N9-Substituted N6-[(3-methylbut-2-en-1-yl)amino]purine derivatives and their biological activity in selected cytokinin bioassays

Article abstract of DOI:10.1016/j.bmc.2011.09.052

Rational design is one of the latest ways how to evaluate particular activity of signal molecules, for example cytokinin derivatives. A series of N⁶-[(3-methylbut-2-en-1-yl)amino]purine (iP) derivatives specifically substituted at the N9 atom of purine moiety by tetrahydropyran-2-yl, ethoxyethyl, and C2-C4 alkyl chains terminated by various functional groups were prepared. The reason for this rational design was to reveal the relationship between specific substitution at the N9 atom of purine moiety of iP and cytokinin activity of the prepared compounds. The synthesis was carried out either via 6-chloro-9-substituted intermediates prepared originally from 6-chloropurine, or by a direct alkylation of N9 atom of N⁶-[(3- methylbut-2-en-1-yl)amino]purine. Selective reduction was implemented in the preparation of compound N⁶-[(3-methylbut-2-en-1-yl)amino]-9-(2- aminoethyl-amino)purine (12) when 6-[(3-methylbut-2-en-1-yl)amino]-9-(2- azidoethyl)purine (7) was reduced by zinc powder in mild conditions. The prepared derivatives were characterized by C, H, N elemental analyses, thin layer chromatography (TLC), high performance liquid chromatography (HPLC), melting point determinations (mp), CI+ mass spectral measurement (CI+ MS), and by ¹H NMR spectroscopy. Biological activity of prepared compounds was assessed in three in vitro cytokinin bioassays (tobacco callus, wheat leaf senescence, and Amaranthus bioassay). Moreover, the perception of prepared derivatives by cytokinin-sensitive receptor CRE1/AHK4 from Arabidopsis thaliana, as well as by the receptors ZmHK1 and ZmHK3a from Zea mays, was studied in a bacterial assay where the response to the cytokinin treatment could be specifically quantified with the aim to reveal the way of the perception of the above mentioned derivatives in two different plant species, that is, Arabidopsis, a model dicot, and maize, a model monocot. The majority of cytokinin derivatives were significantly active in both Amaranthus as well as in tobacco callus bioassay and almost inactive in detached wheat leaf senescence assay. N9-Substituted iP derivatives remained active in both in vitro bioassays in a broad range of concentrations despite the fact that most of the derivatives were unable to trigger the cytokinin response in CRE1/AHK4 and ZmHK1 receptors. However, several derivatives induced low but detectable cytokinin-like activation in maize ZmHK3a receptor. Compound 6-[(3-methylbut-2-en-1-yl)amino]- 9-(tetrahydropyran-2-yl)purine (1) was also recognized by CRE1/AHK4 at high concentration ≥50 μM.

Full text of DOI:10.1016/j.bmc.2011.09.052

Bioorganic & Medicinal Chemistry 19 (2011) 7244–7251
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
N9-Substituted N6-[(3-methylbut-2-en-1-yl)amino]purine derivatives
and their biological activity in selected cytokinin bioassays
a
a,
⇑
a
a
b
c
ˇ
ˇ
Václav Mik , Lucie Szücová , Lukáš Spíchal , Ondrej Plíhal , Jaroslav Nisler , Lenka Zahajská ,
a
a
ˇ
Karel Dolezal , Miroslav Strnad
a Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacky´ University in Olomouc, Šlechtitelu˚ 11, CZ 783 71 Olomouc, Czech Republic
b Laboratory of Growth Regulators, Institute of Experimental Botany AS CR, Šlechtitelu˚ 11, CZ 783 71 Olomouc, Czech Republic
c
ˇ
Isotope Laboratory of Institute of Experimental Botany AS CR, Vídenská 1083, CZ 142 20 Prague 4, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 July 2011
Revised 5 September 2011
Accepted 8 September 2011
Available online 5 October 2011
Rational design is one of the latest ways how to evaluate particular activity of signal molecules, for exam-
ple cytokinin derivatives. A series of N6-[(3-methylbut-2-en-1-yl)amino]purine (iP) derivatives specifi-
cally substituted at the N9 atom of purine moiety by tetrahydropyran-2-yl, ethoxyethyl, and C2–C4
alkyl chains terminated by various functional groups were prepared. The reason for this rational design
was to reveal the relationship between specific substitution at the N9 atom of purine moiety of iP and
cytokinin activity of the prepared compounds. The synthesis was carried out either via 6-chloro-9-substi-
tuted intermediates prepared originally from 6-chloropurine, or by a direct alkylation of N9 atom of
N6-[(3-methylbut-2-en-1-yl)amino]purine. Selective reduction was implemented in the preparation of
compound N6-[(3-methylbut-2-en-1-yl)amino]-9-(2-aminoethyl-amino)purine (12) when 6-[(3-methyl-
but-2-en-1-yl)amino]-9-(2-azidoethyl)purine (7) was reduced by zinc powder in mild conditions. The
prepared derivatives were characterized by C, H, N elemental analyses, thin layer chromatography
(TLC), high performance liquid chromatography (HPLC), melting point determinations (mp), CI+ mass
spectral measurement (CI+ MS), and by 1H NMR spectroscopy. Biological activity of prepared compounds
was assessed in three in vitro cytokinin bioassays (tobacco callus, wheat leaf senescence, and Amaranthus
bioassay). Moreover, the perception of prepared derivatives by cytokinin-sensitive receptor CRE1/AHK4
from Arabidopsis thaliana, as well as by the receptors ZmHK1 and ZmHK3a from Zea mays, was studied
in a bacterial assay where the response to the cytokinin treatment could be specifically quantified with
the aim to reveal the way of the perception of the above mentioned derivatives in two different plant spe-
cies, that is, Arabidopsis, a model dicot, and maize, a model monocot. The majority of cytokinin derivatives
were significantly active in both Amaranthus as well as in tobacco callus bioassay and almost inactive in
detached wheat leaf senescence assay. N9-Substituted iP derivatives remained active in both in vitro
bioassays in a broad range of concentrations despite the fact that most of the derivatives were unable
to trigger the cytokinin response in CRE1/AHK4 and ZmHK1 receptors. However, several derivatives
induced low but detectable cytokinin-like activation in maize ZmHK3a receptor. Compound 6-[(3-meth-
ylbut-2-en-1-yl)amino]-9-(tetrahydropyran-2-yl)purine (1) was also recognized by CRE1/AHK4 at high
Keywords:
