N-(Cycloalkylamino)acyl-2-aminothiazole Inhibitors of Cyclin-Dependent Kinase 2. N-[5-[[[5-(1,1-Dimethylethyl)-2-oxazolyl]methyl]thio]-2-thiazolyl] -4-piperidinecarboxamide (BMS-387032), a Highly Efficacious and Selective Antitumor Agent

Article abstract of DOI:10.1021/jm0305568

N-Acyl-2-aminothiazoles with nonaromatic acyl side chains containing a basic amine were found to be potent, selective inhibitors of CDK2/cycE which exhibit antitumor activity in mice. In particular, compound 21 {N-[5-[[[5-(1,1-dimethylethyl)-2- oxazolyl]m

Full text of DOI:10.1021/jm0305568

J . Med. Chem. 2004, 47, 1719-1728
1719
N-(Cycloa lk yla m in o)a cyl-2-a m in oth ia zole In h ibitor s of Cyclin -Dep en d en t Kin a se
2. N-[5-[[[5-(1,1-Dim eth yleth yl)-2-oxa zolyl]m eth yl]th io]-2-th ia zolyl]-4-
p ip er id in eca r boxa m id e (BMS-387032), a High ly Effica ciou s a n d Selective
An titu m or Agen t
Raj N. Misra,* Hai-yun Xiao, Kyoung S. Kim, Songfeng Lu, Wen-Ching Han, Stephanie A. Barbosa,
J ohn T. Hunt, David B. Rawlins,† Weifang Shan, Syed Z. Ahmed,‡ Ligang Qian, Bang-Chi Chen, Rulin Zhao,
Mark S. Bednarz,† Kristen A. Kellar,† J anet G. Mulheron,† Roberta Batorsky, Urvashi Roongta, Amrita Kamath,
Punit Marathe, Sunanda A. Ranadive, J ohn S. Sack, J ohn S. Tokarski, Nikola P. Pavletich,| Francis Y. F. Lee,
Kevin R. Webster,§ and S. David Kimball†
Bristol-Myers Squibb Pharmaceutical Research Institute, PO Box 4000, Princeton, New J ersey 08543-4000
Received November 4, 2003
N-Acyl-2-aminothiazoles with nonaromatic acyl side chains containing a basic amine were found
to be potent, selective inhibitors of CDK2/cycE which exhibit antitumor activity in mice. In
particular, compound 21 {N-[5-[[[5-(1,1-dimethylethyl)-2-oxazolyl]methyl]thio]-2-thiazolyl]-4-
piperidinecarboxamide, BMS-387032}, has been identified as an ATP-competitive and CDK2-
selective inhibitor which has been selected to enter Phase 1 human clinical trials as an
antitumor agent. In a cell-free enzyme assay, 21 showed a CDK2/cycE IC50 ) 48 nM and was
10- and 20-fold selective over CDK1/cycB and CDK4/cycD, respectively. It was also highly
selective over a panel of 12 unrelated kinases. Antiproliferative activity was established in an
A2780 cellular cytotoxicity assay in which 21 showed an IC50 ) 95 nM. Metabolism and
pharmacokinetic studies showed that 21 exhibited a plasma half-life of 5-7 h in three species
and moderately low protein binding in both mouse (69%) and human (63%) serum. Dosed orally
to mouse, rat, and dog, 21 showed 100%, 31%, and 28% bioavailability, respectively. As an
antitumor agent in mice, 21 administered at its maximum-tolerated dose exhibited a clearly
superior efficacy profile when compared to flavopiridol in both an ip/ip P388 murine tumor
model and in a sc/ip A2780 human ovarian carcinoma xenograft model.
In tr od u ction
Cyclin-dependent kinases (CDKs) are a family of
serine\ threonine protein kinases which play key roles
in the normal growth and life cycle of eukaryotic cells.1
Specifically, CDKs, along with their activating subunit
cyclins or inhibitory subunit CDKi (e.g., p16, p21, p27),
are responsible for cellular response to external stimuli
or insults. Together they provide for the orderly coor-
dination of cellular events through the cell cycle and
ensure compliance of the genetic integrity of new cells.
Because of their key role as drivers of cell growth and
division, their misregulation in a number of cancers,2
and the strong positive correlation between CDK2
activity and poor clinical prognosis in cancer patients,3
oncology drug discovery programs have directed major
efforts toward the identification of small molecule
inhibitors of CDKs as potential antitumor therapeutic
F igu r e 1. Structures of CDK inhibitors flavopiridol (1) and
aminothiazole 2.
agents.4
Flavopiridol (1), a synthetic flavone, was described as
inhibitor a number of years ago and has progressed into
advanced clinical trials as an antitumor agent.5 We
recently described N-phenylacetyl-2-aminothiazole 2 as
a potent CDK2-selective inhibitor with in vivo antitumor
activity superior to 1.6 In this disclosure we describe
the SAR, metabolism, pharmacokinetics, and solid-state
structural studies of a series related to 2, the N-(cy-
cloalkylamino)acyl-2-aminothiazoles.7 It is from this
series that our clinical development candidate has
emerged. In addition, a preliminary report on the in vivo
a benchmark broad-spectrum ATP-competitive CDK
* To whom correspondence should be addressed. Raj N. Misra,
Ph.D., 12 Eaton Place, Hopewell, NJ 08525. E-mail: raj_n_misra@
hotmail.com.
† Current address: Lexicon Pharmaceuticals, 350 Carter Rd., Prin-
ceton, NJ 08540.
‡ Current address: FMC Corporation, PO Box 8, Princeton, NJ
08543.
§ Current address: Astra-Zeneca R&D Boston, 35 Gatehouse Dr.,
Waltham, MA 02451.
| Affiliation: Memorial Sloan-Kettering Cancer Center, New York,
NY.
10.1021/jm0305568 CCC: $27.50 © 2004 American Chemical Society
Published on Web 03/02/2004

1720 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Misra et al.
Sch em e 1a
a
Reaction conditions. (a) NaN3, acetone, 25 °C, 100%; (b) Pd/C, H2, aq HCl, MeOH, 25 °C, 91%; (c) Et3N/CH2Cl2, -10 °C, 98%; (d)
POCl3, 105 °C, 80%; (e) Br2, KSCN, MeOH, 26%; (f) NaBH4 (2 equiv), EtOH, 25 °C followed by chloride 6 (1.1 equiv), reflux, 94%; (g)
RCOOH, EDAC (2 equiv), DMAP (1 equiv), DMF, CH2Cl2, 25 °C.
Sch em e 2a
a
Reaction conditions. (a) (tBoc)2O, aq NaOH, dioxane, CH3CN, 25 °C, 81%; (b) amine 8, EDAC (2 equiv), DMAP (0.5 equiv), DMF,
CH2Cl2, 25 °C, 97%; (c) 4 N HCl in dioxane, CHCl3, 45-55 °C, 83%.
antitumor activity of selected analogues including our
clinical development candidate is disclosed.
analogues 9. A specific example for the preparation of
21 is shown in Scheme 2. Compounds 22-24 were
prepared from 21 by elaboration of the amino function-
ality using standard known synthetic methods.
Syn th etic Ch em istr y Meth od s. A convergent syn-
thetic route which involved the coupling of chlorom-
ethyloxazole 6 with thiazole 7 to access key intermediate
amine 8 was utilized to rapidly prepare acylated ana-
logues of general structure 9 (specific examples 12-27).
Thus, as shown in Scheme 1, commercially available
R-bromopinacolone 3 was converted smoothly to R-ami-
no derivative 4 by treatment with sodium azide followed
by catalytic hydrogenation. Acylation of 4 with R-chlo-
roacetyl chloride afforded keto-amide 5 which was
cyclized to key chloromethyloxazole 6 in refluxing
phosphorus oxychloride. The thiazole core was elabo-
rated by treatment of commercially available 2-ami-
nothiazole with bromine and potassium thiocyanate to
give 7 in a low yield but moderately scalable process.8
Reduction of 7 by exposure to sodium borohydride in
methanol followed by alkylation of the resulting thiolate
with chloromethyloxazole 6 gave key 2-aminothiazole
intermediate 8.
Resu lts a n d Discu ssion
In Vitr o Biologica l Eva lu a tion . Compounds were
initially evaluated in a cell-free enzyme assay for
inhibition of CDK2/cyc E induced phosphorylation of RB
protein and subsequently for antiproliferative effects in
a whole cell 72 h cytotoxicity assay. An ovarian cancer
cell line, A2780, was utilized in the cellular cytotoxity
assay. Detailed protocols for both assays were described
previously and are included in the Experimental Sec-
tion.6 The results of these assays are included in Table
1 as part of the SAR study for this series.
Str u ctu r e-Activity Stu d ies. Summarized in Table
1 is the SAR of a series of N-acyl-2-aminothiazoles in
which a nonaromatic side chain containing a basic
amine has been introduced on the acyl substituent.
