The antimicrobial activity of annona emarginata (Schltdl.) H. Rainer and most active isolated compounds against clinically important bacteria

Article abstract of DOI:10.3390/molecules23051187

Annona emarginata (Schltdl.) H. Rainer, commonly known as “arachichú”, “araticú”, “aratigú”, and “yerba mora”, is a plant that grows in Argentina. Infusions and decoctions are used in folk medicine as a gargle against throat pain and for calming toothache; another way to use the plant for these purposes is chewing its leaves. Extracts from bark, flowers, leaves, and fruits from A. emarginata were subjected to antibacterial assays against a panel of Gram (+) and Gram (?) pathogenic bacteria according to Clinical and Laboratory Standards Institute protocols. Extracts from the stem bark and leaves showed moderate activity against the bacteria tested with values between 250–1000 μg/mL. Regarding flower extracts, less polar extracts (hexane, dichloromethane) showed very strong antibacterial activity against methicillin-sensitive Staphylococcus aureus ATCC 25923 and methicillin-resistant S. aureus ATCC 43300 with values between 16–125 μg/mL. Additionally, hexane extract showed activity against Klebsiella pneumoniae (MIC = 250 μg/mL). The global methanolic extract of the fruits (MeOHGEF) was also active against the three strains mentioned above, with MICs values 250–500 μg/mL. Bioassay-guided fractionation of MeOHGEF led to the isolation of a new main compound—(R)-2-(4-methylcyclohex-3-en-1-yl)propan-2-yl (E)-3-(4-hydroxyphenyl)acrylate (1). The structure and relative configurations have been determined by means of 1D and 2D NMR techniques, including COSY, HMQC, HMBC, and NOESY correlations. Compound 1 showed strong antimicrobial activity against all Gram (+) species tested (MICs = 3.12–6.25 μg/mL). In addition, the synthesis and antibacterial activity of some compounds structurally related to compound 1 (including four new compounds) are reported. A SAR study for these compounds was performed based on the results obtained by using molecular calculations.

Full text of DOI:10.3390/molecules23051187

molecules
Article
The Antimicrobial Activity of Annona emarginata
(Schltdl.) H. Rainer and Most Active Isolated
Compounds against Clinically Important Bacteria
Juan G. Dolab 1 ID , Beatriz Lima 2, Ewelina Spaczynska 3, Jiri Kos 4, Natividad H. Cano 2,
Gabriela Feresin 2, Alejandro Tapia 2, Francisco Garibotto 1,5, Elisa Petenatti 1
,
ID
Monica Olivella 1, Robert Musiol 3, Josef Jampilek 4,
*
and Ricardo D. Enriz 1,5,
*
Faculty of Chemistry Biochemistry and Pharmacy, National University of San Luis, Chacabuco 917,
5700 San Luis, Argentina; juandolab@gmail.com (J.G.D.); fgaribotto@gmail.com (F.G.);
elipete@unsl.edu.ar (E.P.); olivellamonica@gmail.com (M.O.)
Institute of Biotechnology-Institute of Basic Sciences, National University of San Juan, Av. Libertador
General San Martin 1109, 5400 San Juan, Argentina; blima@unsj.edu.ar (B.L.);
natividadhc17@hotmail.com (N.H.C.); gferesin@unsj.edu.ar (G.F.); atapia@unsj.edu.ar (A.T.)
Institute of Chemistry, University of Silesia, 75 Pulku Piechoty 1, 41500 Chorzow, Poland;
ewelina.spaczynska@gmail.com (E.S.); robert.musiol@us.edu.pl (R.M.)
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Comenius University, Odbojarov 10,
83232 Bratislava, Slovakia; jirikos85@gmail.com
ID
1
2
3
4
5
Multidisciplinary Institute of Biological Research IMIBIO-CONICET, Chacabuco 917,
5700 San Luis, Argentina
*
Correspondence: josef.jampilek@gmail.com (J.J.); denriz@unsl.edu.ar (R.D.E.); Tel.: +421-250-117-229 (J.J.);
+54-0266-4520300 (R.D.E.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 24 April 2018; Accepted: 10 May 2018; Published: 16 May 2018
Abstract: Annona emarginata (Schltdl.) H. Rainer, commonly known as “arachichú”, “araticú”,
“aratigú”, and “yerba mora”, is a plant that grows in Argentina. Infusions and decoctions are used
in folk medicine as a gargle against throat pain and for calming toothache; another way to use
the plant for these purposes is chewing its leaves. Extracts from bark, flowers, leaves, and fruits
from A. emarginata were subjected to antibacterial assays against a panel of Gram (+) and Gram ( )
−
pathogenic bacteria according to Clinical and Laboratory Standards Institute protocols. Extracts from
the stem bark and leaves showed moderate activity against the bacteria tested with values between
250–1000
very strong antibacterial activity against methicillin-sensitive Staphylococcus aureus ATCC 25923 and
methicillin-resistant S. aureus ATCC 43300 with values between 16–125 g/mL. Additionally, hexane
extract showed activity against Klebsiella pneumoniae (MIC = 250 g/mL). The global methanolic
extract of the fruits (MeOHGEF) was also active against the three strains mentioned above, with MICs
values 250–500 g/mL. Bioassay-guided fractionation of MeOHGEF led to the isolation of a new
main compound—(R)-2-(4-methylcyclohex-3-en-1-yl)propan-2-yl (E)-3-(4-hydroxyphenyl)acrylate
). The structure and relative configurations have been determined by means of 1D and 2D NMR
techniques, including COSY, HMQC, HMBC, and NOESY correlations. Compound 1 showed strong
antimicrobial activity against all Gram (+) species tested (MICs = 3.12–6.25 g/mL). In addition, the
synthesis and antibacterial activity of some compounds structurally related to compound (including
µg/mL. Regarding flower extracts, less polar extracts (hexane, dichloromethane) showed
µ
µ
µ
(1
µ
1
four new compounds) are reported. A SAR study for these compounds was performed based on the
results obtained by using molecular calculations.
Keywords: Annona emarginata; (R)-2-(4-methylcyclohex-3-en-1-yl)propan-2-yl (E)-3-(4-hydroxyphenyl)-
acrylate; antibacterial activity; Staphylococcus aureus; structure-activity relationships
Molecules 2018, 23, 1187; doi:10.3390/molecules23051187
www.mdpi.com/journal/molecules

