N2-substituted O6-cyclohexylmethylguanine derivatives: potent inhibitors of cyclin-dependent kinases 1 and 2.

Article abstract of DOI:10.1021/jm0311442

The adenosine 5'-triphosphate (ATP) competitive cyclin-dependent kinase inhibitor O(6)-cyclohexylmethylguanine (NU2058, 1) has been employed as the lead in a structure-based drug discovery program resulting in the discovery of the potent CDK1 and -2 inhibitor NU6102 (3, IC(50) = 9.5 nM and 5.4 nM vs CDK1/cyclinB and CDK2/cyclinA3, respectively). The SAR for this series have been explored further by the synthesis and evaluation of 45 N(2)-substituted analogues of NU2058. These studies have confirmed the requirement for the hydrogen bonding N(2)-NH group and the requirement for an aromatic N(2)-substituent to confer potency in the series. Additional potency is conferred by the presence of a group capable of donating a hydrogen bond at the 4'-position, for example, the 4'-hydroxy derivative (25, IC(50) = 94 nM and 69 nM vs CDK1/cyclinB and CDK2/cyclinA3, respectively), 4'-monomethylsulfonamide derivative (28, IC(50) = 9 nM and 7.0 nM vs CDK1/cyclinB and CDK2/cyclinA3, respectively), and 4'-carboxamide derivative (34, IC(50) = 67 nM and 64 nM vs CDK1/cyclinB and CDK2/cyclinA3, respectively). X-ray crystal structures have been obtained for key compounds and have been used to explain the observed trends in activity.

Full text of DOI:10.1021/jm0311442

3710
J . Med. Chem. 2004, 47, 3710-3722
Articles
N2-Su bstitu ted O6-Cycloh exylm eth ylgu a n in e Der iva tives: P oten t In h ibitor s of
Cyclin -Dep en d en t Kin a ses 1 a n d 2
Ian R. Hardcastle,†,* Christine E. Arris,‡ J ohanne Bentley,‡ F. Thomas Boyle,§ Yuzhu Chen,‡ Nicola J . Curtin,‡
J ane A. Endicott,∇ Ashleigh E. Gibson,† Bernard T. Golding,† Roger J . Griffin,† Philip J ewsbury,§
J erome Menyerol,† Veronique Mesguiche,† David R. Newell,‡ Martin E. M. Noble,∇ David J . Pratt,∇
Lan-Zhen Wang,‡ and Hayley J . Whitfield†
Northern Institute for Cancer Research, Bedson Building, School of Natural Sciences, University of Newcastle,
Newcastle upon Tyne, NE1 7RU, UK, Northern Institute for Cancer Research, Paul O’Gorman Building,
Medical School, Framlington Place, Newcastle Upon Tyne, NE2 4HH, UK, Laboratory of Molecular Biophysics and
Department of Biochemistry, University of Oxford, Oxford, OX1 3QU, UK, and AstraZeneca Pharmaceuticals,
Alderley Park, Cheshire, SK10 4TG, UK
Received December 16, 2003
The adenosine 5′-triphosphate (ATP) competitive cyclin-dependent kinase inhibitor O6-
cyclohexylmethylguanine (NU2058, 1) has been employed as the lead in a structure-based drug
discovery program resulting in the discovery of the potent CDK1 and -2 inhibitor NU6102 (3,
IC50 ) 9.5 nM and 5.4 nM vs CDK1/cyclinB and CDK2/cyclinA3, respectively). The SAR for
this series have been explored further by the synthesis and evaluation of 45 N2-substituted
analogues of NU2058. These studies have confirmed the requirement for the hydrogen bonding
N2-NH group and the requirement for an aromatic N2-substituent to confer potency in the
series. Additional potency is conferred by the presence of a group capable of donating a hydrogen
bond at the 4′-position, for example, the 4′-hydroxy derivative (25, IC50 ) 94 nM and 69 nM vs
CDK1/cyclinB and CDK2/cyclinA3, respectively), 4′-monomethylsulfonamide derivative (28, IC50
) 9 nM and 7.0 nM vs CDK1/cyclinB and CDK2/cyclinA3, respectively), and 4′-carboxamide
derivative (34, IC50 ) 67 nM and 64 nM vs CDK1/cyclinB and CDK2/cyclinA3, respectively).
X-ray crystal structures have been obtained for key compounds and have been used to explain
the observed trends in activity.
In tr od u ction
UCN01,10 flavonoids exemplified by flavopiridol,11,12 and
flavopiridol mimics such as 2-benzylidenebenzofuran-
3-ones.13 The 6-benzylamino- and 6-anilino-substituted
purines form a large class of inhibitors,14-19 a group that
includes olomoucine, roscovitine, and the purvalanols.
Other bicyclic nitrogen heterocycle-based inhibitors
have also been described, of which oxindoles,20,21 indoli-
nones,22 pyrrolopyrazines,23 pyridopyrimidines,24 and
quinazolines25,26 are well studied examples. Other no-
table inhibitors include pyrimidine-based compounds,27-30
as well as tricyclic and more complex molecules,31-39
some of which are natural products. First generation
CDK inhibitors have entered clinical trials,40 and it is
already apparent that significant complexities will be
encountered in the development of this drug class.41 In
particular, inhibitor specificity for CDKs over other
kinases and selectivity for a specific target CDK within
the CDK family of enzymes remain major challenges.
The cyclin-dependent kinases (CDKs) play an es-
sential role in regulating eukaryotic cell-cycle progres-
sion.1,2 Sequential activation of CDK4/6 and CDK2
controls progression from G1 into S phase, and activa-
tion of CDK1 is essential for progression from G2 into
M phase. Many cell-cycle regulatory mechanisms are
altered in tumors. For example, mutation, deletion, or
epigenetic silencing of the p16INK4a CDK inhibitor
protein, loss of p53 and hence reduced p21Cip1 expres-
sion, lowered expression of p27Kip1, overexpression of
cyclin D, and mutation of CDK4 have all been re-
ported.3-5 These alterations can result in the loss of
checkpoint control and so give rise to unregulated cell
growth. Additionally, the cell cycle machinery may be
overdriven in response to many oncogenic signaling
pathways.
A large number of ATP-competitive CDK inhibitors
from a variety of chemical classes have been identified.6-9
Examples include staurosporine derivatives including
We have previously reported that compounds based
on O6-cyclohexylmethylpurine (NU2058, 1) are competi-
tive inhibitors of both CDK1 and CDK2 with respect to
ATP, which also display good selectivity over CDK4.27
Analysis of the X-ray structure of 1 bound to monomeric
CDK227 revealed that 1 forms a triplet of hydrogen
bonds within the CDK2 ATP binding site. NH-9 acts as
a hydrogen bond donor to the backbone carbonyl group
* Corresponding author. UK. Tel: +44 191 222 6645; fax +44 191
222 8591; e-mail I.R.Hardcastle@ncl.ac.uk.
† School of Natural Sciences, Northern Institute for Cancer Re-
search.
‡ Medical School, Northern Institute for Cancer Research.
3 University of Oxford.
§ AstraZeneca Pharmaceuticals.
10.1021/jm0311442 CCC: $27.50 © 2004 American Chemical Society
Published on Web 06/10/2004

Inhibitors of Cyclin-Dependent Kinases 1 and 2
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 15 3711
Sch em e 1a
of Glu81, and N-3 and 2-NH2 accept and donate a
hydrogen bond to the backbone carbonyl and amide
groups, respectively, of Leu83. The O6-cyclohexyl-
methyl group of 1 occupies the CDK2 ribose-binding
pocket.27,42 Comparison of the structures of 1 and
olomoucine bound to monomeric CDK2 revealed that 1
represents a different class of inhibitor to olomoucine
as the two compounds bind in different orientations.27
Extending this observation, the X-ray structure of 1
bound to fully active Thr160 phosphorylated CDK2/
cyclinA (T160pCDK2/cyclin A) has also been deter-
mined.43 This structure has been used to guide the
design of more potent inhibitors that exploit the “speci-
ficity surface” of the CDK2 C-terminal domain beyond
the ATP binding cleft. Examples of this compound series
include the 2-anilinopurine derivative (NU6094, 2)
which displays IC50 values of 1.6 ( 0.1 and 0.97 ( 0.03
µM against CDK1/cyclin B1 and CDK2/cyclin A3, re-
spectively, and the 2-(4-sulfanilyl)purine (NU6102, 3)
which exhibits nanomolar potency against CDK1 and
CDK2 (IC50 values of 9.5 ( 0.1 and 5.4 ( 1.0 nM).43 The
greatly increased potency of 3 arises primarily from the
formation of two additional hydrogen bonds to Asp86
of CDK2, which facilitate optimum hydrophobic packing
of the anilino group with the specificity surface of CDK2.
Cellular studies with 3 demonstrated inhibition of
MCF-7 cell growth and target protein phosphorylation,
consistent with CDK1 and CDK2 inhibition.43
a
Reagents and conditions: (a) cyclohexylmethanol, sodium, 90
°C; (b) ethanolamine, DMF, 90 °C.
Sch em e 2a
a
Reagents and conditions: (a) HBF4, NaNO2, H2O, -12 °C; (b)
RNH2, EtOH, reflux.
Sch em e 3a
a
Reagents and conditions: (a) either ArNH2, glycerol, n-BuOH,
reflux; ArNH2, TFA, n-BuOH, reflux; ArNH2, TFA, DMSO, 60 °C;
or ArNH2, TFA, TFE, reflux.
Sch em e 4a
a
Reagents and conditions: (a) RNH2, n-BuOH, reflux.
of primary and secondary aliphatic amines and amino
alcohols in refluxing ethanol to give the N2-substituted
purines 7-14 in modest to poor yields.
Initial attemps to substitute the 2-fluoro group of 6
with anilines under the same conditions met with
failure (Scheme 3). However, the use of higher temper-
atures (120 °C; n-butanol) resulted in some reaction at
prolonged reaction times. Optimization of these condi-
tions resulted in the observation that the reaction was
assisted by the addition of trifluoroacetic acid (TFA).45
In many cases the poor solubility of the aniline was
problematic. To overcome this problem, 2,2,2-trifluoro-
ethanol (TFE) with TFA was employed as the solvent
resulting in a significant improvement in the reaction
yields. The use of TFE as solvent is considered to be
optimal for substitution reactions of this type.45
The thiophenemethylamino and hydrazinosulfanilyl
derivatives (43, 44, and 46) were prepared by reaction
of the corresponding amines with fluoropurine 6 in
n-butanol at 120 °C (Scheme 4). The 4-methoxyphenoxy
compound (45) was synthesized by heating 4-methoxy-
phenol with 6 in DMSO at 140 °C in the presence of
potassium fluoride on alumina and catalytic 18-crown-6
(Scheme 5).