Previous SAR and structural studies in this series had
established that the acyl side chain extends toward the
exterior of the protein and was amenable to significant
structural modification.6 This finding was exploited to
manipulate the physiochemical properties of the inhibi-
tor and resulted in the identification of inhibitor 2 which
contains an aromatic acyl side chain with a basic amino
group. The basic amine was introduced into the inhibitor
In general, amine 8 was coupled either directly to the
appropriate carboxylic acid or when necessary the N-t-
Boc protected carboxylic acid using 1-(3-dimethylami-
nopropyl)-3-ethylcarbodiimide (EDAC), optionally in the
presence of 4-(dimethylamino)pyridine (DMAP) and/or
N-hydroxybenzotriazole, followed by removal of the
protecting group to afford the desired acylated amino

Inhibitors of Cyclin-Dependent Kinase 2
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7 1721
Ta ble 1. Structure-Activity Relationship Studies of
Aminothiazoles 12-27a
the side chain affords a potent enzyme inhibitor with
only a 4-fold decrease in CDK2 inhibitory activity but
a larger 10-fold loss of potency in the cellular antipro-
liferation assay. Subsequently we found that by increas-
ing the lipophilicity of amino analogue 13 and constrain-
ing the amine group within or onto a ring we were able
to restore cellular antiproliferative activity (see ana-
logues 19-23, vide infra). Analogues in which a meth-
ylene group is present between the carbonyl group and
the cyclic substituent (14 vs 15, 16 vs 17, 20 vs 21)
exhibited a 4-7-fold increase in enzyme potency versus
analogues in which a methylene spacer was absent.
This, however, was not necessarily translated into an
increase in cellular antiproliferative activity (e.g., 20 vs
21). We speculate that the allowed orientations of a
cyclic ring attached directly to the carbonyl group are
sufficiently restricted that it does not allow for simul-
taneous optimal binding of the side chain and the
aminothiazole ring. Introduction of a methylene spacer
provides the necessary flexibility to optimize binding of
both the side chain and aminothiazole ring. Comparison
of phenyl analogues 14 and 15 with their cyclohexyl
analogues 16 and 17 indicated that the saturated
carbocycles were ∼15-fold less potent than their phenyl
counterparts. The reduced enzyme activity of 16 and 17
relative to the phenyl analogues may be due to the
increased steric bulk of the cyclohexyl group and/or its
increased lipohilicity. Comparison of the cyclohexyl
analogues 16 and 17 with their piperidinyl counterparts
20 and 21, however, revealed a 5 to 6-fold increase in
enzyme activity for the piperidinyl analogues. Amines
20 and 21 were only 2 to 3-fold less potent than phenyl
analogues 14 and 15. This comparison supports the
hypothesis that there is not a steric issue with the
cyclohexyl ring since the piperidinyl ring is essentially
the same size. The basis for the diminished activity of
16 and 17 seems to be the high lipophilicity (i.e., low
aqueous solubility) of the cyclohexyl side chain. The SAR
is consistent with the solid-state structural data which
indicates that the acyl side chain extends into the
hydrophilic environment at the interface of the protein
and aqueous external environment. Highly lipophilic
groups would be expected to be poorly accommodated
in this region. Because of their acceptable enzyme and
cellular antiproliferative activities, increased solubility,
and reduced protein binding, we focused more closely
on the exploration of analogues containing an ami-
nocycle in the side chain and employed 21 as our
benchmark inhibitor. Ring size and substitution were
explored through analogues 18 and 19. Thus, the
racemic proline analogue 18 showed similar enzyme
inhibitory potency to 21 although it was 16-fold less
potent in cells. The racemic 3-piperidinyl analogue 19,
however, was only 2-fold less potent than 21 in both the
enzyme and cellular antiproliferation assays but carried
the synthetic liability of a chiral center which could not
be readily accessed through a commercially purchasable
intermediate. The effect of substitution on the piperidi-
nyl nitrogen was explored through analogues 22-25.
N-Methyl, N-hydroxyethyl, and sulfonamide analogues
(22-24) showed similar activity to 21 in both enzyme
and cells assays while the N-tert-butoxycarbonyl ana-
logue 25 was significantly less potent in both of these
assays. As noted previously with cyclohexyl analogues
a
See ref 6 and or Experimental Section for description of assay
b
conditions. Assay was performed employing 25 µM [ATP].
to increase solubility and reduce both protein binding
and metabolic transformation. Importantly, this modi-
fication was a key to improving in vivo antitumor
efficacy. This report is the conclusion of this work in
which we have focused on examining lower molecular
weight nonaromatic acyl side chains with basic amines
and has led to the identification of our clinical develop-
ment candidate. Initial comparison of analogues 12 and
13 indicated that introduction of a primary amine into

1722 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Misra et al.
Ta ble 2. Metabolism and Pharmacokinetic Evaluation of
Aminothiazole Analogues in Micea
microsomal
stability
(nmol/min/mg
protein)
dose
Cmax Tmax AUC T1/2 MRT
compd
(µmol/kg) (µM) (h) (µM‚h) (h) (h)
2
19
21
22
23
26b
0.22
0.05
18
26
23
25
24
23
0.8 1.0
3.5 0.5
1.3 0.5
1.1 0.5
1.1 0.5
13.6 0.1
2.7 1.8 2.9
12.1 2.2 3.5
4.0 2.5 3.5
1.2 0.7 1.1
1.8 1.9 2.4
11.8 2.2 2.2
0.07
a
b
See ref 6 for description of assay conditions. Compound was
dosed iv.
toward the thiazole core and into the ribose pocket
rather than extending toward Phe-80. No clear hydro-
gen bonding interactions are seen between the oxazole
ring and the protein. Important hydrogen bonds be-
tween the amide backbone atoms of Leu-83 and both
the thiazole nitrogen and exocyclic amide proton are
clearly discernible. Finally, the piperidinyl ring extends
toward the exterior of the protein consistent with the
SAR which established that variable substitution at this
position of the inhibitor is tolerated. The origins of the
CDK2 selectivity of this series are not readily apparent
from the structural data. For comparison, the lower
panel of Figure 2 is an overlay which shows the relative
orientation of inhibitor 21 bound to CDK2 with that of
ATP bound to CDK2.9
Meta bolism a n d P h a r m a cok in etics. Selected ana-
logues were assayed for mouse serum protein binding
using an equilibrium dialysis protocol (Table 1) and for
metabolic stability toward mouse liver microsomes
(Table 2). Inhibitors with suitable in vitro CDK2 inhibi-
tory potency in both enzyme and cell assays (IC50 < 100
nM) and low to moderate protein binding (65-85%)
were selected, and pharmacokinetic (PK) parameters
were determined. Each compound was dosed intraperi-
toneally (ip) at a dose of 10 mg/kg in mice, with the data
shown in Table 2. Although the compounds were all
similar in structure, cumulative drug exposure over 6
h as measured by AUC varied 6- to 7-fold. In particular,
N-methyl analogue 22 showed significantly poorer PK
parameters within the group as indicated by lower AUC
and, both shorter half-life (T1/2) and mean-residence
time (MRT) while 19, 21, and 26 exhibited relatively
high exposures and longer half-lives. We speculate that
the poor PK of 22 could be due to either oxidation or
demethylation of the tertiary amine.
In Vivo An titu m or Activity. On the basis of their
in vitro potency and favorable in vivo exposure, com-
pounds 19 (homochiral isomer, see Experimental Sec-
tion), 21, 23, and 26 were evaluated for in vivo antitu-
mor efficacy in mice using P388 murine leukemia
tumors and A2780 human ovarian carcinoma xe-
nografts.10 In the P388 model, drugs were dosed intra-
peritonially (ip) once a day for 7 days (qdx7) in immu-
nocompetent mice immediately after the tumors were
implanted ip. The antitumor efficacy was measured as
an increase in lifespan, and the data are expressed as
a ratio of lifespan of drug-treated group (T) versus
control group (C). In the A2780 xenograft model, tumors
were implanted subcutaneously (sc) in nude mice, and
compounds were dosed ip once a day for 8 days (qdx8)
starting when tumors had grown to a median weight of
F igu r e 2. Upper panel: Solid-state structure of piperidinyl
aminothiazole 21 bound in the ATP-pocket of CDK2 (no cyclin).
The inhibitor carbon atoms are shown in green, the nitrogen
atoms are shown in blue, oxygen atoms are shown in red, and
the sulfur atoms are shown in yellow. The protein carbons are
in gray. Hydrogen bonds are shown by the magenta dotted
lines. Lower panel: Relative binding mode of inhibitor 21
(carbon atoms in light blue, nitrogen atoms in dark blue,
oxygen atoms in red and sulfur atoms in yellow) bound to
CDK2 with that of ATP (carbon atoms in green, nitrogen atoms
in blue, oxygen atoms in red, and phosphorus atoms in
magenta) bound to CDK2.
