Synthesis, characterisation and antitubercular activities of a series of pyruvate-containing aroylhydrazones and their Cu-complexes

Article abstract of DOI:10.1039/c2dt30322a

A series of eight pyruvate-based aroylhydrazones was synthesised and characterised. The reaction of the sodium salts of the aroylhydrazones with one equivalent of copper(ii) chloride allowed the isolation of neutral 1:1 complexes in which the hydrazones occupy three basal coordination sites of a square pyramidal Cu(ii)-centre, with two solvent molecules completing the coordination sphere. Structural details were obtained through the determination of the crystal structures of two representative pyruvate-based aroylhydrazones and three Cu(ii) complexes. The evaluation of the antimycobacterial activity of the sodium salts of the eight pryruvate hydrazones showed that the compounds are essentially inactive in their anionic form. The corresponding neutral Cu(ii) complexes, however, exhibit promising antimycobacterial activities if tested under high iron (8 μg Fe per mL) conditions. As observed for the related antimycobacterial agent isoniazid, the activity of the complexes decreases if the M. tuberculosis cells are grown under low iron (0.02 μg Fe per mL) conditions. The Cu(ii) complexes may thus have a similar mode of action and may require an iron-containing heme-dependent peroxidase for activation.

Full text of DOI:10.1039/c2dt30322a

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PAPER
Synthesis, characterisation and antitubercular activities of a series of
pyruvate-containing aroylhydrazones and their Cu-complexes†
Abeda Jamadar,a Anne-K. Duhme-Klair,*a Kiranmayi Vemuri,b Manjula Sritharan,*b Prasad Dandawatec
and Subhash Padhye*c
Received 13th February 2012, Accepted 17th May 2012
DOI: 10.1039/c2dt30322a
A series of eight pyruvate-based aroylhydrazones was synthesised and characterised. The reaction of the
sodium salts of the aroylhydrazones with one equivalent of copper(II) chloride allowed the isolation of
neutral 1 : 1 complexes in which the hydrazones occupy three basal coordination sites of a square
pyramidal Cu(II)-centre, with two solvent molecules completing the coordination sphere. Structural details
were obtained through the determination of the crystal structures of two representative pyruvate-based
aroylhydrazones and three Cu(II) complexes. The evaluation of the antimycobacterial activity of the
sodium salts of the eight pryruvate hydrazones showed that the compounds are essentially inactive in their
anionic form. The corresponding neutral Cu(II) complexes, however, exhibit promising antimycobacterial
activities if tested under high iron (8 μg Fe per mL) conditions. As observed for the related
antimycobacterial agent isoniazid, the activity of the complexes decreases if the M. tuberculosis cells are
grown under low iron (0.02 μg Fe per mL) conditions. The Cu(II) complexes may thus have a similar
mode of action and may require an iron-containing heme-dependent peroxidase for activation.
Introduction
Tuberculosis (TB) is a highly infectious airborne disease caused
by the pathogenic bacterium Mycobacterium tuberculosis. It is
estimated that TB affects approximately one third of the world’s
population.1 Whilst the antitubercular drugs that are presently
available for treatment are effective against actively growing
Scheme 1 General structure of acyl hydrazones and their iminol
drug-susceptible bacteria, they often fail to eradicate resistant
tautomers.
and persistent strains.2,3
In the search for new antitubercular agents with improved
efficacy, a variety of substituted acyl hydrazones (Scheme 1)
derived from the front-line antitubercular agents isoniazid (iso-
nicotinyl hydrazide, INH, R′ = 4-pyridyl) and pyrazinamide
(PZA, R′ = 2-pyrazinyl) have been investigated for their activity
against M. tuberculosis.4–9 The most promising strategies in the
design of INH-based hydrazones aim to improve the potency of
the parent antimicrobial agent by increasing its bioavailabilty.
Approaches include the enhancement of the diffusion of the
drug through the waxy mycobacterial cell wall by introducing
hydrophobic substituents4,5,10 and the prevention of metabolic
acetylation of INH by protecting the free amino group through
hydrazone formation.6,11 INH acetylation is catalysed by both
mammalian and mycobacterial N-arylaminoacetyl transferases
(NATs) and leads to drug deactivation.12,13
In addition, metal-bound hydrazone derivatives of INH have
been reported to possess excellent in vitro antimycobacterial
activities,14–16 with some complexes even retaining activity
against resistant strains.17 It is well established that acyl hydra-
zones form stable chelate complexes with transition metal
cations by utilising both their oxygen and imine nitrogen as
donor atoms.14,18 Importantly, acyl hydrazones allow additional
donor sites to be introduced (via R′′ and R′′′, Scheme 1) in order
to increase the denticity of the resulting ligands and hence their
affinity for biologically relevant metal ions, such as Cu(II),
Mn(II) and Fe(II).19–21
aDepartment of Chemistry, University of York, Heslington, York YO10
5DD, UK. E-mail: anne.duhme-klair@york.ac.uk;
Fax: (+44) (0)1904 322516
bDepartment of Animal Sciences, School of Life Sciences, University of
Hyderabad, Hyderabad 500046, India. E-mail: srimanju@yahoo.com;
Fax: (+91) 40 23010307
cISTRA, Department of Chemistry, Abeda Inamdar College, University
of Pune, Pune 411001, India. E-mail: sbpadhye@hotmail.com;
Fax: (+91) 20 26446970
A factor that may contribute to the observed enhanced anti-
mycobacterial activity of metal-bound acyl hydrazones is that
metal coordination to the deprotonated anionic form of the
†Electronic supplementary information (ESI) available. CCDC reference
numbers 866682 & 866686. For ESI and crystallographic data in CIF or
other electronic format see DOI: 10.1039/c2dt30322a
9192 | Dalton Trans., 2012, 41, 9192–9201
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ligand can give rise to neutral complexes with enhanced lipophi-
licity, thus facilitating drug transport through the mycobacterial
cell wall.14,16 Since certain mycobacterial resistance mechanisms
are modulated by intracellular oxidative stress22 the redox
activity of the metal centres in metallodrugs of this type may be
significant. Free intracellular Cu(I) or Fe(II) cations, for example,
cause oxidative stress, which in turn initiates the expression of
the catalase-peroxidase KatG23 involved in the activation of the
antitubercular prodrug INH. One of us (M. S.) has previously
shown that the effectiveness of INH is associated with the iron
status of the organisms since the peroxidase activity required for
activation of INH is considerably lowered upon iron deficiency.
In contrast, iron-sufficient M. tuberculosis cells showing notable
peroxidase activity are susceptible to INH treatment.24,25
Recently it was also reported that M. tuberculosis is more sus-
ceptible to the toxicity of free copper than other bacteria and it
was suggested that the mammalian immune system utilizes Cu(I)
as a defense mechanism against M. tuberculosis.26
By considering the factors discussed above, we have designed
a series of aroyl hydrazones and their Cu complexes (Scheme 2).
In order to gain an element of diversity, the intensely studied
INH framework was replaced with a series of mono- and di-sub-
stituted aroyl analogues. Our decision to move away from the
INH framework was encouraged by recent reports that confirm
that hydrazones derived from fluoro-, chloro- and nitrobenzoyl
hydrazides or even benzoyl hydrazide can exhibit promising
antimycobacterial activities.27,28 The finding that benzoyl hydra-
zide forms an NAD adduct similar to the INH-NAD adduct
responsible for the antitubercular activity of INH suggests that
benzoyl hydrazide may have the same mode of action.29 We
anticipate that upon removal of the Cu-centre from the ligands,
for example due to Cu-sequestration by mycobacterial metal-
lothionein,26 the hydrazone ligands are released and sub-
sequently hydrolyse to give their respective parent hydrazides
and pyruvate. Antitubercular activity then arises from a combi-
nation of the activity of the parent hydrazide and copper toxicity.
