New strigolactone mimics as exogenous signals for rhizosphere organisms

Article abstract of DOI:10.3390/molecules22060961

The importance of strigolactones in plant biology prompted us to synthesize simplified strigolactone mimics effective as exogenous signals for rhizosphere organisms. New strigolactone mimics easily derived from simple and available starting materials in significant amounts were prepared and fully characterized. These compounds contain an aromatic or heterocyclic ring, usually present in various bioactive molecules, connected by an ether link to a furan-2-one moiety. The new synthesized strigolactone mimics were confirmed to be active on plant pathogenic fungi and parasitic weed seeds.

Full text of DOI:10.3390/molecules22060961

molecules
Article
New Strigolactone Mimics as Exogenous Signals for
Rhizosphere Organisms
1
2
3
4
Florin Oancea , Emilian Georgescu , Radoslava Matusova , Florentina Georgescu ,
Alina Nicolescu 5 , Iuliana Raut , Maria-Luiza Jecu , Marius-Constantin Vladulescu ,
Lucian Vladulescu 4 and Calin Deleanu
,6
1
1
4
5,6,
*
1
National Research & Development Institute for Chemistry & Petrochemistry–ICECHIM,
Spl. Independentei 202, RO-060021 Bucharest, Romania; Florin.Oancea@icechim.ro (F.O.);
iulia_rt@yahoo.com (I.R.); Jecu.Luiza@icechim.ro (M.-L.J.)
Research Center Oltchim, St. Uzinei 1, RO-240050 Ramnicu Valcea, Romania; g_emilian@yahoo.com
Plant Science and Biodiversity Center SAS, Institute of Plant Genetics and Biotechnology, P.O. Box 39A,
2
3
9
5007 Nitra, Slovakia; Radka.Matusova@savba.sk
4
Research Department Teso Spec S. R. L., Str. Muncii 53, RO-915200 Fundulea, Romania;
florentina_fg@yahoo.com (F.G.); marius.vladulescu@tesospec.com (M.-C.V.);
lucian.vladulescu@tesospec.com (L.V.)
Centre of Organic Chemistry, Romanian Academy, Spl. Independentei 202B, RO-060023 Bucharest, Romania;
alina220382@yahoo.com
5
6
Institute of Macromolecular Chemistry, Romanian Academy, Aleea Grigore Ghica Voda 41-A,
7
00487 Iasi, Romania
*
Correspondence: calin.deleanu@yahoo.com; Tel.: +40-744-340-456
Academic Editor: Kamal Kumar
Received: 28 April 2017; Accepted: 7 June 2017; Published: 9 June 2017
Abstract: The importance of strigolactones in plant biology prompted us to synthesize simplified
strigolactone mimics effective as exogenous signals for rhizosphere organisms. New strigolactone
mimics easily derived from simple and available starting materials in significant amounts were
prepared and fully characterized. These compounds contain an aromatic or heterocyclic ring, usually
present in various bioactive molecules, connected by an ether link to a furan-2-one moiety. The new
synthesized strigolactone mimics were confirmed to be active on plant pathogenic fungi and parasitic
weed seeds.
Keywords: strigolactones; strigolactone mimics; natural-products like molecules; exogenous signals
1
. Introduction
More than 25 natural strigolactones (SLs) have been detected and isolated from root
exudates of mono- and dicotyledonous plants. SLs are apocarotenoids [
plant hormones [4,5], endogenous signals which regulate plant response to stress [
ecomones/semiochemicals, exogenous signals involved into exchange of information in the plant
rhizosphere [ 10]. Strigol, the first member of the SLs family, was isolated from cotton root
1
–3
] functioning as both
6,7
], and
8–
exudates and identified as a germination stimulant of the seed of the parasitic plant Striga lutea [11].
The function of germination stimulants of parasitic weed seeds in the rhizosphere is only the first of SLs’
recognized functions [12
symbiosis with mycorrhizae (AM) fungi [15
plants [18 21]. Conflicting data regarding the influence of SL analogues on fungal pathogen growth
patterns were reported [22 26]. Reactive oxygen species (ROS) and mitochondria have been recently
reported as emerging mediators of SLs’ actions on plant pathogenic fungi [23]. SLs were recognized
–
14]. SLs have been also identified as signaling molecules involved in plants’
–17] and in the symbiosis between rhizobia and leguminous
–
–
Molecules 2017, 22, 961; doi:10.3390/molecules22060961
www.mdpi.com/journal/molecules

