Synthesis and biological activities of C-2, N-9 substituted 6- benzylaminopurine derivatives as cyclin-dependent kinase inhibitor

Article abstract of DOI:10.1002/(SICI)1521-4184(19996)332:6<187::AID-ARDP187>3.0.CO;2-D

In this study, C-2, N-9 substituted 6-benzylaminopurine derivatives were synthesized and their inhibitory effects on cyclin-dependent kinase (CDK2) were evaluated. The effect of substituents at the C-2 and N-9 positions of substituted purine was investigated. Among the compounds tested, compound 7b- iii (6-benzylamino-2-thiomorpholinyl-9-isopropylpurine) was the most active inhibitor (IC₅₀ = 0.9 μM). Compound 7b-iii showed 10-fold higher activity compared to olomoucine and almost the same activity as roscovitine. Results from structure-activity relationship studies should allow the design of more potent and selective CDK inhibitors, which may provide an effective therapy for cancer or other CDK dependent diseases.

Full text of DOI:10.1002/(SICI)1521-4184(19996)332:6<187::AID-ARDP187>3.0.CO;2-D

Full Papers
Synthesis and Biological Activities of C-2, N-9 Substituted
6-Benzylaminopurine Derivatives as Cyclin-Dependent Kinase Inhibitor
a)
a)
a)
d)
b)
b)
c)
Chang-Hyun Oh , Su-Chul Lee , Ki-Soo Lee , Eun-Rhan Woo , Chang Yong Hong , Boem-Seok Yang , Dae Jin Baek ,
a)
and Jung-Hyuck Cho *
a) Medicinal Chemistry Research Center, Korea Institute of Science and Technology, Seoul 130-650, Korea
b) Biotech Research Institute, LG Chemical Ltd., Taejon 305-380, Korea
c) Dept. of Chem., Hanseo University, Seosan 352-820, Korea
d) College of Pharmacy, Chosun University, Kwangju 501-759, Korea
Key Words: CDK2 inhibitor; 6-benzylaminopurine; cell cycle regulation; antiproliferative effect
area of the ATP-binding pocket occupied by the ribose in the
CDK2/ATP complex.
Summary
Olomoucine, a purine derivative, displays quite a narrow
selectivity: among 35 kinases tested, it only inhibits cdc2 (cell
division cycle 2, CDK1), CDK2 and CDK5 and ERK-l (en-
doplasmic reticulum kinase-1) to a lesser extent . The po-
sition of olomoucine in the ATP binding pocket of CDK2 has
In this study, C-2, N-9 substituted 6-benzylaminopurine deriva-
tives were synthesized and their inhibitory effects on cyclin-de-
pendent kinase (CDK2) were evaluated. The effect of substituents
at the C-2 and N-9 positions of substituted purine was investigated.
Among the compounds tested, compound 7b-iii (6-benzylamino-
2-thiomorpholinyl-9-isopropylpurine) was the most active inhibi-
tor (IC50 = 0.9 µM). Compound 7b-iii showed 10-fold higher
activity compared to olomoucine and almost the same activity as
roscovitine. Results from structure-activity relationship studies
should allow the design of more potent and selective CDK inhibi-
tors, which may provide an effective therapy for cancer or other
CDK dependent diseases.
[8]
been determined by analysis of olomoucine/CDK2 co-crys-
[11]
tal
. Recently, olomoucine was found to stimulate massive
apoptosis in cells which have been arrested in G2 by the use
[12]
of DNA-damaging agents . A synthesis of olomoucine,
roscovitine, and structurally related compounds as well as
[13]
their SAR study was recently reported
. These encourag-
ing results led us to investigate other C-2, C-6, and N-9-sub-
stituted purines as potential CDK inhibitors.
Introduction
Most of the extracellular signals are amplified and trans-
duced inside cells by a cascade of various kinases. Protein
kinases are involved in essentially all intracellular regulatory
pathways. Cyclin-dependent kinase (CDK) is one of the
cellular kinases which control the cell division cycle (cdc).
