2-Anilino-4-(thiazol-5-yl)pyrimidine CDK Inhibitors: Synthesis, SAR Analysis, X-ray Crystallography, and Biological Activity

Article abstract of DOI:10.1021/jm0309957

Following the identification through virtual screening of 4-(2,4-dimethyl-thiazol-5-yl)pyrimidin-2-ylamines as moderately potent inhibitors of cyclin-dependent kinase-2 (CDK2), a CDK inhibitor analogue program was initiated. The first aims were to optimize potency and to evaluate the cellular mode of action of lead candidate molecules. Here the synthetic chemistry, the structure-guided design approach, and the structure-activity relationships (SARs) that led to the discovery of 2-anilino-4-(thiazol-5-yl)pyrimidine ATP-antagonistic CDK2 inhibitors, many with very low nM K_is against CDK2, are reported. Furthermore, X-ray crystal structures of four representative analogues from our chemical series in complex with CDK2 are presented, and these structures are used to rationalize the observed biochemical SARs. Finally results are reported that show, using the most potent CDK2 inhibitor compound from the current series, that the observed antiproliferative and proapoptotic effects are consistent with cellular CDK2 and CDK9 inhibition.

Full text of DOI:10.1021/jm0309957

1662
J . Med. Chem. 2004, 47, 1662-1675
2-An ilin o-4-(th ia zol-5-yl)p yr im id in e CDK In h ibitor s: Syn th esis, SAR An a lysis,
X-r a y Cr ysta llogr a p h y, a n d Biologica l Activity
Shudong Wang,‡ Christopher Meades,‡ Gavin Wood,‡ Andrew Osnowski,‡ Sian Anderson,‡ Rhoda Yuill,‡
Mark Thomas,‡ Mokdad Mezna,‡ Wayne J ackson,‡ Carol Midgley,‡ Gary Griffiths,‡ Ian Fleming,‡ Simon Green,‡
Iain McNae,† Su-Ying Wu,† Campbell McInnes,‡ Daniella Zheleva,‡ Malcolm D. Walkinshaw,† and
Peter M. Fischer*,‡
Cyclacel Limited, J ames Lindsay Place, Dundee DD1 5J J and Structural Biochemistry, The University of Edinburgh,
Michael Swann Building, King’s Buildings, Edinburgh, EH9 3J R, Scotland, UK
Received August 7, 2003
Following the identification through virtual screening of 4-(2,4-dimethyl-thiazol-5-yl)pyrimidin-
2-ylamines as moderately potent inhibitors of cyclin-dependent kinase-2 (CDK2), a CDK
inhibitor analogue program was initiated. The first aims were to optimize potency and to
evaluate the cellular mode of action of lead candidate molecules. Here the synthetic chemistry,
the structure-guided design approach, and the structure-activity relationships (SARs) that
led to the discovery of 2-anilino-4-(thiazol-5-yl)pyrimidine ATP-antagonistic CDK2 inhibitors,
many with very low nM Kis against CDK2, are reported. Furthermore, X-ray crystal structures
of four representative analogues from our chemical series in complex with CDK2 are presented,
and these structures are used to rationalize the observed biochemical SARs. Finally results
are reported that show, using the most potent CDK2 inhibitor compound from the current
series, that the observed antiproliferative and proapoptotic effects are consistent with cellular
CDK2 and CDK9 inhibition.
In tr od u ction
activity than normal cells, and it has in fact been shown
that tumor cells are selectively sensitive to inhibition
of cyclin A/CDK2.4 Finally, entry and progression
through mitosis are controlled by cyclin B/CDK1 activi-
ties. Furthermore, there exist several additional cyclin/
CDK complexes, many of them with activities related
to the cell cycle, but also in the regulation of transcrip-
tion. Thus CDKs 7, 8, and 9 have all been implicated in
modulation of RNA elongation by phosphorylation of the
C-terminal domain of RNA polymerase II.5
Many ATP-antagonistic CDK inhibitor molecules
have been discovered (reviewed in refs 6-8), and some
of them have now reached clinical evaluation.9,10 It
nevertheless remains somewhat unclear what selectivity
profile a CDK inhibitor should have for optimal anti-
cancer activity. For example, a dominant-negative form
of CDK2 devoid of catalytic activity was shown to
prevent growth of cells in culture.11,12 On the other
hand, certain osteosarcomas and pRb-negative cervical
cancers were shown to continue to proliferate after
depletion of CDK2,13 and CDK2-knockout mice appear
to develop normally.14,15 Our work has been guided by
the most selective clinical CDK inhibitor, i.e. CYC202
(R-roscovitine), whose main targets are CDK2 and
CDK9,16 as well as by the hypothesis that CDK2
inhibition may provide a way of interfering selectively
with cancer cells.17 We recently reported the identifica-
tion of 2-anilino-4-(thiazol-5-yl)pyrimidines as CDK2
inhibitors using virtual screening methods.18 Here we
describe our results to date with compounds from this
new pharmacophore.
The fidelity of progression through the human cell
cycle is interrogated at various checkpoints in order to
ensure the integrity of daughter cells. If errors in DNA
replication are detected, normal proliferating cells
undergo programmed cell death (apoptosis). Trans-
formed cells, however, gain a proliferative advantage
by bypassing or overriding these checkpoints. CDKs are
intimately associated with the regulation of cell cycle
progression.1 Cyclins, the activating subunits of CDKs,
are expressed specifically during certain phases of the
cycle. Thus D-type cyclins appear in G1 and associate
with CDKs 4 and 6. Later cyclin E is expressed and
forms an active complex with CDK2. The action of these
complexes on various substrates, and the retinoblas-
toma tumor suppressor protein (pRb), in particular,
permits passage into S phase by dissociating the com-
plex between pRb and E2F transcription factors. Next
in turn is cyclin A, which associates with CDK2 and
maintains pRb hyperphosphorylation. Later in S phase,
however, cyclin A/CDK2 fulfills another important func-
tion, i.e. phosphorylation and deactivation of E2F.2 This
activity is necessary for progression and inappropriate
E2F transcriptional activity in late S phase constitutes
an apoptotic trigger.3 This suggests that inhibition of
cyclin A/CDK2 during S phase would lead to S-phase
arrest, inappropriately persistent E2F-1 activity, and
apoptosis. Tumor cells with disrupted pRb regulation
would have higher levels of active E2F, which may leave
them more vulnerable to the effects of persistent E2F
* To whom correspondence should be addressed. Cyclacel Limited,
J ames Lindsay Place, Dundee DD1 5J J , Scotland, UK. Tel: +44-1382-
206062. Fax: +44-1382-206067. E-mail: pfischer@cyclacel.com.
‡ Cyclacel Limited.
Ch em istr y
2-Amino-4-(thiazol-5-yl)pyrimidines (Scheme 1) were
synthesized by the general pyrimidine condensation
† University of Edinburgh.
10.1021/jm0309957 CCC: $27.50 © 2004 American Chemical Society
Published on Web 02/07/2004

2-Anilino-4-(thiazol-5-yl)pyrimidine CDK Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7 1663
Sch em e 1a
a
Reagents: (a) pyridine, MeOH; (b) N,N-dimethylformamide dimethyl acetal, 120 °C; (c) HNO3, aq cyanamide, 100 °C; (d) NaOH,
2-methoxyethanol, 125 °C.
method of Bredereck.19 Unless commercially available,
5-acetyl-thiazoles 3 were prepared from the appropriate
thioureas 1 and 3-chloro-2,4-pentadione 2 and were
converted to the enaminones 4 by heating in N,N-
dimethylformamide dimethyl acetal. Enaminones 4
were then condensed to the desired pyrimidines at
elevated temperature in alcoholic alkali with guanidine
carbonate. When it was found that introduction of
anilino groups at the pyrimidine C2 position dramati-
cally increased the biological activity of the resulting
compounds in both enzymatic and cell-based assays, a
number of 2-phenylamino-4-(thiazol-5-yl)pyrimidines 7
were designed and prepared. The anilino guanidines
were derived from the corresponding anilines 5 by
treatment of their nitrate or hydrochloride salts with
cyanamide.20 Synthesis of 4-N,N-dimethylamino-3-chlo-
rophenyl guanidine was conducted by the sequence in
Scheme 1, starting from 3-chloro-4-fluoronitrobenzene.
Selective displacement of the fluorine was achieved by
heating this compound with N,N-dimethylamine hydro-
chloride in DMSO in the presence of potassium carbon-
ate in a sealed tube. The nitro group in the product was
subsequently reduced with Fe/HOAc to the primary
amine and further treated with cyanamide to afford the
corresponding guanidine. Similarly, 3-nitro-4-N,N-di-
methylamino anilino guanidine was prepared by nu-
cleophilic displacement at C4 of 4-fluoro-3-nitroaniline.
From the acetylthiazole 3b, which contains a primary
amino group, the enaminone was obtained in the imino
form 4b. This compound could be used directly in the
following pyrimidine ring formation reactions, during
which the amino group (7, R1 ) NH2) was regenerated.
Resu lts a n d Discu ssion
In itia l SAR a n d Design . We recently reported the
discovery of a new 2-amino-4-heteroarylpyrimidine ATP-
antagonist CDK inhibitor pharmacophore through vir-
tual screening and structure-based design.18 One of the
lead compounds from the screening was the dimeth-
ylthiazoloaminopyrimidine 8 (Table 1), which possessed
modest CDK2 inhibitory potency. Inspection of the
binding mode of this compound in the ATP pocket of a
CDK2 complex crystal structure18 revealed that it bound
in a similar way to ATP itself (Figure 1). In both cases
a group of three H-bonds between the ligand and the
backbone of CDK2 residues Glu81 and Leu83 was
observed, interactions that are conserved in most kinase
inhibitor complexes. In the ATP structure21 the guanine
amino group donates a H-bond to the carbonyl of Glu81,
whereas N1 and H2 of the purine system accept and
donate H-bonds to the amide NH and carbonyl of Leu83,
respectively. Compound 8 formed similar H-bonding
contacts through the NH2 (Glu81 CO), pyrimidine N1
(Leu83 NH), and H6 (Leu83 CO). The dimethylthiazole
ring overlaps roughly with the space occupied by the
ribose system in ATP. As expected from this binding
mode, we found that bismethylation of the primary

1664 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Ta ble 1. Inhibition of CDK Activity and Tumor Cell Proliferation
Wang et al.
Ki (µM)a
compd
formula
R1
R2
R3
R4
R5
R6
CDK2
CDK4
16 ( 9
>20
>20
2.6 ( 0.2
>20
9.4 ( 0.1
72-h MTT IC50 (µM)b
8
9
I
I
I
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
-
-
-
-
-
H
H
H
-
6.5 ( 3.0
>10
Me
Me
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
H
Cl
H
H
F
-
-
>20
>20
0.08 ( 0.02
>20
>10
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
-
-
>10
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
4.9 ( 1.1
>10
H
Cl
H
H
F
H
H
CF3
H
OH
OH
OH
H
H
H
NO2
NO2
NO2
NO2
NO2
NO2
NO2
H
NH2
CN
H
NO2
Cl
0.67 ( 0.10
1.7 ( 2.2
>10
Cl
2.5 ( 1.5
>20
H
H
F
H
1.2 ( 0.4
1.2 ( 0.7
0.9
>20
>20
>20
>20
0.21 ( 0.16
0.37 ( 0.02
>20
>10
H
H
CF3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0.10 ( 0.05
0.04 ( 0.03
2.0 ( 0.9
6.8 ( 1.9
>10
>20
>20
H
>10
CF3
H
0.29 ( 0.24
0.06 ( 0.03
0.11 ( 0.10
2.3 ( 1.7
1.2 ( 0.6
1.6 ( 1.1
>10
H
H
NHMe
NHEt
Me
NHMe
NH2
Me
>10
OH
OH
OH
H
0.14 ( 0.12
0.07 ( 0.06
0.030 ( 0.005
0.11 ( 0.03
0.32 ( 0.18
0.32 ( 0.27
0.32 ( 0.21
2.0 ( 2.6
1.0 ( 0.2
0.2 ( 0.2
1.0 ( 0.7
>10
>20
>20
H
H
H
H
H
H
Me
>20
NMe2
NHMe
NHAll
NH2
pyrid-2-yl
Me
0.06 ( 0.02
5.3 ( 2.9
2.8 ( 0.5
3.4 ( 0.8
0.5 ( 0.0
0.3 ( 0.1
>10
0.80 ( 0.55
1.6 ( 0.1
0.16 ( 0.07
7.1 ( 4.1
0.0020 ( 0.0006
0.053 ( 0.005
>20
>20
>20
10.64 ( 0.05
NO2
H
4.1 ( 1.4
>10
Me
Me
Me
Me
0.40 ( 0.02
0.30 ( 0.45
0.22 ( 0.21
0.020 ( 0.025
0.10 ( 0.08
0.70 ( 0.25
2.1 ( 1.4
1.5 ( 0.9
0.6 ( 0.5
0.6 ( 0.4
0.2 ( 0.2
2.3 ( 0.2
H
0.48 ( 0.11
0.96 ( 0.69
0.27 ( 0.13
1.0 ( 0.5
NMe2
NMe2
NMe2
NMe2
Me
H
NH2
0.90 ( 0.26
a
b
Average values from at least 4 independent determinations (( 1 standard deviation). anti-proliferative activities (72-h MTT IC50
values ( 1 standard deviation) quoted are the averages from measurements using the human tumor cell lines A549, HT29, and SaOS-2
(3 independent determinations each).
F igu r e 1. H-Bonding interactions of ATP (a) and compounds 8 (b), and 20 (c) with the backbone of CDK2 residues Glu81 and
Leu83.
amino group on the pyrimidine ring had a detrimental
effect on binding and in fact abolished biological activity
completely (10). Surprisingly, however, monomethyla-
tion (9) had the same effect, despite the fact that one

