Antimycobacterial in vitro activity of cobalt(II) isonicotinoylhydrazone complexes. Part 10

Article abstract of DOI:10.1016/S0960-894X(00)00648-X

Octahedral cobalt(II) complexes of isonicotinoylhydrazones, which were obtained from the primary antituberculous agent isoniazid, have been synthesised and characterised. Their antimycobacterial in vitro activity has been evaluated against Mycobacterium t

Full text of DOI:10.1016/S0960-894X(00)00648-X

Bioorganic & Medicinal Chemistry Letters 11 (2001) 301±303
Antimycobacterial In Vitro Activity of Cobalt(II)
Isonicotinoylhydrazone Complexes. Part 10
B. Bottari, R. Maccari, F. Monforte, R. Ottana
Á
,a E. Rotondob
a
a
a
a,
and M. G. Vigorita *
aDipartimento Farmaco-chimico, FacoltaÁ di Farmacia, UniversitaÁ di Messina, Vl. SS. Annunziata, 98168 Messina, Italy
bDipartimento Ch. Inorg., Anal. e Ch.-®s, FacoltaÁ di Scienze MMFFNN, UniversitaÁ di Messina, Salita Sperone 31,
9
8166 Messina, Italy
Received 24 July 2000; revised 13 October 2000; accepted 9 November 2000
AbstractÐOctahedral cobalt(II) complexes of isonicotinoylhydrazones, which were obtained from the primary antituberculous
agent isoniazid, have been synthesised and characterised. Their antimycobacterial in vitro activity has been evaluated against
Mycobacterium tuberculosis H37Rv: they exhibit MIC values ranging from <0.1 to 0.39 mg/mL, showing them to be generally more
active than previously reported analogous Cu(II) and Ni(II) complexes. # 2001 Elsevier Science Ltd. All rights reserved.
Tuberculosis (TB) is presently considered as the most
dangerous infective disease world-wide and one of the
major AIDS-associated infections. According to alarming
data furnished by the World Health Organization
ability to cross the mycobacterial cell wall. In fact, the
polar donor atoms of ISNE, as enolate, co-ordinate the
metal ion while the hydrophobic moieties form an outer
lipophilic envelope that should facilitate the di€usion
through biomembranes. Consequently, these chelates
could be considered as repositories for the active ligands
and potentially long-acting antituberculous agents.
(
WHO), more than a third of the entire world popula-
tion is a‚icted with TB and it is believed it could a€ect
00 million people within the next 10 years. The simulta-
3
neous presence of HIV infection, the spreading of drug-
resistant strains of Mycobacterium tuberculosis and the
scarce compliance with the lengthy complex therapies
often complicate the treatment of TB.1 Therefore, the
search for new antituberculous agents is required, in
spite of the availability of e€ective drugs such as iso-
niazid (INH) and rifampin (RMP).
In fact, a renewed interest in metal-based chemotherapies
has stemmed from the possibility of designing slow release
,2
4
drugs. In addition, co-ordination to a metal ion has
often been reported to enhance the antiinfective and
antiproliferative properties of several ligands.5
�10
All these considerations prompted us to extend our
research to analogous cobalt(II) complexes 2a±g (Scheme
1), obtained by the reaction of ISNEs 1 with Co(II)
nitrate, in molar ratio 2:1, at 50 C in methanol solution.
The brown microcrystalline powders were separated
Recently we have reported copper(II) and nickel(II) chel-
ates (MeL (H O) ) of isonicotinoylhydrazones (ISNE=
2
2
2
ꢀ
L), which were obtained from INH and ¯uorinated
carbonyl compounds. Most of them display signi®cant
antimycobacterial in vitro activity and low cytotoxi-
from the stirred solutions by dripping 0.1 N CH ONa.
3
3
city. The ligands are e€ective against M. tuberculosis
After 2 h they were ®ltered at room temperature, washed
with methanol and dried in vacuo to constant weight.
Complexes 2 are scarcely soluble in methanol, ethanol and
water and moderately soluble in DMSO and DMF. Their
structures were characterised by means of elemental
analyses, IR and UV spectroscopies and magnetic mea-
surements.
H37Rv and also, to some extent, against M. avium com-
plex, the latter being unsusceptible to parent-drug INH.3
We have suggested that the antituberculous activity of
these complexes should be ultimately due to the slow
release of the active ligands, while the metal ion should
play a role mainly connected with the enhanced cap-
The formation of neutral 1:2 complexes was monitored
by means of the electronic spectra of the reagents,
according to Job's method of continuous variation.
*
3
Corresponding author. Tel.: +39-090-6766469; fax: +39-090-
55613; e-mail: vigorita@pharma.unime.it
1
1,12
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00648-X

