1,3-Azoles from ortho-naphthoquinones: Synthesis of aryl substituted imidazoles and oxazoles and their potent activity against Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.bmc.2012.08.041

Twenty-three naphthoimidazoles and six naphthoxazoles were synthesised and evaluated against susceptible and rifampicin- and isoniazid-resistant strains of Mycobacterium tuberculosis. Among all the compounds evaluated, fourteen presented MIC values in the

Full text of DOI:10.1016/j.bmc.2012.08.041

Bioorganic & Medicinal Chemistry 20 (2012) 6482–6488
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
1,3-Azoles from ortho-naphthoquinones: Synthesis of aryl substituted
imidazoles and oxazoles and their potent activity against Mycobacterium
tuberculosis
Kelly C. G. Moura a, Paula F. Carneiro a, Maria do Carmo F. R. Pinto a, José A. da Silva b, Valéria R. S. Malta b,
Carlos A. de Simone c, Gleiston G. Dias d, Guilherme A. M. Jardim d, Jéssica Cantos e, Tatiane S. Coelho e,
d,
Pedro E. Almeida da Silva e, , Eufrânio N. da Silva Jr.
⇑
⇑
a Núcleo de Pesquisas de Produtos Naturais, UFRJ, 21944-971 Rio de Janeiro, RJ, Brazil
b Instituto de Química e Biotecnologia, UFAL, 57072-970 Maceió, AL, Brazil
c Departamento de Física e Informática, Instituto de Física, USP, 13560-970 São Carlos, SP, Brazil
d Laboratório de Química Sintética e Heterocíclica, Instituto de Ciências Exatas, Departamento de Química, UFMG, 31270-901 Belo Horizonte, MG, Brazil
e Laboratório de Micobacterias, Faculdade de Medicina, FURG, 96200-190 Rio Grande, RS, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Twenty-three naphthoimidazoles and six naphthoxazoles were synthesised and evaluated against sus-
Received 17 June 2012
Revised 13 August 2012
Accepted 20 August 2012
Available online 31 August 2012
ceptible and rifampicin- and isoniazid-resistant strains of Mycobacterium tuberculosis. Among all the com-
pounds evaluated, fourteen presented MIC values in the range of 0.78 to 6.25 lg/mL against susceptible
and resistant strains of M. tuberculosis. Five structures were solved by X-ray crystallographic analysis.
These substances are promising antimycobacterial prototypes.
Ó 2012 Elsevier Ltd. All rights reserved.
This paper is dedicated to the memory of
Professor Antonio V. Pinto who passed away
in 2010, a great scientist and our mentor in
all aspects.
Keywords:
Imidazoles
Oxazoles
b-Lapachone
Mycobacterium tuberculosis
1. Introduction
nisms of action against resistant and susceptible bacterial strains.
Although a large number of molecules have been developed as po-
Tuberculosis (TB) is one of the most widespread diseases in the
world, responsible for approximately 1.3 million deaths annually.1
Although the currently available clinical treatments can cure most
cases of TB with drug susceptible, problems such as the long dura-
tion of treatment, the need for multiple drug therapy, antimicro-
bial resistance, and co-infection with HIV necessitate the
discovery of new drugs to treat TB.2 Furthermore, the complex
pathobiology of M. tuberculosis, which enables the organism to per-
sist in a dormant stage for years, makes treatment even more
difficult.3
tential new drugs, none have emerged for clinical use in the last
45 years.4,5 However, there are a wide variety of research resources
and strategies aimed at developing new drugs for TB treatment,
some of which involve the screening of prototypes of synthetic
molecules and natural products.2,6 Biodiversity is an important fac-
tor in the discovery of active substances, leading to further studies
of chemical structural modification to enhance their biological
activity.
Recently, several imidazole and oxazole compounds (Fig. 1)
were described as having antimycobacterial activity, and novel
substances were designed based on this important nitrogenated
class of heterocycles.7
It is important to develop new drug prototypes that are both
more active and more effective and that have alternative mecha-
Quinones represent a wide and diverse family of plant metabo-
lites.8 In addition to their anti-inflammatory and cytotoxic proper-
ties, the activity of quinones against viruses and pathogenic
microorganisms has been reported.9 The main deleterious action
of quinones in biological systems is oxidative stress promotion, a
⇑
Corresponding authors. Tel.: +55 53 32338895; fax: +55 53 32338863
(P.E.A.d.S.); tel.: +55 31 34095710; fax: +55 31 34095700 (E.N.d.S.).
E-mail addresses: pedrefurg@gmail.com (P.E.A. da Silva), eufranio@ufmg.br
(E.N. da Silva).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.08.041

K. C. G. Moura et al. / Bioorg. Med. Chem. 20 (2012) 6482–6488
6483
Br
O
N
N
H
N
Br
NH2
N
N
H
N
O
O
H
O
O
H
N
O
O
H
N
N
Br
N
O
N
N
H
N
NO2
N
NH2
O
N
H
N
N
N
Figure 1. Synthetic drugs already tested against Mycobacterium tuberculosis.7
harmful process that causes irreversible damage by altering vital
cellular components.10 One consequence of this mechanism is
the destruction of biological membranes, resulting from peroxida-
tion of the lipid components. Moreover, quinones can interfere
with a variety of biosynthetic pathways, including the destruction
of proteins, damage to the structures of nucleic acids and DNA
strand breaks.11
heterocyclic substitutions, such as quinolinic, isoquinolinic or
pyridinic core structures.
