Synthesis and antimicrobial evaluation of promising 7-arylamino-5,8-dioxo-5,8-dihydroisoquinoline-4-carboxylates and their halogenated amino compounds for treating Gram-negative bacterial infections

Article abstract of DOI:10.1039/c7ra00825b

Pathogenic bacteria may cause serious infections, such as pneumonia, which can be fatal especially to immunosuppressed individuals. Hospitalized patients are particularly susceptible to antibiotic-resistant infections, which are worsened when caused by resistant Gram-negative pathogens due to there being few therapeutic options available. Thus, this work describes the synthesis and in vitro antimicrobial profile of 7-arylamino-5,8-dioxo-5,8-dihydroisoquinoline-4-carboxylates and their halogenated aminoquinones against Gram-positive and Gram-negative bacteria. Interestingly, these bioactive substances have shown promising activity against Gram-negative pathogenic strains. Among these derivatives, two non-halogenated amino compounds exhibited promising MIC and MBC values (MIC = MBC = 1-2 μg mL^?1) against Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853, two Gram-negative strains of clinical importance. In addition, mono- and di-brominated aminoquinones were also effective in preventing the growth of E. coli (MIC = MBC = 2-4 μg mL^?1). The in vitro hemocompatibility evaluation showed a low toxicity profile for the active aminoquinones in hemolysis assays. These results suggest that these substances have potential for exploring the design of new antimicrobial prototypes against Gram-negative bacteria.

Full text of DOI:10.1039/c7ra00825b

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PAPER
Synthesis and antimicrobial evaluation of promising
7-arylamino-5,8-dioxo-5,8-dihydroisoquinoline-4-
carboxylates and their halogenated amino
compounds for treating Gram-negative bacterial
infections†
Cite this: RSC Adv., 2017, 7, 18311
Juliana S. Novais,a Vinicius R. Campos,b Ana Carolina J. A. Silva,a Maria C. B. V. de
Souza,b Vitor F. Ferreira,b Vitor G. L. Keller,b Matheus O. Ferreira,b Flaviana R. F. Dias,b
Maıra I. Vitorino,a Plınio C. Sathler,c Marcos V. Santana,a Jackson A. L. C. Resende,de
´
´
b
Helena C. Castroa and Anna C. Cunha
*
Pathogenic bacteria may cause serious infections, such as pneumonia, which can be fatal especially to
immunosuppressed individuals. Hospitalized patients are particularly susceptible to antibiotic-resistant
infections, which are worsened when caused by resistant Gram-negative pathogens due to there being
few therapeutic options available. Thus, this work describes the synthesis and in vitro antimicrobial
profile of 7-arylamino-5,8-dioxo-5,8-dihydroisoquinoline-4-carboxylates and their halogenated
aminoquinones against Gram-positive and Gram-negative bacteria. Interestingly, these bioactive
substances have shown promising activity against Gram-negative pathogenic strains. Among these
derivatives, two non-halogenated amino compounds exhibited promising MIC and MBC values (MIC ¼
MBC ¼ 1–2 mg mLꢀ1) against Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853,
two Gram-negative strains of clinical importance. In addition, mono- and di-brominated aminoquinones
were also effective in preventing the growth of E. coli (MIC ¼ MBC ¼ 2–4 mg mLꢀ1). The in vitro
hemocompatibility evaluation showed a low toxicity profile for the active aminoquinones in hemolysis
assays. These results suggest that these substances have potential for exploring the design of new
antimicrobial prototypes against Gram-negative bacteria.
Received 19th January 2017
Accepted 22nd February 2017
DOI: 10.1039/c7ra00825b
rsc.li/rsc-advances
Center for Disease Control and Prevention (CDC) has revealed
that about 2 million patients in the United States (US) develop
bacterial infections each year and 23 000 deaths are directly
related to bacterial infections.3
1. Introduction
According to the World Health Organization,1 bacterial infec-
tions are among the top ten causes of morbidity and mortality
worldwide. The bacterial resistance phenomenon and indis-
criminate antibiotic use have contributed to the restriction of
the therapeutic options to treat these infections.2 Data from the
Based on these facts and due to the high degree of resistance
of Gram-negative bacteria with the development of multidrug-
resistant (MDR – resistant to three or more classes of antimi-
crobials) and extensively drug-resistant (XDR – resistant to one
or two classes of antimicrobials) strains, and the threatening
risk of the development of pan-drug resistant (PDR – resistant to
all classes of antimicrobials) strains, researchers have paid
particular attention to the infection rate caused by this group.4,5
Usually, Gram-negative bacteria are commonly associated with
intra-abdominal infections (IAIs), bacteremia, ventilator-
associated pneumonia (VAP) and urinary tract infections
(UTIs). The main pathogens involved in these infections are
Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeru-
ginosa. Together, these species account for 70% of all Gram-
negative bacteria causing health care associated infections in
the US.6
a
´
˜
ˆ
Programa de Pos-Graduaçao em Ciencias e Biotecnologia, Universidade Federal
