Discovery of aminothiazole inhibitors of cyclin-dependent kinase 2: Synthesis, x-ray crystallographic analysis, and biological activities

Article abstract of DOI:10.1021/jm0201520

High throughput screening identified 2-acetamido-thiazolylthio acetic ester 1 as an inhibitor of cyclin-dependent kinase 2 (CDK2). Because this compound is inactive in cells and unstable in plasma, we have stabilized it to metabolic hydrolysis by replacing the ester moiety with a 5-ethyl-substituted oxazole as in compound 14. Combinatorial and parallel synthesis provided a rapid analysis of the structure-activity relationship (SAR) for these inhibitors of CDK2, and over 100 analogues with IC₅₀ values in the 1-10 nM range were rapidly prepared. The X-ray crystallographic data of the inhibitors bound to the active site of CDK2 protein provided insight into the binding modes of these inhibitors, and the SAR of this series of analogues was rationalized. Many of these analogues displayed potent and broad spectrum antiproliferative activity across a panel of tumor cell lines in vitro. In addition, A2780 ovarian carcinoma cells undergo rapid apoptosis following exposure to CDK2 inhibitors of this class. Mechanism of action studies have confirmed that the phosphorylation of CDK2 substrates such as RB, histone H1, and DNA polymerase α (p70 subunit) is reduced in the presence of compound 14. Further optimization led to compounds such as water soluble 45, which possesses a favorable pharmacokinetic profile in mice and demonstrates significant antitumor activity in vivo in several murine and human models, including an engineered murine mammary tumor that overexpresses cyclin E, the coactivator of CDK2.

Full text of DOI:10.1021/jm0201520

J . Med. Chem. 2002, 45, 3905-3927
3905
Discover y of Am in oth ia zole In h ibitor s of Cyclin -Dep en d en t Kin a se 2:
Syn th esis, X-r a y Cr ysta llogr a p h ic An a lysis, a n d Biologica l Activities
Kyoung Soon Kim,*,† S. David Kimball,‡ Raj N. Misra,† David B. Rawlins,† J ohn T. Hunt,§ Hai-Yun Xiao,†
Songfeng Lu,† Ligang Qian,† Wen-Ching Han,† Weifang Shan,† Toomas Mitt,† Zhen-Wei Cai,| Michael A. Poss,|
Hong Zhu,| J ohn S. Sack, J ohn S. Tokarski, Chieh Ying Chang, Nikola Pavletich,# Amrita Kamath,∇
William G. Humphreys,∇ Punit Marathe,∇ Isia Bursuker,O Kristen A. Kellar,§ Urvashi Roongta,§
Roberta Batorsky,§ J anet G. Mulheron,§ David Bol,§ Craig R. Fairchild,§ Francis Y. Lee,§ and Kevin R. Webster∧
Departments of Oncology Chemistry, Oncology Drug Discovery, New Leads Chemistry, Structural Biology and Modeling,
Metabolism and Pharmacokinetics, and High Throughput Screening, Bristol-Myers Squibb Pharmaceutical Research Institute,
Princeton, New J ersey 08543-4000
Received April 9, 2002
High throughput screening identified 2-acetamido-thiazolylthio acetic ester 1 as an inhibitor
of cyclin-dependent kinase 2 (CDK2). Because this compound is inactive in cells and unstable
in plasma, we have stabilized it to metabolic hydrolysis by replacing the ester moiety with a
5-ethyl-substituted oxazole as in compound 14. Combinatorial and parallel synthesis provided
a rapid analysis of the structure-activity relationship (SAR) for these inhibitors of CDK2,
and over 100 analogues with IC50 values in the 1-10 nM range were rapidly prepared. The
X-ray crystallographic data of the inhibitors bound to the active site of CDK2 protein provided
insight into the binding modes of these inhibitors, and the SAR of this series of analogues was
rationalized. Many of these analogues displayed potent and broad spectrum antiproliferative
activity across a panel of tumor cell lines in vitro. In addition, A2780 ovarian carcinoma cells
undergo rapid apoptosis following exposure to CDK2 inhibitors of this class. Mechanism of
action studies have confirmed that the phosphorylation of CDK2 substrates such as RB, histone
H1, and DNA polymerase R (p70 subunit) is reduced in the presence of compound 14. Further
optimization led to compounds such as water soluble 45, which possesses a favorable
pharmacokinetic profile in mice and demonstrates significant antitumor activity in vivo in
several murine and human models, including an engineered murine mammary tumor that
overexpresses cyclin E, the coactivator of CDK2.
In tr od u ction
point. Checkpoints serve to maintain the proper se-
quence of cell cycle events and allow the cell to respond
to insults or to proliferative signals, while the loss of
proper checkpoint control in cancer cells contributes to
tumorigenesis.4 The CDK2 pathway influences tumori-
genesis at the level of tumor suppressor function (e.g.,
p53, RB, and p27) and oncogene activation (cyclin E).
Many reports have demonstrated that both the coacti-
vator, cyclin E,5 and the inhibitor, p27,6 of CDK2 are
either over- or underexpressed, respectively, in breast,
colon, nonsmall cell lung, gastric, prostate, bladder, non-
Hodgkin’s lymphoma, ovarian, and other cancers. Their
altered expression has been shown to correlate with
increased CDK2 activity levels and poor overall sur-
vival.6,7 This observation makes CDK2 and its regula-
tory pathways compelling targets for the development
of novel chemotherapeutic agents. During the past few
years, a number of adenosine 5′-triphosphate (ATP)
competitive small organic molecules8,9 as well as pep-
tides10 have been reported in the literature as CDK
inhibitors for the potential treatment of cancers. Cur-
rently, the nonselective CDK inhibitor flavopiridol is
undergoing human clinical trials.11
The cyclin-dependent kinases (CDKs) are serine/
threonine protein kinases, which are the driving force
behind the cell cycle and cell proliferation.1,2 Individual
CDKs perform distinct roles in cell cycle progression and
can be classified as either G1, S, or G2/M phase
enzymes. Uncontrolled proliferation is a hallmark of
cancer cells, and misregulation of CDK function occurs
with high frequency in many important solid tumors.3
CDK2 and CDK4 are of particular interest because their
activities are frequently misregulated in a wide variety
of human cancers. CDK2 activity is required for pro-
gression through G1 to the S phase of the cell cycle, and
CDK2 is one of the key components of the G1 check-
* To whom correspondence should be addressed. Tel: (609)252-5181.
Fax: (609)252-6601. E-mail: kyoung.kim@bms.com.
† Department of Oncology Chemistry, Bristol-Myers Squibb Phar-
maceutical Research Institute.
‡ Lexicon Pharmaceuticals, East Windsor, NJ .
§ Department of Oncology Drug Discovery, Bristol-Myers Squibb
Pharmaceutical Research Institute.
| Department of New Leads Chemistry, Bristol-Myers Squibb
Pharmaceutical Research Institute.
Department of Structural Biology and Modeling, Bristol-Myers
Squibb Pharmaceutical Research Institute.
#
Memorial Sloan-Kettering Cancer Center, New York, NY.
∇ Department of Metabolism and Pharmacokinetics, Bristol-Myers
Squibb Pharmaceutical Research Institute.
High throughput screening at Bristol-Myers Squibb
for inhibitors of CDKs led to the identification of several
new chemotypes. Among the most interesting of these
was the 2-amino-5-thio-substituted thiazole 1 (Figure
O Department of High Throughput Screening, Bristol-Myers Squibb
Pharmaceutical Research Institute.
∧ Astra Zeneca R&D Boston Inc., Waltham, MA.
10.1021/jm0201520 CCC: $22.00 © 2002 American Chemical Society
Published on Web 08/02/2002

3906 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18
Kim et al.
CDK2. In this paper, we describe the discovery and
optimization of aminothiazole-based CDK2 inhibitors,
modeling studies based on X-ray crystallographic data,
and in vitro and in vivo biological activities.
Ch em istr y
2-Amino-5-thiocyanatothiazole 2 was prepared by
reacting 2-aminothiazole with sodium thiocyanate and
bromine (Scheme 1).12 Acylation of the 2-amino-5-
thiocyanatothiazole with acetic anhydride gave key
intermediate 3 on a multigram scale. Reduction of 3 by
dithiothreitol (DTT) or sodium borohydride afforded the
intermediate thiolate, which was alkylated in situ by
an electrophile such as tert-butyl bromoacetate to give
ester 5. Alternatively, compound 5 could be obtained
from commercially available 2-acetylamino-5-acetylth-
iothiazole 4. In this case, treatment of 4 with alkoxide
to cleave the thioester to the thiolate followed by
reaction with tert-butyl bromoacetate provided 5 di-
rectly. This thiolate intermediate could be reacted with
various electrophiles to produce analogues of 1 (7-10).
Coupling of acid 6 with methylethylamine or benzyl
alcohol provided amide 11 and benzyl ester 12, respec-
tively (Scheme 1).
F igu r e 1.
1). While flavopiridol inhibits CDK1, CDK2, and CDK4
with minimal selectivity, the 2-amino-5-thiothiazole 1
is 10-fold selective vs CDK1 and 100-fold selective vs
CDK4. The small molecular size combined with potency,
structural simplicity, and modular synthesis made the
compound 1 series a particularly interesting starting
point for research. A robust structure-activity relation-
ship (SAR) quickly evolved from both solution phase and
solid phase chemistry, and X-ray crystallographic struc-
tures of the inhibitor-CDK2 complex provided insight
into the binding of these molecules to the active site of
The 5-ethyl-substituted oxazole analogue 14 was
prepared from acid 6 by coupling with 1-amino-2-
Sch em e 1a
a
Conditions: (a) NaSCN, Br2, MeOH. (b) Ac2O, pyridine, CH2Cl2. (c) DTT, MeOH. (d) tert-Butyl bromoacetate. (e) KOtBu or NaOEt.
(f) TFA. (g) Ethyl bromoacetate. (h) Dess-Martin periodinane. (i) 2-tert-Butoxyethyl methanesulfonate. (j) Phenethyl bromide. (k) HOBt,
EDCI, ethylmethylamine. (l) Diethyl azodicarboxylate, PPh3, benzyl alcohol.

Aminothiazole Inhibitors of Cyclin-Dependent Kinase 2
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18 3907
Sch em e 2a
a
Conditions: (a) EDCI, HOBt, 1-amino-2-butanone, CH2Cl2. (b) Ac2O, H2SO4, NaOAc. (c) Et3N, chloroacetyl chloride. (d) Oxalyl chloride,
Et3N, DMSO. (e) POCl3, 90 °C. (f) KOtBu, compound 4. (g) NBS, benzoyl peroxide, 76 °C.
Sch em e 3
butanone followed by cyclodehydration of 13 using acetic
anhydride and concentrated sulfuric acid (Scheme 2).
To explore the SAR of the oxazole substitution, 4-ethyl-
substituted oxazole regioisomer 18 and 4,5-disubstituted
oxazole analogue 20 were prepared. Dehydrocyclization
of aldehyde 16 with POCl3 followed by the coupling of
the resulting chloromethyl oxazole 17 with the thiolate
intermediate from 4 provided regioisomer 18. Treatment
of 2,4,5-trimethyl oxazole with N-bromosuccinimde
produced 2-bromomethyloxazole 19, which led to ana-
logue 20 upon coupling with the thiolate from 4 (Scheme
2).
of diazoketone 21 with boron trifluoride etherate in the
presence of chloroacetonitrile (Scheme 3).13 This meth-
odology allowed the synthesis of oxazole analogues with
various substituents at C-5 of the oxazole ring.
A wide variety of derivatives with different substit-
uents on the C-2 amino group of the thiazole were easily
prepared from amine 28 by reacting with activated acid
derivatives to give amide analogues 29-33, with chlo-
roformates to give carbamates 34 and 35, with sulfonyl
chlorides to give sulfonamide 36, and with isocyanates
to give ureas 38-41 (Scheme 4). Methylation of the
amide nitrogen of 29 was accomplished by reacting 29
with methyl iodide in the presence of potassium carbon-
ate to obtain 37.
Alternatively, 2-chloromethyl oxazoles with various
substituents at C-5, 22, were prepared by the treatment

3908 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18
Kim et al.
Sch em e 4a
a
Conditions: (a) Aqueous NaOH, EtOH, 100 °C. (b) RCOCl, pyridine, or RCOOH, HOBt, EDCI, or ROCOCl, pyridine, DMAP, or
PhSO2Cl, pyridine. (c) RNCO, DMF, 55 °C. (d) MeI, K2CO3, DMF.
Sch em e 5a
a
Conditions: (a) EDCI, CH2Cl2. (b) THF-DMF, room temperature. (c) Aqueous NaOH, EtOH, 100 °C. (d) RCOCl, pyridine, or RCOOH,
HOBt, EDCI.
Acyl aminothiazoles with more polar functional groups
were explored. The serinol functional group was intro-
duced by the treatment of benzylbromo derivative 43
with serinol 44 to obtain 45. Alternatively, the pyridyl-
substituted 5-ethyloxazole analogue 47 was prepared by
coupling with 3-pyridylacetic acid (Scheme 5).
Alternative heterocycles and sulfur oxidation products
were also studied (Scheme 6). Thiadiazole analogue 48
and the regioisomer of the 2-amino-5-thiothiazole nucleus,
compound 50, were synthesized from 2-mercapto-5-
aminothiadiazole and 2-mercapto-5-aminothiazole,14 re-
spectively. Oxidation of sulfide 29 with mCPBA pro-
vided sulfoxide 51 and sulfone 52.
Scheme 7. The synthesis was initiated by conversion of
SASRIN resin 5315 to its correspondering chloromethyl
derivative 54 with triphosgene and PPh3.16 After unsuc-
cessful attempts to directly couple 2 to benzyl chloride
resin 54, the aminothiazole was acylated with trifluo-
roacetic anhydride to give 55, which was alkylated with
i
benzyl chloride resin 54 in the presence of Pr2NEt to
form a resin-bound thiocyanate 56. Thiocyanate 56 was
then reduced to a resin-bound thiol 57 with DTT. The
resulting resin-bound thiol 57 was alkylated with a
variety of chlorides or bromides in the presence of DBU
to obtain a series of analogues 58. Deprotection of the
trifluoroacetyl group using sodium borohydride in etha-
nol followed by acylation of the secondary amine of 59
provided amide 60 with a variety of R′′ groups. Cleaving
Libraries of 2-amino-5-thio-substituted thiazoles were
prepared on solid phase using the methods shown in

Aminothiazole Inhibitors of Cyclin-Dependent Kinase 2
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18 3909
Sch em e 6a
a
Conditions: (a) Ac2O, pyridine. (b) tert-Butyl bromoacetate. (c) CH3COCl, pyridine. (d) KOtBu, compound 22d , THF. (e) mCPBA,
CH2Cl2.
Sch em e 7a
a
Conditions: (a) Triphosgene, PPh3, CH2Cl2. (b) TFAA, 2,6-lutidine. (c) i-Pr2EtN, CH2Cl2. (d) DTT, THF/MeOH. (e) DBU, DMF, 70 °C.
(f) NaBH4, EtOH/THF. (g) i-Pr2EtN, CH2Cl2, 0 °C to room temperature. (h) 50% TFA/CH2Cl2.
the product from solid support with 50% trifluoroacetic
acid (TFA)/CH2Cl2 provided the final products, some of
which are shown in Table 7.
ceptors such as alcohol 7, ketone 8, ether 9, and amide
11 exhibit only weak activity, at best. The combination
of a finely tuned hydrophobic moiety and a hydrogen-
bonding acceptor within the ester moiety appears to be
needed for good inhibitory activity although we still
cannot rule out stabilization of the bioactive conforma-
tion by the ester or its involvement in other interactions.
Despite its potent CDK2 inhibitory activity, compound
1 lacks cell activity (IC50 > 25 µM) in a cell proliferation
assay (ovarian tumor cell line A2780). We hypothesized
that this is due to metabolism of the ester to the
carboxylic acid 6 (Table 1), which is completely devoid
of CDK2 inhibitory activity. With the metabolic liability
of the ester function in mind, a metabolically stable
ester surrogate17 was sought to replace the ester in 1.
SAR a n d Str u ctu r a l Stu d ies
The initial screening hit compound 1 is a relatively
selective inhibitor of CDK2 (Table 1). The bulkier tert-
butyl ester group of 5 increases the activity as compared
to the original ethyl ester 1, but there appears to be a
limit to the allowable size of the ester as benzyl ester
12 shows reduced activity (5 vs 12). A simple hydro-
phobic substituent as in 10 has diminished activity,
which implies the possible involvement of the ester
function in hydrogen bonding with the protein in the
binding site. However, potential hydrogen-bonding ac-

