Synthesis and in vitro biological evaluation of 2,6,9-trisubstituted purines targeting multiple cyclin-dependent kinases

Article abstract of DOI:10.1016/j.ejmech.2012.06.036

Several inhibitors of cyclin-dependent kinases (CDKs), including the 2,6,9-trisubstituted purine derivative roscovitine, are currently being evaluated in clinical trials as potential anticancer drugs. Here, we describe a new series of roscovitine derivatives that show increased potency in vitro. The series was tested for cytotoxicity against six cancer cell lines and for inhibition of CDKs. For series bearing 2-(hydroxyalkylamino) moiety, cytotoxic potency strongly correlated with anti-CDK2 activity. Importantly, structural changes that increase biochemical and anticancer activities of these compounds also increase elimination half-life. The most potent compounds were investigated further to assess their ability to influence cell cycle progression, p53-regulated transcription and apoptosis. All the observed biological effects were consistent with inhibition of CDKs involved in the regulation of cell cycle and transcription.

Full text of DOI:10.1016/j.ejmech.2012.06.036

European Journal of Medicinal Chemistry 61 (2013) 61e72
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and in vitro biological evaluation of 2,6,9-trisubstituted purines
targeting multiple cyclin-dependent kinases
,
b
a
b
a
,
b
a
Marek Zatloukal a , Radek Jorda , Tomás Gucký , Eva Reznícková , Jirí Voller , Tomás Pospísil ,
ꢀ
*
ꢀ
c
ꢀ
b
ꢀ
ꢀ
ꢀ
a
a,b
ꢀ
Veronika Malínková , Helena Adamcová , Vladimír Krystof , Miroslav Strnad
Centre of the Region Haná for Biotechnological and Agricultural Research, Department of Growth Regulators, Faculty of Science, Palacký University, Slechtitelu 11,
a
ꢀ
ꢁ
783 71 Olomouc, Czech Republic
b
ꢀ
ꢁ
Laboratory of Growth Regulators, Faculty of Science, Palacký University & Institute of Experimental Botany, Slechtitelu 11, 78371 Olomouc, Czech Republic
c
ꢀ
ꢁ
BioPatterns s.r.o., Slechtitelu 21, 78371 Olomouc, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
Several inhibitors of cyclin-dependent kinases (CDKs), including the 2,6,9-trisubstituted purine deriva-
tive roscovitine, are currently being evaluated in clinical trials as potential anticancer drugs. Here, we
describe a new series of roscovitine derivatives that show increased potency in vitro. The series was
tested for cytotoxicity against six cancer cell lines and for inhibition of CDKs. For series bearing
2-(hydroxyalkylamino) moiety, cytotoxic potency strongly correlated with anti-CDK2 activity. Impor-
tantly, structural changes that increase biochemical and anticancer activities of these compounds also
increase elimination half-life. The most potent compounds were investigated further to assess their
ability to influence cell cycle progression, p53-regulated transcription and apoptosis. All the observed
biological effects were consistent with inhibition of CDKs involved in the regulation of cell cycle and
transcription.
Received 19 January 2012
Received in revised form
7 June 2012
Accepted 16 June 2012
Available online 23 June 2012
Keywords:
Cyclin-dependent kinase
Inhibitor
Roscovitine
Cancer
Apoptosis
Ó 2012 Elsevier Masson SAS. All rights reserved.
Cell cycle
1. Introduction
To date, more than 20 of the most potent inhibitors have been
registered for clinical trials in cancer patients [2]. These compounds
The cell division cycle is driven by sequential activation of
cyclin-dependent kinases (CDKs), enzymes activated primarily by
binding to phase-specific protein cyclins. When complexed with
cyclins, CDKs are involved in, e.g., changes in the expression of cell
cycle-specific genes in the G1 phase required for cell cycle entry,
duplication of chromosomes and centrosomes during the S phase,
mitotic spindle formation, nuclear membrane breakout, and chro-
matin condensation in the M phase [1]. CDKs and cyclins, as well as
many other proteins that interact with them, are frequently
deregulated in cancer cells, causing the cell cycle control mecha-
nisms to be dismantled and hyperactivation of CDKs [1]. These
observations have provided a basis for the development of CDK
inhibitors as novel anticancer drugs.
are often classified according to their selectivity towards CDKs. The
majority of known inhibitors exhibit activity towards a broad range
of CDKs, which may be therapeutically advantageous as genetic
studies have shown that CDK 2, 4 and 6 are dispensable for the
cycling of most cell types, whereas CDK1 has been shown to be
absolutely essential for cell proliferation, at least in mice [3e6].
One of the clinically evaluated compounds is the trisubstituted
purine derivative roscovitine, which inhibits CDK 1, 2, 5, 7, and 9
[7,8]. By targeting CDK1 and CDK2, roscovitine arrests the cell cycle,
while inhibition of transcriptional CDK7 and CDK9 induces
apoptosis in cancer cells [9,10]. Roscovitine is currently being
evaluated in patients diagnosed with non-small cell lung cancer
and nasopharyngeal cancer [11].
Roscovitine is orally bioavailable but displays a strong first-pass
effect and rapid clearance [12]. Moreover, it is a relatively low
potency drug in comparison with other clinically evaluated CDK
inhibitors, such as AT7519 or dinaciclib [13,14]. Clinical studies
suggest that b.i.d. dosing is necessary to maintain therapeutically
effective concentrations [15]. The aim of the present work was to
prepare novel derivatives of roscovitine that possessed enhanced
Abbreviations: CDK, cyclin-dependent kinase; DIAD, diisopropyldiazadicarbox-
ylate; NMP, N-methylpyrrolidone; PARP, poly(ADP-ribose)polymerase; TLC, thin
layer chromatography.
* Corresponding author. Tel.: þ420 585634953; fax: þ420 585634870.
E-mail address: marek.zatloukal@upol.cz (M. Zatloukal).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.06.036

62
M. Zatloukal et al. / European Journal of Medicinal Chemistry 61 (2013) 61e72
anti-kinase and cytotoxic activities as well as structural features
that could increase their elimination half-lives. Replacement of the
primary hydroxy group in the side-chain with an amino group, or
a secondary or tertiary hydroxy moiety was conducted to prevent
formation of the carboxylate, which is a dominant and relatively
inactive roscovitine metabolite [16,17]. Furthermore, introduction
of a basic moiety was expected to increase the affinity of the
compounds for tissues [18e20].
temperature was kept within the range of 80e100 ꢀC. The reaction
time varied from 3 to 6 h, depending on the reaction temperature
and amine reactivity. Crude intermediates were purified by crys-
tallization from isopropanol, if necessary. The yields were satis-
factory (70e85%) in all cases.
The final step was accomplished by previously described
method [7,22,25,26]. The appropriate aminoalcohol or diamine for
the SN2 substitution at C2 was used in excess (5e20 equiv.)
and Hunig’s base was employed as an auxiliary base in several
cases. When a C2 substituent was a rare sterically hindered ami-
ne (4-amino-2-methylbutan-2-ol, 1-amino-2-methylpropan-2-ol,
(2RS,3R)-3-aminopentan-2-ol, (S)-3-amino-2-methylbutan-2-ol),
only a small excess of amine (5e7 equiv.) was used, in the presence
of an auxiliary base (DIPEA), and N-methylpyrrolidone was used as
a solvent. The reaction temperature was kept at 150e160 ꢀC (sealed
tube) and the reaction time varied from several hours in case of
reactive amines (all C2 e roscovitine amine derivatives, 3f, 3n, 3o,
piperazine derivatives) to 72 h in cases of less reactive sterically
hindered aminoalcohols (3bee, 3q, 3r, 3t). Yields varied from 60 to
80% in cases of usage of reactive roscovitine type aminoalcohols to
20e40% in cases of sterically hindered secondary and tertiary
aminoalcohols.
Crude products were purified by crystallization from diethyl
ether and finally re-crystallized from mixture of ethyl acetate and
hexanes (1:2). The majority of final compounds were crystallized
with exception of 3c, 3e, 3n, 3q, 3r, 3t, which had to be purified by
flash chromatography. The reaction conditions including yields
were optimized in some cases for the future possibility of a large
scale production.
2. Results and discussion
2.1. Synthesis
In this paper, a series of novel 2,6,9-trisubstituted purine CDK
inhibitors was successfully synthesized through an improved
procedure (see Experimental section 4.3., Scheme 1). The identity
of all final compounds was confirmed by 1H NMR, 1He1H COSY, 13
C
NMR and 1He13C HSQC spectrometry, mass spectrometry (ESI-MS)
and elemental analysis.
The straightforward three-step synthesis of 2,6,9-trisubstituted
purines started from commercially available 2,6-dichloropurine,
which was in the first step alkylated by isopropyl alcohol via Mit-
sunobu alkylation [21] at N9 to give 2,6-dichloro-9-isopropylpurine
(1). A former conventional method for the preparation of this
key intermediate used an isopropyl halide as an alkylating
agent [22e24], but the disadvantage of this method was a poor
regioselectivity as a considerable amount of by-product, which is
N7 e isomer, was also formed. The crude product has to be purified
by multiple crystallizations or by column chromatography. More-
over, the method uses a toxic and cancerogenic isopropyl halo-
genide. For this reason we decided to use the Mitsunobu alkylation
reaction, which employs isopropanol as an alkylating agent [21].
The reaction temperature was kept within the range of 20e25 ꢀC to
minimize the formation of undesired N7 e isomer, on the contrast
to Weibing Lu et al. [21], who carried out the alkylation at 70 ꢀC. We
have found that the regioselectivity of the alkylation decreased
with an increase of the reaction temperature. This method pro-
ceeded very smoothly and was much more regioselective in
comparison with the halide method. We also employed a lower
excess of DIAD (1.2 eq.) and triphenyl phosphine (1.2 eq.), and
shorter reaction time (1e2 h) than Weibing Lu, who used 2.1 eq. of
both reactants and longer reaction time (6 h). The crude alkyl
derivative was purified from contaminating by-products (triphenyl
phosphine oxide and traces of N7 e isomer) by crystallization from
lower alcohol or flash chromatography.
2.2. Structureeactivity relationships in biological assays
From a structural point of view, the prepared compounds differ
from roscovitine by substitutions at positions 2 and 6. As in
roscovitine, all compounds bear an isopropyl substitution at posi-
tion 9, which was previously shown to be optimal for purine CDK
inhibitors [7,27,28]. All newly synthesized purines were tested for
cytotoxicity against various cancer cell lines (MCF7, K562, HOS,
CEM, HCT-116, and G361) and for CDK2/5/7/9 kinase inhibition,
according to established protocols [25,29]. The resulting data are
presented in Table 1 and clearly show that most of new compounds
are significantly more potent CDK2 inhibitors than the reference
compound roscovitine, and about half of them also had higher
cytotoxicity. These results confirmed previous data with another
library of purine inhibitors where correlation between inhibition of
CDK and antiproliferative activity was shown [32]. On the other
hand, cytotoxic activities for a number of C2-piperazine derivatives
(3gei, 3m) that have not shown reliable anti-kinase activity could
be probably explained by additional off-targets as documented e.g.
for roscovitine with pyridoxalkinase.
The second step, preparation of 2-chloro-6-(subst. benzyla-
mino)-9-isopropylpurines (2ae2i, see Experimental section 4.3.2.)
was a nucleophilic substitution at C6 purine position with appro-
priate substituted benzylamine [22,25,26]. The reaction was carried
out in n-propanol and triethylamine, or N,N-diisopropyl-N-ethyl
amine (Hunig’s base) was used as an auxiliary base. The reaction
R4
R4
R3
R2
R5
R6
R3
R2
R5
R6
R4
R3
R2
R5
R6
amines
Cl
N
Cl
N
HN
N
HN
N
160 °C
H2N
(X = OH, NH2)
N
N
N
N
N
N
N
2-propanol
N
N
N
N
Ph3P, DIAD
THF, rt
Et3N/n-PrOH
90 °C
N
H
Cl
Cl
Cl
R1
1
3a - 3u
Y = 30-80%
2a - 2i
Y=75-90%
Y= ca 60%
Scheme 1. Synthesis of 2,6,9-trisubstituted purine CDK inhibitors. The numbering of substituents (R1eR6) corresponds to the description of structures given in Table 1.