Molecules 2018, 23, 1187
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1. Introduction
Some genera from the family Annonaceae are an important natural source of products with
antifungal [ ], bactericidal [ ], pesticidal [ ], herbicidal [ ], antimalarial [ ], antiprotozoal [ ], and
cytostatic [ ] activity, especially some chemically isolated components (primarily from seeds, leaves,
1
2
3
4
5
6
7,8
and bark). These compounds include flavonoids, alkaloids, and acetogenins. This family is widely
distributed in tropical and subtropical regions around the world in diversity of habitats (forests, scrub,
and grassland), with higher prevalence in rain forests. In general, representatives of the family are
shrubs, but it is also possible to find dwarf shrubs or small trees and, rarely, canopy trees or lianas
among them. The Annona genus comprises ca. 160 species from the tropical and subtropical areas,
from both hemispheres. Four species grow mainly in Argentina: Annona emarginata (Schltdl.) H.
Rainer (=Rollinia emarginata Schltdl.), A. neosalicifolia H. Rainer (=Rollinia salicifolia Schltdl.), A. nutans
(R.E. Fr.), and A. rugulosa (Schltdl.) H. Rainer. Previous studies related to ethnopharmacological use
have shown that the infusions and decoctions of the leaves of A. emarginata, commonly known
as “arachichú”, “araticú”, “aratigú”, and “yerba mora”, are used in folk medicine as a gargle
against throat pain and for calming the toothache. Another way it is used is by absorbing juice
produced by chewing the leaves [9,10]. Antiprotozoal [11] and antifeedant [12] properties have been
reported. Regarding its chemical composition, rolliniastatin-1, sylvaticin, squamocin, rollidecin B,
lirioresinol B, liriodenine [11]; vomifoliol, dehydrovomifoliol, blumenol C, loliolide, 7-epiloliolide,
vanillin, dihydroactinolide [12]; ursolic acid, oleanolic acid, 8-trans-p-coumaroyloxy-
α
-terpineol,
-L-rhamnopyranoside [13]; alkaloids including two
)-asimilobine and one benzyltetrahydroisoquinoline, (+)-reticuline [14],
quercetin-3-O-
aporphines (
α
-L-arabinopyranosyl(1
→
2)-α
−
)-anonaine, (
−
and cyanogenic glycosides [15] have been previously isolated from its aerial parts. Resistance to
antimicrobials is a problem that increasingly threatens the world population [16]. The World Health
Organization established as “priority pathogens” those resistant to antibiotics, which include 12
families of bacteria most dangerous to human health, the so-called “superbugs”. Among them,
especially the threat posed by Gram ( ) bacteria resistant to multiple antibiotics is highlighted. Among
−
the Gram (+) bacteria, methicillin-resistant Staphylococcus aureus (MRSA) [17] is one of the most
important. Thus, the development of new molecules, alternative strategies, and prevention and control
measures is necessary to avoid this problem.
The main objective of this work is to determine the antibacterial effect of different extracts of
A. emarginata (Schltdl.) H. Rainer, which is widely used in folk medicine in the northeast part of
Argentine. Another goal is to try to determine which active compound is responsible for such activity.
Thus, this work has been done in several stages. In the first step, the antimicrobial properties of
extracts of A. emarginata were studied, and the bioassay-guided isolation of its most active compound
was made. The compound was tested against an extended panel of both standardized and clinical
opportunistic pathogenic bacteria. The species were selected to include Gram (+) and Gram ( )
−
species of the most common human pathogens. Once the structure of the compound responsible for
the antibacterial activity was determined, some structurally related compounds were synthesized.
Finally, based on molecular calculations, we performed a study of the structure-activity relationships
of antibacterial compounds of the different series reported here.
2. Results and Discussion
2.1. Antimicrobial Evaluation of A. emarginata Extracts and Its Active Ingredient
The extracts from the stem bark and leaves of A. emarginata showed moderate activity against the
bacteria tested, with values between 250 and 1000 µg/mL (Table 1). Regarding flower extracts, the
less polar (hexane, dichloromethane) extracts, HFlE and DCMFlE, showed very strong antibacterial
activity against methicillin-sensitive S. aureus (MSSA) ATCC 25923 and methicillin-resistant S. aureus
(MRSA) ATCC 43300, with values between 16 and 125
µg/mL. Additionally, hexane extract showed
activity against Klebsiella pneumoniae (MIC = 250 g/mL). The global methanolic extract from the fruits
µ

Molecules 2018, 23, 1187
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(MeOHGFE) was also active against the three strains mentioned above, with minimum inhibitory
concentration (MIC) values between 250–500 g/mL. In order to identify bioactive compounds, a
µ
bioassay-guided fractionation of the MeOHGFE was performed.
Table 1. Antibacterial activity (MIC (µg/mL)) of extracts of A. emarginata (Annonaceae).
MIC (µg/mL)
Extracts
E. coli ATCC
MSSA
MRSA
E. coli LM2
K. pneumoniae
Salmonella sp.
25922
HBE
>1000
1000
500
>1000
>1000
1000
500
250
500
500
500
>1000
500
>1000
1000
>1000
>1000
1000
250
500
DCMBE
EtAcOBE
MeOHBE
Stem bark
Leaves
500
1000
1000
1000
HLE
500
500
500
250
500
500
>1000
>1000
>1000
500
>1000
1000
>1000
500
>1000
1000
250
500
500
500
500
DCMLE
EtAcOLE
MeOHLE
>1000
>1000
500
HFlE
125
62.5
250
125
16
250
>1000
>1000
1000
>1000
>1000
>1000
1000
250
1000
500
1000
>1000
>1000
>1000
DCMFlE
EtAcOFlE
MeOHFlE
Flowers
Fruits
>1000
>1000
1000
>1000
MeOHGFE
500
0.5
500
0.5
>1000
1.9
>1000
0.5
250
15
>1000
12.5
cefotaxime
HBE: hexane bark extract; DCMBE: dichloromethane bark extract; EtOAcBE: ethyl acetate bark extract; MeOHBE:
methanol bark extract; HLE: hexane leaf extract; DCMLE: dichloromethane leaf extract; EtOAcLE: ethyl acetate leaf
extract; MeOHLE: methanol leaf extract; HFlE: hexane flower extract; DCMFlE: dichloromethane flower extract;
EtOAcFlE: ethyl acetate flower extract; MeOHFlE: methanol flower extract; MeOHGFE: methanol global fruit extract.
Tables 2 and 3 show the antimicrobial activity of fractions and subfractions of the MeOHGFE.
Fraction F3 displayed the strongest antibacterial activity (Table 2) against MSSA, MRSA, Escherichia coli
ATCC 25922 (MIC = 500 µg/mL), and K. pneumoniae (MIC = 125 µg/mL). To gain further information
about their activity spectrum against Gram (+) strains, we added a panel of clinical isolates obtained
from human patients (coagulase negative Staphylococcus aureus 502, Streptococcus pyogenes and
Streptococcus agalactiae) (Tables 3 and 4). Fraction F3 was further percolated on Sephadex LH-20
affording 5 subfractions (I–V). One of them, subfraction FIV, showed strong antibacterial activity
with MIC = 16
aureus 502 (MIC = 31.2
250 µg/mL, respectively). On the basis of the previous results, subfraction FIV was successively
purified by Sephadex LH-20 and silica gel column, affording the main antibacterial compound
(Figure 1). As we expected, compound showed stronger antimicrobial activity against both Gram (+)
ATCC strains with MICs = 3.12–6.25 g/mL, and the expanded panel strains were also sensitive to this
g/mL (Table 4). In contrast, compound (see below) was
µg/mL against all the Gram (+) bacteria tested, except for coagulase negative S.
µ
g/mL), Escherichia coli LM2 (MIC = 125 g/mL), and K. pneumoniae (MIC
µ
=
1
1
µ
compound with MICs value equal 3.12
practically inactive.
µ
2

