Discovery of a novel class of 2-aminopyrimidines as CDK1 and CDK2 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.05.081

A series of new 2-(2-aminopyrimidin-4-yl)phenol derivatives were synthesized as potential antitumor compounds. Substitution with pyrrolidine-3,4-diol at the 4-position of phenol provided potent inhibitory activity against CDK1 and CDK2. X-ray crystal structural studies were performed to account for the effect of the substituent on both the enzymatic and cell growth inhibitory activities.

Full text of DOI:10.1016/j.bmcl.2011.05.081

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4203–4205
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of a novel class of 2-aminopyrimidines as CDK1 and CDK2 inhibitors
Jinho Lee a, , Kyoung-Hee Kim b, Shinwu Jeong c
⇑
a Department of Chemistry, Keimyung University, Daegu 704-701, South Korea
b LG Life Sciences, Science Town, Taejon 305-380, South Korea
c USC/Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, CA 90089, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new 2-(2-aminopyrimidin-4-yl)phenol derivatives were synthesized as potential antitumor
compounds. Substitution with pyrrolidine-3,4-diol at the 4-position of phenol provided potent inhibitory
activity against CDK1 and CDK2. X-ray crystal structural studies were performed to account for the effect
of the substituent on both the enzymatic and cell growth inhibitory activities.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 7 March 2011
Revised 17 May 2011
Accepted 23 May 2011
Available online 27 May 2011
Keywords:
CDK
2-Aminopyrimidine
Pyrrolidine-3,4-diol
Inhibitor
The cell division cycle is regulated by cyclin dependent kinases
(CDKs). CDKs are serine/threonine kinases that are regulated pre-
cisely by the binding of their regulatory subunits. CDKs are acti-
vated by cyclin binding and phosphorylation, and deactivated by
either removal of the cyclin or binding with CDK inhibitors (CDKIs)
such as Cip/Kip and INK families. Cell cycle deregulation is fre-
quently accompanied by anomalous CDK activity in numerous hu-
man cancers, caused by abnormally high expression of cyclin and/
or downregulation of CDKIs.1–3 This suggests that CDKs are attrac-
tive pharmacological targets for the treatment of cancer.4 The
majority of drug discovery efforts have focused on the invention
of ATP competitive inhibitors.5
inhibitory activity against both CDK1 and CDK2. With our finding
that pyrrolidine-3,4-diol had this effect, further modifications were
performed at the 6-position of the pyrimidine ring of 2-(2-amino-
pyrimidin-4-yl)phenol.
The synthetic route for the preparation of (3S,4S)-1-(3-(2-amino-
pyrimidin-4-yl)-4-hydroxyphenyl)pyrrolidine-3,4-diol is shown in
Scheme 1.
Table 1
CDK inhibitory activities of 2-(aminopyrimidin-4-yl)phenol derivativesa
2-Aminopyrimidines have been widely used as pharmaco-
phores for drug discovery. Among those compounds having potent
anticancer activity and CDK inhibitory activity,6–9 2-(2-aminopyr-
imidin-4-yl)phenols showed very potent enzyme inhibitory activ-
ity and anti-proliferative activity.8 As a part of our search for
CDK inhibitors, a 2-(2-aminopyrimidin-4-yl)phenol scaffold was
chosen and structural variations were performed. This report sum-
marizes the synthesis of these compounds and the effect of struc-
tural variations on both enzymatic and cellular activities.
First, the search for new substituents at the 4-position of phenol
was tried. Introduction of cyclic amines reduces the inhibitory
activity drastically against both CDK1 and CDK2 (Table 1). How-
ever, substitution of nitrogen with oxygen improved the potency
up to the submicromolar range (compounds 3 and 4). The intro-
duction of pyrrolidine-3,4-diol (compound 8) provided very strong
NH2
N
N
OH
R
b
Compds
R
IC50
CDK1
(
l
M)
CDK2
1
2
3
4
5
6
7
8
Piperidin-1-yl
>10
>10
>10
4-Ethylpiperazin-1-yl
Morpholino
1,1-Dioxidothiomorpholino
4-Oxopiperidin-1-yl
4-Hydroxypiperidin-1-yl
3-Hydroxypyrrolidin-1-yl
(3S,4S)-3,4-Dihydroxypyrrolidin-1-yl
7.94
2.24
7.5
0.79
3.98
>10
3.76
2.11
0.021
>10
10.0
2.99
0.015
a
The CDK inhibitory assays were performed as described in Ref. 10.