M. Zatloukal et al. / European Journal of Medicinal Chemistry 61 (2013) 61e72
63
Table 1
Structures and biological activity of prepared compounds.
General structure
Compound
R1
R2
H
R3
H
R4
H
R5
H
R6
H
IC50
CDK2 (nM)
Cytotoxicitya
22.5
(mM)
R4
R3
Roscovitine
180
50
R5
R6
R2
Olomoucine II
OH
H
H
H
H
H
H
H
H
10.6
31.9
HN
N
N
N
R1
N
3a
H2N
20
3b
3c
NH2
NH2
H
H
H
H
H
H
H
H
20
17.1
100
790
3d
NH2
H
H
H
H
70
23.2
3e
3f
OH
OH
H
H
H
H
H
H
H
H
H
H
H
260
220
340
51.6
16.7
9.2
H
3g
OH
3h
3i
H
H
H3CO
H
H
890
890
7.7
He
He
H3COe
He
He
12.6
3j
H3COe
OHe
H3COe
H3COe
H3COe
He
He
He
He
He
He
He
He
He
31
60
59
40.5
22.2
9.0
3k
3l
OHe
(continued on next page)

64
M. Zatloukal et al. / European Journal of Medicinal Chemistry 61 (2013) 61e72
Table 1 (continued )
General structure
Compound
R1
R2
R3
R4
R5
R6
IC50
CDK2 (nM)
Cytotoxicitya
20.3
(mM)
3m
OHe
H3COe
He
He
He
630
3n
3o
3p
OHe
OHe
OHe
He
He
He
He
He
He
Cle
Cle
Cle
He
He
He
61
25
9.4
6.0
2.4
4.4
3q
3r
OHe
He
He
He
He
Cle
Cle
He
He
19.0
13.1
4.1
2.4
OHee
3s
OHe
He
He
Cle
He
24.3
1.8
3t
OHe
OHe
He
He
He
He
Fe
Fe
He
He
11.8
19.0
2.1
4.6
3u
a
Cytotoxicity averaged from measurements on a panel of 6 cancer cell lines. Full results available in the Supplementary data.
Several reports in the literature have demonstrated that
a hydroxy substituent on the benzyl ring enhances CDK inhibitory
activity [25,26,30,31]; an example of such a compound is olo-
moucine II, which has a 2-hydroxybenzyl side chain [25,32]. Except
of derivatives bearing hydroxy group on the benzyl ring (3eeg) we
introduced also compounds with amino group (3aed) as well as
some derivatives with methoxy group (3h, 3i). CDK activity of
compounds bearing this type of substituents at position 2 and
different at position 6 (3c vs 3e, R2 ¼ NH2 vs OH; 3g vs 3h, R2 ¼ OH
vs OCH3) demonstrates this positive effect of hydroxy substituent
(see Table 1). An addition of methoxy group at meta position
(3je3l) sustained high anti-CDK2 activity of 2-substituted
compounds. A positive effect on CDK inhibition has also been
shown for halogenated benzyl derivatives [26,27]. Our results
indicate that the combination of both structural motives may be
advantageous because substitution of hydrogen in the olomoucine
II R5 position with a halide group at R5 further improved inhibitory
activity (3p, 3u). Overall, the 2-hydroxy-5-chloro derivatives 3oes
and 2-hydroxy-5-fluoro derivatives 3t and 3u were the most potent
among the synthesized novel compounds, both in terms of their
anti-CDK2 and cytotoxic activity.
Next, we explored changes at the purine position
2 in
compounds where the position 6 was occupied by substituted
benzylamine. We introduced alkylamino (3f), aminoalkylamino
(3gei, 3m, 3o) and hydroxyalkylamino group with primary (3a,
3jel, 3p, 3u), secondary (including hydroxycycloalkyl) (3n, 3r, 3t)
or tertiary hydroxyls (3bee; 3q; 3s) at position 2.
A high
activity against CDK2 was observed for the structurally diverse
2-hydroxyalkylamino derivatives, which clearly correlated with
antiproliferative activity (rank correlation
r
¼ 0.72, p < 0.01). While
branching of hydroxyalkylamino substituents at position 2 did not
have a pronounced effect (3u vs. 3t; 3p vs. 3r vs. 3q), an increase in
the distance between the N2 nitrogen and the hydroxy group
resulted in a loss of the activity for the compound pairs 3bec and
(to lesser extent) 3pen. The high activity of 3o in both the kinase
and cytotoxicity assays suggests that not only the terminal hydroxyl
(3n) but also the terminal amino group (3o) may take part in
favorable binding interactions within the active site of CDKs. In

