Preparation and biological activity of 6-benzylaminopurine derivatives in plants and human cancer cells

Article abstract of DOI:10.1016/j.bmc.2005.09.004

To study the structure-activity relationships of aromatic cytokinins, the cytokinin activity at both the receptor and cellular levels, as well as CDK inhibitory and anticancer properties of 38 6-benzylaminopurine (BAP) derivatives were compared in various

Full text of DOI:10.1016/j.bmc.2005.09.004

Bioorganic & Medicinal Chemistry 14 (2006) 875–884
Preparation and biological activity of 6-benzylaminopurine
derivatives in plants and human cancer cells
a
a
a,b
a
Karel Dolezˇal,a, Igor Popa, Vladimır Krystof, Lukas Spıchal, Martina Fojtıkova,
´ˇ
*
´
ˇ
Jan Holub, Rene Lenobel, Thomas Schmulling and Miroslav Strnad
¨
´
´
´
a
a
b
a
´
aLaboratory of Growth Regulators, Palacky University and Institute of Experimental Botany AS CR,
ˇ
Slechtitelu 11, 783 71 Olomouc, Czech Republic
bFree University of Berlin, Institute of Biology/Applied Genetics, Albrecht-Thaer-Weg 6, D-14195 Berlin, Germany
Received 18 February 2005; accepted 6 September 2005
Available online 7 October 2005
Abstract—To study the structure–activity relationships of aromatic cytokinins, the cytokinin activity at both the receptor and cel-
lular levels, as well as CDK inhibitory and anticancer properties of 38 6-benzylaminopurine (BAP) derivatives were compared in
various in vitro assays. The compounds were prepared by the condensation of 6-chloropurine with corresponding substituted ben-
zylamines. The majority of synthesised derivatives exhibited high activity in all three of the cytokinin bioassays employed (tobacco
callus, wheat senescence and Amaranthus bioassay). The highest activities were obtained in the senescence bioassay. For some com-
pounds tested, significant differences of activity were found in the bioassays used, indicating that diverse recognition systems may
operate and suggesting that it may be possible to modulate particular cytokinin-dependent processes with specific compounds. Posi-
tion-specific steric and hydrophobic effects of different phenyl ring substituents on the variation of biological activity were con-
firmed. In contrast to their high activity in bioassays, the BAP derivatives were recognised with much lower sensitivity than
trans-zeatin in both Arabidopsis thaliana AHK3 and AHK4 receptor assays. The compounds were also investigated for their effects
on cyclin-dependent kinase 2 (CDK2) and for antiproliferative properties on cancer and normal cell lines. Several of the tested com-
pounds showed stronger inhibitory activity and cytotoxicity than BAP. There was also a significant positive correlation of the inhib-
itory effects on human and plant CDKs with cell proliferation of cancer and cytokinin-dependent tobacco cells, respectively. This
suggests that at least a part of the antiproliferative effect of the new cytokinins was due to the inhibition of CDK activity.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
effects might be based on the development and examina-
tion of BAP derivatives that do not exhibit unwanted
side effects. For example, the high biological activity
of 6-(3-hydroxybenzylamino)purine (meta-topolin, mT)
in different bioassays has been described by several
authors,3,4 The positive effect of mT versus BAP was lat-
er shown in the in vitro shoot and root production and
post vitro rooting of Spathiphyllum floribundum. Plants
grown on media containing 10 lM of mT rooted better
during acclimatization compared with those developed
on an equimolar concentration of BAP. This fact can
be explained by differences in the metabolism of BAP
and mT.2 In addition, it was shown that the endogenous
occurrence of hydroxylated BAP analogues in Zantedes-
chia aethiopica fruits is correlated with the self-inhibition
of fruit senescence.5 The aim of this study was to pre-
pare a large family of other BAP derivatives, substituted
on the phenyl ring, and to compare the cytokinin activ-
ity of prepared compounds in three different bioassays,
based on the stimulation of tobacco callus growth, the
6-Benzylaminopurine (BAP) and its derivatives are
active and easily obtainable plant growth promoting
substances. They have long been thought to be purely
synthetic compounds. However, some of them have also
been detected and identified in different plant tissues (for
details, see1).
BAP is also one of the most effective and affordable cyt-
okinins used in many micropropagation systems. Never-
theless, it has disadvantages in some crops, including
acclimatization problems, heterogenity in growth and
rooting inhibition.2 One way to eliminate the side
Keywords: 6-Benzylaminopurine; Cytokinin; Receptor; Bioassay;
Cyclin-dependent kinase; Cytotoxicity.
*
Corresponding author. Tel.: +420 585634940; fax: +420
585634870; e-mail: dolezal@risc.upol.cz
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.09.004

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K. Dolezˇal et al. / Bioorg. Med. Chem. 14 (2006) 875–884
retention of chlorophyll in excised wheat leaves and the
dark induction of betacyanin synthesis in Amaranthus
cotyledons. The new cytokinin derivatives have also
been screened for their capacity to be recognised by
the recently discovered cytokinin receptor proteins
AHK3 and AHK4.6–8
2. Results and discussion
2.1. Synthesis
Thirty-eight analogues of BAP with various substituents
attached to the phenyl ring had been synthesised (Table
1) in order to determine their structure–activity relation-
ships. The prepared compounds were characterised by
elemental analysis, TLC, melting point, ES + MS (Table
Although cytokinins regulate many cellular processes,
the control of cell division is crucial and is considered
diagnostic for this class of phytohormones; hence, the
generic name is ÔcytokininsÕ.9 Cytokinins are involved
in the regulation of both G1/S and G2/M transitions
of the cell cycle. They elevate the expression of the
CYCD3 gene, which encodes a D-type cyclin.10,11 In ani-
mal cells, D-cyclins are regulated by a wide variety of
growth factors and play a key role in regulating the pas-
sage through the cell cycle restriction point in G1. It has
also been shown that the constitutive expression of
CYCD3 can bypass the cytokinin requirement for cell
proliferation, thus causing the cytokinin-independent
growth of Arabidopsis thaliana calli.10
2) as well as H and 13C NMR (Supplementary data).
1
The previously published syntheses of BAPs, monosub-
stituted on the benzyl ring with CH3, NH2, NO2, OH,
Table 1. Structures of prepared compounds
R4
R5
R6
R3
R2
HN
N
Cytokinins are also important for the regulation of the
G2/M transition. In tobacco BY-2 cells, which are cyto-
kinin-autonomous, endogenous zeatin cytokinins peak
around S and M phases.12 The application of lovastatin,
an inhibitor of mevalonic acid synthesis, inhibits cytoki-
nin accumulation and mitosis, respectively. Applied
trans-zeatin (tZ) overrides the lovastatin block. This
indicates that tZ is indispensable for the G2/M transi-
tion.13 The regulation of the G2/M transition is medi-
ated by activated cyclin-dependent kinases (CDK). In
pea root tissues, mRNA of mitotic CDK was induced
within 10 min by auxin application. High levels of an
inactive CDC2-like protein were also found in tobacco
pith cultured on auxin medium.14 The cytokinin effect
has been linked to the activation of a Cdc25-like phos-
phatase which dephosphorylates plant homologue of
CDK1 on Thr 14/Tyr15 residues.15 This process pro-
vides one of the potential links between cytokinin and
auxin effects on the regulation of the cell cycle.
