Allylic thiocyanates as a new class of antitubercular agents

Article abstract of DOI:10.1016/j.bmcl.2012.08.048

TB is a global public health emergency in which new drugs are desperately needed. Herein we report on the synthesis of a diverse panel of 41 aryl allylic azides, thiocyanates, isothiouronium salts, and N,N′-diacetylisothioureas that were evaluated for their in vitro activity against replicating and non-replicating Mycobacterium tuberculosis (Mtb) H₃₇Rv and toxicity to VERO cells. We found a selective group of new and promising compounds having good (micromolar) to excellent (sub-micromolar) potency against replicating Mtb H₃₇Rv. Allylic thiocyanates bearing halophenyl (halo = 2-Br, 4-Br, 4-Cl, 4-F), 4-methylphenyl and 2-naphthyl moieties were the most active as antitubercular agents. In particular, the 2-bromophenyl-substituted thiocyanate showed MIC = 0.25 μM against replicating Mtb, MIC = 8.0 μM against non-replicating Mtb and IC₅₀ = 32 μM in the VERO cellular toxicity assay.

Full text of DOI:10.1016/j.bmcl.2012.08.048

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6486–6489
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Allylic thiocyanates as a new class of antitubercular agents
Gustavo P. Silveira a, Misael Ferreira b, Luciano Fernandes b, , Garrett C. Moraski c, Sanghyun Cho d,
Changhwa Hwang d, Scott G. Franzblau d, Marcus M. Sá b,
⇑
a Departamento de Química, Instituto de Química, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS 91501-970, Brazil
b Departamento de Química, Universidade Federal de Santa Catarina, Florianópolis, SC 88040-900, Brazil
c Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA
d Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, IL 60612, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 May 2012
Revised 31 July 2012
Accepted 13 August 2012
Available online 21 August 2012
TB is a global public health emergency in which new drugs are desperately needed. Herein we report on
the synthesis of a diverse panel of 41 aryl allylic azides, thiocyanates, isothiouronium salts, and N,N0-diac-
etylisothioureas that were evaluated for their in vitro activity against replicating and non-replicating
Mycobacterium tuberculosis (Mtb) H37Rv and toxicity to VERO cells. We found a selective group of new
and promising compounds having good (micromolar) to excellent (sub-micromolar) potency against rep-
licating Mtb H37Rv. Allylic thiocyanates bearing halophenyl (halo = 2-Br, 4-Br, 4-Cl, 4-F), 4-methylphenyl
and 2-naphthyl moieties were the most active as antitubercular agents. In particular, the 2-bromophenyl-
Keywords:
Anti-tuberculosis agents
Morita–Baylis–Hillman
Allylic azide
Allylic thiocyanate
Isothiouronium salt
substituted thiocyanate showed MIC = 0.25
lM against replicating Mtb, MIC = 8.0
lM against non-repli-
cating Mtb and IC50 = 32 M in the VERO cellular toxicity assay.
l
Ó 2012 Elsevier Ltd. All rights reserved.
Tuberculosis (TB) is caused by the bacterium Mycobacterium
tuberculosis (Mtb). This dreadful disease has been responsible for
the deaths of over one billion people and currently infects one-
third of the world’s population.1 In 1993, when an estimated 7–8
million cases and 1.3–1.6 million deaths were occurring, the World
Health Organization (WHO) declared TB a global public health
emergency. In 2010, there were still an estimated 8.5–9.2 million
cases and 1.2–1.5 million deaths (including deaths from TB among
HIV-positive people)1 indicating improvements have been made
but the battle is far from won. The spread of TB was temporarily
interrupted by DOTS (directly observed treatment, short course)
of isoniazid, rifampicin, ethambutol and pyrazinamide in combina-
tion.2 However, the increase of TB/HIV co-infection continues to
grow among people living with HIV, accounting for about 13% of
TB cases registered in 2010.1 Therefore, there is an urgent need
to develop new antitubercular drugs to combat the spread of this
virulent disease.3
thiocyanate functionality is sparsely documented.8,9 On the other
hand, isothiouronium salts have attracted the attention of the sci-
entific community due to their unique and wide spread chemical
and biological properties.10 They have been explored as agonists
of GABA-type receptors,11 as prodrugs of the aldehyde dehydroge-
nase inhibitor cyanamide,12 as local anesthetics,13 and as antibac-
terial agents.14 While the structurally related thiosemicarbazones
are known to possess pronounced antituberculosis activity associ-
ated with low cytotoxicity,15 much less attention has been devoted
to investigate the screening of isothiouronium salts and analogs
against Mtb.16
Herein we present our preliminary results involving the screen-
ing of aryl allylic azides 1, thiocyanates 2, isothiouronium salts 3
and 4, and N,N0-diacetylisothioureas 5 (Scheme 1) against Mtb
H37Rv for the development of possible new antitubercular agents.
