Synthesis and in vitro anti Mycobacterium tuberculosis activity of a series of phthalimide derivatives

Article abstract of DOI:10.1016/j.bmc.2009.04.042

New phthalimide derivatives were easily prepared through condensation of phthalic anhydride and selected amines with variable yields (70-90%). All compounds (3a-l) were evaluated against Mycobacterium tuberculosis H₃₇Rv using Alamar Blue susceptibility. The compounds 3c, 3i, and 3l have the minimum inhibitory concentrations (MICs) of 3.9, 7.8, and 5.0 μg/mL, respectively, and could be considered new lead compounds in the treatment of tuberculosis and multi-drug resistant tuberculosis. Crown Copyright

Full text of DOI:10.1016/j.bmc.2009.04.042

Bioorganic & Medicinal Chemistry 17 (2009) 3795–3799
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and in vitro anti Mycobacterium tuberculosis activity of a series
of phthalimide derivatives
a
a
b
Jean L. Santos a, , Paulo R. Yamasaki , Chung Man Chin , Célio H. Takashi ,
*
Fernando R. Pavan b, Clarice Q. F. Leite b
a Lapdesf—Laboratório de Pesquisa e Desenvolvimento de Fármacos, Faculdade de Ciências Farmacêuticas, Universidade Estadual Paulista ‘Júlio de Mesquita Filho’,
Rod. Araraquara-Jaú Km.01 s/n, Campus, 1480 1-902, Araraquara, SP, Brazil
b Departamento de Ciências Biológicas, Faculdade de Ciências Farmacêuticas, Universidade Estadual Paulista ‘Júlio de Mesquita Filho’, Rod. Araraquara-Jaú Km.01 s/n,
Campus, 14801-902, Araraquara, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
New phthalimide derivatives were easily prepared through condensation of phthalic anhydride and
selected amines with variable yields (70–90%). All compounds (3a–l) were evaluated against Myco-
bacterium tuberculosis H37Rv using Alamar Blue susceptibility. The compounds 3c, 3i, and 3l have
the minimum inhibitory concentrations (MICs) of 3.9, 7.8, and 5.0 lg/mL, respectively, and could
be considered new lead compounds in the treatment of tuberculosis and multi-drug resistant
Received 6 March 2009
Revised 17 April 2009
Accepted 21 April 2009
Available online 3 May 2009
tuberculosis.
Keywords:
Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved.
Phthalimide derivatives
Antimycobacterial
Antitubercular activity
Cytotoxicity
1. Introduction
tericidal activity highlight the need for new, safer, and more effec-
tive antimycobacterial compounds.
Tuberculosis (TB) has become an important problem worldwide :
about 2 million people die each year, particularly in developing
countries; it is estimated that about one-third of the world popula-
tion is currently infected with the bacillus in its latent form and that
nearly 9 million new cases develop each year.1 According to WHO,
multiresistant tuberculosis is responsible for approximately 460
thousand new cases per year and for about 740 thousand new
patients infected by both Mycobacterium tuberculosis and HIV/AIDS.
Recent estimates show that 10% of all new TB infections are resistant
to at least one anti-TB drug.2
To treat an infection, a cocktail of drugs including, for example,
isoniazid, rifampin, ethambutol, and pyrazinamide is prescribed
for 2 months followed by a continuation phase in which isoniazid
and rifampin are taken. Long-term therapies lasting between 6
and 9 months have frequently led to patient non-compliance and,
in turn, contributed to the emergence of multi-drug resistant TB
(MDR-TB).3
In the last 10 years the research on M. tuberculosis and possible
drug candidates has undergone much progress with genome unre-
vealed and the discovery of different biological targets.4,5 The po-
tential activities of several natural and synthetic compounds
have been described against M. tuberculosis.