2-Anilino-4-(thiazol-5-yl)pyrimidine CDK Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7 1665
would expect that the methylamino function should still
be able to form a similar H-bond with the CDK2 Glu81
carbonyl group as the primary amino group of compound
8. In any case, introducing an aromatic substituent to
this amino function is not only tolerated, but actually
results in a significant potency increase. Thus the
aniline derivative 11 is almost 100-fold more potent in
inhibition of substrate phosphorylation by CDK2 than
the corresponding amine 8. Furthermore, during the
SAR studies addressing the anilino part of the phar-
macophore, we succeeded in obtaining a CDK2 complex
crystal structure of a relevant compound (20). Prior to
this a pharmacophore hypothesis was proposed for the
aniline derivatives based on molecular dynamics dock-
ing techniques and was used in the initial design
phases. The structural solution confirmed this hypoth-
esis and showed a different binding mode with respect
to the pyrimidine ring compared to compound 8.18 In
this alternative pose the H-bonds referred to above now
were as follows: pyrimidine H6 (Glu81 CO), N1 (Leu83
NH), and anilino NH (Leu83 CO); i.e., the pyrimidine
ring in the CDK complex of 20 was in the same plane
but flipped through 180° compared to 8 (Figure 1). As a
result, the phenyl group of 20 pointed out toward the
entrance of the binding cleft in an area that is not
occupied by ATP.
Finally amino (35) and nitrile (36) substituents in the
anilino meta-position also furnished potent compounds.
We next turned our attention to modification of the
thiazole system. Whereas the thiazol-4-yl methyl group
makes near-optimal hydrophobic contacts with Phe80
of CDK2 (Figure 2), the thiazol-2-yl methyl group is
positioned in the vicinity of several polar residues,
particularly Asn132. We therefore expected that intro-
duction of groups capable of H-bonding should be
favorable. We investigated this in the context of the
optimal m-/p--hydroxy- and m-nitroanilino pyrimidin-
2-yl systems. However, replacement of the thiazol-2-yl
methyl group with a methylamino group (compounds
22, 25, and 30) did not change potency appreciably.
Placement of bulkier secondary amino groups, e.g.
ethylamino (23) or pyrid-2-yl-amino (33), was not toler-
ated, with the exception of the allylamino group in the
context of the m-nitroanilino system (31). A primary
amino group in place of the thiazol-2-yl methyl group
preserved activity (26); again the m-nitroanilino system
provided a different context, and here the primary
amino derivative 32 was a particularly potent CDK2
inhibitor, although this potency gain was not accompa-
nied in equal measure by increased cellular activity. The
fact that a dimethylamino (29) replacement preserved
activity indicated that a H-bond acceptor group was
favorable in place of the thiazol-2-yl methyl group.
Op tim iza tion of th e In Vitr o P oten cy. Examina-
tion of the initial docked ligands and the subsequent
crystal structures provided insight for the design of
more potent analogues. To pick up potential additional
interactions with any of the numerous polar residues
lining the rim of the ATP binding cleft, we envisaged
substitution of the phenyl ring and modification of the
thiazol-2-yl methyl group in, for example, compound 11.
We first investigated the effect of substituents on the
aniline moiety while leaving the dimethylthiazole ring
unchanged. In general we found that only electron-
withdrawing substituents preserved or enhanced CDK2
inhibitory potency. Furthermore, this effect was only
observed when such groups were introduced in the
meta- or para-position of the aniline, whereas the
corresponding ortho-substituted analogues were gener-
ally less active or inactive. For example, of the chloro-
anilines 12-14 the meta-isomer 13 was somewhat less
potent than the unsubstituted aniline 11, whereas the
para-isomer 14 was less potent and the ortho-isomer
possessed only marginal activity. A similar situation
was seen with the more potent fluoroanilines 15-17;
except here the para-isomer 17 was most potent. Of the
corresponding trifluoromethyl compounds 18-20, only
the para-isomer 20 displayed significant biological
activity. When a hydroxyl group was introduced, this
was found to maintain potency with respect to the
unsubstituted analogue 11, both in the para- (24) and
particularly in the meta-isomer 21. Furthermore, cel-
lular antiproliferative potency was now also enhanced.
The m-nitro compound 27 was somewhat less effective
than the reference compound 11 as a CDK2 inhibitor,
but displayed about 5-fold higher cellular activity. The
corresponding p-nitro isomer 34, on the other hand, was
practically devoid of activity. To reconfirm the impor-
tance of the anilino NH function as a H-bond donor, we
prepared and tested an N-methyl derivative of com-
pound 27; as expected, this analogue 28 was inactive.
As discussed above, a comparatively poor correlation
between the biochemical and cellular potency of the
various test compounds was observed. Nevertheless, the
fact that compounds with poor potency or no activity
against isolated CDK enzymes were also devoid of
cytotoxic activity strongly suggests that the antiprolif-
erative effects are a consequence of cellular CDK
inhibition. It cannot be ruled out, however, that inhibi-
tion of other cellular (protein kinase) targets contributes
to the antiproliferative activities observed. Unlike bio-
chemical activity, cellular potency is affected by such
factors as membrane permeability, protein binding,
solubility, and stability in physiological medium. These
factors, as well as metabolism, also affect in vivo
absorption and disposition and are therefore of prime
importance in terms of bioavailability. Overall the
compounds discussed thus far were found to be com-
paratively lipophilic and poorly water-soluble. This
situation was improved with compounds that contained
a p-dimethylamino substituent in the anilino portion
(37-40). Combination of this group in 37 with otherwise
favorable meta-anilino substituents such as chloro (39),
and nitro (38), led to soluble nM CDK2 inhibitor
compounds with good cellular activity. Combination
with the thiazol-2-yl primary amino group (40) dis-
cussed above, on the other hand, did not improve
potency. As previously, antiproliferative potency did not
follow biochemical CDK2 inhibition potency gains in the
p-(dimethylamino)aniline series 37-40, either. We are
currently evaluating compounds from this series for
their in vivo antitumor effects. Furthermore, we have
chosen our most potent CDK2 inhibitor, i.e., compound
32, to verify that the antiproliferative effects observed
correlate with cellular CDK inhibition (see below).
X-r a y Cr ysta llogr a p h y. Structural data for the four
new inhibitor/CDK2 complexes are summarized in Table
2 and Figure 2. In each case, structure refinement gave

1666 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Wang et al.
F igu r e 2. Inhibitor/CDK2 complex crystal structures. Electron density for all four ligands in the CDK2 crystal complexes solved.
A stereoimage of the CDK2/32 is shown at the top. The H-bonds are indicated with red broken lines. The two positions of the
nitro group are distinguished from each other by one of the rings being colored green. Asn132 is shown in the position that
interacts with the NH2 group.
Ta ble 2. X-ray Data Collection and Structure Refinement
CDK2/21
CDK2/25
CDK2/32
CDK2/37
resolution (Å)
(outer shell)
Unit cell
25-2.53
(2.69-2.53)
a ) 53.489
b ) 71.987
c )72.362
91355
25-2.5
(2.66-2.5)
a ) 52.896
b ) 70.002
c ) 71.709
102882
9345
15-1.96
(2.07-1.96)
a ) 53.491
b ) 72.232
c ) 72.578
226048
20604
23-2.3
(2.44-2.3)
a ) 53.337
b ) 71.794
c ) 71.414
78546
P212121
(R ) â ) γ ) 90°)
total reflections observed
unique reflections
multiplicity
9820
9.30
12686
3.4
11.0
10.97
R
merge % (outer shell)
11.7 (50.7)
12.8 (3.38)
99.9 (100)
22.1
8.9 (49.0)
13.8 (2.76)
96.4 (96.7)
23.0
5.9 (16.5)
22.4 (7.7)
99.4 (99.9)
21.8
5.0 (14.8)
3.8 (4.9)
98.7 (96.7)
23.0
I/σ(I) (outer shell)
completeness % (outer shell)
Rwork
Rfree
%
%
28.0
27.7
23.7
27.3
RMS bonds (Å);
0.007/
0.010/
0.006/
0.006/
RMS angles (deg)
1.3
1.5
1.3
1.2
refined ligand occupancy %
97
98
80
80
rise to electron density maps that permitted unambigu-
ous placement of the ligand structure in the active site
of CDK2. The binding poses in the ATP pocket are
similar for the four ligands. In each case, the charac-
teristic H-bonding network involving the Glu81 and
Leu83 backbone is observed. Furthermore, the Leu134
side chain forms strong hydrophobic interactions with
the pyrimidine and anilino rings and the thiazol-4-yl
methyl groups always pack up against the Phe80 phenyl
ring. The Asp145 carboxyl group forms a polar interac-
tion with the ligand thiazole N atom in each complex;
in the case of 21, 25, and 37, this carboxyl also interacts
directly with the Lys33 side chain amino function. In
all four CDK-ligand structures the conformation of the
side chain of Lys33 adopts a different conformation to
that found in the native CDK structure and the T-loop
(residues 147-165) is disordered. This observation fits
with the suggested mechanism for ligand-induced dis-
ordering of the activation loop.18
The Ki values for the four compounds in this chemi-
cally related series generally correlate with the number
of observed H-bonds, van der Waals interactions, and

2-Anilino-4-(thiazol-5-yl)pyrimidine CDK Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7 1667
Ta ble 3. Selectivity of Kinase Inhibition by Compound 32
protein kinase
Ki (nM)a
CDK1/cyclin B
CDK2/cyclin E
CDK4/cyclin D1
CDK7/cyclin H
CDK9/cyclin T1
ERK-2
PKCR
SAPK2A (p38)
CaMKII
Abl
CKII
p70 S6
Akt/PKB
Plk1
80 ( 40
2.0 ( 0.6
53 ( 5
70 ( 10
4 ( 3
>1000
>1000
>1000
>1000
160 ( 10
F igu r e 3. Western blot analysis of A549 cells 6 and 24 h
posttreatment with (1) DMSO control; (2) 0.5 µM compound
32 (72-h MTT IC50); (3) 2 µM compound 32. Reduced phos-
phorylation of pRb at Thr821 and Ser249/Thr252 is observed,
indicating cellular inhibition of CDK2 and CDK4, respectively.
Phosphorylation is also reduced at the RNA polymerase II
Ser-2 and Ser-5 sites, a possible indication of inhibition of
CDK7 and CDK9. (The 6-h analysis was only performed for
the pRB Thr821 phosphorylation).
>1000
>1000
>1000
>1000
>1000
PKA
GSK3â
20 ( 10
a
Average values from at least four independent determinations
(( one standard deviation).
polar interactions. The fact that both meta- and para-
substituted anilino moieties are tolerated in the various
thiazolopyrimidine inhibitors is consistent with the
observed H-bonding interactions of the hydroxyl group
with either the side chains of Asp86 (21) or Lys89 (25).
Replacement of the thiazol-2-yl methyl group with
NHMe (25) does not result in additional H-bonding
interactions; however, for the most potent CDK2 inhibi-
tor 32, the presence of the primary NH2 group leads to
increased electrostatic interactions with the side chains
of Asp145 and Asn132. The side chain of Asn132 is
found to adopt two alternative conformations, only one
of which interacts with the NH2 group (Figure 2). The
significantly tighter binding of 32 may also be explained
by additional interactions involving the nitro substitu-
ent. Careful refinement showed that the nitrophenyl
group was in fact disordered. In both orientations
observed, the nitro group makes weak but favorable
electrostatic interactions. Particularly notable is the
interaction between the carboxyl group of Asp86 to the
NO2 nitrogen of 3.48 Å and a close contact with the
backbone carbonyl of Gln131. In the alternative confor-
mation there is a weak H-bond between the nitro group
and the side chain of Lys89 (Figure 2). It has been
observed from structures of CDK2/cyclin A that Asp145
undergoes a significant rearrangement upon cyclin
binding.22 The inhibitor complex structures presented
here are with monomeric CDK2, and therefore the
possibility cannot be excluded that the thiazole NH2 in
compound 32 makes more favorable contacts in the
activated kinase. Overlay of the CDK2 complex of 32
with the CDK2/cyclin A complex (PDB accession code
1J ST) suggests that this may indeed is the case, as
Asp145 is significantly closer to the amino group. This
observation could also explain the markedly lower
potency of 32 against CDK1/cyclin B1 (Table 3). As the
ATP clefts of CDK2 and CDK1 are highly conserved,
large differences in inhibition would not be expected. It
is conceivable, however, that activation of CDK1 by
cyclin B leads to a different conformation of Asp146
(residue corresponding to Asp145 in CDK2), and per-
haps of the ATP binding site in general, and hence could
result in less favorable contacts in this context.
other CDKs, particularly CDK9, were also inhibited at
somewhat reduced potency. Unlike other small-molecule
ATP-antagonist CDK inhibitors,23 compound 32 did not
have a significant effect on the catalytic activity of the
structurally related ERK-2 MAP kinase. GSK3, on the
other hand, which is also closely related to CDKs, was
inhibited by 32 with sub-micromolar potency. Of the
other protein kinases assayed, only the Ableson protein
tyrosine kinase was inhibited appreciably.
The retinoblastoma tumor suppressor proteins (pRb,
p107, p130) are the main physiological substrates for
the G1/S CDK complexes (CDK4/cyclin D1, CDK2/cyclin
E, and CDK2/cyclin A). The entry of cells into S phase
requires CDK-mediated phosphorylation of pRb, releas-
ing and activating transcription factors of the E2F
family, which are held in inactive pRb/E2F complexes
during the G0 and M phases.1 We therefore sought to
demonstrate inhibition of CDK2 activity in live cells by
examining the effects on the phosphorylation state of
pRb upon exposure of the cells to compound 32. We
found that when A549 (a lung adenocarcinoma cell line)
cells were treated with compound 32, a time- and dose-
dependent decrease in phosphorylation of pRb at Thr821,
a CDK2-preferential phosphorylation site of pRb,24 was
observed (Figure 3). Treatment with compound 32 also
resulted in decreased phosphorylation at the Ser249/
Thr252 residues of pRb, sites preferential for cyclin
D/CDK4 phosphorylation,24 and at the Ser-2 and Ser-5
sites of RNA polymerase II, sites potentially phospho-
rylated by CDKs 1, 7, 8, and 9.25 Such a reduction in
pRb phosphorylation demonstrates an inhibitory effect
of compound 32 on cellular CDK2 and CDK4 activities,
while the reduced phosphorylation at Ser-2 and Ser-5
of RNA polymerase II suggests potential inhibition of
CDKs 9, 8, or 7. Such cellular inhibitions are in
agreement with the in vitro kinase Ki values presented
in Table 3 and would indicate a mode of action of
compound 32 involving multiple CDKs.
To examine the effects of compound 32 on the cell
cycle, A549 cells were synchronized at the G1/S bound-
ary by sequential block and release using thymidine and
mimosine.26 Compound 32 was added at 0.5 µM con-
centration on the final release and subsequent progress
through the cell cycle was monitored by fixing cells at
several time points and analyzing DNA and cyclin B
Bioch em istr y a n d Biology. The kinase selectivity
of prototype compound 32 was investigated (Table 3).
We found that potency was highest against CDK2, but