Molecules 2018, 23, 1187
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Table 2. Antibacterial activity (MIC (µg/mL)) of fractions F1-F6 from MeOHGEF.
MIC (µg/mL)
E. coli LM2
>1000
Extracts
E. coli ATCC
MSSA
MRSA
K. pneumoniae
Salmonella sp.
25922
MeOHGFE
500
500
>1000
250
>1000
F1
F2
F3
F4
F5
F6
>1000
500
500
1000
500
250
>1000
250
500
1000
500
500
>1000
>1000
500
1000
1000
250
>1000
>1000
>1000
125
250
250
>1000
>1000
125
1000
125
>1000
>1000
>1000
>1000
>1000
>1000
MeOHGFE
Fractions
>1000
cefotaxime
0.5
0.5
1.9
0.5
15
12.5
MeOHGFE: methanol global extract fruit; F1 (200 mg; hex 100%), F2 (115 mg; 50:50% H:DCM), F3 (1200 mg; 90:10%
DCM:EtOAc), F4 (187.7 mg 80:20% DCM:EtOAc), F5 (155 mg; 70:30% DCM:EtOAc), and F6 (137 mg; EtOAc100%).
Table 3. Antibacterial activity (MIC (µg/mL)) of subfractions FI-V from F3.
MIC (µg/mL)
Extracts
MSSA MRSA
SA
SP
SAG
EC
ELM
KP
Ssp
500
500
500
250
250
500
>1000
125
>1000
MeOHGFE Fraction F3
Subfraction I
>250
>250
>250
16
>250
>250
>250
16
>250
>250
>250
31.2
>250
>250
>250
16
>250
>250
>250
16
>250
250
>250
>250
>250
>250
>250
250
125
>250
>250
>250
>250
250
>250
>250
>250
>250
>250
Subfraction II
Subfraction III
Subfraction IV
Subfraction V
F3
subfractions
62.5
62.5
62.5
62.5
62.5
>250
cefotaxime
0.5
0.5
0.5
0.25
0.25
1.9
0.5
15
12.5
MSSA = methicillin-sensitive Staphylococcus aureus ATCC 25923, MRSA = methicillin-resistant Staphylococcus aureus
ATCC 43300, SA = coagulase negative Staphylococcus aureus 502, SP = Streptococcus pyogenes, SAG = Streptococcus
agalactiae, EC = Escherichia coli ATCC 25922, ELM = Escherichia coli LM2, KP = Klebsiella pneumoniae, Ssp =
Salmonella sp.
Table 4. Antibacterial activity (MIC [
µg/mL]) of compound 1 (isolated from subfraction FIV) and
compounds 2–7 obtained by synthesis.
MIC (µg/mL)
Comp.
MSSA
MRSA
SA
SP
SAG
EC
ELM
KP
1
2
3
4
5
6
7
CEF
4
>50
50
>50
50
25
6.25
>50
50
>50
>50
50
3.12
>50
50
>50
25
50
12.5
0.5
3.12
>50
50
>50
25
50
25
0.25
3.12
>50
50
>50
50
50
12.5
0.25
>50
>50
>50
>50
>50
>50
>50
1.9
50
>50
>50
>50
>50
25
50
>50
>50
>50
>50
>50
>50
15
12.5
0.5
50
0.5
12.5
0.5
MSSA = methicillin-sensitive Staphylococcus aureus ATCC 25923, MRSA = methicillin-resistant Staphylococcus aureus
ATCC 43300, SA = coagulase negative Staphylococcus aureus 502, SP = Streptococcus pyogenes, SAG = Streptococcus
agalactiae, EC = Escherichia coli ATCC 25922, ELM = Escherichia coli LM2, KP = Klebsiella pneumoniae, CEF = cefotaxime.
Figure 1. Structural features of compounds studied. Torsional angles are shown for compound 1.

Molecules 2018, 23, 1187
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2.2. Natural Lead Structure
The strongest antimicrobial subfraction IV was successively chromatographed on silica gel to
afford pure compound
, which was identified by its spectroscopic data, including 1H, 13C-NMR, and
HR-EIMS spectra as (R)-2-(4-methylcyclohex-3-en-1-yl)propan-2-yl (E)-3-(4-hydroxyphenyl)acrylate
), see Figure 1 and spectra in Supplementary Material. The relevant NOESY correlations (Figure 2), a
strong positive Cotton effect observed in the CD spectrum and spectroscopic data, which are in partial
agreement with those previously published for the compound 9-trans-p-coumaroyloxy- -terpineol,
1
(1
α
isolated from Haplopappus taeda [18], support the structure and absolute configuration proposed in
Figure 2. In addition, (DFT) (B3LYP/6-31G (d)) calculations suggest that this structure is 0.46 Kcal/mol
more stable than its isomeric form, giving additional support to the experimental data.
Figure 2. Key NOESY correlations of compound 1.
2.3. Synthetic Analogues
Observing the structure of compound
the aromatic ring corresponds to a structure very similar to that of p-coumaric and caffeic
acids. Previously, it was reported that analogues of p-coumaric and caffeic acids have
significant antibacterial and antifungal activities [19 20]. In fact, several phenolic compounds
1, it is interesting to note that the moiety having
,
and derivatives possessing antifungal effects have been reported. For example, gallic acid
has been reported to prevent aflatoxin biosynthesis by Aspergillus flavus [21]. Two compounds
of natural origin, 1-(30-methoxypropanoyl)-2,4,5-trimethoxybenzene and 2-(2Z)-(3-hydroxy-3,7-
dimethylocta-2,6-dienyl)-1,4-benzenediol, isolated from the root bark of Cordia alliodora, have been
also reported as antifungal and larvicidal compounds [22]. In turn, Kim et al. [23] reported antifungal
effects of several phenolic compounds, including cinnamic, m-coumaric, p-coumaric, and caffeic acids;
therefore, the significant antibacterial activity found for compound 1 should not be so surprising.
At this stage of the study, efforts were focused on finding new compounds structurally related
to lead compound
propan-2-yl (E)-3-(4-ethoxyphenyl)acrylate (
see Scheme 1. Interestingly, compound 2 was devoid of antibacterial activity displaying MICs values
>50 g/mL. It should be noted that the only structural difference between compounds and is the
ethylation of the hydroxyl group of compound 1.
1
that may have antibacterial activity. First, (R)-2-(4-methylcyclohex-3-en-1-yl)-
2) was obtained by a simple modification of compound
1,
µ
1
2

Molecules 2018, 23, 1187
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Scheme 1.
(R)-2-(4-Methylcyclohex-3-en-1-yl)propan-2-yl (E)-3-(4-ethoxyphenyl)acrylate (2)—
etherified product of (R)-2-(4-methylcyclohex-3-en-1-yl)propan-2-yl (E)-3-(4-hydroxyphenyl)-acrylate
(1). Reagents and conditions: (a) t-BuOK, DMSO, CH3CH2I, 30 min.
Then, compounds
while the second part (4-hydroxyphenyl) of the molecule was replaced by a fused ring (rings A
and B) of the quinoline scaffold (see Figure 1). Styrylquinoline derivatives and were prepared
by condensation of the quinoline derivative with an appropriate aldehyde in a mixture of acetic
anhydride/acetic acid. 2-[2-(4-Hydroxyphenyl)vinyl]quinolin-8-ol ( ) was hydrolysed in methanol
with K2CO3, then precipitated by concentrated HCl and crystallized from ethanol, see Scheme 2.
3–5 were synthesized, in which the first moiety of compound 1 was maintained,
3
4
5
Scheme 2. Synthesis of compound
and N2 atmosphere; (b) MeOH, K2CO3, and HCl.
3–5
. Reagents and conditions: (a) Ac2O, 80% AcOH, 18 h at 130 ◦C,
After synthesis of compounds
compound showed moderate antibacterial activity, compounds
to note that the only structural difference between compound
replacement of the OH group by OAc and OCH3, respectively. These results are in accord with those
obtained for compounds and , since the ethylation of the OH group of produced significant loss of
3–5
, their potential antibacterial activities were evaluated. While
and were inactive. It is interesting
and compounds and is the
5
3
4
5
3
4
1
2
1
antibacterial activity. From these results, it appears that the presence of a proton donor group in the
ring A of these compounds could play an important role in the antibacterial effect, e.g., compare Gonec
et al. [24] with Kos et al. [25,26]. In order to understand better this behaviour, a conformational and
electronic study was performed for these compounds with the aim to determine their stereo-electronic
characteristics. Details about the type of molecular calculations and the methodology used are
explained in the Section 3.4. In addition, details about the conformational analysis of the compounds
are also given in the same section. Knowledge of the stereo-electronic attributes and properties of these
compounds will contribute significantly to the elucidation of the structural requirements to produce
antibacterial activity. Molecular electrostatic potentials (MEP) are of particular value, because they
allow the visualization and assessment of the capacity of a molecule to interact electrostatically with
a putative-binding site [27–29]. Thus, once the conformational behaviours of compounds reported
here had been analysed, an electronic study of their respective preferred conformations by using
Electrostatic Potentials Maps (EPMs) was performed. Figure 3 gives the Molecular Electrostatic
Potentials (MEP) obtained for compounds
1 and 3. The MEP obtained for compound 1 (Figure 3a)
exhibit three characteristic regions: a clear minimum value (deep red zone with V(r)min ≈ −0.1559

