Biaryl purine derivatives as potent antiproliferative agents: Inhibitors of cyclin dependent kinases. Part I

Article abstract of DOI:10.1016/j.bmcl.2009.10.025

The introduction of an aryl ring onto the 4-position of the C-6 benzyl amino group of the Cdk inhibitor roscovitine (2), maintained the potent Cdk inhibition demonstrated by roscovitine (2) as well as greatly improving the antiproliferative activity. A series of C-6 biarylmethylamino derivatives was prepared addressing modifications on the C-6 biaryl rings, N-9 and C-2 positions to provide compounds that displayed potent cytotoxic activity against tumor cell lines. In particular, derivative 21h demonstrated a >750-fold improvement in the growth inhibition of HeLa cells compared to roscovitine (2).

Full text of DOI:10.1016/j.bmcl.2009.10.025

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6608–6612
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Biaryl purine derivatives as potent antiproliferative agents: Inhibitors
of cyclin dependent kinases. Part I
a
a
a
a
Michael P. Trova a, Keith D. Barnes a, , Curt Barford , Travis Benanti , Mark Bielaska , Lori Burry ,
John M. Lehman b, Christine Murphy a, Harold O’Grady a, Denise Peace b, Susan Salamone b,
Jennifer Smith a, Patricia Snider a, Joseph Toporowski a, Steven Tregay a, Alison Wilson a,
Michael Wyle a, Xiaozhang Zheng a, Thomas D. Friedrich b
*
a AMRI, Medicinal Chemistry Department, 26 Corporate Circle, PO Box 15098, Albany, NY 12212-5098, USA
b Albany Medical College, Center for Immunology and Microbial Disease, 47 New Scotland Avenue, Albany, NY 12208-3479, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The introduction of an aryl ring onto the 4-position of the C-6 benzyl amino group of the Cdk inhibitor
roscovitine (2), maintained the potent Cdk inhibition demonstrated by roscovitine (2) as well as greatly
improving the antiproliferative activity. A series of C-6 biarylmethylamino derivatives was prepared
addressing modifications on the C-6 biaryl rings, N-9 and C-2 positions to provide compounds that dis-
played potent cytotoxic activity against tumor cell lines. In particular, derivative 21h demonstrated a
>750-fold improvement in the growth inhibition of HeLa cells compared to roscovitine (2).
Ó 2009 Elsevier Ltd. All rights reserved.
Received 21 August 2009
Revised 2 October 2009
Accepted 5 October 2009
Available online 12 October 2009
Keywords:
Purines
Cdk Inhibitor
Antiproliferative agent
The cyclin dependent kinases (Cdks) are a family of serine–thre-
onine protein kinases which play key roles in eukaryotic cell divi-
sion, a process regulated by a series of events defined as the cell
cycle.1–5 The activity of the kinases as their name implies is depen-
dent on association with cyclin partners. The Cdks drive the cell
cycle through the individual phases of the process with different
Cdk/cyclin pairs active during each phase of the cell cycle. Cell
cycle dysfunction is a characteristic feature of tumor cells, with
Cdk-related events among the most common genetic changes
found in human tumors.6,7 The observations of the connection be-
tween Cdks and the molecular pathology of cancer suggest the
Cdks as potential therapeutic targets for the treatment of prolifer-
ative diseases.
A variety of chemical classes have been identified as Cdk inhibi-
tors. Examples of these include staurosporine derivatives,8 flavo-
noids,9,10 oxindoles,11,12 indolinones,13 pyrrolopyrazines,14 pyrido-
pyrimidine,15 quinazolines,16 indenopyrazole,17 and purines.18–22
The 2,6,9-substituted purines form a class of inhibitors which in-
clude olomoucine (1)23 and roscovitine (2).24 Olomoucine (1), which
contains a benzylamino group in the 6-position, was one of the first
purine Cdk inhibitors discovered which demonstrated modest Cdk
inhibitory activity and selectivity with respect to a number of ki-
nases. Optimization of this structuregave rise to a second generation
purine, roscovitine (2), which had increased steric bulk at the 2- and
9-positions. Roscovitine (2) demonstrated an increase in potency for
a number of Cdks while also maintaining selectivity toward other
kinases.