2. Results and discussion
2.1. Chemistry
Our research group has described the synthesis and antimyco-
bacterial activity of naphthoquinoidal and heterocyclic derivatives
from lapachol.12 This quinone is an important natural compound
isolated from the hard core of trees of the bignoniaceous family,
found mainly in the tropical areas of the Western Hemisphere.13
Initially, phenazines from lapachones were obtained, and the com-
pound derived from allyl-b-lapachone was identified as a potent
All of the naphthoimidazoles (Fig. 3) and naphthoxazoles
(Fig. 4) were obtained by the reaction of a quinonoid compound
(b-lapachone) with aromatic aldehydes in the presence of ammo-
nium acetate, as previously described.16,17 In summary, the sub-
stances were separated by column chromatography using eluents
of varying polarity: oxazoles with 94:6 hexane:ethyl acetate and
imidazoles with 90:10 hexane:ethyl acetate.
Some aryl-substituted imidazoles were also obtained from nor-
b-lapachone by the method described in Scheme 1. Nor-lapachol
was obtained from lapachol by the Hooker oxidation method18
and was then reacted with H2SO4 to obtain the cyclised product,
nor-b-lapachone. The unpublished 1,3-azoles (Scheme 1) were ob-
tained through the reaction of nor-b-lapachone, different substi-
tuted aldehydes and a nitrogenating reagent (AcONH4) in acetic
acid. The reaction conditions allowed us to obtain a mixture of
products (imidazoles and oxazoles). In some cases both com-
pounds were obtained, while in other cases, only the imidazole
or oxazole was obtained.
Compound 30 was obtained from C-allyl lawsone (29), as previ-
ously described19 and was used to prepare the respective oxazole
derivative 31, described herein for the first time (See Scheme 2).
Compounds 1–21 were synthesised as previously described,16,17
and substances 22–28 and 31, described for the first time, were
isolated and characterised by 1H and 13C NMR, infrared, and elec-
tron-impact mass spectrometry. For some of the compounds, 13C
NMR spectra could not be collected due to their low solubility in
most of the organic solvents tested. For compounds 1, 26, 27, 28
and 31, suitable crystals were obtained, and the structures were
re-confirmed by X-ray crystallographic study.
substance (Fig. 2) with MIC values equal to 0.78 lg/mL against
pan-susceptible and rifampicin-resistant strains of M. tuberculo-
sis.12 More recently, we reported a phenazine obtained from a lapa-
chol derivative that was considered highly active (MIC 63 lg/mL)
against M. tuberculosis H37Rv, a pan-susceptible strain. Against
ATCC 35822 strains, this compound was less active, with an MIC
value of 12.5 mg/mL.14 Nor-lapachol also presented an
MIC = 3.12 l
g/mL against a rifampicin-resistant strain (Fig. 2).14
Imidazole compounds are important drug prototypes for TB.15
Different synthetic strategies have been used to obtain imidazoles
with diverse patterns of substitution, and these substances present
low MIC values, proving the importance of imidazole moieties for
activity against M. tuberculosis.7,15
In the last few years, a series of manuscripts showed that the
functionalisation of the quinoidal moieties of lapachol and b-lapach-
one into naphthoimidazoles and naphthoxazoles is an important
strategy in the development of new, active compounds.16
In this context, we synthesised naphthoimidazoles and naphth-
oxazoles that were evaluated against pan-susceptible M. tuberculo-
sis strains that were resistant to isoniazid and rifampicin. Our
strategy was to examine imidazoles and oxazoles with electron-
donating and electron-withdrawing aryl substituents or that had
Phenazine from a reduced lapachol
≤
MIC 3 µg/mL for H37Rv
O
O
N
O
OH
N
N
NH
Nor-lapachol
MIC = 3.12 µg/mL for RIFr
O
Phenazine from Allyl-β-lapachone
MIC = 0.78 µg/mL for H37Rv and RIFr
Figure 2. Lapachol derivatives with TB activity.

6484
K. C. G. Moura et al. / Bioorg. Med. Chem. 20 (2012) 6482–6488
1 - R1 = R2 = R3 = R4 = R5 = H
2
- R2 = R3 = R4 = R5 = H, R1 = Br
N
N
3 - R1 = R3 = R4 = R5 = H, R2 = Br
4 - R1 = R2 = R4 = R5 = H, R3 = Br
5 - R1 = R3 = R4 = R5 = H, R2 = F
6 - R1 = R2 = R4 = R5 = H, R3 = F
7 - R1 = R2 = R4 = R5 = H, R3 = Cl
8 - R1 = R3 = R4 = R5 = H, R2 = CN
18 R =
19 R =
R4 R3
N
R
R5
N
R2
N
NH
R1
9
- R1 = R2 = R4 = R5 = H, R3 = CN
10 - R2 = R3 = R4 = R5 = H, R1 = NO2
11
NH
N
20 R =
- R1 = R3 = R4 = R5 = H, R2 = NO2
12 - R1 = R2 = R4 = R5 = H, R3 = NO2
13
O
- R2 = R3 = R4 = R5 = H, R1 = OMe
14 - R2 = R3 = R4 = R5 = H, R1 = CH3
15
O
21 R =
- R2 = R3 = R4 = R5 = H, R1 = CF3
16 - R1 = R3 = R4 = R5 = H, R2 = CF3
17
- R1 = R2 = R4 = R5 = H, R3 = CF3
Figure 3. Naphthoimidazoles 1–21 obtained from b-lapachone.
