Synthesis, characterization and in vitro anti-Trypanosoma cruzi and anti-Mycobacterium tuberculosis evaluations of cyrhetrenyl and ferrocenyl thiosemicarbazones

Article abstract of DOI:10.1016/j.jorganchem.2013.12.049

To study the electronic influence of the organometallic moieties in thiosemicarbazones, two short libraries of cyrhetrenyl thiosemicarbazone and ferrocenyl thiosemicarbazone hybrids were synthesized and tested for their antichagasic and antitubercular act

Full text of DOI:10.1016/j.jorganchem.2013.12.049

Journal of Organometallic Chemistry 755 (2014) 1e6
Contents lists available at ScienceDirect
Journal of Organometallic Chemistry
journal homepage: www.elsevier.com/locate/jorganchem
Synthesis, characterization and in vitro anti-Trypanosoma cruzi and
anti-Mycobacterium tuberculosis evaluations of cyrhetrenyl and
ferrocenyl thiosemicarbazones
,
a
,
b
b
c
Rodrigo Arancibia a , A. Hugo Klahn , Michel Lapier , Juan D. Maya , Andrés Ibañez ,
*
*
Maria Teresa Garland c, Séverine Carrère-Kremer d, Laurent Kremer d e, Christophe Biot f
,
a Instituto de Química, Pontificia Universidad Católica de Valparaíso, Casilla, 4059 Valparaíso, Chile
b Programa de Farmacología Clínica y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile
c Departamento de Física, Facultad de Ciencias Físicas y Matemáticas, Universidad de Chile, Santiago, Chile
d Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, UMR 5235 CNRS, Université Montpellier 2I, Place Eugène Bataillon,
34095 Montpellier Cedex 05, France
e INSERM, DIMNP, Place Eugène Bataillon, 34095 Montpellier Cedex 05, France
f Université Lille1, Unité de Glycobiologie Structurale et Fonctionnelle, CNRS UMR 8576, IFR 147, 59650 Villeneuve d’Ascq Cédex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
To study the electronic influence of the organometallic moieties in thiosemicarbazones, two short li-
braries of cyrhetrenyl thiosemicarbazone and ferrocenyl thiosemicarbazone hybrids were synthesized
and tested for their antichagasic and antitubercular activities. The unreported cyrhetrenyl thio-
Received 28 October 2013
Received in revised form
26 December 2013
semicarbazone derivatives of the form [(
h
5-C5H4)eC(R1) ¼ NNHC(S)NHR2]Re(CO)3 (R1 ¼ H, CH3; R2 ¼ H,
Accepted 27 December 2013
CH3, CH2CH3, C6H5) were prepared from cyrhetrenylcarbaldehyde (1a) or acetylcyrhetrene (1b) and the
corresponding thiosemicarbazide. The 1H and 13C NMR spectra indicate that these compounds have the
anti-(E) conformation in solution, and the X-ray crystal structure of formylcyrhetrene 4-methyl-thio-
semicarbazone (2b) confirms that this complex also adopts the anti-(E) form in the solid state. The new
cyrhetrenyl thiosemicarbazones and their ferrocene analogues were screened in vitro against Trypano-
soma cruzi and Mycobacterium tuberculosis. The anti-T. cruzi evaluation showed that the ferrocenyl de-
rivatives were more efficient trypanocidal agents compared to their cyrhetrenyl counterparts. The
incorporation of any organometallic fragment into thiosemicarbazone scaffold showed moderate anti-
tuberculosis activity against mc27000 strain.
Keywords:
Organometallic thiosemicarbazones
Cyrhetrene
Ferrocene
Trypanocidal
Anti-Mycobacterium tuberculosis activity
Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction
and their transition metal complexes as potential antichagasic
and antitubercular agents, with the goal of finding new and
Thiosemicarbazones (TSCs) are a class of small molecules that
have been extensively studied for several decades because they
allow a great number of substitution patterns and because of their
capability to act as a ligand for metal ions in several bonding modes
[1e4].