3
02
B. Bottari et al. / Bioorg. Med. Chem. Lett. 11 (2001) 301±303
Scheme 1.
The IR spectra of 2 lack the strong carbonyl absorption
�
Tuberculosis Antimicrobial Acquisition and Coordinating
17�19
1
at 1705±1660 cm typical of the free ligands. The eno-
late structure is also supported by (a) a medium band
Facility (TAACF) screening program.
�
1
between 1630 and 1600 cm due to the nitrogen co-
�
All tested complexes 2 produce 98±99% inhibition of M.
tuberculosis H37Rv growth with MICs ranging between
<0.1 and 0.39 mg/mL (with the exception of 2g) (Table 1).
1
ordinated CˆN stretching, (b) a band at 1050 cm due
to the enolic C±O stretching, and (c) a broad absorption
centred at about 3350 cm attributable to the presence
of water in the co-ordination sphere.13
�
1
The in vitro cytotoxicity in VERO cells, expressed as
IC50 (mg/mL), is generally 10 mg/mL and the selectivity
The magnetic moments, m , per metal ion grammoa-
indexes (SI=IC /MIC) are 74 for 2b and >50 for 2d
50
and 2e.
e€
14
tom, range between 3.9 and 4.0 BM. These values are
7
consistent with d low spin electronic con®guration in
octahedral environment without any metal±metal
In comparison with their respective ligands, complexes 2
3
1
5
interaction.
exhibit similar (2a, 2b, 2d, 2g) or lower (2e, 2f) potency.
Compound 2g, obtained from a,a,a-tri¯uoroacetophe-
none ISNE, is the least e€ective among the tested Co(II)
chelates, similar to the Cu(II) one; the ligand itself is less
active than other ISNEs.
Therefore, we propose for Co(II) complexes 2 the same
octahedral co-ordination geometry evidenced for the ana-
logous Cu(II) and Ni(II) chelates, with four equatorial co-
ordination sites occupied by two ISNE enolates and the
axial sites occupied by two molecules of water (Scheme 1).
The comparison with previously reported complexes3
shows that compounds 2 are always more potent than
their Ni(II) analogues and often than the Cu(II) ones. In
particular, 2c/2f and 2a are 8- and 4-fold more active
than the corresponding Ni(II) complexes, respectively.
2a is moderately more active and 2d is 4-fold more
e€ective than the respective Cu(II) analogues.
The behaviour of complexes 2 in MeOH/H O 1:1 solu-
2
tion, in the pH range 3.5±12.0, is very similar to that of
3
the Cu(II) and Ni(II) analogues. Maximum absorption
is detected at 300 nm at pH between 6.0 and 7.0 and no
breakdown is evidenced up to pH 12.0. The stability has
also been veri®ed in conditions mimicking those
employed for antimycobacterial assays (phosphate buf-
Therefore, the replacement of Cu(II) or Ni(II) with Co(II)
as co-ordinating metal ion represents an improvement in
the antimycobacterial activity of these isonicotinoyl-
hydrazone metal complexes, on the whole. The pharma-
cological data so far collected and the comparison with
the free ligands seem to support the hypothesis that they
should act like a repository of the active ISNEs, simi-
ꢀ
16
fer: pH 6.5/6.8; 37 C; 10 days).
In contrast, the complexes undergo breakdown at pH
.0±5.0.
4
The antimycobacterial in vitro activity of Co(II) complexes
has been evaluated according to the protocols of the
3
2
larly to the reported Cu(II) and Ni(II) analogues.
17
Table 1. Antimycobacterial in vitro activity of 2a±g, expressed as MIC (mg/mL), against M. tuberculosis H37Rv
Compound
% Inhibition
MIC (mg/mL)
Compound
% Inhibition
MIC (mg/mL)
2a
2b
2c
2d
2e
99
99
99
98
99
<0.1
ꢁ 0.1
0.2
2f
2g
RMP
INH19
99
99
0.39
6.25-12.5
0.06-0.25
0.025-0.05
19
ꢁ 0.2
0.2
RMP=Rifampin; INH=Isoniazid

