H NMR (600 MHz,
DMSO-d6) δ: 4.71 (s, 2H), 7.04 (t, J＝9 Hz, 2H), 7.24 (t,
J＝7.2 Hz, 1H), 7.33 (t, J＝7.8 Hz, 2H), 7.38 (d, J＝7.8
Hz, 2H), 7.68 (s, 2H), 8.14 (s, 1H), 8.32 (s, 1H), 9.16 (s,
1H), 12.93 (s, 1H); HRMS calcd for C18H15FN6
335.1420, found 335.1350. Anal. RP-HPLC tR＝14.0
1
208－210 ℃. H NMR (400 MHz, DMSO-d6) δ: 0.92
－1.00 (m, 2H), 1.57－1.21 (m, 3H), 1.61－1.73 (m,
6H), 3.23 (s, 2H), 7.94 (s, 1H), 8.08 (s, 1H), 12.85 (s,
1H); HRMS: m/z calcd for C12H16ClN5 [M ＋ 1] ＋
Chin. J. Chem. 2013, 31, 1181—1191
© 2013 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.cjc.wiley-vch.de
1187

Wang et al.
FULL PAPER
266.1172, found 266.1177.
General synthesis of N6-(cyclohexylmethyl)-N2-
substitued phenyl-9H-purine-2,6-diamine (11a, 11h)
DMSO-d6) δ: 0.93－1.02 (m, 2H), 1.13－1.24 (m, 3H),
1.63－1.78 (m, 6H), 3.34 (s, 2H), 7.10 (t, J＝8.8 Hz,
2H), 7.73－7.77 (m, 2H), 8.23 (s, 2H), 9.30 (s, 1H),
12.32 (s, 1H); HRMS calcd for C18H21FN6 341.1890,
found 341.1876. Anal. RP-HPLC tR ＝ 8.1 min
[acetonitrile∶water (containing 0.1% formic acid)＝
35∶65; purity＝99.4%].
A mixture of sulfanilamide (1.56 g, 9 mmol), com-
pound 10 (0.6 g, 2.25 mmol), TFA (1.7 mL, 22.6 mmol)
and 2,2,2-trifluoroethanol (15 mL) was heated to reflux
for 15 h. After cooling to room temperature, solvent was
removed in vaccum and the residue was purified with
chromatography to afford the target compounds.
N6-(Cyclohexylmethyl)-N2-p-tolyl-9H-purine-2,6-dia-
mine (11e)
1
Yield 60%. m.p.＞300 ℃; H NMR (400 MHz,
General synthesis of N6-(cyclohexylmethyl)-N2-sub-
stitued phenyl-9H-purine-2,6-diamine (11b－11g)
DMSO-d6) δ: 0.96－1.02 (m, 2H), 1.13－1.21 (m, 3H),
1.63－1.78 (m, 6H), 2.25 (s, 3H), 3.36 (s, 2H), 7.07 (d,
J＝8 Hz, 2H), 7.62 (d, J＝8 Hz, 2H), 8.15 (s, 1H), 8.24
(s, 1H), 9.20 (s, 1H), 13.13 (s, 1H); HRMS calcd for
C19H24N6 337.2140, found 337.2139. Anal. RP-HPLC tR
＝9.3 min [acetonitrile∶water (containing 0.1% formic
acid)＝35∶65; purity＝100%].
To a stirred suspension of 10 (0.6 g, 2.25 mmol) in
n-butanol (15 mL), various substituted aniline (15.8
mmol) and TFA (0.1 mL) were added successively. The
reaction mixture was heated to reflux at 130 ℃ for 6 h.
After cooling to room temperature, the precipitate was
filtered and washed with CH2Cl2 to afford correspond-
ing compounds.
N6-(Cyclohexylmethyl)-N2-(4-methoxyphenyl)-9H-
purine-2,6-diamine (11f)
1
Yield 61%. m.p.＞300 ℃; H NMR (400 MHz,
4-(6-(Cyclohexylmethyl-amino)-9H-purine-2-yl-
amino)benzenesulfonamide (11a)
DMSO-d6) δ: 0.93－1.02 (m, 2H), 1.13－1.24 (m, 3H),
1.63－1.77 (m, 6H), 3.35 (s, 2H), 3.73 (s, 3H), 6.86 (d,
J＝8.8 Hz, 2H), 7.60 (d, J＝8.8 Hz, 2H), 8.08 (s, 1H),
8.17 (s, 1H), 9.06 (s, 1H), 13.01 (s, 1H); HRMS calcd
for C19H24N6O 353.2090, found 353.2078. Anal. RP-
HPLC tR＝17.7 min [acetonitrile∶water (containing
0.1% formic acid)＝28∶72; purity＝99.3%].