N
N
N
H
Compound
R2
R3
R4
R5
R6
1
2
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3O
H
H
H
H
F
H
F
H
H
3
H
Cl
H
F
H
4
H
Cl
H
H
5
H
H
Cl
H
6
H
H
H
7
Br
H
H
Br
H
H
8
H
Br
H
9
H
H
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
H
I
H
H
CH3
H
H
H
H
CH3
H
H
H
H
CH3
H
H
CH3O
H
H
H
CH3O
H
H
H
H
CH3O
H
H
Recently, another important property of cytokinin ana-
logues has been proved. The natural isoprenoid and aro-
matic cytokinins were found to inhibit several human
protein kinases in a non-specific manner, including
CDKs. On the other hand, the screening of chemically
synthesised cytokinin analogues revealed selective
CDK inhibitor olomoucine, that is, the BAP derivative
6-benzylamino-2-(2-hydroxyethylamino)-9-methylp-
urine. Small modifications of the BAP molecule led to
the specific inhibition of several important protein ki-
nases like CDK1/cyclin B, CDK2/cyclin A, CDK2/cy-
clin E, brain CDK5/p35 and ERK1/MAP kinase.16
Because of the strong anticancer properties of these
compounds (olomoucine, roscovitine, etc.),17,18 the new-
ly prepared BAP analogues have also been tested in an
in vitro CDK2/cyclin E kinase inhibition assay and also
for their possible cytotoxic activity against selected can-
cer cell line lines and normal mouse fibroblasts (NIH/
3T3). In this report, we are also trying, for the first time,
to correlate the relationships between the molecular
mode of action with appropriate cellular effects.
H
NO2
H
H
H
Cl
NO2
Cl
H
H
Cl
H
H
Cl
H
H
CH3O
CH3O
H
CH3O
H
H
CH3O
CH3O
H
H
CH3O
CH3O
H
H
H
CH3O
H
CH3O
H
CH3O
CH3O
F
CH3O
H
CH3O
H
F
H
F
H
F
F
F
F
H
F
F
H
H
F
Cl
H
F
F
H
H
H
H
H
H
H
H
H
Cl
F
H
H
F
H
OH
OH
OH
H
CH3O
H
H
H
CH3O
H
H
OH
OH
CH3O
H
H
OH
OH
MeO
H

K. Dolezˇal et al. / Bioorg. Med. Chem. 14 (2006) 875–884
877
Table 2. Elemental analyses, ES+ mass spectrometry analyses and melting points of prepared substituted 6-benzylaminopurines
Compound
Elemental analysis calculated/found
%H
Mp (ꢁC)
ES–MS [M+H+]
%C
%N
1
2
59.3/59.1
59.3/59.1
59.3/59.2
55.5/55.7
55.5/55.2
55.5/55.3
47.4/47.6
47.4/47.1
47.4/47.8
41.0/41.4
65.2/65.0
65.2/64.8
65.2/65.8
61.2/60.7
53.3/53.1
53.3/53.1
49.0/49.4
49.0/49.1
58.9/59.3
58.9/59.3
58.9/59.7
58.9/59.7
57.1/57.3
57.1/57.2
55.2/55.2
55.2/54.4
51.6/52.2
51.6/51.2
51.6/50.6
51.9/51.8
51.9/51.8
57.6/57.8
57.6/57.1
56.0/55.4
56.0/56.4
55.8/56.1
4.1/3.8
4.1/3.9
4.1/4.0
3.8/4.2
3.8/3.9
3.8/3.8
3.3/3.5
3.3/3.4
3.3/3.4
2.8/2.8
5.5/5.8
5.5/6.0
5.5/5.4
5.1/5.3
3.7/3.6
3.7/3.5
3.1/3.1
3.1/3.2
5.3/5.5
5.3/4.9
5.3/5.6
5.3/5.3
5.4/5.3
5.4/5.2
3.5/3.4
3.5/3.2
2.9/2.7
2.9/2.7
2.9/2.8
3.3/3.2
3.3/3.3
4.8/4.8
4.8/4.0
4.3/4.8
4.3/4.0
5.0/5.0
28.8/27.8
28.8/27.7
28.8/27.9
27.0/26.0
27.0/26.2
27.0/26.6
23.0/22.5
23.0/23.1
23.0/22.3
20.0/19.5
29.3/28.8
29.3/29.1
29.3/27.5
27.4/26.7
31.1/30.8
31.1/30.9
23.8/23.1
23.8/23.0
24.5/23.5
24.5/23.5
24.5/23.8
24.5/23.4
22.2/21.9
22.2/21.9
26.8/26.2
26.8/26.4
25.1/24.5
25.1/24.5
25.1/24.5
25.2/24.9
25.2/24.7
25.8/25.5
25.8/24.9
27.2/26.6
27.2/27.5
23.2/22.9
245–246
247–248
267–268
227–228
252–253
277–278
222–223
256–257
292–293
248–249
267–268
232–235
288–290
263–265
356–357
335–337
309–311
332–333
229–230
189–191
262–263
311–313
208–210
227–229
255–256
261–262
280–281
280–281
274–275
172–173
262–263
253–254
253–254
256–257
273–274
247–248
244
244
244
260
260
260
304
304
304
352
240
240
240
256
271
271
295
295
286
286
286
286
316
316
262
262
280
280
280
279
279
272
272
258
258
302
3
4
5
6
7
8
9
10
11
12
13
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
CH3O and Cl, were achieved by condensing 6-(methyl-
mercapto)purine with 2–3 molecular equiv of corre-
sponding amines.20 However, this method usually gives
lower yields, compared with our results, which were
achieved using 6-chloropurine. The melting points,
reported for the compounds prepared using an earlier
method, are also somewhat lower. The preparation of
some BAP derivatives, substituted by single halogen
atom on the phenyl ring, using 6-(methylmercapto)pu-
rine has been previously reported by other authors.28,29
However, no physico-chemical data to validate the iden-
tity and purity of the prepared compounds are present in
these reports. Moreover, because different assays were
used to evaluate their biological activity, it is difficult
to compare the results.
Derivatives 1 and 2 containing a fluorine atom in the
ortho and/or meta position of the phenyl ring topped
BAP activity in the Amaranthus bioassay by 16% and
40%, respectively. Among the monosubstituted BAP
derivatives, any substituent at the para-position (com-
pounds 3, 6, 9, 13, 16 and 18) reduced the activity dras-
tically in this bioassay. In contrast, all tested di- and
trifluoro derivatives 27–31 were found to be very active
(in all three biotests), even those containing the substitu-
ent in the para position (27, 29 and 31). However, di-
and trisubstituted hydroxy/methoxy derivatives (21–26
and 33–37) have been found to be inactive in this assay.
