Quinonoid and phenazine compounds: Synthesis and evaluation against H 37Rv, rifampicin and isoniazid-resistance strains of Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.ejmech.2011.07.026

Several quinonoid and phenazine compounds were synthesized in moderate to high yields and showed activity against H₃₇Rv, rifampicin and isoniazid-resistance strains of Mycobacterium tuberculosis. The cytotoxity of the compounds were evaluated against human peripheral blood mononuclear cells (PBMC) and these substances emerge as promising antitubercular prototypes.

Full text of DOI:10.1016/j.ejmech.2011.07.026

European Journal of Medicinal Chemistry 46 (2011) 4521e4529
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Quinonoid and phenazine compounds: Synthesis and evaluation against H Rv,
37
rifampicin and isoniazid-resistance strains of Mycobacterium tuberculosis
a
a
b
c
c
Paula F. Carneiro , Maria do Carmo F.R. Pinto , Tatiane S. Coelho , Bruno C. Cavalcanti , Cláudia Pessoa ,
d,
e
d,
e
f
f
Carlos A. de Simone , Isabelle K.C. Nunes , Nathalia M. de Oliveira , Renata G. de Almeida ,
a
,
1
a
b,
f,
**
*
Antonio V. Pinto , Kelly C.G. de Moura , Pedro A. da Silva , Eufrânio N. da Silva Júnior
a
Núcleo de Pesquisas de Produtos Naturais, UFRJ, 21941-971, Rio de Janeiro, RJ, Brazil
Laboratório de Micobacterias, Faculdade de Medicina, FURG, 96200-190 Rio Grande, RS, Brazil
Departamento de Fisiologia e Farmacologia, UFC, Campus do Porangabussu, 60430-270 Fortaleza, CE, Brazil
Instituto de Química e Biotecnologia, UFAL, Tabuleiro do Martins, 57072-970 Maceió, AL, Brazil
Departamento de Física e Informática, Instituto de Física, USP, 13560-970 São Carlos, SP, Brazil
Laboratório de Química Sintética e Heterocíclica, Instituto de Ciências Exatas, Departamento de Química, UFMG, 31270-901 Belo Horizonte, MG, Brazil
b
c
d
e
f
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 May 2011
Received in revised form
Several quinonoid and phenazine compounds were synthesized in moderate to high yields and showed
activity against H37Rv, rifampicin and isoniazid-resistance strains of Mycobacterium tuberculosis. The
cytotoxity of the compounds were evaluated against human peripheral blood mononuclear cells (PBMC)
and these substances emerge as promising antitubercular prototypes.
6
July 2011
Accepted 15 July 2011
Available online 23 July 2011
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
Lapachol
Phenazines
Mycobacterium tuberculosis
Antitubercular
1. Introduction
Clofazimine, an important phenazine compound, was originally
reported in 1957 as a potent antituberculosis agent and has been
Tuberculosis (TB) is a very aggressive elderly lung disease
caused by Mycobacterium tuberculosis [1] and still represents
nowadays a cause of globally death [2]. According to WHO, 9.27
million new cases of TB occurred in 2007 and about these cases,
estimates that, 1.37 million (14.8%) were HIV-positive [3]. The
emergence of the M. tuberculosis in the immunocompromised
population represents a problem that suggest continuing search of
new compounds against this pathogen [4]. Moreover, in the
absence of an effective vaccine, treatment is the main tool for
control ling the dissemination of TB, but the length of treatment
used as prototype to developed new drugs with antimicrobial
activities [6]. Phenazine-1-carboxylic acids, also known as tuber-
mycins present potent activity against M. tuberculosis, and repre-
sent a class of phenazines used to obtain new actives substances by
chemical modifications (Fig. 1).
Quinonoid compounds have been described as important
structures with pharmacological diversity [7]. Recently, our
research group has investigated the antitumor [8,9], trypanocidal
[10,11] and antibacterial [12] activity of a large number of
substances obtained from lapachol (1) as part of our study to search
new heterocyclic compounds [13] with potential activity against
neglected diseases. In this context, some of us have synthesized and
evaluated a group of the macrolactones and phenazine compounds
identifying new actives substances against M. tuberculosis [14].
Following our program to developed new compounds with
potent antimycobacterial activities, herein, we present the
synthesis and evaluation of quinones and new phenazine
compounds against M. tuberculosis pan-susceptible and resistants
strains.
(
usually 6 months) makes it difficult for patients to comply with
treatment [5].
*
Corresponding author. Tel.: þ55 53 32338895; fax: þ55 53 32338863.
*
* Corresponding author. Tel.: þ55 31 34095710; fax: þ55 31 34095700.
E-mail addresses: pedre@furg.br (P.A. da Silva), eufranio@ufmg.br, eufranio.
junior@yahoo.com.br (E. N. da Silva Júnior).
1
Dedicated to the memory of Professor Antonio V. Pinto who has been a great
inspiration to us over the years.
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.07.026

4522
P.F. Carneiro et al. / European Journal of Medicinal Chemistry 46 (2011) 4521e4529
Cl
N
(AeB) and that plane passing through the atoms of benzene ring
(
ꢀ
D) is 2.3 (3) , therefore, all rings of the molecule are almost planar
as showing in Fig. 3a. The torsion angle between atoms
ꢀ
C6eC13eC14eC15 is 47.5 (2) .The packing of the crystal structure
CH3
CH
N
is show in Fig. 3b.
N
R
R
3
3. Results and discussion
N
N
NH
R = OH or COOH
Tubermycin compounds
The minimum inhibitory concentrations (MICs) for all the
phenazine compounds and precursor quinones were evaluated
Clofazimine
against M. tuberculosis H37Rv, M. tuberculosis rifampicin resistance
Cl
(ATCC 35338) and M. tuberculosis isoniazid resistance (ATCC 35822)
(
Table 2). The toxicity of compounds toward a normal proliferating
Fig. 1. Antimycobacterial phenazine compounds.
cell was investigated using the Alamar Blue assay performed with
peripheral blood mononuclear cells (PBMC) after 72 h of drug
exposure. The compounds were classified by us according to their
2
. Chemistry
activity against M. tuberculosis as highly active (MIC ꢁ 3
lowly active (3 g/mL < MIC < 100 g/mL and), or inactive
(MIC > 100 g/mL).
mg/mL),
Lapachol (1) was extracted from the heartwood of Tabebuia sp.
Tecoma) and purified by a series of recrystallizations [15]. From this
m
m
(
m
quinone, nor-lapachol (11) was obtained by Hooker oxidation
method [16].
Lapachol (1), an important naphthoquinone precursor presents
lowly active against H37Rv and ATCC 35338 strains and for ATCC
35822 strains the substance was inactive.
Compounds 2, 9, 10, 15, 18, 19, 26, 28, 30 and 31 were considered
37
inactive when MIC >100 mg/mL for H Rv, ATCC 35338 and ATCC
Initially, from lapachol (1) the phenazine 2 was prepared in two
steps as previously described by us [17]. Lapachol was reduced by
catalytic reduction with Pd/C as catalyst and the substances 3 and 4
were prepared [18] and used to synthesized the phenazine
compounds 5 and 7 in good yields and the ether derivatives 6 and 8,
respectively. Finally, the ether phenazines 9 and 10 were obtained
from the substances 6 and 8, as yellow solids (Scheme 1).
Nor-lapachol (11) was submitted to reaction with ortho-phe-
nylenediamine in acetic acid to obtain the phenazine 12 and from
the reaction of the catalytic reduction the substances 13 and 14
were also obtained from this naphthoquinone. In order, to obtain
new phenazine compounds 15 and 18 from 13 and 14 the same
reaction for form the phenazinic ring was employed (Scheme 2).
35822 strains.
