Synthesis and biological evaluation of N,N′-di(thiopheneacetyl) diamines series as antitubercular agents

Article abstract of DOI:10.1080/10426500802049803

The series of new N,N′-di(thiopheneacetyl)diamines derivatives, 8-17, were synthesized and evaluated for their in vitro antibacterial activity against Mycobacterium tuberculosis(TB), and the activity expressed as the minimum inhibitory concentration (MIC) in μg/mL. Compound 13 was the only one determined to be active and exhibited a MIC 50 μg/mL, indicating that the alkyl chain-length of the diamines is critical for biological activity. This class of compound has not been evaluated before, and it could be a good starting point to find new lead compounds in the fight against multi-drug resistant TB. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/10426500802049803

This article was downloaded by: [Pennsylvania State University]
On: 16 May 2013, At: 11:34
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954
Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH,
UK
Phosphorus, Sulfur, and Silicon
and the Related Elements
Publication details, including instructions for
authors and subscription information:
http://www.tandfonline.com/loi/gpss20
Synthesis and Biological
Evaluation of N,N′-
di(thiopheneacetyl)diamines
Series as Antitubercular Agents
Marcus Vinícius Nora de Souza a , Maria Cristina
Silva Lourenço b , Mônica Amado Peralta a , Raoni
Schroeder Borges Gonçalves a , Thais Cristina
Mendonça Nogueira a , Camilo Henrique da Silva
Lima a , Marcelle de Lima Ferreira a & Emerson
Teixeira da Silva a
a FioCruz-Fundação Oswaldo Cruz, Instituto
de Tecnologia em Fármacos-Far Manguinhos,
Manguinhos, Rio de Janeiro-RJ, Brazil
b Instituto de Pesquisas Clínica Evandro Chagas
(IPEC), Rio de Janeiro, RJ, Brazil
Published online: 03 Nov 2008.
To cite this article: Marcus Vinícius Nora de Souza , Maria Cristina Silva Lourenço ,
Mônica Amado Peralta , Raoni Schroeder Borges Gonçalves , Thais Cristina
Mendonça Nogueira , Camilo Henrique da Silva Lima , Marcelle de Lima Ferreira
& Emerson Teixeira da Silva (2008): Synthesis and Biological Evaluation of N,N′-
di(thiopheneacetyl)diamines Series as Antitubercular Agents, Phosphorus, Sulfur, and
Silicon and the Related Elements, 183:12, 2990-2997
To link to this article: http://dx.doi.org/10.1080/10426500802049803
PLEASE SCROLL DOWN FOR ARTICLE

Full terms and conditions of use: http://www.tandfonline.com/page/terms-
and-conditions
This article may be used for research, teaching, and private study purposes.
Any substantial or systematic reproduction, redistribution, reselling, loan,
sub-licensing, systematic supply, or distribution in any form to anyone is
expressly forbidden.
The publisher does not give any warranty express or implied or make any
representation that the contents will be complete or accurate or up to
date. The accuracy of any instructions, formulae, and drug doses should be
independently verified with primary sources. The publisher shall not be liable
for any loss, actions, claims, proceedings, demand, or costs or damages
whatsoever or howsoever caused arising directly or indirectly in connection
with or arising out of the use of this material.