B. Bottari et al. / Bioorg. Med. Chem. Lett. 11 (2001) 301±303
303
However, further pharmacological evaluation against
INH-resistant strains of M. tuberculosis and in TB-infec-
ted macrophages as well as against M. avium is ongo-
8. Sa
J. A. J. Med. Chem. 1996, 39, 1095.
. Ramadan, A. M. J. Inorg. Biochem. 1997, 65, 183.
Â
nchez-Delgado, R. A.; Navarro, M.; Pe
Â
rez, H.; Urbina,
9
1
8,19
10. Malhotra, R.; Singh, J. P.; Dudeja, M.; Dhindsa, K. S. J.
Inorg. Biochem. 1992, 46, 119.
1. Foley, R. T.; Anderson, R. C. J. Am. Chem. Soc. 1948, 70,
195.
2. Job, P. Ann. Chim. 1928, 9, 113; 1936, 11, 97.
3. Infrared spectra were recorded as Nujol mulls in the range
4000±300 cm using CsI disks on a Perkin±Elmer FT-IR
model 1730 spectrophotometer.
ing.
A comprehensive pro®le of these transition
metal complexes as antituberculous agents showing
some advantages, if any, compared to INH, is expected
to be acquired in the near future.
1
1
1
1
�
1
Acknowledgements
1
4. Magnetic measurements were performed according to the
procedure described in ref 3.
5. Cotton, F. A.; Wilkinson, G. In Advanced Inorganic
Chemistry, 5th ed.; Wiley: New York, 1966.
6. pH measurements were performed on a Metrohm 632 pH-
Meter calibrated on CH OH/H O 1:1 solutions of bu€er
The Authors gratefully acknowledge the Sta€ of the
Tuberculosis Antimicrobial Acquisition and Coordinat-
ing Facility (TAACF), directed by the U.S. National
Institute of Allergy and Infectious Diseases for the
antimycobacterial assays. This work was supported by
1
1
3
2
®
nancial assistance from Ministero Universita Ricerca
Á
phosphate. UV spectra were recorded in the thermostated cell
compartment of a Cary 219 or a Perkin±Elmer Lambda 3
spectrophotometer.
Scienti®ca e Tecnologica (MURST, Rome) and from
Consiglio Nazionale Ricerche (CNR, Italy).
1
7. Compounds are initially screened against M. tuberculosis
H37Rv (ATCC 27294, susceptible both to rifampin, RMP and
isoniazid, INH), at 6.25 mg/mL, in BACTEC 12B medium,
using a broth microdilution assay, the Microplate Alamar
Blue Assay (MABA). Compounds exhibiting ¯uorescence are
tested in the BACTEC 460 radiometric system. Compounds
demonstrating at least 90% inhibition are re-tested at lower
concentrations against M. tuberculosis H37Rv to determine
the actual minimum inhibitory concentration (MIC), using
MABA. The MIC is de®ned as the lowest concentration
e€ecting a reduction in ¯uorescence of 90% relative to con-
trols. Concurrent with the determination of MICs, compounds
are tested for cytotoxicity (IC50) in VERO cells at concentra-
tions ꢁ62.5 mg/mL or 10Â the MIC for M. tuberculosis
H37Rv. After 72 h exposure, viability is assessed on the basis
of cellular conversion of MTT into a formazan product using
the Promega CellTiter 96 Non-radioactive Cell Proliferation
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