Synthesis, in vitro antiproliferative and anti-mycobacterium tuberculosis activities of novel β-carboline derivatives

Article abstract of DOI:10.5935/0103-5053.20160062

A series of β-carboline derivatives with amino or guanidinium were synthesized and evaluated in vitro against anti-Mycobacterium tuberculosis and for antiproliferative activities against nine human cancer cell lines. The compounds 1-(4-hydroxyphenyl)-3-carboxamide(ethylamine) β-carboline (24.9 μg mL^-1) and 1-(4-methoxyphenyl)-3-carboxamide(ethylamine) β-carboline (26.9 μg mL^-1) were the most active against M. Tuberculosis (MTB). Compounds 1-(4-hydroxyphenyl)-3-carboxamide(ethylamine) β-carboline and 1-(4-methoxyphenyl)-3-carboxamide(propylamine) β-carboline, which had the same substituted groups, inhibited the growth of all human tumor cell lines with growth inhibitory activity (GI₅₀) values from 1.37 to 9.20 mmol L^-1. Also in this series, compounds 1-(4-hydroxyphenyl)-3-carboxamide(propylamine) β-carboline and 1-(3-nitrophenyl)-3-carboxamide(propylamine) β-carboline demonstrated significant activity against NCI/ADR cells. Among compounds with a terminal guanidine group, compounds 1-(4-hydroxyphenyl)-3-carboxamide(ethyl)guanidine β-carboline (27.8 μg mL^-1) and 1-(3-nitrophenyl)-3-carboxamide(ethyl) guanidine β-carboline (37.4 μg mL^-1) demonstrated the greatest activity against MTB. Additionally, compounds 1-(4-methoxyphenyl)-3-carboxamide(ethyl)guanidine β-carboline (GI₅₀ = 0.45 mmol L^-1) effectively inhibited growth and was highly selective against NCI/ADR. The in silico study revealed that 1-(4-hydroxyphenyl)-3-carboxamide(ethylamine) β-carboline, 1-(4-methoxyphenyl)-3-carboxamide(ethylamine) β-carboline, 1-(4-hydroxyphenyl)-3-carboxamide(propylamine) β-carboline, 1-(4-methoxyphenyl)-3-carboxamide(propylamine) β-carboline and 1-(3-nitrophenyl)-3-carboxamide(propylamine) β-carboline compounds follow the rules established by Lipinski, suggesting that this compound has no problems with oral bioavailability.

Full text of DOI:10.5935/0103-5053.20160062

http://dx.doi.org/10.5935/0103-5053.20160062
J. Braz. Chem. Soc., Vol. 27, No. 8, 1398-1405, 2016.
Printed in Brazil - ©2016 Sociedade Brasileira de Química
0103 - 5053 $6.00+0.00
Article
Synthesis, in vitro Antiproliferative and Anti-Mycobacterium tuberculosis Activities
of Novel β-Carboline Derivatives
Flora M. F. Moreira,a Julio Croda,a Maria H. Sarragiotto,b Mary A. Foglio,c
Ana L. T. G. Ruiz,c João E. Carvalhoc and Anelise S. N. Formagio*,d
aFaculdade de Ciências da Saúde and dFaculdade de Ciências Agrárias, Universidade Federal da
Grande Dourados, Rodovia Dourados - Itahum, Km 12, 79804-970 Dourados-MS, Brazil
bDepartamento de Química, Universidade Estadual de Maringá, Avenida Colombo, 5790,
Jardim Universitário, 87020-900 Maringá-PR, Brazil
cCentro Pluridisciplinar de Pesquisas Químicas, Biológicas e Agrícolas, Universidade Estadual de
Campinas, CP 6171, 13083-970 Campinas-SP, Brazil
A series of β-carboline derivatives with amino or guanidinium were synthesized and evaluated in
vitro against anti-Mycobacterium tuberculosis and for antiproliferative activities against nine human
cancer cell lines. The compounds 1-(4-hydroxyphenyl)-3-carboxamide(ethylamine) β-carboline
(24.9 μg mL-1) and 1-(4-methoxyphenyl)-3-carboxamide(ethylamine) β-carboline (26.9 μg mL-1)
were the most active against M. Tuberculosis (MTB). Compounds 1-(4-hydroxyphenyl)-3-
carboxamide(ethylamine) β-carboline and 1-(4-methoxyphenyl)-3-carboxamide(propylamine)
β-carboline, which had the same substituted groups, inhibited the growth of all human tumor cell
lines with growth inhibitory activity (GI50) values from 1.37 to 9.20 mmol L-1. Also in this series,
compounds 1-(4-hydroxyphenyl)-3-carboxamide(propylamine) β-carboline and 1-(3-nitrophenyl)-
3-carboxamide(propylamine) β-carboline demonstrated significant activity against NCI/ADR
cells. Among compounds with a terminal guanidine group, compounds 1-(4-hydroxyphenyl)-3-
carboxamide(ethyl)guanidine β-carboline (27.8 μg mL-1) and 1-(3-nitrophenyl)-3-carboxamide(ethyl)
guanidine β-carboline (37.4 μg mL-1) demonstrated the greatest activity against MTB. Additionally,
compounds 1-(4-methoxyphenyl)-3-carboxamide(ethyl)guanidine β-carboline (GI50 = 0.45 mmol L-1)
effectively inhibited growth and was highly selective against NCI/ADR. The in silico study
revealed that 1-(4-hydroxyphenyl)-3-carboxamide(ethylamine) β-carboline, 1-(4-methoxyphenyl)-
3-carboxamide(ethylamine) β-carboline, 1-(4-hydroxyphenyl)-3-carboxamide(propylamine)
β-carboline, 1-(4-methoxyphenyl)-3-carboxamide(propylamine) β-carboline and 1-(3-nitrophenyl)-
3-carboxamide(propylamine) β-carboline compounds follow the rules established by Lipinski,
suggesting that this compound has no problems with oral bioavailability.