Phosphorus, Sulfur, and Silicon, 183:2990–2997, 2008
Copyright © Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426500802049803
Synthesis and Biological Evaluation of
N,N’-di(thiopheneacetyl)diamines Series as
Antitubercular Agents
1
´
Marcus Vinıcius Nora de Souza, Maria Cristina Silva
2
1
ˆ
Lourenc¸o, Monica Amado Peralta, Raoni Schroeder
Borges Gonc¸alves,1 Thais Cristina Mendonc¸a Nogueira,1
Camilo Henrique da Silva Lima,1 Marcelle de Lima
Ferreira,1 and Emerson Teixeira da Silva1
1FioCruz-Fundac¸a˜o Oswaldo Cruz, Instituto de Tecnologia em
Fa´rmacos-Far Manguinhos, Manguinhos, Rio de Janeiro-RJ, Brazil
2Instituto de Pesquisas Cl´ınica Evandro Chagas (IPEC), Rio de
Janeiro-RJ, Brazil
The series of new N,N’-di(thiopheneacetyl)diamines derivatives, 8–17, were synthe-
sized and evaluated for their in vitro antibacterial activity against Mycobacterium
tuberculosis(TB), and the activity expressed as the minimum inhibitory concentra-
tion (MIC) in µg/mL. Compound 13 was the only one determined to be active and
exhibited a MIC 50 µg/mL, indicating that the alkyl chain-length of the diamines
is critical for biological activity. This class of compound has not been evaluated
before, and it could be a good starting point to find new lead compounds in the fight
against multi-drug resistant TB.
Keywords Antimycobacterial activity; thiophene; tuberculosis (TB)
INTRODUCTION
Tuberculosis (TB) is becoming again an important public health prob-
lem worldwide, declared by WHO (World Health Organization) as a
global health emergency in 1993.1 This contagious disease is transmit-
ted through the air and it is caused by the bacterium Mycobacterium
tuberculosis, which is responsible for 1.7–2.0 millions of deaths each
year. 2 However, in spite of the worldwide problem caused by TB, it has
Received 12 March 2008; accepted 25 March 2008.
Address correspondence to Marcus Vin´ıcius Nora de Souza, FioCruz-Funda c¸a˜o Os-
waldo Cruz, Instituto de Tecnologia em Fa´rmacos-Far Manguinhos. Rua Sizenando
Nabuco, 100, Manguinhos, 21041-250, Rio de Janeiro-RJ, Brasil. E-mail: marcos souza@
far.fiocruz.br
2990

N,N’-di(thiopheneacetyl)diamines as Antitubercular Agents
2991
H2N
NH2
5
H
H
S
Cl
N
S
N
S
4
n
1
2 .
n
O
THF
98-83%
O
O
2
3
1
2 n = 2;
3 n = 3; 4 n = 4;
8 n = 2;
9 n = 3; 10 n = 4;
5 n = 6; 6 n = 8; 7 n = 10
11 n = 6; 12 n = 8; 13 n = 10
SCHEME 1 Synthesis of aliphatic N,N’-di(thiopheneacetyl)diamines.
been nearly 30 years since a novel drug has been introduced to treat TB
(with exception of Rifapentine approved by FDA in 1998). In this con-
text, there is an urgent need for new drugs to fight against this disease.
Considering that, the thiophene nucleus represents a very important
field in drug discovery, and is present in many natural and synthetic
products with a wide range of pharmacological activities. 3,4
There are three basic factors involved in the development of new TB
drugs; to reduce the total duration of treatment, to improve the MDR
TB (Multidrug-resistant TB) and to provide more effective treatment
of latent TB infection. Due to TB problem worldwide and the lack of
new drugs nowadays, the aim of this article is to present a series of
news N,N’-di(thiopheneacetyl)diamines 8–17 derivatives, which have
been synthesized, see Schemes 1 and 2, and evaluated for their in vitro
antibacterial activity against Mycobacterium tuberculosis.
RESULTS AND DISCUSSION
Chemistry
The synthesis of di(thiopheneacetyl)diamines compounds 8–17 in-
volved the reaction between appropriate 2-thiopheneacetyl chloride 1
and the respective diamines 2–7, 14 and 16 (Schemes 1 and 2), as de-
scribed in the general procedure leading to the desire compounds 8–17
in 77–98% yields. All the compounds were identified by spectral data.
In general, IR spectra showed the C=O peak at 1650–1675 cm−1. The
1H NMR spectrum showed the amide (NH) proton as a large singlet
at 8.15–7.24 ppm, CH2CO proton as a singlet at 3.87–3.53 ppm, and
NHCH2 proton as a triplet or quartet at 3.04–3.50 ppm. The 13C NMR
spectrum showed the C=O signals at 170.0–168.6, CH2CO signals at
39.0–37.8 and aliphatic carbons at the region of 26.5–36.5 ppm.