3910 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18
Ta ble 1. SAR of Thiomethylene Substitution
Kim et al.
IC50 (µM)
CDK2/
cyc E
CDK1/ CDK4/
compd no.
R
cyc B1
cyc D1
1
5
6
7
8
COOC2H5
COOC(CH3)3
COOH
CH(OH)CH2C(CH3)3
C(O)CH2C(CH3)3
CH2OC(CH3)3
CH2C6H5
C(O)N(CH3)C2H5
COOCH2C6H5
0.17
0.05
>25
1.87
0.44
23.03
5.38
2.81
1.84
1.74
9
10
11
12
12.58
>25
0.34
2.38
19.06
Ta ble 2. SAR of Oxazole Substitution
F igu r e 2. ATP binding site residues of the X-ray structure
of CDK2 in complex with compound 5. Hydrogen-bonding
interactions are shown. The oxygen atoms are colored red,
nitrogen atoms are colored blue, protein carbon atoms are
colored gray, and ligand carbon atoms are colored green.
IC50 (µM)
CDK2/ CDK1/ CDK4/ cytotox
cyc E cyc B1 cyc D1 A2780
compd no.
R1
C2H5
H
CH3
R2
14
18
20
23
24
25
26
27
H
0.02
0.30
2.25
1.40
C2H5 0.20
CH3
H
H
H
H
0.36
CH3
0.09
5.16
1.07
0.69
0.64
4.51
CH(CH3)2
C(CH3)3
CH2-c-hexyl
C6H5
0.006
0.005
0.004
0.35
0.06
0.04
0.03
0.20
0.05
1.54
H
This led to key 5-ethyloxazole-substituted thiazole 14.
The oxazole 14, which is metabolically stable against
esterases, maintained CDK2 selectivity and displayed
increased kinase inhibitory potency (14 vs 1) as well as
potent antiproliferative activity with an IC50 of 1.4 µM
in the 72 h ovarian cancer cell A2780 proliferation assay
(Table 2).
The solid state structure of CDK2 with compound 5
bound in the ATP binding site was determined by single-
crystal X-ray diffraction (Figure 2). Interestingly, the
thiazole ester 5 adopts a folded conformation in the
CDK2-bound complex, while the X-ray structure of 5
alone reveals an extended conformation (data not
shown). In this folded conformation, as in all of the
subsequent structures of the 2-amino-5-thioalkyl-sub-
stituted thiazoles bound to CDK2, a pair of reciprocal
hydrogen bonds is formed in the CDK2 hinge region of
the ATP binding site between the 2-amino hydrogen and
the carbonyl of Leu83, as well as the 3-nitrogen of the
aminothiazole ring and the NH of Leu83. The thio-
methylene moiety occupies a hydrophobic pocket formed
by Ala31, Val64, Phe80, and Ala144 of CDK2, and the
alkyl group of the ester resides in a region containing
hydrophobic residues such as Val18, Leu134, and the
CR and Câ atoms of Asn132. In addition, the 2-amide
carbonyl oxygen of 5 forms a hydrogen bond with a
water molecule, an interaction that was not always
found in the structures of related analogues.
F igu r e 3. ATP binding site residues of the X-ray structure
of CDK2 in complex with compound 46.
ATP binding site, it is observed that in most of the solid
state X-ray structures the density of the Lys33 side
chain was insufficient to assign its position unambigu-
ously. The ester carbonyl oxygen of 5 is directed toward
the Lys33 side chain but is 4.4 Å from the side chain
terminal amino nitrogen and 3.4 Å from the side chain
Cꢀ. In only a few cases, such as the CDK2 complex with
46, is the oxazole ring nitrogen and the terminal side
chain amino nitrogen of Lys33 within a reasonable
hydrogen-bonding distance (3.25 Å) as shown in Figure
3. For most of the structures, in which the analogue
binds in a folded conformation, the geometry reveals an
efficient packing of the oxazole ring with residues such
as Val18 and/or the hydrocarbon portion of the Lys33
side chain. The 5-oxazole substituent makes similar
interactions with CDK2 as shown above for the tert-
Although we anticipated that the ester carbonyl or
oxazole heteroatom(s) would be involved as a hydrogen-
bonding acceptor with the nearby residue Lys33 of the

Aminothiazole Inhibitors of Cyclin-Dependent Kinase 2
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18 3911
Ta ble 3. SAR of 2-Amino Substitution
IC50 (µM)
CDK2/
cyc E
CDK1/
cyc B1
CDK4/
cyc D1
cytotox
A2780
compd no.
R1
R2
25
29
30
31
32
33
34
35
36
37
38
39
40
41
45
C(O)CH3
H
H
H
H
H
H
H
H
H
CH3
H
H
H
0.005
0.005
0.017
0.026
0.006
0.06
0.025
0.15
4.28
0.04
0.03
0.09
0.69
0.03
3.48
0.96
3.13
0.05
0.05
0.25
0.84
0.9
C(O)CH(CH3)2
C(O)C(CH3)3
C(O)C6H5
C(O)-(R)-CH(CH3)C6H5
C(O)-(S)-CH(CH3)C6H5
C(O)OCH(CH3)2
C(O)OCH2C6H5
SO2C6H5
C(O)CH(CH3)2
C(O)NHC2H5
0.018
2.99
0.64
1.04
2.86
>25
0.05
0.12
0.90
0.24
1.5
C(O)NH-(4-F)- C6H5
C(O)NH-(2,6-di-F)- C6H3
C(O)NH-(2,6-di-Cl)- C6H3
C(O)CH2-(4-CH2NHCH(CH2OH)2)-C6H4
0.011
0.002
0.003
0.003
0.019
0.010
0.03
0.90
0.39
>1
H
H
0.03
0.10
0.03
butyl group of ester 5. While the 5-substituent of the
oxazole interacts favorably with hydrophobic residues
in a location that preferably accommodates a sizable
group, the C-4 position of the oxazole points toward
buried residues such as Lys33 and Asp145 of the ATP
binding site. Therefore, anything larger than a hydrogen
atom would be disruptive to binding. This is consistent
with the diminished activity of regioisomer 4-ethylox-
azole 18 and 4,5-dimethyl-substituted oxazole 20 as
compared to 14 (Table 2).
Increasing the bulk of the 5-oxazole substituent by
incorporating a branched sp3 carbon center increases
potency in vitro (24-26 vs 23) against CDK2/cyclin E
(Table 2). However, substitution by a phenyl ring (27)
decreases activity. Apparently, the 5-oxazole substituent
binding pocket can accommodate a large substituent as
evidenced by the good activity of 26. Modeling studies
of 27 in the active site of CDK2 reveal that in the folded
conformation both the 2-amide carbonyl oxygen of the
thiazole and the backbone carbonyl oxygen of Gln131
point toward the C-5 phenyl of the oxazole. The interac-
tion of the π system of the C-5 phenyl with the carbonyl
oxygens of the 2-amide group of the thiazole or of
Gln132 would be unfavorable. In addition, because the
phenyl ring is more rigid, it may not be able to adjust
to fit the enzyme pocket as productively as the ana-
logues with more flexible sp3-hybridized carbons at the
C-5 position of the oxazole.
Because the 5-tert-butyl oxazole appears nearly op-
timal in its activity against CDK2/cyclin E and in the
A2780 cell proliferation assay, most of the SAR that is
discussed in this paper is restricted to analogues in
which this moiety is held constant. Using solution phase
parallel synthesis as well as individual compound
synthesis, we rapidly prepared a series of 5-tert-butyl-
oxazolylmethylthio thiazoles that further explored the
SAR of the 2-amino substitution.
While there are clear differences in the enzyme
selectivity and cell activity among amides and carbam-
ates (25 and 29-35), these substitutions are broadly
active against CDK2/cyclin E (Table 3). Carbamates (34
and 35) are generally less potent inhibitors of the CDK2/
cyclin E complex than are the amides. Interestingly,
F igu r e 4. (a) ATP binding site residues of the X-ray structure
of CDK2 in complex with compound 32. (b) The ATP binding
site of the X-ray structure of CDK2 (shown as a solid molecular
surface) in complex with compound 32. The surface of CDK2
is colored according to elevation, i.e., blue indicates deepest
pockets, green indicates more shallow pockets, and orange
indicates peaks.
carbamate 34 lost the selectivity of CDK2 inhibitory
activity over CDK1. It is noteworthy that there is a 10-
fold activity difference between the two stereoisomers
of 32 and 33. Figure 4a,b shows the solid state structure
of 32 bound to the active site of CDK2. Modeling studies
of (S)-isomer 33 in the X-ray structure of CDK2 (not
shown) revealed that the phenyl ring of the 2-amino

3912 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18
Kim et al.
F igu r e 5. ATP binding site residues of an X-ray structure of
CDK2 in complex with compound 41.
substituent would likely form significant steric contacts
with the side chains of Lys89 and Asp86, the latter
residue being involved in an intramolecular hydrogen
bond with the backbone amino nitrogen of Lys89, an
interaction found in all of the in-house crystal struc-
tures. Furthermore, the favorable interactions of the
phenyl ring with Phe82 and Ile10 observed with the (R)-
isomer 32 would be lost with the (S)-isomer, thereby
providing a possible explanation for the decrease in
activity of (S)-isomer 33.
A sulfonamide 36 is much less active, and consistent
with the structural information in Figure 3, N-methy-
lation of the 2-amide (37) destroys activity in vitro.
Therefore, the hydrogen-bonding donor NH of the amide
(and the NH(s) of the urea, see below) to Leu83 is
essential to potent activity against CDK2/cyclin E (29
vs 37).
Phenyl-substituted ureas (39-41) are more potent
inhibitors than the simple ethyl urea 38. The X-ray
structure of the complex of CDK2 and the 2,6-dichlo-
rophenyl analogue 41 reveals that the phenyl ring of
41 interacts favorably with the side chains of Ile10 and
Phe82, which may explain why phenyl-substituted
ureas are more potent inhibitors than the simple ethyl
urea (see Figure 5). The diortho substitution on the
phenyl ring helps orient the ring out-of-plane with the
urea group in such a way that it optimally interacts with
Ile10. In addition, both urea NHs are within hydrogen-
bonding distance (2.60 and 2.88 Å) to the carbonyl of
Leu83. The X-ray structure complexes of CDK2 and 41
with and without cyclin A were obtained (Figure 6a,b).
It is observed that the binding mode and ligand confor-
mation are virtually identical in both complexes. How-
ever, the T loop of CDK2 is in different locations in the
two structures. The T loop is oriented further away from
the ATP binding site in the ternary complex of CDK2,
41, and cyclin A than is found in the complex without
cyclin A. This observation is similar to the findings of
Pavletich et al.18
F igu r e 6. (a) Schematic drawing of the CDK2-41 complex.
CDK2 is shown in a ribbon representation with the R-helices
in red, â-sheets in green, and loops in yellow. Compound 41 is
shown in stick representation. (b) The CDK2-41-cyclin A
ternary complex. Cyclin A is colored purple.
correlate with differences in activity. On the other hand,
as the 2-amino substituent extends further away from
the thiazole ring and toward solvent, the interactions
with the CDK2 become less likely. In as much as the
2-amino substituent is directed toward the solvent-
exposed hinge region, we felt that substitution at the
2-amino position would be key for optimizing the physi-
cal and pharmacokinetic properties of these molecules
As shown in the X-ray structure of Figure 4b, the
2-amide side chain of the thiazole 32 in the binding
pocket of CDK2 points toward the solvent-exposed
region. However, because the portion of the 2-substitu-
ent closest to the thiazole nucleus is still within the
binding site and able to interact with the protein,
functional group changes near the 2-amino tether may

Aminothiazole Inhibitors of Cyclin-Dependent Kinase 2
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18 3913
Ta ble 6. SAR of the Sulfur Oxidation Analogues
Ta ble 4. CDK Activity Data of the Ethyloxazole Analogue
IC50 (µM)
IC50 (µM)
CDK2/ CDK1/ CDK4/ cytotox
CDK2/
cyc E
CDK1/
cyc B1
CDK4/
cyc D1
compd no.
R
cyc E
cyc B1 cyc D1 A2780
compd no.
n
46
47
CH(CH3)2
CH2-3-pyridyl
0.005
0.005
0.060
0.08
1.07
1.09
0.20
0.24
29
51
52
0
1
2
0.005
0.14
0.25
0.03
0.14
>0.25
0.90
>25
Ta ble 5. CDK Activity Data of the Analogue
Biologica l Eva lu a tion
Kin a se Selectivity. To establish that compounds
from this series were specific for the CDK class of serine/
threonine protein kinases, a panel of assays was estab-
lished. Using a diverse set of kinase proteins, we were
able to track changes in the structures that conferred
specificity for CDKs vs a number of unrelated serine/
threonine kinases, such as protein kinase C (PKC)
family members, as well as nonreceptor and receptor
tyrosine kinases such as Lck, EMT, Zap70, and Her
family and IGF-1 enzymes. More importantly, assays
for CDK1 and CDK4 allowed us to establish which
compounds were selective for CDK2 relative to other
close family members. As can be seen in Table 8,
compounds such as the 5-ethyloxazole- or 5-tert-butyl-
oxazole-substituted thiazoles 14 and 45 were relatively
selective for CDK2, being 10-fold more potent against
this enzyme than CDK1/cyclin B complex. The CDK2
inhibitory activity observed was superior to that of the
clinical compound flavopiridol, which was less selective
among all CDK complex activities studied, as well as
inhibiting some PKC family members. Improvements
in aqueous solubility by introduction of the polar moiety
in 45 were compatible with improving potency and
retaining selectivity for CDK2. This compound was 10-
and 30-fold selective for CDK2 vs CDK1 and CDK4 and
3-5 orders of magnitude more potent against CDK2
than all other non-CDKs tested (Table 8).
CDK Su bstr a te P h osp h or yla tion . Confirmation
that these compounds were inhibiting CDK2 enzyme
activity in cells was obtained through examination of
CDK substrate phosphorylation states. Actively prolif-
erating A2780 ovarian carcinoma cells were treated with
32P-orthophosphate in the presence or absence of 3 µM
14. Subsequent immunoprecipitation of the CDK sub-
strates RB, histone H1, and DNA polymerase R with
specific antibodies showed reduced labeling with 32P in
the presence of compound 14, as compared to untreated
cells (Figure 7). In each case, there was an obvious
reduction in the level of phosphorylation, suggesting
that CDK2 function was inhibited in the cellular
context. Equal loading of substrate was verified by
parallel immunoprecipitation followed by western blot
with the same antibody (data not shown).
CDK2/cyc E
IC50 (µM)
compd no.
X
R
48
50
N
CH
R1
R2
>25
>25
while still retaining good CDK2 inhibitory activity. With
these thoughts in mind, a polar amino alcohol group as
well as a pyridyl moiety were introduced as in 45 and
47 to improve the aqueous solubility. In concordance
with our expectations, the more water soluble, yet
higher molecular weight compound 45 retained potent
activity in both CDK2/cyclin E enzyme and cell prolif-
eration assays. Pyridyl-substituted 5-ethyloxazole ana-
logue 47 also exhibited potent inhibitory activity against
CDK2/cyclin E, but it was less active than amino
alcohol-substituted 5-tert-butyloxazole analogue 45 in
the cytotoxic assay (Tables 3 and 4).
In an effort to understand the binding requirements
for activity against CDK2/cyclin E, several compounds
were prepared that incorporate modifications of the
central thio-aminothiazole ring (Table 5). Incorporation
of an additional nitrogen into the heterocycle as in
thiodiazole 48 or moving the position of the ring
nitrogen of the aminothiazole (50) led to a complete loss
of activity against the enzyme. Modeling studies suggest
that substitution of the thiazole CH with N introduces
an unfavorable N‚‚‚O lone pair electron repulsion with
the carbonyl oxygen of Glu81. Furthermore, a favorable
interaction found with the parent aminothiazole ring,
which participates in an aromatic CH‚‚‚OdC interaction
with the carbonyl oxygen of Glu81, an interaction also
observed with CDK2 complexes of other chemotypes,19
is absent in 48. The inactivity of 50 can be explained
by the observations from the solid state structure that
the 3-nitrogen of the parent thiazole ring participates
in a critical hydrogen bond with the amide NH of Leu83.
Potential oxidative metabolism of the sulfur atom led
to the exploration of sulfoxide 51 and sulfone 52. These
oxidized compounds show diminished activity in vitro
(Table 6). However, studies of the oxidative metabolism
of the sulfur atom in vivo indicated that sulfur oxidation
was not a major problem since the amount of oxidative
metabolism of sulfur was minimal.
Cell Cycle An a lysis. Treatment of A2780 cells with
CDK2 inhibitors, as described in the Experimental
Section, resulted in an altered cell cycle distribution
relative to untreated cells. The cell cycle effects of 14
and 47 are compared to flavopiridol at equipotent
concentrations (determined in the 72 h cell proliferation
assay with A2780 cells) in Table 9. It is clear that
exposure to either compound 14 or compound 47 results
Only a few examples from the solid phase synthesis
are shown in Table 7. As seen in this table, the
5-alkyloxazoles were optimal as other heterocycles (e.g.,
61, 64-66, etc.) were much less active.