16 and 17, this may be the result of introducing a highly
lipophilic group into a hydrophilic space. Finally, the
isomeric cis and trans exocyclic amino analogues 26 and
27 were prepared and examined. The cis isomer, 26, was
7-fold more potent than trans isomer 27 in the enzyme
assay but only 2-fold more potent in the cellular anti-
proliferation assay. The origin for the differences in
enzyme activity between the two isomers were not
apparent. In both of the assays, 26 compared favorably
to our benchmark inhibitor 21.
Solid -Sta te Str u ctu r e Stu d y. The three-dimen-
sional solid-state structure of 21 in complex with CDK2
was determined by X-ray crystallography (see Support-
ing Information for summary of crystallographic data).
Crystals were obtained by incubating inhibitor 21 (72
h) with crystalline protein in the absence of cyclin. The
crystal structure, as shown in the upper panel of Figure
2, confirmed that 21 binds in the ATP-binding site and
the inhibitor adopts the same orientation and “folded”
conformation previously described with this series.6 In
this conformation the tert-butyl oxazole ring wraps back

Inhibitors of Cyclin-Dependent Kinase 2
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7 1723
Ta ble 3. In Vivo Antitumor Activity of Aminothiazoles against
a cytochrome P450 panel which included CyP1A2,
CyP2C9, CyP2C19, CyP2D6, and CyP3A4.
The antitumor efficacy of 21 in the A2780 human
ovarian carcinoma xenograft model was evaluated in
greater detail along with flavopiridol for comparative
purposes
P388 and A2780 Tumors in Micea
P388b
MTDd dose
A2780c
MTDd dose
compd
(mg/kg)
%T/C
(mg/kg)
LCKe
1
2
7.5
32
20
22.5
22.5
8.0
110
140
152
140
140
179
7.5
26
22
0.6-1.5
3.3
1.6
(Figure 3). Tumors were implanted sc in nude mice
and grown to 100 mg, and then animals were dosed with
21 once a day ip for 8 days at 18, 36, and 48 mg/kg
(MTD). Groups of eight mice were utilized for each dose.
Aminothiazole 21 was found to have antitumor activity
at all three doses (i.e., LCK > 1.0). At the low 18 mg/kg
dose, 21 showed minor tumor regression with a growth
delay of 2.1 LCK. At the higher 36 and 48 mg/kg doses,
21 showed rapid, significant tumor regression and
growth delays of >5.6 LCK and >6.5, respectively. In
addition, after extended times at the two high doses,
cures were seen in 9 of 16 animals as determined by no
measurable tumor at day 68. At autopsy the cured
animals were found to be free of tumor cells. Signifi-
cantly, 21 was found to be active not only at its MTD
but also at both sub-MTD doses (18 and 36 mg/kg),
indicating the potential for an enhanced safety window.
For comparison, flavopiridol showed a growth delay of
0.4 LCK (inactive in this model, LCK<1.0) with no
regression or cures at its MTD (7.5 mg/kg).
Selection of F in a l F or m . Because of the low aque-
ous solubility of free base 21 (0.28 mg/mL) and an
anticipated clinical plan which included an iv dosing
protocol, a number of mineral and organic acid salts of
21 were examined with the goal of increasing aqueous
solubility and identifying a final form with acceptable
physical and stability properties. The 1:2 L-tartrate salt
(acid:21) was selected as the final form due to its overall
favorable physical properties which included examina-
tion of polymorph, hydrate, solvate issues, ease, and
reproducibility of crystallization, hygroscopicity, solution
stability, and aqueous solubility (5.5 mg/mL). On the
basis of its physical properties, selectivity, pharmaco-
kinetics, and antitumor activity, 21 (BMS-387032) as
the 1/2-L-tartrate salt, was selected to progress into
clinical development as an antitumor agent.
19f
21
23
26
48
>3.6-5.0
8.0
1.7
a
b
See refs 6 and 10 for experimental protocols. P388 dosing
schedule, ip/qdx7. c A2780 dosing schedule, ip/qdx8. MTD )
d
maximum tolerated dose. e See ref 10 for definition of LCK.
f Homochiral isomer, see Experimental Section.
100 mg. The antitumor efficacy was measured as a
growth delay and expressed in log cell kill units (LCK).10
The data are summarized in Table 3. All compounds
were active in the P388 model showing %T/C values
between 140 and 179, where %T/C > 125 was defined
as active. In the A2780 xenograft model, 3-piperidinyl
analogue 19 and 4-aminocyclohexyl analogue 26 pro-
duced a growth delay of 1.6 and 1.7 LCK, respectively.
Both compounds were considered comparable in activity
to flavopiridol but significantly less efficacious than
aminothiazole 2 despite their superior exposure num-
bers. 4-Piperidinyl analogue 21, which had shown
intermediate exposure, however, produced a growth
delay between 3.6 and 5.0 LCK and was significantly
more efficacious than either flavopiridol (1) or amino-
thiazole 2. The enhanced efficacy of 21 will be the
subject of future reports.
Biology Su m m a r y for 21 (BMS-387032). On the
basis of its superior antitumor efficacy, we focused our
studies on 4-piperidinyl analogue 21. Closer examina-
tion of the CDK selectivity (Table 4) confirmed that 21
exhibited a similar CDK2-selective profile as reported
for earlier analogues in the aminothiazole series. Specif-
ically, 21 was 10-fold selective for CDK2/cycE relative
to CDK1/cycB and 20-fold selective relative to CDK4/
cycD. In addition, 21 showed remarkable selectivity over
a panel of unrelated kinases. It exhibited IC50 values
>40 µM for PKCR, PKCâ, PKCγ, Chk1, IKK, EMT, Lck,
FAK, ZAP70, and IC50 values >25 µM for HER1, HER2,
and IGF-1R. In both mouse and human serum, 21
showed low to moderate protein binding indicating high
exposure to free drug.
Su m m a r y
Aminothiazole 21 (BMS-387032, N-[5-[[[5-(1,1-dim-
ethylethyl)-2-oxazolyl]methyl]thio]-2-thiazolyl]-4-pipe-
ridinecarboxamide) has been identified as a CDK2-
selective inhibitor which has been selected to enter
clinical development as an antitumor agent. Compound
21 has shown superior antitumor efficacy to both
flavopiridol and previously reported analogues in this
series. The key modification from our previously re-
ported aminothiazole analogues was replacement of a
substituted aromatic acyl side chain with a nonaromatic
amino acyl side chain. This substitution reduced mo-
lecular weight, protein binding, and in vitro measured
metabolism in liver microsomes while at the same time
it increased aqueous solubility. SAR studies were con-
sistent with the solid-state structure of the inhibitor
bound to CDK2 protein, which indicated that the acyl
side chain of the molecule extended into the hydrophilic
extraprotein space and was amenable to modification.
Optimization of potency, pharmacokinetics, and evalu-
ation in both an ip/ ip P388 murine tumor model and
The pharmacokinetic parameters for 21 administered
in mouse, rat, and dog are shown in Table 5. The
compound exhibits a moderate half-life (T1/2) of between
5 and 7 h across the three species. The T1/2 is consistent
with in vitro studies which indicated a low rate of
metabolism (oxidation and glucuronidation) in liver
microsomes. In addition, 21 was well distributed into
tissues as indicated by a high volume of distribution
(Vss). Examination in three species indicated 21 is orally
bioavailable in all three species, although less so in rat
and dog than mouse. Metabolism studies in rats indi-
cated that dosed iv (8.9 mg/kg) 28% of parent drug is
recovered unchanged in urine and 11% in bile 9 h
postadministration. Minor metabolites resulting from
cleavage of the amide bond, oxidation of the thioether
linker, and hydroxylation of the tert-butyl group were
identified. Finally, 21 showed IC50 values >40 µM across

1724 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Ta ble 4. Summary of in Vitro Evaluation of Aminothiazole 21
Misra et al.
A2780 cellular
cytotoxicity
IC50 (nM)
CDK1/cycB
IC50 (nΜ)
CDK2/cycE
IC50 (nM)
CDK4/cycD
IC50 (nM)
% protein
binding mouse
% protein
binding human
480
48
925
95
69
63
Ta ble 5. Pharmacokinetic Parameters for Aminothiazole 21 in
Mouse, Rat, and Dog
All compounds exhibited spectra consistent with their assigned
structures. All new compounds were homogeneous (>98%) by
HPLC analysis. All previously unreported final analogues
showed acceptable (+0.4%) elemental analysis and/or high-
resolution mass spectrum (HRMS). Melting points (mp) are
uncorrected. Flash chromatography was performed using E.