Keywords: synthesis, β-carboline, Mycobacterium tuberculosis, antiproliferative activity
Introduction
resistant strains and given high toxicity of anti-tuberculosis
drugs, to the need to develop new drugs that are more
effective and less toxic than current drugs, which would
reduce time and complexity of treatment, is urgent.3,4 The
discovery of new drugs is also necessary for the treatment
of cancer, because most chemotherapeutic agents exhibit
severe toxicity and cause many undesirable side effects;
additionally, current agents are very expensive, mutagenic,
carcinogenic and teratogenic.5 Tuberculosis, caused by
Mycobacterium tuberculosis (MTB),1 and cancer6 have
affected human health for thousands of years and remain
a major cause of diseases affecting public health around
the world, and the possible interaction of mycobacterial
Tuberculosis (TB) exhibits high morbidity and
mortality. The long-term treatment regimen can cause
patients to be non-compliant in completing the treatment,
thus leading to emergence of multidrug-resistant (MDR-
TB) and extensively drug-resistant (XDR-TB) TB strains.
Infections caused by MDR-TB and XDR-TB do not
respond to first-line drugs that are used to treat TB, and
alternative treatment regimens include mostly injected
drugs and prolonged treatments.1,2 Due to the appearance of
*e-mail: aneliseformagio@ufgd.edu.br

Vol. 27, No. 8, 2016
Moreira et al.
1399
pathogens with cancer cells may be influenced by genetic
alterations in the tumor cells.
phenyl)-3-ethylamine-carboxamide β-carboline 2a-c
were obtained by reaction of the methyl esters 1a-c
(2.0 mmol L-1) with 1,2-ethylenediamine (6 mmol L-1)
at room temperature, stirred for 36 h. The formed solids
were collected by filtration. The 1-(substituted-phenyl)-3-
prophylamine-carboxamide β-carboline 3a-c were obtained
by reaction of the methyl esters 1a-c (1.7 mmol L-1) with
1,3-propanediamine (5 mmol L-1) in 25 mL MeOH/CHCl3
was refluxed for 32 h. The formed solids were collected
by filtration.26
To a solution of S-methylisothiourea sulfate
(0.5 mmol L-1) in water (1.5 mL) and 2 mmol L-1 NaOH
(0.5 mL) was added a suspension of β-carboline-3-
ethylamine-carboxamide 2a-c at 0 °C. The mixture was
kept under stirring, at room temperature by 1 h and
later under reflux for 24 h, was again added a solution
of S-methylisothiourea sulfate (0.5 mmol L-1) in water
(1.5 mL) and 2 mol L-1 NaOH (0.5 mL) and reflux for
24 h. The formed solids were collected by filtration and
washed with cold water for obtained 1-(substituted phenyl)-
3-carboxamide-ethylguanidine β-carboline (4a-c).26 The
characterization data of the compounds obtained are given
bellow that corroborates with correlation spectroscopy
(COSY), H1 detected multiquantum coherence (HMQC)
and heteronuclear multiple-bond correlation (HMBC)
two-dimensional NMR spectra.
Synthetic β-carboline derivatives exhibit a wide range
of pharmacological activities, including antitumor and anti-
tuberculosis activities.7-14 Our research group demonstrated
that β-carboline derivatives with various substituents at
positions-1, 3 and 9 of the β-carboline skeleton presented
significant in vitro antitumoral, antiviral, antitrypanosomal
and antileishmanial activities.15-23 Other studies have shown
that β-carboline derivatives with a methyl-substituted group
at position-1 and a guanidinium group-terminated side chain
at C-3 exhibited anti-HIV-1 activity in MT4 cells by hindering
the essential interaction of the regulatory protein Tat with
trans-activation response region (TAR).24,25 Some results
cited above indicated that β-carboline derivatives containing
4-hydroxyphenyl, 4-methoxyphenyl or 3-nitrophenyl group
at C-1 showed potent anticancer activity for some of the
human cancer cell lines tested. This led us to study novel
β-carboline analogs that might serve as antitumoral and anti-
tuberculosis agents as part of our ongoing research program.
Based on the idea that the addition of appropriate
substituents at positions-1 and -3 might result in more
potent compounds, we synthesized novel 1-substituted-
phenyl-β-carboline with an amino or guanidinium group-
terminated side chain at C-3 and evaluated the in vitro anti-
tuberculosis, antiproliferative properties and in silico study.
Experimental
1-(4-Hydroxyphenyl)-3-carboxamide(ethylamine)
β-carboline (2a)
General procedure
Yield 68%; mp 171-174 °C; IR (KBr) νmax / cm-1
3090, 1684, 1480, 1460; 1H NMR (300 MHz, DMSO-d6)
d 2.74 (t, 2H, J 6.0 Hz, CH2-ethyl), 3.07 (t, 2H, J 6.0 Hz,
CH2-ethyl), 7.01 (d, 2H, J 8.7 Hz, Phenyl-H); 7.28 (t, 1H,
J 7.0 Hz, H-6), 7.57 (td, 1H, J 7.7, 5.0 Hz, H-7), 7.67 (d,
1H, J 8.4 Hz, H-8), 7.99 (d, 2H, J 8.7 Hz, Phenyl-H),
8.37 (d, 1H, J 7.8 Hz, H-5); 8.73 (s, 1H, H-4); 13C NMR
(75.5 MHz, DMSO-d6) d 41.2, 41.9, 112.1, 112.7, 115.7,
120.3, 121.5, 121.8, 128.5, 129.2, 129.7, 130.0, 134.8,
138.7, 141.4, 141.8, 157.9, 167.1.
1H and 13C nuclear magnetic ressonance (NMR)
spectra were recorded on a Varian Mercury Plus (Palo
Alto, EUA) spectrometer operating at 300 and 75.5 MHz,
respectively, using deuterated dimethyl sulfoxide
(DMSO-d6), chloroform (CDCl3) and methanol (CD3OD)
as solvent, and tetramethylsilane (TMS) as internal
reference. Infrared (IR) spectra were recorded as potassium
bromide pellets on a BOMEM spectrometer model MB-
100 (Houston, USA). Melting points were determined in
a Micro-Química apparatus MQAPF-301 model (Palhoça,
Brazil) and are uncorrected. The reactions were monitored
by thin layer chromatography (TLC) conducted on Merck
TLC plates (Silica Gel 60 F254, Darmstadt, Germany).
All reagents were purchased from commercial suppliers.