In addition, the pharmacological properties of TSCs and their
metal complexes have been of interest since 1946 [5]. At present,
there is a large amount of literature that is entirely dedicated to
their broad range of biological and therapeutic applications, such as
their antiviral [6], antibacterial [7], antifungal [8] and antitumoral
properties [9]. Recently, there has been greater interest in TSCs
more effective therapies to decrease toxic effects and the growing
incidence of drug resistance against clinically established drugs
[10e18].
Several organic TSCs as well as TSCemetal complexes have been
studied with regard to their trypanocidal activity toward the par-
asites Trypanosoma brucei and Trypanosoma cruzi [19]. Although the
mechanism of their activity remains unclear, the nonpeptidic na-
ture of these compounds, coupled with their low cost of synthesis,
makes this class of reversible covalent inhibitors very promising
candidates for the development of new antitrypanosomal chemo-
therapy [19e21]. For this reason, considerable attention has been
focused on aromatic and nitroheterocyclic thiosemicarbazones for
designing new anti-T. cruzi prodrugs [22,23]. Coordination com-
pounds based on TSC also appear to be a promising alternative in
the search for a pharmaceutical solution to Chagas disease [24e28].
* Corresponding authors. Tel.: þ56 32 2274922.
E-mail address: hklahn@ucv.cl (A.H. Klahn).
0022-328X/$ e see front matter Ó 2014 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jorganchem.2013.12.049

2
R. Arancibia et al. / Journal of Organometallic Chemistry 755 (2014) 1e6
Recently, Gambino and co-workers reported the synthesis and
trypanocidal evaluation of the first organoruthenium compounds
containing coordinated TSCs [28b,c]. To the best of our knowledge,
organometallic TSCs have not been explored previously with regard
to their antitrypanosomal activity.
2.2.1. Formylcyrhetrene thiosemicarbazone (2a)
White solid, yield: 90% (54 mg, 0.12 mmol). IR (CH2Cl2, cmꢀ1):
2027 (s) (
3120 (m) (
837 (m) (
C]S). 1H NMR (CDCl3):
n
CO), 1935 (s) (
NH), 2025 (s) (
n
CO), 1605 (w) (
n
C]N). IR (KBr, cmꢀ1):
n
n
CO), 1915 (s) (nCO), 1582 (w) (nC]N),
n
d
5.38 (t, 2H, J ¼ 2.2 Hz, C5H4);
Among a wide spectrum of bioactivities already mentioned,
TSCs and related analogues have also been extensively used against
Mycobacterium tuberculosis (MTB), the pathogenic agent of tuber-
culosis (TB) [29]. Since Domagk’s first report on the antituberculosis
activity of TSCs, a large number of organic compounds that contain
thiosemicarbazones have been reported and evaluated against
MTB, both in vitro and in vivo [30]. One of them, p-acet-
amidobenzaldehyde thiosemicarbazone, is currently being used for
the treatment of TB in Africa and South America and is commer-
cially available as thiacetazone. The rise of multidrug resistance in
MTB has complicated and prolonged treatment, and for that reason,
new strategies have emerged to develop therapeutics for TB, which
can reduce the duration of treatment and provide a more effective
therapy against active and latent TB. To this end, ferrocenyl thio-
semicarbazones and their metal complexes have been demon-
strated to have promising properties as anti-TB agents [31e33].
Taking into account the promising reports that involve organ-
ometallic groups being incorporated into various trypanocidal and
antitubercular agents, we present a study on the synthesis and
characterization of new cyrhetrenyl thiosemicarbazones (2aeh).
We also include in this report the anti-T. cruzi and anti-
M. tuberculosis evaluation of the new compounds and their ferro-
cene analogues (2iep).
5.80 (t, 2H, J ¼ 2.2 Hz, C5H4); 6.40 (bs, 1H, NH2); 7.03 (bs, 1H,
NH2); 7.58 (s, 1H, CH]N); 9.76 (s, 1H, NH). 13C NMR (CDCl3):
d
84.5 (C5H4); 84.6 (C5H4); 97.1 (C5H4ipso); 134.3 (CH]N); 178.2
(C]S); 192.7 (ReeCO). Mass spectrum m/z: 438 [Mþ]; 409
[Mþ ꢀ CO]; 381 [Mþ ꢀ 2CO]; 353 [Mþ ꢀ 3CO]. Anal. (%) Calc. for
C
10H8N3O3SRe: C, 27.52; H, 1.85 and N, 9.63; found: C, 27.59; H,
1.86 and N, 9.62.
