Synthesis and antimicrobial evaluation of amino sugar-based naphthoquinones and isoquinoline-5,8-diones and their halogenated compounds

Article abstract of DOI:10.1016/j.ejmech.2018.06.050

Antibiotic resistance has emerged as a serious global public health problem and lately very few antibiotics have been discovered and introduced into clinical practice. Therefore, there is an urgent need for the development of antibacterial compounds with new mechanism of action, especially those capable of evading known resistance mechanisms. In this work two series of glycoconjugate and non-glycoconjugate amino compounds derived from of isoquinoline-5,8-dione and 1,4-naphthoquinone and their halogenated derivatives were synthesized and evaluated for antimicrobial activity against Gram-positive (Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 25923, S. epidermidis ATCC 12228, S. simulans ATCC 27851) and Gram-negative bacteria (E. coli ATCC 25922, Proteus mirabilis ATCC 15290, K. pneumoniae ATCC 4352 and P. aeruginosa ATCC 27853) strains of clinical importance. This study revealed that glycoconjugate compounds derived from halogeno-substituted naphthoquinones were more active against Gram-negative strains, which cause infections whose treatment is even more difficult, according to the literature. These molecules were also more active than isoquinoline-5,8-dione analogues with minimum inhibitory concentration (MIC = 4–32 μg/mL) within Clinical and Laboratory Standard Institute MIC values (CLSI 0.08–256 μg/mL). Interestingly the minimal bactericidal concentration (MBC) values of the most active compounds were equal to MIC classifying them as bactericidal agents against Gram-negative bacteria. Sixteen compounds among eighteen carbohydrate-based naphthoquinones tested showed no hemolytic effects on health human erythrocytes whereas more susceptibility to hemolytic cleavage was observed when using non-glycoconjugate amino compounds. In silico Absorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) evaluation also pointed out that these compounds are potential for oral administration with low side effects. In general, this study indicated that these compounds should be exploited in the search for a leading substance in a project aimed at obtaining new antimicrobials more effective against Gram-negative bacteria.

Full text of DOI:10.1016/j.ejmech.2018.06.050

Accepted Manuscript
Synthesis and antimicrobial evaluation of amino sugar-based naphthoquinones and
isoquinoline-5,8-diones and their halogenated compounds
Flaviana R.F. Dias, Juliana S. Novais, Talita A. do Nascimento Santos Devillart,
Wanderson Amaral da Silva, Matheus O. Ferreira, Raquel de S. Loureiro, Vinícius R.
Campos, Vitor F. Ferreira, Maria C.B.V. de Souza, Helena C. Castro, Anna C. Cunha
PII:
S0223-5234(18)30541-5
10.1016/j.ejmech.2018.06.050
EJMECH 10520
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 8 May 2018
Revised Date: 19 June 2018
Accepted Date: 21 June 2018
Please cite this article as: F.R.F. Dias, J.S. Novais, T.A. do Nascimento Santos Devillart, W.A. da
Silva, M.O. Ferreira, R.d.S. Loureiro, Viní.R. Campos, V.F. Ferreira, M.C.B.V. de Souza, H.C. Castro,
A.C. Cunha, Synthesis and antimicrobial evaluation of amino sugar-based naphthoquinones and
isoquinoline-5,8-diones and their halogenated compounds, European Journal of Medicinal Chemistry
(2018), doi: 10.1016/j.ejmech.2018.06.050.
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Graphical Abstract
This work describes the synthesis and in vitro antimicrobial evaluation of carbohydrate-
based isoquinoline-5,8-diones and 1,4-naphthoquinones and their halogenated
derivatives against Gram-positive and Gram-negative bacteria. Several halogenated
quinones exhibited promising MIC and MBC values (MIC=MBC = 4-16 µg/mL)
against two Gram Negative bacteria strains of clinical importance.

ACCEPTED MANUSCRIPT
Synthesis and antimicrobial evaluation of amino sugar-based
naphthoquinones and isoquinoline-5,8-diones and their halogenated
compounds
Flaviana R. F. Diasa, Juliana S. Novaisb, Talita A. do Nascimento Santos Devillartc,
Wanderson Amaral da Silvad, Matheus O. Ferreiraa, Raquel de S. Loureirob, Vinícius R.
Camposa, Vitor F. Ferreirae, Maria C. B. V. de Souzaa, Helena C. Castrob and Anna C.
Cunhaa*
aUniversidade Federal Fluminense, Instituto de Química, Departamento de Química Orgânica, Programa de Pós-
Graduação em Química, 24020-141, Niterói, Rio de Janeiro, Brazil.
b Universidade Federal Fluminense, PPBI, Instituto de Biologia, Departamento de Biologia Celular e Molecular,
24020-150, Niterói, Rio de Janeiro, Brazil.
c Universidade Federal Fluminense, Departamento de Patologia, Programa de Pós-Graduação em Patologia, Hospital
Universitário Antônio Pedro, 24033-900, Niterói, Rio de Janeiro, Brazil
dCentro Federal de Educação Tecnológica Celso Suckow da Fonseca, Coordenação de Licenciatura em Física, 25620-
003, Petrópolis, Rio de Janeiro, Brazil
eUniversidade Federal Fluminense, Departamento de Tecnologia Farmacêutica, Faculdade de Farmácia, Santa Rosa,
24241-002, Niterói, Rio de Janeiro, Brazil.
*Corresponding authors: Anna C. Cunha (Telephone number: +552126292148; e-mail: annac@vm.uff.br) and
Helena C. Castro (Telephone number: +5526299954; e-mail: hcastrorangel@yahoo.com.br).
Abstract
Antibiotic resistance has emerged as a serious global public health problem and lately
very few antibiotics have been discovered and introduced into clinical practice.
Therefore, there is an urgent need for the development of antibacterial compounds with
new mechanism of action, especially those capable of evading known resistance
mechanisms. In this work two series of glycoconjugate and non-glycoconjugate amino
compounds derived from of isoquinoline-5,8-dione and 1,4-naphthoquinone and their
halogenated derivatives were synthesized and evaluated for antimicrobial activity
against Gram-positive (Enterococcus faecalis ATCC 29212, Staphylococcus aureus
ATCC 25923, S. epidermidis ATCC 12228, S. simulans ATCC 27851) and Gram-
negative bacteria (E. coli ATCC 25922, Proteus mirabilis ATCC 15290, K. pneumoniae

