Structure-based design of 2-arylamino-4-cyclohexylmethyl-5-nitroso-6-aminopyrimidine inhibitors of cyclin-dependent kinases 1 and 2

Article abstract of DOI:10.1016/S0960-894X(03)00651-6

A series of O⁴-cyclohexylmethyl-5-nitroso-6-aminopyrimidines bearing 2-arylamino substituents was synthesised and evaluated for CDK1 and CDK2 inhibitory activity. Consistent with analogous studies with O ⁶-cyclohexylmethylpurines, 2-

Full text of DOI:10.1016/S0960-894X(03)00651-6

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3079–3082
Structure-Based Design of 2-Arylamino-4-cyclohexylmethyl-5-
nitroso-6-aminopyrimidine Inhibitors of
Cyclin-Dependent Kinases 1 and 2
Kerry L. Sayle,a Johanne Bentley,b F. Thomas Boyle,c A. Hilary Calvert,b Yuzhu Cheng,b
Nicola J. Curtin,b Jane A. Endicott,d Bernard T. Golding,a Ian R. Hardcastle,a
Philip Jewsbury,c Veronique Mesguiche,a David R. Newell,b Martin E. M. Noble,d
Rachel J. Parsons,a David J. Pratt,d Lan Z. Wangb and Roger J. Griffina,*
aNorthern Institute for Cancer Research, School of Natural Sciences-Chemistry, Bedson Building, University of Newcastle,
Newcastle Upon Tyne NE17RU, UK
bNorthern Institute for Cancer Research, Medical School, Framlington Place, Newcastle upon Tyne NE2 4HH, UK
cAstraZeneca Pharmaceuticals, Alderley Park, Cheshire SK10 4TG, UK
dLaboratory of Molecular Biophysics and Department of Biochemistry, University of Oxford, Oxford OX13QU, UK
Received 3 April 2003; accepted 11 May 2003
Abstract—A series of O4-cyclohexylmethyl-5-nitroso-6-aminopyrimidines bearing 2-arylamino substituents was synthesised and
evaluated for CDK1 and CDK2 inhibitory activity. Consistent with analogous studies with O6-cyclohexylmethylpurines, 2-aryla-
minopyrimidines with a sulfonamide or carboxamide group at the 40-position were potent inhibitors, with IC50 values against
CDK2 of 1.1ꢀ0.3 and 34ꢀ8 nM, respectively. The crystal structure of the 40-carboxamide derivative, in complex with phospho-
Thr160 CDK2/cyclin A, confirmed the expected binding mode of the inhibitor, and revealed an additional interaction between the
carboxamide function and an aspartate residue.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
tional cytotoxic treatments. However, currently avail-
able inhibitors are sub-optimal with regard to one or
more of the following properties: selectivity for CDKs
over other kinases, specificity for individual CDKs, and
potency against tumour cells both in vitro and in
vivo.5ꢁ8 The challenge in the area of CDK inhibitor
development is thus to identify agents with improved
kinase-specificity, coupled with greater potency against
tumour cell lines. We have previously reported the
purine NU2058 (1) as an ATP-competitive CDK inhi-
bitor that exhibits reasonable inhibitory activity against
CDK1 (Ki=5 mM) and CDK2 (Ki=12 mM), but which
is essentially inactive against CDK4.9 Elucidation of the
crystal structure of 1 in complex with both monomeric
CDK2, and fully active CDK2-cyclin A, revealed a
binding orientation distinct from that of ATP and the
benchmark 6-aminopurine CDK inhibitors, including
roscovitine and purvalanol. Structure–activity relation-
ship (SAR) studies conducted with 1 have demonstrated
that a six-membered alicyclic group at the purine O6-
position affords maximum inhibitory potency, whereas
The cyclin-dependent kinase (CDK) family of enzymes
play a pivotal role in the control of cell-cycle progres-
sion, particularly at cell-cycle checkpoints. Aberrant cell
cycle control, arising from tumour suppressor gene
malfunction or oncogene activation, is associated with
increased CDK/cyclin activity in human tumours.1,2
CDKs are thus recognised as important therapeutic
targets in the treatment of cancer and non-neoplastic
proliferative diseases, and inhibitors of CDKs have
recently entered clinical trials as prospective antitumour
agents.3,4 Studies to date with small molecule CDK
inhibitors, which invariably target the ATP-binding
domain of the enzymes, have demonstrated single-agent
antitumour activity in preclinical tumour models, as
well as synergistic activity in combination with conven-
*Corresponding author. Tel.: +44-191-222-8591; fax: +44-191-222-
8591; e-mail: r.j.griffin@ncl.ac.uk
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00651-6

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K. L. Sayle et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3079–3082
Scheme 1. General synthesis of N2-arylaminopyrimidines. Reagents and conditions: (i) n-BuBr, NaOH, EtOH, 50 ꢂC; (ii) C6H11CH2OH, DIAD,
PPh3, THF, 25 ꢂC; (iii) m-CPBA, DCM, 25 ꢂC; (iv) RC6H4NH2, TFE, TFA, reflux; (v) NaNO2, AcOH (30% aq), 80 ꢂC.