The studies described in this paper elaborate struc-
ture-activity relationships within the 2-substituted O6-
cyclohexylmethylpurine series. Compounds were de-
signed with a knowledge of the details of the binding of
3 to T160pCDK2/cyclinA. In particular, the effect of
modulating the potential to form hydrogen bonds with
Asp86 was explored.
Ch em istr y
The N2-substituted 6-cyclohexylmethoxypurines used
in this study were prepared by following the routes
described in Schemes 1-4. As illustrated in Scheme 1,
compound 4 was prepared in good yield by the reaction
of 2,6-dichloropurine with sodium cyclohexylmethoxide.
Reaction of 4 with ethanolamine in DMF gave the
dimethylamino compound 5 in good yield.44
The key fluoro intermediate 6 was prepared from
2-amino-6-cyclohexylmethoxypurine (1) via a Balz-
Schiemann reaction employing fluoroboric acid, to give
the product in 44% yield (Scheme 2). The 2-fluoro group
of 6 was then substituted by reaction with a selection
Resu lts a n d Discu ssion
SAR for CDK1 a n d CDK2 In h ibition . Previous
studies in which 58 O6-substituted guanines were

3712 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 15
Hardcastle et al.
Sch em e 5a
derivative 5 retained activity against CDK2, as previ-
ously reported for the C2-unsubstituted purine.27 In
contrast, the N2-monosubstituted (methyl, 7; ethyl, 8;
isopropyl, 9; and 2-hydroxyethyl, 10) derivatives were
more potent as both CDK1 and CDK2 inhibitors than
the parent 1. This increase in potency was most marked
against CDK2 where the compounds were ca. 10-fold
more potent than 1. However, larger alkyl groups such
as R- or S-(1′-hydroxymethyl)propyl or R- or S-(1′-
hydroxymethyl-2′-methyl)propyl (11-14) were not toler-
ated. The latter compounds were essentially devoid of
kinase inhibitory activity at the maximum concentration
that could be tested, due to solubility constraints (10
µM). Together, these data indicate that the hydrogen
bond donated from the 2-NH to the backbone carbonyl
moiety of Leu83 confers activity, particularly against
CDK1, and that small hydrophobic substituents at N2
can increase activity against CDK1 and more dramati-
cally against CDK2.
a
Reagents and conditions: (a) 4-methoxyphenol, KF on alu-
mina, DMSO, 140 °C.
Ta ble 1. Inhibition of CDKs 1 and 2 by N2-Substituted
O6-Cyclohexymethylpurines
C2-An ilin o-O6-cycloh exylm eth ylp u r in es (Table 2).
As described previously,43 the 2-anilino compound 2
inhibited CDK1 and CDK2 with IC50 values of 1.6 (
0.1 and 0.97 ( 0.03 µM, respectively. The crystal
structure of 2 in complex with T160pCDK2/cyclin A343
revealed an extensive hydrophobic interaction between
the phenyl ring of the anilino substituent and the
hydrophobic surface of the enzyme. Taking 2 as a
starting point, the effect of substitution at the 3′- and
4′-positions on the aniline ring was investigated.
3′-Su bst it u t ed C2-a n ilin o-O6-cycloh exylm et h yl-
p u r in es. The X-ray structure of purvanalol B bound to
CDK214 suggests that a favorable interaction between
the inhibitor’s 3′-chloro group and the enzyme is formed.
The analogous anilino compound 15 was prepared but
its activity was not greater than that of the parent 2.43
Similarly, the 3′-fluoro (16), 3′-bromo (17), and 3′,5′-
dichloro (18) derivatives were no more active that 2,
with 15 being the most active of the compounds. The
3′-ethyl derivative 19 was again markedly less active
than 2. However, the 3′-hydroxymethyl derivative 20
was more potent than 2 against both CDK 1 and 2.
Given the poor activity of 19, the most persuasive
explanation for the enhanced activity of 20 is that it is
able to form a hydrogen bond with CDK2, presumably
at a site conserved in CDK1 and CDK2, and potentially
Asp86 as in the case of 3. Consistent with the need for
a hydrogen bond donor at the 3′-position for enhanced
activity, both the 3′-methoxyanilino and 3′-methyl-
mercaptoanilino compounds, 21 and 22, displayed simi-
lar activity to the 3′-chloro compound 15 and the parent
aniline 2.
4′-Su bstitu ted C2-An ilin o-O6-cycloh exylm eth yl-
p u r in es. As observed at the 3′-position, the introduction
of a 4′-fluoro substituent (23) reduced activity relative
to the parent aniline 2. 3′-Cl-4′-F-substitution (24)
improved activity over 23 but not to the level seen with
2. In contrast, 4′-hydroxy substitution (25) generated a
compound which was substantially more potent than
the parent aniline 2, with IC50 values of e100 nM for
both CDK1 and CDK2. As with 20, the result with 25
strongly suggests that there is a hydrogen bond acceptor
in the region of the protein accessed by the 2-anilino
group. Asp86, a residue that is conserved in CDK 1 and
CDK2, is an obvious candidate.
synthesized and evaluated identified the O6-cyclohexyl-
methoxy group as the optimal substituent for binding
in the ribose pocket of CDK2.42 Therefore this group was
retained in all the compounds described here.
Non a r om a t ic C2-Su b st it it u t ed -O6-cycloh exyl-
m eth ylp u r in es (Table 1). To confirm that the increased
potency of 2 and 3 was due in part to hydrophobic
packing of the anilino group with the specificity surface
of CDK2, compounds lacking an aromatic substituent
at the 2-position were prepared. In comparison to 1,
compounds lacking an N-H at the 2-position (4-6) were
less active against CDK1, although the dimethylamino

Inhibitors of Cyclin-Dependent Kinases 1 and 2
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 15 3713
Ta ble 2. Inhibition of CDKs 1 and 2 by N2-anilino-O6-cyclohexymethylguanines
The structure of the 25/T160pCDK2/cyclin A complex
confirmed this prediction and showed that the binding
mode of 25 to T160pCDK2/cyclin A is similar to that of
3 (Figure 1).43 The lengths of the hydrogen bonds
between the purine ring and CDK2 backbone residues
within the hinge region, and the values of the purine/
anilino twist angle and the anilino/backbone angle, are
all very similar for each inhibitor in the two complexes.
However, in the 25/T160pCDK2/cyclin A structure the
ø2 angle of Asp86 changes by 44° so that the carboxylate
moiety forms a weak interaction with the 4-hydroxy
group of the inhibitor (25 O23-Asp86 OD2 ) 3.3 Å).
The hydroxy group of 25 also interacts with Asp86 via
a water-mediated hydrogen bond (water molecules
included in the atomic coordinates file as the Z chain)
(25 O23-Z143-Asp86 OD2 ) 2.8 Å/3.0 Å, Figure 2).
To probe the requirement for a hydrogen bond donor
at the 4′-position, the 4′-methoxyanilino derivative 26
was synthesized, and, as predicted, this compound was
markedly less active than 25, showing approximately
10-fold less activity against both CDK1 and CDK2. The
corresponding 4′-methylmercaptoanilino derivative 27
was essentially inactive at 10 µM. The corresponding
3′-methoxyanilino and 3′-methylmercaptoanilino deriva-

3714 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 15
Hardcastle et al.
F igu r e 1. Binding of 25, 34, and 33 to T160pCDK2/cyclinA3. Selected CDK2 residues are labeled and drawn in ball-and-stick
representation. The final 2Fo - Fc density for each of the inhibitors is shown as a blue grid. Hydrogen bonds are indicated by
dashed lines.
more detail the contributions of these two hydrogen
bonds to activity against CDK1 and CDK2. The N-
monomethyl derivative 28 displayed similar activity to
3, whereas the N,N-dimethylsulfonamide (29) was an
order of magnitude less potent. These results strongly
suggest that a single sulfonamide N-H is sufficient for
optimal binding to the enzyme. The 4-methyl sulfone
derivative 30, which would also be unable to donate a
hydrogen bond to a side-chain oxygen of Asp86, dis-
played similar activity to 29, again consistent with the
hypothesis that the activity of 3 is due to the two
H-bonds formed with Asp86. The 4′-methyl sulfoxide 31
was less active again than 30, suggesting that in
addition to the loss of the NH hydrogen bond, the
remaining sulfonyl oxygen can no longer adopt an
optimal geometry for binding to the backbone NH of
Asp86. Lack of optimal H-bonding geometry presumably
also underlies the greatly reduced activities of 32 and
33 against both CDKs, relative to 3.
The structure of 15 in complex with T160pCDK2/
cyclin A revealed that both conformations which the
3-chlorophenyl group would be predicted to adopt, one
directed toward Asp86, the other directed toward His84,
were present in the complex, each with approximately
50% occupancy.43 However, the sulfonamide group of 33
bound to T160pCDK2/cyclin A is directed toward His84,
and there is no indication that the compound binds in
the alternative conformation with the sulfonamide
directed toward Asp86 (Figure 1). The increased bulk
of the sulfonamide moiety compared to that of the
chlorine atom probably accounts for the lack of this
second conformation. The sulfonamide group in 33 forms
only one direct hydrogen bond with the protein (33
N26-His84 CO ) 2.8 Å). However, three potential
water-mediated hydrogen bonds between 33 N26-Z66-
Gln85 NE2, 33 O24-Z13-Glu8 OE2, and 33 O24-Z13-
Lys20 NZ, of lengths 3.3 Å/3.2 Å, 2.9 Å/2.9, and 2.9 Å/2.8
Å, respectively, are also observed. Notably, CDK2 Glu8
has not been previously targeted in this series, and it
is one of the sequence positions at which CDK2 and
CDK4 differ; the equivalent residue in CDK4 is Ala10.