2.2.2. Formylcyrhetrene 4-methyl-thiosemicarbazone (2b)
Yellow crystal, yield: 90% (56 mg, 0.12 mmol). IR (CH2Cl2,
cmꢀ1): 2027 (s) (
n
CO), 1933 (s) (
cmꢀ1): 3128 (m) (
NH), 2025 (s) ( CO), 1581 (w)
C]N), 828 (m) ( d 3.23 (d, 3H,
C]S). 1H NMR (CDCl3):
n
CO), 1604 (w) (
nC]N). IR (KBr,
n
n
n
CO), 1913 (s) (
(
n
n
J ¼ 4.9 Hz, CH3); 5.39 (t, 2H, J ¼ 2.0 Hz, C5H4); 5.78 (t, 2H,
J ¼ 2.0 Hz, C5H4); 7.51 (s, 1H, CH]N); 9.29 (bs, 1H, NHCH3). 13C
NMR (CDCl3):
d 31.4 (CH3); 84.4 (C5H4); 84.5 (C5H4); 97.1
(C5H4ipso); 134.5 (CH]N); 178.1 (C]S); 192.5 (ReeCO). Mass
spectrum m/z: 451 [Mþ]; 423 [Mþ ꢀ CO]; 367 [Mþ ꢀ 3CO]. Anal.
(%) Calc. for C11H10N3O3SRe: C, 29.33; H, 2.24 and N, 9.33; found:
C, 29.31; H, 2.25 and N, 9.32.
2.2.3. Formylcyrhetrene 4-ethyl-thiosemicarbazone (2c)
Yellow solid, yield: 85% (54 mg, 0.11 mmol). IR (CH2Cl2, cmꢀ1):
2027 (s) (
3130 (m) (
828 (m) (
C]S). 1H NMR (CDCl3):
n
CO), 1933 (s) (
NH), 2025 (s) (
n
CO), 1604 (w) (
n
C]N). IR (KBr, cmꢀ1):
2. Experimental
n
nCO), 1913 (s) (nCO), 1590 (w) (nC]N),
n
d
1.29 (t, 3H, J ¼ 4.2 Hz, CH3); 3.71
2.1. Materials
(m, 2H, CH2); 5.38 (t, 2H, J ¼ 2.0 Hz, C5H4); 5.80 (t, 2H, J ¼ 2.0 Hz,
C5H4); 7.20 (pst, 1H, NHC2H5); 7.54 (s, 1H, CH]N); 9.74 (s, 1H, NH).
All manipulations were conducted under an N2 atmosphere
13C NMR (CDCl3):
d 14.3 (CH3); 39.4 (CH2); 84.4 (C5H4); 84.7 (C5H4);
using Schlenk techniques. The complexes (
h
5-C5H4CHO)Re(CO)3
97.2 (C5H4ipso); 134.7 (CH]N); 176.8 (C]S); 192.7 (ReeCO). Mass
spectrum m/z: 465 [Mþ]; 437 [Mþ ꢀ CO]; 381 [Mþ ꢀ 3CO]. Anal. (%)
Calc. for C12H12N3O3SRe: C, 31.03; H, 2.60 and N, 9.05; found: C,
31.10; H, 2.59 and N, 9.07.
(1a) [34], (
h
5-C5H4COCH3)Re(CO)3 (1b) [35] and the ferrocenyl
thiosemicarbazones (Fc-TSCs) (2iep) [36] were prepared according
to published procedures. Ferrocenecarboxaldehyde (98%), ace-
tylferrocene (95%), thiosemicarbazide (98%), 4-methyl-thio-
semicarbazide (98%), 4-ethyl-thiosemicarbazide (98%), and 4-
phenyl-thiosemicarbazide (98%) were obtained from Aldrich. Sol-
vents such as dichloromethane (CH2Cl2), hexane, acetone, ethanol
(EtOH), dimethyl sulfoxide (DMSO) and tetrahydrofuran (THF) were
obtained commercially and purified using standard methods.