Merck silica gel 60 under medium nitrogen pressure eluting
with the solvent systems indicated. Concentration in vacuo
refers to the use of a rotorary evaporator then oil pump
vacuum for removal of volatile components. Compounds 2, 6,
8, 12 and 15 were prepared previously.6 Biological assays,
metabolism and pharmacokinetic studies, and solid state
structural studies were conducted utilizing previously pub-
lished protocols.6,10
mousea
rata
24
5.3
3.7
6.3
64
15
0.52
4.7
31
dogb
dose (µmol/kg)
1/2 (h), iv
24
4.8
2.8
4.8
81
1.2
7.0
6.3
2.1
9.4
3.6
0.33
0.4
28
T
MRT (h), iv
AUCtot (µM‚h), iv
clearance (mL/min/kg), iv
Vss (L/kg), iv
Cmax (µM), po
Tmax (h), po
14
1.4
2.0
100
% oral bioavailability
a
b
Dosed in 1:9 EtOH/water vehicle. Dosed in 1:19 dextrose/
water, po dose 2.4 µmol/kg.
P r ep a r a tion of 2-Am in o-5-[[[5-(1,1-d im eth yleth yl)-2-
oxa zolyl]m eth yl]th io]th ia zole (8). To a 2L three-necked
round-bottom flask fitted with a mechanical stirrer was added
R-bromopinacolone (134 g, 747 mmol, 1.0 equiv), acetone (1.2
L), and sodium azide (63.2 g, 971 mmol, 1.3 equiv). The
reaction mixture was stirred at room-temperature overnight
and then filtered, and the solids were washed with acetone (2
× 100 mL). The filtrate was concentrated in vacuo to provide
1
R-azidopinacolone (105.0 g, 100%) as an oil. H NMR (CDCl3)
δ 1.17 (s, 9H), 4.07 (s, 2H). The crude material was used in
the next step without further purification.
To a 2 L three-necked round-bottom flask fitted with a
mechanical stirrer were added R-azidopinacolone (28.6 g, 203
mmol, 1.0 equiv), methanol (1145 mL), concentrated HCl (18
mL), and 10% Pd/C (3.5 g, 50% water wet). The reaction
mixture was stirred under hydrogen at 20 psi for 2 h, the
mixture was filtered through a pad of Celite, and the residue
rinsed with methanol (2 × 50 mL). The filtrate was concen-
trated under reduced pressure at a temperature below 40 °C.
The resulting wet solid was azeotroped with 2-propanol (2 ×
100 mL), anhydrous ether (100 mL) was added, and the slurry
which formed was stirred for 5 min. The solid product was
collected by filtration, and the cake was washed with diethyl
ether (2 × 30 mL) and then dried in vacuo to give R-aminopi-
nacolone hydrochloride 4 (28.0 g, 91%), mp 205 °C (lit. 199-
200 °C).11 1H NMR (DMSO-d6) δ 1.13 (s, 9H), 4.06 (s, 2H), 8.34
(s, 3H).
To a 1 L three-necked round-bottom flask fitted with a
mechanical stirrer were added R-aminopinacolone hydrochlo-
ride 4 (15.2 g, 100 mmol, 1.0 equiv) and CH2Cl2 (350 mL). The
slurry was cooled to -5 °C, and triethylamine (35 mL, 250
mmol, 2.5 equiv) was added. The resulting mixture was stirred
and cooled to -10 °C. A solution of R-chloroacetyl chloride (8.8
mL, 110 mmol, 1.1 equiv) in CH2Cl2 (20 mL) was added
dropwise over 15 min while keeping the reaction temperature
below -5 °C. The reaction was stirred for 1 h and then
quenched with 1 N aq HCl (200 mL). The phases were
separated, and the organic phase was washed with 1 N aq HCl
(200 mL), and water (50 mL), dried (Na2SO4), and concentrated
in vacuo to afford R-N-2-(chloroacetylamino)pinacolone 5 (18.9
g, 98%) as a white solid, mp 50-52 °C. 1H NMR (CDCl3) δ
1.21 (s, 9H), 4.09 (s, 2H), 4.30 (s, 2H), 7.35 (s, 1H). 13C NMR
(CDCl3) δ 210.13, 166.62, 45.23, 43.51, 42.79, 27.45. Anal.
(C8H14NO2Cl): C, H, N, Cl.
F igu r e 3. Comparative antitumor efficacy of aminothiazole
21 (BMS-387032) and flavopiridol (1) in a A2780 human
ovarian carcinoma xenograft nude mouse model.
an A2870 human ovarian cancer xenograft tumor mouse
model led to the identification of the 4-piperidinyl
analogue, 21, as our clinical development candidate.
Detailed biochemical, antitumor efficacy, and clinical
studies will be the subject of future disclosures.
Exp er im en ta l Section
All air- and moisture-sensitive reactions were conducted
under an argon atmosphere. All reagents and starting materi-
als were obtained from commercial sources and used without
further purification unless indicated or referenced. DMF refers
to N,N-dimethylforamide, TFA refers to trifluoroacetic acid,
DMAP refers to 4-(dimethylamino)pyridine, and EDAC refers
to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochlo-
ride. 1H NMR (400 or 500 MHz) and 13C (100 or 125 MHz)
spectra were recorded on J EOL or Bruker spectrometers and
chemical shifts are reported in parts per million downfield from
internal trimethylsilane. Mass spectra (LCMS) were measured
using a Shimadzu HPLC 10A system linked to a Micromass
MS detector in positive chemical ionization mode. Analytical
high performance liquid chromatography (HPLC) was per-
formed on Shimadzu 10A systems fitted with YMC S5 ODS
4.6 × 50 mm columns, using a UV detector at 220 or 254 nM.
To a 100 mL rounded bottom flask fitted with a magnetic
stirrer were added 5 (18.9 g, 98.6 mmol, 1 equiv) and POCl3
(38 mL, 407 mmol, 4.1equiv). The reaction mixture was heated

Inhibitors of Cyclin-Dependent Kinase 2
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7 1725
to 105 °C and stirred for 1 h. After being cooled to room
temperature, the reaction mixture was poured carefully into
ice (180 g). The mixture was extracted with ether (6 × 150
mL). The organic extracts were combined and neutralized to
pH 7-8 with saturated sodium bicarbonate (∼700 mL). The
organic phase was separated and washed successively with
saturated sodium bicarbonate (100 mL), water (100 mL), and
brine (50 mL), dried (MgSO4), and concentrated in vacuo. The
crude material was distilled under reduced pressure to give
5-tert-butyl-2-chloromethyloxazole, 66 (13.6 g, 80%), as a
[M + H]+. HRMS FAB [M + H]+ calcd for C19H21N3O2S2
387.1075; found 388.1167.
P r epar ation of N-[5-[[[5-(1,1-Dim eth yleth yl)-2-oxazolyl]-
m e t h y l ]t h i o ]-2 -t h i a z o l y l ]c y c l o h e x y l m e t h a n e c a r -
boxa m id e (16). To a stirred mixture of cyclohexylacetic acid
(53 mg, 0.37 mmol), amine 8 (100 mg, 0.37 mmol), N-
hydroxybenzotriazole hydrate (60 mg, 0.44 mmol), and DMAP
(45 mg, 0.37 mmol) in DMF (1.4 mL) were added diisopropy-
lethylamine (0.16 mL, 0.89 mmol) and EDAC (71 mg, 0.37
mmol) at room temperature. This mixture was stirred at room
temperature for 6 h and then heated to 80 °C for 16 h. The
reaction mixture was cooled to room temperature. This was
purified by preparative HPLC (YMC ODS S5 20 × 100 mm
column, 30% to 100% gradient over 10 min with 0.1%TFA in
MeOH-water solvent system, 20 mL/min) to give the TFA salt
of 16 (73 mg, 50%) as a white solid. 1H NMR(CDCl3) δ 7.32 (s,
1H), 6.60 (s, 1H), 3.97 (s, 2H), 2.36 (d, 7.2 Hz, 2H), 1.94-1.88
(m, 1H), 1.78-1.70 (m, 5H), 1.28-1.25 (m, 12H), 1.04-0.97
(m, 2H). LCMS: 394 [M + H]+. HRMS FAB [M + H]+ calcd
for C19H27N3O2S2 393.1545; found 394.1621.
1
colorless oil, bp 38-40 °C (0.25 mm). H NMR (CDCl3) δ 1.32
(s, 9H), 4.60 (s, 2H), 6.70 (s, 1H). 13C NMR (CDCl3) δ 28.92,
31.91, 36.57, 120.87, 158.03, 162.90.
To a solution of thiocyanate 78 (10.0 g, 63.3 mmol) in
absolute EtOH (600 mL) was added NaBH4 (4.8 g, 120 mmol)
portionwise at room temperature. The mixture was stirred for
1 h, and then acetone (300 mL) was slowly introduced. After
1 h, a solution of oxazole chloride 6 (12.0 g, 69 mmol) in EtOH
(100 mL) was added, and the resulting dark reaction mixture
heated to reflux for 1 h. The resulting mixture was cooled,
concentrated in vacuo, and then partitioned between EtOAc
and brine. The organic phase was separated, dried (MgSO4),
and concentrated in vacuo to give a crude solid. The crude
material was triturated with diethyl ether/hexane to provide
amine 86 (16.0 g, 94%) as a pale red-brown solid.
P r epar ation of N-[5-[[[5-(1,1-Dim eth yleth yl)-2-oxazolyl]-
m eth yl]th io]-2-th ia zolyl]cycloh exa n eca r boxa m id e (17).