Cyclin-dependent kinases constitute a family of highly con-
served protein kinases playing essential roles in cell cycle
We here report the synthesis of a new potent and selective
CDK2 inhibitor, 6-benzylamino-2-thiomorpholinyl-9-iso-
propylpurine (7b-iii). The structure-activity relationship of
the synthesized compounds as well as cytotoxicity using
human cell line is reported.
[1,2]
regulation
. The CDKs are activated through binding to a
family of regulatory proteins called cyclins and phosphoryla-
tion on a specific threonine residue. The crystal structures of
[3]
[4]
CDK2
and CDK2/cyclinA
have recently been deter-
mined, allowing a very precise understanding of the mecha-
nisms of enzyme activation and activity. Numerous examples
Chemistry
of CDK deregulation in human primary tumors and in tumor
[5,6]
cell lines have been described recently
. It has already
Adenine (1) is readily oxidized with hydrogen peroxide in
aqueous acetic acid to give adenine 1-N-oxide. The deamina-
tion of N-oxide (2) was carried out with sodium nitrite in
acetic acid. 2,6-Dichloropurine (4) was obtained by treating
3 with phosphoryl chloride in the presence of an organic base.
The 2,6-chloropurine derivative was reacted with benzyl-
amine in n-butanol at 100 °C to yield 6-benzylaminopurine
(5). N-9 Alkylation of the purine (5) was carried out withalkyl
halide in the presence of sodium hydride. The final compound
(7) was synthesized by the reaction of 6 with cyclic amino
been shown in animal models that different inhibitors of CDK
[7]
show antitumor activity . The search for potent inhibitors of
CDKs has led to the discovery of a family of C-2, C-6,
N-9-substituted purines with high CDK selectivity. The most
[8]
studied members of this family, olomoucine
scovitine
and ro-
[9,10]
[9,11]
, have been co-crystallised with CDK2
.
Both molecules interact with CDK2 at the ATP binding site.
Analysis of the CDK2/olomoucine and CDK2/roscovitine
crystal structures reveals that the C-2 substituents bind to an
Arch. Pharm. Pharm. Med. Chem.
© WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1999
0365-6233/99/0606/0187 $17.50 +.50/0

188
Cho and co-workers
alcohol (4-hydroxypiperidine, 2-hydroxymethylpyrrolidine, tried to replace the aliphatic alcoholic group on the C-2
etc.) and thiomorpholine in n-butanol at 120 °C.
position of purine by cyclic alcohol moieties such as 4-hy-
droxylpiperidine and 2-(hydroxymethyl)pyrrolidine. lt is in-
teresting that replacement of the hydroxy moiety at the C-2
position of the purine by a thiomorpholine led to highly potent
inhibition. It may be possible that lone pair electrons on the
sulfur atom in the thiomorpholine could be provided for the
hydrogen bonding to the residue of CDK2 enzyme.
Biological Evaluation
Enzymatic Activity
For CDK2/cyclin A enzyme inhibition studies, CDK2 and
cyclin A gene were subcloned into pBacPaK8 vector (Clon
Tech, USA) respectively. CDK2 gene was tagged with hexa-
histidine at the N-terminal for affinity purification of
CDK2/cyclin A complex. Active CDK2/cyclin A was puri-
fied using His-Resin(Novagen, USA) from sf21 insect cells
cotransfected with a 1:1 ratio of CDK2 and cyclin A bacu-
loviral stocks. Enzyme assays were done in 20 µ1 reaction
mixture containing 100 ng of purified CDK2/cyclin A en-
Cellular Activity
Cytotoxic activity of the anticancer drugs against human
cancer cell lines were investigated using the MTT assay.