The conformation of Gln85 is also different to that
observed in all other structures for this series of
compounds bound to T160pCDK2/cyclin A determined
to date. The twist angle between the purine and anilino
rings of compound 33 is between that of 2 and 3 at 41°,
F igu r e 2. Hydrogen bonds between the inhibitors, CDK2, and
crystallographic waters. All distances are in angstroms.
tives (21 and 22) were equipotent but less active than
the 4′methoxy derivative 26.
As previously described for 3, the NH2 group of the
sulfonamide donates a hydrogen bond to a side-chain
oxygen of Asp86 (3 N26 to Asp86 OD2 ) 2.9 Å) and one
sulfonamide oxygen accepts a hydrogen bond from the
backbone nitrogen of Asp86 (3 O24 to Asp86 NH ) 3.1
Å). Hence compounds were synthesized to define in

Inhibitors of Cyclin-Dependent Kinases 1 and 2
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 15 3715
CDK inhibitory activity similar to that of 29 and 30,
suggesting that 34 was forming only a single hydrogen
bond with either CDK1 or CDK2.
The geometry of the 4′-carboxamide group precludes
the possibility of it making both the interactions ob-
served between Asp86 and the 4′-sulfonamide moiety
of 3, although the formation either one is possible. The
structure of benzamide 35, the pyrimidine analogue of
compound 34, bound to T160pCDK2/cyclin A has been
solved recently.47 The resolution of this latter structure
was not sufficient to determine unambiguously whether
the amide carbonyl was interacting with the backbone
nitrogen of Asp86, or alternatively if the amide nitrogen
was interacting with the carboxylate moiety of Asp86.
Taking into consideration the predicted hydrogen bond
lengths associated with each binding mode, compound
35 was modeled with the carboxamide moiety twisted
36° relative to the anilino ring so that a hydrogen bond
of 3.3 Å results between 35 N25 and Asp86 OD2.
F igu r e 3. The structure of the 4-sulfamoylaniline used as a
test compound to model sulfamoyl conformational energy.
and the angle between the anilino ring and plane of the
peptide backbone between His84 and Gln85 is 27°. This
observation suggests that the stacking interactions
between the anilino ring of 33 and CDK2 are more
favorable than those between 3 and CDK2.
To probe these results further, the preferred confor-
mation of a sulfamoyl group attached to an aniline ring
was analyzed. The Cambridge Structural Database
(CSD) contains only 20 unique entries for this substruc-
ture. To derive a more complete picture of the sulfamoyl
group conformational energy profile, quantum mechan-
ical calculations were carried out using 4-sulfamoyl-
aniline as a model compound for 3. An all atom model
was built that included hydrogen atoms (Figure 3) and
minimized with the 6-31G** basis set in Gaussian.44 All
distances, angles, and torsions were allowed to be
refined, except the C3-C4-S1-N2 torsion angle, which
was fixed at a range of values. The relative energies of
the various conformations, with respect to the C3-C4-
S1-N2 and C3-C4-S1-O1 torsion angles, are shown
in Figure 4. The overall minimized structure of 4′-
sulfamoylaniline with the 6-31G** basis set shows that
the preferred C3-C4-S1-O1 torsion angle is 8.6°.
Assuming that 4-sulfamoylaniline is a valid model for
3 and that 3-sulfamoyl groups show similar behavior
to 4-sulfamoyl groups, then the conformation of the 33
sulfamoyl group is energetically very favorable, with a
torsion angle C21-C20-S23-O24 of 3°.
The resolution of the structure of the 34/T160pCDK2/
cyclin A complex was slightly higher at 2.4 Å than that
of 35/T160pCDK2/cyclin A, but still does not provide
sufficient detail to make an unambiguous determina-
tion. The carboxamide in the 34/T160pCDK2/cyclin A
structure has been modeled in the same conformation
as the carboxamide of 35, with a hydrogen bond between
34 N25 and Asp86 OD2 of 3.1 Å and a twist of the
carboxamide of 35° relative to the anilino ring. The
mean twist angle of a carboxamide group bound to a
phenyl ring is 18° (std dev ) 10°; CSD), a result that
implies that the conformations of the carboxamide
groups in both the CDK2-bound 34 and 35 structures
lead to a slight energy penalty. The twist of the anilino
ring of 34 relative to the purine and to the peptide
backbone between His84 and Gln85 is very similar to
that of 3, while the purine ring is shifted slightly closer
to the hinge backbone. Finally, the carbonyl moiety of
34 forms a weak water-mediated hydrogen bond with
Glu8 (Figure 2) that is not seen in the 35/T160pCDK2/
cyclin A complex structure.
The above studies defined SARs for the anilino-
sulfonamide group in detail and provided a strong
structural rational for the results of the enzyme inhibi-
tion assays. Analogous studies were therefore under-
taken with 4′-carboxamide derivatives, the carboxamide
substituent being potentially capable of forming a
similar pattern of hydrogen bonds with CDK2 as the
sulfonamide 3. The 4′-carboxamide derivative 34 had
The N-monomethyl derivative 36 was less active than
the parent 34, and of similar potency to 37, the N,N-
dimethylcarboxamide, whereas the acetophenone de-
rivative 38 had equivalent activity to 37. Together these
data strongly suggest that the 4′-carboxamide deriva-
tives form only a single H-bond with CDK1 and CDK2,
F igu r e 4. The energy profile of the 4-sulfamoylaniline compound with respect to the C3-C4-S1-N2 and C3-C4-S1-O1 torsion
angles.

3716 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 15
Hardcastle et al.
Ta ble 3. Inhibition of CDKs 1 and 2 by
O6-Cyclohexymethylguanines with Non-anilino Aromatic and
Heterocyclic N2-Substituents
IC50 ) 600 nM), phospholipid dependent kinase 1
(PDK1, IC50 ) 800 nM), and dual-specificity tyrosine
phosphorylated and regulated kinase 1A (DYRK1A, IC50
) 900 nM) at an assay ATP concentration of 100 µM.
These results indicate that 3 has at least 10-fold
selectivity for the target kinases, CDK1 and -2.
Con clu sion s
The targeting of Asp86 in CDK2 by hydrogen bonding
groups has been observed in a number of other series
of CDK inhibitors. In the quinazoline series H-bonding
between a 3-anilino group and the side-chain carboxyl
group of Asp 86 was observed.26 Interestingly, the
oxindoles show a different pattern of activity to the
anilino-purines.20 In this case, the unsubstituted sul-
fonamide and mono- and disubstituted sulfonamides
were found to be equipotent while cyclic sulfone deriva-
tives suffered a small drop in activity, suggesting that
a single H-bond formed between an aryl sulfonamide
SdO to the backbone NH of Asp 86 was responsible for
the gain in potency observed. The Asp86 residue has
also been targeted in the flavopiridol mimicking benz-
ilidinebenzofuran-3-one series, where a 10-fold gain in
potency was observed on the introduction of a sulfon-
amide group at the position suggested by molecular
modeling.13
In general, the gains in potency observed for the
anilino-purine series described here results from favor-
able hydrophobic packing between the aromatic aniline
ring and an area outside the ATP binding site of the
CDK enzyme. Additional gains in potency are dependent
on the formation of hydrogen bonds between carbox-
amide and sulfonamide groups, and Asp86, resulting in
the identification of inhibitors with nanomolar potency.
No direct interactions with Lys89, the residue originally
targeted in the design of compound 3, have yet been
seen. However, interactions with other areas of the
protein that show sequence differences between CDK2
and CDK4 have been observed (e.g., 15 and 33), offering
further avenues to explore for gains in potency and
selectivity beyond the often-targeted Asp86/Lys89 pair.
In conclusion, we have identified highly potent CDK1
and CDK2 inhibitors based on a 6-cyclohexylmethoxy-
purine scaffold by the introduction of substituted 2-
anilino groups. Further studies, including investigation
of the cellular activies, broader kinase specificities, and
additional structure-activity relationships are ongoing.
and that this bond is donated by one of the NH
hydrogens, presumably to the side-chain oxygen of
Asp86.
Compared to the acetophenone derivative, the corre-
sponding benzoic acid 39 has 2-fold reduced activity,
while methylation of the benzoic acid to yield the methyl
ester 40 produced a further 10-fold decrease in potency.
The carboxamide homologue 41 was 2-fold less active
than the 4′-carboxamide 34, and the corresponding 4′-
cyanomethyl derivative 42 was 4-fold less active.
O6-Cycloh exylm eth ylp u r in es w ith Non -a n ilin o
Ar om a tic a n d Heter ocyclic C2 Su bstitu en ts (Table
3). As shown in Table 3, a small series of derivatives
were prepared in which the C2-anilino group was
replaced with an aralkyl or heteroaralkyl group. The
only compound of note was the thiophene derivative 43
that was of similar potency to the parent aniline 2.
However, the sulfonamide 44 was 1000-fold less potent
than 3, presumably because the sulfonamide group was
not correctly oriented toward Asp 86. The inactivity of
the phenoxy derivative 45 presumably reflects the
importance of the hydrogen bond donated by the N2-
amino group, and the poor activity of the hydrazine 46
again presumably reflects the importance of the interac-
tion with Leu83.
The data presented suggest that the 2-anilino sub-
stituent can have a modest effect on CDK2 versus CDK1
selectivity, for example, the sulfone 30 and 3-methoxy-
sulfonamide 32 display some selectivity for CDK2 over
CDK1. Further studies with human CDK1, as opposed
to the starfish CDK1 used in the current experiments,
have shown that 2-anilinoguanines are selective CDK2
inhibitors with, for example, 3 being 46-fold more active
against CDK2 than CDK1 (data not shown).
Exp er im en ta l Section
Melting points were obtained on a Stuart Scientific SMP3
apparatus and are uncorrected. Infrared spectra (IR) were
recorded as KBr disks on a Nicolet 20 PC Fourier Transform
spectrometer or neat on an Excalibur series BioRad Spectro-
photometer. Ultraviolet (UV) spectra were recorded in MeOH
or EtOH on a U-2001 Hitachi Spectrophotometer. LC-MS
analysis was conducted on a Waters/Micromass Platform LC
instrument, LC chromatography used a Waters Symmetry
column (50 × 4.6 mm) with a 10 min methanol/0.05% formic
acid gradient, UV detection was achieved with a Waters 996
detector scanning from 240 to 400 nM, and MS was measured
under electrospray mode in positive and negative ion mode.