The key allylic bromides 6 participate in halide displacement by
nucleophiles in aqueous medium to give different classes of substi-
tuted allylic derivatives in good to high yields according to previ-
ous reports5–7,17 (Scheme 1). Therefore, allylic bromides 6, which
Our laboratory has had
a long research interest in the
Morita–Baylis–Hillman (MBH) reaction,4–7 resulting in the prepa-
ration of various allylic azides, thiocyanates, isothiouronium salts,
and N,N’-diacetylisothioureas in aqueous medium as versatile scaf-
folds for synthesis (Scheme 1). The biological activity related to the
are readily available in multigram scale by treating a-methylene-
b-hydroxyesters (MBH adducts)18–20 with LiBr/H2SO4 in acetoni-
trile at room temperature,21 were the common precursors for
azides 1, thiocyanates 2, and isothiouronium salts 3 and 4 depend-
ing on the nucleophile employed.
⇑
Corresponding author. Tel.: +55 48 37216844; fax: +55 48 37216850.
E-mail address: marcus.sa@ufsc.br (M.M. Sá).
The synthesis of allylic azides 1 was performed by reacting bro-
mides 6 with sodium azide in aqueous acetone at room tempera-
ture for 10–15 min.7,17,22 Similarly, allylic thiocyanates 2 were
Present address: Coordenação de Engenharia Química, Universidade Tecnológica
Federal do Paraná, Ponta Grossa, PR 84016-210, Brazil.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.048

G. P. Silveira et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6486–6489
6487
O
NaG
1 G = N3 (t = 10 min)
2 G = SCN (t = 1 h)
R
OCH3
Acetone:H2O
25 oC
O
G
O
R
OCH3
S
7a,b
O
S
Br
6
R1HN NH2
Ac2O, CH2Cl2
R
R
OCH3
OCH3
O
Acetone:H2O
or CH3CN
NaOH, H2O
25 oC, 1-3 h
O
S
R = Alk, Ar
1 = H, Ph
25 oC, 1 h
R1 = H
R1HN
N
H
N
5
R
NH.HBr
3,4
Scheme 1. Synthetic routes for the preparation of allylic azides 1, thiocyanates 2, isothiouronium salts 3 and 4, and N,N0-diacetylisothioureas 5 from bromides 6.
obtained by mixing the bromide 6 with NaSCN in the same condi-
tions,23 but in these cases a longer period (1 h) was required for the
reaction to attain >95% conversion to the expected product.6 All
azides 1 and thiocyanates 2 were stable compounds readily iso-
lated in high yields after flash chromatography.
groups (2h,i), as well as most of the halo-substituted derivatives
(2j,m–o) showed very potent inhibition against Mtb in sub-micro-
molar MIC range (Table 1).
Table 1
Potency of allylic azides 1, thiocyanates 2, isothiouronium salts 3 and 4, and N,N0-
Isothiouronium salts 3 were also readily prepared from 6 by ha-
lide displacement in aqueous acetone, in this case employing thio-
urea (7a) as an ambident nucleophile5 to give salts 3 as crystalline
solids. The synthesis of N-phenyl-substituted isothiouronium salts
4 from bromides 6 and phenylthiourea (7b) was also achieved with
excellent chemoselectivity, providing that aqueous acetone is re-
placed by acetonitrile as the reaction solvent. Under these condi-
tions, S-alkylated salts 4 were formed as crystalline solids that
were readily collected by filtration without any further purifica-
tion. Considering that either the sulfur or one of the nitrogen atoms
in 7b is able to participate in a nucleophilic attack on one of the
several electrophilic sites available in the allylic bromides 6, the
selective formation of isothiouronium salt 4 as the sole product
in high yields is noteworthy.
diacetylisothioureas 5 against replicating Mtb (MIC in l
M) in GAS mediuma,b
O
R
OCH3
G
#
G
R
MIC (l
M)
1a
1b
1c
1d
1e
1f
1g
1h
1i
N3
N3
N3
N3
N3
N3
N3
N3
N3
N3
SCN
SCN
SCN
SCN
SCN
SCN
4-NO2C6H4
2-NO2C6H4
(E)-C6H5CH@CH
3,4-(OCH2O)C6H3
4-CH3OC6H4
C6H5
2-C10H7
4-ClC6H4
2-ClC6H4
2,4-Cl2C6H3
4-NO2C6H4
2-NO2C6H4
(E)-C6H5CH@CH
3,4-(OCH2O)C6H3
4-CH3OC6H4
3,4-(CH3O)2C6H3
C6H5
4.0
64
64
64
>128
>128
>128
>128
>128
>128
>128
Finally, the N,N’-diacetyl isothioureas 5 were obtained by acet-
ylation of the corresponding salts 3 in aqueous alkaline medium5
(Scheme 1) in up to 3 h to give the diacetylated products as crystal-
line solids after recrystallization.
