Synthesis, characterization and biological activity of ring-substituted 6-benzylamino-9-tetrahydropyran-2-yl and 9-tetrahydrofuran-2-ylpurine derivatives

Article abstract of DOI:10.1016/j.bmc.2009.01.041

In an attempt to improve specific biological functions of cytokinins routinely used in plant micropropagation, 33 6-benzylamino-9-tetrahydropyran-2-ylpurine (THPP) and 9-tetrahydrofuran-2-ylpurine (THFP) derivatives, with variously positioned hydroxy and methoxy functional groups on the benzyl ring, were prepared. The new derivatives were prepared by condensation of 6-chloropurine with 3,4-dihydro-2H-pyran or 2,3-dihydrofuran and then by the condensation of these intermediates with the corresponding benzylamines. The prepared compounds were characterized by elemental analyses, TLC, HPLC, melting point determinations, CI+ MS and ¹H NMR spectroscopy. The cytokinin activity of all the prepared derivatives was assessed in three classical cytokinin bioassays (tobacco callus, wheat leaf senescence and Amaranthus bioassay). The derivatives 6-(3-hydroxybenzylamino)-9-tetrahydropyran-2-ylpurine (3) and 6-(3-hydroxybenzylamino)-9-tetrahydrofuran-2-ylpurine (23) were selected, because of the high affinity of their parent compound meta-topolin (mT, 6-(3-hydroxybenzylamino)purine) to cytokinin receptors, as model compounds for studying their perception by the receptors CRE1/AHK4 and AHK3 in a bacterial assay. Both receptors perceived these two derivatives less well than they perceived the parent compound. Subsequently, the susceptibility of several new derivatives to enzyme degradation by cytokinin oxidase/dehydrogenase was studied. Substitution of tetrahydropyran-2-yl (THP) at the N⁹ position decreased the turnover rates of all new derivatives to some extent. To provide a practical perspective, the cytotoxicity of the prepared compounds against human diploid fibroblasts (BJ) and the human cancer cell lines K-562 and MCF-7 was also assayed in vitro. The prepared compounds showed none or marginal cytotoxicity compared to the corresponding N⁹-ribosides. Finally, the pH stability of the two model compounds was assessed in acidic and neutral water solutions (pH 3-7) by high-performance liquid chromatography (HPLC).

Full text of DOI:10.1016/j.bmc.2009.01.041

Bioorganic & Medicinal Chemistry 17 (2009) 1938–1947
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, characterization and biological activity of ring-substituted
6-benzylamino-9-tetrahydropyran-2-yl and 9-tetrahydrofuran-2-ylpurine
derivatives
a,
a
a
a
a
b
*
ˇ
ˇ
Lucie Szücová , Lukáš Spíchal , Karel Dolezal , Marek Zatloukal , Jarmila Greplová , Petr Galuszka ,
a
a
a
c
c
a
ˇ
Vladimír Kryštof , Jirí Voller , Igor Popa , Frank J. Massino , Jan-Elo Jørgensen , Miroslav Strnad
a Laboratory of Growth Regulators, Palacky University & Institute of Experimental Botany AS CR, Šlechtitelu˚ 11, 783 71 Olomouc, Czech Republic
b Department of Biochemistry, Palacky University, Šlechtitelu˚ 11, 783 71 Olomouc, Czech Republic
c Senetek PLC, 831A Latour Court, 94558 Napa, CA, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
In an attempt to improve specific biological functions of cytokinins routinely used in plant micropropa-
gation, 33 6-benzylamino-9-tetrahydropyran-2-ylpurine (THPP) and 9-tetrahydrofuran-2-ylpurine
(THFP) derivatives, with variously positioned hydroxy and methoxy functional groups on the benzyl ring,
were prepared. The new derivatives were prepared by condensation of 6-chloropurine with 3,4-dihydro-
2H-pyran or 2,3-dihydrofuran and then by the condensation of these intermediates with the correspond-
ing benzylamines. The prepared compounds were characterized by elemental analyses, TLC, HPLC, melt-
ing point determinations, CI+ MS and 1H NMR spectroscopy. The cytokinin activity of all the prepared
derivatives was assessed in three classical cytokinin bioassays (tobacco callus, wheat leaf senescence
and Amaranthus bioassay). The derivatives 6-(3-hydroxybenzylamino)-9-tetrahydropyran-2-ylpurine
(3) and 6-(3-hydroxybenzylamino)-9-tetrahydrofuran-2-ylpurine (23) were selected, because of the high
affinity of their parent compound meta-topolin (mT, 6-(3-hydroxybenzylamino)purine) to cytokinin
receptors, as model compounds for studying their perception by the receptors CRE1/AHK4 and AHK3
in a bacterial assay. Both receptors perceived these two derivatives less well than they perceived the par-
ent compound. Subsequently, the susceptibility of several new derivatives to enzyme degradation by
cytokinin oxidase/dehydrogenase was studied. Substitution of tetrahydropyran-2-yl (THP) at the N9 posi-
tion decreased the turnover rates of all new derivatives to some extent. To provide a practical perspective,
the cytotoxicity of the prepared compounds against human diploid fibroblasts (BJ) and the human cancer
cell lines K-562 and MCF-7 was also assayed in vitro. The prepared compounds showed none or marginal
cytotoxicity compared to the corresponding N9-ribosides. Finally, the pH stability of the two model com-
pounds was assessed in acidic and neutral water solutions (pH 3–7) by high-performance liquid chroma-
tography (HPLC).
Received 16 October 2008
Revised 15 January 2009
Accepted 20 January 2009
Available online 23 January 2009
Keywords:
Cytokinins
Antisenescence
Receptor
Stability
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
used in plant micropropagation; in particular, BAP is currently re-
garded as one of the most effective and affordable cytokinins for
Cytokinins belong to a group of plant growth hormones in-
volved in the regulation of almost all stages of plant growth and
development.1 Naturally occurring cytokinins are based structur-
ally on N6-substituted adenine. Members of this group are classi-
fied as isoprenoid (ISCK) and aromatic (ARCK) according to the
substituent on the N6-atom of adenine.1,2 Although ISCKs such as
isopentenyladenine (iP), trans-zeatin (tZ) or dihydrozeatin have at-
tracted the most attention,3 ARCKs may also occur naturally and
have been isolated, for example, from poplar leaves.4–7 Two repre-
sentatives of ARCKs, 6-benzylaminopurine (BAP) and 6-(3-hydrox-
ybenzylamino)purine (meta-topolin, mT) have been increasingly
routine use.8–16 Nevertheless, the application of BAP has some dis-
advantages in the propagation of many crop species, including
problems with heterogeneity of growth and rooting inhibition.16,17
One possible way to eliminate the undesirable side effects of BAP
whilst simultaneously retaining its beneficial organogenic activity
is the development of new BAP derivatives.16,17
Various modifications of BAP moiety by substitution, especially
at the N9 atom of the adenine, directly influence the invigorating
effects of cytokinins on plant growth and are associated with the
reinforcement of targeted cytokinin effects.17,18 The most effective
N9-substituted derivatives developed so far are 6-benzylamino-9-
tetrahydropyran-2-ylpurine (PBA) and 6-benzylamino-9-tetrahy-
drofuran-2-ylpurine (FBA), which have both proved to be consider-
ably more active than BAP in evoking of several growth
* Corresponding author. Tel.: +420 585634940; fax: +420 585634870.
