Structure-activity relationships of N-phenyl-N′-benzothiazol-6-ylurea synthetic derivatives: Cytokinin-like activity and adventitious rooting enhancement

Article abstract of DOI:10.1016/j.phytochem.2011.10.012

Some years ago we demonstrated the cytokinin-like activity of the synthetic N-phenyl-N′-benzothiazol-6-ylurea (PBU) and a relevant adventitious rooting adjuvant activity of symmetric urea derivatives devoid of any cytokinin- or auxin-like activity per se. Here we report the synthesis and the biological activity evaluation of nine symmetric or asymmetric ureas/thioureas, structurally related to PBU. None of them show cytokinin-like activity, while we demonstrate for the first time that PBU interacts with Arabidopsis cytokinin receptor CRE1/AHK4 in a heterologous bioassay system. Among the PBU derivatives, all the symmetric ureas/thioureas show an adventitious rooting adjuvant activity in various bioassays, confirming that this activity is strictly dependent on their chemical structure.

Full text of DOI:10.1016/j.phytochem.2011.10.012

Phytochemistry 74 (2012) 159–165
Contents lists available at SciVerse ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Structure–activity relationships of N-phenyl-N0-benzothiazol-6-ylurea synthetic
derivatives: Cytokinin-like activity and adventitious rooting enhancement
Enrico Rolli a,1, Matteo Incerti b,1, Federica Brunoni a, Paola Vicini b, Ada Ricci a,
⇑
a Dipartimento di Biologia Evolutiva e Funzionale, Viale G.P.Usberti 11/A, 43124 Parma, Italy
b Dipartimento Farmaceutico, Viale G.P.Usberti 27/A, 43124 Parma, Italy
a r t i c l e i n f o
a b s t r a c t
Some years ago we demonstrated the cytokinin-like activity of the synthetic N-phenyl-N0-benzothiazol-
6-ylurea (PBU) and a relevant adventitious rooting adjuvant activity of symmetric urea derivatives devoid
of any cytokinin- or auxin-like activity per se. Here we report the synthesis and the biological activity
evaluation of nine symmetric or asymmetric ureas/thioureas, structurally related to PBU. None of them
show cytokinin-like activity, while we demonstrate for the first time that PBU interacts with Arabidopsis
cytokinin receptor CRE1/AHK4 in a heterologous bioassay system. Among the PBU derivatives, all the
symmetric ureas/thioureas show an adventitious rooting adjuvant activity in various bioassays, confirm-
ing that this activity is strictly dependent on their chemical structure.
Article history:
Received 22 August 2011
Received in revised form 23 October 2011
Available online 22 November 2011
Keywords:
N,N0-Diarylureas
N,N0-Diarylthioureas
Symmetric ureas
Ó 2011 Elsevier Ltd. All rights reserved.
Asymmetric ureas
Synthesis
Cytokinin-like activity
Cytokinin receptor CRE1/AHK4
Adventitious rooting adjuvants
1. Introduction
chloro-4-pyridyl)urea (CPPU) and the N-phenyl-N0-(1,2,3-thia-
diazol-5-yl)urea (thidiazuron, TDZ), showing a cytokinin-like activ-
As a result of competitive researches carried on by the middle of
the 1950s, Miller and co-workers demonstrated that the 6-furfury-
laminopurine (kinetin) is a growth promoter in plant tissue culture
(Miller et al., 1955, 1956), while Shantz and Steward showed that
the N,N0-diphenylurea (DPU) is able to stimulate cell division in
mature carrot phloem cell cultures (Shantz and Steward, 1955).
Miller’s work gave rise to the discovery of cytokinins, naturally
occurring N6-substituted adenine derivatives containing an iso-
prenoid or aromatic side chain (Mok and Mok, 2001 and Strnad,
1997, respectively). These plant hormones are involved in many
essential developmental processes, such as cell division, senes-
cence, nutrient mobility, chlorophyll biosynthesis, as well as sym-
biosis, pathogenesis, abiotic stress response (Mok and Mok, 2001;
Werner et al., 2001; Haberer and Kieber, 2002; Werner and
Schmülling, 2009). On the other hand, although it has been later
demonstrated that DPU is a rather weak cytokinin-like synthetic
compound (Miller, 1960; Bottomley et al., 1963), the fortuitous
finding of DPU activity was the starting point for the discovery of
much more potent urea derivatives, as the N-phenyl-N0-(2-
ity sometimes exceeding that of the adenine-type derivatives
(Takahashi et al., 1978; Mok et al., 1982; Shudo, 1994). Their high
cytokinin-like activity was connected to their extreme stability in
plant tissue, as it has been demonstrated that the TDZ molecule
remains intact in tissue culture systems and that its metabolism,
consisting in glucosides formation, is extremely slow (Mok and
Mok, 1985). Moreover, it has been demonstrated that they strongly
inhibit the activity of cytokinin oxidases, the plant enzymes that
catalyse the degradation of adenine-type cytokinins by selectively
cleaving unsaturated N6-isoprenoid side chain (Victor et al., 1999;
Mok and Mok, 2001 and references herein). Thus, their high
cytokinin-like activity could be the result of indirect effects on
the regulation of the endogenous cytokinin metabolism. At the
same time, the similarity in the biological activity of these two
structurally unrelated classes of compounds, the naturally occur-
ring adenine derivatives and the synthetic ureas, has posed one
of the most interesting problems in cytokinin structure–activity
relationships, i.e. whether they can work through interaction with
a common cellular target, the cytokinin receptor. Finally, three
membrane-bound hybrid sensor histidine kinases, CRE1/AHK4,
AHK2 and AHK3, have been identified as cytokinin receptors in
Arabidopsis thaliana (Inoue et al., 2001; Suzuki et al., 2001; Ueguchi
et al., 2001). They mediate signal transduction in a two-component
⇑
Corresponding author. Tel.: +39 0521 906056; fax: +39 0521 905403.