´
Fluminense, Niteroi, Rio de Janeiro, Brazil
b
´
ˆ
Universidade Federal Fluminense, Departamento de Quımica Organica, Programa de
´
˜
´
˜
˜
´
Pos-Graduaçao em Quımica, Outeiro de Sao Joao Batista, 24020-141 Niteroi, RJ,
Brazil. E-mail: annac@vm.uff.br; Fax: +55 21 26292145; Tel: +55 21 26292148
c
´
Universidade Federal do Rio de Janeiro, Faculdade de Farmacia, LabTIF, 21941-909,
Rio de Janeiro, RJ, Brazil
d
´
˜
Universidade Federal Fluminense, Laboratorio de difraçao de raios X, Programa de
´
˜
´
´
Pos-Graduaçao em Quımica, 24020-141, Niteroi, RJ, Brazil
e
´
Universidade Federal do Mato Grosso, Centro Universitario do Araguaia, Instituto de
ˆ
Ciencias Exatas e da Terra, 78600-000, Barra do Garças, MT, Brazil
† Electronic supplementary information (ESI) available. CCDC 1528385 and
1528399. For ESI and crystallographic data in CIF or other electronic format see
DOI: 10.1039/c7ra00825b
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These pathogens present resistance mechanisms that
involve: (a) the expression of antibiotic-inactivating enzymes,
such as carbapenemases, that usually confer resistance to b-
lactams,7 and (b) non-enzymatic mechanisms, such as low
permeability of the outer membrane and the presence of an
efficient efflux system.8 The most severe clinical cases of infec-
tions caused by Gram-negative pathogens are oen associated
with health care acquired infections caused by extended-
spectrum b-lactamase (ESBL) producing Enterobacteriaceae
(e.g. E. coli), carbapenem-resistant Enterobacteriaceae (CRE)
(e.g. K. pneumoniae), MDR Acinetobacter baumannii and MDR P.
aeruginosa.9
In nosocomial infections, an important factor for the path-
ogenicity and persistence potential of Gram-negative bacteria is
their ability to form a biolm.10 Bacteria that produce biolms
become more resistant to antibiotics than those in planktonic
form as the biolm acts as a physical barrier.11 P. aeruginosa and
E. coli are the most common Gram-negative bacteria able to
form biolms in medical devices.12,13 P. aeruginosa is also
capable of forming biolms in the lungs of cystic brosis
patients,14 whereas E. coli is frequently associated with urinary
catheter-related infections.15
Fig. 1 Examples of quinone derivatives as antibacterial agents.
All of these issues compromise the current strategies for the
treatment of infections caused by Gram-negative bacteria,
especially in view of the few therapeutic alternatives presently
available.16 There are few antibacterial agents on the market, and
combining antimicrobials, such as ceolozane–tazobactam, is
the current option for treating infections caused by resistant
strains. The ceolozane–tazobactam combination is effective
against resistant Gram-negative bacteria such as P. aeruginosa
and Enterobacteriaceae, including extended-spectrum b-lacta-
mase (ESBL) producers.17 Meanwhile, ceazidime, a broad-
spectrum cephalosporin, and avibactam, a b-lactamase inhib-
itor, are combined to treat resistant infections caused by ESBL
and Klebsiella pneumoniae carbapenemase (KPC).18
Based on this scenario and considering the prevalence of
infections caused by Gram-negative pathogens resistant to many
or all antibacterial agents available, the discovery of new agents
with potent activity against these strains is urgently required.19,20
Naturally occurring quinones are well-known as clinically
useful drugs for treating cancer.21 Although most of the quinone
derivatives show a potential cytotoxic prole against a number
of cancer cell lines,22 they are still associated with other phar-
macological activities, such as antibacterial,23 antifungal,24
antiviral,25 anti-inammatory,26 and antimalarial.27
Fig. 2 Two homologous series of aminoquinone compounds, 9a–
d and 10a–d, which are structurally related to the reported fungicidal
agents 10c–d (see ref. 34).
process with interference of endogenous vitamin K or ubiquinone
in the electron transfer chain.33
Recently Ryu, Song and Hong34 described 7-arylamino-5,8-
dioxo-5,8-dihydroisoquinoline-4-carboxylates as antifungal
agents with similar or better proles than uconazole, a current
therapeutic drug. Interestingly, these authors suggested that
the inhibition of the fungal respiratory electron-transfer
pathway was a feasible mechanism for active aminoquinone
derivatives. Since this pathway is also present in Gram-negative
and Gram-positive bacteria,35 in this article we report the
synthesis of two homologous series of 7-arylamino-5,8-dioxo-
5,8-dihydroisoquinoline-4-carboxylates (9a–d and 10a–b),
structurally related to the fungicidal agents 10c–d (Fig. 2),34 and
tested them against bacterial strains of clinical importance.
In this work, we also analyzed their mechanism of action
(bactericidal and/or bacteriostatic), the hemocompatibility
prole and the contribution of different substituents on the
phenylamino ring (9a–d), also including the bromo group at the
C-6 carbon of the aza-quinone nucleus (10a–d).
The literature about the rational design of antimicrobials
describes several aminoquinone compounds28,29 (1–6) and halo-
1,4-naphtoquinone derivatives30 (7–8) with promising inhibitory
effects against pathogenic bacteria (Fig. 1).