Molecules 2018, 23, 1187
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el/au3) in the vicinity of the OH group of the ring A, a positive region (deep blue zone with V(r)max
0.20 el/au3) located in the linker chain, and an extended hydrophobic zone (deep and light green
zone with an almost neutral potential V(r)med ≈ −0.0011 el/au3) in the zone near to the ring B.
≈
Figure 3. Electrostatic potential-encoded electron density surfaces of core structures of compounds
1
(
a
), ), and ). The colouring represents electrostatic potential, with red indicating the strongest
3
(
b
6 (c
attraction to a positive point charge and blue indicating the strongest repulsion. The electrostatic
potential is the energy of interaction of the positive point charge with the nuclei and electrons of a
molecule. It provides a representative measure of overall molecular charge distribution.
It is interesting to note that the MEP of compound 3 (Figure 3b) is similar to that obtained for 1.
However, for compound 3, both the most electronegative zone (zone with red and orange tones) and
the electropositive part of the molecule (area with bluish tonalities) are significantly more tenuous
with values of V(r)min ≈ −0.120 el/au3 and V(r)max
≈
0.110 el/au3, respectively. These results are in
agreement with the experimental data, considering that compound 3 displayed lower antibacterial
activity than compound 1.
A
series
6-hydroxy-N-phenylnaphthalene-2-carboxamide (
naphthalene-2-carboxamide ( ) (see Figure 1), was reported recently [25]. They were prepared
of
carboxanilides
with
antimycobacterial
properties,
including
6
) and 6-hydroxy-N-(3-trifluoromethylphenyl)
7
according to Scheme 3 by modified microwave-assisted synthesis in one step with excellent yields. At
first, the carboxyl group was activated with phosphorus trichloride. The final amide was immediately
formed by aminolysis of acyl chloride by a ring-substituted aniline in dry chlorobenzene. All the crude
target compounds were recrystallized from ethanol.
NH2
O
R
COOH
a
N
H
+
R
HO
HO
6
7
R = H ( ), 3-CF3 ( )
Scheme 3. Synthesis of ring-substituted 6-hydroxynaphthalene-2-carboxanilides
conditions: (a) PCl3, chlorobenzene, and microwave irradiation (MW) [25].
6 and 7. Reagents and
It should be noted that these compounds are naphthalene analogues (carbon isosteres) of the
quinoline scaffold, i.e., they also possess fused rings in their structure (rings A and B), which gives a

Molecules 2018, 23, 1187
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certain structural similarity to the first portion of compound
1. Next, a conformational and electronic
study of compounds
the rest of compounds studied here. Figure 3c indicates that compound 6 displays MEP that are very
similar to those obtained for compound (compare Figure 3a,c). In this case, the values obtained for
V(r)max and V(r)min are very similar to those observed in compound . A very similar result was
obtained for compound . Based on these results, it was decided to test these compounds as potential
antibacterial agents. Compounds and , especially compound , showed significant antibacterial
6 and 7 was performed, and the results were compared with those obtained for
1
1
7
6
7
7
effects against the different strains studied here (Table 4). These experimental results fully agree with
the molecular calculations and give additional support to the proposed pharmacophoric pattern. Once
again, the presence of a proton donor group at ring A appears to be an essential structural requirement
for antibacterial effect.
3. Materials and Methods
3.1. Extractions and Isolation
3.1.1. General Methods
1H and 13C-NMR spectra were recorded on a High Resolution Spectrometer Advance 400
(working frequency 400 MHz and 100 MHz, respectively) at ambient temperature in CDCl3 (Aldrich,
St. Louis, MO, USA). Carbon spectra assignments are supported by DEPT-135 spectra, 13C-1H (HMQC)
and 13C-1H (HMBC), and NOESY correlations. High-resolution mass spectrums were measured on
Bruker Micro TOF Q II equipment (Bruker, Karlsruhe, Germany), operated with an ESI source in the
positive mode. Optical rotations were measured with a Jasco polarimeter DIP-1000 P-1010 34 (Jasco,
Easton, MD, USA). The CD (Circular Dichroism) and UV-VIS spectra of methanol solutions of
1 and 2
were recorded simultaneously using a Jasco J-810 spectropolarimeter, model J-810 150S and a 100 lm
quartz cuvette with resolution 1 nm, time constant 1 s, scan speed 100 nm/min, and 3 accumulations,
employing standard sensitivity and a measurement range of 700–190 nm.
3.1.2. Plant Material
The aerial parts (leaves, flowers, and fruits) of A. emarginata were collected in San Cosme, province
of Corrientes (Argentina) at 27.36667 lat. S; 58.51667 long. W, in the summer of 2013 and were
authenticated by Dra. Elisa Petenatti and L.A. Del Vitto. A voucher specimen (L.A. Del Vitto & E.M.
Petenatti # 9490) has been deposited at the Herbarium, National University of San Luis, Argentina.
3.1.3. Preparation of Extracts
Finely ground A. emarginata fruits (100 g) were extracted at room temperature using methanol
MeOH (200 mL
×
3-fold
×
96 h each), filtered and concentrated under reduced pressure to afford
the corresponding crude global methanolic extract (MeOHGFE; 17.7 g, yield 17.7% w/w). Dried and
finely ground A. emarginata flowers (100 g) were successively extracted at room temperature (200
mL
×
3-fold
×
96 h, each 3
×
24 h) with hexane (H), dichloromethane (DCM), ethyl acetate (EtOAc),
and methanol (MeOH). Then, solvents were evaporated under vacuum to give flower extracts HFlE,
DCMFlE, EtOAcFlE, and MeOHFlE with yields (w/w) of 4.3%, 1.9%, 0.66%, and 5.9%, respectively.
Finely ground dried A. emarginata stem bark (100 g) was successively extracted at room temperature
(200 mL
×
3-fold
×
96 h, each 3
×
24 h) with H, DCM, EtOAc, and MeOH. The solvents were
evaporated under vacuum to afford bark extracts HBE, DCMBE, EtOAcBE, and MeOHBE with yields
(w/w) of 0.92%, 0.27%, 0.34%, and 4.23%, respectively. Finely ground dried A. emarginata leaves (100 g)
were successively extracted at room temperature (200 mL
EtOAc, and MeOH. Then, solvents were evaporated under vacuum to produce leaf extracts HLE,
×
3-fold
×
96 h, each 3
×
24 h) with H, DCM,
DCMLE, EtOAcLE, and MeOHLE, affording 0.63%, 1.54%, 0.75%, and 1.02% yield (w/w), respectively.