2992
M. V. N. de Souza et al.
S
O
O
S
HN
NH
NH2
H2N
14
S
Cl
15
84%
2 .
O
1
NH2
H2N
S
O
O
16
S
HN
NH
80%
17
SCHEME 2 Synthesis of cyclic N, Nꢀ−di(thiopheneacetyl)diamines.
Biology Evaluation
In the biological evaluation of the series of news N,N’-di(thiophe
neacetyl)diamines derivatives 8–17, only the compound 13 exhibited
activity with a MIC of 50 µg/mL against M. tuberculosis infection,
when compared to first line drugs as isoniazid (INH) and rifampin (RIP)
(Table I). All others compounds synthesized (8–12 and14–17) were in-
active against M. tuberculosis. This result indicated that the length of
the alkyl chain is critical for the biological activity. Additionally, com-
pound 13 was not cytotoxic to host cells at the same concentration
(Table II).
EXPERIMENTAL
General Procedures
Melting points were determined on a Buchi apparatus and are un-
corrected. Infrared spectra were recorded on a Thermo Nicolet Nexus
670 Spectrometer as potassium bromide pellets and frequencies are
expressed in cm−1. Mass spectra (CG/MS) were recorded on an Agi-
lent Technologies 6890/5972A Mass Spectrometer. NMR spectra were

N,N’-di(thiopheneacetyl)diamines as Antitubercular Agents
2993
TABLE I Antimycobacterial Activities of
N,N’-di(thiopheneacetyl)diamines
Compound
M.p. (oC)
Yield (%)
MIC (µg/mL)
8
9
145–150
118–120
146–148
110–112
216–220
115–117
102–104
102–107
98
90
88
83
92
77
84
80
Resistant
Resistant
Resistant
Resistant
Resistant
50
10
11
12
13
15
17
Resistant
Resistant
recorded on a Bruker Avance 500 Spectrometer operating at 500.00
MHz (1H) and 125.0 MHz (13C), in deuterated dimethylsulfoxide. Chem-
ical shifts are reported in ppm (δ) relative to tetramethylsilane. Proton
and carbon spectra were typically obtained at room temperature. TLC
plates coated with silica gel were developed in ethyl acetate and spots
were visualized under UV light.
General Procedures for the Synthesis
of N,N ’-di(thiopheneacetyl)diamines Derivatives (8–17)
The N,Nꢀ-di(thiopheneacetyl)diamines derivatives (8–17) were pre-
pared by reaction between the appropriate diamines 2–7, 14, and 16
(1.0 equiv.) with thiophenylacetylchloride (2.2 equiv.) in tetrahydrofu-
ran (20 mL), under nitrogen atmosphere (Scheme 1). After stirring for
2–3 h at room temperature the reaction mixture was quenched with
30 mL of water and extracted with ethylacetate (2 × 20 mL). The com-
bined organic layers were washed with saturated aqueous NaHCO3 and
brine, dried (MgSO4), filtered, and concentrated in vacuo to produce the
desired diamines without further purification.
N,N’-di(thiopheneacetyl)ethylenediamine (8)
Yield: 98% m.p.: 145–150oC
CG/MS: m/z [M]+.: 308
1H NMR [500.00 MHz (FIDRES 0.15 Hz), DMSO-d6] δ: 8.10 (s,
2H; NH); 7.37 (dd, 2H; J = 5.0 and 2.0 Hz; H4); 6.94 (dd, 2H; J = 5.0
and 2.0 Hz; H3); 6.89 (ls, 2H; H2); 3.87 (s, 4H, CH2CO); 3.2 (t; 4H; J =
7.0 Hz; NCH2CH2N) ppm. 13C NMR (125.0 MHz, DMSO-d6) δ: 169.0;
137.0; 126.1; 125.8; 124.7; 38.6; 36.4 ppm.
IVνmax(cm−1; KBr pellets): 1657 (CO).