1668 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Wang et al.
F igu r e 4. Compound 32 blocks cells in G1/S and prevents induction of cyclin B and entry into G2/M. Human A549 cells were
synchronized using a sequential thymidine/mimosine block. Upon release of the block, cells were treated with compound 32, and
their synchronous passage through the cell cycle was followed up to 9 h by FACS analysis. Panel A: untreated cells labeled with
propidium iodide to detect DNA content; panel B: untreated cells labeled with FITC-conjugated anti-cyclin B antibody; panel C:
cells treated with 0.5 µM compound 32 at t ) 0; labeled with propidium iodide to detect DNA content; panel D: cells treated with
0.5 µM compound 32 at t ) 0, labeled with FITC-conjugated anti-cyclin B antibody.
content. On release the majority of untreated cells had
a 2n DNA content and a low level of cyclin B (t ) 0 in
Figure 4) indicating synchronization at G1/early S. Over
the following 6-7 h, the DNA content gradually in-
creased, shown in panel A of Figure 4 as an increase in
FL3 fluorescence, so that at 9 h the majority of cells
had a 4n DNA content and high cyclin B levels (panel
B in Figure 4), indicating that the cells had reached G2/
M. However, the cells treated with compound 32 did not
show the same increase in DNA content (panel C in
Figure 4), and there was no increase in cyclin B levels
(panel D in Figure 4), indicating that they were arrested
in G1/early S and unable to exit S phase.
The effects of compound 32 on S-phase transition are
consistent with the inhibition of CDK2. The generally
accepted view is that cyclin D-dependent kinases initiate
pRb phosphorylation in G1, and that cyclin E/CDK2
then completes the process, allowing activation of E2F-
dependent transcription and coordinated expression of
a number of genes required for DNA synthesis and
subsequent S-phase progression. In addition, cyclin
E/CDK2 and cyclin A/CDK2 phosphorylate other sub-
strates, which promote the initiation of replication
complex assembly, and stimulate DNA synthesis.27-31
Furthermore, appropriately timed deactivation of E2F
is critical for S-phase progression, and this is also, at
least partly, mediated by cyclin A/CDK2. E2F-1 forms
stable complexes with cyclin A/CDK2, which phospho-
rylate bound DP-1, resulting in inhibition of DNA-
binding by E2F-1. If E2F-1 activity is allowed to persist
inappropriately into S phase, as might be the case in
the presence of a CDK2 inhibitor, the result is S-phase
arrest and/or apoptosis.3 Completion of S phase must
also be coordinated with the onset of mitosis, a process
controlled by cyclin B-dependent kinases. Cyclin B1
protein levels increase prior to the onset of mitosis due
to inactivation of the proteolytic anaphase promoting
complex (APC). The mechanism of APC inactivation in
turn involves cyclin A/CDK2-dependent phosphorylation
of its activating subunit, cdh1.32 Consequently, the
failure of cells treated with compound 32 to progress
through S phase, or to accumulate cyclin B1, are an
indication of efficient cellular CDK2 inhibition. Ad-
ditionally, there may be a role for inhibition of other
CDKs, particularly CDK9, which is inhibited by com-
pound 32 with similar potency as CDK2. Previous
reports have suggested that roscovitine33 and flavopiri-
dol,34 i.e. CDK inhibitors with similar selectivity profiles,
may inhibit general RNA synthesis by blocking the
phosphorylation of the carboxy-terminal domain (CTD)
of RNA polymerase II. Positive transcription elongation
factor b (P-TEFb), which is comprised of CDK9/cyclin
T1 or T2 complexes, phosphorylates the CTD of RNA
polymerase II, an event which is essential for the
transition from transcriptional initiation to elongation
in human cells. Progression through the cell cycle would
be affected by the lack of transcription of essential genes
as a result of the inhibition of CTD phosphorylation.
To look at the effect of compound 32 on unsynchro-
nized cells, HeLa (a cervical carcinoma cell line) cells
were incubated for 24 h with 0.1 or 0.5 µM compound
32 and then stained to reveal tubulin (anti-R-tubulin

2-Anilino-4-(thiazol-5-yl)pyrimidine CDK Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7 1669
(250 × 22 mm) columns, respectively. Linear gradient elution
using H2O/MeCN systems (containing 0.1% CF3COOH) at flow
rates of 1 mL/min (analytical) and 9 mL/min (preparative) was
performed. Purity was assessed by integration of chromato-
grams (λ ) 254 nm). Silica gel (EM Kieselgel 60, 0.040-0.063
mm, Merck) or ISOLUTE pre-packed columns (J ones Chro-
matography Ltd. UK) were used for flash chromatography.
3-Dim et h yla m in o-1-(2,4-d im et h ylt h ia zol-5-yl)p r op e-
n on e (4a , R ) Me). A solution of 1-(2,4-dimethylthiazol-5-
yl)ethanone (3a , R ) Me; 10 g, 64 mmol) in dimethoxymethyl-
dimethylamine (10 mL, 8.97 g, 75 mmol) was refluxed under
N2. After 21 h, the mixture was cooled and evaporated to
i
dryness. The solid residue was recrystallized from Pr2O/CH2-
Cl2 to afford the product as an orange solid (9.94 g, 79%); mp
96-98 °C. Anal. RP-HPLC: tR 7.1 min (10-70% MeCN, purity
100%). 1H NMR (CDCl3): δ 2.66 (s, 6H, CH3), 2.70 (s, 6H, CH3),
5.37 (d, 1H, J ) 12.2 Hz, CH), 7.66 (d, 1H, J ) 12.2 Hz, CH).
13C NMR (DMSO-d6): δ 18.19, 19.52, 37.77, 45.19, 94.50,
134.01, 153.62, 154.38, 165.64, 179.93. MS (ESI+) m/z 211.01
(M + H)+. Anal. (C10H14N2OS) C, H, N.
F igu r e 5. Compound 32 reduces mitotic index and induces
cell death in unsynchronized cultured HeLa cells. (A) Cells
treated for 24 h with two concentrations of compound 32 were
stained with DAPI (blue), anti-R-tubulin (red), and anti-γ-
tubulin (green). (B) Percentages of mitotic, multinuclear, and
dying cells with fragmented nuclei.
N′-[5-(3-Dim et h yla m in oa cr yloyl)-4-m et h ylt h ia zol-2-
yl]-N,N-d im eth ylfor m a m id in e (4b). A mixture of thiourea
(1, R ) H; 5.18 g, 68 mmol) in MeOH (20 mL) was cooled in
an ice bath. Pyridine (2 mL) was added, followed by dropwise
addition of 3-chloropenta-2,4-dione (2; 9.15 g, 68 mmol). The
mixture was allowed to warm to room temperature and was
stirred for 4 h. The resulting precipitates were filtered and
washed with EtOAc to afford 1-(2-amino-4-methylthiazol-5-
yl)ethanone 3b as a white solid in a quantitative yield. Mp
183-185 °C. Anal. RP-HPLC: tR 4.8 min (10-70% MeCN,
antibody), centrosomes (anti-γ-tubulin antibody), and
DNA (DAPI) (Figure 5A). In each sample >1000 cells
were counted and the mitotic index was determined by
calculating the percentage of cells observed to be in
mitosis (prophase, metaphase, anaphase, or telophase,
Figure 5B). There was a decrease in mitotic index after
drug treatment, suggesting that unsynchronized cells
are also unable to enter mitosis and supporting the
previous observation that cells are arrested at an earlier
point in the cycle. There was also a large increase in
cell death after treatment of HeLa cells with 0.5 µM
compound 32 (Figure 5B), consistent with apoptosis
being triggered by inappropriate E2F-1 activity. A549
cells treated the same way showed a similar reduction
of mitotic cells, but not the massive increase in cell
death (results not shown) seen with HeLa. This may
indicate differences between tumor cell lines in whether
their response to S-phase delay results in S-phase arrest
or apoptosis.
Con clu sion s. We have developed the 2-anilino-4-
(thiazol-5-yl)pyrimidine CDK inhibitor pharmacophore
using structure-based design methods. A prototype
compound from the series (32) with particularly high
biochemical potency against CDKs 2 and 9 was used to
demonstrate a cellular mode of action in human tumor
cell lines consistent with inhibition of functions of these
kinase complexes. We are currently evaluating selected
compounds using in vivo tumor efficacy models, as well
as further optimizing the pharmaceutical properties in
analogues of the inhibitor compound group discussed
here.
1
purity 100%). H NMR (DMSO-d6): δ 2.31 (s, 3H, CH3), 2.39
(s, 3H, CH3), 7.88 (s, 1H, NH). 13C NMR (DMSO-d6): δ 18.81,
30.12, 121.91, 157.77, 171.09, 188.97. MS (ESI+) m/z 156.89
(M + H)+. This material (3.35 g, 21 mmol) was treated with
dimethoxymethyl-dimethylamine (4.49 g, 38 mol). After heat-
ing at 120 °C for 6 h, the mixture was evaporated to dryness.
The residue was triturated with EtOAc, and the precipitates
were collected and washed with EtOAc/PE (5:1). The title
compound was obtained after crystallization from EtOAc/PE
(2:1) as an orange solid (4.4 g, 79%): mp 198-199 °C. Anal.
RP-HPLC: tR 7.1 min (10-70% MeCN, purity 100%). 1H NMR
(CDCl3): δ 2.64 (s, 3H, CH3), 3.08 (s, 6H, CH3), 3.11 (s, 6H,
CH3), 5.35 (d, 1H, J ) 12.2 Hz, CH), 7.67 (d, 1H, J ) 12.2 Hz,
CH), 8.23 (s, 1H, CH). 13C NMR (DMSO-d6): δ 18.78, 35.25,
60.44, 94.62, 126.84, 152.95, 153.48, 157.11, 173.75, 180.15.
MS (ESI+) m/ z 267.15 (M + H)+. Anal. (C12H18N4OS) C, H, N.
3-Dim eth yla m in o-1-(4-m eth yl-2-m eth yla m in oth ia zol-
5-yl)p r op en on e (4c, R ) Me). A solution of 3-chloropentane-
2,4-dione (2; 2.5 g, 19 mmol) in MeOH (10 mL) was treated
with methyl thiourea (1, R ) Me; 1.67 g, 19 mmol) and
pyridine (1.5 mL). The mixture was stirred at room temper-
ature for 2 h. Precipitated 1-(4-methyl-2-methylaminothiazol-
5-yl)ethanone (3c, R ) Me; 2.1 g, 65%) was filtered, washed
with Et2O, and dried: mp 201-203 °C. Anal. RP-HPLC: tR
5.5 min (10-70% MeCN, purity 100%). 1H NMR (CDCl3): δ
2.47 (s, 3H, CH3), 2.61 (s, 3H, CH3), 3.18 (s, 3H, CH3). 13C NMR
(DMSO-d6): δ 19.16, 30.05, 31.54, 49.28, 121.78, 158.54,
188.81. MS (ESI+) m/z 170.87 (M + H)+. This material
(2.05 g, 12 mmol) was suspended in dimethoxymethyldimeth-
ylamine (5.3 mL, 4.80 g, 40 mmol), and the mixture was heated
at 120 °C with stirring for 16 h. On cooling, the reaction
mixture was evaporated to dryness. The residue was crystal-
lized from EtOAc to afford the title product as an orange solid
(1.82 g, 67%): mp 209-211 °C. Anal. RP-HPLC: tR 7.1 min
(10-70% MeCN, purity 100%). 1H NMR (CDCl3): δ 2.55 (s,
3H, CH3), 2.94 (s, 3H, CH3), 3.41 (s, 6H, CH3), 5.30 (d, 1H,
J ) 12.2 Hz, CH), 7.64 (d, 1H, J ) 12.2 Hz, CH). 13C NMR
(DMSO-d6): δ 18.94, 31.41, 54.99, 94.32, 122.05, 152.96,
153.83, 169.41, 179.89. MS (ESI+) m/z 226.11 (M + H]). Anal.
(C10H15N3OS.MeOH) C, H, N.
Exp er im en ta l Meth od s
Anhydrous reaction solvents and other reagents were
obtained from commercial sources and were used without
further purification. Melting points were determined with a
Leica testo-720 electrothermometer instrument and are uncor-
rected. NMR spectra were obtained using Bruker DPX-300 or
Varian INOVA-500 instruments. Chemical shifts are reported
in parts per million relative to internal tetramethylsilane
standard. Mass spectra were obtained using a Waters ZQ2000
single quadrupole mass spectrometer with electrospray ioniza-
tion (ESI). Elemental analyses were performed by the Analyti-
cal Group, Department of Chemistry, The University of
Edinburgh, UK. Results obtained were within 0.4% of calcu-
lated values. Analytical and preparative RP-HPLC was per-
formed using Vydac 218TP54 (250 × 4.6 mm) and 218TP1022
3-Dim eth yla m in o-1-(2-(eth yla m in o)-4-m eth ylth ia zol-5-
yl)p r op en on e (4c, R ) Et). To a solution of ethyl thiourea
(5.20 g, 50 mmol) in MeOH (25 mL) were added dropwise
pyridine (4 mL) and then 3-chloropenta-2,4-dione (6.73 g, 50