M. Zatloukal et al. / European Journal of Medicinal Chemistry 61 (2013) 61e72
65
Table 2
contrast, introduction of a piperazine-1-yl group at position 2 of the
purine ring (3gei, 3m) led to a marked decrease in activity against
CDK2. Since the latter compounds were all highly active against the
studied cancer cell lines, their effect is probably mediated by
interference with targets other than CDKs. Comparable activities of
3h and its N-methylated derivative 3i suggest that the terminal
amino group acts as a basic center rather than a hydrogen bond
donor in this interaction.
We, and others, have previously found that oxidation of the
terminal hydroxyl at the purine position 2 to carboxylate is a major
route of metabolic inactivation of roscovitine and related inhibitors
in vivo [8,16,33], and protection against oxidation contributes
to lower metabolic clearance [17]. Overall, the observed
structureeactivity relationships demonstrate that compounds in
which this oxidizable group is replaced by a secondary or tertiary
hydroxy or even an amino group, which do not readily convert to
carboxylate, retain strong anti-CDK activity. This is in agreement
with findings published recently by Wilson et al. [17], who studied
similar CDK inhibitors with oxidizable group at position and
showed increased stability towards mouse microsomes and
promising in vivo PK properties and efficacy in vivo following oral
Biochemical and cellular activities of selected compounds. CDK inhibitory selectivity
was determined in biochemical kinase assays; maximum activation of p53 was
determined in a cellular reporter system.
Compound
IC50 (nM)
CDK2
Maximum p53
activation (mM)
CDK5
CDK7
CDK9
Roscovitine
Olomoucine II
180
51
20
790
70
260
220
61
25
9
19
13
1080
270
740
>5000
675
4485
2360
550
125
150
69
793
n.a.
1200
n.a.
290
n.a.
n.a.
540
160
n.a.
435
97
2694
815
1240
>5000
845
>5000
3130
910
39
285
n.a.
165
24.5
11.4
16.8
>100
24.0
50.0
>100
14.6
3.4
10.0
4.1
2.1
1.8
2.5
3b
3c
3d
3e
3f
3n
3o
3p
3q
3r
3s
3t
71
n.a.
67
240
24
11
19
n.a.
180
n.a.
n.a.
100
106
3u
6.6
n.a. e not available.
compared to control cells after the treatment with 5
mM 3r. Staining
administration for candidate compound aSbR-21 [17] 2. The anal-
treated HCT-116 and K562 cells with 5-bromo-20-deoxyuridine
(BrdU) also revealed a decrease in DNA replication (Fig. 1C) in
a dose-dependent manner.
ogous compounds in our study, 3b and 3qet, belong to the most
active in the described series of compounds, with IC50 values below
20 nM.
We next monitored levels of phosphorylation of RNA poly-
merase II, which is a substrate of CDK7 and CDK9, in cells treated
with 3r. Immunoblotting analysis revealed a rapid decrease in
phosphorylation at serines 2 and 5 (Fig. 2A, B), confirming cellular
inhibition of these two kinases. Several recent reports have sug-
gested that inhibition of CDK9 (and inhibition of transcription in
general) in cells leads to accumulation of the tumor suppressor
p53 in an active form [34,35]. Therefore, we investigated the effect
of treating colorectal carcinoma HCT-116 cells with 3r. We found
that 3r rapidly increased the expression of p53 and p53-regulated
Several recent reports have suggested that CDK inhibition in
cells causes accumulation of tumor suppressor p53 in an active
form [34,35]. A positive relationship between inhibition of CDK and
p53-regulated transcription is clear also for this series of
compounds; rank correlation between IC50 for CDK 2, 5, 9 and
concentration inducing p53 in cell reporter system
r > 0.71
(p < 0.05). The most potent CDK inhibitors 3oer, 3u and 3t induced
p53-regulated transcription at low micromolar concentrations, as
assessed by a cellular reporter assay. Molecular mechanisms of
induced p53-dependent transcription are not yet fully understood,
but our recent studies suggest that inhibition of CDK9 (an essential
transcriptional activator) leads to down-regulation of HDM2 (a
negative regulator of p53), which in turn stabilizes p53 [36,37].
Therefore, in the present study we investigated the inhibitory
activity of the most potent compounds towards CDKs other than
CDK2, including CDK5, CDK7 and CDK9. As shown in Table 2, all
compounds that potently inhibited CDK2 also exhibited a strong
activity towards CDK5 and CDK9, as expected based on the high
sequence and structural similarities of their active sites [38].
Interestingly, comparison of the selectivity profiles and effects on
p53-regulated transcription (Table 2) suggest that activation of p53
may not be caused only by inhibition of CDK9, but also CDK2, and
perhaps other kinases that were not screened. This can be seen by
comparing 3r and 3t with 3o, which shows that the latter produces
2-fold lower and 2-fold higher CDK2 and CDK9 inhibition, respec-
tively. Increased potency and selectivity of 3o towards CDK9 was
however not reflected by an increased ability to activate p53.
p21WAF1 at concentrations of 1
mM and higher (Fig. 2C). Accu-
mulation of p53 was accompanied by decreased expression
of Mdm-2, which is a negative regulator of p53. We also observed
the same effect in a reporter assay using cell line Arn-8. 3r exer-
ted a dose-dependent effect on p53 transcriptional activation,
with the maximum effect obtained at a concentration of 2
(Fig. 2D).
mM
The strong cytotoxicity of many of the observed potent CDK
inhibitors towards different cancer cell lines prompted us to
analyze the mechanism of cell death induced by 3r in HCT116 and
K562 cells. The results of an immunoblotting analysis of several
proteins involved in apoptotic cell death are shown in Fig. 3A and B.
While expression of PUMA, Bcl-2 and caspase-3 remained largely
unchanged in both cell lines treated with 3r, the level of anti-
apoptotic protein Mcl-1 showed a large dose-dependent decrease.
Another typical apoptotic marker, an 89 kDa fragment of poly(ADP-
ribose)polymerase (PARP), was detected in treated cells.
A
fluorimetry-based caspase-3/7 activity assay of lysates of HCT-116
and K562 cells treated with 3r revealed potent dose-dependent
activation of the caspase in K562 but only weak activation in
HCT-116 cells (Fig. 3C).
2.3. Cellular effects of selected compounds
Compound 3r was selected for this study as the most potent
inhibitor among the series of compounds showing over 13-fold
higher activity versus roscovitine in terms of CDK inhibition that
corresponds with over 9-fold increase in cytotoxicity on a panel of
six cancer cell lines (see Table 1). The antiproliferative activity of 3r,
was measured in an asynchronously growing colon carcinoma cell
line, HCT-116, and a chronic myeloid leukemia cell line, K562. As
shown in Fig. 1, inhibitor 3r potently arrested cells in late S and G2/
M phases. This effect was particularly evident in HCT-116 cells,
where about 15% more cells were in the G2/M phase of the cell cycle
3. Conclusions
This study involved the synthesis and modification of the bio-
logical activity of novel purine CDK inhibitors derived from rosco-
vitine by changing the moieties at positions 2 and 6. Many of the
prepared compounds proved to be more potent at limiting the
proliferation of the tested cancer cell lines than roscovitine and
olomoucine II. For the 2-hydroxyalkylamino derivatives studied,

66
M. Zatloukal et al. / European Journal of Medicinal Chemistry 61 (2013) 61e72
not abolish biological activity. Example compound 3r blocked cell
cycle progression and induced apoptosis in cells as a result of
transcriptional perturbations due to reduced phosphorylation of
Ser-2 and Ser-5 in the C-terminal domain of RNAP-II, caused by
inhibition of CDK9 and CDK7. Pharmacokinetic studies of selected
derivatives are currently underway.
4. Experimental
4.1. General procedures
The elemental contents of the prepared compounds were
determined using an EA1108 CHN analyzer (Thermo Finnigan). Their
melting points were determined using a Büchi Melting Point B-540
apparatus. Thin layer chromatography (TLC) was performed using
silica gel 60 WF254 plates (Merck) with
a mobile phase
(CHCl3:MEOH:conc. NH4OH, 8:2:0.2, v/v/v). Flash chromatography
was performed using a VersaFlash purification station (Supelco)
coupled to a 2110 Fraction Collector (Bio-Rad). Compounds were
separated on VersaPak Cartridges (25 ꢁ 100 mm, Supelco) con-
taining 23 g of spherical silica and eluted with a mobile phase
(CHCl3:MeOH, 90:10, v/v). To determine their HPLC purity, samples
were dissolved in HPLC mobile phase (initial conditions), applied to
an RP-column (150 mm ꢁ 4.6 mm, 5
mm, Microsorb C18; Varian) and
the separated constituents were eluted with a linear methanolic
gradient (10e90% over 30 min, pH adjusted to 4 using ammonium
formate) at a flow rate of 0.6 ml/min. Eluting compounds were
detected by scanning the UV absorbance of the eluate between 240
and 300 nm. CIþ and EIþ mass spectra were recorded using a Polaris
Q (Finnigan) mass spectrometer equipped with a Direct Insertion
Probe (DIP). The compounds were heated in an ion source with
a 40e450 ꢀC temperature gradient, the mass monitoring interval
was 50e1000 am, and spectra were collected using 1.0 s cyclical
scans, applying 70 eV electron energy. In the CIþ ionization mode,
isobutane was used as a reagent gas at a flow rate of 2 l/h. The mass
spectrometer was directly coupled to an Xcalibur data system. NMR
spectra were acquired using a Bruker Avance AV 300 spectrometer
operating at a temperature of 300 K and a frequency of 300.13 MHz
(1H). Samples were prepared by dissolving compounds in DMSO-d6
and tetramethylsilane (TMS) was used as the internal standard.
4.2. Chemicals
2,6-Dichloropurine and 4-aminomethylphenol were obtained
from OlChemim. Triphenylphosphine, diisopropyldiazadicarbox-
ylate (DIAD), 2-aminobenzylamine, 4-aminobenzylamine, 4-
methoxybenzylamine, R,S-2-amino-1-butanol, S-valinol, 1,4-trans-
diaminocyclohexane, azacycloheptane, piperazine, N-methyl-
piperazine were purchased from SigmaeAldrich. Variously
substituted benzylamines were prepared via reduction of oximes of
corresponding substituted benzaldehydes [39]. Secondary and
tertiary aminoalcohols, as (2RS, 3R)-3-aminopentan-2-ol, (S)-3-
amino-2-methylbutan-2-ol, 1-amino-2-methylpropan-2-ol, 4-
amino-2-methylbutan-2-ol and 2,4-dimethyl-3-aminopentan-2-
ol were synthesized according the described procedure [40].
Lach-Ner supplied methanol, 2-propanol, chloroform, diethyl ether,
dimethylformamide, ethyl acetate, anhydrous magnesium sulfate.
Milli-Q water was used throughout. Solvents and chemicals used
were all of standard p.a. quality.
Fig. 1. Effect of 3r on the cell cycle of HCT-116 and K562 cancer cell lines following 24 h
treatment. Flow cytometric analysis of cell cycle after propidium iodide staining in (A)
HCT-116 and (B) K562 cell lines, and (C) quantification of actively-replicating BrdU
positive S phase cells in both cell lines.
4.3. Synthesis of 2,6,9-trisubstituted purines
cytotoxic potency strongly correlated with anti-CDK2 activity.
However, highly cytotoxic compounds with piperazin-1-yl group at
position 2 were only weak CDK2 inhibitors. Importantly, structural
changes that were expected to increase the elimination half-life did
4.3.1. 2,6-Dichloro-9-isopropylpurine (1)
2,6-Dichloropurine (1.89 g; 0.01 mol) was dissolved under
a nitrogen atmosphere in a mixture of tetrahydrofuran (40 ml) and

M. Zatloukal et al. / European Journal of Medicinal Chemistry 61 (2013) 61e72
67
Fig. 2. Effect of 3r on transcription and activation of p53 in HCT-116 and K562 cell lines following 24 h treatment. (A, B) Immunoblotting analysis of phosphorylation of RNA
polymerase II at serines 2 and 5. Tubulin levels were detected to verify equal protein loading. (C) Immunoblotting of p53, p21WAF1 and Mdm-2 in HCT-116 cells; PCNA levels were
detected to verify equal protein loading. (D) Analysis of relative p53-dependent transcriptional activity by b-galactosidase reporter assay of Arn-8 cell line.
Fig. 3. Effect of 3r on induction of apoptosis in HCT-116 and K562 cell lines following 24 h treatment. (A, B) Immunoblotting analysis of selected apoptotic markers. PCNA and
tubulin levels were detected to verify equal protein loading. (C) Fluorimetric caspase-3,7 activity assay based on the cleavage of a specific Ac-DEVD-AMC peptide substrate.

