Natural Abenquines and Their Synthetic Analogues Exert Algicidal Activity against Bloom-Forming Cyanobacteria

Article abstract of DOI:10.1021/acs.jnatprod.6b00629

Abenquines are natural quinones, produced by some Streptomycetes, showing the ability to inhibit cyanobacterial growth in the 1 to 100 μM range. To further elucidate their biological significance, the synthesis of several analogues (4f-h, 5a-h) allowed us to identify some steric and electronic requirements for bioactivity. Replacing the acetyl by a benzoyl group in the quinone core and also changing the amino acid moiety with ethylpyrimidinyl or ethylpyrrolidinyl groups resulted in analogues 25-fold more potent than the natural abenquines. The two most effective analogues inhibited the proliferation of five cyanobacterial strains tested, with IC₅₀ values ranging from 0.3 to 3 μM. These compounds may be useful leads for the development of an effective strategy for the control of cyanobacterial blooms.

Full text of DOI:10.1021/acs.jnatprod.6b00629

Article
pubs.acs.org/jnp
Natural Abenquines and Their Synthetic Analogues Exert Algicidal
Activity against Bloom-Forming Cyanobacteria
†
,†,‡
‡
́
́
Celia Regina Alvares Maltha,
Amalyn Nain-Perez, Luiz Clau
́
dio Almeida Barbosa,*
,§
and Giuseppe Forlani*
†
Department of Chemistry, Universidade Federal de Minas Gerais, Avenida Pres. Anto
1270-901, Belo Horizonte, MG, Brazil
̂
nio Carlos, 6627, Campus Pampulha, CEP
3
‡
Department of Chemistry, Federal University of Vico
̧
sa, 36570-000, Vico
Department of Life Science and Biotechnology, University of Ferrara, Via L. Borsari 46, I-44121 Ferrara, Italy
S Supporting Information
̧ sa, MG, Brazil
§
*
ABSTRACT: Abenquines are natural quinones, produced by some Streptomycetes, showing the ability to inhibit cyanobacterial
growth in the 1 to 100 μM range. To further elucidate their biological significance, the synthesis of several analogues (4f−h, 5a−
h) allowed us to identify some steric and electronic requirements for bioactivity. Replacing the acetyl by a benzoyl group in the
quinone core and also changing the amino acid moiety with ethylpyrimidinyl or ethylpyrrolidinyl groups resulted in analogues
2
5-fold more potent than the natural abenquines. The two most effective analogues inhibited the proliferation of five
cyanobacterial strains tested, with IC50 values ranging from 0.3 to 3 μM. These compounds may be useful leads for the
development of an effective strategy for the control of cyanobacterial blooms.
7
yanobacteria, also referred to as blue-green algae, are the
climate changes, the development of efficient methods for
C
only prokaryotes able to perform oxygenic photosyn-
thesis. Widely distributed even in strikingly different and harsh
habitats, they play a major role in both carbon and nitrogen
bloom control is urgently required.
1
8
9
With this aim, physical and chemical strategies have been
exploited. However, since the former are too costly to be
applied on a large scale and the latter can cause further
pollution, a biological approach to the problem would be a
global cycling, since several free-living strains can accomplish
biological N fixation. In freshwater bodies and coastal waters
under favorable conditions, e.g., warm temperature, sunlight,
and increased N and P availability, some cyanobacteria are
capable of blooming, rapidly multiplying and forming a dense
surface scum. This often leads to the establishment of anoxic
conditions, altering the ecological structure and contributing to
the ecosystem’s decline. Moreover, with the only exception of
the genus Spirulina/Arthrospira, cyanobacteria produce a variety
of potentially harmful toxins, targeting either the liver, the
gastrointestinal apparatus, or the nervous system. The release
of high concentrations of toxins in cyanobacterial blooms
causes concern for public safety and human health, deteriorates
freshwater quality affecting drinking water supplies, and
threatens aquaculture and fisheries, potentially causing serious
2
2
10
possibly better choice. Under natural conditions, cyanobacte-
rial proliferation seems to be limited by some algicidal bacteria
that are believed to contribute to the control and termination of
3
11
blooms. Many bacteria showing inhibitory properties in vitro
against harmful cyanobacteria have been recently isolated
whose activity was shown to rely on production and release of
4
12−16
active substances.