E-mail address: ada.ricci@unipr.it (A. Ricci).
1
These authors contributed equally to this work.
0031-9422/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2011.10.012

160
E. Rolli et al. / Phytochemistry 74 (2012) 159–165
signalling phosphorelay cascade similar to the procariotic one (To
and Kieber, 2008) and are all required for cytokinin perception in
planta, even if they act redundantly (Higuchi et al., 2004; Riefler
et al., 2006). Interestingly, by a cytokinin heterologous assay
system with microbial cells, it has been demonstrated that TDZ is
also able to exert a direct cytokinin activity through the cytokinin
receptors (Yamada et al. 2001; Spíchal et al., 2004; Romanov et al.,
2006). As result of structure–activity relationship studies of
synthetic N,N0-diaryl substituted ureas, we reported the cytoki-
nin-like activity of N-phenyl-N0-benzothiazol-6-ylurea (PBU),
structurally related to thidiazuron, and the unexpected adventi-
tious rooting adjuvant activity of some symmetric urea derivatives,
showing any biological activity per se (Ricci and Bertoletti, 2009
and references herein; Rolli and Ricci, 2009; Rolli et al., 2011). With
the aim of exploring the dependence of the cytokinin-like activity
and the adventitious rooting enhancement showed by the N,N0-dia-
ryl substituted urea derivatives on their chemical structure, we
herein report the synthesis and the biological evaluation of a
number of compounds rationally related to PBU and to symmetric
ureas previously described by some of us (Ricci et al., 2001a,b).
Starting from the lead compound PBU, we synthesized nine ureas
(Table 1) designed on the basis of the benzothiazolyl substituent
positional isomerism (compounds 1–3), of the oxygen–sulphur
isosteric replacement in the urea skeleton (compounds 6–9) and
on the basis of the N,N0-symmetric introduction of the benzothia-
zole substituent (compounds 4, 5 and 8, 9). The hypothetical
cytokinin-like activity of the compounds was analysed by different
bioassays based on various biological effects of cytokinins: the
regeneration of tomato cotyledon explants and the Amaranthus
bioassay. As ultimate test to corroborate a direct cytokinin-like
activity, we investigated the ability of test compounds to interact
with the Arabidopsis cytokinin receptor CRE1/AHK4. On the other
hand, their effect on adventitious root formation was analysed by
adventitious rooting of Arabidopsis etiolated hypocotyls. Further,
three more adventitious rooting assays (Vigna radiata hypocotyl,
apple cutting and apple slice bioassays) were performed only with
the compounds that positively affected the previous Arabidopsis
etiolated hypocotyl bioassay.
2. Results and discussion
2.1. Chemistry
Compounds 1–9 and PBU (Table 1) were prepared by
a
three-step procedure starting from the commercial suitable chlo-
ronitroanilines that, refluxed with formic acid to give the formyl
derivatives, upon following heterocyclization in the presence of so-
dium sulphide, afforded the target nitrobenzothiazoles, previously
reported by Vel’tnan, 1960. The only 6-nitrobenzothiazole was
more conveniently synthesized through direct nitration of benzo-
thiazole, commercially available, following the procedure of Galla-
gher et al. (1980). The obtained nitrobenzothiazoles were then
reduced by iron in acetic acid, affording in good yields the corre-
sponding amines. The aminobenzothiazoles, described elsewhere
(Spieler and Prijs, 1950; Shimidzu and Uno, 1973; Gallagher
et al., 1980) were finally heated with phenylisocyanate, BOC
anhydride or phenylisothiocyanate to afford the target ureas and
thioureas (Fig. 1). N-phenyl-N0-benzothiazol-5-ylurea (1) and thio-
ureas 6 and 7 have already been described (El-Kerdawy et al.,
1999; Chakrabarty et al., 2010, respectively) and the data of the
compounds synthesized for the present study were consistent with
the characterization literature data. The new compounds 2–5 and
8–9 were characterized by elemental analyses and NMR spectral
data. These data, detailed in the experimental part, are consistent
with the suggested structures.
Table 1
Structures of the compounds.