Infrared spectra were recorded in solution (CH2Cl2) or solid state
(KBr disc) on a PerkineElmer FT-1605 spectrophotometer. 1H and
13C NMR spectra were measured on a Bruker AVANCE 400 spec-
trometer. 1H NMR chemical shifts were referenced using the
chemical shifts of residual solvent resonances, and 13C chemical
shifts were referenced to solvent peaks. Elemental analyses were
measured on a PerkineElmer CHN Analyzer 2400. Mass spectra
were obtained at the Laboratorio de Servicios Analíticos, Uni-
versidad Católica de Valparaíso, and masses are quoted in reference
to 187Re.
2.2.4. Formylcyrhetrene 4-phenyl-thiosemicarbazone (2d)
Yellow solid, yield: 85% (60 mg, 0.11 mmol). IR (CH2Cl2, cmꢀ1):
2027 (s) (
3126 (m) (
830 (m) (
C]S). 1H NMR (CDCl3):
n
CO), 1932 (s) (
NH), 2025 (s) (
n
CO), 1594 (w) (
n
C]N). IR (KBr, cmꢀ1):
n
n
CO), 1912 (s) (nCO), 1587 (w) (nC]N),
n
d
5.38 (t, 2H, J ¼ 2.2 Hz, C5H4);
5.83 (t, 2H, J ¼ 2.2 Hz, C5H4); 7.29 (m, 1H, C6H5); 7.41 (t, 2H,
J ¼ 7.8 Hz, C6H5); 7.58 (d, 2H, J ¼ 7.8 Hz, C6H5); 7.67 (s, 1H, CH]N);
8.93 (s, 1H, NHC6H5); 10.55 (s, 1H, NH). 13C NMR (CDCl3):
d 84.5
(C5H4); 85.1 (C5H4); 96.5 (C5H4ipso); 124.9 (C6H5); 126.6 (C6H5);
128.9 (C6H5); 135.8 (CH]N); 137.5 (C6H5); 175.8 (C]S); 192.9 (Ree
CO). Mass spectrum m/z: 513 [Mþ]; 485 [Mþ
ꢀ
CO]; 429
[Mþ ꢀ 3CO]. Anal. (%) Calc. for C16H12N3O3SRe: C, 37.49; H, 2.36 and
N, 8.20; found: C, 37.48; H, 2.36 and N, 8.22.
2.2.5. Acetylcyrhetrene thiosemicarbazone (2e)
2.2. Synthesis of cyrhetrenyl thiosemicarbazones. General
procedure
White solid, yield: 50% (30 mg, 0.1 mmol). IR (CH2Cl2, cmꢀ1):
2025 (s) (
3151 (m) (
(m) (
C]S). 1H NMR (CDCl3):
J ¼ 2.2 Hz, C5H4); 5.76 (t, 2H, J ¼ 2.2 Hz, C5H4); 6.40 (bs, 1H, NH2);
7.03 (bs, 1H, NH2); 8.75 (s, 1H, NH). 13C NMR (CDCl3):
30,8 (CH3);
n
CO), 1933 (s) (
n
CO), 1606 (w) (
CO), 1913 (s) ( CO),1590 (w) (
2.03 (s, 3H, CH3); 5.37 (t, 2H,
n
C]N). IR (KBr, cmꢀ1):
n
NH), 2023 (s) (
n
n
nC]N), 819
The cyrhetrenyleTSCs were prepared following the same pro-
cedure as for their ferrocenyl analogues [36]. Equimolar amounts of
1a or 1b and the corresponding thiosemicarbazide were dissolved
in anhydrous ethanol (25 mL) and refluxed for 24 h, under nitrogen
atmosphere. After this time, the solvent was removed under vac-
uum and the solid obtained was purified by crystallization from
CH2Cl2/hexane (1:5) at ꢀ18 ꢁC.
n
d
d
84.3 (C5H4); 85.7 (C5H4); 96.9 (C5H4ipso); 138.3 (C]N); 178.5 (C]S);
192.9 (ReeCO). Mass spectrum m/z: 451 [Mþ]; 423 [Mþ ꢀ CO]; 367
[Mþ ꢀ 3CO]. Anal. (%) Calc. for C11H10N3O3SRe: C, 29.33; H, 2.24 and
N, 9.33; found: C, 29.31; H, 2.25 and N, 9.32.

