Design of antibacterial agents: Alkyl dihydroxybenzoates against xanthomonas citri subsp. citri

Article abstract of DOI:10.3390/ijms19103050

Xanthomonas citri subsp. citri (Xcc) causes citrus canker, affecting sweet orange-producing areas around the world. The current chemical treatment available for this disease is based on cupric compounds. For this reason, the objective of this study was to design antibacterial agents. In order to do this, we analyzed the anti-Xcc activity of 36 alkyl dihydroxybenzoates and we found 14 active compounds. Among them, three esters with the lowest minimum inhibitory concentration values were selected; compounds 4 (52 μM), 16 (80 μM) and 28 (88 μM). Our study demonstrated that alkyl dihydroxybenzoates cause a delay in the exponential phase. The permeability capacity of alkyl dihydroxybenzoates in a quarter of MIC was compared to nisin (positive control). Compound 28 was the most effective (93.8), compared to compound 16 (41.3) and compound 4 (13.9) by percentage values. Finally, all three compounds showed inhibition of FtsZ GTPase activity, and promoted changes in protofilaments, leading to depolymerization, which prevents bacterial cell division. In conclusion, heptyl dihydroxybenzoates (compounds 4, 16 and 28) are promising anti-Xcc agents which may serve as an alternative for the control of citrus canker.

Full text of DOI:10.3390/ijms19103050

International Journal of
Molecular Sciences
Article
Design of Antibacterial Agents: Alkyl
Dihydroxybenzoates against Xanthomonas citri
subsp. citri
Ana Carolina Nazaré 1,
* ,
, Carlos Roberto Polaquini 1 , Lúcia Bonci Cavalca 2,3
Daiane Bertholin Anselmo 1, Marilia de Freitas Calmon Saiki 4 , Diego Alves Monteiro 4,
Aleksandra Zielinska 3, Paula Rahal 4, Eleni Gomes 4, Dirk-Jan Scheffers 3
Henrique Ferreira 2 and Luis Octavio Regasini 1,
,
*
1
Department of Chemistry and Environmental Sciences, Institute of Biosciences, Humanities and Exact
Sciences, São Paulo State University, São José do Rio Preto, SP 15054-000, Brazil;
carlos_polaquini@hotmail.com (C.R.P.); daiane_bertholin@hotmail.com (D.B.A.)
Department of Biochemistry and Microbiology, Biosciences Institute, São Paulo State University,
Rio Claro, SP 13506-900, Brazil; l.bonci@hotmail.com (L.B.C.); henrique.ferreira@unesp.br (H.F.)
Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute,
University of Groningen, 9747 AG Groningen, The Netherlands; a.u.zielinska@rug.nl (A.Z.);
d.j.scheffers@rug.nl (D.-J.S.)
Department of Biology, Institute of Biosciences, Humanities and Exact Sciences, São Paulo State University,
IBILCE, São José do Rio Preto, SP 15054-000, Brazil; macal131@gmail.com (M.d.F.C.S.);
diego8monteiro@hotmail.com (D.A.M.); prahal@ibilce.unesp.br (P.R.); eleni@ibilce.unesp.br (E.G.)
Correspondence: acarolnazare@gmail.com (A.C.N.); luis.regasini@unesp.br (L.O.R.);
Tel.: +55-17-3221-2362 (L.O.R.)
2
3
4
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 24 August 2018; Accepted: 1 October 2018; Published: 6 October 2018
Abstract: Xanthomonas citri subsp. citri (Xcc) causes citrus canker, affecting sweet orange-producing
areas around the world. The current chemical treatment available for this disease is based on cupric
compounds. For this reason, the objective of this study was to design antibacterial agents. In order to
do this, we analyzed the anti-Xcc activity of 36 alkyl dihydroxybenzoates and we found 14 active
compounds. Among them, three esters with the lowest minimum inhibitory concentration values
were selected; compounds
4 (52 µM), 16 (80 µM) and 28 (88 µM). Our study demonstrated that
alkyl dihydroxybenzoates cause a delay in the exponential phase. The permeability capacity of alkyl
dihydroxybenzoates in a quarter of MIC was compared to nisin (positive control). Compound 28
was the most effective (93.8), compared to compound 16 (41.3) and compound
4 (13.9) by percentage
values. Finally, all three compounds showed inhibition of FtsZ GTPase activity, and promoted changes
in protofilaments, leading to depolymerization, which prevents bacterial cell division. In conclusion,
heptyl dihydroxybenzoates (compounds 4, 16 and 28) are promising anti-Xcc agents which may serve
as an alternative for the control of citrus canker.
Keywords: citrus canker; antimicrobial; plant disease; membrane disruption; phenolic acids
1. Introduction
Citrus canker is caused by Xanthomonas citri subsp. citri (Xcc), which is responsible for a severe
form of the disease (Asian citrus canker) [1–3]. Typical symptoms of this plant disease are brownish
eruptive lesions on their aerial parts, which may be surrounded by chlorotic halos. Bacteria infect
leaves, stems, and fruit, which leads to defoliation, twig dieback and early fruit drop. As a consequence,
the disease brings significant economic losses to the citrus sector [3–6].
Int. J. Mol. Sci. 2018, 19, 3050; doi:10.3390/ijms19103050
www.mdpi.com/journal/ijms