3914 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18
Kim et al.
Ta ble 7. CDK Activity Data of the Analogue from Solid Phase Synthesis

Aminothiazole Inhibitors of Cyclin-Dependent Kinase 2
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18 3915
Ta ble 10. Intraperitoneal Dose in Mice
Ta ble 8. In Vitro Kinase Inhibition, IC50 (nM)
protein kinase
flavopiridol
14
297
21
2250
45
dose
dose
Cmax Tmax AUCtot T1/2 MRT
compd
(mg/kg) (µmol/kg) (µM) (h) (µM‚h) (h) (h)
CDK1/cyclin B
CDK2/cyclin E
CDK4/cyclin D1
PKCR
30
330
150
30
3
flavopiridol
50
10
36
114
18
100
6.9 0.5
13.2
2.7
23
1.1 2.0
1.8 2.9
1.1 0.8
102
>45 000
>45 000
45 000
45
47
0.8
40
1
0.5
2800
PKCâ
>45 000
PKCδ
890
480
Ta ble 11. Compound 45: IV and Oral Dose in Mice
PKCꢀ
5600
PKCú
>45 000
>100 000
14 850
11 000
>50 000
7600
35 000
28 000
29 000
>40 000
>50 000
>25 000
>25 000
24 000
IV dose
oral (PO) dose
IKK
dose (mg/kg)
dose (nmol/kg)
Cmax (nM)
10
18 971
8272
0.08
4984
2.2
1.8
67
6.9
10
18 971
634.6
0.33
1201
1.4
EMT
Lck
ZAP70
Her1
Her2
>10 000
Tmax (h)
AUCtot (nM‚h)
15 800
13 000
>25 000
>25 000
T
1/2 (h)
IGFR
MRT (h)
clearance (mL/min/kg)
Vss (L/kg)
4.1
bioavailability (%)
24
human normal fibroblast cells, as well as mouse tumor
and fibroblast cells and bovine normal endothelial cells.
The data are expressed as IC50 values and visually as a
mean bar graph, which is derived from the IC50 results
(Figure 8). In a mean bar graph, bars to the right
indicate cell lines that are more sensitive than the mean
while bars that project to the left represent cell lines
that are less sensitive than the mean.20 As shown in
Figure 8, the cell panel mean IC50 value for flavopiridol
was 0.055 and 1.24 µM for compound 14, which was
chosen as a representative example. Examination of the
individual IC50 values or the mean bar graph pattern
indicates that these compounds have potent cytotoxic
activity against a wide variety of cell types (ovarian,
breast, prostate, colon, lung, and leukemia) with a
modest difference (7-fold) between the most sensitive
and the most resistant cell lines. Interestingly, a similar
pattern of cell line selectivity is observed between these
two compounds even though compound 14 is a more
selective CDK2 inhibitor than flavopiridol.
F igu r e 7. Immunoprecipitation of CDK substrate proteins
following 32P-orthophosphate labeling.
Ta ble 9. Cell Cycle Analysis of A2780 Cells Treated with CDK
Inhibitors (IC Values against CDK2/cyc E)
P h ar m acokin etics an d Metabolism in Mice. Phar-
macokinetic parameters of flavopiridol, 45, and 47 in
mice after intraperitoneal dosing are summarized in
Table 10. The half-life and the mean residence time
(MRT) of 45 after ip dosing were longer than 47 and
flavopiridol. Compound 45 was also administered in a
second study by the IV and oral routes at a dose of 10
mg/kg; the estimated half-life and the MRT were 2.2
and 1.8 h, respectively, with a large Vss (Table 11 and
Figure 9). The oral bioavailability of 45 is 24%. Human
serum protein binding for 45 and 47 was 78 and 82%,
respectively, and the extent of binding of 45 to plasma
protein was similar in human (78%) and mouse (79%),
while the protein binding of flavopiridol was found to
be 91 and 63%, respectively. The rate of oxidation of 45
in vitro in mouse and human microsomes was moderate
(Table 12).
In Vivo An titu m or Activity. To determine the in
vivo antitumor effects of CDK inhibition, compounds
were evaluated against a panel of four tumor models
including the P388 murine leukemia model, a murine
mammary carcinoma originated from a strain of trans-
genic mice overexpressing cyclin E (Br-CycE),21 human
ovarian carcinoma (A2780), and human squamous cell
carcinoma (A431). Figure 10 shows the effects of 45 in
the ip/ip P388 leukemia model. Drug treatment was
14
IC50 IC90 IC50 IC90 IC50
(20 (295 (5 (29 (330 (2980
control nM) nM) nM) nM) nM)
47
flavopiridol
IC90
cell cycle
position
nM)
G1
S
G2/M
sub-G1
apoptotic
63
16
18
3
58
11
29
2
62
6
25
7
71
7
20
2
50
7
18
25
69
60
16
23
1
61
12
25
2
3
6
16
4
1
9
in a clear decrease in the S phase cell population as well
as a substantial increase in apoptotic cells (measured
as either sub-G1 or <2 N DNA content determined by
propidium iodide staining or as tunnel positive cells).
Response to flavopiridol is more modest after 24 h of
compound exposure. While there is a significant level
of apoptosis induced, there is a less dramatic impact on
the S phase cell population (Table 9). It is known that
the CDK2 peptide inhibitors also induce the apoptosis
of the transformed cells.10
In Vitr o Cytotoxicity. Flavopiridol and many thia-
zole series CDK inhibitors demonstrated potent in vitro
cytotoxicity against the ovarian tumor cell line A2780/
DDP-S with IC50 values of 0.05-1.5 µM (Tables 2-4).
In addition, some compounds were tested against a cell
line panel consisting of mostly human tumors but also

3916 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18
Kim et al.
F igu r e 8. In vitro cytotoxicity of CDK inhibitors against a panel of tumor and normal cell lines. IC50 values were determined
after 72 h drug exposure.
F igu r e 10. Effects of compound 45 treatment on the lifespan
of mice inoculated ip with the leukemia P388. Mice received
F igu r e 9. Plasma concentration vs time profiles of 45 in mice
treatment daily for 7 consecutive days (8, 16, and 32 mg/kg/
after IV and oral dosing.
inj), beginning 1 day posttumor inoculation. Each symbol
represents an individual mouse, each treatment group con-
sisted of six mice, and the control group contained eight mice.
Ta ble 12. In Vitro Data of Flavopiridol, 45, and 47
% serum protein
binding
metabolic rate
(nmol/min/mg protein)
Ta ble 13. In Vivo Antitumor Activity against P388 Murine
Leukemia
compd
human
mouse
human
mouse
compd
dose (mg/kg) (MTD)
route/schedule
%T/C
flavopiridol
45
47
91
78
82
63
79
0.07
0.21
0.03
0.22
flavopiridol
7.5
32
120
ip/qdx7
ip/qdx7
ip/qdx7
110
140
115
45
47
initiated on day 1, ip, every day for 7 days (q1d × 7).
Treatment with compound 45 at its maximum tolerated
dose (MTD) of 32 mg/kg/inj resulted in a significant
increase of lifespan (%T/C ) 140) (Figure 10 and Table
13). Both flavopiridol and 47 were inactive at their
MTDs (6 and 120 mpk) in this model, yielding %T/C
values of 110 and 115%, respectively (Table 13).
producing 3.3 log cell kill (LCK) at its MTD (36 mpk,
ip, qd × 8) (Figure 11). In comparison, both flavopiridol
and 47 were considerably less active, producing only 1.5
and 0.4 LCK, respectively, in this model at their MTDs
(Table 14).
Compound 45 demonstrated antitumor activity in two
other tumor models. In the Br-CycE mammary carci-
noma, it yielded 1.1 LCK at its MTD (24 mpk, ip, q1d
Compound 45 was highly active against the human
A2780 ovarian carcinoma implanted sc in nude mice,

Aminothiazole Inhibitors of Cyclin-Dependent Kinase 2
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18 3917
binatorial and parallel synthesis provided a means of
generating a rapid SAR for these inhibitors of CDK2,
and many analogues in this series exhibit potent CDK2
inhibitory activity with >10-100-fold selectivity over
CDK1 and CDK4 as well as other kinases. The solid
state X-ray crystallographic data of these inhibitors
bound to the CDK2 protein, combined with modeling
studies, provided insight into the binding mode of these
inhibitors at the active site of CDK2 and rationale for
interpreting the SAR. Many of these analogues dis-
played potent activity in the 72 h ovarian cancer cell
A2780 proliferation assay. Compound 14 exhibited
potent cytotoxicity in vitro against a variety of cell lines
and reduced the phosphorylation of CDK2 substrates
such as RB and histone H1 in in vitro A2780 cells. Cell
cycle analysis of 47 at its IC90 concentration in A2780
cells showed a reduction of cell population in S phase
and a large increase in the apoptotic fraction. Optimiza-
tion of the physical properties such as solubility and
pharmacokinetics led to compound 45, which demon-
strated good efficacy in vivo in several human cancer
cell line xenograft models as well as a murine tumor
line driven by cyclin E overexpression. Further studies
with this class of compound will be reported in the
future.
F igu r e 11. Comparative antitumor activity of flavopiridol and
compound 45 on the human ovarian carcinoma A2780 grown
in nude mice.
Ta ble 14. In Vivo Antitumor Activity against A2780 Human
Ovarian Carcinoma
compd
dose (mg/kg) (MTD)
route/schedule
LCK
flavopiridol
45
47
7.5
36
75
ip/qdx8
ip/qdx8
ip/2qdx9
0.6-1.5
3.3
0.4
× 8). Concomitantly tested flavopiridol yielded 0.8 LCK
at its MTD (5.0 mpk, ip, q1d × 8). It was gratifying to
see the in vivo efficacy of the CDK2 selective compound
45 in this cyclin E overexpressed model, which is
directly related to CDK2 activity. In the human squa-
mous cell carcinoma A431, compound 45 elicited a
tumor response of 1 LCK whereas flavopiridol produced
only 0.5 LCK at their respective MTDs (24 and 5.0 mpk,
ip, q1d × 8). We believe that the efficacy of 45 in vivo
stems from its potent CDK2 inhibitory activity, cell
activity, and favorable pharmacokinetic profile, particu-
larly its high serum concentration over time (Figure 9).
Exp er im en ta l Section
All new compounds were homogeneous by thin-layer chro-
matography (TLC) and reversed-phase high-performance liq-
uid chromatography (HPLC, >99%). Flash chromatography
was carried out on E. Merck Kieselgel 60 silica gel (230-400
mesh). All of the reaction solvents used such as tetrahydro-
furan (THF), N,N-dimethylformamide (DMF), and dichlo-
romethane (CH2Cl2) were anhydrous solvents, which were
purchased from Aldrich Chemical Co. EDCI and HOBt refer
to ethyl dimethylaminopropylcarbodiimide hydrochloride salt
and 1-hydroxybenztriazole, respectively. Preparative HPLC
was run on YMC OD S-10 50 mm × 500 mm column eluting
with a mixture of solvents A and B (starting from 10% of
solvent B to 100% solvent B over 30 min gradient time; solvent
A: 10% MeOH/90% H2O/0.1% TFA; solvent B: 90% MeOH/
10% H2O/0.1% TFA; flow rate, 84 mL/min; UV 254 nm). 1H
NMR and 13C NMR spectra were obtained on a J EOL 400 MHz
Con clu sion
Metabolically stable oxazole-containing aminothiazole
CDK2 inhibitors were discovered and optimized starting
from the initial high throughput screening hit 1. Com-
Ta ble 15. Summary of the Crystallographic Data Collection, Reduction, and Refinement
5
32
41
41 with cyclin A
46
space group
P212121
53.46
71.49
72.05
1.8
1.91-1.8
64 507
21 369
4.9 (18.3)
24.5 (4.9)
82.4 (40.1)
25.2 (31.3)
28.7 (35.0)
27.7
20.1
298
2537
2397
18
122
0.006
1.2
P212121
53.58
71.62
72.17
2.0
2.12-2.0
106 091
18 817
7.4 (25.3)
22.0 (3.4)
98.2 (78.8)
25.6 (28.2)
29.6 (33.7)
19.9
32.0
298
2496
2397
27
72
0.006
1.2
P212121
53.22
71.45
72.34
2.0
2.12-2.0
49 063
17 556
7.3 (35.9)
12.7 (1.6)
82.3 (49.9)
32.0 (44.2)
37.8 (46.2)
25.1
35.4
298
2483
2397
28
58
0.008
1.3
P6222
179.21
179.21
213.29
2.6
2.76-2.6
204 401
52 703
6.1 (36.8)
16.2 (2.3)
86.5 (71.8)
26.8 (43.0)
32.2 (47.0)
41.9
33.4
1118
9203
8994
56
153
0.007
1.3
P212121
53.58
71.62
72.17
2.0
2.12-2.0
49 067
16 965
13.5 (29.3)
19.8 (4.5)
89.2 (66.6)
27.0 (32.6)
32.1 (37.1)
28.2
21.4
298
2480
2397
20
63
0.016
1.96
a (Å2)
b (Å2)
c (Å2)
max obsvd resolution (Å)
highest resolution shell (Å)
no. of observations
no. of unique reflections
Rsymm (on F) (%)a
mean I/σ(I)a
completeness (%)a
R-factor (%)a
R-free (%)a
avg B-factor (protein) (Å2)
avg B-factor (ligand) (Å2)
residues in final model
total no. of atoms
no. of protein atoms
no. of ligand atoms
no. of solvent atoms
rms (bonds) (Å2)
rms (angles) (deg)
rms (impropers) (deg)
0.75
0.78
0.88
0.96
1.72
a
Numbers in parentheses represent the values in the highest resolution shell.

