Novel potent inhibitors of A. thaliana cytokinin oxidase/dehydrogenase

Article abstract of DOI:10.1016/j.bmc.2008.09.008

The synthesis of a new group of 2-X-6-anilinopurines, including compounds with potential cytokinin-like activities, with various substitutions (X = H, halogen, amino, methylthio or nitro) on the phenyl ring is described. The prepared compounds have been characterized using standard physico-chemical methods, and the influence of individual substituents on biological activity has been compared in three different bioassays, based on the stimulation of tobacco callus growth, retention of chlorophyll in excised wheat leaves and the dark induction of betacyanin synthesis in Amaranthus cotyledons. The biological activity of the prepared compounds was also assessed in receptor assays, in which the ability of the compounds to activate the cytokinin receptors AHK3 and AHK4/CRE1 was studied. Finally, the interactions of the compounds with the Arabidopsis cytokinin oxidase/dehydrogenase AtCKX2 (heterologously expressed) were investigated. Systematic testing led to the identification of two very potent inhibitors of AtCKX2: 2-chloro-6-(3-methoxyphenyl)aminopurine and 2-fluoro-6-(3-methoxyphenyl)aminopurine.

Full text of DOI:10.1016/j.bmc.2008.09.008

Bioorganic & Medicinal Chemistry 16 (2008) 9268–9275
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel potent inhibitors of A. thaliana cytokinin oxidase/dehydrogenase
*
ˇ
ˇ
Marek Zatloukal , Markéta Gemrotová , Karel Dolezal , Libor Havlícek, Lukáš Spíchal, Miroslav Strnad
´
Laboratory of Growth Regulators, IEB AS CR & Palacky University Olomouc, 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:
The synthesis of a new group of 2-X-6-anilinopurines, including compounds with potential cytokinin-like
activities, with various substitutions (X = H, halogen, amino, methylthio or nitro) on the phenyl ring is
described. The prepared compounds have been characterized using standard physico-chemical methods,
and the influence of individual substituents on biological activity has been compared in three different
bioassays, based on the stimulation of tobacco callus growth, retention of chlorophyll in excised wheat
leaves and the dark induction of betacyanin synthesis in Amaranthus cotyledons. The biological activity
of the prepared compounds was also assessed in receptor assays, in which the ability of the compounds
to activate the cytokinin receptors AHK3 and AHK4/CRE1 was studied. Finally, the interactions of the
compounds with the Arabidopsis cytokinin oxidase/dehydrogenase AtCKX2 (heterologously expressed)
were investigated. Systematic testing led to the identification of two very potent inhibitors of AtCKX2:
2-chloro-6-(3-methoxyphenyl)aminopurine and 2-fluoro-6-(3-methoxyphenyl)aminopurine.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 23 April 2008
Revised 28 August 2008
Accepted 4 September 2008
Available online 7 September 2008
Keywords:
Cytokinins
6-Anilinopurines
Cytokinin oxidase/dehydrogenase
1. Introduction
inhibition and other potential cytokinin-like activities were exam-
ined. These compounds were prepared via condensation of 2-
Adenine derivatives, bearing various substituents at N6-posi-
tion, are an important group of biologically active molecules which
can be used in both medicine and plant biotechnology.1,2
Systematic testing of purines with different substituents in var-
ious assays, including receptor tests, has recently led to several
general conclusions regarding structure/activity relationships of
the plant hormones cytokinins.1,2 The chemical nature of the sub-
stituents and their positions influence their recognition as ligands
by specific receptors and/or as substrates of degradative enzymes
(histidine kinase receptors and cytokinin oxidase/dehydrogenase,
respectively, in this context). Cytokinin dehydrogenase (CKX) is in-
volved in the regulation of endogenous cytokinin contents in
plants by oxidative removal of the side chain and seven distinct
genes, AtCKX1 to AtCKX7, encode the enzyme in Arabidopsis thali-
ana.3 Isolation of the pure protein is difficult because of its extre-
mely low concentration in plant tissues. However, the CKX2 gene
from A. thaliana4 has recently been cloned in yeast S. cerevisiae,
allowing us to obtain large amounts of homogenous, active protein
from the yeast culture media, and to test the ability of a library of
novel 6-anilinopurine derivatives to inhibit the activity of the
enzyme.
substituted-6-chloropurines with corresponding anilines (SN2
mechanism) and characterized by standard physico-chemical
methods. The synthesis and subsequent purification of the 6-anil-
inopurines was sometimes tedious, in comparison to purifying cor-
responding 6-benzylaminopurines, due to the relatively low
nucleophilicity of the anilines and large amounts of by-products
arising from side reactions. Due to the structure similarity to aro-
matic cytokinins, the prepared derivatives were tested for their
biological activity in classical cytokinin bioassays. Subsequently,
the influence of individual substituents on the activation of cytoki-
nin receptors (AHK3, CRE1/AHK4) and on the interaction with the
key enzyme involved in cytokinin degradation in A. thaliana (cyto-
kinin oxidase/dehydrogenase 2, AtCKX2) was also examined. This
screening led to the discovery that 2-chloro-6-(3-methoxy-
phenyl)aminopurine and 2-fluoro-6-(3-methoxyphenyl)aminopu-
rine are highly potent AtCKX2 inhibitors with decreased ability
to activate cytokinin receptors.