According to the literature, quinones may present different
antimicrobial mechanisms.31,32 These substances may act through
redox reactions producing reactive oxygen species (ROS), which
can lead to cellular damage, oxidative stress and DNA damage.31
Quinone compounds are also able to inhibit DNA topoisomerase,
an enzyme that catalyzes changes in DNA topology and is essential
2. Results and discussion
2.1 Chemistry
for bacterial replication.32 Another mechanism involves inhibition The 7-arylamino-5,8-dioxo-5,8-dihydroisoquinoline-4-carboxylates
of bacterial cell growth via a competitive electron transport 9a–d were prepared using the synthetic sequence illustrated in
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Scheme 1. The reaction between 2,5-dihydroxyacetophenone (11) bromine in a buffer solution of sodium acetate in glacial acetic
and methyl 3-aminocrotonate (12) afforded methyl 5,8-dihydroxy- acid, as shown in Scheme 2. This reaction afforded a mixture of
1,3-dimethylisoquinoline-4-carboxylate (13), which was subjected di-brominated and tri-brominated compounds (17 and 18,
to oxidation with MnO2 to furnish isoquinoline-5,8-dione 14 in respectively).
80% yield.36
Compounds 17 and 18 were not isolated due to further
Aromatic amines 15a–d were transformed into the corre- degradation during chromatographic processing. For this
sponding amino compounds 9a–d (Scheme 1) by ultrasound- reason, the crude product, which essentially consists of two
accelerated Michel addition of anilines to electrophilic major compounds, 17 and 18, was then submitted to nucleo-
quinone compound 14, under cerium catalysis, according to the philic substitution reaction with arylamines 15a–d, furnishing
procedure described in the literature34,36 with minor a mixture of the corresponding aminoquinone compounds 10a–
modications.
d and 19a–d (Scheme 3).
The regioselective introduction of aniline substituents at the
The X-ray crystallographic analysis of the amino compounds
C-7 position of 14 was conrmed based on the 2D HMBC 10d and 19d (Fig. 4) allowed the molecular structures of 17 and
spectrum of the aminoquinone derivative 9d. For example, the 18 to be conrmed (Scheme 2).
proton from the N–H bond (7.67 ppm) showed long-range
All compounds, except 19a and 19b, were isolated as pure
correlations (3JCH) with the carbon resonance for C-6 (102.8 materials by silica gel column chromatography using
ppm), C-20/C-60 (124.6 ppm) and C-8 (181.6 ppm). This latter chloroform/acetone (9.5 : 0.5) as an eluent mixture. The struc-
carbon showed correlation with the hydrogen H-6 (6.28 ppm). tures of the novel aminoquinone derivatives 9d, 10a–b and 19c–
The observed regioselectivity for products 9a–d is in agreement d were conrmed by spectral data, including one- and two-
with that reported by Ryu, Song and Hong.34 The carbonyl dimensional 1H and 13C NMR spectra [1H, 13C-APT, COSY-1H
1
carbons C-5 and C-8 were differentiated based on the resonance ꢁ H, HSQC and HMBC], IR and mass analysis.
effect caused by the presence of the amino group bonded at the
C-7 position of the quinone-5,8-dione moiety of 9a–d, which
stabilizes the C-5 carbonyl carbon making it less electrophilic
2.2 Biological assay
(Fig. 3).
The reaction of compounds 9a–d with N-bromosuccinimide
(NBS) led to halogenated derivatives 10a–d in poor yields aer
purication by silica gel column chromatography.
An alternative to the preparation of substances 10a–d con-
sisted of the bromination reaction of 14 with molecular
Initially, the antibacterial prole of the aza-quinone derivatives
9a–d, 10a–d, 14 and 19c–d was evaluated in vitro by measuring
the inhibition zone against Gram-positive (Enterococcus faecalis
ATCC 29212, Staphylococcus aureus ATCC 25923, Staphylococcus
epidermidis ATCC 12228, and Staphylococcus simulans ATCC
27851) and Gram-negative bacteria (Enterobacter cloaceae ATCC
23355, Serratia marcescens ATCC 14756, Escherichia coli ATCC
25922, Proteus mirabilis ATCC 15290, Klebsiella pneumoniae
ATCC 4352 and Pseudomonas aeruginosa ATCC 27853).
The disk diffusion data showed an inhibition zone (8–18
mm) for these compounds with different patterns. Only
compounds 9c and 9d were active against both Gram-positive
(S. aureus ATCC 25923, S. epidermidis ATCC 12228 and S.
Scheme 2 Preparation of brominated compounds 17 and 18.
Scheme 1 Synthesis of aminoquinones 9a–d.
Fig. 3 Canonic structures of compounds 9a–d.
Scheme 3 Preparation of quinone derivatives 10a–d and 19a–d.
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the second bromine at the benzylic position was responsible for
the absence of activity. On the other hand, the dibromo
compound 19d, containing a chlorine substituent on the
benzene ring of the aniline group, showed antimicrobial activity
against S. simulans ATCC 27851, E. coli ATCC 25922 and P.
aeruginosa ATCC 27853. This fact is in agreement with previous
results that indicate the chlorine atom substituent as a modu-
lating parameter for the antibacterial prole.38
Aer the disk diffusion analyses, the active compounds were
analyzed using an MIC assay to determine the lowest concen-
tration capable of inhibiting visible bacterial growth. The MIC
values listed in Table 2 revealed that the non-brominated
compounds 9c–d had promising antimicrobial proles against
Gram-negative strains (E. coli ATCC25922 and P. aeruginosa
ATCC27853) with MICs (1–2 mg mLꢀ1) close to that observed for
ciprooxacin, an antibacterial currently in use, and within CLSI
values (0.05–256 mg mLꢀ1) (Table 2).