Among this, compounds containing phthalimide subunit have
been described as an scaffold (biophoro) to design new prototypes
drug-candidates with different biological activities and are used in
different diseases as, for example, leprosy, AIDS, tumor, diabetes,
multiple myeloma, convulsion, inflammation, pain, bacterial infec-
tion among others.6–8
Several reports in the literature demonstrate the antimicrobial
potential of phthalimide derivative.9–11 Some of these derivatives
with N-substituted phthalimide moieties possess minimal inhibi-
tory concentrations (MICs) comparable with those of clinically
used antibiotics.12
Previous results from our group demonstrated that molecular
hybridization between phthalimide subunit present in thalido-
mide and the sulfonamide drugs, as for example dapsone, leads
to antimycobacterial compounds with activity against Mycobac-
terium leprae. These compounds also showed immunomodula-
tory properties (unpublished results). All these results
encourage us to evaluate these same compounds against the
M. tuberculosis.
The ever-increasing drug resistance, toxicity, and side effects of
currently used antituberculosis drugs, and the absence of their bac-
* Corresponding author. Tel.: +55 16 3301 6962; fax: +55 16 3322 0073.
E-mail addresses: jeanleandrosantos@yahoo.com.br, chungmc@fcfar.unesp.br
(J.L. Santos).
0968-0896/$ - see front matter Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.04.042

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J. L. Santos et al. / Bioorg. Med. Chem. 17 (2009) 3795–3799
The new phthalimide derivatives (3a–l) were evaluated for
positions or changed by isosteric replacement the activity against
in vitro anti-TB activity against M. tuberculosis H37Rv and the cyto-
toxicity of those compounds was analyzed utilizing macrophage
cell line (J774).
M. tuberculosis decreases. The compound 3i inhibited the growth
at 7.8 lg/mL. When the amino group of compound 3i was dupli-
cated and substituted by another phthalimide ring (3j) there was
decrease in activity.
Molecular modifications in pyridine ring of compound 3l de-
crease anti-tubercular activity. Furthermore, the result of com-
pound 3l is consistent with the structure–activity relationship
described for isoniazid in which the substitution of N-2 can lead
to active compounds, but less than isoniazid. The introduction of
phthalimide group by molecular hybridization strategy does not
improve the activity of parental drug-isoniazid.
Antimicrobial activity assay of those three compounds (3c, 3i,
and 3l) provide evidence that they are potential against tuberculo-
sis agent. Although the MIC values obtained here are larger than
2. Results and discussion
2.1. Chemistry
The compounds were prepared through condensation reaction
between phthalic anhydride and amines previously selected in
acetic acid under reflux during 2–3 h (Scheme 1). After cooling
the compounds were filtered and recrystallized in ethanol in 70–
80% yields, except compound 3i that was purified by preparative
column chromatography.
those of isoniazid (0.02–0.03 lg/mL), these compounds have stron-
The purity of synthesized compounds was checked by thin layer
chromatography (TLC) and elemental analyses, and the structures
were identified by spectral data. The IR spectra of compounds
(3a–j), the bands representing carbonyl group of imide phthali-
mide ring appeared at 1782 cmꢀ1 and 1713 cmꢀ1. The sulfonyl
group appeared at 1350 cmꢀ1 and 1140 cmꢀ1. The nuclear mag-
netic resonance spectra (1H NMR) of 3(a–j) showed multiple sig-
nals corresponding to resonances of phthalimide protons at 7.9–
8.0 ppm and phenyl ring linked to phthalimide subunit at
7.6 ppm and 8.0 ppm. The nuclear magnetic resonance spectra
ger in vitro activity than pyrazinamide (MIC of 50–100
lg/mL), an-
other first line antitubercular drug.13
2.3. In vitro cytotoxicity evaluation
The compounds were tested for cytotoxicity (IC50) in J774 cells
at concentrations until 150ꢁ the MIC for M. tuberculosis H37Rv. Ta-
ble 2 shows the cytotoxicity to host cells of compounds 3c, 3i, and
3l. The selective index was calculated as ratio of MIC in lg/mL and
cytotoxicity (IC50).14
(
13C NMR) of 3(a–j) showed the signals corresponding to reso-
The selective index showed that J774 cells containing com-
pounds (3c, 3i, and 3l) have higher activity to M. tuberculosis than
eukaryotic cells.
nances of phthalimide protons at 123.7, 134.8, 131.4, and
166.2 ppm. The 13C NMR showed the signals of carbon resonance
of phenyl ring linked to phthalimide subunit at 132, 126, 128,
and 137 ppm.