[ATP] = 100 M.
The values displayed correspond to the mean average of three experiments.
⇑
l
Corresponding author.
E-mail address: jinho@kmu.ac.kr (J. Lee).
b
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.081

4204
J. Lee et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4203–4205
Bn
Bn
O
O
O
O
O
O
OH O
O
O
O
R
O
O
O
(a)
(b)
N
(a)
(b)
R
HN
NH2
HN
O
HN
O
NH2
NH2
HN
NH2
NH2
OH N
N
NH2
N
Bn
O
N
N
OH N
N
(c)
(d)
OH N
(d)
(c)
R
R
N
HN
N
Boc
HN
O
HO OH
HO
OH
Scheme
1. Synthesis
of
(3S,4S)-1-(3-(2-aminopyrimidin-4-yl)-4-hydroxy-
phenyl)pyrrolidine-3,4-diol 8. Reagents and conditions: (a) (i) Ac2O, TEA, DCM,
89%; (ii) AlCl3, CS2, AcCl, CH3CN, 85%; (iii) BnBr, K2CO3, DMF, 98%; (b) DMFDEA, 98%;
(c) (i) Boc2O, DMAP, TEA, 77%; (ii) 2 N NaOH, 96%; (iii) NH2CNHNH2, NaOEt, 98%; (d)
(i) TFA, DCM, 80%; (ii) (2R,3R)-ICH2CH(OMOM)CH(OMOM)CH2I, TEA, n-BuOH, 44%;
(iii) BBr3, DCM, 89%.
Scheme 3. Synthesis of substituted (3S,4S)-1-(3-(2-aminopyrimidin-4-yl)-4-
hydroxyphenyl)pyrrolidine-3,4-diol. Reagents and conditions: (a) (i) AC2O, TEA,
DCM, 89%; (ii) AlCl3, CS2, AcCl, CH3CN, 85%; (iii) RCOCl, TEA, THF, 22–96%; (b) NaH,
HMPA, THF, 23–80%; (c) NH2CNHNH2ÁHCl, KOBu-t, t-BuOH, 27–49%; (d) (i) BnBr,
K2CO3, DMF, 88–98%; (ii) Boc2O, DMAP, TEA, then 2 N NaOH, 66–79%; (iii) TFA,
DCM, 95–98%; (iv) (2R,3R)-ICH2CH(OMOM)CH(OMOM)CH2I, i-Pr2NEt, n-BuOH, 21–
68%; (v) BBr3, DCM, 43–87%.
After an acetyl group was introduced through Fries rearrange-
ment, aminopyrimidine was synthesized via reaction with N,
N-dimethylformamide diethylacetal followed by reaction with
guanidine. Formation of pyrrolidine was performed with MOM-
protected 2,3-dihydroxy-1,4-diiodobutane, which was prepared
Table 2
Classical complement inhibition, cytotoxicity, and apoptosis assay results for
compounds 1–5 and 9–19a
R
IC50
(
l
M)
Cytotoxicityb
(
l
M)
from diethyl L
-tartarate.11 Modifications of the substituent at
CDK1
CDK2
HCT116
A549
EJ
the 6-position of 2-aminopyrmidine were performed via two
methods, as shown in Schemes 2 and 3. Sukuki coupling with
2-amino-4,6-dichloropyrmidine followed by reaction with corre-
sponding amines provided amine substituted compounds (17–
24) as shown in Scheme 2.