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ATCC 4352 and P. aeruginosa ATCC 27853) strains of clinical importance. This study
revealed that glycoconjugate compounds derived from halogeno-substituted
naphthoquinones were more active against Gram-negative strains, which cause
infections whose treatment is even more difficult, according to the literature. These
molecules were also more active than isoquinoline-5,8-dione analogs with minimum
inhibitory concentration (MIC = 4-32 µg/mL) within Clinical and Laboratory Standard
Institute MIC values (CLSI 0.08-256 µg/mL). Interestingly the minimal bactericidal
concentration (MBC) values of the most active compounds were equal to MIC
classifying them as bactericidal agents against Gram-negative bacteria. Sixteen
compounds among eighteen carbohydrate-based naphthoquinones tested showed no
hemolytic effects on health human erythrocytes whereas more susceptibility to
hemolytic cleavage was observed when using non-glycoconjugate amino compounds. In
silico Absorption, Distribution, Metabolism, Excretion and Toxicity (ADMET)
evaluation also pointed out that these compounds are potential for oral administration
with low side effects. In general, this study indicated that these compounds should be
exploited in the search for a leading substance in a project aimed at obtaining new
antimicrobials more effective against Gram-negative bacteria.
Keywords: Quinone, Isoquinoline-5,8-dione, Antimicrobial, Glycoconjugate.
Introduction
In the past few decades, the discovery and development of antibiotics have
successfully led to the drastic reduction in human mortality.[1] However, the overuse
and incorrect prescription of antimicrobials have resulted in hospital and community-
acquired multi-drug resistant pathogens.[2] The rising drug resistance against first line
drugs had requested more costly second and third line drugs that are often unaffordable
or unavailable to most of developing and poor countries.[3] Recently, bacterial resistance
to oral ciprofloxacin was associated to the use of substandard and spurious quality drugs
in the developing countries.[4]
According to the Infectious Diseases Society of America (IDSA), few
substances have been discovered and/or developed against resistant Gram-negative
bacteria, such as Enterobacter spp., Pseudomonas aeruginosa, Klebsiella pneumoniae
and Acinetobacter spp.[5] The development of antimicrobial agents is far from being

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considered profitable for the pharmaceutical industries, different from those used to
treat such as chronic illness (e.g., diabetes, high blood pressure, psychiatric disorders
and asthma).[6]
Important antibiotic-resistance genes currently involve K. pneumoniae
carbapenemase (KPC), methicillin-resistant Staphylococcus aureus (MRSA),
carbapenemase-producing Enterobacteriaceae (CPE), penicillin-resistant Streptococcus
pneumoniae (PRSP), vancomycin-resistant enterococci (VRE) and extended-spectrum
β-lactamase producing Enterobacteriaceae (ESBL).[7,8] These bacteria are responsible
for causing serious life-threating infections such as hospital-acquired pneumonia,
bloodstream infections, urinary tract infections from catheters, abdominal infections and
cerebro-spinal meningitis.[7,9,10] The prevalence of infections caused by Gram-negative
bacteria has increased worldwide. They are more resistant to several or all antibacterial
agents currently available than Gram-positive bacteria[11-13] this resistance profile due to
outer membrane of the Gram-negative bacterial cell wall that acts as a barrier to many
substances, including antibiotics.[14,15]
Quinones are widely distributed in different families of plants, fungi and some
animals. Many of them have been described as cytotoxic agents for anticancer
therapy[16-19] whereas others play a role in vital biochemical processes.[20,21] Ubiquinone
(1, UQ), also called coenzyme Q, and menaquinone (2, MK), a fat-soluble vitamin K,
are essential components of the electron-transfer bacterial pathway (Figure 1).[22-25]
Figure 1. Examples of quinone derivatives as antibacterial agents.
The quinones can be cytotoxic through several mechanisms of action, including
redox cycle that affects the electron transport systems in cells.[19] As part of our research

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program on the synthesis of structurally modified quinones with biologically
antibacterial activity for medical research[26], we explored the synthesis and antibacterial
evaluation of sugar-based naphthoquinones and isoquinoline-5,8-diones. According to
the literature, representative examples of biologically active naphthoquinones include
amino compounds 3-4 (Figure 1)[26], which showed promising antimicrobial profile with
Minimum Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration
(MBC) values in range of 1.0 to 2.0 µg/ml against Escherichia coli ATCC25922 and P.
aeruginosa ATCC27853, two Gram-negative strains of clinical importance. Mono- and
di-brominated aminoquinones 5 and 6 were also effective in preventing E. coli growth
(MIC = MBC = 2-4 µg/mL).[26] Other classes of aminoquinone analogs 7-8[27,28] and
halogenated derivatives 9-11[29,30] (Figure 1) were also reported to exhibit significant
antimicrobial activity against pathogenic bacteria.
The current literature pointed the generation of active oxygen species (ROS) by
redox cycling, intercalation in the DNA double helix or alkylation of biomolecules as
inhibitory mechanisms of quinone compounds.[19,31-33] In all of them, the biological
profile requires bioreduction of the quinone nucleus as the first activating step.[19] The
reduction process of the quinones to hydroquinones involving their redox properties is
very important in many biological systems and may be related to efficacy and
selectivity profiles of these antibiotics.[33] Quinones and related analogues are also
known by their irreversibly complexation with nucleophilic amino acids in proteins,
leading to inactivation and loss of proteins function. In this case, their biological activity
is probably due to their ability to interact with cellular polypeptides, membrane-bound
enzymes and surface-exposed adhesins of pathogenic bacteria.[34,35]
Aminoglycosides represent another class of antimicrobial agents that has been
extensively studied. Carbohydrate-based antibiotics such as streptamine and
epistreptamine[36] are representative members of this family. Carbohydrates are
important as signaling molecules and for cellular recognition events, being explored for
the development of new biologically active sugar-based compounds.[37,38]
Based on our experience in the field of synthetically modified quinones[16-18,26]
we present the synthesis of two classes of quinones containing amino sugar groups
(12a-c and 15a-c), related halogen compounds (13a-c, 14a-c, 16a-c and 17a-c) and the
antimicrobial evaluation of these substances against Gram-positive and Gram-negative
bacteria of clinical importance (Figure 2).

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We also explored the synthesis and antibacterial profile of the non-halogenated
amino compounds 12d and 15d and of the halogenated derivatives 13d, 14d, 16d and
17d, to evaluate the effect of the sugar chain attached to the naphtoquinone ring on their
biological activity. Finally, we also determined the in silico ADMET parameters and in
vitro hemocompatibility of the most active compounds.
Figure 2. Structures of amino sugar-based isoquinoline-5,8-diones 12a-d and naphthoquinones
15a-d and their halogenated compounds 13a-d, 14a-d, 16a-d and 17a-d.
Results and Discussion
Chemistry
The synthesis of the derivatives 22a-c was performed as shown in the following
scheme (Scheme 1).[16] The compounds 18a-c were converted to the acetonides 19a-b
and 23 by reaction with acetone under acidic conditions.[39] Selective desprotection of
the 3,5-acetonide moiety of 23, in aqueous solution of hydrochloric acid, afforded the
monoacetonide structure 19c.[40] Reaction of the carbohydrate derivatives 19a-c with
tosyl chloride, in pyridine, furnished the corresponding tosyl esters 20a-c in good
yields.[41,42] The latter derivatives were transformed into the corresponding azide
carbohydrates 21a-c by reaction with sodium azide in DMF. Catalytic reduction (H2,
10% Pd/C) of sugar-azides 21a-c gave the aminocarbohydrates 22a-c[43-45], as outlined
in Scheme 1.