1670 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Wang et al.
1
mmol). The mixture was stirred at room temperature for 4 h.
The precipitates were collected, filtered, and washed several
times with Me2CO to afford 1-(2-(ethylamino)-4-methylthiazol-
5-yl)ethanone (3c, R ) Et) as a white solid (9.2 g, 100%): mp
211-213 °C. 1H NMR (CDCl3): δ 1.17 (t, 3H, J ) 7.3 Hz, CH3),
2.42 (s, 3H, CH3), 2.54 (s, 3H, CH3), 3.42 (m, 2H, CH2). This
material (1.83 g, 10 mmol) was suspended in dimethoxymeth-
yldimethylamine (2 mL, 1.79 g, 15 mmol), and the mixture
was heated at 115-120 °C for 22 h. After cooling, the reaction
mixture was evaporated to dryness. A minimal amount of
EtOAc was added, and the resulting precipitates were filtered.
After crystallization from EtOAc/MeOH, the title product was
obtained as a light yellow solid (1.61 g, 42%): mp 204-205
°C. Anal. RP-HPLC: tR 7.3 min (10-70% MeCN, purity 100%).
1H NMR (CDCl3): δ 1.25 (t, 3H, J ) 6.0 Hz, CH3), 2.49 (s, 3H,
CH3), 3.20 (s, 6H, CH3), 3.28 (m, 2H, CH2), 5.35 (d, 1H, J )
11.5 Hz, CH), 7.67 (d, 1H, J ) 11.5 Hz, CH). 13C NMR (DMSO-
d6): δ 14.92, 18.95, 31.41, 54.99, 94.34, 121.81, 152.92, 153.74,
168.49, 179.90. MS (ESI+) m/z 240.17 (M + H)+.
3-Dim et h yla m in o-1-(2-a llyla m in o-4-m et h ylt h ia zol-5-
yl)p r op en on e (4c, R ) All). A solution of allyl thiourea (1,
R ) All; 3.95 g, 34 mmol) in MeOH (10 mL) was treated with
3-chloropenta-2,4-dione (2; 4.58 g, 34 mmol) and pyridine (3
mL). The mixture was refluxed for 20 min, cooled to room
temperature, and stirred for 24 h. After concentration, the
resulting precipitates were filtered and washed several times
with Et2O to afford 1-(2-allylamino-4-methylthiazol-5-yl)etha-
none (3c, R ) All) as a white solid (6.0 g, 90%): mp 136-138
°C. Anal. RP-HPLC: tR 7.7 min (10-70% MeCN, purity 100%).
1H NMR (DMSO-d6): δ 2.43 (s, 3H, CH3), 3.89 (m, 2H, CH2),
5.13-5.24 (m, 2H, CH2), 5.84 (m, 1H, CH), 8.84 (sb, 1H, NH).
13C NMR (CDCl3): δ 17.14, 93.95, 115.84, 121.52, 133.64,
153.89, 154.29, 170.04, 181.99. MS (ESI+) m/z 197.02 (M +
H)+. Anal. (C9H12N2OS.2Me2CO) C, H, N. This material (1.24
g, 6 mmol) was treated with dimethoxymethyldimethylamine
(5.3 mL, 4.8 g, 40 mmol). After refluxing for 12 h, the mixture
was cooled and concentrated. The resulting precipitates were
collected and washed with EtOAc/MeOH (10:1) to afford the
title product as a light yellow solid (1.0 g, 65%): mp 136-138
°C. Anal. RP-HPLC: tR 7.7 min (10-70% MeCN, purity 100%).
1H NMR (DMSO-d6): δ 2.46 (s, 3H, CH3), 3.22 (s, 6H, CH3),
3.87 (m, 2H, CH2), 5.11-5.28 (m, 3H, CH2 & CH), 5.89 (m,
1H, CH), 7.54 (d, 1H, J ) 12.0 Hz, CH). 13C NMR (DMSO-d6):
δ 18.93, 47.00, 55.03, 94.29, 116.66, 122.28, 135.16, 152.99,
153.42, 168.46, 179.89. MS (ESI+) m/z 252.13 (M + H)+. Anal.
(C12H17N3OS.MeOH) C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of N-P h en yl-
gu a n id in e Nitr a tes (6). To an ice-cooled mixture of the
appropriate aniline 5 (25 mmol) in EtOH (6 mL) was added
nitric acid (1.8 mL of 70% solution in H2O) dropwise with
stirring. After complete addition, cyanamide (5 mL of 50%
solution in H2O) was added, and the mixture was heated at
100 °C for 16-18 h under N2. After cooling to room temper-
ature, it was poured into excess Et2O and was basified with
aq NaOH solution. The ethereal layer was separated, and the
aqueous phase was extracted with Et2O several times. The
combined organic phases were washed with brine, dried on
MgSO4, filtered, and evaporated. The resulting residue was
purified by crystallization or flash chromatography in ap-
propriate mixtures of EtOAc/PE or EtOAc/MeOH.
Gen er a l P r oced u r e for th e P r ep a r a tion of [4-(Th ia zol-
5-yl)p yr im id in -2-yl]p h en yla m in es (7). A mixture of 3-(di-
methylamino)-1-thiazol-5-yl-propenone (4; 1 equiv), the ap-
propriate N-phenylguanidine nitrate (6; 2 equiv), and NaOH
(1 equiv) in 2-methoxyethanol (0.2 mL/mmol) was heated at
125 °C for 22 h under N2. After cooling, the solvent was
evaporated and the residue was purified by flash chromatog-
raphy using appropriate mixtures of EtOAc/hexane as the
eluant. The products 7 were further purified by crystallization
from EtOAc/MeOH.
purity 100%). H NMR (DMSO-d6): δ 2.57 (s, 3H, CH3), 2.60
(s, 3H, CH3), 6.66 (bs, 2H, NH2), 6.79 (d, 1H, J ) 5.3 Hz, Py-
H), 8.26 (d, 1H, J ) 5.5 Hz, Py-H). 13C NMR (DMSO-d6): δ
18.44, 19.58, 107.20, 131.43, 152.05, 158.91, 159.70, 164.02,
166.41. MS (ESI+) m/z 207.21 (M + H)+. Anal. (C9H10N4S) C,
H, N.
[4-(2,4-Dim et h ylt h ia zol-5-yl)-p yr im id in -2-yl]m et h yl-
a m in e (9). From 3-(dimethylamino)-1-(2,4-dimethylthiazol-5-
yl)propenone (4a , R ) Me) and N-methylguanidine nitrate.
Buff solid (60%): mp 112-113 °C. Anal. RP-HPLC: tR 10.2
min (0-60% MeCN, purity 100%). 1H NMR (DMSO-d6): δ 2.59
(s, 3H, CH3), 2.79 (s, 3H, CH3), 2.80 (s, 3H, CH3), 6.77 (bs, 1H,
NH), 7.11 (bs, 1H, Py-H), 8.29 (bs, 1H, Py-H). 13C NMR
(DMSO-d6): δ 18.50, 19.57, 28.44, 106.77, 131.41, 152.23,
158.66, 159.54, 163.18, 166.43. MS (ESI+) m/z 221.27 (M +
H)+. Anal. (C10H12N4S‚0.33H2O) C, H, N.
[4-(2,4-Dim eth ylth ia zol-5-yl)-p yr im id in -2-yl]d im eth yl-
a m in e (10). From 3-(dimethylamino)-1-(2,4-dimethylthiazol-
5-yl)propenone (4a , R ) Me) and N,N-dimethylguanidine
nitrate. Brown solid (57%): mp 75-76 °C. Anal. RP-HPLC:
tR 10.5 min (0-60% MeCN, purity 100%). 1H NMR (DMSO-
d6): δ 2.60 (s, 3H, CH3), 3.11 (s, 6H, CH3), 6.77 (d, 1H, J ) 5.5
Hz, Py-H), 8.34 (d, 1H, J ) 5.5 Hz, Py-H). 13C NMR (DMSO-
d6): δ 18.57, 19.57, 37.15, 106.11, 131.47, 152.36, 158.42,
159.37, 162.18, 166.57. MS (ESI+) m/z 235.22 (M + H)+.
[4-(2,4-Dim et h ylt h ia zol-5-yl)-p yr im id in -2-yl]p h en yl-
a m in e (11). From 3-(dimethylamino)-1-(2,4-dimethylthiazol-
5-yl)propenone (4a , R ) Me) and phenylguanidine nitrate (6,
R1 ) R2 ) R3 ) H). Pale pink solid (48%): mp 153-155 °C;
Anal. RP-HPLC: tR 17.7 min (0-60% MeCN, purity 100%).
1H NMR (DMSO-d6): δ 2.61 (s, 3H, CH3), 2.64 (s, 3H, CH3),
6.95 (m, 1H, Ph-H), 7.06 (d, 1H, J ) 5.0 Hz, Py-H), 7.28 (m,
2H, Ph-H), 7.77 (m, 2H, Ph-H), 8.49 (d, 1H, J ) 5.5 Hz, Py-
H). 13C NMR (DMSO-d6): δ 18.57, 19.67, 37.15, 109.10, 119.67,
122.25, 129.16, 131.46, 140.98, 152.66, 158.59, 159.73, 160.37,
167.10. MS (ESI+) m/z 283.19 (M + H)+. Anal. (C15H14N4S‚
0.33H2O) C, H, N.
(2-Ch lor op h en yl)-[4-(2,4-d im eth ylth ia zol-5-yl)p yr im i-
d in -2-yl]a m in e (12). From 3-(dimethylamino)-1-(2,4-dimeth-
ylthiazol-5-yl)propenone (4a, R ) Me) and 2-chlorophenylguani-
dine nitrate (6, R1 ) Cl, R2 ) R3 ) H). Pale yellow solid
(95%): mp 190-191 °C. Anal. RP-HPLC: tR 20.3 min (0-60%
MeCN, purity 100%). 1H NMR (DMSO-d6): δ 2.55 (s, 3H, CH3),
2.60 (s, 3H, CH3), 7.04 (d, 1H, J ) 5.3 Hz, Py-H), 7.16 (m, 1H,
Ph-H), 7.34 (m, 1H, Ph-H), 7.49 (m, 1H, Ph-H), 7.76 (m, 1H,
Ph-H), 8.43 (d, 1H, J ) 5.1 Hz, Py-H), 8.85 (bs, 1H, NH). 13C
NMR (DMSO-d6): δ 18.47, 19.63, 109.57, 126.18, 126.74,
128.04, 128.24, 130.12, 130.91, 136.98, 152.95, 158.86, 159.85,
160.77, 167.08. MS (ESI+) m/z 316.51 (M + H)+. Anal. (C15H13
ClN4S) C, H, N.
-
(3-Ch lor op h en yl)-[4-(2,4-d im eth ylth ia zol-5-yl)p yr im i-
d in -2-yl]a m in e (13). From 3-(dimethylamino)-1-(2,4-dimeth-
ylthiazol-5-yl)propenone (4a, R ) Me) and 3-chlorophenylguani-
dine nitrate (6, R1 ) H, R2 ) Cl, R3 ) H). Light yellow solid
(52%): mp 191-192 °C. Anal. RP-HPLC: tR 19.8 min (10-
70% MeCN, purity 100%). 1H NMR (DMSO-d6): δ 2.52 (s, 3H,
CH3), 2.64 (s, 3H, CH3), 6.99 (m, 1H, Ph-H), 7.13 (d, 1H, J )
5.1 Hz, Py-H), 7.30 (m, 1H, Ph-H), 8.04 (s, 1H, Ph-H), 8.55 (d,
1H, J ) 5.1 Hz, Py-H), 9.88 (bs, 1H, NH). 13C NMR (DMSO-
d6): δ 18.59, 19.69, 109.76, 117.88, 121.67, 130.77, 131.25,
133.70, 142.56, 152.96, 158.62, 159.84, 160.05, 167.08. MS
(ESI+) m/z 316.51 (M + H)+. Anal. (C15H13ClN4S) C, H, N.
(4-Ch lor op h en yl)-[4-(2,4-d im eth ylth ia zol-5-yl)p yr im i-
d in -2yl]a m in e (14). From 3-(dimethylamino)-1-(2,4-dimeth-
ylthiazol-5-yl)propenone (4a, R ) Me) and 4-chlorophenylguani-
dine nitrate (6, R1 ) R2 ) H, R3 ) Cl). Light yellow solid
(30%): mp 291-292 °C. Anal. RP-HPLC: tR 13.5 min (10-
70% MeCN, purity 100%). 1H NMR (DMSO-d6): δ 2.42 (s, 3H,
CH3), 3.08 (s, 3H, CH3), 6.89 (d, 1H, J ) 5.5 Hz, Py-H), 7.28
(m, 2H, Ph-H), 7.77 (m, 2H, Ph-H), 8.32 (d, 1H, J ) 5.3 Hz,
Py-H), 9.55 (bs, 1H, NH). 13C NMR (DMSO-d6): δ 19.14,
107.96, 118.57, 120.93, 120.96, 125.25, 128.86, 140.34, 152.96,
158.31, 159.39, 159.92, 169.57. MS (ESI+) m/z 318.04 (M +
H)+.
4-(2,4-Dim et h ylt h ia zol-5-yl)p yr im id in -2-yla m in e (8).
From 3-(dimethylamino)-1-(2,4-dimethylthiazol-5-yl)propenone
(4a , R ) Me) and guanidine carbonate. Yellow solid (33%): mp
177-178 °C. Anal. RP-HPLC: tR 8.6 min (0-60% MeCN,