Electron impact (EI) mode mass spectra were determined on
a Kratos MS80 spectrometer. Proton (1H) and carbon (13C)
nuclear magnetic resonance (NMR) spectra were recorded at
300 and 75.5 MHz, respectively, on a Bruker Avance 300
spectrometer, using the deuterated solvent as internal stand-
ard. Unless indicated otherwise, spectra were recorded in
To confirm the selectivity of 3, the compound has been
independently tested as an inhibitor of 28 kinases.43 In
addition to CDK2 (IC50 ) 30 nM), other kinases inhib-
ited by 3 were Rho-dependent protein kinase II (ROCKII,

Inhibitors of Cyclin-Dependent Kinases 1 and 2
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 15 3717
DMSO-d6 as solvent. NH signals appeared as broad singlets
(br s) exchangeable with D2O. Chemical shift values are quoted
in parts per million (ppm) and coupling constants (J ) in hertz
(Hz). Key: t ) triplet, s ) singlet, q ) quartet, d ) doublet,
dd ) double doublet, m ) multiplet. The TLC systems
employed Merck 1.05554 aluminum sheets precoated with
Kieselgel 60F254 (0.2 mm) as the adsorbent and were visualized
with UV light at 254 and 365 nm. Column chromatography
was conducted under medium pressure on silica (Kieselgel 60,
240-400 mesh). Elemental analyses were performed in house
appropriate solvent or column chromatography gave the
desired product.
Gen er a l P r oced u r e C. A mixture of 6, the appropriate
aniline (4 equiv) and trifluoroacetic acid (10 equiv) and either
n-BuOH or a mixture of n-BuOH and glycerol was heated to
reflux for the specified time, cooled, and concentrated in vacuo.
The residue was diluted with water, neutralized (NaHCO3),
and extracted (EtOAc). The combined organic layers were dried
(MgSO4) and concentrated in vacuo. Recrystallization from the
appropriate solvent gave the desired product.
Gen er a l P r oced u r e D. A mixture of 6, the appropriate
aniline (2 equiv), TFA (5 equiv), glycerol, and n-BuOH was
heated to reflux for the specified time, cooled, and concentrated
in vacuo. The residue was diluted with water, neutralized
(NaHCO3), and extracted (EtOAc). The combined organic
layers were dried (MgSO4) and concentrated in vacuo. Column
chromatography gave the desired product.
Gen er a l P r oced u r e E . A mixture of 6, the appropriate
aniline derivative (7 equiv), TFA (0.4 equiv), glycerol, and
either n-BuOH or EtOH, was heated to reflux for the specified
time, cooled, and concentrated in vacuo. The residue was
diluted with water, neutralized (NaHCO3), and extracted
(EtOAc). The combined organic layers were dried (MgSO4) and
concentrated in vacuo. Column chromatography gave the
desired product.
Gen er a l P r oced u r e F . A mixture of 6, the appropriate
aniline (2 equiv), TFA (5 equiv), and 2,2,2-trifluoroethanol (4
mL) was heated to 100 °C for the specified time, cooled, and
concentrated in vacuo. The residue was diluted with water,
neutralized (NaHCO3), and extracted (EtOAc). The combined
organic layers were dried (MgSO4), and concentrated in vacuo.
Column chromatography gave the desired product.
on
a Carlo-Erba Instrumentazione 1106 analyzer, or by
Butterworth Laboratories, Middlesex, UK, and are within
(0.4% of theory unless otherwise specified. Reagents were
purchased from Aldrich Chemical Co., Gillingham, UK, or
Lancaster Synthesis and used as received unless otherwise
stated. Ethanol and methanol were dried using Mg/I2 and
stored over 4 Å molecular sieves. Diethyl ether (predried over
CaCl2) and tetrahydrofuran (predried over KOH) were distilled
from sodium/benzophenone. Acetonitrile was predried over
potassium carbonate and distilled from CaH2. Petroleum ether
refers to that fraction in the boiling range 40-60 °C. Organic
solvents from separations were dried using anhydrous MgSO4.
Reactions were routinely performed under an atmosphere of
N2 or Ar.
2-Ch lor o-6-cycloh exylm eth oxyp u r in e (4). Sodium (0.18
g, 7.94 mmol) was disolved in cyclohexylmethanol (10 mL, 79.0
mmol) at 90 °C. 2,6-Dichloropurine (0.50 g, 2.64 mmol) was
added and heating continued at 90 °C for 1.5 h. The mixture
was cooled to room temperature, neutralized (AcOH), and
concentrated in vacuo. The residue was triturated with water
and filtered giving 4 as a white solid (0.60 g, 85%), mp 234-
240 °C. υmax/cm-1 3420 (NH2), 3114 (NH), 2929 (CH2), 1602
(CdC); 1H NMR (200 MHz, DMSO-d6) δppm 1.26 (5H, m,
cyclohexyl), 1.83 (6H, m, cyclohexyl), 4.33 (2H, d, OCH2, J )
6.12 Hz), 7.99 (1H, s, C(8)H); MS (EI) m/z 266 (M+).
2-An ilin o-6-cycloh exylm eth oxyp u r in e (2). A mixture of
6 (0.20 g, 0.8 mmol), aniline (0.37 g, 4.0 mmol, 5 equiv), and
n-BuOH (3.5 mL) was heated to 120 °C for 16 h and then
cooled, and the solvent was removed in vacuo. The residues
were disolved in DCM and filtered, and the filtrate was
purified by chromatography (5% MeOH, DCM) giving 2 as a
6-Cycloh exylm eth oxy-2-d im eth yla m in op u r in e (5). A
mixture of 4 (0.15 g, 0.56 mmol), ethanolamine (0.12 mL, 1.95
mmol), and DMF (3.0 mL) was heated at 90 °C for 3 days and
then concentrated in vacuo. The residues were purified by
chromatography (10% MeOH, DCM) and recrystallized (EtOAc)
to give 5 as a white solid (0.10 g, 63%). 1H NMR (200 MHz,
DMSO-d6) δppm 1.27 (5H, m, cyclohexyl), 1.99 (6H, m, cyclo-
hexyl), 3.20 (6H, s, N(CH3)2), 4.36 (2H, d, OCH2, J ) 6.12 Hz),
7.95 (1H, s, C(8)H); MS (EI) m/z 275 (M+). C, H, N. C14H21N5O
requires C 59.15, H 7.75, N 24.65; found C 60.02, H 7.40, N
24.29.
O6-Cycloh exylm eth yl-2-flu or op u r in e (6). To a stirred
solution of 50% aqueous hydrofluoroboric acid (13 mL, 210
mmol) at 0 °C was added O6-cyclohexylmethylguanine (2.6 g,
10.5 mmol) in one portion. To this was added a solution of
sodium nitrite (1.45 g, 21.0 mmol) in water (20 mL) dropwise,
ensuring that the reaction temperature did not exceed 10 °C.
The reaction mixture was allowed to warm to room temper-
ature and stirred for 18 h before being neutralized with 15%
(w/v) sodium carbonate solution. The resulting precipitate was
collected via filtration and stirred in ethyl acetate. The filtrate
was concentrated under reduced pressure to yield a white
crystalline solid (1.41 g, 53.6%): mp 172-173 °C; 1H NMR (300
MHz, DMSO-d6) δ 1.21 (5 H, m), 1.79 (6 H, m), 4.32 (2 H, d, J
) 6.25 Hz), 6.57 (1 H, s), 8.39 (1 H, s). 13C NMR (300 MHz,
DMSO-d6) δppm 25.4, 26.2, 29.3, 37.0, 72.6, 156.0, 158.8.
1
white solid (0.12 g, 46%). mp 196-199 °C (MeOH); H NMR
(200 MHz, DMSO-d6) δppm 1.26 (m, 5H, cyclohexyl), 1.93 (m,
6H, cyclohexyl), 4.34 (d, 2H, OCH2), 7.02 (t, 1H, ArHC), 7.35
(d, 2H, ArH), 7.81 (d, 2H, ArH), 8.04 (s, 1H, H8), 9.50 (s, 1H,
D2O exch. N9H); HRMS (EI) 323.175270 (M+); Anal. (C18H21N5O)
C, H, N.
6-Cycloh exylm eth oxy-2-(4′-su lfam oylan ilin o)pu r in e (3).
General Procedure C: a mixture of n-BuOH, glycerol, and
sulfanilamide, reflux 4.5 days, washing (Et2O, petroleum
ether), followed by recrystallization (H2O) gave NU6102, white
solid (60%), mp 152-154 °C. 1H NMR (200 MHz, DMSO-d6)
δppm 1.23 (5H, m), 1.91 (6H, m), 4.46 (2H, d), 7.27 (s, 2H), 7.81
(d, 2H), 8.08 (d, 2H), 8.19 (s, 1H), 9.82 (s, 1H). MS (EI) m/z
402 (M+); Anal. (C18H22N6O3S + 0.5 CF3CO2H) C, H, N.
2-Meth yla m in o-6-cycloh exylm eth oxyp u r in e (7). Gen-
eral Procedure A: 6 (0.107 g, 0.43 mmol) and methylamine
(10 mL, 8.03 M in ethanol), 18 h, gave 7 as a pale yellow solid
1
(62 mg, 70%), mp 159-160 °C. H NMR (200 MHz, D2O) δppm
1.2-1.5 (m, 5H, cyclohexyl) 1.7-2.1 (m, 6H), 2.9 (d, 3H,
CH3NH), 4.3 (2H, d, OCH2), 6.8 (1H, m, NHCH3, ex), 7.9 (1H,
s, C8H), 12.5 (1H, brs, N9H, ex); HRMS (EI) m/z 261.158669
(M+). C, H, N (C13H16N5O) C, H; N 24.65; found 24.08.
6-Cycloh exylm eth oxy-2-(eth yla m in o)p u r in e (8). Gen-
eral Procedure A: 6 (0.49 g, 1.95 mmol) and ethylamine (10
mL; 2.0 M in EtOH), 20 h. Chromatography (10% MeOH,
DCM) followed by recrystallization (petroleum ether, EtOAc,
MeOH) gave 8 as a cream solid (0.0171 g, 3%), mp 165-166
°C. 1H NMR (200 MHz, DMSO-d6) δppm 1.0-2.0 (17H, m,
cyclohexyl and CH(CH3)2), 4.1 (1H, m, CH(CH3)2), 4.4 (2H, d,
OCH2), 6.65 (1H ex, br t, NHCH2), 7.9 (1H, s, C8H), 12.6 (1H
ex, br s, N9H). HRMS (EI) m/z 289.191032 [M]+. C, H, N
(C14H21N5O) C, H; N calcd N 25.45; found N 24.74.