N6-[(3-Methylbut-2-en-1-yl)amino]purine
Isopentenyladenine derivatives
N9-Substituted derivatives
Cytokinins
Cytokinin receptors
Rational design
concentration P50 lM.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
derivative has high cytokinin activity.2,3 Despite of previous discov-
eries of iP hydroxylated analogs cis-zeatin (cZ), trans-zeatin (tZ), and
dihydrozeatin (DHZ) in plants, a free base of iP was found in auton-
omous strains of tobacco tissue only in the year 1972.4 Isoprenoid
cytokinin derivatives were often modified with the aim to find a
new, perspective plant growth regulator, especially after the finding
that isoprenoid cytokinins were preferably recognized by cytokinin
receptors AHK3 and CRE1/AHK4 present in a dicot plant Arabidopsis
thaliana used currently as an experimental model.5 Isoprenoid cyto-
kinins also trigger signaling pathway in known Zea mays receptors
ZmHK1, ZmHK2, and ZmHK3a/ZmHK3b.6 The majority of synthetic
derivatives of isoprenoid cytokinins prepared so far has been mainly
Isoprenoid cytokinins, among which N6-[(3-methylbut-2-en-1-
yl)amino]purine belongs, are a group of naturally occurring signal-
ing molecules structurally based on purine that are able to influence
physiological processes such as plant growth and development as
well as cell differentiation.1 N6-[(3-Methylbut-2-en-1-yl)amino]-
purine, also known as isopentenyladenine (iP), was originally syn-
thesized by Leonard et al. in 1965 and it was discovered that the
⇑
Corresponding author. Tel.: +420 585634940; fax: +420 585634870.
E-mail address: lucie.szucova@upol.cz (L. Szücová).
ˇ
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.09.052

V. Mik et al. / Bioorg. Med. Chem. 19 (2011) 7244–7251
7245
mimetic compounds with serious changes of N6-side chain or
Table 1
heterocyclic moiety such as 6-alkenyl and 6-alkynylpurines,7
N6-alkylaminopurines,8 7-acylaminoimidazo[4,5-b]pyridines or 4-
acylaminoimidazo[4,5-c]pyrimidines,9 5-amino substituted 7-
chloro-imidazo[1,2-c]pyrimidines,10 or N6-ethoxyaminopurines.11
Unfortunately, their cytokinin activity has never exceeded the activ-
ity of parent iP. In the last 15 years, after the discovery of benzylami-
nopurine (BA) derivatives in plants,12 the majority of interest has
been paid to synthesis and biological activity of fully or partly
aromatic cytokinin derivatives, such as to (Z)- and (E)-b-substituted
6-styrylpurines,13 benzyl ring substituted benzylaminopurines,14
aromatic N9-(tetrahydropyran-2-yl)purines and N9-(tetrahydrofu-
ran-2-yl)purines,15 N9-substituted kinetin derivatives,16 and aro-
matic N9-ribosides.17 Even though many of the above mentioned
aromatic cytokinin derivatives were highly active in various cytoki-
nin bioassays, with the exception of meta-topolin, they usually did
not trigger cytokinin signaling pathway of the above mentioned
AHK3 and CRE1/AHK4 receptors.4 Therefore, we have attempted to
prepare molecules that are not mimetic but direct derivatives of
the isoprenoid cytokinin with the aim to obtain active derivatives
that are able to trigger cytokinin signaling pathway. Today, combi-
natorialorganicchemistryallowsus topreparepreciselysubstituted
molecules, for example, N6-[(3-methylbut-2-en-1-yl)amino]purine
substituted in C2, N1, N3, N7, C8 as well as at N9 position of purine
moiety. Therefore, we specifically focused on N9 position of iP as
Structures of the prepared compounds and their abbre-
viations. Wavy line represents a position of connection
to the N9 atom of the purine moiety
Compound
R
1
O
2
3
O
O
O
Br
4
5
6
7
8
9
Cl
OH
N3
Cl
CN
O
10
O
OH
11
12
the position that is, due to 9-b-
D
-glucopyranylation, naturally occu-
O
NH2
pied in the cytokinin metabolism in plants.1,18 Generally, N9-gluco-
sides are considered as nonactive or weakly active cytokinin
forms.1,19 For that reason, the preparation of N9-substituted cytoki-
nin derivatives as the compounds prospective for plant growth is
rather controversial. Isopentenyladenine derivatives substituted at
N9 by 2-carboxyethyl, 2-carbo-t-butoxyethyl, 2-nitriloethyl were
prepared by Corse et al. in 1989 and they were tested for growth re-
sponse in the soybean callus assay. They all reduced the biological
activity of the parent compound even though they all still remained
considerably active.20 Corse’s finding contributed to the widely
spread assumption that N9-substituted derivatives are less active
than their free bases. On the other hand, the N9 analogs of aromatic
cytokinins with certain substituents very often maintained cytoki-
nin activity, for example, N9-(tetrahydropyran-2-yl) deriva-
tives15,21,22 or N9-methyl derivatives23 and sometimes were even
more effective than their free bases. Conversely, cytokinins substi-
tuted by 9-methoxymethyl, 9-cyclohexyl or 9-(3-hydroxypropyl)
groups were less active in tobacco callus assay than the unsubstitut-
ed ones.24 9-(4-Chlorobutyl) derivative of benzylaminopurine was
substantially more effective in the ability to cause chlorophyll reten-
tion in intact soybean leaves.25 N9-Halogenoalkyl and other N9-
substituted derivatives of kinetin showed strong anti-senescent
activities under both dark and light conditions and in addition, the
compounds were able to protect membrane lipids against the nega-
tive action of reactive oxygen species that accumulate in tissues dur-
ing leaf senescence.16 Young and Letham modified iP with the
substitution in N9 by the addition of tetrahydropyran-2-yl (THP)
group and the substitution markedly enhanced the activity of iP in
tobacco leaf senescence assay.19 Tetrahydropyran-2-yl derivative
of iP can serve as a good example of how controversial can be the re-
sults from the testing of various N9 cytokinin derivatives in available
cytokinin assays – substituent in N9 may probably play as important
role as the chosen testing system. In addition, N9 substitution of
cytokinins offers the easiest way how to prepare direct, not mimetic
analogs of free cytokinin bases. Therefore, we prepared a small li-
brary of variously alkylated N9-iP derivatives with a number of ter-
minal groups such as hydroxy-, chloro-, bromo-, amino-, azido-,
cyano-, carboxy-, etc. These functional groups were placed at the
end of the carbon or carbon/oxygen spacers with modifiable length.