X
R1
R2
C
N
N
H
H
Compound
PBU
X
O
R1
R2
N
S
N
1
2
O
O
S
N
S
3
O
N
S
N
S
O
C
4
5
6
7
8
9
O
O
S
N
N
N
S
a
N
H
N
H
NH2
S
N
S
N
PBU, 1, 2, 3
b
N
S
N
S
O
C
S
S
N
c
N
H
N
H
S
N
4, 5
S
N
N
S
N
S
S
C
S
N
C
S
N
S
S
N
N
N
N
H
H
H
H
S
N
S
N
8, 9
6, 7
S
Fig. 1. Scheme of synthesis; reagents and conditions: (a) PhNCO, PhCH3, reflux 1 h,
(b) di-t-butyl dicarbonate (BOC2O), pyridine, 110 °C (sealed vessel) 18 h, (c) PhNCS,
PhCH3, reflux 6 h.
S
S

E. Rolli et al. / Phytochemistry 74 (2012) 159–165
161
2.2. Activity in cytokinin bioassays
once more this is not a strange behaviour. In fact, it has been re-
cently reported that some kinetin derivatives, highly active in cyto-
kinin bioassays, are not able to activate CRE1/AHK4 (Mik et al.,
2011). However, as the PBU cytokinin-like activity previously re-
ported (Ricci et al., 2001b; Carra et al., 2006; Torelli et al., 2006;
Rolli et al., 2011) and its slight CRE1/AHK4 activation, here demon-
strated for the first time, seem inconsistent results, an indirect
effect through a perturbation of the endogenous cytokinin metab-
olism cannot be excluded. A hypothetical mode of action could be
the interference with the N- or O-glucosylation of endogenous
cytokinins as well as the inhibition of the cytokinin oxidase, as
previously reported for other urea derivatives (Mok et al., 2005;
Taking advantage of the tomato explant regeneration assay, the
cytokinin-like activity of PBU has been already reported (Ricci
et al., 2001b), thus the same bioassay was used to compare the bio-
logical activity of its derivatives. Then, all the test compounds were
tested in another classical cytokinin bioassay, the Amaranthus one.
These two bioassays have been developed based on the various
biological effects of cytokinins, i.e. to assess the induction of organ-
ogenesis with a long assay time and the stimulation of a biochem-
ical event with a short assay time, respectively. Finally, the
capacity of the test compounds to interact with the Arabidopsis
cytokinin receptor CRE1/AHK4 was analysed in the heterologous
bacterial assay. This was the only one cytokinin receptor we used
in this study rather than either of the other two Arabidopsis cytoki-
nin receptors, AHK2 and AHK3, because it has been reported that
CRE1/AHK4 provokes a severalfold higher response in Escherichia
coli than AHK3, even if they showed different ligand specificity
(Spíchal et al., 2004). It has also been demonstrated that in planta
transmission of a cytokinin signal through AHK3 and AHK2 was
several fold lower than through CRE1/AHK4 (Hwang and Sheen,
2001). None of the PBU derivatives showed any cytokinin-like
activity in these bioassays (data not shown), suggesting that only
N-phenyl-N0-benzothiazol-6-ylurea (PBU) possesses the desired
chemical features for the tested activity. All the changes, based
on positional isomerism, O = S isosterism and symmetry, intro-
duced in the designed analogues 1–9, have completely cancelled
the cytokinin-like activity related to PBU. The lacking of activity
obtained by the thiourea derivatives was the only result that we
could expect, as it has already been reported that HNCSNH bridge
lowers the biological activity of urea derivatives (Bruce and Zwar,
1966). Strangely, also PBU was inactive in the Amaranthus assay,
confirming the different sensitivity of the bioassays, even if stable
synthetic cytokinins should display similar activities in different
bioassays (Mok, 1994). On the other hand, in the heterologous bac-
terial assay, PBU was able to directly interact with the Arabidopsis
cytokinin receptor CRE1/AHK4, as it was possible to detect the
b-galactosidase activity in a concentration dependent manner.
PBU exerted its activity to a lower extent and at concentration
ˇ
´
Kopecny et al., 2010).