1
Yield 11%. m.p. 258 ℃ (decomposition point); H
NMR (400 MHz, DMSO-d6) δ: 1.00－1.05 (m, 2H),
1.18－1.23 (m, 3H), 1.64 (s, 1H), 1.70 (s, 3H), 1.79－
1.82 (m, 2H), 3.39 (s, 2H), 7.16 (s, 2H), 7.70 (d, J＝8.4
Hz, 2H), 7.96 (d, J＝8.4 Hz, 2H), 8.22 (s, 1H), 8.39 (s,
1H), 9.65 (s, 1H), 13.32 (s, 1H); HRMS calcd for
C18H23N7O2S 402.1712, found 402.1688. Anal. RP-
HPLC tR＝12.4 min [acetonitrile∶water (containing
0.1% formic acid)＝25∶75; purity＝99.2%].
N2-(4-Bromophenyl)-N6-(cyclohexylmethyl)-9H-pu-
rine-2,6-diamine (11g)
1
Yield 59%. m.p.＞300 ℃; H NMR (400 MHz,
N6-(Cyclohexylmethyl)-N2-phenyl-9H-purine-2,6-
DMSO-d6) δ: 1.00－1.03 (m, 2H), 1.16－1.21 (m, 3H),
1.63－1.79 (m, 6H), 3.36 (s, 2H), 7.42 (d, J＝8.8 Hz,
2H), 7.76 (d, J＝8.8 Hz, 2H), 8.20 (s, 1H), 8.32 (s, 1H),
9.41 (s, 1H), 13.35 (s, 1H); HRMS calcd for
C18H21BrN6 401.1089, found 401.1114. Anal. RP-HPLC
tR ＝ 45.7 min [methanol ∶ water (containing 0.1%
formic acid)＝60∶40; purity＝95.4%].
diamine (11b)
1
Yield 65%. m.p.＞300 ℃; H NMR (400 MHz,
DMSO-d6) δ: 0.95－1.03 (m, 2H), 1.13－1.21 (m, 3H),
1.63－1.80 (m, 6H), 3.36 (s, 2H), 6.94 (t, J＝7.2 Hz,
1H), 7.26 (t, J＝8 Hz, 2H), 7.75 (d, J＝8 Hz, 2H), 8.20
(s, 2H), 9.27 (s, 1H), 13.13 (s, 1H); HRMS calcd for
C18H22N6 323.1984, found 323.1987. Anal. RP-HPLC
tR ＝16.7 min [acetonitrile∶water (containing 0.1%
formic acid)＝30∶70; purity＝96.9%].
N6-(Cyclohexylmethyl)-N2-(4-(methylsulfonyl)phe-
nyl)-9H-purine-2,6-diamine (11h)
1
Yield 6%. m.p. 250－253 ℃; H NMR (400 MHz,
DMSO-d6) δ: 1.01－1.03 (m, 2H), 1.17－1.22 (m, 3H),
1.63－1.83 (m, 6H), 3.14 (s, 3H), 3.41 (s, 2H), 7.78 (d,
J＝8.8 Hz, 2H), 8.05 (d, J＝8.8 Hz, 2H), 8.34 (s, 1H),
8.41 (s, 1H), 9.81 (s, 1H), 13.16 (s, 1H); HRMS calcd
for C19H24N6O2S 401.1759, found 401.1762. Anal.
RP-HPLC tR＝6.5 min [acetonitrile∶water (containing
0.1% formic acid)＝35∶65; purity＝97.3%].
N6-(Cyclohexylmethyl)-N2-(4-hydroxyphenyl)-9H-
purine-2,6-diamine (11c)
1
Yield 51%. m.p. 285 ℃ (decomposition point); H
NMR (400 MHz, DMSO-d6) δ: 0.93－0.99 (m, 2H),
1.15－1.20 (m, 3H), 1.63－1.77 (m, 6H), 3.31 (s, 2H),
6.66 (d, J＝8.8 Hz, 2H), 7.52 (d, J＝8.8 Hz, 2H), 7.63
(br s, 1H), 7.84 (s, 1H), 8.60 (s, 1H), 8.90 (s, 1H), 12.48
(s, 1H); HRMS calcd for C18H22N6O 339.1933, found
339.1936. Anal. RP-HPLC tR＝6.6 min [acetonitrile∶
water (containing 0.1% formic acid)＝25∶75; purity＝
100%].