Human lung (A549), human ovarian (SKOV-3), human
melanoma (SKMEL-2), human CNS (XF-498), and human
colon (HCT15) cancer cell lines were supplied from the
College of Medicine, Seoul National University. All cell lines
zyme, 4 µg of histone H1 as substrate, 100 µM ATP, 0.2 µCi
32
γ-P -ATP, 10 mM MgCl , 1 mM DPT in 50 mM Tris-HCI were grown in RPMI 1640 (Gibco BRL) supplemented with
2
(pH 7.5) buffer. The reaction was continued for 10 min at 10% (v/v) heat inactivated Fetal Bovine Serum (FBS) and
30 °C in the presence of inhibitor and stopped by adding 80 µl maintained at 37 °C in a humidified atmosphere with 5%
of 12% phosphoric acid. The stopped mixtures were trans- CO . MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
2
ferred to a 96-well PVDF filter (Millipore, USA) prewetted zolium bromide) was purchased from Sigma. The cells (3–
3
with 50% EtOH and drained with mild suction. Each well was 4 × 10 cells/well) were seeded into a 96-well plate. Various
washed five times with 10 mM Tris-HCl (pH 8.0) containing concentrations of samples were added to each well in dupli-
32
0.1 M NaCl to remove free γ-P -ATP. The filter was briefly cate, then incubated at 37 °C with 5% CO for three days such
2
dried and exposed to Phosphoimager (Molecular Dynamics, that cells are in the exponential phase of growth at the time
USA) to measure radioactivity.
of drug addition. Then 50 µl of 0.2% MTT solution was added
In this study, C-2, N-9 substituted 6-benzylaminopurine and incubated in the dark for 4 h at 37 °C. After 4 h incuba-
derivatives were synthesized and their structures and enzyme tion, the medium and MTT were removed from the wells, and
inhibition activities are listed in Table 1.
The effect of substituents at the C2 and N9 positions of the in 15 µl DMSO (dimethyl sulfoxide). The plates were then
purine was investigated. Among the compounds tested, com-
the water-insoluble MTT-formazan crystals were dissolved
vigorously shaken in order to ensure solubilization of the blue
pound 7b-iii was the most active inhibitor (IC = 0.9 µM). formazan. The optical density was measured using a micro-
50
Compound 7b-iii showed 10-fold increased activity com- plate reader (Vmax, Molecular Devices) with a 540 nm wave-
pared to olomoucine and almost the same activity as ro- length and the anticancer affective concentration was
[14–15]
scovitine. With regard to substituents at the N-9 position of expressed as an IC
.
50
purine, it was shown that the isopropyl derivatives are 4–10
As shown in Table 1, in general, compounds( 7a-iii, 7a-iv,
times more potent than their corresponding analogues, such 7b-iii, and 7b-iv) which were potent inhibitors of CDK, also
as methyl, allyl, and cyclopropylmethyl, respectively. We showed antiproliferative activity, whereas derivatives which
Arch. Pharm. Pharm. Med. Chem. 332, 187–190 (1999)

Substituted 6-Benzylaminopurines
189
Table 1. Inhibitory activity of 6-benzylamino-purine derivatives against CDK-2 and various human cancer cell lines (IC50, µM).
temperature. The precipitate was collected and washed with water to give 2
as a white solid. Yield 15.7 g (70.1%) mp >300 °C (dec.), mp (lit.)
300 °C(dec.)
were inactive at the enzyme level showed only marginal
antiproliferative activity. Compound 7b-iii was most inhibi-
tory to CDK activity, an effect which could be translated into
an antiproliferative effect.
Hypoxanthine 1-N-Oxide (3)
Adenine 1-N-oxide (7.19 g, 0.053 mmol) was suspended in a solution
containing 33.07 g of NaNO2 (0.48 mol) in 500 ml of water. The mixture
was cooled to 10 °C in an ice bath, and 300 ml of 30% aqueous acetic acid
was added dropwise with stirring over a period of 30 min. After the addition
of acid was complete, the solution was heated at 70–80 °C for 2 h, then cooled
to room temperature and allowed to stand for 4 days. The precipitate was
collected and washed with water, alcohol, and ether to afford 3 as a yellow
solid. Yield 5.3 g (66.2%), mp >300 °C(dec.) mp (lit.) 300 °C (dec.)
Experimental Part
Melting points (mp) were determined on a Thomas Hoover apparatus and
1
are uncorrected. H-NMR spectra: Varian Gemini 300 spectrometer, tetra-
methylsilane (TMS) as an internal standard. The mass spectrometer was a
Hewlett Packard Model HP5989A MS Engine (Palo Alto, CA, USA) spec-
trometer interfaced with a HP Model 59987A electrospray system.