2.3. Activity in adventitious rooting bioassays
Adventitious rooting is a postembryonic organogenic process in
which new root meristems are induced from a regulated sequence
of cell proliferation and differentiation at position where roots do
not normally originate. A new hormonal balance is required to en-
able certain somatic differentiated cells, retaining an intrinsic com-
petence to form adventitious roots, to switch their fate into
multipotent cells by remarkable changes in gene expression pat-
tern (Busov et al., 2009; Ludwig-Müller, 2009; Abarca and Diaz-
Sala, 2009). Auxins are the main inducers of adventitious rooting
in plants and their role in the process has been widely studied
(Pop et al., 2011 and references herein), whereas cytokinins,
usually considered auxin-antagonists, are described as negative
regulators of root meristem activity and of lateral root formation
(Werner et al., 2003; Werner and Schmülling, 2009). As to the
adventitious rooting process, their obvious effect is inhibition
and it has been reported that TDZ and 6-benzylaminopurine
(BAP) are the most effective (De Klerk et al., 2001). On the other
hand, the same Authors demonstrated that the inhibiting effect is
strictly related to the strength of the cytokinin, as ‘‘weak’’ cytoki-
nins may even stimulate adventitious rooting in a concentration
dependent manner. In fact, during the early stages of the adventi-
tious rooting process, cytokinins may stimulate initial cell divisions
that are required to achieve adventitious roots; afterwards, they
become inhibitory (De Klerk et al., 2001). Keeping in mind the
results obtained in the above mentioned cytokinin bioassays and
according to the previously reported capacity of some urea deriva-
tives to enhance the adventitious root formation depending on
their chemical structure even without showing any cytokinin-like
activity (Ricci and Bertoletti, 2009 and references herein), we
decided to analyse the effect of PBU and its derivatives on adven-
titious rooting. Firstly, using a simple, fast and reliable bioassay,
we screened their capacity to enhance adventitious root formation
in Arabidopsis etiolated hypocotyls (Sorin et al., 2005). In the
absence of exogenous auxin, PBU, 1–3, 6 and 7 were completely
ineffective in enhancing adventitious root formation, while the
symmetric compounds 4, 5, 8 and 9 were differently able to
interact with endogenous auxin (Table 2). In fact, compound 4
significantly enhanced the mean adventitious root number at all
the concentrations tested, compounds 5 and 8 were ineffective
and compound 9 significantly enhanced the mean adventitious
100-fold higher (100 lM) than that required for receptor activation
by TDZ (Fig. 2). Thus, from these results, we can infer that PBU is
not a very good ligand of this cytokinin receptor, as the weak direct
response, significantly different from that of DMSO used as control
solvent, is induced only at the highest concentration tested, but
5000
4500
TDZ
4000
3500
3000
2500
2000
1500
1000
500
PBU
DMSO
root number at 4 and 16
lM. In the simultaneous presence of
0.1 indole-3-butyric acid (IBA), as exogenous auxin, the
lM
0
asymmetric compounds 1–3, 6 and 7 were once more completely
ineffective. On the contrary, PBU behaved as a ‘‘weak’’ cytokinin-
like compound, significantly enhancing the mean adventitious root
0
1
10
100
Concentration (M)
number at 4
concentration (16
l
M and significantly inhibiting it at the highest
M). Thus, it was confirmed that ‘‘weak’’ ade-
Fig. 2. Activation of cytokinin receptor CRE1/AHK4 by PBU. b-galactosidase activity
l
is
expressed
as
Miller
units
by
the
formula
1000 ꢀ [OD420
/
nine- and urea-type cytokinins promote adventitious root forma-
tion at relatively low concentrations whereas they inhibit the
process at supraoptimal concentrations (De Klerk et al., 2001; Ricci
(OD600 ꢀ 20
l
l ꢀ 120 min)].⁄ The difference between each mean and the control
(DMSO) is significantly different, Student’s t-test, p < 0.05. Error bars show SE
(n = 6).

162
E. Rolli et al. / Phytochemistry 74 (2012) 159–165
Table 2
Effect of selected compounds on adventitious rooting.
Compound
Arabidopsis bioassaya,b
Mung bean bioassaya,c
Apple slice bioassaya,d
Comp. concentration (
Comp. concentration (
l
M)
Comp. concentration (
l
M)
lM)
1
4
16
0.01
0.1
1
10
1
4
16
+IBA
+IBA
29.9*
75.3*
41.0*
58.4*
43.5*
+IBA
e
PBU
4
5
ꢂ2.8
40.8*
0.3
16.1
17.9
12.3
ꢂ2.1
41.2
13.1
ꢂ6.9
63.5*
5.3
ꢂ19.5*
108.8*
34.4*
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
9.1
45.4*
-
ꢂ63.6*
-
40.8*
ꢂ9.6
ꢂ8.7
50.2*
-
61.0*
9.5
42.1*
52.1*
52.5*
27.8*
51.8*
15.2*
8
9
ꢂ10.8
9.6
103.6*
51.3*
19.7
58.6*
19.5*
40.7*
ꢂ27.9
ꢂ27.1
ꢂ0.9
a
The results are expressed as a percentage of the control by the formula [(T ꢂ C/C) ꢀ 100].
b
Effect on adventitious rooting of Arabidopsis hypocotyls in the absence or in the simultaneous presence (+IBA) of 0.1
M IBA, respectively).
lM IBA. The results are expressed as a percentage of
the control (hormone free or 0.1
l
c
Effect on adventitious rooting of Vigna radiata hypocotyls.
Effect on adventitious rooting of apple slices in the simultaneous presence of 1
Ineffective.
d
e
lM IBA.