68
M. Zatloukal et al. / European Journal of Medicinal Chemistry 61 (2013) 61e72
isopropanol (4 ml; 0.06 mol), and then triphenyl phosphine (3.14 g;
0.012 mol) and DIAD (2.4 ml; 0.012 mol; dropwise) were added.
The temperature of reaction mixture was maintained at 20e25 ꢀC
during addition of DIAD. The reaction mixture was the stirred for
2 h at 20e25 ꢀC. TLC (mobile phase: ethyl acetate e toluene; 1:1)
after 2 h indicated that the reaction had gone to completion. The
reaction mixture was then evaporated to a thick yellow residue,
which was dissolved in hot (60 ꢀC) methanol (30 ml). After crys-
tallization in a refrigerator (ꢂ18 ꢀC) overnight, the crystalline mass
was collected by filtration, washed with cold (ꢂ10 ꢀC) methanol
(2 ꢁ 5 ml) and dried in the vacuum drying oven (60 ꢀC) to constant
weight. Yield: 1.20 g of almost white crystalline powder (52%). The
purity (HPLC): >98%.
(m, 6H, 2 ꢁ CH3), 4.50 (s(br), 2H, CH2), 4.58 (sep, J ¼ 6.0,1H, CH), 6.67
(t, J ¼ 8.2, 1H, ArH), 6.75e6.88 (m, 2H, ArH), 7.50 (s(br), 1H, NH), 7.78
(s, 1H, CH).
4.3.2.7. 2-[(2-Chloro-9-isopropyl-9H-purin-6-ylamino)-methyl]-6-
methoxyphenol (2g). 1H NMR (DMSO-d6): 1.49 (d, J ¼ 6.6, 6H,
CH3), 3.77 (s, 3H, CH3), 4.44 (d, J ¼ 6.7, 2H, CH2), 4.68 (sep, J ¼ 6.6,
1H, CH), 6.68e6.88 (m, 3H, ArH), 8.28 (s, 1H, CH), 8.67 (t, J ¼ 6.7,
1H, NH), 9.32 (s, 1H, OH).
4.3.2.8. 4-Chloro-2-[(2-chloro-9-isopropyl-9H-purin-6-ylamino)-
methyl]-phenol (2h). 1H NMR (DMSO-d6): 1.47 (d, J ¼ 6.6, 6H,
CH3), 4.53 (d, J ¼ 5.6, 2H, CH2), 4.62 (sep, J ¼ 6.8, 1H, CH), 6.79 (d,
J ¼ 8.5, 1H, ArH), 7.02e7.09 (m, 2H, ArH), 8.28 (s, 1H, CH), 8.61 (t,
J ¼ 5.6, 1H, NH), 9.92 (s, 1H, OH).
M.p. ¼ 149e152 ꢀC. Physico-chemical data including 1H NMR
were in accordance with those published [21e24]. The second crop
(up to 10%) of product of lower purity could be obtained from
mother liquor.
4.3.2.9. 2-[(2-Chloro-9-isopropyl-9H-purin-6-ylamino)-methyl]-4-
fluorophenol (2i). 1H NMR (DMSO-d6): 1.50 (d, J ¼ 6.5, 6H, CH3),
4.56 (d, J ¼ 4.7, 2H, CH2), 4.68 (sep, J ¼ 6.3, 1H, CH), 6.75e6.92 (m,
3H, ArH), 8.31 (s, 1H, CH), 8.61 (t, J ¼ 4.7, 1H, NH), 9.63 (s, 1H, OH).
4.3.2. 2-Chloro-6-(subst. benzylamino)-9-isopropylpurines (2ae2i)
2,6-Dichloro-9-isopropylpurine (0.05 mol), appropriate substituted
benzylamine (0.05 mol), n-propanol (230 ml) and triethylamine
(0.15 mol) were placed into a reaction bulb, under nitrogen. The reac-
tion mixturewas warmed upto 90 ꢀC, and stirred fora period of 4 h. The
contents of the bulb were then evaporated on rotary vacuum evapo-
rator to give a semisolid residue, which was then treated with water
(200 ml) for 20 min. The precipitatewas filtered off, washed with water
(3 ꢁ 20 ml) and dried in the vacuum oven to constant weight. Yield:
75e90%, according to the type of benzylamine used. The purity (HPLC):
min. 97%. The crude product can be purified by crystallization from
isopropyl alcohol if required.
4.3.3. Substitution at C2 (3ae3u; final compounds)
2-Chloro-6-(subst. benzyl)amino-9-isopropylpurine (0.01 mol)
and appropriate aminoalcohol or diamine (0.1e0.2 mol) were stir-
red under nitrogen at 160 ꢀC for 3e12 h, the time depending on the
reactivity of amine used and on the character of substituent at C6.
The course of the reaction was monitored by TLC (mobile phase:
chloroformemethanol 85:15 þ trace of ammonia). After the reac-
tion had gone to completion, the reaction mixture was evaporated
on a rotary vacuum evaporator. The residue was partitioned
between water (100 ml) and ethyl acetate (100 ml). The organic
layer was separated and the water phase was extracted with ethyl
acetate (2 ꢁ 50 ml). The combined organic layers were washed
with water (30 ml), dried with anhydrous magnesium sulfate,
and evaporated to dryness. The sticky residue was treated
with diethyl ether (75 ml). The product gradually crystallized. The
crude substance was purified by crystallization from ethyl
acetateehexane (1:1). In some cases a purification by flash chro-
matography of products was necessary. Yield: 30e80%, according to
the type of amine used; purity (HPLC: >98%). When an expensive
aminoalcohol or diamine were employed, the reaction conditions
could be modified by adding a suitable solvent (e.g. NMP) and an
auxiliary base (e.g. N,N-diisopropyl-N-ethyl amine).
According to this procedure the following compounds were
prepared:
4.3.2.1. (4-Aminobenzyl)-(2-chloro-9-isopropyl-9H-purin-6-yl)-amine
(2a). 1H NMR (DMSO-d6): 1.49 (d, J ¼ 6.6, 6H, CH3), 4.44 (d, J ¼ 6.7,
2H, CH2), 4.66 (sep, J ¼ 6.6, 1H, CH), 5.21 (s(br), 2H, NH2), 6.49 (t,
J ¼ 7.0, 1H, ArH), 6.62 (d, J ¼ 7.8, 1H, ArH), 6.94 (t, J ¼ 7.0, 1H, ArH),
7.08 (d, J ¼ 7.8, 1H, ArH), 8.28 (s, 1H, CH), 8.67 (t, J ¼ 6.7, 1H, NH).
4.3.2.2. (2-Aminobenzyl)-(2-chloro-9-isopropyl-9H-purin-6-yl)-amine
(2b). 1H NMR (DMSO-d6): 1.49 (d, J ¼ 6.6, 6H, CH3), 4.44 (d, J ¼ 6.7,
2H, CH2), 4.66 (sep, J ¼ 6.6, 1H, CH), 5.21 (s(br), 2H, NH2), 6.49 (t,
J ¼ 7.0, 1H, ArH), 6.62 (d, J ¼ 7.8, 1H, ArH), 6.94 (t, J ¼ 7.0, 1H, ArH),
7.08 (d, J ¼ 7.8, 1H, ArH), 8.28 (s, 1H, CH), 8.67 (t, J ¼ 6.7, 1H, NH).
4.3.4. (R)-2-[6-(4-Aminobenzylamino)-9-isopropyl-9H-purin-2-
ylamino]-butan-1-ol (3a)
4.3.2.3. 2-[(2-Chloro-9-isopropyl-9H-purin-6-ylamino)-methyl]-phenol
(2c). 1H NMR (DMSO-d6): 1.64 (d, J ¼ 6.8, 6H, CH3), 4.68 (d, J ¼ 5.3, 2H,
CH2), 4.92 (sep, J ¼ 6.8, 1H, CH), 6.86 (t, J ¼ 7.5, 1H, ArH), 6.97 (d,
J ¼ 8.2,1H, ArH), 7.20 (t, J ¼ 8.2, 1H, ArH), 7.28 (d, J ¼ 7.5, 1H, ArH), 8.44
(t, J ¼ 5.3, 1H, NH), 8.59 (s, 1H, CH), 9.92 (s, 1H, OH).
The compound was prepared from the intermediate 2a and (R)-
2-amino-butan-1-ol by heating at 160 ꢀC for 3 h. Yield: 72%; white
solid. M.p. ¼ 126e128 ꢀC. 1H NMR (DMSO-d6): 0.86 (t, J ¼ 6.5, 3H,
CH3), 1.46 (d, J ¼ 6.0, 6H, CH3), 1.54e1.69 (m, 2H, CH2), 3.42e3.55
(m, 2H, CH2), 3.85 (sex, J ¼ 6.2, 1H, CH), 4.40 (s(br), 2H, CH2), 4.49
(sep, J ¼ 6.0, 1H, CH), 4.57 (t, J ¼ 6.0, 1H, OH), 4.92 (s(br), 2H, NH2),
5.83 (d, J ¼ 6.2, 1H, NH), 6.47 (d, J ¼ 7.4, 2H, ArH), 7.11 (d, J ¼ 7.4, 2H,
ArH), 7.48 (s(br), 1H, NH), 7.78 (s, 1H, CH). 13C NMR (DMSO-d6):
11.50, 22.35, 23.04, 24.43, 48.01, 54.00, 61.13, 111.05, 117.22, 120.37,
128.64, 135.91, 151.37, 154.34, 154.99, 159.39. MS m/z (ESI) 370.2
(M þ H)þ. Anal.: Calcd for C19H27N7O: C, 61.77; H, 7.37; N, 26.54.
Found: C, 61.39; H, 7.38; N, 26.25.
4.3.2.4. 4-[(2-Chloro-9-isopropyl-9H-purin-6-ylamino)-methyl]-phenol
(2d). 1H NMR (DMSO-d6): 1.46 (d, J ¼ 6.0, 6H, CH3), 4.49 (d, J ¼ 5.4, 2H,
CH2), 4.57 (sep, J ¼ 6.0, 1H CH), 6.69 (d, J ¼ 7.50, 2H, ArH), 7.13 (d,
J ¼ 7.50, 2H, ArH), 7.85 (t, J ¼ 5.4, 1H, NH), 8.15 (s, 1H, CH), 10.15 (s(br),
1H, OH).
4.3.2.5. (2-Chloro-9-isopropyl-9H-purin-6-yl)-(4-methoxybenzyl)-
amine (2e). 1H NMR (DMSO-d6): 1.49 (d, J ¼ 6.2, 6H, CH3), 3.72
(s, 3H, CH3), 4.50 (s(br), 2H, CH2), 4.65 (sep, J ¼ 6.2, 1H, CH), 6.83
(d, J ¼ 7.75, 2H, ArH), 7.29 (d, J ¼ 7.75, 2H, ArH), 7.80 (s(br), 1H,
NH), 7.95 (s, 1H, CH).
4.3.5. 1-[6-(2-Aminobenzylamino)-9-isopropyl-9H-purin-2-
ylamino]-2-methylpropan-2-ol (3b)
The compound was prepared from the intermediate 2b and
1-amino-2-methylpropan-2-ol by heating at 160 ꢀC in the presence
of N,N-diisopropyl ethyl amine for 48 h. Yield: 80%; white solid.
M.p. ¼ 181e183 ꢀC. 1H NMR (DMSO-d6): 1.10 (s, 6H, CH3), 1.45
4.3.2.6. (2-Chloro-9-isopropyl-9H-purin-6-yl)-(2,3-dimethoxybenzyl)-
amine (2f). 1H NMR (DMSO-d6): 1.46 (d, J ¼ 6.0, 6H, CH3), 3.75e3.77