The substances 3, 4, 12, 13, 14, 20, 21, 22, 23 and 25 were lowly
active (12.5 g/mL for H37Rv, ATCC 35338 and
g/mL ꢁ MIC ꢁ 100
ATCC 35822 strains). With MIC value of the 25, 12.5 and 25 g/mL
for the M. tuberculosis H Rv, M. tuberculosis rifampicin resistance
m
m
m
3
7
(ATCC 35338) and M. tuberculosis isoniazid resistance (ATCC 35822)
the reduced quinones 4 and 14 are important prototypes for new
derivation for the achievement of compounds more actives.
The substance 7 was considered highly active [MIC ꢁ 3
(9.75 M)] against M. tuberculosis H Rv. For ATCC 35822 strain the
compound was less active with MIC value of 12.5 mg/mL and for
mg/mL
m
37
The Hooker’s reaction with PbO
2
[19] with minor modifications
[
13,20] was used to produce the ether derivatives 16 and 17 from
ATCC 35822 strain was inactive, this could be related to the
molecular basis of resistance of these strains. This compound also
the reduced quinones 13 and 14 and subsequently these substances
were used to obtain the ether phenazine derivatives 19 and 20 with
the side chain and aromatic portions reduced (Scheme 2).
From C-allyl lawsone (21) [21] was obtained the reduced
products 22 and 25 and the ether derivative 24. The phenazinic
compounds 23 and 26 were obtained from C-allyl lawsone and
reduced quinone 25, respectively. From ether quinones 24, 27 and
was not cytotoxic against normal cells [IC50 > 25
(>78.02 M)] and represents an important prototype for develop-
ment of new drugs against TB.
Nor-lapachol (11) showed promising activity against ATCC
35338 strains MIC [3.12 g/mL (13.7 M)] but lowly active for H Rv
and not active for ATCC 35822 strains (MIC ¼ 100
previously described nor-lapachol (11) was not cytotoxic against
normal cells (IC50 > 25 g/mL [>109.53 M]) and this substance
mg/mL
m
m
m
37
mg/mL). As
2
9 the ether phenazine compounds 28, 30 and 31 were synthesized
using the reaction with the diamine in acetic acid (Scheme 3).
All the compounds were purified by silica gel column chroma-
tography and in the case of the ether phenazines the re-
crystallization were preferred to avoid the cleavage of the CeOeC
bonds as previously described by us [18] and in general, the
substances were obtained in excellent yields.
m
m
appears as excellent prototype against resistant to rifampicin
strains.
4. Conclusions
The structures of the compounds were confirmed by techniques
Some phenazine compounds were synthesized and evaluated
against M. tuberculosis and the compounds 7 and 11 presented
important activity and could be considered new antimycobacterial
prototypes. The substances were inactive against human peripheral
blood mononuclear cells (PBMC) in a concentrate of 25 mg/mL and
present great selectivity against the agent that cause TB.
1
13
such as H and C NMR, IR, high-resolution (electrospray ioniza-
13
tion) mass spectra. For the compounds 12 and 23 the C NMR
spectra were not obtained due the low solubility in different
solvents in appropriate concentration to get these spectra. The
substance 23 was obtained in your crystalline form and the struc-
ture was solved by crystallographic methods.
Ortep-3 diagram of the molecule is shown in Fig. 2, and Table 1
lists main crystallographic parameters. In the unit cell there are
four independent molecules not related by symmetry and for the
sake of clarity in Fig. 2 we are showing only one of four molecules.
Bond lengths and angles are available in supporting information
and are in good agreement with the expected values reported in the
literature [22]. The dihedral angle formed between the least
squares plane passing through the atoms of benzophenazine ring
5. Experimental
5.1. Chemistry
Melting points were obtained on Thomas Hoover and are
uncorrected. Analytical grade solvents were used. Column chro-
matography was performed on silica gel (Acros Organics
0.035e0.070 mm, pore diameter ca 6 nm). Infrared spectra were

P.F. Carneiro et al. / European Journal of Medicinal Chemistry 46 (2011) 4521e4529
4523
NH
2
N
O
NH
2
NH
AcOH,AcONa
iii
O
OH
5, 90%
i
O
3
NH
2
2
O
O
O
N
O
O
O
N
NH
O
O
AcOH
O
6
O
,60%
O
O
O
N
9
OH
O
N
O
1
O
2
NH
2
N
O
NH
2
NH
O
AcOH,AcONa
iii
OH
7,
80%
ii
O
4
O
O
N
O
O
NH
NH
AcOH
2
O
N
O
O
O
2
O
O
8
1
0, 40%
Scheme 1. Obtention of phenazine compounds 2, 5, 7, 9 and 10. Reagents and conditions: (i) THF, 10 mol% Pd/C, 30 psi of H
AcOH, PbO
2
, 15 min; (ii) AcOH, 10 mol% Pd/C, 55 psi of H
2
, 6 h; (iii)
2
.
1
recorded on an FTIR Spectrometer IR Prestige-21 e Shimadzu. H
mass spectra (electrospray ionization) were obtained using
a MicroTOF Ic e Bruker Daltonics. In some cases the spectra were
obtained using a gas chromatograph mass spectrometer GCMS-
QP2010 PLUS Shimadzu with column DB-5MS and GCMS-QP5000
(70 eV). The fragments were described as a relation between
atomic mass units and the charge (m/z) and the relative abundance
13
and
a VNMRSYS-500, Varian MR 400 instrument, Varian Mercury Plus
00 instrument and Bruker AVANCE DRX200, in the solvents
indicated, with TMS as internal standard. Chemical shifts ( ) are
given in ppm and coupling constants (J) in Hertz. High resolution
C NMR were recorded at room temperature using
3
d
NH
NH
2
2
N
NH
AcOH, AcONa
O
15, 85%
O
O
O
O
O
N
O
O
NH
NH
2
2
OH
iii
O
O
O
N
NH
NH
2
2
O
O
O
N
O
OH
i
NH
AcOH
1
3
16
19, 45%
AcOH
AcONa
O
11
ii
1
2, 85%
O
O
O
NH
2
2
OH
iii
O
O
O
O
N
NH
N
O
O
14
O
O
AcOH
O
1
7
20, 90%
NH
NH
AcOH, AcONa
2
N
2
NH
O
18, 95%
2 2
Scheme 2. Obtention of phenazine compounds 12, 15, 18, 19 and 20. Reagents and conditions: (i) THF, 10 mol% Pd/C, 30 psi of H , 15 min; (ii) AcOH, 10 mol% Pd/C, 55 psi of H , 6 h;
(
iii) AcOH, PbO
2
.

4524
P.F. Carneiro et al. / European Journal of Medicinal Chemistry 46 (2011) 4521e4529
O
O
O
O
O
NH2
NH
AcOH
N
O
i
OH
iii
O
O
O
N
2
O
O
O
22
27
30, 55%
N
O
NH2
NH
NH
2
a
N
O
cO
,
H
A
23, 70%
OH
O
c
A
O 21
iii
O
O
O
NH2
O
O
N
N
O
NH
2
O
O
O
8, 60%
O
4
AcOH
2
2
O
O
O
O
NH
NH
2
O
N
ii
OH
O
O
iii
O
N
2
O
O
O
29
AcOH
O
25
31, 40%
NH2
NH
AcOH, AcONa
N
O
2
NH
2
6, 75%
2 2
Scheme 3. Obtention of phenazine compounds 23, 26, 28, 30 and 31. Reagents and conditions: (i) THF, 10 mol% Pd/C, 30 psi of H , 15 min; (ii) AcOH, 10 mol% Pd/C, 55 psi of H , 6 h;
(
iii) AcOH, PbO
2
.
in percentage of the base peak intensity. All the compounds were
nominated using the program CS ChemDraw Ultra version 10.0.