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These phytopathogens enter the plant through stomata and injuries caused during crop
management or by the citrus leaf miner (Phyllocnistis citrella). They colonize the intercellular space
in the apoplast and break down the leaf epidermis as a result of cell hyperplasia [
wind and movement of infected seedlings moving are the causes for this bacterial dissemination [
6
]. Rainfall,
].
7
Under current legislation, the state of São Paulo in Brazil was has been declared a Risk Mitigation
System (RMS) area [8]. This is a control that aims to reduce citrus canker inoculum potential.
The chemical control of citrus canker is carried out with copper-based antibacterials [
Copper applications reduce the intensity of the disease and the number of inoculations into the plant,
because bacterial survival is reduced [10 16]. Although copper sprays reduce crop loss and help to
control citrus canker, they may also negatively affect the environment [17]. Copper may accumulate in
9–11].
–
the soil, and can cause adverse effects on root growth and on nutrient uptake by citrus trees [18
–22].
In addition, the emergence of copper-resistant bacterial strains is a reality [23,24].
Phenolic compounds have been evaluated as an alternative to current citrus canker treatment.
In our previous studies, potent antibacterial activities of a homologous series of alkyl gallates
(3,4,5-trihydroxybenzoates) [25] were described. The antibacterial mechanism of these compounds
involves action on the membrane and they promote cell death by a combination of mechanisms:
FtsZ inhibition, membrane permeability, and possibly another activity [26]. In order to improve
the anti-Xcc activity of alkyl gallates, four monoacetylated gallates were synthesized and tested
against Xcc [27]. As expected, the insertion of the acetyl group on the alkyl gallates conferred an
increase of lipophilicity on these compounds, and made them more potent than the pattern phenolic
compounds [25,27]. Altogether, these data suggest that modifications in hydroxyl groups may result
in increased anti-Xcc potency.
Therefore, our research group has investigated dihydroxybenzoic acids and their alkyl esters as
antimicrobials against Gram-positive and Gram-negative bacteria and yeasts [28–30]. Besides their
antimicrobial activity, we selected these compounds in order to investigate the relevance of the number
and position of the hydroxyls to anti-Xcc activity. According to Nihei, Nihei and Kubo, in order to
elicit activity as the hydrophilic ‘head’ portion, two hydroxyl groups are essential [29].
In our continuing search for anti-Xcc agents from phenolic natural products-based compounds, we
synthesized three series of alkyl esters from
activity of these esters was evaluated against X. citri subsp. citri strain 306 (IBSBF 1594) to derive
preliminary structure-activity relationships. The three most active compounds ( 16 and 28) were
β-resorcilic, gentisic and protocatechuic acids. The anti-Xcc
4,
investigated regarding their toxicity against eukaryotic cell lines and their effects on the growth of Xcc.
In addition, their ability to act on membrane and cell division by inhibition of the GTPase activity of
FtsZ were carried out.
2. Results
2.1. Synthesis, Purification and Identification of Alkyl Dihydroxybenzoates
The new antibacterial agents were synthesized in three different series with 12 alkyl
dihydroxybenzoates each: series I (
1
–
12), series II (13
–
24) and series III (25
–36). Each series
had carboxylic acids as precursors; they were the 2,4 dihydroxybenzoic (or
β-resorcilic) acid (I),
the 2,5 dihydroxybenzoic (or gentisic) acid (II) and the 3,4 dihydroxybenzoic (or protocatechuic)
acid (III). Different purification methods were used in order to obtain the pure compounds;
low temperature hexane washes, hexane and acetone crystallization techniques, and normal phase
column chromatography. The purity degree analysis was carried out with high-performance
liquid chromatography (HPLC), and was measured by integrating the peaks of the chromatograms.
The values varied from 76.5% to 99.9% (Table 1), thus validating our methodologies for purifying and
obtaining the compounds. The alkyl dihydroxybenzoates structures were determined by 1H and 13
NMR analyses. Spectra and chromatograms are presented as supplementary material.
C