3918 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18
Kim et al.
CPF-270 spectrometer operating at 270 or 67.5 MHz, respec-
tively, and are reported as parts per million downfield from
an internal tetramethylsilane standard. The abbreviations of
qn. and sx. in H NMR refer to quintet and sextet, respectively.
Melting points are uncorrected.
1H), 3.81-3.73 (m, 1H), 2.80-2.67 (m, 2H), 2.29 (s, 3H), 1.53-
1.32 (m, 2H), 0.93 (s, 9H). 13C NMR (CD3OD): δ 169.5, 160.9,
142.8, 123.1, 67.6, 49.9, 47.0, 29.9, 29.4, 21.4. MS (ESI): 288.9
(M + H)+, 287.2 (M - H)-. Anal. (C12H20N2O2S2) C, H, N, S.
1
N-[5-[(4,4-Dim eth yl-2-oxop en tyl)th io]-2-th ia zolyl]a ce-
ta m id e (8). To a stirred solution of alcohol 7 (40.2 mg, 0.14
mmoL) in CH2Cl2 (1.2 mL) was added Dess-Martin periodi-
nane (211 mg, 0.5 mmol)22 at room temperature and stirred
at room temperature overnight. The reaction mixture was
diluted with CH2Cl2 (120 mL) and washed with 0.1 N NaOH
(2 × 30 mL) followed by saturated NaHCO3 (30 mL) and brine
(30 mL). The organic layer was dried over MgSO4, filtered,
and concentrated in vacuo. The residue was chromatographed
on silica gel eluting with 2% CH3OH in CH2Cl2 to give the
partially purified product (28.8 mg). This was further purified
by a preparative TLC (SiO2) eluting with 4% CH3OH in CH2-
Cl2 to obtain desired product 8 as a light yellow solid (13.2
2-Aceta m in o-5-th iocya n a toth ia zole (3). To a mixture of
2 (15.7 g, 100 mmol)11 and pyridine (12 g, 150 mmol) in CH2-
Cl2 (100 mL) was added acetic anhydride (1.2 g, 120 mmol) at
room temperature. After it was stirred for 6 h, the mixture
was concentrated to dryness and MeOH (50 mL) was added
to the residue. The precipitates were collected, washed with
water, dried, and recrystallized from MeOH to afford 2 (15.2
g, 76%) as a light yellow solid; mp 212 °C (literature11b 212-
1
214 °C). H NMR (CD3OD): δ 7.79 (s, 1H), 2.23 (s, 3H).
[[2-(Acetyla m in o)-5-th ia zolyl]th io]a cetic Acid 1,1-Di-
m eth yleth yl Ester (5). To a mixture of 3 (5.97 g, 30 mmol)
in MeOH (360 mL) under argon atmosphere was added DTT
(9.26 g, 60 mmol) at room temperature. The mixture was
stirred for 2 h and then concentrated in vacuo to afford a solid.
The solid was dissolved in DMF (30 mL), and to this solution
were added tert-butyl bromoacetate (5.85 g, 30 mmol) and
potassium carbonate (5.0 g, 36 mmol) at room temperature.
The mixture was stirred at room temperature for 2 h, and then,
H2O (200 mL) was added to the reaction mixture. The
precipitates were collected, washed with water, dried, dissolved
in CH2Cl2 (100 mL) and MeOH (10 mL), and filtered through
a silica pad. The filtrate was concentrated in vacuo to afford
the desired product 5 (7.5 g, 87%) as a light yellow solid; mp
162-163 °C. 1H NMR (CDCl3): δ 12.2 (s, 1H), 7.48 (s, 1H),
3.37 (s, 2H), 2.32 (s, 3H), 1.45 (s, 9H). MS (ESI): 289 (M +
H)+, 287 (M - H)-. Anal. (C11H16N3O3S2) C, H, N, S.
1
mg, 33%); mp 163-164 °C. H NMR (CDCl3): δ 7.42 (s, 1H),
3.51 (s, 2H), 2.45 (s, 2H), 2.30 (s, 3H), 1.01 (s, 9H). MS (ESI):
287.12 (M + H)+. Anal. (C12H18N2O2S2‚0‚25H2O) C, H, N, S.
N-[5-[[2-(1,1-Dim eth yleth oxy)eth yl]th io]-2-th ia zolyl]-
a ceta m id e (9). To a stirred mixture of 2-tert-butoxyethanol
(3.0 g, 25.3 mmol) and Et3N (35.5 mmol) in CH2Cl2 (40 mL) at
0 °C was added methanesulfonyl chloride (2.4 mL, 30 mmol)
slowly. The reaction mixture was stirred at 0 °C for 2 h, diluted
with CH2Cl2 (160 mL), and then was washed with 1 N HCl
solution (3 × 40 mL) followed by saturated NaHCO3 (2 × 40
mL) and brine (40 mL). The organic layer was dried (MgSO4),
filtered, and concentrated in vacuo to give crude 2-tert-
butoxyethyl methanesulfonate in a quantitative yield as an
oil, which was used for the next step without any further
purification.
[[2-(Acetyla m in o)-5-th ia zolyl]th io]a cetic Acid (6). A
solution of ester 5 (4.32 g, 15 mmol) in CH2Cl2 (30 mL) and
TFA (20 mL) was stirred at room temperature overnight and
concentrated. To the residue was added Et2O (50 mL). The
solid was collected, washed with Et2O, and dried to afford the
To a stirred mixture of thioacetate 4 (185 mg, 0.86 mmol)
in THF (10 mL) under argon at room temperature was added
KOtBu (0.94 mL of 1 M solution in THF, 0.94 mmol). To this
mixture was added methanesulfonyl compound obtained above
(250 mg, 1.28 mmol) in THF (1 mL). The reaction mixture was
purged with argon for 2 min and heated at 55 °C for 16 h. The
mixture was cooled to room temperature, diluted with EtOAc
(100 mL), and washed with water (30 mL) followed by
saturated NaHCO3 (2 × 30 mL) and brine (30 mL). The organic
layer was dried over MgSO4, filtered, and concentrated in
vacuo. This was chromatographed on silica gel eluting with
2% CH3OH in CH2Cl2 to obtain 9 as a light yellow solid (120
1
desired product (3.38 g, 97%) as a solid; mp 210 °C. H NMR
(CD3OD): δ 7.48 (s, 1H), 3.47 (s, 2H), 2.20 (s, 3H). MS (ESI):
231 (M - H)-. Anal. (C7H8N2O3S2) C, H, N, S.
[[2-(Acet yla m in o)-5-t h ia zolyl]t h io]a cet ic Acid E t h yl
Ester (1). It was prepared following the same procedure as
in 5 using ethyl bromoacetate instead of tert-butyl bromo-
1
acetate. H NMR (CDCl3): δ 7.44 (s, 1H), 4.11 (q, J ) 6.7 Hz,
2H), 3.40 (s, 2H), 2.27 (s, 3H), 1.20 (t, J ) 6.7 Hz, 3H). 13C
NMR (CDCl3): δ 168.9, 168.1, 162.8, 141.6, 121.8, 61.8, 40.1,
23.1, 14.1. Anal. (C9H12N2O3S2) C, H, N, S.
1
mg, 51%); mp 119-121 °C. H NMR (CDCl3): δ 7.35 (s, 1H),
3.50 (t, J ) 6.4 Hz, 3H), 2.84 (t, J ) 6.4 Hz, 3H), 2.28 (s, 3H),
1.13 (s, 9H). 13C NMR (CDCl3): δ 168.1, 162.1, 139.1, 124.4,
73.6, 60.8, 39.0, 27.5, 23.1. MS (ESI): 275.2 (M + H)+. Anal.
(C11H17N2O2S2) C, H, N, S.
N-[5-(2-Hyd r oxy-4,4-d im eth yl-p en tylsu lfa n yl)th ia zol-
2-yl]a ceta m id e (7). To a stirred solution of 5,5-dimethyl-1-
hexene (3.45 g, 35.2 mmol) in CH2Cl2 (40 mL) at room
temperature was added m-chloroperoxybenzoic acid (75%
purity, 8.49 g, 49.2 mmol). The reaction mixture was stirred
at room temperature for 2.5 h. This mixture was washed with
0.1 N NaOH (3 × 30 mL), then diluted with CH2Cl2 (15 mL),
and washed with 0.1 N NaOH (20 mL) and brine (20 mL). The
organic layer was dried (MgSO4) and filtered to give a solution
of 1,2-epoxy-5,5-dimethylhexane in CH2Cl2, which was used
in the next reaction directly without any further purification.
To a stirred mixture of thioacetate 4 (0.65 g, 3.0 mmol, Applied
Chemical Laboratory, Inc.) in EtOH (10 mL) under argon
atmosphere at room temperature was added slowly 21%
NaOEt solution in EtOH (3.30 mmol). The reaction mixture
was purged with argon for 5 min. To this mixture was then
added 1,2-epoxy-5,5-dimethylhexane (4.48 mmol) in CH2Cl2
(7.0 mL) obtained above. The reaction mixture was again
purged with argon for 5 min and stirred at room temperature
for 19 h. The reaction mixture was concentrated in vacuo and
mixed with saturated NaHCO3 (100 mL), and the product was
extracted with EtOAc (2 × 120 mL) and CH2Cl2 (120 mL). The
combined organic extracts were washed with 5% KHSO4 (40
mL), saturated NaHCO3 (40 mL), and brine (40 mL). The
organic layer was dried over MgSO4 and concentrated in vacuo
to give a crude oil, which was purified by flash column
chromatography on silca gel eluting with 25% EtOAc in CH2-
Cl2 to obtain 7 (0.20 g, 23%). 1H NMR (CD3OD): δ 7.35 (s,
N-[5-[(2-P h en yleth yl)th io]-2-th ia zolyl]a ceta m id e (10).
A solution of KOtBu in THF (0.51 mL of 1 M solution, 0.51
mmol) was added to a stirred mixture of 4 (100 mg, 0.46 mmol)
in dry THF (5 mL) under argon atmosphere. After it was
stirred for 10 min, a solution of phenethyl bromide (103 mg,
0.56 mmol) in THF (1 mL)was added and the mixture was
heated at 55 °C for 7 h. It was cooled to room temperature
and concentrated in vacuo, and the residue was purified by
column chromatography on silica gel eluting with CH2Cl2 to
obtain 10 (104 mg, 81%) as a light yellow solid; mp 172-173
°C. 1H NMR (CDCl3): δ 7.40-7.17 (m, 5H), 3.00 (t, J ) 5.6
Hz, 2H), 2.91 (t, J ) 5.6 Hz, 2H), 2.33 (s, 3H). 13C NMR
(CDCl3): δ 168.0, 162.0,142.0, 139.5, 128.6, 126.6, 123.3, 39.6,
36.0, 23.2. HRMS FAB (M + H)+ calcd for C13H14N2OS2,
279.0626; found, 279.0628.
2-[[2-(Acetylam in o)-5-th iazolyl]th io]-N-eth yl-N-m eth yl-
a ceta m id e (11). A mixture of 6 (15.0 mg, 0.065 mmol), HOBt
(10.0 mg, 0.065 mmol), and EDCI (25 mg, 0.13 mmol) in DMF
(0.5 mL) was stirred at 0 °C for 0.5 h, and then, neat
N-ethylmethylamine (0.1 mL) was added. The mixture was
stirred at 0 °C for 2 h and overnight at room temperature.
The product was purified directly by preparative HPLC to
obtain 11 after lyophilizing (12 mg, 67%) as a light yellow solid
of the mixture of 1:1 ratio of rotamers; mp 138-140 °C. 1H
NMR (CDCl3): δ (a mixture of 1:1 ratio of rotamers) 7.48 and

Aminothiazole Inhibitors of Cyclin-Dependent Kinase 2
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18 3919
7.47 (s, 1H), 3.63 and 3.61 (s, 2H), 3.43 and 3.41 (q, J ) 7.0
Hz, 2H), 3.00 and 2.93 (s, 3H), 2.28 (s, 3H), 1.20 and 1.14 (t,
J ) 7.0 Hz, 3H). MS (ESI): 274 (M + H)+, 272 (M - H)-.
HRMS FAB (M + H)+ calcd for C10H16N3O2S2, 274.0684; found,
274.0692.
anhydride (100 mL) at room temperature was added concen-
trated sulfuric acid (10 mL). The mixture was stirred at 55-
60 °C for 2 h, sodium acetate (15 g, 180 mmol) was added,
and it was concentrated in vacuo. To the residue was added
cold water (100 mL), the precipitated solid was collected,
washed with water, and dried, and the product was purified
by flash chromatography on silica gel (CH2Cl2:MeOH/100:5)
to afford 14 (4.2 g, 43%) as a light yellow solid; mp 147-148
°C. 1H NMR (CDCl3): δ 12.47 (s, 1H), 7.29 (s, 1H), 6.61 (s,
1H), 3.91 (s, 2H), 2.64 (q, J ) 7.6 Hz, 2H), 2.25 (s, 3H), 1.21 (t,
J ) 7.6 Hz, 3H). 13C NMR (CDCl3): δ 168.1, 163.1, 158.9, 155.3,
143.6, 122.0, 120.9, 34.9, 23.0, 19.1, 11.8. MS (ESI): 284 (M
+ H)+. Anal. (C11H13N3O2S2) C, H, N, S.
2-Ch lor o-N-[1-(h yd r oxym eth yl)p r op yl]a ceta m id e (15).
To a mixture of 2-amino-1-butanol (5.0 mL, 53 mmol) and Et3N
(15.0 mL, 111 mmol) in CH2Cl2 (20 mL) at -70 °C was added
chloroacetyl chloride (4.6 mL, 58 mmol) dropwise. The reaction
mixture was stirred at -70 °C for 15 min, warmed to room
temperature, diluted with EtOAc (50 mL), and quenched with
water (50 mL). The organic layer was separated, and the
aqueous layer was extracted with EtOAc (3 × 30 mL). The
combined organic layers were dried over MgSO4 and concen-
trated to afford 15 (8.6 g, 98%) as a brown solid. 1H NMR
(CDCl3): δ 6.75 (bs, 1H), 4.03 (s, 2H), 3.90 (m, 1H), 3.68 (m,
2H), 2.98 (bs, 1H), 1.60 (m, 2H), 0.97 (t, J ) 7.8 Hz, 3H).
2-Ch lor o-N-(1-for m ylp r op yl)a ceta m id e (16). To a solu-
tion of oxalyl chloride (14.5 mL of 2 M solution in CH2Cl2, 29.0
mmol) in CH2Cl2 (30 mL) at -78 °C DMSO (2.75 mL, 38.8
mmol) was added dropwise over 5 min. The reaction mixture
was stirred at -78 °C for 10 min, and then, 15 (4.0 g, 24 mmol)
in CH2Cl2 (20 mL) was added dropwise over 15 min. After the
reaction mixture was stirred at -78 °C for 40 min, Et3N (9.4
mL, 68 mmol) was added dropwise over 5 min and the reaction
mixture was allowed to warm to room temperature and stirred
for 2 h. The precipitated solid was removed by filtration and
washed with EtOAc. The organic phase was washed with 1 N
HCl (2 × 100 mL) and saturated NaHCO3 (10 mL), dried over
MgSO4, concentrated, and purified by flash chromatography
on silica gel (CH2Cl2:MeOH/100:7) to afford 16 (3.7 g, 95%) as
a brown oil. 1H NMR (CDCl3): δ 9.60 (s, 1H), 7.16 (broad, 1H),
4.52 (m, 1H), 4.12 (s, 2H), 2.05 (m, 1H), 1.80 (m, 1H), 0.97 (t,
J ) 7.8 Hz, 3H).
[[2-(Acetyla m in o)-5-th ia zolyl]th io]a cetic Acid Ben zyl
Ester (12). A mixture of 6 (34.8 mg, 0.15 mmol), benzyl alcohol
(24.3 mg, 0.225 mmol), diethyl azodicarboxylate (32.0 mg, 0.18
mmol), and triphenylphosphine (59.0 mg, 0.225 mmol) in THF
(1.5 mL) was stirred at 0 °C for 1 h, warmed to room
temperature, and was allowed to stay overnight. The mixture
was concentrated in vacuo, and the residue was purified by
preparative HPLC. The fraction containing the desired product
was collected, concentrated, and lyophilized to afford 12 as a
beige-colored solid (36.0 mg, 75%); mp 142 °C. 1H NMR
(CDCl3): δ 7.39 (s, 1H), 7.26-7.33 (m, 5H), 5.30 (s, 2H), 3.50
(s, 2H), 2.26 (s, 3H). MS (ESI): 323 (M + H)+, 321 (M - H)-.
Anal. (C14H14N2O3S2) C, H, N, S.
[[2-(Acet yla m in o)-5-t h ia zolyl]t h io]-N-(2-oxob u t yl)a c-
eta m id e (13). (A) 1-[(Ben zyloxyca r bon yl)a m in o]-2-bu -
ta n ol. A mixture of 1-amino-2-butanol (5.5 g, 61.8 mmol),
benzyl chloroformate (11.5 g, 67.6 mmol), and sodium carbon-
ate (7.16 g, 67.7 mmol) in water (50 mL) was stirred at 0 °C
for 3 h. Water (50 mL) was added to the reaction mixture, the
product was extracted with CH2Cl2 (3 × 20 mL), dried over
Na2SO4, and concentrated, and the residue was purified by a
flash chromatography on SiO2 eluting with hexanes:EtOAc/
10:1 followed by EtOAc to afford 1-benzyloxycarbonylamino-
2-butanol (13.9 g, 100%) as an oil. 1H NMR (CDCl3): δ 7.30
(m, 5H), 5.45 (s, 1H), 5.06 (s, 2H), 3.57 (s, 1H), 3.31 (m, 1H),
3.04 (m, 1H), 2.91 (m, 1H), 1.43 (m, 2H), 0.91 (t, J ) 7.6 Hz,
3 H).
(B) 1-[(Ben zyloxyca r bon yl)a m in o]-2-bu ta n on e. To a
solution of oxalyl chloride (37 mL of 2 M solution in CH2Cl2,
74 mmol) in CH2Cl2 (60 mL) at -78 °C under argon was added
dimethyl sulfoxide (DMSO, 7.8 g, 100 mmol). The mixture was
stirred at -78 °C for 20 min, and to this mixture was added a
solution of 1-benzyloxycarbonylamino-2-butanol (13.9 g, 61.8
mmol) in CH2Cl2 (40 mL). The mixture was stirred at -78 °C
for 1 h, and then, Et3N (21 mL) was added to the mixture.
The mixture was warmed to room temperature and washed
with 1 N hydrochloric acid followed by aqueous sodium
bicarbonate solution. The organic solution was dried over
MgSO4 and concentrated to afford 1-benzyloxycarbonylamino-
2-butanone (11.2 g, 82%) as a solid, which was enough pure
2-(Ch lor om eth yl)-4-eth yloxa zole (17). To a solution of
16 (3.7 g, 23 mmol) in toluene (10 mL) at room temperature
was added POCl3 (6.3 mL, 68 mmol). The reaction mixture
was heated at 90 °C for 1 h under nitrogen. After it was cooled
to room temperature, the reaction mixture was poured into
ice water (10 mL) and the pH was adjusted to 7 with 5 N
NaOH. The organic layer was separated, and the aqueous layer
was extracted with CH2Cl2 (3 × 20 mL). The combined organic
layers were concentrated and distilled (105 °C/760 mm Hg) to
1
for the next reaction. H NMR (CDCl3): δ 7.32 (m, 5H), 5.50
(s, 1H), 5.06 (s, 2H), 4.07 (s, 2H), 2.43 (q, J ) 7.6 Hz, 2H),
1.06 (t, J ) 7.6 Hz, 3H).
(C) 1-Am in o-2-bu ta n on e. A solution of 1-benzyloxycarbon-
ylamino-2-butanone (9.30 mg, 42 mmol) in ethanol (50 mL)
and 1 N hydrochloric acid solution (46 mL) was stirred under
hydrogen gas (1 atm) in the presence of Pd/C (1.5 g, 10%) at
room temperature for 4 h. The mixture was filtered, and the
filtrate solution was concentrated. The residue was triturated
with Et2O to afford 1-amino-2-butanone (5.29 g, 100%) as a
white solid of hydrochloride salt. 1H NMR (CD3OD): δ 3.97
(s, 2H), 2.60 (q, J ) 7.6 Hz, 2H), 1.08 (t, J ) 7.6 Hz, 3H). 13C
NMR (CD3OD): δ 203.2, 46.5, 32.6, 6.06.
(D) [[2-(Acetyla m in o)-5-th ia zolyl]th io]-N-(2-oxobu tyl)-
a ceta m id e (13). A mixture of acid 6 (9.0 g, 39 mmol), HOBt
(5.94 g, 39 mmol), and EDCI (11.16 g, 58 mmol) in DMF (50
mL) was stirred at 0 °C for 0.5 h. To this mixture was added
1-amino-2-butanone hydrochloride (5.27 g, 42.7 mmol) followed
by Et3N (15 mL, 107.5 mmol). The mixture was stirred at 0
°C for 0.5 h and at room temperature for 1 h. Water (200 mL)
was added, and the product was extracted with CH2Cl2
containing 10% MeOH (5 × 100 mL). The combined extracts
were dried over Na2SO4 and concentrated, and the residue was
triturated with water to obtain 13 as a solid (10.5 g, 90%); mp
195-196 °C. 1H NMR (CDCl3): δ 7.53 (s, 1H), 4.14 (s, 2H),
3.46 (s, 2H), 2.50 (q, J ) 7.6 Hz, 2H), 2.25 (s, 3H), 1.12 (t, J )
7.6 Hz, 3H). MS (ESI): 302 (M + H)+.
1
afford 17 (1.1 g, 31%) as a colorless liquid. H NMR (CDCl3):
δ 7.30 (s, 1H), 4.50 (s, 2H), 2.50 (q, J ) 7.3 Hz, 2H), 1.22 (t,
J ) 7.3 Hz, 3H).
N-[5-[[(4-E t h yl-2-oxa zolyl)m et h yl]t h io]-2-t h ia zolyl]-
a ceta m id e (18). To a solution of 4 (10 mg, 0.050 mmol) in
THF (5 mL) at room temperature was added KOtBu (0.05 mL
of 1.0 M solution in THF, 0.05 mmol). The reaction mixture
was stirred at room temperature for 15 min, and then, 17 (15
mg, 0.10 mmol) was added. After it was stirred for 3 h,
saturated NaHCO3 solution (5 mL) was added. The organic
layer was separated, and the aqueous layer was extracted with
CH2Cl2 (3 × 10 mL). The combined organic layers were
concentrated, and the residue was purified by flash chroma-
tography on SiO2 (MeOH:CH2Cl2/1:20) to afford 18 (5 mg, 36%)
as a white solid. 1H NMR (CDCl3): δ 11.25 (s, 1H), 7.34 (s,
1H), 7.31 (s, 1H), 3.95 (s, 2H), 2.50 (q, J ) 6.7 Hz, 2H), 2.27
(s, 3H), 1.19 (t, J ) 6.7 Hz, 3H). MS (ESI): 284 (M + H)+.
HRMS FAB (M + H)+ calcd for C11H13N3O2S2, 284.0528; found,
284.0529.
2-(Br om om eth yl)-4,5-d im eth yloxa zole (19). A mixture
of 2,4,5-trimethyloxazole (0.50 mL, 4.3 mmol, Aldrich Chemical
Co.), N-bromosuccinimide (0.77 g, 4.3 mmol), and benzoyl
peroxide (0.21 g, 0.86 mmol) in CCl4 (4 mL) was heated at 76
N-[5-[[(5-Eth yl-2-oxa zolyl)m eth yl]th io]-2-th ia zolyl]a c-
eta m id e (14). To a solution of 13 (10.5 g, 34.8 mmol) in acetic