Gen er a l P r oced u r e A. A mixture of 6 and the appropriate
amine in ethanol (10 mL) was heated to reflux for the specified
time and then concentrated in vacuo. The residues were
suspended in water (5 mL) and extracted (EtOAc, 3 × 10 mL).
The combined organic extracts were dried (MgSO4) and dried
in vacuo. Column chromatography and/or recrystallization
from the appropriate solvent gave the desired product.
Gen er a l P r oced u r e B. A mixture of 6, the appropriate
aniline (7 equiv), and trifluoroacetic acid (0.4 equiv), and either
EtOH or n-BuOH was heated to reflux for the specified time
and then cooled, and the desired product precipitated by
addition of either MeOH or DCM. Recrystallization from the
6-Cycloh exylm eth oxy-2-(1-m eth yleth yla m in o)p u r in e
(9). General Procedure A: 6 (0.18 g, 0.72 mmol) and isopro-
pylamine (0.21 g, 3.59 mmol), 28 h. Chromatography (EtOAc,
petroleum ether; 1:1) followed by recrystallization (petroleum

3718 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 15
ether, EtOAc, MeOH) gave 9 as a cream solid (0.096 g, 42%),
Hardcastle et al.
(6H, m), 4.46 (2H, d), 6.82 (1H, d.t), 7.38 (1H, q), 7.59 (1H, d),
8.04 (1H, d), 8.25 (1H, s), 9.70 (1H, s). 19F NMR (500 MHz,
DMSO-d6) δppm 81.03. MS (EI) m/z 341 (M+). Anal. (C18H20N5-
OF + 0.25 CF3CO2H) C, H, N.
2-(3′-Br om oa n ilin o)-6-cycloh exylm eth oxyp u r in e (17).
General Procedure B: 3-bromoaniline and n-BuOH, reflux 18
h, precipitation (MeOH). Chromatography (silica; DCM:MeOH;
95:5), gave 17, white solid (35%), mp 204-207 °C. 1H NMR
(200 MHz, DMSO-d6) δppm 1.26 (5H, m), 1.93 (6H, m), 4.45 (2H,
d), 7.18 (1H, d), 7.32 (1H, t), 7.77 (1H, d, 8.16 (1H, s), 8.40,
(1H, s), 9.50 (1H, s). MS (EI) m/z 401 (M+); Anal. (C18H20BrN5O
+ 0.5 MeOH) C, H, N.
1
mp 165-166 °C. H NMR (200 MHz, DMSO-d6) δppm 1.0-2.0
(17H, m, cyclohexyl and CH(CH3)2), 4.1 (1H, m, CH(CH3)2),
4.4 (2H, d, OCH2), 6.65 (1H ex, br t, NHCH2), 7.9 (1H, s, C8H),
12.6 (1H ex, br s, N9H). HRMS (EI) m/z 289.191032 [M]+. Anal.
(C15H23N5O + 0.25 CH3OH) C, H, N.
6-Cycloh e xylm e t h oxy-2-(2-h yd r oxye t h yla m in o)p u -
r in e (10). General Procedure A: 6 (0.10 g, 0.4 mmol) and
ethanolamine (0.122 g, 2.0 mmol), 16 h, gave 10 as a cream
solid (55 mg, 47%), mp 108-109 °C. 1H NMR (200 MHz,
DMSO-d6) δppm 1.2-1.6 (5H, m, cyclohexyl), 1.8-2.1 (6H, m,
cyclohexyl), 3.45 (2H, m, CH2OH), 3.6 (2H, m, CH2N), 4.3 (2H,
d, OCH2), 4.75 (1H ex, t, OH), 6.7 (1H ex, br t, NHCH2), 7.9
(1H, s, C8H), 12.4 (1H ex, br s, N9H). HRMS (EI) m/z
291.170555 (M+). Anal. (C14H21N5O2) C, H, N.
2-(3,5-Dich lor oa n ilin o)-6-cycloh e xylm e t h yloxyp u -
r in e (18). General Procedure C: 6 (0.20 g, 0.8 mmol), 3,5-
dichloroaniline (0.52 g, 3.2 mmol), reflux 6.5 h. Chromatog-
raphy (silica; 5% MeOH, DCM) gave 18, white solid (0.100 g,
32%), 218-220 °C (ex MeOH). ν (cm-1), 3418 (NH stretch),
(R)-6-Cycloh exylm et h oxy-2-(1-et h yl-2-h yd r oxyet h yl-
a m in o)p u r in e (11). General Procedure A: 6 (0.44 g, 1.75
mmol) and (R)-2-aminobutanol (0.78 g, 8.76 mmol), 3 days.
Chromatography (10% MeOH, DCM) followed by recrystalli-
zation (petroleum ether, EtOAc) gave 11 as a cream solid
1
1435 (OCH2), 947 (cyclohexane), 650, 560 (purine); H NMR
(200 MHz, DMSO-d6) δppm 1.23 (5H, m, cyclohexyl), 1.86 (6H,
m, cyclohexyl), 4.41 (2H, d, OCH2), 7.12 (1H, s, ArH), 8.01 (2H,
s, ArH), 8.16 (1H, s, H8), 9.82 (1H, s, N9H); MS (ESI) m/z )
392 [M + H]+; Anal. (C18H19N5OCl2) C, H; N requires 17.89;
found 16.90.
6-Cycloh exylm et h oxy-2-(3′-et h yla n ilin o)p u r in e (19).
General Procedure B: 3-ethylaniline and EtOH, reflux 1 h,
precipitation on cooling. Recrystallization (MeOH) gave 19,
white solid (34%), mp 180-183 °C. 1H NMR (200 MHz, DMSO-
d6) δppm 1.23 (5H, m), 1.22 (3H, t), 1.91 (6H, m), 2.63 (2H, q),
6.75 (1H, d), 7.14 (1H, t), 7.55 (1H, d), 7.68 (1H, s), 8.25 (1H,
s), 9.70 (1H, br s). MS (EI) m/z 351 (M+).
[3-(6-Cycloh exylm eth oxy-9H-p u r in -2-yla m in o)p h en yl]-
m eth a n ol (20). General Procedure C: a mixture of n-BuOH/
glycerol and (3-aminophenyl)methanol, reflux 48 h, trituration
(Et2O) gave 20 as a white solid (59%), 174-177 °C. λmax (EtOH)
237.0 and 308.0 nm; ν (cm-1) 3292 (OH), 3107 (NH), 1490
(OCH2), 1038, 1009 (cyclohexane), 650, 560 (purine); 1H NMR
(200 MHz, DMSO-d6) δppm 1.01 (5H, m, cyclohexyl), 1.74 (6H,
m, cyclohexyl), 4.27 (2H, d, OCH2), 4.39 (2H, s, CH2OH), 6.80
(1H, d, ArH), 7.13 (1H, t, ArH), 7.57 (1H, d, ArH), 7.73 (1H, s,
ArH), 8.04 (1H, s, H8), 9.26 (1H, s, N9H); HRMS (EI) M+
353.185555 (C19H23N5O2 calcd as 353.185175); Anal. (C19H23N5O2
+ 0.2 EtOAc, 0.8 CF3CO2H) C, H, N.
6-Cycloh exylm eth oxy-2-(3′-m eth oxyan ilin o)pu r in e (21).
General Procedure B: 3-methoxyaniline and n-BuOH, reflux
2 h, precipitation (DCM). Recrystallization (MeOH) gave 21,
white solid (40%), mp 186-189 C. 1H NMR (200 MHz, DMSO-
d6) δppm 1.23 (5H, m), 1.91 (6H, m), 3.86 (3H, s), 4 46 (2H, d),
6.62 (1H, d.t), (7.28 (1H, q), 7.41 (1H, d), 7.73 (1H, d), 8.40
(1H, s), 9.70 (1H, s). MS (EI) m/z 353 (M+); Anal. (C19H23N5O2
+ 1.0 CF3COOH, 0.5 H2O) C, H, N.
6-Cycloh exylm et h oxy-2-(3′-m et h ylm er ca p t oa n ilin o)-
p u r in e (22). General Procedure B: 3-methylmercaptoaniline
and EtOH, reflux 16 h, precipitation on cooling. Recrystalli-
zation (MeOH) gave 22, white solid (44%), mp 190-192 °C.
1H NMR (200 MHz, DMSO-d6) δppm 1.23 (5H, m), 1.91 (6H,
m), 2.60 (3H, s), 4 45 (2H, d), 6.92 (1H, d), 7.32 (1H, t), 7.63
(1H, d), 7.98 (1H, s), 8.42 (1H, s), 9.70 (1H, s). MS (EI) m/z
369 (M+).
6-Cycloh exylm eth oxy-2-(4′-flu or oa n ilin o)p u r in e (23).
General Procedure B: 4-fluoroaniline and n-BuOH, reflux 16
h, precipitation (MeOH). Recrystallization (MeOH) gave 23,
off-white solid (25%), mp 207-209 °C. 1H NMR (200 MHz,
DMSO-d6) δppm 1.33 (5H, m), 1.90 (6H, m), 4.43 (2H, d), 7.22
(2H, d), 7.93 (2H, d), 8.23 (1H, s), 9.44 (s, 1H). MS (EI)
341.165825 (C18H20FN5O calcd as 341.165189) M+ 341 (15%),
57 (100%); Anal. (C18H20FN5O + 1.25 CF3COOH) C, H, N.
6-Cycloh exylm et h oxy-2-(3′-ch lor o-4′-flu or oa n ilin o)-
p u r in e (24). General Procedure B: 3-chloro-4-fluoroaniline
and n-BuOH, reflux 16 h, precipitation (MeOH). Recrystalli-
zation (MeOH) gave 24, off-white solid (21%), mp 225-226 °C.