Molecules 2018, 23, 1187
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3.1.4. Bioassay-Guided Fractionation of Annona emarginata Fruit Extracts (MeOHGFE)
MeOHGFE (11 g) was applied onto a column (column length 70 cm, i.d. 5 cm) containing silica
gel (0.063–0.2 mesh, Merck 60, Merck, Darmstadt, Germany) and eluted with H, DCM, and EtOAc
gradients (100% H to 100% EtOAc). Six fractions of 1 L each were obtained, which concentrated
under reduced pressure to afford F1 (200 mg; H 100%), F2 (115 mg; 50:50% H/DCM), F3 (1200 mg;
90:10% DCM/EtOAc), F4 (187.7 mg 80:20% DCM/EtOAc), F5 (155 mg; 70:30% DCM/EtOAc), and
F6 (137 mg; EtOAc 100%) fractions. Fractions F1–F6 were tested for antimicrobial activity. Fraction
F3 (1100 mg) permeated through Sephadex LH-20 (column length 23 cm, i.d. 4 cm; petroleum ether
(PE)/MeOH/DCM 2:1:1). Some 21 fractions of 15 mL each were obtained. After TLC comparison (silica
gel, PE/EtOAc 8:2) as the mobile phase, detection under UV light and spraying with p-anisaldehyde
sulfuric acid, the fractions with similar TLC patterns were combined as follows: I (477 mg), II (142 mg),
III (98 mg), IV (295 mg), and V (84 mg).
3.1.5. Isolation and Characterization of (R)-2-(4-Methylcyclohex-3-en-1-yl)propan-2-yl
(E)-3-(4-hydroxyphenyl)acrylate (1)
Separation of the fraction IV (295 mg) using column chromatography on silica gel (column length
40 cm, internal diameter 2 cm; 50 g silica gel 0.063–0.2 mesh, Merck 60); (100% H to 100% EtOAc)
provided 200 mg of pure compound
CDCl3)
1
: oil; [α]2D3 + 29 (c = 0.17 g/100 mL, CHCl3), 1H-NMR (400 MHz,
δ
1.50 (s, 3H, H-90), 1.53 (s, 3H, H100), 1.67 (s, 3H, H-70), 1.85–1.35 (m, 2H, H-30), 2.12 (m, 1H,
H-40), 2.16–1.86 (m, 4H, H-20, H-50), 5.39 (s, 1H, H-60), 6.22 (d, J = 16 Hz, 1H, H-8), 6.85 (d, J = 8Hz, 2H,
H-3 and H-5), 7.38 (d, J = 8 Hz, 2H, H-2 and H-6), 7.51 (d, J = 16 Hz, 1H, H-7); 13C-NMR (100 MHz,
CDCl3)
115.9 CH
δ 23.3 CH3, δ
23.4 CH3, 23.5 CH3, 24.0 CH2, 26.5 CH2, 30.9 CH2, 42.8 CH (C-40), 85.2 C (C-80),
×
2 (C-3 and C-5), 117.2 CH (C-8), 120.3 CH (C-60), 127.0 (C-1), 129.9 CH
×
2 (C-2 and C-6),
134.0 (C-10), 143.8 (C-7), 158.4 (C-4), 167.8 C (C=O), HR-EIMS 323.1618, (calcd for C19H24O3 (M + Na)
323.1618). (See spectra in Supplementary Material).
3.2. Synthesis
3.2.1. General Methods
All of the reagents were purchased from Aldrich. Kieselgel 60, 0.040–0.063 mm (Merck,
Darmstadt, Germany) was used for the column chromatography. TLC experiments were performed
on alumina-backed silica gel 40 F254 plates (Merck, Darmstadt, Germany). The plates were illuminated
under UV (254 nm) and evaluated in iodine vapour. The melting points were determined on an
Optimelt MPA-100 apparatus (SRS, Stanford, CA, USA). The purity of the final compounds was
checked using HPLC. Detection wavelengths of 210 and 250 nm were chosen for detection. The purity
of individual compounds was determined from the area peaks in the chromatogram of the sample
solution in order to ensure >95% purity. All 1H-NMR spectra were recorded on a Bruker AM-400
(400 MHz for 1H and 101/126 MHz for 13C), BrukerBioSpin Corp., Rheinstetten, Germany. Chemical
shifts are reported in ppm (δ) against the internal standard, Si(CH3)4. Easily exchangeable signals
were omitted when they were diffused. Signals are designated as follows: s, singlet; d, doublet; dd,
doublet of doublets; m, multiplet. The high-resolution mass spectra of compounds 3–5 were measured
using a high-performance liquid chromatograph Dionex UltiMate®3000 (Thermo Scientific, Waltham,
MA, USA) coupled with a LTQ Orbitrap XLTM Hybrid Ion Trap-Orbitrap Fourier Transform Mass
Spectrometer (Thermo Scientific) with injection into HESI II in the positive mode.
3.2.2. Synthesis of (R)-2-(4-Methylcyclohex-3-en-1-yl)propan-2-yl (E)-3-(4-ethoxyphenyl)acrylate (2)
In a Schlenk tube equipped with a magnetic stirrer, t-BuOK (0.118 mmol) was added to 2 mL
of degassed DMSO under nitrogen atmosphere. After 10 min, when no more solid was present,
compound
1 (0.098 mmol) was added. The reaction mixture was stirred for 30 min until the formation
of the corresponding ions. The reaction was then quenched by adding excess ethyl iodide (0.196 mmol)