2-Anilino-4-(thiazol-5-yl)pyrimidine CDK Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7 1671
(2-F lu or op h en yl)-[4-(2,4-d im eth ylth ia zol-5-yl)p yr im i-
d in -2-yl]a m in e (15). From 3-(dimethylamino)-1-(2,4-dimeth-
ylthiazol-5-yl)propenone (4a, R ) Me) and 2-fluorophenylguani-
dine nitrate (6, R1 ) F, R2 ) R3 ) H). Grey solid (100%): mp
182-183 °C. Anal. RP-HPLC: tR 17.4 min (10-70% MeCN,
113.30, 116.29, 116.50, 120.51. MS (ESI+) m/z 351.42 (M +
H)+. Anal. (C16H13F3N4S) C, H, N.
3-[4-(2,4-Dim et h ylt h ia zol-5-yl)p yr im id in -2-yla m in o]-
p h en ol (21). From 3-(dimethylamino)-1-(2,4-dimethylthiazol-
5-yl)propenone (4a , R ) Me) and N-(3-hydroxyphenyl)-
guanidine nitrate (6, R1 ) R2 ) OH, R3 ) H). Orange solid
(36%): mp 235 °C (dec). Anal. RP-HPLC: tR 12.1 min (10-
70% MeCN, purity 100%). 1H NMR (DMSO-d6): δ 2.62 (s, 3H,
CH3), 2.64 (s, 3H, CH3), 6.37 (m, 1H, Ph-H), 7.04 (m, 2H, Py-H
& Ph-H), 7.21 (m, 2H, Ph-H), 8.48 (d, 1H, J ) 5.1 Hz, Py-H),
9.21 (s, 1H, OH/NH), 9.50 (s, 1H, NH/OH). 13C NMR (DMSO-
d6): δ 18.51, 19.65, 106.98, 109.05, 109.59, 110.76, 129.72,
131.41, 141.97, 152.61, 158.19, 158.67, 159.48, 160.29, 167.16.
MS (ESI+) m/z 299.29 (M + H)+.
3-[4-(4-Meth yl-2-m eth yla m in oth ia zol-5-yl)p yr im id in -2-
yla m in o]p h en ol (22). From 3-(dimethylamino)-1-(2-methyl-
amino-4-methylthiazol-5-yl)propenone (4c, R ) Me) and N-(3-
hydroxyphenyl)guanidine nitrate (6, R1 ) R2 ) OH, R3 ) H).
Yellow solid (19%): mp 251 °C (dec). Anal. RP-HPLC: tR 16.3
min (0-60% MeCN, purity 100%). 1H NMR (DMSO-d6): δ 2.86
(s, 3H, CH3), 3.24 (s, 3H, CH3), 6.36 (d, 1H, J ) 4.9 Hz, Ph-H),
6.89 (d, 1H, J ) 5.5 Hz, Py-H), 7.03 (t, 1H, J ) 8.7 Hz, Ph-H),
7.24 (bs, 1H, Ph-H), 8.06 (bs, 1H, OH), 8.33 (d, 1H, J ) 4.5
Hz, Py-H), 9.22 (s, 1H, NH), 9.31 (s, 1H, NH). 13C NMR
(DMSO-d6): δ 19.33, 31.42, 106.75, 107.45, 109.15, 110.54,
118.49, 129.59, 142.37, 152.92, 158.15, 158.17, 159.33, 160.20,
169.96. MS (ESI+) m/z 314.02 (M + H)+. Anal. (C15H15N5OS)
C, H, N.
1
purity 100%). H NMR (DMSO-d6): δ 2.56 (s, 3H, CH3), 2.60
(s, 3H, CH3), 7.03 (d, 1H, J ) 5.1 Hz, Py-H), 7.15-7.25 (m,
3H, Ph-H), 7.69 (m, 1H, Ph-H), 8.44 (d, 1H, J ) 5.3 Hz, Py-
H), 9.12 (bs, 1H, NH). 13C NMR (DMSO-d6): δ 18.46, 19.60,
109.40, 116.20, 124.76, 125.60, 126.10, 127.80, 131.01, 152.86,
154.96, 156.91, 158.82, 159.73, 160.79, 166.99. MS (ESI+) m/z
301.10 (M + H)+. Anal. (C15H13FN4S‚0.5H2O) C, H, N.
(3-F lu or op h en yl)-[4-(2,4-d im eth ylth ia zol-5-yl)p yr im i-
d in -2-yl]a m in e (16). From 3-(dimethylamino)-1-(2,4-dimeth-
ylthiazol-5-yl)propenone (4a, R ) Me) and 3-fluorophenylguani-
dine nitrate (6, R1 ) H, R2 ) F, R3 ) H). Grey solid (69%):
mp 205 °C (dec). Anal. RP-HPLC: tR 18.0 min (10-70% MeCN,
1
purity 100%). H NMR (DMSO-d6): δ 2.63 (s, 3H, CH3), 2.66
(s, 3H, CH3), 6.75 (m, 1H, Ph-H), 7.13 (d, 1H, J ) 5.1 Hz, Py-
H), 7.30 (m, 1H, Ph-H), 7.48 (m, 1H, Ph-H), 7.81 (m, 1H, Ph-
H), 8.55 (d, 1H, J ) 5.5 Hz, Py-H), 9.89 (bs, 1H, NH). 13C NMR
(DMSO-d6): δ 18.58, 19.70, 106.00, 108.35, 109.69, 115.28,
130.68, 142.90, 152.89, 158.62, 159.81, 160.09, 162.06, 163.97,
167.25. MS (ESI+) m/z 300.47 (M + H)+. Anal. (C15H13FN4S)
C, H, N.
(4-F lu or op h en yl)-[4-(2,4-d im eth ylth ia zol-5-yl)p yr im i-
d in -2-yl]a m in e (17). From 3-(dimethylamino)-1-(2,4-dimeth-
ylthiazol-5-yl)propenone (4a, R ) Me) and 4-fluorophenylguani-
dine nitrate (6, R1 ) R2 ) H, R3 ) F). Grey solid (89%): mp
199-200 °C. Anal. RP-HPLC: tR 16.4 min (10-70% MeCN,
3-[4-(2-Eth yla m in o-4-m eth ylth ia zol-5-yl)p yr im id in -2-
yla m in o]p h en ol (23). From 3-(dimethylamino)-1-(2-(ethyl-
amino)-4-methylthiazol-5-yl)propenone (4c, R ) Et) and N-(3-
hydroxyphenyl)guanidine nitrate (6, R1 ) R2 ) OH, R3 ) H).
Light yellow solid. Anal. RP-HPLC: tR 17.7 min (0-60%
1
purity 100%). H NMR (DMSO-d6): δ 2.61 (s, 3H, CH3), 2.63
(s, 3H, CH3), 7.06 (d, 1H, J ) 5.2 Hz, Py-H), 7.13 (m, 2H, Ph-
H), 7.75 (m, 2H, Ph-H), 8.49 (d, 1H, J ) 5.3 Hz, Py-H), 9.66
(bs, 1H, NH). 13C NMR (DMSO-d6): δ 18.55, 19.65, 109.11,
115.55, 121.35, 121.38, 131.38, 137.34, 152.69, 156.94, 158.60,
158.83, 159.70, 160.28, 167.05. MS (ESI+) m/z 300.41(M + H)+.
Anal. (C15H13FN4S) C, H, N.
1
MeCN, purity 100%). H NMR (DMSO-d6): δ 1.15 (t, 3H, J )
7.5 Hz, CH3), 2.49 (s, 3H, CH3), 3.25 (m, 2H, CH2), 6.33 (d,
1H, J ) 8.3 Hz, Ph-H), 6.86 (d, 1H, J ) 5.4 Hz, Py-H), 7.00 (t,
1H, J ) 7.7 Hz, Ph-H), 7.21 (m, 2H, Ph-H and OH), 8.10 (t,
1H, J ) 5.9 Hz, Ph-H), 8.29 (d, 1H, J ) 5.4 Hz, Py-H), 9.19 (s,
1H, NH), 9.28 (s, 1H, NH). 13C NMR (DMSO-d6): δ 14.97,
19.34, 106.72, 106.74, 107.47, 109.13, 110.53, 118.21, 129.58,
142.38, 152.85, 158.15, 159.34, 160.20, 169.02. MS (ESI+) m/z
328.05 (M + H)+.
[4-(2,4-Dim eth ylth ia zol-5-yl)p yr im id in -2-yl]-(2-tr iflu o-
r om eth ylp h en yl)a m in e (18). From 3-(dimethylamino)-1-
(2,4-dimethylthiazol-5-yl)propenone (4a , R ) Me) and N-(2-
trifluoromethylphenyl)guanidine nitrate (6, R1 ) CF3, R2
)
R3 ) H). Yellow solid (31%): mp 178-180 °C. Anal. RP-
1
HPLC: tR 18.6 min (10-70% MeCN, purity 100%). H NMR
4-[4-(2,4-Dim eth yl-th ia zol-5-yl)-p yr im id in -2-yla m in o]-
p h en ol (24). From 3-(dimethylamino)-1-(2,4-dimethylthiazol-
5-yl)propenone (4a , R ) Me) and N-(4-hydroxyphenyl)-
guanidine nitrate (6, R1 ) R2 ) H, R3 ) OH). Orange solid
(81%): mp 230 °C (dec). Anal. RP-HPLC: tR 10.9 min (10-
70% MeCN, purity 100%). 1H NMR (CDCl3): δ 2.59 (s, 3H,
CH3), 2.62 (s, 3H, CH3), 6.69 (d, 1H, J ) 8.8 Hz, Ph-H), 6.96
(d, 1H, J ) 5.1 Hz, Py-H), 7.47 (d, 1H, J ) 8.9 Hz, Ph-H), 8.41
(d, 1H, J ) 5.1 Hz, Py-H), 9.03 (bs, 1H, NH/OH), 9.29 (bs, 1H,
OH/NH). 13C NMR (DMSO-d6): δ 18.52, 19.62, 108.25, 115.60,
121.96, 131.57, 132.48, 152.42, 153.13, 158.54, 159.58, 160.63,
166.85. MS (ESI+) m/z 299.23 (M + H)+. Anal. (C15H14N4OS‚
0.33H2O) C, H, N.
4-[4-(4-Meth yl-2-m eth yla m in oth ia zol-5-yl)p yr im id in -2-
yla m in o]p h en ol (25). From 3-(dimethylamino)-1-(2-methyl-
amino-4-methylthiazol-5-yl)propenone (4c, R ) Me) and N-(4-
hydroxyphenyl)guanidine nitrate (6, R1 ) R2 ) H, R3 ) OH).
Orange solid (60%): mp 213-214 °C. Anal. RP-HPLC: tR 9.7
min (10-70% MeCN, purity 100%). 1H NMR (DMSO-d6): δ
2.53 (s, 3H, CH3), 2.98 (s, 3H, CH3), 6.77 (d, 1H, J ) 8.8 Hz,
Ph-H), 6.87 (d, 1H, J ) 5.6 Hz, Py-H), 7.44 (d, 1H, J ) 8.8 Hz,
Ph-H), 8.21 (d, 1H, J ) 5.4 Hz, Py-H). 13C NMR (DMSO-d6):
δ 19.29, 31.36, 106.71, 115.54, 116.84, 118.69, 121.62, 128.22,
132.87, 152.77, 152.78, 158.27, 159.23, 160.39, 169.81. MS
(ESI+) m/z 313.61.
(DMSO-d6): δ 2.07 (s, 3H, CH3), 2.58 (s, 3H, CH3), 6.99 (d,
1H, J ) 5.3 Hz, Py-H), 7.41 (m, 1H, Ph-H), 7.67 (m, 1H, Ph-
H), 7.73 (m, 1H, Ph-H), 8.39 (d, 1H, J ) 5.3 Hz, Py-H), 8.84
(bs, 1H, NH). 13C NMR (DMSO-d6): δ 18.55, 19.62, 109.76,
115.32, 118.24, 123.99, 126.16, 130.23, 131.29, 141.84, 153.00,
158.56, 159.88, 160.04, 167.22. MS (ESI+) m/z 351.31 (M +
H)+. Anal. (C16H13F3N4S) C, H, N.
[4-(2,4-Dim eth ylth ia zol-5-yl)p yr im id in -2-yl]-(3-tr iflu o-
r om eth ylp h en yl)a m in e (19). From 3-(dimethylamino)-1-
(2,4-dimethylthiazol-5-yl)propenone (4a , R ) Me) and N-(3-
trifluoromethylphenyl)guanidine nitrate (6, R1 ) H, R2 ) CF3,
R3 ) H). Orange solid (60%): mp 194-195 °C. Anal. RP-
1
HPLC: tR 20.3 min (10-70% MeCN, purity 100%). H NMR
(DMSO-d6): δ 2.63 (s, 3H, CH3), 2.64 (s, 3H, CH3), 7.16 (d,
1H, J ) 5.3 Hz, Py-H), 7.29 (s, 1H, Ph-H), 7.51 (m, 1H, Ph-H),
8.35 (s, 1H, Ph-H), 8.57 (d, 1H, J ) 5.3 Hz, Py-H), 10.03 (s,
1H, NH). 13C NMR (DMSO-d6): δ 18.39, 19.59, 109.40, 123.50,
125.50, 126.46, 127.05, 130.54, 133.64, 138.06, 152.96, 158.90,
159.79, 161.88, 166.94. MS (ESI+) m/z 350.86. Anal. (C16H13
F3N4S‚0.5H2O) C, H, N.
-
[4-(2,4-Dim eth ylth ia zol-5-yl)p yr im id in -2-yl]-(4-tr iflu o-
r om eth ylp h en yl)a m in e (20). From 3-(dimethylamino)-1-
(2,4-dimethylthiazol-5-yl)propenone (4a , R ) Me) and N-(4-
trifluoromethylphenyl)guanidine nitrate (6, R1 ) R2 ) H,
R3 ) CF3). Orange solid (38%): mp 183-185 °C. Anal. RP-
4-[4-(2-Am in o-4-m eth ylth ia zol-5-yl)p yr im id in -2-yla m i-
n o]p h en ol (26). From N′-[5-(3-dimethylaminoacryloyl)-4-
methylthiazol-2-yl]-N,N-dimethylformamidine (4b) and N-(4-
hydroxyphenyl)guanidine nitrate (6, R1 ) R2 ) H, R3 ) OH).
Yellow solid (43%): mp 212-213 °C. Anal. RP-HPLC: tR 10.5
min (0-60% MeCN, purity 100%). 1H NMR (DMSO-d6): δ 2.40,
(s, 3H, CH3), 6.65 (m, 2H, Ph-H), 6.77 (d, 1H, J ) 5.5 Hz, Py-
1
HPLC: tR 21.1 min (10-70% MeCN, purity 100%). H NMR
(DMSO-d6): δ 2.73 (s, 3H, CH3), 2.75 (s, 3H, CH3), 7.06 (d,
1H, J ) 5.3 Hz, Py-H), 7.28 (s, 1H, Ph-H), 7.40 (bs, 1H, NH),
7.65 (m, 1H, Ph-H), 7.83 (m, 1H, Ph-H), 8.49 (d, 1H, J ) 5.3
Hz, Py-H). 13C NMR (DMSO-d6): δ 47.96, 47.54, 52.60, 91.95,
96.57, 98.20, 99.16, 99.21, 99.28, 100.13, 102.50, 108.08,