ˇ
E-mail address: lucie.szucova@upol.cz (L. Szücová).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.01.041

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L. Szücová et al. / Bioorg. Med. Chem. 17 (2009) 1938–1947
1939
responses.18 Skoog et al. observed that PBA is exceptionally potent
in chlorophyll retention, while Arena et al. described the growth-
promoting effect of PBA on adventitious shoot formation in com-
parison to kinetin or iP.19,20 In addition, PBA has, for a long time,
been used successfully to control flowering and inflorescence
development in various plants.21–23 It has been suggested, how-
ever, that the observed increased activity of these N9-substituted
derivatives could be a consequence of their ability to release free
bases, thus supplying desirable concentrations over a prolonged
period.18 Thus, susceptibility to enzymatic cleavage is probably
the critical factor determining the biological activity of N9-substi-
tuted cytokinins. Hence, less active compounds are probably not
susceptible to cleavage of N9-substituents and exhibit low or zero
activity because of their stability.18,24,27–30 These data are some-
what controversial because of the fast release of free aglycone after
the application of much less active 9-methyl-BAP that was ob-
served by Fox et al.24 These authors studied the metabolism of 9-
methyl-BAP, considered to exhibit low activity, in tobacco and soy-
bean callus tissue and demonstrated rapid conversion to several
products. The metabolites were not identified definitively,
although it was proposed that conversion to free BAP occurred.24
Pietraface and Blaydes examined the metabolism of 9-methyl-
BAP in germinating lettuce seed and, based on chromatographic
data, suggested that BAP riboside and nucleotide formation was
occurring.25 Nevertheless, free BAP was not detected. Although free
BAP has not been found as a metabolite, the conversion to BAP 9-
glucoside and 7-glucoside (inactive metabolites) has been estab-
lished unequivocally by mass spectrometry.25 The formation of
these glucosides presumably requires them to be converted to BAP.
Zhang and Letham studied the metabolism of several 9-substi-
tuted BAP derivatives and suggested that another reason for their
enhanced antisenescence properties might originate from differ-
ences in the catabolism of BAP,18 PBA and possibly FBA. Their
study established that PBA and FBA were subject to both N6-deb-
enzylation and N9-substituent breakage in soybean leaves. How-
ever, debenzylation was more prominent in the metabolism of
PBA than in the metabolism of BAP. Besides, these compounds
were less likely to form inactive or toxic metabolites.26 With re-
spect to the different types of cytokinin derivatives mentioned
here, the most remarkable seemed to be 9-tetrahydropyranyl
and 9-tetrahydrofuranyl BAPs. We, therefore, prepared new series
of 6-(hydroxy- and 6-(methoxy-benzylamino)purines substituted
by tetrahydropyran-2-yl (THP) and tetrahydrofuran-2-yl (THF)
groups at the N9 position of purine, in order to improve their bio-
logical functions and maintain or even decrease their already low
cytoxicity. Both these groups are cyclic ethers in which the ether
oxygen is attached to the carbon linked to the N9 atom of the
purine moiety. THP and THF were originally described as protec-
tive groups in organic chemistry that are easily removed under
acidic conditions.31,32 Hence, we examined the stability of se-
lected model derivatives, specifically 6-(3-hydroxybenzylamino)-
9-tetrahydropyran-2-ylpurine (3) and 6-(3-hydroxybenzylami-
no)-9-tetrahydropyran-2-ylpurine (23), in acidic or neutral water
solutions (pH 3–7) to be sure that breakage of THP or THF did not
occur in the media used for the biological and receptor assays.
Furthermore, using the model compounds in a bacterial assay,
we studied the effect of the THP and THF groups on the ability
of Arabidopsis cytokinin receptors AHK3 and CRE1/AHK4 to detect
the compounds.33
in excised wheat leaves and the dark induction of betacyanin syn-
thesis in Amaranthus cotyledons. To investigate possible differ-
ences in in vivo stability, the newly prepared derivatives were
also tested as substrates for cytokinin oxidase/dehydrogenase. This
enzyme catalyzes irreversible cleavage of the N6-substituent from
the cytokinin molecule, leading to the total loss of biological activ-
ity, and seems to be one of the main mechanisms by which cytoki-
nin homeostasis is maintained in plant tissues.3
It has been suggested recently that cytokinin derivatives substi-
tuted by ribose in the N9 position exhibit significant cytotoxic
activity.36 In order to exclude cytotoxicity of newly prepared com-
pounds with potential agricultural and cosmetic use their cytotoxic
effect against human diploid fibroblasts (BG) and cancer cell lines
(MCF-7 and K-532) was evaluated. The cytotoxic activity of se-
lected THP and THF derivatives was then compared to the corre-
sponding riboside analogues.
2. Results and discussion
2.1. Synthesis
Thirty-three ring-substituted derivatives of benzylamino-9-
tetrahydropyran-2-ylpurine and 9-tetrahydrofuran-2-ylpurine
were synthesized according to Scheme 1 (Table 1, Scheme 2). Pre-
pared compounds were characterized by elemental analyses, thin
layer chromatography (TLC), melting point determinations, CI+
MS and 1H NMR spectroscopy. For better lucidity, Scheme 2
shows the schematic representation of newly prepared tetrahy-
dropyran-2-yl and tetrahydrofuran-2-yl derivatives. The purity
of prepared derivatives was confirmed by high-performance li-
quid chromatography (HPLC). Table 2 shows C, H, N elemental
analysis data, mp, CI+ MS and HPLC purity data, whilst 1H NMR
spectral data are given in Supplementary data. Compounds PBA
(1) and FBA (21) were prepared using a slightly modified version
of a method previously published in the literature.18 The melting
points and 1H NMR spectral data for compounds 1 and 21 pre-
pared in our laboratory were consistent with the data found in
the literature.18 The preparation of new derivatives is described
in greater detail in Section 4.3.