The analysis of the antibacterial mechanism of these deriv-
atives showed signicant minimal bactericidal concentration
(MBC) values (Table 3). The MBC is complementary to the MIC,
since whereas MIC demonstrates the lowest level of antimi-
crobial necessary to inhibit growth, MBC represents the lowest
level of antimicrobial agent that results in microbial death.
According to the ratio of MBC/MIC, it is possible to identify
Fig. 4 Asymmetric unit representation of quinone derivatives 10d and
19d (ellipsoids at the 50% probability level).
simulans ATCC 27851) and Gram-negative (E. coli ATCC 25922
and P. aeruginosa ATCC 27853) bacteria (Table 1), suggesting
that the substitution pattern on the aniline ring is a funda-
mental requirement for the antimicrobial activity.
Differently, compound 10d was effective against S. aureus
ATCC 25923 and S. simulans ATCC 27851, whereas the related
brominated compounds 10a and 10c showed antimicrobial
effects against Gram-negative strains (E. coli ATCC 25922 and P.
aeruginosa ATCC 27853) (Table 1). The derivative 10b was active
against S. simulans ATCC 27851, E. coli ATCC 25922 and P.
aeruginosa ATCC 27853.
The selectivity prole of the halogen compounds 10a and 10c
was not observed for the non-brominated analogues 9a and 9c,
indicating the importance of this type of substituent for the
antibacterial activity.
Isoquinoline-5,8-dione (14) exhibited broad spectrum
activity (14–25 mm) against Gram-positive and Gram-negative
strains. These results showed the importance of the quino-
noid ring for antibacterial activity.37
a
compound's antibacterial prole (bacteriostatic and/or
bactericidal). A ratio of MBC/MIC #2 indicates bactericidal
activity, whereas a ratio of MBC/MIC $4 denes a bacteriostatic
effect.39
Interestingly, the MBC and MIC values of the active deriva-
tives 9c–d, 10c, 14 and 19d were identical against E. coli ATCC
25922 and P. aeruginosa ATCC 27853. This suggests that these
compounds can be classied as bactericidal agents against
Gram-negative bacteria, which is a more interesting prole.
19c, a dibromo analogue of compound 10c, showed no
antimicrobial activity, which suggested that the introduction of
Table 1 Antimicrobial evaluation of quinone derivatives 9a–d, 10a–d, 14 and 19c–d against Gram-positive and Gram-negative bacterial strains
of clinical importance using a disc diffusion testa
Inhibition zone (mm)
Gram-positive
Gram-negative
S. aureus
S. epidermidis
S. simulans
E. coli
P. aeruginosa
Compound
ATCC 25923
ATCC 12228
ATCC 27851
ATCC 25922
ATCC 27853
9a
0
0
0
0
0
9b
0
0
0
0
0
9c
9d
0
12 ꢂ 2
11 ꢂ 1
10 ꢂ 2
0
18 ꢂ 2
15 ꢂ 1
0
8 ꢂ 1
8 ꢂ 2
0
15 ꢂ 1
12 ꢂ 2
9 ꢂ 2
9 ꢂ 2
0
9 ꢂ 2
10 ꢂ 2
10a
10b
10c
10d
14
19c
19d
Cip
Van
DMSO
0
0
0
0
0
0
0
10 ꢂ 3
0
12 ꢂ 1
11 ꢂ 2
16 ꢂ 2
0
0
15 ꢂ 1
14 ꢂ 1
25 ꢂ 1
23 ꢂ 1
0
0
0
0
0
0
8 ꢂ 2
9 ꢂ 2
34 ꢂ 2
—
10 ꢂ 2
39 ꢂ 3
—
—
—
—
14 ꢂ 2
15 ꢂ 2
12 ꢂ 2
0
0
0
0
0
a
Cip ¼ ciprooxacin and Van ¼ vancomycin (positive controls); DMSO ¼ dimethyl sulphoxide (negative control).
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Table 2 Comparison of the minimum inhibitory concentrations (MICs) of quinone compounds 9a–d, 10a–d, 14 and 19c–d against Gram-
positive and Gram-negative bacteriac
MIC (mg mLꢀ1
)
S. aureus
S. epidermidis
S. simulans
E. coli
P. aeruginosa
Compound
ATCC 25923
ATCC 12228
ATCC 27851
ATCC 25922
ATCC 27853
9a
9b
9c
9d
10a
10b
10c
10d
14
19c
19d
Cipa
Vanb
ND
ND
ND
ND
ND
ND
ND
32
ND
ND
ND
—
ND
ND
ND
ND
ND
ND
ND
ND
128
ND
ND
—
ND
ND
ND
ND
ND
64
ND
16
128
ND
32
ND
ND
1
ND
ND
2
1
2
ND
128
4
ND
4
ND
2
0.12
—
32
128
ND
ND
8
ND
16
0.5
—
—
2
2
2
a
Ciprooxacin: positive control used for Gram-negative strains. b Vancomycin: positive control used for Gram-positive strains. c ND: no detected
activity at 256 mg mLꢀ1
.