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Table 1. Antibacterial activities against Xanthomonas citri subsp. citri, purity and log Po/w of
dihydroxybenzoates 1–36.
−1
MIC µg mL
MBC µg
mL
Entry
R
Purity
log Po/w
−1
(MIC in µM)
2,4 DHB acid
H
85.7
87.6
85.6
81.7
83.8
79.6
99.0
99.5
>99.9
2.6
2.8
3.9
5.0
5.1
6.1
6.2
6.9
7.0
>100 (>648)
>100 (>548)
39.8 (189)
20.0 (84)
13.2 (52)
>100 (>375)
>100 (>356)
>100 (>340)
>100 (>310)
>100
>100
50
50
25
>100
>100
>100
>100
1
2
3
4
5
6
7
8
CH2CH3
(CH2)3CH3
(CH2)5CH3
(CH2)6CH3
(CH2)7CH3
(CH2)8CH3
(CH2)9CH3
(CH2)11CH3
Series I
OH
O
R
O
HO
9
93.2
2.6
>100 (>510)
>100
10
>99.9
2.2
>100 (>450)
>100
11
>99.9
4.0
40.5 (171)
100
2,5 DHB acid
H
97.3
98.3
82.0
80.1
99.3
79.8
>99.9
76.5
>99.9
2.8
2.4
3.4
4.5
5.0
5.2
5.7
6.3
6.8
>100 (>648)
>100 (>549)
>100 (>475)
33.5 (140)
>100
>100
>100
50
13
14
15
16
17
18
19
20
CH2CH3
(CH2)3CH3
(CH2)5CH3
(CH2)6CH3
(CH2)7CH3
(CH2)8CH3
(CH2)9CH3
(CH2)11CH3
20.2 (80)
25
Series II
>100 (>375)
>100 (>357)
>100 (>340)
>100 (>310)
>100
>100
>100
>100
OH
O
R
O
21
>99.9
2.2
>100 (>510)
>100
OH
22
>99.9
3.0
83.5 (375)
100
23
24
>99.9
>99.9
3.5
3.1
90.4 (382)
59.7 (231)
100
100
3,4 DHB acid
H
99.6
99.1
99.1
99.1
98.5
98.5
97.9
91.3
>99.9
0.8
1.7
2.6
3.7
4.3
4.9
5.5
6.0
6.9
>100 (>648)
>100 (>549)
>100 (>475)
44.4 (186)
22.2 (88)
31.2 (117)
26.7 (95)
>100 (>340)
>100 (>310)
>100
>100
>100
50
50
50
50
100
>100
25
26
27
28
29
30
31
32
CH2CH3
(CH2)3CH3
(CH2)5CH3
(CH2)6CH3
(CH2)7CH3
(CH2)8CH3
(CH2)9CH3
(CH2)11CH3
Series III
O
HO
HO
R
O
33
>99.9
1.4
>100 (>510)
>100
34
>99.9
2.2
>100 (>450)
>100
35
36
>99.9
>99.9
2.7
2.4
>100 (>423)
89.1 (345)
>100
100
copper
oxychloride
Kanamycin
43.1
-
-
19.9 (40)
MIC—minimal inhibitory concentration; MBC—minimal bactericidal concentration.

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2.2. Partition Coefficient Study
The partition coefficients were measured by HPLC method according to OECD 117 – Guidelines
for the Testing of Chemicals [31] to all compounds. The log Po/w values of the alkyl dihydroxybenzoates
and carboxyl acids were ranged from 0.8 to 7.0 (Table 1).
2.3. Determination of MIC and MBC against Xanthomonas citri subsp. citri Assay
The antibacterial potential of carboxylic acids and their esters were compared to copper
oxychloride (a commercial active often used to combat citrus canker). Minimal inhibitory concentration
(MIC) values were determined by REMA assay, which measures cellular respiratory activity.
We assayed the carboxylic acids (2,4 dihydroxybenzoic acid, 2,5 dihydroxybenzoic acid and 3,4
dihydroxybenzoic acid) which demonstrated they were inactive since they did not inhibit Xcc growth
(MIC > 100 µ
g mL−1). However, 14 esters showed potential anti-Xcc activity, with MIC values lower
than 100 µg mL−1 (2–4, 11, 15, 16, 22–24, 27–30 and 36).
The minimal bactericidal concentration (MBC) was determined by observing the bacterial growth
in Petri dishes transferred from the REMA plates. We observed bactericidal action for compounds 11
22 24 31 and 36 at 100 15 27 30 at 50 and 16
g mL−1, compounds g mL−1 and compounds
at 25 µg mL−1
,
–
,
µ
2
,
3
,
,
–
µ
4
.
2.4. Cytotoxicity Assay
The cytotoxicity of the three most potent compounds (
4, 16 and 28) was evaluated using the
MTT test. This test is based on the reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-tetrazole
bromide by viable cells, with formation of violet insoluble formazan crystals. Five cell lines were used
in order to evaluate the cytotoxic effect of the compounds as a whole.
Thus, it can be seen in Table 2 that compounds
4, 16 and 28 had low cytotoxic potential because
they showed IC50 values above 100 M for MRC-5 (human lung fibroblast cell line), VERO (monkey
µ
kidney epithelial cell line) and ACHN (human renal adenocarcinoma cell line). In contrast, HaCaT
(human keratinocyte cell line) and Huh-7.5 (human hepatoma cell line) were more sensitive to selected
compounds, with IC50 values in the range of 70
µM, with the exception of compound 16 (20.60
µM) for
Huh-7.5. The kanamycin (control) and copper oxychloride showed IC50 values above 200
µg mL−1, respectively.
µM and 200
Table 2. IC50 value of compounds 4, 16 and 28 in µM against cell lines.
Compounds
HaCaT
Huh-7,5
MRC-5
VERO
ACHN
4.59 b,c,h
111.4 ± 8.34 c,f,h
93.55 ± 1.93 f
92.31 ± 9.34 f,g
>200 i
152.9 ± 7.07 d
140.35 ± 7.56 b,d
114.05 ± 9.97 g
>200 i
4
16
28
64.86 ± 8.91 a
69.81 ± 4.87 a
74.02 ± 11.74 a,g
>200 i
70.63 ± 5.52 a
20.60 ± 0.23 e
64.84 ± 14.91 a
>200 i
127.45
±
144.8 ± 6.29 b,d
128.7 ± 2.12 b,c,h
>200 i
Kanamycin
copper oxychloride
>200 i
>200 i
>200 i
>200 i
>200 i
Different letters indicate statistical significance p < 0.05 in Tukey’s multiple comparisons test.
2.5. Growth Curve
According to the growth curves of the positive control (PC) and vehicle control (DMSO), it was
observed that Xcc reaches the maximum growth phase in a period of 15 h. The growth rates of
Xcc under the effects of dihydroxybenzoic acids (DA)
4
,
16 and 28 were measured at concentrations
1
equivalent to MIC and MIC. The early growth inhibition (lag phase), which happens in the first hours
2
of contact (period of the first cellular divisions) can be a dynamic process. The lag phase delay was
observed in the three compounds tested, and it showed that the higher the concentration, the higher
the death rate. This can be explained because the compounds have bacteriostatic action; that is, as the
time passes, the cells become able to recover and restart doubling. Another important finding is that
the lag phase consists in the cell adaptation to the new medium conditions and the bacterial population