Using 2,6,9-trisubstituted purines as a starting scaffold, we25–28
found that the installation of a C-6 biarylmethylamino group onto
the purine nucleus resulting in compound 3a, gave Cdk inhibition
similar to roscovitine, but afforded much greater antiproliferative
activity as evidenced by a 50-fold improvement in the inhibition
of the cell growth (GI50) of HeLa cells (see Fig. 1). Based on these
findings, a systematic structure–activity relationship study investi-
gating purines containing biarylmethylamino groups at C-6 was
undertaken. The focus of this study was directed toward examining
the effects of substitution at C-2, N-9 as well as the C-6 aminom-
ethylene linker and C-6 biphenyl substitution. A full accounting
of these results is presented herein. Subsequently, others29 have
also noted that several C-6 biarylmethylamino roscovitine ana-
logues demonstrated moderate antiproliferative properties.
The synthesis of a series of C-2 modified analogues is shown in
Scheme 1. Commercially available 2,6-dichloropurine (4) was trea-
ted with 4-phenyl benzyl amine to afford 5 followed by alkylation
of the N-9 with 2-iodopropane. Nucleophilic displacement of the
C-2 chloro with (R)-(À)-2-amino-1-butanol afforded 3a. Analogues
3b–o, maintaining the C-6 p-biphenylmethylamino and N-9 iso-
propyl functionalities of 3a were readily prepared by displacement
of the C-2 chloro of 6 with a variety of amines.
* Corresponding author.
E-mail address: keith.barnes@amriglobal.com (K.D. Barnes).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.025

M. P. Trova et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6608–6612
6609
NH
NH
NH
N
N
N
N
N
N
N
N
N
HN
N
HN
N
HN
N
OH
OH
OH
3a
Olomoucine (1)
Roscovitine (2)
µ
µ µ
IC50 = 4.6 M (Cdk2/cyclin A) IC50 = 2.0 M (Cdk2/cyclin A)
IC50 = 0.52 M (Cdk2/cyclin E) IC50 = 0.60 M (Cdk2/cyclin E)
IC50 = 10 M (Cdk2/cyclin A)
µ
µ
µ
IC50 = 10 M (Cdk2/cyclin E)
µ
µ
µ
GI50 = 65 M (HeLa cells)
GI50 = 20 M (HeLa cells)
GI50 = 0.40 M (HeLa cells)
Figure 1.
Cl
N
NH
N
NH
NH
N
b
c
N
N
N
a
N
N
N
N
N
N
H
Cl
N
H
X
Cl
N
N
Cl
N
6
4
5
3a–o
a:
X = ( )-NHCH(CH CH )CH OH
R
m:
n:
HN
OH
X =
2
3
2
i:
N
N
C(O)NH2
CH2NH2
X =
X =
b: X = NHCH2CH2NH2
f
c: X = NHCH2CH2CH2NH2
X = HN
j:
d: X = NHCH2CH2CH2CH2NH2
e: X = ( )-NHCH CH(NHBoc)CH
S
f: X = (S)-NHCH22CH(NH2)CH3
3
d
NH2
NH2
HN
NH2
k:
l:
X =
o:
N
X =
g:
X =
NCbz
HNCH2
e
HN
H2N
X =
h:
X =
NH
HNCH2
Scheme 1. Reagents and conditions: (a) 4-phenyl benzyl amine (1.01 equiv), EtN(i-Pr)2 (1.95 equiv), water, reflux, 5 h; (b) i-PrI (4.0 equiv), K2CO3 (8.0 equiv), DMSO, rt,
overnight; (c) amine (5–20 equiv), EtOH, 150–190 °C (sealed tube), 2–60 h; (d) HCl, Et2O, CH2Cl2, MeOH, 16 h, rt; (e) Pd/C, NH4HCO2, MeOH, reflux, 4 h; (f) BH3, THF, reflux,
2 h.
Alternatively the C-2 amine derivatives 3p and 3q, maintaining
the C-6 p-biphenylmethylamino and N-9 isopropyl functionalities
can be prepared has shown in Scheme 2. The C-6 chloro was dis-
placed with 4-bromo benzyl amine to afford 7, which was con-
verted to the N-9 isopropyl intermediate 8, followed by reaction
with the appropriate amines to yield 9a and 9b. Suzuki reaction
of 9a or 9b with phenyl boronic acid gave the corresponding biphe-
nyl derivatives 3p and 3q, respectively.
group of 3k was derivatized. These modifications were directed to-
ward modulation of the solubility and permeability of the mole-
cules, adjustment of the basicity of this group and the
introduction of steric bulk. Reductive amination of a series of alde-
hydes, provided mixtures of the mono-alkyl and bis-alkyl deriva-
tives which were separated by chromatography and converted to
their respective HCl salts 10a–h. Acylations of 3k afforded 11a–c
and sulfonylations gave 11d and 11e.