R4 R3
Ueq of the carbon or nitrogen atom to which they are attached. The
final difference Fourier map synthesis showed the highest peak
and deepest hole between (0.480 eÅꢀ3) and (ꢀ0.427 eÅꢀ3), respec-
tively, for all compounds. The ORTEP-3 program for Windows20
was used for graphic presentations, and the material for publica-
tion was prepared using WinGX-Routine.21 Figure 5 shows the
ORTEP-3 diagrams for five of the structures.
R5
N
R2
R1
O
22 - R2 = R3 = R4 = R5 = H, R1 = CN
23 - R1 = R3 = R4 = R5 = H, R2 = CN
24 - R1 = R2 = R4 = R5 = H, R3 = CN
O
Figure 4. Naphthoxazoles 22–24 obtained from b-lapachone.
2.3. Biological assays
2.2. X-ray analysis
The minimum inhibitory concentrations (MIC) for all of the
compounds were evaluated against M. tuberculosis H37Rv (pan-
susceptible), rifampicin-resistant (RIFr, ATCC 35338) and isonia-
zid-resistant (INHr, ATCC 35822) strains (Table 1). Eighteen
compounds, 1–3, 5–7, 10, 12–16, 18, 20 and 22–24, displayed MIC
Hydrogen atoms bound to carbon and nitrogen were located at
their idealised positions using appropriate HFIX instructions in
SHELXL (43 for the aromatic and 33 for the terminal –CH3 methyl
groups) and were included in subsequent refinement cycles in
the riding-motion approximation with isotropic thermal displace-
ments parameters (Uiso) fixed, respectively, at 1.2 or 1.5 times the
values between 0.78 and 100
lg/mL against all strains. The sub-
stances 25–31 were considered mildly active or inactive with MIC
P100
lg/mL for all three strains.
O
O
1. Na2CO3, H2O2
THF, 60 ºC
OH
OH
2. NaOH/Na2CO3
CuSO4
O
O
THF
Nor-lapachol
H2SO4
Lapachol
R
O
O
O
N
O
H
Z
25 - Z = NH, R = H
26 - Z = NH, R = Me
R
27
28
- Z = O, R = NO2
- Z = O, R = Me
AcONH4
AcOH
O
Nor-β-lapachone
Scheme 1. Naphthoimidazoles and naphthoxazoles 25–28 obtained from nor-b-lapachone.
NO2
O
O
O
O
H
N
O
OH
O
O
H2SO4
O2N
AcONH4
AcOH
O
C-allyl Lawsone (29)
(30)
31
(
)
Scheme 2. Naphthoxazole 31 obtained from substance 30.

K. C. G. Moura et al. / Bioorg. Med. Chem. 20 (2012) 6482–6488
6485
Figure 5. An ORTEP-3 projection of molecules 1, 26, 27, 28 and 31, showing the atom-numbering and displacement ellipsoids at the 50% probability level.
The insertion of an aryl-substituted ring into the imidazole group
enhanced its microbicidal activity and is an important prototype for
novel compounds against TB. For example, ortho-substituted
m-CF3 (16), with MICs below 18.0
(11) compounds were inactive. Substances 3 (m-Br, MIC = 15.3
and 16 (m-CF3, MIC = 4.0 M) were the most active imidazoles in
l
M. The m-CN (8) and m-NO2
l
M)
l
compounds 2 (o-Br, MIC values of 7.6
for ATCC 35338 and ATCC 35822 strains) and 10 (o-NO2, MIC values
of 267.8, 33.5 and 16.7 M against the H37Rv, RIFr and INHr strains,
respectively) were more potent than the naphthoimidazole 1 (no
substituent on the phenyl ring, MIC >304.5 M for all strains). Com-
pound 15 (o-CF3, MICvalues = 4.0, 2.0 and 4.0 M against H37Rv, RIFr
and INHr strains, respectively) was more active than 1 and its pre-
cursor, b-lapachone (MIC = 6.43 M for H37RV and ATCC 35338)
indicating that the insertion of –CF3 group at the ortho-position in
the phenyl ring enhances the activity. Substances 13 (o-OMe) and
14 (o-CH3), which were substituted with electron donating groups,
were highly active with MIC values in the range of 4.5–17.4 lM.
Among the meta-substituted phenyl derivatives, those more
active against the M. tuberculosis were m-Br (3), m-F (5) and
l
M for H37RV and 15.3
l
M
this series for all of the M. tuberculosis strains. Regarding the pres-
ence of substituents at the para position, the naphthoimidazole 12
(p-NO2, MIC = 4.2 lM) was the most active against INHr but was
l
inactive against the RIFr strain. The p-Br (4), p-CN (9) and p-CF3
(17) compounds were inactive, demonstrating that the insertion
of substituents in this position decreases the activity. Although
compounds 1–17 possess substituents with different sizes, sterics
and electronic properties, no consistent structure–activity relation-
ships could be established.