A similar pattern of relative activity was also obtained in
the callus bioassay, except for the high activity of 3.
In contrast, the senescence bioassay revealed responses
different from those of the other two biotests. As we pre-
viously reported for endogenously occurring 2- and 3-
methoxy derivatives of BAP,19 their activity was either
comparable or slightly lower in the Amaranthus and
tobacco callus bioassays, but double that of BAP in
the senescence assay. This indicates that these com-
pounds may preferentially affect physiological processes
associated with the impact of different stress factors on
2.2. Cytokinin activity in bioassays
The effect of introduced substituents on BAP has been
investigated in three cytokinin bioassays, based on the
stimulation of different physiological processes in plants
(Table 3). As a comparison, BAP itself, a highly active
and widely practically used cytokinin, was employed
as standard compound.

878
K. Dolezˇal et al. / Bioorg. Med. Chem. 14 (2006) 875–884
Table 3. Relative cytokinin bioassay activity of prepared 6-benzylaminopurine derivatives at optimal concentration compared with the activity of 6-
benzylaminopurine (BAP) (100% means 10À5 M BAP for the Amaranthus betacyanin bioassay, 10À4 M BAP in the case of the senescence bioassay
and 10À6 M BAP for the tobacco callus bioassay)
Compound
Amaranthus bioassay
Senescence bioassay
Tobacco callus bioassay
Optimal concentration Relative activity Optimal concentration Relative activity Optimal concentration Relative
(mol lÀ1
)
(%)
(mol lÀ1
)
(%)
(mol lÀ1
)
activity (%)
1
2
10À4
10À4
10À5
10À5
10À5
10À5
10À4
10À4
10À5
10À5
10À5
10À5
10À5
10À4
10À4
10À4
10À4
10À4
10À4
10À5
10À4
10À4
10À4
10À4
10À4
10À4
10À4
10À4
10À4
10À4
10À5
116 ( 3)
140 ( 5)
44 ( 4)
109 ( 8)
96 ( 5)
35 ( 7)
94 ( 6)
71 ( 5)
17 ( 7)
79 ( 3)
98 ( 22)
84 ( 14)
26 ( 10)
23 ( 2)
66 ( 7)
25 ( 2)
19 ( 8)
63 ( 10)
22 ( 3)
12 ( 2)
2 ( 1)
10À4
10À4
10À4
10À4
10À4
10À4
10À4
10À4
10À5
10À4
10À4
10À4
10À4
10À4
10À4
10À4
10À5
10À4
10À4
10À7
10À4
10À4
ntÀ4
10
169 ( 20)
200 ( 25)
95.5 ( 3.5)
116.5 ( 6.5)
82 ( 2)
10À6
10À5
10À6
10À6
10À5
10À6
10À5
10À6
10À6
10À5
10À6
10À6
10À6
10À6
nt
111 ( 21)
135 ( 8)
122 ( 12)
93 ( 4)
94 ( 6)
64 ( 8)
102 ( 5)
85 ( 11)
15 ( 9)
76 ( 8)
118 ( 3)
79 ( 5)
52 ( 8)
39 ( 17)
nt
3
4
5
6
64.5 ( 13.5)
52 ( 14)
48 ( 6)
7
8
9
30 ( 15)
83.5 ( 23)
158 ( 29)
111 ( 16)
35 ( 23)
79 ( 6)
10
11
12
13
16
17
18
19
20
21
22
23
24
25
26
27
28
29
31
33
34
37
60 ( 15)
83 ( 9)
nt
nt
0
nt
nt
117 ( 22)
109 ( 5)
26 ( 11)
16 ( 1)
nt
nt
10À6
nt
7 ( 3)
nt
nt
nt
27 ( 6)
3 ( 3)
2 ( 2)
43 ( 17)
nt
25 ( 2)
nt
nt
nt
nt
nt
nt
88 ( 7)
107 ( 2)
107 ( 5)
108 ( 1)
91 ( 7)
19 ( 3)
43 ( 9)
10À5
10À5
10À5
ntÀ5
10
10À5
10À4
139 ( 2)
156 ( 4)
131 ( 22)
nt
10À6
ntÀ6
10
95 ( 1)
nt
76 ( 4)
101 ( 1)
90 ( 1)
nt
10À5
10À6
nt
141 ( 20)
34 ( 5)
134 ( 12)
10À6
39 ( 6)
the photosynthetic apparatus. In the present study, we
found this tendency to be more or less general among
aromatic cytokinins (Table 3). Moreover, 6-(2,3-dim-
ethoxybenzylamino)purine (21), an inactive compound
in the two other biotests, was still able to exceed the
activity of BAP in the senescence bioassay. Strong differ-
ences in activities of the same cytokinin compounds
(e.g., 3, 18 and 21) in the different bioassays indicate that
it may be possible to design-specific compounds that can
be used to modulate cytokinin-dependent processes in a
targeted fashion. This may also indicate that another
receptor and/or signalling system is involved in mediat-
ing the influence of cytokinins on the senescence process
than in mediating their influence on cell growth and
division.
to systematically investigate the relative activities of nat-
ural cytokinin metabolites.24 However, in contrast with
our bioassay results, only few BAP derivatives tested
were able to activate AHK4 significantly (namely 2, 3
and 15), although with much lower sensitivity (below
10%), when compared to trans-zeatin (Fig. 1). Most of
the prepared compounds were recognised by the
AHK3 receptor (1–5, 14, 17, 20, 27 and 29), but also
only with low sensitivity, between 10% and 32% of
trans-zeatin activity (Fig. 1). The differences of activities
in bioassays and the receptor assay may indicate that
either AHK2. the cytokinin receptor that was not tested
in this study, preferentially recognises BAP derivatives
or that another recognition system, also able to interact
with BAP derivatives, exists in plants.