2994
M. V. N. de Souza et al.
N,N’-di(thiopheneacetyl)propylenediamine (9)
Yield: 90% m.p.: 118–120oC
CG/MS: m/z [M]+.: 322
1H NMR [500.00 MHz (FIDRES
0.15 Hz), DMSO-d6] δ: 7.24
(dd, 2H; J = 5.0 and 1.5 Hz; H4); 6.99 (t, 2H; J = 5.0 Hz; H3); 6.94 (ls,
2H; H2); 6.31 (ls, 2H; NH); 3.76 (s, 4H, CH2CO); 3.2 (q, 4H; J = 7.0 Hz;
NCH2CHCH2N); 3.2 (quint., 2H; J = 7.0 Hz; NCH2CH2CH2N) ppm.
13C NMR (125.0 MHz, DMSO-d6) δ: 170.5; 136.1; 127.3; 127.2; 125.5;
124.7; 37.6; 36.1; 29.6 ppm.
IVνmax(cm−1; KBr pellets): 1670 (CO).
N,N’-di(thiopheneacetyl)butylenediamine (10)
Yield: 88% m.p.: 146–148oC
CG/MS: m/z [M]+.: 336
1H NMR [500.00 MHz (FIDRES 0.15 Hz), DMSO-d6] δ: 8.02 (ls,
2H; NH); 7.32 (dd, 2H; J = 5.0 and 1.5 Hz; H4); 6.99 (dd, 2H; J = 5.0
Hz and 1.5 Hz; H3); 6.88 (ls, 2H; H2); 3.60 (s, 4H, CH2CO); 3.04 (t, 4H;
J = 10 Hz; NCH2(CH2)2CH2N); 1.42–1.36 (m, 4H; NCH2(CH2)2CH2N )
ppm. 13C NMR (125.0 MHz, DMSO-d6) δ: 168.8; 137.8; 126.5; 125.8;
125.5; 124.6; 39.0; 36.5; 26.5 ppm.
IVνmax(cm−1; KBr pellets): 1666
N,N’-di(thiopheneacetyl)hexylenediamine (11)
Yield: 83% m.p.: 110–112oC
CG/MS: m/z [M]+.: 364
1H NMR [400.00 MHz (FIDRES 0.12 Hz), DMSO-d6] δ: 8.03 (ls,
2H; NH); 7.33 (dd, 2H; J = 5.2 Hz and 1.2 Hz; H4); 6.93 (dd, 2H; J =
5.2 Hz and 1.2 Hz; H3); 6.89 (ls, 2H; H2); 3.64 (s, 4H, CH2CO); 3.50 (q,
4H; J = 6.8 Hz; NCH2(CH2)4CH2N); 1.39−1.35 and 1.23−1.17 (m, 8H;
NCH2(CH2)4CH2N ) ppm. 13C NMR (100.0 MHz, DMSO-d6) δ: 168.8;
137.9; 126.5; 125.8; 124.6; 38.5; 36.5; 28.9; 26.0 ppm.
IVνmax(cm−1; KBr pellets): 1655
N,N’-di(thiopheneacetyl)octylenediamine (12)
Yield: 92% m.p.: 212–220oC
CG/MS: m/z [M]+.: 392
1H NMR [500.00 MHz (FIDRES
0.15 Hz), DMSO-d6] δ: 7.86
(ls, 2H; NH); 7.32 (dd, 2H; J = 5.0 and 1.5 Hz; H4); 6.91 (dd, 2H; J =
5.0 Hz and 1.5 Hz; H3); 6.83 (ls, 2H; H2); 3.53 (s, 4H, CH2CO); 3.50 (t,
4H; J = 11 Hz; NCH2(CH2)6CH2N); 1.77–1.63 and 1.23–1.20 (m, 12H;
NCH2(CH2)6CH2N ) ppm. 13C NMR (125.0 MHz, DMSO-d6) δ: 168.6;
137.7; 126.4; 125.8; 124.6; 51.9; 36.5; 31.8; 24.3 ppm.