Figure 1. Structures of NU2058 (1), NU6027 (2) and NU6102 (3), indicating hydrogen bond interactions with the backbone of amino acid residues
within the ATP-binding site of CDK2.
substitution at the N-9 position essentially abolishes
activity.10 Interestingly, the purine core structure is not
a prerequisite for CDK inhibitory activity and compar-
able potency was observed with the corresponding
‘purine-mimetic’ nitrosopyrimidine derivative NU6027
(2; Ki=2.5 and 1.3 mM for CDK1 and CDK2, respec-
tively).9 The crystal structure of 2 in complex with
CDK2 showed that the binding mode of the pyrimidine
was nearly identical to that of the purine 1, both inhi-
bitors making a triplet of hydrogen bond interactions
within the ATP-binding site as represented in Figure 1.
Consistent with this observation, SARs for the O4-
position of 2 closely parallel those observed for the O6-
position of 1, with cyclohexylmethyl again proving
optimal.11
structure-based design approach enabled the eventual
identification of NU6102 (3), which proved 1000-fold
more potent than the parent O6-alkylguanine (Ki=9
and 6 nM for CDK1 and CDK2, respectively). Impor-
tantly, the increased potency of 3 could be fully ratio-
nalised on the basis of additional protein–ligand
interactions observed in the inhibitor–CDK2 complex,
most notably the formation of two additional hydrogen
bonds between the sulfonamide group and Asp 86 (Fig.
1).12 The close similarity of binding between 1 and 2,
and the remarkable increase in potency observed for 3
compared with 1, clearly offered the opportunity to
improve activity in the pyrimidine series through analo-
gous modifications at the pyrimidine 2-position. In this
communication, we report the results of these studies.
A comparison of the binding mode of 1 with that of the
6-aminopurine CDK inhibitors olomoucine and purva-
lanol A, suggested that an aryl substituent at the purine
2-position of 1 would make favourable interactions with
the ATP-binding domain and improve potency. This
Chemical Synthesis
The required N2-substituted pyrimidines were synthe-
sised from the 2-n-butylsulfonylpyrimidine (7), which

K. L. Sayle et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3079–3082
3081
2-arylaminopyrimidines 8 were prepared by reaction of
7 with the appropriate aniline in 2,2,2-trifluoroethanol
(TFE), in the presence of trifluoroacetic acid (TFA).
Final nitrosation at the pyrimidine C5-position pro-
ceeded smoothly on treatment with NaNO2 in AcOH,
to furnish the target pyrimidines 9 in good yield.13
Results and Discussion
A structure-based inhibitor design approach, utilising
the O6-alkylguanine NU2058 (1), has previously
enabled identification of the 2-arylamino derivative
NU6102 (3) as a nanomolar CDK1/CDK2 inhibitor.