5.2. General procedures to prepare the phenazine compounds
The respective quinone (1.0 mmol), 7.9 mmol of crystalline
sodium acetate, 2.3 mmol of ortho-phenylenediamine and 5 mL of
glacial acetic acid were heated on steam-bath for a short time and
monitored by silica gel TLC (eluted with hexane and ethyl acetate
and dichloromethane 8:1:1). After this time, the solution was
poured on ice and filtered under vacuum. The phenazine
compounds obtained from lapachol (1), nor-lapachol (11), C-allyl
lawsone (21) and the reduced derivatives were purified by column
chromatography eluted with an increasing polarity gradient
mixture of hexane and ethyl acetate.
5.2.1. 6-Isopentylbenzo[a]phenazin-5(7H)-one (5)
The reaction of 2-hydroxy-3-isopentylnaphthalene-1,4-dione
(3), (244 mg, 1 mmol), 7.9 mmol of crystalline sodium acetate,
2.3 mmol of ortho-phenylenediamine in 5 mL of glacial acetic acid
yielded product 5, (287 mg, 0.9 mmol, 90% yield) as an red solid;
ꢀ
mp 79e80 C; IR (KBr) 3466, 3352, 3057, 2953, 2918, 2868, 2851,
1661, 1647, 1583, 1580, 1545, 1530, 1499, 1468, 1458, 1435, 1413,
1
9
d
381,1362,1346,1335,1312,1277,1246, 1220,1111, 1040, 1030, 1015,
ꢂ1
1
64, 934, 770, 756, 723 cm
;
3
H NMR (400 MHz, CDCl )
9.49e9.23 (m, 1H), 8.38e8.05 (m, 3H), 7.86e7.71 (m, 4H),
Fig. 2. An ORTEP-3 projection, showing the atom-numbering and displacement
ellipsoids at the 50% probability level of the compound 23.
3.46e3.18 (m, 2H),1.83e1.72 (m,1H),1.66e1.59 (m, 2H),1.07 (s, 3H),

P.F. Carneiro et al. / European Journal of Medicinal Chemistry 46 (2011) 4521e4529
4525
Table 1
38.5, 28.3, 24.5, 24.4, 22.6, 22.3, 22.2, 21.5; MS [70 eV, m/z] (%): 320
9), 305 (5), 277 (22), 264 (100), 249 (22), 235 (12), 219 (11), 205 (6),
193 (3), 181 (2), 128 (2), 115 (4), 103 (4), 91 (3), 77 (8), 65 (93), 43 (5).
Crystal data and structure refinement.
(
Empirical formula
Formula weight
Temperature
Wavelength
Crystal system
Space group
19 14 2
C H N O
286.33 g/mol
295(2) K
0.71073 Å
Triclinic
5.2.3. 2-Isopentyl-3-(6-isopentyl-5-oxo-5,6-dihydrobenzo[a]
phenazin-6-yloxy)naphthalene-1,4-dione 9
The reaction of 3-isopentyl-3-(3-isopentyl-1,4-dioxo-1,4-dihydr-
onaphthalen-2-yloxy)naphthalene-1,2,4(3H)-trione (6), (486 mg,
Pꢂ1
ꢀ
Unit cell dimensions
a ¼ 9.4877(5) Å
b ¼ 17.7654(13) Å
c ¼ 18.0763(12) Å
a
b
g
¼ 67.679(3)
ꢀ
ꢀ
¼ 89.733(1)
1
mmol), 2.3 mmol of ortho-phenylenediamine in 5 mL of glacial
¼ 90.077(4)
acetic acid yielded product 9, (340 mg, 0.6 mmol, 61% yield) as an
3
Volume
Z
2818.4(3) Å
ꢀ
yellow solid; mp 160e162 C; IR (KBr) 3070, 2953, 2930, 2870, 2855,
8
3
Density (calculated)
Absorption coefficient
F(000)
Crystal size
Theta range for
data collection
Index ranges
1.354 Mg/m
0.085 mm
1699, 1655, 1647, 1597, 1574, 1490, 1460, 1433, 1287, 1267, 1221, 1202,
ꢂ1
ꢂ1 1
1157, 1117, 1016, 1078, 1050, 1016, 954, 934, 764, 718 cm ; H NMR
400 MHz, CDCl 8.95 (d, 1H), 8.24 (d, 1H), 8.15 (d, 1H), 7.99e7.86
(m, 3H), 7.76e7.63 (m, 3H), 7.52 (t, 1H), 7.43e7.38 (d, 1H), 7.34 (t, 1H),
.99e2.91 (t, 2H), 2.27e2.16 (m,1H), 2.14e2.03 (m,1H),1.90e1.79 (m,
2H), 1.75e1.64 (m, 1H), 1.49e1.35 (m, 2H), 1.33e1.23 (m, 1H),
1208
(
3
) d
3
(0.23 ꢃ 0.14 ꢃ 0.04) mm
ꢀ
2.0 to 27.5
2
ꢂ10 ꢁ h ꢁ 12, ꢂ18 ꢁ k ꢁ 22,
ꢂ16 ꢁ l ꢁ 23
13
1
1
1
.15e1.06 (dd, 6H), 0.76 (dd, 6H); C NMR (100 MHz, CDCl
84.8, 181.1, 153.6, 152.5, 141.6, 144.1, 141.1, 135.5, 134.6, 133.6, 133.5,
32.4, 131.7, 131.4, 131.3, 130.6, 130.0, 129.9, 129.3, 129.1, 127.7, 126.2,
3
) d 192.8,
Reflections collected
Independent reflections
Completeness to
18586
12305 [R(int) ¼ 0.088]
98.7%
ꢀ
125.9,125.7, 88.1, 40.4, 37.0, 31.8, 28.8, 28.1, 22.7, 22.6, 22.4, 22.3, 22.2;
theta ¼ 27.42
þ
þ
Absorption correction
Refinement method
none
EI/HRMS (m/z) [M þ H] 559.2604. Calcd for [C36
34 2 4
H N O H] :
2
Full-matrix least squares on F
7435/0/793
1.05
559.2596.
Data/restraints/parameters
Goodness-of-fit on F2
5.2.4. 2-Isopentyl-3-(6-isopentyl-5-oxo-1,2,3,4,5,6-hexahydrobenzo
Final R indices [I > 2sigma(I)] R1 ¼ 0.0862, wR2 ¼ 0.1003
R indices (all data)
R1 ¼ 0.1003, wR2 ¼ 0.1612
[a]phenazin-6-yloxy)-5,6,7,8-tetrahydronaphthalene-1,4-dione 10
ꢂ3
Largest diff. peak and hole
0.280 and ꢂ0.326 e.Å
The reaction of 3-isopentyl-3-(3-isopentyl-1,4-dioxo-1,4-
dihydronaphthalen-2-yloxy)naphthalene-1,2,4(3H)-trione
(8),
(494 mg, 1 mmol), 2.3 mmol of ortho-phenylenediamine in 5 mL of
glacial acetic acid yielded product 10, (226 mg, 0.4 mmol, 40% yield)
as an yellow solid; mp 105e107 C; IR (KBr) 3040, 2953, 2935, 2868,
ꢀ
1
1
1
.07 (s, 3H); 13C NMR (50 MHz, DMSO-d
41.10, 141.14, 135.07, 135.01, 131.8, 131.2, 130.7, 129.9, 128.9, 128.7,
28.6, 126.8, 125.3, 80.4, 32.0, 27.0, 27.0, 22.0, 22.7, 22.1; MS [70 eV,
m/z] (%): 316 (12), 301 (3), 260 (100), 246 (6), 231 (27), 218 (5), 204
3), 190 (3), 177 (3), 164 (1), 140 (4), 127 (9), 115 (12), 102 (10), 89 (4),
6
)
d
198.6, 155.5, 143.8,
1680, 1643, 1605, 1500, 1470, 1460, 1449, 1429, 1412, 1325, 1287,
ꢂ1
1
1267, 1213, 1159, 1509, 1126, 1047, 916, 840, 766 cm
(400 MHz, CDCl 8.08 (dd, 1H), 7.98e7.94 (m, 1H), 7.74e7.66 (m,
; H NMR
3
) d
(
7
2H), 3.34e3.23 (m, 1H), 2.99e2.87 (m, 1H), 2.78e2.64 (m, 3H),
2.49e2.39 (m, 1H), 2.35e2.28 (m, 2H), 2.21e2.05 (m, 1H),
7 (26), 63 (4), 43 (32), 41 (25).