*
The difference between each mean and the control is significantly different, Student’s t-test, p < 0.05.
et al., 2005). All the symmetric compounds, both ureas 4 and 5, and
thioureas 8 and 9, significantly enhanced the mean adventitious
root number to a different extent when supplemented at 4 and
Among the PBU derivatives, only the symmetric ones were able
to enhance adventitious rooting to a different extent in various
bioassays, with a not fully understood mechanism. However, it
has been once more confirmed that there is a close connection
between the chemical structure of some urea derivatives and their
behaviour as adventitious rooting adjuvants. It is noteworthy that
the linkage NHCSNH that negatively affects the cytokinin-like
activity, does not cancel the adventitious rooting adjuvant activity.
16 lM. According to the results obtained, only the compounds that
enhanced the formation of adventitious roots in Arabidopsis etio-
lated hypocotyls, the symmetric ones and PBU, were used in the
following bioassays: rooting of V. radiata hypocotyls, rooting of ap-
ple cuttings and rooting of apple slices. Compound 9 was able to
enhance the adventitious root formation in V. radiata hypocotyls
when supplemented at the lowest concentrations (0.01 and
4. Experimental
0.1 lM) while all the other compounds were ineffective (Table
2). In the adventitious rooting of apple cuttings all the compounds
were ineffective (data not shown). Then, the adventitious rooting
adjuvant activity of PBU, of compounds 4, 5, 8 and 9 was assayed
using the apple stem slice test, an endogenous growth regula-
4.1. General experimental procedures
Melting points (mp °C) were determined with a Buchi 512
apparatus and are uncorrected. Elemental analysis was performed
on a ThermoQuest (Italia) FlashEA 1112 Elemental Analyser, for C,
H, N, and S. The values found for C, H, N, S were within 0.4% of the
theoretical ones. 1H NMR spectra of the newly synthesised com-
pounds, in DMSO-d6 solutions, were recorded on a Bruker AV
300 instrument at 298 K. Chemical shifts are reported as d (ppm)
relative to TMS as internal standard; coupling constants J are
expressed in Hz. The progress of the reactions was monitored by
thin layer chromatography with F254 silica-gel precoated sheets
(Merck, Darmstadt, Germany). UV light was used for detection.
For all target ureas: TLC eluent = CH2Cl2/MeOH 99:1.
tors-free system performing
highly medium-dependent (Van der Krieken et al., 1993), either
alone or in the presence of 1 M IBA as exogenous auxin. When
a reproducible rooting response
l
the compounds were supplemented alone, it was impossible to
detect any biological activity, the slices browning as in hormone
free condition. When supplemented in the simultaneous presence
of IBA, PBU showed the same behaviour previously reported in
Arabidopsis etiolated hypocotyls, enhancing at 4
lM then inhibiting
at 16 M the percentage of rooted slices. Compound 4 was
l
completely ineffective. Compound 5 enhanced the percentage of
rooting at all the concentrations tested. Compounds 8 and 9
enhanced the percentage of rooting at 4 and 16 lM (Table 2).
4.2. Chemicals
Solvents, unless otherwise specified, were of analytical reagent
grade or of the highest quality commercially available. Synthetic
starting material, reagents and solvents were purchased from
Aldrich Chemical Co. and from Fluka. IBA, TDZ and 6-benzylamino-
purine (BAP) were purchased from Duchefa (The Netherlands).
3. Conclusions
A few years ago we reported the cytokinin-like activity of
N-phenyl-N0-benzothiazol-6-ylurea (PBU), a synthetic urea deriva-
tive structurally related to thidiazuron. Here we report for the first
time that PBU is able to interact with the cytokinin receptor CRE1/
AHK4 at high concentrations, allowing us to confirm PBU as a
‘‘weak’’ urea-type cytokinin, in comparison to TDZ that interacts
at lower concentrations with the same receptor. It is clear that
the cytokinin-like activity of PBU is dependent on selective interac-
tions with its biological target as it is shown by the dramatic abo-
lition of any cytokinin activity upon even minor structural
modifications (see compounds 1–9). On the other hand, our data
suggest that further investigations are needed to elucidate the
mode of action of PBU, as a possible interaction with AHK2 or/
and AHK3 cytokinin receptors, or a hypothetical inhibition of cyto-
kinin oxidase. Moreover, a higher affinity for the CRE1/AHK4 recep-
tor could be obtained on the basis of structure-based modelling
studies.
4.3. Synthesis
4.3.1. General procedure for the synthesis of 4-, 5-, and 7-
nitrobenzothiazoles
The appropriately substituted 2-chloronitroaniline (170 mmol)
was refluxed in a mixture of formic acid – acetic anhydride 1:1
(23 ml) for 3 h. The mixture was cooled to room temperature and
the solid residue was filtered off. Na2Sꢁ9H2O (246 mmnol) was
added to the solid suspended in EtOH (40 ml) and the mixture
was again refluxed for 3 h. The solvent was evaporated under
reduced pressure and the residue was acidified with conc. HCl to
obtain a yellow precipitate. The crude product was purified by
flash chromatography (SiO2, CH2Cl2).