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is smaller in relation to the exponential phase facilitates the action of the compounds in the first hours
(Figure 1) [32]. Observing the exposure of Xcc to compounds 16 and 28 it can be seen that there was a
delay in the exponential phase, prolonging the time taken for the bacteria to reach the stationary phase.
4,
B
1.5
A
1.5
Control
DMSO
Control
DMSO
4 MIC
4 1/2MIC
16 MIC
16 1/2MIC
1.0
0.5
0.0
1.0
0.5
0.0
0
12
24
36
48
60
72
0
12
24
36
48
60
72
Time (h)
Time (h)
D
C
1.5
1.0
0.5
0.0
1.5
Control
DMSO
CO MIC
CO 1/2MIC
Control
DMSO
28 MIC
1.0
0.5
0.0
28 1/2MIC
0
12
24
36
48
60
72
0
12
24
36
48
60
72
Time (h)
Time (h)
1
Figure 1. Growth curve profiles of Xanthomonas citri subsp. citri with treatments in MIC and MIC
2
values of alkyl dihydroxybenzoates for 72 h.
A Compound 4, B compound 16, C compound 28, and D
copper oxychloride (CO) in comparison to control (untreated) and DMSO (vehicle control).
When we evaluated the concentration-response behavior of the compounds, we obtained a
1
decrease in the curves with the concentration of the MIC. Compared to the PC and DMSO, it was more
2
evident in the MIC curve, which showed that for all three compounds, the higher the concentration,
the greater the influence on Xcc.
2.6. Cellular Membrane Permeability of Xcc Using the Live/Dead Kit
Membrane integrity was measured by using the commercial assay Live/Dead BacLight® bacterial
viability kit (Invitrogen, Carlsbad, CA, USA). We observed cells with intact membrane in fluorescent
blue (SYTO 9) and cells with disturbance or affected membrane integrity in red or pink (propidium
iodide). The data analyses were plotted as the percentage of cells permeated by propidium iodide
(Figure 2).
The nisin was used as a positive control for causing pores in the membrane [26]. When we
1
observed the permeability capacity of alkyl dihydroxybenzoates in MIC and compared these to nisin
4
(79.47%), we noticed that compound 28 was the most effective, because its permeation ability reached
93.8%, while for compound 16 it was 41.3%, and for compound 4, 13.9%.
Permeation percentages were obtained by fluorescence optical microscopy (Figure 3). Compound
28 showed high permeability, evidencing the entry of propidium iodide (PI) into cells. This behavior
is similar to that of the positive control (nisin). For this reason, the target of compound 28 is
membrane- related.

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****
100
80
60
40
20
0
****
****
ns
4
ns
NC DMSO nisin
16
28
Figure 2. Cellular permeability evaluation in the presence of alkyl dihydroxybenzoates (**** p < 0.0001,
ANOVA with Dunnett’s post-test), ns = not significant.
A
B
C
D
E
F
1
Figure 3. Optical microscopy of fluorescence images at MIC of alkyl dihydroxybenzoates for
4
15 min. (A) Negative control (untreated), (B) vehicle control (DMSO), (C) positive control (nisin),
(D) compound 4, (E) compound 16, and (F) compound 28.