We tested their biological properties in cytokinin bioassays such as
tobaccocallus, detachedwheatleafsenescence,andAmaranthusbio-
assay in order to clarify ambiguous situation that persists in the
structure and activity relationship of N9-substituted cytokinins. In
addition, using these compounds we also studied the cytokinin per-
ception in A. thaliana receptor CRE1/AHK4 and maize ZmHK1 and
ZmHK3a receptors to compare the results in two different model
plants.
2. Results and discussion
2.1. Synthesis
The compounds described in this paper are shown in the Scheme
1 while the substituents in N9 position of the purine moiety are
HN
N
N
N
R
N
Scheme 1. Schematic representation of N6-isopentenyladenine derivatives.

7246
V. Mik et al. / Bioorg. Med. Chem. 19 (2011) 7244–7251
Cl
N
HN
N
N
a
c
N
N
N
N
b
N
(1)
N
O
O
Cl
N
Cl
N
HN
N
N
N
N
b
N
N
(2)
N
H
N
N
O
O
HN
b
N
N
N
H
N
h
f
j
d
g
e
i
HN
HN
HN
HN
HN
HN
HN
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
O
O
O
(3)
(10)
OH (6)
(4)
(5)
CN (9)
(8)
Br
Cl
O
Cl
k
l
HN
HN
HN
m
N
N
N
N
N
N
N
N
N
N
N
N
OH
NH
N
(11)
(7)
2
(12)
3
O
Scheme 2. Reaction scheme for the synthesis of N9-substituted cytokinin derivatives. (a) 3,4-Dihydropyran, EtOAc, TFA, rt, 2 h; (b) 3-methylbut-2-en-1-amine HCl, Et3N,
PrOH, 100 °C, 4 h; (c) ethyl vinyl ether, EtOAc, TFA, rt, 2 h; (d) sodium acetate, DMF, 100 °C, 3 h; (e) 1,2-dibromoethane, K2CO3, DMSO, rt, 12 h; (f) 1-bromo-2-chloroethane,
K2CO3, DMSO, rt, 12 h; (g) ethylene carbonate, NaOH, DMF, 154 °C, 2 h; (h) 4-chlorobutyronitrile, K2CO3, DMF rt, 12 h; (i) 1-bromo-4-chlorobutane, K2CO3, DMSO, rt, 12 h; (j)
ethyl 4-bromobutyrate, K2CO3, DMF, rt, 12 h; (k) 0.5 M NaN3 in DMSO, 80 °C, 1 h; (l) NaOH, 50% ethanol, rt, 12 h; (m) Zn, NH4Cl, EtOH, H2O, rt, 2 h.
listed in Table 1. From Scheme 2 it is obvious that the compounds
were prepared either via the appropriate 6-chloro-9-substituted
derivative (1 and 2) made of 6-chloropurine that afterward reacted
with 3-methylbut-2-en-1-yl)amine, or by a direct alkylation of
6-[(3-methylbut-2-en-1-yl)amino]purine (3–12) in the presence
of potassium carbonate in DMSO or DMF followed by purification
from arising N7 derivative using flash chromatography. 6-[(3-
Methylbut-2-en-1-yl)amino]purine was prepared according to
slightly modified procedure described by Robins et al. in the litera-
ture.26 Compound 2 was prepared according to a paper previously

V. Mik et al. / Bioorg. Med. Chem. 19 (2011) 7244–7251
7247
published in literature.27 We used one of the suggested methods
that was reaction of 6-chloropurine with ethyl vinyl ether and sub-
sequently with 3-methylbut-2-en-1-ylamine as given in Scheme 2.
