The design and synthesis of purine inhibitors of CDK2. III

Article abstract of DOI:10.1081/NCN-100002493

Cyclin-dependent kinases (CDKs) belong to a class of enzymes that control the ability of a cell to enter into and proceed through the cell division cycle. Using purine as a scaffold, we have synthesized a number of nanomolar inhibitors of CDK-2/cyclin E. In this report, the synthesis of a series of piperidine-substituted purine analogs will be presented, as well as some of their in vitro and in vivo biological effects.

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THE DESIGN AND SYNTHESIS OF PURINE INHIBITORS
OF CDK2. III
P. W. Shum a , N. P. Peet a , P. M. Weintraub a , T. B. Le a , Z. Zhao a , F. Barbone a , B.
Cashman a , J. Tsay a , S. Dwyer a , P. C. Loos a , E. A. Powers a , K. Kropp a , P. S. Wright
a , A. Bitonti a , J. Dumont a & D. R. Borcherding a
a Aventis Pharmaceuticals Inc. , Rt. 202-206, Bridgewater, New Jersey, 08807, U.S.A.
Published online: 07 Feb 2007.
To cite this article: P. W. Shum , N. P. Peet , P. M. Weintraub , T. B. Le , Z. Zhao , F. Barbone , B. Cashman , J. Tsay , S.
Dwyer , P. C. Loos , E. A. Powers , K. Kropp , P. S. Wright , A. Bitonti , J. Dumont & D. R. Borcherding (2001) THE DESIGN
AND SYNTHESIS OF PURINE INHIBITORS OF CDK2. III, Nucleosides, Nucleotides and Nucleic Acids, 20:4-7, 1067-1078, DOI:
10.1081/NCN-100002493
To link to this article: http://dx.doi.org/10.1081/NCN-100002493
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(4–7), 1067–1078 (2001)
THE DESIGN AND SYNTHESIS OF PURINE
INHIBITORS OF CDK2. III
P. W. Shum,∗ N. P. Peet, P. M. Weintraub, T. B. Le,
Z. Zhao, F. Barbone, B. Cashman, J. Tsay, S. Dwyer,
P. C. Loos, E. A. Powers, K. Kropp, P. S. Wright, A. Bitonti,
J. Dumont, and D. R. Borcherding
Aventis Pharmaceuticals Inc., Rt. 202-206, Bridgewater, New Jersey 08807
ABSTRACT
Cyclin-dependent kinases (CDKs) belong to a class of enzymes that control the
ability of a cell to enter into and proceed through the cell division cycle. Using
purine as a scaffold, we have synthesized a number of nanomolar inhibitors of
CDK-2/cyclin E. In this report, the synthesis of a series of piperidine-substituted
purine analogs will be presented, as well as some of their in vitro and in vivo
biological effects.
Cyclin-dependent kinases (CDKs) belong to an important class of enzymes
that are responsible for entry into and regulation of the cell division cycle (1,2).
CDKs are activated by interaction with members of the cyclin family through the
formation of heterodimeric complexes (1,2). Normal cell growth is regulated by
changes in the balance between activators and endogenous inhibitors of CDKs.
In cancer, the cells have lost this balance. Tumor cells exhibit mutations, aberrant
expression levels, or altered activities of one or more of the regulatory proteins for
the cell cycle checkpoints (within the G1, S, and G2/M phases). These regulatory
components include the CDK inhibitors (such as p16, p21, and p27), the cyclin-
CDK pairs (such as CDK-4/cyclin D1, CDK-2/cyclin E, and CDK-1/cyclin B), and
tumor suppressors (such as p53 or pRb). Inhibitors of CDKs complexes like CDK-
2/cyclin E, involved in the transition from G1 to S phase, may have therapeutic
∗Corresponding author.
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Copyright 2001 by Marcel Dekker, Inc.
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potential against tumor cell proliferation. Recent in vitro studies by Chen et al.
(3) have suggested that selective killing of transformed cells over non-transformed
cells may be accomplished with CDK2/cyclin E antagonists. Small molecule CDK
inhibitors, including the purine analog olomoucine have antimitotic activity and
been shown to inhibit cancer cell proliferation (4,5).
MDL 106,327DA was identified through database mining using olomoucine
astheleadstructureandfoundtoinhibitCDK-2/cyclinEwithanIC50 valueof50nM
(Fig. 1). This lead was then systematically modified to find the optimum inhibitory
activity for CDK-2/cyclin E (6–8). One of the unique features of MDL 106,327DA
is the trans-1,4-diaminocyclohexyl group in the 2-position of its purine ring. In
an effort to optimize the affinity of this lead structure by replacing the substituent
at the 2-position with a variety of other diamines, we found that the trans-1,4-
diaminocyclohexylgroupwastheonlysubstituenttogivelownanomolaraffinityfor
CDK-2/cyclin E (6). The 9-position generally accommodates relatively small alkyl
groups (7). When the cyclopentyl group of MDL 106,327DA was replaced with an
isopropylgroup, theIC50 valuewas180nM(7). Smallcycloalkylgroups, suchasthe
cyclopentyl group, gave the best affinity of all substituents at 11 nM against CDK-
2/cyclin E. The 6-position was very amenable to modification without significant
loss of activity, because this position is exposed to the solvent when bound to human
CDK-2 (6,8). Modifications of the imidazole in the purine ring also gave a loss of
activity (9). When the N7 position was replaced with a CH group or the C8 position
was replaced with a nitrogen atom, a reduction in activity was observed.
Figure 1. Adenine scaffold lead from database mining around olomoucine.