2
.00e1.66 (m, 8H), 1.59e1.30 (m, 8H), 1.22e1.11 (m, 1H), 1.03 (dd,
13
5.2.2. 6-Isopentyl-1,2,3,4-tetrahydrobenzo[a]phenazin-5(7H)-one
3
6H), 0.79 (dd, 6H); C NMR (100 MHz, CDCl ) d 194.9, 187.6, 183.5,
(
7)
154.4, 150.6, 147.4, 145.8, 141.8, 141.3, 140.2, 139.0, 138.0, 129.9,
129.5, 129.4, 128.9, 110.8, 87.3, 40.9, 37.0, 32.0, 28.7, 28.1, 24.9, 23.3,
The reaction of 2-hydroxy-3-isopentyl-5,6,7,8-tetrahydronaph-
þ
thalene-1,4-dione (4), (248 mg, 1 mmol), 7.9 mmol of crystalline
sodium acetate, 2.3 mmol of ortho-phenylenediamine in 5 mL of
glacial acetic acid yielded product 7, (262 mg, 0.8 mmol, 82% yield) as
22.7, 22.6, 22.6, 22.4; EI/HRMS (m/z) [M þ H] 567.3215. Calcd for
þ
[C34
38
H N
2
O
4
H] : 567.32231.
ꢀ
an yellow solid; mp 125e127 C; IR (KBr) 3372, 3354, 3331, 3300,
060, 2949, 2848, 2828, 1627, 1601, 1475, 1460, 1433, 1416, 1370,
5.2.5. 6-(2-Methylprop-1-enyl)benzo[a]phenazin-5(7H)-one 12
The reaction of 2-hydroxy-3-(2-methylprop-1-enyl)naphtha-
lene-1,4-dione (11), (228 mg, 1 mmol), 7.9 mmol of crystalline
sodium acetate, 2.3 mmol of ortho-phenylenediamine in 5 mL of
glacial acetic acid yielded product 12, (210 mg, 0.7 mmol, 70% yield)
3
ꢂ1
1
1
360, 1337, 1221, 1190, 1174, 1138, 1035, 758 cm
500 MHz, CDCl 8.18 (t, 2H), 7.77e7.66 (m, 2H), 5.41 (s, 1H), 3.41
s, 2H), 3.34e3.27 (t, 2H), 2.88 (s, 2H), 1.97 (s, 4H), 1.73 (m, 1H), 1.57
;
H NMR
(
(
(
3
) d
13
ꢀ
dd, 2H), 1.05 (s, 3H), 1.05 (s, 3H); C NMR (125 MHz, CDCl
3
)
d
152.9,
as an yellow solid; mp 178e180 C; IR (KBr) 3280, 3200, 3080, 2930,
142.4, 142.2, 140.6, 139.8, 134.0, 132.0, 129.6, 129.3, 129.0, 128.2, 116.5,
2914, 2860, 1585, 1500, 1408, 1358, 1320, 1300, 1246, 1217, 1067,
Fig. 3. (a): A view of the molecule parallel to planes of the rings and (b): The packing of the molecules, viewed down the c axis.

4526
P.F. Carneiro et al. / European Journal of Medicinal Chemistry 46 (2011) 4521e4529
Table 2
MIC (minimum inhibitory concentration) of the tested compounds against Mycobacterium tuberculosis H37Rv, M. tuberculosis rifampicin resistance (ATCC 35338) and
M. tuberculosis isoniazid resistance (ATCC 35822). Alamar Blue Assay was performed with human peripheral blood mononuclear cells (PBMC) after 72 h of drug exposure. Nd:
not determined.
Compound
MIC H37Rv
50 (206.6)
Inactive (>180.2)
50 (205.0)
m
g/mL (
m
M)
MIC 35338
m
g/mL (
m
M)
MIC 35822
100 (413.2)
Inactive (>180.2)
50 (205.0)
m
g/mL (
m
M)
PBMC mg/mL (mM)
1
2
3
4
50 (206.06)
100 (180.2)
50 (205.0)
12.5 (50.4)
>25 (>103.19)
>25 (>45.07)
>25 (>102.34)
20.18 (81.26)
25 (100.8)
25 (100.8)
1
7.81e23.64 (71.72e95.20)
16.08 (50.82)
4.27e19.34 (45.10e61.12)
>25 (>78.02)
>25 (>44.74)
>25 (>44.90)
>25 (>109.53)
19.75 (65.75)
5
6.25 (19.8)
12.5 (39.5)
12.5 (39.1)
Inactive (>178.9)
100 (176.4)
3.12 (13.7)
12.5 (39.5)
1
7
9
ꢁ3.12 (9.75)
Inactive (>178.9)
100 (176.4)
50 (219.3)
Inactive (>312.5)
Inactive (>178.9)
Inactive (>176.4)
100 (438.6)
10
11
12
25 (83.3)
25 (83.3)
25 (83.3)
1
8.61e22.68 (61.96e75.51)
21.53 (93.50)
0.18e23.49 (87.64e102.01)
17.81 (73.20)
5.62e19.24 (64.20e79.08)
1
3
4
50 (217.4)
25 (106.7)
25 (108.7)
50 (217.4)
25 (106.7)
2
1
12.5 (53.35)
1
15
18
19
20
21
22
Inactive (>331.1)
Inactive (>326.8)
Inactive (>188.3)
50 (92.8)
100 (467.3)
100 (462.5)
Inactive (>331.1)
Inactive (>326.8)
Inactive (>188.3)
25 (46.4)
25 (116.8)
50 (231.25)
Inactive (>331.1)
Inactive (>326.8)
Inactive (>188.3)
25 (46.4)
100 (467.3)
100 (462.5)
>25 (>82.68)
>25 (>81.59)
>25 (>47.11)
>25 (>46.41)
>25 (>116.70)
23.64 (109.32)
2
1.15e25.37 (97.81e117.32)
21.19 (74.0)
8.42e23.17 (64.33e80.92)
22.47 (102.01)
9.62e25.09 (89.07e113.91)
2
3
5
25 (87.4)
50 (174.8)
25 (113.5)
12.5 (43.7)
25 (113.5)
1
2
50 (227.0)
1
2
2
3
3
6
8
0
1
Nd
Nd
Nd
>25 (>85.5)
>25 (>50.14)
>25 (>49.74)
>25 (>48.96)
Inactive (>200.8)
Inactive (>198.8)
Inactive (>195.7)
0.1 (0.12)
Inactive (>200.8)
Inactive (>198.8)
100 (195.7)
>2 (2.43)
Inactive (>200.8)
Inactive (>198.8)
Inactive (>195.7)
Rifampicin
Isoniazid
0.02 (0.145)
>0.2 (1.45)
66 cm ; 1H NMR (400 MHz, CDCl
ꢂ1
)
d
9.43e9.37 (m, 1H),
2864, 2837, 2525,1624,1603,1524,1458,1431,1381,1364,1335,1231,
7
8
3
2
3
ꢂ1
1
.43e8.21 (m, 3H), 7.80 (m, 4H), 6.67 (s, 1H), 6.27 (s, 1H), 2.19 (s,
H), 1.77 (s, 3H); MS [70 eV, m/z] (%): 300 (33), 285 (100), 268 (4),
55 (10), 242 (3), 231 (6), 217 (2), 207 (2), 190 (1), 177 (1), 165 (1),
1200, 1175, 1152, 1134, 1111, 1028, 945, 840, 756 cm
; H NMR
(400 MHz, CDCl 8.24e8.12 (m, 2H), 7.77e7.63 (m, 2H), 5.56e5.40
3
) d
(m, 1H), 3.47e3.36 (m, 2H), 3.22e3.13 (m, 2H), 2.91e2.83 (m, 2H),
2.26e2.11 (m, 1H), 2.00e1.91 (m, 4H), 1.03e1.01 (s, 3H), 1.01e0.99 (s,
1
42 (24), 128 (14), 115 (8), 102 (8), 89 (4), 77 (21), 63 (8), 51 (9).