R. Arancibia et al. / Journal of Organometallic Chemistry 755 (2014) 1e6
3
2.2.6. Acetylcyrhetrene 4-methyl-thiosemicarbazone (2f)
37 ꢁC in Sauton’s medium, supplemented with 20
m
g mlꢀ1 of
Yellow solid, yield: 60% (37 mg, 0.1 mmol). IR (CH2Cl2, cmꢀ1):
pantothenic acid. The susceptibility of M. tuberculosis mc27000 to
the various compounds was determined as reported previously
[39]. In brief, the Middlebrook 7H10 solid medium that contains
2025 (s) (
3145 (m) (
(m) (
C]S). 1H NMR (CDCl3):
n
CO), 1931 (s) (
n
CO), 1604 (w) (
CO),1911 (s) ( CO),1583 (w) (
2.02 (s, 3H, CH3); 3.23 (d, 3H,
n
C]N). IR (KBr, cmꢀ1):
n
NH), 2023 (s) (
n
n
nC]N), 820
n
d
oleic-albumin-dextrose-catalase
enrichment
(OADC)
and
J ¼ 4.9 Hz, CH3); 5.37 (t, 2H, J ¼ 2.0 Hz, C5H4); 5.77 (t, 2H, J ¼ 2.0 Hz,
20
m
g mlꢀ1 of pantothenic acid was supplemented with increasing
C5H4); 7.40 (pst, 1H, NHCH3); 8.59 (s, 1H, NH). 13C NMR (CDCl3):
concentrations of the chemical analogues. Serial 10-fold dilutions
of each actively growing culture were plated and incubated at 37 ꢁC
for 2e3 weeks. The Minimum Inhibitory Concentration (MIC) was
defined as the minimum concentration required to inhibit 99% of
the growth.
d
14.3 (CH3); 31.4 (CH3); 84.5 (C5H4); 85.6 (C5H4); 97.1 (C5H4ipso);
138.6 (C]N); 178.6 (C]S); 193.0 (ReeCO). Mass spectrum m/z: 465
[Mþ]; 437 [Mþ ꢀ CO]; 381 [Mþ ꢀ 3CO]. Anal. (%) Calc. for
C
12H12N3O3SRe: C, 31.03; H, 2.60 and N, 9.05; found: C, 31.04; H,
2.62 and N, 9.07.
2.5. X-ray crystal structure determinations
2.2.7. Acetylcyrhetrene 4-ethyl-thiosemicarbazone (2g)
Yellow pale solid, yield: 70% (44 mg, 0.1 mmol). IR (CH2Cl2,
A suitable X-ray single crystal of the compound 2b was obtained
as described above and was mounted on top of glass fibres in a
random orientation. Crystal data, data collection, and refinement
parameters are given in the Supplementary material. Compound 2b
was studied at 150(2) K, on a Bruker Smart Apex diffractometer
cmꢀ1): 2025 (s) (
n
CO), 1931 (s) (
cmꢀ1): 3144 (m) (
NH), 2023 (s) (
C]N), 822 (m) ( d
C]S). 1H NMR (CDCl3):
n
CO), 1606 (w) (
n
C]N). IR (KBr,
CO), 1583 (w)
1.29 (t, 3H, J ¼ 4.2 Hz,
n
n
CO), 1911 (s) (n
(n
n
CH3); 2.01 (s, 3H, CH3); 3.73 (m, 2H, CH2); 5.38 (t, 2H, J ¼ 2.0 Hz,
C5H4); 5.76 (t, 2H, J ¼ 2.0 Hz, C5H4); 7.33 (pst, 1H, NHC2H5); 8.48 (s,
equipped with a bidimensional CCD detector, using graphite-
1H, NH). 13C NMR (CDCl3):
d
14.1 (CH3); 31.3 (CH3); 39.4 (CH2); 84.4
monochromated Mo-K
a
radiation (l ¼ 0.71073 A). The diffraction
ꢀ
(C5H4); 84.7 (C5H4); 97.1 (C5H4ipso); 138.7 (C]N); 178.7 (C]S);
193.1 (ReeCO). Mass spectrum m/z: 479 [Mþ]; 451 [Mþ ꢀ CO]; 395
[Mþ ꢀ 3CO]. Anal. (%) Calc. for C13H14N3O3SRe: C, 32.63; H, 2.95 and
N, 8.78; found: C, 32.62; H, 2.96 and N, 8.80.
frames were integrated using the SAINT package [40] and were
corrected for absorption with SADABS [41]. The structures were
solved using XS in SHELXTL-PC [42] by the Patterson method and
completed (non-H atoms) by use of difference Fourier techniques.