Compound 3k demonstrated outstanding antiproliferative
Compounds in which the C-6 p-biphenylmethylamino and C-2
trans-4-aminocyclohexylamine were maintained and the N-9 alkyl
group varied were prepared as shown in Scheme 4. Alkylation of 5
activity with a GI50 = 0.077
lM, therefore a series of analogues
was prepared as shown in Scheme 3, wherein the distal amino
Cl
N
NH
NH
N
NH
N
9a:
9b:
X = HN
H2N
HN
N
b
c
N
N
a
N
N
Br
N
N
N
Br
d
N
Br
N
N
H
Cl
N
H
Cl
N
X
Cl
4
8
7
X =
OH
3p:
3q:
X = HN
NH
N
N
N
H2N
HN
X
N
X =
OH
Scheme 2. Reagents and conditions: (a) 4-bromo benzyl amine (2.15 equiv), EtN(i-Pr)2 (4.0 equiv), 1:1 water/EtOH, reflux, overnight; (b) i-PrI (3.0 equiv), K2CO3 (5.0 equiv),
DMSO, rt, overnight; (c) amine (7–15 equiv), 150–190 °C, 25–60 h; (d) PhB(OH)2 (3.6 equiv), Pd(PPh3)4 (0.27 equiv), Na2CO3, DME, water, reflux, 18 h.

6610
M. P. Trova et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6608–6612
X
N
NH
N
NH
N
Cl
NH
N
N
N
N
N
N
N
N
b
N
a, b
N
a
Br
N
H
HN
HN
N
Cl
Br
N
N
H
Cl
4
14a,b
•HCl
X
NH2
NH
N
R1
R
3k
X
N
10a–h
NH
N
N
N
Br
N
d
1
c
N
N
10a
10b
: R = H, R = CH2CH3
1
c
HN
: R = H, R = (CH2)2CH3
Cl
10c: R = H, R1 = (CH2)3CH3
10d: R = H, R1 = CH - -C H
2 c
3
5
1
15a,b
10e:
R = R = CH2CH3
16a,b
10f: R1 = R = (CH2)2CH3
NH
N
NH2
X
10g: R1 = R = (CH2)3CH3
N
N
NH
N
X
1
NH
10h:
R = R = CH - C H
c-
2
3
5
N
N
Br
N
HN
HN
e, f
N
N
11a:
11b:
R = COCH3
R = COCH2CH3
N
N
HN
HN
N
11c: R = COCH - -C H
c
2
3
5
11d: R = SO2CH3
11e: R = SO2CF3
11a–e
R
•HCl
17a,b
NHBoc
Scheme 3. Reagents and conditions: (a) the appropriate aldehyde (0.9–1.1 equiv),
Na(OAc)3BH (1.25–1.55 equiv), 1,2-dichloroethane, rt, 1.5–3 h; (b) 2 N HCl, EtOAc,
rt; (c) the appropriate acyl chloride or sulfonyl chloride (1.1–3.0 equiv), pyridine or
EtN(i-Pr)2, CH2Cl2, rt, 1–18 h.
NH2 18a,b
a:
b:
X = CH2CH2
X = CH(CH3)
Scheme 5. Reagents and conditions: (a) the appropriate amine (1.1 equiv), EtN(i-
Pr)2 (2 equiv), water, EtOH, reflux; (b) i-PrI (4.0 equiv), K2CO3 (8.0 equiv), DMSO, rt,
overnight; (c) trans-1,4-diaminocyclohexane, EtOH, 140–160 °C, 48–60 h; (d)
Boc2O, NaHCO3, THF, water; (e) PhB(OH)2 (3.6 equiv), Pd(PPh3)4 (0.27 equiv),
Na2CO3, DME, water; (f) HCl, MeOH or 1:1 TFA/water.