13
3
6
H); C NMR (100 MHz, DMSO-d ) d 153.4, 142.9, 142.1, 140.5, 139.8,
5
.2.6. 6-Isobutylbenzo[a]phenazin-5(7H)-one 15
The reaction of 2-hydroxy-3-isobutylnaphthalene-1,4-dione
134.2,132.0,129.6,129.3,129.0,128.1,115.2, 32.8, 29.0, 24.5, 24.4, 22.8,
22.4, 22.2; MS [70 eV, m/z] (%): 306 (40), 291 (24), 275 (3), 263 (100),
249 (11), 235 (22), 219 (16), 205 (14), 193 (8), 181 (4), 167 (3), 140 (3),
132 (5), 115 (8), 109 (13), 89 (4), 77 (20), 65 (8), 43 (38), 41 (32).
(13), (230 mg, 1 mmol), 7.9 mmol of crystalline sodium acetate,
2
.3 mmol of ortho-phenylenediamine in 5 mL of glacial acetic acid
yielded product 15, (263 mg, 0.8 mmol, 87% yield) as an red solid;
ꢀ
mp 120e124 C; IR (KBr) 3439, 3374, 3100, 2951, 2920, 2866, 1655,
5.2.8. 2-Isobutyl-3-(6-isobutyl-5-oxo-5,6-dihydrobenzo[a]
phenazin-6-yloxy)naphthalene-1,4-dione 19
1
1
643, 1624, 1591, 1460, 1369, 1350, 1331, 1279, 1236, 1179, 1163,
ꢂ1
1
040,1020, 970, 910, 780, 760, 727 cm ; H NMR (400 MHz, CDCl
9.22e9.15 (m, 1H), 8.30e8.25 (m, 1H), 8.18e8.12 (d, 1H), 7.95 (d,
H), 7.75e7.69 (m, 2H), 7.65 (m, 2H), 3.05 (d, 2H), 2.18 (m, 1H), 1.03
152.6, 144.7,
41.9, 139.1, 139.2, 130.0, 129.7, 129.03, 129.0, 128.65, 128.61, 127.6,
3
)
The reaction of 3-isobutyl-3-(3-isobutyl-1,4-dioxo-1,4-dihydro-
naphthalen-2-yloxy)naphthalene-1,2,4(3H)-trione (16), (458 mg,
1 mmol), 2.3 mmol of ortho-phenylenediamine in5mL of glacialacetic
acid yielded product 19, (212 mg, 0.4 mmol, 40% yield) as an yellow
d
1
(
s, 3H), 1.03 (s, 3H); 13C NMR (75 MHz, DMSO-d
6
)
d
ꢀ
1
solid; mp 173e175 C; IR (KBr) 3040, 2957, 2926, 2868, 1707, 1659,
1
2
24.6, 122.8, 115.7, 32.0, 28.2, 22.5; MS [70 eV, m/z] (%): 302 (26),
87 (17), 271 (7), 259 (100), 246 (10), 231 (36), 218 (8), 204 (4), 190
1643, 1595, 1574, 1491, 1458, 1429, 1363, 1333, 1308, 1296, 1261, 1234,
1204, 1180, 1155, 1142, 1128, 1115, 1070, 1045, 970, 955, 923, 772, 762,
ꢂ1
1
(
(
2), 176 (2), 164 (1), 151 (2), 127 (11), 115 (11), 102 (11), 89 (4), 71
29), 63 (2), 41 (8).
3
721, 708 cm ; H NMR (400 MHz, CDCl ) d 8.94 (d, 1H), 8.24 (d, 1H),
8.14 (d,1H), 7.97 (d,1H), 7.89 (dd, 2H), 7.71 (t, 2H), 7.64 (dd,1H), 7.52 (t,
1
H), 7.41 (d, 1H), 7.34 (dd, 1H), 2.88 (d, 2H), 2.56e2.42 (m, 1H),
13
5.2.7. 6-Isobutyl-1,2,3,4-tetrahydrobenzo[a]phenazin-5(7H)-one 18
2.20e2.07 (m, 2H), 2.02e1.93 (m, 1H), 1.18 (dd, 6H), 0.81 (t, 6H);
C
The reaction of 2-hydroxy-3-isobutyl-5,6,7,8-tetrahydronaphth-
3
NMR (100 MHz, CDCl ) d 192.7, 185.1, 181.0, 153.5, 153.0, 144.1, 141.5,
alene-1,4-dione (18), (234 mg, 1 mmol), 7.9 mmol of crystalline
sodium acetate, 2.3 mmol of ortho-phenylenediamine in 5 mL of
glacial acetic acid yielded product 18, (294 mg, 0.9 mmol, 96% yield)
141.0, 135.4, 134.5, 133.6, 132.4, 132.1, 131.5, 131.3, 130.7, 130.0, 129.8,
129.3, 129.0, 127.8, 126.2, 125.9, 125.8, 88.8, 50.7, 32.8, 27.9, 24.4, 24.1,
þ
23.7, 23.3, 23.1; EI/HRMS (m/z) [M þ H] 531.2273. Calcd for
ꢀ
þ
as an yellow solid; mp 131e133 C; IR (KBr) 3399, 3057, 2949, 2930,
[C34
H
30
N
2
O
4
H] : 531.22841.

P.F. Carneiro et al. / European Journal of Medicinal Chemistry 46 (2011) 4521e4529
4527
5.2.9. 2-Isobutyl-3-(6-isobutyl-5-oxo-1,2,3,4,5,6-hexahydrobenzo
13C NMR (100 MHz, CDCl
3
)
d
192.2, 184.3, 181.2, 152.7, 144.2, 141.6,
[
a]phenazin-6-yloxy)-5,6,7,8-tetrahydronaphthalene-1,4-dione 20
The reaction of 3-isobutyl-3-(3-isobutyl-1,4-dioxo-1,4,5,6,7,8-
141.1, 135.5, 134.7, 134.0, 133.8, 132.6, 131.6, 131.4, 131.3, 130.5, 130.3,
130.1, 129.4, 129.1, 127.7, 126.3, 125.9, 125.8, 120.0, 116.6, 87.9, 46.8,
þ
þ
22 2 4
H N O H] :
hexahydronaphthalen-2-yloxy)-5,6,7,8-tetrahydronaphthalene-
,2,4(3H)-trione (17), (458 mg, 1 mmol), 2.3 mmol of ortho-phe-
28.2; EI/HRMS (m/z) [M þ H] 499.1649. Calcd for [C32
1
499.16581.