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Scheme 1. Preparation of the amino-compound 22a-c.
The reaction between 2,5-dihydroxyacetophenone (24) and methyl 3-
aminocrotonate (25) (Scheme 2) furnished 5,8-dihydroxy-1,3-dimethyl-isoquinoline-4-
carboxylate (26), which was subsequently oxidized to isoquinoline-5,8-dione (27) with
MnO2 in CH2Cl2.[46]
Scheme 2. Preparation of functionalized isoquinolinedione 27.
The Michael addition reaction, under ultrasound, between the
aminocarbohydrates 22a-c and the quinones 27-28 led to the formation of the respective
sugar-based quinones 12a-c and 15a-c, as shown in Scheme 3. These compounds were
purified by silica gel column chromatography and their structures were confirmed using

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13
1
one- and two-dimensional NMR techniques [1H, C-APT, COSY-1H x H, HSQC and
HMBC nJCH (n = 2 and 3)], IR spectroscopy and elemental analysis.
Scheme 3. Synthesis of amino sugar quinones 12a-c and 15a-c.
Major 2D HMBC correlations observed in the spectra of 27 and 12a confirmed
the regioselectivity of the addition reaction. In the spectrum of isoquinoline-5,8-dione
27 long range correlations from the methyl protons (3.04ppm) led to the assignments of
C-1 (159.9ppm) and C-8a (121.0ppm). It was also observed long-range connectivity
from H-7 (7.26ppm) to C-8a. In both spectra there were observed long range
correlations from H-6 (5.70ppm, 7.26ppm) to C-4a (138.2ppm, 135.4ppm), C-8
(181.4ppm, 185.3ppm) and C-5 (180.6ppm, 184.4ppm). The resonances of the carbonyl
carbons, C-5 and C-8, were assigned on the basis of analysis of the resonance effect of
the amino group attached to the C-7 position of the quinone ring of 12a-d, which
renders the C-5 carbon less electrophilic.
In the Scheme 4, the addition reaction of sodium azide to unsaturated carbonyl
compounds 27 and 28 gave the corresponding azidohydroquinones 30 and 31, which
were converted into the desired amino compounds 12d and 15d.[47]

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Scheme 4: Synthesis of non-substituted aminoquinones 12d and 15d.
The reactions of the quinones 12a-d and 15a-d with N-chlorosuccinimide (NCS)
and N-bromosuccinimide (NBS) gave the corresponding chlorinated compounds 13a-d,
16a-d and the brominated derivatives 14a-d, 17a-d, in good yields (Scheme 5).
Scheme 5: Preparation of quinone derivatives 13a-d, 16a-d, 14a-d and 17a-d.
Biological analysis
Aminoquinones 12a–d, 13a–d, 14a-d, 15a-d, 16a-d and 17a-d were initially
screened for antimicrobial activity against Gram-positive (Enterococcus faecalis ATCC
29212, Staphylococcus aureus ATCC 25923, S. epidermidis ATCC 12228, S. simulans
ATCC 27851) and Gram-negative bacteria (E. coli ATCC 25922, Proteus mirabilis

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ATCC 15290, K. pneumoniae ATCC 4352 and P. aeruginosa ATCC 27853) of clinical
importance. The disc diffusion test is a qualitative susceptibility assay that revealed 14
active compounds among the 24 tested, with inhibition zones against both Gram-
positive and Gram-negative bacteria (7-30 mm) similar to those of ciprofloxacin and
vancomycin (Table 1). Interestingly, within the homologous series of 7-amino-
isoquinoline-5,8-diones (12a-d), only quinone glycoconjugates 12b-c were active
(Table 1). These results suggested that the two types of sugar groups, galactopyranosyl
and xilofuranosyl, respectively, are an important requirement for the selective
antimicrobial activity of these compounds against S. epidermidis ATCC 12228 strain.
Compounds 13b-d, resulting from the incorporation of chlorine substituent in
the structure of their respective analogues 12b-d, showed different antibacterial profiles
(Table 1). The aminoquinone derivative 13b was inactive against all microorganisms
tested and 13c produced a discrete halo of inhibition against the Gram-positive
bacterium S. epidermidis ATCC 12228. Whereas 13d showed growth inhibitory halos
against all bacteria of the genus Staphylococcus similar to vancomycin. Brominated
glycoconjugate derivative 14a and non-glycosylated compound 14d showed broad
spectrum of activity against Gram-positive and Gram-negative strains. Interestingly,
similar to vancomycin and ciprofloxacin, 14d was active against four Gram-positive
bacteria (S. aureus ATCC 25923, S. epidermidis ATCC 12228, S. simulans ATCC
27851 and S. aureus MRSA (BMB9393), and two Gram-negative bacteria (E. coli
ATCC 25922 and P. aeruginosa ATCC 27853).

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Table 1: Results of antimicrobial evaluation of compounds 12a-d, 13a-d, 14a-d, 15a-d, 16a-d and 17a-d against Gram-positive and Gram-
negative bacterial strains of clinical importance using disc diffusion test
.
Inhibition zone (mm)
Gram-positive
Gram-negative
E.
faecalis
S.
S.
S.
S.
aureus
E.
coli
P.
K.
P.
Compound
aureus epidermidis
simulans
mirabilis penumoniae
aeruginosa
ATCC
29212
ATCC
25923
ATCC
12228
0
9±1
10±2
0
ATCC
27851
MRSA
(BMB9393)
ATCC ATCC
ATCC
4352
ATCC
27853
25922
15290
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
12a
12b
12c
0
0
0
0
0
0
0
12d
13a
13b
13c
13d
14a
14b
14c
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
8±1
18±2
11±2
0
15±1
13±
0
16±1
0
0
12±2
12±2
0
0
17±1
0
0
17±2
0
0
0
7
0
0
0
0
0
0
0
0
0
0
0
0
0
14d
15a
15b
15c
23±1
0
0
22±1
0
0
17±2
0
0
22±1
0
0
0
30±1
0
0
28±2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
15d
16a
16b
16c
16d
17a
17b
17c
15±1
0
17±1
9±1
8±1
15±1
21±1
10±1
0
10±1
12±1
0
18±2
7±1
0
13±2
14±1
8±1
0
9±1
17±
0
17±1
0
0
0
20±1
0
0
0
20±1
10±2
12±1
17±1
10±1
16±2
0
12±1
8±1
0
12±2
9±1
13±1
21±3
15±1
13±1
0
16±1
14±1
0
9±1
14±1
12±1
8±1
0
8±1
10±1
0
0
0
-
0
0
0
0
17d
DMSO
Cip
16±1
0
-
-
-
-
35±2
-
28±1
-
27±1
-
36±1
-
Van
16±1
16±1
15±2
16±1
14±1
Cip = ciprofloxacin and Van = vancomycin (positive controls); DMSO = dimethyl sulphoxide (negative control).