(2-Amino-6-chloro-9H-purine-9-yl)methylpivalate
(12)
6-Chloro-9H-purine-2-amine (5, 8.5 g, 50 mmol),
K2CO3 (20.7 g, 150 mmol) and chloromethyl pivalate
(Pom-Cl, 8.7 mL, 60 mmol) were disloved in 180 mL
DMSO and the mixture was reacted at room
temperature overnight. Then the mixture was diluted
with ethyl acetate and washed with brine, then dried
N6-(Cyclohexylmethyl)-N2-(4-fluorophenyl)-9H-
purine-2,6-diamine (11d)
1
Yield 65 %. m.p.＞300 ℃; H NMR (400 MHz,
1188
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© 2013 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2013, 31, 1181—1191

Design, Synthesis and Preliminary Biological Evaluation of Purine-2,6-diamine Derivatives
over MgSO4 and concentrated under reduced pressure.
The residue was purified with recrystallization from
ethanol to obtain target compound 12 (3.7 g, 68%). m.p.
THF, then 3 mol/L NaOH (0.3 mL, 0.9 mmol) was
added. After vigorous stirring for at least 1 h, the sol-
vent was removed under reduced pressure. Then 2
mol/L HCl was used to adjust pH＝4. The precipitate
was collected and washed with methanol to get target
compounds.
1
160－162 ℃; H NMR (300 MHz, CDCl3) δ: 1.18 (s,
9H), 5.18 (s, 2H), 6.00 (s, 2H), 8.01 (s, 1H); ESI-MS
m/z: 284.3 [M＋1]＋.
(2-Amino-6-((cyclohexylmethyl)amino)-9H-purine-9-
N-(6-((Cyclohexylmethyl)amino)-9H-purine-2-yl)-4-
yl)methyl pivalate (13)
fluorobenzenesulfonamide (15a)
1
A mixture of 12 (5.7 g, 20 mmol), aminomethyl-
cyclohexane (3.2 mL, 24 mmol) and TEA (4.2 mL, 30
mmol) in 50 mL EtOH was refluxed for 7 h. The reac-
tion was cooled down. The precipitate was collected and
washed with EtOH, then dried in vacuo to obtain 13 (4.9
g, 67%). m.p. 159－161 ℃; 1H NMR (600 MHz, CDCl3)
δ: 0.98－1.06 (m, 2H), 1.17 (s, 9H), 1.20－1.27 (m, 3H),
1.57－1.82 (m, 6H), 3.48 (br s, 2H), 4.81 (s, 2H), 5.75
(br s, 1H), 5.96 (s, 2H), 7.69 (s, 1H); ESI-MS m/z: 361.5
[M＋1]＋.
Yield 78%. m.p. 256 ℃ (decomposition point); H
NMR (600 MHz, DMSO-d6) δ: 0.75－0.77 (m, 2H),
1.11－1.16 (m, 3H), 1.46－1.63 (m, 6H), 3.03－3.05
(m, 2H), 7.26－7.45 (m, 2H), 7.74－8.14 (m, 4H),
11.17 (br s, 1H), 12.70 (br s, 1H); HRMS calcd for
C18H21FN6O2S 405.1509, found 405.1502. Anal.
RP-HPLC tR＝8.1 min [acetonitrile∶water (containing
0.1% formic acid)＝40∶60; purity＝98.7%].
N-(6-((Cyclohexylmethyl)amino)-9H-purine-2-yl)-4-
nitrobenzenesulfonamide (15b)
General synthesis of (6-((cyclohexylmethyl)amino)-
2-(substitued phenylsulfonamido)-9H-purine-9-yl)-
methyl pivalate (14a－14c)
1
Yield 85%. m.p. 246 ℃ (decomposition point); H
NMR (600 MHz, DMSO-d6) δ: 0.67－0.73 (m, 2H),
1.00－1.08 (m, 3H), 1.51－1.59 (m, 6H), 2.95 (br s,
2H), 8.08 (d, J＝7.8 Hz, 2H), 8.17－8.21 (m, 2H), 8.37
(d, J＝7.8 Hz, 2H); HRMS calcd for C18H21N7O4S
432.1454, found 432.1447. Anal. RP-HPLC tR＝6.4 min
[acetonitrile∶water (containing 0.1% formic acid)＝
40∶60; purity＝95.5%].