The complete structure was then refined by the full matrix least-
squares procedures on the reflection intensities (F2) [43]. All non-
hydrogen atoms were refined with anisotropic displacement co-
efficients, and all hydrogen atoms were placed in idealized
locations.
2.2.8. Acetylcyrhetrene 4-phenyl-thiosemicarbazone (2h)
Brown solid, yield: 60% (42 mg, 0.1 mmol). IR (CH2Cl2, cmꢀ1):
2025 (s) (
3133 (m) (
(m) (
C]S). 1H NMR (CDCl3):
n
CO), 1930 (s) (
n
CO), 1605 (w) (
CO),1910 (s) ( CO),1580 (w) (
2.03 (s, 3H, CH3); 5.38 (t, 2H,
n
C]N). IR (KBr, cmꢀ1):
n
NH), 2023 (s) (
n
n
nC]N), 827
n
d
J ¼ 2.2 Hz, C5H4); 5.83 (t, 2H, J ¼ 2.2 Hz, C5H4); 7.29 (m, 1H, C6H5);
3. Results and discussion
7.41 (t, 2H, J ¼ 7.8 Hz, C6H5); 7.58 (d, 2H, J ¼ 7.8 Hz, C6H5); 8.73 (s,
1H, NHC6H5); 9.11 (s, 1H, NH). 13C NMR (CDCl3):
d
84.4 (C5H4); 85.5
3.1. Design and synthesis
(C5H4); 97.0 (C5H4ipso); 124.9 (C6H5); 126.6 (C6H5); 128.9 (C6H5);
138.5 (CH]N); 137.5 (C6H5); 178.6 (C]S); 193.0 (ReeCO). Mass
spectrum m/z: 527 [Mþ]; 499 [Mþ ꢀ CO]; 443 [Mþ ꢀ 3CO]. Anal. (%)
Calc. for C17H14N3O3SRe: C, 38.77; H, 2.68 and N, 7.98; found: C,
38.80; H, 2.69 and N, 7.99.
In view of the large amount of literature that addresses organic-
TSCs and their applications as potential antitrypanosomal [24e28]
and antituberculosis [29] agents, it is surprising that organome-
tallic analogues with these biological targets have not been
extensively studied. Recently, Chibale et al. reported the incorpo-
ration of ferrocenyl TSCs into the structure of metallodendrimers
[32] and gold (III) complexes [31] and their biological evaluations
against malaria and TB.
To continue our studies on the electronic influence of the
organometallic moiety on bioconjugates [44e47], two short li-
braries of unreported cyrhetrenyl-thiosemicarbazones and their
previously reported ferrocenyl-thiosemicarbazone hybrids were
synthesized.
2.3. In vitro antitrypanosomal activity
Trypanocidal activity of the organometallic TSC derivatives was
evaluated against the T. cruzi epimastigote stages (Dm28c strain).
The compounds were dissolved in DMSO and were added to
3 ꢂ 106 epimastigotes/mL suspensions. Epimastigotes were incu-
bated (28 ꢁC, 24 h) in Diamond’s monophasic medium, supple-
mented with 4 mM hemin and 4% inactivated bovine calf serum.
Afterwards, trypanocidal activity was measured through the MTT
assay as described elsewhere [37]. Briefly, MTT was added at a final
concentration of 0.5 mg/mL and incubated at 37 ꢁC for 4 h. Parasites
were solubilized with 10% sodium dodecyl sulphate and 0.1 mM
HCl and incubated overnight. Formazan formation was measured at
570 nm in a multiwell reader (Asys Expert PlusÓ, Austria). The final
concentration of DMSO was less than 0.1% v/v. The trypanocidal
Nifurtimox was added as a drug control. We determined the IC50
value of viable parasites (IC50 is the drug concentration needed to
reduce by 50% the parasite viability) at 24 h after compound
addition by non-lineal regression analysis from the log of the
concentration vs the percentage of viable cells curve.