For the compound 6, we adopted preparation published by Rosen-
gren et al. for 9-(2-hydroxyethyl)adenine synthesis28 but the yield
of 6 was not as high as for 9-(2-hydroxyethyl)adenine. Particular
syntheses are given in greater detail in Section 4.3. Prepared com-
pounds were characterized by elemental analyses, thin layer chro-
matography (TLC), melting point determinations (mp), CI+ MS, and
1H NMR spectroscopy. The purity of the prepared substances was
confirmed by high-performance liquid chromatography (HPLC). Ta-
ble 2 shows C, H, N elemental analysis data, melting points, MS and
HPLC purity data. 1H NMR spectral data are given in Supplementary
data.
launched with the polarity and steric effect of particular substitu-
ents.20 The compounds prepared within the frame of this study
were subjected to three cytokinin bioassays (tobacco callus, wheat
senescence, and Amaranthus bioassay)17 and the results are sum-
marized in Table 3. The achieved activities of new compounds were
compared to those of iP that represents free base without any N9
substitution. The effects of novel compounds on the proliferation
of cytokinin-dependent tobacco callus were tested in the callus bio-
assay. Derivatives 2, 3, and 7–9 showed very high activity exceeding
110% of the activity of parent compound (iP), when used at their
optimal concentrations (Table 3). The activity of compounds 5, 10,
and 12 almost equaled the activity of iP and, interestingly, also
the other derivatives 1, 4, 6, 11 reached only slightly lower activity
then their parent compound. Cytokinins are known for their stimu-
latory effects on cell proliferation in their optimal concentrations,
2.2. Cytokinin bioassays
however typically in the concentrations exceeding 10 lM their
effect on cell division turns into inhibitory.31 The compound 1, be-
The preparation of N9-substituted cytokinins as the compounds
prospective for plants is rather controversial due to unlike results
obtained for N9-substituted derivatives in some cytokinin bioas-
says29 as well as for the known fact that glucosylation on N9 posi-
tion of the purine based cytokinins may turn active free cytokinin
into their inactive forms: N9-glucosides. On the other hand, some
N9 analogs of aromatic cytokinins very often maintained cytokinin
activity15,22,23,25 while some others were reported to be less effec-
tive than free bases24 or reduced the biological activity of parent
compound even when still maintained considerable activity.20
However, Corse emphasis the fact that the activity is probably
haved as classical cytokinin and, in a manner similar to iP, inhibited
cell proliferation at concentrations exceeding 10 lM (Fig. 1A). In
contrast, many other compounds, for example, 2–4 and 6–8 showed
no such activity, instead maintaining a strong stimulatory effect on
cell division, even at the highest concentration tested (100 lM,
Fig. 1A). In Amaranthus bioassay, some of new compounds were
found to be highly active again. The most active substances
(compounds 2 and 4) exceeded 120% of iP activity (Table 3;
Fig. 1B) followed by compounds 1, 5, and 10 that were still more
active than iP. Other compounds were less active and their
activities fluctuated from approximately 80% to 30% (Table 3). In
Table 2
Elemental analyses, melting points, CI+ MS spectral data and HPLC purity of the prepared compounds
Compound
Elemental analyses calculated/found
%H
Mp (°C)
CI+ MS [M+H+]
HPLC (%)
%C
%N
1
2
3
4
5
6
7
8
9
62.7/62.5
61.1/60.6
62.7/53.9
46.5/46.3
54.2/54.6
58.3/57.7
52.9/52.2
57.2/56.4
62.2/59.1
60.6/60.4
58.1/56.1
58.5/58.5
7.37/7.5
7.7/7.8
7.4/6.5
5.2/5.2
6.1/6.2
6.9/6.7
5.9/5.9
6.9/6.9
6.7/7.0
7.3/7.4
6.6/6.5
7.4/7.5
24.4/24.4
25.4/25.6
24.4/22.8
22.6/22.5
26.4/26.2
28.3/24.2
41.2/43.3
23.8/22.7
31.1/28.1
22.1/20.7
24.2/26.0
34.1/35.2
85.7–89.5
44.1–45.5
55.7–57.1
120.2–121.5
130.2–132.3
73.1–76.2
92.4–94.9
141.1–142.7
59.5–61.3
55.5–57.8
152.6–154.7
164–169
288.3
276.3
290.3
311.2
266.1
248.3
273.1
294.8
271.2
318.2
290.2
247.5
98.5
99.4
98.8
99.5
99.8
98.5
99.9
99.0
98.8
99.9
99.5
98.5
10
11
12
Table 3
Relative cytokinin activity of the prepared N6-(2-isopentenyl)-adenine (iP) derivatives in classical cytokinin bioassays. The effect in optimal concentration is compared with the
activity of parental compound iP (100% means 10ꢀ5 M iP for the Amaranthus betacyanin bioassay, 10ꢀ4 M iP for the senescence bioassay and 10ꢀ5 M iP for the tobacco callus
bioassay). The values represent means SD of at least two independent assays each performed in five technical replicates
Compound
Amaranthus bioassay
Relative activity
Senescence bioassay
Relative activity
Tobacco callus bioassay
Relative activity
(%)
79.3 6.1
Optimal concentration
Optimal concentration
Optimal concentration
(mol lꢀ1
)
(%)
(mol lꢀ1
)
(%)
(mol lꢀ1
)
1
2
3
4
5
6
7
8
9
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
109.3 24
122.8 0.1
82.1 32
120.8 4.1
115.0 19
29.0 1.8
82.3 7.3
59.9 7.0
61.2 3.2
102.2 21
47.8 5.4
33.7 8.3
10ꢀ4
10ꢀ4
10ꢀ5
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
53.0 3.9
11.0 1.8
66.5 0.8
37.7 14.6
23.2 6.6
47.0 14
n.a.
39.5 5.9
15.5 7.8
53.2 4.3
n.a.
10ꢀ5
10ꢀ5
10ꢀ5
10ꢀ5
10ꢀ5
10ꢀ5
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ5
10ꢀ5
10ꢀ5
118.0 11
110.5 7.8
80.9 30
98.7 19
82.7 1.0
122.5 6.3
115.15 10
119.5 0.1
92.6 23
10
11
12
79.7 10
95.0 18
n.a.
n.a. means not active.