2.2. Stability in acidic solutions
Tetrahydropyran-2-yl (THP) and tetrahydrofuran-2-yl (THF)
groups have been commonly used in organic chemistry as protec-
tive groups, readily removable under acidic conditions.31 There-
fore we verified that THP or THF groups did not break off under
our bioassay conditions, since their removal would turn them into
the corresponding free bases thereby influencing the cytokinin
activity. 6-(3-Hydroxybenzylamino)-9-(tetrahydropyran-2-yl)pur-
ine (3) and 6-(3-hydroxybenzylamino)-9-(tetrahydrofuran-2-yl)-
purine (23) were chosen as the model compounds. We
performed HPLC stability measurements in 10ꢀ4 M stock solu-
tions with pH decreasing from 7 to 3. The percentage of the THPP
and THFP derivatives 3 and 23 together with the pH- and time-
dependent release of the free base (m-topolin), as determined
by HPLC, is given in Table 3. Both tested compounds were stable
at pH 7 and 6 even 24 h after sample preparation. Compound 23
started to decompose after 24 h at pH 5 (9% of the free base re-
leased). A significant decomposition of 3 and 23 occurred after
24 h at pH 4 and increased significantly at pH 3 (Table 3). It
can, therefore, be concluded that the prepared THPP and THFP
cytokinin derivatives are not entirely stable at pH < 4 and under
more acidic conditions they decompose to their free bases. Since
the pH of the media in the assays used within the framework of
this study varied between 6 and 7, we could be sure that the ori-
ginal compounds were those being tested.
Several papers about ARCK derivatives have mentioned the
relationship between benzyl ring substitutions and cytokinin
activity.33–36 Therefore, we also studied the differences in biologi-
cal activity of 6-(hydroxy- and 6-(methoxybenzylamino)-9-tetra-
hydropyran-2-ylpurine derivatives in classical cytokinin
bioassays. Cytokinin activity was compared in three tests—the
stimulation of tobacco callus growth, the retention of chlorophyll

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L. Szücová et al. / Bioorg. Med. Chem. 17 (2009) 1938–1947
R
Cl
HN
N
N
N
N
R-NH2
N
N
propanol, reflux
O
N
N
Cl
N
O
O
N
CF3COOH
N
ethylacetate
N
H
R
Cl
HN
O
N
N
R-NH2
N
N
propanol, reflux
N
N
N
N
R
R3
O
O
R
R4
R5
2
R2 - R6 are variously positioned -OH and/or -OMe groups
R6
Scheme 1. Schematic representation of the syntythetic pathway of THPP and THFP derivatives described in the study.
2.3. Cytokinin activity in bioassays
suggests that different receptor and/or signaling systems are in-
volved in transduction of different cytokinin-dependent physiolog-
ical responses.
Cytokinin activity of all the prepared compounds was deter-
mined by three classical cytokinin bioassays (tobacco callus, wheat
senescence and Amaranthus bioassays) and the results are pre-
sented in Table 4. The activities of the compounds were compared
to those of BAP, which represents a highly active, commonly used
cytokinin. Figure 1A–C compares the activity of 6-(ortho-, 6-(meta-
and 6-(para-hydroxybenzylamino)tetrahydropyran-2-ylpurines in
all three bioasays, showing a decrease in cytokinin activity be-
tween the substituents, as follows: meta > ortho ꢁ para. These re-
sults are in agreement with those previously published in the
literature related to ortho-, meta-, and para-topolin.7,34,35
Almost all THPP and THFP derivatives containing two or more
hydro or methoxy groups on the benzyl ring were inactive in the
tobacco callus bioassay; the exceptions were compounds 11, 12
and 29, although these exhibited only half the activity of BAP (Ta-
ble 4). None of di- or tri- substituted compounds exceeded the
activity of BAP either in the senescence or Amaranthus bioassays
(Table 4). This fact supports the recently published fact that di-
and trihydroxy (methoxy) BAPs are not strongly active in cytokinin
assays.35
For 6-(methoxybenzylamino)tetrahydropyran-2-ylpurine deriva-
tives, the cytokinin activity decreased in the substituent order
ortho > meta ꢁ para; this is also consistent with the data found in
the literature (Fig. 1D–F).35,36 Moreover, it was found that substitu-
tion in the para-position caused the loss of activity in senescence as
well as in tobacco callus bioassay. This finding has been reported
recently for similar compounds,36 differing in their N9-substitution
by ribose, and supported by the fact that compounds 4, 7, 18, 24,
27, 30, 32, 34 and 35, which are substituted in the para position
by a methoxy group, were inactive in all three bioassays (Table 4,
Fig. 1). We also compared the activity of THPP and THFP deriva-
tives in tobacco callus, senescence and Amaranthus biotests with
the corresponding free base. This test used compounds 3 and 23
and their parent compound 6-(3-hydroxybenzylamino)purine
(meta-topolin, mT). The results presented in Figure 2 show that
the antisenescence activity of the THPP derivative was slightly
higher than that of the free base and the THFP derivative
(Fig. 2A). The activities of the THPP and THFP derivatives were
comparable in the tobacco callus assay and did not differ signifi-
cantly from the activity exhibited by the corresponding base
(Fig. 2B). A different result was found in the Amaranthus bioassay,
where both THPP and THFP derivatives were less active, although
their activities were of a similar order to that in the senescence as-
say (Fig. 2C). Moreover, the results of Amaranthus and tobacco cal-
lus bioassay are similar to the values obtained for the
corresponding ribotides.7,34,36 However, the activity of methoxyb-
enzyladenosines in senescence bioassay is twice higher in compar-
ison with those for THPP and THFP derivatives. This, again,
2.4. Recognition by cytokinin receptors
In our previous work we have shown that, in contrast to activity