M. Zatloukal et al. / European Journal of Medicinal Chemistry 61 (2013) 61e72
69
(d, J ¼ 6.4, 6H, CH3), 3.27 (d, J ¼ 5.6, 2H, CH2), 4.46 (s(br), 2H, CH2),
4.54 (sep, J ¼ 6.4, 1H, CH), 4.84 (s(br), 1H, OH), 5.21 (s(br), 2H, NH2),
6.09 (t, J ¼ 5.6, 1H, NH), 6.45 (t, J ¼ 7.1, 1H, ArH), 6.58 (d, J ¼ 7.1, 1H,
ArH), 6.91 (t, J ¼ 6.9, 1H, ArH), 7.12 (d, J ¼ 7.1, 1H, ArH), 7.66 (s(br),
1H, NH), 7.80 (s, 1H, CH). 13C NMR (DMSO-d6): 22.07, 27.53, 42.10,
45.63, 52.41, 69.99, 113.61, 114.42, 115.45, 122.86, 127.46, 129.53,
135.04, 146.25, 154.41, 159.47. MS m/z (ESI) 370.2 (M þ H)þ. Anal.:
Calcd. for C19H27N7O: C, 61.77; H, 7.37; N, 26.54. Found: C, 61.83; H,
7.59; N, 27.02.
(DMSO-d6): 0.85 (d, J ¼ 6.6, 6H, CH3), 1.46 (d, J ¼ 6.7, 6H, CH3), 1.84
(sep, J ¼ 6.6, 1H, CH), 3.05 (t, J ¼ 6.3, 2H, CH2), 4.47e4.60 (m, 3H,
CH2, CH), 6.39 (t, J ¼ 6.3, 1H, NH), 6.71 (t, J ¼ 7.4, 1H, ArH), 6.75
(d, J ¼ 7.4, 1H, ArH), 7.04 (t, J ¼ 7.4, 1H, ArH), 7.14 (d, J ¼ 7.4, 1H, ArH),
7.55 (s(br), 1H, NH), 7.79 (s, 1H, CH), 9.94 (s(br), 1H, OH). 13C NMR
(DMSO-d6): 20.26, 21.90, 27.75, 45.62, 48.78, 52.5, 113.41, 114.1,
115.17, 118.60, 126.10, 127.65, 128.66, 135.10, 154.90, 158.92. MS m/z
(ESI) 355.4 (M þ H)þ. Anal.: Calcd. for C19H26N6O: C, 64.38; H, 7.39;
N, 23.71. Found: C, 64.23; H, 7.41; N, 23.55.
4.3.6. 4-[6-(2-Aminobenzylamino)-9-isopropyl-9H-purin-2-
ylamino]-2-methylbutan-2-ol (3c)
4.3.10. 4-[(9-Isopropyl-2-piperazin-1-yl-9H-purin-6-ylamino)-
methyl]-phenol (3g)
The compound was prepared from 2b and 1-amino-3-
methylbutan-3-ol by heating at 160 ꢀC in NMP in the presence of
N,N-diisopropyl ethyl amine for 72 h. The crude product was
purified by flash chromatography. Yield: 27%; off white amorphous
solid. 1H NMR (DMSO-d6): 1.11 (s, 6H, CH3), 1.51 (d, J ¼ 6.5, 6H, CH3),
3.35 (q, J ¼ 6.9, 2H, CH2), 4.61 (sep, J ¼ 6.5, 1H, CH), 4.68 (s(br), 2H,
CH2), 4.86 (s(br), 1H, OH), 5.21 (s(br), 2H, NH2), 6.11 (t, J ¼ 6.9, 1H,
NH), 7.18e7.43 (m, 4H, ArH), 7.67 (s(br),1H, NH), 7.80 (s,1H, CH). 13C
NMR (DMSO-d6): 22.07, 26.30, 27.53, 42.20, 44.92, 52.50, 70.03,
113.61, 114.42, 115.45, 122.86, 127.46, 129.53, 135.04, 146.25, 154.41,
159.47. MS m/z (ESI) 384.2 (M þ H)þ. The compound was trans-
The compound was prepared from the intermediate 2d and
piperazine by heating at 160 ꢀC for 20 h. Yield: 51%; white solid.
M.p. ¼ 156e158 ꢀC. 1H NMR (DMSO-d6): 1.46 (d, J ¼ 6.0, 6H, CH3),
2.84e2.95 (m, 4H, CH2), 3.69e3.80 (m, 4H, CH2), 4.49 (s(br), 2H,
CH2), 4.57 (sep, J ¼ 6.0, 1H, CH), 6.70 (d, J ¼ 7.5, 2H, ArH), 7.16
(d, J ¼ 7.5, 2H, ArH), 7.85 (s, 1H, CH), 8.15 (s, 1H, NH), 9.50 (s(br), 1H,
NH), 10.15 (s(br), 1H, OH). 13C NMR (DMSO-d6): 21.97, 43.40, 44.07,
45.70, 54.93, 113.41, 114.74, 128.67, 130.76, 132.58, 135.81, 150.37,
154.00, 155.97, 157.91. MS m/z (ESI) 368.0 (M þ H)þ. Anal.: Calcd. for
C19H25N7O: C, 62.11; H, 6.86; N, 26.68. Found: C, 62.23; H, 6.90; N,
26.55.
ferred
into
its
corresponding
dihydrochloride
salt.
M.p. ¼ 196e199 ꢀC; white solid. Anal.: Calcd for C20H29N7O. 2 HCl:
4.3.11. (9-Isopropyl-2-piperazin-1-yl-9H-purin-6-yl)-(4-
methoxybenzyl)-amine (3h)
C, 52.63; H, 6.85; N, 21.48. Found: C, 52.41; H, 6.92; N, 21.14.
The compound was prepared from the intermediate 2e and
piperazine by heating at 160 ꢀC for 20 h. Yield: 45%; white solid.
M.p. ¼ 132e134 ꢀC. 1H NMR (DMSO-d6): 1.49 (d, J ¼ 6.2, 6H, CH3),
2.64e2.72 (m, 4H, CH2), 3.56e3.65 (m, 4H, CH2), 3.72 (s, 3H, OCH3),
4.50 (s(br), 2H, CH2), 4.65 (sep, J ¼ 6.2, 1H, CH), 6.83 (d, J ¼ 7.6, 2H,
ArH), 7.29 (d, J ¼ 7.6, 2H, ArH), 7.80 (1H, s, CH), 7.95 (s(br), 1H, NH),
10.15 (s(br),1H, NH). 13C NMR (DMSO-d6): 21.95, 44.85, 45.25, 45.61,
54.47, 59.65, 64.81, 113.38, 128.66, 132.68, 135.52, 150.54, 156.00,
157.94, 158.33. MS m/z (ESI) 382.1 (M þ H)þ. Anal.: Calcd. for
C20H27N7O: C, 62.97; H, 7.13; N, 25.70. Found: C, 62.43; H, 7.13; N,