2. Results and discussion
2.1. Chemistry
Here, we describe the preparation of a series of new 2-X-6-anil-
inopurines (X = H, halogen, amino, methylthio or nitro) with vari-
ous substitutions on the phenyl ring. Both their AtCKX2
Nineteen substituted 6-anilinopurines (Table 1) were prepared
according to the synthetic procedures outlined above. The identity
and purity of the prepared compounds were verified by 1H NMR,
mass spectrometry (chemical ionization), HPLC-UV and elemental
analysis (Table 2b). Synthesis of some of the substituted 6-anilin-
opurines has been previously described.5–9 The parent molecule,
* Corresponding author. Tel.: +420585634940; fax: +420585634870.
ˇ
E-mail address: karel.dolezal@upol.cz (K. Dolezal).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.09.008

M. Zatloukal et al. / Bioorg. Med. Chem. 16 (2008) 9268–9275
9269
Table 1
2-Chloro-6-(x-halogenoanilino)purines (3–6) were prepared
using a similar method to that described by Imbach et al.,9 reacting
corresponding halogenoanilines with 6-chloropurine in dimethyl-
formamide at 80–100 °C in the presence of triethylamine as a base.
2-Chloro-6-(x-halogenoanilino)purines have been previously used
as intermediates for preparing very potent CDK inhibitors of the
purvalanol type, with strong anti-proliferative properties.9,11 Using
this approach we successfully prepared chloro- and fluoro-anilin-
opurines in meta- and para-positions, while attempts to prepare
ortho-derivatives failed (these compounds have not yet been de-
scribed in the literature to date). The synthesis, which was carried
out in n-propanol or n-butanol at 90 °C under a nitrogen atmo-
sphere, was complicated by the large amounts of impurities that
arose from side reactions of halogenoanilines. Furthermore, purifi-
cation by crystallization or flash chromatography of the crude
products, especially 6-(3-fluoroanilino) and 6-(3-chloroanilino)-
purines, was also essential in these cases.
Two methoxyanilinopurines have also been synthesised and
used as precursors for the synthesis of CDK inhibitors. Imbach
et al.9 synthesised 2-chloro-6-(3-methoxyanilino)purine (11) in
n-pentanol without use of tertiary amine, while Qu et al.12 very re-
cently prepared the corresponding para-isomer (12), by reacting
2,6-dichloropurine with para-anisidine without use of either sol-
vent or auxiliary base. Preparation of 2-chloro-6-(3-methoxyanili-
no)purine using dimethylformamide as solvent and triethylamine
as a base, at reaction temperatures of 80–100 °C, has also been pre-
viously described,8 but the yield was low (56%). On the other hand,
synthesis of the corresponding ortho-isomer has not been previ-
ously reported. We synthesised all three isomers as hydrochlorides
by reacting 2,6-dichloropurine and the corresponding anisidine in
refluxing n-propanol with high yields (80–90%) and high purity
(>98% HPLC). Free bases were obtained from hydrochlorides by
treatment with diluted aqueous ammonia. The same method was
used to prepare all three isomers of hydroxyanilinopurines (7–9),
which have not been previously reported in the literature;
although the yields were somewhat lower compared with the pre-
vious case.
Structure and abbreviations of prepared compounds
Compound
R
R1
R2
R3
R4
R5
R6
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
F
NO2
NH2
CH3S
Cl
H
H
H
H
H
H
OH
H
H
OCH3
H
H
H
H
H
H
H
H
H
H
Cl
H
F
H
H
OH
H
H
OCH3
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
H
Cl
H
F
H
H
OH
H
H
OCH3
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
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
H
H
H
H
H
H
H
H
CH3
Isopropyl
Cl
H
2-chloro-6-anilinopurine (2), previously prepared by Thompson
et al. by condensation of 2,6-dichloropurine with aniline in n-pro-
panol at 100 °C in the presence of N,N-diisopropylethylamine as an
auxiliary base,5 is an important intermediate for the synthesis of
2,6,9-trisubstituted purines, which have been found to be potent
CDK inhibitors.10 Unsubstituted 6-anilinopurine (1) was first syn-
thesized (by Elion et al.7) by direct reaction of 6-methylsulfanylp-
urine and aniline at a higher temperature and later (by Daly et al.6)
by condensation of 6-chloropurine with aniline without either
solvent or auxiliary base. In our study, both anilinopurines were
prepared using a slightly different method, by reacting 6-chloro
or 2,6-dichloropurine with aniline in n-propanol in the presence
of N,N-diisopropylethylamine at a somewhat lower temperature
(90 °C), for a prolonged time (2 days) with very good yield
(84.9%) and satisfactory purity for testing biological activity. Both
of these anilinopurines were tested for CKX inhibitory activity,
but were found to be almost inactive (Table 3).