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V. Mik et al. / Bioorg. Med. Chem. 19 (2011) 7244–7251
Figure 1. The influence of the N9-substituents of iP, particularly tetrahydropyran-2-yl (1) and 2-bromoethyl (4), on biological activity in cytokinin bioassays, particularly on
the stimulation of cytokinin-dependent tobacco callus growth (A), the dark induction of betacyanin synthesis in Amaranthus cotyledons (B) and on the retention of
chlorophyll in excised wheat leaves (C).
all cases, the maximal activities were reached in 10-times higher
concentration (100 lM) than in the case of free iP control. The new-
ly prepared compounds were further tested in detached wheat
leaves senescence bioassay where the effects of the compounds
on the retention of chlorophyll in detached wheat leaves in the dark
was observed. Despite of the recently published results on N9-
substituted kinetin derivatives16 N9-iP derivatives did not show
significant antisenescence activity as obvious from Table 3. The
overall low activity of iP derivatives in the senescence assay is not
surprising due to the fact that iP itself is only weakly active in this
assay compared to other cytokinins such as BA or tZ. Although we
claim that the structural motifs for senescence delay are rather
localized in the N6 part of the purine molecule, we showed again
that short alkylhalogen substitutions (2-chloroethyl, 2-bromoeth-
yl) in N9 part of the molecule can also significantly improve cytoki-
nin properties of the compound and, additionally, can prevent
negative effects on cell proliferation, which are commonly caused
by high concentrations of exogenously applied cytokinins.
2.3. CRE/AHK4, ZmHK1, and ZmHK3a receptor bioassays
Previous tests of various N9-substituted cytokinins showed that
majority of aromatic cytokinins are probably not very good sub-
strates for CRE1/AHK4 receptor.5,15,16 Therefore, we chose isopren-
oid cytokinin isopentenyladenine (iP) as a joint structural motif of
newly prepared derivatives, because iP triggers signaling pathway
in all the intended receptors very effectively.5,6 We compared the
response of CRE1/AHK4 receptor of a dicot model plant Arabidopsis
with the response in the receptors of a model monocot maize, par-
ticularly in ZmHK1 and ZmHK3a, using a bacterial assay we have
adapted previously,5 to reveal if N9 isopentenyladenine derivatives
perception may differ among different plants. We were also inter-
ested in the fact if the response is dependent on structural motif of
the substituent in N9 position and/or polarity of terminal group,
and if the perception in one plant differs from one receptor to an-
other. We therefore tested the interactions between our new iP
derivatives and the Arabidopsis cytokinin receptor CRE1/AHK4,
using a transformed Escherichia coli strain expressing the receptors
and the cytokinin-activated reporter gene cps::lacZ (fusion gene of
capsular polysaccharide synthesis operon and reporter gene coding
for b-galactosidase.5,32 The risk of metabolic conversion of the
tested cytokinin derivative was eliminated due to the short incuba-
tion time in this assay. With the exception of compound 1 which
activated the CRE1/AHK4 receptor to approximately 25% in
Figure 2. The perception of N9-substituted compounds 1, 4, 5, and 8 by cytokinin
recognizing receptor CRE1/AHK4 (A) and of the compounds 4, 5, and 8 by cytokinin
recognizing receptor ZmHK3a (B), both by cytokinin-induced b-galactosidase assay.
iP was used as a positive control. Error bars show SEM for three to six replicates.
ZmHK3a for selected derivatives with N9-(2-bromoethyl)
(compound 4), N9-(2-chloroethyl) (compound 5), and N9-(4-chlo-
robutyl) (compound 8) side chains. While neither of the com-
pounds was able to activate ZmHK1 receptor, both of them (5
and 8) triggered signaling pathway via ZmHK3a receptor approxi-
50 lM concentration, all other N9-substituted iP derivatives that
were otherwise also highly active in bioassays (i.e., compounds 2,
7, and 8) were not able to activate CRE1/AHK4 (Fig. 2A. Therefore,
we performed receptor tests also in maize receptors ZmHK1 ad
mately at the concentration above 10 lM and higher (given in
Figure 2B). Despite the fact that the perception via ZmHK3a recep-
tor was lower than in the case of iP control, it contributed the fact

V. Mik et al. / Bioorg. Med. Chem. 19 (2011) 7244–7251
7249
that N9-halogenoalkyls of cytokinins maintain their cytokinin
activity at high concentrations.16 The length of the spacer did not
make a difference in the cytokinin perception, but the terminal
substitution proved to be critically important in the subsequent
receptor activation. While N9-(2-chloroethyl) and N9-(2-chlorobu-
tyl) showed measurable activation of the receptor, 2-bromoethyl
derivative did not show any cytokinin-like activity (Fig. 2B) This
can be caused by the fact that bromine atom is markedly bigger
than chlorine and that dimensions of the terminal group might
thus play role in the interaction with the receptor domain.
silica gel 60 WF254 plates (Merck Co.). CHCl3:MeOH (4:1, v:v) or
ethyl acetate/toluene (2:1, v:v) were used as mobile phase. CI+
MS were recorded using Polaris Q (Finnigan) 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 am, and spectra were col-
lected using 1.0 s cyclical scans, applying 70 eV electron energy.
In the CI+ ionization mode, isobutane was used as the reagent
gas at a flow-rate of 2 L/h. The mass spectrometer was directly
coupled to an Xcalibur data system. The purity of synthesized com-
pounds was confirmed by high performance liquid chromatogra-
phy (Beckman Gold system). To determine their HPLC purity, the
samples were dissolved in HPLC mobile phase (initial conditions),
applied to an RP-column (150 mm ꢁ 4.6 mm, 5 mL, Microsorb
C18, Varian) and the separated constituents were eluted with a lin-
ear methanolic gradient (10–90% over 30 min, pH adjusted to four
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. 1H NMR spectra were recorded
on a Bruker Avance 300 spectrometer operating at a temperature
of 300 K and a frequency of 300.13 MHz. Samples were prepared
by dissolving the substances in DMSO-d6. Tetramethylsilane (TIR)
was used as the internal reference standard.