In addition, the hydrazide–hydrazone derived from pyrazinoic
acid was investigated. Pyrazinoic acid is of particular interest
since this hydrolysis product of the antitubercular agent pyrazi-
namide is able to target persisting M. tuberculosis. Only very
recently, pyrazinoic acid was found to inhibit trans-translation, a
process important in stress survival and recovery from nutrient
starvation.30
Scheme 2 Preparation of pyruvate-based hydrazones NaHL1–NaHL8
and their copper(II) complexes [Cu(L1)(H2O)2]–[Cu(L8)(H2O)2]. Con-
ditions: (a) methanol–water (7 : 3), reflux for 2 h; (b) methanol, 25 °C,
2 h.
pyruvate with isoniazid-analogues and binding the resulting
hydrazones to Cu(II) may result in prodrugs that after activation
do not only kill actively growing bacteria but also show activity
against persistent strains.
Within this study, a total of eight pyruvate hydrazones and
their Cu(II)-complexes were synthesised, characterised and tested
against M. tuberculosis grown initially in Middlebrook
7H9 medium and then compounds exhibiting low MIC
values were screened under high (8 μg Fe per mL) and low
(0.02 μg Fe per mL) iron conditions.
Results and discussion
Synthesis and characterisation
The pyruvate-based hydrazone salts NaHL1–NaHL8 were syn-
thesised in good yields by Schiff base condensation of commer-
cially available hydrazides with sodium pyruvate (Scheme 2).
After recrystallisation from ethanol, NaHL1–NaHL8 were
obtained as white to pale yellow powders and characterized by
IR, 1H and 13C NMR spectroscopy, mass spectrometry and
elemental analysis. Details are given in the ESI.†
Acyl hydrazones can potentially form amide/iminol-tautomers
and E/Z-isomers,33 as indicated in Schemes 1 and 2, respectively.
For biologically active thiosemicarbazones, which share key
structural features with acyl hydrazones, detailed recent studies
have shown that E/Z isomerisation and tautomerisation processes
affect the metal-chelating properties of these ligands.34,35
In order to investigate which E/Z-isomers and amide/iminol-
tautomers are formed by the sodium salts of the acyl hydrazones
in the solid state, the crystal structure of NaHL1 was determined.
Single crystals were obtained by slow evaporation of a concen-
trated solution of NaHL1 in ethanol. Selected bond angles and
distances are given in Table 1. As shown in Fig. 1, NaHL1 crys-
tallises as the Z-isomer and amide-tautomer. The amide hydro-
gen H(2) could be located in the difference Fourier map.
A strong intramolecular hydrogen-bond is formed between H(2)
and the deprotonated carboxylate oxygen O(1) of the pyruvate
unit (dN(2)–O(1) = 2.5772(14) Å). The C(3)–N(2) bond length of
1.3522(16) Å is consistent with a single bond whilst the hydrazi-
nic C(3)vO(3) bond length of 1.2330(15) Å shows double bond
character.36 However, the similarity of the bond angles at N(1) of
The pyruvate moiety was introduced in order to prevent meta-
bolic acetylation of the parent hydrazides and to provide an
additional oxygen donor for copper binding. Since the pyruvate
moiety is deprotonated at physiological pH, it balances a positive
charge of the bound metal cations and thus allows the pre-
paration of neutral complexes to facilitate diffusion into the
mycobacterial cell. In addition, a recent computational study
showed that pyruvate-based hydrazones can bind to and poten-
tially inhibit the active site of isocitrate lyase (ICL).31 Since ICL
plays a crucial role in the persistence of M. tuberculosis, it is
considered an attractive target for novel antitubercular drugs.32
The docking study found that the binding affinities of the pyru-
vate hydrazones investigated were comparable to the affinity
obtained for pyruvate. The higher binding affinity obtained for
some of the hydrazone ligands may be due to additional hydro-
gen bond formation by nitrogen atom.21 Hence, combining
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Table 1 Selected bond lengths (Å) and angles (°) for NaHL1 and
4-methylbenzoyl hydrazide38 and 4-chlorobenzoyl hydrazide.39
These three neutral pryruvic acid-derivatives crystallise as the
E-isomers and lack the intramolecular hydrogen bond and
sodium counter ion observed in the structure of NaHL1.
In NaHL1 the sodium counter ion interacts with the hydrazinic
carbonyl oxygen, thereby slightly lengthening the hydrazinic
carbonyl bond. The carboxylate donor of an adjacent symmetry-
generated HL1(1−) anion and four bridging aqua ligands, two of
which are symmetry-generated, complete the octahedral coordi-
nation sphere of the sodium cation (Fig. S2a and b†).
H2L8
NaHL1
H2L8
C(1)–O(1)
C(1)–O(2)
C(3)–O(3)
C(2)–N(1)
C(3)–N(2)
N(1)–N(2)
N(1)⋯O(4)
N(2)⋯O(1)
C(3)–N(2)–N(1)
C(2)–N(1)–N(2)
Na(1)–O(3)
Na(1)–O(4)
Na(1)–O(5)
Na(1)–O(1)#2
Na(1)–O(4)#1
Na(1)–O(5)#1
1.2743(16)
1.2380(16)
1.2330(15)
1.2897(17)
1.3522(16)
1.3835(15)
2.9249(15)
2.5772(14)
119.46(11)
117.29(11)
2.3482(10)
2.3162(11)
2.3952(11)
2.4222(11)
2.3459(11)
2.4232(11)
C(7)–O(2)
C(7)–O(3)
C(5)–O(1)
C(6)–N(4)
C(5)–N(3)
N(3)–N(4)
O(2)...O(4)
1.3163(17)
1.2151(17)
1.2209(17)
1.2831(18)
1.3616(18)
1.3745(16)
2.5642(14)
In order to investigate which isomer is adopted in solution, the
1H NMR spectrum of NaHL1 was recorded immediately after
dissolving the solid product in d6-dmso. The initial spectrum
obtained indicated the presence of two species in a 1 : 5.7 ratio
(Fig. S1, ESI†). The major species shows a characteristic singlet
resonance exceptionally downfield at 16.2 ppm, which could be
due to an iminol–OH40 or a strongly hydrogen-bonded amide
proton.33,35 In view of the strong intramolecular hydrogen bond
observed in the crystal structure of NaHL1 the latter seems more
likely and is consistent with the amide Z-isomer observed in the
solid state also dominating in solution. The corresponding amide
NH-proton of the minor species is more shielded and resonates
at 10.9 ppm, a chemical shift consistent with the NH proton of
the E-isomer, which lacks intramolecular hydrogen bonding.33
Upon heating to 378 K, the minor E-isomer is readily converted
into the more stable Z-isomer and remains as such upon sub-
sequent cooling to 298 K. Further support for the assignment of
the more stable species as the Z-isomer is provided by the
13C NMR data. Palla et al.33 investigated a series of esters of
puruvic acid hydrazones and reported that in d6-dmso a chemical
shift of around 20 ppm for the pyruvic methyl carbon is
diagnostic for the Z-isomer whilst a shift of 13 ppm is character-
istic for the E-isomer.37 In the 13C NMR spectra of fully equili-
brated solutions of NaHL1–NaHL8 in d6-dmso, the pyruvic
methyl carbon resonance is observed between 21.1 and
21.6 ppm, further confirming the assignment as Z-isomer.