SAR studies conducted on the 2-arylamino ring of 3,
indicate that substituents possessing dual hydrogen
bond donor-acceptor functions, for example SO2NH2
or CONH2, confer potent inhibitory activity,14 con-
sistent with additional interactions observed within the
ATP-binding site of inhibitor–CDK2 complexes.
Although NU6027 (2) has a very similar binding mode
to 1, the O4-alkylpyrimidine is a more potent inhibitor,
and preliminary results suggest that 2 may have
favourable cellular properties compared with 1. The
introduction of appropriately substituted 2-arylamino
groups onto the pyrimidine scaffold was investigated in
the expectation that such compounds would combine
nanomolar potency with enhanced cellular penetration.
Figure 2. (A) Crystal structure of compound 9d bound to phospho-
Thr160 CDK2/cyclin A. Phospho-CDK2 is shown in red and cyclinA
in green. Compound 9d is drawn in ball and stick mode with carbon,
nitrogen and oxygen atoms coloured green, blue and red, respectively;
(B) interaction of compound 9d with the active-site, showing hydro-
gen-bonds between the inhibitor and the protein.
Table 1. Inhibition of CDK1 and CDK2 by selected 2-arylamino-
pyrimidine derivatives9
The inhibitory activity of the 2-arylamino-5-
nitrosopyrimidines (9a–9e) against CDK1 and CDK2
was determined as described previously,9 and the results
are shown in Table 1. Pyrimidine intermediates (8a and
8b), which lack a 5-nitroso substituent, are included for
comparative purposes, together with NU6027 (2) and
the purines NU2058 (1) and NU6102 (3). In all cases,
the introduction of a substituted 2-arylamino group
onto the pyrimidine template improved potency against
both kinases compared with NU6027, activity varying
widely with the nature of the aryl substituent. Thus,
whereas a 3-bromophenyl group (9a) gave only a mod-
est improvement in activity, the introduction of
4-methoxyphenyl (9b) or 4-methylthiophenyl (9c) resul-
ted in an approximate ten-fold increase in potency
compared with 2. These results closely parallel those
observed for the 2-arylaminopurine series, where com-
parable increases in potency were observed relative to
the parent inhibitor 1 (results not shown).
No.
R1
R2
IC50 (mM)a
CDK1
CDK2
1 (NU2058)b
—
—
—
H
—
—
—
7.0ꢀ0.7c
2.9ꢀ0.1
17ꢀ2
2 (NU6027)b
2.2ꢀ0.6
3 (NU6102)d
0.0095ꢀ0.0013 0.0054ꢀ0.001
8a
8b
9a
9b
9c
9d
9e
4-CONH2
4-SO2NH2
3-Br
4-OMe
4-SMe
63ꢀ3
59ꢀ31
2.9ꢀ0.4
H
5.3ꢀ0.9
NO
NO
NO
0.8ꢀ0.1
0.5ꢀ0.2
0.23ꢀ0.023
0.22ꢀ0.03
0.07ꢀ0.005
0.22ꢀ0.05
0.12ꢀ0.03
0.034ꢀ0.008
NO 4-CONH2
NO 4-SO2NH2
0.005ꢀ0.0015 0.0011ꢀ0.0003
a12.5 mM ATP concentration.
bRef 9.
cData are meanꢀstandard deviation of three or more individual
experimental determinations.
dRef 12.
As predicted, and consistent with the results obtained in
the purine series, the introduction of a carboxamide (9d)
or sulfonamide (9e) group at the 4-position of the pyri-
midine 2-arylamino ring resulted in a dramatic increase
in activity over the parent nitrosopyrimidine 2. Indeed,
in keeping with the relative potencies of 1 and 2, the
sulfonamide derivative (9e) proved to be approximately
2-fold and 5-fold more active than the corresponding
purine (3) against CDK1 and CDK2, respectively.