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None strains were sensitive to naphthoquinone glycoconjugates 15a-c in contrast to
four Gram-positive and two Gram-negative bacteria that showed sensitivity to the
related non-substituted amino compound 15d (Table 1).
The replacement of a hydrogen atom by electronegative halogen substituents (Cl
and Br) in inactive molecules 15a-c had a positive effect on the antimicrobial activity of
corresponding chlorinated compounds 16a-c and brominated compound 17a (Table 1).
These substances showed higher inhibition zones against Gram-positive and Gram-
negative strains (7-21 mm). Based on the positive biological results of the chlorinated
derivatives 16a-c as well as of the brominated compound 17a which have different
monosaccharide units in their structures and also in the fact that the non-halogenated
precursors 15a-c have been inactive against the bacterial strains employed in the
biological evaluation, we suggest that the presence of the halogen substituent is
essential for the antimicrobial profile of these substances. The aminonaphthoquinone
derivative 15d, which lacks the sugar moiety in its structure, and its halogenated
analogues 16d and 17d showed similar activity profiles against most bacterial strains.
Interestingly, these molecules showed activity against S. aureus MRSA (16-20mm),
which causes severe infection and is a difficult to treat strain (Table 1).
After disk diffusion analyses, the active quinone derivatives were submitted to MIC
assays to determine the lowest concentration capable of inhibit bacterial visible growth.
Promisingly, according to our MIC evaluation, the compounds 13c-d, 14a-d, 15d, 16a-
d and 17a-d presented an activity level (4-256 µg/mL) within the CLSI (CLSI 2015)
range (0.08-256 µg/mL) (Table 2).
The quantitative analysis revealed that among isoquinoline-5,8-dione derivatives
12b-c and their halogenated analogues 13c-d and 14a-d, only chlorinated amino
compound 13c exhibited a good active profile against Gram-positive bacteria, S. aureus
ATCC 25923, S. epidermidis ATCC 12228 and S. simulans ATCC 27851 (MIC = 32 to
64 µg/mL). Interestingly, two brominated compounds, 14a and 14d, inhibited the
growth of two Gram-negative bacteria, E. coli ATCC 25922 (8 and 8 µg/mL) and P.
aeruginosa ATCC 27853 (8 and 32 µg/mL). These results suggested that the
ribofuranosyl ring present in the 14a structure contributed to the increase of the
antibacterial activity in relation to P. aeruginosa.

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Table 2. Comparison of Minimum Inhibitory Concentration (MIC) of quinone compounds 12b-c, 13c-d, 14a-d, 16a-c and 17a against Gram-
positive and Gram-negative bacteria.
MIC (µg/mL)
Gram-positive
Gram-negative
Compound
S.
S. aureus
ATCC
25923
S. simulans
ATCC
S. aureus
MRSA
(BMB9393)
epidermidis
ATCC
E. coli ATCC
P. aeruginosa ATCC
25922
27853
27851
12228
12b
12c
13c
13d
14a
14c
14d
15d
16a
16b
16c
16d
ND
ND
ND
64
ND
ND
ND
32
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
8
ND
ND
ND
ND
8
256
256
32
32
ND
ND
ND
ND
64
ND
ND
ND
ND
ND
32
8
32
8
32
64
32
64
ND
128
ND
64
ND
64
ND
ND
ND
64
ND
ND
ND
ND
ND
128
ND
ND
64
16
4
128
32
8
16
17a
17c
17d
Cip
Van
ND
ND
64
ND
ND
128
ND
2
ND
ND
128
ND
2
ND
ND
64
32
16
64
16
64
32
ND
2
ND
2
0.25
ND
0.25
ND
Cip = ciprofloxacin and Van = vancomycin (positive controls). ND = no detected activity at 256 µg/mL.

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The structural analysis based on the antibacterial profile of the non-halogenated
1,4-naphthoquinones 15a-d pointed to the direct relationship of the selectivity with the
free amino group in the quinonoid ring, since only 15d inhibited bacterial strains Gram-
positive and Gram-negative. The correlated chlorinated compounds, 16a-c and 17a-c,
showed promising effects on two Gram-negative bacteria, E. coli ATCC 25922 and P.
aeruginosa ATCC 27853, with MIC in the range 4-128 µg/mL, within the values of
CLSI. The introduction of a halogen substituent on the naphthoquinone ring appears to
affect the selective inhibitory profile of compounds 16a-c and 17a-c against Gram-
negative bacteria. These data are in accordance with previous results which indicate the
halogen substituent as a modulator parameter for the antibacterial profile.[26] The
compounds 16d and 17d, containing free amino group, exhibited antibacterial profile
against Gram-positive strains, including S. aureus ATCC 25923 and S. aureus MRSA
(BMB9393). The antibacterial profile of compounds 16d and 17d against MRSA
contrasted with the absence of activity of their glycoconjugate analogs 16a-c and 17a-c
which suggested that the presence of the carbohydrate moiety is important to these
results. The sensitivity of MRSA to quinones 15d, 16d and 17d is very important as this
strain is a major public health problem worldwide over the past 20 years causing several
serious diseases such as toxic shock syndrome, endocarditis, soft tissue infections, and
necrotizing pneumonia.[48] All naphthoquinones tested (15d, 16a-d and 17a-d)
displayed a significant antibacterial profile against two Gram-negative bacteria (E. coli
and P. aeruginosa). Interestingly, the halogenated derivatives containing xylofuranosil
group 16c and 17c were selective and 2- to 32-fold more potent than their corresponding
parent compounds 16a-b and 17a-b. Apparently the enhanced antimicrobial activity of
these derivatives can be related to the chemical structure of the furanose side chain (e.g.,
conformation and intermolecular interactions). Reports in the literature[30] indicate that
2-halo-1,4-naphthoquinones are capable of inhibiting the growth of Gram-positive and
Gram-negative bacteria. They suggest that the antimicrobial effect occurs through a
competitive electron transport pathway with vitamin K or menaquinones[49], which are
essential naphthoquinone structures for the survival of several bacteria. Therefore, in the
case of the new molecules here shown, possibly the sensitive strains initially recognized
these naphthoquinone derivatives as quinones of their own biological systems, leading
to a negative interference in the metabolic pathways involved with bacterial growth.
The analysis of the antibacterial profile of compounds 12b-c, 13c-d, 14a, 14c-d,
15d, 16a-c and 17c also included evaluation of Minimum Bactericidal Concentration

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(MBC) (Table 3). This parameter defines the lowest concentration of an antibacterial
agent needed to kill the bacteria. The MBC/MIC ratio indicates the antibacterial
(bacteriostatic and/or bactericidal) profile of the compound. The MBC / MIC ratio ≤ 2
indicates bactericidal activity, while the ratio MBC / MIC ≥4 defines the bacteriostatic
effect.[50] According to our data, the MBC and MIC values of the most active
compounds 14a, 14d, 16a-b, 16c and 17a were the same, classifying them as
bactericidal agents against Gram-negative bacteria (Table 3). This is an interesting and
promising profile since this type of bacteria causes more difficult to treat infections hard
to eradicate.[51]
In this work, the naphthoquinone derivatives (15d, 16a-d, 17a and 17c-d) were
more active than those derived from the isoquinoline-5,8-diones (14a and 14d) against
Gram-negative bacteria E. coli ATCC 25922 and P. aeruginosa ATCC 27853. The
naphthoquinone derivatives 15d, 16d and 17d showed promising antibacterial activity
against two important gram positive bacteria S. aureus ATCC 25923 and S. aureus
MRSA (BMB 9393). Thus these results indicate that this type of naphthoquinone
compounds may contribute for the rational planning of the synthesis of new, more
potent molecules against bacteria of great clinical importance.