3920 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18
Kim et al.
°C under nitrogen for 3 h. After it was cooled to room
temperature, the solid was removed by filtration, the filtrate
was washed with saturated NaHCO3 (20 mL) and concen-
trated, and the residue was purified by flash chromatography
on SiO2 (hexanes:EtOAc/4:1) to afford 19 (64 mg, 8% yield) as
(CDCl3): δ 11.55 (s, 1H), 7.32 (s, 1H), 6.61 (s, 1H), 3.96 (s,
2H), 2.94 (hept, J ) 6.8 Hz, 1H), 2.28 (s, 3H), 1.24 (d, J ) 6.8
Hz, 6H). 13C NMR (CDCl3): δ 167.86, 162.55, 159.23, 158.83,
143.89, 120.94, 34.91, 26.04, 23.08, 20.64. MS (ESI): 298 (M
+ H)+. HRMS FAB (M + H)+ calcd for C12H16N3O2S2, 298.0684;
found, 298.0687.
N-[5-[[[5-(Cycloh exylm eth yl)-2-oxa zolyl]m eth yl]th io]-
2-th ia zolyl]a ceta m id e (26). Yield, 55%; mp 151-153 °C. 1H
NMR (CDCl3): δ 7.24 (s, 1H), 6.62 (s, 1H), 3.96 (s, 2H), 2.48
(d, J ) 7.0 Hz, 2H), 2.31 (s, 3H), 1.50-1.72 (m, 5H), 1.10-
1.30 (m, 4H), 0.85-0.95 (m, 2H). HRMS FAB (M + H)+ calcd
for C16H21N3O2S2, 352.1154; found, 352.1167.
N-[5-[[[5-P h en yl-2-oxa zolyl]m eth yl]th io]-2-th ia zolyl]-
a ceta m id e (27). Yield, 49%; mp 193-195 °C. 1H NMR
(CDCl3): δ 7.53 (s, 1H), 7.51 (s, 1H), 7.18-7.35 (m, 5H), 4.00
(s, 2H), 2.18 (s, 3H). 13C NMR (CDCl3): δ 168.1, 162.9, 159.4,
152.4, 141.1, 129.0, 128.8, 127.5, 124.2, 122.3, 121.3, 34.7, 23.0.
HRMS FAB (M + H)+ calcd for C15H13N3O2S2, 332.0528; found,
332.0513.
1
a yellow oil. H NMR (CDCl3): δ 4.4 (s, 2H), 2.25 (s, 3H), 2.05
(s, 3H).
N-[5-[[(4,5-Dim eth yl-2-oxazolyl)m eth yl]th io]-2-th iazolyl]-
a ceta m id e (20). To a solution of 19 (0.050 g, 0.23 mmol) in
THF (10 mL) at room temperature KOtBu (0.25 mL of 1.0 M
solution in THF, 0.25 mmol) was added. After the reaction
mixture was stirred at room temperature for 15 min, 19 (0.064
g, 0.34 mmol) was added. The reaction mixture was stirred at
room temperature for 3 h. Saturated NaHCO3 solution (20 mL)
was added, the organic layer was separated, and the aqueous
layer was extracted with CH2Cl2 (3 × 20 mL). The combined
organic layers were concentrated, and the residue was purified
by flash chromatography on silica gel (MeOH:CH2Cl2/1:20) to
1
afford 20 (15 mg, 23%) as a yellow solid. H NMR (CDCl3): δ
11.78 (s, 1H), 7.38 (s, 1H), 3.90 (s, 2H), 2.30 (s, 3H), 2.22 (s
3H), 2.05 (s, 3H). MS (ESI): 284 (M + H)+. HRMS FAB (M +
H)+ calcd for C11H13N3O2S2, 284.0528; found, 284.0526.
5-[[[5-t er t -Bu t yl-2-oxa zolyl]m e t h yl]t h io]-2-t h ia zol-
a m in e (28). A solution of 25 (5.56 g 17.9 mmol) and sodium
hydroxide solution (1 M in water, 36 mL, 36 mmol) in ethanol
(46 mL) was heated at reflux temperature under argon for 7
h, additional sodium hydroxide solution (1 M in water, 5 mL,
5 mmol) was added, and the mixture was heated for 2 more
hours. It was concentrated, and hydrochloric acid solution (1
M in water, 20 mL, 20 mmol) was added (pH ∼13) to the
residue. The precipitated solid was filtered, washed with
water, and purified by a flash column on silica gel (EtOAc,
and then EtOAc:MeOH/95:5) to obtain 28 (3.63 g, 75%) as a
yellow solid; mp 118-121 °C. 1H NMR (CDCl3): δ 6.99 (s, 1H),
6.59 (s, 1H), 5.01 (s, 2H), 3.89 (s, 2H), 1.27 (s, 9H). 13C NMR
(CDCl3): δ 171.78, 162.04, 158.65, 141.99, 120.02, 115.49,
34.60, 31.49, 28.59. MS (ESI): 270 (M + H)+. Anal. (C11H15N3-
OS2) C, H, N, S.
1-Dia zo-3,3-d im eth yl-2-bu ta n on e (21, R ) tBu ). To a
mixture of 40% aqueous KOH solution (15 mL) and Et2O (50
mL) at 0 °C was added N-methyl-N′-nitro-N-nitrosoguanidine
(5 g, 68 mmol) in portions with stirring. The mixture was
stirred at 0 °C for 0.5 h. The organic phase was decanted into
a dry flask and dried over solid KOH pellets to give diaz-
omethane solution (50 mL, ca. 0.5 M by titrating with acetic
acid). To the diazomethane solution obtained above at 0 °C
was added a solution of trimethylacetyl chloride (1.23 mL, 10
mmol) in Et2O (1 mL) dropwise with stirring. The mixture was
kept at 0 °C for 16 h. The solution was purged with argon to
remove the excess diazomethane, and then, Et2O was removed
under reduced pressure to give crude 21 (1.33 g, 10 mmol,
100%) as a yellow liquid, which was used directly in the next
step.
N-[5-[[[5-ter t-Bu tyl-2-oxazolyl]m eth yl]th io]-2-th iazolyl]-
2-isop r op yla m id e (29). To a solution of aminothiazole 28
(0.97 g, 3.6 mmol) and pyridine (0.37 g, 4.7 mmol) in CH2Cl2
was slowly added isobutyryl chloride (0.43 g, 4.0 mmol) at ice
bath temperature. The reaction mixture was warmed to room
temperature and stirred for 4 h. It was diluted with CH2Cl2
and washed with saturated aqueous NaHCO3, and the organic
layer was collected, dried over Na2SO4, and concentrated to
obtain the solid residue, which was recrystallized from EtOAc/
hexane (1:3) to give 29 as a white solid (1.14 g, 93%); mp 182-
2-(Ch lor om eth yl)-5-ter t-bu tyloxa zole (22d ). To a solu-
tion of boron trifluoride etherate (2 mL, 16 mmol) in chloro-
acetonitrile (20 mL, 320 mmol) at 0 °C was added a solution
of 1-diazo-3,3-dimethyl-2-butanone (1.33 g, 10 mmol) in chlo-
roacetonitrile (5 mL, 80 mmol) dropwise. The resulting solution
was stirred at 0 °C for 0.5 h, and it was poured onto saturated
aqueous sodium bicarbonate solution to neutralize the acid.
Product was extracted with CH2Cl2 (3 × 50 mL), and the
combined extracts were dried, concentrated, and purified by
flash chromatography on silica gel eluting with CH2Cl2 to give
22d (1.1 g, 6.4 mmol, 64% from the acid chloride) as a yellow
1
183 °C. H NMR (CDCl3): δ 11.76 (br, 1H), 7.29 (s, 1H), 6.59
(s, 1H), 3.96 (s, 2H), 2.67 (m, 1H), 1.28 (d, J ) 6.6 Hz, 6H),
1.25 (s, 9H). 13C NMR (CDCl3): δ 175.09, 162.97, 161.76,
158.81, 143.68, 120.81, 125.13, 35.25, 34.86, 31.41, 28.53,
19.17. MS (ESI): 340 (M + H)+. Anal. (C15H21N2O2S2) C, H,
N, S.
1
liquid. H NMR (CDCl3): δ 1.30 (s, 9H), 4.58 (s, 2H), 6.68 (s,
1H). 13C NMR (CDCl3): δ 28.56, 31.54, 36.20, 120.51, 157.69,
162.56. MS (ESI): 174 (M + H)+.
N-[5-[[(5-ter t-Bu tyl-2-oxa zolyl)m eth yl]th io]-2-th ia zol-
yl]a ceta m id e (25). To a solution of 4 (50 mg, 0.23 mmol) in
THF (10 mL) was added KOtBu solution (0.25 mL of 1 M
solution, 0.25 mmol) at room temperature under argon. The
heterogeneous mixture was stirred for 15 min, and then, a
solution of 22d (59 mg, 0.34 mmol) in THF (1 mL) was added.
It was stirred at room temperature for 16 h, concentrated, and
purified by flash chromatography on silica gel (EtOAc:hexanes/
1:1 and then EtOAc) to give 25 (44 mg, 0.14 mmol, 61%) as a
white solid; mp 141-142 °C. 1H NMR (CDCl3): δ 11.03 (broad
s, 1H), 7.31 (s, 1H), 6.59 (s, 1H), 3.95 (s, 2H), 2.27 (s, 3H), 1.27
(s, 9H). 13C NMR (CDCl3): δ 167.94, 162.45, 162.12, 159.04,
144.35, 121.24, 120.36, 35.15, 31.71, 28.83, 23.34. MS (ESI):
312 (M + H)+. Anal. (C13H17N3O2S2) C, H, N, S.
N-[5-[[(5-Meth yl-2-oxa zolyl)m eth yl]th io]-2-th ia zolyl]-
a ceta m id e (23). Yield, 57%; mp 159-160 °C. 1H NMR
(CDCl3): δ 11.72 (s, 1H), 7.34 (s, 1H), 6.63 (s, 1H), 3.95 (s,
2H), 2.31 (s, 3H), 2.30 (s, 3H). 13C NMR (CDCl3): δ 167.93,
162.60, 158.94, 149.79, 143.82, 123.24, 120.93, 34.86, 23.03,
10.94. MS (ESI): 270 (M + H)+. Anal. (C10H11N3O2S2‚0‚4H2O)
C, H, N, S.
N-[5-[[[5-ter t-Bu tyl-2-oxazolyl]m eth yl]th io]-2-th iazolyl]-
2,2-ter t-bu tyla m id e (30). mp 133-134 °C. 1H NMR
(CDCl3): δ 9.66 (br, 1H), 7.32 (s, 1H), 6.60 (s, 1H), 3.92 (s,
2H), 1.29 (s, 9H), 2.25 (s, 9H). 13C NMR (CDCl3): δ 176.24,
161.90, 161.64, 144.61, 120.85, 120.13, 39.08, 34.90, 31.37,
28.65, 28.51, 27.08. MS (ESI): 354 (M + H)+. Anal. (C16H23
-
N2O2S2) C, H, N, S.
N-[5-[[[5-ter t-Bu tyl-2-oxazolyl]m eth yl]th io]-2-th iazolyl]-
ben za m id e (31). Yield, 80%; mp 149-150 °C. 1H NMR (CD3-
OD): δ 7.99 (d, J ) 7.5 Hz, 2H), 7.64 (t, J ) 7.5 Hz, 1H), 7.54
(t, J ) 7.5 Hz, 2H), 7.40 (s, 1H), 6.68 (s, 1H), 4.02 (s, 2H), 1.25
(s, 9H). MS (ESI): 374 (M + H)+. HRMS FAB (M - H)- calcd
for C18H19N3O2S2, 372.0840; found, 372.0837.
(rR)-N-[5-[[[5-ter t-Bu tyl-2-oxa zolyl]m eth yl]th io]-2-th i-
a zolyl]-r-m eth ylben zen ea ceta m id e (32) a n d (rS)-N-[5-
[[[5-ter t-Bu tyl-2-oxa zolyl]m eth yl]th io]-2-th ia zolyl]-r-m e-
t h ylb en zen ea cet a m id e (33). Compounds 32 and 33 were
prepared by coupling with (R)- and (S)-mandelic acid to obtain
the products as glassy material in 50 and 53% yields, respec-
tively. Compound 32:1H NMR (CDCl3): δ 7.29-7.19 (m, 5H),
7.18 (s, 1H), 6.49 (s, 1H), 3.86 (s, 2H), 3.72 (q, J ) 7.0 Hz,
1H), 1.52 (d, J ) 7.0 Hz, 3H), 1.15 (s, 9H), NH of amide: too
broad to assign. 13C NMR (CDCl3): δ 179.7, 172.1, 162.2, 161.7,
N-[5-[[[5-Isop r op yl-2-oxa zolyl]m eth yl]th io]-2-th ia zol-
yl]a ceta m id e (24). Yield, 79%; mp 123-124 °C. 1H NMR