1672 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Wang et al.
H), 7.43 (bs, 2H, NH2), 7.48 (d, 1H, J ) 8.9 Hz, Ph-H), 8.23 (d,
1H, J ) 5.3 Hz, Py-H), 9.01 (bs, 1H, NH2), 9.05 (bs, 1H, OH).
13C NMR (DMSO-d6): δ 19.08, 106.75, 106.76, 115.52, 115.54,
118.92, 121.55, 132.83, 152.31, 152.83, 158.27, 158.28, 159.27,
160.38, 169.40. MS (ESI+) m/z 300.07 (M + H)+.
H), 7.54 (t, 1H, J ) 8.2 Hz, Ph-H), 7.77 (m, 1H, Ph-H), 8.40
(d, 1H, J ) 5.5 Hz, Py-H), 8.85 (m, 1H, Ph-H), 9.90 (bs, 1H,
NH). MS (ESI+) m/z 369.52 (M + H)+.
[4-(2-Am in o-4-m eth ylth ia zol-5-yl)p yr im id in -2-yl](3-n i-
tr op h en yl)a m in e (32). From 3-(dimethylamino)-1-(2-amino-
4-methylthiazol-5-yl)propenone (4c, R ) H) and 3-nitrophen-
ylguanidine nitrate (6, R1 ) H, R2 ) NO2, R3 ) H); Light yellow
solid (80%): mp 270-272 °C; Anal. RP-HPLC: tR 13.5 min
(10-70% MeCN, purity 100%).1H NMR (CDCl3) δ: 2.50 (s, 3H,
CH3), 6.98 (d, 1H, J ) 5.5 Hz, Py-H), 7.61 (m, 2H, Ph-H), 7.79
(m, 1H, Ph-H), 8.10 (m, 1H, Ph-H), 8.40 (d, 1H, J ) 5.4 Hz,
Py-H), 8.82 (bs, 2H, NH2). 13C NMR (DMSO-d6): δ 19.07,
108.72, 113.10, 116.10, 118.30, 125.23, 130.30, 142.62, 148.77,
153.18, 158.33, 159.50, 159.72, 169.75. MS (ESI+) m/z 329.44
(M + H)+; Anal. (C14H12N6O2S.HCl) C, H, N.
[4-(4-Meth yl-2-p yr id in -2-ylth ia zol-5-yl)p yr im id in -2-yl]-
(3-n itr op h en yl)a m in e (33). To a solution of thionicotinamide
(5.02 g, 38 mmol) and pyridine (3.0 g, 38 mmol) in MeOH (10
mL) was added 3-chloropenta-2,4-dione (5.12 g, 38 mmol)
dropwise. After heating at 75 °C for 4 h the mixture was
concentrated. The resulting precipitates were collected and
washed with EtOAc several times to afford a yellow solid of
1-(4-methyl-2-pyridin-3-ylthiazol-5-yl)ethanone (4.33 g, 52%).
An aliquot of this material (2.0 g, 9.2 mmol) was treated with
dimethoxymethyldimethylamine (4 mL, 30 mmol) at 80 °C for
22 h. The mixture was concentrated and the resulting pre-
cipitates were collected and washed with EtOAc/PE (1:5)
several times. The dark brown residue was crystallized from
EtOAc/PE (1:2) to afford 3-(dimethylamino)-1-(4-methyl-2-
pyridin-3-ylthiazol-5-yl)propenone (2.05 g, 82%). 1H NMR
(CDCl3): δ 2.94 (s, 3H, CH3), 2.96 (s, 3H, CH3), 3.50 (s, 3H,
CH3), 5.48 (d, 1H, J ) 12.0 Hz, CH), 7.38 (m, 1H, pyridyl-H),
7.77 (d, 1H, J ) 12.1 Hz, CH), 8.28 (m, 1H, pyridyl-H), 8.66
(m, 1H, pyridyl-H), 9.16 (m, 1H, pyridyl-H). 13C NMR (DMSO-
d6): δ 18.49, 37.93, 45.34, 55.02, 94.30, 124.94, 129.36, 134.29,
135.85, 147.59, 151.87, 154.88, 163.16, 179.42.
2-[N-(3-Nitr op h en yl)]-4-(2,4-d im eth ylth ia zol-5-yl)p y-
r im id in ea m in e (27). From 3-(dimethylamino)-1-(2,4-dimeth-
ylthiazol-5-yl)propenone (4a , R ) Me) and 3-nitrophenylguani-
dine nitrate (6, R1 ) H, R2 ) NO2, R3 ) H). Yellow solid
(72%): mp 176-178 °C. Anal. RP-HPLC: tR 18.2 min (10-
70% MeCN, purity 100%). 1H NMR (CDCl3): δ 2.64 (s, 3H,
CH3), 2.65 (s, 3H, CH3), 7.19 (d, 1H, J ) 5.3 Hz, Ph-H), 7.58
(t, 1H, J ) 8.2 Hz, Ph-H), 7.80 (m, 1H, Ph-H), 8.05 (m, 1H,
Ph-H), 8.60 (d, 1H, J ) 5.2 Hz, Py-H), 8.91 (m, 1H, Ph-H),
10.20 (br. s, 1H, NH). 13C NMR (DMSO-d6): δ 18.58, 19.66,
110.04, 113.12, 116.41, 125.36, 130.36, 131.18, 142.33, 148.80,
153.10, 158.65, 159.81, 159.87, 167.28. MS (ESI+) m/z 327.62
(M + H)+. Anal. (C15H13N5O2S) C, H, N.
[4-(2,4-Dim eth ylth ia zol-5-yl)-p yr im id in -2-yl]m eth yl-(3-
n itr op h en yl)a m in e (28). Compound 27 (0.5 g, 1.53 mmol)
was dissolved in anhydrous DMF (5 mL), and sodium hydride
(95%, 0.043 g, 1.68 mmol) was added in portions. The mixture
was stirred for 30 min before the dropwise addition of
iodomethane (0.115 mL, 1.68 mmol). After strirring for 1 h,
the mixture was poured into water (70 mL) and extracted with
EtOAc. The extract was washed with brine, dried on Na2SO4
and the solvent was evaporated to leave a yellow residue,
which was purified by flash chromatography (4:1 EtOAc/
heptane). Recrystallization from EtOAc afforded the title
product as yellow crystals (0.33 g, 62%): mp 145-146 °C. Anal.
RP-HPLC: tR 20.1 min (0-60% MeCN, purity 100%). 1H NMR
(CDCl3): δ 2.53 (s, 3H, CH3), 2.59 (s, 3H, CH3), 3.56 (s, 3H,
CH3), 7.05 (d, 1H, J ) 4.9 Hz, Py-H), 7.67 (t, 1H, J ) 8.3 Hz,
Ph-H), 7.88 (m, 1H, Ph-H), 8.04 (d, 1H, J ) 8.3 Hz, Ph-H),
8.28 (s, 1H, Ph-H), 8.46 (d, 1H, J ) 5.4 Hz, Py-H). 13C NMR
(DMSO-d6): δ 18.50, 19.61, 38.29, 109.25 120.22, 121.46,
130.48, 130.51, 133.28, 146.56, 148.56, 153.09, 158.60, 159.56,
161.22, 167.17. MS (ESI+) m/z 342.17 (M + H)+. Anal.
(C15H13N5O2S) C, H, N.
The title product was obtained from 3-(dimethylamino)-1-
(4-methyl-2-pyridin-3-ylthiazol-5-yl)propenone (0.27 g, 1.0 mmol)
and 3-nitrophenylguanidine nitrate (0.24 g, 1.0 mmol; 6, R1 )
H, R2 ) NO2, R3 ) H) as yellow crystals (0.15 g, 39%): mp
215-217 °C. Anal. RP-HPLC: tR 20.2 min (10-70% MeCN,
[4-(2-N,N-Dim eth yla m in o-4-m eth ylth ia zol-5-yl)p yr im i-
d in -2-yl]-(3-n itr op h en yl)a m in e (29). From 3-(dimethylami-
no)-1-(2-N,N-dimethylamino-4-methylthiazol-5-yl)propenone (4a,
R ) NMe2) and 3-nitrophenylguanidine nitrate (6, R1 ) H,
R2 ) NO2, R3 ) H). Yellow solid (47%): mp 250-252 °C. Anal.
RP-HPLC: tR 14.8 min (10-70% MeCN, purity 100%). 1H
NMR (CDCl3): δ 2.52 (s, 3H, CH3), 3.11 (s, 6H, CH3), 7.02 (d,
1H, J ) 5.4 Hz, Py-H), 7.54 (m, 1H, Ph-H), 7.79 (m, 1H, Ph-
H), 7.97 (m, 1H, Ph-H), 8.40 (d, 1H, J ) 5.3 Hz, Py-H), 8.45
(s, 1H, NH), 9.05 (m, 1H, Ph-H), 10.02 (s, 1H, NH). 13C NMR
(DMSO-d6): δ 19.43, 108.08, 112.78, 116.19, 119.75, 125.32,
130.32, 142.65, 148.94, 154.09, 158.59, 159.12, 159.70, 170.41.
MS (ESI+) m/z 357.05 (M + H)+.
1
purity 100%). H NMR (CDCl3): δ 2.82 (s, 3H, CH3), 7.24 (d,
1H, J ) 5.2 Hz, Py-H), 7.53 (m, 2H, pyridyl-H), 7.82 (m, 1H,
pyridyl-H), 8.35 (m, 1H, pyridyl-H), 8.62 (d, 1H, J ) 5.4 Hz,
Py-H), 8.68 (m, 1H, Ar-H), 9.21 (m, 2H, pyridyl-H), 10.24 (sb,
1H, NH). 13C NMR (DMSO-d6): δ 18.89, 55.06, 110.00, 113.06,
116.64, 119.44, 125.05, 125.55, 129.33, 139.49, 131.17, 131.91,
142.26, 147.63, 148.92, 152.13, 156.54, 159.89, 164.54. MS
(ESI+) 391.02 [M + H]+; Anal. (C19H14N6O2S) C, H, N.
[4-(2,4-Dim et h ylt h ia zol-5-yl)p yr im id in -2-yl](4-n it r o-
p h en yl)a m in e (34). From 3-(dimethylamino)-1-(2,4-dimeth-
ylthiazol-5-yl)propenone (4a , R ) Me) and 4-nitrophenylguani-
dine nitrate (6, R1 ) H, R2 ) H, R3 ) NO2). Grey solid: mp
302-304 °C. Anal. RP-HPLC: tR 19.97 min (10-70% MeCN,
[4-(4-Met h yl-2-m et h yla m in ot h ia zol-5-yl)p yr im id in -2-
yl](3-n itr op h en yl)a m in e (30). From 3-(dimethylamino)-1-
(2-methylamino-4-methylthiazol-5-yl)propenone (4c, R ) Me)
and 3-nitrophenylguanidine nitrate (6, R1 ) H, R2 ) NO2,
R3 ) H). Orange solid (22%): mp 302-304 °C. Anal. RP-
1
purity 100%). H NMR (CDCl3): δ 2.66 (s, 3H, CH3), 2.68 (s,
3H, CH3), 7.28 (d, 1H, J ) 5.2 Hz, Py-H), 8.05 (d, 2H, J ) 9.3
Hz, Ph-H), 8.23 (d, 2H, J ) 9.3 Hz, Ph-H), 8.64 (d, 1H, J ) 5.3
Hz, Py-H), 10.49 (sb, 1H, NH). 13C NMR (DMSO-d6): δ 18.61,
19.70, 110.00, 110.91, 118.53, 125.66, 125.67, 131.02, 141.18,
147.65, 153.30, 158.84, 159.62, 159.97, 167.62. MS (ESI+) m/z
328.08 [M + H]+.
1
HPLC: tR 14.8 min (10-70% MeCN, purity 100%). H NMR
(CDCl3): δ 2.88 (s, 3H, CH3), 3.09 (s, 3H, CH3), 7.02 (d, 1H,
J ) 5.4 Hz, Py-H), 7.56 (t, 1H, J ) 7.8 Hz, Ph-H), 7.79 (m,
1H, Ph-H), 8.02 (m, 1H, Ph-H), 8.17 (bs, 1H, NH), 8.42 (d, 1H,
J ) 5.2 Hz, Py-H), 8.98 (m, 1H, Ph-H). 13C NMR (DMSO-d6):
δ 19.36, 31.64, 108.33, 112.94, 116.10, 118.31, 125.26, 130.27,
142.67, 148.86, 153.79, 158.36, 159.34, 159.72, 170.50. MS
(ESI+) m/z 342.98 (M + H)+. Anal. (C15H14N6O2S‚1.5H2O) C,
H, N.
N-[4-(2,4-Dim eth ylth ia zol-5-yl)pyr im id in -2-yl]ben zen e-
1,3-d ia m in e (35). Compound 27 (0.5 g, 1.53 mmol) was
suspended in EtOH (5 mL) and glacial acetic acid (2.5 mL).
The mixture was heated to reflux, and iron powder (325 mesh,
0.85 g, 15.3 mmol) was added in potions. Heating was
continued for 30 min, and then the hot mixture was filtered
through a pad of Celite. The filtrate was cooled, evaporated,
treated with saturated aq NaHCO3 solution (75 mL), and
extracted with EtOAc. The extract was washed with brine,
dried on Na2SO4, and filtered. The solvent was evaporated to
leave an orange solid, which was purified by flash chroma-
tography (50:1 CH2Cl2:MeOH.). Crystallization from EtOAc
afforded the title product as pale yellow crystals (211 mg,
[4-(2-Allyla m in o-4-m eth ylth ia zol-5-yl)p yr im id in -2-yl]-
(3-n itr op h en yl)a m in e (31). From 3-(dimethylamino)-1-(2-
allylamino-4-methylthiazol-5-yl)propenone (4c, R ) All) and
3-nitrophenylguanidine nitrate (6, R1 ) H, R2 ) NO2, R3
)
H); orange solid (33%): mp 307-308 °C. Anal. RP-HPLC: tR
1
13.3 min (10-70% MeCN, purity 100%). H NMR (CDCl3): δ
2.47 (s, 3H, CH3), 3.89 (m, 1H, CH), 5.13 (m, 1H, CH), 5.24
(m, 1H, CH), 5.88 (m, 1H, CH), 6.99 (d, 1H, J ) 5.7 Hz, Py-