Compounds 14–19 (see Table 1) have also not been previously
described. We prepared them using a conventional method (see
Table 2a
Detailed reaction conditions
Compound Starting
purine
Base Solvent
Temperature (°C)
Time (h)
Auxiliary base Yield (%)/HPLC purity
(%)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
A
B
B
B
B
B
B
B
B
B
B
B
A
C
D
a
a
b
c
d
e
f
g
h
i
j
k
j
j
j
n-Propanol
n-Propanol
n-Butanol
n-Propanol
n-Butanol
n-Butanol
n-Butanol
n-Butanol
n-Butanol
n-Butanol
n-Pentanol
n-Butanol
n-Butanol
n-Propanol
(1) n-Propanol(2) Methanolic HCl(3) 3%
NH4OH
n-Propanol
n-Propanol
n-Propanol
n-Propanol
90
98–100
110
98–100
100–110
100–110
90
90
90
90
100
20
4
3
4
3
3
3
4
4
4
4
4
3
4
DIPEA
DIPEA
84.9/98.0+
Triethylamine 65.8/98.0+*
DIPEA
65.0/98.0+
Triethylamine 69.6/98.0+*
Triethylamine 19.0/98.0+**
Triethylamine 75.3/98.0+
DIPEA
DIPEA
DIPEA
84.1/98.0+
83.0/99.0+
64.0/98.0+
Triethylamine 77.8/98.0+
Triethylamine 88.8/99.0+
Triethylamine 82.9/98.0+
Triethylamine 61.0/99.0+
100
110
90
(1) -5–0; then rt(2) 50(3) (1) 12(2) 12(3)
rt
98–100
98–100
98–100
90
—
34.9/99.0+
1
3
4
4
4
16
17
18
19
E
F
G
H
j
j
j
j
—
—
—
—
85.0/99.0+ (as HCl salt)
69.0/99.0+ (as HCl salt)
46.0/99.0 (as HCl salt)
80.0/98.7 (as HCl salt)
A, 6-chloropurine; B, 2,6-dichloropurine; C, 2-fluoro-6-chloropurine; D, 2-nitro-6-chloropurine; E, 2-amino-6-chloropurine; F, 2-methylthio-6-chloropurine; G, 2,6-dichloro-
9-methylpurine; H, 2,6-dichloro-9-isopropylpurine; a, aniline; b, 3-chloroaniline; c, 4-chloroaniline; d, 3-fluoroaniline; e, 4-fluoroaniline; f, 2-aminophenol; g, 3-amino-
phenol; h, 4-aminophenol; i, 2-anisidine; j, 3-anisidine; k, 4-anisidine; DIPEA, N,N-diisopropyl-N-ethyl amine; rt, room temperature.
*
Purified by flash chromatography.
Purified by crystallization from 2-propanol.
**

9270
M. Zatloukal et al. / Bioorg. Med. Chem. 16 (2008) 9268–9275
Table 2b
Chemical characteristics of prepared compounds
Compound
Melting points (°C)
Melting points (°C)—literature
MS-CI
Calculated%
Found%
H
(M+H)+
C
H
N
C
N
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
279
320
279–2826
211(EI)
246
280
280
264
264
262
262
262
276
276
276
242
260
287
257
288
290
318
62.5
53.8
47.0
47.0
50.1
50.1
50.5
50.5
50.5
52.3
52.3
52.3
59.7
55.6
50.3
56.2
54.3
53.9
56.7
4.3
3.3
2.9
2.9
2.7
2.7
3.1
3.1
3.1
3.7
3.7
3.7
3.7
3.9
3.5
4.7
4.6
4.2
5.1
33.2
28.5
24.9
24.9
26.6
26.6
26.8
6.8
26.8
25.4
25.4
25.4
29.0
27.0
29.4
32.8
24.4
24.2
22.0
62.1
53.4
46.8
46.9
49.9
49.9
49.9
49.8
50.5
51.9
51.8
52.0
59.4
55.2
50.0
56.1
54.1
53.3
56.2
4.2
3.3
2.9
3.0
3.0
2.9
3.1
3.1
3.1
3.8
3.5
3.7
3.5
3.9
3.6
4.7
4.6
4.3
5.0
32.9
28.3
24.6
24.7
26.5
26.0
26.7
26.8
26.8
25.5
25.7
25.2
29.0
26.8
29.1
32.4
24.2
23.9
21.8
32019
>350
>350
>350
>350
>350
>350
>350
258
238
>300
226
195
280
Not given9
—
Not given8,9
Not given9
—
—
—
—
Not given8,9
Not given20
—
—
—
—
—
—
—
>300
261
225
>300
Table 3
of the crude product by column chromatography. Using this meth-
od we prepared one of the strongest CKX inhibitors obtained in this
study; 2-fluoro-6-(3-methoxyanilino)purine (compound 14) (Table
3). This compound is also a very important precursor for the solid
phase synthesis of 2,6,9-trisubstituted purine CDK inhibitors,23 be-
cause of the high reactivity of the C2-position, activated by the
fluorine atom.