1j
2a
2b
2c
2d
2e
2f
2g
2h
2i
8.0
MICs against replicating Mtb H37Rv were determined by the
microplate Alamar Blue assay (MABA).24 MICs against non-repli-
cating (NR) Mtb were determined using the low-oxygen-recovery
assay (LORA).25 Toxicity was measured against VERO cells26 and
reported as the half maximal inhibitory concentration (IC50). A to-
tal of 41 derivatives were synthesized for this study and evaluated
for their in vitro antitubercular activity and toxicity. There is about
a twofold error within any whole-cell screen and we take this into
consideration as we describe the SAR trends presented in Table 1.
We spent considerable synthetic effort to develop an understand-
ing of the structure–activity relationship (SAR) of the each scaffold
(and the analogs generated) within this whole-cell screening
paradigm.
>128
>128
16
16
1.0
SCN
SCN
SCN
SCN
2-C10H7
4-CH3C6H4
4-ClC6H4
0.25
0.50
0.25
2j
2k
2l
SCN
SCN
SCN
SCN
2-ClC6H4
2,4-Cl2C6H3
2-BrC6H4
4-BrC6H4
4-FC6H4
4-NO2C6H4
3,4-(OCH2O)C6H3
4-CH3OC6H4
C6H5
>128
>128
0.25
2m
2n
2o
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
4f
0.25
0.50
8.0
SCN
SC(NH)NH2ꢀHBr
SC(NH)NH2ꢀHBr
SC(NH)NH2ꢀHBr
SC(NH)NH2ꢀHBr
SC(NH)NH2ꢀHBr
>128
32
64
Most of the allylic azides
(MIC > 128 M). The only exception was the azide 1a bearing a
4-nitrophenyl group (MIC = 4.0 M). It is known that DprE1, a
1 were inactive against Mtb
2-ClC6H4
>128
l
SC(NC6H5)NH2ꢀHBr
SC(NC6H5)NH2ꢀHBr
SC(NC6H5)NH2ꢀHBr
SC(NC6H5)NH2ꢀHBr
SC(NC6H5)NH2ꢀHBr
SC(NC6H5)NH2ꢀHBr
SC(NC6H5)NH2ꢀHBr
SC(NC6H5)NH2ꢀHBr
SC(NC6H5)NH2ꢀHBr
SC(NCOCH3)NHCOCH3
SC(NCOCH3)NHCOCH3
4-NO2C6H4
3-NO2C6H4
2-NO2C6H4
3,4-(OCH2O)C6H3
4-CH3OC6H4
C6H5
4-CH3C6H4
4-ClC6H4
4-FC6H4
4.0
l
16
16
16
16
16
16
8.0
8.0
32
32
mycobacterial nitroreductase involved in the cell-wall biogenesis,
can be inhibited by some aromatic nitro compounds through a
FAD-dependent mechanism where the nitro group is reduced to
the corresponding nitroso function.27 This process might be related
to the moderate activity observed for the nitro-substituted allylic
azide 1a.
4g
4h
4i
5a
5b
However, allylic thiocyanates 2 exhibited an opposite trend in
comparison with azides 1. The 4-nitrophenyl thiocyanate 2a was
C6H5
2-ClC6H4
inactive (MIC > 128 lM) whilst its 2-substituted isomer 2b and ana-
logs bearing electron-releasing groups (2c–f) were also poorly ac-
tive. Gratifyingly, thiocyanates substituted with weakly-donating
a
Values reported are the average of three individual measurements and rounded
to nearest dilution.
b
GAS = glycerol-alanine-salts medium.