E. Rolli et al. / Phytochemistry 74 (2012) 159–165
163
4.3.2. Procedure for the synthesis of 6-nitrobenzothiazole
Benzothiazole (53 mmol) was added slowly, dropwise, to 30 ml
of concentrated sulphuric acid, previously chilled to 0 °C, then
15 ml of concentrated nitric acid (333 mmol) were added dropwise
maintaining the temperature below 0 °C. Finally the reaction was
allowed to warm up to room temperature and stirred for 12 h.
The reaction mixture was poured into ice water and crushed ice
to produce a yellow precipitate, constituted of 6-nitroderivative
as main product, that was filtered under vacuum and crystallized
from EtOH (75%).
4.3.6. Synthesis of thioureas (6–9)
To a boiling solution of the appropriate amine (3.3 mmol) dis-
solved in anhydrous toluene (20 ml), phenylisothiocyanate
(3.65 mmol) was added in one batch. The mixture was stirred un-
der reflux for 6 h. The solvent was removed under reduced pres-
sure and the crude product purified by flash chromatography
(SiO2, CH2Cl2/MeOH 99:1) afforded a first fraction (major) consti-
tuted of N-phenyl benzothiazolylthiourea (55%) and a second frac-
tion (minor) constituted of N-N0-di(benzothiazolyl)thiourea.
4.3.6.1. 1,3-Di(benzo[d]thiazol-6-yl)thiourea (8). Yield: 38%; mp
202–203 °C (EtOH/H2O). 1H NMR (DMSO-d6 d, ppm): 10.10 (s,
2H, NH); 9.33 (s, 1H, CH); 8.30 (d, 2H, J = 2.1, Ar); 8.03 (d, 2H,
J = 9.0, Ar); 7.58 (dd, 2H, J = 2.4 J = 8.7, Ar); Anal. Calcd. for
4.3.3. General synthesis of aminobenzothiazoles
A stirred mixture of the suitable nitrobenzothiazole (6 mmol),
iron powder (21 mg-atom), HOAc (2.5 ml) and EtOH (20 ml) was
heated under reflux for 3 h, and then poured in water and crushed
ice. The water layer was extracted with CH2Cl2; the organic phase
was washed with a solution of 10% NaOH, water and then dried
over anhydrous sodium sulphate. Evaporation of the solvent under
reduced pressure afforded a green solid.
C15H10N4S3 (342.69): C, 52.61; H, 2.94; N, 16.36; S, 28.09. Found
C, 52.61; H, 3.38; N, 15.36; S, 27.95%.
4.3.6.2. 1,3-Di(benzo[d]thiazol-5-yl)thiourea (9). Yield: 35%; white
crystals, mp 191–193 °C (EtOH/H2O). 1H NMR (DMSO-d6 d, ppm):
1H NMR (DMSO-d6 d, ppm): 10.13 (s, 2H, NH); 9.40 (s, 2H, CH);
8.24 (d, 2H, J = 1.5 Ar); 8.30 (d, 2H, J = 2.1 Ar); 8.11 (d, 2H, J = 8.4
Ar); 7.57 (dd, 2H, J = 2.4 J = 8.7 Ar). Anal. Calcd. for C15H10N4S3
(342.69): C, 52.61; H, 2.94; N, 16.36; S, 28.09. Found C, 52.61; H,
3.38; N, 15.36; S, 27.95%.
4.3.4. Synthesis of N-phenyl-N0-benzothiazolylureas (PBU, 1–3)
The target ureas were prepared according to the procedure
described for PBU by Ricci et al. (2001a) starting from the appropri-
ate aminobenzothiazole. Briefly, to a boiling solution of the amine
(5.3 mmol) dissolved in anhydrous toluene (20 ml), phenylisocya-
nate (7.3 mmol) was added in one batch. The mixture was stirred
under reflux for 1 h, then filtered and the collected white solid
was recrystallised from the appropriate solvent.
4.4. Cytokinin and adventitious rooting bioassays
All the tested compounds were dissolved in dimethylsulphox-
ide (DMSO) and the final concentration of DMSO in the culture
medium or in the aqueous solutions did not exceed 0.2% (Schmitz
and Skoog, 1970).
4.3.4.1. 1-(Benzo[d]thiazol-4-yl)-3-phenylurea(2). Yield: 40%; mp
184–185 °C (EtOH). 1H NMR (DMSO-d6 d, ppm): 9.57 (s, 1H, NH);
9.38 (s, 1H, NH); 9.22 (s, 1H, CH); 8.32 (d, 2H, J = 8.1, Ar); 7.70 (d,
1H, J = 7.5, Ar); 7.50 (d, 2H, J = 8.1, Ar); 7.42 (t, 2H, J = 8.1, Ar);
7.30 (d, 1H, J = 8.1, Ar) 6.99 (t, 1H, J = 7.2, Ar). Anal. Calcd. for
4.4.1. Tomato explant regeneration assay
Twelve cotyledon explants, obtained from in vitro cultured
seedlings, were plated on MS medium (Murashige and Skoog,
C14H11N3SO (269.33): C, 62.43; H, 4.12; N, 15.60; S, 11.90. Found
C, 62.17; H, 4.10; N, 15.52; S, 11.92%.