Despite the large number of organisms
5
with algicidal activity, the development of effective biological
strategies for bloom control requires isolation, characterization,
and inexpensive chemical synthesis of these active com-
17
pounds.
6
economic damage. Since the frequency of cyanobacterial
Received: July 7, 2016
blooms is increasing as a consequence of both pollution and
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.6b00629
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
During the last decades the exploitation of microbial
products and their synthetic analogues has proved a promising
source of new bioactive compounds for agriculture, medicine,
effective compounds was evaluated against various cyanobacte-
rial strains, some heterotrophic microorganisms, and plants at
either the seedling or the undifferentiated tissue level, pointing
out a selectivity for photosynthetic bacteria.
18−21
and the food industry.
Within the plethora of active
principles identified so far, quinones are an important class of
22−25
naturally occurring and synthetic compounds
with a
RESULTS AND DISCUSSION
■
variety of biological properties, such as antibacterial, antifungal,
Synthesis of Natural Abenquines and Some Ana-
logues. Abenquines (4a−d) and their analogues (4e−h; 5a−
h) were prepared as shown in Scheme 1.
2
6−28
and antitumor activity.
Owing to their electron transfer
2
9
properties, they also have the ability to interfere with the
photosynthetic electron transport chain, resulting in phytotoxic
In order to explore the ability of abenquines to inhibit
cyanobacterial growth, a number of potentially active
compounds were desired. Two parts of the molecule were
modified, replacing either of the substituents in the core of the
benzoquinone group (Figure 2). Initially the amino acid moiety
was substituted with different amines, maintaining the
acetamide group as part of the abenquine scaffold. The
preparation of analogues (4e−h) involved the reaction of an
3
0−32
effects.
Among natural quinones are the abenquines, a group of
benzoquinones bearing both an N-acetyl and an amino acid
moiety (Figure 1). These compounds were recently isolated
34,40
equivalent amount of the different amines and quinone 3a.
Further modification on the abenquine structure involved
replacement of the acetyl moiety by the bulkier group benzoyl
(
Figure 2). To obtain the analogues 5a−h, the required
intermediate quinone 3b was prepared employing the same
chemical procedure used for the synthesis of 3a. Initially N-
(
2,5-dimethoxyphenyl)benzamide (2b) was prepared in
Figure 1. Structure of naturally occurring abenquines.
quantitative yield by reaction of commercial 2,5-dimethoxyani-
line (1) and benzoyl chloride. Further oxidation of 2b with
phenyl iodine diacetate (PIDA) afforded the benzoquinone 3b
(Scheme 1). The benzoyl analogues of natural abenquines
from the fermentation broth of a Streptomyces sp. found in the
33
Chilean highland of the Atacama Desert and were synthesized
34
for the first time by our group. Abenquines were shown to
(
5a−d) were then synthesized by reacting 3b with different
possess only mild inhibitory properties against bacteria,
dermatophytic fungi, and phosphodiesterase type 4b.
3
3
amino acids. The amino analogues (5e−h) were synthesized in
the same way by reaction of 3b with different amines.
The structures of compounds 4a−h and 5a−h were
confirmed by extensive spectroscopic analysis. In the IR
spectra, the typical signals corresponding to stretch vibration
34
However, abenquines A, B2, and D in the concentration
range of 10−6 to 10−4 M were found to inhibit the growth of a
model cyanobacterial strain, Synechococcus elongatus PCC
3
4
6
301.