Adenine 1-N-Oxide (2)
2,6-Dichloropurine (4)
Adenine (20.0 g, 0.15 mol) was suspended in 120 ml of acetic acid and
was refluxed for 1 h. After the solid was completely dissolved, the mixture
was cooled to room temperature. To this solution was added dropwise 74 ml
of 30% H2O2 and then the solution was allowed to stand for 3 days at room
Hypoxanthine 1-N-oxide (2.4 g, 0.016 mol) was suspended in a mixture of
120 ml of phosphoryl chloride and 4 ml of triethylainine and was refluxed
for 3 h under N2. After the mixture was cooled to room temperature, excess
phosphoryl chloride was distilled off under reduced pressure. The residue
Arch. Pharm. Pharm. Med. Chem. 332, 187–190 (1999)

190
Cho and co-workers
was dissolved in 100 ml of water, and was extracted with ethyl ether
(5 × 100 ml). The organic solvent was evaporated in vacuo to give a crude
oil, which was chromatographed on silica gel using ethyl acetate/n-hexane
(1:1) as an eluent to give 4 as a white solid. Yield 1.2 g (39.9%). mp
177–178°C, mp (lit.) 177 °C.
4.77(d, 2H, -NHCH2-), 6.39(bs, 1H, -NHCH2-), 7.22–7.44 (m, 6H, phenyl,
C8-H).– MS m/z 369 (M+ C19H25N6S).
7b-iv, mp 58–61 °C.– 1H-NMR(CDCl3);
δ (ppm) =1.53 (d,
6H,CH(CH3)2), 1.65 (m, 1H, -NCHCH2CH2), 1.89 (m, 2H, -NCHCH2CH2-),
2.14 (m, 1H, -NCHCH2CH2), 3.57–3.87 (m, 4H, -CH2NCHCH2OH), 4.28
(m, 1H, -CH2OH), 4.59 (m, 1H. CH(CH3)2), 4.79 (d, 2H, -CH2Ph), 6.12 (bs,
1H, -NHCH2-), 7.26–7.39 (m, 6H, phenyl), 7.47 (s, 1H, C8-H).– MS m/z 367
(M+ C20H27N6O).
6-Benzylamino-2-chloropurine (5)
7c-i, mp 130–133 °C.– 1H-NMR(CDCl3); δ (ppm) = 2.58 (m, 6H,
-CH2N(CH2)2-), 3.69 (t, 2H, -CH2OH), 3.85 (m, 4H, -N(CH2)2-), 4.66 (d,
2H, J = 8.8 Hz, -CHCH2-), 4.79 (d, 2H, J = 5.0 Hz, -NHCH2-), 5.19–5.29
(dd, 2H, -NCH2-), 5.87–6.08 (m, 2H, NHCH2-, -CH=CH2), 7.26–7.39 (m,
5H, Phenyl), 7.45 (s, 1H, C8-H).– MS m/z 394 (M* C21H28N7O).
7c-ii, mp 184–188 °C.– 1H-NMR (DMSO-d6); δ (ppm) = 1.23 (m, 2H,
-CH2CHCH2-) 1.69 (m, 2H, -CH2CHCH2-), 3.07 (m, 2H, -CHNCH2-), 3.63
(m, 1H, -CHOH), 4.26 (m, 2H, 4.61 (d, 4H, -CHCH2, -NHCH2-), 5.07–5.25
(dd, 2H, -NCH2-), 6.03(m, 1H, -CHCH2), 7.19–7.37 (m, 5H, phenyl), 7.70
(s, 1H, C8-H).– MS m/z 365 (M+ C20H25N6O).
Benzylamine (0.87 ml, 8.0 mmol) and 2,6-dichloropurine (0.5 g,
2.7 mmol) were dissolved in n-BuOH (15 ml) and the solution was heated at
100 °C for 5 h. The resulting solution was evaporated and diluted with ethyl
acetate. The organic layer was washed with water, 1N HCl, and brine.