Alkyl groups were introduced at the 6-position of 2-aminopy-
rmidine, as shown in Scheme 3. The acylated 2-hydroxyacetophe-
none, upon treatment with sodium hydride in the presence of
HMPA, underwent the Baker–Venkataraman rearrangement to give
the diketone. Reaction with guanidine provided the 4,6-disubsti-
tuted 2-aminopyrimidine.
The inhibitory activity of the compounds was dependent on the
substituent at the 6-position of the 2-aminopyrimidine (Table 2).
Short alkyl chains were well tolerated, while long alkyl chains gave
about one order of magnitude lower potency against both CDK1
and CDK2 (9, 10 vs 12). Cycloalkyl groups also showed good po-
tency, except for cyclopropyl which showed a drastic drop in inhib-
itory activity (13 vs 14–16). The cycloheptyl group showed the
8
9
H
Me
Et
i-Pr
0.015
0.020
0.031
0.094
0.26
0.021
0.042
0.050
0.068
0.45
2.5
0.4
1.0
0.9
0.5
nd
5.5
2.0
3.0
1.8
0.7
nd
4.0
0.9
1.5
1.5
0.8
nd
0.9
1.2
0.7
>10
nd
nd
nd
nd
nd
4.5
2.0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
n-Hex
Cyclopropyl
Cyclopentyl
Cyclohexyl
Cycloheptyl
3-Piperidinyl
4-Piperidinyl
Dimethylamino
Morpholino
4-Methylpiperazin-1-yl
2-Morpholinoethylamino
Cyclopentylamino
Cyclohexylamino
>10
>10
0.023
0.045
0.031
0.22
0.34
0.28
1.0
0.42
0.79
0.019
0.020
0.042
0.052
0.090
0.36
0.20
0.25
0.94
0.30
0.45
0.015
0.060
0.4
0.8
0.3
>10
>10
>10
>10
>10
>10
3.0
0.9
2.0
1.2
0.6
>10
nd
nd
nd
nd
nd
7.0
1.5
a
The values displayed correspond to the mean average of three experiments
(nd = not determined)
b
Exponentially growing cells were treated with test compounds at various
concentrations for 48 h, and then the cell numbers were measured. The compound
concentration with 50% growth inhibition activity was determined.12
Bn
Bn
O
N
O
N
OH
B
OH
Bn
Br
O
most potent anti-proliferative activity against three cancer cell
lines. Introduction of amino substituents reduced the inhibitory
activity about 10-fold (17–22). However, amino groups substituted
with cycloalkyl groups restored the activity, making it similar to
that seen for alkyl groups (23, 24).
OH
(a)
(b)
(c)
Br
HN
O
NH2
MOM O O MOM MOM O O MOM
X-ray structures showed that the substitutions at the 6-position
of the 2-aminopyrimidine ring cause the compounds to rotate in
the ATP-binding pocket and the extents of rotation are dependent
on the nature of the substitutions (Fig. 1).13
NH2
NH2
N
Bn
O
N
N
N
OH N
R1
R
(d)
(e)
N
The substitutions at the 6-position of the pyrimidine are located
in a pocket formed by the side chains of Gln85, Asp86 and Lys89.
The size of this pocket limits the size of the substituent, as ob-
served in the structure–activity relationship. Compound 15, with
its bulky cyclohexyl group rotates further toward the hinge region,
compared to compound 11 with the isopropyl group. Despite these
significant movements, the 2-aminopyrimidine core continues to
hydrogen bond with the hinge region. Another interesting feature
is the orientation of pyrrolidine-3,4-diol. Without the substituent
at the 6-position, it is in the same plane as the phenol ring. How-
ever, in the presence of the substituent, the pyrrolidine-3,4-diol
MOM O
O MOM
HO OH
R1 : CH3
Cl
9
17-24
Scheme 2. Synthesis of substituted (3S,4S)-1-(3-(2-aminopyrimidin-4-yl)-4-
hydroxyphenyl)pyrrolidine-3,4-diol. Reagents and conditions: (a) (i) Br2, AcOH,
89%; (ii) BnBr, K2CO3, 99%; (iii) Boc2O, DMAP, TEA, 95%; (iv) 2 N NaOH, 93%; (v) TFA,
DCM, 93%; (b) (2R,3R)-ICH2CH(OMOM)CH(OMOM)CH2I, TEA, n-BuOH, 30%; (c) n-
BuLi, (CH3O)3B, THF, 24%; (d) Pd(PPh3)4, K2CO3, DMF, 2-amino-4,6-dichloropyrim-
idine, 40% or 4-chloro-6-methylpyrimidin-2-amine, 86%; (e) (i) R3R4NH, DMF, 13–
80% for 17–24; (ii) HBr, AcOH, 26–97%.