The naphthoimidazoles 8 and 9 and the naphthoxazoles 22–24
with a cyano group showed diverse activity for all of the M. tuber-
culosis strains; the naphthoimidazoles were inactive, while the
naphthoxazoles exhibited the opposite effect. For the heterocyclic
compounds 18–21, substance 20 with a quinoline group was the
l
l
l

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K. C. G. Moura et al. / Bioorg. Med. Chem. 20 (2012) 6482–6488
Table 1
4. Experimental protocols
MIC (minimum inhibitory concentration) of the tested compounds against Mycobac-
terium tuberculosis H37Rv, rifampicin-resistant M. tuberculosis (RIFr) and isoniazid-
resistant M. tuberculosis (INHr)
4.1. General procedures
Compd No.
CMI
lg/mL (lM)
Melting points were determined in a capillary Thomas Hoover
apparatus (Thomas Co., Philadelphia, PA, USA) and are uncorrected.
Analytical grade solvents were used. Column chromatography was
performed on silica gel (Acros Organics 0.035–0.070 mm, pore diam-
eter ca 6 nm). Infrared spectra wererecorded on a Perkin–Elmer FT-IR
spectrometer (Perkin–Elmer Inc., Wellesley, MA, USA). 1H and 13C
NMR spectra were recorded at room temperature using a VNMR-
SYS-500, Varian MR 400 instrument and Gemini 200-MHz spectrom-
eter (Varian, Palo Alto, CA, USA) in the solvents indicated with TMS as
an internal reference. Chemical shifts (d) are given in ppm, and
coupling constants (J) are reported in Hertz. The mass spectra were
obtained at 70 eV in a VG Autospec apparatus. The fragments are
described as a relationship between atomic massunits and the charge
(m/z) and the relative abundance in percentage. All the compounds
were nominated using the program ChemBioDraw Ultra 12.0.
H
37Rv (ATCC27294) RIFr (ATCC 35338) INHr (ATCC 35822)
1
2
3
4
5
6
7
8
>100 (>304.5)
3.12 (7.6)
6.25 (15.3)
Na
3.12 (9.0)
25 (72.2)
12.5 (34.4)
Na
>100 (>304.5)
6.25 (15.3)
6.25 (15.3)
Na
6.25 (18.0)
6.25 (18.0)
6.25 (17.2)
Na
12.5 (38.0)
6.25 (15.3)
6.25 (15.3)
Na
6.25 (18.0)
6.25 (18.0)
6.25 (17.2)
Na
9
Na
Na
Na
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
100 (267.8)
Na
12.5 (33.5)
Na
Na
6.25 (17.4)
1.56 (4.5)
0.78 (2.0)
1.56 (4.0)
Na
6.25 (19.0)
Na
60.78 (62.0)
Na
6.25 (17.6)
6.25 (17.6)
1.56 (4.4)
100 (318.0)
100 (304.5)
Nd
6.25 (16.7)
Na
100 (267.8)
6.25 (17.4)
3.12 (9.0)
1.56 (4.0)
1.56 (4.0)
Na
25 (75.9)
Na
6.25 (16.4)
Na
12.5 (35.2)
12.5 (35.2)
6.25 (17.6)
>100 (>318.0)
>100 (>304.5)
>100 (>277.5)
>100 (>303.6)
100 (466.8)
3.12 (14.5)
>100 (>288.7)
1.56 (4.2)
6.25 (17.4)
1.56 (4.5)
1.56 (4.0)
1.56 (4.0)
Na
6.25 (19.0)
Na
60.78 (62.0)
Na
6.25 (17.6)
6.25 (17.6)
3.12 (8.8)
>100 (>318.0)
>100 (>304.5)
>100 (>277.5)
>100 (>303.6)
100 (466.8)
Na
4.2. General procedure for the preparation of the imidazoles
and oxazoles
To a solution of b-lapachone (1.1 mmol) in acetic acid (6 mL),
the desired aldehyde (2.5 mmol) was added, and the mixture was
heated to 70 °C; at this point, ammonium acetate (16.5 mmol)
was slowly added, and reflux was maintained for a determined
time. All the reactions were monitored by thin layer chromatogra-
phy. At the end of the reaction, after addition into water, the pre-
cipitate was purified by column chromatography using as eluent
a mixture of hexane/ethyl acetate, with a gradient of increasing
polarity, as previously described.16
Nd
25 (116.7)
Na
100 (288.7)
>4 (>4.8)
60.06 (60.4)
>100 (>288.7)
60.125 (60.1)
1 (7.3)
Rifampicin 60.125 (60.1)
Isoniazid 60.06 (60.4)
4.3. 2-(6,6-Dimethyl-5,6-dihydro-4H-benzo[7,8]chromeno[6,5-
d]oxazol-2-yl)benzonitrile (22)
Using 2-formylbenzonitrile (327.5 mg, 2.5 mmol), 22 was
obtained as a yellow solid (53.1 mg, 0.15 mmol, 15% yield, mp
181–183 °C).
most active, with an MIC 62.0
resistant M. tuberculosis.
lM for rifampicin- and isoniazid-
We have previously described a strategy to modify the C-ring of
the b-lapachone (pyran ring versus furan ring) that was success-
fully used to obtain trypanocidal compounds.22 Substances 25–28
and 31, which have a dihydrofuran ring, were synthesised and
evaluated against all of the M. tuberculosis strains to determine
the effectiveness of this approach against TB. None of these sub-
stances with the dihydrofuran ring were active against M.
tuberculosis.