Three membrane-located sensor histidine kinases of A.
thaliana have been identified recently to function as
cytokinin receptors. The AHK4 protein (identical to
CRE1 and WOL)6–8 was shown to bind directly cytoki-
nins8,23 and two of its homologues, AHK2 and AHK3,
have also been found to be cytokinin-binding recep-
tors.23,31 We employed DrcsC Escherichia coli strains
that express AHK4 and AHK37,23 to study the relative
sensitivity of these receptors to the prepared com-
pounds. This system was previously successfully used
2.3. Antitumour and kinase assays
We also tested the prepared compounds for their antit-
umour activity against cell lines derived from human
T-lymphoblastic leukaemia (CEM), promyelocytic leu-
kaemia (HL-60), human malignant melanoma (G-361),
human chronic myelogenous leukaemia (K-562), human
osteogenic sarcoma (HOS), breast carcinoma (MCF-7)
and mouse melanoma (B16). The cytotoxicity of selected
compounds for NIH-3T3 (normal mouse fibroblasts)

K. Dolezˇal et al. / Bioorg. Med. Chem. 14 (2006) 875–884
879
650
600
550
500
450
400
350
300
250
200
150
100
50
AHK3
0
2200
2000
1800
CRE1/AHK4
350
300
250
200
150
100
50
0
Figure 1. Comparison of the sensitivities of CRE1/AHK4 and AHK3 to different cytokinin derivatives in the Escherichia coli assay. Concentration of
tested compounds was 1 lM. The b-galactosidase activity in non-induced strains (control) is indicated by the dotted line. Error bars show SD (n = 3).
tZ means trans-zeatin.
was examined as well. The data obtained from a calcein
AM viability/cytotoxicity assay are presented in Table 4.
The obtained IC50 values show interesting cytotoxic
properties of compound 34 (IC50 = 26.6 lmol/l for
MCF-7), in contrast to other very similar derivatives
(e.g., compounds 35 and 36), which displayed no activity
in this assay. Weak cytotoxicity of several other mono-
substituted halogen-, methyl- and methoxy-BAPs (1, 3,
4, 13, 15 and 16) was also confirmed (Table 4). The cyto-
toxic properties as well as the cytokinin activity of relat-
ed ribosides will be described elsewhere (Dolezˇal et al.,
manuscript in preparation).
been previously reported.32–36 Ribosides have been
shown to be potent inducers of apoptosis. In contrast,
cytokinin free bases did effectively induce HL-60 cell
differentiation and transformation into mature
granulocytes.
It is also known that natural cytokinins are weak and
non-specific inhibitors of various protein kinases.16
However, systematic screening of purine derivatives of
cytokinin origin led to the discovery of 2,6,9-trisubsti-
tuted purines and revealed a large number of highly
active CDK inhibitors such as olomoucine and roscovi-
tine.16,17 To verify the anticancer properties of BAP
analogues, they were screened for the ability to
inhibit human recombinant CDK2 (summarised in
The growth-inhibiting ability of several well-known cyt-
okinins as well as their ribosides on the HL-60 cells has

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K. Dolezˇal et al. / Bioorg. Med. Chem. 14 (2006) 875–884
Table 4. Cytotoxicity of prepared BAP derivatives in calcein AM assay (IC50, lmol/l)
Compound
Cell line/IC50 (lmol/l)
HOS
HL 60
K-562
MCF7
NIH-3T3
G-361
B16
CEM
BAP
1
>166.7
>166.7
>166.7
>166.7
122.6
>166.7
nt
>166.7
147.1
>166.7
59.2
138.9
132.6
105.2
>166.7
>166.7
nt
nt
nt
>166.7
111.4
>166.7
66.4
nt
116.0
>166.7
>166.7
56.6
79.1
nt
2
nt
3
>166.7
95.7
>166.7
146.2
nt
nt
4
109.6
>166.7
>166.7
133.3
>166.7
>166.7
88
>166.7
nt
16.9
nt
58.4
5
>166.7
>166.7
94.7
>166.7
>166.7
>166.7
>166.7
102.5
148.6
nt
>166.7
>166.7
124.6
>166.7
>166.7
72.1
6
nt
nt
10
11
12
13
14
15
16
17
18
24
25
27
28
29
31
33
34
35
36
37
38
>166.7
>166.7
>166.7
>166.7
>166.7
>166.7
>166.7
nt
nt
>166.7
>166.7
>166.7
160.4
nt
156.6
>166.7
72.8
nt
nt
nt
164.1
nt
>166.7
115.8
158.1
nt
>166.7
>166.7
>166.7
>166.7
>166.7
>166.7
153.4
>166.7
nt
>166.7
121.7
142.4
nt
134.9
41.5
84.7
nt
107.8
>166.7
>166.7
>166.7
>166.7
>166.7
99.5
>166.7
>166.7
nt
124.7
>166.7
nt
>166.7
>166.7
>166.7
>166.7
>166.7
>166.7
>166.7
>166.7
>166.7
>166.7
26.6
>166.7
>166.7
>166.7
>166.7
>166.7
>166.7
>166.7
>166.7
>166.7
>166.7
>166.7
>166.7
>166.7
>166.7
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
>166.7
nt
nt
nt
nt
139.7
nt
32.6
nt
nt
nt
nt
nt
>166.7
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
>166.7
64.3
nt
nt
>166.7
69
124.4
46.3
nt
nt
nt
nt
nt
>166.7
>166.7
>166.7
>166.7
nt
nt
nt
nt
>166.7
>166.7
>166.7
nt
nt
>166.7
>166.7
>166.7
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
The following cell lines were used: CEM—human T-lymphoblastic leukaemia, HL-60—promyelocytic leukaemia, G-361—human malignant mel-
anoma, K-562—human chronic myelogenous leukaemia, HOS—human osteogenic sarcoma, MCF-7—human breast carcinoma, B 16—mouse
melanoma and NIH/T3T—mouse normal fibroblasts.
Table 5). The results showed that the attachment of one
halogen (compounds 1–10), methyl (11 and 13) or meth-
oxy (14–16) group at any position of the benzyl ring led
to an approximately 2-fold increase in inhibitory activity
compared to BAP. Nitro derivatives (17 and 18) were
however less potent. Certain losses of inhibitory activity
were also observed after the introduction of more than
one hydroxy and/or methoxy substituents to the benzyl
moiety (21, almost completely inactive 23 and 26). On
the other hand, difluoro, trifluoro and corresponding
chloro derivatives (27–32) still led to a slight increase
in activity. Some recent studies have also suggested that
different substituents on the benzyl ring (Cl and OCH3)
may further enhance the ability of purines to block
CDK1 activity.37,38 Although none of the 6-substituted
purines tested here was found to be a very potent
CDK2/cyclin E inhibitor, we assume that our results
may also be useful for the future design and synthesis
of the next successful class of related trisubstituted pur-
ine-based inhibitors.
okinins. While this is valid for many of the compounds
tested, it is not so for all of them (nitro derivatives)
(Fig. 2). Using high concentrations of different BAP
derivatives, a good correlation between callus growth
inhibition and the ability to inhibit human recombinant
CDK2/cyclin E kinase has been found. This was further
verified by testing selected BAP derivatives in a plant ki-
nase inhibition assay. Figure 3 shows that several of the
cytokinins tested (isopentenyladenine (iP), BAP, 4) also
have the ability to inhibit plant CDKs (in concentrations
higher than 50 lM). On the other hand, 6-(3-nitro-
benzylamino)purine 17, the compound, which did not
show any inhibition of callus growth, was also inactive
in the plant CDK assay (see Figs. 2 and 3 for comparison).