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Table 3. Bactericidal and / or bacteriostatic profiles of active compounds 12b-c, 13c-d, 14a, 14c-d, 15d, 16a-d, 17a and 17c-d based on the
calculation of the Minimal Bactericidal Concentration (MBC) / Minimum Inhibitory Concentration (MIC)
S. aureus ATCC
S. epidermidis
S. simulans ATCC
S. aureus MRSA
E. coli ATCC
P. aeruginosa
25923
ATCC 12228
27851
(BMB9393)
25922
ATCC 27853
Compound
MBC
(µg/mL) /MIC
MBC
MBC
(µg/mL) /MIC
MBC
MBC
(µg/mL)
MBC/
MIC
MBC
(µg/mL)
MBC/
MIC
MBC
(µg/mL)
MBC/
MIC
MBC
(µg/mL) /MIC
MBC
ND
ND
ND
64
-
-
256
256
32
1
1
1
1
-
ND
ND
ND
32
-
-
ND
ND
ND
ND
ND
ND
ND
64
-
-
ND
ND
ND
ND
8
-
-
ND
ND
ND
ND
8
-
-
12b
12c
13c
13d
14a
14c
14d
15d
16a
16b
16c
16d
17a
17c
17d
-
-
-
-
-
1
-
32
1
-
-
-
-
ND
ND
ND
64
ND
64
ND
ND
ND
64
-
1
-
1
-
-
1
1
1
-
-
-
ND
8
ND
32
64
32
32
8
-
32
-
-
1
1
1
1
1
1
1
1
1
1
1
-
1
-
128
ND
ND
ND
ND
ND
ND
128
1
-
1
-
32
8
ND
ND
ND
64
ND
ND
ND
128
ND
ND
128
ND
ND
ND
64
-
-
-
-
16
4
1
1
1
1
1
1
-
-
-
-
1
-
-
1
-
1
-
128
32
16
64
16
34
16
32
ND
ND
64
-
ND
ND
64
-
-
-
-
1
1
1
1
ND = no detected activity at 256 µg/mL. MBC/MIC≤2 = bactericidal activity; MBC/MIC≥4 = bacteriostatic activity.

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This study also investigated the hemocompatibility of the quinone derivatives
12a-d, 13a-d, 14a-d, 15a-d, 16a-d and 17a-d using the hemolysis test (Graphic 1).
These compounds (200 µg/mL) were added to the solution of healthy human
erythrocytes to check whether or not erythrocyte membrane lysis would occur, with
consequent release of hemoglobin. Among the 24 molecules tested, the quinone
compounds containing free amino group 12d and 15d and glycoconjugated derivatives
12a-d, 13a-b, 14a-c, 15a-c, 16a-b, 17a and 17c showed a percentage of lysis of
erythrocytes less than 10% which classified them as nonhemolytic, according to the
literature.[52] The results showed that non-halogenated isoquinoline-5,8-diones 12b-c
were less toxic than their chlorinated 13b-c and brominated compounds 14a-c. An
opposite effect occurred with the naphthoquinone series where the halogenated
derivatives 16a-c, 17a and 17c were less toxic than their non-halogenated analogues of
quinone. The quinone compounds containing free amino group 13d, 14d, 15d, 16d and
17d and three glycoconjugated derivatives 13c, 16c and 17b showed a discrete
hemolytic profile (10.08-14.43%).
Graphic 1. Hemocompatibility profile of glycoconjugate and non-glycoconjugate
amino compounds derived from of isoquinoline-5,8-dione and 1,4-naphthoquinone
through hemolytic assay. Non-hemolytic ≤10%. Van = vancomycin; Cip =
ciprofloxacin; C- = negative control (1% DMSO) and C+ = positive control (1% Triton
X-100).

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Finally, we analyzed the theoretical Absorption, Distribution, Metabolism and
Excretion (ADMET) profiles of the derivatives 12a-d, 13a-d, 14a-d, 15a-d, 16a-d and
17a-d and the results revealed all with high gastrointestinal (GI) absorption profile. This
is an important an expected parameter for oral administration medications.[53] According
to the in silico data, only non-glyconconjugate amino compounds derived from 1,4-
naphthoquinone (15d, 16d and 17d) showed cross blood-brain barrier (BBB) features.
This result suggested that the sugar groups may influence the polarity, lipophilicity and
volume of these molecules, leading to the reduction in the ability to overcome the
barrier.[54] BBB is known as avoiding the brain uptake of most pharmaceuticals[55]
including some antibiotics of clinical use such as ciprofloxacin, vancomicyn,
nitrofurantoin and cefoxitin (Table 4). Molecules that overcome this barrier may be
used in the treatment of nervous system infections (e.g., meningitis).[56,57]
Cytochrome P4503A4 is the most commonly isoform in human liver,
responsible for metabolization about 60% of the therapeutics drugs.[58] The interaction
with CYP3A4 can interfere in drug metabolism and enhance toxicological effects.
Antibiotics such as macrolides (e.g., erythromycin and clarithromycin), used to treat
respiratory tract infections, skin infections and syphilis, are moderate/potent inhibitors
of CYP3A4. This feature increases the risk of drug-drug interactions and severe toxicity
when co-administrated with CYP3A4 substrates, (e.g., sildenafil, midazolam and
omeprazole).[59,60] In this work we analyzed whether the derivatives could act as
inhibitors of the CYP3A4 isoenzyme by using the software SwissADME.
Table 4. Absorption, Distribution, Metabolism and Excretion (ADME) and theoretical
toxicity parameters, calculated by SwissADME and admetSAR software, respectively.
Comparison among glycoconjugate and non-glycoconjugate amino compounds and
antibiotics (ciprofloxacin, vancomycin, nitrofurantoin and cefoxitin) of clinical use.
ADMET parameters
Compounds
GI
BBB
CYP3A4 inhibitor Carcinogenic Mutagenic
High
High
High
High
High
High
High
High
-
-
-
-
-
-
-
-
-
-
-
-
-
+
-
12a-c
12d
-
-
13a-d
14a-d
15a-c
15d
-
-
-
-
-
-
+
-
+
-
+
-
16a-c
16d
+
+
+