2-Nitro-6-chloropurine is a compound that has been described
very recently by Rodenko et al.24 It was synthesised using 6-
chloro-9-t-butoxycarbonylpurine (prepared from 6-chloropurine
and t-butyl bicarbonate24) via nitration by trifluoroacetyl nitrate,
generated in situ from trifluoroacetic anhydride and tetra-n-butyl-
ammonium nitrate, at the temperature À5 °C. The authors ex-
plored the reaction mechanism, which is rather complex, using
detailed 1H, 13C, 15N, and 19F NMR analyses.24 The presence of
the strongly electron-withdrawing nitro group enables easy substi-
tution by nucleophiles, which can be introduced at the C6 position
by displacement of the chlorine atom at low temperatures (below
0 °C), and thus a large number of biologically active compounds
can be prepared from this intermediate.24 Finally, the protecting
t-butoxycarbonyl (t-Boc) group can be removed in acidic media
either before or after condensation of this precursor with various
amines.24
Biological activity of novel 6-anilinopurines: activation of cytokinin receptors AHK4/
CRE1 and AHK3 expressed as EC50 values, and inhibition of recombinant AtCKX2 as
IC50 values
Compound
EC50
CRE1/AHK4
(
lM)
IC50 (lM)
AHK3
AtCKX2
1
2
3
4
5
6
7
8
na
na
>50
>50
10.7
15.1
6
50
19
13
16.5
33
ni
80
3.9
25.4
7.5
7.5
>50
24.8
na
>50
3.75
4.6
na
7
na
31
19
na
na
na
na
na
1.1
1
50
>100
3.75
>100
>100
1.9
>100
12.4
1
14
27
>100
61
>100
ni
9
10
11
12
13
14
15
16
17
18
19
tZ
TDZ
21
33.1
2.3
19
na
na
na
na
na
0.7
0.95
29
It could be very interesting for future preparation of 6-arylam-
inopurines to use Hartwig–Buchwald coupling of electron deficient
halopurines and anilines. This method requires usage of special
catalysts—Pd complexes with phosphine derivatives, and 1,3-dial-
kyl or 1,3-diarylimidazolium halogenides. The reported yields are
relatively high, but purification of raw product by column chroma-
tography is required (removal of traces of heavy metal catalysts
and other impurities).21
Our method for the preparation of 6-arylaminopurines—aro-
matic nucleophilic substitution—is cheap and without presence
of expensive Pd catalysts, therefore it can be used for large scale
preparations. The raw products can be purified by simple crystalli-
zation, if needed. The yields are relatively high in most cases, as
well.
The natural cytokinin trans-zeatin and synthetic cytokinin thidiazuron were used as
controls. na, no activation; ni, no inhibition.
text above) from corresponding precursors (2-fluoro-, 2-nitro-, 2-
amino and 2-methylthio-6-chloropurines). Substances 18 and 19
were prepared from m-anisidine and 2,6-dichloro-9-methylpurine
or 2,6-dichloro-9-isopropylpurine, respectively. These precursors
were prepared via alkylation of 2,6-dichloropurine by methyl io-
dide and isopropylbromide, respectively, in alkaline media in a po-
lar aprotic solvent (dimethylformamide or dimethylsulfoxide) as
previously described.25,26
2-Fluoro-6-chloropurine was synthesised from 2-amino-6-
chloropurine by diazotization using t-butyl nitrite and subsequent
Schiemann reaction of the diazonium salt with tetrafluoroboric
acid in dimethylformamide at À15 °C (modified method according
to Ref. 22,23) with satisfactory yield (65%). Our method eliminates
the previous requirement for tedious separation of the desired
product from large amounts of inorganic salts,22 and purification
2.2. Biological activities
Biological effects of prepared compounds were compared to
those of thidiazuron, a well known synthetic cytokinin with high
biological activities mediated by strong activation of cytokinin

M. Zatloukal et al. / Bioorg. Med. Chem. 16 (2008) 9268–9275
9271
receptors,1,28 and CKX inhibition,13 as well as classical isoprenoid
and aromatic cytokinins trans-zeatin (tZ) and 6-benzylaminopu-
rine (BAP), respectively. To explore the cytokinin-like effects of
moval of the methylene group between the ring and the amino
group at the 6-position or lengthening of the bridge reduces the
compound’s cytokinin activity.14,15 Thus, 6-anilinopurine (2), 6-
(2-phenylethylamino)purine and 6-[(2-furan-2-yl-ethyl)amino]-
purine have been reported to be either as active or less active than
BAP and kinetin. However, our results from comparison of biolog-
ical activity of BAP and prepared 6-anilinopurine derivatives in the
tobacco and Amaranthus bioassays show that the benzyl group can
be replaced by a phenyl ring without loss of cytokinin activity
(Table 4). In good agreement with results of the receptor binding
assays, meta-substituted derivatives (compounds 3, 5, 11, 13 and
14) showed high activity in both tests. On the other hand, in con-
trast to the AHK assay, non-substituted 6-anilinopurine (1), as well
as compounds 2 (2-chloro derivative) and 4, which also carry a
chlorine atom in the para-position of the phenyl ring, also highly
actively induced betacyanin synthesis and stimulated tobacco
callus growth.
The position of the hydroxyl group on the side of the cytokinin
ring usually has a significant effect on the biological activity of
hydoxylated BAP derivatives. Hydroxylation of the benzyl group
in meta-position has been found to increase cytokinin activity
while ortho- and para-hydroxylation strongly reduces it.30,16
However, surprisingly, both meta- and para-hydroxy derivatives
of 2-chloro-6-anilinopurine (8,9) were much less active in both
the tobacco and Amaranthus bioassays, compared with unsubsti-
tuted 2-chloro-6-anilinopurine (Table 4).
the prepared anilinopurines E. coli strains with
Drcs and cps::lacZ
mutant backgrounds that express AHK3 and AHK4 cytokinin recep-
tors were used.1,28,29 Interactions of the 6-anilinopurines with
AtCKX2 were investigated using an assay based on the coupled re-
dox reaction of PMS and MTT resulting in the formation of a forma-
zan dye.27 The effects of substitution position on the phenyl ring
and various C2 and N9 substitutions on receptor activation and
CKX inhibition were assessed by determining and comparing
EC50 and IC50 values, respectively, of the prepared compounds.