Molecules 2017, 22, 961
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as a new class of plant hormones [
4,
5,27
–
31], which regulate plants’ architecture [32
–
34], being also
related to plants’ response to stress [6,7,35].
Naturally occurring SLs have a tricyclic lactone skeleton (ABC rings), attached by an enol ether
bond to an -unsaturated furanone part (the D-ring). There are two naturally occurring SL families
depending on the BCD part stereochemistry: one in which the stereochemistry is the same as in
α,β
(
(
+)-strigol (
Figure 1).
I
) and the other one having the stereochemistry found in natural ( )-orobanchol (II) [36,37]
−
The structure–activity relationship of SLs as exogenous signals that stimulate the germination of
parasitic plant seeds, revealed that the bioactivity is located in the CD part of the SL molecule [37–39],
0
and the presence of the methyl group at C-4 of the D-ring and the presence of an enol ether
combined with a carbonyl group of a ketone or of an ester, linked to the D-ring are essential for
their bioactivity [37]. The substitution of the ether oxygen by nitrogen or sulfur atom does not appear
to affect the bioactivity.
1
O
1
O
C
2
2
O
O
8
b
8
b
8
8
5
8a
8
a
C
3
7
6
3
7
6
A
B
6'
A
B
6'
3a
3a
1'
1
O
D
'
4
a
4a
O
O
4
5
4
O 5'
D
5
'
OH
OH
2'
2'
3'
O
O
3
'
4'
4'
I
II
Figure 1. Naturally occurring SL structures.
Natural SLs can be obtained in minute amounts from plant root exudates or by long multistep
chemical syntheses. Simplified synthetic SL analogues and mimics were developed as natural
product-like small molecules, in order to investigate the structure–activity relationship for their
various activities in plants. SL analogues have a simplified structure that retained the bioactophore
CD part of natural SLs [40–44]. Among these, GR24 (Figure 2), prepared firstly by Johnson et al. [43], is
considered as the standard for SL analogues and its synthetic procedure was improved and further
developed later by other groups [44].
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
GR 24
GR 5
GR 7
O
O
Cl
Br
OMe
N
O
O
S
O
O
O
O
O
O
O
Nijmegen-1
5-[(4-chlorophenyl)thio]-
,4-dimethylfuran-2-(5H)-one
4 BD
3
Figure 2. Chemical structures of several synthetic SL analogues and mimics.

Molecules 2017, 22, 961
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SL mimics are an important group of simple compounds that mimic the activity of SLs. SL mimics
do not have the typical SL structural requirements of ABC rings attached by an enol ether bond to
a D-ring. Usually, SL mimics have only an aryloxy-, arylthia- or aroyloxy-substituent at C-5 of the
D-ring (Figure 2) [30,31,37,45–50]. The mode of action was explained by the interaction of these SL
mimics with a protein receptor which led probably to the enzymatic hydrolytic elimination of the
D-ring as HO-D. This first step generates a sequence of reactions at the receptor site and triggers the
signal transmission [50
–
53]. However, the protein receptors of AM fungi have not been yet isolated
and identified.
SLs represent an important research field which may lead to agricultural innovation [35
,49,54].
The SLs importance in plant biology, both as exo- and endo-signals, prompted us to synthesize
simplified SL mimics easily accessible in significant amounts. Our group is interested in the synthesis
of new potentially bioactive compounds [55–57] and as part of our ongoing research related to such
molecules, we report here the synthesis of some new SL mimics, derived from simple and available
starting materials, containing an aromatic or heteroaromatic nucleus connected by an ether link to
a furan-2-one moiety. We also report their biological activity as exogenous signals for rhizosphere
organisms, parasitic weed seeds and plant pathogenic fungi.
2
. Results and Discussion
2
.1. Synthesis of New Strigolactone Mimics
Our new SL mimics, derived from simple and available starting materials, contain an aromatic or
heteroaromatic ring, present in various bioactive molecules, linked by an ether group to a furan-2-one
moiety. The common features of new synthesized compounds are a bioactive (hetero)aromatic core
and the presence of a D ring that can increase the pre-existing activity.
The SL mimic 3-methyl-5-(2-pyrimidin-4-yl-phenoxy)-5H-furan-2-one (
coupling reaction of 4-(2-hydroxyphenyl)pyrimidine ( ) with 5-bromo-3-methyl-5H-furan-2-one
) in basic medium. The starting 4-(2-hydroxyphenyl)pyrimidine ( ) was prepared for the first
3) was obtained by the
1
(2
1
time, according to a previously reported method [58], by treating 2-hydroxyacetophenone with
trisformylaminomethane (Scheme 1).
Br
O
O
OH
O
p-TSA
O
O
O
OH
2
CH3
HC(NHCHO)3
+
N
N
N
N
1
3
Scheme 1. Synthesis of SL mimic 3 derived from 4-(2-hydroxyphenyl)pyrimidine (1).
The other two synthetic SLs were obtained by treating commercially available cyclic
hydroxyl-ketones in DMF with 5-bromo-3-methyl-5H-furan-2-one ( ) in the presence of K CO
Scheme 2). Thus, the coupling reaction of 2-hydroxy-benzo[de]isoquinoline-1,3-dione (
-bromo-3-methyl-5H-furan-2-one ( ) led to the SL mimic 3-methyl-5-(benzo[de]isoquinoline-
,3-dione-2-yloxy)-5H-furan-2-one ( ), bearing a naphthalimide core linked by an ether group
to the furan-2-one ring. Numerous compounds containing a N-substituted phthalimide nucleus
are known as plant growth regulating chemicals [59 61], as well as compounds regulating the
activity of gibberellins A4 and A7 [62 63]. In the same way, by treating a cyclic hydroxyketone,
2
2
3
(
4) with
5
2
1
5
–
,