IR m
max(cmꢀ1, KBr): 3061 (Ar-CH), 2225 (C„N), 1599 (C@N),
1237 (C–O–C), 1119 (C–O–C). 1H NMR (200 MHz, DMSO-d6) d:
8.46–8.28 (2H, m), 8.20 (1H, d, J = 8.2 Hz), 8.02 (1H, d, J = 7.4 Hz),
7.88 (1H, t, J = 7.4 Hz), 7.76–7.62 (2H, m), 7.56 (1H, d, J = 8.2 Hz),
3.05 (2H, t, J = 6.4 Hz), 1.98 (2H, t, J = 6.4 Hz), 1.44 (s, 6H). 13C
NMR (50 MHz, DMSO-d6) d: 157.9, 148.0, 135.0, 134.0, 133.3,
130.7, 128.7, 128.5, 127.0, 124.8, 124.5, 123.4, 122.6, 122.2,
121.1, 117.4, 108.7, 101.8, 75.6, 30.6, 26.2, 16.6. MS (70 eV, m/z)
(%): [M+1] 355 (8), 354 (38), 299 (24), 298 (100), 162 (5), 130
(43), 115 (6), 114 (7), 102 (15), 41 (4).
For compounds 1–11, 20 and 25, the toxicity to human periph-
eral blood mononuclear cells (PBMC) after 72 h of drug exposure
was evaluated by the Alamar Blue assay. For these substances,
the IC50 values were higher than 25.0 lg/mL. The described azoles
represent an important series of compounds with potent activity
against TB with low cytotoxicity.
4.4. 3-(6,6-Dimethyl-5,6-dihydro-4H-benzo[7,8]chromeno[6,5-
d]oxazol-2-yl)benzonitrile (23)
Using 3-formylbenzonitrile (327.5 mg, 2.5 mmol), 23 was ob-
tained as a yellow solid (35.0 mg, 0.1 mmol, 10% yield, mp 217–
220 °C).
3. Conclusions
IR m
max(cmꢀ1, KBr): 3076 (Ar-CH), 2225 (C„N), 1601 (C@N),
To discover antimicrobial compounds, we describe fourteen
1237 (C–O–C), 1118 (C–O–C). 1H NMR (400 MHz, CDCl3) d: 8.52–
8.43 (2H, m), 8.32–8.30 (1H, m), 7.85 (1H, dd, J = 0.7, 7.8 Hz),
7.73 (1H, td, J = 1.1, 7.8 Hz), 7.64 (1H, td, J = 1.1, 7.8 Hz), 7.53 (2H,
ddd, J = 1.1, 7.8, 15.2 Hz), 3.16 (2H, t, J = 6.6 Hz), 2.00 (2H, t,
J = 6.6 Hz), 1.50 (6H, s). 13C NMR (50 MHz, DMSO-d6) d: 157.9,
148.4, 147.0, 132.9, 130.3, 129.8, 129.5, 129.0, 127.0, 125.2,
124.7, 124.0, 122.6, 121.5, 118.1, 113.1, 101.4, 75.3, 31.5, 26.6,
17.20. MS (70 eV, m/z) (%): [M+1] 355 (10), 354 (38), 299 (26),
298 (100), 162 (8), 130 (50), 115 (7), 114 (7), 102 (18), 41 (4).
substances with MIC values in the range of 0.78 to 6.25
against strains of M. tuberculosis. Substance 20, with a quinoline
group, was the most active, with an MIC value 60.78 g/mL for rif-
ampicin-resistant M. tuberculosis (RIFr) and isoniazid-resistant M.
tuberculosis (INHr) strains. Therefore, this substance emerges as
an important derivative for the design of novel active compounds.
The research described here is an important contribution in the
field of biologically active heterocyclic compounds against TB, a
neglected disease and an epidemic in many developing countries.
lg/mL
l

K. C. G. Moura et al. / Bioorg. Med. Chem. 20 (2012) 6482–6488
6487
4.5. 4-(6,6-Dimethyl-5,6-dihydro-4H-benzo[7,8]chromeno[6,5-
d]oxazol-2-yl)benzonitrile (24)
4.10. 5-Methyl-2-(4-nitrophenyl)-4,5-dihydrofuro[30,20:3,4]
naphtho[1,2-d]oxazole (31)
Using 4-formylbenzonitrile (327.5 mg, 2.5 mmol), 24 was
obtained as a yellow solid (71.0 mg, 0.2 mmol, 20% yield, mp
230–234 °C).
Using compound 30 (428 mg, 2 mmol) and p-NO2 benzaldehyde
(679.5 mg, 4.5 mmol), 31 was obtained as a red solid (138 mg,
0.40 mmol, 20% yield, mp 234–237 °C).