In continuation of our interest in using natural products as a
model for the development of new synthetic bioactive
+
−1
of the NH3 group in the region νmax = 3554−2794 cm was
evident in the case of compounds bearing amino acid groups
(4a−d and 5a−d), whereas for the analogues carrying amine
groups (4e−h and 5e−h) the stretching vibration of N−H in
3
5−39
compounds,
we further investigated the potential of
abenquines as algicides. With this aim, variations were
introduced in their scaffold by replacing either the amino acid
substituent with different amines or adding a bulky group
through the substitution of the acetyl for a benzoyl group
−1
41
1
the range ν = 3379−3238 cm was observed. In the H
̅
NMR spectra, all acetamide compounds (4a−h) showed a
signal as a singlet around δ = 2.06−2.22, corresponding to the
methyl in the acetamide moiety. The signals of the aromatic
hydrogen atoms corresponding to the benzoyl group in
compounds 5a−h were observed in the range δ = 7.50−7.90.
In addition, all signals corresponding to residues of amines or
amino acids in the high shielded region were confirmed in the
(
Figure 2). Here we report that, with the exception of
abenquine C, both natural and synthetic abenquines were able
to inhibit cyanobacterial growth, with some derivatives showing
a 25-fold increased effectiveness. The activity of the two most
13
C NMR spectra. Moreover, the carbonyls of the amide and
quinone were detected at δ = 165.83−171.62 and δ = 178.01−
84.48, respectively. The assignments were possible by means
1
of 2D NMR techniques when required, and the data were fully
consistent with the proposed structures (see Supporting
1
13
Information for selected H and C NMR spectra and
spectroscopic data).
Both Natural and Synthetic Abequines Are Able to
Inhibit Cyanobacterial Growth (Table 1). When added at
micromolar concentrations to the culture medium, abenquines
A (4a), B2 (4b), and D (4d) inhibited the growth of the
cyanobacterium Synechococcus elongatus PCC 6301 (IC ≈ 10
5
0
μM). Abenquine C (4c) was less effective, causing a 40%
inhibition at 100 μM (Figure S1).
Analogues 5a−d, in which the acetyl moiety had been
replaced with a benzoyl group, showed similar activity.
However, compound 5c, an analogue of the poorly active
Figure 2. Variations introduced in the abenquine scaffold. Analogues
were obtained by either replacing the amino acid substituent with
different amines in the core of the benzoquinone group or adding a
bulky group through the substitution of the acetamide moiety with a
benzamide group.
34
B
DOI: 10.1021/acs.jnatprod.6b00629
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Journal of Natural Products
Article
Scheme 1. Synthesis of Natural Abenquines (4a−d) and Their Analogues (4e−h, 5a−h)
abenquine C (4c), was also inhibitory. Analogues containing a
benzoyl group were slightly less active than their acetyl
counterparts when bearing aromatic amino acids (5a, 5d), but
the activity increased or did not change when an aliphatic
amino acid was present (5b, 5c). Compound 5e, with an
aromatic group in both sides, presented an IC50 > 100 μM,
whereas its acetyl analogue (4e) was more active (IC50 = 2.0
μM).
Table 1. Concentrations of Natural and Synthetic
Abenquines Able to Inhibit by 50% (IC ) the Growth of the
5
0
Model Cyanobacterial Strain Synechococcus elongatus PCC
6301
b
compound
IC50 (μM)
compound
IC50 (μM)
a
4
4
4
4
4
4
4
4
a
9.6 ± 2.2
13.8 ± 1.9
>100
5a
5b
5c
5d
5e
5f
27.7 ± 3.0
13.5 ± 1.0
25.9 ± 1.8
26.2 ± 1.6
>100
a
b
a
a
c
Interestingly, when the amino acid moieties were replaced
with some amino groups, the biological activity of abenquines
was significantly enhanced. In particular, the presence of groups
such as ethylpyrimidinyl and ethylpyrrolidinyl (as in com-
pounds 4f−g) caused a 10-fold increase of the inhibitory
potential (Figure S1), with IC values lower than 1 μM (Table
d
11.7 ± 2.6
2.0 ± 0.2
0.8 ± 0.1
0.6 ± 0.1
a
e
f
<0.50
g
h
5g
5h
<0.50
3.8 ± 0.6
3.1 ± 0.2
5
0
a
b
Data quoted from ref 34. The IC values in μg/mL are presented in
1
). The more aliphatic benzamide analogues 5f−g showed a
50
Table S2.
higher effectiveness, causing almost complete suppression of
cyanobacterial growth at 1 μM (Figure S1).