Molecules 2017, 22, 961
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namely 2-hydroxy-1,4-naphthoquinone (6), with 5-bromo-3-methyl-5H-furan-2-one (2) the SL mimic
3-methyl-5-(1,4-naphthoquinone-2-yloxy)-5H-furan-2-one (7) was obtained.
O
O
OH
N
O
O
O
N
2
O
O
4
5
O
O
OH
O
O
2
O
O
O
6
7
Scheme 2. Synthesis of synthetic SLs derived from cyclic hydroxy ketones.
The newly synthesized compounds were structurally characterized by their chemical and spectral
data. The synthesized compounds have only one asymmetric carbon atom, thus there are no particular
stereochemical issues to be solved. All compounds produced in the above described reactions were
obtained as racemates.
1
The progress of the coupling reactions may be followed in the H-NMR spectra of the synthesized
compounds by the disappearance of the characteristic OH group signal from 10–13 ppm and the
appearance of the three signals corresponding to the 5-bromo-3-methyl-5H-furan-2-one moiety.
The detailed NMR data (chemical shifts and coupling constants) for each of the synthesized compounds
are presented in the Materials and Methods Section, while the NMR spectra are presented as
Supplementary Materials.
2
.2. The Biological Activity of Synthesized Compounds
2
.2.1. The Effect on Parasitic Weed Seeds
The effect of the synthesized SLs on the induction of parasitic weed seed germination was tested.
The germination bioassays were carried out according to the previously described method [64] on
Phelipanche ramosa, Phelipanche aegyptiaca, Orobanche cumana and Striga hermonthica seeds. The results
clearly demonstrated that compounds 3, 5, 7 and reference synthetic SL analogue GR24 (racemic
mixture), induced germination of the seeds. Results also showed the different activity of the compounds
on different parasitic weed seeds (Figure 3). The applied doses covered a broad range of responses
−
4
from no germination to toxic effects in the case of analogue 7 (10 M) for S. hermonthica.
Low, but significant (11%) spontaneous germination for P. aegyptiaca seeds was observed
(demineralized water, or demineralized water plus 3% acetone as negative controls). Spontaneous
germination was rarely observed for P. ramosa, O. cumana and S. hermonthica. The synthetic mimic
− −5
8
3
induced germination at concentrations ranging from 10 M for P. aegyptiaca to 10 M for
S. hermonthica. Compound
high dose of compound (10 M), in comparison to the dose which is necessary to induce 50% of
maximum germination [GS]50 by the reference analogue GR24 (5.7
3
induced 55% germination of S. hermonthica seeds, but with relatively
−
4
−
9
×
10 M). The lowest germination
activity of was observed for O. cumana seeds, with maximal germination induced (Rmax = 28%) and
3

Molecules 2017, 22, 961
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−
6
[
9
GS]50 1.44
×
×
10 M. The compound
3
was more potent for P. ramosa with Rmax = 96% and [GS]50
−
7
−7
10 M and P. aegyptiaca with Rmax = 93% and [GS]50
3
×
10 M. The synthetic mimic
5
was an
−
8
−9
10 M for
active germination stimulant for broomrapes, with [GS] 1.7
P. aegyptiaca and 6.7 × 10− M for O. cumana.
×
10 M for P. ramosa, 5.3
×
50
9
Figure 3. Effect of new synthetic compounds 3, 5, 7 and reference synthetic analogue GR24
on germination rate of Phelipanche ramosa, P. aegyptiaca, Orobanche cumana and Striga hermonthica.
Germination assays were performed in triplicate, over a range of SL concentrations. Data are
the mean
Hill coefficient.
±
SE. Logistic dose-response curves were gained by nonlinear regression, with variable
Compound
germination ability of GR24. For S. hermonthica, a higher concentration of compound (10 M) was
needed to reach maximal seed germination. Synthetic compound was the least potent among all
analogues tested at inducing germination of P. ramosa, S. hermonthica and O. cumana, with maximal
5 induced maximal germination in these species, comparable to the maximal
−
6
7
−
4
germination induced by
7 reaching 61%, 47% and 28% for these species. At high dose (10 M) 7
greatly reduced germination of P. ramosa seeds (21%) and caused no germination of S. hermonthica
seeds. Our results indicate different affinity of the compounds to the putative receptor(s) which is
expressed by [GS]50 values. In general,
00,000 times less active then GR24 in the species studied—while
induction of parasitic weed seed germination out of new synthetic SL mimics tested.
3
exhibits the lowest affinity to the receptor(s)—about 10 to
1
5
is the most potent compound for
2
.2.2. The Effect on Plant Pathogenic Fungi
The synthetic SL mimics inhibited the growth of the tested fungal plant pathogens, the effect
−
6
being visible from the lowest concentration (3
×
10 M, Figure 4). Generally, compounds 3 and 5
display a similar response as GR24 on S. sclerotiorum (DSM 1946) and M. phaseolina (DSM 62744) as
the responses of these tested fungi to compounds and are on the same level with those reported
already for the strains of the same genera treated with GR24 [22]. Compound is less effective than
other compounds on F. graminearum (DSM 4527). However, it is the most active compound on R. solanii
3
5
3