The cyrhetrenyl TSCs were prepared following the same pro-
cedure that was described for their ferrocenic analogues [36], that
is, the reaction of cyrhetrenecarboxaldehyde (1a) or acetylcyrhe-
trene (1b) with the corresponding thiosemicarbazide in anhydrous
EtOH (Scheme 1). In all cases, the products were isolated as solids
after crystallization with the CH2Cl2ehexane mixture. These
products are air-stable and are soluble in most organic solvents.
The IR spectra of all compounds showed the expected absorp-
tion band for the nC]N, nC]S and nN-H stretchs in the range of
1594e1606 cmꢀ1, 819-837 cmꢀ1 and 3120-3151 cmꢀ1, respectively,
in a CH2Cl2 solution or in solid state (KBr). Similar frequencies
values have been reported for thiosemicarbazones derived from
ferrocene [48,49]. In addition, the spectra show that the nCO bands
2.4. In vitro anti-tubercular activity
of the carbonyls bound to rhenium are shifted to lower wave-
number than the bands for their cyrhetrene precursors [50]. The
stoichiometry of the complexes was established by elemental
analysis and mass spectrometry. The mass spectra for compounds
Bacterial strains and growth conditions: M. tuberculosis
mc27000, an unmarked version [38] of mc26030, was grown at

4
R. Arancibia et al. / Journal of Organometallic Chemistry 755 (2014) 1e6
Scheme 1. Synthesis of cyrhetrenyl and ferrocenyl thiosemicarbazones.
2aeh all showed a strong molecular ion and fragments that
correspond to the successive loss of three CO groups.
3.2. X-ray crystallography
For all complexes, the 1H NMR spectra showed the presence of a
single compound. In the case of 2aed, a sharp singlet was observed
near 7.5e7.7 ppm and was assigned to the iminic proton. In addi-
tion, a signal due to the methyl protons of the eC(CH3)]N-frag-
ment was also observed at approximately 2.0 ppm for compounds
2eeh. These results are in agreement with the values reported for
ferrocenyl TSCs [48,49]. For all complexes, proton NMR spectra
showed two triplets in the region of 5.3e5.8 ppm, which are
ascribed to the two types of protons of the cyrhetrenyl moiety
[50,51]. It is important to note that the TSCs are capable of exhib-
iting the thione or the thiol tautomeric forms [3]. However, the 1H
NMR spectra of all cyrhetrenyl TSCs exhibit the eNHe resonance as
a broad singlet in the range of 8.5e10.5 ppm, indicating that they
remain solely as the thione form in solution, just like their ferro-
cene counterparts do [52].
With the aim of comparing the structural parameters of
cyrhetrenyl TSCs with the crystallographic data reported for their
ferrocenic analogues, we undertook a crystallographic study of 2b.
Fig. 1 shows an ORTEP representation of 2b and the most relevant
bond lengths and angles. The structure confirms the E configuration
tentatively assigned by NMR.
The crystallographic data obtained for 2b did not show any
remarkable differences from the structures previously reported for
the ferrocenyl analogues [57e59]. However, the internal CeC bond
distances of the cyclopentadienyl ring are slightly different than
those measured by Fun for 2k [58], so some degree of delocalization
13C NMR data were also in accordance with the existence of a
single compound. The most important feature of these spectra is
the presence of a low field resonance (134e138 ppm), which was
assigned to the iminyl carbon [C]N]. The carbon chemical shift of
this group, compared with that of their ferrocene analogues (144e
150 ppm), shows a clear dependence on the presence of an
organometallic fragment. The upfield shift observed for the
cyrhetrenyl TSC vs ferrocenyl TSC (Dd ¼ 10.0 ppm) can be related to
the opposite electronic effects of these organometallic fragments
[53,54]. We previously reported similar results for Schiff bases and
chalcones that contain ferrocenyl and cyrhetrenyl moieties [44,45].