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Figure 2. MDL 108,522.
We have synthesized a number of very active inhibitors of CDK-2/cyclin E
using rational drug design and parallel synthesis approaches. Another inhibitor with
an extended substituent at C6 nitrogen MDL 108,522 (1) was shown to have good
CDK-2/cyclin E activity, in vitro and in vivo antitumor activity (Fig. 2). Structure
activity studies around this new structure using parallel synthesis was then initiated.
In this report we will describe the synthesis, SAR, CDK-2/cyclin E activity, in vitro
and in vivo antitumor activity for a number of compounds related to MDL 108,522.
Chemistry
MDL 108,522 and its analogs (8–46) were prepared by parallel synthesis.
However, very few 1-substituted-4-aminopiperidines are commercially available.
The 1-substituted-4-aminopiperidines were prepared according to Scheme 1 by
alkylating piperidine-4-carboxyamide 2 using Cs2CO3 and potassium iodide in 3-
pentanone to give 3 in yields ranging from 17 to 74%. The Hoffmann rearrangement
(10) of compound 3 was accomplished by using [bis(trifluoroacetoxy)iodo]benzene
in acetonitrile/water to afford 4 in 64% to quantitative yields. The final compounds,
shown in Scheme 2, were prepared using parallel synthesis techniques. Compound
6 was prepared from 5 as previously described (6) and treated with the appro-
priately substituted piperidine 4 in EtOH at reflux to give >90% yields of the
first intermediate (7) for 8–46. The EtOH was removed by evaporation, trans-
1,4-diaminocyclohexane was added neat and the mixture was heated to 150◦C for
24 hours to yield 8–46 in 50 to 85% yields. The final compounds (8–46) were
purified using 2 gram silica gel solid phase extractors and purities (>85%) were
established by LC/MS.

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Scheme 1. Synthesis of 4-Amino-1-benzylpiperidines.
Biology
MDL 108,522 (1) was previously described as an inhibitor of CDK-2 and
tumor cell proliferation (6). In an attempt to determine the SAR and the scope of
the biological activity of MDL 108,522, a series of analogs were prepared. The
compounds (1 and 8–46) were tested against human CDK-2 and CDK-4 obtained
from SF9 cell lysates and IC50 values were measured (Table 1) (6). It was deter-
mined that substituted benzyl groups on the piperidine ring had minimal effects on
biological activity with the IC50 values ranging from 0.071 to 2.1 µM. Therefore,
these compounds only afforded a difference in activity of 30 fold. Compound 8
was the most potent against CDK-2, having an IC50 value of 0.071 µM, while
compound 42 was the most selective, having a CDK-4/CDK-2 ratio of 89. Most
compounds had an IC50 below 0.5 µM for CDK-2. This is not totally unexpected
since known x-ray structures for this type of compound indicates that the piperidine
group would be exposed to solvent when bound to CDK-2. Thus, substitution on
the piperidine ring nitrogen may not have a large effect on the binding affinity of
these compounds for CDK-2 (8).
Scheme 2. Synthesis of MDL 108,522 and its analogs (via parallel synthesis).