Molecules 2017, 22, 961
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(DSM 22842). Compound
5
has an almost similar response to GR24 on F. graminearum (DSM 4527)
inhibition and is the least active compound on R. solanii (DSM 22842). Compound
7 is very active
on R. solanii (DSM 22842), being more active than the reference compound GR24. On M. phaseolina
−
6
(
DSM 62744) compound
7
is the most active compound at concentrations higher than 15
×
10
M
and on F. graminearum (DSM 4527) is displays a response like GR24, however, it is the least active from
the tested SL mimics on S. sclerotiorum (DSM 1946).
Figure 4. Effect of new synthetic SL mimics
growth of water–agar 1.8% of F. graminearum (DSM 4527), S. sclerotiorum (DSM 1946), R. solanii
DSM 22842) and M. phaseolina (DSM 62744). Least-significant differences (LSD) were calculated and
means were compared. The different letters indicate significant differences (p < 0.05). M —water-agar
3, 5, 7 and reference synthetic analogue GR24 on radial
(
0
1
.8%; M —water agar containing 0.5% (w/v) acetone.
1
There are conflicting data in the literature regarding the influence of synthetic SL mimics on
fungal plant pathogen development patterns. Thus, GR24 was reported to inhibit radial fungal growth
when embedded into agar media [22,23]. However, GR24 spread from an acetone solution on the
surface of the agar media [24], or which is diffusing from small holes in agar medium [25] or from
small fiberglass discs deposited on the surface of the agar media [26] were reported to have no effects
on the growth of fungal plant pathogens.
Experimental conditions thus clearly influence the results, and availability of the rather
hydrophobic SL chemical analogues on rather hydrophilic (agar) medium being essential for better
understanding the interactions between synthetic SLs and/or the naturally occurring SLs and plant
fungal pathogens. A better understanding of SLs bioavailability on hydrophilic/amphiphilic media
could support also further optimization of SLs application as soil treatment. The tested compounds
produced an increase of branching activities (Table 1).
−
6
The tested SL synthetic mimics, at the highest concentration (75
×
10 M) determine the most
significant increase in hyphal branching on S. sclerotiorum (DSM 1946). This is similar to the result
reported for another S. sclerotiorum strain as response to GR24 [22]. A lower hyphal branching was

Molecules 2017, 22, 961
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determined by the tested synthetic SL compounds on R. solanii (DSM 22842) and F. graminearum
DSM 4527). However, M. phaseolina (DSM 62744) has an opposite response to the tested synthetic
SL mimics, the hyphal branching being depressed by all tested compounds. The responses of tested
fungal strains to compound are quite similar to the response to GR24. Compound determines a
less significant hyphal branching response and compound 3 determines an intermediate response.
(
5
7
−
6
Table 1. The effect of the tested SL synthetic mimics, at 75
pathogen branching.
×
10 M concentration, on fungal plant
Tested Fungi
Water Control
Acetone Control
GR24
3
5
7
Fusarium graminearum
1
2
4
.67 (a)
.83 (a)
.50 (a)
1.33 (a)
5.83 (c)
4.50 (bc)
5.16 (c)
2.83 (ab)
(
DSM 4527)
Sclerotinia sclerotiorum
DSM 1946)
Rhizoctonia solanii
DSM 22842)
Macrophomina phaseolina
DSM 62744)
3.33 (a)
3.67 (a)
20.66 (c)
6.83 (ab)
3.60 (c)
13.83 (b)
6.67 (ab)
5.33 (c)
21.33 (c)
10.67 (b)
6.67 (bc)
12.33 (b)
7.83 (ab)
7.33 (bc)
(
(
1
7.83 (a)
12.33 (ab)
(
On each primary branch, the number of hyphal branches, total and branches of different orders, from second till
fifth, on a total length of 1000 m were determined. As negative controls, water agar and water agar with 0.5%
v/v) solvent (acetone) have been used. Least-significant differences (LSD) were calculated and compared with the
µ
(
differences between the means, based on Tukey–Kramer method (p < 0.05). Numbers followed by the same letter in
brackets do not significantly differ (p < 0.05).
The effects on plant pathogenic fungi were reported till now only for SL analogues (i.e., GR24).
We report here the first evidence of the effects for SL mimics on plant pathogenic fungi. This suggests
the existence on plant pathogenic fungi of a SL perception system able to realize the enzymatic
hydrolytic elimination of the D-ring as HO-D.
2
.3. Chemical Stability
The stability of our SL mimics was verified in a known test in aqueous solution, acetone-
ethanol-water [30]. This aqueous solution test demonstrated the following order
Figure 5), our SL mimics showing a good stability.
5
> 3 > 7 > GR24
(
1
1
20
00
8
6
4
2
0
0
0
0
0
GR24
3
5
7
0
2
4
6
8
Time (days)
◦
and (+/ )GR24 at 2 C, pH 6.7
g/mL, done by dilution of acetone stock solution into an ethanol:water
Figure 5. Chemical stability of SL mimics, (+/
on aqueous solutions of 50
mixture (7:30, v/v). Data are represented as means ± SE (n = 3).
−
)
3
, (+/
−
)
5
, (+/
−
)7
−
µ