J. Lee et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4203–4205
4205
Education, Science and Technology (the Regional Core Research
Program/Anti-aging and Well-being Research Center)
References and notes
1. Sausville, E. A.; Zaharevitz, D.; Gussio, R.; Meijer, L.; Louarn-Leost, M.; Kunick,
C.; Schultz, R.; Lahusen, T.; Headlee, D.; Stinson, S.; Arbuck, S. G.; Senderowicz,
A. Pharmacol. Ther. 1999, 82, 285.
2. Schwartz, G. K. Cell Cycle (Georgetown, Tex) 2002, 1, 122.
3. Shapiro, G. I. J. Clin. Oncol. 2006, 24, 1770.
4. Malumbres, M.; Barbacid, M. Nat. Rev. Cancer 2009, 9, 153.
5. McInnes, C. Drug Discovery Today 2008, 13, 875.
6. Xie, F.; Zhao, H.; Zhao, L.; Lou, L.; Hu, Y. Bioorg. Med. Chem. Lett. 2009, 19, 275.
7. Jones, C. D.; Andrews, D. M.; Barker, A. J.; Blades, K.; Byth, K. F.; Finlay, M. R. V.;
Geh, C.; Green, C. P.; Johannsen, M.; Walker, M.; Weir, H. M. Bioorg. Med. Chem.
Lett. 2008, 18, 6486.
8. Seong, Y. S.; Min, C.; Li, L.; Yang, J. Y.; Kim, S. Y.; Cao, X.; Kim, K.; Yuspa, S. H.;
Chung, H. H.; Lee, K. S. Cancer Res. 2003, 63, 7384.
9. Vilchis-Reyes, M. A.; Zentella, A.; Martínez-Urbina, M. A.; Guzmán, A.; Vargas,
O.; Apan, M. T. R.; Gallegos, J. L. V.; Díaz, E. Eur. J. Med. Chem. 2010, 45, 379.
10. Kitagawa, M.; Higashi, H.; Takahashi, I. S.; Okabe, T.; Ogino, H.; Taya, Y.;
Hishimura, S.; Okuyama, A. Oncogene 1994, 9, 2549.
11. Procedure for the synthesis of (3S,4S)-pyrrolidine-3,4-diol:
Figure 1. Comparison of the X-ray structures of compounds 8 (purple), 11 (green)
and 15 (grey) in the ATP pocket of CDK2.
A
mixture of diethyl L-tartarate (50.0 g, 0.242 mol), MOMCl (46.0 ml,
0.605 mol), and DIPEA (127.0 mL, 0.726 mol) in DCM was stirred for 10 h at
rt. The solvent was removed under reduced pressure, and the remaining
residue was dissolved in EA. The organic layer was washed with water and
brine, dried over MgSO4, filtered, and concentrated under reduced pressure.
The residue was purified by column chromatography on silica gel using Hex/EA
(1:1, v/v) as eluent to give (2R,3R)-diethyl 2,3-bis(methoxymethoxy)succinate
in a yield of 99%. Conventional reduction of ester using LiAlH4 provided (2S,3S)-
is rotated about 90° to make it perpendicular to the plane of the
phenol ring. In both orientations, the pyrrolidine-3,4-diol makes
similar interactions with Lys33 and Asp145.