Because these results again confirmed our previous
hypothesis, it might be concluded that the tobacco callus
growth inhibition, which is caused by excessive cytokinin
concentrations, is at least partially due to the cytokinin
inhibition of CDK activity. However, many other en-
zymes besides CDKs, involved in regulation of cell
growth and division, could also be inhibited.16
In along-term experiment, the treatment oftobaccocallus
with cytokinin stimulates the rate of cell division. The
optimal cytokinin concentration for cell proliferation is
typically 10À9 to 10À5 M. At higher concentrations (above
10À5 M) cytokinins become less effective; above about
10À4 M they become inhibitory. This is shown in Figure
2 where the growth curve falls below the dashed line,
which represents growth in the absence of exogenous cyt-
3. Conclusions
In summary, a group of 6-benzylaminopurine deriva-
tives with different phenyl ring substituents has been
prepared. Many of them have been found to be very
active in different cytokinin bioassays. In contrast, only

K. Dolezˇal et al. / Bioorg. Med. Chem. 14 (2006) 875–884
Table 5. Inhibition of CDK2/cyclin E by BAP derivatives at 50 lM
881
concentration, with iP and olomoucine, 2-(2-hydroxyethylamino)-6-
benzylamino-9-methylpurine, as controls
Compound
Residual CDK2 activity (%)
1
36.0 ( 1.5)
37.4 ( 1.2)
40.8 ( 1.8)
27.3 ( 0.3)
36.2 ( 1.1)
39.6 ( 2.4)
26.2 ( 0.6)
27.8 ( 3.2)
37.4 ( 3.1)
33.6 ( 3.1)
34.0 ( 1.9)
47.6 ( 2.8)
68.3 ( 5.0)
48.7 ( 3.7)
34.4 ( 3.9)
78.7 ( 5.9)
67.8 ( 7.2)
34.9 ( 9.4)
47.9 ( 3.8)
56.4 ( 8.0)
92.6 ( 9.8)
99.1 ( 2.4)
44.1 ( 1.9)
47.5 ( 4.3)
43.9 ( 3.3)
62.0 ( 4.6)
50.6 ( 5.0)
7.1 ( 1.8)
2
3
4
5
6
7
8
9
10
11
13
14
15
16
17
Figure 3. Inhibition of histone kinase activity by purine derivatives.
CDK-like protein kinases were purified from Arabidopsis thaliana
callus by binding to p13suc1-Sepharose and assayed in the presence of
increasing concentrations of BAP, 4 and 17. Effects of isopentenylad-
enine (iP) and roscovitine, 2-{[(1-hydroxymethyl)propyl]amino}-6-
benzylamino-9-isopropylpurine, were evaluated as controls in the
same conditions.
18
19
20
21
23
26
27
29
On the other hand, some of the prepared compounds
displayed (in higher concentrations) cytotoxic activity
against both animal and plant cells. This effect may
be, at least partially, caused by the inhibition of CDK
activity in these cells. This would provide a mechanistic
explanation for the dose–response curves of the callus
growth test to cytokinins. The results from the antitu-
mour assay and human recombinant CDK2 inhibition
assay can also help to design novel trisubstituted com-
pounds potent as CDK inhibitors and apoptosis induc-
ers in cancer cells.
34
BAP
iP
Olomoucine
All values are averaged from three independent experiments.
a few were able to interact with the cytokinin AHK
receptors. One tentative conclusion is that a different
sensing mechanism for aromatic cytokinins may exist
in plants.
16
14
12
10
8
BAP
16
BAP
4
17
5
14
18
6
12
10
8
29
control
control
6
6
4
4
2
2
0
0
1,00E-09 1,00E-08 1,00E-07 1,00E-06 1,00E-05 1,00E-04 1,00E-03
1,00E-09 1,00E-08 1,00E-07 1,00E-06 1,00E-05 1,00E-04 1,00E-03
Concentration [mol/l]
Concentration [mol/l]
Figure 2. Typical dose–response curves for cytokinin-induced growth of cytokinin-dependent tobacco callus. Error bars show standard deviations of
the mean for 10 replicate determinations. Dashed line indicates the value for the control treatment without any cytokinin, 4.0 0.4 g.

882
K. Dolezˇal et al. / Bioorg. Med. Chem. 14 (2006) 875–884
4. Materials and methods
general procedures for the preparation of 6-benzylamin-
opurines were described earlier.4,19,20 In brief, 6-chlo-
ropurine was heated with the appropriate primary
amine to 90 ꢁC for 4 h in n-butanol containing an exces-
sive amount of triethylamine. After cooling, the precip-
itated product was filtered off, washed with cold water
and n-butanol and crystallised from dimethylformam-
ide. The identity and purity of the synthesised com-
pounds were confirmed by elemental and melting point
analyses, analytical thin-layer chromatography, high
performance liquid chromatography, MS (Table 2)
and NMR (Supplementary data).
4.1. General procedures
The melting points were determined on a Boetius stage
apparatus and are uncorrected. Analytical thin-layer
chromatography (TLC) was carried out using silica gel
60 WF254 plates (Merck). Elemental analyses (C, H
and N) were performed using an EA1108 CHN analyser
(Fissons Instruments). ES + mass spectra were recorded
using direct probe on Waters ZMD 2000 mass spec-
trometer. The mass monitoring interval was 10–
1500 amu. The spectra were collected using 3.0 s cyclical
scans and applying sample cone voltage 25 V at source
block temperature 150 ꢁC, desolvation temperature
80 ꢁC and desolvation gas flow rate 200 l/h . The mass
spectrometer was directly coupled to a MassLynx data
system. NMR spectra were measured in a Bruker
Avance AV 300 spectrometer operating at a temperature
of 300 K and a frequency of 300.13 MHz (1H) and
75.48 MHz (13C), respectively. Samples were prepared
by dissolving the compounds in DMSO-d6. Tetrameth-
ylsilane (TMS) was used as the internal standard.
4.4. Preparation of 3,5-dihydroxybenzylamine
hydrobromide
3,5-Dimethoxybenzylamine was converted to its hydrox-
ylated analogue as previously described.21,22 Acetanhy-
dride (15 ml) was added, by syringe, to 3,5-
dimethoxybenzylamine solution in 47% HBr under
nitrogen. The stirred reaction mixture was subsequently
heated to 107 ꢁC for 5 h. After evaporation of the acid in
vacuo, ethanol (15 ml) was added to the residue, and the
solvent was again evaporated. The crude product (1.8 g)
was recrystallised from EtOH. Elemental analysis (C, H
and N) (Found: C, 37.9; H, 4.7; N, 6.6. C7H10NO2Br
calcd: C, 38.2; H, 4.6; N, 6.4); TLC (chloroform:metha-
nol:ammonia (8:2:0.1, v/v/v) as the mobile phase): single
spot; mp 197–198 ꢁC; ES + MS: m/z 140 [MH]+; 1H
NMR (DMSO-d6, 300 MHz): 9.45 (NH2), 8.10 (OH),
6.29d (2H, 6H), 6.23tr (4H), 3.82s (7H); 13C NMR
(DMSO-d6, 75 MHz): 159.03 (3C, 5C), 136.05 (1C),
107.18 (2C, 6C), 102.89 (4C), 42.70 (7C).