Molecules 2017, 22, 961
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Synthetic SLs with good stability in aqueous solution, compared to GR24, were already
reported [30 31]. However, these synthetic SLs, reported as stable in aqueous solution, have a low
,
activity as exo-signals in the rhizosphere [31]. Our SL analogues and mimics, stable in aqueous
solution, on the other hand, have a significant activity as exogenous signals for rhizosphere organisms.
3
. Materials and Methods
3
.1. General Information
The melting points have been determined on a Boetius apparatus (Carl Zeiss, Jena, Germany)
and are uncorrected. A Nicolet Impact 410 spectrometer (Thermo Scientific, Waltham, MA, USA) was
used for recording the IR spectra in KBr pellets. The NMR analysis was performed on an Advance III
4
00 spectrometer (Bruker BioSpin, Rheinstetten, Germany), operating at 400.1, 100.6 and 40.6 MHz
1
13
15
for H, C and N, respectively. The 1D and 2D spectra were recorded with a 5 mm multinuclear
inverse detection z-gradient probe. Unambiguous 1D NMR signal assignments were made based on
2D NMR homo—and heteronuclear correlations. H-H COSY, H-C HSQC and H-C HMBC spectra
were recorded using standard pulse sequences in the version with z-gradients, as delivered by Bruker
with TopSpin 2.1 PL6 spectrometer control and processing software. The 15N chemical shifts were
obtained as projections from the 2D indirectly detected H-N HMBC spectra, employing a standard
pulse sequence in the version with z-gradients as delivered by Bruker (TopSpin 2.1 PL6). Chemical
shifts are reported in
δ units (ppm) and were referenced to the residual solvent signals (for CDCl
3
1
H at 7.26 ppm and C at 77.0 ppm and for DMSO-d6 1H at 2.51 ppm and C at 39.4 ppm). The
13
13
15
N
chemical shifts are referenced to liquid ammonia (0.0 ppm) using nitromethane (380.2 ppm) as external
standard. Satisfactory microanalyses for all new compounds were obtained: C 0.20, H 0.16,
0.26. All other reagents, aside from the starting material which preparation is given below, and
the common intermediate 5-bromo-3-methyl-5H-furan-2-one ( ), which was obtained in good yield
81%) by the bromination of 3-methyl-5H-furan-2-one with N-bromosuccinimide in CCl4 [65] and was
±
±
N
±
1
2
(
used as crude reaction product, were commercial products (Sigma-Aldrich, Steinheim, Germany) used
without any additional purification.
3
.2. Preparation of 4-(2-Hydroxyphenyl)pyrimidine (1)
Starting material was prepared by treating 2-hydroxyacetophenone with trisformyl-
1
aminomethane according to a previously reported method for the synthesis of 4-phenylpyrimidine [58].
◦
Pale yellow solid, mp 116–118 C. Yield 61%. Anal. Calcd. for C H N O (172.18): C, 69.75; H, 4.68;
10
8
2
N, 16.27%. Found: C, 69.92; H, 4.75; N, 16.20%. FT-IR (KBr): 3053, 1581, 1538, 1504, 1480, 1417, 1284,
− 0 0
240, 1157 cm . H-NMR (DMSO-d ), δ (ppm): 6.96–7.01 (m, 2H, H-3 and H-5 ), 7.43 (td, J = 6.9 Hz,
6
1
1
1
1
(
1
(
0
0
.6 Hz, 1H, H-4 ), 8.11 (dd, J = 7.8 Hz, 1.4 Hz, 1H, H-6 ), 8.26 (dd, J = 5.6 Hz, 1.3 Hz, 1H, H-6), 8.90
1
3
d, J = 5.6 Hz, 1H, H-5), 9.25 (d, J = 1.1 Hz, 1H, H-2), 12.80 (s, 1H, OH). C-NMR (DMSO-d ),
δ
(ppm):
6
0
0
0
0
0
17.3 (CH-6), 117.9 (CH-3 ), 118.0 (C-1 ), 119.4 (CH-5 ), 128.4 (CH-6 ), 133.2 (CH-4 ), 156.7 (CH-2), 158.0
CH-5), 159.3 (C-2 ), 162.9 (C-4). N-NMR (DMSO-d ), δ (ppm): 268.1 (N-3), 289.5 (N-1).
0
15
6
3
.3. Typical Procedure for the synthesis of SL mimics
To a solution of a hydroxy derivative or
1.52 g, 11 mmol), followed by the slow addition under stirring at room temperature of the crude
-bromo-3-methyl-5H-furan-2-one ( , 2.21 g, 12.5 mmol). The resulting mixture was continuously
agitated at room temperature for the next 24 h. The reaction mixture was then poured into water
80 mL) and extracted with CH Cl (3 75 mL). The combined extracts were washed with an equal
volume of water and dried over anh.Na SO . The solvent was partly removed under reduced pressure
1,
4
6 (10 mmol) in DMF (15 mL) was added K CO
2 3
(
5
2
(
×
2
2
2
4
and the solid formed was filtered and recrystallized to obtain SL compounds
For the separation of compound the solvent was removed under reduced pressure, the residue was
chromatographed on a SiO -packed column eluting with EtOAc:hexane 25% and finally recrystallized.
3 and 7, respectively.
5
2

