Synthesis and antimicrobial activity of some benzoxazinoids derivatives of 2-nitrophenol and 3-hydroxy-2-nitropyridine

Article abstract of DOI:10.1080/00397911.2018.1554146

Benzoxazinoids (BXs), alkaloids frequently found in Gramineae species, are natural defensives that can potentially be exploited to the development of novel antimicrobial agents. Here, BXs analogs were synthesized from 2-nitrophenol (benzoxazinone series)

Full text of DOI:10.1080/00397911.2018.1554146

Synthetic Communications
An International Journal for Rapid Communication of Synthetic Organic
Chemistry
ISSN: 0039-7911 (Print) 1532-2432 (Online) Journal homepage: http://www.tandfonline.com/loi/lsyc20
Synthesis and antimicrobial activity of some
benzoxazinoids derivatives of 2-nitrophenol and 3-
hydroxy-2-nitropyridine
William Gustavo Lima, Flávio José dos Santos, Adriana Cristina Soares,
Francisco A. Macías, José M. G. Molinillo, Jaqueline Maria Siqueira Ferreira &
João Máximo de Siqueira
To cite this article: William Gustavo Lima, Flávio José dos Santos, Adriana Cristina
Soares, Francisco A. Macías, José M. G. Molinillo, Jaqueline Maria Siqueira Ferreira & João
Máximo de Siqueira (2019): Synthesis and antimicrobial activity of some benzoxazinoids
derivatives of 2-nitrophenol and 3-hydroxy-2-nitropyridine, Synthetic Communications, DOI:
10.1080/00397911.2018.1554146
To link to this article: https://doi.org/10.1080/00397911.2018.1554146
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SYNTHETIC COMMUNICATIONSV
https://doi.org/10.1080/00397911.2018.1554146
Synthesis and antimicrobial activity of some
benzoxazinoids derivatives of 2-nitrophenol
and 3-hydroxy-2-nitropyridine
b
c
William Gustavo Limaa , Flavio Jose dos Santos , Adriana Cristina Soares ,
ꢀ
ꢀ
d
d
ꢀ
ꢀ
Francisco A. Macıas , Jose M. G. Molinillo
,
Jaqueline Maria Siqueira Ferreiraa , and Joao Maximo de Siqueira
b
~
ꢀ
a
ꢀ
ꢀ
~
Laboratorio de Microbiologia Medica, Campus Centro-oeste Dona Lindu, Universidade Federal de Sao
b
~
ꢀ
ꢀ
ꢀ
Joao del-Rei, Divinopolis, Brazil; Laboratorio de Farmacognosia e Quımica de Produtos Naturais,
~
~
ꢀ
Campus Centro-oeste Dona Lindu, Universidade Federal de Sao Joao del-Rei, Divinopolis, Brazil;
c
ꢀ
~
Laboratorio de Farmacologia da Dor e Inflamac¸ao, Campus Centro-oeste Dona Lindu, Universidade
d
~
~
ꢀ
Federal de Sao Joao del-Rei, Divinopolis, Brazil; Allelopathy Group, Department of Organic Chemistry,
Institute of Biomolecules (INBIO), School of Science, University of Cadiz, Puerto Real, Spain
ABSTRACT
ARTICLE HISTORY
Received 10 July 2018
Benzoxazinoids (BXs), alkaloids frequently found in Gramineae species,
are natural defensives that can potentially be exploited to the devel-
opment of novel antimicrobial agents. Here, BXs analogs were synthe-
sized from 2-nitrophenol (benzoxazinone series) and 3-hydroxy-2-
nitropyridine (pyridoxazinone series) and tested against fungal and
bacteria of medical interest. The starting materials were submitted to
adequate nucleophilic substitution in order to functionalize of analogs,
followed by a reductive cyclization catalyzed by palladium on carbon.
Next, the biological assays showed that pyridoxazinone serie has a
good antibacterial activity, especially against Enterococcus faecalis
KEYWORDS
Benzoxazinones; pyridoxazi-
nones; antifungal activity;
antibacterial activity;
cytotoxicity; chemo-
informatics
(Minimum inhibitory concentration—MIC: 7.8–15.6 lg.mLꢀ1
) and
Acinetobacter baumannii (MIC 31.25–125 lg.mLꢀ1). Antifungal activity,
in turn, was related to compound 2e which showed a MIC of
62.5 lg.mLꢀ1 against Candida albicans, Candida glabrata, and Candida
tropicalis. All analogs complied with Lipinski’s rules and were pre-
dicted to have a low toxicity.