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2.7. Inhibition Assays of GTPase Activity
The cell division process is mediated by the formation of the Z ring, coordinated by GTP hydrolysis
activity of FtsZ [33 35]. Because of this, we evaluated the inhibition of GTPase activity of purified
–
FtsZ from Xcc, incubated with compounds
assay [36].
4
,
16 and 28 using the continuous substrate regeneration
When evaluating the behavior of compounds 4,
16 and 28, we could see decay in the GTPase
activity of FtsZ. These data suggest that all three compounds promoted changes in protofilaments,
leading to depolymerization, which reduces bacterial cell division.
3. Discussion
Xanthomonas is one of the most important genera among the phytopathogens. It causes diseases in
economically important cultivars such as rice, beans, tomatoes, oranges, peppers, passion fruit, grapes
and others [5].
In this study, we have focused our work on Xcc, which is the main cause of citrus canker.
The chemical control of citrus canker is mainly through application of cupric compounds, but the
emergence of copper-resistant strains has encouraged research into new compounds [37–39].
Recently, other effective compounds against Xcc have been reported, such as bismerthiazol [40
]
and 3-indolylacetonitrile [41], which are promising agents against citrus canker.
The 2,4 DHB, 2,5 DHB and 3,4 DHB acids were inactive (MIC > 100
µ
g mL−1). Then,
we evaluated three ester homologous series containing ethyl, butyl, hexyl, octyl, decyl and dodecyl
dihydroxybenzoates. In general, hexyl and octyl dihydroxybenzoates displayed potent activity against
Xcc. Thus, we decided to evaluate heptyl and nonyl esters. Heptyl dihydroxybenzoates 4, 16 and 28
exhibited the lowest MIC values. Subsequently the branched, cyclic and aromatic esters were evaluated
to analyze the hypothetical effects of stereoelectronic parameters on the anti-Xcc effect. However,
clear conclusions about the relationship between structure and activity were not established. Copper
oxychloride showed anti-Xcc activity, with the MIC value of 43.1 µg mL−1
.
In our previous study, we described the anti-Xcc activity of alkyl gallates (alkyl 3,4,5-
trihydroxybenzoates). In order to investigate the relevance of number and position of hydroxyl groups
on anti-Xcc activity, alkyl dihydroxybenzoates were evaluated. Heptyl dihydroxybenzoates (
16—80 M and 28—88 M) were more active than heptyl gallate (MIC = 116 M) [25]. This set of data
indicated that reduction in the number of hydroxyls increased anti-Xcc activity. Comparisons among
MIC values of compounds 16 and 28 suggested the hydroxyls at positions 2 and 4 (compound 4)
4—52 µM,
µ
µ
µ
4
,
increased anti-Xcc activity when compared to positions 2 and 5 (compound 16), as well as 3 and 4
(compound 28).
This was corroborated using other data regarding the dihydroxybenzoic acids (DA) and their esters
that had antimicrobial activity described, against Gram-positive and Gram-negative bacteria [42–48]
and yeasts [29]. Some DAs, such as 2,3; 2,4; 2,5; 2,6 and 3,4 show activity against Campylobacter jejuni,
Escherichia coli, Listeria monocytogenes and Salmonella enterica [30].
Merkl and collaborators studied the antimicrobial activity of the phenolic acids and their alkyl
esters, against E. coli, Bacillus cereus, L. monocytogenes, Saccharomyces cerevisiae and Fusarium culmorum
and reported significant differences between values of MIC of acids and their esters. In general,
butyl esters were twice as potent as their acids. In addition to this, the authors associated the increase
of antibacterial activity with the size of the side alkyl chain [28].
With the increase of the alkyl chain, a considerable decrease in solubility occurs in aqueous
medium (culture medium). This favors the formation of micelles, which are not able to cross bacterial
cells. The amphiphilic character of the alkyl dihydroxybenzoates allows the micellar formation due to
the hydrophilicity of the hydroxyl groups on the phenolic ring, and the long carbonic chain is highly
hydrophobic [49].
The compound with the highest lipophilicity was the dodecyl 2,4 dihydroxybenzoate (
8), and the
compound with the lowest lipophilicity was the 3,4 dihydroxybenzoic acid (3,4-DHB acid). As the