Molecules 2017, 22, 961
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3
.4. Product Characterization
◦
-Methyl-5-(2-pyrimidin-4-yl-phenoxy)-5H-furan-2-one (3). Pale yellow crystals, m.p. 215–217 C (from
3
CHCl /MeOH). Yield 39% (1.05 g). Anal. Calcd. for C H N O (268.27): C, 67.16; H, 4.51; N, 10.44%.
3
15 12
2
3
Found: C, 67.29; H, 4.55; N, 10.52%. IR (KBr): 1765, 1726, 1575, 1538, 1477, 1416, 1388, 1242, 1209, 1097,
−
1 1
1
6
017 cm . H-NMR (CDCl ),
δ
(ppm): 1.97 (t, J = 1.4 Hz, 3H, CH ), 6.30 (quintet, J = 1.2 Hz, 1H, H-5),
3
3
0
.89 (quintet, J = 1.5 Hz, 1H, H-4), 7.21 (td, J = 7.3 Hz, 1.0 Hz, 1H, H-5 ), 7.38 (dd, J = 8.1 Hz, 0.5 Hz, 1H,
0
0
00
H-3 ), 7.47 (td, J = 7.4 Hz, 1.7 Hz, 1H, H-4 ), 7.73 (dd, J = 5.3 Hz, 1.4 Hz, 1H, H-6 ), 7.86 (td, J = 7.7 Hz,
0 00 00 13
.6 Hz, 1H, H-6 ), 8.70 (d, J = 5.3 Hz, 1H, H-5 ), 9.25 (d, J = 1.2 Hz, 1H, H-2 ). C-NMR (CDCl ), δ
3
1
(
1
(
0 00 0 0 0
ppm): 10.6 (CH ), 99.4 (CH-5), 116.6 (CH-3 ), 121.8 (CH-6 ), 124.2 (CH-5 ), 127.6 (C-1 ), 131.1 (CH-6 ),
3
0 0 00 00
31.9 (CH-4 ), 134.7 (C-3), 141.9 (CH-4), 154.4 (C-2 ), 156.4 (CH-5 ), 158.6 (CH-2 ), 162.5 (C-4”), 170.9
CO). 15N-NMR (CDCl ), δ (ppm): 281.4 (N-3 ), 290.6 (N-1 ).
00
00
3
3
-Methyl-5-(benzo[de]isoquinoline-1,3-dione-2-yloxy)-5H-furan-2-one (
5
). Beige solid, yield 78%, 2.41 g,
◦
m.p. 236–238 C (from acetone + MeOH). Anal. Calcd. for C H NO (309.28): C, 66.02; H, 3.58; N,
1
1
7
11
5
−
4
.53%. Found: C, 66.16; H, 3.51; N, 4.47%. IR (KBr, νmax): cm 1789, 1726, 1688, 1583, 1355, 1341, 1231,
1
1
(
188, 1097, 1016. H-NMR (CDCl ),
δ
(ppm): 2.02 (s, 3H, CH ), 6.48 (s, 1H, H-5), 7.18 (s, 1H, H-4), 7.79
3
3
0 0 0
t, J = 7.8 Hz, 2H, H-6 ), 8.26 (d, J = 8.1 Hz, 2H, H-7 ), 8.64 (d, J = 7.3 Hz, 2H, H-5 ). C-NMR (CDCl ),
3
13
0
0
0
0
δ
(ppm): 10.8 (CH ), 103.2 (CH-5), 122.5 (2
×
C-4 ), 127.1 (2
×
CH-6 ), 127.5 (C-9 ), 131.8 (C-8 ), 132.1
3
0
0
0
(2 × CH-5 ), 134.9 (2 × CH-7 ), 136.0 (C-3), 139.9 (CH-4), 160.8 (2 × CO-3 ), 170.7 (CO-2).
3
-Methyl-5-(1,4-naphthoquinone-2-yloxy)-5H-furan-2-one (
7
). Beige solid, yield 51%, 1.38 g, m.p.
◦
1
53–155 C (from CHCl /MeOH). Anal. Calcd. for C H O (270.24): C, 66.67; H, 3.73%. Found: C,
3
15 10
5
−
1
6
6.75; H, 3.78%. IR (KBr, νmax): cm 3096, 3049, 1785, 1679, 1657, 1614, 1593, 1349, 1331, 1303, 1266,
1
0
244, 1205, 1091, 1017. H-NMR (CDCl ), δ (ppm): 2.03 (s, 3H, CH ), 6.35 (s, 1H, H-5), 6.63 (s, 1H, H-3 ),
3 3
1
7
8
0 0 0
.12 (s, 1H, H-4), 7.73 (t, J = 7.2 Hz, 1H, H-8 ), 7.77 (t, J = 7.2 Hz, 1H, H-7 ), 8.08 (d, J = 6.8 Hz, 1H, H-6 ),
0
13
0
δ (ppm): 10.7 (CH ), 96.7 (CH-5), 114.6 (CH-3 ), 126.3
3
.12 (d, J = 7.1 Hz, 1H, H-9 ). C-NMR (CDCl ),
3
0 0 0 0 0 0
(
1
CH-6 ), 126.8 (CH-9 ), 130.8 (C-10 ), 131.7 (C-5 ), 133.7 (CH-8 ), 134.5 (CH-7 ), 135.4 (C-3), 141.2 (CH-4),
0 0 0
56.9 (C-2 ), 170.1 (CO-2), 179.3 (CO-1 ), 184.4 (CO-4 ).
3
.5. Biological Activity Tests
3
.5.1. Germination Bioassay
Preparation of Seeds
The biological activity of the new synthetic SL mimics was assayed on P. ramosa, P. aegyptiaca,
O. cumana and S. hermonthica seeds. The seeds of broomrapes and witchweed require pre-
conditioning/warm stratification. Seed pre- conditioning was done under sterile conditions, according
to Matusova et al. [64]. Seeds were briefly surface sterilized, by immersion for 5 min in 2% sodium
hypochlorite, which contained 0.02% (v/v) Tween 20, and then they were repeatedly washed with
sterile water. Seeds were dried on laminar air flow. Around 100 seeds were distributed on 8 mm
(diameter) discs, made from glass fibre filter paper (GFFP). Twelve discs with parasitic weed seeds
were placed on previously prepared 9 cm Petri dishes. These prepared Petri dishes were obtained by
layering 2 sheets of Whatman filter paper and humidifying them with 3 mL of sterile demineralized
water. The Petri dishes with GFFP discs seeds were sealed with Parafilm and incubated. The incubation,
◦
necessary to break the dormancy, was done in darkness at 21 C for 12 days for broomrape seeds and
in darkness at 30 C for 10 days for S. hermonthica seeds [64].
◦
Preparation of Test Solutions
All compounds were dissolved in acetone to make 3 mM stock solutions and diluted with sterile
demineralized water to get appropriate concentrations. All test solutions were prepared just before use.