in the classical cytokinin bioassays, the position of ARCK benzyl
ring substituents do not improve the recognition of a compound
by cytokinin receptors in a bacterial assay.35,36 Here we used a bio-
assay employing transformed E. coli strains expressing the cytoki-
nin receptors AHK3 and CRE1/AHK4 with the cytokinin-activated
reporter gene cps::lacZ to investigate the effect of THP and THF
groups on the ability of meta-topolin, as a model compound, to
activate the cytokinin signaling pathway through these recep-
tors.33,37,38 As shown in Figure 3, mT itself was recognized well
by both receptors, although to a lesser extent than the trans-zeatin
used as a control. In contrast, the THPP and THFP derivatives were
not detected by CRE1/AHK4 at all (Fig. 4A). Partial receptor activa-
tion occurred only with AHK3 at the highest concentration, corrob-
orating the sensitivity of AHK3 to N9-substituted cytokinins
(Fig. 3).33 The experiment showed that THP and THF ARCK deriva-
tives are only weak ligands of the cytokinin receptors. This fact
means that, like other aromatic cytokinins, a different recognition
system that is able to interact with BAP derivatives may exist in
plants.35
2.5. Enzymatic degradation in vitro
Cytokinin oxidase/dehydrogenase in higher plants is encoded
by small gene families, and the enzymes are distinguished by di-
verse subcellular compartmentalization as well as by different spa-

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L. Szücová et al. / Bioorg. Med. Chem. 17 (2009) 1938–1947
1941
Table 1 (continued)
Table 1
Structures of the prepared compounds and their abbreviations
Compound
R2 (o)
R3 (m)
R4 (p)
R5 (m)
R6 (o)
R1
Compound
R2 (o)
R3 (m)
R4 (p)
R5 (m)
R6 (o)
R1
18
OCH3
H
OCH3
OCH3
H
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
PBA (1)
H
H
H
H
H
O
O
O
O
O
O
O
O
O
O
O
O
O
19
OCH3
H
OCH3
OCH3
H
H
OCH3
2
OH
H
H
H
H
H
20
H
OCH3
OCH3
H
3
OH
H
H
H
H
FBA (21)
22
H
H
H
H
4
H
OH
H
H
H
OH
H
H
H
H
H
5
OCH3
H
H
H
23
OH
H
H
H
H
6
H
OCH3
H
H
H
24
H
OH
H
H
H
7
H
H
OCH3
H
H
25
OCH3
H
H
H
8
OH
H
OCH3
OCH3
H
H
H
H
26
H
OCH3
H
H
H
9
OH
OCH3
H
H
H
27
H
H
OCH3
OCH3
H
H
H
10
11
12
13
OCH3
OCH3
OCH3
H
H
H
28
H
OCH3
OCH3
H
H
H
H
OCH3
H
29
H
OCH3
H
H
H
H
OCH3
30
OCH3
OCH3
H
OCH3
H
H
OCH3
OCH3
H
H
31
H
H
OCH3
H
14
15
H
OCH3
OH
H
H
OCH3
H
H
O
O
32
OCH3
OCH3
OH
OCH3
H
H
OH
H
33
OCH3
H
H
16
17
H
OH
H
OH
H
H
H
O
O
34
35
OCH3
H
H
OCH3
OCH3
OCH3
OCH3
H
H
O
O
OCH3
OCH3
OCH3
OCH3

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L. Szücová et al. / Bioorg. Med. Chem. 17 (2009) 1938–1947
1942
R 4 (p)
medium through the developing roots and are then distributed
via the xylem bundles.
R5
R6
(m) R3
(m)
(o)
ARCKs are, in general, poor substrates for cytokinin oxidase/
dehydrogenase activity; this probably increases their stability
in vivo compared to isoprenoid cytokinins. None of the seven
isoenzymes of cytokinin oxidase/dehydrogenase from Arabidopsis
thaliana prefer aromatic cytokinins.42 It has been shown that
aromatic rings act as spherical obstacles to the formation of sub-
strate/product intermediates. Thus, the catalytic reaction cannot
be speeded up even in the presence of appropriate electron
acceptors within ISCK, which should transfer electrons from
the reduced FAD cofactor of the enzyme and thus dramatically
increase the rate of degradation.43 Kinetic constants of maize
cytokinin oxidase/dehydrogenase for selected THPP derivatives
compared to their free bases, BAP, BAPR and PBA are given in
Table 5. N9-substitution by THP decreases turnover rates of all
aromatic cytokinins to some extent. Furthermore, the lower sus-
ceptibility to degradation by ZmCKX1 of these derivatives is sus-
(o)
R2
HN
N
N
N
N
R1
Scheme 2. Schematic representation of tetrahydropyran-2-yl and tetrahydrofuran-
2-yl derivatives; (o) indicates ortho-, (m) meta- and (p) para- position in the benzyl
ring.
tial and temporal expression patterns.39 In this study, the recombi-
nant maize enzyme Zea mays cytokinin oxidase/dehydrogenase 1
(ZmCKX1) was used to study the ability of selected compounds,
especially those that bioassays showed to be more active than their
corresponding free bases, to be cleaved as substrates by this en-
zyme. This protein has previously been localized in the apoplast
of vascular bundles, and exhibits its peak expression there in ger-
minated seeds and young roots.40,41 Thus, it can contribute to the
degradation of exogenous cytokinins in micropropagation systems
where these, and other additives, are absorbed from the growing
tained by higher Km values, decreasing the total efficiency (kcat
/
Km) by a factor of 7–33. The higher activity of THPP derivatives
in bioassays may therefore be explained by their greater resis-
tance to enzymatic breakdown by CKX, which increases their
stability and prolongs their lifespan in plant tissues. Previous
studies of the metabolism of several 9-substituted BAP deriva-
tives have suggested that the reason for their enhanced cytoki-
nin activity might originate from differences in the catabolism
of BAP, PBA and possibly FBA.18 This attribute can even over-
come the poorer recognition of THPP and THFP derivatives by
the known cytokinin receptors.
2.6. Cytotoxicity
Table 2
Elemental analyses, melting points and CI+ MS and HPLC purity of the prepared
compounds
The substitution of certain BAP derivatives with ribose moiety
in the N9 position is known to significantly increase their cytotox-
icity against human cells.35,36 Therefore, we compared the cyto-
toxic effects of corresponding ribosyl and tetrahydropyran-2-yl
derivatives in vitro against cancer human cell lines (breast carci-
noma, MCF-7, and chronic myelogenous leukaemia, K-562) by Cal-
cein AM viability assay. The results summarizing the IC50 values for
all the tested compounds are presented in Table 6.