IR
m
max(cmꢀ1, KBr): 3046 (Ar-CH), 2226 (C„N), 1606 (C@N),
IR m
max(cmꢀ1, KBr): 1600 (C@N), 1258 (C–O–C), 1130 (C–O–C).
1203 (C–O–C), 1120 (C–O–C). 1H NMR (200 MHz, DMSO-d6) d:
8.40–7.80 (8H, m), 3.12 (2H, t, J = 6.7 Hz), 2.02 (2H, t, J = 6.7 Hz),
1.52 (6H, s). 13C NMR (50 MHz, DMSO-d6) d: 158.0, 148.5, 147.0,
138.5, 132.7, 132.4, 131.5, 129.7, 126.9, 126.7, 118.3, 117.5,
117.3, 113.0, 101.4, 75.3, 31.4, 26.5, 17.1. MS (70 eV, m/z) (%):
[M+1] 355 (10), 354 (39), 299 (26), 298 (100), 162 (6), 130 (56),
115 (7), 114 (7), 102 (22), 41 (5).
1H NMR (400 MHz, CDCl3) d: 8.52 (1H, d, J = 8.2 Hz), 8.44–8.32
(4H, m), 8.07 (1H, d, J = 8.2 Hz), 7.68–7.64 (1H, m), 7.56–7.52
(1H, m), 5.41–5.26 (1H, m), 3.75 (1H, dd, J = 15.2, 9.2 Hz), 3.23
(1H, dd, J = 15.2, 7.4 Hz), 1.65 (3H, s). MS (70 eV, m/z) (%): 346
(92), 316 (19), 300 (20), 228 (7), 155 (24), 141 (34), 127 (23),
115 (61), 102 (31), 88 (29), 76 (100), 63 (31), 43 (77).
4.11. Minimum inhibitory concentration determination
4.6. 5,5-Dimethyl-2-phenyl-4,5-dihydro-3H-furo[30,20:3,4]
naphtho[1,2-d]imidazole (25)
The experiment was performed using Mycobacterium tuberculo-
sis H37Rv (ATCC27294), a pan-susceptible strain, RIFr with a His-
526?Tir mutation in the rpoB gene and INHR with a Ser-315?Tir
mutation in the katG gene. The strains were maintained in Oga-
wa-Kudoh media for 14 days at 37 °C.
The determination of antimycobacterial activity was performed
by REMA (Resazurin Microtitre Assay), as previously described.23 In
brief, the bacterial suspensions were homogenized by vortex
agitation, and the turbidity was adjusted in agreement with tube
one of the scale 1.0 of McFarland (3.2 ꢁ 106 cfu/mL). The inoculum
was prepared by diluting the bacterial suspension 1:20 in Middle-
brook 7H9 OADC media (4.7 g Middlebrook 7H9 base; Difco, Becton
Dickinson). The 7H9 medium consisted of 0.5 g of ammonium sul-
Using nor-b-lapachone (228 mg, 1 mmol) and benzaldehyde
(265 mg, 2.5 mmol), 25 was obtained as a yellow solid (157 mg,
0.5 mmol, 50% yield, mp 265–266 °C).
IR
m
max(cmꢀ1, KBr): 3100 (Ar-CH), 1606 (C@N), 1110 (C–O–C).
1H NMR (200 MHz, DMSO-d6) d: 8.47 (1H, d, J = 8 Hz), 8.2 (2H,
m), 7.88 (1H, d, J = 8 Hz), 7.66–7.13 (5H, m), 2.5 (s, 2H); 1.5 (s,
6H). MS (70 eV, m/z) (%): 314 (100); 299 (75); 272 (8); 245 (3);
210 (4); 196 (11); 169 (8); 149 (11); 115 (8); 77 (6); 57 (8).
4.7. 5,5-Dimethyl-2-(p-tolyl)-4,5-dihydro-3H-furo[30,20:3,4]
naphtho[1,2-d]imidazole (26)
fate, 0.5 g of L-glutamic acid, 0.1 g of sodium citrate, 0.001 g of pyr-
Using nor-b-lapachone (456 mg, 2 mmol) and 4-metyl benzal-
dehyde (540 mg, 4.5 mmol), 26 was obtained as a white solid
(98 mg, 0.30 mmol, 15% yield, mp 275–278 °C).
idoxine, 0.0005 g of biotin, 2.5 g of disodium phosphate, 1.0 g of
monopotassium phosphate; 0.04 g ferric ammonium citrate,
0.05 g of magnesium sulfate, 0.0005 g of calcium chloride, 0.001 g
of Zinc sulfate, 0.001 g of copper sulfate, 2 mL of glycerol, 900 mL
of H2O, and 10% OADC (oleic acid, albumin, dextrose, catalase).
The assay was performed in 96-well microplates, with a concentra-
IR m
max(cmꢀ1, KBr): 3140 (Ar-CH), 1606 (C@N), 1123 (C–O–C).