25.11.
4.3.7. (S)-3-[6-(2-Aminobenzylamino)-9-isopropyl-9H-purin-2-
ylamino]-2,4-dimethylpentan-2-ol (3d)
The compound was prepared from 2b and 1-amino-2,4-
dimethylpentan-2-ol by heating at 160 ꢀC in NMP in the presence
of N,N-diisopropyl ethyl amine for 72 h. Yield: 28%; white solid.
M.p. ¼ 198e200 ꢀC. 1H NMR (DMSO-d6): 0.87e0.90 (d, J ¼ 6.90, 6H,
CH3), 1.04 (s, 3H, CH3),1.18 (s, 3H, CH3), 1.45 (d, J ¼ 6.6, 6H, CH3), 2.11
(sep, J ¼ 6.9, 1H, CH), 3.93e3.97 (d, J ¼ 6.9, 1H, CH), 4.33 (s(br), 1H,
OH), 4.47 (s(br), 2H, CH2), 4.53 (sep, J ¼ 6.6, 1H, CH), 5.21 (s(br), 2H,
NH2), 5.56 (d, J ¼ 6.9, 1H, NH), 6.44 (t, J ¼ 7.4, 1H, ArH), 6.59
(d, J ¼ 7.4, 1H, ArH), 6.91 (t, J ¼ 7.4, 1H, ArH), 7.14 (d, J ¼ 7.4, 1H, ArH),
7.57 (s(br), 1H, NH), 7.76 (s, 1H, CH). 13C NMR (DMSO-d6): 17.64,
21.89, 22.79, 28.01, 28.44, 61.27, 67.65, 72.57, 114.35, 115.34, 127.42,
129.73, 134.90, 146.22, 150.60, 154.35, 160.17. MS m/z (ESI) 412.2
(M þ H)þ. Anal.: Calcd. for C22H33N7O: C, 64.21; H, 8.08; N, 23.82.
Found: C, 63.98; H, 8.11; N, 23.55.
4.3.12. [9-Isopropyl-2-(4-methylpiperazin-1-yl)-9H-purin-6-yl]-(4-
methoxybenzyl)-amine (3i)
The compound was prepared from 2e and N-methylpiperazine
by heating at 135 ꢀC for 4 h. Yield: 56%; almost white solid.
M.p. ¼ 129e130 ꢀC. 1H NMR (DMSO-d6): 1.46 (d, J ¼ 6.0, 6H, CH3),
2.17 (s, 3H, OCH3), 2.26e2.37 (m, 4H, CH2), 3.63e3.72 (m, 6H, CH2),
4.57 (sep, J ¼ 6.0, 1H, CH), 6.85 (d, J ¼ 7.5, 2H, ArH), 7.30 (d, J ¼ 7.5,
2H, ArH), 7.82 (s, 1H, CH), 7.95 (s(br), 1H, NH), 10.25 (s(br), 1H, H19).
13C NMR (DMSO-d6): 21.67, 42.45, 43.95, 45.61, 54.84, 64.81, 133.36,
128.64, 132.62, 135.54, 150.48, 153.99, 157.95, 158.20. MS m/z (ESI)
396.2 (M þ H)þ. Anal.: Calcd. for C21H29N7O: C, 63.77; H, 7.39; N,
24.79. Found: C, 63.63; H, 7.40; N, 24.55.
4.3.8. 2-{[2-(3-Hydroxy-3-methylbutylamino)-9-isopropyl-9H-
purin-6-ylamino]-methyl}-phenol (3e)
The compound was prepared from the intermediate 2c and
1-amino-3-methylbutan-3-ol by heating at 160 ꢀC for 72 h. The
crude product was purified by flash chromatography. Yield: 43%;
white solid. M.p. ¼ 72e75 ꢀC. 1H NMR (DMSO-d6): 1.11 (s, 6H, CH3),
1.46 (d, J ¼ 6.7, 6H, CH3), 1.65 (t, J ¼ 6.5, 2H, CH2), 3.29 (q, J ¼ 6.5, 2H,
CH2), 4.28 (s, 1H, OH), 4.50e4.57 (m, 3H, CH, CH2), 6.30 (t, J ¼ 6.5,
1H, NH), 6.69e6.78 (m, 2H, ArH), 7.05 (t, J ¼ 7.3, ArH), 7.14 (d, J ¼ 7.3,
1H, ArH), 7.56 (s(br), 1H, NH), 7.79 (s, 1H, CH), 9.99 (s(br), 1H, OH).
13C NMR (DMSO-d6): 22.02, 29.47, 37.56, 42.53, 45.74, 68.57, 84.60,
115.35, 118.73, 122.30, 126.27, 127.82, 128.85, 135.22, 139.85, 150.47,
155.03, 158.81. MS m/z (ESI) 385.3 (M þ H)þ. Anal.: Calcd. for
C20H28N6O2: C, 62.48; H, 7.34; N, 21.86. Found: C, 62.20; H, 7.31; N,
21.55.
4.3.13. (R)-2-[6-(2,3-Dimethoxybenzylamino)-9-isopropyl-9H-
purin-2-ylamino]-butan-1-ol (3j)
The compound was prepared by heating of 2f in (R)-2-amino-
butan-1-ol at 160 ꢀC for
3 h. Yield: 65%; white solid.
M.p. ¼ 102e105 ꢀC. 1H NMR (DMSO-d6): 0.81 (t, J ¼ 7.0, 3H, CH3),
1.42e1.45 (m, 1H, CH2a), 1.46 (d, 6H, J ¼ 7.0), 1.47e1.49 (m, 1H,
CH2b), 3.35e3.45 (m, 2H, CH2), 3.77 (s, 3H, OCH3), 3.79 (s, 3H,
OCH3), 4.45e4.54 (m, 2H, 2 ꢁ CH), 4.57 (s(br), 2H, CH2), 5.79
(d, J ¼ 6.2,1H, NH), 6.82 (d, J ¼ 6.9,1H, ArH), 6.85e6.97 (m, 2H, ArH),
7.45 (s(br), 1H, NH), 7.78 (s, 1H, CH). 13C NMR (DMSO-d6): 10.63,
22.00, 22.10, 23.86, 45.65, 54.00, 55.63, 55.68, 62.95, 111.26, 119.64,
124.15, 133.85, 146.00, 152.14, 154.61, 158.99. MS m/z (ESI) 415.1
(M þ H)þ. Anal.: Calcd. for C21H30N6O3: C, 60.85; H, 7.30; N, 20.27.
Found: C, 60.61; H, 7.32; N, 20.09.
4.3.9. 2-[(2-Isobutylamino-9-isopropyl-9H-purin-6-ylamino)-
methyl]-phenol (3f)
The compound was prepared from 2c and isobutyl amine by
heating at 160 ꢀC in NMP in the presence of N,N-diisopropyl ethyl
amine for 12 h. Yield: 45%; white solid. M.p. ¼ 83e85 ꢀC. 1H NMR