42
The Most Effective Abenquine Analogues Show
Selective Toxicity for Photosynthetic Bacteria. Com-
pounds 5f and 5g were evaluated against a wide range of
cyanobacteria. Data, summarized in Table 2, showed that all
strains were highly susceptible to these quinones, with IC50
values ranging from 0.3 to 3.0 μM. Interestingly, the three
unicellular, non-nitrogen-fixing strains belonging to the order
Chroococcales were significantly more sensitive than the two
filamentous, nitrogen-fixing strains of the order Nostocales. The
growth of Microcystis aeruginosa, which is believed to be the
most dangerous toxin-producing species, was inhibited at
concentrations about 1 order of magnitude lower than those
found effective in the case of some natural inhibitors recently
described, comprising two algicidal diketopiperazines produced
15
by a Chryseobacterium sp., the active 3-methylindole isolated
16
from an algicidal strain of Aeromonas sp., and the pyrazine-
1
4
1,4-diones purified from an algicidal Bacillus sp.
29
Due to their electron transfer properties, the toxicity of
these abenquines could depend on their ability to interfere with
the photosynthetic electron transport chain, as previously
C
DOI: 10.1021/acs.jnatprod.6b00629
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Journal of Natural Products
Article
Table 2. Concentrations of 5f and 5g Able to Inhibit by 50%
the Growth of Various Organisms
abenquines 4a−d and their analogues 4e−h and 5a−h (Table
3).
5
f
5g
Table 3. Physicochemical Properties of Natural Abenquines
and Their Analogues
a
species
Nostoc sp. PCC 7524-1
IC50 (μM)
IC50 (μM)
2.4 ± 0.1
3.1 ± 0.3
0.8 ± 0.1
0.4 ± 0.0
0.4 ± 0.0
2.6 ± 0.2
1.7 ± 0.2
0.4 ± 0.0
0.4 ± 0.0
0.3 ± 0.0
>100
H-
Anabaena sp. PCC 7120
compound
mol weight
328.32
log P
H-acceptors
donors nRotb
Microcystis aeruginosa PCC 7941
Synechocystis sp. PCC 6803
4
4
4
4
4
4
4
4
5
5
5
5
5
a
b
c
d
e
f
−0.26
−0.44
−0.95
0.20
7
3
3
6
6
294.31
280.28
367.36
270.29
277.32
291.35
285.30
390.39
356.38
342.35
429.43
332.36
339.39
353.42
355.39
7
Synechococcus elongatus PCC 6301
Oryza sativa L., seedlings
7
3
5
>100
8
4
6
Oryza sativa L., cultured cells
>100
>100
0.89
5
2
4
Brassica napus L., seedlings
>100
>100
−0.08
0.43
6
2
5
Brassica napus L., cultured cells
Escherichia coli, mineral medium
Escherichia coli, organic medium
Lactobacillus casei, mineral medium
Lactobacillus casei, organic medium
Saccharomyces cerevisiae, mineral medium
Saccharomyces cerevisiae, organic medium
nonmalignant mouse fibroblasts (NIH 3T3)
>100
>100
g
h
a
b
c
d
e
6
2
5
13.4 ± 0.7
>100
22.7 ± 1.2
>100
0.12
6
2
5
1.41
7
3
7
62.7 ± 8.9
>100
83.1 ± 12.2
>100
1.23
7
3
7
0.73
7
3
6
>100
>100
1.87
8
4
7
>100
>100
2.56
5
2
5
2.0
2.1
a
5f
1.59
6
2
6
The IC50 values in μg/mL are presented in Table S3.
5
5
g
2.10
6
2
6
h
1.04
7
2
6
30−32
shown for other quinones.