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side chain increased, the lipophilicity of these compounds increased; that is, the interaction of the
compounds with the C18 column was more intense, culminating in longer retention times, being
slightly different for the branched and cyclic compounds. Compound
(meta) positions, compound 16 has two hydroxyls at the 2.5 (para), and compound 28 has another
two at the 3,4 (ortho) positions. The log Po/w values of 5.1 (compound ), 5.0 (compound 16) and 4.3
4 has two hydroxyls at the 2,4
4
(compound 28) obtained through the partition coefficient are relatively close, with compound
4
being
more lipophilic.
Branched compounds
9 (2.6), 21 (2.2) and 33 (1.4) presented smaller than their isomers 2
(3.9), 14
(3.4) and 26 (2.6). This is due to the fact that branching reduces the size of the molecule, decreasing the
surface area and making it difficult to solvate in the non-polar phase [50], similar to cyclic compounds.
In this work, we demonstrated that heptyl dihydroxybenzoates had no cytotoxicity. Our results
corroborate those of Medina-Alarcón and colleagues (2017), who reported that protocatechuates did
not show cytotoxicity in MRC-5 and HepG2 cell lines [51].
When we observed the growth curves treated with the compounds, we noticed a delay in the
exponential phase, which prolonged the time taken for the bacterium to reach the stationary phase.
This delay is more evident when increasing the concentration of the compounds; the greater the
concentration, the greater the influence on Xcc. The ester interactions with Xcc showed different
curves, which is an interesting fact, since the esters have the same side carbonic chain size. Therefore,
this made us consider the influence of hydroxyl positions on kinetics growth. Concerning the growth
curve, it was observed that, compared to the PC and the DMSO, Xcc bacterial growth suffered a delay
of approximately 18 h to reach the stationary phase. However, when comparing the compounds, 16
demonstrated a greater inhibition of bacterial growth, decreasing the height of the curve significantly;
that is, with a lower optical density. Therefore, it can be inferred that its action on Xcc is greater than
the action of compound 4. The profile of compound 28 showed increased bacterial growth delay of
20 to 30 h, and even beyond this time it continued to inhibit the growth of Xcc more effectively than
compound 4, but with less killing capacity than compound 16 (Figure 1).
When we evaluated copper oxychloride, we found an MIC value of 43.1 µ
g mL−1 to potential
anti-Xcc activity on cell lines tested that did not demonstrate cytotoxicity. On the growth curves
treated with the copper oxychloride, we noticed that the curve pattern did not change and the copper
oxychloride was inactive under the conditions tested. Behlau and colleagues (2017), described copper
oxychloride as an insoluble copper formulation (ICuF) (syn. fixed copper), and being the most studied
and commonly used form of copper for the control of citrus canker [12
activity of copper oxychloride in our trials.
,17], this could explain the low
The minimal bactericidal concentration was determined by observing the bacterial growth in Petri
dishes transferred from the REMA plates. We obtained bactericidal action in compounds 11 22–24,
,
31 and 36 at the concentration of 100
µ
g mL−1, in compounds
2
,
3
,
15
,
27
–
30 at the concentration of
50 µg mL−1 and in compounds 4 and 16 at the concentration of 25 µg mL−1
.
Finally, we investigated the alkyl dihydroxybenzoates for interference in the modulation of FtsZ
cell-mediated Xcc cell division [52]. FtsZ is strongly related to -tubulin in three-dimensional
structure [53], the possession of GTPase activity [35 54] and the ability to polymerize in a
nucleotide-dependent manner in vitro 34 55]. The continuous substrate regeneration assay was
performed to characterize GTPase activity similar to bacterial self-assembling tubulin, FtsZ [56].
The compounds 16 and 28 showed decay in the GTPase activity of FtsZ. These data suggest that all
α/β
,
[
,
4,
three compounds promoted changes in protofilaments, leading to depolymerization, which prevents
bacterial cell division.
All compounds affected membrane integrity (Figure 2), but only compound 28 showed
high permeability similar to nisin. Therefore, similar to alkyl gallates [26], compounds 4, 16
and 28 possibly promote cell death by some combination of the FtsZ inhibition and membrane
permeabilization mechanisms.

Int. J. Mol. Sci. 2018, 19, 3050
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In summary, three series of alkyl dihydroxybenzoates were designed and synthesized as part
of our ongoing search for anti-Xcc agents based on natural phenolic compounds. The heptyl
dihydroxybenzoates (compounds 4, 16 and 28) displayed potent anti-Xcc activity. Furthermore,
due to preliminary low toxicity against eukaryotic cells, as well as the ability to act on membrane and
cell division, these compounds are promising agents against citrus canker.
4. Methods
4.1. Synthesis of Alkyl Dihydroxybenzoates
The esters of dihydroxybenzoic acids were prepared by Fischer’s Esterification (Scheme 1)
methodology with modifications [25,57]. 6 mmol of carboxylic acids were dissolved in 18 mmol of
alkyl alcohols (ethanol, 1-butanol, 1-hexanol, 1-heptanol, 1-octanol, 1-nonanol, 1-decanol, 1-dodecanol,
2-propanol, cyclopentanol, cyclohexanol and 2-phenylethanol) under constant magnetic stirring.
After complete dissolution, catalytic amounts of sulfuric acid (1–3 drops) were added. The reactions
were maintained at temperatures close to boiling point for the respective alcohols and were conducted
for a period of 1–3 days. The progress of the reaction was checked by thin layer chromatography
(TLC) [7:3 n-hexane: ethyl acetate]. After the conversion, the residue was partitioned 3 times with
ethyl acetate. Later, it was washed with distilled water (3 times) and 1 mol L−1 sodium bicarbonate
solution (3 times). The organic phase was dried using a rotary evaporator. The crude products
were purified with a low temperature hexane washes, crystallized with hexane or acetone and over
normal phase column chromatography (silica gel 0.06–0.20 mm, ACROS Organics, New Jersey, USA).
All compounds were identified with the use of NMR spectra on a Bruker Avance III 14.1 T, Bruker
Avance III 9.4 T and Bruker Fourier 7.1 T spectrometer, using deuterated chloroform (CDCl3) or
deuterated dimethylsulfoxide (DMSO-d6) as solvents. Compounds were analyzed by HPLC for
determination of their purity.
R1
O
R1
O
R1 = R3 = OH; R2 = R4 = H (I)
R1 = R4 =OH; R2 = R3 = H (II)
R1 = R4 =H; R2 = R3 = OH (III)
Alkyl dihydroxybenzoates
R2
R3
R2
R3
R
H2SO4
O
R-OH
HO
+
R4
R4
R =
Series I (2,4 dihydroxybenzoic acid), II (2,5 dihydroxybenzoic acid), III (3,4 dihydroxybenzoic
acid).
Scheme 1. Preparation of alkyl dihydroxybenzoates by Fischer’s Esterification.
4.2. Partition Coefficient Determination Log Po/w by HPLC
The log Po/w were determined using the HPLC procedure according to OECD Guidelines for the
Testing of Chemicals [31,58] on a system equipped with a binary pump, automatic sampler column
(Agilent® Technologies, Santa Clara, CA, USA, model 1220 Infinity), and photodiode array system
Zorbax Eclipse Plus C-18 column (4.6
×
250 mm, 5
µ
m, Agilent® Technologies, Santa Clara, CA, USA).
−1
The analyses were performed with the use of an isocratic flow in methanol: water (3:1) at 1.0 mL min
We injected 20.0
phenol, acetophenone, benzoic acid, benzene, toluene, biphenyl, fluoranthene and triphenylamine
.
µL and analyses were performed at 254 nm. We used as reference compounds thiourea,