70
M. Zatloukal et al. / European Journal of Medicinal Chemistry 61 (2013) 61e72
4.3.14. 2-{[2-((R)-1-Hydroxymethylpropylamino)-9-isopropyl-9H-
purin-6-ylamino]-methyl}-6-methoxyphenol (3k)
(m, 4H), 1.46 (d, J ¼ 6.6, 6H, CH3), 1.70e1.86 (m, 4H), 2.40 (qui,
J ¼ 5.7, 1H, CH), 3.55 (sex, J ¼ 7.1, 1H, CH), 4.05 (s(br), 2H, NH2),
4.45e4.61 (m, 3H, CH2, CH), 6.08 (d, J ¼ 7.1, 1H, NH), 6.79 (d, J ¼ 8.5,
1H, ArH), 7.02e7.08 (m, 2H, ArH), 7.62 (s(br), 1H, NH), 7.80 (s, 1H,
CH), 10.12(s(br), 1H, OH). 13C NMR (DMSO-d6): 15.71, 22.60, 31.84,
35.82, 46.26, 50.18, 50.52, 65.46, 117.14, 122.34, 127.50, 127.93,
129.38, 135.80, 154.70, 155.00, 158.84. MS m/z (ESI) 430.3 (M þ H)þ.
Anal.: Calcd. C21H28ClN7O: C, 58.67; H, 6.56; N, 22.80. Found: C,
58.29; H, 6.61; N, 22.53.
The compound was prepared by heating of 2g in (R)-2-amino-
butan-1-ol (excess) at 160 ꢀC for 3 h. Yield: 43%; almost white solid.
M.p. ¼ 100e103 ꢀC. 1H NMR (DMSO-d6): 0.86 (t, J ¼ 6.9, 3H, CH3),
1.46 (d, J ¼ 6.5, 6H, CH3), 1.63 (sex, J ¼ 6.2, 1H, CH), 2.54 (s(br), 1H,
OH), 3.39e3.54 (m, 2H, CH2), 3.77 (s, 3H, OCH3), 3.83 (sep, J ¼ 6.9,
1H, CH), 4.48e4.58 (m, 2H, CH2), 4.63 (s(br), 2H, CH2), 5.85 (d, 1H,
J ¼ 6.2, NH), 6.68e6.88 (m, 3H, ArH), 7.50 (s(br), 1H, NH), 7.78 (s, 1H,
CH), 9.32(s(br), 1H, OH). 13C NMR (DMSO-d6): 10.56, 15.07, 21.86,
21.95, 45.76, 54.01, 55.75, 62.92, 64.83, 110.73, 113.55, 118.43,
120.65, 126.78, 135.21, 144.01, 147.57, 150.55, 154.42, 158.74. MS m/z
(ESI) 401.2 (M þ H)þ. Anal.: Calcd. for C20H28N6O3: C, 59.98; H, 7.05;
N, 20.98. Found: C, 59.63; H, 7.05; N, 20.59.
4.3.19. 4-Chloro-2-{[2-((R)-1-hydroxymethylpropylamino)-9-
isopropyl-9H-purin-6-ylamino]-methyl}-phenol (3p)
The compound was prepared from 2h and (R)-2-amino-butan-
1-ol (excess) by heating at 160 ꢀC for 3 h. Yield: 65%; white solid.
M.p. ¼ 180e183 ꢀC. 1H NMR (DMSO-d6): 0.82 (t, J ¼ 7.2, 3H, CH3),
1.46 (d, J ¼ 6.6, 6H, CH3), 3.34e3.48 (m, 2H, CH2), 3.79 (sex, J ¼ 7.3,
1H, CH), 4.46e4.60 (m, 4H, CH, CH2, OH), 5.89 (d, J ¼ 7.3, 1H, NH),
6.79 (d, J ¼ 8.4, 1H, ArH), 7.05 (d, J ¼ 8.4, 1H, ArH), 7.10 (s, 1H, ArH),
7.65 (s(br), 1H, NH), 7.81 (s, 1H, CH), 10.11 (s(br), 1H, OH). 13C NMR
(DMSO-d6): 11.14, 22.51, 22.61, 24.43, 46.35, 54.62, 63.49, 114.13,
117.22, 122.78, 127.67, 128.21, 129.27, 135.91, 151.37, 154.34, 154.99,
159.39. MS m/z (ESI) 405.1 (M þ H)þ. Anal.: Calcd for C19H25ClN6O2:
C, 56.36; H, 6.22; N, 20.76. Found: C, 56.29; H, 6.31; N, 20.53.
4.3.15. 2-{[2-((S)-1-Hydroxymethylpropylamino)-9-isopropyl-9H-
purin-6-ylamino]-methyl}-6-methoxyphenol (3l)
The compound was prepared by heating of 2g in (S)-2-amino-
butan-1-ol (excess) at 160 ꢀC for 3 h. Yield: 40%; almost white solid.
M.p. ¼ 100e102 ꢀC. 1H NMR (DMSO-d6): 0.86 (t, J ¼ 6.9, 3H, CH3),
1.46 (d, J ¼ 6.5, 6H, CH3), 1.63 (sex, J ¼ 6.2, 1H, CH), 2.54 (s(br), 1H,
OH), 3.39e3.54 (m, 2H, CH2), 3.77 (s, 3H, OCH3), 3.83 (sep, 1H,
J ¼ 6.9, CH), 4.48e4.58 (m, 2H, CH2), 4.63 (s(br), 2H, CH2), 5.85
(d, J ¼ 6.2,1H, NH), 6.68e6.88 (m, 3H, ArH), 7.50 (s(br),1H, NH), 7.78
(s, 1H, CH), 9.32 (s(br), 1H, OH). 13C NMR (DMSO-d6): 10.56, 15.07,
21.86, 21.95, 45.76, 54.01, 55.75, 62.92, 64.83, 110.73, 113.55, 118.43,
120.65, 126.78, 135.21, 144.01, 147.57, 150.55, 154.42, 158.74. MS m/z
(ESI) 401.2 (M þ H)þ. Anal.: Calcd. for C20H28N6O3: C, 59.98; H, 7.05;
N, 20.98. Found: C, 59.63; H, 7.05; N, 20.59.
4.3.20. 4-Chloro-2-{[2-(2-hydroxy-2-methylpropylamino)-9-
isopropyl-9H-purin-6-ylamino]-methyl}-phenol (3q)
The compound was prepared from 2h and 1-amino-2-
methylpropan-2-ol by heating at 160 ꢀC in NMP in the presence
of N,N-diisopropyl ethyl amine for 24 h. The crude product was
purified by flash chromatography. Yield: 40%; white solid.
M.p. ¼ 183e186 ꢀC 1H NMR (DMSO-d6): 1.06 (s, 6H, CH3), 1.46
(d, J ¼ 6.6, 6H, CH3), 3.23 (d, J ¼ 5.7, 2H, CH2), 4.47e4.58 (m, 3H, CH2,
CH), 4.75 (s(br), 1H, OH), 6.07 (t, J ¼ 5.7, 1H, NH), 6.79 (d, J ¼ 8.3, 1H,
ArH), 7.06e7.12 (m, 2H, ArH), 7.75 (s(br), 1H, NH), 7.84 (s, 1H, CH),
10.17 (s(br),1H, OH). 13C NMR (DMSO-d6): 17.76, 22.59, 26.88, 28.03,
46.30, 52.91, 70.51, 115.26, 117.25, 122.75, 127.70, 128.20, 136.03,
154.33, 154.37, 159.89. MS m/z (ESI) 405.2 (M þ H)þ. Anal.: Calcd for
C19H25ClN6O2: C, 56.36; H, 6.22; N, 20.76. Found: C, 56.10; H, 6.28;
N, 20.49.
4.3.16. 2-[(9-Isopropyl-2-piperazin-1-yl-9H-purin-6-ylamino)-
methyl]-6-methoxyphenol (3m)
The compound was prepared by heating of 2g in molten
piperazine (excess) at 160 ꢀC for 3 h. Yield: 65%; almost white solid.
M.p. ¼ 158e160 ꢀC. 1H NMR (DMSO-d6): 1.45 (d, J ¼ 6.90, 6H, CH3),
2.55e2.73 (m, 4H), 3.45e3.59 (m, 4H), 3.76 (s, 3H, eOCH3), 4.54
(sep, J ¼ 6.90, 1H, CH), 4.61 (s(br), 2H, CH2), 6.66 (t, J ¼ 7.7, 1H, ArH),
6.75e6.83 (m, 2H, ArH), 7.55 (s(br), 1H, NH), 7.83 (s, 1H, CH). 13C
NMR (DMSO-d6): 21.29, 22.58, 45.87, 46.06, 46.27, 56.31, 60.30,
110.96, 114.07, 118.94, 121.03, 127.34, 136.35, 144.38, 147.92, 151.01,
154.76, 158.94. MS m/z (ESI) 398.1 (M þ H)þ. Anal.: Calcd. for
C20H27N7O2: C, 60.44; H, 6.85; N, 24.67. Found: C, 60.25; H, 6.87; N,
24.73.
4.3.21. 4-Chloro-2-{[2-((R)-1-ethyl-2-hydroxypropylamino)-9-
isopropyl-9H-purin-6-ylamino]-methyl}-phenol (3r)
The compound was prepared from 2h and (2RS, 3R)-3-
aminopentan-2-ol by heating at 160 ꢀC in NMP in the presence of
N,N-diisopropyl ethyl amine for 72 h. The crude product was
purified by flash chromatography. Yield: 29.5%; off white solid. The
product is a mixture of diastereoisomers. M.p. ¼ 86e91 ꢀC. 1H NMR
(DMSO-d6): 0.78 (t, J ¼ 6.9, 3H, CH3), 0.97 (t, J ¼ 5.5, 3H, CH3), 1.43
(d, J ¼ 6.7, 6H, CH3), 3.69 (qui, J ¼ 6.9, 2H, CH2), 4.41e4.72 (m, 4H,
CH2, CH, CH), 5.92 (d, J ¼ 6.6, 1H, NH), 6.76 (d, J ¼ 8.5, 1H, ArH),
7.02e7.08 (m, 2H, ArH), 7.62 (s(br), 1H, NH), 7.77 (s, 1H, CH), 10.12
(s(br), 1H, OH). 13C NMR (DMSO-d6): 10.78, 20.30, 21.88, 22.53,
45.63, 57.82, 66.94, 68.77, 113.19, 116.53, 122.09, 127.00, 127.57,
128.58, 135.21, 151.25, 153.66, 154.29, 159.22. MS m/z (ESI) 419.2
(M þ H)þ. Anal.: Calcd for C20H27ClN6O2: C, 57.34; H, 6.50; N, 20.06.
Found: C, 57.31; H, 6.52; N, 19.89.
4.3.17. 4-Chloro-2-{[2-(4-hydroxycyclohexylamino)-9-isopropyl-
9H-purin-6-ylamino]-methyl}-phenol (3n)
The compound was prepared by heating of 2h in excess of 4-
aminocyclohexanol at 160 ꢀC for 12 h. The crude product was
purified by flash chromatography. Yield: 70%; white solid.
M.p. ¼ 201e204 ꢀC. 1H NMR (DMSO-d6): 1.11e1.18 (m, 4H), 1.24
(d, J ¼ 6.6, 6H, CH3), 1.46e1.84 (m, 4H), 3.39 (qui, J ¼ 7.0, CH), 3.57
(s(br), 1H, OH), 4.44 (sex, J ¼ 7.7, 1H, CH), 4.48e4.57 (m, 3H, CH2,
CH), 6.06 (d, J ¼ 7.7, 1H, NH), 6.79 (d, J ¼ 8.4, 1H, ArH), 7.04e7.08
(m, 2H, ArH), 7.58 (s(br), 1H, NH), 7.80 (s, 1H, CH), 10.01 (s(br),
1H, OH). 13C NMR (DMSO-d6): 22.51, 30.93, 34.81, 42.03, 46.13,
49.91, 69.04, 111.12, 114.10, 117.88, 120.60, 126.71, 135.21, 139.82,
147.57, 150.55, 154.42, 158.74. MS m/z (ESI) 431.3 (M þ H)þ. Anal.:
Calcd. for C21H27ClN6O2: C, 58.53; H, 6.32; N, 19.50. Found: C, 58.22;
H, 6.31; N, 19.55.
4.3.22. 4-Chloro-2-{[2-((S)-2-hydroxy-1,2-dimethyl-propylamino)-
9-isopropyl-9H-purin-6-ylamino]-methyl}-phenol (3s)
The compound was prepared from 2h and (S)-3-amino-2-
methylbutan-2-ol by heating at 160 ꢀC in NMP in the presence of
N,N-diisopropyl ethyl amine for 24 h. The crude product was
purified by flash chromatography. Yield: 47%; white amorphous
solid (foam). 1H NMR (DMSO-d6): 1.25e1.31 (m, 9H, CH3), 1.53
(d, J ¼ 6.6, 6H, CH3), 4.02 (qui, J ¼ 7.0, 1H, CH), 4.50e4.55 (s(br), 2H,
4.3.18. 2-{[2-(4-Aminocyclohexylamino)-9-isopropyl-9H-purin-6-
ylamino]-methyl}-4-chlorophenol (3o)
The compound was prepared from 2h and 1,4-trans-dia-
minocyclohexane (excess) by heating at 160 ꢀC for 4 h. Yield: 38%;
white solid. M.p. ¼ 100e103 ꢀC. 1H NMR (DMSO-d6): 0.97e1.21