Despite the fact that these types of compounds can adopt two
different forms (E- or Z-) [55,56], their 1H and 13C NMR spectra,
which agreed with those previously reported for the related fer-
rocenyl thiosemicarbazones, revealed that only one isomer (the E-
form) was present in solution. Further proof was provided by the X-
ray crystal structure determination of 2b (see below).
Fig. 1. ORTEP representation of the asymmetrical unit of 2b. Relevant bond lengths
ꢀ
(A): C(4)eC(9) 1.455(8); C(9)eN(1) 1.287(7); N(1)eN(2) 1.391(6); N(2)eC(10) 1.377(7);
C(10)eSC(1) 1.681(6); Cp(centroid)eRe(1) 2.297(6) and angles (ꢁ): C(4)eC(9)eN(1)
119.5(6); C(9)eN(1)eN(2) 114.6(5); N(1)eN(2)eC(10) 117.3(5); N(2)eC(10)eS(1)
118.8(5).

R. Arancibia et al. / Journal of Organometallic Chemistry 755 (2014) 1e6
5
Table 1
ferrocenic TSCs, these complexes adopt an anti configuration for the
iminyl moiety and a thione tautomeric form, both in solution and in
the solid state. The electron-donating (ferrocenyl) and electron-
withdrawing (cyrhetrenyl) capability of the organometallic frag-
ment on the imine moiety was correlated properly with the 13C
shift of the carbon nuclei of the C]N group. The results of an
in vitro antitrypanosomal assay of the compounds against T. cruzi
(Dm28c strain) indicate that the ferrocenyl TSCs were more active
than their cyrhetrene analogues, most likely due to the redox
properties of the ferrocenic entity. Within the ferrocenyl series, the
replacement of a hydrogen atom by a methyl group on the imine
carbon produced a two-fold decrease in the trypanocidal activity.
The incorporation of any organometallic fragment into thio-
semicarbazone scaffold showed moderate antituberculosis activity
against mc27000 strain.
In vitro anti-T. cruzi and antitubercular activity against the T. cruzi and M. tuberculosis
strains.
Compound
T. cruzi (IC50
/
mM)
M. tuberculosis (MIC/mM)
Dm28c strain
mc27000 strain
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
>100
>100
>100
>100
>100
>100
>100
>100
17.9 ꢃ 4.0
15.8 ꢃ 1.1
9.1 ꢃ 1.2
20.8 ꢃ 1.6
30.8 ꢃ 2.6
29.4 ꢃ 1.1
27.5 ꢃ 1.6
32.7 ꢃ 2.1
17.4 ꢃ 5.1
e
45e115
111
>107
>98
e
e
>105
95
174
66e166
63e159
>138
166
>159
>152
>133
e
Acknowledgements
2p
A.H.K. acknowledges FONDECYT-Chile (Project 1110669) and D.I.
Pontificia Universidad Católica de Valparaíso. C.B. thanks
FONDECYT-Chile the financial support for a research stay in Val-
paraiso. R.A. acknowledges MECESUP and CONICYT for a Doctoral
scholarship and D.I.-PUCV and FONDECYT-Chile (Project 3120091)
for a postdoctoral position.
Nfx
Rifampicin
0.06
of electron density between the CH]N group and the cyrhetrenyl
fragment can be considered. On the other hand, within the cyrhe-
trenyl group, the average ReeC(O) distance and the ReeCeO angle
are concordant with the distances and angles reported for the related
tricarbonyl cyclopentadienyl rhenium (I) complexes [51]. In addition,
the bond distances of the N(1)eN(2)eC(10)eS(1) fragment are
slightly shorter than those reported for the ferrocenyl analogue [59]
and may suggest some contribution of the thione form.
Appendix A. Supplementary material
CCDC 963290 contains the supplementary crystallographic data
for this paper. These data can be obtained free of charge from The
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif.
3.3. Biological evaluations
Appendix B. Supplementary data
3.3.1. In vitro anti-T. cruzi activity
The anti-T. cruzi activities of the cyrhetrenyl TSCs (2aeh) and the
ferrocenyl TSCs (2iep) are shown in Table 1, along with IC50 values
for the standard trypanocidal drug Nifurtimox (Nfx). Ferrocenyl
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.jorganchem.2013.12.049.
TSCs (IC50 ¼ 9.1e31.0
m
M) were more active than their analogues
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