C(5)–N(3)–N(4)
N(3)–N(4)–C(6)
117.71(12)
116.30(12)
Symmetry transformations used to generate equivalent atoms: #1 2/3 −
y, −2/3 + x − y, 1/3 + z; #2 5/3 − x, 1/3 − y, 7/3 − z.
Owing to its relevance to the antitubercular agent pyrazina-
mide, the crystal structure of the hydrazide–hydrazone derived
from pyrazinoic acid was also determined. Crystals of H2L8
were obtained through evaporation of a slightly acidic solution
of the compound in aqueous methanol. The molecular structure
of H2L8 is shown in Fig. 1 and selected bond angles and dis-
tances are given in Table 1. Like the neutral pyruvic acid hydra-
zones reported in the literature,37–39 H2L8 adopts the E-isomer in
the solid state. Again, the amide tautomer is formed with a
hydrazinic C(5)vO(1) bond length of 1.2209(17) Å and a C(5)–
N(3) bond length of 1.3616(18) Å. The proton on N(3) could be
located in the difference Fourier map and O(1) accepts a weak
hydrogen bond from an adjacent water molecule (dO(1)–O(4)
=
2.9683(16) Å). On the whole, the bond distances and angles are
consistent with those reported for other pyruvic acid-based
hydrazones.
Fig. 1 ORTEP plots (50% probability ellipsoids) of the asymmetric
units of NaHL1·2H2O (top) and H2L8·H2O (bottom).
The copper(II) complexes [Cu(L1)(H2O)2]–[Cu(L8)(H2O)2]
were prepared by reacting a solution of the respective sodium
salts of the ligands in methanol with one equivalent of copper(II)
chloride dihydrate. Slow evaporation of the resulting solutions at
room temperature yielded green microcrystalline powders, which
were purified by washing with water, followed by recrystallisa-
tion from methanol. The complexes were obtained in moderate
117.29(11)° and N(2) of 119.46(11)° indicates the potential for
electron delocalisation.
Overall, the bond distances and angles listed in Table 1 are
similar to those reported for the protonated neutral pyruvic acid-
based hydrazones (H2L) derived from salicyloyl hydrazide,37
9194 | Dalton Trans., 2012, 41, 9192–9201
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yields and characterised by IR spectroscopy, mass spectrometry
and elemental analysis.
The imine ν(CvN), amide and carboxylato ν(CvO) bands,
which are reasonably well resolved in the solid state IR spectra
of the free ligands, merge upon complex formation to give a
broad band in the range between 1622 and 1649 cm−1. The
centre of the broad band is shifted by 10–30 cm−1 to lower
frequencies if compared with the respective highest frequency
ν(CvO) band in the IR spectrum of the free ligand, consistent
with a weakening of the bond upon coordination and shift
towards the iminolate tautomer.41
The positive ion ESI mass spectra of methanolic solutions of
complexes [Cu(L1)(H2O)2]–[Cu(L8)(H2O)2] are interesting since
the observed molecular ion peaks for all but one ([Cu(L4)-
(H2O)2]) of the complexes appear at one m/z unit higher than
expected (Fig. S5†). In principle, the higher mass could be
explained by either the addition of a hydrogen atom or the
addition of a proton with concomitant reduction of Cu(II) to
Cu(I) to account for the positive charge. The detection of ions of
composition [M + 2Na]+ in the spectra of the copper complexes
of L1, L5 and L6 (Fig. S5†) provides support for the latter, as
these ions show the addition of a cation which requires the
charge to be balanced by a reductive process, most likely the
reduction of Cu(II) to Cu(I). The reduction of Cu(II) to Cu(I) in
positive ion ESI mass spectra of Schiff base complexes has pre-
viously been reported and attributed to the transfer of an electron
from a solvent methanol molecule to Cu(II) in the gas phase.42
In order to examine the structure of the Cu(II)-complexes
formed in more detail, the solid state structures of three represen-
tative examples were determined. The ORTEP plot of the asym-
metric unit of [Cu(L1)(H2O)2] is shown in Fig. 2, selected bond
lengths and angles are listed in Table 2. The ORTEP plots,
selected bond lengths and bond angles for [Cu(L2)(MeOH)2]
and [Cu(L4)(MeOH)2] can be found in the ESI (Fig. S3, S4 and
Table S1†).
Fig. 2 ORTEP plot (50% probability ellipsoids) of the asymmetric
unit of [Cu(L1)(H2O)2].
Table 2 Selected bond lengths (Å) and angles (°) for [Cu(L1)(H2O)2]
Complex 1
Complex 2
C(1)–O(1)
C(1)–O(2)
C(3)–O(3)
C(2)–N(1)
C(3)–N(2)
N(1)–N(2)
Cu(1)–N(1)
Cu(1)–O(4)
1.291(3)
1.226(3)
1.301(3)
1.286(3)
1.326(3)
1.372(2)
1.8988(19)
1.958(2)
1.9873(16)
1.9877(16)
2.218(2)
156.67(9)
79.98(7)
105.71(8)
81.46(7)
90.13(8)
161.11(7)
108.83(8)
93.73(9)
92.16(7)
97.10(7)
2.702(3)
2.634(3)
2.750(2)
C(12)–O(6)
C(12)–O(7)
C(14)–O(8)
C(13)–N(3)
C(14)–N(4)
N(3)–N(4)
Cu(2)–N(3)
Cu(2)–O(10)
1.282(3)
1.244(3)
1.295(3)
1.289(3)
1.328(3)
1.371(2)
1.9065(19)
1.971(2)
1.9939(16)
1.9940(16)
2.152(2)
152.75(9)
78.91(7)
101.09(8)
81.66(7)
94.46(8)
160.22(7)
111.51(9)
95.72(9)
89.83(8)
100.79(8)
2.714(3)
2.778(3)
2.744(2)
Cu(1)–O(3)
Cu(1)–O(1)
Cu(1)–O(5)
Cu(2)–O(8)
Cu(2)–O(6)
Cu(2)–O(9)
The asymmetric unit obtained for [Cu(L1)(H2O)2] contains
two crystallographically independent complexes with slightly
different coordination geometries. Both are square pyramidal,
with τ-indices43 of 0.074 (complex 1) and 0.125 (complex 2)
indicating that the latter deviates slightly more from perfect
square pyramidal geometry. In both complexes, L1(2−) is doubly
deprotonated and acts as a tridentate ligand, forming two 5-mem-
bered chelate rings by using the hydrazinic carbonyl oxygen, a
pyruvate carboxylate oxygen and the imine nitrogen atom as
donors. Two coordinated aqua ligands complete the coordination
sphere. Whilst in both complexes, the aqua ligand in apical pos-
ition is only weakly coordinated, as expected for Jahn–Teller dis-
torted Cu(II) complexes, the apical Cu(1)-O(5) contact of
2.218(2) Å in complex 1 is even longer than the equivalent
Cu(2)–O(9) contact of 2.152(2) Å in complex 2. Whilst the
apical aqua ligands hydrogen bond with the pyruvate moieties of
adjacent symmetry-generated complexes, each basal aqua ligand
donates a hydrogen bond to the hydrazinic carbonyl oxygen of
the other complex in the asymmetric unit (Fig. 2).