N,N’-di(thiopheneacetyl)diamines as Antitubercular Agents
2995
IVνmax(cm−1; KBr pellets): 1675
N,N’-di(thiopheneacetyl)decyldiamine (13)
Yield: 71% m.p.: 115–117oC
CG/MS: m/z [M]+.: 420
1H NMR [500.00 MHz (FIDRES
0.15 Hz), DMSO-d6] δ: 7.96
(ls, 2H; NH); 7.32 (d, 2H; J = 5.0 Hz; H4); 6.93 (dd, 2H; J = 5.0
Hz and 1.5 Hz; H3); 6.88 (ls; 2H; H2); 3.60 (s, 4H, CH2CO); 3.50 (q,
4H; J = 7.0 Hz; NCH2(CH2)8CH2N); 1.77–1.63 and 1.40–1.23 (m, 16H;
NCH2(CH2)8CH2N ) ppm. 13C NMR (125.0 MHz, DMSO-d6) δ: 168.7;
137.9; 126.4; 125.8; 124.6; 38.6; 36.6; 28.9; 28.8; 28.6; 26.3 ppm.
IVνmax(cm−1; KBr pellets): 1650
N,N’-di(thiopheneacetyl)(1R,2S)-1,2-cyclohexanediamine (15)
Yield: 84% m.p.: 102–104oC
CG/MS: m/z [M]+.: 362
1H NMR [500.00 MHz (FIDRES
0.12 Hz), DMSO-d6] δ: 8.05
(ls, 2H; NH); 7.33 (dd, 2H; J = 5.0 Hz and 1.5 Hz; H4); 6.93 (dd, 2H;
J = 5.0 Hz and 1.5 Hz; H3); 6.88 (ls, 2H; H2); 3.60 (s, 4H, CH2CO);
3.50 (q, 4H; J = 7.0 Hz; NCH2CH2N); 1.38−1.36 and 1.22 (ls, 8H;
NCH2(CH2)4CH2N ) ppm. 13C NMR (125.0 MHz, DMSO-d6) δ: 168.8;
137.9; 126.5; 125.9; 124.7; 38.6; 36.6; 28.9; 28.6; 26.6 ppm.
IVνmax(cm−1; KBr pellets): 1665
N,N’-di(thiopheneacetyl)phenylenediamine (17)
Yield: 80% m.p.: 102–107oC
CG/MS: m/z [M]+.: 356
1H NMR [500.00 MHz (FIDRES 0.12 Hz), DMSO-d6] δ: 8.15 (ls,
2H; NH); 7.31−7.28 (m, 4H; Harom.); 7.09 (dd, 2H; J = 7.0 Hz and 3.5
Hz; H4); 7.03 (t, 2H; J= 7.0 Hz; H3); 6.97 (ls, 2H; H2); 3.84 (s, 4H,
CH2CO) ppm. 13C NMR (125.0 MHz, DMSO-d6) δ: 168.8; 135.5; 130.1;
126.5; 127.8; 127.5; 126.5; 125.9; 125.4; 125.3; 120.4; 116.9; 37.8 ppm.
IVνmax(cm−1; KBr pellets): 1673
Biological Test
The antimycobacterial N,N’-di(thiopheneacetyl)diamines derivatives
8–17 were assessed against M. tuberculosis ATTC 27294,5 using the
micro plate Alamar Blue assay (MABA)6 (Table I). This methodology
is nontoxic, uses thermally–stable reagent and shows good correla-
tion with proportional and BACTEC radiometric methods.7,8 Briefly,
two hundred microliters its of sterile deionized water was added to