nylenediamine in 5 mL of glacial acetic acid yielded product 20,
ꢀ
(
425 mg, 0.7 mmol, 79% yield) as an yellow solid; mp 134e138 C;
5.2.13. 2-(5-Oxo-6-propyl-5,6-dihydrobenzo[a]phenazin-6-yloxy)-
3-propylnaphthalene-1,4-dione 30
IR (KBr) 3040, 2955, 2868, 1694, 1645, 1633, 1622, 1605, 1489, 1464,
ꢂ1
1
1
(
(
2
4
447, 1326, 1265, 1219, 1157, 1130, 1045, 910, 770 cm ; H NMR
400 MHz, CDCl 8.06 (d,1H), 7.91 (d,1H), 7.69 (m, 2H), 3.33e3.24
m, 1H), 2.97e2.87 (m, 1H), 2.79e2.70 (m, 1H), 2.62 (d, 2H),
The reaction of 3-(1,4-dioxo-3-propyl-1,4-dihydronaphthalen-
2-yloxy)-3-propylnaphthalene-1,2,4(3H)-trione (27), (430 mg,
1 mmol), 2.3 mmol of ortho-phenylenediamine in 5 mL of glacial
acetic acid yielded product 30, (261 mg, 0.5 mmol, 50% yield) as an
3
) d
.48e2.39 (m, 1H), 2.36e2.30 (m, 1H), 2.25 (m, 1H), 2.07e1.93 (m,
H), 1.91e1.83 (m, 4H), 1.79e1.69 (m, 1H), 1.64e1.59 (m, 1H),
ꢀ
yellow solid; mp 163e165 C; IR (KBr) 3071, 2957, 2928, 2870, 1694,
1
CDCl
.55e1.34 (m, 4H), 1.08 (dd, 6H), 0.79 (dd, 6H); 13C NMR (100 MHz,
1659, 1647, 1597, 1574, 1491, 1458, 1337, 1261, 1236, 1204, 1107, 1091,
ꢂ1
1
3
)
d
194.7, 187.8, 183.4, 154.3, 151.1, 147.2, 145.8, 141.7, 141.2,
1044, 1018, 959, 947, 926, 762, 717 cm ; H NMR (400 MHz, CDCl
3
)
1
2
40.1, 139.0, 138.0, 129.9, 129.4, 128.9, 127.9, 87.8, 51.6, 32.3, 27.9,
d 8.95 (d,1H), 8.23 (d,1H), 8.14 (d,1H), 8.00e7.85 (m, 3H), 7.75e7.62
4.9, 24.4, 24.1, 23.5, 23.4, 23.2, 23.0, 22.6, 22.0, 21.7, 21.6, 20.9, 20.9;
(m, 3H), 7.51 (t,1H), 7.41 (d, 1H), 7.37e7.30 (t,1H), 2.98e2.88 (t, 2H),
2.25e2.05 (m, 3H), 2.02e1.81 (m, 2H), 1.60e1.31 (m, 2H), 1.19 (t,
þ
þ
38 2 4
H N O H] :
EI/HRMS (m/z) [M þ H] 539.2901. Calcd for [C34
13
5
39.29101.
3H), 0.81 (t, 3H); C NMR (100 MHz, CDCl
3
) d 188.9, 181.0, 177.2,
149.5, 148.8, 140.3, 131.6, 130.7, 129.7, 129.1, 128.5, 127.7, 127.5, 127.4,
5
.2.10. 6-Allylbenzo[a]phenazin-5(7H)-one 23
126.7, 126.1, 126.0, 125.4, 125.2, 123.7, 122.3, 122.0, 121.8, 84.2, 40.5,
þ
The reaction of 2-allyl-3-hydroxynaphthalene-1,4-dione (21),
228 mg,1 mmol), 7.9 mmol of crystalline sodium acetate, 2.3 mmol
22.3, 17.6, 12.9, 10.7, 10.0; EI/HRMS (m/z) [M þ H] 503.1976. Calcd
þ
(
for [C32H
26
N
2
O
4
H] : 503.19711.
of ortho-phenylenediamine in 5 mL of glacial acetic acid yielded
product 23, (200 mg, 0.7 mmol, 70% yield) as an red solid; mp
5.2.14. 2-(5-Oxo-6-propyl-1,2,3,4,5,6-hexahydrobenzo[a]phenazin-
6-yloxy)-3-propyl-5,6,7,8-tetrahydronaphthalene-1,4-dione 31
The reaction of 3-(1,4-dioxo-3-propyl-1,4,5,6,7,8-hexahy-
dronaphthalen-2-yloxy)-3-propyl-5,6,7,8-tetrahydronaphthalene-
1,2,4(3H)-trione (29), (438 mg, 1 mmol), 2.3 mmol of ortho-phenyl-
enediamine in 5 mL of glacial acetic acid yielded product 31, (117 mg,
ꢀ
158e160 C; IR (KBr) 3372, 3248, 3156, 3076, 3021, 2980, 2924,
2
888,1640, 1618,1600, 1591,1576,1533,1510, 1477, 1463,1431, 1410,
ꢂ1
1
1385, 1364, 1346, 1317, 1250, 1229, 770 cm ; H NMR (400 MHz,
C
8
5
D
5
N)
d 9.75e9.55 (d, 1H), 8.84e8.77 (d, 1H), 8.49 (d, 1H),
.43e8.31 (m, 1H), 7.94e7.82 (m, 2H), 7.80e7.77 (m, 1H), 7.65e7.50
ꢀ
(
4
(
1
m, 1H), 6.54e6.34 (m, 1H), 5.41e5.28 (d, 1H), 5.17e5.08 (d, 1H),
.57 (m, 2H); MS [70 eV, m/z] (%): 286 (35), 271 (100), 255 (8), 242
7), 233 (10), 217 (4), 164 (1), 142 (16), 142 (16), 128 (21), 121 (20),
02 (14), 88 (5), 77 (26), 63 (8), 44 (36).
0.2 mmol, 23% yield) as an yellow solid; mp 161e162 C; IR (KBr)
3080, 2940, 2932, 2872,1676,1643,1603,1480,1456,1420,1317,1265,
ꢂ1
1
1234, 1217, 1159, 1128, 1103, 1082, 960, 918, 766 cm
(400 MHz, CDCl 8.07 (d, 1H), 7.96 (d, 1H), 7.74e7.64 (m, 2H),
.34e3.22 (m, 1H), 2.99e2.86 (m, 1H), 2.77e2.61 (m, 3H), 2.48e2.38
(m, 1H), 2.36e2.28 (m, 2H), 2.19e1.69 (m, 10H), 1.56e1.38 (m, 4H),
; H NMR
3
) d
3
5.2.11. 6-Propyl-1,2,3,4-tetrahydrobenzo[a]phenazin-5(7H)-one 26
13
The reaction of 2-hydroxy-3-propyl-5,6,7,8-tetrahydronaph-
1.34e1.22 (m, 2H),1.10 (t, 3H), 0.82 (t, 3H); C NMR (100 MHz, CDCl
3
)
thalene-1,4-dione (25), (220 mg, 1 mmol), 7.9 mmol of crystalline
sodium acetate, 2.3 mmol of ortho-phenylenediamine in 5 mL of
glacial aceticacidyielded product 26, (218 mg, 0.7 mmol, 75% yield)as
d
194.9, 187.7, 183.6, 154.2, 150.8, 147.6, 145.9, 141.8, 141.3, 140.2, 138.9,
137.9,129.9,129.5,129.4,129.0,128.9, 87.2, 45.0, 25.5, 24.9, 23.3, 22.6,
þ
21.9, 21.7, 21.6, 21.5, 20.9, 20.9,16.9,14.6,14.0;EI/HRMS (m/z)[Mþ H]
ꢀ
þ
an yellow solid; mp 147e148 C; IR (KBr) 3403, 3080, 2949, 2938,
511.2595. Calcd for [C32
H
34
N
2
O
4
H] : 511.2597.