GRAPHICAL ABSTRACT
ꢀ
ꢀ
Laboratorio de Farmacognosia e Quımica
de Produtos Naturais, Campus Centro Oeste Dona Lindu, Universidade Federal de Sao Joao Del-Rei, Rua Sebastiao
Gonc¸alves Coelho, no 400, Chanadour, Divinopolis, Minas Gerais CEP: 35501-293, Brazil.
CONTACT William Gustavo Lima
williamgustavofarmacia@hotmail.com
~
~
~
ꢀ
Supplemental data for this article can be accessed on the publisher’s website.
ß 2018 Taylor & Francis Group, LLC

2
W. GUSTAVO LIMA ET AL.
Figure 1. Chemical structures of the benzoxazinoids most commonly found in Graminaceae species.
Benzoxalinones: benzoxazolin-2-one (BOA) and 6-methoxy-benzoxazolin-2-one (MBOA); Lactams: 2-hydroxy-
1,4-benzoxazin-3-one (HBOA) and 2-hydroxy-7-methoxy-1,4-benzoxazin-3-one (HMBOA); Hydroxamic acids:
2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) and 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA).
Introduction
Natural compounds are currently the main search of antimicrobial agents.[1] Most agents
now clinically used in the treatment of infection are natural or have been obtained by
semi-synthesis, such as beta-lactams, aminoglycosides, and macrolides.[2] Secondary metab-
olites involved in defense processes in plants have shown great potential in the production
of new anti-infective agents.[1,2] Among them stand out the benzoxazinoids (BXs), which
are alkaloids widely distributed among monocotyledons of the Gramineae family.[3] BXs
are characterized by the presence of a 2-hydroxy-2H-1,4-benzoxazin-3(4H)–one skeleton
and can be divided into three groups: benzoxalinones, represented by benzoxazolin-2-one
(BOA) and 6-methoxy-benzoxazolin-2-one (MBOA); lactams represented by 2-hydroxy-
1,4-benzoxazin-3-one
(HBOA)
and
2-hydroxy-7-methoxy-1,4-benzoxazin-3-one
(HMBOA); and hydroxamic acids represented by 2,4-dihydroxy-1,4-benzoxazin-3-one
(DIBOA) and 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) (Figure 1).[3]
Glycosidic forms of lactams and hydroxamic acids are also frequently found.[4]
Benzoxazinoids potentially act as anti-inflammatories, antinociceptives, stimulants of
reproductive and immune systems, antitumor agents, antiretrovirals, and appetite sup-
pressors.[3,5] Antibacterial and antifungal activities have also been described.[5] Thus, in
view of the potential antimicrobial of benzoxazinoids and with the aim to maximize
this pharmacological property, our group has developed and synthesized a new series of
benzoxazinoids analogs. Specifically, a set of C2-substituted compounds were produced
from 2-nitrophenol (benzoxazinone series) and 3-hydroxy-2-nitropyridine (pyridoxazi-
none series).
Results and discussion
The benzoxazinone series (1a–e) was successfully obtained by treatment of 2-nitrophe-
nol with potassium hydroxide (KOH) diluted in ethanol followed with nucleophilic sub-
stitution and reductive cyclization, in accordance with Macias et al.[6] Pyridoxazinone
series (2a–e), in turn, was synthesized in same reaction conditions of benzoxazinones,
differing only in starting material that, in this case, was the 3-hydroxy-2-nitropyridine.

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3
To the functionalization of compounds with the addition of different moieties at C2
position, the starting materials employed were substituted in adequate positions by an
aromatic nitration reaction.