To a solution of amine 8 (1.00 g, 3.70 mmol), cyclohexylcar-
boxylic acid (711 mg, 5.55 mmol), and DMAP (138 mg, 1.11
mmol) in DMF (4.5 mL) and CH2Cl2 (13.5 mL) was added
EDAC (2.12 g, 11.1 mmol) at room temperature. The reaction
mixture was stirred overnight then poured into EtOAc and
water to which were added aq sodium bicarbonate and brine.
The precipitate that formed was collected by filtration, washed,
and dried to give 17 (844 mg, 60%) as a white solid, mp 189-
P r epar ation of N-[5-[[[5-(1,1-Dim eth yleth yl)-2-oxazolyl]-
m eth yl]th io]-2-th iazolyl]-2-am in oeth an ecar boxam ide Hy-
d r och lor id e (13). To a stirred solution of amine 8 (1.20 g,
4.45 mmol) and N-tBoc-â-alanine (2.10 g, 11.1 mmol) in
anhydrous DMF (10 mL) was added EDAC in several portions
(2.55 g, 13.3 mmol) over 5 min at room temperature. The
mixture was stirred for 1.5 h and then partitioned between
EtOAc (75 mL) and 1 M aq HCl (75 mL). The organic phase
was separated and washed successively with 1 M HCl (75 mL),
5% aq sodium bicarbonate (75 mL), and brine (50 mL), dried
(Na2SO4) and concentrated in vacuo to give an oil. The crude
material was purified by flash chromatography (EtOAc) to
afford N-[5-[[[5-(1,1-dimethylethyl)-2-oxazolyl]methyl]thio]-2-
thiazolyl]-2-(tert-butyloxycarbonyl)aminoethanecarboxamide
(1.93 g, 98%) as a solid white foam. 1H NMR (CDCl3) δ 11.9
(s, 1H), 7.35 (s, 1H), 6.62 (s, 1H), 5.28 (br s, 1H), 4.09 (s, 2H),
3.54 (m, 2H), 2.72 (m, 2H), 1.43 (s, 9H), 1.27 (s, 9H). LCMS:
441 [M + H]+.
To a solution of the above t-Boc intermediate (1.82 g, 4.13
mmol) in CH2Cl2 (10 mL) was added TFA (5 mL) rapidly at
room temperature. After 1 h, the solution was concentrated
in vacuo. The residue was partitioned between EtOAc (25 mL)
and water (35 mL) and then neutralized by addition of solid
sodium bicarbonate. The organic layer was separated, washed
successively with 5% aq sodium bicarbonate (25 mL) and brine
(25 mL), dried (Na2SO4), and concentrated in vacuo to give
the free amine (1.26 g) as a solid foam. The free amine was
solubilized with 1 M aq HCl (3.7 mL, 1 equiv) and water (5
mL) and then lyophilized to afford the hydrochloride salt of
13 (1.37 g, 88%) as an off-white amorphous solid foam. 1H
NMR (DMSO-d6) δ 12.5 (br s, 1H), 8.02 (br s, 2H), 7.39 (s, 1H),
6.71 (s, 1H), 4.06 (s, 2H), 3.08 (m, 2H), 2.84 (t, J ) 7, 2H),
1.20 (s, 9H); LCMS: 341 [M + H]+. HRMS FAB [M + H]+ calcd
for C14H20N4O2S2 340.1028; found 341.1095.
1
192 °C. H NMR (CDCl3) δ 7.29 (s, 1H), 6.57 (s, 1H), 3.92 (s,
2H), 2.50 (m, 1H), 2.1-1.2 (br m, 10H), 1.23 (s, 9H). LCMS:
380 [M + H]+. HRMS FAB [M + H]+ calcd for C18H27N3O2S2
379.1388; found 380.1463.
P r ep a r a tion of (()-N-[5-[[[5-(1,1-Dim eth yleth yl)-2-ox-
a zolyl]m eth yl]th io]-2-th ia zolyl]-2-p yr r olid in eca r boxa m -
id e (18). To a stirred solution of amine 8 (0.50 g, 1.86 mmol)
and N-tBoc-DL-proline (0.80 g, 3.71 mmol) in CH2Cl2 (20 mL)
at room temperature was added EDAC (0.71 g, 3.71 mmol).
The reaction mixture was stirred for 1 h and then diluted with
CH2Cl2 (50 mL). The organic phase was washed with aq
NaHCO3 solution twice and brine once, dried (MgSO4), and
concentrated in vacuo. The crude material was purified by
flash chromatography (EtOAc/hexane 1:1) to give N-tBoc-18
1
as a light color solid (0.71 g, 82%). H NMR (CDCl3) δ 7.33 (s,
1H), 6.58 (s, 1H), 4.12 (m, 1H), 3.93 (s 2H,), 1.94 (m, 2H), 1.44
(m, 4H), 1.26 (s, 9H). LCMS: 467 [M + H]+.
To a stirred solution of N-tBoc-18 (0.70 g, 1.50 mmol) in
CH2Cl2 (2 mL) at room temperature was added TFA (2.5 mL).
The reaction mixture was stirred for 1.5 h and then concen-
trated in vacuo. The resulting residue was made basic by
addition of aq NaHCO3 solution and extracted with EtOAc (3
× 50 mL). The organic extracts were dried (MgSO4) and
concentrated in vacuo to afford a light foam. The foam was
treated with 1 N aq HCl (1.45 mL) and lyophilized to give the
hydrochloride of 18 as a white solid (0.54 g, 89%), mp 118-
1
120 °C. H NMR (CDCl3) δ 10.50 (s, 1H), 9.2 (s, 1H), 7.19 (s,
1H), 6.62 (s, 1H), 4.8 (m, 1H), 4.01 (s, 2H), 3.56 (m, 2H), 2.63
(m, 1H), 2.14 (m, 3H) 1.30 (s, 9H). LCMS: 367 [M + H]+.
HRMS FAB [M + H]+ calcd for C16H22N4O2S2 366.1184; found
367.1266.
P r epar ation of N-[5-[[[5-(1,1-Dim eth yleth yl)-2-oxazolyl]-
m eth yl]th io]-2-th iazolyl]ph en ylm eth an ecar boxam ide Tr i-
flu or oa cetic Acid Sa lt (14). To a stirred mixture of pheny-
lacetic acid (50 mg, 0.37 mmol), amine 8 (100 mg, 0.37 mmol),
N-hydroxybenzotriazole hydrate (60 mg, 0.44 mmol), and
DMAP (45 mg, 0.37 mmol) in DMF (1.4 mL) were added
diisopropylethylamine (0.16 mL, 0.89 mmol) and EDAC (71
mg, 0.37 mmol) at room temperature. The reaction mixture
was cooled to room temperature. This was purified by prepara-
tive HPLC (YMC ODS S5 20 × 100 mm column, 30% to 100%
gradient over 10 min with 0.1%TFA in MeOH-water solvent
system, 20 mL/min) to give the TFA salt of 14 (40 mg, 28%)
P r ep a r a tion of (()-N-[5-[[[5-(1,1-Dim eth yleth yl)-2-ox-
a zolyl]m eth yl]th io]-2-th ia zolyl]-3-p ip er id in eca r boxa m -
id e (19) a n d Sep a r a tion of En a n tiom er s. Nipecotic acid
(1.3 g, 10 mmol, 1 equiv) was combined with dioxane (10 mL),
acetonitrile (2 mL), water (10 mL), and 1 N aqueous NaOH
(10 mL, 1 equiv). Di-tert-butyl dicarbonate (3.3 g, 15 mmol,
1.5 equiv) was added, the reaction was stirred at room-
temperature overnight and then concentrated in vacuo, and
10% aqueous citric acid was added. The mixture was extracted
with EtOAc (3 × 100 mL). The organic extracts were dried
(Na2SO4), filtered through silica gel, and concentrated in vacuo
to give a solid. The resulting crude material was recrystallized
1
as a white solid. H NMR (CDCl3) δ 7.49-7.25 (m, 6 H), 6.54
(s, 1 H), 3.93 (s, 2 H), 3.79 (s, 2 H), 1.21 (s, 9 H). LCMS: 388

1726 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Misra et al.
(EtOAc/hexanes) to provide N-tert-butoxycarbonylnipecotic
P r epar ation of N-[5-[[[5-(1,1-Dim eth yleth yl)-2-oxazolyl]-
m eth yl]th io]-2-th ia zolyl]-4-p ip er id in eca r boxa m id e (21),
Sch em e 2. To a mixture of amine 8 (9.60 g, 35.6 mmol, 1
equiv), N-tert-butoxycarbonylisonipecotic acid 11 (12.6 g, 55
mmol, 1.5 equiv), DMAP (2.0 g, 16 mmol, 0.45 equiv) in DMF
(36 mL), and CH2Cl2 (100 mL) was added EDAC (13.8 g, 72
mmol, 2 equiv) at room temperature. The reaction mixture was
stirred for 3.5 h. Water (300 mL) and EtOAc (200 mL) were
added, and the resulting precipitate was collected by filtration.