2.2.1. Receptor binding assay
The affinity of the cytokinin receptors for the prepared 6-anilin-
opurines was compared to their affinity for the natural ligand
trans-zeatin (tZ) and synthetic cytokinin thidiazuron (TDZ). tZ
and TDZ were recognized by both receptors with high affinity
(EC50 ca. 1 lM in each case), while the non-substituted 6-anilinop-
urine (1) was not recognized by either of the receptors, and substi-
tuted 6-anilinopurines were recognized to varying degrees (Table
3). Histidine-kinase CRE1/AHK4 recognized only meta-substituted
derivatives, with preferences for hydroxy (8) and methoxy (11)
groups, followed by the halogens chlorine (3) and fluorine (5),
reaching EC50 values from 3.75 to 19 lM (Table 3). An interesting
exception was compound 9, which harbours a hydroxy group at
the para-position of aniline and showed strong activation of
CRE1/AHK4, equalling that of the meta-analog. It is clear from Table
3 that the strongest binding was shown by the compounds bearing
a halogen group at C2 of the purine ring, while the presence of
other groups at C2, and substitution of the N9 led to loss of affinity.
Receptor AHK3 bound the tested compounds with higher affinity
than CRE1/AHK4, again with highest preference for meta-substi-
tuted derivatives, but their EC50 values were not much lower than
those of derivatives with substituents at the para- and ortho-posi-
tions (Table 3). The findings that there were no strict relationships
between either the types or positions of substituents and the abil-
ity of the 6-anilinopurine to activate the AHK3 signalling pathway
confirms previous findings indicating that AHK3 has broader ligand
specificity than AHK4/CRE1.1,2
In contrast with the results of the two bioassays described
above, and the activity of the corresponding 6-benzylaminopu-
rines,2 all tested 6-anilinopurine derivatives were virtually inactive
in the senescence bioassay (Table 4).
Similar biological activity was found with meta- and para-fluo-
rine derivatives (5,6). Interestingly, in the callus bioassay the meta-
derivative (6) embodied maximum of biological activity in broad
concentration range and, in contrary to control cytokinin BAP, even
the highest concentration applied (100 lM) was not toxic and did
not inhibited callus growth (Fig. 1A). The same activity of the high-
est concentration of compound 6 was observed also in Amaranthus
assay (Fig. 1B).
Interestingly, in contrast with the results of the two bioassays
described above, and the activity of the corresponding 6-benzy-
laminopurines,2 all tested 6-anilinopurine derivatives were virtu-
ally inactive in the senescence bioassay (Table 4 and Fig. 1C).
Finally, we compared biological activity of compounds 11 and
14, which are interesting due to their interaction with CKX (as de-
scribed bellow), to the activity of highly active synthetic cytokinin
TDZ. As shown in Figure 2A EC50 of the compound 11 was about
1000-fold higher compared to TDZ in the callus assay. In the Ama-
ranthus assay, EC50 of TDZ was again much lower than of the com-
pound 11 (about 100-fold), however the compound 11 showed
2.2.2. Cytokinin bioassays
Most of the tested compounds retained high cytokinin activity
in bioassays, based on the dark induction of betacyanin synthesis
in Amaranthus cotyledons and stimulation of cytokinin-dependent
tobacco callus growth, comparable to the activity of corresponding,
previously described 6-benzylaminopurines2 (Table 4). The ring-
substituted aminopurine with the strongest cytokinin activity is
6-benzylaminopurine, according to various reports.14,15 Either re-
Figure 1. Dose–response curves for: cytokinin-induced dark betacyanin synthesis in Amaranthus caudatus cotyledon-hypocotyl explants (A), chlorophyll retention in excised
wheat leaf tips (B), and growth of cytokinin-dependent tobacco callus (C). Error bars show standard deviations of the mean for five replicate determinations. Dashed lines
indicate values obtained for the control treatment without any cytokinin.

9272
M. Zatloukal et al. / Bioorg. Med. Chem. 16 (2008) 9268–9275
Figure 2. Biological activity of CKX inhibitor 2-chloro-6-(3-methoxyphenyl)aminopurine (compound 11) in classical cytokinin bioassays compared to synthetic cytokinins
thidiazuron (TDZ) and 6-benzylaminopurine (BAP). (A) Effect on growth of cytokinin-dependent tobacco callus; (B) effect on dark betacyanin synthesis in Amaranthus
caudatus cotyledon-hypocotyl explants. Error bars show standard deviations of the mean for five replicate determinations. Dashed lines indicate values obtained for the
control treatment without any cytokinin.
very high maximal activity in the concentration that is toxic for
TDZ (Fig. 2B). Similar results were obtained with compound 14 (Ta-
ble 4, graphs not shown).
(20–30 lM), that of the disubstituted anilinopurine 11 was much
lower, submicromolar (Fig. 3A, Table 3). With the methoxy group
placed on the phenyl ring, the ability of the compound to inhibit
CKX activity decreased in the order meta > >para P ortho
(Fig. 3B). C2 substituents influenced the strength of inhibition in
the order F > Cl > NO2 > NH2 > SCH3, indicating that electron-with-
drawing substituents enhance the inhibitory activity of these com-
pounds (Fig. 3C). Similar effects have been described amongst
fluorine-containing derivatives of natural cytokinins, for which it
has been proposed that the fluorine substituents form hydrogen
bonds with electron donors of cytokinin receptors.17,18 The pres-
ence of the third substituent at the N9 position of the purine ring,
i.e. methyl or isopropyl, significantly decreased the compounds’
potency to inhibit AtCKX2 (Fig. 3D and Table 3).