After the reaction completed, the products were efficiently purified by crystallization
and column chromatography on silica gel. All the compounds and their intermediates
1
were also successfully characterized using Infrared (IR), H and 13C Nuclear Magnetic
Resonance (NMR) and High-resolution Mass Spectrometry (HRMS). The benzoxazi-
none series (1a–e) was synthesized yield ranging of 51–71% and their spectral data
already has been described in the literature.[6] Pyridozaxinone analogs showed better
yield with a range of 59–84%. The IR spectrum of compounds 2a–e showed a strong
absorption band at 1621–1762 cmꢀ1 due to carbonyl group (C=O) in benzoxazin-3-one
ring. Stretching frequency at 3415 cmꢀ1 indicates the presence of secondary amine
(–NH) and hydroxyl (–OH) group. In addition, absorption band at 1244 cmꢀ1 indicated
the C–O–C linkage present in compounds of this series. In 1H NMR spectra, the
appearance of doublet peaks in compounds 2a–e at region d = 7.07–8.04 ppm was due
to three protons in pyridine ring (C4–H, C5–H, and C6–H). Protons at position C10–H
of benzoxazin-3-one ring is observed as a multiplet at d = 4.69–4.89 ppm, and the pro-
ton attached with O of hydroxyl group gave a singlet at d = 9.83–10.55 ppm. The 13C
NMR spectra of pyridozaxinone compounds (2a–e) have showed characteristic signals
at d = 119.71–142.62, which is related to the carbon atoms that form pyridine ring
(C2–C6). In turn, the carbon atoms of benzoxazin-3-one ring displayed peaks in the
range d = 161.44–163.94 ppm and d = 67.89–79.81 due to C = O and C–O–C, respect-
ively. Finally, the signals of carbons from side chains of compounds 2a–e vary from to
d = 9.04–33.11. The HMRS analyses showed that the molecular ions peaks of pyridoxa-
zinone compounds is in agreement with the calculated value of the proposed structure
and polarimetric studies have showed that the products are a racemic mixture. The
1
details of spectral data for all the compounds of this series, as well as 13C and H ana-
lysis, are presented in the supplementary file.
Biologic activity
The minimal inhibition concentration (MIC) of all the compounds was determined by
broth microdilution method against several bacterial and yeast of medical interest. In
antibacterial assays, the Gram-positive microorganisms were most susceptible, particu-
larly Enterococcus faecalis and Staphylococcus aureus (Table 1). Among the pyridoxazi-
nones analogs, the derivative 2e showed a high antibacterial activity against E. faecalis
(MIC 7.8 lg.mLꢀ1) and S. aureus (MIC 31.2 lg.mLꢀ1) and was moderately active
against Streptococcus agalactiae (MIC 125 lg.mLꢀ1). However, the acetyl group in the
compound 2c increased the activity for S. agalactiae (MIC 62.5 lg.mLꢀ1), making them
more active towards this species than the antibiotic chloramphenicol (CHL). The com-
pounds 2e (S. aureus MBC 62.5 lg.mLꢀ1 and E. faecalis MBC 15.6 lg.mLꢀ1), 2d
(E. faecalis and S. aureus MBC 125 lg.mLꢀ1) and 2 b (S. agalactiae MBC 250
lg.mLꢀ1), in addition to bacteriostatic activity, showed a bactericidal effect. In turn, the
benzoxazinone derivatives were inactive against S. agalactiae, but maintained their pat-
tern of activity for S. aureus and E. faecalis, with MICs in the range of 62.5–250

4
W. GUSTAVO LIMA ET AL.
Table 1. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
of benzoxazinoids analogs against Gram-positive and Gram-negative bacteria.
Bacterial strains, MIC (MBC) (in lg.mLꢀ1 a
)
Gram-positives
Gram-negatives
E.
S.
S.
K.
A.
P.
E.
faecalis
19433
aureus
29213
agalactiae pneumoniae baumannii aeruginosa
29106 43816 19606 15442
cloacae
23355
E.