The filtrate was extracted with EtOAc. The organic extracts
were dried (MgSO4) and concentrated in vacuo to provide a
yellow solid which was combined with the precipitate. The solid
was heated to reflux in a mixture of ethanol, acetone, and
water for 20 min. The solid was filtered, washed with an
ethanol/water mixture, and dried under vacuum to give crude
N-tBoc-25 (16.6 g, 97%) as a white solid.
acid12 (2.2 g, 96%) as a white solid.
To a mixture of amine 8 (2.7 g, 10 mmol, 1 equiv), N-tert-
butoxycarbonylnipecotic acid (3.4 g, 1.5 mmol, 1.5 equiv) in
DMF (10 mL), and CH2Cl2 (30 mL) was added EDAC (3.8 g,
20 mmol, 2 equiv) at room temperature. The reaction mixture
was stirred for 4 h. The resulting black solution was concen-
trated in vacuo, diluted with water (90 mL), and extracted with
EtOAc (100 mL, and then 2 × 75 mL). The organic extracts
were dried (Na2SO4), concentrated in vacuo, and purified by
flash chromatography (50-100% EtOAc in hexanes gradient)
to provide (()-N-[5-[[[5-(1,1-dimethylethyl)-2-oxazolyl]methyl]-
thio]-2-thiazolyl]-3-(N-tert-butoxycarbonyl)piperidinecar-
1
boxamide (3.80 g, 79%) as a yellow solid. H NMR (CDCl3) δ
7.31 (s, 1H), 6.57 (s, 1H), 3.98-3.92 (m, 1H), 3.92 (s, 2H), 3.72
(br m), 3.35 (br m) 3.09 (br), 2.54 (br m), 1.95 (br, 2H), 1.7-
1.3 (br), 1.45 (s, 9H), 1.23 (s, 9H). LCMS: 481 [M + H]+.
A magnetically stirred suspension of crude N-tBoc-25 (20.2
g, 42.1 mmol) in 200 mL of CHCl3 was warmed until homo-
geneous, and then a solution of HCl (31 mL, 4 N in dioxane)
was added at 55 °C. Gas was evolved, and a precipitate formed
within a few minutes. After 7 h, HPLC indicated the reaction
was about 2/3 complete. Additional HCl solution (10 mL) was
introduced, and the reaction mixture was stirred at 60 °C for
1 h. A third portion of HCl solution (10 mL) was added and
the reaction mixture stirred at 45 °C for 6 h. The resultant
heavy suspension was cooled in an ice-bath, and saturated aq
sodium bicarbonate solution (200 mL) was added slowly. Gas
was evolved during the addition. The heavy suspension became
homogeneous and then formed a light suspension. The light
suspension was treated with solid sodium carbonate (6 g),
heated at 60 °C for 20 min, and then diluted with CHCl3 (100
mL). The aqueous phase was separated and extracted with
CHCl3 (2 × 100 mL). The organic extracts were combined,
washed with brine (100 mL), dried (Na2CO3/Na2SO4), and
concentrated in vacuo to give a crude yellow solid. The yellow
solid was recrystallized (50% aq EtOH, 400 mL) to afford 21
(13.3 g, 83%) as a white solid, mp 171-173 °C. 1H NMR
(DMSO-d6) δ 7.38 (s, 1H), 6.72 (s, 1H), 4.05 (s, 2H), 2.97 (d, J
) 12 Hz, 2H), 2.6-2.4 (m, 3H, overlapped with solvent peak,
but discernible), 1.67 (d, J ) 12 Hz, 2H), 1.47 (dq, J ) 4 Hz,
12 Hz, 2H), 1.17 (s, 9H). 13C NMR (DMSO-d6) δ 174.36, 161.90,
161.14, 159.11, 145.48, 120.47, 118.63, 45.62, 42.38, 34.32,
31.26, 29.24, 28.63. LCMS: 381 [M + H]+. HPLC: HI > 99%
at 3.12 min (YMC S5 ODS column 4.6 × 50 mm, 10-90%
aqueous methanol over 4 min containing 0.2% phosphoric acid,
4 mL/min, monitoring at 220 nm). Anal. (C17H24N4O2S2) C, H,
N, S.
To a solution of the above t-Boc intermediate (355 mg, 0.74
mmol, 1 equiv) in CH2Cl2 (3 mL) was added TFA (3 mL), and
the mixture was stirred at room temperature for 20 min. The
solution was concentrated in vacuo and neutralized with
saturated aqueous NaHCO3. The resulting mixture was ex-
tracted with EtOAc. The organic extracts were dried (Na2SO4),
concentrated in vacuo, and recrystallized (EtOAc) to provide
racemic 19 (142 mg, 50%) as a white solid, mp 175-177 °C.
1H NMR (DMSO-d6) δ 7.23 (s, 1H), 6.69 (s, 1H), 3.96 (s, 2H),
3.2-2.8 (b, 2H), 2.7-2.6 (m, 1H), 2.5-2.3 (m, 2H), 1.87-1.78
(m, 1H), 1.60-1.50 (m, 2H), 1.38-1.26 (m, 1H), 1.19 (s, 9H).
LCMS: 381 [M + H]+. HRMS FAB [M + H]+ calcd for
C
17H24N4O2S2 380.1341; found 381.1429.
For in vivo evaluation, homochiral 19 was obtained as
follows. The racemic t-Boc-protected intermediate from above
was separated into the individual enantiomers by chiral
preparative HPLC (Chiral Pak AD column, 5 × 50 cm, 20 µm,
10% 0.1% triethylamine/2-propanol in hexanes; 45 mL/min,
detection at 254 nm, loading 300 mg of racemic 19 in 5 mL of
2-propanol). The two individual tBoc-protected enantiomers
were separately deprotected using TFA and isolated as the free
bases as described above. They were then converted to their
hydrochloride salts for in vivo evaluation. The homochiral
isomer of 19 obtained from the slower eluting t-Boc intermedi-
ate was evaluated in vivo. The absolute configuration was not
determined.
P r epar ation of N-[5-[[[5-(1,1-Dim eth yleth yl)-2-oxazolyl]-
m eth yl]th io]-2-th ia zolyl]-4-m eth ylp ip er id in eca r boxa m -
id e (20). To a stirred solution of amine 8 (0.20 g, 0.74 mmol)
and N-tBoc-piperidine-4-acetic acid (0.34 g, 1.46 mmol) in CH2-
Cl2 (10 mL) at room temperature was added EDAC (0.28 g,
1.46 mmol). The reaction mixture was stirred overnight and
then concentrated in vacuo. The crude material was purified
by flash chromatography (EtOAc/hexane 1:1 to 3:2) to give
To a warm solution of 21 (1.75 g, 4.6 mmol) in absolute
ethanol (70 mL) was added a solution of L-tartaric acid (345
mg, 2.3 mmol, 0.5 equiv) in absolute ethanol (5 mL). A
precipitate started to form after several minutes. The mixture
was allowed to stand for 4 h at room temperature, and then
the solid precipitate was collected on a Buchner funnel, washed
with absolute ethanol, and dried in vacuo at 85 °C to afford
the 1/2-L-tartrate salt of 21 (326 mg, 94%) as off-white crystals,
mp 234-236 °C. Anal. (C17H24N4O2S2‚0.5-L-Tartaric acid) C,
H, N, S.
1
N-tBoc-20 as a white solid (0.32 g, 89%). H NMR (CDCl3) δ
11.1 (s, 1H), 7.28 (s, 1H), 6.57 (s,1H), 4.09 (m, 1H), 3.94 (s,
2H), 2.71 (m, 2H), 2.37 (d, J ) 7 Hz, 2H), 2.05 (m, 1H), 1.71
(m, 2H), 1.43 (s, 9H), 1.23 (s, 9H), 1.17 (m, 2H); 13C NMR
(CDCl3) δ 169.5, 161.8, 158.8, 154.7, 143.9, 121.2, 120.1, 79.5,
42.9, 34.9, 33.2, 31.9, 31.6, 28.6, 28.4. LCMS: 495 [M + H]+.
P r ep a r a tion of 1-Meth yl-N-[5-[[[5-(1,1-d im eth yleth yl)-
2-oxa zolyl]m eth yl]th io]-2-th ia zolyl]-4-p ip er id in eca r box-
a m id e (22). To a suspension of amine 21 (700 mg, 1.80 mmol)
and sodium triacetoxyborohydride13 (1.70 g, 8.1 mmol) in 1,2-
dichloroethane (45 mL) was added aq formaldehyde solution
(37%, 0.61 mL, 8.1 mmol) at room temperature. The reaction
mixture was stirred overnight, sat aq sodium bicarbonate
solution was added, and the mixture was extracted with CH2-
Cl2. The organic extracts were dried (Na2SO4) and concentrated
in vacuo. The crude material was purified by flash chroma-
tography (1:2:17 MeOH/Et3N/EtOAc) to afford 22 (530 mg,
75%) as a white solid, mp 177-180 °C. 1H NMR (CDCl3) δ 9.10
(br s, 1H), 7.29 (s, 1H), 6.57 (s, 1H), 3.91 (s, 2H), 2.91 (d, J )
11.4 Hz, 2H), 2.28 (s, 3H), 2.33-2.26 (b, 1H overlapped with
2.28 peak), 1.99 (t, J ) 11.4 Hz, 2H), 1.83-1.93 (m, 4H), 1.23
(s, 9H). LCMS: 395 [M + H]+. Anal. (C18H26N4O2S2) C, H, N,
S.