M. Zatloukal et al. / European Journal of Medicinal Chemistry 61 (2013) 61e72
71
CH2), 4.59 (sep, J ¼ 6.9, 1H, CH), 5.01 (s, 1H, OH), 6.84 (d, J ¼ 8.4, 1H,
ArH), 6.99 (s(br), 1H, NH), 7.13e7.15 (m, 2H, ArH), 7.52 (s, 1H, CH).
13C NMR (DMSO-d6): 17.02, 22.53, 24.58, 28.30, 40.91, 46.96, 56.72,
74.10, 114.22, 119.47, 124.25, 126.93, 129.56, 130.62, 135.28, 150.11,
154.24, 154.81, 158.70. MS m/z (ESI) 419.3 (M þ H)þ. Anal.: Calcd for
C20H27ClN6O2: C, 57.34; H, 6.50; N, 20.06. Found: C, 57.10; H, 6.43;
N, 19.91.
Ascent microplate reader (Labsystems). IC50 (the drug concentra-
tion that reduced the number of viable cells to 50%) values were
determined from the doseeresponse curves.
4.4.3. Kinase inhibition assays
CDK2/Cyclin E kinase was produced in Sf9 insect cells via
baculoviral infection and purified on a NiNTA column (Qiagen).
CDK5/p35, CDK7Cyclin H/MAT1 and CDK9/Cyclin T1 were
purchased from ProQinase GmbH. The kinase reactions were
assayed with 1 mg/ml histone H1 (for CDK2 and CDK5) or
(YSPTSPS)2KK peptide (for CDK7 and CDK9) in the presence of 15/
4.3.23. 2-{[2-((R)-1-Ethyl-2-hydroxypropylamino)-9-isopropyl-
9H-purin-6-ylamino]-methyl}-4-fluorophenol (3t)
The compound was prepared from 2i and (2RS, 3R)-3-
aminopentan-2-ol by heating at 160 ꢀC in NMP in the presence of
N,N-diisopropyl ethyl amine for 72 h. The crude product was
purified by flash chromatography. Yield: 26%; an off white amor-
phous solid. The product is a mixture of diastereoisomers. The free
base was transferred into hydrochloride salt; m.p. ¼ 100e106 ꢀC;
yellowish solid. 1H NMR (DMSO-d6): 0.81 (t, J ¼ 6.9, 3H, CH3),
0.99 (t, J ¼ 5.5, 3H, CH3), 1.46 (d, J ¼ 6.7, 6H, CH3), 3.60e3.72 (m, 2H,
CH2), 4.41e4.65 (m, 4H, CH2, CH, CH), 5.94 (d, J ¼ 6.6, 1H, NH), 6.76
(m, 1H, ArH), 6.83e6.88 (m, 2H, ArH), 7.62 (s(br), 1H, NH), 7.79 (s,
1H, CH), 9.88 (s(br), 1H, OH). 13C NMR (DMSO-d6): 10.81, 20.00,
22.12, 22.53, 45.63, 55.58, 66.94, 65.25, 112.58, 113.25, 115.00,
115.17, 116.38, 116.48, 122.69, 128.70, 129.12, 129.01, 129.82, 136.00,
151.27, 152.50, 154.42, 155.03, 157.52, 160.00. MS m/z (ESI) 403.4
(M þ H)þ. Anal. Calcd. for C20H27FN6O2: C, 59.69; H, 6.76; N, 20.88.
Found: C, 59.38; H, 6.82; N, 20.63.
0.15/1.5/1.5
ATP and of the test compound in a final volume of 10
a reaction buffer (60 mM HEPES-NaOH, pH 7.5, 3 mM MgCl2, 3 mM
MnCl2, 3 M Na-orthovanadate,1.2 mM DTT, 2.5 g/50 l PEG20.000).
The reactions were stopped by adding 5 l of 3% aq. H3PO4. Aliquots
mM ATP (for CDK2/CDK5/CDK7CDK9), 0.05
mCi [
g-
33P]
ml, all in
m
m
m
m
were spotted onto P-81 phosphocellulose (Whatman), washed 3ꢁ
with 0.5% aq. H3PO4 and finally air-dried. Kinase inhibition was
quantified using a FLA-7000 digital image analyzer (Fujifilm). The
concentration of the test compounds required to decrease the CDK
activity by 50% was determined from doseeresponse curves and
designated as IC50 [29,36].
4.4.4. Immunoblotting
Immunoblotting analysis was performed as described earlier
[29,36]. Briefly, cellular lysates were prepared by harvesting cells
in Laemmli sample buffer. Proteins were separated on SDS-
polyacrylamide gels and electroblotted onto nitrocellulose
membranes. After blocking, the membranes were incubated with
specific primary antibodies overnight, washed and then incubated
with peroxidase-conjugated secondary antibodies. Finally, peroxi-
dase activity was detected with ECLþ reagents (AP Biotech) using
a CCD camera LAS-4000 (Fujifilm).
4.3.24. 4-Fluoro-2-{[2-((R)-1-hydroxymethylpropylamino)-9-
isopropyl-9H-purin-6-ylamino]-methyl}-phenol (3u)
The compound was prepared from 2i and (R)-2-amino-butan-1-
ol (excess) by heating at 160 ꢀC for 3 h. Yield: 82%; white solid. Re-
crystallized from ethyl acetate. M.p. ¼165e167 ꢀC. 1H NMR (DMSO-
d6): 0.84 (t, J ¼ 6.9, 3H, CH3), 1.46 (d, J ¼ 6.0, 6H, CH3), 3.40e3.46
(m, 2H, CH2), 3.76 (sex, J ¼ 6.9, 1H, CH), 4.49e4.58 (m, 4H, CH,
CH2, OH), 5.79 (d, J ¼ 6.9, 1H, NH), 6.75e6.93 (m, 3H, ArH), 7.51
(s(br), 1H, NH), 7.79 (s, 1H, CH), 9.75 (s(br), 1H, OH). 13C NMR
(DMSO-d6): 11.13, 22.49, 22.59, 24.43, 46.36, 54.62, 63.50, 113.90,
114.19, 114.85, 115.15, 116.38, 116.48, 122.69, 128.53, 128.73, 128.77,
128.82, 135.90, 151.27, 151.57, 154.42, 155.03, 157.52, 159.40. MS m/z
(ESI) 389.5 (M þ H)þ. Anal.: Calcd for C19H25FN6O2: C, 58.75; H,
6.49; N, 21.63. Found: C, 58.62; H, 6.51; N, 21.44.
4.4.5. Cell cycle analysis
Sub-confluent cells were treated with test compounds at
different concentrations for 24 h. The cultures were pulse-labeled
with 10 m
M 5-bromo-20-deoxyuridine (BrdU) for 30 min at 37 ꢀC
prior to harvesting. The cells were then washed in PBS, fixed with
70% ethanol, and denatured in 2 M HCl. Following neutralization,
the cells were stained with anti-BrdU fluorescein-labeled anti-
bodies, washed, stained with propidium iodide and analyzed by
flow cytometry using a 488 nm laser (Cell Lab Quanta SC, Beckman
Coulter) as described previously [29,36].
4.4. Biological assays
4.4.1. Antibodies
Specific antibodies were purchased from SigmaeAldrich (anti-
4.4.6. p53-Dependent transcriptional activity
a
-tubulin, clone DM1A, peroxidase-labeled secondary antibodies),
To measure p53-dependent transcriptional activity, b-galacto-
Santa Cruz Biotechnology (anti-Mcl-1, clone S-19; anti-PARP, clone
F-2; anti-Mdm-2, clone SMP14; anti-Bcl-2), DAKO Cytomation
(anti-caspase-3), Roche Applied Science (anti-5-bromo-20-deoxy-
uridine-fluorescein, clone BMC 9318), Jackson ImmunoResearch
Laboratory (fluorescein-conjugated Goat Anti-Mouse IgG), Bethyl
Laboratories (anti-phospho RNA polymerase II (S5); anti-phospho
RNA polymerase II (S2)), Millipore (anti-RNA polymerase II, clone
ARNA-3), Cell Signaling (anti-PUMA) or were a generous gift from
sidase activity was determined in the human melanoma cell line
Arn-8, stably transfected with a p53-responsive reporter construct
pRGC
with the inhibitors, the cells were permeabilized with 0.3% Triton
X-100 for 15 min, and then 4-methylumbelliferon-ß- -galactopyr-
anoside was added as a substrate to a final concentration of 80 M.
DfoslacZ as described before [29,36]. After 24 h incubation
D
m
After 1 h, the fluorescence was measured at 355/460 nm (ex/em)
with a Fluoroskan Ascent microplate reader (Labsystems).
Dr. B. Vojtesek (anti-p53, clone DO-1; anti-p21waf1, clone 118; anti-
ꢀꢀ
PCNA).
4.4.7. Caspase-3/7 assay
The cells were homogenized in an extraction buffer (10 mM
KCl, 5 mM HEPES, 1 mM EDTA, 1 mM EGTA, 0.2% CHAPS, inhibitors
of proteases, pH 7.4) on ice for 20 min. The homogenates were
clarified by centrifugation at 10,000 ꢁ g for 30 min at 4 ꢀC, and
then the proteins were quantified and diluted to equal concen-
4.4.2. Cell maintenance and cytotoxicity assays
The cytotoxicity of the studied compounds was determined
using cell lines of different histological origin as described earlier
[29,36]. Briefly, the cells were assayed with compounds using
three-fold dilutions in triplicate. Treatment lasted for 72 h, followed
by addition of Calcein AM solution, and measurement of the fluo-
rescence of live cells at 485 nm/538 nm (ex/em) with a Fluoroskan
trations. Lysates were then incubated for 1 h with 100
mM
Ac-DEVD-AMC as a substrate in the assay buffer (25 mM PIPES,
2 mM EGTA, 2 mM MgCl2, 5 mM DTT, pH 7.3). To serve as negative

72
M. Zatloukal et al. / European Journal of Medicinal Chemistry 61 (2013) 61e72
pharmacokinetics of the cyclin-dependent kinase inhibitor R-roscovitine in
the mouse, Mol. Cancer Ther. 4 (2005) 125e139.
controls, the lysates were supplemented with 100 mM Ac-DEVD-
CHO, which is a caspase-3/7 inhibitor. The fluorescence of the
product was measured using a Fluoroskan Ascent microplate
reader (Labsystems) at 355/460 nm (ex/em) as described previ-
ously [29].
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Rank correlation coefficients were calculated in R.
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Acknowledgment
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This work was supported by the Czech Science Foundation
(GACR 305/12/0783, GACR 301/08/1649) and Ministry of Education,
Youth and Sports, Czech Republic (ED0007/01/01 e Centre of the
Region Haná for Biotechnological and Agricultural Research).
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