4.2. Chemicals
6-Benzylaminopurine, 6-chloropurine, 4-methyl umbel-
liferyl galactoside, 4-methylumbelliferone, DMEM, tryp-
sin and DMSO were purchased from Sigma. Aldrich
supplied 2-fluorobenzylamine, 3-fluorobenzylamine, 4-
fluorobenzylamine, 3-chlorobenzylamine, 4-chloroben-
zylamine, 2-bromobenzylamine hydrochloride, 3-bro-
mobenzylamine hydrochloride, 4-bromobenzylamine
hydrochloride, 2-iodobenzylamine, 2-methylbenzyl-
amine, 3-methylbenzylamine, 4-methylbenzylamine,
3-methoxylbenzylamine, 3-nitrobenzylamine hydrochlo-
ride, 4-nitrobenzylamine hydrochloride, 2,4-dichloro-
benzylamine, 3,4-dichlorobenzylamine, 2,3-dimethoxyl-
benzylamine, 3,4-dimethoxylbenzylamine, 3,5-dim-
ethoxylbenzylamine, 2,4-dimethoxylbenzylamine hydro-
chloride, 3,4,5-trimethoxylbenzylamine and 2,4,6-
trimethoxylbenzylamine hydrochloride. Fluka supplied
2-methoxylbenzylamine, 4-methoxylbenzylamine, 2-
chlorobenzylamine, 2,4-difluorobenzylamine, 3,5-difluo-
robenzylamine, 2,4,5-trifluorobenzylamine, 2,3,6-trifluo-
robenzylamine, 2,4,5-trifluorobenzylamine, 2-chloro-4-
fluorobenzylamine and 3-chloro-4-fluorobenzylamine
were purchased from Fluorochem. Merck supplied casa-
mino acids, calcein AM was purchased from Molecular
Probes. [c-33P]ATP was provided by MP Biomedicals.
NiNTA column was purchased from Qiagen. 6-(3,3-Dim-
ethylallylamino)purine (isopentenyladenine, iP), 6-((E)-
4-hydroxy-3-methylbut-2-enylamino)purine (trans-zea-
tin, tZ), olomoucine and bohemine were obtained from
Olchemim (Olomouc, Czech Republic). Milli-Q water
was used throughout. The other solvents and chemicals
used were all of standard pa quality.
4.5. Preparation of 2-hydroxy-3-methoxybenzylamine
hydrobromide and 2,3-dihydroxybenzylamine
hydrobromide
The compounds were prepared from 2,3-dimethoxyben-
zylamine (1.5 ml; 10 mmol) using a procedure similar to
that for 3,5-dihydroxybenzylamine. After refluxing
(107 ꢁC) for 4 h, the reaction mixture was kept in a
freezer (À20 ꢁC) for 48 h. The colourless crystals of
2-hydroxy-3-methoxybenzylamine hydrobromide were
filtered off, washed with acetone and diethyl ether, dried
and recrystallised from acetone. Elemental analysis (C,
H and N) (Found: C, 41.0; H, 5.4; N, 5.9. C8H12NO2Br
calcd: C, 41.1; H, 5.2; N, 6.0); TLC (chloroform:metha-
nol:ammonia (8:2:0.1, v/v/v) as the mobile phase): single
spot; mp 209–211 ꢁC; ES + MS: m/z 154 [MH]+; 1H
NMR (DMSO-d6, 300 MHz): 9.42 (NH2), 8.28 (OH),
7.00kv (5H), 6.92kv (6H), 6.80tr (4H), 3.95s (7H),
3.80s (–OCH3); 13C NMR (DMSO-d6, 75 MHz):
147.92 (3C), 144.17 (2C), 128.45 (1C), 122.29 (5C),
120.81 (6C), 112.77 (4C), 56.44 (–OCH3), 37.88 (7C).
The remaining filtrate was evaporated in vacuo, ethanol
(10 ml) was added to the residue and the solvent was
again evaporated. Ether (20 ml) was subsequently add-
ed, the solid residue was dried and recrystallised from
ethanol to give 0.25 g of 2,3-dihydroxybenzylamine hyd-
robromide. Elemental analysis (C, H and N) (Found: C,
38.2; H, 4.1; N, 6.1. C7H10NO2Br calcd: C, 38.2; H, 4.6;
4.3. Synthesis of 6-benzylaminopurines
The preparation and cytokinin activity of 6-(2-meth-
oxybenzylamino)purine 14 and 6-(3-methoxybenzylami-
no)purine 15 have been described elsewhere.19 The

K. Dolezˇal et al. / Bioorg. Med. Chem. 14 (2006) 875–884
883
N, 6.4); TLC (chloroform:methanol:ammonia (8:2:0.1,
v/v/v) as the mobile phase): single spot; mp
153–154 ꢁC; ES + MS: m/z 140 [MH]+; 1H NMR
(DMSO-d6, 300 MHz): 9.55 (NH2), 8.15 (OH), 6.85kv
(5H), 6.78kv (6H), 6.65tr (4H), 3.92s (7H); 13C NMR
(DMSO-d6, 75 MHz): 145.59 (3C), 145.05 (2C), 129.46
(1C), 121.15 (5C), 120.83 (6C), 116.29 (4C), 38.09 (7C).
ed. The cultures were further grown at 25 ꢁC. Incuba-
tion times of 17 h and 28 h were found to be optimal
for CRE1/AHK4 and AHK3, respectively. The cultures
were centrifuged and 50 ll aliquots of the supernatant
were transferred to microtitre plate containing 2 ll of
50 mM 4-methyl umbelliferyl galactoside, which was
subsequently incubated for 1 h at 37 ꢁC. The reaction
was stopped by adding 100 ll 0.2 M of Na2CO3. Fluo-
rescence was measured using a Fluoroscan Ascent
(Labsystems, Finland) at the excitation and emission
wavelengths of 365 and 460 nm, respectively. The
OD600 of remaining culture was determined and b-galac-
tosidase activity was calculated as nanomole 4-
4.6. Preparation of 4-hydroxy-3,5-dimethoxybenzylamine
hydroiodide
3,4,5-Trimethoxybenzylamine was dissolved in 55% HI
and acetanhydride was added by syringe. The reaction
mixture was subsequently magnetically stirred at room
temperature for 4 h. The crude product was recrystal-
lised from ethanol. Elemental analysis (C, H and N)
(Found: C, 34.9; H, 4.2; N, 4.8. C9H14NO3I calcd: C,
34.8; H, 4.5; N, 4.5); TLC (chloroform:methanol:ammo-
nia (8:2:0.1, v/v/v) as the mobile phase): single spot; mp
213–314 ꢁC; ES + MS: m/z 196 [MH]+; 1H NMR
(DMSO-d6, 300 MHz): 8.55 (NH2), 8.02 (OH), 6.75s
(2H, 6H), 3.75s (–OCH3), 3.92s (7H); 13C NMR
(DMSO-d6, 75 MHz): 148.34 (3C, 5C), 136.13 (4C),
124.01 (1C), 107.13 (2C, 6C), 56.64 (–OCH3), 43.04
(7C).
methylumbelliferone · ODÀ1 · hÀ1
.