Aminothiazole Inhibitors of Cyclin-Dependent Kinase 2
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18 3921
158.7, 143.6, 139.4, 119.1, 127.8, 127.5, 121.0, 120.1, 46.7, 45.7,
34.8, 31.4, 28.5, 18.4. MS (ESI): 402 (M + H)+. Anal.
(C20H23N3O2S2) C, H, N, S.
3.32 (q, J ) 6.7 Hz, 2H), 1.21 (s, 9H), 1.17 (t, J ) 6.7 Hz, 3H).
13C NMR (CDCl3): δ 165.57, 161.74, 158.91, 154.33, 144.65,
120.00, 118.35, 34.98, 34.80, 31.37, 28.51, 15.14. MS (ESI): 341
(M + H)+. Anal. (C14H20N4O2S2) C, H, N, S.
N-[5-[[[5-ter t-Bu tyl-2-oxazolyl]m eth yl]th io]-2-th iazolyl]-
N′-(4-flu or op h en yl)u r ea (39). Yield, 83%. 1H NMR (acetone-
d6): δ 7.58 (dd, J 1 ) 4.9 Hz, J 2 ) 4.2 Hz, 2H), 7.25 (s, 1H),
7.11 (dd, J 1 ) J 2 ) 8.8 Hz, 2H), 6.65 (s, 1H), 4.01 (s, 2H), 1.26
(s, 9H). 13C NMR (acetone-d6): δ 164.7, 161.9, 160.4, 159.1,
158.0, 151.9, 144.6, 133.3, 121.8, 119.6, 115,6, 115.4, 34.7, 31.3,
28.6. MS (ESI): 407.09 (M + H)+. Anal. (C18H19FN4O2S2) C,
H, N, S, F.
N-(2,6-Diflu or op h en yl)-N′-[5-[[[5-ter t-bu tyl-2-oxa zolyl]-
m eth yl]th io]-2-th ia zolyl]u r ea (40). To a stirred mixture of
aminothiazole 28 (50 mg, 0.19 mmol) in DMF (1.2 mL) was
added 2,6-difluorophenylisocyanate (57.8 mg, 0.37 mmol). The
mixture was heated at 90 °C for 3.5 h, additional 2,6-
difluorophenylisocyanate (15 mg, 0.01 mmol) was added, and
the reaction mixture was further heated at 90 °C for 3 h. It
was concentrated in vacuo and purified by preparative HPLC
to give the desired compound in 51 mg (65%) as a white solid;
mp 133-134 °C. 1H NMR (CD3OD): δ 7.35-7.60 (m, 1H), 7.24
(s, 1H), 7.06 (t, J ) 7.9 Hz, 2H), 6.67 (s, 1H), 4.85 (s, 2H), 1.24
(s, 9H). MS (ESI): 425.9 (M + H)+, 423.1 (M - H)-. Anal.
(C18H18F2N4O2S2) C, H, N, S.
[5-[[[5-ter t-Bu tyl-2-oxa zolyl]m eth yl]th io]-2-th ia zolyl]-
isop r op yl Ca r ba m a te (34). A solution of 28 (162 g, 0.6
mmol), isopropyl chloroformate (1 M in toluene, 0.72 mL, 0.72
mmol), pyridine (0.2 mL, 2.5 mmol), and 4-(dimethylamino)-
pyridine (26 mg, 0.21 mmol) in CH2Cl2 (10 mL) was stirred at
room temperature overnight. Water (20 mL) and EtOAc (20
mL) were added to the reaction mixture. The organic solution
was separated, and the aqueous layer was washed with EtOAc
(30 mL). The combined organic solutions were dried (Na2SO4)
and concentrated, and the residue was purified by flash
chromatography on silica gel (EtOAc:hexanes/1:2, and then
EtOAc:hexanes/1:1) to afford 34 (194 mg, 91%) as a white solid;
mp 100-101 °C. 1H NMR (CDCl3): δ 12.48 (s, 1H), 7.20 (s,
1H), 6.56 (s, 1H), 5.03 (hept, J ) 6.2 Hz, 1H), 3.91 (s, 2H),
1.31 (d, J ) 6.2 Hz, 6H), 1.20 (s, 9H). 13C NMR (CDCl3): δ
165.07, 161.54, 158.81, 153.21, 144.29, 120.02, 119.44, 70.14,
34.78, 31.29, 28.43, 21.83. MS (ESI): 356 (M + H)+. Anal.
(C15H21N3O3S2‚0‚2H2O) C, H, N, S.
[5-[[[5-ter t-Bu tyl-2-oxa zolyl]m eth yl]th io]-2-th ia zolyl]-
ben zyl Ca r ba m a te (35). Yield, 93%; mp 126-127 °C. 1H
NMR (CDCl3): δ 12.67 (s, 1H), 7.35-7.40 (m, 5H), 7.11 (s, 1H),
6.55 (s, 1H), 5.24 (s, 2H), 3.89 (s, 2H) 1.19 (s, 9H). 13C NMR
(CDCl3): δ 164.70, 161.54, 158.81, 153.45, 144.43, 135.21,
128.62, 128.58, 128.25, 120.00, 119.84, 67.94, 34.72, 31.25,
28.41. MS (ESI): 404 (M + H)+. Anal. (C19H21N3O3S2) C, H, N, S.
N-(2,6-Dich lor op h en yl)-N′-[5-[[[5-ter t-bu tyl-2-oxa zolyl]-
m eth yl]th io]-2-th ia zolyl]u r ea (41). Yield, 59%; mp 88-89
1
°C. H NMR (CDCl3): δ 7.39 (d, J ) 8.3 Hz, 2H), 7.23 (t, J )
8.3 Hz, 1H), 6.63 (s, 1H), 3.98 (s, 2H), 1.27 (s, 9H). MS (ESI):
N-[5-[[[5-ter t-Bu tyl-2-oxazolyl]m eth yl]th io]-2-th iazolyl]-
ben zen esu lfon a m id e (36). To a solution of 28 (162 mg, 0.6
mmol) in pyridine (1 mL) was added benzenesufonyl chloride
(0.9 mmol) under argon with stirring. The reaction mixture
was stirred at 50 °C for 2.5 h. Additional benzenesufonyl
chloride (0.9 mmol) was added dropwise with stirring at 50
°C, and the mixture was stirred at 60 °C for 5 h. To the mixture
was added EtOAc (50 mL) and washed with water (5 mL),
aqueous HCl (1 M solution, 5 mL), and water. The organic
solution was separated, dried (Na2SO4), concentrated, and
purified by a flash chromatography on silica gel (EtOAc:
heptane/1:1, and then EtOAc) to afford a yellow solid, which
was recrystallized (EtOAc:heptane) to give 36 (143 mg, 58%)
as a white solid; mp 189-190 °C. 1H NMR (DMSO-d6): δ 12.88
(s, 1H), 7.78 (d, J ) 7.1 Hz, 2H), 7.62 (t, J ) 7.4 Hz, 1H), 7.56
(t, J ) 7.4 Hz, 2H), 7.38 (s, 1H), 6.75 (s, 1H), 4.11 (s, 2H), 1.19
(s, 9H). 13C NMR (DMSO-d6): δ 169.36, 160.76, 158.05, 141.73,
132.01, 131.36, 128.75, 125.36, 119.84, 112.27, 32.665, 30.68,
28.01. MS (ESI): 410 (M + H)+. Anal. (C17H19N3O3S3) C, H,
N, S.
458, 459 (M + H)+.
4-(Br om om eth yl)-N-[5-[[[5-ter t-bu tyl-2-oxa zolyl]m eth -
yl]th io]-2-th ia zolyl]ben zen ea ceta m id e (43). To a stirred
mixture of 28 (8.01 g, 29.7 mmol) and EDCI (6.83 g, 35.6 mmol)
in CH2Cl2 (100 mL) at room temperature under nitrogen was
added 42 (10.26 g, 44.8 mmol, Aldrich Chemical Co.). The
reaction mixture was stirred at room temperature for 30 min,
diluted with EtOAc (300 mL), and washed with H2O (60 mL),
saturated NaHCO3 solution (2 × 60 mL), and brine (2 × 60
mL). The organic solution was dried over MgSO4 and concen-
trated, and the residue solid was triturated with ethyl ether
to obtain 43 (11.0 g, 77%). 1H NMR (CDCl3): δ 7.39 (d, J )
8.36 Hz, 2H), 7.30 (s, 1H), 7.27 (d, J ) 8.36 Hz, 2H), 6.55 (s,
1H), 4.49 (s, 2H), 3.94 (s, 2H), 3.80 (s, 2H), 1.23 (s, 9H). MS
(ESI): 481.98 (M + H)+.
N-[5-[[[5-ter t-Bu tyl-2-oxazolyl]m eth yl]th io]-2-th iazolyl]-
4-[[[b is (h y d r o x y m e t h y l)m e t h y l]a m in o ]m e t h y l]b e n -
zen ea ceta m id e Hyd r och lor id e Sa lt (45). To a mixture of
serinol 44 (5.0 g, 55 mmol) in THF (20 mL) and DMF (20 mL)
at room temperature under nitrogen atmosphere was added
43 (1.5 g, 3.1 mmol) in portions over 10 min, and it was stirred
at room temperature overnight. The reaction mixture was
diluted with EtOAc (180 mL), washed with 10% citric acid
solution and 10% LiCl solution, dried over MgSO4, and
concentrated to an yellow oil, which was purified by flash
chromatography on silica gel (15% MeOH in EtOAc with 0.6%
NH4OH) to yield the pure product as a free amine (1.03 g,
62.8%). The free amine was mixed with 1 N HCl (2.1 mL) to
give its HCl salt after drying; mp 132-134 °C. 1H NMR
(DMSO-d6): δ 12.56 (s, 1H), 8.89 (s, 1H), 7.50 (d, J ) 8.14 Hz,
1H), 7.39 (s, 1H), 7.37 (d, J ) 8.14 Hz, 1H), 6.70 (s, 1H), 4.20
(m, 2H), 4.05 (s, 2H), 3.79 (s, 2H), 3.66 (d, J ) 2.1 Hz, 4H),
3.05 (q, J ) 2.1 Hz, 1H). 13C NMR (DMSO-d6): δ 169.5, 161.5,
161.1, 159.0, 145.1, 135.5, 130.8, 130.4, 129.6, 120.2, 119.2,
N-[5-[[[5-ter t-Bu tyl-2-oxazolyl]m eth yl]th io]-2-th iazolyl]-
N-m eth yl-2-isop r op yla m id e (37). To a mixture of 29 (102
mg, 0.30 mmol) and methyl iodide (86 mg, 0.61 mmol) in DMF
(2 mL) was added K2CO3 (80 mg, 0.58 mmol) at 0 °C. The
mixture was stirred at 0 °C and then at room temperature for
7 h. EtOAc (60 mL) and H2O (40 mL) were added to the
reaction mixture. The organic solution was separated, and the
aqueous solution was washed with ethyl acetate (50 mL). The
combined organic solutions were dried (Na2SO4) and concen-
trated, and the crude product was purified by flash chroma-
tography on silica gel (EtOAc:hexanes/1:2, and then EtOAc:
1
hexanes/1:1) to afford 37 (66 mg, 62%) as a yellow liquid. H
NMR (CDCl3): δ 7.37 (s, 1H), 6.58 (s, 1H), 3.93 (s, 2H), 3.70
(s, 3H), 3.07 (hept, J ) 6.7 Hz, 1H), 1.26 (d, J ) 6.7 Hz, 6H),
1.24 (s, 9H). 13C NMR (CDCl3): δ 176.81, 163.42, 161.97,
159.40, 144.81, 121.84, 120.48, 35.29, 34.57, 32.45, 31.78,
28.90, 19.42. MS (ESI): 354 (M + H)+. HRMS FAB (M + H)+
calcd for C16H24N3O2S2, 354.1310; found, 354.1296.
N-[5-[[[5-ter t-Bu tyl-2-oxazolyl]m eth yl]th io]-2-th iazolyl]-
N′-eth ylu r ea (38). To a solution of 28 (162 mg, 0.6 mmol) in
THF (10 mL) was added ethyl isocyanate (0.12 mL, 1.4 mmol)
under argon at room temperature, and the mixture was stirred
at 55 °C for 68 h. It was concentrated and purified by flash
chromatography on silica gel (EtOAc:heptane/1:1, and then
EtOAc) to afford 38 (98 mg, 48%) as a yellow glassy material.
1H NMR (CDCl3): δ 7.15 (s, 1H), 6.57 (s, 1H), 3.91 (s, 2H),
59.6, 57.4, 47.9, 41.4, 34.2, 31.2, 28.5. MS (ESI): 491.3 (M+
H). Anal. (C23H30N4O4S2‚HCl) C, H, N, S, Cl.
+
N-[5-[[(5-Eth yl-2-oxa zolyl)m eth yl]th io]-2-th ia zolyl]-1-
1
isop r op yla m id e (46). Yield, 91%; mp 143-144 °C. H NMR
(CDCl3): δ 11.71 (s, 1H), 7.30 (s, 1H), 6.64 (1s, 1H), 3.95 (s,
2H), 2.65 (m, 3H), 1.28 (d, J ) 7.0 Hz, 6H), 1.23 (t, J ) 7.6
Hz, 3H). 13C NMR (CDCl3): δ 176.1, 163.9, 159.9, 156.2, 144.8,
123.0, 121.9, 36.3, 35.9, 20.2, 20.1, 12.8. Anal. (C13H17N3O2S2)
C, H, N, S.
N-[5-[[(5-Eth yl-2-oxa zolyl)m eth yl]th io]-2-th ia zolyl]-3-
p yr id in ea ceta m id e (47). Yield, 49%; mp 166-167 °C. 1H

3922 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18
Kim et al.
NMR (DMSO-d6): δ 8.92 (s, 1H), 8.88 (d, J ) 5.72 Hz, 1H),
8.55 (d, J ) 7.92 Hz, 1H), 8.08 (m, 1H), 7.43 (s, 1H), 6.73 (s,
1H), 4.16 (s, 2H), 4.05 (s, 2H), 2.60 (q, J ) 7.48 Hz, 2H), 1.13
(t, J ) 7.48 Hz, 1H). 13C NMR (DMSO-d6): δ 167.8, 160.6,
158.5, 154.3, 147.3, 144.9,141.9, 139.8, 134.5, 126.6, 121.6,
119.0, 37.5, 33.8, 18.1, 11.5. MS (ESI): 361.5 (M + H)+. Anal.
(C16H16N4O2S2‚2HCl‚0‚5H2O) C, H, N, S, Cl.
mg, 0.147 mmol) and m-chloroperoxybenzoic aicd (100 mg,
0.319 mmol) in CH2Cl2 (5 mL) was stirred at room temperature
overnight. The reaction mixture was diluted with CH2Cl2,
washed with aqueous NaHCO3 solution, dried over MgSO4,
and purified by flash column on silica gel (EtOAc/Hexane 1:3)
1
to give 52 as a white solid (12 mg, 22%); mp 217-218 °C. H
NMR (CDCl3): δ 10.35 (s, 1H), 7.81 (s, 1H), 6.70 (s, 1H), 4.68
(s, 2H), 2.70 (hept, J ) 6.84 Hz, 1H), 1.31 (d, J ) 6.84 Hz,
6H), 1.26 (s, 9H). MS (ESI): 372.1 (M + H)+. HRMS FAB (M
- H)- calcd for C15H21N3O4S2, 370.0895; found, 370.0886.
Gen er a l P r oced u r e for Solid P h a se Syn t h esis of
Am in oth ia zole CDK Kin a se In h ibitor s: Syn th esis of
N-[5-[[(5-ter t-Bu tyl-2-oxa zolyl)m eth yl]th io]-2-th ia zolyl]-
ter t-bu tyla m id e (30). N-[(5-Th iocya n a to)-2-th ia zolyl]tr i-
flu or oa ceta m id e (55). To a mixture of 5-thiocyanato-2-
aminothiazole (4.7 g, 30 mmol) and 2,6-lutidine (3.75 g, 35
mmol) in THF (25 mL) and CH2Cl2 (50 mL) at -78 °C under
argon was slowly added trifluoroacetic anhydride (6.86 g, 33
mmol). The mixture was allowed to warm to room temperature
and stirred overnight, diluted with CH2Cl2 (100 mL), and
washed with 5% aqueous citric acid, followed by brine. The
organic layer was dried (MgSO4) and passed through a pad of
silica gel. The filtrate was concentrated to afford a light brown
[[5-(Acetylam in o)-1,3,4-th iadiazol-2-yl]th io]acetic Acid,
ter t-Bu tyl Ester (48). A mixture of 5-aminothiadiazole-2-
mercaptan (1.33 g, 10 mmol, Aldrich Chemical Co.), acetic
anhydride (1.1 mL), and pyridine (5 mL) in CH2Cl2 (15 mL)
was stirred at room temperature for 7 h. To the reaction
mixture CHCl3 (50 mL) and water (100 mL) were added. The
precipitated solid was filtered, washed with water, and dried
to obtain the thiol (0.62 g) contaminated by its symmetric
disulfide. A partially heterogeneous mixture of this mercaptan
and disulfide compound in MeOH (30 mL) and DMF (5 mL)
was stirred in the presence of DTT (0. 62 g) for 1.5 h. Most of
the MeOH was removed in vacuo, and K2CO3 (0.6 g) was added
to the residue in DMF followed by tert-butyl bromoacetate (0.9
mL). After it was stirred for 1 h at room temperature, water
(100 mL) and CHCl3 (100 mL) were added to the reaction
mixture. The organic solution was separated, dried over
MgSO4, and concentrated, and the remaining solid was
triturated with Et2O to obtain 48 as a yellow solid (0.91 g,
1
solid 55 (5.3 g, 70%). H NMR (CDCl3): δ 12.4 (br, 1H), 7.83
(s, 1H). MS (ESI): 254 (M + H)+.
1
38% overall yield); mp 149-151 °C. H NMR (CDCl3): δ3.90
4-(Ch lor om eth yl)-3-m eth oxyp h en oxy Mer r ifield Resin
(54). To a solution of PPh3 (17 g, 65 mmol) in CH2Cl2 (200
mL) at 0 °C was slowly added triphosgene (9.2 g, 31 mmol)
portionwise over 30 min. After this was added, the reaction
mixture was stirred at 0 °C for 10 min. The solvent was
removed in vacuo, the residue was then dissolved in CH2Cl2
(200 mL), and here was added SASRIN resin 53 (12 g, 12
mmol). The resulting mixture was agitated for 4 h, and
the resin was washed with dry CH2Cl2 (6×) and dried in
vacuo.
(s, 2H), 2.43 (s, 3H), 1.43 (s, 9H). 13C NMR (CDCl3): δ 168.7,
166.8,1, 82.9, 36.7, 27.9, 23.0. MS (ESI): 290 (M + H)+. Anal.
(C10H15N3O3S2) C, H, N, S.
[[5-(Acetyla m in o)-2-th ia zolyl]th io]a cetic Acid , Meth yl
Ester (49). To a stirred mixture of 5-aminothiazole-2-mer-
captan (304 mg, 2.29 mmol, Aldrich Chemical Co.) and
pyridine (728 mg, 9.20 mmol) in CH2Cl2 (4 mL) under argon
at 0 °C was added acetyl chloride (0.34 mL, 4.58 mmol). The
reaction mixture was purged with argon for 20 min and stirred
at room temperature for 17 h. The reaction mixture was
diluted with EtOAc (120 mL) and washed with 1 N HCl (2 ×
20 mL), saturated NaHCO3 (20 mL), and brine (20 mL). The
organic layer was dried (MgSO4), filtered, and concentrated
in vacuo. The residue was chromatographed on silica gel
eluting with 50% EtOAc in hexanes (120 mL), 70% EtOAc in
hexanes (100 mL), and 80% EtOAc in hexanes (100 mL) to
3-Met h oxy-4-[[(5-t h iocya n a t o-2-t h ia zolyl)(t r iflu or o-
a cetyl)a m in o]m eth yl]p h en oxy Mer r ifield Resin (56). A
mixture of resin-bound benzyl chloride 54 (15 g), thiazole 55
(14 g, 55.3 mmol), and diisopropylethylamine (7.8 mL, 45
mmol) in DMF (50 mL) and CH2Cl2 (100 mL) was agitated
overnight. The resin 56 was washed with DMF (2×), MeOH
(2×), and CH2Cl2 (4×) and dried in vacuo.
1
give desired thioacetate 49 (78.4 mg, 16%) as an oil. H NMR
4-[[(5-Mer ca p t o-2-t h ia zolyl)(t r iflu or oa cet yl)a m in o]-
m eth yl]-3-m eth oxyp h en oxy Mer r ifield Resin (57). A mix-
ture of resin-bound thiocyanate 56 (18.5 g) and DTT (12 g, 78
mmol) in THF (100 mL) and MeOH (100 mL) was agitated
overnight. The resin 57 was washed with DMF (2×), MeOH
(2×), and CH2Cl2 (4×), dried in vacuo, and stored under argon
at -20 °C.
(CDCl3): δ 6.51 (s, 1H), 2.23 (s, 3H), 2.10 (s, 3H).
N-[2-[[(5-ter t-Bu tyl-2-oxa zolyl)m eth yl]th io]-5-th ia zol-
yl]a ceta m id e (50). To a stirred mixture of thioacetate 49 (78.4
mg, 0.36 mmol) in THF (3 mL) under argon was added KOtBu
(0.40 mL of 1 M solution in THF, 0.40 mmol). The resulting
mixture was purged with argon and was added to a solution
of 2-chloromethyl-4-tert-butyl-oxazlole 22d (69.2 mg, 0.40
mmol). After it was stirred at room temperature for 16 h, the
reaction mixture was diluted with saturated NH4Cl (60 mL)
solution and extracted with EtOAc (3 × 80 mL). The combined
EtOAc extracts were dried (MgSO4) and concentrated, and the
residue was chromatographed on silica gel eluting with 4%
CH3OH in CH2Cl2 to give 50 (81.1 mg, 72%) as an oil. 1H NMR
(CDCl3): δ 7.11 (s, 1H), 6.58 (s, 1H), 4.32 (s, 2H), 2.17 (s, 3H),
1.28 (s, 9H). 13C NMR (CDCl3): δ 167.3, 162.4, 158.9, 151.4,
137.4,127.7,119.3,31.6,31.4,28.4,22.6.MS (ESI): 312.2(M +H)+.
N-[5-[[(5-ter t-Bu tyl-2-oxa zolyl)m eth yl]su lfin yl]-2-th ia -
zolyl]-2-isop r op yla m id e (51). A mixture of compound 29 (20
mg, 0.059 mmol) and m-chloroperoxybenzoic acid (75% purity,
20 mg, 0.064 mmol) in CH2Cl2 (5 mL) was stirred at room
temperature for 2 h. The reaction mixture was diluted with
CH2Cl2, washed with aqueous NaHCO3 solution, dried over
MgSO4, and purified by flash column on silica gel (EtOAc/
Hexane 4:1 to 9:1) to give 51 as a colorless oil (8 mg, 34%). 1H
4-[[[5-[[(5-ter t-Bu tyl-2-oxa zolyl)m eth yl]th io]-2-th ia zol-
yl](t r iflu or oa cet yl)a m in o]m et h yl]-3-m et h oxyp h en oxy
Mer r ifield Resin (58). A stream of argon was bubbled
through a mixture of resin-bound thiol 57 (500 mg), 22 (2.0
mmol, R ) tBu), and DBU (0.23 mL, 1.5 mmol) in DMF (3
mL) for 5 min, and then, it was heated at 80 °C for 2 h. The
resin was washed with DMF (2×), MeOH (2×), and CH2Cl2
(4×) and dried in vacuo.
4-N-[5-[[(5-ter t-Bu t yl-2-oxa zolyl)m et h yl]t h io]-2-t h ia -
zolyl]m eth yl-3-m eth oxyp h en yloxy Mer r ifield Resin (59).
A mixture of resin-bound trifluoroacetyl amide 58 (500 mg)
and sodium borohydride (4 mmol) in THF (2 mL) and ethanol
(2 mL) was agitated overnight. The resin 59 was washed with
50% DMF/H2O (2×), DMF (2×), MeOH (2×), and CH2Cl2 (4×)
and dried in vacuo.
4-[[[5-[[(5-ter t-Bu tyl-2-oxa zolyl)m eth yl]th io]-2-th ia zol-
yl](2,2-d im eth yl-1-oxop r op yl)a m in o]m eth yl]-3-m eth oxy-
p h en oxy Mer r ifield Resin (60). To a mixture of resin-bound
amine 59 (100 mg) and diisopropylethylamine (1.2 mmol) in
CH2Cl2 (2 mL) in a polypropylene tube fitted into an aluminum
block that is cooled with circulating 0 °C coolant was added
trimethylacetyl chloride (1 mmol). The mixture was agitated
at room temperature overnight. The resin was washed with
DMF (2×), MeOH (2×), and CH2Cl2 (4×) and used in the next
step without drying.
NMR (CDCl3): δ 7.62 (s, 1H), 6.66 (s, 1H), 4.55 (AB q, J 1
)
28.5 Hz, J 2 ) 9.5 Hz, 2H), 2.70 (hept, J ) 6.84 Hz, 1H), 1.30
(d, J ) 6.84 Hz, 6H), 1.16 (s, 9H). 13C NMR (DMSO): δ 175.7,
164.6, 163.0, 153.0, 140.9, 133.0, 121.3, 56.2, 35.8, 31.8, 28.7,
19.4. MS (ESI): 356.4 (M + H)+.
N-[5-[[(5-ter t-Bu tyl-2-oxa zolyl)m eth yl]su lfon yl]-2-th ia -
zolyl]-2-isop r op yla m id e (52). A mixture of compound 29 (50