1H NMR (400 MHz, DMSO-d6) d: 8.48 (1H, d, J = 8.0 Hz), 8.14–
8.06 (2H, m), 7.89 (1H, d, J = 8.0 Hz), 7.57 (1H, t, J = 7.5 Hz), 7.44
(1H, t, J = 7.5 Hz), 7.38–7.34 (2H, m), 3.37 (2H, s), 2.39 (3H, s),
1.58 (6H, s). MS (70 eV, m/z) (%): 328 (64), 313 (55), 299 (5), 285
(7), 196 (6), 167 (8), 140 (14), 130 (9), 115 (22), 102 (12), 91
(27), 77 (14), 65 (15), 41 (100).
tion of 100
well. The microplate was incubated at 37 °C for 7 days. After this,
30 L of resazurin was added to each well, and the plate was
lg/mL in the first well and up to 0.19 lg/mL in the last
l
incubated for 2 days at 37 °C. The reading was recorded from the
oxi-reduction of the resazurin, noting the change of color when
cellular growth was taking place.
4.8. 5,5-Dimethyl-2-(4-nitrophenyl)-4,5-dihydrofuro
[30,20:3,4]naphtho[1,2-d]oxazole (27)
4.12. Inhibition of PBMC proliferation
Using nor-b-lapachone (456 mg, 2 mmol) and p-NO2 benzalde-
hyde (679.5 mg, 4.5 mmol), 27 was obtained as a red solid
(410 mg, 1.14 mmol, 57% yield, mp 224–226 °C).
To investigate the selectivity of compounds toward a normal
proliferating cell, the Alamar blue assay was performed with
peripheral blood mononuclear cells (PBMC) after 72 h of drug
IR m
max(cmꢀ1, KBr): 1600 (C@N),1553, 1316 (NO2), 1256 (C–O–
C), 1105 (C–O–C). 1H NMR (400 MHz, CDCl3) d: 8.52 (1H, d,
J = 8.2 Hz), 8.45–8.34 (4H, m), 8.08 (1H, d, J = 8.2 Hz), 7.69–7.62
(1H, m), 7.57–7.50 (1H, m), 3.43 (2H, s), 1.67 (6H, s). MS (70 eV,
m/z) (%): 360 (75), 330 (8), 318 (11), 197 (11), 169 (20), 47 (100).
exposure. After 24 h, compounds (0.048 to 25
in DMSO (0.1%) were added to each well and incubated for 72 h.
Twenty-four h before the end of the incubation, 10 L of stock
lg/mL) dissolved
l
solution (0.312 mg/mL) of the Alamar Blue (Resazurin, Sigma–Al-
drich Co) was added to each well. The absorbance was measured
using a multiplate reader (DTX 880 Multimode Detector, Beckman
CoulterÒ) and the drug effect was quantified as the percentage of
control absorbance at 570 nm and 595 nm. The absorbance of Ala-
mar Blue in culture medium is measured at a higher wavelength
and a lower wavelength. The absorbance of the medium is also
measured at the higher and lower wavelengths. The absorbance
of the medium alone is subtracted from the absorbance of medium
plus Alamar Blue at the higher wavelength. This value is called
AOHW. The absorbance of the medium alone is subtracted from
the absorbance of medium plus Alamar Blue at the lower wave-
length. This value is called AOLW. A correction factor, R0, can be
calculated from AOHW and AOLW, where R0 = AOLW/AOHW. The
4.9. 5,5-Dimethyl-2-(p-tolyl)-4,5-dihydrofuro[30,20:3,4]
naphtho[1,2-d]oxazole (28)
Using b-nor-lapachone (456 mg, 2 mmol) and p-methyl benzal-
dehyde (540 mg, 4.5 mmol), 28 was obtained as a white solid
(86 mg, 0.26 mmol, 13% yield, mp 198–200 °C).
IR m
max(cmꢀ1, KBr): 1612 (C@N), 1258 (C–O–C), 1065 (C–O–C).
1H NMR (400 MHz, CDCl3) d: 8.44 (1H, d, J = 8.2 Hz), 8.08 (2H, d,
J = 7.9 Hz), 7.97 (1H, d, J = 8.2 Hz), 7.56–7.48 (1H, m), 7.44–7.36
(1H, m), 7.24 (2H, d, J = 7.9 Hz), 3.33 (2H, s), 2.35 (3H, s), 1.56
(6H, s). MS (70 eV, m/z) (%): 329 (100), 314 (8), 300 (2), 287 (16),
197 (15), 169 (16), 141 (10), 139 (7), 119 (33), 91 (23).

6488
K. C. G. Moura et al. / Bioorg. Med. Chem. 20 (2012) 6482–6488
Moraski, G. C.; Markley, L. D.; Chang, M.; Cho, S.; Franzblau, S. C.; Hwang, C. H.;
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Boshoff, H. I. M.; Franzblau, S. G.; Barry, C. E.; Copp, B. R. Tetrahedron Lett. 2005,
46, 7355.
percent Alamar Blue reduced is then expressed as follows:% redu-
ced = ALW—(AHW ꢁ R0) ꢁ 100.
8. Thomson, R. H. In Naturally Occuring Quinones: IV Recents Advances; Champman,
Hall, Eds.; Champman & Hall: London, 1997.
4.13. X-ray crystallographic analysis
9. (a) da Silva, E. N., Jr.; Souza, M. C. B. V.; Pinto, A. V.; Pinto, M. C. F. R.; Ferreira, V.