Molecules 2018, 23, 1187
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to obtain compound
2. The solution was extracted with ethyl acetate and water. The organic extract
was analysed by GC-MS. The reaction was then quenched by adding excess ethyl iodide (0.196 mmol)
to obtain a brown oil that was purified by column chromatography on silica gel (column length 40
cm, internal diameter 2 cm; 50 g silica gel (0.063–0.2 mesh, Merck 60) using a solvent gradient (100%
PE to 100% EtOAc) to afford 110 mg of pure compound 2
: oil (yield 95%); [α]2D3 + 23 (c = 0.11 g/100
0 0
δ 1,42 (t, 3H, CH3 ethyl), 1.50 (s, 3H, H-9 ), 1.53 (s, 3H, H10 ),
mL, CHCl3), 1H-NMR (400 MHz, CDCl3)
1.65 (s, 3H, H-70), 1.85–1.35 (m, 2H, H-30), 2.12 (m, 1H, H-40) 2.16–1.86 (m, 4H, H-20, H-50), 4.05 (q, 2H,
CH2 ethyl), 5.39 (s, 1H, H-60), 6.23 (d, J = 16 Hz, 1H, H-8), 6.87 (d, J = 8Hz, 2H, H-3, H-5), 7.44 (d J = 8
Hz, 2H, H-2, H-6), 7.52 (d, J = 16 Hz, 1H, H-7); 13C-NMR (100 MHz, CDCl3)
δ 14.7 CH3 (ethyl) 23.3
0
CH3, 23.4 CH3, 23.5 CH3, 24.0 CH2, 26.5 CH2, 30.9 CH2, 42.7 CH (C-4 ), 63.5 (s, 2 H, CH2 ethyl), 84.9 C
(C-80), 114,7 CH (C-3, C-5), 117.6 CH (C-8), 120.4 CH (C-60), 127.2 (C-1), 129.6 CH (C-2 and C-6), 134.0
(C-10), 143.3 (C-7), 160.0 CH (C-4), 168.9 C (C=O), HREIMS 351.1935, (calcd for C21H28O3 (M + Na)
351.1931). (See spectra in Supplementary Material).
3.2.3. General Synthesis of Styrylquinoline Derivatives 3–5
The appropriate quinoline derivative (2.5 mmol) in acetic anhydride with 80% acetic acid was
◦
thoroughly mixed with one equivalent aldehyde and heated in an inert gas atmosphere (N2) at 130 C
for 18 h. Then, the mixture was evaporated to dryness, and a solid was recrystallized from ethanol.
The appropriate styrylquinoline derivative (2.5 mmol) in methanol was thoroughly mixed with 2.5
equivalent K2CO3 at room temperature for 2 h. Then, concentrated HCl was added, and the resulting
precipitate was filtered and washed with H2O. The crude product was crystallized from ethanol.
2-[2-(4-Methoxyphenyl)vinyl]quinolin-8-yl acetate (
3
)
): yield 20%, light brown solid, m.p. 97
−
100 ◦C;
HPLC purity 95.82%; 1H-NMR (400 MHz, DMSO-δ6
δ
8.38 (d, J = 8.6 Hz, 1H), 7.90–7.82 (m, 2H), 7.78
(d, J = 16.3 Hz, 1H), 7.70 (d, J = 8.5 Hz, 2H), 7.57–7.47 (m, 2H), 7.30 (d, J = 16.3 Hz, 1H), 7.02 (d, J =
8.5 Hz, 2H), 3.82 (s, 3H), 2.50 (s, 3H); 13C-NMR (126 MHz, DMSO-δ6
172.49, 160.23, 154.26, 153.29,
)
δ
138.60, 136.84, 134.57, 129.58, 129.39, 129.11, 127.97, 127.24, 126.14, 122.09, 121.25, 118.02, 114.87, 111.58,
55.69, 21.52. HR-MS: for C20H18NO3 [M + H]+ calculated 320.1281 m/z, found 320.1284 m/z.
◦
2-[2-(4-Acetoxyphenyl)vinyl]quinolin-8-yl acetate (
purity 95.83%; 1H-NMR (400 MHz, CDCl3)
(m, 3H), 7.19–7.12 (m, 2H), 2.58 (s, 3H), 2.35 (s, 3H); 13C-NMR (101 MHz, CDCl3)
4
): yield 67%, yellow solid, m.p. 157–159 C; HPLC
δ
8.15 (dd, J = 8.5, 5.1 Hz, 1H), 7.73–7.60 (m, 5H), 7.55–7.37
169.84, 169.33,
δ
155.65, 150.91, 147.38, 140.94, 136.41, 134.25, 133.71, 131.21, 129.10, 128.60, 128.32, 125.75, 125.54, 121.95,
121.68, 120.19, 21.17, 21.03. HR-MS: for C21H18NO4 [M + H]+ calculated 348.1230 m/z, found 348.1239
m/z.
◦
2-[2-(4-Hydroxyphenyl)vinyl]quinolin-8-ol (
98.38%; 1H-NMR (400 MHz, DMSO-δ6
8.9 Hz, 1H), 8.22 (d, J = 16.2 Hz, 1H), 7.79 (d, J = 16.3 Hz, 1H), 7.67–7.56 (m, 4H), 7.47 (dd, J = 6.1, 2.6
5
): yield 63%, orange solid, m.p. 156–157 C; HPLC purity
)
δ 11.81 (s, 1H), 10.38 (s, 1H), 8.84 (d, J = 8.9 Hz, 1H), 8.42 (d, J =
Hz, 1H), 6.95 (d, J = 8.6 Hz, 2H); 13C-NMR (126 MHz, DMSO-δ6
) δ 161.19, 153.30, 148.85, 144.14, 143.99,
143.72, 130.85, 129.57, 128.20, 126.55, 119.16, 118.74, 116.83, 116.50. HR-MS: for C17H13NO2 [M + H]+
calculated 264.1019 m/z, found 264.1028 m/z.
6-Hydroxy-N-phenylnaphthalene-2-carboxamide (6) and 6-hydroxy-N-(3-trifluoromethylphenyl)-
naphthalene-2-carboxamide (7) were synthesized and characterized recently [25].
3.3. Antibacterial Activity
For the antibacterial evaluation, methicillin-sensitive Staphylococcus aureus ATCC 25923,
methicillin-resistant S. aureus ATCC 43300, Escherichia coli ATCC 25922, and clinical isolates from
human patients—coagulase negative Staphylococcus aureus 502, Streptococcus pyogenes, Streptococcus
agalactiae, Escherichia coli LM2, Salmonella sp. and Klebsiella pneumoniae—provided by Laboratorio de
Microbiologia, Hospital Marcial Quiroga, San Juan, Argentina, were used.