Aminothiazole Inhibitors of Cyclin-Dependent Kinase 2
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18 3923
N-[5-[[(5-ter t-Bu tyl-2-oxazolyl)m eth yl]th io]-2-th iazolyl]-
ter t-bu tyla m id e (30). A resin-bound amide 60 (100 mg) was
treated with 60% TFA/CH2Cl2 (2 mL) in a polypropylene tube
fitted with a polyethylene frit and a luer stopcock for 4 h. The
solution was drained in a tube and combined with CH2Cl2
rinsing of the resin. The solution was concentrated in Speed
Vac. The residue was purified by preparative HPLC to afford
30 (11.3 mg) as a white solid; mp 133-134 °C. 1H NMR
(CDCl3): δ 9.66 (br, 1H), 7.32 (s, 1H), 6.60 (s, 1H), 3.92 (s,
2H), 2.25 (s, 9H), 1.29 (s, 9H). 13C NMR (CDCl3): δ 176.24,
161.90, 161.64, 144.61, 120.85, 120.13, 39.08, 34.90, 31.37,
28.65, 28.51, 27.08. MS (ESI): 354 (M + H)+. Anal. (C16H23N2-
O2S2) C, H, N, S.
for 45 min at 30 °C and stopped by the addition of cold TCA
to a final concentration of 15%. TCA precipitates were collected
onto GF/C unifilter plates (Packard Instrument Co.) using a
Filtermate universal harvester (Packard Instrument Co.), and
the filters were quantitated using a TopCount 96 well liquid
scintillation counter (Packard Instrument Co.). Dose response
curves were generated to determine the concentration required
inhibiting 50% of kinase activity (IC50). Compounds were
dissolved at 10 mM in DMSO and evaluated at six concentra-
tions, each in triplicate. The final concentration of DMSO in
the assay equaled 2%. IC50 values were derived by nonlinear
regression analysis and have a coefficient of variance (SD/
mean, n ) 6) ) 14%.
Cr ysta l Str u ctu r e of Hu m a n CDK2. Baculovirus insect
cells, expressing CDK2 at a level of 100 mg/L of culture, were
grown and collected by centrifugation. The CDK2 protein was
purified by subjecting the cell lysate to a high-speed ultracen-
trifuge spin, a fast-flow Q-sepharose column, and an ATP-
agarose affinity column. Crystals of CDK2 were grown at room
temperature by the hanging drop vapor diffusion method using
35% poly(ethylene glycol) 5000 monomethyl ester, 0.2 M
ammonium acetate, 0.1 M N-(2-hydroxyethyl)piperazine-N′-
ethanesulfonic acid (HEPES), pH 7.0, as a reservoir solution.
The hanging drop consisted of 10λ of the concentrated protein
(13 mg/mL) mixed with an equal volume of reservoir solution.
Prior to data collection, crystals were transferred to a vial
containing the mother liquor with 2.4-3.0 nM ligand added
and allowed to soak for 3 days. For X-ray crystallographic data
collection, the crystal was transferred to a vial containing the
mother liquor with 15% glycerol added. The crystal was then
flash-cooled by transferring the crystal on a nylon loop to a
stream of liquid nitrogen at 100 K for data collection. Data
were collected on a Siemens Hi-Star area detector system using
mirror-focused Cu K radiation from a Rigaku RU-2000 rotating
anode. The X-ray intensity data were reduced using program
XENGEN and refined by the method of simulated annealing
using program XPLOR. The crystallographic data collection,
reduction, and refinement are summarized in Table 15.
Cr ysta l Str u ctu r e of Hu m a n CDK2/Cyclin A Com p lex.
Crystals of CDK2 complex with cyclin A and 41 were grown
at 4 °C by the hanging drop vapor diffusion method as
previously reported.17 The hanging drop consisted of 10λ of the
concentrated CDK2/Cyclin A complex mixed with an equal
volume of reservoir solution containing 21% saturated (NH4)2-
SO4‚0.5 M KCl, 5 mM DTT, 40 mM HEPES, pH 7.0. For data
collection, a crystal, 0.3 mm × 0.15 mm × 0.15 mm, was placed
in a cryoprotectant containing 6 M Na Formate in 25 mM
Hepes, pH 7.5. The crystal was frozen, and data were collected
as described above.
CDK4/Cyclin D1 Kin a se Assa y. Kinase reactions con-
sisted of 150 ng of baculovirus expressed GST-CDK4, 280 ng
of Stag-cyclin D1, 0.5 µg of GST-RB fusion protein (amino acids
776-928 of retinoblastoma protein), 0.2 µCi 33P γ-ATP, and
25 µM ATP in 50 µL of kinase buffer (50 mM HEPES, pH 8.0,
10 mM MgCl2, 1 mM EGTA, 2 mM DTT). Reactions were
incubated for 1 h at 30 °C and stopped by the addition of cold
TCA to a final concentration of 15%. TCA precipitates were
collected onto GF/C unifilter plates (Packard Instrument Co.)
using a Filtermate universal harvester (Packard Instrument
Co.), and the filters were quantitated using a TopCount 96
well liquid scintillation counter (Packard Instrument Co.).
Dose response curves were generated to determine the con-
centration required inhibiting 50% of kinase activity (IC50).
Compounds were dissolved at 10 mM in DMSO and evaluated
at six concentrations, each in triplicate. The final concentration
of DMSO in the assay equaled 2%. IC50 values were derived
by nonlinear regression analysis and have a coefficient of
variance (SD/mean, n ) 6) ) 18%.
HER1/HER2 Kin a se Assa y. Kinase reactions consisted of
10 ng of baculovirus expressed GST-HER1 or 100 ng of HER2,
100 ng/mL poly(Glu/Tyr) (Sigma), 0.2 µCi 33P γ-ATP, and 1
µM ATP in 50 µL of kinase buffer (50 mM Tris, pH 7.5, 10
mM MnCl2, 0.5 mM DTT). Reactions were incubated for 1 h
at 27 °C and stopped by the addition of cold TCA to a final
concentration of 15%. TCA precipitates were collected onto
GF/C unifilter plates (Packard Instrument Co.) using a Fil-
termate universal harvester (Packard Instrument Co.), and
the filters were quantitated using a TopCount 96 well liquid
scintillation counter (Packard Instrument Co.). Dose response
curves were generated to determine the concentration required
to inhibit 50% of kinase activity (IC50). Compounds were
dissolved at 10 mM in dimethyl sulfoxide (DMSO) and evalu-
ated at six concentrations, each in triplicate. The final
concentration of DMSO in the assay equaled 1%. IC50 values
were derived by nonlinear regression analysis and have a
coefficient of variance (SD/mean, n ) 6) ) 16%.
CDK1/Cyclin B1 Kin a se Assa y. Kinase reactions con-
sisted of 100 ng of baculovirus expressed GST-CDK1/cyclin B1
complex, 1 µg histone H1 (Boehringer Mannheim, Indianapo-
lis, IN), 0.2 µCi 33P γ-ATP, 25 µM ATP in 50 µL of kinase buffer
(50 mM Tris, pH 8.0, 10 mM MgCl2, 1 mM EGTA, 0.5 mM
DTT). Reactions were incubated for 45 min at 30 °C and
stopped by the addition of cold trichloroacetic acid (TCA) to a
final concentration of 15%. TCA precipitates were collected
onto GF/C unifilter plates (Packard Instrument Co., Meriden,
CT) using a Filtermate universal harvester (Packard Instru-
ment Co.), and the filters were quantitated using a TopCount
96 well liquid scintillation counter (Packard Instrument Co.).
Dose response curves were generated to determine the con-
centration required to inhibit 50% of kinase activity (IC50).
Compounds were dissolved at 10 mM in DMSO and evaluated
at six concentrations, each in triplicate. The final concentration
of DMSO in the assay equaled 2%. IC50 values were derived
by nonlinear regression analysis and have a coefficient of
variance (SD/mean, n ) 6) ) 16%.
P KC Assa ys. The procedure is a modification of Amer-
sham’s Serine/Threonine kinase SPA assay (Cat. no. RPNQ
0200). The in vitro kinase reaction assay is based on the
phosphorylation of a serine residue on the biotinylated, PK-
Cepsilon specific pseudosubstrate (BES) with the following
sequence Biot-ERMRPRKRQGSVRRRV-OH. The reaction
buffer for PKC assays contained 50 mM MOPS (pH 7.2), 1 µM
ATP, 5 mM MgCl2, 1-2 µM BES, 1 µCi/mL of 33P-γ-ATP, 0.04
mg/mL phosphatidyl serine, and 0.02 mg/mL diacylglycerol.
The following enzyme concentrations (ng/mL) were 200 (ꢀ), 400
(δ), 600 (ú), 200 (R), and 200 (â-1). The PKC R and â-1 assays
were supplemented with 10 µM CaCl2. The reaction was
started by the addition of enzyme and the reaction buffer to
Optiplates (Packard Instruments) containing the compound.
After 30 min at 37 °C, the reaction was stopped by adding 150
µL of stop solution (50 µM ATP, 50 mM ethylenediaminetet-
raacetic acid (EDTA), 0.1% Triton-X 100, and 2.5 mg/mL
streptavidin-coated PVT-SPA beads; Amersham Cat. no.
RPNQ0007, in 1 × phosphate-buffered saline (PBS)). The SPA
beads were allowed to settle overnight, and the plates were
counted in a Packard Topcount.
CDK2/Cyclin E Kin a se Assa y. Kinase reactions consisted
of 5 ng of baculovirus expressed GST-CDK2/cyclin E complex,
0.5 µg GST-RB fusion protein (amino acids 776-928 of
retinoblastoma protein), 0.2 µCi 33P γ-ATP, and 25 µM ATP
in 50 µL of kinase buffer (50 mM HEPES, pH 8.0, 10 mM
MgCl2, 1 mM EGTA, 2 mM DTT). Reactions were incubated
IKK Assa y. Kinase activity was measured with a fluores-
cence polarization assay using baculovirus-expressed GST-
IKK1. Kinase reactions consisted of 40 ng of GST-IKK and