The compound 3h was obtained through condensation reaction
between phthalyc anhydride and isoniazid. The IR spectra showed
that the carbonyl group of imide phthalimide ring appeared at
1782 cmꢀ1 and 1713 cmꢀ1 and the hydrazide carbonyl group ap-
peared at 1662 cmꢀ1. The nuclear magnetic resonance spectra
(1H NMR) of 3h showed multiple signals corresponding to reso-
nances of phthalimide protons at 8.0 ppm and of pyridinic protons
at 8.85 ppm and 7.88 ppm. The 13C NMR showed the signals of
phthalimide ring at 124, 135, 131, and 164 ppm and of the isonia-
zid subunit derivative at 165, 139, 121, and 150 ppm.
3. Conclusions
All compounds were obtained with good yields (37–90%) from
commercially available materials.
These compounds 3c, 3i, and 3l showed to be more potent than
the other phthalimide derivatives in inhibition of M. tuberculosis
growth with MICs of 3.9, 7.8, and 5.0 lg/mL, respectively. These
compounds are not cytotoxic to host cell at the concentrations
effective in inhibiting M. tuberculosis infection. The lipophilicity is
an important physico-chemical property related to the capacity
of the compounds across the membrane. All compounds presented
lipophilicity higher than drugs such as isoniazide and pyrazina-
mide used in the treatment. These compounds could be considered
new lead compounds in the treatment of tuberculosis, multi-drug
resistant, and latent tuberculosis.
The elemental analysis results were within 0.4% of the theoret-
ical values. The lipophilicities of the synthesized compounds (3a–l)
were calculated using CHEMDRAW ultra 8.0 (Table 1). All compounds
presented lipophilicity higher than drug such as isoniazid.
2.2. Antimycobacterial activity in vitro determination
4. Experimental
The compounds (3a–l) were evaluated against M. tuberculosis
H37Rv (ATTC 27294) using Microplate Alamar Blue Assay (MABA).
Isoniazid was used as control drug in the tests. The MIC (minimal
inhibition concentration) was defined as the lowest drug concen-
tration required to complete inhibition of bacterial growth. The
MICs of the compounds were reported in Table 1.
Melting points were taken with an electrothermal melting point
apparatus (SMP3 Bibby Stuart Scientific) in open capillary tubes
and are uncorrected. Infrared spectra (KBr disc) were run on a
FTIR-8300 Shimadzu and the frequencies were expressed in
cmꢀ1 1H NMR and 13C NMR spectra were scanned on a Bruker
.
The compounds 3c and 3l inhibited M. tuberculosis growth with
DRX-400 (400 MHz) NMR spectrometer using DMSO-d6 as solvent.
Chemical shifts were expressed in ppm (parts per million) relative
to tetramethylsilane. Elemental analyses (C, H, and N) were per-
formed on Perkin Elmer model 240C analyzer and the data were
within 0.4% of the theoretical values. Clog P values were calcu-
lated using CHEM DRAW ultra 8.0 software.
MICs of 3.9 and 5 lg/mL, respectively. Structure–activity relation-
ship showed that if pyrimidine ring (3c–f) is substituted in any
NH2
O
O
a
O
+
N
S
O
O
HN
4.1. General procedure for the synthesis of phthalimide
derivatives (3a–l)
O
O
R
O
O
S
R
1
2 (a-j)
3 (a-j)
A mixture of selected amine (2 a–l) (4,0 mmol), phthalic
anhydride (0.5 g, 3.4 mmol), and 20 mL of glacial acetic acid
Scheme 1. Reagents and conditions: acetic acid, reflux, 2 h, 37–90%.