N(1)–Cu(1)–O(4)
N(1)–Cu(1)–O(3)
O(4)–Cu(1)–O(3)
N(1)–Cu(1)–O(1)
O(4)–Cu(1)–O(1)
O(3)–Cu(1)–O(1)
N(1)–Cu(1)–O(5)
O(4)–Cu(1)–O(5)
O(3)–Cu(1)–O(5)
O(1)–Cu(1)–O(5)
O(4)⋯O(8)
N(3)–Cu(2)– (10)
N(3)–Cu(2)–O(8)
O(10)–Cu(2)–O(8)
N(3)–Cu(2)–O(6)
O(10)–Cu(2)–O(6)
O(8)–Cu(2)–O(6)
N(3)–Cu(2)–O(9)
O(10)–Cu(2)–O(9)
O(8)–Cu(2)–O(9)
O(6)–Cu(2)–O(9)
O(10)⋯O(3)
O(5)⋯O(1)#1
O(5)⋯O(7)#2
O(9)⋯O(6)#2
O(9)⋯O(2)#1
Symmetry transformations used to generate equivalent atoms: #1 −x +
2,−y + 1,−z + 1; #2 −x + 1,−y,−z + 1.
C (14)–N(4) distances of 1.326(3) and 1.328(3) Å in complexes
1 and 2, respectively, are significantly shorter than the C(3)–N(2)
distance of 1.3522(16) Å in NaHL1. Upon complexation L1(2−)
hence adopts an intermediate structure that lies in between the
structures to be expected for the amide and iminol tautomers.
To date, the only other crystal structure of a Cu(II) complex of
a pyruvate-based aroylhydrazone that has been deposited in the
The bond lengths of the hydrazinic carbonyl groups are
1.301(3) and 1.295(3) Å in complexes 1 and 2, respectively.
Both are slightly longer than the equivalent C(3)–O(3) bond
length of 1.2330(15) Å in NaHL1, however, both still have sig-
nificant double bond character. Conversely, the C(3)–N(2) and
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Cambridge Crystallographic Data Centre (CCDC) is the 1 : 2
complex [Cu(C10H9N2O4)2] reported by He et al. (Fig. 3).44
In [Cu(C10H9N2O4)2] the ligand remained mono-protonated. The
hydrazinic CvO bond lengths in [Cu(C10H9N2O4)2] are charac-
teristic of a double bond and resemble those of NaHL1.
As expected, the Cu-donor bond distances in the six-coordinate
complex [Cu(C10H9N2O4)2], are slightly longer than those in the
five-coordinate complex [Cu(L1)(H2O)2].
Whilst the pyruvate-based hydrazones and their parent hydra-
zides were found to be inhibitory, their minimum inhibitory con-
centration (MIC) values were significantly higher than the MIC
of INH (Table 3). Once coordinated to Cu(II), however, the pyru-
vate-based hydrazones were highly potent, with the complexes
[Cu(L1)(H2O)2]–[Cu(L8)(H2O)2] exhibiting MIC values in the
range <0.5–2 μg mL−1 when evaluated against M. tuberculosis
grown under high iron conditions (Table 3). The lower antibac-
terial activity of the pyruvate-based hydrazones may be rational-
ised by the fact that they are likely to be anionic at physiological
pH (the pKa value of pyruvic acid is 2.49)45 and hence less able
to diffuse through lipophilic cell membranes. In contrast, the
copper(II) complexes under investigation are neutral and may
facilitate the cellular uptake of their components. Removal of the
copper inside the mycobacterial cell, for example through
capture by metallothioneins, could therefore lead to trapping of
the hydrophilic hydrazones inside the mycobacterial cell.
Antimycobacterial activity testing
The pyruvate-based hydrazones NaHL1–NaHL8, their parent
hydrazides and the copper complexes [Cu(L1)(H2O)2]–[Cu(L8)-
(H2O)2] were screened against M. tuberculosis under both low
and high iron conditions. INH was used as the reference drug.
If grown under iron-deficient conditions, INH and the com-
plexes [Cu(L1)(H2O)2]–[Cu(L8)(H2O)2] were less effective. INH
is a prodrug that, upon entry into the pathogen is converted into
the active form by KatG, a dual enzyme with catalase and per-
oxidase activities. The latter, involving an iron-containing heme
centre in KatG, is responsible for the generation of free radicals
from INH.46 In earlier studies, one of us (M. S.)24,25 found a
higher MIC (minimum inhibitory concentration) of INH against
iron-limited M. tuberculosis cells than the routinely used MIC of
0.2 μg mL−1 for INH, which applies to higher iron levels in the
growth medium. The failure to activate INH under iron-limiting
conditions was associated with the low peroxidase activity that
was possibly due to decreased heme availability. The observation
Fig. 3 The 1 : 2 complex [Cu(C10H9N2O4)2] reported by He et al.44
Table 3 Influence of iron concentrations in the growth medium on the minimum inhibitory concentrations (MICs) of: (a) INH and [Cu(L1)(H2O)2]–
[Cu(L8)(H2O)2]; (b) NaHL1–NaHL8 and their parent hydrazides (low iron: 0.02 μg Fe per mL; high iron: 8 μg Fe per mL, FeSO4 as iron source)
(a)
MIC of the copper complexes/μg mL−1
Compound
X, Rn
7H9 medium
High Fe
Low Fe
Fold difference in MIC
INH
0.25
4
1
4
8
8
8
16
4
0.125
<0.5
2
32
256
>256
128
64
>256
>256
128
32
[Cu(L1)(H2O)2]
[Cu(L2)(H2O)2]
[Cu(L3)(H2O)2]
[Cu(L4)(H2O)2]
[Cu(L5)(H2O)2]
[Cu(L6)(H2O)2]
[Cu(L7)(H2O)2]
[Cu(L8)(H2O)2]
CH, 4-Me
CH, 4-NO2
CH, 4-(CH3)3
CH, 4-NO2, 3-Me
CH, 4-CF3
CH, 3-F
128
256
64
128
128
64
1
<0.5
<0.5
0.5
1
<0.5
CH, 3-OH
N, H
32
256
>512
(b)
MIC of the parent hydrazide/μg mL−1
MIC of pyruvate hydrazone ligands/μg mL−1
Compound
High Fe
Low Fe
Fold difference
High Fe
Low Fe
Fold difference
NaHL1
NaHL2
NaHL3
NaHL4
NaHL5
NaHL6
NaHL7
NaHL8
32
16
32
32
32
64
32
32
256
256
>256
>256
256
256
>256
256
8
16
8
16
8
16
16
32
8
256
256
256
128
256
256
256
256
16
32
16
16
16
16
8
16
<8
<8
8
4
<8
8
32
9196 | Dalton Trans., 2012, 41, 9192–9201
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that the antimycobacterial activity of complexes [Cu(L1)-
(H2O)2]–[Cu(L8)(H2O)2], NaHL1–NaHL8 and their parent
hydrazides also depend on the availability of iron suggests a
similar mode of action. If this is the case, at least part of the
observed antibacterial activity should be due to the parent hydra-
zides produced by dissociation of the Cu-complexes and
hydrolysis of the hydrazone ligands inside the cell. In the case of
[Cu(L8)(H2O)2], the intracellularly released hydrazide would be
a prodrug, which requires further hydrolysis to produce its active
component, pyrazinoic acid. Interestingly, the parent hydrazides
have lower antimycobacterial activities than the hydrazones
NaHL1–NaHL8 (Table 3b) when added to the growth medium
under the same conditions. These lower activities, however, do
not necessarily correspond to lower intracellular potencies since
the lower activities of the hydrazides could be due to restricted
cellular uptake.