Int. J. Mol. Sci. 2018, 19, 3050
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in order to construct the curve log K
was determined from their retention times and interpolation in linearity curve log K
We performed the analyses in duplicate.
×
log Po/w. The lipophilicity of alkyl dihydroxybenzoates
×
log Po/w.
4.3. Determination of MIC and MBC against Xanthomonas citri subsp. citri Assay
The antibacterial activity of alkyl dihydroxybenzoates and the determination of minimum
inhibition concentration (MIC) were performed by the fluorimetric method Resazurin Microtiter
Assay (REMA) with some modifications [25
(IBSBF 1594) [60] was grown in 20 mL in Nitrogen Yeast Glycerol (NYG) at 29 C in constant agitation
of 180–200 rpm. After 12 h, the Xcc inoculum was dispersed in NYG medium and distributed into
a 96 well microtiter plate to a final volume of 100
tested compounds at 10 mg mL−1 were prepared in dimethyl sulfoxide (DMSO) and dispersed in
NYG medium to obtain 1% DMSO and the final compound concentration range of 0.78–100
g mL−1
The negative control was constituted of 100 L culture medium. In addition, the copper oxychloride
was tested as a comparison to alkyl dihydroxybenzoates and kanamycin (20
g mL−1), which was
used as a control antibiotic. The plate was subsequently incubated at 29 ◦C for 12 h, and 15 g mL−1
,
59]. An overnight culture of X. cit◦ri subsp. citri strain 306
µ
L per well (105 CFU per well). Stock solutions of
µ
.
µ
µ
µ
freshly prepared resazurin solution in water was added to each well, and the plate was incubated for
2 h. The fluorescence was determined on a Synergy H1 (BioTek, Winooski, VT, USA) microplate reader
using the excitation and emission filters at wavelengths of 530 and 590 nm respectively. After the
REMA assay, the minimum bactericidal concentration (CBM) assay was performed using a 96-pin
stainless steel plate replicator, accurately transferring the wells from the REMA plate to Petri dishes
◦
Petri dishes were incubated in oven B.O.D. at 29 C for 24 to 48 h [61]. The experiments were performed
in triplicate.
4.4. Determination of Cytotoxicity
The cytotoxicity of compounds 4, 16, 28 and copper oxychloride was determined by MTT
assay [62]. The cell lines used were HaCaT (human keratinocyte cell line—CLS 300493), Huh-7.5
(human hepatoma cell line—ATCC® PTA-8561), MRC-5 (human lung fibroblast cell line—ATCC
CCL-171), VERO (monkey kidney epithelial cell line—ATCC® CCL81) and ACHN (human renal
adenocarcinoma cell line—ATCC® CRL1611). The cells were cultured in appropriate medium
recommended by the American Type Culture Collection.
The assay was performed with ◦the concentration of 104 cells for both cell lines, distributed in
96-well plates and incubated at 37 C in a 5% CO2 controlled atmosphere oven for 24 h. After
the incubation period, the cells were treated with the compounds with a range of 1.56–200
L−1 and incubated for 24 h. The supernatant was removed, then 100
L of 3-(4,5-dimethyl-2-
thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) salt at 1 mg mL−1 diluted in DMEM medium
Gibco Life Technologies, Gaithersburg, MD, USA) was added and incubated for 30 min. Subsequently,
the MTT was removed, then 100 L of DMSO under agitation for 5 min at 60 rpm was added.
µmol
µ
(
µ
The plates were read at 570 nm in a FLUOstar Omega/BMG LABTECH plate reader, Offenburg,
BW, DE. We carried out the experiments in triplicate and three independent events for statistical
analysis (GraphPad Software, San Diego, CA, USA). The MTT assay is based on the tetrazolium salt
(yellow-colored) reduction to formazan (purple-colored) by metabolically active cells [63].
4.5. Growth Curve
An isolated colony of Xanthomonas citri subsp. citri was inoculated in NYG medium for 12 h at
29 C. After this, the optical density (OD 600 nm) was measured and adjusted to ~0.1. Positive controls
◦
(cells and culture medium) and cells with treatments of compounds
4
,
16 and 28 were then prepared
1
in MIC and MIC values. Optical density reading was measured on a Synergy H1-BioTek plate reader
2
every 30 min for 72 h. The growth curves were plotted with GraphPad Prism software. At the same
time, the degradation assay was performed, solubilizing the compounds in DMSO, which were later