We discovered that the alternation of ribose by a THP or THF
group in the N9 position can markedly decrease cytotoxicity. Figure
4 shows a comparison of dose–response curves for 6-(2-hydroxy-
3-methoxybenzylamino)-9-tetrahydropyran-2-ylpurine (8) and 6-
(2,3-dihydroxy-benzylamino)-9-tetrahydropyran-2-ylpurine (15)
and their analogues with ribose at N9. While cytotoxic effects of
both THPP derivatives against the MCF-7 cell line were negligible,
their riboside analogues 6-(2-hydroxy-3-methoxybenzylamino)-9-
Compound
Elemental analyses calculated/
found
Mp (°C)
CI+ MS HPLC (%)
[M+H+]
%C
%H
%N
PBA,1
2
3
4
5
6
7
8
9
66.0/66.1
62.8/62.7
62.8/62.4
62.8/62.8
63.7/63.9
63.7/63.9
63.7/63.9
60.8/60.7
60.8/60.6
61.8/61.8
61.8/61.7
61.8/61.8
61.8/61.9
61.8/61.9
59.8/59.9
59.8/59.9
60.1/60.2
60.1/60.2
60.1/60.0
60.1/59.9
65.1/65.1
61.7/61.9
61.7/61.7
61.7/61.8
62.8/62.9
62.8/62.9
62.8/62.9
60.8/60.9
60.8/60.7
60.8/60.7
60.8/60.6
59.8/60.0
59.2/59.1
59.2/59.2
59.2/59.3
6.2/6.1
5.9/5.8
5.9/5.9
5.9/5.9
6.2/6.3
6.2/6.3
6.2/6.2
5.9/5.9
5.9/6.0
6.3/6.3
6.3/6.4
6.3/6.3
6.3/6.3
6.3/6.4
5.6/5.6
5.6/5.7
6.3/6.2
6.3/6.4
6.3/6.4
6.3/6.3
5.8/5.8
5.5/5.5
5.5/5.5
5.5/5.6
5.9/5.8
5.9/6.0
5.9/5.8
5.9/5.9
5.9/5.7
5.9/5.9
5.9/5.8
5.6/5.7
6.0/6.0
6.0/5.9
6.0/6.0
22.6/22.5
21.5/21.6
21.5/21.3
21.5/21.3
20.6/20.7
20.6/20.6
20.6/20.6
19.7/19.8
19.7/19.8
18.9/18.7
18.9/18.9
18.9/18.9
18.9/18.8
18.9/18.9
20.5/20.5
20.5/20.4
17.5/17.5
17.5/17.6
17.5/17.4
17.5/17.8
23.7/23.8
22.5/22.3
22.5/22.6
22.5/22.5
21.5/21.5
21.5/21.5
21.5/21.5
19.7/19.8
19.7/19.6
19.7/19.6
19.7/19.7
20.5/20.5
18.2/18.2
18.2/18.2
18.2/18.3
110–112 310
172–175 326
120–121 326
173–174 326
106–108 340
134–135 340
137–138 340
187–188 356
189–190 356
479–180 370
150–151 370
170–171 370
156–157 370
145–146 370
174–175 342
212–214 342
142–143 400
128–129 400
178–179 400
127–128 400
100–102 296
135–136 312
124–126 312
182–183 312
>98
>98
>99
>99
>98
>98
>99
>98
>98
>98
>98
>98
>98
>98
>98
>99
>98
>98
>98
>98
>98
>98
>98
>98
>98
>98
>98
>98
>98
>98
>98
>98
>98
>98
>98
10
11
12
13
14
15
16
17
18
19
20
FBA,21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
b-
-ribofuranosylpurine exhibited significant cytotoxicity with IC50
values of 20.2 mol/L and 5.2 mol/L, respectively. Similar results
D-ribofuranosylpurine and 6-(2,3-dihydroxybenzylamino)-9-b-
D
l
l
were obtained for K-562 cells where the replacement of ribose
moiety with a THP group led to an increase in the IC50 value from
27.9
92
bases were above 100
l
mol/L to >100
mol/L (compound 15). IC50 values for the corresponding free
mol/L.35
lmol/L (compound 8) and from 27.9 to
l
l
The cytotoxicity of the prepared compounds was also evaluated
in human diploid fibroblasts (BJ) by MTT viability assay. Almost all
the tested THPP and THFP compounds were only marginally toxic
or were non-toxic, even at the highest concentration tested
97–99
87–88
326
326
182–183 326
131–132 356
99–100
356
(100 lmol/L or solubility limit). Their toxicity profile was compara-
125–126 356
103–104 356
140–142 342
140–141 386
ble or better than the profile of the corresponding cytokinin bases
under the assay conditions.35 We, therefore, conclude that tetra-
hyropyranylation or tetrahydrofuranylation of cytokinins can lead
to a general reduction in cytotoxicity, a useful property in agricul-
tural applications and cosmetics.
99–100
386
118–119 386

ˇ
L. Szücová et al. / Bioorg. Med. Chem. 17 (2009) 1938–1947
1943
The prepared compounds were subjected to three cytokinin bioas-
says. The N9 substitution by a THP or THF group either did not
change or improved the biological activity of free bases recorded
in classical cytokinin bioassays, although it negatively influenced
the recognition of these compounds by cytokinin receptors. The
improved activity of the THP cytokinins could be explained by their
higher resistance to enzymatic breakdown by CKX. The resistance
to degradation, and thus probably their prolonged lifespan
in vivo, can be enhanced by the presence of hydroxy- or methoxy
groups in the meta position of the benzyl ring. The specificity con-
stant (kcat/Km) of 6-(3-hydroxybenzylamino)-9-tetrahydropyran-2-
ylpurine toward ZmCKX1 is 25-fold and 530-fold lower than that
for 6-benzylaminopurine and 6-(3-hydroxybenzylamino)purine,
respectively. However, in vivo metabolic studies of the newly pre-
pared compounds are required to explain in more detail the mode
of action of THPP and THFP derivatives in plants. Such a study is
underway in our laboratory.
The hydroxy- and methoxybenzyl THFPs and THPPs with high
levels of cytokinin activity, along with the majority of newly pre-
pared compounds, were found to be non-toxic in the cytotoxicity
tests performed in vitro on different cancer and normal human cell
lines. HPLC assaying of the stability of meta-topolin THPP and THFP
derivatives at various pHs (3–7) showed that the compounds were
stable in the pH range of the media used for the cytokinin bioas-
says. The fact that almost all the substances exhibited no or only
slight cytotoxicity even at high concentrations is very encouraging
and may mean that these cytokinins can be used not only in the
3. Conclusions
We prepared and characterized thirty-three hydroxy and/or
methoxy ring-substituted 6-benzylamino-9-tetrahydropyran-2-
ylpurine and 6-benzylamino-9-tetrahydrofuran-2-yl derivatives.