J. L. Santos et al. / Bioorg. Med. Chem. 17 (2009) 3795–3799
3797
Table 1
Physical constants and in vitro antimycobacterial activities of compounds 3a–l
O
O
S O
R
N
O
Compound
R
Yields (%)
90
Mp (°C)
Clog Pa
MIC (
l
M)
3a
H
303–306
0.91
>250
l
g/mL
H
N
N
N
N
N
3b
3c
3d
73
77
76
277–280
308–310
275–278
2.31
1.57
2.07
>250
l
g/mL
H
N
3.9 lg/mL
N
N
N
H
N
CH3
CH3
>250
>250
>250
l
l
l
g/mL
H
N
3e
74
297–301
2.57
g/mL
g/mL
CH3
N
H
N
3f
71
70
74
262–265
269–271
215–218
2.12
0.24
2.04
N
OCH3
NH
3g
3h
125
125
l
g/mL
N
H
NH2
O
N
CH3
l
g/mL
N
H
3i
3j
37
83
217–220
306–310
2.36
3.84
7.8 lg/mL
NH2
O
>250 lg/mL
N
O
O
77
—
133–136
—
ꢀ0.44
ꢀ1.15
5 lg/mL
N
NH
O
N
O
3l
Isoniazid
Calculated on CHEM DRAW ULTRA 8.0 software.
0.02 lg/mL
a
was stirred under nitrogen at 130 °C for 2 h. After that the mix-
ture was cooled and 30 mL of cold water was added. The prod-
uct was filtered and washed with cold water. The product was
recrystallized in ethanol without further purification to lead to
compounds with variable yields (37–90%).
Table 2
Cytotoxicity and selective index
Compound
MIC (lg/mL)
Cytotoxicity (lg/mL)
Selective index
3c
3i
3l
3.9
7.8
5.0
312.5
312.5
625
80
40
125
—
4.1.1. 4-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)benzene-
sulfonamide (3a)
Isoniazid
0.015–0.03
—
Yield: 90%, mp: 303–306 °C.

3798
J. L. Santos et al. / Bioorg. Med. Chem. 17 (2009) 3795–3799
IR mmax (cmꢀ1; KBr pellets):1779 and 1715 (C@O imide); 1312
(2H; d; J = 8.7 Hz; H6); 3.79 (3H; s) ppm; 13C NMR
and 1137 (S@O).
(400 MHz, DMSO-d6) d: 167; 151.4; 150.2; 145.2; 136; 132;
134.8; 131.9; 129.1; 127.7; 124; 56.7 ppm. Calcd for
C19H14N4O5S: C, 55.6; H, 3.44; N, 13.65. Found: C, 55.5; H,
3.1; N, 13.2.
NMR 1H (400 MHz, DMSO-d6) d: 8.1 (2H; d; J = 8.5 Hz; H7); 8.0
(2H; m; H2); 7.9 (2H; m; H1); 7.8 (2H; d; J = 8,5 Hz; H6) ppm; 13C
NMR (400 MHz, DMSO-d6) d: 167.4; 136.7; 135.6; 132.7; 130.7;
128.4; 128.0; 124.4 ppm. Calcd for C14H10N2O4S: C, 55.62; H,
3.33; N, 9.27. Found: C, 55.2; H, 3.11; N, 9.53.
4.1.7. N-[Amino(imino)methyl]-4-(1,3-dioxo-1,3-dihydro-2H-
isoindol-2-yl)benzenesulfonamide (3g)
4.1.2. 4-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)-N-pyridin-2-
yl benzenesulfonamide (3b)
Yield: 70%, mp: 269–271 °C.
IR mmax (cmꢀ1; KBr pellets): 3495 (N–H); 1797 and 1713 (C@O
imide); 1367 and 1134 (S@O).
Yield: 73, mp: 277–280 °C.
IR mmax (cmꢀ1; KBr pellets): 1782 and 1713 (C@O imide); 1311
NMR 1H (400 MHz, DMSO-d6) d: 7.99 (2H; m; H2); 7.93 (2H;
m; H1); 7.90 (2H; d; J = 8.2 Hz; H7); 7.60 (2H; d; H6); 6.77 (4H;
N–H; s) ppm; 13C NMR (400 MHz, DMSO-d6) d: 166.6; 158.2;
143.7; 134.8; 136.2; 131.4; 127.2; 126.2; 123.5 ppm. Calcd for
C15H12N4O4S: C, 52.32; H, 3.51; N, 16.27. Found: C, 52.77; H,
3.69; N, 16.6.
and 1139 (S@O).