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High
High
High
Low
High
Low
-
+
-
-
+
-
-
-
-
-
-
-
-
+
-
17a-c
17d
Ciprofloxacin
Vancomycin
Nitrofurantoin
Cefoxitin
-
-
-
-
+
+
+
-
-
GI = Gastrointestinal; BBB = Blood-brain barrier; CYP3A4 = Cytochrome P450 3A4.
The results predicted that 7-amino-isoquinoline-5,8-dione glycoconjugates 12a-
c, 13a-c and 14a-c and non-glycoconjugate derivatives 12d, 13d and 14d did not inhibit
CYP3A4. However, the non-glycoconjugate compounds derived from 1,4-
naphthoquinone (15d, 16d and 17d) showed inhibitory activity suggesting that the
presence of carbohydrate substituent in the parent structures 15a-c, 16a-c and 17a-c
seems to be important to avoid enzyme inhibition.
Theoretical toxicity analysis predicted glycoconjugate quinones 12a-c, 13a-c,
14a-c, 15a-c, 16a-c and 17a-c with no carcinogenic or mutagenic profiles, similar to
ciprofloxacin, vancomycin and cefoxitin (Table 4). The results highlighted the
importance of the sugar groups in contributing to the non-mutagenic effect observed for
these derivatives, since those non-glycoconjugated compounds 12d, 15d, 16d and 17d
presented a risk of mutagenic effect. It is important to note that far from assuming the
absence of risk and side effects profiles, the in silico data reinforced the promising
potential of carbohydrate-conjugated quinones for further exploring in vitro and in vivo
studies and to priorize the molecules for these evaluations.
Experimental Section
In vitro biological assay
Bacterial strains
The species used in this study were supplied by Fiocruz and obtained by the
American type culture collection (ATCC), of which 5 Gram-positives: Enterococcus
faecalis INCQS 00234 (ATCC 29212), S. aureus INCQS 00015 (ATCC 25923), S.
epidermidis INCQS 00016 (ATCC 12228), S. simulans INCQS 00254 (ATCC 27851),
and 4 Gram-negative, Proteus mirabilis INCQS 00095 (ATCC 15290), Pseudomonas

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aeruginosa INCQS 00099 (ATCC 27853), E. coli INCQS 00033 (ATCC 25922),
Klebsiella pneumoniae INCQS 00083 (ATCC 4352). We also used a multiresistant
MRSA strain, BMB9393, from the lineage ST239 of multilocus sequencing type
(MLST), which is highly disseminated in Brazilian hospitals. This variant was isolated
in 1993 from a case of nosocomial bloodstream infection in Rio de Janeiro[61], and its
complete genome was sequenced by Costa and coworkers.[62] All strains were kept in
Müller Hinton Broth (MHB) supplemented with 10% (v/v) glycerol at -70°C and were
routinely cultivated on MHB.
Disc Diffusion Test
The screening of the antibacterial activity of quinones was perfomed by the method
initially describes by Kirby-Bauer and according by the Clinical and Laboratory
Standards Institute CLSI.[63] Bacterial strains were carried out in Mueller Hinton
medium (HIMEDIA) and adjusted to 108 UFC/mL (0.5 McFarland standard) and was
uniform spread with swab at petri plate containing Agar Mueller Hinton (HIMEDIA).
The compounds were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich) 99.9%
and applied in paper disks (Laborclin) in the concentration of 5mg/mL. After the
incubation period at 37ºC between 18-24h the interpretation of result occurred by the
measurement of the inhibition zone forming around the disk. The negative control used
was DMSO. The positive controls utilized were vancomicyn (by Gram-positive strains)
and ciprofloxacin (by Gram-negative strains). The assays were realized in three
independent triplicates.
Minimum Inhibitory Concentration (MIC) using microdilution method
The MIC is defined as the lowest concentration of the substance that prevents visible
bacterial growth. The active molecules detected in disk diffusion method were diluted in
a 96-well microplate with 100 µL of liquid growth medium in geometrically increasing
concentrations (0.125 µg/mL to 256 µg/mL). Then 100 µL of bacteria suspension which
the inoculum containing 106 UFC/mL was added in each well. The highest DMSO
concentration used in this system was 5%, which had no effect on bacterial growth.
Ciprofloxacin and vancomycin were used as positive control against Gram-negative and
Gram-positive strains, respectively. After 24 hours of incubation at 37°C, the presence
of turbidity or a sediment indicates growth of microorganism and the MIC was

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determined by the lower concentration that led to no turbidity. Experiments were
performed in triplicate.
Minimum bactericidal concentration – MBC
The MBC assay was performed through the transference of the culture medium of
each well in MIC microplate with no visible growth (10 µL) to agar plates. These plates
were incubated during 24 h at 37°C and the MBC was determined as the minimum
concentration of the compounds capable of inhibiting 99.9% of bacterial growth. The
experiment was carried out in triplicate. A colorimetric assay using resazurin was also
performed and compared to the conventional plate method to reinforce the quantitative
data. An aliquot of 20 µL of 0.01% resazurin (Sigma-Aldrich) was added into each well
of the 96-well microtiter plates used for the MIC assay. The cells metabolically active
were identified by changing the color of the resazurin from blue to pink after incubation
for 2 hours at 37°C. The ratio of MBC/MIC was used to classify if the mechanism of
action of the derivatives was bactericidal (MBC/MIC≤2) or bacteriostatic
(MBC/MIC≥4).[50]
Hemocompatibility – hemolysis assay
The blood samples were donated from healthy human subjects in compliance with
ethics committee and institutional guidelines, document number 621196 approved in
May 2014 with an expiration date of May 2018. The blood was donated after reading
and signing the consent form terms. Erythrocytes were washed collected in citrate tube
and washed 3 times with PBS (pH 7.4) by centrifugation and suspended in the same
buffer. All derivatives (200 µg/mL) were incubated with the erythrocyte suspension for
3 h at 37°C. The interaction with the erythrocytes membrane and possible release of
hemoglobin was quantified by spectrophotometric reading of the supernatant at 545 nm.
The experiments were performed three times in independent way. Complete hemolysis
(positive control) was determined by using 1% Triton X-100. Hemolysis less than 10%
represented hemocompatibility and non-toxicity against erythrocyte membranes, as
described elsewhere.[52]

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In silico ADMET properties
Toxicological profile
The in silico analysis involved toxicology parameters of two series of glycoconjugate
and non-glycoconjugate amino compounds derived from of isoquinoline-5,8-dione and
1,4-naphthoquinone and their halogenated derivatives calculated by using admetSAR@
LMMD software (lmmd.ecust.edu.cn:8000/). The data were compared with the profiles
calculated for four antibacterial (cefoxitin, ciprofloxacin, nitrofurantoin and
vancomycin). The carcinogenic and mutagenic properties of the molecules were
calculated based on a data bank with over 210,000 pieces of toxicological information
for more than 96,000 compounds with 45 ADMET-associated properties in scientific
literature.[64]
ADME parameters
The Absorption, Distribution, Metabolism and Excretion (ADME) properties
were calculated using SwissADME web tool (www.swissadme.ch). The analysis was
based on molecular fingerprint that uses a sequence of bits that consider all fragments of
the molecular structure, allowing to access models for ADME behaviors in a large
virtual screening. In this work, we analyzed interaction with blood-brain barrier (BBB),
gastrointestinal absorption and inhibition of CYP450 3A4 (CYP3A4).[64]
Chemistry
Melting points were determined with a Fisher-Johns/Melting Point Apparatus
instrument and are uncorrected. Infrared (IR) spectra were recorded on a Perkin-Elmer
FT-IR, model 1600 series spectrometer in KBr pellets, and frequencies were expressed
in cm−1. NMR spectra were recorded on a Varian Unity Plus 300 MHz or 500 MHz
spectrometer, in the specified solvents. Chemical shifts (δ) are reported in ppm, and the
coupling constants (J) are expressed in Hertz. Mass spectra were recorded on a high-
resolution TOF apparatus – QTOF/Micro from Waters-MicroMass. The fragments are
described as the relation between atomic mass units and the load (m/z). Column
chromatography was performed on silica gel flash from Acros. The reactions were
routinely monitored by thin layer chromatography (TLC) on silica gel pre-coated F254