If so, plants and eukaryotic
Tice’s rule ≥150 and ≤500 ≤3.5
≥2 and ≤12
≤3
≤12
algae would be most likely susceptible to their activity,
representing a drawback for the use in the environment to
control cyanobacterial blooms. Thus, the effects of 5f and 5g on
the growth of rice (a monocotyledon) and rapeseed (a
dicotyledonous species) were evaluated under either photo-
autotrophic (seedlings) or heterotrophic (suspension cultured
cells) conditions. The results showed a negligible effect at 3 and
In general, the molecular mass should range between 150
43
and 500. Increasing molecular mass has frequently been
observed to correlate with lower solubility and poorer
penetration through membranes. In Tice’s rule, the logarithm
of the octanol/water partition coefficient (log P) should be
between 1.2 and 2.4; log P values for compounds 4e−h are
lower than 1.0, but those of compounds 5f and 5g fall in the
suitable range. All the compounds present suitable numbers of
hydrogen-bond acceptors and rotatable bonds (Table 3), and
most abenquines and their analogues have a suitable number of
hydrogen-bond donors. The most effective compounds, 5f and
5g, present molecular structure descriptors and lipophilicity
values that seem on the whole compatible with a potential use
as pesticides.
1
0 μM for seedling and cultured cells, respectively. Concen-
−
7
−6
trations in the range from 10 to 3 × 10 M, levels at which
the growth of cyanobacteria is progressively reduced, did not
exert any phytotoxic effect.
Compounds 5f and 5g were evaluated against Escherichia coli,
Lactobacillus casei, and Saccharomyces cerevisiae. Data showed
that the growth of the eukaryotic microorganism in a mineral
medium was substantially unaffected up to 100 μM, whereas
that of E. coli was strongly inhibited in the 1 to 100 μM range,
with IC50 values of 13 and 23 μM for 5f and 5g, respectively.
The growth of L. casei was also affected, although to a lesser
extent (Table 2). As often observed in the case of antimetabolic
compounds, when the experiment was repeated growing the
bacteria in organic media, the sensitivity was much lower, with
only a slight growth inhibition at 100 μM.
CONCLUSIONS
■
The replacement in the quinone core of the acetyl with a
benzoyl moiety and of the amino acid with an ethylpyrimidinyl
or an ethylpyrrolidinyl group increased the inhibitory potential
of natural abenquines up to 25-fold, and the most active
analogues exerted their effects in the 0.1 to 1 μM range.
Therefore, these substances appear as promising tools toward
the control of cyanobacterial blooms.
Moreover, preliminary data showed a significant cytotoxicity
of some abenquines against nonmalignant NIH 3T3 mouse
fibroblasts (Table 2). However, in the case of the most active
compounds 5f and 5g a significant selectivity index was evident
(
SI = 5.5 and 7.0, respectively, Table S1) between the effect on
EXPERIMENTAL SECTION
■
Synechococcus elongatus PCC 6301 and that on mouse cells.
The Physicochemical Properties of the Most Effective
Abenquines Comply with Tice’s Rule-of-Five. To be
developed as a new pesticide, a bioactive compound should
General Experimental Procedures. All reagents were purchased
from Sigma-Aldrich (Milwaukee, WI, USA) and were used without any
purification; solvents were acquired from Fluka (Rio de Janeiro,
Brazil). Analytical thin layer chromatography (TLC) was performed
on silica gel 60 F254 0.2 mm thick plates (Merck). Flash column
chromatography was performed using silica gel 230−400 mesh. All
43−45
have some specific properties.
Pesticides must be absorbed
by pests, be transported to the target site, and finally interact
with the molecular target. All these steps may be greatly
influenced by solubility, lipophilicity, stability, and the acid−
base character of a compound. To help characterize the
suitability of a substance as a potential pesticide, a set of rules
based on key molecular structure descriptors and lipophilicity
1
13
compounds were fully characterized by EIMS, H NMR, and
C
NMR spectroscopy. Mass spectra were recorded on a Finnigan MAT
LCQ 7000 or Sciex API2000 under electrospray ionization (ESI).
Elemental analyses were measured on a Foss-Heraeus Vario EL unit.