ꢁ
Compounds
coli 25922
1a
125 (125)
125 (250) >250 (ND) >250 (ND) 250 (>250) >250 (ND) >250 (ND) >250 (ND)
1b
1c
62.5 (62.5) 62.5 (125) >250 (ND) >250 (ND) >250 (ND) >250 (ND) 125 (>250) 250 (>250)
62.5 (125) 125 (125) >250 (ND) >250 (ND) >250 (ND) >250 (ND) 250 (>250) 250 (>250)
1d
1e
2a
2b
250 (250) 250 (>250) >250 (ND) >250 (ND) >250 (ND) >250 (ND) >250 (ND) >250 (ND)
125 (250) 250 (>250) >250 (ND) >250 (ND) 250 (>250) >250 (ND) 250 (>250) >250 (ND)
>250 (ND) >250 (ND) >250 (ND) 250 (>250) 62.5 (>250) 250 (>250) 250 (>250) 250 (>250)
>250 (ND) 125 (>250) 250 (250) 125 (>250) 31.2 (250) >250 (ND) 125 (>250) 125 (250)
>250 (ND) 250 (>250) 62.5 (>250) 125 (>250) 31.2 (250) 250 (>250) 125 (>250) 125 (>250)
2c
2d
15.6 (125)
125 (125) 125 (>250) 125 (>250) 31.2 (250) >250 (ND) 125 (250)
62.5 (250)
2e
7.8 (15.6) 31.2 (62.5) 125 (>250) >250 (ND) 125 (>250) >250 (ND) 125 (>250) 125 (>250)
CHL
AMO
Small MICs
that
CHL/
AMO#
3.9 (ND)
0.9 (0.9)
0/0
3.9 (7.8) 125 (>250) 3.9 (>250) 250 (>250) 15.6 (250) 0.9 (31.25)
0.9 (7.8)
>250 (ND) 125 (250) >250 (ND) 250 (>250) 3.9 (15.6)
0/4 6/5 0/2 0/8 0/0
0.2 (0.4)
0/0
0.9 (3.9)
1/0
CHL: Chloramphenicol; AMO: Amoxicillin; ND: Not determined.
aꢁValues are the average of three readings.
Due to fastidious growth and low turbidity generated by E. faecalis the reading, in this case, was realized spectro-
photometrically at 600 nm.
#It shows the amount of compound that showed lower MICs values that the reference drugs.
lg.mLꢀ1. In addition, a bactericidal effect was found among these series, with MBC
ranging from 62.5 to 250 lg.mLꢀ1
.
Gram-negative bacteria were considerably less susceptible to benzoxazinone analogs.
A moderate antibacterial activity was observed only for 1c against Enterobacter cloacae
(MIC 125 mg.mLꢀ1). However, pyridoxazinones showed a high activity especially against
Acinetobacter baumannii, with the compounds containing methyl (1b), acetyl (1c), and
propyl (1d) groups in C2 being the most active ones (MIC 31.2 mg.mLꢀ1). The antibac-
terial activity of these compounds against A. baumannii was eight and four times higher
than that of CHL (MIC 250 lg.mLꢀ1) and amoxicillin (MIC 125 mg.mLꢀ1), respectively.
The same analogs also showed a good activity against E. coli (MIC in the range of 62.5
to 125 mg.mLꢀ1). However, Pseudomonas aeruginosa showed a significant resistance to
all compounds tested. Medical problems associated with this pathogen are often difficult
to control because of the intrinsic resistance that P. aeruginosa shows to various clinic-
ally used antibacterial agents is notable.[7] Thus, this feature helps to explain the absence
of an activity against P. aeruginosa.
In terms of the antifungal effect, pyridoxazinone compounds showed greater activity
(Table 2). The species C. albicans was the most sensitive, showing a considerable sus-
ceptibility to 2e (MIC 62.5 lg.mLꢀ1), which was two times more active than ketocon-
azole (KTZ; MIC 125 lg.mLꢀ1). Furthermore, the butyl group in the 2e compound
conferred a good antifungal activity against Candida glabrata (MIC 62.5 lg.mLꢀ1) and
Candida tropicalis (MIC 62.5 lg.mLꢀ1) and a moderate activity against Candida krusei
(MIC 125 lg.mLꢀ1). The effect of this substituent is predominantly fungicidal, with
MFC in the range of 62.5–125 mg.mLꢀ1. Herein, the microbicidal activity is of

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5
Table 2. Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of
benzoxazinoids analogs against Candida species.