NH
NH
N
a
N
N
N
N
N
H
Cl
N
Cl
N
R
5
12a–f
b
NH
NH
N
NH
N
N
N
N
N
N
N
Y
N
a
: R = CH3
a
b:
R = CH2CH3
c: R = (CH2)2CH3
N
HN
N
HN
HN
N
X
R
d: R = CH2CH=CH2
e: R = -C H
c
f: R = CH(CH93)CH2CH3
5
19:
20: Y = I
Y = Br
NH2 21a–k
NH2
NH2
13a–f
a:
b:
c:
d:
e:
f:
g:
h:
i:
X = 2-Cl
X = 3-Cl
X = 4-F
X = 3-OCH3
X = 3-CO2H
X = 3,5-di-CH3
X = 3,4-di-CH3
Scheme 4. Reagents and conditions: (a) the appropriate alkyl halide (4.0 equiv),
K2CO3 (8.0 equiv), DMSO, rt, 18–48 h; (b) trans-1,4-diaminocyclohexane (10 equiv),
EtOH, 140–160 °C (sealed tube), 24–72 h.
4-
X = Cl
j:
X = 3-CH3
X = 4-CH3
X = 3-F
k:
with the corresponding alkyl halides, gave compounds 12a–f
which upon reaction with trans-1,4-diaminocyclohexane gave
13a–f, respectively.
Scheme 6. Reagents and conditions: (a) the appropriate substituted phenyl boronic
acid (3.6 equiv), Pd2(dba)3 (0.04 equiv), PPh3 (0.6 equiv), Na2CO3, DME, water,
reflux, 12–20 h.
C-6 Aminomethylene modified analogues were prepared as
shown in Scheme 5. Reaction of 4 with 2-(4-bromophenyl)ethan-
amine and 1-(4-bromophenyl)ethanamine afforded 14a and 14b,
respectively. N-Alkylation with isopropyl iodide and displacement
of the C-2 chloro with trans-1,4-diaminocyclohexane gave 16a,b
which were converted to the respective biphenyl derivatives
17a,b by Suzuki coupling with phenyl boronic acid. Treatment with
HCl gave the salts 18a,b.
Analogues investigating substitution on the distal phenyl ring of
3k were prepared as shown in Scheme 6, introducing the various
substitutions by Suzuki reactions of 19 or 20 with substituted phe-
nyl boronic acids.
The data in Table 1 summarizes the in vitro Cdk2/cyclinA and
Cdk2/cyclinE inhibitory concentrations (IC50) and the growth inhibi-
tion (GI50) of HeLa cells for the compounds prepared. The series of
compounds, 3a–q, in which a variety of acyclic and cyclic amines
were introduced at the C-2 position while maintaining the C-6
biphenylmethylamino and N-9 isopropyl groups, showed that
compound 3k, in which the trans-4-aminocyclohexylamine group
was introduced at C-2 was the best compound in this series with re-
spect to both the Cdk inhibition and HeLa cell growth inhibitory
activity. Antiproliferative activity of 3k was fivefold greater than 3a.
Derivatization of the distal amino group of 3k afforded com-
pounds 10a–h and 11a–e. Mono-alkyl derivatives 10a–d demon-
strated good antiproliferative activities with bis-alkylation 10e–h
resulting in a decrease in activity. The introduction of an acetyl
group, compound 11a, maintained potent HeLa cell inhibitory
activity, which decreased with increasing size of the acyl group
11b and 11c. The methylsulfonyl analogue 11d showed moderate
activity, but the trifluoromethylsulfonyl compound 11e was much
less potent against the Cdks and HeLa cells.
Of the series of compounds 13a–f, in which the N-9 substituent
was varied, the N-ethyl derivative, 13b, demonstrated antiprolifer-
ative activity similar to the N-isopropyl compound 3k. Compounds

M. P. Trova et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6608–6612
6611
Table 1
In vitro inhibition of Cdk2 and effect of cell proliferation for compounds 3a–q, 10a–h, 11a–e, 13a–f, 18a, 18b and 21a–k
Compds
Cdk2/cyclinA32 IC50
,
lM
Cdk2/cyclinE32 IC50
,
l
M
HeLa33 GI50
,
l
M
Compds
Cdk2/cyclinA IC50
,
lM
Cdk2/cyclinE IC50
,
l
M
HeLa GI50 lM
3a
3b
3c
3d
3e
3f
3h
3i
3j
3k
3l
3m
3n
3o
2
0.6
3
3
1
0.6
0.5
0.4
3
0.8
0.9
2.5
>5
1
0.1
0.4
0.4
0.9
3
0.9
0.6
1
0.4
0.3
0.3
3
NT
NT
NT
0.40
0.20
0.43
2.50
0.20
0.35
2.5
11a
11b
11c
11d
11e
13a
13b
13c
13d
13e
13f
18a
18b
21a
21b
21c
21d
21e
21f
0.5
0.5
0.6
0.5
>5
1
NT
NT
NT
0.1
NT
1
0.3
0.2
0.3
0.2
>5
0.8
NT
NT
NT
0.09
NT
0.8
4
0.6
0.4
1
0.2
0.2
0.6
0.4
0.15
1
0.097
0.30
0.30
0.20
3.0
0.12
0.048
0.47
0.43
0.40
1.0
2
4
>5
4
0.4
0.9
0.4
1
3
2
2
3
4.0
0.47
0.077
0.20
0.35
1.5
5.0
3.5
5
1
0.5
0.40
0.084
10.5
0.054
2.0
0.15
1.0
0.026
10
3p
3q
1.07
0.43
1.1
0.097
0.40
0.30
3.50
1.26
3.0
0.4
0.8
0.3
0.4
1
0.5
0.35
6
10a
10b
10c
10d
10e
10f
10g
10h
0.5
1
0.9
6
21g
21h
21i
21j
21k
NTa
NT
NT
0.4
0.5
0.2
0.25
0.19
2.5
3.0
a
NT = not tested.