3. Conclusions
We prepared twelve N9-substituted isopentenyladenine deriva-
tives with the aim to bring more light on structure and activity
relationship of N9-substituted cytokinin derivatives. The substitu-
tion of iP in N9 position of the purine with ethoxyethyl-, acetoxy-,
azido-, 4-chlorobutyl-, and 3-cyanopropyl-groups significantly im-
proved their biological activity in proliferation of plant cells in to-
bacco callus assay in comparison to iP, although it negatively
influenced the recognition of these compounds by cytokinin recep-
tors CRE1/AHK4, ZmHK1, and ZmHK3a. Due to the fact that all the
above mentioned groups have high polarity and the substituents
consist of at least three spacer atoms, we can state that polarity
and length of the N9-substituent chain has significant impact on
compound activity in tobacco callus assay. Majority of prepared
compounds also showed high activity in Amaranthus bioassay. Iso-
pentenyladenine derivatives substituted on N9 position are not,
despite of their similarity to the kinetin derivatives that possess
significant activities, effective retardants of plant leaves senes-
cence as showed in detached wheat leaves senescence assay.
Almost all the compounds were active cytokinins even at high con-
centrations where free cytokinins are generally no longer effective.
Receptor assay showed that these N9 derivatives are not recog-
nized via Arabidopsis CRE1/AHK4 receptor and only slightly via
maize ZmHK3a receptor at high concentrations. The result why
these derivatives still show significant cytokinin activity in tobacco
callus and Amaranthus bioassay can rather contribute to the previ-
ously published observations that other ways for N9 cytokinins
may exist that enable them to effectively transmit the signal into
the cell and to control the cell growth and development.
4.3. Syntheses
Reaction procedures of all the below mentioned substances are
given in Scheme 2, particular steps are marked with a–m letters
and explained under the scheme.
4.3.1. Synthesis of 6-[(3-methylbut-2-en-1-yl)amino]-9-(tetrahy
dropyran-2-yl)purine (1)
The substance was prepared according to the slightly modified
procedure described previously in the literature.21 In the first step,
6-chloro-9-(tetrahydropyran-2-yl)purine (a) was prepared from
6-chloropurine.15,26 The intermediate (1 g, 4.2 mmol) was then re-
fluxed with the appropriate amount of (3-methylbut-2-en-1-yl)a-
mine hydrochloride (0.61 g, 5 mmol) in n-propanol (30 mL) in the
presence of triethylamine (1.06 g, 10.5 mmol, 1.46 mL) for 4 h (b).
The reaction mixture was evaporated to dryness. The residue was
dissolved in water (50 mL) and extracted into EtOAc (3 ꢁ 10 mL).
The combined ethyl acetate phases were dried (Na2SO4) and con-
centrated under reduced pressure. The pure product was obtained
using flash chromatography (CHCl3:MeOH, 9:1). C, H, N elemental
analyses, CI+ MS and HPLC purity data as well as mp are given in
Table 2 while 1H NMR data are given in Supplementary data.
4. Experimental
4.1. Chemicals
(3-methylbut-2-en-1-yl)amine hydrochloride, 6-[(3-methyl-
but-2-en-1-yl)amino]purine and 6-chloropurine were purchased
from Olchemim, triethylamine, ethylacetate, n-propanol, n-buta-
nol, diethyl ether, petrolether, hexane, dimethylsulfoxide (DMSO),
N,N0-dimethylformamide (DMF), and other common organic
solvents used for synthesis were purchased from Litolab and
Lachema. 1-bromo-2-chloroethane, 1-bromo-4-chlorobutane, 1,2-
dibromoethane, and ethyl vinyl ether were purchased from Sig-
ma–Aldrich. 3,4-dihydro-2H-pyran, 4-chlorobutyronitrile, ethyl-
4-bromobutyrate, sodium iodide were purchased from Fluka.
NaOH, sodium azide, CaCl2, acetic acid, Zn powder, NH4Cl, NH3,
K2CO3, and ethylene carbonate were purchased from Litolab.
4.3.2. Synthesis of 6-[(3-methylbut-2-en-1-yl)amino]-9-(ethoxy
ethyl)purine (2)
In the first step, 6-chloro-9-(ethoxyethyl)purine was prepared
according to the data found in the literature (c).30 The intermediate
(1 g, 4.4 mmol) was refluxed with 3-methylbut-2-en-1-yl)amine
hydrochloride (0.64 g, 5.3 mmol) in n-propanol (25 mL) in the
presence of triethylamine (1.34 g, 13.5 mmol, 1.84) for 4 h (b).
After the evaporation of the solvent, the crude product was treated
with H2O (30 mL) and extracted into EtOAc (20 mL). The organic
phase was dried (Na2SO4) and concentrated under reduced pres-
sure. Pure white waxy substance was obtained by flash chromatog-
raphy (mobile phase EtOAc:hexane, 1:2). C, H,
N elemental
analyses, CI+ MS and HPLC purity data as well as mp are given in
Table 2 while 1H NMR data are given in Supplementary data.
4.2. General procedures
Elemental analyses (C, H, and N) were determined of an EA1112
Flash analyzer (Thermo-Finnigan). The melting points were deter-
mined on Büchi Melting Point B-540 apparatus and are uncor-
rected. Thin layer chromatography (TLC) was carried out using
4.3.3. 6-[(3-Methylbut-2-en-1-yl)amino]-9-(acetoxy)purine (3)
6-[(3-Methylbut-2-en-1-yl)amino]purine (0.5 g, 2.5 mmol) was
mixed with sodium acetate (0.32 g, 3.9 mmol) in DMF (20 mL) and
stirred for 3 h at 95–100 °C and subsequently, 12 h at 25 °C (d). A

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V. Mik et al. / Bioorg. Med. Chem. 19 (2011) 7244–7251
crude product obtained after the evaporation of the solvent was
poured onto trash ice and extracted to EtOAc (20 mL). The organic
phase was dried (Na2SO4) and concentrated under reduced pres-
sure. Pure white crystalline product was obtained after the crystal-
lization from methanol. C, H, N elemental analyses, CI+ MS, and
HPLC purity data as well as mp are given in Table 2 while 1H
NMR data are given in Supplementary data.
