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

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,^aQuande Wang,^aLiangren Zhang,^band Hao Fang*^,a

^aDepartment of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University,

Jinan, Shandong 250012, China

^bState 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

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

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

TEA, n-butanol

N

H

Cl

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

H

Cl

NH

11a - 11h

N

NH₂

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

N

O

N

Cl

N

K₂CO₃, DMSO

F

N

O

K₂CO₃, DMSO

N

H

H₂N

N

5

H₂N

3

O

12

NH

N

NMP, amino acid, DBU, N₂

or amino alcohol, DMSO, DIEA, N₂

N

R

NH

arylsulfonyl chloride

pyridine

N

NH₂

TEA, EtOH

H

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

N

NH

N

HBF₄, NaNO₂

H₂O

benzylamine, TEA

N

R

O

n-butanol

N

R

O

N

H

H₂N

N

O

N

H

S

H₂N

N

S

N

5

N

O

H

6

O

H

O

14a - 14c

15a - 15c

NH

ArNH₂, TFA, n-butanol

NH

N

or ArNH₂, TFA, TFE

affinities to CDK1. Only compounds 4e and 4f have the

IC₅₀value about 5 μmol/L, but still are not good as

Roscovitine (Table 1).

N

R

N

H

F

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 R¹substitution, one is benzyl group

and the other is cyclohexylmethylene fragment. For the

target compounds with benzyl group in R¹position (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 R¹position, 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

R²

N

H

R³

Compd

R¹

R²

R³

Compd

R¹

R²

R³

H

−

1

−

1

IC₅₀^a/(μmol•L )

OH

SO₂NH₂

9.54

2.74

4a

8g

8h

O

S

OH

＞10

H

＞10

4b

4c

O

OCH₃

OH

8i

OH

SO₂NH₂

＞10

5.13

5.45

11a

11b

11c

H

0.35

＞10

1.05

4d

4e

4f

OH

F

OH

＞10

11d

11e

H

5.37

4g

4h

O

OH

CH₃

＞10

Br

OCH₃

Br

H

＞10

3.34

H

＞10

1.06

8a

8b

8c

8d

8e

8f

11f

11g

11h

15a

15b

15c

OCH₃

CH₃

O

S

O

S

＞10

F

O

OH

F

S

NO₂

OCF₃

O

S

＞10

O

(R,S)-Roscovitine

2.54

^aIC₅₀values 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

IC₅₀^a,b/(μmol•L⁻¹)

Compd

5.38±1.8 8g

Compd

IC₅₀^a,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

(R,S)-Roscovitine

^aIC₅₀values were the mean values of three repeated experiments. ^bIC₅₀values 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 IC₅₀^a,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

^aIC₅₀values were the mean values of three repeated experiments. ^bIC₅₀values represent the concentration which results in 50% decrease

in cell growth after 48 h incubation.

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Chin. J. Chem. 2013, 31, 1181—1191

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 N₂at 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 MgSO₄and 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 t_R＝ 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 t_R＝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

N₂at 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 MgSO₄and then purified by

column chromatography to afford the target compounds.

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C₁₈H₃₀N₆O₂: 363.2508, found 363.2536. Anal. RP-

HPLC t_R＝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 t_R＝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

t_R＝ 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 C₁₂H₁₂N₆241.1201, 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 t_R＝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 C₁₂H₁₀FN₅244.0998, 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 t_R＝ 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 C₁₈H₂₈N₆O₃377.2301, found

377.2319. Anal. RP-HPLC t_R＝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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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 t_R＝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 t_R＝ 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 t_R＝ 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 t_R＝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 C₁₉H₁₈N₆331.1671, found 331.1668.

Anal. RP-HPLC t_R＝ 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 NaHCO₃and 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 t_R＝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 C₁₈H₁₆N₆317.1514, found

317.1521. Anal. RP-HPLC t_R＝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 t_R＝ 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 t_R＝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

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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 C₁₈H₂₁FN₆341.1890,

found 341.1876. Anal. RP-HPLC t_R＝ 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

C₁₉H₂₄N₆337.2140, found 337.2139. Anal. RP-HPLC t_R

＝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 CH₂Cl₂to 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 t_R＝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 t_R＝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

C₁₈H₂₁BrN₆401.1089, found 401.1114. Anal. RP-HPLC

t_R＝ 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

C₁₈H₂₂N₆323.1984, found 323.1987. Anal. RP-HPLC

t_R＝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 t_R＝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 t_R＝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,

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Chin. J. Chem. 2013, 31, 1181—1191

Design, Synthesis and Preliminary Biological Evaluation of Purine-2,6-diamine Derivatives

over MgSO₄and 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 t_R＝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 t_R＝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 MgSO_4,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 t_R＝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 IC₅₀values 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

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% CO₂humidified 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 IC₅₀values 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 IC₅₀values 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 IC₅₀values

were calculated from inhibition%, which was calculated

according to the equation: Compound inhibition rate%

＝[(“compound reading”−ZPE)/(HPE− ZPE)]×100.

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1191

Article Doi

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

DOI: 10.1002/cjoc.201300420

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Article abstract of DOI:10.1002/cjoc.201300420

Full text of DOI:10.1002/cjoc.201300420

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