1-(4-Methoxyphenyl)-3-carboxamide(ethylamine)
β-carboline (2b)
Yield 74%; mp 180-182 °C; IR (KBr) νmax / cm-1
1
3354, 3106, 1686, 1590, 860; H NMR (300 MHz,
CDCl3/CD3OD) d 2.84 (t, 2H, J 6.0 Hz, CH2-ethyl), 3.87
(t, 2H, J 6.0 Hz, CH2-ethyl); 3.95 (s, 3H, OCH3), 7.17 (d,
2H, J 8.1 Hz, Phenyl-H), 7.36 (ddd, 1H, J 7.8, 7.5, 2.1 Hz,
H-6), 7.58 (d, 1H, J 7.5 Hz, H-8), 7.61 (ddd, 1H, J 7.8, 7.5,
2.1 Hz, H-7), 8.02 (d, 2H, J 8.1 Hz, Phenyl-H), 8.24 (d, 1H,
J 7.8 Hz, H-5), 8.83 (s, 1H, H-4); 13C NMR (75.5 MHz,
CDCl3/CD3OD) d 37.6, 39.3, 55.4, 113.1, 114.6, 115.1,
Preparation of 1-(substituted phenyl)-3-carboxamide-
ethylguanidine-β-carboline (4a-c)
The 1-(substituted phenyl)-3-carbomethoxy-β-carboline
were obtained as previously reported.21 The 1-(substituted-

1400
Synthesis, in vitro Antiproliferative and Anti-Mycobacterium tuberculosis Activities of Novel b-Carboline Derivatives
J. Braz. Chem. Soc.
121.0, 122.4, 122.8, 130.8, 131.6, 132.3, 132.9, 133.6,
139.5, 141.9, 142.0, 144.3, 162.5.
(d, 1H, J 7.8 Hz, H-8), 7.91 (t, 1H, J 7.8 Hz, H-7), 8.33
(dd, 1H, J 8.1, 1.5 Hz, Phenyl-H), 8.45 (d, 1H, J 7.8 Hz,
Phenyl-H), 8.64 (d, 1H, J 8.1 Hz, H-5), 8.86 (s, 1H, H-4);
13C NMR (75.5 MHz, CD3OD) d 32.8, 36.8, 37.0, 112.6,
113.7, 120.4, 121.2, 122.2, 123.5, 123.6, 128.9, 130.2,
130.4, 134.7, 135.1, 138.2, 138.9, 139.6, 141.6, 166.5.
1-(3-Nitrophenyl)-3-carboxamide(ethylamine) β-carboline
(2c)
Yield 66%; mp 172-175 °C; IR (KBr) νmax / cm-1 3190,
1
1636, 1555, 1496, 1420; H NMR (300 MHz, CDCl3/
CD3OD) d 2.98 (t, 2H, J 6.0 Hz, CH2-ethyl), 3.63 (t, 2H,
J 6.0 Hz, CH2-ethyl), 7.37 (m, 1H, H-6), 7.60 (m, 2H,
H-8, Phenyl-H), 7.81 (t, 1H, J 7.8 Hz, H-7), 8.23 (dd, 1H,
J 7.8 Hz, H-5), 8.37 (dd, 1H, J 7.0, 2.0 Hz, Phenyl-H),
8.40 (d, 1H, J 7.0 Hz, Phenyl-H), 8.86 (sl, 1H, Phenyl-H),
8,89 (s, 1H, H-4); 13C NMR (75.5 MHz, CDCl3/CD3OD) d
39.9, 40.7, 112.4, 114.3, 120.8, 121.6, 121.7, 123.4, 123.5,
129.1, 129.9, 131.1, 134.7, 135.0, 138.6, 138.9, 139.4,
141.8, 148.5, 167.1.
1-(4-Hydroxyphenyl)-3-carboxamide(ethyl)guanidine
β-carboline (4a)
Yield 30%; mp 176-178 °C; IR (KBr) νmax / cm-1
3260, 1670, 1544, 1424, 1460, 1236;1H NMR (300 MHz,
DMSO-d6) d3.22 ( t, 2H, J 6.5 Hz, CH2-ethyl), 3.49 (t, 2H,
J 6.5 Hz, CH2-ethyl), 5.50 (s, 2H, NH2), 6.14 (s, 1H, NH),
6.63 (s, 1H, NH), 7.05 (d, 2H, J 8.4 Hz, Phenyl-H), 7.28
(t, 1H, J 7.5 Hz, H-6), 7.56 (t, 1H, J 7.5 Hz, H-7), 7.67 (d,
1H, J 8.1 Hz, H-8), 8.05 (d, 2H, J 8.4 Hz, Phenyl-H), 8.37
(d, 1H, J 7.8 Hz, H-5), 8.72 (s, 1H, H-4), 8.81 (s, 1H, NH);
13C NMR (75.5 MHz, DMSO-d6) d 37.3, 40.5, 112.4, 113.0,
116.0, 120.5, 121.5, 122.1, 128.6, 128.8, 129.9, 130.5,
134.1, 139.6, 141.3, 141.7, 158.6, 159.6, 163.1.
1-(4-Hydroxyphenyl)-3-carboxamide(propylamine)
β-carboline (3a)
Yield 72%; mp 180-182 °C; IR (KBr) νmax / cm-1 3226,
1640, 1550, 1496, 1440; 1H NMR (300 MHz, DMSO-d6) d
1.65 (q, 2H, J 6.3 Hz, CH2-prophyl), 2.65 (t, 2H, J 6.3 Hz,
CH2-prophyl), 3.45 (q, 2H, J 6.0 Hz, CH2-prophyl), 7.00 (d,
2H, J 8.1 Hz, Phenyl-H), 7.29 (t, 1H, J 7.2 Hz, H-6), 7.57
(t, 1H, J 7.2 Hz, H-7), 7.67 (d, 1H, J 8.1 Hz, H-8), 8.02 (d,
2H, J 8.1 Hz, Phenyl-H), 8.36 (d, 1H, J 8.1 Hz, H-5), 8.72
(s, 1H, H-4); 13C NMR (75.5 MHz, DMSO-d6) d 32.8, 36.9,
39.5, 112.0, 112.6, 115.6, 120.1, 121.3, 121.9, 128.3, 128.4,
129.6, 130.1, 133.8, 139.7, 140.9, 141.5, 158.5, 164.9.