To the solution of 13 (0.36 g, 1 mmol) in 4 mL an-
hydrous pyridine, substituted benzenesulfonyl chloride
(1.5 mmol) was added. Then the mixture stayed for at
least 24 h till completion of the reaction. The mixture
was diluted with ethyl acetate and washed with 10%
citric acid, then dried over MgSO4, and then purified by
chromatography to get corresponding compounds.
N-(6-((Cyclohexylmethyl)amino)-9H-purine-2-yl)-4-
(trifluoromethoxy)benzenesulfonamide (15c)
(6-((Cyclohexylmethyl)amino)-2-(4-fluorophenylsul-
1
fonamido)-9H-purine-9-yl)methyl pivalate (14a)
Yield 82%. m.p. 261 ℃ (decomposition point); H
1
NMR (600 MHz, DMSO-d6) δ: 0.76－0.78 (m, 2H),
1.09－1.16 (m, 3H), 1.45－1.65 (m, 6H), 2.98－3.01
(m, 2H), 7.53 (d, J＝6.6 Hz, 2H), 7.79 (s, 1H), 7.99－
8.20 (m, 3H); HRMS calcd for C19H21F3N6O3S
471.1426, found 471.1425. Anal. RP-HPLC tR＝34.0
min [methanol∶water (containing 0.1% formic acid)＝
60∶40; purity＝96.2%].
Yield 71%. m.p. 182－183 ℃; H NMR (600 MHz,
CDCl3) δ: 0.92－0.98 (m, 2H), 1.17 (s, 9H), 1.19－1.31
(m, 3H), 1.66－1.75 (m, 6H), 3.28 (br s, 2H), 5.93－
6.03 (m, 3H), 7.17 (t, J＝8.4 Hz, 2H), 7.87 (s, 1H), 8.16
－8.18 (m, 2H); ESI-MS m/z: 519.4 [M＋1]＋.
(6-((Cyclohexylmethyl)amino)-2-(4-nitrophenylsul-
fonamido)-9H-purine-9-yl)methyl pivalate (14b)
1
Biological assay
Yield 45%. m.p. 168－169 ℃; H NMR (600 MHz,
CDCl3) δ: 0.93－1.00 (m, 2H), 1.16 (s, 9H), 1.21－1.30
(m, 3H), 1.69－1.78 (m, 6H), 3.28 (br s, 2H), 5.82 (br s,
1H), 6.00 (s, 2H), 8.02 (s, 1H), 8.35－8.38 (m, 4H);
ESI-MS m/z: 546.4 [M＋1]＋.
Biological activities of the target compounds include
two parts: enzyme inhibition assays and cell inhibition
assays.
CDK1/cyclin B enzyme inhibition assays
(6-((Cyclohexylmethyl)amino)-2-(4-(trifluorometho-
xy)phenylsulfonamido)-9H-purine-9-yl)methyl piva-
late (14c)
The IC50 values of tested compounds in inhibiting
CDK1/cyclin B enzyme activities were measured by a
homogeneous time-resolved fluorescence (HTRF) assay
in a 384-well format. The enzymatic reaction buffer
contained 250 mmol/L Hepes, 0.5 mmol/L Orthova-
nadate, 5 mmol/L MgCl2, 1 mmol/L DTT, 0.1% NaN3
and 0.05% BSA. HTRF detection buffer contained 50
mmol/L Hepes, 0.8 mol/L KF, 20 mmol/L EDTA and
0.1% BSA. ATP as follows: 10 μmol/L. For all test
compounds, 100 µmol/L was used as the starting point.
Three folds, ten serial dilutions, the concentrations
tested were from 100 to 5.08 nmol/L. Positive control
was subtracted and activities were expressed in % of the
1
Yield 38%. m.p. 126－127 ℃; H NMR (600 MHz,
CDCl3) δ: 0.95－0.98 (m, 2H), 1.17 (s, 9H), 1.22－1.31
(m, 3H), 1.68－1.74 (m, 6H), 3.29 (br s, 2H), 5.97 (s,
2H), 7.25 (br s, 1H), 7.33 (d, J＝8.4Hz, 2H), 7.88 (s,
1H), 8.22 (d, J＝8.4 Hz, 2H); ESI-MS m/z: 585.4 [M＋
1]＋.