Since ferrous sulphate was used as the iron source in the
activity tests, it is also conceivable that Fe(II) binds to NaHL1–
NaHL8 or the parent hydrazides in the growth medium under
high iron conditions, however, if the resulting iron complexes
are formed, they are less potent than the corresponding Cu(II)
complexes. The copper component of the complexes clearly
exerts an additional inhibitory effect, as expected since copper is
known to be highly toxic to M. tuberculosis.26
Finally, the pyruvate moiety may also be important due to
its significance in the glyoxylate shunt cycle, a response of
M. tuberculosis to nutrient starvation in the phagosome.32
Amongst the known inhibitors of the glyoxylate shunt cycle in
E. coli are the pyruvate compounds that activate the isocitrate
dehydrogenase enzyme of the tricarboxylic acid (TCA) cycle
and in turn inactivate the glyoxylate shunt pathway.47
Fig. 4 Absorption spectra recorded in PBS buffer at pH 7.4 and 37 °C
for NaHL5 in comparison with its parent hydrazide (top) and [Cu(L5)-
(H2O)2] (bottom), immediately after dissolution and after the indicated
time-intervals given in hours.
Hydrolytic stability study
In order to investigate if the dissociation of the Cu(II) complexes
accelerates the hydrolysis of the hydrazones to form their parent
hydrazides, the stabilities of NaHL5 and [Cu(L5)(H2O)2] in
phosphate buffered saline (PBS) were compared. PBS is isotonic
with human plasma and has previously been used in hydrolysis
studies on related hydrazones.48 Both compounds were dissolved
in DMSO and the solutions diluted to a final concentration of
0.05 mM with PBS. The solutions (pH 7.4) were then main-
tained at 37 °C whilst the progress of hydrolysis was monitored
spectrophotometrically at defined time-intervals.
the solution due to the limited water solubility of the compound.
Consequently, Cu(II)-binding leads to a considerable stabilisation
of the ligand towards hydrolysis.
Summary and conclusions
Analysis of the UV-vis spectra obtained revealed the gradual
hydrolysis and degradation of NaHL5 (Fig. 4). Hydrolysis was
evidenced by a decrease in intensity of the absorption band
at 270 nm, as expected for the formation of the parent hydrazide,
the spectrum of which is shown for comparison (dotted line).
Up to a time delay of 14 hours an isosbestic point is observed at
248 nm. However, an increase in absorbance at around 330 nm
indicates further degradation or oxidation processes leading to
the formation of additional species so that in spectra recorded
between 53 and 120 hours after dissolution of NaHL5 an iso-
sbestic point is no longer observed. In contrast, the overall shape
of the UV-vis spectra recorded for [Cu(L5)(H2O)2] changed very
little. The slight decrease in intensity of the band at 325 nm can
be explained by slow precipitation of some of the complex from
In summary, a series of eight pyruvate-based aroylhydrazones
HL− and their Cu(II)-complexes [Cu(Ln)(H2O)2] were syn-
thesised, characterised and the crystal structures of two pyruvate-
based aroylhydrazones and three Cu(II) complexes determined.
Whilst the sodium salts of the eight pryruvate hydrazones were
inactive, their neutral Cu(II) complexes exhibited promising anti-
mycobacterial activities if tested under high iron conditions.
As observed for INH, the activity of the complexes decreases
significantly if the M. tuberculosis cells are grown under iron-
deficient conditions. Consequently, the Cu(II) complexes may, at
least partially, exhibit a similar mode of action, requiring dis-
sociation, hydrolysis and activation of the released parent hydra-
zide by an iron-dependent peroxidase. A hydrolysis study
performed with a representative ligand confirmed that the
This journal is © The Royal Society of Chemistry 2012
Dalton Trans., 2012, 41, 9192–9201 | 9197

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dissociation of its Cu(II) complex leads to the hydrolysis of the
hydrazone ligand to give its biologically active parent hydrazide.
In case of the pyrazinamide-derivative [Cu(L8)(H2O)2], the
released hydrazide could undergo further hydrolysis to produce
the active component pyrazinoic acid.
(1.72 mmol, 95%, m.p.: 204–205 °C. Anal. calcd for
C10H8N3O5Na1·2.2H2O: C, 38.40; H, 4.00; N, 13.28. Found:
C, 38.69; H, 3.87; N, 13.43. HR ESI-MS: Calcd for
C10H9N3O5Na1 ([M + H]+) 274.0434, found: 274.0429, differ-
1
ence: 0.5 mDa. H NMR (DMSO-d6, 400 MHz) 2.01 (s, 3H,
Considering that pyrazinamide, which has a MIC value as
high as 400 μg mL−1 under certain conditions,49 is included in
the current multi-drug regimen for the treatment of TB, com-
plexes [Cu(L1)(H2O)2]–[Cu(L8)(H2O)2] represent interesting
potential prodrug candidates that merit further investigations.
CH3), 8.03 (d, 2H, JH–H = 8.8 Ar–H), 7.55, 8.34 (d, 2H, JH–H =
8.8 Hz Ar–H), 16.7 (1H, br, NH); 13C NMR (DMSO-d6,
100.5 MHz): 166.1, 160.2, 151.9, 149.2, 139.6, 128.4, 124.0,
21.2. Significant IR bands (KBr, ν cm−1): 1699, 1668, 1635,
1600, 1521, 1467, 1402, 1374, 1352.
Preparation of sodium 2-[(4-tert-butyl-benzoyl)-hydrazono]-
propionate (NaHL3). NaHL3 was synthesised using 4-tert-
butyl-benzoyl hydrazide 0.35 g (1.82 mmol) to give NaHL3,
0.23 g (1.23 mmol, 67%), m.p.: 224–225 °C. Anal. calcd for
C14H17N2O3Na1·1.75H2O: C, 53.24; H, 6.54; N, 8.87. Found:
C, 53.45; H, 6.41; N, 8.64. HR ESI-MS: Calcd for C14H19N2O3
([M − Na + 2H]+) 263.1390, found: 263.1391, difference:
Experimental
Materials and methods
The hydrazones and their Cu(II)-complexes were synthesised and
characterised in the Chemistry Department, University of York.
Chemicals were purchased from the following suppliers:
sodium pyruvate (Fluka), hydrazides, pyrazine-2-carboxylate
and CuCl2·2H2O (Sigma), analytical grade solvents (Fischer
Scientific). Pyrazinoyl hydrazide was prepared according to a
published procedure.50 1H and 13C NMR spectra were recorded
1
−0.1 mDa. H NMR (DMSO-d6, 400 MHz) 1.29 (s, 9H, Ar–C
(CH3)3), 1.99 (s, 3H, CH3), 7.74 (d, 2H, JH–H = 8.0 Hz Ar–H),
7.51 (d, 2H, JH–H = 8.0 Ar–H), 7.55 (m, 1H, Ar–H-8),
16.17 (1H, br, NH); 13C NMR (DMSO-d6, 100.5 MHz): 166.1,
161.7, 154.4, 150.4, 131.1, 126.7, 125.5, 34.6, 30.9, 21.2. Sig-
nificant IR bands (KBr, ν cm−1): 1652, 1634, 1610, 2963, 3426.
on
a
Jeol ECS 400 instrument (1H NMR 400 MHz,
13C 100.6 MHz). Chemical shifts (δ) are given in ppm refer-
enced to the residual DMSO solvent peak. Infrared spectra were
recorded on an Avatar 370 FT-IR Thermo Nicolet instrument
using KBr discs. ESI-MS spectra were recorded on a Bruker
microTOF electrospray mass spectrometer. Melting points were
obtained on a Stuart Scientific SMP3 melting point apparatus.