Molecules 2018, 23, 1187
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Minimal inhibitory concentration (MIC) values were determined using the broth microdilution
method according to the protocols of the Clinical and Laboratory Standards Institute [30]. The assay
was executed in Mueller–Hinton broth (MHB), using strains 24 h old. The inoculum employed was
1–5
×
105 CFU/mL. Stock solutions of extracts in DMSO (50 mg/mL) were diluted to give serial
2-fold dilutions that were added to each medium to obtain final concentrations ranging from 1000
to 1 µg/mL. Stock solutions of compounds in DMSO (5 mg/mL) were diluted to give serial 2-fold
dilutions that were added to each medium to obtain final concentrations ranging from 50 to 1 µg/mL.
The final concentration of DMSO in the assay did not exceed 1%. Cefotaxime Argentia® (Bristol-Myers
Squibb, Buenos Aires, Argentina) was included in the assays as a positive control, while MHB and
DMSO (1%) were used as negative controls. The plates were incubated for 24 h at 37 ◦C. Activity was
evaluated at 620 nm using a Multiskan
™ FC Microplate Photometer (Thermo Fisher Scientific, Vantaa,
Finland). The MIC values were defined as the lowest extract concentrations showing no bacterial
growth after the incubation time. Tests were done in triplicate.
3.4. Conformational and Electronic Study
All calculations were carried out using the Gaussian 09 program (Gaussian, Wallingford, CT,
USA) [31]. The search for low-energy conformations on the potential energy surface for compounds
1, 3, 6, and 7 was carried out by using combined ab initio (RHF/3-21G) and density functional
theory (DFT) (B3LYP/6-31G(d)) calculations. Final DFT geometries were obtained from the geometry
optimisation jobs. Minima were characterized through harmonic frequency analysis calculated at DFT
level. Correlations effects were included using DFT and the functional of Lee [32] as proposed and
parameterized by Becke [33
simple conformational problem, in which three torsional angles (
rotations in this molecule. Our calculations predict that the torsional angle
,
34] (RB3LYP) and 6-31G (d) basis set. Compound
1
φ
looks like a relatively
3, Figure 1) are main
1 possesses only two
φ
1, 2, and
φ
φ
possible quasi-planar conformations: one of them near to 0◦, and the opposite position giving values
near to 180◦. The energy barrier obtained for this rotation is more than 10 Kcal/mol, suggesting a
significantly restricted torsional angle (see Figure S1 in Supplementary Material). It should be noted
that the cis form possesses about 1 Kcal/mol above the trans form; therefore, the conformational
analysis of compound
B are mainly described by the dihedral angles
computational method to evaluate how the local energy minima and the transition states are linked
together requires an exploration of the complete potential energy surface (PES) generated by 2 vs.
3. This PES was calculated using a 12 12 grid generated by rotating through 2 and
3 in 30◦
increments from
180 to 180◦. At each point, a complete geometry optimization was performed with
2 and 3 frozen at their respective grid values. The graphical presentation of the PES was generated
using the program AXUN 5.0 (MathSoft, Cambridge, MA, USA), plotting the total energy values as a
dependent variable generated by double scans over independent variables 2 and 3. The optimum
coordinates of 2 and 3 for all possible minima were then visually estimated from the level contours
diagrams (Figure S2 in Supplementary Material). Five different conformations (conformations 1–5)
were obtained for ; such low-energy conformers might be well appreciated in this surface. The global
1
considering
φ
1 near to 180◦ was performed. The orientations of the ring
2 and 3, and probably the most comprehensive
φ
φ
φ
φ
×
φ
φ
−
φ
φ
φ
φ
φ
φ
1
minimum (conformer 1) corresponds to a partially extended form (see Table S1 in Supplementary
Material). On the other hand, the conformational analysis suggests planar forms as energetically
preferred structures for compounds
characteristics of their connecting chains. In compound
compounds and , it is a peptide bond. The electronic study of compounds
3
,
6
, and
7
. This is an expected result, considering the structural
, it is a highly delocalized system, while in
, and was carried
3
6
7
1,
3,
6
7
out using molecular electrostatic potentials (MEP) [27]. MEP have been shown to provide reliable
information both on the interaction sites of the molecules with point charges and on the comparative
reactivities of such sites [28,29]. These MEP were calculated using B3LYP/6-311G (d,p) wave functions
from the MOLEKEL 4.0. program (CSCS, Manno, Switzerland) [35].

Molecules 2018, 23, 1187
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4. Conclusions
Strong antibacterial activities found for some of the extracts studied here fully justify the use of
infusions and decoctions of Annona emarginata (Schltdl.) H. Rainer in folk medicine for the treatment
of some infections. First, it is interesting to remark that the less polar flower extracts (hexane,
dichloromethane) showed very strong antibacterial activity against methicillin-sensitive S. aureus
ATCC 25923 and methicillin-sensitive S. aureus ATCC 43300. Additionally, the hexane extract showed
activity against K. pneumoniae, while the methanol extract from the fruits was also active against the
three strains mentioned above. Such activity might be explained at least in part by the content of the
new main compound (R)-2-(4-methylcyclohex-3-en-1-yl)propan-2-yl (E)-3-(4-hydroxyphenyl)acrylate
(1). This compound showed strong antimicrobial activity against all Gram (+) species tested. The
demand for new antibacterial agents and the new trend towards the use of characterized natural
extracts or compounds isolated as medicines instead of pure drugs might open an alternative way for
the development of new antibacterial agents, trying to avoid the serious problem of resistance to the
antibiotics in use today. On the other hand, the molecular calculations allowed for the establishment
of structure-activity relationships between the antibacterial compounds reported here, which gives
important information about a possible pharmacophoric pattern of these compounds. This information
may be of great interest for the search and procurement of new structurally related antibacterial agents.
Supplementary Materials: The Supplementary materials are available online.
Author Contributions: Ricardo D. Enriz, Josef Jampilek, and Alejandro Tapia conceived and designed the
experiments. Juan G. Dolab, Beatriz Lima, Natividad H. Cano, Gabriela Feresin, and Alejandro Tapia performed
and analyzed the experiments, isolation, synthesis, the spectroscopic data of compounds
1 and 2, and the
antibacterial activity. Francisco Garibotto and Monica Olivella performed the molecular modelling. Ewelina
Spaczynska and Robert Musiol performed synthesis and characterization of compounds . Jiri Kos and Josef
. Elisa Petenatti identified the plant. All
3–5
Jampilek performed synthesis and characterization of compounds
authors wrote, read, and approved the final manuscript.
6 and 7
Acknowledgments: This research was partially supported by grants from Universidad Nacional de San Luis
and PIP 444-CONICET. J.D. thanks a fellowship from CONICET. E.S. and R.M. appreciate National Science
Centre grant No 2013/09/B/NZ7/00423. J.K. and J.J. were supported by the grant of the Faculty of Pharmacy of
Comenius University in Bratislava No. FaF UK/37/2018 and by SANOFI-AVENTIS Pharma Slovakia, s.r.o. The
authors thank Luis A. Del Vitto (UNSL) for his help in the botanical classification and Marcos Maiocchi (UNNE)
for the help in the collection of the plant material. B.L., G.E.F. and A.T. thank CICITCA-UNSJ.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
Moreira, I.C.; Lago, J.H.G.; Young, M.C.M.; Roque, N.F. Antifungal aromadendrane sesquiterpenoids from
the leaves of Xylopia brasiliensis. J. Braz. Chem. Soc. 2003, 14, 828–831. [CrossRef]
Takahashi, J.A.; Pereira, C.R.; Pimenta, L.P.; Boaventura, M.A.; Silva, L.G. Antibacterial activity of eight
Brazilian Annonaceae plants. Nat. Prod. Res. 2006, 20, 21–26. [CrossRef] [PubMed]
Santos Pimenta, L.P.; Pinto, G.B.; Takahashi, J.A.; Silva, L.G.F.; Boaventura, M.A.D. Biological screening of
Annonaceous Brazilian Medicinal Plants using Artemia salina (Brine Shrimp Test). Phytomedicine 2003, 10,
209–212. [CrossRef] [PubMed]
4.
5.
6.
Novaes, P.; Bezerra Torres, P.; Alves, D.Y.; dos Santos, C. Biological activities of Annonaceae species extracts
from Cerrado. Braz. J. Bot. 2016, 39, 131–137. [CrossRef]
Frausin, G.; Lima, R.B.S.; Hidalgo, A.F.; Maas, P.; Pohlit, A.M. Plants of the Annonaceae traditionally used as
antimalarials: A review. Rev. Bras. Frutic. 2014, 36, 315–337. [CrossRef]
Waechter, A.I.; Cave, A.; Hocquemiller, R.; Bories, C.; Munoz, V.; Fournet, A. Antiprotozoal activity of
aporphine alkaloids isolated from Unonopsis buchtienii (Annonaceae). Phytotherapy Res. 1999, 13, 175–177.
[CrossRef]
7.
Chen, Y.; Xu, S.S.; Chen, J.W.; Wang, Y.; Xu, H.Q.; Fan, N.B.; Li, X. Anti-tumor activity of Annona squamosa
seeds extract containing annonaceous acetogen in compounds. J. Ethnopharmacol. 2012, 142, 462–466.
[CrossRef] [PubMed]