General synthesis of N-(6-((cyclohexylmethyl)-
amino)-9H-purine-2-yl)-substituted benzenesulfona-
mide (15a－15c)
The material 14 (0.3 mmol) was dissloved in 3 mL
Chin. J. Chem. 2013, 31, 1181—1191
© 2013 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.cjc.wiley-vch.de
1189

Wang et al.
FULL PAPER
maximal activity, i.e., in the absence of inhibitors.
Negative controls experiments were performed with no
enzyme in the plate and the inhibition was 100%. The
reaction was started by the addition of 4 μL compound
to the reaction well first, then 2 μL CDK1 enzyme (8
ng/ well, Carna Biosciences) was added and incubated
with compound for 15 min at room temperature, later 2
μL substrate Rb (Ser780; Cell Signaling Technology, 1
mmol/L) biotinylated peptide and 2 μL ATP were added.
The reaction plate was incubated at room temperature
for 70 min. At last, Streptavidin-XL665 (0.0625 mol/L)
and Eu-anti-P-Rb (30 to 50 K cpm per well) (each 5 μL)
were added, the plate was sealed and incubated for 1 h
at room temperature. Finally, read the plate on an Infi-
nite® F500 instrument and do data analysis. The IC50
values were calculated from inhibition% which was de-
termined based on the ratio of readings at 665 nm and
normalized for Europium readings at 620 nm. Ratio was
calculated according to the equation: Ratio＝[665 nm
counts/620 nm counts]×10000. Then inhibition was
calculated by the equation based on ratio above: Inhibi-
tion %＝[(positive control ratio−compound ratio)/
(positive control ratio−negative control ratio)]×100.
and incubated in RPMI1640 medium containing 10%
FBS overnight at 37 ℃ in a 5% CO2 humidified incu-
bator. The cells were treated with compounds and
Roscovitine at final concentrations ranging from 1.5625
to 100 μmol/L, while control cells were treated with
equal volume DMSO. After 48 h treatment, 0.5% MTT
(Amresco, USA) solution was added to each well, and
further incubated for 4 h, then cells were centrifuged at
2500 r/min for 12 min, the culture medium was re-
moved, and 100 μL DMSO was added to dissolve the
formazan. After mixing for 2 min, optical density was
detected at 570 nm on a microplate reader (Thermo,
USA), and the IC50 values were calculated according to
the inhibition ratios.
Acknowledgement
This work was supported by the National Natural
Foundation Research Grant (No. 21172133), the Shan-
dong Provincial Natural Science Foundation (No.
ZR2010HM028), the Program for New Century Excel-
lent Talents in University (No. NCET-12-0337), Inde-
pendent Innovation Foundation of Shandong University
(IIFSDU, No. 2012JC003), and the Open Research
Fund of State Key Laboratory of Natural and Biomi-
metic Drugs in Peking University (No. K20100107).
CDK2/cyclin A enzyme inhibition assays
A Kinase-Glo Luminescent Kinase Assay was used
to test the IC50 values of the target compounds in inhib-
iting CDK2/cyclin A enzyme activities. Assay reagent
included: assay buffer (25 mmol/L HEPES, 10 mmol/L
MgCl2, 0.01% Triton X-100, 100 μg/mL BSA, 2.5
mmol/L DTT, pH7.4), kinase-glo reagent (Promega,
Cat#V6711), CDK2/Cyclin A2 (Signalchem, Cat#C22-
18G), substrate (Histone H derived peptide: HDB,
H09-19T) and ATP (Sigma, Cat#A7699) final concen-
tration 10 μmol/L.
For all test compounds, 20 µmol/L was used as the
starting point. Five folds, six serial dilutions, the con-
centrations tested were from 20 µmol/L to 6.4 nmol/L.
HPE (hundred percent effect): No compound, no en-
zyme, but containing ATP and substrate; ZPE (zero
percent effect): No compound, but containing ATP,
substrate and kinase. The reaction was started by the
addition of 1 μL compound to the reaction well first,
then 2 μL CDK2/cyclin A2 enzyme was added and in-
cubated with compound for 5 min at room temperature,
later 2 μL substrate and 5 μL ATP were added. The re-
action plate was incubated at 30 ℃ for 1 h. To the re-
action mixture, 10 μL of the appropriate kinase-glo re-
agent was added and incubated at 27 ℃ for 20 min.
Read the assay plate on Envision at last. The IC50 values
were calculated from inhibition%, which was calculated
according to the equation: Compound inhibition rate%
＝[(“compound reading”−ZPE)/(HPE− ZPE)]×100.
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