Table 3 Analysis of the mechanism of action (bactericidal and/or bacteriostatic) through minimal bactericidal concentration (MBC) and MBC/
MIC ratio determination of the active compounds 9c–d, 10a–d, 14 and 19da
S. epidermidis ATCC
P. aeruginosa ATCC
S. aureus ATCC 25923
12228
S. simulans ATCC 27851
E. coli ATCC 25922
27853
MBC
MBC
MBC
MBC
MBC
Compound
(mg mLꢀ1
)
MBC/MIC
(mg mLꢀ1
)
MBC/MIC
(mg mLꢀ1
)
MBC/MIC
(mg mLꢀ1
)
MBC/MIC
(mg mLꢀ1
)
MBC/MIC
9c
9d
ND
ND
ND
ND
ND
32
—
—
—
—
—
1
ND
ND
ND
ND
ND
ND
128
ND
—
—
—
—
—
—
1
ND
ND
ND
64
ND
16
—
—
—
1
—
1
1
1
1
1
—
1
1
—
1
2
2
1
1
1
1
—
—
1
10a
10b
10c
10d
14
ND
128
4
ND
4
32
128
ND
ND
8
ND
ND
—
—
128
32
1
1
19d
—
2
1
16
1
a
ND: no detected activity at 256 mg mLꢀ1. A ratio of MBC/MIC #2 indicates bactericidal activity, whereas a ratio of MBC/MIC $4 denes
a bacteriostatic effect.
It is known that Gram-negative bacteria are more resistant to a selective mechanism for those effective against Gram-negative
antimicrobial agents than Gram-positive bacteria due to their strains (e.g. 9c–d). Among the mono-brominated compounds
different morphological features. These bacteria present a more tested, the derivative 10b containing methoxy groups at the
complex biological barrier that avoids the penetration of ortho- and para-positions of the aniline group exhibited the
different antibiotics, whereas their periplasmic space has highest MIC (128 mg mLꢀ1), whereas monomethoxylated related
proteins and enzymes able to break down foreign molecules.40 compound 10c showed 32 times lower MIC (4 mg mLꢀ1) and
Therefore, the identication of active molecules/lead MBC (4 mg mLꢀ1) against E. coli ATCC 25922 (Table 2). The
compounds against Gram-negative bacteria, such as those comparison between non-brominated compound 9c and
identied in this work, is extremely relevant for designing new brominated compound 10c indicated the negative effect of the
antimicrobials for medical use. This is even more important in bromine atom, which reduced the antibacterial activity against
the case of difficult-to-treat infections, like those involving Gram-negative bacteria at least four-fold (Table 2).
resistant microorganisms. Thus bactericidal compounds that
A selective antibacterial prole against Gram-negative
are able to kill microorganisms lead to a faster end of the strains was observed when comparing mono-10d and di-19d
bacterial infection, also reducing the rates of re-incidence and brominated compounds. Apparently, the second bromine atom
resistance.41
had a great effect on the bacterial activity of 19d (Table 2). This
Most of the quinone analogues showed high MIC values molecule was more potent and selective against E. coli ATCC
(>256 mg mLꢀ1) against Gram-positive strains, suggesting 25922 and P. aeruginosa ATCC 27853 than derivative 10d.
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Compound 14 is a synthetic intermediate that revealed worldwide, especially in immunosuppressed individuals such
broad-spectrum activity, detected in the disk diffusion test as those infected with human immunodeciency virus (HIV).46
(Table 1). Interestingly, the results of the MIC assay showed This situation has worsened due to the increase of multi-drug
better activity against Gram-negative strains (4–8 mg mLꢀ1), resistant pathogen strains (e.g. fungi, bacteria and viruses),
indicating that the isoquinoline-5,8-dione is an important which require more and new treatment options.47,48 Therefore,
medicinal moiety that can be modulated for targeting Gram- the discovery of molecules with antifungal and antibacterial
negative strains. Additionally, a Michael 1,4-addition type proles such as 10c–d, as well as exploring the dual mechanism
reaction between 14 and p-chloroaniline or p-methoxyaniline in new antibacterial derivatives such as 9c–d for treating
led to the formation of the corresponding compounds 9c–d, immunosuppressed individuals such as those with HIV or
which exhibited the best antibacterial activity among all of the cancer is interesting.
molecules studied, with MIC values of 1.0 mg mLꢀ1 and 2.0 mg
mLꢀ1 against E. coli ATCC 25922 and P. aeruginosa ATCC 27853.