1962) containing the PBU derivatives at 1, 5 and 10
lM, both alone
and in the presence of 20 M 1,2-benzisoxazole-3-acetic acid
l
4.3.4.2. 1-(Benzo[d]thiazol-7-yl)-3-phenylurea (3). Yield: 55%; mp
248–250 °C (EtOH). 1H NMR (DMSO-d6 d, ppm): 9.37 (s, 1H, CH);
8.99 (s, 1H, NH); 8.81 (s, 1H, NH); 7.88 (d, 1H, J = 8.4, Ar); 7.80
(d, 1H, J = 8, 1 Ar); 7.53–7.48 (m, 3H, Ar); 7.31 (d, 2H, J = 7.6, Ar);
7.00 (t, 1H, J = 7.2, Ar). Anal. Calcd. for C14H11N3SO (269.33): C,
62.43; H, 4.12; N, 15.60; S, 11.90. Found C, 62.20; H, 4.10; N,
15.61; S, 11.93%.
(BOAA), as auxin (Ricci et al., 1996). Control was performed by cot-
yledon explants cultured in the presence of TDZ, as cytokinin-like
urea derivative, in the same culture conditions. After 2 week incu-
bation at light intensity of 27 l
mol mꢂ2 sꢂ1 at 26 °C under 16 h
photoperiod, the cotyledon explants were transferred to a hor-
mone free medium and the number of explants that regenerated
shoots was checked 2 weeks later. The experiments were carried
out in triplicate, and repeated three times.
4.3.5. Synthesis of N-N0-di(benzothiazolyl)ureas (4, 5)
A solution of the appropriate aminobenzothiazole (6.6 mmol)
and di-t-butyl dicarbonate (BOC2O), (3.33 mmol) in 20 ml of
anhydrous pyridine placed in a sealed vessel was heated at
115 °C for 18 h. The solvent was removed under reduced pressure
and the crude product was purified by flash chromatography (SiO2,
CH2Cl2/MeOH 95:5).
4.4.2. Amaranthus assay
The Amaranthus caudatus L. bioassay was performed as previ-
ously described (Ricci et al., 2006). Four different concentrations
10 nM, 100 nM, 1 lM and 10 lM of the test compounds were
assayed. Control was performed with phosphate buffer alone
(hormone free, HF). For each experiment the same concentrations
of 6-benzylaminopurine (BAP) were also tested and the experi-
ment was considered ‘‘valid’’ only in the presence of a classical
BAP concentration-course variation. The experiments were carried
out in triplicate and repeated three times. The results were
expressed as a percentage of the control (phosphate buffer alone)
by the formula (tested-control/control) ꢀ 100 [(T ꢂ C/C) ꢀ 100].
4.3.5.1. 1,3-Di(benzo[d]thiazol-6-yl)urea (4). Yield: 45%; mp
300–301 °C (DMF/H2O). 1H NMR (DMSO-d6 d, ppm):.9.22 (s, 2H,
CH); 9.05 (s, 2H, NH); 8.38 (d, 2H, J = 1.5, Ar); 8.01 (d, 2H, J = 9.0,
Ar); 7.52 (dd, 2H, J = 2.1 J = 8.7, Ar); 7.30 (d, 1H, J = 8.1, Ar). Anal.
Calcd. for C15H10N4OS2 (326.40): C, 55.20.51; H, 3.09; N, 17.18; S,
19.65. Found C, 54.96; H, 3.11; N, 16.91; S, 19.41%.
4.4.3. Heterologous bacterial assay
4.3.5.2. 1,3-Di(benzo[d]thiazol-5-yl)urea (5). Yield: 40%; mp 306–
308 °C (DMF/H2O). 1H NMR (DMSO-d6 d, ppm): 9.35 (s, 2H, CH);
8.99 (s, 2H, NH); 8.34 (s, 2H, Ar); 8.05 (d, 2H, J = 8.7, Ar); 7.51 (d,
E. coli strain KMI001, harbouring vector pINIII-AHK4 expressing
the Arabidopsis cytokinin receptor CRE1/AHK4, that is capable of
signalling the downstream YojN ? RcsB ? cps::lacZ pathway in
response to external cytokinins, was used in the experiments
(Suzuki et al., 2001; Yamada et al., 2001). Bacterial strains were
kindly provided by dr. T. Mizuno (Nagoya, Japan). Bacteria were
2H, J = 9.0, Ar). Anal. Calcd. for
C15H10N4OS2 (326.40): C,
55.20.51; H, 3.09; N, 17.17; S, 19.65. Found C, 60.43; H, 3.44; N,
12.15; S, 19.11%.