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Analysis of the data showed that 3,5-bis(trifluoromethyl) (42), 4-trifluoro-
methyl (38), 4-chloro (23) and 3,5-dichloro (11) substitution on the benzyl group
gave the best CDK-4/CDK-2 selectivity ratios of 89, 42, 34 and 25, respectively.
This indicates that substitution on the piperidine might effect CDK-4 binding more
significantly, since CDK-4 binding affinities were more directly effected. Other
3,5-disubstituted compounds (32) and (36) which have difluoro or dimethoxy
groups gave selectivity with CDK-4/CDK-2 ratios of 9 and 11, respectively. Com-
pounds with electron withdrawing groups (25 and 43) in the 4-position which
have fluoro and trifluoromethoxy groups gave selective compounds with CDK-
4/CDK-2 ratios of 11 and 8, respectively. Compounds 13 and 20, which have
methoxy and methyl electron donating groups in the 4-position of the benzyl
group, were determined to be almost equally potent against CDK-2 and CDK-4,
with IC50 values below 0.5 µM. This small set of compounds would indicate
that electron donating groups in the 4-position provide compounds with greater
CDK-4 binding affinities. The 2,4-difluorobenzyl substituted compound 12 also
showed little selectivity, but had IC50 values for both CDK-2 and CDK-4 below
0.5 µM.
A SAR pattern emerged for the trifluoromethyl substituted series of com-
pounds, where the 3-trifluoromethylbenzyl substituted compound 8 gave most po-
tent IC50 value of 0.071 µM for CDK-2. Trifluoromethyl substitution at the 4- or
3,5-positions (38 and 42) gave decreased affinity for CDK-2 with IC50 values of
0.48 and 0.72 µM, respectively. A larger decrease in IC50 values for the CDK-4
activity was also observed for these compounds. When a trifluoromethyl group was
placed in either the 2- or 3,4-position of the benzyl group as in compounds 45 and
46, there resulted the least active compounds in this study having IC50 values of 1.1
and 2.1 µM, respectively. Compounds that had chloro or fluoro group substitution
in general gave the best affinity for CDK-2, regardless of the substitution patterns.
Monochloro- and dichloro-substitution on the benzyl group provided compounds
with IC50 values of 0.34 µM or better, with the exception of compound 44 (CDK-2
IC50 = 1.0 µM), which had the 2,6-dichloro substitution pattern. Monofluoro- and
difluoro-substituted benzyl groups all gave IC50 values for CDK-2 of less than
0.5 µM. When the phenyl group was replaced by napthyl as in compounds 31 and
35, IC50 values for CDK-2 were 0.26 and 0.43 µM, indicating large groups were
well-tolerated. Also, when the phenyl group was replaced by a cyclohexyl group
(9) or a cyclopropyl group (17) good affinity for CDK-2 was maintained with IC50
values of 0.11 and 0.19 µM, respectively.
The compounds were then tested against seven tumor cell lines for antiprolif-
erative effects. All compounds demonstrated potent activity against breast, colon,
lung and prostrate tumor cells lines. The in vitro antiproliferation IC50 values gen-
erally correlated with the IC50 values generated for CDK-2 rather than CDK-4.
The most selective compounds (11, 23, 38 and 42) showed submicromolar activity
against the tumor cells, which correlated well with the CDK-2 IC50 values. These
data would indicate that CDK-2, in this study, was responsible for the antiprolifer-
ative effects of these compounds since compounds like 38 and 42, which have IC50
values for CDK-4 of 20 and 64 µM, respectively, still showed significant activity.