2-Anilino-4-(thiazol-5-yl)pyrimidine CDK Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7 1673
46%): mp 163-164 °C. Anal. RP-HPLC: tR 10.8 min (0-60%
MeCN, purity 98%). 1H NMR (DMSO-d6): δ 2.61 (s, 3H, CH3),
2.63 (s, 3H, CH3), 4.92 (bs 2H, NH2), 6.20 (d, 1H, J ) 8.3 Hz,
Ph-H), 6.90-6.92 (m, 2H, Py-H and Ph-H), 7.00-7.01 (m, 2H,
Ph-H), 8.45 (d, 1H, J ) 4.9 Hz, Py-H), 9.32 (bs, 1H, NH). 13C
NMR (DMSO-d6): δ 18.55, 19.67, 105.61, 108.20, 108.68,
108.84, 129.38, 131.53, 141.49, 149.44, 152.53, 158.56, 159.56,
160.52, 167.00. MS (ESI+) m/z 298.20 (M + H)+. Anal.
(C15H15N5S) C, H, N.
3-[4-(2,4-Dim et h ylt h ia zol-5-yl)p yr im id in -2-yla m in o]-
ben zon itr ile (36). From 3-(dimethylamino)-1-(2,4-dimeth-
ylthiazol-5-yl)propenone (4a , R ) Me) and N-(3-cyanophenyl)-
guanidine nitrate (6, R1 ) H, R2 ) CN, R3 ) H). Light yellow
crystals (28%): mp 162-163 °C. Anal. RP-HPLC: tR 17.1 min
(10-70% MeCN, purity 97%). 1H NMR (CDCl3): δ 2.59 (s, 3H,
CH3), 2.60 (s, 3H, CH3), 7.12 (d, 1H, J ) 5.0 Hz, Py-H), 7.35
(m, 1H, Ph-H), 7.45 (t, 1H, J ) 8.0 Hz, Ph-H), 7.93 (m, 1H,
Ph-H), 8.28 (bs, 1H, NH), 8.53 (d, 1H, J ) 5.0 Hz, Py-H). 13C
NMR (DMSO-d6): δ 18.59, 19.68, 110.08, 112.06, 119.75,
121.95, 124.00, 125.42, 130.62, 141.91, 153.08, 158.67, 159.90,
159.94, 167.28. MS (ESI+) m/z 308.13 (M + H)+.
N-[4-(2,4-Dim eth ylth ia zol-5-yl)p yr im id in -2-yl]N′,N′-d i-
m eth ylben zen e-1,4-d ia m in e (37). From 3-(dimethylamino)-
1-(2,4-dimethylthiazol-5-yl)propenone (4a , R ) Me) and N,N-
dimethylaminophenyl guanidine nitrate (6, R1 ) H, R2 ) H,
R3 ) NMe2). Yellow crystals (72%): mp 236-238 °C. Anal. RP-
HPLC: tR 11.2 min (0-60% MeCN, purity 97%). 1H NMR
(DMSO-d6): δ 2.60 (s, 3H, CH3), 2.62 (s, 3H, CH3), 2.82 (s, 6H,
CH3x2), 6.70 (d, 1H, J ) 8.8 Hz, Ph-H), 6.94 (d, 1H, J ) 5.3
Hz, Py-H), 7.53 (d, 1H, J ) 9.0 Hz, Ph-H), 8.40 (d, 1H, J ) 5.3
Hz, Py-H), 9.27 (s, 1H, NH). 13C NMR (CDCl3): δ 16.47, 17.71,
39.52, 106.51, 111.77, 120.38, 127.53, 129.73, 145.76, 150.50,
156.83, 157.40, 158.75, 165.03. MS (ESI+) m/z 326.20 (M +
NH)+. Anal. (C17H19N5S‚0.15H2O) C, H, N.
followed by ether to give 19.0 g of 4-N,N-dimethylamino-3-
nitrophenylguanidine nitrate (6, R1 ) H, R2 ) NO2, R3 ) NMe2)
as a tan solid. This was stirred with potassium carbonate
(15.04 g, 108.8 mmol) in 2-methoxyethanol (250 mL) for 10
min before addition of 3-(dimethylamino)-1-(2,4-dimethylthi-
azol-5-yl)propenone (4a , R ) Me, 9.53 g, 45.33 mmol). The
mixture was heated at 125 °C for 18 h, then concentrated,
diluted with EtOAc, filtered through a pad of silica, and
evaporated to leave dark oil, which was purified by flash
chromatography (EtOAc). Recrystallization from toluene yielded
the title product as a reddish solid (7.3 g, 43%).
2-Ch lor o-N4-[4-(2,4-d im eth ylth ia zol-5-yl)p yr im id in -2-
yl]-N1,N1-d im eth ylben zen e-1,4-d ia m in e (39). A solution of
3-chloro-4-fluoronitrobenzene (3.0 g, 17.1 mmol), dimethyl-
amine hydrochloride (1.53 g, 18.8 mmol), and potassium
carbonate (4.96 g, 35.9 mmol) in dimethyl sulfoxide (20 mL)
was heated in a sealed tube at 105 °C for 18 h. On cooling,
the reaction mixture was poured into water (200 mL) and was
extracted with EtOAc. The extract was washed with brine,
dried on MgSO4, filtered, and evaporated to afford 3.47 g of
3-chloro-4-(dimethylamino)nitrobenzene as a yellow solid. An
aliquot (3.4 g, 16.95 mmol) of this material was dissolved in
of ethanol/acetic acid (20 mL) with warming. Iron powder (325
mesh, 9.5 g, 170 mmol) was added in portions. The mixture
was then heated on a steam bath for 30 min, cooled and filtered
through a pad of Celite, and the filtratewas evaporated to give
3.33 g of 3-chloro-4-(dimethylamino)aniline as a dark solid. A
solution of this material in ethanol (10 mL) was treated with
nitric acid (69%, 2.6 mL, 40.6 mmol) dropwise, followed by
cyanamide (50% solution in water, 5.3 mL, 67.78 mmol). After
heating for 18 h at reflux, the reaction mixture was cooled to
room temperature, poured into ether (100 mL), and basified
with sodium hydroxide solution (2 M, 100 mL). The ethereal
layer was separated, and the aqueous phase was extracted
with ether. The combined organic phases were washed with
brine, dried on MgSO4, filtered, and evaporated to give dark-
brown oil, which solidified on standing to afford of 4-N,N-
dimethylamino-3-chlorophenylguanidine nitrate (1.6 g, 34%;
N4-[4-(2,4-Dim et h ylt h ia zol-5-yl)p yr im id in -2-yl]N1,N1-
dim eth yl-2-n itr oben zen e-1,4-diam in e (38). Nitric acid (69%,
24 µL, 0.36 mmol) was added dropwise to acetic anhydride (1
mL) at room-temperature, keeping the internal temperature
below 25 °C. The mixture was stirred at room temperature
for 15 min before cooling to -5 °C in an ice-methanol bath.
Compound 38 (50 mg, 0.15 mmol) in acetic anhydride (1 mL)
was added dropwise to the cooled solution of acetyl nitrate.
After stirring with cooling for 1 h and a further 2 h at room
temperature, the mixture was poured into ice-water (20 mL)
and the pH was adjusted to 7-8 by the addition of saturated
aq NaHCO3 solution. The mixture was extracted with EtOAc
and washed with brine, dried on MgSO4, and filtered. The
solvent was evaporated, and the residue was purified by flash
chromatography (heptane/EtOAc) to afford the title product
as a pale reddish solid (32 mg, 57%). Anal. RP-HPLC: tR
1
6, R1 ) H, R2 ) Cl, R3 ) NMe2). H NMR (DMSO-d6): δ 2.71
(s, 6H, CH3), 7.12 (dd, 1H, J ) 8.5, 2.4 Hz, Ph-H), 7.18 (d, 1H,
J ) 8.8 Hz, Ph-H), 7.25 (d, 1H, J ) 2.4 Hz, Ph-H), 7.57 (bs,
4H, NH).
In an alternative preparation 3-chloro-4-fluoronitrobenzene
(5.0 g, 28.48 mmol) was dissolved in ethanol (150 mL) and
dimethylamine (5.6 M solution in ethanol, 80 mL, 448 mmol)
was added in a steady stream. The mixture was heated
overnight at reflux. It was concentrated in vacuo and water
was added. The resulting precipitate was filtered, washed with
water, and dried under vacuum at 40 °C to afford of 3-chloro-
4-(dimethylamino)nitrobenzene (5.65 g) as a yellow solid.
1
12.7min (10-70% MeCN, purity >95%): mp 192-194 °C. H
The title product was obtained 3-(dimethylamino)-1-(2,4-
dimethylthiazol-5-yl)propenone (4a , R ) Me) and 4-N,N-
dimethylamino-3-chlorophenyl guanidine nitrate (6, R1 ) H,
R2 ) Cl, R3 ) NMe2): mp 131-132 °C; RP-HPLC: tR 9.4 min
NMR (DMSO-d6): δ 2.61 (s, 3H, CH3), 2.63 (s, 3H, CH3), 2.74
(s, 6H, CH3), 7.07 (d, 1H, J ) 5.5 Hz, Py-H), 7.22 (d, 1H, J )
9.0 Hz, Ph-H), 7.77 (dd, 1H, J ) 8.7, 2.7 Hz, Ph-H), 8.38 (d,
1H, J ) 2.7 Hz), 8.51 (d, 1H, J ) 5.1 Hz, Py-H), 9.79 (bs, 1H,
NH). 13C NMR (CDCl3): δ 18.55, 19.68, 43.56, 109.30, 115.73,
120.53, 125.38, 131.32, 133.38, 140.65, 141.38, 152.87, 159.80,
159.81, 160.07, 167.11. MS (ESI+) m/z 393.32 (M + Na). Anal.
(C17H18N6O2S) C, H, N.
1
(10-70% MeCN, purity > 95%). H NMR (DMSO-d6): δ 2.95
(s, 3H, CH3), 2.97 (s, 3H, CH3), 3.00 (s, 6H, CH3), 7.38 (d, 1H,
J ) 5.2 Hz, Py-H), 7.45 (d, 1H, J ) 8.9 Hz, Ph-H), 7.93 (m,
1H, Ph-H), 8.29 (s, 1H, Ph-H), 8.82 (d, 1H, J ) 5.2 Hz, Py-H).
13C NMR (DMSO-d6): δ 18.58, 19.68, 44.35, 109.16, 118.96,
121.08, 121.16, 127.87, 131.39, 136.73, 144.82, 152.77, 158.56,
159.74, 160.12, 167.08. MS (ESI+) m/z 359.94 (M). Anal.
(C17H18ClN5S) C, H, N.
In an alternative preparation, 4-fluoro-3-nitroaniline (20 g,
128 mmol) was dissolved in ethanol (300 mL), and dimethyl-
amine (5.6 M solution in ethanol, 360 mL, 2.02 mol) was added
in a steady stream. After refluxing for 18 h, the reaction
mixture was cooled and water (100 mL) was added. The
ethanol was removed by evaporation, and the residue was
extracted with ether (3 × 100 mL). The combined organics
were washed with brine and filtered, and the solvent was
evaporated to give 4-(dimethylamino)-3-nitroaniline (22.8 g)
as black oil. This was dissolved in ethanol (80 mL), and nitric
acid (69%, 18.5 mL, 22.1 mmol) was added dropwise, followed
by cyanamide (50% w/v in water, 37 mL, 476 mmol). The
mixture was heated at reflux for 18 h. On cooling, the mixture
was poured into ether (1 L). The ethereal supernatant was
decanted, and the residue was treated with propan-2-ol,
N-[4-(2-Am in o-4-m et h yl-t h ia zol-5-yl)-p yr im id in -2-yl]-
N′,N′-d im eth ylben zen e-1,4-d ia m in e (40). From N′-[5-(3-
dimethylaminoacryloyl)-4-methylthiazol-2-yl]-N,N-dimethyl-
formamidine (4b) and N,N-dimethylaminophenylguanidine
nitrate (6, R1 ) H, R2 ) H, R3 ) NMe2): mp 204-206 °C; RP-
HPLC: tR 9.8 min (0-60% MeCN, purity > 95%). 1H NMR
(DMSO-d6): δ 2.40 (s, 3H, CH3), 3.15 (s, 3H, CH3), 3.16 (s, 3H,
CH3), 4.08 (bs, 1H, NH), 6.68, (d, 2H, J ) 8.5 Hz, Ph-H), 6.76
(d, 1H, J ) 5.5 Hz, Py-H), 7.42 (sb, 2H, NH2), 7.54 (d, 2H, J )
8.5 Hz, Ph-H), 8.22 (d, 1H, J ) 5.5 Hz, Py-H), 9.04 (sb, 1H,
NH). 13C NMR (DMSO-d6): δ 14.73, 19.06, 21.40, 60.44, 106.61,
113.57, 119.04, 121.16, 131.41, 146.68, 152.22, 158.20, 159.30,