Yeasts, MIC (MFC) (lg.mLꢀ1 a
)
Compounds
C. albicans 10231
C. krusei 34135
C. glabrata 2001
C. tropicalis 28707
1a
1b
1c
1d
1e
2a
2b
2c
2d
2e
KTZ
NYS
>250 (ND)
>250 (ND)
>250 (ND)
250 (>250)
125 (250)
250 (125)
250 (>250)
>250 (ND)
125 (>250)
62.5 (62.5)
125 (>250)
4(4)
>250 (ND)
>250 (ND)
>250 (ND)
>250 (ND)
>250 (ND)
250 (>250)
>250 (ND)
250 (>250)
>250 (ND)
125 (250)
7.8 (7.8)
>250 (ND)
>250 (ND)
>250 (ND)
125 (>250)
62.5 (>250)
250 (>250)
250 (>250)
>250 (ND)
125 (250)
62.5 (125)
125 (>250)
4(4)
>250 (ND)
>250 (ND)
>250 (ND)
>250 (ND)
250 (250)
250 (250)
250 (>250)
>250 (ND)
250 (>250)
62.5 (250)
31.2 (62.5)
2(2)
8(8)
0/0
Small MIC that KTZ/NYS#
4/0
2/0
0/0
KTZ: Ketoconazole; NYS: Nystatin; ND: Not determined.
aValues are the average of three readings.
#It shows the amount of compound that showed lower MICs values then the references drugs.
paramount importance because substances able to kill pathogens are presented as strong
candidates for clinical use.[8] Among the benzoxazinone analogs, the derivatives butyl-
substituted in C2 (1e) stand out, revealing the importance of low-polar groups in deter-
mining the antifungal activity. In this case, C. glabrata showed the highest sensitivity
(MIC 62.5 mg/mL) although the absence of nitrogen in the aromatic nucleus of benzox-
azinoids reduced the fungicidal effect (MFC range from 250 to >250 mg.mLꢀ1).
The cytotoxicity of the synthesized compounds was performed via the MTT method.
The CC50 against Vero and BHK-21 cells was above the highest concentration tested
(100 mg.mLꢀ1) for all compounds. Subsequently, the selectivity index (SI) was calculated
through the ratio between the geometric mean of MICs values and the CC50 of the
compounds (Table 3).[9] The analogs 1d (1.36) and 1e (1.62) showed selectivity to bac-
terial pathogens in relation to mammalian cells. Selectivity to fungal cells was observed
only with the compound 2e (SI 1.36). In addition, the investigation of the computa-
tional chemistry of these compounds (Table S1) revealed a low systemic toxicity, sug-
gesting that benzoxazinone and pyridoxazinone analogs have a low and absence of
tumorigenic capacity, respectively. The toxic effect on the reproductive system was pre-
dicted to be absent in both compound series.
Structure–activity relationship (SAR) study has revealed that the presence of a pyri-
dine ring (in the pyridoxazinone series) is directly associated with an antibacterial activ-
ity against Gram-negative microorganisms (Figure 2). At physiological pH levels, it is
suggested that this nitrogen is protonated, ensuring a positive charge of the pyridine
analogs. The outer membrane (OM) of Gram-negative bacteria is chemically character-
ized by the presence of lipopolysaccharides, rich in negative charges.[10] Thus, the posi-
tively charged nitrogen in pyridoxazinones increases the affinity to the negatively
charged OM of Gram-negatives, justifying in this way the great activity against these
microorganisms. In addition, different substituents at C2 contribute directly to a spe-
cies-specific effect (Figure 2). Among benzoxazinone analogs, the methyl (1b) group
selects for anti-staphylococcal and anti-streptococcal activities. The butyl group (1e), in
turn, selects the C. glabrata within the antifungal profile of this series. Once inside the

6
W. GUSTAVO LIMA ET AL.
Table 3. Cytotoxic concentration (CC50) and selective index (SI) of benzoxazinoids analogs.