1-(4-Methoxyphenyl)-3-carboxamide(ethyl)guanidine
β-carboline (4b)
Yield 45%; mp 190-194 °C; IR (KBr) νmax / cm-1 3320,
1670, 1550, 1520, 1448; 1H NMR (300 MHz, DMSO-d6)
d3.25 (q, 2H, J 6.0 Hz, CH2-ethyl), 3.45 (q, 2H, J 6.0 Hz,
CH2-ethyl), 3.96 (s, 3H, OCH3), 5.54 (s, 2H, NH2), 6.18 (t,
1H, J 7.5 Hz, NH), 7.22 (d, 2H, J 8.7 Hz, Phenyl-H), 7.34
(t, 1H, J 7.0 Hz, H-6), 7.60 (t, 1H, J 7.5 Hz, H-7), 7.70
(d, 1H, J 8.0 Hz, H-8), 8.20 (d, 2H, J 8.7 Hz, Phenyl-H),
8.41 (d, 1H, J 8.1 Hz, H-5), 8.78 (s, 1H, H-4), 8.84 (t, 1H,
J 7.5 Hz, NH), 11.81 (s, 1H, NH); 13C NMR (75.5 MHz,
DMSO-d6) d 38.6, 40.3, 55.4, 112.3, 112.6, 114.2, 120.8,
121.2, 121.9, 129.7, 129.9, 130.1, 133.8, 138.4, 139.6,
140.5, 141.5, 159.0, 159.9, 165.1.
1-(4-Methoxyphenyl)-3-carboxamide(propylamine)
β-carboline (3b)
Yield 60%; mp 188-193 °C; IR (KBr) νmax / cm-1
3200, 1678, 1550, 1520, 1448, 1010; 1H NMR (300 MHz,
DMSO-d6) d 1.64 (qt, 2H, J 6.6 Hz, CH2-prophyl), 2.64
(t, 2H, J 6.6 Hz, CH2-prophyl), 3.44 (q, 2H, J 6.6 Hz,
CH2-prophyl), 3.88 (s, 3H, OCH3), 7.18 (d, 2H, J 8.7 Hz,
Phenyl-H), 7.30 (t, 1H, J 7.2 Hz, H-6), 7.58 (t, 1H, J 8.1 Hz,
H-7), 7.67 (d, 1H, J 8.1 Hz, H-8), 8.14 (d, 2H, J 8.7 Hz,
Phenyl-H), 8.38 (d, 1H, J 7.2 Hz, H-5), 8.75 (s, 1H, H-4);
13C NMR (75.5 MHz, DMSO-d6) d 33.0, 36.9, 37.0, 55.4,
112.3, 112.6, 114.2, 120.1, 121.3, 121.9, 128.4, 129.7,
130.0, 130.1, 133.9, 139.8, 140.4, 141.6, 159.9, 164.5.
1-(3-Nitrophenyl)-3-carboxamide(ethyl)guanidine
β-carboline (4c)
Yield 38%; mp 192-194 °C; IR (KBr) νmax / cm-1
1
3204, 1668, 1530, 1520, 1448; H NMR (300 MHz,
DMSO-d6) d3.22 (m, 2H, CH2-ethyl), 3.42 (q, 2H, J 6.5 Hz,
CH2-ethyl), 5.49 (s, 2H, NH2), 6.15 (t, 1H, J 7.0 Hz, NH),
7.34 (t, 1H, J 7.0 Hz, H-6), 7.60 (d, 1H, J 7.8 Hz, H-8),
7.62 (t, 1H, J 7.8 Hz, Phenyl-H), 7.95 (t, 1H, J 8.0 Hz,
H-7), 8.39 (d, 1H, J 7.7 Hz, H-5), 8.45 (d, 1H, J 8.1 Hz,
Phenyl-H), 8.65 (d, 1H, J 7.5 Hz, Phenyl-H), 8.86 (s, 1H,
Phenyl-H), 8.89 (s, 1H, H-4), 8.90 (s, 1H, NH), 12.02 (s,
1H, NH); 13C NMR (75.5 MHz, DMSO-d6) d 38.7, 40.5,
112.6, 113.8, 114.2, 120.4, 121.2, 122.3, 123.4, 123.6,
128.9, 130.4, 130.5, 134.4, 135.5, 138.1, 138.9, 140.1,
141.7, 148.3, 159.01, 164.9.
1-(3-Nitrophenyl)-3-carboxamide(propylamine) β-carboline
(3c)
Yield 55%; mp 178-180 °C; IR (KBr) νmax / cm-1 3160,
1686, 1534, 1474, 1440; 1H NMR (300 MHz, CD3OD) d
1.65 (qt, 2H, J 6.5 Hz, CH2-prophyl), 2.65 (t, 2H, J 6.0 Hz,
CH2-prophyl), 3.46 (q, 2H, J 6.0 Hz, CH2-prophyl), 7.33
(t, 1H, J 7.5 Hz, H-6), 7.63 (m, 2H, H-8, Phenyl-H), 7.72

Vol. 27, No. 8, 2016
Moreira et al.
1401
Anti-Mycobacterium tuberculosis activity
The tests were performed using the colorimetric method
with sulforhodamine B according to the National Cancer
Institute (NCI) standard protocol; doxorubicin was used as
a positive control.29 Assays were performed in a 96-well
plate using four serial 10-fold dilutions (0.25-250 μg mL-1)
for each test compound. The anticancer activity was
determined based on concentration-response curves, and
three concentration response parameters, growth inhibitory
activity (GI50), growth inhibition (TGI) and cytotoxic
activity (LC50) were calculated. The response parameter
GI50 refers to the drug concentration that produces 50%
reduction of cell growth when compared to untreated
control cells. The TGI and LC50 parameters refer to the
drug concentrations required for total growth inhibition and
for 50% cell mortality, respectively. Compounds with GI50
values < 100 μmol L-1 were considered active.