Table 3
Stability of 6-(3-hydroxybenzylamino)-9-tetrahydropyran-2-ylpurine (3) and 6-(3-
hydroxybenzylamino)-9-tetrahydropyran-2-ylpurine (23) in acidic solutions
pH
Compound peak area (%)
3
Free base
23
Free base
(A) 1 h After sample preparation
7
6
5
4
3
99.9
99.8
99.5
99.5
98.8
0.1
0.2
0.5
0.5
1.2
99.7
99.6
99.5
97.2
63.8
0.3
0.4
0.5
2.8
36.2
(B) 24 h After sample preparation
7
6
5
4
3
99.8
99.3
99.2
93.9
39.6
0.2
0.7
0.8
6.1
60.4
99.7
99.4
90.8
37.5
0.3
0.3
0.6
9.2
62.5
99.7
The pH stability of compounds 3 and 23 in 10ꢀ4 M water solutions with pHs
decreasing from 7 to 3 measured 1 h (A) and 24 h (B) after sample preparation. The
percentage of the released free base 6-(3-hydroxybenzylamino)purine (mT) was
determined by HPLC.
Table 4
Relative cytokinin bioassay activity of the prepared derivatives at the optimal concentration compared with the activity of BAP (100% means 10ꢀ6 M BAP for the tobacco callus
bioassay, 10ꢀ5 M BAP for the Amaranthus betacyanin bioassay and 10ꢀ4 M BAP for the senescence bioassay)
Compound
Tobacco callus bioassay
Relative
Amaranthus bioassay
Relative activity
Senescence bioassay
Optimal concentration Relative activity
Optimal concentration
Optimal concentration
(mol lꢀ1
)
activity(%)
(mol lꢀ1
)
(%)
(mol lꢀ1
)
(%)
PBA (1)
2
3
4
5
6
7
8
9
10ꢀ6
10ꢀ5
10ꢀ6
10ꢀ5
10ꢀ6
10ꢀ6
10ꢀ6
10ꢀ4
10ꢀ4
n.a.
102 ( 2)
37 ( 2)
95 ( 8)
20 ( 9)
93 ( 9)
90 ( 1)
10 ( 6)
12 ( 4)
8.7 ( 2)
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10-4
114 ( 7)
46 ( 9)
100 ( 6)
34 ( 9)
84 ( 2)
76 ( 2)
18 ( 5)
18 ( 7)
26 ( 0.2)
9 ( 4)
28 ( 5)
13 ( 1)
8 ( 3)
9 ( 2)
8 ( 3)
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
n.a.
104 ( 1)
31 ( 10)
140 ( 10)
4 ( 8)
104 ( 5)
72 ( 5)
n.a.
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
n.a.
7 ( 3)
10
11
12
13
14
15
16
17
18
19
20
FBA (21)
22
23
24
25
26
27
28
29
30
31
32
33
34
36
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
25 ( 1)
19 ( 3)
14 ( 3)
8 ( 0.3)
7 ( 4)
10ꢀ5
10ꢀ4
n.a.
42 ( 10)
57 ( 5)
n.a.
10ꢀ4
10ꢀ5
n.a.
3,2 ( 0.1)
36 ( 1)
33 ( 6)
4 ( 3)
3 ( 2)
10ꢀ4
10ꢀ4
n.a.
15 ( 8)
7 ( 4)
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ6
10ꢀ4
10ꢀ6
10ꢀ5
10ꢀ5
10ꢀ6
10ꢀ6
10ꢀ6
10ꢀ4
10ꢀ6
10ꢀ5
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
11 ( 3)
2 ( 1)
14 ( 2)
19 ( 6)
109 ( 9)
41 ( 7)
82 ( 5)
10 ( 5)
107 ( 10)
123 ( 8)
25 ( 6)
5 ( 2)
18 ( 3)
14 ( 2)
41 ( 9)
17 ( 6)
15 ( 2)
14 ( 2)
17 ( 3)
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
3 ( 2)
7 ( 4)
17 ( 9)
95 ( 3)
58 ( 8)
87 ( 11)
19 ( 1)
91 ( 7)
96 ( 2)
7 ( 3)
107 ( 8)
25 ( 8)
131 ( 7)
8 ( 6)
72 ( 1)
82 ( 11)
16 ( 4)
7 ( 1)
8 ( 0.1)
18 ( 9)
41 ( 2)
4 ( 5)
3 ( 2)
14 ( 6)
3 ( 0.1)
61 ( 7)
13 ( 2)
7 ( 1)
9 ( 4)
9 ( 1)
10 ( 4)
14 ( 4)
4 ( 2)
n.a. Means not active.

ˇ
L. Szücová et al. / Bioorg. Med. Chem. 17 (2009) 1938–1947
1944
Figure 1. The influence of hydroxy or methoxy group position in the benzyl ring of 6-benzyl-9-tetrahydropyran-2-yl-purines on their biological activity in classical cytokinin
bioassays. Comparison of the impact of hydroxy (A–C) and methoxy (D–F) groups substituted in ortho- (circles), meta- (triangles), and para- (diamonds) positions,
respectively, on: the retention of chlorophyll in excised wheat leaves (A, D); the stimulation of cytokinin-dependent tobacco callus growth (B, E); and the dark induction of
betacyanin synthesis in Amaranthus cotyledons (C, F). The graphs show representative examples of results obtained in individual bioassays. BAP was used as a positive control
(squares), the dashed line shows the solvent control (0.2% DMSO). Error bars represent SD (n = 5).
regulation of plant growth and development, agriculture and plant
biotechnology, but also, potentially, in cosmetics, where the use of
cytotoxic substances is prohibited.
organic solvents used for syntheses and subsequent crystallization
of the products were purchased from Sigma–Aldrich and Lachema,
and used as received. 2,3-Dihydrofuran (98%) was purchased from
Across Organics; 3,4-dihydro-2H-pyran and trifluoracetic acid
were obtained from Fluka; furfurylamine, 2-methoxybenzylamine,
3-methoxybenzylamine, 4-methoxy-benzylamine, 2,4-dimethoxy-
benzylamine, 2,5-dimethoxybenzylamine, 2,6-dime-thoxybenzyl-
amine, 3,4-dimethoxybenzylamine, 3,5-dimethoxybenzylamine,
4. Experimental
4.1. Chemicals
2,4,6-trimethoxybenzylamine,
aniline, 4-methoxyaniline, 4-aminophenol were obtained from Al-
drich; 2-hydroxybenzylamine, 3-hydroxybenzylamine, 4-hydroxy-
3,4,5-trimethoxybenzylamine,
6-Chloropurine was purchased from Olchemim, triethylamine
(TEA), ethyl acetate (EtOAc), n-propanol, n-butanol, isopropanol,
diethyl ether, hexane, N,N0-dimethylsulfoxide and other common
Figure 2. The influence of 9-tetrahydropyran-2-yl and 9-tetrahydrofuran-2-yl groups on the biological activity of 6-benzyl-(3-hydroxybenzylamino)purine in classical
cytokinin bioassays. (A) The effect on chlorophyll retention in excised wheat leaf tips; (B) the growth of cytokinin-dependent tobacco callus; (C) the effect on dark betacyanin
synthesis in Amaranthus caudatus cotyledon-hypocotyl explants. The dashed line shows the solvent control (0.2% DMSO). Error bars represent SD (n = 5).