600
4.9. Antitumour activity testing
Human T-lymphoblastic leukaemia (CEM), promyelo-
cytic leukaemia (HL-60), human malignant melanoma
(G-361), human chronic myelogenous leukaemia (K-
562), human osteogenic sarcoma (HOS), human breast
carcinoma (MCF-7), mouse melanoma (B16) and mouse
normal fibroblast (NIH/3T3) cell lines (ATCC, Rockvill,
Maryland, USA) were used for a cytotoxicity determina-
tion of the prepared compounds by a calcein AM assay,
as we have previously described.26 Briefly, the cells were
maintained in plastic tissue culture flasks and were
grown on DulbeccoÕs modified EagleÕs cell culture medi-
um (DMEM) at 37 ꢁC in a 5% CO2 atmosphere and
100% humidity. The cells were redistributed into 96-well
microtitre plates (Nunc, Denmark). After 12 h of prein-
cubation, the tested compounds (in the 0.5–170 lM final
concentration range) were added. Incubation lasted for
72 h. At the end of this period, the cells were incubated
for 1 h with calcein AM and the fluorescence of the living
cells was measured at 485/538 nm (ex/em) with a Fluoro-
scan Ascent reader (Labsystems, Finland). IC50 values,
the compound concentrations lethal to 50% of the tu-
mour cells, were estimated. All experiments were repeat-
ed in quadruplicate with a maximum deviation of 15%.
Because of their limited solubility in water, all the com-
pounds tested were dissolved in DMSO and then diluted
with water to a final DMSO concentration of 0.6%.
4.7. Testing for cytokinin activity
The cytokinin activity of all the prepared derivatives was
tested in three standard bioassays based on the stimula-
tion of tobacco callus growth, the retention of chloro-
phyll in excised wheat leaves and the dark induction of
betacyanin synthesis in Amaranthus cotyledons, as pre-
viously described.5,30 Prior to testing, stock solutions
of cytokinins in DMSO were prepared and further dilut-
ed in the media used for each biotest. The final concen-
tration of DMSO in the media did not exceed 0.5%.
Activity testing was done over the 10À4 to 10À8 M con-
centration range. Five replicates were prepared for each
cytokinin concentration and the entire test was repeated
at least three times. From these data, the concentration
with the highest biological response and the relative
activity at this concentration for each compound were
calculated (Table 3). The activity of BAP at the optimal
concentration was set at 100 and the activities of the
tested compounds were related to that of BAP. The opti-
mal BAP concentrations, used for calculations, were
10À5 M for the Amaranthus betacyanin bioassay,
10À4 M in the case of the senescence bioassay and
10À6 M for the tobacco callus bioassay.
4.10. Human kinase assay
Human CDK2/cyclin E kinase assay was performed
according to a published method.37 The Sf9 culture
co-infected with the appropriate baculoviruses was har-
vested 70 h post infection, incubated in lysis buffer
(50 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA,
20 mM NaF, 1% Tween 20, 1 mM DTT, 0.1 mM
PMSF, 0.5 lg/ml leupeptin and 1 lg/ml aprotonin) for
30 min on ice and the soluble fraction was recovered
by centrifugation at 20.000g for 10 min. The enzyme
was purified on a NiNTA column (Qiagen), stored at
4 ꢁC and used within a week. To carry out the enzyme
inhibition assays under linear conditions, the final point
test system for kinase activity measurement was used.
Kinase was added to the reaction mixture in such a
way as to obtain linear activity both with respect to
the concentration of the enzyme and time. The kinase
inhibition determination involved the use of 1 mg/ml
4.8. Bacterial cytokinin assay
The preparation of Escherichia coli strain KMI001 har-
bouring the plasmid pIN-III-AHK4 or pSTV28-AHK3
and the bacterial cytokinin assay were performed as de-
scribed,7,23,24 albeit with slight modifications. The E. coli
strains were grown overnight at 25 ꢁC in M9 media en-
riched with 0.1% casamino acids25 to OD600 ꢀ 1. The
preculture was diluted 1:600 in 200 ll M9 medium con-
taining 0.1% casamino acids and 1 ll stock solution of
either the tested compound or solvent control was add-

884
K. Dolezˇal et al. / Bioorg. Med. Chem. 14 (2006) 875–884
7. Suzuki, T.; Miwa, K.; Ishikawa, K.; Yamada, H.; Aiba,
H.; Mizuno, T. Plant Cell Physiol. 2001, 42, 107.
8. Ueguchi, C.; Sato, S.; Kato, T.; Tabata, S. Plant Cell
Physiol. 2001, 42, 751.
9. Skoog, F.; Miller, C.O. In Molecular and Cellular Aspects
of Development, Bell, E. Ed.; Harper and Row: New York,
1965; pp 481–494.
10. Soni, R.; Carmichael, J. P.; Shah, Z. H.; Murray, J. A. H.
Plant Cell 1995, 7, 85.
11. Riou-Khamlichi, C.; Huntley, R.; Jacqmard, A.; Murray,
J. A. H. Science 1999, 283, 1541.
histone H1 (type III-S, Sigma) in the presence of 15 lM
ATP, 0.05 lCi [c-33P]ATP and of the tested compound
in a final volume of 10 ll, all in reaction buffer
(50 mM HEPES, 10 mM MgCl2, 5 mM EGTA,
10 mM 2-glycerolphosphate, 1 mM NaF and 1 mM
DTT, pH 7.4). All the compounds tested were dissolved
in DMSO at 5 mM, diluted to 250 lM with 5 mM HCl
and assayed immediately at a final concentration of
50 lM. After 10 min incubation, reactions were stopped
by the addition of 5 ll of 3% H3PO4, aliquots were spot-
ted on P-81 phosphocellulose (Whatman, USA) which
was subsequently washed 3 · with 5% H3PO4 and finally
air-dried. The measurements of kinase inhibition em-
ployed digital image analyser BAS-1800 (Fujifilm, Ja-
pan). Kinase activity was expressed as percentage of
maximum activity (i.e., without an inhibitor).
´
12. Redig, P.; Shaul, O.; Inze, D.; Van Montagu, M.; Van
Onckelen, H. FEBS Lett. 1996, 391, 175.
13. Laureys, F.; Dewitte, W.; Witters, E.; Van Montagu,
M.; Inze, D.; Van Onckelen, H. FEBS Lett. 1998, 426,
29.