M. tuberculosis H37Rv (ATCC27294) strains were
grown in Ogawa-Kudoh (OK) medium for 10 days at
37 °C. For testing, aliquots were removed and cultured
in Middlebrook 7H9 broth (Difco, Sparks, USA)
supplemented with oleic acid, bovine serum albumin,
dextrose and catalase (OADC enrichment BBL/Becton-
Dickinson); 0.5% glycerol was added as a carbon source
and 0.5% Tween 80 was added to prevent the appearance
of lumps. The culture was maintained for 15 days at 37 ºC.
The bacterial suspensions were prepared and adjusted to
the No. 1 of the McFarland scale.
Stock solutions of the test compounds were
solubilized in dimethyl sulfoxide (DMSO) (Sigma-
Aldrich, St. Louis, USA) and diluted in Middlebrook 7H9
broth (Difco, Sparks, USA) supplemented with OADC
enrichment BBL/Becton Dickinson. Rifampicin and
isoniazid were solubilized according to the manufacturer’s
recommendations (Sigma-Aldrich, St. Louis, USA) and
used as positive controls.
Antimycobacterial activity was determined using the
resazurin microtiter assay (REMA).27 Briefly, 100 μL of
supplemented Middlebrook 7H9 broth (Difco, Sparks,
USA) was dispensed into each well of a sterile flat-bottom
96-well plate; then, serial dilutions of the test compounds
(0.98-250 μg mL-1) and reference drugs (0.004-1 μg mL-1)
were prepared. One hundred microliters of bacterial
suspension (5 × 105 UFC mL-1) was then added to each
well. Plates were incubated for 7 days at 37 °C, after was
added 30 μL of resazurin (Sigma-Aldrich, St. Louis, USA)
in sterile water (0.01%) in whole plate, and the samples
were incubated for 24 h at 37 °C.
In silico study
The in silico computational study of compounds were
performed to determine Lipinski’s rules of five30 (hydrogen
bond donors ≤ 5; hydrogen bond acceptors ≤ 10; molecular
weight ≤ 500; the Log P is ≤ 5), topological polar surface
area (TPSA) and percentage of absorption (%ABS).
Calculations were performed using Molinspiration
online property calculation toolkit software31 and
OSIRIS property explorer software.32 The percentage of
absorption was estimated using the following equation:
%ABS = 10 – [0.345 × TPSA].
Results and Discussion
Chemistry
The change in absorbance, at 492 nm wavelength, was
measured using a microplate reader TP-Reader (Thermo
Plate®, Männedorf, Switzerland). Each compound was
analyzed in triplicate on alternate days. The minimum
inhibitory concentration (MIC) was defined as the lowest
concentration that resulted in 90% inhibition of the growth
of M. tuberculosis.28 MIC values were used to classify a
compound’s activity as follows: inactive, > 150 μg mL-1;
moderate, between > 10 and < 100 μg mL-1; and active,
< 10 μg mL-1.
The synthetic pathway for the preparation of
1,3-disubstituted β-carbolines is presented in Scheme 1. The
methyl esters in 1a-c were prepared by a Pictet-Spengler
condensation of L-tryptophan with the appropriate aromatic
aldehydes in acidic media, subsequent esterification of
the resulting carboxylic acids with methanol and sulfuric
acid, and oxidation with sulfur in refluxing xylene.22
Compounds 2a-c and 3a-c were obtained by the reaction
of β-carboline methyl ester with 1,2-ethylenediamine
and 1,3-propanediamine, respectively, and resulted in an
amino group-terminated side chain at C-3. Finally, the
coupling of β-carboline carboxamide derivatives 2a-c
with S-methylisothiourea yielded compounds 4a-c, which
include a terminal guanidinium group.
In vitro antiproliferative assay
The tested compounds were evaluated in vitro against
a nine-cell line panel comprising melanoma UACC-62,
breast MCF7, lung NCI-460, leukemia K-562, ovarian
OVCAR, prostate PCO-3, colon HT29, renal 786-0 and
adriamycin drug-resistant ovarian cancer NCI/ADR cells.
The novel compounds 2a-c, 3a-c and 4a-c were
1
characterized using H and 13C NMR spectroscopy, as
detailed in the Experimental section. The 1H NMR spectra

1402
Synthesis, in vitro Antiproliferative and Anti-Mycobacterium tuberculosis Activities of Novel b-Carboline Derivatives
J. Braz. Chem. Soc.
Scheme 1. Synthetic route for the preparation of the β-carbolines derivatives. Reagents and conditions: (a) 1,2-ethylenediamine, at room temperature,
36 h or 1,3-propanediamine, CHCl3/MeOH, reflux, 32 h (55-72%); (b) S-methylisothiourea, 2N NaOH, 4 °C to room temperature, reflux, 48 h (30-45%).
of carboxamides 2a-c and 3a-c showed additional signals at
dH 1.64-3.95 (reflecting the integration of two protons) and
at dH 7.01-8.94 (corresponding to aromatic hydrogens). The
presence of the ethylamine or propilamine carboxamide
in position C-3 group was confirmed by 13C NMR, which
showed signals at dc 32.8-41.9 (CH2) and dc 162.5-167.1
(C=ON). Derivatives 4a-c was characterized by the
presence of an additional signal at dc 159.01-159.90,
corresponding to the guanidinium group.
compounds, which demonstrated potent antitubercular
activity against M. tuberculosis, aminopyrimidine
derivatives exhibit moderate to potent anti-MTB activity,
with MIC values ranging from 12.5-3.12 μg mL-1.33
The introduction of an ethylguanidinium group at the
upper rim resulted in high antimycobacterial activities
for the unsubstituted, 5,5’-dimethyl-2,2’-bipyridyl and
4,4’-dimethyl-2,2’-bithiazolyl analogs, with MIC values
of 1.51 and 2.69 μg mL-1, respectively, values that were
similar to those of current commercially available anti-
tuberculosis agents.34
Anti-Mycobacterium tuberculosis activity (MTB)
Derivatives 2a-c, 3a-c and 4a-c were evaluated
in vitro for their antimycobacterial activity against
M. tuberculosis H37Rv (ATCC 27294) using the REMA
method.27 The MIC values (μg mL-1 and μmol L-1) were
measured with respect to two standard antitubercular
drugs, isoniazid (INH) and rifampicin (RFP), and the
screening results are presented in Table 1. Among
the nine compounds evaluated against MTB, seven
presented moderate activity, with MIC values ranging
from 58.3-24.9 μg mL-1; in particular, compounds 2a
(24.9 μg mL-1), 2b (26.9 μg mL-1), 4a (27.8 μg mL-1)
and 4c (37.4 μg mL-1) presented interesting activity.