F.; Menna-Barreto, R. F. S.; Silva, R. S. F.; Teixeira, D. V.; de Simone, C. A.; de
Castro, S. L. Eur. J. Med. Chem. 2008, 43, 1774; (b) da Silva, E. N., Jr.; de Deus, C.
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Moraes, M. O.; Pinto, M. C. F. R.; de Simone, C. F.; Ferreira, V. F.; Goulart, M. O.
F.; Andrad, C. K. Z.; Pinto, A. V. J. Med. Chem. 2010, 53, 504; (c) da Silva, E. N., Jr.;
de Souza, M. C. B. V.; Pinto, A. V.; Pinto, M. C. F. R.; Goulart, M. O. F.; Pessoa, C.;
Costa-Lotufo, L.; Montenegro, R. C.; Morae, M. O.; Ferreira, V. F. Bioorg. Med.
Chem. 2007, 15, 7035; (d) Lourenço, A. L.; Abreu, P. A.; Leal, B.; Da Silva, E. N., Jr.;
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2009, 50, 1575.
Data for each compound were collected at room temperature on
an Enraf Nonius FR590 charge-coupled device (CCD) area-detector
diffractometer (Mo
K
graphite-monochromated radiation,
a
k = 0.71073 Å) controlled by the COLLECT software package.24
Images were processed using the Denzo and Scalepack software
packages.25 The structures were solved using direct methods with
SHELXS-9726 and refined by full-matrix least squares on F2 using
SHELXL-97.26 All non-hydrogen atoms were successfully refined
using anisotropic displacement parameters.
Crystallographic data (excluding structure factors) for the struc-
tures reported in this paper have been depositedwith the Cambridge
Crystallographic Data Centre as supplementary publication No.
CCDC 814214, 814215, 882547, 882549 and 882552. Copies of the
data can be obtained free of charge on application to CCDC, 12 Union
Road, Cambridge CB2 2EZ, UK (Fax: (+44) 1223 336033; e-mail:
deposit@ccdc.cam.ac.uk).
Acknowledgments
This work was supported by CNPq (National Council of Research
of Brazil), CAPES, UFAL, UFRJ, FURG and UFMG. The authors are also
indebted to the PRONEX-FAPERJ (E-26/110.574/2010). Dr. E.N. da
Silva Júnior. thanks the Programa Institucional de Auxílio à Pesqu-
isa de Doutores Recém-Contratados—UFMG (Edital 08/2010),
Universidade Federal de Minas Gerais (Edital PRPq—12/2011) and
FAPEMIG (APQ-04166-10). Dr. C.A. de Simone is grateful to the
crystallography group at IFSC-USP for allowing the use of a Kap-
pa-CCD diffractometer. The authors would also like to thank
Dr. Bruno C. Cavalcanti and Professor Dr. Cláudia Pessoa (Federal
University of Ceará) by performing the Alamar Blue assay and prof.
Dr. Solange L. de Castro (Instituto Oswaldo Cruz, FIOCRUZ-RJ) for
stimulating discussions and consistently excellent advice.
14. Carneiro, P. F.; Pinto, M. C. F. R.; Coelho, T. S.; Cavalcanti, B. C.; Pessoa, C.; de
Simone, C. A.; Nunes, I. K. C.; de Oliveira, N. M.; de Almeida, R. G.; Pinto, A. V.;
de Moura, K. C. G.; da Silva, P. A.; da Silva, E. N., Jr. Eur. J. Med. Chem. 2011, 46,
4521.
15. (a) Pandey, J.; Tiwari, V. K.; Verma, S. S.; Chaturvedi, V.; Bhatnagar, S.; Sinha, S.;
Gaikwad, A. N.; Tripathi, R. P. Eur. J. Med. Chem. 2009, 44, 3350; (b) Lu, X.; Liu,
X.; Wan, B.; Franzblau, S. G.; Chen, L.; Zhou, C.; You, Q. Eur. J. Med. Chem. 2012,
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Napiórkowska, A. Bioorg. Med. Chem. 2012, 20, 137.
16. (a) de Moura, K. C. G.; Salomão, K.; Menna-Barreto, R. F. S.; Emery, F. S.; Pinto,
M. C. F. R.; Pinto, A. V.; de Castro, S. L. Eur. J. Med. Chem. 2004, 39, 639; (b) de
Moura, K. C. G.; Emery, F. S.; Pinto, C. N.; Pinto, M. C. F. R.; Dantas, A. P.;
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de Castro, S. L.; de Castro, S. L. Arzneim-Forsch. 1997, 47, 74; (b) Neves-Pinto, C.;
Dantas, A. P.; Moura, K. C. G.; Emery, F. S.; Polequevitch, P. F.; Pinto, M. C. F. R.;
de Castro, S. L.; Pinto, A. V. Arzneim-Forsch. 2000, 50, 1120.
18. Fieser, L. F.; Fieser, M. J. Am. Chem. Soc. 1948, 70, 3215.
19. Silva, R. S. F.; Costa, E. M.; Trindade, U. L. T.; Teixeira, D. V.; Pinto, M. C. F. R.;
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Filho, J. D.; Valença, W. O.; Camara, C. A.; de Oliveira, R. N.; de Araujo, A. S.;
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