2.2.3. Inhibition of cytokinin oxidase/dehydrogenase
The ability of the novel 6-anilinopurines to inhibit the activity
of CKX was compared with that of thidiazuron, a well known inhib-
itor of CKX. As shown in Figure 3A, substitution of the phenyl side
chain and/or C2 substitution at the purine ring were found to be
essential for the inhibition of AtCKX2. The combination of these
two substitutions led to the discovery of the most potent com-
pounds; 2-chloro-6-(3-methoxyphenylamino)purine (11) and
2-fluoro-6-(3-methoxyphenylamino)purine (14). While monosub-
stituted derivatives 2 and 13 had similar IC50 values to TDZ
Figure 3. Effects of substituents on the purine ring on inhibition of AtCKX2 activity. (A) Key substitutions of the 6-anilinopurine moiety leading to the potent inhibitor 2-
chloro-6-(3-methoxyphenylamino)purine (11). (B) Effect of the methoxy group’s position on the phenyl side chain. (C) Effects of substitutions at the C2 position. (D) Effects of
substitutions at N9.

M. Zatloukal et al. / Bioorg. Med. Chem. 16 (2008) 9268–9275
9273
R2
3. Conclusion
R3
R4
R1
In summary, a group of 6-anilinopurine derivatives with differ-
ent substitutions on the phenyl moiety, as well as with C2 and N9
substitutions, have been prepared. Biological testing showed that
these compounds can be classified as a new generation of pur-
ine-derived CKX inhibitors. Compared to thidiazuron, a synthetic
cytokinin with strong biological activity resulting from strong
activation of cytokinin receptors and CKX inhibition, most of the
6-anilinopurines inhibited CKX much more strongly, but their
sensing by cytokinin receptors was much weaker.
NH
R5
R
N
N
N
N
R6
Scheme 1. General structure of 6-anilinopurines.
4.3. Synthesis of N6-anilinopurines
4.3.1. General procedure
4. Materials and methods
4.1. General procedures
Variously substituted 6-anilinopurines (Scheme 1) were pre-
pared by reacting corresponding 2-substituted 6-chloropurines or
6-methylthiopurines with appropriate anilines in various solvents,
preferably alcohols (C2–C5), or dimethylformamide, in the presence
(or absence, depending on the basicity of the aniline used) of base
(tertiary amine, potassium carbonate, sodium hydride, and sodium
methoxide) (Scheme 2 and Table 2a). The reaction temperatures
varied from sub-zero to 110 °C, depending on the reactivity of the
substituted 6-chloro- or 6-methylthiopurine used and the basicity
of the selected substituted aniline (Table 2a). Basic chemical charac-
teristics of the synthesised compounds are presented in Table 2b.
NMR data are available in the supplementary material.
The elemental contents of the prepared compounds were deter-
mined using an EA1108 CHN analyser (Thermo Finnigan). Their
melting points were determined using a Büchi Melting Point B-
540 apparatus (uncorrected values are presented here), and they
were separated from reaction by-products and reactants by analyt-
ical thin layer chromatography (TLC) using silica gel 60 WF254
plates (Merck) with a mobile phase of 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 Collec-
tor (Bio-Rad). Compounds were separated on VersaPak Car-
tridges (25 Â 100 mm, Supelco) containing 23 g of spherical
silica and eluted with a mobile phase containing CHCl3/MeOH
(90:10, v/v).
We describe here a few examples to illustrate key synthetic pro-
cedures used throughout the study:
4.3.1.1. 2-Chloro-6-anilinopurine (compound 2). N,N-Ethyldi-
isopropylamine (6.44 g; 0.05 mol) and aniline (4.66 g; 0.05 mol)
To determine their HPLC purity, samples were dissolved in
HPLC mobile phase (initial conditions), applied to an RP-column
were added to
a suspension of 2,6-dichloropurine (5.67 g;
(150 mm  4.6 mm, 5
lm, Microsorb C18; Varian) and the sepa-
0.03 mol) in n-propanol (60 ml). The reaction mixture was stirred
at 100 °C for 4 h. After cooling to room temperature, the precipitate
was filtered off, washed with cold n-propanol (2Â 10 ml) and water
(3Â 10 ml) then dried at 60 °C to constant weight. Yield: 6.26 g yel-
lowish substance (84.9 %). TLC (chloroform/methanol 85:15): one
single spot; free of the starting material. HPLC purity: 98+%.
rated constituents were eluted with a linear methanolic gradient
(10–90% 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.
CI+ and EI+ mass spectra were recorded using a Polaris Q (Finn-
igan) mass spectrometer equipped with a Direct Insertion Probe
(DIP). The compounds were heated in an ion source with a 40–
450 °C temperature gradient, the mass monitoring interval was
50–1000 amu, and spectra were collected using 1.0 s cyclical scans,
applying 70 eV electron energy. In the CI+ ionization mode, isobu-
tane was used as a reagent gas at a flow rate of 2L/h. The mass
spectrometer was directly coupled to an Xcalibur data system.
NMR spectra were acquired using a Bruker Avance AV 300 spec-
trometer operating at a temperature of 300 K and a frequency of
300.13 MHz (1H). Samples were prepared by dissolving the com-
pounds in DMSO-d6, and tetramethylsilane (TMS) was used as
the internal standard.