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Figure 3.
It cannot be ruled out that CDK-1 or another kinase is responsible for part or all
of the activity observed. However, CDK-4 activity does not appear to be a critical
component for the activity of these compounds. It should pointed out that the least
selective compounds (12, 13 and 20) also showed good activity against these tumor
cells as well. However, the compounds displayed IC50 values below 0.33 µM for
both CDK-2 and CDK-4.
MDL 108,522 (1) was selected for in vivo testing in a xenograft tumor model
in nude mice (6). Shown in Figure 3 are the in vivo effects of MDL 108,552 for
the inhibition of PC-3 prostate tumor growth in nude mice. When dosed orally at
1 mg/kg, MDL 108,522 showed minimal activity; however, at 3 mg/kg the com-
pound showed significant activity against PC-3 tumor cell growth.
In thisreport, wehaveshownthatinhibitionofCDK-2withsubstitutedpurines
can be correlated with the antiproliferative activity effects in the seven different
tumor cell lines. These effects can translate into good in vivo activity, indicating
that CDK-2 is a good target for the development of therapies where it is desirable
to control unregulated cell growth such as that seen in cancers.
EXPERIMENTAL
General Procedure for the Preparation of 4-amino-1-alkylpiperidine
4-Amino-1-(2,4-dichlorobenzyl)piperidine
To a mixture of isonipecotamide (5.0 g, 39 mmol), cesium carbonate (8.0 g,
24 mmol), and potassium iodide (2 spatula tips, cat.) in 3-pentanone (25 mL) was
added 2,4-dichlorobenzyl chloride (6.5 mL, 47 mmol). The mixture was heated

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at 100◦C (oil bath) for 5 h. The mixture was filtered hot, and the filter cake was
washed thoroughly with hot acetone. The filtrate was concentrated, and the re-
sulting slurry was recrystallized from acetone to give 8.2 g (74%) of 4-[1-(2,4-
dichlorobenzyl)piperidine]-carboxamide.
A solution of bis(trifluoroacetoxy)iodobenzene (3.6 g, 8.4 mmol) in acetoni-
trile (20 mL) and water (15 mL) was added to 4-[1-(2,4-dichlorobenzyl)piperidine]
carboxamide (2.0 g, 7.0 mmol). This mixture was heated at 65◦C (oil bath)
overnight. Water (60 mL) was then added, and the mixture was cooled in an ice
bath. Concentrated HCl (ca. 10 mL) was added, and the mixture was washed twice
with diethyl ether. The aqueous layer was concentrated in vacuo, and the residue
was dissolved in water (ca. 40 mL). The solution was saturated with solid potassium
carbonate. This mixture was extracted with dichloromethane (2X). The combined
organic extracts were dried over anhydrous sodium sulfate, filtered, and concen-
trated in vacuo to yield 1.35 g (74%) the title compound as a yellow oil. Other
4-amino-1-alkylpiperidines were prepared via the same procedure.
General Procedure for the Parallel Synthesis
of Purine Scaffold Molecules
Toa4or20mLvialwasadded9-(cyclopentyl)-2,6-dichloropurine(0.20mmol),
TEA or DIEA (200 mg), reacting amine (0.2 mmol), ethanol or toluene (2 mL), and
a stir bar. The vial was capped and the mixture was stirred at reflux for overnight,
after which the solution was cooled to room temperature. The solution was con-
centrated, and trans-1,4-diaminocyclohexane (200 mg) was added. The vial was
capped and heated at 150◦C for 24 hours. After cooling the material was passed
through a prepacked 1 gram or 2 gram silica gel column (Fischer or Alltech) pre-
equilibrated with hexane, then the product was eluted with of CH2Cl2 (4 mL) then
with of CH2Cl2:MeOH 4:1 (15 mL). The fractions which appeared to have the
product based upon TLC analysis, were concentrated, and repurified by passing
through another prepacked silica gel column pre-equilibrated with hexane, this
time beginning with CH2Cl2, followed by CH2Cl2:MeOH 9:1. The concentrated
fractions with pure product were dissolved in EtOH (2 mL). Six drops of 1N HCl
were added, and the solutions were concentrated to give hydrochloride salts of the
desiredproducts. LC/MSwasusedtodeterminepurityandmass. TheRf valueswere
determined by an AQ 4 × 50 column (YMC) with a linear gradient from 100% C
to 100% D in four minutes with a two minute hold at 100% D, where C is 5:95
acetonitrile:water with 0.1% TFA, and D is 95:5 acetonitrile:water with 0.085%
TFA. Molecular ion determinations were made using a Finnigan MAT SSQ-710
mass spectrometer.
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