6488
G. P. Silveira et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6486–6489
Table 2
In the case of the 2-naphthyl-derived thiocyanate 2h (MIC =
0.25 M) and the 4-methylphenyl analog 2i (MIC = 0.50 M), their
slightly higher potency compared with that observed for the
unsubstituted phenyl thiocyanate 2g (MIC = 1.0 M) suggests the
Anti-TB activity and selectivity of the best leads
l
l
O
l
hypothesis that having a tethered hydrophobic group on the aro-
matic ring is beneficial to the activity. The excellent activity of both
2- and 4-bromo-substituted allylic thiocyanates 2m and 2n
R
OCH3
G
(MIC = 0.25 lM) corroborates the existence of favorable hydropho-
bic effects. Conversely, substitution with the more electronegative
chlorine at the 2-position (2k and 2l) led to a dramatic loss in the
#
G
R
MIC (
l
M) versus Mtb
IC50 (lM)
Replicatinga
NRb
VERO
1a
2h
2i
N3
4-NO2C6H4
2-C10H7
>32
32
8
8
8
16
16
64
>32
16
16
16
8
>64
32
32
16
32
32
32
NT
>64
activity against Mtb (MIC > 128 lM). However, the introduction of
a chlorine solely on the 4-position (as in the derivative 2j)
still maintained the inhibition at sub-micromolar figures (MIC =
SCN
SCN
SCN
SCN
SCN
SCN
IBc
4-CH3C6H4
4-ClC6H4
2-BrC6H4
4-BrC6H4
4-FC6H4
2j
0.25
l
M). The 4-fluoro substituted thiocyanate 2o also showed a
M), which is probably re-
2m
2n
2o
3a
4a
4
high level of Mtb inhibition (MIC = 0.50
l
16
16
NT
>64
lated to the stereoelectronic effects that are particularly prominent
in governing the behavior of fluorinated organic compounds.28
Because of the much more pronounced activity against Mtb
observed for the allylic thiocyanates 2 compared with azides 1,
we turned our attention on other allylic scaffolds that contain
variants of the S–C–N moiety. Therefore, representative isothiouro-
nium salts and acetylated derivatives were screened against
replicating Mtb (Table 1).
4-NO2C6H4
4-NO2C6H4
PIBd
a
Cultured in Middlebrook 7H12 medium.
NR = non-replicating Mtb using the Low oxygen recover assay (LORA).
VERO = African green monkey kidney cell line. NT = not tested. Values reported are
the average of three individual measurements and rounded to nearest dilution.
b
c
IB = isothiouronium bromide [SC(NH)NH2ꢀHBr].
d
PIB = N-phenylisothiouronium bromide [SC(NC6H5)NH2ꢀHBr].
In all but one case (3a, MIC = 8.0
salts were only modestly effective against Mtb (MIC’s of
32–128 M), while the N-phenyl-substituted isothiouronium salts
4a–j presented homogeneously good activity (MIC’s of 4.0–16 M)
lM), the allylic isothiouronium
3
In conclusion, our laboratories identified various small allylic
thiocyanates and isothiouronium salts that are active against
Mtb. We reported five new and promising compounds having good
(micromolar) to excellent (sub-micromolar) potency against repli-
cating Mtb H37Rv. Two of the most potent compounds (2j and 2m)
also displayed moderate activity against non-replicating (NR) Mtb
l
l
for the nine compounds tested. Nevertheless, none compared
favorably with the results observed for the allylic thiocyanates 2,
in particular, the isothiouronium salts 4 substituted with haloaryl
groups (F and Cl) were significantly less potent than the corre-
sponding thiocyanate analogs 2.
in the low oxygen recovery assay with MICs of 8.0 and 4.0 lM,
respectively. Additionally, evaluation in the VERO cellular toxicity
Acetylation of both amino groups of the allylic isothioureido
moiety of 3 did not bring any advantage in terms of improving
the activity against Mtb (Table 1, compounds 5a and 5b, MIC =
assay revealed a good selectivity index in some cases, particularly
with the two potent compounds 2j and 2m (IC50 of 16 and 32 lM,
respectively). We are currently synthesizing and screening other
halogenated thiocyanates as well as conformationally constrained
and unconstrained derivatives in order to gain more information
associated with this new class of antitubercular agents, and the re-
sults will be reported in due course.
32
lM).
Antibacterial agents are often prioritized on the basis of their
in vitro activity as well as other parameters. However, the apparent
inhibitory activity of new leads can be misleading because most
culture media do not reproduce an environment relevant to infec-
tion in vivo. Glycerol has been known to be the preferred source of
energy and carbon for M. tuberculosis under in vitro conditions. As
such, glycerol is present in most standard M. tuberculosis growth
media.26
To further validate the potency of the allylic derivatives studied,
the most potent compounds were additionally screened in a cul-
ture medium (7H12) which lacks glycerol.25 The 7H12 medium
contains palmitic acid as its carbon source while the GAS medium
consists of glycerol–alanine–salts. Selected results are displayed in
Table 2. It is noteworthy that compounds 2h–j and 2m–o all de-
creased in activity in the glycerol-free medium (MIC’s in the range
Acknowledgments
This research was supported in part by Grant 2R01AI054193
from the National Institutes of Health (NIH). The excellent techni-
cal assistance of Baojie Wan and Yuehong Wang with anti-TB as-
says at UIC is greatly appreciated. The authors wish to thank the
Central de Análises (Departamento de Química, UFSC) for spectro-
scopic analysis. G.P.S. thanks FAPERGS for supporting the short
training in microbiology assays, and to Jed Fisher for regular and
lasting scientific discussions. L.F. and M.F. are grateful to CNPq
and CAPES (Brazilian Research Councils) for fellowships. M.M.S. is
grateful to CNPq for a research fellowship. Financial support from
INCT-Catalysis/CNPq is also gratefully acknowledged.