4.3.1.2. 2-Chloro-6-(4-chloroanilino)purine (compound 4). N,N-
Ethyldiisopropylamine (6.44 g; 0.05 mol) and 4-chloroaniline
(6.38 g; 0.05 mol) was added to a suspension of 2,6-dichloropurine
(5.67 g; 0.03 mol) in n-propanol (60 ml). The reaction mixture was
stirred at 100 °C for 4 h. After cooling to room temperature, the
precipitate was filtered off, washed with cold n-propanol (2Â
10 ml) and water (3Â 10 ml) then dried in an oven at 60 °C to con-
stant weight. Yield: 5.46 g yellowish substance (65%). TLC (chloro-
form/methanol 85:15): one single spot; free of the starting
material. HPLC purity: 98+%.
4.3.1.3. 2-Chloro-6-(3-methoxyanilino)purine (compound 11). To
a suspension of 2,6-dichloropurine (3.78 g; 0.02 mol) and m-anisi-
4.2. Chemicals
Y
2,6-Dichloropurine, 2-amino-6-chloropurine, and 2-methyl-
thio-6-chloropurine were obtained from Olchemim, aniline, o-,m-
,p-anisidine, 3-chloroaniline, 4-chloroaniline, diisopropylethyl-
amine, 3-fluoroaniline, 4-fluoroaniline and tetrafluoroboric acid
from Fluka, o-, m- and p-aminophenol from Baker. 2-fluoro-6-chlo-
ropurine was synthesised from 2-amino-6-chloropurine by a previ-
ously published method.22,23 Lach-Ner supplied n-butanol, n-
propanol, diethyl ether, dimethylformamide, ethyl acetate, n-pro-
panol and anhydrous sodium sulphate. Milli-Q water was used
throughout. The other solvents and chemicals used were all of
standard p.a. quality.
Cl
N
NH2
NH
N
N
N
Et3N or DIPEA
N
N
N
N
+
Y
alcohol C3-C5
X
90 -110°C; 3 - 4 hrs
X
R
R
R = H, CH3, isopropyl
X = H, Cl, F, NO 2, NH2, CH3S
Y = H, Cl, F, CH 3O, OH
Scheme 2. Reaction scheme for the preparation of 6-arylaminopurines.

9274
M. Zatloukal et al. / Bioorg. Med. Chem. 16 (2008) 9268–9275
Table 4
Relative cytokinin bioassay activity of prepared 6-anilinopurine derivatives at optimal concentration compared with activity of 6-benzylaminopurine (BAP) (100% means 10À6 M
BAP for the tobacco callus bioassay, 10À5 M BAP for the Amaranthus betacyanin bioassay, and 10À4 M BAP in the case of the senescence bioassay)
Compound
Tobacco callus bioassay
Relative activity
Amaranthus bioassay
Relative activity
Senescence bioassay
Optimal concentration Relative activity
Optimal concentration
Optimal concentration
(mol lÀ1
)
(%)
(mol lÀ1
)
(%)
(mol lÀ1
)
(%)
tZ
TDZ
1
2
3
4
5
6
10À5
10À7
10À4
10À6
10À5
10À5
10À6
10À6
104.6 ( 7)
102.3 ( 11)
47 ( 2)
103 ( 4)
95 ( 23)
77 ( 11)
101 ( 7)
99 ( 6)
10À4
10À7
10À4
10À4
10À5
10À4
10À4
10À4
129.6 ( 10)
121.1 ( 9)
52 ( 2)
139 ( 16)
119 ( 15)
139 ( 6)
10À4
10À4
10À4
10À4
10À4
10À4
10À4
10À4
122 ( 8)
108.7 ( 5)
14 ( 5)
23 ( 1)
11.5 ( 1)
10 ( 3)
15 ( 5)
5 ( 3)
147 ( 14)
92 ( 27)
7
8
9
nt
nt
nt
10À5
10À4
10À5
10À5
10À5
10À5
10À5
10À4
10À5
10À5
10À5
10À5
111 ( 5)
60 ( 8)
100 ( 10)
103 ( 1)
76 ( 7)
42 ( 18)
74 ( 17)
35.5 ( 4)
71 ( 22)
91 ( 10)
76 ( 10)
38 ( 23)
10À4
10À4
10À4
10À4
10À4
10À4
10À4
10À4
10À4
10À4
10À4
10À4
52 ( 12)
47.5 ( 2)
63 ( 19)
149 ( 17)
60 ( 29)
124.5 ( 31)
107 ( 31)
43 ( 25)
116 ( 3)
101 ( 35)
69 ( 9)
10À4
10À4
10À4
10À4
10À4
10À4
10À4
10À4
10À4
10À4
10À4
10À4
6 ( 3)
14 ( 8)
29 ( 4)
11 ( 1)
19 ( 14)
13 ( 2)
13.5 ( 1)
15 ( 2)
16 ( 1)
2 ( 3)
10
11
12
13
14
15
16
17
18
19
30 ( 16)
5 ( 16)
44 ( 14)
dine (3.69 g; 0.03 mol) in n-pentanol (40 ml), triethylamine (7 ml;
0.05 mol) was added. The reaction mixture was stirred at 100 °C for
4 h and subsequently allowed to cool to room temperature. The
white precipitate was collected, washed with cold isopropanol
(2Â 10 ml) and water (3Â 10 ml). The crude product was purified
by crystallization from methanol in the presence of activated char-
coal to give almost white crystals. Yield: 4.36 g (77.8%). TLC (chlo-
roform/methanol 85:15): one single spot, free of starting material.
HPLC purity: 98+%.
yellow precipitate was filtered off, rinsed with water (3Â 10 ml),
and dried to constant weight. Yield: 100 mg (34.9%) of yellow crys-
talline powder. HPLC purity: 99+%.