3. Conclusions
Hemolysis tests on erythrocytes performed with compounds
9c–d, 10a–d, 14 and 19d showed that these quinone derivatives In summary, we have reported the efficient synthesis and in
had no hemolytic effect aer 3 hours of incubation (0.65–8.6%) vitro antimicrobial activity of 7-arylamino-5,8-dioxo-5,8-
(Chart 1). Thus, these compounds can be identied as hemo- dihydroisoquinoline-4-carboxylates 9a–d and their haloge-
compatible and non-cytotoxic to erythrocyte membranes, nated amino derivatives 10a–d and 19c–d. Among these ami-
according to Fisher and co-workers (lysis rate <10%).42
noquinone compounds, the non-halogenated substances 9c–
It is important to highlight that the aminoquinones 10c– d showed promising antimicrobial proles with MIC and MBC
d were recently described due to their antifungal effect against values in the range of 1.0 to 2.0 mg mLꢀ1 against Gram-negative
pathogenic fungi strains.34 The results from these studies strains (E. coli ATCC25922 and P. aeruginosa ATCC27853). In
showed that the substituted phenylamino group at the quinone addition, the brominated compounds 10c and 19d and their
nucleus plays an important role in the antifungal activity, similar synthetic precursor 14 showed selective antimicrobial activity
to what we observed in the case of antibacterial activity. The against E. coli ATCC25922, with both MIC and MBC values in
authors34 suggested that 5,8-dioxo-5,8-dihydroisoquinolines the range of 2.0 to 4.0 mg mLꢀ1
related to the compounds 10c–d can act as inhibitors of the
Our results suggest that the antibacterial effect with
.
cytochrome b complex through the blockade of mitochondrial a bactericidal prole of these compounds against Gram-
electron transport in fungi. Considering that this is a pathway negative bacteria is of importance, since the emergence of
also present in bacteria and that quinones and related analogs resistant strains continues and this type of bacteria is of great
may act as redox-active molecules, this is also a possible mech- concern. The derivatives 9c–d, 10c, 14 and 19d had no hemo-
anism of action for the active compounds 9c–d, 10a–d, 14 and lysis prole when tested on erythrocyte cultures, and constitute
19d. The structures of these substances are in agreement with an promising prototypes for further analysis on antimicrobial
electron-acceptor prole, which corroborates this hypothesis. therapy.
The use of orthogonal assays to further explore quinone deriv-
atives as inhibitors of the mitochondrial respiratory chain
enzyme cytochrome b will be extremely useful, and will ratio-
nally assist the selection of these molecules for further in vivo
and in vitro assays.43–45
4. Experimental
4.1 In vitro biological assay
Bacterial strains. Bacterial strains were obtained from the
According to the World Health Organization (WHO), the American Type Culture Collection (ATCC) including Gram-
incidence of fungal and bacterial infections is a serious problem positive (Enterococcus faecalis ATCC 29212, Staphylococcus
aureus ATCC 25923, S. epidermidis ATCC 12228, and S. simulans
ATCC 27851) and Gram-negative (Enterobacter cloaceae ATCC
23355, Serratia marcescens ATCC 14756, Escherichia coli ATCC
25922, Proteus mirabilis ATCC 15290, Klebsiella pneumoniae
ATCC 4352 and Pseudomonas aeruginosa ATCC 27853) bacteria.
All strains were maintained in 10% glycerol stocks and were
ꢃ
stored at ꢀ20 C until use.
Disk diffusion method. Initially, the antibacterial prole of
the isoquinoline derivatives was evaluated using the disk
diffusion method based on Kirby–Bauer's description and
according to the recommendations of the Clinical and Labora-
tory Standards Institute.49 Briey, we prepared a bacterial
suspension (1.0 x 108 colony-forming units (CFU mLꢀ1)), which
corresponded to McFarland standard 0.5. This suspension was
¨
inoculated on a Petri dish containing Muller Hinton agar
Chart 1 Hemolytic profile through hemolysis assay. Values below 10%
are considered non-hemolytic. 1% DMSO (Cꢀ) and Triton X-100 (C+)
are the negative and positive controls, respectively.
(HIMEDIA) and aer drying, paper disks (6 mm in diameter –
Laborclin®) with 30 mg per disk of derivative dissolved in 100%
18316 | RSC Adv., 2017, 7, 18311–18320
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dimethyl sulfoxide (DMSO – Sigma-Aldrich) were applied onto
them. Aer 24 h at 37 ꢃC, the inhibition zone formed around the
disks was measured in mm. Vancomycin and ciprooxacin were
used as positive controls, whereas DMSO (100%) was used as
4.2 Synthesis of 7-arylaminoisoquinolinequinone
derivatives 9a–d
Into a 50 mL round-bottom ask were added 245 mg (1.0 mmol)
of isoquinoline-5,8-dione (14), 2.0 mmol of the required aryl-
amine 15a–d, 18.5 mg (0.05 mmol) of CeCl3$7H2O and 20 mL of
anhydrous ethanol. The reaction mixture was stirred at room
temperature for 5 hours. Aer removal of the solvent under
reduced pressure, the aminoquinone derivatives 9a–d were
puried using a silica gel chromatographic column, using
chloroform/acetone (9.5 : 0.5) as the eluent.
a
negative control. The experiments were performed in
triplicate.
Minimum inhibitory concentration (MIC) by the micro-
dilution method. Active compounds detected by the disk
diffusion assays were subjected to MIC analyses to determine
the lowest concentration that inhibits bacterial growth. First,
a three-fold derivative dilution (256–0.125 mg mLꢀ1) was
prepared in a 96-well microplate, with a volume of 100 mL in
each well. The highest DMSO concentration used was 5% with
no detectable effect on bacterial growth. Secondly, 100 mL of
bacterial inoculum containing 106 CFU mLꢀ1 was added. A
negative control was prepared using the inoculum without
derivative, whereas vancomycin and ciprooxacin were used as
Methyl 7-(4-chlorophenyl)amino-1,3-dimethyl-5,8-dioxo-5,8-
dihydroisoquinoline-4-carboxylate (9d). The substance 9d was
ꢃ
obtained as an orange solid, mp: 284–286 C, in 85% yield.