GM (mg.mLꢀ1 b
)
SIc
Compound
CC50 (mg.mLꢀ1 a
)
Bacteria
Fungi
Bacteria
Fungi
1a
1b
1c
1d
1e
2a
2b
2c
2d
2e
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
189.46
111.33
113.19
74.30
–
–
–
0.53
0.91
0.89
1.36
1.62
0.64
0.96
0.57
0.40
0.40
–
–
–
0.57
0.81
0.40
0.81
0.40
0.64
1.36
176.78
125.00
250.00
124.93
250.00
157.49
74.32
62.46
157.49
105.11
176.78
250.00
250.00
aCC50 is the concentration necessity to kill 50% of cells in relation to the growth control.
bGeometric mean (GM) of MICs values of the benzoxazinoids against bacteria and fungi (for the calculate, the values of
MIC >250 ware disregarded).
cSelectivity index (SI) is a ratio of is a ratio of CC50 to GM of MICs values. For the fact of all CC50 were higher than the
highest concentration tested drug, for the purpose of calculating the SI, the value used was 100 in all cases. Large SI
indicates a more security compound.
Figure 2. Schematic representation of pharmacophoric scaffold detected in benzoxazinoids analogs.
pyridine analogs, the presence of methyl (2b), acetyl (2c), and propyl (2d) substituents
increases the selectivity for A. baumannii, restricting the antibacterial spectrum for this
species. The methyl group also selects the anti-Candida activity, being considerably
higher against C. albicans.
Finally, the pharmaceutical potential of the compounds was predicted by determin-
ation of the drug-likeness, using the software package DataWarrior version 4.2.2.
Druglikeness is an indicator that combines topological descriptors (such as fragments of
commercialized drugs with associated drug-likeness scores) and physical-chemical prop-
erties (such as cLogP and molecular weights) to determines quantitatively the potential

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SYNTHETIC COMMUNICATIONSV
7
therapeutic of a molecule. Many of the clinically available drugs have a drug-likeness
score in the range of 0.5–4, whereas pharmacologically inert compounds generally have
negative values.[11] The presence of a pyridine ring did not affect the drug-likeness of
benzoxazinoids analogs, as evidenced by identical values among the series with and
without nitrogen in the aromatic nucleus. In addition, the more lipophilic derivatives
(1e and 2e: Rbutyl ꢀ3.30) showed negative values of drug-likeness, which predicts a
lower potential on the pharmaceutical market. The analogs 1b and 2b (Rmethyl: 1.69), 1c
and 2c (Rethyl: 3.20), and 1d and 2d (Rpropyl: 0.59) had drug-likeness values within the
therapeutic range. In addition, we determined the compliance of compounds with the
Lipinski’s rule of five (Ro5), aiming to evaluate whether they are permeable or poorly
absorbed via oral administration.[12] As shown in Table S1, all compounds tested are
suggesting to a good oral bioavailability.
Conclusion
We have accomplished benzoxazinone and pyridoxazinone analogs through the nucleo-
philic substitution followed by reductive cyclization of starting material 2-nitrophenol
and 3-hydroxy-2-nitropyridine, respectively. Our results lead us to infer that the ben-
zoxazinoids analogs in this study seem to function as potential prototypes of new anti-
microbials, based on their good activity and low toxicity. The pyridine ring is an
important pharmacophoric scaffold to antibacterial activity against Gram-negative spe-
cies, such as A. baumannii. In addition, the compound 1e showed a high antibacterial
activity against E. faecalis and was also effective against C. albicans.
Experimental
Chemistry
Chemical compounds employed in the synthesis (e.g., solvent, reagents and catalyst)
were commercially acquired from Sigma-Aldrich Co. (Germany) in adequate purity and
used without previous treatment. Spectrometric analysis in infrared (IR) was determined
on an IR Affinity-1 (Shimadzu) spectrophotometer in the range of 400–4000 cmꢀ1
,
using potassium bromate (KBr) plates as support. The H and 13C nuclear magnetic res-
onance (NMR) spectra were recorded using DMSO-d6 and CDCl3 as solvents in both,
Bruker DPX 200 and Bruker 400 spectrometers. Chemical shifts were reported in parts
per million (d = ppm); the coupling constants (J) were expressed in Hertz (Hz) and sig-
nals were described as singlet (s), doublet (d), triplet (t), and multiplet (m). The reson-
1
1
ance of residual DMSO-d6 for H was set to d 2.50. The solvent peak for 13C was set to
d 39.52 (DMSO-d6) and d 77.0 (CDCl3). Tetramethylsilane (TMS) was used as internal
standard. High-resolution mass (HRM) spectra, in turn, were recorded by means of a
Bruker micro TOF-Q II quadrupole MS/MS detector. The stereochemistry of products
was analyzed in a Bellingham & Stanley ADP220 automatic polarimeter with the optical
ꢂ
path of 1 cm at a temperature of 20 C and using chloroform (CHCl3) as internal con-
trol. All compounds were isolated by crystallization using 10% hydrochloric acid fol-
lowed by filtration on Buchner funnel coated with filter paper. Subsequently, the
crystals were diluted in ethyl acetate and purified by column chromatography (10 mm)