2
864, 2853,1624,1601,1465,1435,1422,1355,13337,1215,1188,1173,
ꢂ1
1
1
d
136, 1105, 1034, 1020, 949, 758 cm ; H NMR (400 MHz, CDCl
8.22e8.08 (m, 2H), 7.80e7.62 (m, 2H), 5.79e5.28 (m,1H), 3.45e3.34
m, 2H), 3.31e3.18 (m, 2H), 2.91e2.82 (m, 2H), 2.00e1.90 (m, 4H),
3
)
5.3. X-ray analysis
(
X-ray diffraction data collection were performed on an Enraf-
Nonius Kappa-CCD diffractometer (95 mm CCD camera on
k-goniostat) using graphite monochromated MoKa radiation
13
1
1
2
(
.81e1.67 (m, 2H), 1.05 (t, 3H); C NMR (50 MHz, DMSO-d
49.1, 147.1, 142.5, 141.8, 135.7, 134.4, 130.8, 130.4, 130.3, 129.9, 118.2,
6.8, 26.4, 25.8, 24.2, 23.7, 23.4,15.8;MS[70 eV, m/z] (%):292 (74), 277
81), 264 (100): 249 (18), 237 (38), 219 (26), 205 (18),193 (12),181 (9),
67 (5), 140 (6), 132 (13), 122 (19), 109 (46), 97 (10), 77 (36), 65 (14),
6
) d 157.8,
(0.71073 Å), at room temperature. Data collection were carried out
ꢀ
using the COLLECT software [23] up to 50 in 2
q. Final unit cell
1
parameters were based on 9578 reflections. Integration and scaling
of the reflections, correction for Lorentz and polarization effects
were performed with the HKL DENZO-SCALEPACK system of
programs [24]. The structure of the compound was solved by direct
methods with SHELXS-97 [25]. The models were refined by full-
4
3 (32).
5.2.12. 2-Allyl-3-(6-allyl-5-oxo-5,6-dihydrobenzo[a]phenazin-6-
yloxy)naphthalene-1,4-dione 28
The reaction of
dihydronaphthalen-2-yloxy)naphthalene-1,2,4(3H)-trione
426 mg, 1 mmol), 2.3 mmol of ortho-phenylenediamine in 5 mL of
2
3-allyl-3-(3-allyl-1,4-dioxo-1,4-
(24),
matrix least squares on F using SHELXL-97 [26]. The program
ORTEP-3 [27] was used for graphic representation and the program
WINGX [28] to prepare materials for publication. All H atoms were
located by geometric considerations placed (CeH ¼ 0.93e0.97 Å;
(
glacial acetic acid yielded product 28, (279 mg, 0.5 mmol, 56% yield)
as an yellow solid; mp 167e168 C; IR (KBr) 3075, 2918, 1710, 1688,
ꢀ
NeH ¼ 0.86 Å) and refined as riding with Uiso(H) ¼ 1.2Ueq
.
1657, 1599, 1593, 1576, 1491, 1460, 1431, 1344, 1308, 1283, 1260,
Crystallographic data for compound 23 have been deposited
with the Cambridge Crystallographic Data Center as Supplemen-
tary Publication No. CCDC 823816. Copies of the data can be
obtained, free of charge, on application to CCDC, 12 Union Road,
Cambridge CH21EZ, UK (fax: þ44 1223 336 033 or e-mail: deposit@
ccdc.cam.ac.uk).
ꢂ1
1
1
220,1202,1160,1120,1078,1045,1033, 961, 920, 768, 725 cm ; H
NMR (400 MHz, CDCl 8.94 (d, 1H), 8.22 (d, 1H), 8.15 (d, 1H), 7.96
t, 2H), 7.90 (t, 3H), 7.76e7.64 (m, 1H), 7.53 (t, 1H), 7.43 (d, 1H), 7.35
t, 1H), 6.27 (m, 1H), 5.80e5.68 (m, 1H), 5.45 (d, 1H), 5.23 (d, 1H),
.99 (d, 1H), 4.90 (d, 1H), 3.72 (d, 2H), 3.03 (dd, 1H), 2.88 (dd, 1H);
3
) d
(
(
4

4528
P.F. Carneiro et al. / European Journal of Medicinal Chemistry 46 (2011) 4521e4529
6
. Biological assay in vitro
post doctoral grant (E-26/101.311/2008). Dr. C.A. de Simone is
thankful to the group of crystallography IFSC-USP for allowing the
use of KappaCCD diffractometer. Dr. E.N. da Silva Júnior thanks the
Programa Institucional de Auxílio à Pesquisa de Doutores Recém-
Contratados e UFMG (Edital 08/2010).
6
.1. Strains and minimum inhibitory concentration (MIC)
determination
The experiment was carried out using M. tuberculosis H37Rv
(
ATCC27294) e a pan-susceptible strain e rifampicin-resistant
Appendix. Supplementary material
strain (ATCC35338) with a His-526-Tir mutation in the rpoB gene
and isoniazid-resistant strain (ATCC35822) with mutation in the
gene katG Ser-315-Tir of M. tuberculosis. The strains were grown in
Supplementary material related to this article can be found
online at doi:10.1016/j.ejmech.2011.07.026.
ꢀ
the Ogawa Kudoh medium for 14 days at 37 C. The determination
of antimicrobacterial activity was performed using the REMA
References
(
Resazurin Microtitre Assay), as previously described [29] In brief,
the bacterial suspensions were homogenized by vortex agitation,
and the turbidity was adjusted according to the McFarland scale
[1] R. Koch, Berl. Klin. Wochenschr. 30 (1882) 221.
[
[
2] R. Fears, S. Kaufmann, V. Meulen, A. Zumla, Tuberculosis 90 (2010) 182.
3] W.H.O. Report, Global Tuberculosis Control 2009 e Epidemiology, Strategy and
Financing (2009), ISBN 9789241563802 WHO/HTM/TB/2009.411; 2009.
6
1
.0 (3.2 ꢃ 10 colony-forming units/mL). The inoculums was
prepared by diluting the bacterial suspension 1:20 in Middlebrook
H9 OADC medium (4.7 g Middlebrook 7H9 base; Difco, Becton
Dickinson).
The MIC assay was performed a 96-well microplates, with
compounds concentration of 100 g/mL in the first well, then
[4] T. Tran, E. Saheba, A.V. Arcerio, V. Chavez, Q.-Y. Li, L.E. Martinez, T.P. Primm,
Bioorg. Med. Chem. 12 (2004) 4809.
7
[
5] P.A. da Silva, J.A. Aínsa, Drugs and drug interactions. in: J.C. Palomino,
S.C. Leão, V. Ritacco (Eds.), Tuberculosis (2007) From Basic Science to Patient
Care. http://www.TuberculosisTextbook.com.
[6] C.E.J. van Rensburg, G.K. Jooné, F.A. Sirgel, N.M. Matlola, J.F. O’Sullivan,
Chemotherapy 46 (2000) 43.
m
serially decreased to 3.12
microplate was incubated for 7 days at 37 C. Following the
m
g/mL in the last well. Each 96-well
[
7] (a) D.M. Hernández, M.A.B.F. de Moura, D.P. Valencia, F.J. González, I. González,
F.C. de Abreu, E.N. da Silva Júnior, V.F. Ferreira, A.V. Pinto, M.O.F. Goulart,
C. Frontana, Org. Biomol. Chem. 6 (2008) 3414;
ꢀ
initial incubation period, 30
m
l of resazurin was added into each
ꢀ
(b) V.F. Ferreira, S.B. Ferreira, F.C. da Silva, Org. Biomol. Chem. 21 (2010) 4793;
well and then incubated for an additional two days at 37 C. The
cell viability observation was done based on the oxi-reduction of
the resazurin by noting the change of color when cellular growth
was occurring.
(c) C.O. Salas, M. Faúndez, A. Morello, J.D. Maya, R.A. Tapia, Curr. Med. Chem.
18 (2011) 144;
(d) A.V. Pinto, S.L. de Castro, Molecules 14 (2009) 4570;
(e) S.B. Ferreira, K. Salomão, F.C. da Silva, A.V. Pinto, C.R. Kaiser, A.C. Pinto,
V.F. Ferreira, S.L. de Castro, Eur. J. Med. Chem. (2011). doi:10.1016/
j.ejmech.2011.03.012.