Evaporation of the solvent in vacuo gave a crude solid, which was washed
with cold acetone to give 5 as a white solid. Yield 0.56 g (80.5%), mp
205–207 °C. 1H-NMR (DMSO-d6); δ (ppm) = 4.43(d, 2H, J = 6.3 Hz,
CH2Ph), 4.64 (br, 1H, -NHCH2-), 6.78 (bs, 1H, N9-H), 7.2-7.35 (m, 5H,
phenyl), 7.62 (s, 1H, C8-H).
1
6-Benzylamino-2-chloro-9-methylpurine (6a)
7d-i mp 135–138 °C.– H-NMR (CDCl3); δ (ppm) = 0.42 (m, 2H, -
CH2CH2-), 0.62 (m, 2H, -CH2CH2-), 1.26 (m, 1H, -CH(CH2)2), 2.63 (m, 6H,
-CH2N(CH2)2-), 3.71 (t, 2H, -CH2OH), 3.87 (m, 6H, -N(CH2)2-, CH(CH2)2),
4.79 (d, 2H, -NHCH2-), 6.03 (bs, 1H, NHCH2-), 7.25–7.38 (rn. 5H, phenyl),
7.54(s, 1H, C8-H).– MS m/z 408 (M+ C22H30N7O).
To a solution of NaH (0.3 g, 10.5 mmol) in dry DMF (100 ml) at 20 °C
was added slowly a solution of 5 (2 g, 8.1 mmol) in DMF under N2 gas. After
1 h methyl iodide (9.1 g, 32.0 mmol) was added dropwise and the reaction
mixture stirred for 24 h. The resulting mixture was diluted with sat. NH4Cl
(30 ml) and ethyl acetate(50 ml). The organic layer was washed with water
(3 × 50 ml), brine, and dried over anhydrous Na2SO4. Removal of the solvent
gave a crude residue, which was chromatographed on silica gel using ethyl
acetate as eluent to give 6a as a pale yellow solid. Yield 1.92 g (90.5%).
1H-NMR (DMSO-d6); δ (ppm) = 3.68 (s, 3H, -NCH3), 4.83 (d, 2H, J = 6.33
Hz, CH2Ph), 7.24–7.35 (m, 5H, phenyl), 8.13 (s, 1H, C8-H), 8.77 (bs, 1H,
-NHCH2).
7d-ii, mp 165–168 °C.– 1H-NMR (CDCl3), δ (ppm) = 0.42 (m, 2H, -
CH2CH2-) 0.63 (m, 2H, -CH2CH2-), 1.26 (m, 1H, -CH(CH2)2), 1.59 (m, 2H,
-CH2CHCH2-) 1.88 (m, 2H, -CH2CHCH2-), 3.76 (m, 2H, -CH2NCH2-) 3.88
(d, 2H, 1.14 Hz, -CH(CH2)2), 4.65 (d, 2H, -CH2NCH2-) 4.79 (d, 2H, -
CH2Ph), 5.13 (m, 1H, -CHOH), 5.83 (br, 1H, -NHCH2-) 7.26–7.36 (m, 5H,
phenyl), 7.55 (s, 1H, C8-H).– MS m/z 379 (M+ C21H27N6O).
References
6-Benzylamino-2-(N-(2-hydroxymethyl)pyrrolidinyl)-9-methylpurine (7a–
iv)
[1] L. Meijer, S. Guidet, M. Philippe, Progress in Cell Research, Plenum
Press, New York 1997, p. 3.
To a solution of compound 6a in n-butanol was added 2-(hydroxymethyl)-
pyrrolidine and the mixture was heated in an evacuated sealed tube at 155 °C
for 3–5 h. After cooling, the solution was diluted with water and ethyl acetate.
and the organic layer was washed with water and brine. Evaporation of the
solvent in vacuo gave a crude solid, which was recrystallized from MeOH/
benzene to give 7–iv as a white solid, 1H-NMR(CDC13); δ (ppm) =1.65 (m,
1H, pyrrolidine H), 1.89 (m, 2H, pyrrolidine H), 2.14 (m, 1H, pyrrolidine H),
3.58 (s, 3H, -NCH3), 3.60–3.88 (bs, 4H), 4.2l (m, lH, pyrrolidine H), 4.73 (d,
2H, J = 6.3 Hz, CH2Ph) 5.98 (bs, 1H, -NHCH2-) 7.26–7.45 (m, 6H, phenyl,
C8-H).– MS: m/z 338 (M+ C18H22N6O).