8
W. GUSTAVO LIMA ET AL.
Figure 3. Schematic representation of reagents and conditions to synthesis of pyridoxazinone series.
(i) Nucleophilic substitution: 3-hydroxy-2-nitropyridine was treated with hydroxide of potassium (KOH;
0.1 M) diluted in ethanol for 1 h. The resulting alkoxide was re-dissolved in dimethylformamide
(50 mL/g) and ethyl-2-bromoalkylate (1.4 M), been that the product formation was monitored by TLC
until the end of the reaction. (ii) Reductive cyclization: The cyclization of resulting organic substance
was performed in palladium (Pd) on carbon (10% Pd and 10% w/w of starting material) by treating
with aqueous solution of 1,4-dioxane (1:1) (100 mL/g starting material) and sodium borohydride (2 M)
with vigorous stirring. The product formation was monitored by TLC until the end of the reaction. To
the synthesis of benzoxazinone series was employed the same reactions conditions, changing only
the starting material (2-nitrophenol).
filled with silica gel (pore size 60 Å, 70–230 mesh). Thin-layer chromatography (TLC)
analysis was performed in Sigma-Aldrich silica gel plates.
General method for the synthesis of pyridoxazinone derivatives (2a–e)
The starting material 3-hydroxy-2-nitropyridine was dissolved in a solution of 0.1 M
potassium hydroxide (KOH) in absolute ethanol (EtOH) (Figure 3(i)). After 1 h, the
solvent was removed at reduced pressure. The resulting alkoxide was re-dissolved in
N,N-dimethylformamide (DMF, 50 mL/g) and treated with 1.4 mol equiv. of ethyl-2-
bromoalkylate, been that the reaction was followed by TLC. Next, the resulting organic
substance was added to ethyl acetate and then washed with five portions of distilled
water. In this step, the organic layers were dried over anhydrous sodium sulfate
(Na2SO4), and the solvent was distilled at reduced pressure. The resultant reaction crude
product was chromatographed (column chromatography, ethyl acetate/hexane, 1:4) to
obtain the ethyl 2-[20-nitropyridine] acetate in quantitative yield.
After, the compound followed for a reaction of reductive cyclization (Figure 3(ii)). It
was performed in palladium (Pd) on carbon using 10% Pd and 10% w/w of starting
material (ethyl 2-[20-nitropyridine] acetate) suspended in an aqueous solution of 1,4-
dioxane (1:1) (100 mL/g starting material). Subsequently, sodium borohydride (NaBH4,
2 mol equiv.) was added, and the solution was vigorously stirred. We then dropwise
added the solution of nitropyridine acetate in 1,4-dioxane (0.5 g/mL). The reaction evo-
lution was controlled via TLC. Once the reaction was completed, the suspension was
vacuum-filtered through Celite to remove the catalyst, and the filtrate was treated with
10% hydrochloric acid until pH 4 was reached. The resulting products were isolated by
filtration, extracted with ethyl acetate, and dried; subsequently, they were further puri-
fied by column chromatography on silica gel to obtain the corresponding product.
General method for the synthesis of benzoxazinone derivatives (1a–e)
To the synthesis of benzoxazinone series, 3-hydroxy-2-nitropyridine was substituted by
2-nitrophenol as starting material. Next, the steps of nucleophilic substitution, reductive

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SYNTHETIC COMMUNICATIONSV
9
cyclization, and purification follow the same experimental conditions described to the
pyridoxazinone analogs. All the spectral data of these compounds have been shown by
Macias and collaborators[6].
Biologic activity
Microorganisms strains
All microorganisms employed in this study are originated from the American Type
Culture Collection (ATCC) and were kindly provided by the Reference Microorganisms
Laboratory of the Oswaldo Cruz Foundation (FIOCRUZ), Rio de Janeiro, Brazil.