6
.2. Inhibition of PBMC proliferation e Alamar Blue assay
[8] (a) E.N. da Silva Júnior, M.C.B.V. de Souza, A.V. Pinto, M.C.F.R. Pinto,
M.O.F. Goulart, F.W.A. Barros, C. Pessoa, L.V. Costa-Lotufo,
R.C. Montenegro, M.O. de Moraes, V.F. Ferreira, Bioorg. Med. Chem. 15
2007) 7035;
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
(b) E.N. da Silva Júnior, M.A.B.F. de Moura, A.V. Pinto, M.C.F.R. Pinto,
M.C.B.V. de Souza, A.J. Araújo, C. Pessoa, L.V. Costa-Lotufo,
R.C. Montenegro, M.O. de Moraes, V.F. Ferreira, M.O.F. Goulart, J. Braz.
Chem. Soc. 20 (2009) 635.
exposure. After 24 h, compounds (0.048e25
DMSO (0.1%) were added to each well and incubated for 72 h.
Doxorubicin (0.01e1.06 M) was used as positive control. Twenty-
four h before the end of the incubation, 10 L of stock solution
0.312 mg/mL) of the Alamar Blue (Resazurin, SigmaeAldrich Co)
mg/mL) dissolved in
[9] E.N. da Silva Júnior, C.F. de Deus, B.C. Cavalcanti, C. Pessoa, L.V. Costa-Lotufo,
R.C. Montenegro, M.O. de Moraes, M.C.F.R. Pinto, C.A. de Simone, V.F. Ferreira,
M.O.F. Goulart, C.K.Z. Andrade, A.V. Pinto, J. Med. Chem. 53 (2010) 504.
10] E.N. da Silva Júnior, R.F.S. Menna-Barreto, M.C.F.R. Pinto, R.S.F. Silva,
D.V. Teixeira, M.C.B.V. de Souza, C.A. de Simone, S.L. de Castro, V.F. Ferreira,
A.V. Pinto, Eur. J. Med. Chem. 43 (2008) 1774.
11] E.N. da Silva Júnior, T.T. Guimarães, R.F.S. Menna-Barreto, M.C.F.R. Pinto,
C.A. de Simone, C. Pessoa, B.C. Cavalcanti, J.R. Sabino, C.K.Z. Andrade,
M.O.F. Goulart, S.L. de Castro, A.V. Pinto, Bioorg. Med. Chem. 18 (2010) 3224.
m
m
[
[
(
was added to each well. The absorbance was measured using
a multiplate reader (DTX 880 Multimode Detector, Beckman
Ò
Coulter ) and the drug effect was quantified as the percentage of
control absorbance at 570 nm and 595 nm. The absorbance of
Alamar 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 wavelength.
This value is called AOLW. A correction factor, R0, can be calculated
from AOHW and AOLW, where R0 ¼ AOLW/AOHW. The percent
[12] A.L. Lourenço, P.A. Abreu, B. Leal, E.N. da Silva Júnior, A.V. Pinto, M.C.F.R. Pinto,
A.M.T. Souza, J.S. Novais, M.B. Paiva, L.M. Cabral, C.R. Rodrigues, V.F. Ferreira,
H.C. Castro, Curr. Microbiol. 62 (2011) 684.
[
[
[
13] (a) E.N. da Silva Júnior, C.A. de Simone, A.C.B. de Souza, C.N. Pinto,
T.T. Guimarães, M.C.F.R. Pinto, A.V. Pinto, Tetrahedron 50 (2009) 1550;
(b) F.S. Emery, M.C.F.R. Pinto, C.A. de Simone, V.R.S. Malta, E.N. da Silva Júnior,
A.V. pinto, Synlett 13 (2010) 1931.
14] (a) R.S.F. Silva, M.C.F.R. Pinto, M.O.F. Goulart, J.D.S. Filho, I. Neves Júnior,
M.C.S. Lourenço, A.V. Pinto, Eur. J. Med. Chem. 44 (2009) 2334;
(b) T.S. Coelho, R.S.F. Silva, A.V. Pinto, M.C.F.R. Pinto, C.J. Scaini, K.C.G. Moura,
P.A. da Silva, Tuberculosis 90 (2010) 293.
15] M.C.F.R. Pinto, A.V. Pinto, C.G.T. Oliveira, An. Acad. Brasil. Ciências 52 (1980)
481.
[
[
16] L.F. Fieser, M. Fieser, J. Am. Chem. Soc. 70 (1948) 3215.
Alamar Blue reduced is then expressed as follows:
%
17] E.N. da Silva Júnior, M.C.F.R. Pinto, K.C.G. de Moura, C.A. de Simone,
C.J. Nascimento, C.K.Z. Andrade, A.V. Pinto, Tetrahedron 50 (2009) 1575.
18] E.N. da Silva Júnior, B.C. Cavalcanti, T.T. Guimarães, M.C.F.R. Pinto, I.O. Cabral,
C. Pessoa, L.V. Costa-Lotufo, M.O. de Moraes, C.K.Z. de Andrade, M.R. dos Santos,
C.A. de Simone, M.O.F. Goulart, A.V. Pinto, Eur. J. Med. Chem. 46 (2011) 399.
19] S.C. Hooker, J. Am. Chem. Soc. 58 (1936) 1168.
20] A.Y. Yakubovskaya, T.Y. Kochergina, V.A. Denisenko, D.V. Berdyshev,
V.P. Glazunov, V.P. Anufriev, Russ. Chem. Bull. Int. Ed. 55 (2006) 301.
21] R.S.F. Silva, E.M. Costa, U.L.T. Trindade, D.V. Teixeira, M.C.F.R. Pinto, G.L. Santos,
V.R.S. Malta, C.A. de Simone, A.V. Pinto, S.L. de Castro, Eur. J. Med. Chem. 41
reduced ¼ ALW ꢂ (AHW ꢃ R0) ꢃ 100.
[
Conflict of interest
Authors declare no conflict of interest.
[
[
Acknowledgments
[
This research was supported by grants from the Conselho
Nacional de Desenvolvimento Científico e Tecnológico (CNPq),
Fundação Carlos Chagas Filho de Amparo à Pesquisa do Rio de
Janeiro (FAPERJ) and PRONEX/E-26/110.574/2010, CAPES, UFRJ,
UFAL, USP, FURG and UFMG. Dr. K.C.G. Moura thanks the FAPERJ for
(
2006) 526.
[22] F.H. Allen, Acta Cryst. B58 (2002) 380.
[
[
23] Enraf-Nonius, COLLECT. Nonius BV, Delft, The Netherlands, 1997-2000.
24] Z. Otwinowski, W. Minor, H.K.L. Denzo, Scalepack, in: C.W. Carter Jr.,
R.M. Sweet (Eds.), Methods in Enzymology, vol. 276, Academic Press, New
York, 1997, pp. 307e326.

P.F. Carneiro et al. / European Journal of Medicinal Chemistry 46 (2011) 4521e4529
4529
[
[
25] G.M. Sheldrick, SHELXS-97. Program for Crystal Structure Resolution.
[27] L.J. Farrugia, J. Appl. Cryst. 30 (1997) 565.
[28] L.J. Farrugia, J. Appl. Crystallogr. 32 (1997) 837.
[29] J.C. Palomino, A. Martin, M. Camacho, H. Guerra, J. Swings, F. Portaels, Anti-
microb. Agents Chemother. 46 (2002) 2720.
University of Göttingen, Göttingen, Germany, 1997.
26] G.M. Sheldrick, SHELXL-97. Program for Crystal Structure Refinement.
University of Göttingen, Göttingen, Germany, 1997.