Compounds 2a and 2b, which had p-hidroxyphenyl and
p-methoxyphenyl substituents, respectively, at position-1
and ethylenediamine moieties at C-3 were the most active
derivatives in this series. The length of the terminated
side chains affected the activities of these compounds.
Substituting the guanidinium group led to reduced
activity (compare compounds 2a and 4a). The effect
of the guanidinium group was particularly significant
when comparing compounds 2c (57.9 μg mL-1) and 4c
(37.4 μg mL-1). The substituents at positions-1 and 3
strongly affected anti-MTB activities of these compounds.
Earlier studies reported the synthesis and investigations
of the antimycobacterial activity, e.g., guanidinium-modified
Table 1. Anti-Mycobacterium tuberculosis H37RV activity of compounds
2a-c, 3a-c and 4a-c
MICa /
(μg mL-1)
MIC /
(μmol L-1)
Compound
R
n
2a
4-OH
4-OCH3
3-NO2
4-OH
4-OCH3
3-NO2
4-OH
4-OCH3
3-NO2
–
2
2
2
3
3
3
2
2
2
–
–
24.9
26.9
57.9
> 250
58.3
> 250
27.8
57.5
37.4
0.05
0.01
75.4
74.6
2b
2c
153.5
> 500
155.7
> 500
74.4
3a
3b
3c
4a
4b
142.6
88.3
4c
Isoniazid
0.3
Rifampicin
–
0.01
aMIC = minimum inhibitory concentration (REMA assay), values quoted
are the means of results for triplicate samples.
Antiproliferative activity
The antiproliferative activities of the synthesized
1,3-disubstituted β-carbolines derivatives (2a-c, 3a-c and
4a-c) were evaluated in vitro against nine human tumor
cell lines. The results for compounds 2a-c and 3a-c,

Vol. 27, No. 8, 2016
Moreira et al.
1403
which were amino group-terminated at C-3, demonstrated
that compounds 2a and 3b, with p-hydroxyphenyl and
p-methoxyphenyl groups, respectively, at position-1,
inhibited growth in all human tumor cell lines with GI50
values rangingfrom 1.37-9.20 μmol L-1.Also in this series,
compounds 3a (GI50 = 0.33 mmol L-1, TGI = 50.28 mmol L-1)
and 3c (GI50 = 0.71 mmol L-1, TGI = 11.08 mmol L-1,
LC50 = 26.62 mmol L-1) showed significant activity and
high selectivity against adriamycin drug-resistant ovarian
cancer cells (NCI/ADR) (Tables 2 and 3).
The compounds with terminal guanidinium
groups, including compound 4b (GI50 = 0.45 mmol L-1;
TGI = 72.09 mmol L-1) effectively inhibited growth and
was highly selective against adriamycin drug-resistant
ovarian cancer cell lines (NCI/ADR) when compared
with compounds 2a-c, which are amino group-terminated.
However, the guanidinium-terminated compounds did not
demonstrate any important interaction that could account
for the cell growth inhibition. Substituting the phenyl group
with electron-donating substituents at position-1 influenced
each series differently.
A previous study evaluated the in vitro antitumor
activities of several benzenesulfonamide derivatives
with various substituted aminoguanidine groups.
Compound 1-allyl-2-[4-chlorophenylcarbamoyl)-2-
methylthiobenzenesulfonyl]-3-(5-nitrofurfurylideneamino)
exhibited remarkable activity against 21 human tumor
cell lines representing leukemia and melanoma and
lung, colon, ovarian, renal, prostate and breast cancers
(GI50 = 0.3-3.0 μmol L-1).35
Table 2. In vitro cell growth inhibition (GI50) of compounds 2a-c, 3a-c and 4a-c against neoplastic cells
GI50a / (μmol L-1)
NCI/ADR
ovarian-
resistant
Compound
R
n
UACC-62
melanoma
MCF7
breast
NCI-460
lung
K-562
leukemia
OVCAR
ovarian
PCO-3
prostate
HT29
colon
786-0
renal
2a
2b
2c
3a
3b
3c
4a
4b
4c
4-OH
4-OCH3
3-NO2
4-OH
2
2
2
3
3
3
2
2
2
5.15
38.75
10.34
25.14
9.66
9.55
44.49
11.72
16.19
6.98
9.64
44.49
88.26
13.59
7.35
2.93
53.07
> 100
13.01
5.20
9.20
17.14
9.36
8.69
4.27
> 100
25.82
23.57
9.02
9.64
33.11
21.64
18.71
4.40
8.18
8.36
2.42
0.33
1.37
0.71
> 100
0.45
1.71
c
*
*
22.75
7.09
23.57
2.39
11.18
–
4-OCH3
3-NO2
4-OH
43.22
> 100
95.93
51.85
23.94
> 100
35.46
99.17
55.16
18.89
–
16.76
> 100
39.94
87.51
27.48
–
27.48
3.61
b
–
4-OCH3
27.17
63.41
15.54
75.64
63.41
3-NO2
> 100
31.03
91.82
25.02
31.03
aGI50 = growth inhibitory activity; bnot determined; cnot tested.