Antibacterial activity was determined against three Gram-positive cocci: E. faecalis
ATCC 19433, S. agalactiae ATCC 29106, and S. aureus ATCC 29213; and five Gram-
negative rods: Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 43816, E. clo-
acae ATCC 23355, Acinetobacter baumannii ATCC 19606, and Pseudomonas aeruginosa
ATCC 15442. In turn, antifungal assay was realized employing four yeasts: Candida
albicans ATCC 10231, C. glabrata ATCC 2001, C. krusei ATCC 34135, and C. tropicalis
ATCC 28707. According to ATCC specifications, C. albicans 10231 is resistant to anidu-
lafungin, voriconazole, itraconazole, fluconazole, and ketoconazole.
Antibacterial activity
The antibacterial effect of benzoxazinoids analogs was determined by minimal inhibition
concentration (MIC) using broth microdiluition method as described in Clinical and
Laboratorial Institute guidelines M07-A9,[13] with modifications.[14] MIC was defined as
the minimum concentration of the compounds inhibiting the visible growth of the
microorganisms. The drugs chloramphenicol (CHL) (Sigma-aldrich, Germany) and
amoxicillin (AMO) (Teuto, Brazil) were used as positive controls, whereas dimethyl
sulfoxide (DMSO at 10% v/v) (Cromato, Brazil) was used as control solvent.
Additionally, the minimum bactericidal concentration (MBC) was determined by pipet-
ting 10 mL of wells that showed no growth in the MIC assay and incubating in Mueller-
Hinton agar (Himedia, Indian) for 24 h, as described previously.[14] All data were
recorded following at least three independent experiments.
Antifungal activity
Fungal susceptibility testing was performed according to norm M27-A3 of CLSI,[15]
with minor modifications.[14] Ketoconazole (Pharma Nostra, Brazil) and nystatin
(Sigma-aldrich, Germany) was included as positive control and DMSO was used at 10%
as vehicle control. For the compounds that showed fungistatic effect was determinate
the minimum fungicidal concentrations (MFC) according with Carvalho and collabora-
tors.[14] 10 mL of wells that not showed visible growth in MICs assays was plated in
Sabouraud-dextrose agar (Acumedia, USA) and incubated to 48 h. The lowest concen-
tration that impaired fungal growth was considered as MFC. All data were recorded fol-
lowing at least three independent experiments.

10
W. GUSTAVO LIMA ET AL.
Cytotoxicity
Kidney epithelial cells of African green monkey (Vero linage ATCC CCL-81) and baby
hamster kidney fibroblasts (BHK-21 linage ATCC CCL-10) were employed in order to
determine the cytotoxicity of the compounds.
The concentration that was cytotoxic to 50% of cells in the culture (CC50) was eval-
uated by the colorimetric method 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) exactly as described by Lima et al.[16] Cell viability was estimated based
on the reduction of the MTT salt from the concentration-effect curves by linear regres-
sion analysis.[16] All assays were carried out in triplicate and results are expressed as the
combined results of three independent experiments.
Acknowledgments
~
ꢁ
W.G.L. is grateful to Funda¸cao de Amparo a Pesquisa de Minas Gerais (FAPMIG) for a graduate
ꢀ
ꢀ
fellowship. JMS acknowledge Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico
(CNPq) for a research grant.
Disclosure statement
The authors declare that they have no conflict of interest. All authors contributed to the develop-
ment, analysis, and drafting of this article.
Funding
~
ꢀ
This work was supported by the Coordena¸cao de Aperfei¸coamento de Pessoal do Nıvel Superior
(CAPES) under Grant 2833-2011.
ORCID
William Gustavo Lima
http://orcid.org/0000-0001-8946-9363
http://orcid.org/0000-0003-4083-6040
http://orcid.org/0000-0002-7844-9401
ꢀ
Francisco A. Macıas
ꢀ
Jose M. G. Molinillo
Jaqueline Maria Siqueira Ferreira
http://orcid.org/0000-0002-1779-1975
~
ꢀ
Joao Maximo de Siqueira
http://orcid.org/0000-0002-2854-9383
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