4.3.8. Synthesis of 6-[(3-methylbut-2-en-1-yl)amino]-9-(4-chlo
robutyl)purine (8)
6-[(3-Methylbut-2-en-1-yl)amino]purine (2 g, 9.8 mmol) was
stirred with 1-bromo-4-chlorobutane (2.02 g, 11.8 mmol,
1.36 mL), and with potassium carbonate (3 g, 21.7 mmol) in DMSO
(50 mL) under CaCl2 drying tube at room temperature for 12 h (i).
The reaction mixture was then poured onto trash ice (200 mL) and
extracted into EtOAc (50 mL). The organic layer was dried (Na2SO4)
and concentrated under reduced pressure. The crude product was
purified using flash chromatography (EtOAc:hexane, 1:2). White
crystals were obtained after the crystallization from diethyl ether.
C, H, N elemental analyses, CI+ MS, and HPLC purity data as well as
mp are given in Table 2 while 1H NMR data are given in Supple-
mentary data.
4.3.4. Synthesis of 6-[(3-methylbut-2-en-1-yl)amino]-9-(2-bro
moethyl)purine (4)
The mixture of 6-[(3-methylbut-2-en-1-yl)amino]purine (1 g,
4.9 mmol), 1,2-dibromoethane (2.31 g, 12.3 mmol, 1.06 mL) and
potassium carbonate (1.7 g, 12.3 mmol) was stirred in DMF
(30 mL) under the CaCl2 drying tube at room temperature for
12 h (e). The reaction mixture was subsequently concentrated in
vaccuo, treated with ice water (50 mL) and extracted into ethyl
acetate (4 ꢁ 10 mL). The organic phases were combined, washed
with water (2 ꢁ 10 mL), brine (2 ꢁ 10 mL), dried over Na2SO4,
and finally concentrated under reduced pressure. The pure product
was obtained after the purification via flash chromatography using
(CHCl3:MeOH, 19:1) as eluent. C, H, N elemental analyses, CI+ MS,
and HPLC purity data as well as mp are given in Table 2 while 1H
NMR data are given in Supplementary data.
4.3.9. 6-[(3-Methylbut-2-en-1-yl)amino]-9-(3-cyanopropyl)
purine (9)
6-[(3-Methylbut-2-en-1-yl)amino]purine (0.5 g, 2.5 mmol) was
mixed with potassium carbonate (0.87 g, 6.25 mmol) in DMF
(15 mL). 4-chlorobutyronitrile (0.31 g, 270 lL, 3 mmol) was slowly
added and the reaction mixture was vigorously stirred at room
temperature under CaCl2 drying tube (h). Another four portions
of 4-chlorobutyronitrile were added during 26 h. The reaction mix-
ture was subsequently evaporated to dryness, treated with trash
ice (50 mL) and extracted into EtOAc (4 ꢁ 10 mL). The combined
organic layers were washed with water (2 ꢁ 10 mL), brine
(2 ꢁ 10 mL), dried (Na2SO4) and finally concentrated under re-
duced pressure. Pure product was obtained by flash chromatogra-
phy using CHCl3:MeOH (19:1) as eluent. Yield 94%: C, H, N
elemental analyses, CI+ MS, and HPLC purity data as well as mp
are given in Table 2 while 1H NMR data are given in Supplementary
data.
4.3.5. Synthesis of 6-[(3-methylbut-2-en-1-yl)amino]-9-(2-chlo
roethyl)purine (5)
6-[(3-Methylbut-2-en-1-yl)amino]purine (2 g, 9.8 mmol) was
stirred with 1-bromo-2-chloroethane (1.4 g, 9.8 mmol, 0.82 mL),
and with K2CO3 (3 g, 21.7 mmol) in DMSO (50 mL) under CaCl2
drying tube at room temperature for 12 h (f). The reaction mixture
was then poured on trash ice (200 mL) and extracted into EtOAc
(50 mL). The organic layer was dried (Na2SO4) a concentrated un-
der reduced pressure. The crude product was purified using flash
chromatography (EtOAc:hexane, 1:2). White crystals were ob-
tained after the crystallization from hexane. C, H, N elemental anal-
yses, CI+ MS, and HPLC purity data as well as mp are given in Table
2 while 1H NMR data are given in Supplementary data.
4.3.10. 6-[(3-Methylbut-2-en-1-yl)amino]-9-(4-ethoxy-4-oxobu
tyl)purine (10)
6-[(3-Methylbut-2-en-1-yl)amino]purine (1 g, 4.9 mmol) was
mixed with potassium carbonate (1.7 g, 12.3 mmol), and DMF
(30 mL) and stirred vigorously at room temperature. Ethyl 4-bro-
4.3.6. Synthesis of 6-[(3-methylbut-2-en-1-yl)amino]-9-(2-hydr
oxyethyl)purine (6)
mobutyrate (1.06 g, 784 lL, 5.4 mmol) was then dropwise added
and the reaction mixture was stirred under CaCl2 drying tube at
room temperature overnight (j). The mixture was evaporated to
dryness and treated with trash ice (150 mL). The white precipitant
was filtered, washed with cold water and dried at room tempera-
ture. The pure product was obtained by flash chromatography
purification using (CHCl3:MeOH, 19:1) as eluent. Yield 74%: C, H,
N elemental analyses, CI+ MS, and HPLC purity data as well as
mp are given in Table 2 while 1H NMR data are given in Supple-
mentary data.
