Heterobiaryl purine derivatives as potent antiproliferative agents: Inhibitors of cyclin dependent kinases. Part II

Article abstract of DOI:10.1016/j.bmcl.2009.10.011

C-6 Biarylmethylamino purine derivatives of roscovitine (1) inhibit cyclin dependent kinases and demonstrate potent antiproliferative activity. Replacement of the aryl rings of the C-6 biarylmethylamino group with heterobiaryl rings has provided compounds

Full text of DOI:10.1016/j.bmcl.2009.10.011

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6613–6617
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Heterobiaryl purine derivatives as potent antiproliferative agents: Inhibitors
of cyclin dependent kinases. Part II
a
a
a
a
Michael P. Trova a, Keith D. Barnes a, , Luis Alicea , Travis Benanti , Mark Bielaska , Joseph Bilotta ,
Brian Bliss a, Thuy Nguyen Duong a, Simon Haydar a, R. Jason Herr a, Yu Hui a, Matthew Johnson a,
John M. Lehman b, Denise Peace b, Matthew Rainka a, Patricia Snider a, Susan Salamone b, Steven Tregay 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:
C-6 Biarylmethylamino purine derivatives of roscovitine (1) inhibit cyclin dependent kinases and demon-
strate potent antiproliferative activity. Replacement of the aryl rings of the C-6 biarylmethylamino group
with heterobiaryl rings has provided compounds with significantly improved activity. In particular, deriv-
atives 18g and 9c demonstrated 1000-fold and 1250-fold improvements, respectively, in the growth inhi-
bition of HeLa cells compared to roscovitine (1).
Received 21 August 2009
Revised 1 October 2009
Accepted 5 October 2009
Available online 12 October 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Purines
Cdk inhibitor
Antiproliferative agent
In the search for potential drug targets to combat cancer, atten-
tion has focused on the cyclin dependent kinases (Cdks) which
drive and control the cell cycle progression.1–5 Uncontrolled cell
proliferation, the trademark of tumor cells is a result of cell cycle
dysfunction. Since Cdk-related events are among the most com-
mon genetic changes found in tumors,6,7 the targeting of the Cdks
may be a means of preventing cell proliferation.
modeling studies (unpublished results) have indicated that the aryl
rings of the C-6 substituent extend into a solvent exposed region of
the Cdk2 enzyme; hence we felt that this would be a suitable site
for the introduction of heteroatoms in order to enhance solubility
and bioavailability. The replacement of the proximal and distal aryl
rings with heteroaryl rings was investigated, as well as the simul-
taneous replacement of both rings with heteroaryl rings.
Numerous scaffolds have been identified as Cdk inhibitors8–17
with the purine18–22 ring system providing a number of potent
inhibitors. Many of these, such as olomoucine23 and roscovitine
(1)24 have also maintained selectivity toward other kinases.
We communicated previously25–28 and reported in detail in the
accompanying paper29 the discovery of the trans-4-aminocyclo-
hexylamino group as a potent C-2 substituent in the C-6 biarylmeth-
ylamino series, affording compounds such as 2 with significantly
improved potencies against Cdks and high antiproliferative activi-
ties, compared to roscovitine (1) (Fig. 1).
The improved in vitro activities realized by the introduction of a
biarylmethylamino group at C-6 suggested the potential for further
activity improvement by variation of this position. Using com-
pound 2 as a starting scaffold, the replacement of the C-6 biaryl
moieties with heterobiaryl moieties was investigated21,22 and fur-
ther details are presented in this Letter. Preliminary molecular
Starting from 2,6-dichloropurine (3), all analogues were pre-
pared by two general routes. The heterobiarylmethylamine groups
at C-6 were in some instances introduced in the first step of the
synthesis by displacement of the C-6 chloro with the appropriate
NH
NH
N
N
N
N
N
N
N
HN
N
OH
HN
Roscovitine (1)
2
NH2
µ
µ
IC50 = 4.6 M (Cdk2/cyclin A) IC50 = 0.40 M (Cdk2/cyclin A)
µ
µ
IC50 = 0.52 M (Cdk2/cyclin E) IC50 = 0.10 M (Cdk2/cyclin E)
µ
µ
GI50 = 0.077 M (HeLa cells)
GI50 = 20 M (HeLa cells)
* Corresponding author.
E-mail address: keith.barnes@amriglobal.com (K.D. Barnes).
Figure 1.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.011