Molecules 2017, 22, 961
10 of 15
Bioassay
GFFP discs with seeds conditioned, for the above indicated period, were aseptically transferred
to new Petri dishes with moistened Whatman filter paper and dried in an air flow cabinet, to remove
excess humidity as we previously described [64]. The tested synthetic SL mimics
analogue GR24 (racemic mixture, Chiralix B.V., Nijmegen, The Netherlands), at concentrations of 100,
0, 1, 0.1, 0.01 and 0.001 M, were added to three replicate disks, in a volume of 40 L per disk. In each
3, 5, 7 and reference
1
µ
µ
bioassay, demineralized water and aqueous solution of 3% acetone were used as negative controls.
Petri dishes were sealed with Parafilm before incubation. The incubation was done in darkness, at
◦ ◦
5 C for 5 days for broomrape seeds and at 30 C for 2 days for witchweed seeds. The number of
2
germinated and nongeminated seeds was determined by counting them, after magnification on a DV4
stereomicroscope (Carl Zeiss) A parasitic weed seed was considered as germinated when the radicle
was easy to observe, because it clearly protruded the seed coat.
Calculation of Logistic Dose–Response Curves.
The calculation of the logistic dose–response curves allows a quantitative description of the
sensitivity of the seeds to the tested SL mimics as germination stimulants. This calculation was
made with non-linear regression and variable Hill coefficient [64] using GraphPad Prism version
7.02 software (GraphPad Software, La Jolla, CA, USA). Three sensitivity parameters were calculated
and used for data analysis: Rmax, the maximum seeds germination; [GS] , the dose necessary to induce
50
5
0% of maximum seeds germination; p, the interaction (Hill) coefficient. The mathematical expression
relating the response of seeds to the dose concentrations is:
Rmax − R
min
R = Rmin
+
ꢀ
ꢁ
(1)
p
[
GS]
50
1
+
[
GS]
where: Rmin is the germination in the absence of the tested germination stimulants (GS); Rmax is the
maximum germination; [GS] is the dose of the tested SL mimics applied on the experiments; [GS]50 is
the dose giving 50% response [64].
3
.5.2. Test on Plant Pathogenic Fungi
Biological Material
The biological activity of the synthetic SL mimics was assayed on the following strains of
soil-borne plant pathogens, purchased from DSMZ (The Leibniz Institute DSMZ—German Collection
of Microorganisms and Cell Cultures, Braunschweig, Germany): Fusarium graminearum Schwabe
(DSM 4527); Sclerotinia sclerotiorum (Libert) De Bary (DSM 1946); Macrophomina phaseolina (Tassi)
Goid (DSM 62744) and Rhizoctonia solani Kühn (DSM 22842). The fungi were initially cultivated
on potato–dextrose agar (PDA) medium (Difco Laboratories, Becton, Dickinson and Company,
Franklin Lakes, NJ, USA). The PDA medium was distributed in 90 mm Petri dishes, inoculated
◦
with fungi and incubated for 4 days at 25 C.
Preparation of Test Solutions
The stock solution of synthetic SL mimics 3, 5 and 7 were prepared by dissolving 5 mg in 1 mL
acetone. From this stock solution, the working solutions were prepared by dilution with acetone.
The resulting synthetic SLs working solutions were further dissolved into sterile, warm and liquid (60
◦
C) water agar 1.8% (Difco Laboratories), 0.5 mL synthetic SLs solution into 99.5 mL warm and liquid
water agar 1.8%. Final concentrations of the SL mimics in agar were: 3, 15, 30, 45, 60, 75 × 10−6 M.