14. John, P. C. L.; Zhang, K.; Don, C.; Diederich, L.;
Wightman, F. Aust. J. Plant Physiol. 1993, 42, 677.
15. Zhang, K.; Letham, D. S.; John, P. C. L. Planta 1996, 200,
2.
4.11. Plant kinase assay
´ˇ
16. Vesely´, J.; Havlıcek, L.; Strnad, M.; Blow, J. J.; Donella-
The kinase inhibition assays with proteins bound to
p13Suc1-Sepharose were performed according to a pub-
lished method.27 Briefly, the cultured A. thaliana cv.
Columbia cells were homogenised, the proteins were
bound to p13Suc1-Sepharose and assayed in the presence
of the tested compound with histone H1 (Sigma type III-
S) and 10 lM ATP + 0.05 lCi of [c-33P]ATP in a final
volume of 20 ll. The reactions were incubated at 30 ꢁC
for 15 min and terminated by 3 · SDS sample buffer.
After separation on 12% SDS PAGE, the dried gels were
analysed by bioimager BAS-1800 (Fujifilm, Japan).
Deana, A.; Pinna, L.; Letham, D. S.; Kato, J. Y.;
Detivaud, L.; Leclerc, S.; Meijer, L. Eur. J. Biochem.
1994, 224, 771.
´ˇ
ˇ
´
17. Havlıcek, L.; Hanus, J.; Vesely, J.; Leclerc, S.; Meijer, L.;
Shaw, G.; Strnad, M. J. Med. Chem. 1997, 40, 408.
´ˇ
18. Hajdu´ch, M.; Kolar, Z.; Novotny, R.; Hanus, J.; Mihal,
V.; Hlobilkova, A.; Noskova, V.; Strnad, M. Anti-Cancer
´
ˇ
´
´
´
Drugs 1997, 8, 1007.
˚
19. Tarkowska, D.; Dolezal, K.; Tarkowski, P.; Astot, C.;
´
Holub, J.; Fuksova, K.; Schmulling, T.; Sandberg, G.;
¨
ˇ
´
Strnad, M. Physiol. Plant 2003, 117, 579.
20. Okumura, F. S.; Kotani, Y.; Ariga, T.; Masumura, M.;
Kuraishi, S. Bull. Chem. Soc. Jpn. 1959, 32, 883.
21. Ikeda, M.; Hirao, K. I.; Okuno, Y.; Numao, N.; Zone-
mitsu, O. Tetrahedron 1977, 33, 489.
22. Lin, A. J.; Driscoll, J. S. J. Pharm. Sci. 1981, 70, 806.
23. Yamada, H.; Suzuki, T.; Terada, K.; Takei, K.; Ishikawa,
K.; Miwa, K.; Yamashino, T.; Mizuno, T. Plant Cell
Physiol. 2001, 42, 1017.
Acknowledgments
This work was supported by the Grant Agency of the
Czech Republic (GA 203/04/1168), Academy of Sciences
of the Czech Republic (S 4055304), Czech Ministry of
Education (MSM 153100008) and the Volkswagen Stif-
´
24. Spıchal, L.; Rakova, N. Y.; Riefler, M.; Mizuno, T.;
Strnad, M.; Romanov, G. A.; Schmulling, T. Plant Cell
¨
´
tung (I/76865). We wish to thank Jarmila Balonova, Eva
´
Physiol. 2004, 45, 1299.
25. Sambrook, J.;Fritsch, E. F.;Maniatis, T. MolecularCloning
A laboratory manual; Cold Spring Harbor: NY, 1989.
´
Friesnerova, Olga Hustakova, Katerina Faksova and
Miloslava Subova for skillful technical assistance.
´
ˇ
´
ˇ
´
ˇ
26. Travnıcek, Z.; Malon, M.; Sindelar, Z.; Dolezal, K.;
´
Rolcık, J.; Krystof, V.; Strnad, M.; Marek, J. J. Inorg.
´ˇ
ˇ
´ˇ
ˇ
ˇ´
Biochem. 2001, 84, 23.
ˇ
Supplementary data
27. Bogre, L.; Zwerger, K.; Meskiene, I.; Binarova, P.;
Csizmadia, V.; Planck, C.; Wagner, E.; Hirt, H.; Heberle-
Bors, E. Plant Physiol. 1997, 113, 841.
28. Sussman, M. R.; Kende, H. Planta 1978, 140, 251.
29. Iwamura, H.; Fujita, T.; Koyama, S.; Koshimizu, K.;
Kumazawa, Z. Phytochemistry 1980, 19, 1309.
30. Kuhnle, J. A.; Fuller, G.; Corse, J.; Mackey, B. E. Physiol.
Plant. 1977, 41, 14.
Supplementary data associated with this article can be
found in the online version at doi:10.1016/
j.bmc.2005.09.004.
References and notes
31. Ueguchi, C.; Koizumi, H.; Suzuki, T.; Mizuno, T. Plant
Cell Physiol. 2001, 42, 231.
32. Honma, Y.; Ishii, Y. Leuk. Lymphoma 2002, 43, 1729.
33. Ishi, Y.; Hori, Y.; Sakai, S.; Honma, Y. Cell Growth
Differ. 2002, 13, 19.
1. Strnad, M. Physiol. Plant 1997, 101, 674.
2. Werbrouck, S. P. O.; Strnad, M.; Van Onckelen, H. A.;
Debergh, P. C. Physiol. Plant 1996, 98, 291.
´
ˇ
3. Kamınek, M.; Vanek, T.; Motyka, V. J. Plant Growth
34. Mlejnek, P.; Kuglik, P. J. Cell Biochem. 2000, 77, 6.
35. Mlejnek, P. J. Cell Biochem. 2001, 83, 678.
Regul. 1987, 6, 113.
ˇ
4. Holub, J.; Hanus, J.; Hanke, D. E.; Strnad, M. Plant
Growth. Regul. 1998, 26, 109.
´
36. Mlejnek, P.; Prochazka, S. Planta 2000, 215, 158.
ˇ
´ˇ
37. Krystof, V.; Lenobel, R.; Havlıcek, L.; Kuzma, M.;
Strnad, M. Bioorg. Med. Chem. Lett. 2002, 12, 3283.
38. Legraverend, M.; Tunnah, P.; Noble, M.; Ducrot, P.;
Ludwig, O.; Grierson, D. S.; Leost, M.; Meijer, L.;
Endicott, J. J. Med. Chem. 2000, 43, 1282.
5. Chaves das Neves, H. J.; Pais, M. S. Biochem. Biophys.
Res. Commun. 1980, 95, 1387.
6. Inoue, T.; Higuchi, M.; Hashimoto, Y.; Seki, M.; Kobay-
ashi, M.; Kato, T.; Tabata, S.; Shinozaki, K.; Kakimoto,
T. Nature 2001, 409, 1060.