ˇ
L. Szücová et al. / Bioorg. Med. Chem. 17 (2009) 1938–1947
1945
Figure 3. Comparison of the perception of THP and THF substituted 6-(3-hydroxybenzylamino)purine (meta-topolin, mT) and mT by selected cytokinin receptors. The effect
of 6-(3-hydroxybenzylamino)-9-tetrahydropyran-2-ylpurine (6(3OHBA)9THPP) and 6-(3-hydroxybenzylamino)-9-tetrahydrofuran-2-ylpurine (6(3OHBA)9THFP) on the
sensing of mT by the cytokinin receptors CRE1/AHK4 (A) and AHK3 (B). trans-Zeatin (tZ) was used as the positive control. The dashed line shows the solvent control (0.5%
DMSO). Error bars show SD (n = 3).
Figure 4. The effect of THP substitution on the cytotoxicity of MCF-7 cells compared to the corresponding ribosides. The compounds 6-(2-hydroxy-3-methoxybenzylamino)-
9-tetrahydropyran-2-ylpurine (8) and 6-(2,3-dihydroxybenzylamino)-9-tetrahydropyran-2-ylpurine (15) did not reduce the number of viable MCF-7 cells. The MCF-7 human
breast cancer cell line was treated for 72 h with increasing concentrations of 8, 15 or their riboside analogues. Then, the number of viable cells was determined by a Calcein
AM assay. Results represent the average SD in three independent experiments.
benzylamine, 4-hydroxy-3-methoxybenzylamine, 2,3-dihydroxyb-
enzylamine, 2,3,4-trimethoxybenzylamine, 2,4,5-trimethoxyben-
zylamine were supplied by Olchemim, The preparation and
characterization of the commercially unavailable 2-hydroxy-3-
methoxybenzylamine hydrobromide and 3,5-dihydroxybenzyl-
amine hydrobromide have been described previously.35 Mili-Q
water was used throughout. The other solvents and chemicals used
were all of standard pa quality. The substances PBA (1) and (21)
were prepared according to slightly modified versions of standard
methods described in the literature.18
Table 5
Kinetic constants of maize cytokinin oxidase/dehydrogenase for selected cytokinins
and their THPP derivatives; enzyme activity was determined by measuring the
amount of aldehyde produced by oxidative cleavage of the side chain of the measured
cytokinins, using the 4-aminophenol assay42
4.2. General procedures
Evaporations were carried out under vacuum rotary oil pump
for n-propanol, n-butanol and ethyl acetate. Elemental analyses
(C, H, N) were determined on an EA1112 Flash analyzer (Thermo-
Finnigan). The melting points were determined on Büchi Melting
Point B-540 apparatus and are uncorrected. Thin layer chromatog-
raphy (TLC) was carried out using silica gel 60 WF254 plates (Merck
Co.). CHCl3:MeOH (9:1, v:v) or ethyl acetate/toluene (8:2, v:v) were
used as solvents. CI+ mass spectra were recorded using a Polaris Q
(Finnigan) mass spectrometer equipped with a Direct Insertion
Probe (DIP). The compounds were heated in an ion source with a
40–450 °C temperature gradient, the mass monitoring interval
was 50–1000 am, and spectra were collected using 1.0 s cyclical
scans, applying 70 eV electron energy. In the CI+ ionization mode,
isobutane was used as a reagent gas at a flow-rate of 2 L/h. The
mass spectrometer was directly coupled to an Xcalibur data sys-
tem. 1H spectra were recorded on a Bruker Avance 300 spectrom-
eter operating at a temperature of 300 K and a frequency of
Compound
kcat (sꢀ1
)
Km
(
l
M)
kcat/Km
iP
0.208
0.225
0.218
0.069
0.244
0.032
0.145
0.006
0.871
0.195
0.476
0.256
1.8
29.6
n.d.
98.9
24.4
125.0
n.d.
n.d.
5.5
33.8
n.d.
n.d.
0.1156
0.0076
n.d.
0.0007
0.0100
0.0003
n.d.
BAP
BAPR
PBA, 1
mT
3
6-fb
6
4-fb
4
n.d.
0.1583
0.0058
n.d.
7-fb
7
n.d.
All data represent mean values of at least two replicates. Deviations between rep-
licates did not exceed 10%; n.d.—not determined.
iP: N6-(2-isopentenyl)adenine, BAP: 6-benzylaminopurine, BAPR: 6-benzylamino-
purine-9-ribosylpurine, fb: free base.

ˇ
L. Szücová et al. / Bioorg. Med. Chem. 17 (2009) 1938–1947
1946
Table 6
1:1.2 ratio in n-propanol or n-butanol (30 mL) in the presence of
triethylamine (4 g, 39.5 mmol, 5 mL) at about 100 °C. The reaction
lasted for 3 h. The reaction mixture was then evaporated to dry-
ness and the residue was treated with 50 mL of ethyl acetate and
50 mL of distilled water. The ethyl acetate phase was separated
and purified using SiO2 and charcoal and then dried over Na2SO4.
Final products were isolated by crystallization using diethyl ether
or hexane and dried in air. C, H, N elemental analyses, CI+ MS
and HPLC purity data and mp for compounds 2–35 are given in Ta-
ble 2 while the yields and 1H NMR are given in Supplementary
data.