Pyrimidines lacking a 5-nitroso function (8a and 8b)
were markedly less potent than their counterparts (9d
and 9e), with a 1000-fold reduction in activity being
observed for 8b compared with 9e. This is consistent
with previous SARs in the nitrosopyrimidine series,11
was prepared in a three-step procedure from commer-
cially available 4-amino-6-hydroxy-2-mercaptopyri-
midine 4 (Scheme 1). Reaction of 4 with n-butyl
bromide in EtOH–NaOH gave the alkyl sulfide 5 in
excellent yield, and subsequent introduction of the 4-
cyclohexylmethyl group was readily achieved under
Mitsunobu conditions, affording pyrimidine 6.
Attempts to effect direct displacement of the n-butyl
sulfide group of 6 with substituted anilines were unsat-
isfactory, giving only intractable mixtures. However, the
corresponding sulfone (7), prepared by oxidation of 6
with m-CPBA, was susceptible to nucleophilic attack at
the pyrimidine 2-position. Accordingly, the required

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K. L. Sayle et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3079–3082
and supports the putative role of the nitroso group in
constraining the 6-amino group of NU6027 into an
optimal binding orientation, by virtue of an
intramolecular hydrogen bond with the pyrimidine
6-amino group.9 Nevertheless, the 2-sulfanilylpyr-
imidine (8b) exhibited potency comparable with the
parent nitrosopyrimidine 2.
Acknowledgements
The authors thank Cancer Research UK, AstraZeneca,
the EPSRC (Studentship to K.L.S.), the BBSRC (Stu-
dentship to D.J.P.), and MRC for financial support. We
also wish to acknowledge the use of the EPSRC Che-
mical Database Service at Daresbury, and to thank the
beamline scientists at Elettra and at the ESRF for
excellent facilities during data collection.
The interaction of the 2-arylaminopyrimidines with
CDK2 was investigated by determining the crystal
structure of the carboxamide derivative (9d) bound to
˚
phospho-Thr160 CDK2/cyclin A complex at 2.6 A (Fig.
References and Notes
2).15 Compound 9d is bound in the ATP-binding site of
the kinase in the expected orientation for this series of
compounds. The pyrimidine moiety binds analogously
to the purine of NU6102, forming three hydrogen bonds
with the hinge region of the protein (rGlu81(CO)-N6=2.7
1. Sherr, C. J. Science 1996, 274, 1672.
2. Malumbres, M.; Barbacid, M. Nat. Rev. Cancer 2001, 1, 222.
3. Senderowicz, A. M.; Sausville, E. A. J. Natl. Cancer Inst.
2000, 92, 376.
4. Meijer, L.; Raymond, E. Acc. Chem. Res. 2003, 36, 417.
5. Sausville, E. A. Trends. Mol. Med. 2002, 8, S32.
6. Knockaert, M.; Greengard, P.; Meijer, L. Trends Pharm.
Sci. 2002, 23, 417.
7. Sielecki, T. M.; Boylan, J. F.; Benfield, P. A.; Trainor, G. L.
J. Med. Chem. 2000, 43, 1.
8. Hardcastle, I. R.; Golding, B. T.; Griffin, R. J. Annu. Rev.
Pharmacol. Toxicol. 2002, 42, 325.
9. Arris, C. E.; Boyle, F. T.; Calvert, A. H.; Curtin, N. J.; End-
icott, J. A.; Garman, E. F.; Gibson, A. E.; Golding, B. T.; Grant,
S.; Griffin, R. J.; Jewsbury, P.; Johnson, L. N.; Lawrie, A. M.;
Newell, D. R.; Noble, M. E. M.; Sausville, E. A.; Schultz, R.;
Yu, W. J. Med. Chem. 2000, 43, 2797.
10. Gibson, A. E.; Arris, C. E.; Bentley, J.; Boyle, F. T.;
Curtin, N. J.; Davies, T. G.; Endicott, J. A.; Golding, B. T.;
Grant, S.; Griffin, R. J.; Jewsbury, P.; Johnson, L. N.; Mes-
guiche, V.; Newell, D. R.; Noble, M. E. M.; Tucker, J. A.;
Whitfield, H. J. J. Med. Chem. 2002, 45, 3381.