Infrared spectra were recorded on a PerkinElmer Paragon 1000 FTIR
spectrophotometer, preparing the samples as potassium bromide disks
4
3
1
13
have been defined. These parameters were thus calculated for
(1% w/w). The H and C NMR spectra were recorded on a Varian
D
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1
Mercury 300 spectrometer at 300 and 75 MHz or on a Bruker Avance
at 400 and 100 MHz. Chemicals shifts (δ) are reported from
tetramethylsilane with the solvent resonance as the internal standard.
Synthesis. Compounds 2a, 3a, and 4a−e were synthesized using
about 1.0 mg L− chlorophyll. Aliquots (2 μL) of suitable dilutions of a
given compound in DMSO were added so as to obtain final
concentrations ranging from 0.1 to 100 μM. A complete randomized
design with four replications was adopted. Cell growth in each well
was followed for 1 week by daily determination of absorbance using a
Ledetect 96 plate reader (Labexim, Lengau, Austria) equipped with an
LED plugin at 660 nm. Following logarithmic transformation of data,
growth constants were calculated and expressed as a percent of the
mean value for controls (no less than 8 replications) treated with the
same volume of DMSO. Mean values ± SE over replicates are
reported. The concentrations causing 50% inhibition (IC50) and their
confidence limits were estimated by nonlinear regression analysis using
Prism 6 for Windows, version 6.03 (GraphPad Software).
34
previously published methods. For the synthesis of analogues 4f−h,
a round-bottom flask was charged with 2-acetamido-1,4-benzoquinone
(3a) (192 mg, 1.1 mmol) and dichloromethane (5 mL) at room
temperature. To this solution was added the appropriate amine (1.1
mmol), and the resultant reaction mixture was stirred for 2 h, followed
by removal of the solvent under reduced pressure. The obtained
residue was purified by silica gel column chromatography (silica gel,
Hex/EtOAc, 1:1 v/v, and EtOAc/MeOH, 1:0.5 v/v). The physical and
spectroscopic data for all compounds are in the Supporting
Information.
Bacteria and Yeast Cultures. E. coli strain BL21(DE3)pLysS was
grown at 37 °C in either standard Luria broth or Davis and Mingioli
medium. The effect of abenquines on its growth was evaluated as
described for cyanobacteria, except that the inoculum was made to an
initial density of about 0.1 Abs (600 nm), and growth was followed for
6 h by determining at 30 min intervals the increase in absorbance using
the plate reader equipped with an LED plugin at 600 nm. L. casei strain
DSM 20011 was grown at 35 °C in either optimum point medium or
in basal minimal medium. Cultures were inoculated to an initial
density of about 0.2 Abs (600 nm), and growth was followed for 10 h
at 1 h intervals, as indicated. Baker’s yeast Ura3 strain S23344C was
grown at 30 °C in either standard yeast carbon base (without amino
acids and with 1.0 g L ammonium sulfate as the nitrogen source and
50 mg L uracil) or yeast extract peptone dextrose medium. Growth
was followed for 10 h by determining every 60 min the absorbance at
600 nm.
Plant Cell Cultures and Seedlings. Rice (Oryza sativa L. cv
Gigante Vercelli) and rapeseed (Brassica napus L., cv. Zeruca)
N-(2,5-Dimethoxyphenyl)benzamide (2b). To a round-bottomed
flask at 0 °C, under magnetic stirring, were added 2,5-dimethoxyaniline
(1) (2.0 g, 13.1 mmol), trimethylamine (2.1 mL, 15.2 mmol), and
dichloromethane (50 mL). To this solution was added benzoyl
chloride (1.8 mL, 15.2 mmol), and the temperature was increased to
2
5 °C. After 2 h the mixture was quenched by addition of water. The
48
organic phase was separated and further washed with saturated
49
aqueous NaHCO solution and brine. After that it was dried over
3
MgSO , filtered, and concentrated under reduced pressure to give the
4
46
−
compound as a brown solid. The residue was purified by column
chromatography on silica gel, eluting with ethyl acetate/hexane (1:3 v/
v), to give the title compound. The physical and spectroscopic data
−1
1
13
−1
and H and C spectra for compound 2b are in the Supporting
Information.