Elemental analyses were performed on an Exeter analytical
CE-440 elemental analyser.
Preparation of sodium 2-[(3-methyl-4-nitro-benzoyl)-hydra-
zono]-propionate (NaHL4). NaHL4 was synthesised using
3-methyl-4-nitro-benzoyl hydrazide 0.35 g (1.82 mmol) to give
NaHL4, 0.43 g (1.5 mmol, 82%), m.p.: 243–245 °C. Anal. calcd
for C11H10N3O5Na1·0.25CH3OH × 1.8H2O: C, 40.95; H, 4.13;
N, 12.45. Found: C, 41.24; H, 4.49; N, 12.82. HR ESI-MS:
Calcd for C11H11N3O5Na1 [M
+
H]+ 288.0596, found:
288.0581, difference: 1.5 mDa. H NMR (DMSO-d6, 400 MHz)
1
2.01 (s, 3H, CH3), 2.56 (s, 3H, Ar–CH3), 7.78 (d,d, 1H, JH–H
1.1, 8.4 Hz Ar–H), 7.87 (s, 1H, Ar–H), 8.09 (d, 1H, JH–H
=
=
General procedure for the preparation of pyruvate-based
aroylhydrazones
8.4 Hz Ar–H), 16.6 (1H, br, NH); 13C NMR (DMSO-d6,
100.5 MHz): 165.9, 160.1, 151.7, 150.5, 137.9, 133.2, 131.4,
125.5, 124.9, 21.1, 19.4. Significant IR bands (KBr, ν cm−1):
1662, 1627, 1586, 1522, 1465, 1368, 1356.
The respective hydrazide (1.82 mmol) and sodium pyruvate
(1.82 mmol) were dissolved in 25 mL of a water–methanol
mixture (methanol–water; 7 : 3). The solution was heated under
reflux for 2 hours. The product was precipitated with diethyl
ether, isolated and recrystallised from ethanol. The purified
product was dried in vacuo.
Preparation of sodium 2-[(4-triflouromethyl-benzoyl)-hydra-
zono]-propionate (NaHL5). NaHL5 was synthesised using
4-triflouromethyl benzoyl hydrazide 0.37 g (1.82 mmol) to give
NaHL5, 0.48 g (1.62 mmol, 89%), m.p.: 125–128 °C. Anal.
calcd for C11H7N2O3F3Na2·1.4CH3OH × 1.7H2O: C, 37.83;
H, 4.10; N, 7.12. Found: C, 37.46; H, 3.73; N, 6.74. HR
ESI-MS: Calcd for C11H9N2O3F3Na1 ([M + H]+) 297.0457,
Preparation of sodium 2-[(4-methyl-benzoyl)-hydrazono]-pro-
pionate (NaHL1). NaHL1 was synthesised using 4-methyl
benzoyl hydrazide 0.27 g (1.82 mmol) to give NaHL1, 0.38 g
(1.57 mmol, 86%), m.p.: 242–244 °C. Anal. calcd for
C11H11N2O3Na1·2.2H2O: C, 46.88; H, 5.51; N, 9.94. Found: C,
46.92; H, 5.41; N, 9.75. HR ESI-MS: Calcd for C11H12N2O3Na1
([M + H]+) 243.0740, found: 243.0738, difference 0.3 mDa.
1H NMR (DMSO-d6, 400 MHz) δ: 2.36 (s, 3H, Ar–CH3), 1.98
(s, 3H, CH3), 7.31 (d, 2H, JH–H = 8.1 Hz Ar–H), 7.71 (d, 2H,
JH–H = 8.1 Ar–H), 16.2 (1H, br, NH); 13C NMR (DMSO-d6,
100 MHz): 166.3, 161.9, 150.5, 141.7, 130.9, 129.3 127.0, 21.2,
21.0. Significant IR bands (KBr, ν cm−1): 1649, 1631, 1610,
1575, 1476, 1368.
1
found: 297.0465, difference: −0.7 mDa. H NMR (DMSO-d6,
400 MHz) 2.01 (s, 3H, CH3), 7.89 (d, 2H, JH–H = 8.3 Hz Ar–H),
7.99 (d 2H, JH–H = 8.3 Hz, Ar–H) 16.6 (1H, br, NH); 13C NMR
(DMSO-d6, 100.5 MHz): 165.9, 160.5, 151.6, 137.8, 131.5,
131.2, 127.8, 125.7, 125.2, 122.5, 21.1. Significant IR bands
(KBr, ν cm−1): 1635, 1632, 1580, 1525, 1472, 1367, 1331.
Preparation of sodium 2-[(3-fluoro-benzoyl)-hydrazono]-pro-
pionate (NaHL6). NaHL6 was synthesised using 3-fluoro-
benzoyl hydrazide 0.27 g (1.82 mmol) to give NaHL6, 0.36 g
(1.46 mmol, 80%), m.p.: 254–257 °C. Anal. calcd for
C10H8N2O3FNa1·0.15CH3OH × 1.1H2O: C, 45.02; H, 4.02;
N, 10.34. Found: C, 45.03; H, 3.92; N, 10.30. HR ESI-MS:
Preparation of sodium 2-[(4-nitro-benzoyl)-hydrazono]-pro-
pionate (NaHL2). NaHL2 was synthesised using 4-nitro-benzoyl
hydrazide 0.33 g (1.82 mmol) to give NaHL2, 0.47 g
9198 | Dalton Trans., 2012, 41, 9192–9201
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Calcd for C10H9N2O3FNa1 ([M + H]+) 247.0489, found:
247.0496, difference: −0.7 mDa. 1H NMR (DMSO-d6,
400 MHz) 1.99 (s, 3H, CH3), 7.40–7.80 (m, 5H, Ar–H) 16.42
(1H, br, NH); 13C NMR (DMSO-d6, 100.5 MHz): 166.2, 163.4,
161.0, 160.6, 160.5, 151.2, 136.4, 136.3, 131.1, 131.0, 122.8,
122.8, 118.7, 118.5, 113.9, 113.7, 21.6. Significant IR bands
(KBr, ν cm−1): 1739, 1635, 1589, 1498, 1471, 1430, 1375,
1271.
41%), m.p.: 250–252 °C. Anal. calcd for Cu1C10H11-
N3O7·1.15H2O: C, 32.51; H, 3.63; N, 11.37. Found: C, 32.67;
H, 3.31; N, 11.05. ESI-MS: Calcd for 63Cu(I)1C10H12N3O7Na1
([M + H + Na]+) 371.99, found: 372.01. Significant IR bands
(KBr, ν cm−1): 1639, 1527, 1493, 1424, 1379, 1366, 1347,
1320.