Molecules 2017, 22, 961
11 of 15
Bioassay
Influence of the new synthetic SL mimics on growth pattern of plant pathogenic fungi was tested
according to Dor et al. [22]. Briefly, 4 mL warm water agar 1.8%, containing new synthetic compounds
were spread on a 47 mm Petri dishes (EMD Millipore, Billerica, MA, USA). The Petri dishes were
cooled. At the centre of each 47 mm Petri dish, with water-agar and various concentrations of the
tested new compounds, it was placed a small piece of mycelium-agar, taken from the edge of each of
◦
the tested fungal plant pathogens colony, developed for 4 days at 25 C on PDA. The inoculated Petri
◦
dishes were incubated at 25 C for 3 days. After 3 days, the developed fungal colonies were observed.
The diameters were measured and the resulting areas of the fungal colonies were calculated. The Petri
dishes were examined on an inverted microscope (Axiovert 40 CFL, Carl Zeiss). On each primary
branch, there were determined the number of hyphal branches, total and branches of different orders,
from second till fifth, on a total length of 1000 µm. As negative controls there were used water agar
and water agar with 0.5% (v/v) of the solvent used for SL analogue and mimics solutions (acetone).
As positive control reference, synthetic SL analogue GR24 was used, in similar final concentrations of
, 15, 30, 45, 60, 75 × 10− M.
6
3
Statistical Analysis
The obtained data were statistically processed by analysis of variance (JMP® v13.0, SAS
Institute Inc., Cary, NC, USA). Least-significant differences (LSD) were calculated and compared
with the differences between the means, based on Tukey–Kramer method (p < 0.05). Each experiment
was repeated three times. Each experiment was done in six replicates. Data presented are the average
of the three experiments.
3
.6. Chemical Stability Tests
The tests for chemical stability of the newly synthesized SL mimics were done in aqueous
solutions, with a final concentration of 50
followed. Stock solution (1 mg/mL) of the tested SL analogues and mimics were prepared in acetone.
From this stock solution, 20 L were distributed into HPLC filter vials through 0.2 m nylon filters
Thomson SINGLE StEP Standard Filter Vials, Restek, Bellefonte, PA, USA), which were further diluted
till 400 L, with 70 L ethanol, 10 L indanol solution 1 mg/mL in acetone (indanol being used as
internal standard) and 300
µg/mL. The method described by Boyer et al. [30] was
µ
µ
(
µ
µ
µ
µ
L water. The pH of the resulted SL analogues and mimics aqueous solution
◦
was 6.7. The HPLC filter vials were incubated at 21 C. Each second day, the SL analogue and mimics
aqueous solutions from sampled vials were filtered and supernatants were assayed by UPLC analysis,
on an Acquity UPLC H-Class System (Waters, Milford, MA, USA) equipped with a Photodiode Array
(
PDA) Detector, using an Acquity UPLC HSS C18 column (1.8
µ
m, 2.1
×
50 mm). The elution phase
for the first 30 s was 5% acetonitrile in water with 0.1% formic acid for 0.5 min. For the next 6 min
a gradient from 5% to 100% acetonitrile in water with 0.1% formic acid was applied, followed by
◦
1
00% acetonitrile with 0.1% formic acid for 3 min. The operating temperature was 40 C and the flow
rate was 0.6 mL/min. The quantification of the non-degraded SL analogues and mimics was done
by peaks integration and comparison with the peak of the stable internal standard, indanol. GR24
(
racemic mixture, Stichting Chemiefonds Paddepoel, Malden, The Netherlands) was used as reference
SL analogue.
4
. Conclusions
New SL mimics containing an aromatic or heteroaromatic ring, present in various bioactive
molecules, connected by an ether link to a furan-2-one moiety were easily synthesized and completely
characterized. These affordable compounds were investigated for their activity as active ingredients
for plant protection products on plant pathogenic fungi and parasitic weed seeds. The new compounds

Molecules 2017, 22, 961
12 of 15
induced germination of seeds of Orobanche, Phelipanche and Striga spp. and proved biological activities
comparable with GR 24 analogue on plant pathogenic fungi.
Supplementary Materials: Supplementary materials are available online. The H- and 13C-NMR spectra for
1
compounds 3, 5, 7 are available as supplementary material.
Acknowledgments: This work was supported by: a grant of the Romanian National Authority for Scientific
Research, CNCS—UEFISCDI, project PN-II-PT-PCCA-2013-4-0846, Contract 159/2014; Scientific Grant Agency
of the Ministry of Education of Slovak Republic and the Slovak Academy of Sciences (VEGA 2/0138/17);
COST Action FA1206 (STREAM, “STRigolactones Enhances Agricultural Methodologies”), supported by COST
(
European Cooperation in Science and Technology). Access to research infrastructure developed in the
“
Petru Poni” Institute of Macromolecular Chemistry through the European Social Fund for Regional Development,
Competitiveness Operational Programme Axis 1, Project InoMatPol (ID P_36_570, Contract 142/10.10.2016, cod
MySMIS: 107464) is gratefully acknowledged.
Author Contributions: E.G. and F.G. conceived and performed the synthesis of strigolactone analogues; A.N.
and C.D. conceived and performed NMR experiments; R.M. conceived, performed and analyzed the experiments
on the germination stimulant activity on parasitic weed seeds; F.O. conceived and designed the experiments
on inhibitor activity on the development of pathogenic fungi; I.R. and M.-L.J. performed the experiments on
inhibitor activity on the development of pathogenic fungi; M.-C.V. and L.V. contributed reagents, analysis tools
and performed chemical characterization of compounds. All authors wrote parts of paper and corrected the
final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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