IR nmax (cmꢀ1; KBr): 3300 (N–H), 1715 and 1669 (C]O), 1074
and 1255 (C–O).
1H NMR (500.00 MHz, CDCl3) d (ppm): 2.61 (s, 3H, CH3), 2.98
(s, 3H, CH3), 4.00 (s, 3H, OCH3), 6.28 (s, 1H, H-6), 7.19 (d, 2H, J ¼
8.5 Hz, H-20 and H-60), 7.39 (d, 2H, J ¼ 8.5 Hz, H-30 and H-50),
7.67 (s, 1H, N–H).
ꢃ
positive controls. The microplates were incubated at 37 C for
24 h and the result was determined by visual reading of the
lowest concentration with no detectable turbidity (growth). The
experiments were performed in triplicate.
13C NMR (125.0 MHz, CDCl3) d (ppm): 23.1 (CH3), 26.2 (CH3),
53.2 (OCH3), 119.9 (C-8a), 124.2 (C-20 and C-60), 125.3 (C-4),
130.1 (C-30 and C-50), 131.8 (C-40), 135.6 (C-10), 137.7 (C-4a),
102.8 (C-6), 145.3 (C-7), 161.2 (C-3), 161.5 (C-1), 169.1 (C-4b),
181.5 (C-5), 181.6 (C-8).
Minimal bactericidal concentration (MBC). The MBC value
was determined by transferring the culture medium of each well
with no visible growth (10 mL) to agar plates. These plates were
incubated for 24 h at 37 ꢃC and the MBC was dened as the
minimum concentration of derivative that showed $99.9%
reduction of the original inoculum. The experiment was
repeated at least three times. A colorimetric assay using resa-
zurin was also performed and compared to the conventional
plate method to reinforce the quantitative data. Briey, 20 mL of
HRMS [M + H]+: m/z calcd for C19H15N2O4: 371.0799; found:
371.0777.
4.3 Synthesis of 7-arylamino-6-bromo-isoquinolinequinone
compounds 10a–d and dibrominated derivatives 19a–d
0.01% resazurin (Sigma-Aldrich) was added into each well of the Into a 50 mL round-bottom ask were added 245 mg (1.0
96-well microtiter plates used for the MIC assay, aer the mmol) of isoquinoline-5,8-dione (14), 660 mg (8.0 mmol) of
previous incubation with the derivative, and re-incubated for 2 NaOAc and 8 mL of HOAc. The reaction mixture was stirred
hours. The MBC was determined in the well treated with the at room temperature for 10 min and then 0.1 mL of bromine
lowest derivative concentration that did not change from blue to was added to the reaction medium. The resulting mixture
pink.50 The ratio of MBC/MIC was used to determine whether was kept in an ultrasonic water bath for 60 minutes. Aer
the mechanism of action of the derivatives was bactericidal this period, the reaction was quenched by addition of 200 mL
(MBC/MIC ¼ 1 or 2) or bacteriostatic (MBC/MIC ¼ 4 or 16).51
of water and the crude residue consisting mainly of a mixture
Hemocompatibility – hemolysis assay. The experiment was of di- and tri-brominated quinones, 17 and 18, respectively,
conducted according to Sathler and collaborators (2014).52 was employed in the next reaction step without prior
Samples were obtained from healthy human subjects in purication.
compliance with laws and institutional guidelines, with the
The products 17 and 18 (200 mg) and the appropriate aryl-
approval of the institutional ethics committee in humans amine (0.15 mmol) were mixed together in EtOH (20 mL). The
research according to the decision 621196 approved in May reaction mixture was stirred at room temperature for 60 min.
2014 with an expiration date of May 2018. The consent terms Aer removal of the solvent under reduced pressure, the mono-
were presented to research participants and signed in all and di-brominated compounds were puried by silica gel
experiments involving humans. Erythrocytes were washed 3 chromatography using chloroform : acetone (9.5 : 0.5) as the
times with PBS (pH 7.4) by centrifugation and suspended in the eluent.
same buffer. All derivatives (200 mg mLꢀ1) were incubated with
Methyl 6-bromo-7-phenylamino-1,3-dimethyl-5,8-dioxo-5,8-
the erythrocyte suspension for 3 h at 37 ꢃC. The lysis of the dihydroisoquinoline-4-carboxylate (10a). The substance 10a
ꢃ
erythrocytes and the release of hemoglobin was determined was obtained as a red solid, mp: 186–188 C, in 63% yield.
from the optical density of the supernatant at 540 nm. The
experiments were performed in triplicate and complete hemo- and 1085 (C–O).