Preparation of [Cu(L3)(H2O)2]. [Cu(L3)(H2O)2] was syn-
thesised using NaHL3, 0.16 g (0.58 mmol) and CuCl2·2H2O
0.1 g (0.58 mmol) to give [Cu(L3)(H2O)2], 0.1 g (0.27 mmol,
47%), m.p.: 244–247 °C. Anal. calcd for Cu1C14H18N2O4·0.25-
CH3OH: C, 48.92; H, 5.47; N, 8.01. Found: C, 49.09; H, 5.38;
N, 7.87. ESI-MS: Calcd for 63Cu(I)1C14H19N2O4Na1 ([M − H2O
+ H + Na]+) 365.05, found: 365.08. Significant IR bands (KBr,
ν cm1): 1651, 1635, 1609, 1575, 1558, 1476, 1436, 1385.
Preparation of sodium 2-[(3-hydroxy-benzoyl)-hydrazono]-
propionate (NaHL7). NaHL7 was synthesised using 3-hydroxy-
benzoyl hydrazide 0.27 g (1.82 mmol) to give NaHL7, 0.39 g
(1.59 mmol, 87%), m.p.: 278–281 °C. Anal. calcd for
C10H9N2O4Na1·1.6H2O: C, 44.00; H, 4.50; N, 10.26. Found: C,
44.16; H, 4.43; N, 10.10. HR ESI-MS: Calcd for
C10H10N2O4Na1 ([M + H]+) 245.0533, found: 245.0533, differ-
Preparation of [Cu(L4)(H2O)2]. [Cu(L4)(H2O)2] was syn-
thesised using NaHL4, 0.17 g (0.58 mmol) and CuCl2·2H2O,
1
ence: 0.0 mDa. H NMR (DMSO-d6, 400 MHz) 2.01 (s, 3H,
CH3), 6.97 (d,d, 1H, JH–H = 2.08, 7.88, Ar–H), 7.78 (m 3H, Ar–
H), 15.9 (1H, br, NH); 13C NMR (DMSO-d6, 100.5 MHz):
166.4, 162.3, 157.9, 150.5, 135.1, 129.8, 118.9, 117.3, 114.1,
21.2. Significant IR bands (KBr, ν cm−1): 1663, 1633, 1612,
1585, 1457, 1373, 1296.
0.10
g
(0.58 mmol) to give [Cu(L4)(H2O)2], 120 mg
(0.31 mmol, 54%), m.p.: 210–212 °C. Anal. calcd for
Cu1C11H13N3O7·0.1H2O: C, 36.06; H, 3.69; N, 11.47. Found: C,
36.06; H, 3.55; N, 11.35. ESI-MS: Calcd for 63Cu(II)1C11H12-
N3O6Na1 ([M − H2O + Na]+) 368.00, found: 368.02. Significant
IR bands (KBr, ν cm−1): 1662, 1524, 1495, 1401, 1360, 1308.
Preparation of sodium 2-[(pyrazine-2-carbonyl)-hydrazono]-
propionate (NaHL8). NaHL8 was synthesised using pyrazinoyl
hydrazide 0.27 g (1.82 mmol) to give NaHL8 0.36 g
(1.46 mmol, 80%), m.p.: decomp. >200 °C. Anal. calcd for
C8H7N4O3Na1·0.15CH3OH × 1.75H2O: C, 36.73; H, 4.20; N,
21.02. Found: C, 36.55; H, 3.90; N, 20.76. HR ESI-MS: Calcd
for C8H9N4O3 ([M − Na + 2H]+) 209.0669, found: 209.0670,
Preparation of [Cu(L5)(H2O)2]. [Cu(L5)(H2O)2] was syn-
thesised using NaHL5, 0.17 g (0.58 mmol) and CuCl2·2H2O
0.1 g (0.58 mmol), to give [Cu(L5)(H2O)2], 0.08 g (0.21 mmol,
36%), m.p.: 214–217 °C. Anal. calcd for Cu1C11H11-
N2O5F3·0.27H2O: C, 34.28; H, 3.27; N, 7.27. Found: C, 34.28;
H, 3.07; N, 6.77. ESI-MS: Calcd for 63Cu(I)1C11H12N2O5F3Na1
([M + H + Na]+) 394.99, found: 395.01. Significant IR bands
(KBr, ν cm−1): 1640, 1626, 1491, 1379, 1365, 1323.
1
difference: −0.1 mDa. H NMR (DMSO-d6, 400 MHz) 2.02 (s,
3H, CH3), 8.72 (d,d 1H, JH–H = 2.5, 1.5 Hz Ar–H), 8.85 (d, 1H,
JH–H = 2.5 Ar–H), 9.21 (d, 1H, JH–H = 1.4 Ar–H); 13C NMR
(DMSO-d6, 100.5 MHz): 165.7, 159.1, 152.0, 147.6, 145.0,
143.9, 143.6, 21.4. Significant IR bands (KBr, ν cm−1): 1670,
1625, 1573, 1486, 1430, 1385, 1275.
Preparation of [Cu(L6)(H2O)2]. [Cu(L6)(H2O)2] was syn-
thesised using NaHL6, 0.14 g (0.58 mmol) and CuCl2·2H2O,
0.10 g (0.58 mmol) to give [Cu(L6)(H2O)2], 0.06 g (0.19 mmol,
32%), m.p.: 232–235 °C. Anal. calcd for Cu1C10H11N2O5-
F1·0.27H2O: C, 45.02; H, 3.56; N, 8.57. Found: C, 45.03;
H, 3.29; N, 8.25. ESI-MS: Calcd for 63Cu(I)1C10H12N2O5F1Na1
([M + H + Na]+) 344.99, found: 345.02. Significant IR bands
(KBr, ν cm−1): 1630, 1581, 1506, 1539, 1484, 1431, 1371,
1347.
General procedure for the preparation of the copper complexes
To a methanolic solution of the respective pyruvate-based aroyl-
hydrazone (0.58 mmol) was added CuCl2·2H2O (0.58 mmol).
The mixture was stirred for 2 hours. The resultant clear green
solution was allowed to evaporate slowly giving green coloured
crystalline powder which was isolated, washed with water and
redissolved in methanol. The resultant solution was allowed to
evaporate slowly at room temperature, giving green coloured
crystals, which were isolated and dried in vacuo.
Preparation of [Cu(L7)(H2O)2]. [Cu(L7)(H2O)2] was syn-
thesised using NaHL7, 0.14 g (0.58 mmol) and CuCl2·2H2O
0.1 g (0.58 mmol) to give [Cu(L7)(H2O)2], 0.06 g (0.2 mmol,
34%), m.p.: 265–268 °C. Anal. calcd for Cu1C10H12N2O6: C,
37.56; H, 3.78; N, 8.76. Found: C, 37.53; H, 3.85; N, 8.23.
ESI-MS: Calcd for 63Cu(I)1C10H11N2O5Na1 ([M − H2O + H +
Na]+) 324.99, found: 325.01. Significant IR bands (KBr,
ν cm−1): 1645, 1588, 1498, 1458, 1376, 1364, 1347, 1302.
Preparation of [Cu(L1)(H2O)2]. [Cu(L1)(H2O)2] was syn-
thesised using NaHL1, 0.14 g (0.58 mmol) and CuCl2·2H2O,
0.10 g (0.58 mmol) to give [Cu(L1)(H2O)2], 0.13 g (0.38 mmol,
65%), m.p.: 223–225 °C. Anal. calcd for Cu1C11H14-
N2O5·0.3H2O: C, 40.88; H, 4.55; N, 8.67. Found: C, 40.54; H,
4.23; N, 8.48. ESI-MS: Calcd for 63Cu(I)1C11H15N2O5Na1
([M + H + Na]+) 341.04, found: 341.02. Significant IR bands
(KBr, ν cm−1): 1622, 1482, 1377, 1363, 1318.