Cytotoxicity IC50 values of the prepared compounds in Calcein AM (K-562 and MCF-7)
and in MTT (BJ) assays
Compound
Cell line/IC50 (lmol/L)
K-562
MCF-7
BJ
1
2
3
4
5
6
7
8
9
>100
>100
81
>100
97
>100
>100
>100
>100
>100
97
>100
>100
>100
>25
>100
>100
>100
>100
>100
>100
>100
>100
>100
>25
>100
>100
>100
>100
63.3
61
>100
>100
>100
>25
16.5
10.3
>100
>100
92
>100
>37.5
>100
>50
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>50
4.4. pH Stability testing of 6-(3-hydroxybenzylamino)-9-
tetrahydropyran-2-ylpurine (3) and 6-(3-
hydroxybenzylamino)-9-tetrahydrofuran-2-ylpurine (23)
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
25.4
>100
>100
>100
>100
>100
>37.5
>100
>50
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>50
The pH stability of compounds 3 and 23 was analyzed by
HPLC-PDA (System Gold; Beckman Instruments, Fullerton, CA,
USA); analytes were monitored at 270 nm. 10ꢀ2 M solutions of
compounds 3 or 23 in methanol were prepared and diluted to
10ꢀ4 M using McIlvaine buffer solution for the appropriate pH
>100
>37.5
>100
>50
(3, 4, 5, 6 or 7).46 One hour after incubation at 25 °C, 5
l
L of
the prepared solution was directly injected onto a reversed phase
column (Symmetry C18; 5
m, 150 ꢂ 2.1 mm; Waters, Milford,
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>50
l
USA). At flow-rate of 0.3 mL/min, the following binary gradient
was used: 0 min, 10% A; 0–25 min, a linear gradient to 90% A; fol-
lowed by 5 min isocratic elution of 90% A, where A was 100%
methanol and B was 15 mM formic acid adjusted to pH 4 with
ammonium. The HPLC measurement of the solutions was re-
peated after a 24 h incubation at 25 °C. The analyses were re-
peated at least three times.
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
4.5. Cytokinin bioassays
All the prepared compounds were tested in three cytokinin
bioassays—the tobacco callus, Amaranthus and senescence bioas-
says—and their activity was compared with BAP. The biological
activities of THP and THF derivatives of 6(3-hydroxybenzylami-
no)purine (3, 23) and 6(3-methoxybenzylamino)purine (6, 26)
were compared to those of free bases. Tested cytokinin deriva-
tives were dissolved in dimethylsulfoxide (DMSO) and diluted
with distilled water to 5 ꢂ 10ꢀ2 M solutions. This stock solution
was further diluted in the media appropriate to each biotest to
concentrations from 10ꢀ8 to 10ꢀ4 M. The final concentration of
DMSO in the media did not exceed 0.2% and thus did not affect
the biological activity of the substance tested in the assay. Five
replicates were prepared for each compound concentration and
the entire tests were repeated at least twice. Detailed descriptions
of the conditions and performance of the tobacco callus bioassay,
Amaranthus bioassay and senescence bioassay are given in Zatlou-
kal et al.47
The maximum concentration tested was 100
17, where lower concentrations (25 and 37
their limited solubility in the culture media.
l
mol/L, except for compounds 11 and
lmol/L, respectively) were used due to
300.13 Hz. Samples were prepared by dissolving the substances in
DMSO-d6. Tetramethylsilane (TMS) was used as the internal refer-
ence standard.
4.3. Synthesis of 9-tetrahydropyran-2-ylpurine (2–20) and 9-
tetrahydrofuran-2-ylpurine (22–35) cytokinin derivatives
The synthesis of the derivatives consisted of two independent
steps, as presented in Scheme 1. In the first step, we employed a
modified version of a method found in the literature.44,45 6-chloro-
purine (10 g, 64.7 mmol) was stirred with 3,4-dihydro-2H-pyrane
(12.4 g, 147 mmol, 14 mL) for 6-chloro-9-tetrahydropyran-2-ylpu-
rine (1a) or with 2,3-dihydrofurane (11.3 g, 162 mmol, 12 mL) for
6-chloro-9-tetrahydrofuran-2-ylpurine (1b), for 10 min in ethyl
acetate (100 mL) and subsequently, trifluoroacetic acid (10.9 g,
84.2 mmol for 1a and 9.6 g, 95.5 mmol for 1b), was added drop-
wise. The reaction mixture was stirred at room temperature for
1 h and then neutralized by the appropriate amount of a mixture
of ammonia and water (2:3). The ethyl acetate phase was sepa-
rated and purified using charcoal and SiO2. Yellowish waxy prod-
ucts were obtained after vacuum evaporation of the solvents. The
products were washed with cyclohexane (1a) or hexane (1b) and
dried in the air. The yields, HPLC purity, MS (CI+) and 1H NMR data,
along with the melting points (mp) are presented in Supplemen-
tary data section. In the second step, 1a (1 g, 4.2 mmol) or 1b
(1 g, 4.4 mmol) was coupled with the appropriate benzylamine in
4.6. Bacterial receptor assay
Escherichia coli KMI001 strains harboring plasmids pIN-III-
AHK4 and pSTV28-AHK3 (Suzuki 2001, Yamada 2001,) were pro-
vided by Dr. T. Mizuno. The strains were used in the bacterial
receptor assays as described, but with a slight modification.33
The assay was optimized to 96-well microtiter plates according
to the literature.35 The preculture was diluted 1:10 and the incu-
bation time was 6 h. Relative activation of cytokinin receptors
was determined by measuring b-galactosidase activity using
the fluorescent substrate 4-methylumbelliferyl-b-D-galactoside
(Sigma) and monitoring the culture density at OD600. The test
was performed in triplicate and the entire test was repeated at
least twice.

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L. Szücová et al. / Bioorg. Med. Chem. 17 (2009) 1938–1947
1947
4.7. Enzymatic cytokinin degradation assay
Supplementary data
The enzyme, recombinant ZmCKX1 produced by the yeast Pichia
pastoris and purified as described previously,48 was obtained from
Dr. Kristin Bilyeu from USDA/ARS Plant Genetics Research Unit,
University of Missouri, Columbia, USA. The end-point assay was
performed according to the literature.49 The reaction mixture for
measuring dehydrogenase activity contained 100 mM McIlvaine
buffer pH 6.0, an aliquot of 10 mM cytokinin substrate stock dis-
solved in DMSO and 0.1 mM 2,6-dichlorophenolindophenol. The fi-
nal concentration of the substrate, when the kcat parameter was
determined, was 0.2 mM. The Michaelis constants were estimated
using a double reciprocal plot with the substrate concentration in
the range 0.05–0.25 mM.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.01.041.
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Acknowledgments
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43. Frébortová, J.; Fraaije, M. W.; Galuszka, P.; Šebela, M.; Pec, P.; Hrbác, J.; Novák,
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This work was financially supported by the Czech Ministry of
Education Youth and Sports (MSM 6198959216 and 1MO6030),
The Grant Agency of the Czech Republic (522/09/1576, 522/08/
0920 and 206/07/0570) and Senetek PLC. We would like to thank
Zdena Kamarádová, JarmilaBalonová, OlgaHustákováand Miloslava
Šubová for skilful technical assistance; and Dr. Kristin Bilyeu for
kindly providing the recombinant enzyme. We would also like to
thank Dr. David Morris and Dr. Janice Martin for helpful suggestions
and critical reading of the manuscript.
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