2,3-bis(methoxymethoxy)butane-1,4-diol in
Bis(methoxymethoxy)butane-1,4-diol (50.0 g, 0.237 mol) in 200 mL THF was
added by dropwise to mixture of PPh3 (137 g, 0.522 mol), I2 (133 g,
a
yield of 98%. 2,3-
The C log P value of the compound can be one factor that affects
both enzymatic and cell growth inhibition.14–16 Compounds having
a low inhibitory activity against both enzymes and cells are substi-
tuted with piperidine, piperazine, and morpholine groups which
provide low C log P values (À0.12, À0.12, 0.03 and 0.44 for 20,
18, 17 and 21, respectively).17 Introduction of n-hexyl and cyclo-
heptyl groups increased the C log P values to 2.8 and provided im-
proved cell growth inhibition. The result that compound 12
showed better cellular activity than 9 and 10 even though 12
was one order of magnitude less potent for both CDK1 and CDK2
supports this speculation. Compounds having short alkyl chains
did not show correlation between enzymatic and cellular inhibi-
tory activity. It can be partially accounted by the offsetting effect
of the binding mode alteration and the lipophilicity change.
In summary, a series of new 2-(2-aminopyrimidin-4-yl)phenol
derivatives were synthesized as potential antitumor compounds.
Pyrrolidine-3,4-diol was found as a new substituent for 2-(2-
aminopyrimidin-4-yl)phenol scaffolds. A series of (3S,4S)-1-(3-(2-
aminopyrimidin-4-yl)-4-hydroxyphenyl)pyrrolidine-3,4-diols was
synthesized and found to be decent CDK1 and CDK2 inhibitors.
a
0.522 mol), and imidazole (72 g, 1.0 mol) in 1.3 L THF. After stirring for 1 h at
rt, the solvent was removed under reduced pressure, and the remaining
residue was dissolved in EA. The organic layer was washed with water and
brine, dried over MgSO4, filtered, and concentrated under reduced pressure.
The residue was purified by column chromatography on silica gel using Hex/EA
(1:1, v/v) as eluent to give (5R,6R)-5,6-bis(iodomethyl)-2,4,7,9-tetraoxadecane
in a yield of 45%. 1H NMR(400 MHz, CDCl3) d (ppm) 3.30 (2H, m), 3.38 (2H, m),
3.43 (6H, s), 3.99 (2H, m), 4.75 (4H, m); ESI MS(m/e) = 430 [M+1].
12. Shoemaker, R. H. Nat. Rev. Cancer 2006, 6, 813.
13. The CDK2 protein was produced and purified following the protocol in the
published paper (Rosenblatt et al. J. Mol. Biol. 1993, 230, 1317). The apo-protein
crystals were grown at 4 °C by the hanging drop vapor diffusion method
against 0.2 M Hepes, pH 7.4. Crystals were soaked in a solution containing
0.5 mM of the compounds for more than 24 h and transferred to the
cryoprotectant solution (0.2 M Hepes, pH 7.4, 35% ethylene glycol). X-ray
data were collected with a MacScience DIP2030 imaging plate area detector
and were processed using the DENZO/SCALEPACK program. Crystals were diffracted
to the 2.3 Å resolution and the binding of the compound was clearly visible in
the electron density map. The structure was refined using the CNX program. The
coordinate of CDK2 and the compound 11 complex has been deposited in the
Protein Data Bank (PDB ID 3S2P).
14. Benites, J.; Valderrama, J.; Taper, H.; Buc Calderon, P. Invest. New Drugs 2010, 1.
15. Hollósy, F.; Seprödi, J.; Örfi, L.; Erös, D.; Kéri, G.; Idei, M. J. Chromatogr., B 2002,
780, 355.
16. Hudgins, W. R.; Shack, S.; Myers, C. E.; Samid, D. Biochem. Pharmacol. 1995, 50,
1273.
Acknowledgments
17. Determined by CSChem software.
This research was supported by a Bisa Research Grant of
Keimyung University and a grant from the Korean Ministry of