11. Mesguiche, V.; Parsons, R. J.; Arris, C. E.; Bentley, J.;
Boyle, F. T.; Calvert, A. H.; Curtin, N. J.; Davies, T. G.; End-
icott, J. A.; Gibson, A. E.; Golding, B. T.; Griffin, R. J.; Jews-
bury, P.; Johnson, L. N.; Newell, D. R.; Noble, M. E. M.; Wang,
L. Z.; Hardcastle, I. R. Bioorg. Med. Chem. Lett. 2003, 13, 217.
12. Davies, T. G.; Bentley, J.; Arris, C. E.; Boyle, F. T.; Curtin,
N. J.; Endicott, J. A.; Gibson, A. E.; Golding, B. T.; Griffin, R. J.;
Hardcastle, I. R.; Jewsbury, P.; Johnson, L. N.; Mesguiche, V.;
Newell, D. R.; Noble, M. E. M.; Tucker, J. A.; Wang, L.; Whit-
field, H. J. Nat. Struct. Biol. 2002, 9, 745.
13. All new compounds exhibited spectral (1H NMR, IR, UV)
and analytical (elemental analysis and/or LC–MS) data fully
consistent with the assigned structures.
14. Hardcastle, I. R.; Arris, C. E.; Bentley, J.; Boyle, F. T,;
Calvert, A. H.; Curtin, N. J.; Davies, T. G.; Endicott, J. A.;
Gibson, A. E.; Golding, B. T.; Griffin, R. J.; Jewsbury, P.;
Johnson, L. N.; Menyerol, J.; Mesguiche, V.; Newell, D. R.;
Noble, M. E. M.; Tucker, J. A.; Wang, L.; Whitfield, H. J.;
Manuscript in preparation.
15. PDB code 10GU. Crystallographic R-factor=20%; free
R-factor=26%.
16. Fletcher, D. A.; McMeeking, R. F.; Parkin, D. J. Chem.
Inf. Comput. Sci. 1996, 36, 746.
˚
˚
˚
A; rLeu83(NH)-N1=3.5 A; rLeu83(CO)-N2=2.7 A). As
expected, the 5-nitroso group forms an intramolecular
hydrogen bond with N6 to stabilise the purine-mimetic
conformation. The N2-aryl substituent also binds simi-
larly to that of NU6102, packing against the peptide
backbone of CDK2 between residues His84 and Gln85.
The Cambridge Structural Database (CSD) for small
molecule crystal structures16 was consulted to determine
the preferred orientation of a carboxamide substituent
on a phenyl ring. The preferred torsion angle between
the plane of the ring and the plane of the carbox-
amide is 18ꢀ10ꢂ. This result was used as a restraint in
the refinement of the crystal structure. Possible inter-
actions close to the carboxamide include the Asp86
side chain and backbone nitrogen. Although electron
density cannot, at this resolution, unambiguously dis-
tinguish the carboxamide nitrogen from the carbox-
amide oxygen, the orientation can be inferred from the
surrounding protein groups. In the most chemically rea-
sonable conformation, the carboxamide nitrogen interacts
˚
with the Asp86 side chain with rN-Asp86(OD2)=3.3 A and
a torsion angle of 41ꢂ. Thus, the carboxamide function
of 9d mimics one of the two hydrogen bonding interac-
tions previously observed for the NU6102 sulfonamide
group.
In summary, an expedient route for the synthesis of 2-
arylamino-4-cyclohexylmethyl-5-nitrosopyrimidine CDK
inhibitors has been developed, and utilised for the pre-
paration of a small series of compounds. The results are
consistent with the known binding mode of NU6027
within the ATP-binding site of CDK2, and with SARs
observed for the analogous purine series. The crystal
structure of 9d in complex with CDK2–cyclinA, reveals
additional protein–ligand interactions compared with 2,
which account for the potent inhibitory activity of the
pyrimidine.