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M. P. Trova et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6613–6617
heterobiarylmethylamine, followed by alkylation of N-9 with iodo-
propane, nucleophilic displacement of the C-2 chloro with trans-
1,4-diaminocyclohexane, and then subsequent treatment with
HCl gave the corresponding salts (Schemes 1, 3 and 6). Alterna-
tively, analogues were prepared by construction of the C-6 hetero-
biarylmethylamine groups in a stepwise fashion (Schemes 2, 4 and
5). The C-6 chloro of 3 was initially displaced with 4-bromo- or 4-
iodobenzylamine or with a bromo substituted heteroarylmethyl-
amine, followed by introduction of the N-9 isopropyl and the C-2
trans-4-aminocyclohexylamine groups. Subsequent Suzuki cou-
plings of aryl/heteroaryl boronic acids with the halogen atoms on
the purine intermediates gave the targeted derivatives.
Analogues wherein the proximal ring is phenyl were prepared
as shown in Schemes 1 and 2. In Scheme 1 the C-6 chloro of 3
was displaced with 4-heteroarylbenzylamines then further elabo-
rated to 4a–d. Treatment of 4c and 4d with HCl gave the corre-
sponding salts 5c and 5d. Following the alternate route (Scheme
2), the C-6 chloro of 3 was displaced with either 4-iodobenzyl-
amine or 4-bromobenzylamine. Late stage Suzuki coupling with
thienylboronic acids gave 4e–i and coupling with 2-furanylboronic
acid gave 4j. Compounds 4f and 4g were converted to their respec-
tive HCl salts 5f and 5g.
Schemes 3 and 4 show the preparations of the analogues in
which the proximal C-6 ring is a heteroaryl ring, while the distal
C-6 ring is phenyl. Analogues with the proximal ring as pyridine
or thiazole were prepared as illustrated in Scheme 3 by initial dis-
placement of the C-6 chloro of 3 with the appropriate phenylpyr-
idylmethylamine or phenylthiazoylmethylamine. Analogues
wherein the proximal ring is thienyl or furanyl were prepared as
shown in Scheme 4. The 5-phenylthiophen-3-yl, 13, and 5-phenyl-
furan-3-yl, 14, analogues were prepared by displacement of the
C-6 chloro of 3 with (5-bromothiophen-3-yl)methylamine and
(5-bromofuran-3-yl)methylamine, respectively, then subsequent
reaction with phenylboronic acid. A series of 5-arylthiophen-2-yl
derivatives, 15a–e, were prepared by displacement of the C-6
chloro of 3 with (5-bromothiophen-2-yl)methylamine, then subse-
quent reaction with a series of substituted phenyl boronic acids.
The preparations of analogues wherein both the proximal and
distal C-6 rings are heteroaryl were prepared as illustrated in
Schemes 5 and 6. As shown in Scheme 5, a series of C-6 5-hetero-
arylthiophen-2-yl analogues, 16a–d, was prepared by reaction of
12 with a series of heteroaryl boronic acids. Alternatively as shown
Cl
NH
N
N
d
a, b, c
N
N
N
X
N
N
H
Cl
N
HN
3
6:
X = Br
NH2
HN
7: X = I
NH
N
NH
N
e
N
N
N
N
R
N
R
N
HN
•HCl
NH2
5f,g
NH2
4e—k
Cl
S
S
S
e:
f:
R =
R =
R =
h:
R =
CH3
CH3
S
i:
j:
R =
R =
S
O
g:
Scheme 2. Reagents and conditions: (a) 4-bromobenzylamine or 4-iodobenzyl-
amine (2.0 equiv), EtN(i-Pr)2 (2.0 equiv), water, reflux, 5–24 h; (b) i-PrI (3.0 equiv),
K2CO3 (5.0 equiv), DMSO, rt, overnight; (c) trans-1,4-diaminocyclohexane (10–
15 equiv), EtOH, 120–190° (sealed tube), 24–60 h; (d) The appropriate heteroaryl
boronic acid (3.0 equiv), Pd2(dba)3 (0.03 equiv), PPh3 (0.5 equiv) Na2CO3, water,
DME, reflux, 24–48 h; (e) HCl, MeOH, rt.
Cl
R
NH
N
N
a, b, c
N
N
N
N
N
H
Cl
N
HN
3
d
8a–e
NH2
R
NH
N
N
N
N
N
a:
b:
c:
R =
R =
R =
R =
Cl
NH
HN
N
a, b, c
N
N
N
N
R
N
N
H
Cl
R
N
HN
N
N
3
•HCl
NH2
9c,d,e
d
F
N
4a—d
d:
e:
NH2
S
NH
N
N
N
N
N
S
a:
b:
c:
R =
R =
R =
R =
R =
HN
N
N
N
N
Scheme 3. Reagents and conditions: (a) the appropriate amine RCH2NH2 (1.1–
2.15 equiv), EtN(i-Pr)2, water, EtOH, reflux, 24 h; (b) i-PrI (3.0 equiv), K2CO3
(5.0 equiv), DMSO, rt, overnight; (c) trans-1,4-diaminocyclohexane (5–30 equiv),
EtOH, 120–150 °C (sealed tube), 24–96 h; (d) HCl, MeOH, rt.
•HCl
NH2
5c,d
d:
N
in Scheme 6, a series 5-heteroarylthiophen-2-yl analogues 17a–c,
as well as a series of 6-heteroarylpyridyl-3-yl analogues, 17d–g,
was prepared by the initial displacement of the C-6-chloro of 3
with the appropriate heterobiaryl amines.
Scheme 1. Reagents and conditions: (a) the appropriate amine (2.15 equiv), 4:1
EtOH/water, reflux, 24 h; (b) i-PrI (3.0 equiv), K2CO3 (5.0 equiv), DMSO, rt, 24 h; (c)
trans-1,4-diaminocyclohexane (15–30 equiv), EtOH, 170–190 °C (sealed tube) 18–
24 h; (d) HCl, MeOH, rt.