1
0
NMR H (400 MHz, DMSO-d6) d: 8.01 (5H; m; H2, H7 e H1 ); 7.77
0
(1H; ddd; J = 8,5 Hz; H3 ); 7.64 (2H; dd; J = 8,73 Hz; H6); 7.24 (1H;
dd; J = 8,5 Hz; H2 ); 6.87 (1H; dd; J = 8,5 Hz; H4 ) ppm; 13C NMR
(400 MHz, DMSO-d6) d: 166.6; 141.1; 136; 134.9; 134.8; 132;
131.4; 129; 128; 124; 123.6; 114.3; 113.3 ppm. Calcd for
C19H13N3O4S: C, 60.15; H, 3.45; N, 11.08. Found: C, 60.55; H,
3.33; N, 11.5.
0
0
4.1.8. 4-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)-N-(5-
methylisoxazol-3-yl)benzenesulfonamide (3h)
Yield: 74%, mp: 215–218 °C.
4.1.3. 4-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)-N-pyrimidin-
2-yl benzenesulfonamide (3c)
IR mmax (cmꢀ1; KBr pellets): 1782 and 1712 (C@O imide); 1380
and 1172 (S@O).
Yield: 77%, mp: 308–310 °C.
NMR 1H (400 MHz, DMSO-d6) d: 8.0 (2H; d; J = 8.3 Hz; H7); 7.99
(2H; m; H2); 7.91 (2H; m; H1); 7.64 (2H; d; J = 8.3 Hz; H6); 6.18
IR mmax (cmꢀ1; KBr pellets): 1786 and 1720 (C@O imide); 1317
and 1141 (S@O).
(1H; s; H1 ) 2.35 (3H; s) ppm; 13C NMR (400 MHz, DMSO-d6) d:
0
NMR 1H (400 MHz, DMSO-d6) d: 8.54 (2H; dd; J = 8,73 Hz; H7);
170.4; 166.4; 143.7; 138.3; 134.8; 131.4; 129.6; 127.6; 124;
119.1; 95.4; 12 ppm. Calcd for C18H13N3O5S: C, 56.4; H, 3.42; N,
10.9. Found: C, 56.5; H, 3.3; N, 11.2.
0
8.13 (2H; dd; J = 8.73 Hz; H2 ); 7.99 (2H; m; H2); 7,92 (2H; m; H1);
0
7.69 (2H; dd; J = 8.73 Hz; H6); 7.07 (1H; dd; J = 8.73 Hz; H3 ) ppm;
13C NMR (400 MHz, DMSO-d6) d: 165.8; 158; 150.2; 136; 135.2;
131.9; 129.3; 124; 122.1; 110 ppm. Calcd for C18H12N4O4S: C,
56.84; H, 3.18; N, 14.73. Found: C, 56.54; H, 3.31; N, 14.5.
4.1.9. 2-{4-[(4-Aminophenyl)sulfonyl]phenyl}-1H-isoindole-
1,3(2H)-dione (3i)
The compound 3i after precipitation was purified by prepara-
tive column chromatography (hexane/ethyl acetate 8:2). Yield:
37%, mp: 217–220 °C.
4.1.4. 4-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)-N-(4-
methylpyrimidin-2-yl)benzenesulfonamide (3d)
Yield: 76%, mp: 275–279 °C.
IR mmax (cmꢀ1; KBr pellets): 3446 and 3530 (N–H); 1786 and
1712 (C@O imide); 1371 and 1142 (S@O).
IR mmax (cmꢀ1; KBr pellets): 1789 and 1712 (C@O imide); 1361
and 1130 (S@O).
NMR 1H (400 MHz, DMSO-d6) d: 8.1 (2H; d; J = 8.5 Hz; H7);
1
0
0
NMR H (400 MHz, DMSO-d6) d: 8.35 (1H; d; J = 8,1 Hz; H1 );
7.99 (2H; m; H2); 7.94 (2H; d; H1 ); 7.91 (2H; m; H1); 7.7
0
8.11 (2H; d; J = 8.3 Hz; H7); 7.98 (2H; m; H2); 7.91 (2H; m; H1);
(2H; d; J = 8.5 Hz; H6); 7.82 (2H; d; H2 ) ppm; 13C NMR
0
7.67 (2H; dd; J = 8.3 Hz; H6); 6.91 (1H; d; J = 8.1 Hz; H2 ) 2.32
(400 MHz, DMSO-d6) d: 166.1; 151.9; 136; 134.8; 132.3; 132;
131.7; 131.4; 129; 128; 123.6; 118.9 ppm. Calcd for
C20H14N2O4S: C, 63.4; H, 3.73; N, 7.4. Found: C, 63.5; H, 3.5;
N, 7.2.