Int. J. Mol. Sci. 2018, 19, 3050
11 of 15
incorporated in NYG medium. Later, we scanned the compounds to measure the absorbance values
from 210 to 700 nm, and the maximum lambda value of each compound was selected after scanning.
After that, punctual readings were carried out every 30 min for 72 h.
4.6. Cellular Membrane Permeability of Xcc Using the Live/Dead Kit
Phase contrast microscopy was used to observe changes in cell shape and signs of cell division
alteration using an objective lens with a magnification of 100 . To evaluate the Xcc cells’ membrane
×
integrity, the BacLight® kit was used. Mixtures of SYTO 9 dyes and propidium iodide (PI). SYTO 9
(blue) can stain nucleic acids in general—with intact or damaged membranes. On the other hand,
PI (red) penetrates only the bacteria with damaged membranes, causing overlap in the fluorescence
of SYTO 9. Therefore, using a mixture of the dyes, the bacteria with disturbed cell membranes are
stained in fluorescent red and the intact cell membranes are stained in fluorescent blue. The Xcc cells at
1
−1
density of 106 cells mL were submitted to 15 min treatments with compounds
4,
16 and 28 at MIC
4
values. Cellular immobilization was performed on slides containing 1% agarose gel in 0.85% saline
buffer. The slides were prepared with 1.0 mL of the agarose solution, which was dispersed in optical
microscopy slides and on each slide another slide was placed so that the gel continued like a thin film
and was of homogeneous height. The slides were dried at room temperature for approximately 20 min
and opened at the time of use, when 10 µL of the cell suspension was deposited in the center and
covered with the cover slip. The cells were visualized using the Olympus BX-61 optical microscope
equipped with OrcaFlash 2.8 monochrome camera (Hamamatsu, Shizuoka, Japan). Cell Sensation
software v11 (Olympus) was used to document and analyze the images.
4.7. Inhibition Assays of GTPase Activity
The evaluation of the inhibition of GTPase activity was measured by the characterization of the
FtsZ from Xcc [36,57]. This methodology is known as continuous assay of substrate regeneration
and consists of the hydrolysis of guanosine triphosphate (GTP) in guanosine diphosphate (GDP) and
inorganic phosphate. The GTP is regenerated by the enzyme pyruvate kinase which, in turn, converts
the phosphoenol pyruvate (PEP) to pyruvate. The pyruvate produced by this reaction is reduced to
lactate by lactate dehydrogenase, using NADH [64].
Reagent stocks were dispersed in Tris 50 buffer (1:1 tris:KCl at pH 7.9). We also prepared
two mixtures: Mixture 1—magnesium chloride (MgCl2), pyruvate kinase/lactate dehydrogenase,
phosphoenol-pyruvate, NADH and GTP, adding or not the test compounds to MIC values; and mixture
2—Xcc-FtsZ. The assay was performed in a 96-well plate, 75
µ
L of mixture 1 was pipetted into a well
◦
and incubated for 5 min at 30 C; then 75
µL of mixture 2 was added and the readings started. A Biotek
Synergy MX plate reader was used. The readings were performed every 5 s, at the wavelength of
340 nm at 30 ◦C, under constant stirring.
The total volume of 150 µL presented the following concentrations: 5 mM MgCl2; pyruvate
kinase/lactate dehydrogenase 20 U mL−1; 2 mM phosphoenol-pyruvate; 1 mM NADH; 400
µM GTP;
Xcc-FtsZ 5 µM.
4.8. Statistical Analysis
All assays were performed in triplicate. The data were analyzed using one-way analysis of
variance (ANOVA) followed by Tukey’s test for Table 2, and Dunnett’s test for Figures 2 and 4.
The data analyses were plotted with GraphPad Prism version 5.0 (GraphPad, San Diego, CA, USA).

Int. J. Mol. Sci. 2018, 19, 3050
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100
80
60
40
20
0
*
**
****
DMSO
4
16
28
Figure 4. Inhibition of GTPase activity of alkyl dihydroxybenzoates in MIC value (* p < 0.04, ** p < 0.001
,
**** p < 0.0001, ANOVA with Dunnett’s post-test).
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/19/10/
3050/s1.
Author Contributions: L.O.R., H.F., D.-J.S., E.G. and P.R. conceived and designed the experiments; A.C.N., C.R.P.,
L.B.C., D.B.A., M.d.F.C.S., D.A.M. and A.Z. performed the experiments; all authors contributed to data analysis.
Funding: This research was funded by Coordination for the Improvement of Higher Education Personnel
(CAPES), Brazilian Council for Scientific and Technological Development (CNPq) (Grants 471129/2013-5 and
306251/2016-7), the São Paulo Research Foundation (FAPESP) (Grants 2013/50367-8, 2014/18330-0, 2015/50162-2
and 2017/50216-0) and “Biobased Economy” from the Netherlands Organisation for Scientific research (NWO,
729.004.005) to D.J.S.
Acknowledgments: A.C.N. thanks FAPESP for research fellowship (2015/08089-6), C.R.P. thanks the National
Institute of Science and Genomics Technology for Citrus Improvement (Citrus-INCT) research fellowship (Grants
FAPESP 2014/50880-0 and 465440/2014-2). The authors gratefully acknowledge the Center for Biomolecular
Innovation (Grants FAPESP 2009/53989-4).
Conflicts of Interest: The authors declare no conflict of interest.
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