To a stirred solution of N-tBoc-20 (0.30 g, 0.62 mmol) in
dioxane (11 mL) at room temperature was added HCl solution
(4 N in dioxane, 3.0 mL). The reaction mixture was stirred
overnight, and then MeOH (4 mL) was added to facilitate
completion of the reaction by solublizing the solids which had
formed. The mixture was concentrated in vacuo, and the
residue was dissolved in water and lyophilized to give the
hydrochloride of 20 as a light yellow hygroscopic solid (0.234
g, 90%), mp 143-145 °C. 1H NMR (DMSO-d6) δ 9.14 (br s,
1H), 8.97 (m,1H), 7.36 (s, 1H), 6.71 (s, 1H), 4.04 (s, 2H), 3.17
(d, J ) 12, 2H), 2.82 (q, J ) 12, 2H), 2.39 (d, J ) 7, 2H), 2.03
(m, 1H), 1.73 (d, J ) 13, 2H), 1.42 (q, J ) 11, 2H), 1.17 (s,
9H). 13C NMR (CDCl3) δ 170.3, 161.2, 161.1, 159.0, 145.3,
120.2, 118.98, 42.9, 41.1, 34.2, 31.1, 30.8, 28.5, 28.1. LCMS:
395 [M + H]+. HRMS FAB [M + H]+ calcd for C18H26N4O2S2
394.1497; found 395.1566.

Inhibitors of Cyclin-Dependent Kinase 2
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7 1727
P r epar ation of N-[5-[[[5-(1,1-Dim eth yleth yl)-2-oxazolyl]-
m eth yl]th io]-2-th ia zolyl]-1-(2-h yd r oxyeth yl)-4-p ip er id i-
n eca r boxa m id e (23). To a solution of amine 21 (1.4 g, 3.7
mmol, 1 equiv) in DMF (30 mL) and THF (100 mL) were added
2-bromoethoxy-tert-butyldimethylsilane (0.79 mL, 3.7 mmol,
1 equiv) and solid sodium bicarbonate (1.8 g) at room temper-
ature. The reaction was stirred at 50 °C for 23 h. Additional
bromide (0.9 mL) was added, and the reaction was stirred at
50 °C for 22 h. The reaction was cooled and concentrated in
vacuo, water (25 mL) was added, and the reaction was
extracted with EtOAc (50 mL). The organic extract was dried
(Na2SO4), concentrated in vacuo, and purified by flash chro-
matography (0-5% triethylamine in EtOAc gradient) to
provide TBDS-protected 23 (1.70 g, 84%) as a yellow solid. 1H
NMR (CDCl3) δ 7.32 (s, 1H), 6.58 (s, 1H), 3.93 (s, 2H), 3.75 (t,
J ) 6.3 Hz, 2H), 3.0 (d, J ) 14 Hz, 2H), 2.53 (t, J ) 6.3 Hz,
2H), 2.36-2.30 (m, 1H), 2.15 (t, J ) 14 Hz, 2H), 1.93-1.79
(m, 4H), 1.24 (s, 9H), 0.89 (s, 9H), 0.06 (s, 6H). LCMS: 539
[M + H]+.
P r ep a r a tion of cis-4-Am in o-N-[5-[[[5-(1,1-d im eth yleth -
yl)-2-oxa zolyl]m eth yl]th io]-2-th ia zolyl]cycloh exa n eca r -
boxa m id e Hyd r och lor id e (26) a n d tr a n s-4-Am in o-N-[5-
[[[5-(1,1-d im e t h y le t h y l)-2-o x a zo ly l]m e t h y l]t h io ]-2-
th ia zolyl]cycloh exa n eca r boxa m id e Hyd r och lor id e (27).
To a solution of 4-aminocyclohexane carboxylic acid (2.86 g,
20 mmol) in 0.5 M aqueous NaOH (40 mL), dioxane (20 mL),
and acetonitrile (4 mL) was added portionwise di-tert-butyl
dicarbonate (6.5 g total, 30 mmol) at room temperature. After
20 h, EtOAc (100 mL) and 10% aqueous citric acid solution
(100 mL) were introduced. The aqueous layer which formed
was separated and extracted with EtOAc (3 × 50 mL). The
organic phases were combined, dried (Na2SO4), and concen-
trated in vacuo to give crude 4-(tert-butoxycarbonylamino)-
cyclohexanecarboxylic acid (6.0 g, 125%) as a colorless oil which
solidified upon standing.
To a solution of crude 4-(tert-butoxycarbonylamino)cyclo-
hexane carboxylic acid (5.0 g, <21 mmol) and amine 8 (3.50 g,
13 mmol) in DMF (13 mL) and CH2Cl2 (36 mL) was added
EDAC (5.0 g, 26 mmol) at room temperature. The reaction
mixture was stirred overnight and diluted with water (100
mL). The aqueous layer was separated and extracted with
EtOAc (2 × 150 mL). The combined organic phases were dried
(Na2SO4) and then filtered through a pad of silica gel. The
filtrate was concentrated in vacuo to afford an orange solid.
The crude material was recrystallized (95% EtOH) to give
tBoc-26/27 (2.78 g, 43%) as a yellow solid.
To a solution of the TBDS intermediate from above (1.45 g,
2.7 mmol, 1 equiv) in acetonitrile (100 mL) was added aq HF
solution (48%, 2.5 mL) at room temperature.14 The reaction
was stirred for 4 h, an additional 2.5 mL of aq HF was added,
and the reaction was stirred overnight. EtOAc (100 mL) and
saturated aq NaHCO3 (50 mL) were slowly added. Additional
solid NaHCO3 was added to make the mixture basic. The
mixture was extracted with EtOAc (2 × 50 mL). The organic
extracts were dried (Na2SO4) and filtered through a pad of
silica gel, and the filtrate was concentrated in vacuo. The
resulting white solid was recrystallized (aq EtOH) to provide
23 (1.60 g, 59%) as a white solid, mp 153-155 °C. 1H NMR
(DMSO-d6) δ 12.20 (s, 1H), 7.38 (s, 1H), 6.72 (s, 1H), 4.35 (t, J
) 5.4 Hz, 1 Hz), 4.05 (s, 2H), 3.48 (q, J ) 5.7 Hz, 2H), 2.89 (d,
J ) 11.3 Hz, 2H), 2.36 (t, J ) 6.0 Hz, 2H), 1.94 (t, J ) 11.3
Hz, 2H), 1.72 (d, J ) 11.3 Hz, 2H), 1.64-1.55 (m, 2H), 1.15 (s,
9H). LCMS: 425 [M + H]+. HRMS FAB (M + H)+ calcd for
To a suspension of the tBoc-protected product (2.6 g, 5.3
mmol) in CH2Cl2 (15 mL) was added TFA (3 mL). The mixture
was stirred at room temperature for 2 h, concentrated in vacuo,
neutralized with base, and extracted with EtOAc. The organic
layer was separated, dried (Na2SO4), and concentrated in vacuo
to give a crude mixture of 26 and 27 (2.7 g). A portion of the
crude mixture (1.2 g) was dissolved in methanol and mixed
with sodium bicarbonate powder to ensure neutralization of
residual TFA. The mixture was stirred for 1 h and filtered to
remove solids, and the filtrate was concentrated in vacuo. The
crude material was purified by flash chromatography (10%
isopropylamine in EtOAc, and then 10% methanol and 20%
isopropylamine in EtOAc) to give cis-product 26 (0.80 g, 16%
from amine 8) as a white solid and trans product 27 (0.08 g,
1.6% from amine 8) as a yellow solid.
C
19H28N4O3S2 424.1603; found 425.1690.
P r epar ation of N-[5-[[[5-(1,1-Dim eth yleth yl)-2-oxazolyl]-
m eth yl]th io]-2-th ia zolyl]-(N-m eth ylsu lfon yl)-4-p ip er id i-
n eca r boxa m id e (24). To a stirred solution of amine 21 (45
mg, 0.12 mmol) in pyridine (0.15 mL) and CH2Cl2 (1 mL) was
added methanesulfonyl chloride (28 µL, 0.36 mmol) at room
temperature. The reaction mixture was stirred for 1 h.