The substance was prepared according to the procedure, found
for 9-(2-hydroxyethyl)adenine in the literature.28 6-[(3-methyl-
but-2-en-1-yl)amino]purine (1 g, 4.9 mmol) was stirred with eth-
ylene carbonate (0.44 g, 5 mmol) and NaOH (0.1 g, 2.5 mmol) in
DMF (20 mL) (g). The reaction lasted for 2 h at 154 °C. The reaction
mixture was poured onto trash ice and extracted into EtOAc
(20 mL). After drying (Na2SO4) and evaporation of the solvent,
the crude product was dissolved in mobile phase and purified by
flash chromatography (CHCl3:MeOH, 95:5). White, flocky sub-
stance was obtained after the crystallization with petroleum ether.
C, H, N elemental analyses, CI+ MS and HPLC purity data as well as
mp are given in Table 2 while 1H NMR data are given in Supple-
mentary data.
4.3.11. 6-[(3-Methylbut-2-en-1-yl)amino]-9-(3-carboxypropyl)
purine (11)
Compound 10 (1 g, 3.2 mmol) was stirred in the solution of
NaOH (0.63 g, 15.8 mmol) in 50% ethanol (50 mL) at room temper-
ature overnight (l). The mixture was cooled in the ice bath and
1.1 mL of concentrated acetic acid was added (pH approximately
5–6). Ethanol was distilled off and the solution was extracted with
ethyl acetate and water. The organic phase was evaporated to ½.of
total volume and stored 2 days at 4 °C. White precipitant that ap-
peared was filtered off, washed with cold ethyl acetate and dried
at room temperature. Pure product was obtained by flash chroma-
tography purification using (CHCl3:MeOH, 9:1) as eluent. Yield
82%: C, H, N elemental analyses, CI+ MS, and HPLC purity data as
well as mp are given in Table 2 while 1H NMR data are given in
Supplementary data.
4.3.7. 6-[(3-Methylbut-2-en-1-yl)amino]-9-(2-azidoethyl)
purine (7)
Compound 4 (2.82 g, 9.1 mmol) was heated with sodium iodide
(0.14 g, 0.9 mmol) in 0.5 M solution of sodium azide in DMSO
(37 mL) at 80 °C for 1 h and after that stirred at room temperature
overnight (k). The mixture was poured onto trash ice (300 mL) and
white precipitate was obtained. The solid was filtered off, washed
with cold water and dried at room temperature. Yield 92%: C, H, N
elemental analyses, CI+ MS, and HPLC purity data as well as mp are
given in Table 2 while 1H NMR data are given in Supplementary
data.

V. Mik et al. / Bioorg. Med. Chem. 19 (2011) 7244–7251
7251
4.3.12. 6-[(3-Methylbut-2-en-1-yl)amino]-9-(2-aminoethyl)
purine (12)
Czech Republic (NAZV QI 92A247). We would like to thank Zdena
Kamarádová, Jarmila Balonová, and Miloslava Šubová for skilful
´
6-[(3-Methylbut-2-en-1-yl)amino]-9-(2-azidoethyl)purine (0.9 g,
3.35 mmol) was dissolved in EtOH (9 mL) and water (3 mL). NH4Cl
(0.41 g, 7.7 mmol) and Zn powder (0.29 g, 4.4 mmol) were then
added to the solution and resulting mixture was vigorously stirred
at room temperature. The reaction was accompanied by the forma-
tion of a white precipitate and by releasing gaseous nitrogen and it
was completed within 2 h (monitored by TLC). The white precipi-
tate was dissolved by the addition of concd. aqueous ammonia
(7.5 mL) and residual Zn powder was filtered off. The volume of
the filtrate was reduced by half by concentration in vacuum. A
white solid formed during concentration was filtered off a clear
filtrate was evaporated in reduced pressure. Yield 92%: C, H, N
elemental analyses, CI+ MS, and HPLC purity data as well as mp
are given in Table 2 while 1H NMR data are given in Supplementary
data.
technical assistance, RNDr. Tomáš Gucky, Ph.D. and Mgr. Jan Walla
for NMR spectral measurement and help with interpretation and to
Mgr. Tibor Béreš for HPLC and CI+ MS measurement.
Supplementary data
Supplementary data associated (1H NMR data for iP derivatives
1–12) with this article can be found, in the online version, at
doi:10.1016/j.bmc.2011.09.052.
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(Nagoya, Japan) and the whole assay was performed as previously
described in Spíchal et al.5 The assay was optimized to 96-well
microtiter plates according to the literature.14 The pre-culture was
diluted 1:10 and the incubation time was 16 h. Relative activation
of the cytokinin receptors was determined by measuring b-galacto-
sidase activity using the fluorescent substrate 4-methylumbellife-
ˇ
ˇ
16. Mik, V.; Szücová, L.; Šmehilová, M.; Zatloukal, M.; Dolezal, K.; Nisler, J.; Grúz, J.;
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ryl-b-D-galactoside (Sigma–Aldrich) and by monitoring the culture
density at OD600. The test was performed in triplicate and the entire
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Acknowledgments
This work was financially supported by the Czech Ministry of
Education Youth and Sports (MSM 6198959216), by Grant No.
ED0007/01/01 Centre of the Region Haná for Biotechnological
and Agricultural Research, by The Grant Agency of the Czech
Republic (522/09/1576) and by The Ministry of Agriculture of the
31. Holub, J.; Hanuš, J.; Hanke, D. E.; Strnad, M. Plant Growth Regul. 1998, 26, 109.
32. Yamada, H.; Suzuki, T.; Terada, K.; Takei, K.; Ishikawa, K.; Miwa, K.; Yamashino,
T.; Mizuno, T. Plant Cell Physiol. 2001, 42, 1017.