1674 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Wang et al.
160.41, 169.40, 171.01. MS (ESI+) m/z 327.12 [M + H]+. Anal.
the solution was filter-sterilized. Medium was removed from
cells followed by a wash with 200 µL/well PBS. MTT solution
was then added at 20 µL/well and incubated in the dark at
37 °C for 4 h. MTT solution was removed and cells were again
washed with 200 µL of PBS. MTT dye was solubilized with
200 µL/well of DMSO with agitation. Absorbance was read at
540 nm and data analyzed using curve-fitting software (Graph-
Pad Prism) to determine IC50 values. The cell lines used were
obtained from the ATCC (American Type Culture Collection,
Manessas, VA).
Im m u n oblottin g. A549 Human lung carcinoma epithelial
cells were seeded at 2 × 105 cells/well of a six-well plate (9.6
cm2 growth area) in DMEM media (Sigma) supplemented with
FCS and P/S. Compound 32 (in DMSO) was added 24 h later,
and cells were harvested at the appropriate time point by
removing media and scraping with 0.7 mL of harvesting buffer.
Harvesting buffer consisted of ice cold PBS, 0.1% protease
inhibitor cocktail (Sigma), 100 mM NaF, and 1 mM Na3VO4.
Cells were spun at 10 000g for 10 min at 4 °C. Cell culture
lysis reagent (5 ×; Promega), diluted with harvesting buffer,
was added to cell pellets, and the mixtures were snap frozen
in liquid N2. Cell lysates were normalized using a protein
estimation protocol (Bio-Rad). Lysate (15 µg) was electropho-
resed on NuPAGE 10% Bis-Tris gels. Proteins were transferred
to a nitrocellulose membrane for immunoprobing. The mem-
brane was probed using antibodies against total pRb (IF-8,
clone produced in-house), pRb pT821, pRb pS249/T252 (Bio-
source), RNAP II Ser-2 (Covance), and RNAP II Ser-5 (Eu-
ropa). Detection was achieved using ECL-plus (Amersham).
Blots were stripped using the recommended protocol (100 mM
2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl pH 6.7 at
50 °C for 30 min) and reprobed.
Syn ch r on iza tion of Cells a t G1/S a n d Cell Cycle
An a lysis by F ACS. A549 cells were synchronized at the G1/S
boundary by sequentially blocking in thymidine, followed by
mimosine. The cells were plated at 5 × 105 cells on 10 cm2
dishes and grown at 37 °C for 24 h. Thymidine (2 mM) was
added to the medium, and the cells were incubated at 37 °C
for 18 h. The cells were released from thymidine block by
removing the medium, washing three times in PBS, and then
adding fresh medium. Cells were incubated for 6 h before
adding 0.5 mM mimosine. After a further 16 h incubation, the
cells were released from mimosine block as described above,
and fresh medium was added containing 0.5 µM compound
32. Cells were fixed for FACS analysis at several time points
after release from block. Cells were trypsinized to release them
from the plate and were fixed for 10 min at 37 °C in 1%
formaldehyde in PBS. They were washed once in PBS, fixed
in 70% ethanol, and stored at -20 °C for at least 24 h.
For FACS analysis, cells were pelleted, washed twice in
PBS/1% BSA, and incubated in PBS, 1% BSA, and 0.25%
Triton X-100 on ice for 15 min. Cells were pelleted and
incubated with FITC-conjugated cyclin B1 antibody (Phar-
mingen 554108) diluted 1:10 for 1 h at room temperature. Cells
were washed twice in 1 mL PBS/1% BSA and incubated in
PBS containing 36 µg/mL RNAse A and 125 µg/mL propidium
iodide for 30 min at 37 °C. Analysis of DNA content measured
by propidium iodide fluorescence (FL3 channel) and cyclin B
levels measured by FITC fluorescence (FL1 channel) was
performed on a BD-FACSCalibur flow cytometer (Becton
Dickinson).
(C16H18N6S) C, H, N.
Cr ysta llogr a p h y. The CDK2 crystals were grown by vapor
diffusion using the hanging drop method. Typically, a 2-µL
solution of CDK2 (7-8 mg/mL in the final purification buffer)
was mixed with 2 µL of precipitant solution containing 15%
PEG-6000 and 100 mM NaHEPES buffer (pH 7.8 ∼ 8.2) and
gave crystals of diffraction quality. Crystals were obtained
after 3-5 days at 4 °C and were about 0.05 mm in length.
Complexes were prepared by soaking native CDK2 crystals
in a solution containing ligand. The soaking solutions used
for 21, 25, and 32 consisted of 5 mM ligand, 20% PEG-6000,
and 100 mM HEPES (pH 7.8). For 32, the soaking solution
was supplemented with 5% EtOH. Crystals were soaked for
up to 2 h. For 37 crystals were transferred to a solution
containing 40% PEG-4000, 0.1 M Na HEPES buffer (pH 7.4)
and 15 mM NaCl from a starting solution of 20% PEG-4000.
PEG-4000 concentration was increased by 5% using 30 min
intervals. The final soaking solution contained 5% DMSO and
0.5 mM, and soaking was performed for 10 d. Crystals were
mounted in 0.05-0.1 mm cryo-loops (Hampton Research). The
crystals were immersed briefly in a cryoprotectant and then
flash-frozen in liquid N2. The cryoprotectant used was 100%
immersion oil, except for 37, where the soaking solution acted
as the cryoprotectant.
All diffraction data (Table 2) were collected at 100 K
(Cryostream, Oxford Cryosystems). Data for CDK2 complexes
with 21, 25, and 32 were collected at the Daresbury (England)
synchrotron facility on stations 14.1 or 9.6 using an ADSC
Quantum4. Data processing was carried out using the pro-
grams DENZO and SCALEPACK.35 Data for 37 were collected
at the European synchrotron radiation facility (ESRF, Greno-
ble, France) on station 14 eh2. Data processing was carried
out using the program MOSFLM.36 The molecular replacement
program MOLREP37 using PDB entries 1HCL or 1CKP as the
search model was used to solve the structures. REFMAC,38
SHELX97,39 and CNS40 were used for structural refinement.
Occupancy refinement of ligands was performed using the
programs SHELX97. A number of rounds of refinement and
model-building with the program O41 were carried out. The
coordinates of the complex structures were deposited with the
Protein Data Bank under the following accession codes: 1PXM
(21), 1PXN (25), 1PXO (32), and 1PXP (37).
Kin a se Assa ys. These were carried out as described.18 CDK
7 and 9 assays: peptide substrate (biotinyl-Ahx-(Tyr-Ser-Pro-
Thr-Ser-Pro-Ser)4-NH2; 1-2 mg/mL) and recombinant human
CDK7/cyclin H or CDK9/cyclin T1 (0.5-2 µg) were incubated
for 45 min at 30 °C in the presence of varying amounts of test
compound in 20 mM MOPS pH 7.2, 25mM â-glycerophosphate,
5 mM EGTA, 1 mM DTT, 1 mM sodium vanadate, 15 mM
MgCl2, and 100 µM ATP (containing a trace amount of
32PγATP) in a total volume of 25 µL in a 96-well microtiter
plate. The reaction was stopped by placing the plate on ice for
2 min. Avidin (50 µg) was added to each well, and the plate
was incubated at room temp for 30 min. The samples were
transferred to a 96-well P81 filter plate and washed (4 × 200
µL per well) with 75 mM phosphoric acid. Microscint 40
scintillation liquid (50 µL) was added to each well, and the
amount of 32P incorporation for each sample was measured
using a Packard Topcount microplate scintillation counter. IC50
values were calculated from dose-response curves (GraphPad
Prism curve-fitting software); from these calculated apparent
inhibition constants (Ki) were obtained using the Cheng-
Prusoff42 equation: Ki ) IC50/(1 + ([ATP]/Km (app) ATP)), where
[ATP] is the ATP concentration used for the IC50 determination
and Km (app) ATP for each kinase was determined experimentally.
Im m u n osta in in g. HeLa cells grown on coverslips and
treated with compound 32 were fixed in cold methanol
(-20 °C) for 3 min, dried, and stored at -80 °C. The coverslips
were washed three times with PBT (PBS, 0.1% Triton X-100)
and once with PBS and then incubated for 2 h at room
temperature with primary antibodies: (rat anti-R-tubulin
(YL1/2 Serotec MCAP77) diluted 1:500, rabbit anti-γ-tubulin
(Sigma DQ-19) diluted 1:1000 in PBS/1% BSA. They were then
washed three times with PBT and incubated for 1 h at room
temperature with secondary antibodies (TRITC-conjugated
donkey anti-rat IgG (J ackson 712-026-150) and Alexafluor
Green 488-conjugated goat anti-rabbit IgG (Molecular Probes
A11008) diluted 1:300. Cells were washed as described above,
Cytotoxicity Assa ys. Standard MTT (thiazolyl blue; 3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assays
were performed.43 In short, cells were seeded into 96-well
plates according to doubling time and incubated overnight at
37 °C. Test compounds were made up in DMSO, and a 3-fold
dilution series prepared in 100 µL of cell medium, added to
cells (in triplicates), and incubated for 72 or 96 h at 37 °C.
MTT was made up as a stock of 5 mg/mL in cell medium, and

2-Anilino-4-(thiazol-5-yl)pyrimidine CDK Inhibitors
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