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Merck plates. The developed chromatograms were viewed under ultraviolet light at 254
nm.
Synthesis of halogenates 2-amino-naphthoquinones derivatives 16a-c and 17a-c
Aminocarbohydrate derivatives 23a-c (1.4 mmol) and N-halosuccinimide (1.35
mmol) were dissolved in 12.5 mL of methanol was kept at room temperature for 24
hours. After the solvent was evaporated under reduced pressure, the products were
purified by column chromatography on silica gel using hexane:ethyl acetate (9:1) as
eluent to yield chlorinated and brominated compounds 16a-c and 17a-c, respectively.
3-chloro-2-(methyl-5’-deoxy-2’,3’-O-isopropylidene-β-D-ribofuranosid-5’-yl)-
0
amino-1,4-naphthoquinone (16a): yield: 91% as a brown solid; m.p.: 165-166 C; IR
1
νmax (cm-1; KBr): 3266 (N-H); 1681 and 1586 (C=O), 1564 (C=C); H NMR (500.00
MHz, CDCl3) δ (ppm): 1.32 (s, 3H, CH3); 1.49 (s, 3H, CH3); 3.86-3.91 (m, 1H, H-5”);
3.43 (s, 3H, OCH3); 4.13-4.18 (m, 1H, H-5’); 4.44-4.47 (m, 1H, H-4’); 4.65 (d, 1H, J =
6,0 Hz, H-3’); 4.68 (d, 1H, J = 6.0 Hz, H-2’); 5.05 (s, 1H, H-1’); 6.46 (s, 1H, N-H); 7.63
(td, 1H, J = 7.5 and 1.0 Hz, H-6); 7.72 (td, 1H, J = 7.5 and 1.0 Hz, H-7); 8.04 (dd, 1H, J
13
= 8.0 and 1.0 Hz, H-5); 8.14 (dd, 1H, J = 7.5 and 1.0 Hz, H-8); C NMR (125.00
MHz, CDCl3) δ (ppm): 25.10 (CH3); 26.59 (CH3); 47.82 (C-5’); 55.80 (OCH3); 82.04
(C-3’); 85.46 (C-4’); 85.71 (C-2’); 110.06 (C-1’); 112.98 (-OCO-); 127.01 (C-8 and C-
5); 130.35 (C-8a); 132.72 (C-6); 132.68 (C-4a); 135.06 (C-7); 149.22 (C-2); 170.72 (C-
4); 179.19 (C-1); HRMS–ESI: m/z: [M + H]+ calcd for C19H20ClNO6, 393.0979; found:
394.1046.
3-Chloro-2-(6’-Deoxy-1’,2’:3’,4’-di-O-isopropylidene-D-galactopiranos-6’-yl)-
amino-1,4-naphthoquinone (16b): yield: 83% as a brown solid; m.p.: 93-950C; IR
1
νmax (cm-1; film): 3330 (N-H), 1602 and 1677 (C=O), 1572 (C=C). H NMR (500.00
MHz, CDCl3) δ (ppm): 1.31 (s, 3H, CH3); 1.36 (s, 3H, CH3); 1.45 (s, 3H, CH3); 1.50
(s, 3H, CH3); 3.89-3.94 (m, 1H, H-6”); 4.03-4.05 (m, 1H, H-5’); 4.21-4.24 (m, 1H, H-
6’); 4.27 (dd, 1H, J = 8.0 and 1.5 Hz, H-4’); 4.33 (dd, 1H, J = 5.0 and 2.5 Hz, H-2’);
4.63 (dd, 1H, J = 8.0 and 2.5 Hz, H-3’); 5.54 (d, 1H, J = 5.0 Hz, H-1’); 6.52 (s, 1H, N-
H); 7.61 (td, 1H, J = 7.5 and 1.5 Hz, H-6); 7.71 (td, 1H, J = 7.5 and 1.5 Hz, H-7); 8.03
13
(dd, 1H, J = 8.0 and 1.0 Hz, H-5); 8.13 (d, 1H, J = 8.0 and 1.0 Hz, H-8). C NMR

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(125.00 MHz, CDCl3) δ (ppm): 24.46 (CH3); 25.03 (CH3); 26.12 (CH3); 45.25 (C-6’);
66.98 (C-5’); 70.68 (C-2’); 71.04 (C-4’); 71.65 (C-3’); 96.53 (C-1’); 108.99 (-OCO-);
109.90 (-OCO-); 126.92 (C-5); 126.99 (C-8); 130.55 (C-8a); 132.59 (C-6); 132.76 (C-
4a); 134.94 (C-7); 147.21 (C-2); 181.79 (C-4); 189.42 (C-1). HRMS–ESI: m/z: [M +
H]+ calcd for C22H24ClNO7: 449.8815; found: 450.1302.
3-chloro-2-(5’-deoxy-1’,2’-O-isopropylidene-D-xilofuranos-5’-yl)-amino-1,4-
naphthoquinone (16c): yield: 80% as a brown solid; m.p.: 99-1010C; IR νmax (cm-1;
1
film): 3307 (N-H), 1598 and 1676 (C=O), 1566 (C=C). H NMR (500.00 MHz,
CDCl3) δ (ppm): 1.32 (s, 3H, CH3); 1.50 (s, 3H, CH3); 4.10-4.17 (m, 2H, H-5’ and H-
5”); 4.34 (d, 1H, J = 2.5 Hz, H-3’); 4.42-4.44 (m, 1H, H-4’) 4.55 (d, 1H, J = 4.0 Hz, H-
2’); 6.00 (d, 1H, J = 3.5 Hz, H-1’); 7.62 (td, 1H, J = 7.5 and 1.5 Hz, H-6); 7.70 (td, 1H,
J = 7.5 and 1.0 Hz, H-7); 8.00 (dd, 1H, J = 7.5 and 1.0 Hz, H-5); 8.12 (dd, 1H, J = 8.0
and 1.0 Hz, H-8). 13C NMR (125.00 MHz, CDCl3) δ (ppm): 26.37 (CH3); 26.91 (CH3);
62.44 (C-5’); 75.65 (C-3’); 79.25 (C-4’); 85.71 (C-2’); 104.77 (C-1’); 112.22 (-OCO-);
127.12 (C-5); 127.78 (C-8); 130.06 (C-8a); 132.34 (C-4a); 135.03 (C-7); 135.64 (C-6);
146.30 (C-2); 171.55 (C-4); 176.94 (C-1). HRMS–ESI: m/z: [M + H]+ calcd for
C18H18ClNO6: 379.0823; found: 380.0883.
3-bromo-2-(methyl-5’-deoxy-2’,3’-O-isopropylidene-β-D-ribofuranosid-5’-yl)-
amino-1,4-naphthoquinone (17a): yield: 85% as a brown solid; m.p.: 124-1250C; IR
1
νmax (cm-1; KBr): 3065 (N-H); 1683 and 1581 (C=O), 1505 (C=C). H NMR (500.00
MHz, CDCl3) δ (ppm): 1.32 (s, 3H, CH3); 1.49 (s, 3H, CH3); 3.83-3.88 (m, 1H, H-5’);
3.42 (s, 3H, OCH3); 4.18-4.23 (m, 1H, H-5”); 4.45-4.47 (m, 1H, H-4’); 4.66 (d, 1H, J =
6.0 Hz, H-3’); 4.68 (d, 1H, J = 6.0 Hz, H-2’); 5.05 (s, 1H, H-1’); 6.45 (s, 1H, N-H); 7.63
(td, 1H, J = 7.5 and 1.0 Hz, H-6); 7.71 (td, 1H, J = 7.5 and 1.0 Hz, H-7); 8.03 (dd, 1H, J
= 8.0 and 1.0 Hz, H-5); 8.14 (dd, 1H, J = 8.0 and 1.0 Hz, H-8). 13C NMR (125.00 MHz,
CDCl3) δ (ppm): 25.10 (CH3); 26.59 (CH3); 48.21 (C-5’); 55.82 (OCH3); 82.05 (C-3’);
85.46 (C-4’); 85.55 (C-2’); 110.11 (C-1’); 112.97 (-OCO-); 127.07 (C-5); 127.22 (C-8);
130.10 (C-8a); 132.66 (C-6); 132.37 (C-4a); 134.95 (C-7); 146.77 (C-2); 176.64 (C-4);
180.22 (C-1). HRMS–ESI: m/z: [M + 2H]+ calcd for C19H20BrNO6, 438.2709; found:
440.0523.