[2] W. G. Dunphy, Methods in Enzymology: Cell Cycle Control, Academic
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Compounds 7a-i–7d-ii were prepared by same procedure as described for
the preparation of 7a-iv.
[7] T. Meyer, U. Regenass, D. Fabbro, E. Alteri, J. Rosel. M. Muller, G.
7a-i, mp 165–167 °C.– 1H-NMR(CDCl3); δ (ppm) = 2.58 (m, 6H,
CH2HN(CH2)2-) 3.69 (q, 5H, -NCH3, -CH2OH), 3.81 (t, 4H, J = 5.0 Hz,
-N(CH2)2-), 4.79 (d, 2H, J = 5.2 Hz,-NHCH2-), 5.87 (bs, 1H, NHCH2-)
7.25–7.43 (m, 6H, -CH2Ph , C8-H).– MS m/z 367(M+ C19H25N7O).
7a-ii, mp 184–187 °C.– 1H-NMR (CDCl3); δ (ppm) = 1.5l (m, 2H),
1.93(m, 2H), 3.25 (m, 2H, -CH2NCH2-), 3.66 (s, 3H, -NCH3), 3.9l (m, 1H,
-CHOH), 4.46 (m, 4H, -CH Ph, -CH2NCH2-), 4.84 (bs, 1H, -NHCH2-),
7.26–7.4 (m, 5H, phenyl), 7.46 (s, 1H, C8-H).– MS: m/z 338 (M+
C18H22N6O).
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M. Inagaki, J.-G. Delcros, J.-P. Moulinoux, Eur. J. Biochem. 1997, 243,
527–536.
7a-iii 1H-NMR (CDCl3); δ (ppm) = 2.61 (d, 4H, -S (CH2)2-), 3.66 (s, 3H,
-NCH3), 4.13 (q, 4H, -N(CH2)2-), 4.77 (d, 2H, -NHCH2-), 6.39 (bs, 1H,
-NHCH2-), 7.22–7.44 (m, 6H, phenyl, C8-H).– MS m/z 340(M+ C17H20N6S).
7b-i, mp 104–107 °C.– 1H-NMR (CDCl3) δ (ppm) = 1.54 (d, 6H,
J = 7.0 Hz, 2CH3) 2.57 (m, 6H, -CH2N(CH2)2-), 3.66 (t, 2H, J = 5.3 Hz,
-CH2OH), 3.82 (t, 4H, -N(CH2)2-), 4.65 (m, 3H, -CH(CH3)2, -CH2Ph),
7.21–7.36 (m, 5H, phenyl), 8.28 (s, 1H, C8-H).– MS m/z 396 (M+
C21H30N7O).
[11] U. Schulze-Gahmen, J. Brandsen, H. D. Jones, D. O. Morgan., L.
Meijer, J. Veseley, S.-H. Kim, Proteins: Structure, Function and Ge-
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7b-ii, mp 184–188 °C.– 1H-NMR (CDCl3); δ (ppm) =1.5l (m, 8H, -
CH2CHCH2-, 2CH3), 1.92 (m, 2H, -CH2CHCH2-), 3.19 (m, 2H, -
CH2NCH2), 3.92 (m, 1H, -CHOH), 4.48 (m, 2H, -CH2NCH2-), 4.68 (m, 1H,
-CH(CH3)2), 4.78 (d, 2H, -CH2Ph), 5.96 (bs, 1H, -NHCH2-), 7.25–7.36 (m,
5H, phenvl) 7.48 (s, 1H. C8-H).– MS m/z 367 (M+ C20H27N6O).
7b-iii, mp 137–141 °C.– 1H NMR(CDCl3); δ (ppm) =1.54 (d, 6H, -2CH3),
2.6l (d, 4H, -S(CH2)2-), 4.13 (q,4H, -N(CH2)2-), 4.63 (m, 1H, -C-H(CH3)2)
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Received: November 6, 1998 [FP344]
Arch. Pharm. Pharm. Med. Chem. 332, 187–190 (1999)