2996
M. V. N. de Souza et al.
TABLE II Cytotoxic Effects of Test Compound on Murine
Macrophage Cells 18 h after the Treatment
% Cell viability
Doses tested (µg/mL)
Compound
0,1
1,0
10
100
90
13
100
100
100
all outer-perimeter wells of sterile 96 well plates (falcon, 3072: Becton
Dickinson, Lincoln Park NJ) to minimize evaporation of the medium
in the test wells during incubation. The 96 plates received 100µL of
the Middlebrook 7H9 broth (Difco laboratories, Detroit, MI, USA) and
a serial dilution of the compounds 9–16 was made directly on the plate.
The final drug concentrations tested were 0.01 to 10.0 µL /mL. Plates
were covered and sealed with parafilm and incubated at 37oC for five
days. After this time, 25 µl of a freshly prepared 1:1 mixture of Alamar
Blue (Accumed International, Westlake Ohio) reagent and 10% tween
80 was added to the plate and incubated for 24 h. A blue color in the
well was interpreted as no bacterial growth; and a pink color was scored
as growth. The MIC (Minimal Inhibition Concentration) was defined
as the lowest drug concentration, which prevented a color change from
blue to pink.
Cell Viability Assay
Cellular viability in the presence and absence of test compound
was determined by Mosmans’s MTT (3-(4,5-dimethylthylthiazol-2yl)-
2,5-dimethyl tetrazolium bromide; Merck) microcultured tetrazolium
assay.9 The cells were plated in flat bottom 96-well plates (2.5 × 106
cells/mL) cultured for 1 h in a controlled atmosphere (CO2 5% at 37oC),
and non-adherent cells were washed by gentle flushing with RPMI
1640. Adherent cells were cultured in the presence of medium alone,
tween 20 (3%) (live and dead controls, respectively) or different con-
centrations of compounds (0.1, 1.0, 10.0, and 100/µg/mL) in a triplicate
assay. After 18 h, stock MTT solution (5 mg/mL of saline; 20 µL/well)
was added to the culture and 4 h later, supernatant was discharged and
DMSO (100 µL/well) was added for formazan crystals solubilization,
and the absorbance was read at 540 nm in a plate reader (Biorad-450).

N,N’-di(thiopheneacetyl)diamines as Antitubercular Agents
2997
CONCLUSION
A series of eight new N,N’-di(thiopheneacetyl)diamines derivatives
8–17 were synthesized and evaluated for their in vitro antibacterial
activity against Mycobacterium tuberculosis. In this series, only the
compound 13 exhibited activity against M. tuberculosis infection, with
a MIC of 50 µg/mL. This result indicates that alkyl chain length is crit-
ical for biological activity. Although this observation was not studied
before, it could be a good starting point to find new lead compounds
in the fight against multi-drug resistant TB. More information about
structure-activity relationship of this compound and their in vivo an-
tibacterial activity test are in progress in our laboratory.
REFERENCES
[1] http://www.who.int/tdr/diseases/tb/default.htm
[2] http://www.webtb.org/
[3] A. Kleemann, J. Engel, B. Kutscher, and D. Reichert, Pharmaceutical Substances
Syntheses Patents Applications. (4th Thieme Stuttgart – New York, 1988).
[4] M. V. N. De Souza, Journal of Sulfur Chemistry, 26, 429–449 (2005).
[5] J. Canetti, E. Rist, and R. Grosset, Pneumology, 27, 217–272 (1963).
[6] S. G. Franzblau, R. S. Witzig, J. C. McLaughlin, P. Torres, G. Madico, A. Hernandez,
M. T. Degnan, M. B. Cook, V. K. Quenzer, R. M. Ferguson, and R. H. Gilman, Journal
of Clinical Microbiology, 36, 362–366 (1998).
[7] J. D. Vanitha, and C. N. Paramasivan, Mycobacteriology, 49, 179–182 (2004).
[8] R. S. Reis, I. Neves, Jr., S. L. S. Lourenc¸o, L. S.Fonseca, and M. C. S. Lourenc¸o,
Journal of Clinical Microbiology, 42, 2247–2248 (2004).
[9] M. C. Souza, A. C. Siani, M. F. S. Ramos, O. M. Limas, Jr., and M. G. M. O. Henrique
Pharmazie, 58 (8), 582–586 (2003).