Table 3. Total growth inhibition (TGI) and lethal concentration (LC50 - in parentheses) of compounds 2a-c, 3a-c and 4a-c (μmol L-1)
NCI/ADR
ovarian-
resistant
UACC-62
melanoma
MCF7
breast
NCI-460
lung
K-562
leukemia
OVCAR
ovarian
PCO-3
prostate
HT29
colon
786-0
renal
Compound
R
n
2
2a
4-OH
15.44
(37.5)
38.27
> 100
34.32
23.87
24.75
(51.7)
24.75
(74.42)
73.52
24.75
(64.37)
24.75
b
2b
2c
4-OCH3
3-NO2
2
2
> 100
> 100
> 100
> 100
44.27
> 100
*
> 100
> 100
> 100
87.41
26.45
79.13
20.35
22.41
19.33
*
(70.93)
(43.42)
(30.85)
a
3a
3b
4-OH
3
3
–
42.55
–
36.01
–
>100
–
–
50.28
4-OCH3
–
15.38
(32.8)
20.83
(51.25)
15.38
(37.3)
21.47
(46.84)
10.56
(35.2)
21.47
(49.15)
16.71
(46.84)
11.97
(28.94)
3c
3-NO2
3
> 100
> 100
–
> 100
> 100
43.09
> 100
> 100
11.08
(26.62)
4a
4b
4c
4-OH
4-OCH3
3-NO2
2
2
2
> 100
–
> 100
> 100
–
> 100
> 100
–
> 100
> 100
55.07
> 100
> 100
–
> 100
> 100
–
> 100
> 100
99.28
> 100
> 100
> 100
> 100
72.09
> 100
90.86
aNot determined; bnot tested.

1404
Synthesis, in vitro Antiproliferative and Anti-Mycobacterium tuberculosis Activities of Novel b-Carboline Derivatives
J. Braz. Chem. Soc.
Table 4. Lipinski’s rule and percentage of absorption (%ABS), topological polar surface area (TPSA), for compounds 2a-c, 3a-c and 4a-c
Lipinsk’s parameter
Compound
%ABSa
TPSAb / Å2
nHBAc,h (nON)
nHBDd (nOHNH)
Log Pe,h
2.02
2.56
2.43
2.29
2.83
2.71
2.08
2.61
2.49
MWf,h
346.39
360.42
375.39
360.42
374.44
389.42
388.43
402.46
417.43
n violationsh
Log Sg,i
–4.23
–4.54
–4.98
–4.50
–4.81
–5.25
–3.87
–4.18
–4.62
2a
2b
2c
3a
3b
3c
4a
4b
4c
73.11
76.91
64.28
73.11
76.91
64.28
60.74
64.53
51.90
104.03
93.04
6
6
5
4
4
5
4
4
7
6
6
0
0
0
0
0
0
1
1
1
129.63
104.03
93.04
8
6
6
129.63
139.91
128.92
165.51
8
8
8
10
a%ABS = 109 – [0.345 × TPSA]; bTPSA = topological polar surface area; cnHBA [number hydrogen bond acceptor (nON)] ≤ 10; dnHBD [number hydrogen
e
f
g
bond donors (OHNH)] ≤ 5; Log P (octanol-water partition coefficient) < 5; MW (molecular weigth) ≤ 500; Log S (solubility) between –1 and –5;
hreference 31; ireference 32.
Lipinski’s rule of five
2a, 2b and 4a were the most active against M. Tuberculosis
H37Rv (ATCC27294). Compound 2a and 3b demonstrated
promising antiproliferative activity for all cancer cell lines.
Eight compounds inhibited the cell growth of adriamycin
drug-resistant ovarian (NCI/ADR), showed activity and
high selectivity for the 3a, 3c and 4b. Compound 2a
demonstrated promising antiproliferative and anti-MTB
activity, in addition to follow as established Lipinski’s rule
of five, suggesting that this compound has no problems with
oral bioavailability, and indicates good permeability a in
the plasma membrane of the cell, which may represent a
precursor to development of new molecules. Further studies
are required to explore the mechanism of action of these
compounds in detail.
The drug-likeness concept helps optimize the
pharmacokinetic properties of a compound, such as
absorption, distribution, metabolism and excretion (ADME)
in the human body.36 Lipinski’s rule of five is a refinement
of drug-likeness and is used to predict whether a chemical
compound will have pharmacological or biological activity
as an orally active drug in humans. This rule was formulated
based on the observation that most medication drugs are
relatively small and lipophilic molecules.33 The results
of the analysis are shown in Table 4 and indicate that the
compounds are in agreement with the values determined by
Lipinski, except 4a-c derivatives, which showed the number
of hydrogen bond donors (nHBD) > 5, in violation of the
Lipinski rules. The calculated percent absorption (%ABS)
of all compounds ranged from 51.90-76.91%, indicating
good cell membrane permeability. Another important
factor is obtained by the volume analysis and TPSA by the
compounds showed lower than 140Ų indicating that these
derivatives have good absorption in the intestine, except the
compound 4c (TPSA = 165.51). The compounds 2a-c, 3a-b
and 4a-c exhibited good solubility (Log S = –3.87 to –4.98),
except the compound 3c which showed a value of Log S
less than –5. Compounds with high solubility are easily
metabolized and eliminated from the body, thus resulting in
a lower probability of adverse effects and bioaccumulation.
Supplementary Information
Supplementary information is available free of charge
at http://jbcs.sbq.org.br, as PDF file.
Acknowledgments
We are grateful to CNPq, FUNDECT and CAPES for
providing financial support and UFGD.
References
1. World Health Organization (WHO); Global Tuberculosis
Report, 20th ed.; Geneva, 2015, p. 1.
Conclusions
2. Gandhi, N. R.; Nunn, P.; Dheda, K.; Schaaf, H. S.; Zignol, M.;
Van Soolingen, D.; Jensen, P.; Bayona, J.; The Lancet 2010,
375, 1830.
Thus, our results showed for the first time the synthesis
and antitumor and anti-MTB activity of compounds with an
amino or guanidinium group-terminated side chain at C-3
of a 1-substituted-phenyl-β-carboline nucleus. Compounds
3. Ma, Z.; Lienhardt, C.; Mcilleron, H.; Nunn, A. J.; Wang, X.;
The Lancet 2010, 375, 2100.

Vol. 27, No. 8, 2016
Moreira et al.
1405
4. Koul, A.; Arnoult, E.; Lounis, N.; Guillemont, J.; Andries, K.;
Nature 2011, 469, 483.
22. Formagio,A. S. N.; Tonin, L. T. D.; Foglio, M.A.; Madjarof, C.;
Carvalho, J. E.; Costa, W. F.; Cardoso, F. P.; Sarragiotto, M. H.;
Bioorg. Med. Chem. 2008, 16, 9660.
5. Jagetia, G. C.; Venkatesh, P.; Baliga, M. S.; Bio. Pharm. Bull.
2005, 28, 58.