of 8.0–32 lM). This is suggestive of possible carbon source depen-
dence29 or some other effect related to the differences in the
culture medium.
Of greater importance, however, are the good activities (MIC’s
from 4.0–16 lM) observed in the LORA assay, which uses the
Supplementary data
Supplementary data (copies of 1H and 13C NMR spectra for the
novel compounds) associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.bmcl.2012.08.048.
7H12 medium under low oxygen conditions (Table 2). The LORA
assay was specifically designed to simulate non-replicating M.
tuberculosis during persistence and/or latency. Given the limited
number of compounds that have shown micromolar potency in
this assay,30 the results achieved with the promising class of allylic
thiocyanates 2 are certainly encouraging.
References and notes
1. (a) Global tuberculosis control WHO report 2011. WHO/HTM/TB/2011.16.; (b)
Kwan, C. K.; Ernst, J. Clin. Microbiol. Rev. 2011, 24, 351.
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Tuberculosis: Pathogenesis; Protection, and Control; ASM Press: Washington, D.C.,
1994.
For the selected compounds tested, VERO screening indicated
low toxicity values with reasonable selectivity indices in most
cases,31 showing a VERO IC50/Mtb MIC ratio higher than 100 for
the more promising compounds 2h, 2m and 2n (Table 2).

G. P. Silveira et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6486–6489
6489
3. (a) Lamichhane, G. Trends Mol. Med. 2011, 17, 25; (b) Nathan, C. Cell Host
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Methyl (Z)-3-(4-chlorophenyl)-2-(thiocyanomethyl)-2-propenoate (2j). Yield
85%; white solid, mp 71.3–71.8 °C. IR (KBr):
m
max/cmꢁ1 3045, 2951, 2149,
1705, 1620, 1584, 1435, 1274, 1199. 1H NMR (400 MHz, CDCl3): d 3.89 (s, 3H),
4.09 (s, 2H), 7.37 (d, J = 8.5 Hz, 2H), 7.44 (d, J = 8.5 Hz, 2H), 7.94 (s, 1H). 13C
NMR (100 MHz, CDCl3): d 31.1, 52.8, 111.8, 126.4, 129.4, 130.6, 132.1, 136.1,
143.5, 166.2.
Methyl (Z)-3-(2-bromophenyl)-2-(thiocyanomethyl)-2-propenoate (2m). Yield
97%; white solid, mp 72.0–73.5 °C. IR (KBr): m
max/cmꢁ1 3060, 2952, 2153,
1717, 1635, 1436, 1289, 1088. 1H NMR (400 MHz, CDCl3): d 3.90 (s, 3H), 3.93 (s,
2H), 7.25–7.29 (m, 1H), 7.37–7.44 (m, 2H), 7.64 (d, J = 7.8 Hz, 1H), 7.96 (s, 1H).
13C NMR (100 MHz, CDCl3): d 31.2, 52.9, 112.0, 123.9, 127.8, 128.0, 129.9,
131.0, 133.2, 134.3, 143.7, 165.9.
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Methyl (Z)-3-(4-bromophenyl)-2-(thiocyanomethyl)-2-propenoate (2n). Yield
87%; white solid, mp 71.5–72.5 °C. IR (KBr):
1714, 1622, 1586, 1488, 1435, 1275, 1159. 1H NMR (400 MHz, CDCl3): d 3.89 (s,
3H), 4.07 (s, 2H), 7.29 (d, J = 8.4 Hz, 2H), 7.59 (d, J = 8.4 Hz, 2H), 7.91 (s, 1H). 13
m
max/cmꢁ1 3062, 2949, 2150,
C
11. Allan, R. D.; Dickenson, H. W.; Johnston, G. A. R.; Kazlauskas, R.; Mewett, K. N.
Neurochem. Int. 1997, 30, 583.
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143.6, 166.2.
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