(3H; s) ppm; 13C NMR (400 MHz, DMSO-d6) d: 166.5; 156.3;
150.2; 139.8; 135.5; 134.8; 131.4; 129; 128.3; 126.9; 123.5;
109.5; 23.1 ppm. Calcd for C19H14N4O4S: C, 57.86; H, 3.58; N,
14.21. Found: C, 57.4; H, 3.29; N, 14.2.
4.1.10. Bis-(phenylsulfonyl-1H-isoindole-1,3(2H)-dione) (3j)
Yield: 83%, mp: 306–310 °C.
4.1.5. N-(4,6-Dimethylpyrimidin-2-yl)-4-(1,3-dioxo-1,3-
dihydro-2H-isoindol-2-yl)benzenesulfonamide (3e)
Yield: 74%, mp: 297–301 °C.
IR mmax (cmꢀ1; KBr pellets): 1784 and 1713 (C@O imide); 1366
and 1137 (S@O).
IR mmax (cmꢀ1; KBr pellets): 1786 and 1718 (C@O imide); 1361
and 1168 (S@O).
NMR 1H (400 MHz, DMSO-d6) d: 8.2 (4H; d; J = 8.6 Hz; H7); 8.0
0
(4H; m; H2); 7.93 (4H; d; H1 ); 7.8 (4H; d; J = 8.6 Hz; H6) ppm;
NMR 1H (400 MHz, DMSO-d6) d: 8.11 (2H; d; J = 8,7 Hz; H7);
7.98 (2H; m; H2); 7.90 (2H; m; H1); 7.64 (2H; d; J = 8.7 Hz; H6);
13C NMR (400 MHz, DMSO-d6) d: 166.4; 135.4; 134.8; 131.4;
129.5; 127.6; 127.1; 113 ppm. Calcd for C28H16N2O6S: C, 66.14;
H, 3.17; N, 5.51. Found: C, 66.4; H, 3.3; N, 5.3.
6.75 (1H; s; H3 ) 2.26 (6H; s) ppm; 13C NMR (400 MHz, DMSO-d6)
0
d: 166.5; 155.9; 135.2; 134.8; 131.6; 131.4; 129.1; 128.5; 126.7;
123.5; 109.5; 23.3 ppm. Calcd for C20H16N4O4S: C, 58.81; H, 3.95;
N, 13.72. Found: C, 58.7; H, 3.77; N, 13.41.
4.1.11. N-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)isonicotina-
mide (3l)
Yield: 77%, mp: 133–136 °C.
4.1.6. 4-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)-N-(5-
methoxypyrimidin-2-yl)benzenesulfonamide (3f)
Yield: 71%, mp: 262–265 °C.
IR mmax (cmꢀ1; KBr pellets): 1783 and 1715 (C@O imide); 1662
(C@O amide).
1
0
NMR H (400 MHz, DMSO-d6) d: 8.85 (2H; d; J = 8.8 Hz; H2 );
IR mmax (cmꢀ1; KBr pellets): 1785 and 1715 (C@O imide); 1367
8.03 (2H; m; H2); 7.98 (2H; m; H1); 7.88 (2H; d; J = 8.8 Hz; H1 )
0
and 1162 (S@O).
ppm; 13C NMR (400 MHz, DMSO-d6) d: 165; 164.2; 150.7; 137.7;
135.4; 129.4; 123.9; 121.4 ppm. Calcd for C14H9N3O3: C, 62.92; H,
3.39; N, 15.72. Found: C, 62.5; H, 3.3; N, 15.41.