M. P. Trova et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6613–6617
6615
S
O
Cl
N
R
NH
N
10:
R =
11:
R =
N
N
N
a, b, c
N
Br
Br
N
Br
S
N
H
Cl
HN
12:
R =
3
10—12
NH2
12
d, e
10
11
d, e
d, e
NH
S
NH
NH
N
S
N
N
X
N
O
N
N
N
a
: X = 2-F
N
N
b
: X = 3-F
HN
N
N
HN
N
HN
c:
d:
e:
X = 4-F
X = 3-CH3
X = 3-OCH3
•HCl
•HCl
NH2
•HCl
NH2
14
NH2
15a—e
13
Scheme 4. Reagents and conditions: (a) the appropriate amine RCH2NH2 (1.1–2.15 equiv), EtN(i-Pr)2, water, EtOH, reflux, 24 h; (b) i-PrI (3.0 equiv), K2CO3 (5.0 equiv), DMSO,
rt, overnight; (c) trans-1,4-diaminocyclohexane (5–30 equiv), EtOH, 120–170 °C (sealed tube), 24–48 h; (d) phenyl boronic acid or the appropriately substituted phenyl
boronic acid (3.6 equiv), Pd(PPh3)4 (0.3 equiv), Na2CO3, DME, water; (e) HCl, MeOH, rt.
The data in Table 1 summarizes the in vitro Cdk2/cyclinA and
Cdk2/cyclinE inhibitory concentrations (IC50) and the growth inhi-
bition (GI50) of HeLa cells for the compounds prepared.
(6-phenylpyridin-3-yl)methylamine derivative 8a demonstrated
antiproliferative activity comparable to 2, and improved Cdk2/cy-
clin inhibition. As we reported in the accompanying Letter,19 intro-
duction of a 3-substituent onto the distal phenyl ring of the C-6
biphenylmethylamino group afforded compounds with improved
antiproliferative activities compared to the unsubstituted ana-
logue. Similarly we found that introduction of a 3-F substituent
onto the distal phenyl ring of 8a to afford 9c, resulted in a 4.4-fold
improvement in antiproliferative activity over 8a, but no improve-
ment in Cdk2/cyclin inhibition.
Maintaining the proximal phenyl ring of the C-6 biphenylmeth-
ylamino moiety, a series of compounds (4a, 4b, 4f, 4h–j, and 5c–e,
5g) was prepared wherein a variety of heterocycles were intro-
duced in place of the distal phenyl ring of 2. A number of these
had very potent HeLa cell growth inhibitory activity with the 2-
pyridyl 4a, 2-thienyl 4f, 3-thienyl 4i, 2-furanyl 4j, and 1-pyrrolyl
5d derivatives demonstrating improved antiproliferative activities
compared to 2. Several substituted 2-thienyl analogues (4h, 5e,
and 5g) were prepared and had weaker HeLa cell growth inhibitory
activities compared to the parent 2-thienyl compound 4f. Although
the 1-pyrrolyl derivative 5d, demonstrated particularly strong
antiproliferative activity, replacement of the 1-pyrrolyl moiety
with a 1-pyrazolyl moiety as in 5c, resulted in an 8.5-fold decrease
in HeLa cell growth inhibitory activity relative to 5d, while simul-
taneously maintaining Cdk2/cyclin inhibitory activity.
Cl
R
NH
N
N
a, b, c
N
N
N
N
N
H
Cl
N
HN
3
d
Compounds 8a, 8b, 9d, 9e, 13, 14, and 15a–e examined the ef-
fects on activity when the distal phenyl ring of 2 is retained and the
proximal phenyl ring is replaced with a heteroaryl ring. Only the
17a—g
NH2
S
S
R
NH
N
N
N
a:
b:
R =
R =
S
N
N
HN
S
S
S
S
NH
N
NH
N
Br
R
N
N
N
N
N
N
•HCl
a,b
N
NH2
18a—d, g
HN
HN
c:
R =
•HCl
16a—d
N
N
NH2
NH2
S
12
f:
d:
e:
R =
R =
R =
R =
N
N
O
S
c:
R =
R =
a:
b:
R =
R =
S
g:
N
O
d:
N
N
N
Scheme 6. Reagents and conditions: (a) the appropriate amine RCH2NH2 (1.1–
2.2 equiv), EtN(i-Pr)2, water, EtOH, reflux, 16–24 h; (b) i-PrI (3.0 equiv), K2CO3
(5.0 equiv), DMSO, rt, overnight; (c) trans-1,4-diaminocyclohexane (10–36 equiv),
EtOH, 120–150 °C (sealed tube), 20–48 h; (d) HCl, MeOH, rt.
Scheme 5. Reagents and conditions: (a) the appropriate heteroaryl boronic acid
(3.6 equiv), Pd2(dba)3 (0.03 equiv), PPh3 (0.5 equiv) Na2CO3, water, DME, reflux, 24–
48 h, (b) HCl, MeOH, rt.