Anhydrous potassium carbonate powder (130 mg, mmol) was
then added, and the mixture was stirred at room temperature
for 1 h. Water (1 mL) was added. The mixture was stirred
vigorously for 30 min. The aqueous solution was extracted with
CH2Cl2 (3 × 1 mL). The combined organic extracts were dried
(Na2SO4), concentrated in vacuo, and purified by flash chro-
matography (50% EtOAc/heptane and then EtOAc) to give 24
(47 mg, 85%) as a white solid, mp 212-214 °C. 1H NMR
(CDCl3) δ 9.11 (s, 1H), 7.29 (s, 1H), 6.57 (s, 1H), 3.92 (s, 2H),
3.79 (dt, J ) 13.3, 4.8 Hz, 2H), 2.89 (dt, J ) 2.7, 11.4 Hz, 2H),
2.48 (m, 1H), 2.06-2.01 (m, 2H), 1.98-1.89 (m, 2H), 1.24 (s,
9H). LCMS: 459 [M + H]+. HRMS FAB (M + H)+ calcd for
Amine 26 was converted to the HCl salt with 1 N aq HCl
(1 equiv) to give a pale yellow solid, mp 224-226 °C. 1H
NMR (CD3OD) δ 7.30 (s, 1H), 6.66 (s, 1H), 3.98 (s, 2H), 3.26
(m, 1H), 2.74 (quintet, J ) 4.8 Hz, 1H), 2.06-2.00 (m, 2H),
1.90-1.74 (m, 6H), 1.24 (s, 9H). LCMS: 359 [M + H]+. Anal.
(C18H27N4O2S2Cl + 1.4 H2O) C, H, N, S, Cl.
Amine 27 was converted to the HCl salt with 1 N aq HCl
(1 equiv) to give a pale yellow solid. 1H NMR (CD3OD) δ
7.32 (s, 1H), 6.68 (s, 1H), 3.99 (s, 2H), 3.12 (tt, J ) 3, 12 Hz,
1H), 2.50 (d, J ) 6 Hz, ∼1H), 2.47 (tt, J ) 3, 12 Hz, ∼1H
overlapped with doublet at 2.50 ppm), 2.13 (d, J ) 12 Hz, 2H),
2.03 (d, J ) 12 Hz, 2H), 1.65 (dq, J ) 3, 12 Hz, 2H), 1.47 (dq,
J ) 3, 12 Hz, 2H), 1.24 (s, 9H). LCMS: 359 [M + H]+. Anal.
(C18H27N4O2S2Cl + 1.03 H2O) C, H, N, S, Cl.
C
18H26N4O4S3 458.1116; found 59.1190.
P r epar ation of N-[5-[[[5-(1,1-Dim eth yleth yl)-2-oxazolyl]-
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
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.). The
filters were quantified using a TopCount 96 well liquid
scintillation counter (Packard Instrument Co.). Dose-response
curves were generated to determine the concentration required
for 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 was 2%. IC50 values were derived by nonlinear
m eth yl]th io]-2-th ia zolyl]-(N-ter t-bu toxyca r bon yl)-4-p ip -
er id in eca r boxa m id e (25). To a mixture of amine 8 (270 mg,
1.10 mmol), N-tert-butoxycarbonylisonipecotic acid (344 mg,
1.50 mmol), DMAP (61 mg, 0.50 mmol), and triethylamine
(0.28 mL, 2.0 mmol) in DMF (1 mL) and CH2Cl2 (3 mL) was
added EDAC (383 mg, 2.00 mmol) at room temperature. The
reaction mixture was stirred for 1.5 h, diluted with EtOAc,
washed sequentially with brine, aq HCl, and aq sodium
bicarbonate, dried (Na2SO4), and concentrated in vacuo. The
crude material was purified by flash chromatography (1:1
EtOAc/hexanes and then EtOAc) to give a yellow solid which
was recrystallized (EtOAc) to afford 25 (363 mg, 76%) as a
1
white solid, mp 216-218 °C. H NMR (CDCl3) δ 8.94 (s, 1H),
7.29 (s, 1H), 6.56 (s, 1H), 4.18 (b, 2H) 3.91 (s, 2H), 2.80 (b,
2H), 2.5-2.41 (m, 1H), 1.88 (d, J ) 15 Hz, 2H), 1.75-1.65 (m,
2H), 1.45 (s, 9H), 1.23 (s, 9H). LCMS: 481 [M + H]+. Anal.
(C22H32N4O4S2) C, H, N, S.

1728 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Misra et al.
(5) (a) Zhai, S.; Senderowicz, A. M.; Sausville, E. A.; Figg, W. D.
Favopiridol, a novel cyclin-dependent kinase inhibitor, in clinical
development. Ann. Pharmacother. 2002, 36, 905-911. (b) Stadler
W. M.; Vogelzang, N. J .; Amato, R.; Sosman, J .; Taber, D.;
Liebowitz, D.; Vokes, E. E. Flavopiridol, a novel cyclin-dependent
kinase inhibitor, in metastatic renal cancer: a University of
Chicago phase II consortium study. J . Clin. Oncol. 2000, 18,
371-375. (c) Senderowicz, A. M. Flavopiridol: the first cyclin-
dependent kinase inhibitor in human clinical trials. Invest. New
Drugs 1999, 17, 313-320. (d) Senderowicz, A. M.; Headlee, D.;
Stinson, S. F.; Lush, R. M.; Kalil, N.; Villalba, L.; Hill, K.;
Steinberg, S. M.; Figg, W. D.; Tompkins, A.; Arbuck, S. G.;
Sausville, E. A. Phase I trial of continuous infusion flavopiridol,
regression analysis and have a coefficient of variance (SD/
mean, n ) 6) of 16%.
72 h An tip r olifer a tion Assa y. In vitro cytotoxicity was
assessed in tissue culture cells by MTS {3-(4,5-dimethylthiazol-
2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfenyl)-2H-tetrazoli-
um, inner salt} assay.15 Depending on the cell line employed,
cells were plated at a density of 3000-6000 cells/well in a 96-
well plate. Drugs were added after 24 h and serial diluted.
The cells were incubated at 37 °C for 72 h at which time the
tetrazolium dye MTS, in combination with phenazine metho-
sulfate, 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.
a
novel cyclin-dependent kinase inhibitor, in patients with
refractory neoplasms. J . Clin. Oncol. 1998, 16, 2986-2999. (e)
Sedlacek, H. H.; Czech, J .; Naik, R.; Kaur, G.; Worland, P.;
Losiewicz, M.; Parker, B.; Carlson, B.; Smith, A.; Senderowicz,
A.; Sausville, E. A. Flavopiridol (L868275; NSC 649890), a new
kinase inhibitor for tumor therapy. Int. J . Oncol. 1996, 9, 1143-
1168. (f) Carlson, B. A.; Dubay, M. M.; Sausville, E. A.; Brizuela,
L.; Worland, P. J . Flavopiridol induces g1 arrest with inhibition
of cyclin-dependent kinase (cdk) 2 and cdk4 in human breast
carcinoma cells. Cancer Res. 1996, 56, 2973-2978. (g) Kaur, G.;
Stetler-Stevenson, M.; Sebers, S.; Worland, P.; Sedlacek, H.;
Myers, C.; Czeck, J .; Naik, R.; Sausville, E. Growth inhibition
with reversible cell cycle arrest of carcinoma cells by flavone
L86-8275. J . Nat. Cancer Inst. 1992, 84, 1736-1740. (f) Kattige,
S. L.; Naik, R. G.; Lakadawalla, A. D.; et al. US Patent 4,900,-
727, February 13, 1990.
Ack n ow led gm en t. We thank Mark Salvati, Zhen-
Wei Cai, Toomas Mitt, David K. Williams, Kenneth
Leavitt, Donald Crews, William G. Humphreys, Robert
A. Kramer, David K. Bol, Deborah Roussell, Tai Wong,
Isia Bursuker, and Chieh Ying Chang for contributions
to this work. We also thank the Bristol-Myers Squibb
Analytical Research and Development Department for
high resolution MS analyses.
Su p p or tin g In for m a tion Ava ila ble: A summary of X-ray
crystallographic data for Figure 2. This material is available
via the Internet at http://pubs.acs.org.
(6) Kim, K. S.; Kimball, S. D.; Misra, R. N.; Rawlins, D. B.; Hunt,
J . T.; Xiao, H.-Y.; Lu, S.; Qian, L.; Han, W.-C.; Shan, W.; Mitt,
T.; Cai, Z.-W.; Poss, M. A.; Zhu, H.; Sack, J . S.; Tokarski, J . S.;
Chang, C.-Y.; Pavletich, N.; Kamath, A.; Humphreys, W. G.;
Marathe, P.; Bursuker, I.; Kellar, K. A.; Roongta, U.; Batorsky,
R.; Mulheron, J . G.; Bol, D.; Fairchild, C. R.; Lee, F. Y.; Webster,
K. R. Discovery of aminothiazole inhibitors of cyclin-dependent
kinase 2. Synthesis, X-ray crystallographic analysis, and biologi-
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J M0305568