Molecules 2018, 23, 1187
13 of 14
8.
9.
Monks, N.R.; Bordignon, S.A.L.; Ferraz, A.; Machado, K.R.; Faria, D.H.; Lopes, R.M.; Mondin, C.A.;
Schwartsmann, G. Anti-tumour screening of Brazilian plants. Pharm. Biol. 2002, 40, 603–616. [CrossRef]
Barboza, G.E.; Cantero, J.J.; Nunez, C.; Pacciaroni, A.; Ariza Espinar, L. Medicinal plants: A general review
and a phytochemical and ethnopharmacological screening of the native Argentine flora. Kurtziana 2009, 34,
7–365.
10. Martinez Crovetto, R. Plantas Utilizadas en Medicina en el NO de Corrientes; Ministry of Culture and Education,
Miguel Lillo Foundation: Tucuman, Argentina, 1981.
11. Fevrier, A.; Ferreira, M.E.; Fournet, A.; Yaluff, G.; Inchausti, A.; Rojas de Arias, A.; Hocquemiller, R.;
Waechter, A.I. Acetogenins and other compounds from Rollinia emarginata and their antiprotozoal activities.
Planta Med. 1999, 65, 47–49. [CrossRef] [PubMed]
12. Colom, O.A.; Popich, S.; Bardon, A. Bioactive constituents from Rollinia emarginata (Annonaceae). Nat. Prod.
Res. 2007, 21, 254–259. [CrossRef] [PubMed]
13. Roth, M.; Araya, J.J.; Timmermann, B.N.; Hagenbuch, B. Isolation of modulators of the liver-specific organic
anion-transporting polypeptides (OATPs) 1B1 and 1B3 from Rollinia emarginata Schltdl. (Annonaceae).
J. Pharmacol. Exp. Ther. 2011, 339, 624–632. [CrossRef] [PubMed]
14. Nieto, M. Alkaloids from Rollinia emarginata. J. Nat. Prod. 1986, 49, 717. [CrossRef] [PubMed]
15. Leboeuf, M.; Cave, A.; Bhaumik, P.K.; Mukherjee, B.; Mukherjee, R. The phytochemistry of the Annonaceae.
Phytochem 1982, 21, 2783–2813. [CrossRef]
16. O’Neill, J. Tackling Drug-Resistant Infections Globally: Final Report and Recommendations; HM Government and
the Wellcome Trust: London, UK, 2016.
17. Gagetti, P.; Corso, A. Actualización en Staphylococcus aureus resistente a meticilina de la comunidad.
Bol. Assoc. Argent. Microbiol. 2011, 193, 7–8.
18. Faini, F.; Labbe, C.; Torres, R.; Rodilla, J.M.; Silva, L.; Delle Monache, F. New phenolic esters from the resinous
exudate of Haplopappus taeda. Fitoterapia 2007, 78, 611–613. [CrossRef] [PubMed]
19. Feresin, G.E.; Tapia, A.; Gimenez, A.; Gutierrez Ravelo, A.; Zacchino, S.; Sortino, M.; Schmeda-Hirschman, G.
Constituents of the Argentinean medicinal plant Baccharis grisebachii and their antimicrobial activity.
J. Ethnopharmacol. 2003, 89, 73–80. [CrossRef]
20. Gianello, J.C.; Giordano, O.S. Constituents from Bacccharis grisebachii. An. Assoc. Quím. Argent. 1987, 75, 1–3.
21. Bisogno, F.; Mascoti, L.; Sanchez, C.; Garibotto, F.; Giannini, F.; Kurina, M.; Enriz, R.D. Structure-antifungal
activity relationship of related cinnamic acid derivatives. J. Agric. Food Chem. 2007, 55, 10635–10640.
[CrossRef] [PubMed]
22. Ioset, J.R.; Marston, A.; Gupta, M.P.; Hostettmann, K. Antifungal and larvicidal compounds from the root
bark of Cordia alliodora. J. Nat. Prod. 2000, 63, 424–426. [CrossRef] [PubMed]
23. Kim, J.H.; Campbell, B.C.; Mahomey, N.E.; Chan, K.L.; Molyneux, R.J. Identification of phenolics for control
of Aspergillus flavus using Saccharomyces cerevisiae in a model target-gene bioassay. J. Agric. Food Chem. 2004
52, 7814–7821. [CrossRef] [PubMed]
,
24. Gonec, T.; Bobal, P.; Sujan, J.; Pesko, M.; Guo, J.; Kralova, K.; Pavlacka, L.; Vesely, L.; Kreckova, E.; Kos, J.;
et al. Investigating the spectrum of biological activity of substituted quinoline-2-carboxamides and their
isosteres. Molecules 2012, 17, 613–644. [CrossRef] [PubMed]
25. Kos, J.; Nevin, E.; Soral, M.; Kushkevych, I.; Gonec, T.; Bobal, P.; Kollar, P.; Coffey, A.; O´Mahony, J.; Liptaj, T.;
et al. Synthesis and antimycobacterial properties of ring-substituted 6-hydroxynaphthalene-2-carboxanilides.
Bioorg. Med. Chem. 2015, 23, 2035–2043. [CrossRef] [PubMed]
26. Kos, J.; Zadrazilova, I.; Nevin, E.; Soral, M.; Gonec, T.; Kollar, P.; Oravec, M.; Coffey, A.; O’Mahony, J.;
Liptaj, T.; et al. Ring-substituted 8-Hydroxyquinoline-2-carboxanilides as potential antimycobacterial agents.
Bioorg. Med. Chem. 2015, 23, 4188–4196. [CrossRef] [PubMed]
27. Polilzer, P.; Truhlar, D.G. Chemical Applications of Atomic and Molecular Electrostatic Potentials; Plenum
Publishing: New York, NY, USA, 1991.
28. Carrupt, P.A.; El Tayar, N.; Karlen, A.; Testa, B. Molecular electrostatic potentials for characterizing
drug-biosystem interactions. Methods Enzymol. 1991, 202, 638–677.
29. Geerlings, P.; Langenaeker, W.; De Proft, F.; Baeten, A. Molecular electrostatic potentials vs. DFT descriptors
of reactivity. In Molecular Electrostatic Potentials: Concepts and Applications. Theoretical and Computational
Chemistry; Politzer, P., Maksic, Z.B., Eds.; Elsevier Science B.V.: Amsterdam, The Netherlands, 1996; Volume 3
,
pp. 587–617.

Molecules 2018, 23, 1187
14 of 14
30. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing;
M100-S22; CLSI: Wayne, PA, USA, 2012.
31. Frisch, M.J.; Trucks, G.W.; Schlegel, H.B.; Scuseria, G.E.; Robb, M.A.; Cheeseman, J.R.; Scalmani, G.; Barone, V.;
Mennucci, B.; Petersson, G.A.; et al. Gaussian 09; Gaussian, Inc.: Wallingford, CT, USA, 2009.
32. Lee, C.; Yang, W.; Parr, R.G. Development of the Colle-Salvetti correlation-energy formula into a functional
of the electron density. Phys. Rev. B 1998, 37, 785–789. [CrossRef]
33. Becke, A.D. Density-functional thermochemistry. III. The role of exact exchange. J. Chem. Phys. 1993, 98,
5618–5652. [CrossRef]
34. Becke, A.D. Density-functional exchange-energy approximation with correct asymptotic behavior.
Phys. Rev. A 1998, 38, 3098–3100.
35. Flukiger, P.; Luthi, H.P.; Portmann, S.; Weber, J. MOLEKEL 4.0; Swiss Center for Scientific Computing: Manno,
Switzerland, 2000.
Sample Availability: Samples of the compounds are available from the authors.
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