4.3.1.6. 2-Amino-6-(3-methoxyanilino)purine (compound
16). To
a
suspension of 2-amino-6-chloropurine (1.69 g;
0.01 mol) in n-propanol (20 ml) m-anisidine (1.23 g; 0.01 mol)
was added. The thick resulting suspension was stirred at 100 °C
for 3 h. TLC analysis showed the absence of the starting material
and the presence of the desired product. The reaction mixture
was then cooled to room temperature. The white precipitate was
filtered off, rinsed with n-butanol (3 Â 10 ml) and water
(3 Â 10 ml), then dried in an oven to constant weight. Yield:
2.19 g (85 %). TLC (chloroform-methanol/NH4OH 4:1:0.05): single
spot, free of starting material. HPLC purity: 99+%.
4.3.1.4. 2-Fluoro-6-(3-methoxyanilino)purine (compound
14). This compound was prepared by reacting 2-fluoro-6-chloro-
purine (1.7 g; 0.01 mol), m-anisidine (1.23 g; 0.01 mol) and triethyl-
amine (3.5 ml; 0.025 mol) in n-propanol (20 ml) at 90 °C for 4 h. The
reaction mixture was then cooled to room temperature. The white
precipitate was filtered off, rinsed with n-butanol (3Â 10 ml) and
water (3Â 10 ml) then dried to constant weight. Yield: 5.55 g (61%)
of yellowish crystalline powder. TLC (chloroform/methanol
85:15): one single spot, free of starting material. HPLC purity: 99+%.
4.3.1.7. 2-Methylthio-6-(3-methoxyanilino)purine (compound
17). This substance was prepared by reacting 2-methylthio-6-
chloropurine (200 mg; 1 mmol) and m-anisidine (132 mg; 1 mmol)
in refluxing n-propanol (100 °C; 4 h). Yield: 220 mg as hydrochlo-
ride (69%); HPLC purity: 99+%.
4.3.1.5. 2-Nitro-6-(3-methoxyanilino)purine (compound 15). 2-
Nitro-6-chloro-9-t-butoxycarbonyl-purine, prepared according to
a previously described method24 (300 mg; 1 mmol), was sus-
pended in n-propanol (10 ml). The suspension was cooled to
À5 °C and m-anisidine (132 mg; 1 mmol) was slowly added while
keeping the temperature of the reaction mixture at 60 °C. The
reaction mixture was then stirred at room temperature for 12 h.
The crystalline mass was filtered off, washed with cold n-propanol
(2Â 5 ml), and dried in a desiccator (in the presence of phosphorus
pentoxide) to constant weight, yielding a yellow solid (290 mg).
This product was found to be a mixture of 2-nitro-6-(3-methoxy-
4.3.1.8. 2-Chloro-6-(3-methoxyanilino)-9-methylpurine (com-
pound 18) and 2-chloro-6-(3-methoxyanilino)-9-isopropylpu-
rine (compound 19). These compounds were prepared from
previously prepared 2,6-dichloro-9-methylpurine25 and 2,6-di-
chloro-9-isopropylpurine,26 respectively, and m-anisidine by pre-
viously described methods. Yields: 46% and 80%, respectively.
Purity: 99% and 98.7%, respectively.
4.4. CKX inhibition measurements
anilino)-9-t-butoxycarbonyl-purine,
2-nitro-6-(3-methoxyanili-
no)purine and unreacted starting compound, 2-nitro-6-chloro-9-
t-butoxycarbonyl-purine. Complete deprotection of t-butoxycar-
bonyl group was achieved by stirring the crude product in metha-
nolic HCl (2 ml conc. HCl and 8 ml methanol) at 50 °C for 12 h to
give the hydrochloride, which was transformed to the free base
using diluted aqueous ammonia (3%; 10 ml) and simultaneously
purified from traces of unreacted starting compound, 2-nitro-6-
chloro-9-t-butoxycarbonyl-purine, which stayed in solution. The
The ability of each of the prepared compounds to inhibit CKX
was evaluated by determining their IC50 values in an assay that
had been previously adapted for screening such activity in ELISA
microtitre plates.27 Each well contained 100
lL of a reaction mix-
ture consisting of 0.1 M KH2PO4 (pH 7.4), 1 mM phenazine metho-
sulfate (MTT) and 0.2 mM 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT), final concentrations, the
tested compound (at various concentrations) and 30 lM of iP as

M. Zatloukal et al. / Bioorg. Med. Chem. 16 (2008) 9268–9275
9275
a substrate. Cell-free growth medium of S. cerevisiae strain 23344c
We thank Jarmila Balonová, Jarmila Greplová, and Miloslava
Šubová for skilful technical assistance.
ura- harbouring the plasmid pYES2-AtCKX2 (100
lL) was used di-
rectly as a source of AtCKX2.4 Plates were incubated in the dark
for 30 min at 37 °C and the enzymatic reaction was stopped by
adding 25 lL of 35% acetic acid per well. The absorbance of the
Supplementary data
mixture in each well at 578 nm was then measured using a Tecan
spectrophotometer (Sunrise, Canada), and the absorbance of sam-
ples with no iP was subtracted. Compounds were tested in two
repetitions and the entire test was repeated at least twice.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2008.09.008.
References and notes
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This work was supported by the Grant Agency of the Czech
Republic (GA 206/07/0570, GA 522/06/0108) and Czech Ministry
of Education (MSM 6198959216, 1M0630, LC06034).