1
0
NMR H (400 MHz, DMSO-d6) d: 8.32 (2H; s; H1 ); 8.1 (2H;
d; J = 8.7 Hz; H7); 7.99 (2H; m; H2); 7.91 (2H; m; H1); 7.68

J. L. Santos et al. / Bioorg. Med. Chem. 17 (2009) 3795–3799
3799
4.2. Biological activity
tions of 625, 312.5, 156.3, 78, 39, 19.5, 9.75, 4.87, 2.43, 1.21, and
0.6 g/mL. Cells were exposed to the compounds for a 24 h period.
l
4.2.1. Antimycobacterial activity in vitro determination
Resazurin solution was added to cell cultures and incubated for a
6-h period. Cell respiration, as an indicator of cell viability, was
determined by resazurin (redox potential), the pink color means
cell viability and the blue color means cell death. Thus, the IC50 va-
lue was defined as the bigger drug concentration at which 50% of
the cells are viable relative to the control and measuring the fluo-
rescence (excitation/emission of 530/590 nm filters, respectively)
in a SPECTRAfluor Plus (Tecan).13
The anti-M. tuberculosis activity of the compounds was deter-
mined using the MABA as analytical method.15 Stock solutions of
the tested compounds were prepared in dimethyl sulfoxide and di-
luted in broth medium Middlebrook 7H9 (Difco), supplemented
with oleic acid, albumin, dextrose, and catalase (OADC enrich-
ment—BBL/Becton-Dikinson, Sparks, MD, USA), to obtain final
drugs with concentration ranges of 0.15–250 lg/mL. The isoniazid
was solubilized with distilled water according to the manufactur-
ers’ recommendations (Difco laboratories, Detroit, MI, USA) and
was used as standard drug. M. tuberculosis H37Rv ATCC 27294
was grown for 7–10 days in Middlebrook 7H9 supplemented with
0.05% Tween 80 OADC to avoid clumps. Suspensions were prepared
and their turbidities matched to a McFarland No. 1 (turbidity stan-
dard). After further dilution of 1:25 in Middlebrook 7H9 supple-
mented with OADC, the inoculum was added to each well of the
96-well microtiter plate (Falcon 3072; Becton Dickinson, Lincoln
Park, NJ) together with the compounds. Samples were set up in
triplicate. Cultures were incubated for 7 days at 37 °C, and after
this resazurin was added for the reading. The minimum inhibitory
concentration (MIC) was defined as the lowest concentration
resulting in 90% inhibition of growth of M. tuberculosis15 measuring
the fluorescence (excitation/emission of 530/590 nm filters,
respectively) in a SPECTRAfluor Plus (Tecan).13 For standard test,
the MIC value of isoniazid was determined each time. The accept-
4.2.3. Selectivity index
A selectivity index (SI) can then be calculated by dividing the
IC50 by the MIC; if the SI is >10, the compound is then evaluated
further.14
Acknowledgment
This study was supported by Fundação de Amparo à Pesquisa do
Estado de São Paulo (FAPESP) ref. Process: 07/54983-4, 07/ 56115-
0 and 08/10390-2. PADC-FCF UNESP Program 07/5-I.
References and notes
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able MIC of isoniazid ranged from 0.015 to 0.05 l
g/mL.13
4.2.2. In vitro cytotoxicity evaluation
In vitro cytotoxicity evaluation was carried out on cultured
murines tumor cell lines J774 as recommended by Ahmed et al.16
The cells were routinely maintained with RPMI medium supple-
mented with 10% fetal bovine serum (FBS), at 37 °C in a humidified
5% CO2 atmosphere. After reaching confluence, the cells were de-
tached and counted. For the cytotoxicity assay, 2 ꢁ 104 cells wellꢀ1
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Gilman, R. H. J. Clin. Microbiol. 1998, 36, 362.
were seeded in 200 lL of complete medium in 96-well plates
(Corning Costar). The plates were incubated at 37 °C in 5% CO2
for 24 h to allow cell adhesion prior to drug testing. The com-
pounds were dissolved in sterile DMSO and diluted to concentra-
16. Ahmed, S. A.; Gogal, R. M.; Walsh, J. E. J. Immunol. Methods 1994, 170, 211.