IR nmax (cmꢀ1; KBr): 3258 (N–H), 1721 and 1671 (C]O), 1296
lysis (positive control) was determined by using 1% Triton X-
1H NMR (500.00 MHz, CDCl3) d (ppm): 2.97 (s, 3H, CH3), 2.62
100. Hemolysis less than 10% represented good hemocompat- (s, 3H, CH3), 4.03 (s, 3H, OCH3), 7.09–7.11 (m, 2H, H-20 and H-
ibility and non-toxicity against erythrocyte membranes, as 60), 7.24–7.27 (m, 1H, H-40), 7.34–7.37 (m, 2H, H-30 and H-50),
described elsewhere.42,53,54
7.98 (s, 1H, N–H).
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13C NMR (125.0 MHz, CDCl3) d (ppm): 22.7 (CH3), 25.6 (CH3), (C-4a), 103.5 (C-6), 144.9 (C-7), 158.5 (C-3), 161.8 (C-1), 167.3 (C-
53.4 (OCH3), 119.6 (C-8a), 125.2 (C-20 and C-60), 126.3 (C-4), 4b), 174.8 (C-5), 179.4 (C-8).
128.7 (C-30 and C-50), 126.6 (C-40), 136.8 (C-10), 137.0 (C-4a),
HRMS [M + H]+: m/z calcd for C20H16Br2N2O5: 522.9504;
104.9 (C-6), 145.0 (C-7), 161.1 (C-3), 161.3 (C-1), 168.2 (C-4b), found: 522.9487.
175.5 (C-5), 179.5 (C-8).
Methyl 6-bromo-3-bromoethyl-7-(4-chlorophenyl)amino-1-
HRMS [M + H]+: m/z calcd for C19H15BrN2O4: 415.0293; methyl-5,8-dioxo-5,8-dihydroisoquinoline-4-carboxylate (19d).
found: 415.0313.
Methyl 6-bromo-7-(2,5-dimethoxyphenyl)amino-1,3-dimethyl- in 30% yield.
5,8-dioxo-5,8-dihydroisoquinoline-4-carboxylate (10b). The
The substance 19d was obtained as a red solid, mp: 160–162 ꢃC,
IR nmax (cmꢀ1; KBr): 3295 (N–H), 1736 and 1679 (C]O), 1300
ꢃ
substance 10b was obtained as a red solid, mp: 138–140 C, in and 1092 (C–O).
52% yield.
1H NMR (500.00 MHz, CDCl3) d (ppm): 2.89 (s, 3H, CH3), 3.95
IR nmax (cmꢀ1; KBr): 3278 (N–H), 1727 and 1677 (C]O), 1316 (s, 3H, OCH3), 4.47 (s, 2H, C3–CH2–Br), 6.95 (d, 2H, J ¼ 8.5 Hz,
and 1157 (C–O).
H-20 and H-60), 7.24 (d, 2H, J ¼ 8.7 Hz, H-30 and H-50), 7.85 (s, 1H,
1H NMR (500.00 MHz, CDCl3) d (ppm): 3.00 (s, 3H, CH3), 2.70 N–H).
(s, 3H, CH3), 3.78 (s, 3H, Ph-OCH3), 3.77 (s, 3H, Ph-OCH3), 4.05
13C NMR (125.0 MHz, CDCl3) d (ppm): 26.2 (CH3), 29.5 (CH2),
(s, 3H, OCH3), 6.64 (d, 1H, J ¼ 2.9 Hz, H-30), 6.77 (dd, 1H, J ¼ 2.9 53.5 (OCH3), 120.8 (C-8a), 126.3 (C-20 and C-60), 125.8 (C-4),
and 9.0 Hz, H-40), 6.82 (d, 1H, J ¼ 9.0 Hz, H-60), 7.73 (s, 1H, N–H). 128.8 (C-30 and C-50), 132.2 (C-40), 135.4 (C-10), 137.9 (C-4a),
13C NMR (125.0 MHz, CDCl3) d (ppm): 21.8 (CH3), 24.4 (CH3), 105.4 (C-6), 144.8 (C-7), 158.6 (C-3), 162.2 (C-1), 167.4 (C-4b),
53.7 (OCH3), 55.8 (Ph-OCH3), 56.2 (Ph-OCH3), 120.5 (C-8a), 175.2 (C-5), 179.4 (C-8).
146.7 (C-20), 111.6 (C-30), 111.9 (C-60), 112.3 (C-40), 153.2 (C-50),
HRMS (M+): m/z calcd for C19H13Br2N2O4: 526.9009; found:
126.6 (C-4), 126.5 (C-10), 137.4 (C-4a), 105.0 (C-6), 145.9 (C-7), 526.9003.
160.2 (C-3), 160.3 (C-1), 167.7 (C-4b), 174.7 (C-5), 178.6 (C-8).
HRMS [M + H]+: m/z calcd for C21H19BrN2O6: 475.0505;
Acknowledgements
found: 475.0485.
Methyl 6-bromo-7-(4-methoxyphenyl)amino-1,3-dimethyl- This work was supported by the Brazilian agency FAPERJ.
5,8-dioxo-5,8-dihydroisoquinoline-4-carboxylate (10c). The Fellowships granted to UFF, by CAPES, CNPq, CNPq-PIBIC and
ꢃ
substance 10c was obtained as a red solid, mp: 136–138 C, in FAPERJ are gratefully acknowledged.
60% yield.
IR nmax (cmꢀ1; KBr): 3303 (N–H), 1732 and 1675 (C]O), 1239
and 1165 (C–O).
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