23. Pedroso, R. B.; Tonin, L. T. D.; Ueda-Nakamura, T.; Dias Filho,
B. P.; Sarragiotto, M. H.; Nakamura, C. V.; Ann. Trop. Med.
Parasitol. 2011, 105, 549.
6. World Health Organization (WHO); World Cancer Report,
1st ed.; Geneva, 2014, p. 1.
7. Ang, K. K.; Holmes, M. J.; Higa, T.; Hamann, M. T.; Kara,
U. A.; Antimicrob. Agents Chemother. 2000, 44, 1645.
8. Cao, R.; Chen, Q.; Hou, X.; Chen, H.; Guan, H.; Ma, Y.;
Peng, W.; Xu, A.; Bioorg. Med. Chem. 2004, 12, 4613.
9. Cao, R.; Chen, H.; Peng, W.; Ma,Y.; Hou, X.; Guan, H.; Liu, X.;
Xu, A.; Eur. J. Med. Chem. 2005, 40, 991.
24. Yu, X.; Lin, W.; Li, J.; Yang, M.; Bioorg. Med. Chem. Lett.
2004, 14, 3127.
25. Yu, X.; Lin, W.; Pang, R.;Yang, M.; Eur. J. Med. Chem. 2005,
40, 831.
26. Ishida, J.; Wang, H.; Bastow, K. F.; Hu, C.; Lee, K.; Bioorg.
Med. Chem. Lett. 1999, 9, 3319.
10. Cao, R.; Peng, W.; Chen, H.; Hou, X.; Guan, H.; Chen, Q.;
Ma, Y.; Xu, A.; Eur. J. Med. Chem. 2005, 40, 249.
11. Wu, Q.; Cao, R.; Feng, M.; Guan, X.; Ma, C.; Liu, J.; Song, H.;
Peng, W.; Eur. J. Med. Chem. 2009, 44, 533.
27. Palomino, J. C.; Martin, A.; Camacho, M.; Guerra, H.;
Swings, J.; Portaels, F.; Antimicrob. Agents Chemother. 2002,
46, 2720.
28. Pavan, F. R.; Poelhsitz, G. V.; Barbosa, M. I.; Leite, S. R.;
Batista, A. A.; Ellena, J.; Sato, L. S.; Franzblau, S. G.;
Moreno, V.; Gambino, D.; Leite, C. Q.; Eur. J. Med. Chem.
2011, 46, 5099.
12. Wu, J.; Li, C.; Zhao, M.; Wang, W.; Wang,Y.; Peng, S.; Bioorg.
Med. Chem. 2010, 18, 6220.
13. Begum, S.; Hassan, S. I.; Siddiqui, B. S.; Nat. Prod. Res. 2004,
18, 341.
29. Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.; Paull, K.;
Vistica, D.; Hose, C.; Langley, J.; Cronise, P.;Vaigro-Wolff,A.;
J. Natl. Cancer Inst. 1991, 83, 757.
14. Begum, S.; Ali, S. N.; Siddiqui, B. S.; US 8.420.660, 2013.
15. Stefanello, T. F.; Panice, M. R.; Ueda-Nakamura, T.; Sarragiotto,
M. H.; Auzely-Velty, R.; Nakamura, C. V.; Antimicrob. Agents
Chemother. 2014, 58, 7112.
30. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J.;
Adv. Drug Delivery Rev. 2001, 46, 3.
16. Savariz, F. C.; Foglio, M.A.; Ruiz,A. L.; Costa, W. F.; Silva, M.;
Santos, J. C.; Figueiredo, I. M.; Meyer, E.; Carvalho, J. E.;
Sarragiotto, M. H.; Bioorg. Med. Chem. 2014, 22, 6867.
17. Savariz, F. C.; Foglio, M.A.; Carvalho, J. E.; Ruiz,A. L.; Duarte,
M. C.; Rosa, M. F.; Meyer, E.; Sarragiotto, M. H.; Molecules
2012, 17, 6100.
31. http://www.molinspiration.com accessed in January 2016.
32. http://www.organic-chemistry.org/prog/peo accessed in January
2016.
33. Singh, N.; Pandey, S. K.; Anand, N.; Dwivedi, R.; Singh, S.;
Sinha, S. K.; Chaturvedi, V.; Jaiswal, N.; Srivastava, A. K.;
Shah, P.; Siddiqui, M. I.; Tripathi, R. P.; Bioorg. Med. Chem.
Lett. 2011, 21, 4404.
18. Barbosa, V. A.; Formagio, A. S.; Savariz, F. C.; Foglio, M. A.;
Spindola, H. M.; Carvalho, J. E.; Meyer, E.; Sarragiotto, M. H.;
Bioorg. Med. Chem. 2011, 19, 6400.
34. Mourer, M.; Dibama, M. H.; Constant, P.; Daffé, M.; Regnouf-
de-Vains, J. B.; Bioorg. Med. Chem. 2012, 20, 2035.
35. Brzozowski, Z.; Sączewski, F.; Sławiński, J.; Eur. J. Med. Chem.
2007, 42, 1218.
19. Savariz, F. C.; Formagio,A. S. N.; Barbosa,V.A.; Foglio, M.A.;
Carvalho, J. E.; Duarte, M. C. T.; Dias Filho, B. P.; Sarragiotto,
M. H.; J. Braz. Chem. Soc. 2010, 21, 288.
36. Vistoli, G.; Pedretti,A.; Testa, B.; Drug Discovery Today 2008,
13, 285.
20. Tonin, L. T.; Panice, M. R.; Nakamura, C. V.; Rocha, K. J.;
Santos, A. O.; Ueda-Nakamura, T.; Costa, W. F.; Sarragiotto,
M. H.; Biomed. Pharmacother. 2010, 64, 386.
Submitted: November 4, 2015
21. Formagio, A. S. N.; Santos, P. R.; Zanoli, K.; Ueda-Nakamura,
T.; Tonin, L. T. D.; Nakamura, C. V.; Sarragiotto, M. H.; Eur.
J. Med. Chem. 2009, 44, 4695.
Published online: February 26, 2016
FAPESP has sponsored the publication of this article.