164
E. Rolli et al. / Phytochemistry 74 (2012) 159–165
grown in Luria broth at 37 °C with ampicillin (50
l
g/ml) with
shoots, 2–2.5 cm in length, were used either for further propaga-
tion or for microcutting rooting experiments or for preparing 1-
mm thick stem slices. Malus M26 microcuttings, 2–2.5 cm in
length excised from 4-week old cultures, were incubated in MP
extensive shaking. Culture density was controlled by measuring
the absorption at 600 nm (OD600). A homogenous bacterial suspen-
sion (OD600 between 0.04 and 0.4) was aliquoted in multiwell
dishes supplemented with 1, 10, and 100
l
M of the test com-
medium in the presence of 1, 4, 8, 16
without any exogenous auxin supplementation, and then cultured
at light intensity of 27
mol mꢂ2 sꢂ1 at 26 °C under 16 h photope-
riod. Control was performed with microcuttings cultured in hor-
mone free (HF) medium under the same culture conditions. In all
the experiments the roots were counted after 4 weeks. For each
experiments 50 microcuttings were used. The results are shown
as number of roots per rooted microcuttings (total induced roots
divided by the number of rooted microcuttings).
lM of the test compounds
pounds. Bacteria grown in the presence of the same concentrations
of TDZ and of the relative volumes of DMSO were used as control.
After 40 h incubation at 25 °C in the dark, the b-galactosidase
activity was detected as described by Zhang and Bremer (1995)
l
with minor modifications. Twenty
mixed into 80 l of permeabilization solution (0.8 mg/ml hexade-
cyltrimethylammonium bromide, 0.4 mg/ml sodium deoxycholate,
100 mM Na2HPO4, 20 mM KCL, 2 mM MgSO4, 5.4 l/ml b-mercap-
toethanol). This mixture was kept at 30 °C for 1.30 h, then 600 l of
30 °C prewarmed substrate solution (60 mM Na2HPO4, 40 mM
NaH2PO4, 1 mg/ml o-nitrophenyl-b- -galactopyranoside, 2.7 l/ml
b-mercaptoethanol) were added to initiate the reaction. After 2 h
at 30 °C, reactions were stopped by the addition of 700 l of 1 M
ll samples of cultures were
l
l
l
4.4.7. Adventitious rooting of apple stem slices
D
l
Malus M26 stem slices were prepared and distributed over dif-
ferent rooting treatments as previously reported (Ricci et al., 2006).
Groups of 16 slices were cultured in Petri dishes with the apical
side on a nylon mesh put on the above described culture medium
l
Na2CO3 and the absorption at 420 nm was recorded for each sam-
ple. Enzyme activities were expressed as Miller units (Miller, 1972)
supplemented with 1, 2, 4 or 8
in the simultaneous presence of 1
l
M of the test compounds alone or
M IBA, as auxin. The dishes
by the formula 1000 ꢀ [OD420/(OD600 ꢀ 20 l ꢀ 120 min)].
l
l
were incubated upside down in the darkness for 6 days. Simply
removing the nylon mesh, all the slices were then transferred to
fresh culture medium lacking of the test compounds and IBA; the
4.4.4. Adventitious rooting of Arabidopsis seedlings
A. thaliana ecotype Columbia (Col-0) seedlings were grown as
previously described (Sorin et al., 2005) with minor modifications.
Seeds were surface sterilized in 70% ethanol for 1 min, followed by
10 min in 50% commercial bleach (equivalent to 2.5% NaOCl),
washed five times in sterile distilled water, sown on 1/4 strength
Murashige and Skoog (MS) medium supplemented with 0.8% agar,
pH 5.8 (germination medium) and kept in the dark at 26 °C. Groups
of 10 three-day-old etiolated seedlings were transferred to Petri
dishes containing full strength MS medium, supplemented with
MS vitamins, 3% sucrose, 0.8% agar, pH 5.8, under different rooting
dishes were incubated at 26 °C at light intensity of 27
mꢂ2 sꢂ1 under 16 h photoperiod. Control was performed with stem
slices cultured in the presence of 1 M IBA alone and slices were
lmol
l
subjected to the same manipulations described above. The number
of rooted slices in relation to total slices in specific treatments and
the induced number of roots were counted after 14 days. The per-
centage of rooting was expressed as a percentage of the control
(1
l
M IBA) by the formula (tested-control/control) ꢀ 100 [(T ꢂ C/
C) ꢀ 100]. All the experiments were carried out in triplicate and
conditions. The medium was supplemented with 1, 2, 4 or 8
the test compounds alone and in the simultaneous presence of
0.1 M IBA, as auxin. Medium containing DMSO or 0.1 M IBA
were used as controls. Petri dishes were placed vertically in a
growth chamber at light intensity of 27
mol mꢂ2 sꢂ1 at 26 °C
lM of
repeated twice.
l
l
Acknowledgments
l
The authors are grateful to Professors C.A Maggiali and G.
Morini for their imput and fruitful discussions to pursue this study.
The authors also acknowledge Dr. Mara Comini for her contribu-
tion to the Experimental Section.
under 16 h photoperiod. Emergent adventitious roots on the
hypocotyls were scored at 7 days after transfer to the light using
a stereomicroscope. The experiments were done in triplicate and
repeated twice. The results were expressed as a percentage of
the control (hormone free or 0.1 lM IBA, respectively) by the
formula (tested-control/control) ꢀ 100 [(T ꢂ C/C) ꢀ 100].
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