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3-bromo-2-(6’-Deoxy-1’,2’:3’,4’-di-O-isopropylidene-D-galactopiranos-6’-yl)-
0
amino-1,4-naphthoquinone (17b): yield: 88% as a brown solid; m.p.: 108-110 C; IR
1
νmax (cm-1; film): 3335 (N-H), 1599 and 1677 (C=O), 1567 (C=C). H NMR (500.00
MHz, CDCl3) δ (ppm): 1.31 (s, 3H, CH3); 1.35 (s, 3H, CH3); 1.45 (s, 3H, CH3); 1.49
(s, 3H, CH3); 3.89-3.95 (m, 1H, H-6’); 4.04-4.05 (m, 1H, H-5’); 4.25-4.27 (m, 1H, H-
6”); 4.28 (dd, 1H, J = 8.0 and 1.5 Hz, H-4’); 4.33 (dd, 1H, J = 5.0 and 2.5 Hz, H-2’);
4.64 (dd, 1H, J = 8.0 and 2.5 Hz, H-3’); 5.52 (d, 1H, J = 5.0 Hz, H-1’); 6.53 (s, 1H, N-
H); 7.57 (td, 1H, J = 14.4 and 8.0 Hz, H-6); 7.70 (td, 1H, J = 14.5 and 8.0 Hz, H-7);
13
8.02 (d, 1H, J = 7.5, H-5); 8.13 (d, 1H, J = 7.5 Hz, H-8). C NMR (125.00 MHz,
CDCl3) δ (ppm): 24.44 (CH3); 25.04 (CH3); 26.15 (CH3); 45.55 (C-6’); 66.86 (C-5’);
70.70 (C-2’); 71.04 (C-4’); 71.64 (C-3’); 96.52 (C-1’); 108.98 (-OCO-); 109.88 (-OCO-
); 127.02 (C-5); 127.14 (C-8); 130.11 (C-8a); 132.52 (C-6); 133.69 (C-4a); 134.83 (C-
7); 148.41 (C-2); 176.58 (C-4); 180.11 (C-1). HRMS–ESI: m/z: [M + 2H]+ calcd for
C22H24ClNO7: 494.0824; found: 496.0806.
3-bromo-2-(5’-Deoxy-1’,2’-O-isopropylidene-D-xilofuranos-5’-yl)-amino-1,4-
naphthoquinone (17c): yield: 68% as a brown solid; m.p.: 85-870C; IR νmax (cm-1;
1
film): 3312 (N-H), 1601 and 1660 (C=O), 1571 (C=C). H NMR (500.00 MHz,
CDCl3) δ (ppm): 1.32 (s, 3H, CH3); 1.49 (s, 3H, CH3); 4.09-4.14 (m, 1H, H-5’); 4.20-
4.23 (m, 1H, OH); 4.33-4.35 (m, 1H, H-3’); 4.35-4.38 (m, 1H, H-5”); 4.42-4.44 (m, 1H,
H-4’) 4.55 (d, 1H, J = 4.0 Hz, H-2’); 6.00 (d, 1H, J = 3.5 Hz, H-1’); 7.62 (td, 1H, J =
8.0 and 1.5 Hz, H-6); 7.70 (td, 1H, J = 7.0 and 1.0 Hz, H-7); 8.01 (d, 1H, J = 7.5 Hz, H-
13
5); 8.12 (d, 1H, J = 7.5 Hz, H-8). C NMR (125.00 MHz, CDCl3) δ (ppm): 26.36
(CH3); 26.92 (CH3); 43.81 (C-5’); 75.66 (C-3’); 79.10 (C-4’); 85.69 (C-2’); 104.96 (C-
1’); 112.19 (-OCO-); 127.07 (C-5); 127.17 (C-8); 130.13 (C-8a); 132.36 (C-4a); 132.63
(C-6); 134.93 (C-7); 147.10 (C-2); 177.24 (C-4); 180.26 (C-1). HRMS–ESI: m/z: [M +
2H]+ calcd for C18H18BrNO6, 424.2415; found: 426.0382.
Conclusion

ACCEPTED MANUSCRIPT
In this work, we synthesized the amino sugar-based isoquinoline-5,8-diones 12a-
c and the naphthoquinones 15a-c, their corresponding halogen compounds 13a-c, 14a-c,
16a-c and 17a-c, 7-amino-isoquinoline-5,8-diones 12d, 13d and 14d and
naphthoquinone derivatives 15d, 16d and 17d. These substances were evaluated for
their antimicrobial activity against Gram-positive and Gram-negative strains and the
isoquinoline-5,8-dione 14a and the naphthoquinone derivatives 16a-c, 17a and 17c
exhibited promising bactericidal activity (MIC and MBC values in the range of 4-64
µg/mL) against Gram-negative bacteria responsible for infections whose treatment is
very difficult.
Only non-glycoconjugate naphthoquinones 15d, 16d and 17d exhibited
important antimicrobial activity against two Gram positive strains S. aureus ATCC
25923 and S. aureus MRSA (BMB9393). The compounds 14a, 16a-c, 17a, 17c and 15d
did not present a hemolytic profile when tested in erythrocyte cultures and constitute
promising molecules for the rational synthetic design of novel antimicrobial agents
against bacterial strains of clinical importance.
Acknowledgements
This work was supported by the Brazilian agency FAPERJ. Fellowships granted to
UFF, by CAPES, CNPq, CNPq-PIBIC and FAPERJ are gratefully acknowledged.
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