6616
M. P. Trova et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6613–6617
Table 1
noted for many of the compounds, and some had significantly im-
proved antiproliferative activities. In particular, derivatives 18g
and 9c demonstrated 1000-fold and 1250-fold improvements
respectively, in the growth inhibition of HeLa cells compared to
roscovitine (1).
In vitro Inhibition of Cdks and effect on cell proliferation for compounds 4a, 4b, 4f,
4h–j, 5c–e, 5g, 8a, 8b, 9c–e, 13, 14, 15a–e, 16a–d, 17e, 17f, and 18a–d, 18g
Compds Cdk2/cyclinA30 IC50
,
Cdk2/cyclinE30 IC50
,
HeLa31 GI50
,
lM
l
M
lM
2
4a
4b
4f
4h
4i
4j
5c
0.40
0.2
0.3
0.1
0.4
0.10
0.1
0.07
0.09
0.3
0.077
0.044
0.14
0.048
0.38
0.04
0.03
0.17
0.02
0.27
0.4
Acknowledgments
The authors thank the NCI and PanLabs for conducting the
kinase panel screens.
0.4
0.2
0.15
0.14
0.2
0.5
0.6
0.09
0.95
0.2
0.2
0.2
5d
5e
5g
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In summary, a series of purines was prepared using 2 as the
starting scaffold in which the biphenylmethylamino group at C-6
was replaced with heterobiarylmethylamino groups. Although
the antiproliferative activity did not correlate well with the Cdk
inhibitory activity, many of the compounds had excellent antipro-
liferative activity. This lack of correlation between Cdk inhibition
and antiproliferative activity may be due to additional mechanisms
of action. Select compounds were screened against a panel of
kinases at PanLabs32 and a panel of kinases by the NCI,33 with no
significant inhibition of any of the kinases observed. A detailed
reporting of this work and other biochemical mechanism of action
studies that were conducted on select compounds will be the sub-
ject of future publications. Additionally, cytotoxicity profiling was
conducted for select compounds in the NCI panel of 60-trans-
formed cell lines as well as in vivo testing. These studies will also
be reported in due course. Typically 10 to 100-fold improvements
in the Cdk inhibitory activities compared to roscovitine (1) were
30. 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

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6617
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 2 Â SDS sample buffer and resolved by SDS–PAGE. H1
phosphorylation was quantified by phosphoimaging. IC50 values were
calculated using GraphPad Prism data analysis software.
Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst.
1990, 82, 1107.
32. The Panlabs kinase panel consisted of PKC alpha, Ca/Calmodulin dependent
PKII, EGF Receptor, ERK1, LCK and PKA.
33. The NCI kinase panel consisted of c-RAF, MEK-1, MAPK2, MKK6, SAPK2b,
31. 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.;
SAPK4, MAPKAP-K2, MKK4, JNK
p70S6K, CHK1, PKB , cSRC, ZAP-70, JNK3, CK1, PKC-d, MAPK1, CHK-2, PRK2,
and AMPK. We thank the NCI for the results of these experiments.
a1, JNKa2, SGK, GSK3b, ROCKII, CK2, LCK,
a