2-Benzamido-1,4-benzoquinone (3b). To a round-bottomed flask
were added (2,5-dimethoxyphenyl)benzamide (2b) (1.0 g, 3.9 mmol),
water (25 mL), and MeOH (625 μL). To the suspension was added
phenyliodine diacetate (1.9 g, 5.9 mmol), and the resultant mixture
was stirred for 1.5 h, before it was diluted with water (50 mL) and
extracted with dichloromethane (3 × 50 mL). The combined extracts
were washed with water and saturated aqueous sodium hydrogen
carbonate solution and dried over Na SO . Then, the solution was
50,51
seedlings and suspension-cultured cells were grown as described.
To measure the effect of abenquines on culture growth, cell samples
were withdrawn from the stock cultures in the early stationary growth
phase and used to inoculate 100 mL culture flasks containing 25 mL of
fresh medium with or without the test compound, to a density of
about 1.0 mg mL (dry weight). After 8 days of incubation, when
untreated controls reached the late exponential phase of growth, cells
were harvested by vacuum filtration, and the dry weight increase was
determined on each sample after drying in an oven at 90 °C for 48 h.
At least four replicates were carried out for each treatment. Data were
expressed as percent values of untreated controls. In the case of
seedling growth, seeds were sown in Magenta GA7 vessels containing
2
4
−1
filtered and the solvent was concentrated under reduced pressure. The
crude product was used without further purification.
For the preparation of analogues 5a−d, to a round-bottomed flask
were added the required amino acid (0.31 mmol) and sodium
bicarbonate (52 mg, 0.62 mmol), dissolved in 5 mL of EtOH. Then, 2-
benzamido-1,4-benzoquinone (3b) (70 mg, 0.31 mmol) was added,
and the mixture was stirred at reflux for 2 h. Then the reaction mixture
was filtered to remove the sodium bicarbonate salt and washed with
ethanol. Therefore, the resulting residual mixture was acidified to pH
5
0 mL of agarized (6‰) half-strength MS medium containing or not
the test compound, 16 seeds per vessel. A randomized complete block
design with three replications was used, each block consisting of 5
vessels of 4 abenquine rates and untreated controls. Destructive
harvest was carried out 14 to 16 days after sowing, when controls had
reached the three-leaf stage, and each seedling was weighed
individually (40 ≤ n ≤ 48, depending on germination rate).
4
−5 with 1 M hydrochloric acid. The mixture was extracted with
EtOAc. The organic extracts were combined, the resulting organic
phase was dried over Na SO4 and filtered, and the solvent was
2
concentrated under reduced pressure. The obtained residue was
purified by silica gel column chromatography eluting at first with Hex/
EtOAc, 1:1 v/v, and then with acetone/methanol, 1:1 v/v.
Analogues 5e−h were synthesized by combination of 2-benzamido-
ASSOCIATED CONTENT
■
1
,4-benzoquinone (3b) (150 mg, 0.6 mmol) in dichloromethane (5
*
S
Supporting Information
mL) and the appropriate amine (0.6 mmol) in a round-bottom flask, at
room temperature with stirring for 2 h. The reaction mixture was
concentrated under reduced pressure, and the obtained residue was
purified by column chromatography (silica gel, Hex/EtOAc, 3:1 v/v).
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.6b00629.
1
13
The physical and spectroscopic data and H and C spectra for all
compounds are in the Supporting Information.
Spectroscopic data and H and 13C NMR spectra for
synthetic abenquines and their effects on model
cyanobacterial strain and plant species (PDF)
1
Cyanobacterial Cultures. Cyanobacterial strains, as listed in
Table 2, were grown in Bg11 mineral medium as previously
4
7
described. Growth was followed by measuring chlorophyll
concentration. Culture aliquots (0.5−1.0 mL) were withdrawn, and
cells were sedimented by centrifugation for 3 min at 14000g. Pellets
were resuspended with 1.0 mL of methanol, and solubilization was
allowed to proceed for 30 min in the dark, with occasional mixing.