References and notes
18a–b wherein the C-6 aminomethylene linkage of the purine to
the diphenyl group is modified, resulted in a significant loss of
HeLa cell growth inhibitory activity.
Compounds 21a–k investigating substitution on the distal phe-
nyl ring demonstrated the best antiproliferative activities occurred
with the 3-substituted derivatives. Within the series of 2-Cl, 3-Cl,
and 4-Cl analogues, compounds 21a–c, respectively, the 3-Cl was
fivefold more potent than the 2-Cl and >100-fold more potent than
the 4-Cl analogue. The 3-CH3 21d and 3-OCH3 21h analogues dem-
onstrated excellent antiproliferative activities.
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Acknowledgments
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The authors thank the NCI and PanLabs for conducting the ki-
nase panel screens.

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M. P. Trova et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6608–6612
PRK2 and AMPK. We thank the NCI for the results of these
26. Trova, M. WO2003022805, 2003.
27. Trova, M. WO2003022219, 2003.
experiments.
28. Trova, M. P.; Friedrich, T. D.; Alicea, L. R.; Barford, C. A.; Barnes, K. D.;
Benanti, T.; Bergeron, M. E.; Bielaska, M.; Bilotta, J. A.; Burry, L. C.; Davisdon,
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Regional Meeting of the American Chemical Society, Saratoga Springs, NY,
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32. The following are the conditions used for the Cdk2/cyclinA and Cdk2/cyclinE
assays. Recombinant Cdk2/CyclinA (15 ng) and Cdk2/CyclinE (5 ng) (Upstate
Biotechnology) assays were carried out in 50 mM Tris–HCl pH 7.4, 10 mM
MgCl2, 1 mM DTT, 0.1 mg/ml histone H1, 0.016 mM ATP, 1 lCi [c
32P]ATP. A
concentration range of each inhibitor dissolved in DMSO was added to the Cdk/
Cyclin complexes in assay buffer in the absence of ATP. DMSO was kept
constant at 0.04% in all reactions. The reaction was initiated by the addition of
ATP and incubated at 30 °C for 30 min. The reactions were terminated by the
addition of 2Â SDS sample buffer and resolved by SDS–PAGE. H1
phosphorylation was quantified by phosphoimaging. IC50 values were
calculated using GraphPad Prism data analysis software.
29. Oumata, N.; Bettayeb, K.; Ferandin, Y.; Demange, L.; Lopez-Giral, A.; Goddard,
M.; Myrianthopoulos, V.; Mikros, E.; Flajolet, M.; Greengard, P.; Meijer, L.;
Galons, H. J. Med. Chem. 2008, 51, 5229.
30. The Panlabs kinase panel consisted of PKC alpha, Ca/Calmodulin dependent
PKII, EGF Receptor, ERK1, lck and PKA.
31. The NCI kinase panel consisted of c-RAF, MEK-1, MAPK2, MKK6, SAPK2b,
33. Growth inhibition (GI50) values were measured with HeLa S-3 cells selected for
growth on plastic. The procedure was based on the sulforhodamine B staining
protocol of Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.;
Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst.
1990, 82, 1107.
SAPK4, MAPKAP-K2, MKK4, JNK
a1, JNKa2, SGK, GSK3b, ROCKII, CK2, LCK,
p70S6 K, CHK1, PKB cSRC, ZAP-70, JNK3, CK1, PKC-d, MAPK1, CHK-2,
a,