1H NMR (200 MHz, DMSO-d6) δppm 1.31 (5H, m), 1.92 (6H,
m), 4.44 (2H, d), 7.43 (1H, t), 7.70 (1H, m), 8.33 (2H, m), 9.67
(s, 1H). MS (EI) 375.126259 (C20H23N5O3 calcd as 375.126216)
1
(0.129 g, 23%), mp 164-165 °C. H NMR (200 MHz, DMSO-
d6) δppm 1.0 (3H, t, CH2CH3), 1.05-2.0 (13H, m, cyclohexyl and
CH2CH3), 3.6 (2H, m, CH2OH), 3.9 (2H, m, CH2N), 4.3 (2H, d,
OCH2), 4.7 (1H ex, t, OH), 6.4 (1H ex, br t, NHCH2), 7.95 (1H,
s, C8H), 12.6 (1H ex, br s, N9H). HRMS (EI) m/z 319.201538
(M+). Anal. (C15H25N5O2) C, H, N.
(S)-6-Cycloh exylm et h oxy-2-(1-et h yl-2-h yd r oxyet h yl-
a m in o)p u r in e (12). A mixture of 6 (0.23 g, 0.92 mmol), (S)-
2-aminobutanol (0.41 g, 4.6 mmol), and n-butanol (10 mL) was
heated to reflux for 24 h and then concentrated in vacuo. The
residues were suspended in water (10 mL) and extracted
(EtOAc, 3 × 10 mL). The combined organic extracts were dried
(MgSO4) and concentrated in vacuo. Chromatography (10%
MeOH, DCM) followed by recrystallization (petroleum ether,
EtOAc) gave 12 as a cream solid (0.010 g, 4%), mp 165-166
1
°C. H NMR (200 MHz, DMSO-d6) δppm 1.0 (3H, t, CH2CH3),
1.1-2.0 (13H, m, cyclohexyl and CH2CH3), 3.5 (2H, m, CH2OH),
3.9 (2H, m, CH2N), 4.3 (2H, d, OCH2), 4.7 (1H ex, t, OH), 6.4
(1H ex, br t, NHCH2), 7.9 (1H, s, C8H), 12.6 (1H ex, br s, N9H).
HRMS (EI) m/z 319.200279 (M+). Anal. (C16H25N5O2) C, H, N.
(R)-6-Cycloh exylm eth oxy-2-(1-h ydr oxym eth yl-2-m eth yl-
p r op yla m in o)p u r in e (13). General Procedure A: 6 (0.42 g,
1.67 mmol) and (R)-2-amino-3-methylbutanol (0.86 g, 8.37
mmol), 3 days. Chromatography (10% MeOH, DCM) followed
by recrystallization (petroleum ether, EtOAc, MeOH) gave 13
as a cream solid (0.231 g, 42%), mp 161-163 °C. 1H NMR (200
MHz, DMSO-d6) δppm 1.0 (6H, d, CH(CH3)2), 1.05-1.5 and 1.6-
2.0 (11H, m, cyclohexyl), 2.1 (1H, m, CH(CH3)2), 3.6 (2H, m,
CH2OH), 3.9 (2H, m, CH2N), 4.4 (2H, d, OCH2), 4.6 (1H ex, t,
OH), 6.4 (1H ex, br t, NHCH2), 7.95 (1H, s, C8H), 12.5 (1H ex,
br s, N9H). HRMS (EI) m/z 333.217804 (M+). Anal. (C17H27N5O2)
C, H, N.
(S)-6-Cycloh exylm eth oxy-2-(1-h ydr oxym eth yl-2-m eth yl-
p r op yla m in o)p u r in e (14). General Procedure A: 6 (0.25 g,
1.00 mmol) and (S)-2-amino-3-methylbutanol (0.52 g, 8.37
mmol), 3 days. Chromatography (10% MeOH, DCM) followed
by recrystallization (hexane, EtOAc) gave 14 as a cream solid
1
(0.0135 g, 4%), mp 157-158 °C. H NMR (200 MHz, DMSO-
d6) δppm 1.0 (6H, d, CH(CH3)2), 1.1-1.5 and 1.7-2.0 (11H, m,
cyclohexyl), 2.1 (1H, m, CH(CH3)2), 3.6 (2H, m, CH2OH), 3.9
(2H, m, CH2N), 4.3 (2H, d, OCH2), 4.6 (1H ex, t, OH), 6.4 (1H
ex, br t, NHCH2), 7.9 (1H, s, C8H), 12.5 (1H ex, br s, N9H).
HRMS (EI) m/z 333.216911 [M]+. Anal. (C17H27N5O2) C, H, N.
2-(3′-Ch lor oa n ilin o)-6-cycloh exylm eth oxyp u r in e (15).
General Procedure B: 3-chloroaniline and n-BuOH, reflux 24
h, precipitation (MeOH). Recrystallization (MeOH) gave 15,
white solid (28%), mp 196-199 °C. 1H NMR (200 MHz, DMSO-
d6) δppm 1.26 (5H, m), 1.93 (6H, m), 4.45 (2H, d), 7.07 (1H, d),
7.39 (1H, t), 7.72 (1H, d), 8.22 (1H, s), 8.56, (1H, s), 9.50 (1H,
s). MS (EI) m/z 357 (M+).
6-Cycloh exylm eth oxy-2-(3′-flu or oa n ilin o)p u r in e (16).
General Procedure B: 3-fluoroaniline and n-BuOH, reflux 1
h, precipitation (MeOH), gave 16, white solid (50%), mp 224-
227 °C. 1H NMR (200 MHz, DMSO-d6) δppm 1.23 (5H, m), 1.91

Inhibitors of Cyclin-Dependent Kinases 1 and 2
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 15 3719
M+ 375 (50%), 279 (100%), Anal. (C20H23N5O3 + 0.8 CF3CO2H,
0.2 H2O) C, H, N.
MeOH, DCM) gave 32 as a yellow solid (0.102 g, 39%), mp
170-173 °C. ν (cm-1) 2924 (NH stretch), 2849, 1315.449 (OMe),
640 (purine ring), 665 (4,5 disubstituted imidazole); 1H NMR
(200 MHz, DMSO-d6) δppm 0.97 (5H, m, cyclohexyl), 1.61 (6H,
m, cyclohexyl), 3.82 (3H, s, OCH3), 4.26 (2H, d, OCH2), 6.79
(2H, s, NH2), 7.31 (1H, d, ArH), 7.50 (1H, d, ArH), 7.80 (1H, s,
ArH), 8.01 (1H, s, H8), 9.52 (1H ex, s, N9H). Anal. (C19H24N6-
SO4 + 0.2 CF3CO2H) C, H, N.
3-(6-Cycloh exylm eth oxy-9H-p u r in -2-yla m in o)ben zen e-
su lfon a m id e (33). General Procedure C: 3-aminobenzene-
sulfonamide (0.55 g, 3.2 mmol). Chromatography (EtOAc) gave
33 (0.130 g, 40%), mp 175-176 °C. λmax (EtOH) 289, 251, 240
nm; ν (cm-1) 3441 (NH2), 3350 (NH2), 2921, 1533, 1391; 1H
NMR (300 MHz, DMSO-d6) δppm 1.18 (5H, m, cyclohexyl) 1.77
(6H, m, cyclohexyl) 4.37 (2H, d, OCH2) 7.37 (d, 1H, ArH) 7.45
(dd, 1H, ArH) 7.88 (d, 1H, ArH) 8.01 (s, 1H, H8) 8.50 (s, 1H,
ex NH) 9.69 (s, 1H, ex NH) 12.86 (s, 1H, ex NH); MS (ESI+)
m/z 403 (M+, 100%); Anal. (C18H22N6O3S) H; calcd C 53.72; N
20.88; found C 53.26; N 21.59.
6-Cycloh exylm eth oxy-2-(4′-h ydr oxyan ilin o)pu r in e (25).
General Procedure B: 4-aminophenol and n-BuOH, reflux 16
h, precipitation (MeOH). Recrystallization (MeOH) gave 25,
off-white solid (51%). 1H NMR (200 MHz, DMSO-d6) δppm 1.31
(5H, m), 1.92 (6H, m), 4.40 (2H, d), 6.76 (2H, d), 7.62 (2H, d),
8.02 (1H, s), 9.04 (2H, s). MS (EI) 339.170807 (calcd as
339.169525) M+ 339 (53%), 243 (100%), Anal. (C18H21N5O2 +
0.1 CF3COOH, 0.1 M MeOH) C, H, N.
6-Cycloh exylm eth oxy-2-(4′-m eth oxyan ilin o)pu r in e (26).
General Procedure B: 4-methoxyaniline and EtOH, reflux 16
h, precipitation on cooling. Recrystallization (MeOH) gave 26,
white solid (41%), mp 189-192 °C. 1H NMR (200 MHz, DMSO-
d6) δppm 1.23 (5H, m), 1.22 (3H, t), 1.91 (6H, m), 2.63 (2H),
6.75 (1H, d), 7.14 (1H, t), 7.55 (1H, d), 7.68 (1H, s), 8.25 (1H,
s), 9.70 (1H, s). MS (EI) m/z 353 (M+).
6-Cycloh exylm et h oxy-2-(4′-m et h ylm er ca p t oa n ilin o)-
p u r in e (27). General Procedure B: 4-methylmercaptoaniline
and n-BuOH, reflux 16 h, precipitation on cooling. Recrystal-
lizations (EtOH and MeOH), gave 27, off-white solid (18%),
mp 159-160 °C. 1H NMR (200 MHz, DMSO-d6) δppm 1.35 (5H,
m), 1.86 (6H, m), 2.60 (3H, s), 4.43 (2H, d), 7.33 (2H, d), 7.89
(2H, d), 8.18 (1H, s), 9.70 (1H, s). MS (EI) 369.161194 (calcd
as 369.162332) M+ 369 (29%), 124 (100%); Anal. (C19H23N5OS
+ 0.9 CF3CO2H) C, H, N.