Samples were then centrifuged again, and chlorophyll content in the
supernatant was estimated spectrophotometrically at 663 nm. Late log-
grown cells were sedimented by centrifugation for 5 min at 4000g and
used to inoculate 96-well plates, 0.2 mL per well, to an initial density of
AUTHOR INFORMATION
■
*
3
Corresponding Authors
Phone (L. C. A. Barbosa): +55 31 38993068. Fax: +55 31
8993065. E-mail: lcab@ufmg.br.
*Phone (G. Forlani): +39 0532 455311. Fax: +39 0532 249761.
E-mail: flg@unife.it.
E
DOI: 10.1021/acs.jnatprod.6b00629
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
ORCID
Luiz Claudio Almeida Barbosa: 0000-0002-5395-9608
́
Notes
Article
(28) Thomson, R. H. Naturally Occurring Quinones IV Recent
Advances; Springer Chapman & Hall, 1997.
29) Nohl, H.; Jordan, W.; Youngman, R. J. Adv. Free Radical Biol.
Med. 1986, 2, 211−279.
30) Omura, S.; Iwai, Y.; Takahashi, Y.; Sadakane, N.; Nakagawa, A.;
(
The authors declare no competing financial interest.
(
Oiwa, H.; Hasegawa, Y.; Ikai, T. J. Antibiot. 1980, 33, 1114−1119.
ACKNOWLEDGMENTS
We are grateful to the following Brazilian agencies: Conselho
Nacional de Desenvolvimento Cientifico e Tecnolo
■
(31) Lotina-Hennsen, B.; Achnine, L.; Ruvalcaba, N. M.; Ortiz, A.;
Hernan
1998, 46, 724−730.
32) Nain-Perez, A.; Barbosa, L. C. A.; Picanco
Forlani, G. Chem. Biodiversity 2016, 13, 1008−1017.
33) Schulz, D.; Beese, P.; Ohlendorf, B.; Erhard, A.; Zinecker, H.;
Dorador, C.; Imhoff, J. F. J. Antibiot. 2011, 64, 763−768.
34) Nain-Perez, A.; Barbosa, L. C. A.; Maltha, C. R. A.; Forlani, G.
Tetrahedron Lett. 2016, 57, 1811−1814.
35) Barbosa, L. C. A.; Rocha, M. E.; Teixeira, R. R.; Maltha, C. R. A.;
Forlani, G. J. Agric. Food Chem. 2007, 55, 8562−8569.
(36) Teixeira, R. R.; Barbosa, L. C. A.; Forlani, G.; Pilo−
de Mesquita Carneiro, J. W. J. Agric. Food Chem. 2008, 56, 2321−2329.
37) Barbosa, L. C. A.; Maltha, C. R. A.; Lage, M. R.; Barcelos, R. C.;
Dona, A.; Carneiro, J. W. M.; Forlani, G. J. Agric. Food Chem. 2012, 60,
0555−10563.
38) Demuner, A. J.; Barbosa, L. C. A.; Mendes Miranda, A. C.;
́ ́
dez, J.; Farfan, N.; Aguilar-Martínez, M. J. Agric. Food Chem.
́
́
gico
(
̧ , M. C.; Giberti, S.;
(
(
(
CNPq, grant 400746-2014) for research fellowships
L.C.A.B.); Fundaca de Amparo a Pesquisa de Minas Gerais
FAPEMIG, grant APQ1557-15) for financial support; and
̧
o
̃
̀
(
́
̃
̧ o de Aperfeic o̧ amento de Pessoal de Nivel Superior
Coordenaca
CAPES) for a research fellowship (A.N.P.). We also thank Dr.
(
(
R. Csuk for mass spectra data and elemental analysis and
cytotoxicity activity experiments. Support from the University
of Ferrara (Fondo di Ateneo per la Ricerca 2013) is also
acknowledged. The authors thank Miss V. Delucca for technical
assistance.
(
́
Veloso, D.;
(
̀
1
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DOI: 10.1021/acs.jnatprod.6b00629
J. Nat. Prod. XXXX, XXX, XXX−XXX