3924 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18
Kim et al.
100 µM GST-IkBR in 20 µL of buffer containing 5 µM ATP, 25
mM Tris, pH 7.5, 7.5 mM MgCl2, 65 µM DTT, 5% glycerol,
and 0.5 mg/mL bovine serum albumin (BSA). Reactions were
incubated for 15 min at room temperature before addition of
5.25 nM FITC-IkB peptide and anti-phospho-IkBR (Santa
Cruz) in Tris-buffered saline, pH 7.6, containing 100 mM
EDTA and 0.1% bovine γ-globulin. After the quenched reaction
reached equilibrium, polarized light was measured with an
LJ L analyst (Molecular Devices Corporation, Sunnyvale, CA).
quantification of the amount of radioactive phosphate trans-
ferred to the poly(Glu/Tyr) substrate. Addition of cold TCA
precipitated the proteins, which were collected onto GF/C
unifilter plates (Packard Instrument Co.) using a Filtermate
universal harvester, and the filters were quantified using a
TopCount 96 well liquid scintillation counter (Packard Instru-
ment Co.). Compounds were dissolved in DMSO to a concen-
tration of 10 mM and were evaluated at six concentrations,
each in triplicate. The final concentration of DMSO added to
the kinase assays was 0.5%, which has been shown to have
no effect on kinase activity. IC50 values were derived nonlinear
regression analysis and have a coefficient of variance (SD/
mean, n ) 6) ) 16%.
Cell Cu ltu r e. Tissue culture cells were maintained in
logarithmic growth in RPMI 1640 medium (Gibco), 5 mM
HEPES buffer, and 10% fetal bovine serum (Gibco). Adult
bovine aortic endothelial (ABAE) cells (a primary cell line)
were grown in RPMI 1640 media containing 2.0 ng/mL basic
fibroblast growth factor. The cell line panel is made up of
human ovarian carcinoma cell lines (A2780/DDP-S, A2780/
DDP-R, A2780/TAX-S, A2780/TAX-R, and OVCAR-3), human
breast carcinoma cell lines (MCF-7 and SKBR-3), human
prostate carcinoma cell lines (LNCAP and PC-3), human colon
carcinoma cell lines (HCT116, HCT116/VM46, HCT116/VP35,
Caco-2, LS 174T, and MIP), human lung carcinoma (A549 and
LX-1), human squamous cell carcinoma cell line (A431), human
leukemia cell lines (CCRF-CEM, HL-60, and K562), human
fibroblast cell line (HS27), ABAE, mouse lung carcinoma cell
line (M109), and mouse lung fibroblast (p53-/-) cell line (MLF)
isolated from p53 knockout mice. The HCT116/VM46 cell line
is a multidrug resistant variant of the parental HCT116 cells
and overexpresses P-glycoprotein.23 The HCT116/VP35 cell line
is a variant that has low topoisomerase II levels and is
resistant to VP-16.23 The A2780/DDP-R cell line is resistant
to cisplatin relative to the parental A2780/DDP-S cells. A
tubulin mutation is present in the A2780/TAX-R, which are
resistant to paclitaxel relative to the parental A2780/TAX-S
cells (provided by Dr. Tito Fojo; NCI, Bethesda, MD).24
72 h P r olifer a tion Assa y. In vitro cytotoxicity was as-
sessed in tissue culture cells by MTS (3-(4,5-dimethylthiazol-
2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfenyl)-2H-tetrazoli-
um, inner salt) assay as previously described.23 Depending
upon the cell line used, cells were plated at a density of 3000-
6000 cells/well, in a 96 well plate, and 24 h later, drugs were
added and serial-diluted. The cells were incubated at 37 °C
for 72 h at which time the tetrazolium dye, MTS in combina-
tion with phenazine methosulfate, was added. After 3 h, the
absorbency was measured at 492 nm, which is proportional
to the number of viable cells. The results are expressed as IC50
values.
Cell Cycle. Log phase A2780 cells were plated overnight
in six well plates before treatment with various concentrations
of compounds and harvested by trypsinization. The cells were
prepared for flow cytometry according to the Apo-Direct
protocol (Pharmingen). Briefly, the cells were fixed in paraform-
aldehyde and ethanol, washed, resuspended in staining solu-
tion of TdT enzyme and FITC-dUTP, washed, and then stained
with PI following RNase treatment.
Em t a n d ZAP -70 Assa ys. The protein substrate for the
kinase reaction was a glutathione S-transferase fusion protein
containing the tyrosine kinase phosphorylation sites of the
SLP-76 protein (Gst-SLP76). Both Emt and ZAP-70 were
generated using a baculovirus expression system. The kinase
reactions (60 µL) containing 25 mM HEPES, pH 7.0, 5 mM
MgCl2, 5 mM MnCl2, 1 µM ATP (0.4 µCi γ-33P-ATP), 0.1 mg/
mL BSA, 83 µg/mL Gst-SLP76, 5 ng of enzyme (Emt or
ZAP70), and various concentrations of tested compound were
carried out at room temperature for 10 min. After it was
terminated by adding 100 µL of 20% TCA and 0.1 mM NaPPi,
the precipitated proteins were harvested on a filter plate and
washed. The radioactivity incorporated was then determined
using a Topcount after adding 30 µL of Microscint scintillation
fluid.
LCK Assa y. Kinase reactions were carried out in a 96 well
format and consisted of 10 ng of baculovirus-expressed GST-
LCK, 5 µg poly(GLU/Tyr), 0.02 µCi of 33P γ-ATP, 1 µM ATP in
50 µL of buffer containing 40 mM HEPES, pH 7.4, 5 mM
MnCl2, 30 µM DTT, and 0.1 mg/mL BSA. Reactions were
incubated for 1 h at 26 °C and stopped by the addition of 2.5
mg/mL BSA in 0.3 M EDTA. The reactions were precipitated
on ice with 100 µL of 3.5 mM ATP in 5% TCA. TCA precipitates
were collected onto GF/C unifilter plates (Packard Instrument
Co.) using a Filtermate universal harvester (Packard Instru-
ment Co.), and the filters were quantitated using a TopCount
96 well liquid scintillation counter (Packard Instrument Co.).
Dose response curves were generated to determine the con-
centration of compounds required to inhibit 50% of kinase
activity (IC50). Compounds were dissolved at 10 mM in DMSO
and evaluated at six concentrations, each in triplicate. The
final concentration of DMSO in the assay equaled 1%. IC50
values were derived by nonlinear regression analysis.
IGF -Recep tor Tyr osin e Kin a se Assa y. The IGF-1 re-
ceptor tyrosine kinase was assayed using the synthetic poly-
mer poly(Glu/Tyr) (Sigma Chemicals) as a phosphoacceptor
substrate. Each reaction mixture consisted of a total volume
of 50 µL and contained 125 ng of baculovirus-expressed
enzyme, 2.5 µg of poly(Glu/Tyr), 25 µM ATP, and 0.1 µCi of
[γ-33P]ATP. The mixtures also contained 20 mM MOPS, pH
7.0, 5 mM MnCl2, 0.5 mM DDT, and 0.1 mg/mL BSA. The
reaction mixtures were incubated at 30 °C for 45 min, and
kinase activity was determined by quantification of the amount
of radioactive phosphate transferred to the poly(Glu/Tyr)
substrate. Addition of cold TCA precipitated the proteins,
which were collected onto GF/C unifilter plates (Packard
Instrument Co.) using a Filtermate universal harvester, and
the filters were quantified using a TopCount 96 well liquid
scintillation counter (Packard Instrument Co.). Compounds
were dissolved in DMSO to a concentration of 10 mM and were
evaluated at six concentrations, each in triplicate. The final
concentration of DMSO added to the kinase assays was 0.5%,
which has been shown to have no effect on kinase activity.
IC50 values were derived from nonlinear regression analysis
and have a coefficient of variance (SD/mean, n ) 6) ) 16%.
F AK Tyr osin e Kin a se Assa y. The focal adhesion kinase
was assayed using the synthetic polymer poly(Glu/Tyr) (Sigma
Chemicals) as a phosphoacceptor substrate. Each reaction
mixture consisted of a total volume of 50 µL and contained
100 ng of baculovirus-expressed enzyme, 2 µg of poly(Glu/Tyr),
1 µM ATP, and 0.2 µCi of [γ-33P]ATP. The mixtures also
contained 40 mM Tris‚HCl, pH 7.4, 1 mM MnCl2, 0.5 mM DTT,
and 0.1 mg/mL BSA. The reaction mixtures were incubated
at 26 °C for 1 h, and kinase activity was determined by
P h osp h op r otein An a lysis. A2780 cells were exposed to
32P-orthophosphate (1 mCi/mL of normal growth media plus
dialyzed fetal bovine serum) for 4 h in the presence or absence
of 3 µM compound 14. Treated A2780S cells were harvested
at approximately 70% confluence, and total protein was
prepared by lysing the cells in RIPA [50 mM Tris (pH 8), 150
mM NaCl, 1% NP-40, 0.5% Na-deoxycolate, 0.1% sodium
dodecyl sulfate (SDS), 0.1% Na3VO4, 0.1 mM NaF, 10 mM
â-glycerophosphate, plus complete protease inhibitors (Boe-
hringer Mannhiem)] buffer. Cell pellets were resuspended at
a density of <2 × 107 cells/mL and incubated for 20 min on
ice followed by a high speed 14 000 rpm centrifugation. The
protein supernatant was then removed from the debris, and
protein content was quantitated using the Micro-BCA assay
(Pierce). Treated extracts (250 µg of total protein/imunopre-
cipitation) were then imunoprecipitated at 4 °C for 1 h using

Aminothiazole Inhibitors of Cyclin-Dependent Kinase 2
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18 3925
5 µg of RB, histone H1, or DNA polymerase R specific
antibodies and 20 µL of Protein A-sepharose (Pharmacia).
Antibodies used were as follows: anti-Rb clone PMG3-245 (BD-
Pharmingen, San Diego, CA), anti-DNA pol-R mixture of clones
SJ K-132-20, SJ K-287-38, SJ K-237-71, and SDK-043 (STK1).
The imunoprecipitated complexes were then washed three
times with cold PBS and separated on a 8 or 10% SDS-
polyacrylamide gel for RB or DNA polymerase R and histone
H1, respectively. The gels were then dried and analyzed by
autoradiography.
and analyzed for the compound. Percent protein bound
was calculated from measured concentrations in buffer and
serum.
In Vivo An titu m or Testin g. Dr u g Ad m in istr a tion . All
CDK inhibitors were first dissolved in a mixture of Cremophor/
ethanol (50:50) and stored at 4 °C; final dilution to the required
dosage strength was made with water so that the dosing
solutions contained Cremophor/ethanol/water at a ratio of 10:
10:80, respectively. Olivomycin was dissolved in saline. The
volume of all compounds injected was 0.01 mL/g of mouse
weight.
P h a r m a cok in etics in Mou se. The pharmacokinetics of
flavopiridol, 45, and 47 were investigated in male Balb-C mice
fasted overnight, following a single dose given intraperito-
neally (ip). The vehicle used was cremophor:ethanol:water (1:
1:8). A total of 15 mice were dosed per compound. Serum
samples were collected from each mouse by cardiac puncture.
Serum levels were monitored for 6 h after ip dosing. Samples
were collected at 0.5, 1, 2, 4, and 6 h after dosing. The mean
body weight of the mice was approximately 20 g.
An im a ls. All rodents were obtained from Harlan Sprague
Dawley Co. (Indianpolis, IN) and maintained in an ammonia-
free environment in a defined and pathogen-free colony. The
animal care program of Bristol-Myers Squibb Pharmaceutical
Research Institute is fully accredited by the American As-
sociation for Accreditation of Laboratory Animal Care (AAA-
LAC).
Mu r in e P 388 Leu k em ia . Female Balb/c × DBA/2J F1
mice, weighing within a 3 g range, were used in these studies.
On day 0, mice were inoculated ip with 106 P388 ascites
leukemic cells. Mice were randomized into treatment groups
and sufficient groups for a bioassay titration of 106 to 104 cells
per mouse. On day 1 postinoculation, mice were given ip
injections of CDK inhibitor on a treatment schedule of every
day for 7 days (qd × 7). A positive reference (olivomycin) was
included in each study. All drugs were administered in uniform
volumes of 0.01 mL/g of mouse weight. The effectiveness of
treatment was assessed by comparison of the median postin-
oculation life span of each group of treated mice (T) with that
of controls (C). The ratio of T to C expressed in percentage
value (i.e., %T/C) provides quantitative indication of efficacy.
A %T/C of 127 or greater is considered an active result.
In a second study, the pharmacokinetics of 45 were inves-
tigated in male Balb-C mice fasted overnight, following a single
dose of 10 mg/kg given either intravenously (IV) or orally by
gavage. The vehicle used was ethanol:water (1:9) for both
routes of administration. The mice were fed 4 h postdose. A
total of 18 mice were dosed; nine by the intravenous route and
nine by the oral route. Three serum samples were collected
from each mouse, the first two samples by retroorbital bleed
(100 µL) and the third sample by cardiac puncture. Serum
levels were monitored for 10 h after dosing. Samples were
collected at 5, 10, and 30 min, 1, 2, 4, 6, 8, and 10 h after IV
dosing and at 10, 20, and 40 min, 1, 2, 4, 6, 8, and 10 h after
oral dosing. The mean body weight of the mice was ap-
proximately 20 g.
Blood samples collected were allowed to clot on ice and then
centrifuged, and the serum was harvested. Samples were
stored at -20 °C until analysis. Samples were analyzed by
LC/MS/MS. Composite serum concentration-time profiles
were constructed for pharmacokinetic analysis. The pharma-
cokinetic parameters were derived by noncompartmental
methods using the KINETICA software program. The Cmax and
Solid Tu m or Xen ogr a fts in Nu d e Mice. The following
tumors were used, A2780 human ovarian carcinoma, Br-cycE
murine breast carcinoma, and A431 human squamous cell
carcinoma. All solid tumors were maintained in Balb/c nu/nu
nude mice. Tumors were propagated as subcutaneous trans-
plants using tumor fragments obtained from donor mice. All
tumor implants for efficacy testing were subcutaneous (sc). The
required number of animals needed to detect a meaningful
response were grouped at the start of the experiment, and each
was given a sc implant of a tumor fragment (∼50 mg) with a
13 gauge trocar. For treatment of early-stage tumors, the
animals were again grouped before distribution to the various
treatment and control groups. For treatment of animals with
advanced-stage disease, tumors were allowed to grow to the
predetermined size window (∼100 mg size, animals with
tumors outside the range were excluded) and animals were
evenly distributed to various treatment and control groups.
Treatment of each animal was based on individual body
weight. Treated animals were checked daily for treatment
related toxicity/mortality. Each group of animals was weighed
before the initiation of treatment (Wt1) and then again
following the last treatment dose (Wt2). The difference in body
weight (Wt2 - Wt1) provides a measure of treatment-related
toxicity. Tumor response was determined by measurement of
tumors with a caliper twice a week, until the tumors reach a
predetermined “target” size of 1 g. Tumor weights (mg) were
estimated from the formula: tumor weight ) (length ×
width2)/2.
T
max values were recorded directly from experimental observa-
tions. The AUCtot values were calculated using a combination
of linear and log trapezoidal rules. The total body clearance
(Cl), MRT, and the steady state volume of distribution (Vss)
were also calculated after IV administration. The oral bio-
availability (expressed as %) was estimated by taking the ratio
of dose-normalized AUC values after oral doses to those after
IV doses.
Meta bolic Sta bility. The compound was incubated with
mouse and human liver microsomes. The mouse microsomes
were prepared by standard methods and were pooled from
livers obtained from a group of animals of the same species (n
) 30). The human microsomes were purchased from In Vitro
Technologies (Baltimore, MD) and were pooled from 10
individual donors. The rates of oxidative metabolism were
measured under the following conditions: compound, 10 µM
final concentration; final microsomal protein concentration
approximately 1 mg/mL; NADPH, 1 mM; pH 7.4 potassium
phosphate buffer, 56 mM. Incubations were performed at 37
°C and were initiated by the addition of the substrate.
Incubations were quenched by the addition of one volume of
acetonitrile after 10 min. Samples were analyzed for the parent
compound. The percent metabolized was calculated based on
the disappearance of the parent compound.
Esta blish m en t of Br -CycE Cyclin E Over exp r essin g
Mu r in e Ma m m a r y Ca r cin om a Xen ogr a ft a n d Cell Lin e.
Each nude mouse was given a subcutaneous implant of a
tumor fragment (≈20 mg) obtained from a donor transgenic
cycE mouse bearing a spontaneously arising mammary tumor.
Implantation was accomplished using a 13 gauge trocar
inserted sc into the dorsal side of the mouse, near the
mammary gland. Nude mice were obtained from Harlan
Sprague Dawley Co. and maintained in an ammonia-free
environment in a defined and pathogen-free colony. Tumor
growth was determined by measurement of tumors with a
caliper 1-2 times a week, until the tumors reached a prede-
P r otein Bin d in g. The extent of protein binding was
determined in mouse and human sera. The method involved
equilibrium dialysis using 0.134 M phosphate buffer (pH 7.4)
and dialysis membranes (Spectrum, Laguna Hills, CA) with
a 12 000-14 000 Da molecular mass cutoff. The dialysis cells
(Spectrum) were rotated at 20 rpm in a water bath maintained
at 37 °C. The extent of binding to serum proteins was
determined at a nominal concentration of 20 µM (n ) 3).
Aliquots of buffer and serum were taken at 4 h after incubation

3926 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18
Kim et al.
in human cancer cell lines. Anti-Cancer Drugs 1997, 8, 623-
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termined target size of 1 g. Tumor weights (mg) were estimated
from the formula: tumor weight ) (length × width2)/2.
Xenograft cycE tumors grown in nude mice were excised and
minced with scissors and were dissociated using an enzyme
cocktail consisting of 0.025% collagenase (Sigma Chemical Co.),
0.05% Pronase (Calbiochem, LaJ olla, CA), and 0.04% DNase
(Sigma) for 1 h at 37 °C. After debris was removed by passing
the cell suspensions through 70 µm nylon screens, the cells
were washed in PBS, counted, and resuspended in complete
RPMI 1640 media supplemented with 10% heat-inactivated
fetal bovine serum (GIBCO). Cells (3 × 10E+5) were then
seeded onto a variety of Biocoat Cellware (Becton Dickinson)
coated separately with the following extracellular matrix:
collagen I, laminin, fibronectin, collagen IV, poly-D-lysine.
Ack n ow led gm en t. Microanalysis and mass spectra
were kindly provided by the Bristol-Myers Squibb
Department of Analysis Research and Development.
Note Ad d ed a fter ASAP P ostin g. This article was
released ASAP on 8/2/2002 with a minor error in Table
9. The correct version was posted on 8/22/2002.
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