4-(6-Cycloh e xylm e t h oxy-9H -p u r in -2-yla m in o)b e n z-
a m id e (34). General Procedure C: n-BuOH and 4-aminobenz-
amide, reflux 16 h. Washing (Et2O, petroleum ether) and
recrystallization (Et2O, EtOH) gave 34, white solid (46%), mp
1
156-158 °C. H NMR (200 MHz, DMSO-d6) δ ppm 1.24 (5H,
m, cyclohexyl), 1.93 (6H, m, cyclohexyl), 4.46 (2H, d, OCH2),
7.27 (1H, s, CONH), 7.88 (1H, s, CONH), 7.90 (2H, d, ArH),
7.99 (2H, d, ArH), 8.10 (1H, s, H8), 9.70 (1H, s, N9H). MS
(ESI+) m/z 367 [M + H]+; Anal. (C19H22N6O2 + 0.4 EtOAc,
0.1 CF3CO2H) C, H, N.
4-(6-Cycloh exylm eth oxy-2-ylam in o)-N-m eth ylben zen e-
su lfon a m id e (28). General Procedure C: n-BuOH and 4-
amino-N-methylbenzenesulfonamide, reflux 48 h, chromatog-
raphy (silica; EtOAc:petroleum ether; 1:1) gave 28, white solid
4-(6-Cycloh exylm eth oxy-9H-pu r in -2-ylam in o)-N-m eth yl-
ben za m id e (36). General Procedure D: 4-amino-N-methyl-
benzamide, reflux 24 h, chromatography (silica; 1-10% MeOH:
DCM) gave 36, pink solid (29%), mp 139-142 °C. 1H NMR
(200 MHz, DMSO-d6) δppm 1.09 (5H, m, cyclohexyl), 1.89 (6H,
m, cyclohexyl), 2.44 (3H, t, CH3), 4.56 (2H, d, OCH2), 7.83 (4H,
q, ArH), 8.35 (1H, s, H8), 9.8 (1H, s, N9H). MS (ESI+) m/z 381
(M + 1); Anal. (C20H24N6O2) C, H, N.
4-(6-Cycloh exylm eth oxy-9H-p u r in -2-yla m in o)-N,N-d i-
m eth ylben za m id e (37). General Procedure C: n-BuOH and
4-amino-N,N-dimethylbenzamide, reflux 48 h, chromatography
(silica; EtOAc:petroleum ether; 2:3) gave 37, beige solid (35%),
mp 184-186 °C. 1H NMR (200 MHz, DMSO-d6) δppm 1.16 (5H,
m, cyclohexyl), 1.90 (6H, m, cyclohexyl), 3.07 (6H, s, 3 × CH3),
4.44 (2H, d, OCH2), 7.45 (2H, d, ArH), 7.98 (2H, d, ArH), 8.13
(1H, s, H8), 9.85 (1H, s, N9H). MS (ESI+) m/z 395.4 (M + 1);
Anal. (C21H26N6O2 0.3 EtOAc) C, H, N.
1
(27%), mp > 250 °C (decomp). H NMR (200 MHz, MeOH-d4)
δ
1.37 (5H, m, cyclohexyl), 2.11 (6H, m, cyclohexyl), 2.71
ppm
(3H, s, CH3), 4.57 (2H, d, OCH2), 7.92 (2H, d, ArH), 8.21 (1H,
s, H8), 8.21 (2H, d, ArH). MS (EI) 416.163048 (calcd as
416.163061), 416 (M, 41%), 320 (M - C7H12, 100%).
4-(6-Cycloh exylm eth oxy-2-yla m in o)-N,N-d im eth ylben -
zen esu lfon a m id e (29). General Procedure C: a mixture of
n-BuOH and glycerol as the solvents and 4-amino-N,N-
dimethylbenzenesulfonamide, reflux 3 days, chromatography
(silica; 1-4% MeOH:DCM) gave 29, brown solid (12%), mp
1
151-153 °C. H NMR (200 MHz, DMSO-d6) δppm 1.18 (5H, m,
cyclohexyl), 1.82 (6H, m, cyclohexyl), 2.82 (3H, s, CH3), 3.93
(2H, d, OCH2), 7.21 (2H, d, ArH), 7.64 (2H, d, ArH), 7.67 (1H,
s, H8), 9.47 (1H, s, N9H). MS (EI) 430.180489 (calcd as
430.178711) 430 (M, 100%), 334 (M - C7H13, 94%), 226 (M -
C7H13 - C2H6NSO2, 73%).
4-(6′-Cycloh e xylm e t h ylp u r in -2-yl)a m in oa ce t op h e -
n on e (38). General Procedure C: n-BuOH and 1-(4-aminophen-
yl)ethanone, reflux 12 h. Washing (hot H2O) gave 38, yellow
powder (61%), mp 236-238 °C. 1H NMR (200 MHz, DMSO-
d6) δppm 1.28 (5H, m, cyclohexyl), 1.90 (6H, m, cyclohexyl), 2.63
(3H, s, CH3), 4.46 (2H, d, OCH2), 8.02 (4H, q, ArH), 8.39 (1H,
s, H8), 9.99 (1H, s, N9H). MS (EI) 365.184105 (calcd as
365.185175) M+ 365 (80%) 269 (M+ - C7H12) 100%; Anal.
(C20H23N5O2 + 0.5 CF3CO2H, 0.1 H2O) C, H, N.
4-(6-Cycloh exylm eth oxyp u r in -2-yla m in o)ben zoic Acid
(39). An ethanolic solution of KOH (2 equiv) was added
dropwise to 40 in THF at 0 °C and the mixture stirred 24 h.
Water was added and the mixture neutralized (HCl; 1 M) and
extracted (DCM). The combined organic layers were dried
(MgSO4) and concentrated in vacuo to give 39 (41%). 1H NMR
(200 MHz, DMSO-d6) δppm 1.31 (5H, m), 1.93 (6H, m), 4 45
(2H, d), 8.02 (4H, q), 8.19 (1H, s), 9.92 (s, 1H), 13.05 (1H, s).
MS (ESI+) m/z 368 (M + 1).
Meth yl 4-N-(6′-Cycloh exylm eth yloxypu r in -2′-yl)am in o-
ben zoa te (40). General Procedure B: methyl 4-aminobenzo-
ate and n-BuOH, reflux for 16 h, precipitation (MeOH).
Chromatography (silica; DCM:MeOH; 9:1) and recrystalliza-
tion (MeOH) gave 40, off-white solid (8%), mp 244-246 °C.
1H NMR (200 MHz, DMSO-d6) δppm 1.31 (5H, m), 1.93 (6H,
m), 3.92 (3H, s), 4 45 (2H, d), 8.02 (4H, q), 8.19 (1H, s), 9.92
(s, 1H). MS (EI) 381.181458 (C20H23N5O3 calcd as 381.180090)
M+ 381 (56%), 185 (100%); Anal. (C20H23N5O3 + 0.2 CF3CO2H,
0.1 MeOH) C, H, N.
4-(6-Cycloh exylm et h oxy-9H -p u r in -2-yl)-(4-m et h a n e-
su lfon ylp h en yl)a m in e (30). General Procedure D: mixture
of n-BuOH/glycerol and 4-methanesulfonylphenylamine, reflux
for 3 days, chromatography (silica; 5% MeOH, DCM) gave 30,
beige solid (4.5%), mp > 270 °C. λmax (MeOH) 210.5, 310.5 nm;
ν (cm-1) 3376 (sulfone), 2924, 2852 (CH), 640 (purine), 665
1
(imidazole); H NMR (200 MHz, DMSO-d6) δppm 1.06 (5H, m,
cyclohexyl), 1.94 (6H, m, cyclohexyl), 3.22 (3H, s, CH3), 4.55
(2H, d, OCH2), 7.89 (2H, d, ArH), 8.08 (2H, d, ArH), 8.36 (1H,
s, H8), 10.20 (1H, br s, N9H, exch in D2O); MS (EI) m/z 402
(100% M+); Anal. (C19H23N5SO3 + 0.6 CF3CO2H) C, H, N.
Meth yl 4-[(6′-Cycloh exylm eth ylpu r in -2-yl)am in o]ph en -
yl Su lfoxide (31). General Procedure C: n-BuOH and 4-(meth-
anesulfinyl)phenylamine, reflux for 48 h. Chromatography
(silica; EtOAc:petroleum ether; 95:5; plus DCM:MeOH; 9:1)
gave 31, brown solid (46%), mp 150-152 °C. 1H NMR (200
MHz, DMSO-d6) δppm 1.22 (5H, m, cyclohexyl), 1.86 (6H, m,
cyclohexyl), 2.80 (3H, s, CH3), 4.42 (2H, d, OCH2), 7.70 (2H, d,
ArH), 8.15 (3H, d, 2 × ArH + H8), 9.90 (1H, s, N9H exch. in
D2O). MS (EI) M+ 385.158508 (calcd as 385.157247) 370 (M+
- CH3, 54%), 274 (M+ - C7H11O, 100%); Anal. (C19H23N5SO2
+ 0.2 CF3CO2H) C, H, N.
4-[6-Cycloh exylm eth oxy-9H-p u r in -2-yla m in o]ben zen e-
(3′-m eth oxy)su lfon a m id e (32). A mixture of 6 (0.15 g, 0.60
mmol), 2-methoxysulfanilamide (0.37 g, 1.84 mmol), trifluoro-
acetic acid (0.46 mL, 6.02 mmol), and DMSO (2 mL) was
heated to 60 °C for 16 h, cooled, neutralized (aq NaHCO3), and
extracted (EtOAc 3 × 20 mL). Chromatography (silica; 10%

3720 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 15
Hardcastle et al.
2-[4-(6-Cycloh exylm eth oxy-9H-pu r in -2-ylam in o)ph en yl]-
a ceta m id e (41). General procedure B: 4-aminophenylacet-
amide (0.53 g, 3.5 mmol), n-BuOH. Recrystallization (Et2O,
EtOAc, EtOH) gave 41 as a beige powder (0.017 g, 5.2%),
233.5-233.8 °C. ν (cm-1) 3346.4, 3200.0 (NH amide stretch),
2918.2 (CH), 2855.3 (ArH), 1676.7 (CO), 1606.5 (Ar), 1128.36
(C-O-C), 633.5 (purine); λmax (EtOH) ) 273, 396 nm; 1H NMR
(200 HMz, DMSO-d6) δppm 1.20-1.08 (5H, m, cyclohexyl), 1.80-
1.60 (6H, m, cyclohexyl), 3.20 (2H, d, CH2), 4.28 (2H, d, OCH2),
6.80 (1H, s, NH), 7.09 (2H, d, ArH), 7.36 (1H, s, NH), 7.65 (2H,
d, ArH), 7.93 (1H, s, H8). LCMS (ESI-) m/z ) 380 [M]+, 379
[M - H]-; Anal. (C20H24N6O2 + 0.1 CH3OH) C, H, N.
6-Cycloh exylm et h oxy-2-(4′-cya n om et h yla n ilin o)p u -
r in e (42). General Procedure B: 4-aminophenylacetonitrile
and n-BuOH, reflux 16 h, precipitation (MeOH). Recrystalli-
zation (MeOH) gave 42, off-white solid (17%). 1H NMR (200
MHz, DMSO-d6) δppm 1.31 (5H, m), 1.92 (6H, m), 4.04 (2H, s),
4 42 (2H, d), 7.34 (2H, d), 7.92 (2H, d), 8.11 (1H, s). MS (EI)
362.186272 (calcd as 362.185510) M+ 362 (55%), 266 (100%);
Anal. (C20H22N6O) C, H, N.
(6-Cycloh exylm et h oxy-9H -p u r in -2-yl)t h iop h en -2-yl-
m eth yla m in e (43). A mixture of 6 (0.20 g, 0.8 mmol), thio-
phene-2-methylamine (0.45 g, 4 mmol), and n-butanol (2 mL)
was heated to 120 °C for 24 h, diluted with water (1 mL),
neutralized with HCl (1 M), and extracted (EtOAc, 3 × 10 mL).
The combined organic extracts were dried (MgSO4) and
concentrated in vacuo. Chromatography (5% MeOH, DCM)
gave 43 (0.070 g, 25%), mp 170-171 °C. ν (cm-1) 3246 (NH),
2922 (CH2), 2848 (OCH2), 1625, 1589 (ArH), 1499 (NH); 1H
NMR (300 MHz, DMSO-d6) δppm 1.04 (2H, m, cyclohexyl), 1.20
(3H, m, cyclohexyl), 1.80 (6H, m, cyclohexyl), 4.29 (2H, s,
OCH2), 4.54 (2H, s, NCH2), 6.97 (1H, m, thiophene H-3′), 7.03
(1H, m, thiophene H-4′), 7.30 (1H, m, thiophene H-5′), 8.14
(1H ex, s, NH), 8.97 (1H, s, H8); MS (EI+) m/z 343 (M+ +1).
En zym e in h ibition stu d ies. Inhibition of starfish oocyte
(Marthasterias glacialis) CDK1/cyclin B1 and human CDK2/
cyclinA was assayed as previously described.27
Exp r ession a n d P u r ifica tion of T160p CDK2/cyclin A3
a n d Cr ysta lliza tion of T160p CDK2/Cyclin A3-In h ibitor
Com p lexes. T160pCDK2/cyclin A was purified as described
previously.48 The protein was concentrated to 10 mg/mL in 0.2
M NaCl, 0.04 M HEPES, pH 7.0, 0.01% monothioglycerol, flash
frozen in 25 µL aliquots in liquid nitrogen, and stored at -80
°C until required. Aliquots were rapidly thawed at room
temperature and then incubated on ice. Prior to setting up
crystallization trials, inhibitor stocks in 100% DMSO (Sigma)
were added to the protein solution to a final concentration of
1 mM or 5 mM, (final DMSO 5% (v/v)). The mixtures were
incubated on ice for 1 h and then spun through a 0.22 µm filter.
Crystals were obtained by sitting drop vapor diffusion, with
the mother liquor consisting of 0.7-0.85 M potassium chloride
(Riedel-de Hae¨n), 1.1-1.25 M ammonium sulfate (Riedel-de
Hae¨n), 40 mM HEPES, pH 7.0 (Sigma), and 5 mM dithio-
threitol (Sigma). Crystal drops were typically composed of 1.5
µL of protein solution and 1 µL of mother liquor. Trays were
set up at room temperature (ca. 21 °C) and then incubated at
4 °C. Crystals took between two weeks and three months to
grow. A variety of morphologies were observed, with dimen-
sions between 100 µm to 1000 µm on the longest edge.
X-r a y Cr ysta llogr a p h y Da ta Collection a n d P r ocess-
in g. Crystals were warmed slowly overnight from 4 °C to room
temperature (ca. 21 °C) before manipulation. Crystals were
briefly immersed in cryoprotectant, 8 M sodium formate, and
then plunge-frozen in liquid nitrogen. All datasets were
collected at 100 K. Data were collected at the ESRF using
MarCCDs on either ID14-EH4 (25) or ID14-EH1 (34 and 33).
Data processing was carried out using MOSFLM,49 SCALA,50
and other programs of the CCP4 suite.51
Str u ctu r e Solu tion a n d Refin em en t. The starting model
in all cases was the structure of NU6102-T160pCDK2/cyclin
A (PDB code 1H1S) with all nonprotein atoms removed. Rigid
body refinement in REFMAC5,52 with data to 3.5 Å, was
applied, first with whole chains as individual rigid bodies and
then with each protein domain as an individual rigid body
(CDK2 residues 0-80, 81-84, 85-296, cyclinA residues 175-
302 and 303-432). Unambiguous electron density for the
inhibitors was visible after rigid body refinement. Inhibitor
models were built using the Monomer Library Sketcher of the
CCP4 suite51 and with reference to the CSD53). Restrained
refinement was then applied to the maximal resolution with
alternative rounds of manual rebuilding in O.54 Toward the
end of refinement, waters were added using ARP55 with
reference to the NU6102 (3) structure.
5-[(6-Cycloh exylm eth oxy-9H-p u r in -2-yla m in o)m eth yl]-
th iop h en e-2-su lfon a m id e (44). A mixture of 6 (0.13 g, 0.53
mmol), 5-aminomethyl-thiophene-2-sulfonamide (0.22 g, 1.06
mmol), and n-butanol (3.5 mL) was heated to 120 °C for 24 h
and then concentrated in vacuo. Chromatography (80% EtOAc,
petroleum ether) gave 44, mp 174 °C. λmax (EtOH) 211, 244,
285; ν (cm-1) 3414, 3267 (NH), 2923 (CH2), 2850 (OCH2), 1635,
1
1589, 1539, 1338, 1147; H NMR (300 MHz, DMSO-d6) δppm
1.12 (5H, m, cyclohexyl), 1.73 (6H, m, cyclohexyl), 4.24 (2H, s,
OCH2), 4.62 (2H, s, NCH2), 6.98 (1H, d, thiophene), 7.36 (1H,
d, thiophene), 7.51 (2H, s, NH2),7.55 (1H, s), 7.85 (1H, s), 12.60
(1H, s, H8); MS (ESI+) m/z 423 [M + H]+.
6-C y c lo h e x y lm e t h o x y -2-(4-m e t h o x y p h e n o x y )-9H -
p u r in e (45). A mixture of 6 (0.20 g, 0.8 mmol), 4-methoxy-
phenol (0.10 g, 0.8 mmol), KF on alumina (0.5 g), 18-crown-6
(0.08 mmol), and DMSO (5 mL) was heated at 140 °C for 48
h, cooled, diluted with water (5 mL), and extracted (EtOAc 3
× 10 mL). The combined organic extracts were dried (Na2SO4)
and concentrated in vacuo. RP-HPLC (J ones C18; MeOH, H2O)
Ack n ow led gm en t. The authors thank Tim Hunt for
the human CDK2 and cyclin A constructs, Carl Mann
for the gift of the Civ1 DNA clone, the beamline
scientists at ID14-EH1 and ID14-EH4, ESRF, Grenoble,
for providing excellent facilities during data collection,
EPSRC Chemical Database Service at Daresbury, and
R. Davison, P. Mackley, I. Taylor, E. F. Garman, and
R. Bryan for technical support. This research was
supported by grants from the Cancer Research UK, The
Royal Society UK, Medical Research Council UK,
BBSRC, Oxford University, and AstraZeneca PLC UK.
gave 45, mp 167 °C. λmax (EtOH); 205, 225, 266 nm; ν (cm-1
)
3445, 3082, 2922, 2852, 1590, 1500, 1448, 1362, 1333, 1197,
1
1125, 1036; H NMR (300 MHz, DMSO-d6) δppm 1.11 (5H, m,
cyclohexyl), 1.72 (6H, m, cyclohexyl), 3.77 (3H, s, OCH3), 4.24
(2H, d, OCH2), 6.97 (2H, d, ArH), 7.13 (2H, d, ArH), 8.16 (1H,
s), 12.83 (s, 1H, D2O exch. NH); MS (ES+) m/z 355; Anal.
(C19H22N4O3 + 0.5 H2O) C, H, N.
4-[N′-(6-Cycloh exylm eth oxy-9H-p u r in -2-yl)h yd r a zin o]-
ben zen esu lfon a m id e (46). A mixture of 6 (0.30 g, 1.2 mmol),
4-hydrazinobenzenesulfonamide (0.53 g, 2.4 mmol), and n-
butanol (3 mL) was heated at 100 °C for 24 h and then
concentrated in vacuo. The residues were partitioned between
saturated sodium bicarbonate solution (10 mL) and EtOAc (10
mL), and then the organic phase was dried (MgSO4) and
concentrated in vacuo. Chromatography (75% EtOAc, petro-
leum ether) gave 46 (0.009 g, 1%), 249-250 °C. λmax (EtOH)
286, 239 nm; 1H NMR (300 MHz, DMSO-d6) δppm 1.12 (5H, m,
cyclohexyl), 1.73 (6H, m, cyclohexyl), 4.22 (2H, d, OCH2), 6.77
(2H, d, ArH), 6.97 (2H, d, NH2), 7.54 (2H, d, ArH), 7.87 (1H,
s), 8.35 (s, 1H, D2O exch. NH), 8.79 (s, 1H, D2O exch. NH),
12.83 (s, 1H, D2O exch. NH); MS (ESI+) m/z 418 [M + H]+.
Su p p or tin g In for m a tion Ava ila ble: Elemental analyses
data. Refinement statistics for published structures. The three
structures solved in this study of T160pCDK2/cyclin A in
complex with the compounds 25, 33 and 34 have been
deposited with the PDB and assigned the codes 1OI9, 1OIU,
1OIY, respectively. The structure of NU2058 (1) solved previ-
ously has been assigned the pdb code 1e1v.27 This material is
available free of charge via the Internet at http://pubs.acs.org.
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