Preparation of [Cu(L8)(H2O)2]. [Cu(L8)(H2O)2] was syn-
thesised using NaHL8, 0.13 g (0.58 mmol) and CuCl2·2H2O,
0.10 g (0.58 mmol) to give [Cu(L8)(H2O)2], 0.11 g (0.36 mmol,
62%), m.p.: 137–139 °C. Anal. calcd for Cu1C8H11N4O5Cl ×
0.1 CH3OH: C, 28.00; H, 3.17; N, 16.03. Found C, 27.81; H,
2.88; N, 15.78. ESI-MS: For 63Cu(I)1C8H9N4O4Na1 ([M − H2O
+ H + Na]+), Calcd: 310.99, found: 311.03. Significant IR bands
(KBr, ν cm−1): 1640, 1614, 1578, 1535, 1471.
Preparation of [Cu(L2)(H2O)2]. [Cu(L2)(H2O)2] was syn-
thesised using NaHL2, 0.16 g (0.58 mmol) and CuCl2·2H2O,
0.10 g (0.58 mmol) to give [Cu(L2)(H2O)2], 0.08 g (0.24 mmol,
This journal is © The Royal Society of Chemistry 2012
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Hydrolytic stability study
(low iron) and 8 μg Fe per mL (high iron), respectively. Ferrous
sulphate was used as the iron source. The iron-regulated cultures
were harvested in the mid-log phase after 10 days of growth. The
iron status of the organisms was confirmed by assaying the side-
rophores mycobactin and carboxymycobactin using established
protocols.25 Care was taken to ensure that the cells were har-
vested before the onset of iron limitation in high iron organisms.
The cells were washed and re-suspended in the respective
medium to get a cell density equivalent to McFarland 1 (3 × 108
cfu mL−1) and further adjusted to get an OD600nm of 0.15 (Bio-
photometer, Eppendorf, USA). This cell suspension was used for
the screening of the compounds.
Accurate weight measurements were conducted on a Mettler AE
240 five figure balance. 0.05 mM stock solutions of the ligands
and Cu(II)-complexes in 90% phosphate buffered saline (PBS)
and 10% spectrophotometric grade DMSO were prepared using
calibrated Eppendorf pipettes. The solutions (pH 7.4) were then
maintained at 37 °C whilst the progress of hydrolysis was moni-
tored by recording absorption spectra at regular time intervals.
The absorption spectra were recorded on an HP 8453 Agilent
Diode Array spectrophotometer using a 5Q quartz UV cell with
path length 10 mm.
X-Ray crystallography
Preparation of the compounds for screening
Diffraction data for NaHL1, [Cu(L1)(H2O)2] and [Cu(L2)-
(MeOH)2] were collected on a Bruker Smart Apex diffractometer
with Mo-Kα radiation (λ = 0.71073 Å) using a SMART CCD
camera. Absorption corrections were applied by SADABS
(v2.10).51 Structures were solved by direct methods using
SHELXS-97 and refined by full-matrix least squares using
SHELXL-97.52 All non-hydrogen atoms were refined anisotropi-
cally. Hydrogen atoms were placed using a riding model and
included in the refinement at calculated positions. Acidic protons
were placed using difference map method.
Stock solutions of the compounds were prepared in HPLC grade
dimethyl sulfoxide (Sigma Chemical Co., St. Louis, MO, USA)
at a concentration of 10 mg mL−1. These were filter-sterilized
(0.22 μm filters, Sartorius) and stored at −80 °C.
Screening of the compounds
The compounds were first screened in the MB/BacT™ 240
Mycobacteria Detection System (BioMerieux/Organon Teknika,
France) followed by the determination of the MIC values by
Microplate Alamar Blue assay (MABA). Finally, the MIC values
were confirmed in the MB/BacT™ 240 system by growth at
MIC and sub-MIC concentrations of the compounds.
24 compounds, including the free ligands, their parent hydra-
zides and Cu-complexes were screened for antimycobacterial
activity in the MB/BacT™240 Mycobacteria Detection System
(BioMerieux/Organon Teknika, France). The detection system
employs standard bottles of 10 mL of culture media to which
0.5 mL of the mycobacterial cell suspension (prepared as
detailed above) was added. The system also included the ‘pro-
portion control’ that was inoculated with 0.5 mL of the cell sus-
pension diluted 1 : 100. The system was standardised for the
screening by adding INH at 0.1–0.5 μg mL−1 final concentration.
All the compounds were added at 256 μg mL−1 and checked for
their effectiveness on M. tuberculosis.
Diffraction data for H2L8 and [Cu(L4)(MeOH)2] were col-
lected on an Oxford Diffraction SuperNova diffractometer with
Mo-Kα radiation (λ = 0.71073 Å) using a EOS CCD camera.
The crystals were cooled with an Oxford Instruments Cryojet.
Face-indexed absorption corrections were applied using
SCALE3 ABSPACK scaling. OLEX253 was used for overall
structure solution, refinement and preparation of computer
graphics and publication data. Within OLEX2, the algorithms
used for structure solution were direct methods using
SHELXS-97 and refinement by full-matrix least-squares used
SHELXL-9752 within OLEX2. All non-hydrogen atoms were
refined anisotropically. Hydrogen atoms were placed using a
riding model and included in the refinement at calculated posi-
tions. Acidic hydrogen atoms were placed using difference map
method.
Crystal data and refinement details are summarised in
Table S2.†
This was carried out essentially as described earlier.25 Briefly,
it included testing of the compounds in 96 clear bottomed,
sterile, microtitre plates (Corning, New York, USA). The outer
perimeter wells were filled with 200 μL of sterile, distilled water
to prevent evaporation from the experimental wells during incu-
bation. 100 μL of 7H9 medium was added to all wells except
those of column 2. 100 μL of the compound(s) to be tested was
added to column 2. This represented the highest concentration of
the compound(s). 100 μL of the compound(s) was added to
column 3 followed by serial dilutions of the compounds from
columns 3–10. Column 11 served as the control with no added
drug. 100 μL of the cell suspension (1 : 50 dilution of the cell
suspension prepared above) was added to all wells and the plate
was incubated at 37 °C for 5 days. 50 μL of Alamar Blue [Invi-
trogen Corporation, Carlsbad, CA, USA; prepared as a 1 : 1 (v/v)
mixture with 10% Tween-80 (Sigma)] was added to the first
control well and observed after 12 h. If the colour changed from
blue to pink, Alamar Blue was added to all the wells and visual
Screening of the compounds for antimycobacterial activity
The hydrazones and their Cu(II)-complexes were screened in the
Department of Animal Sciences, School of Life Sciences, Uni-
versity of Hyderabad.
Mycobacterial strain and growth conditions
Mycobacterium tuberculosis ATCC 27294 was grown in Middle-
brook 7H9 broth medium (pH 7.0, Difco, Detroit, MI, USA)
containing 10% (v/v) albumin-dextrose-catalase (ADC; Difco)
enrichment and 0.2% glycerol and maintained with shaking at
150 rpm at 37 °C.
Iron-regulated growth was performed in Proskauer and
Beck medium (pH 6.8) with iron added at 0.02 μg Fe per mL
9200 | Dalton Trans., 2012, 41, 9192–9201
This journal is © The Royal Society of Chemistry 2012

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Acknowledgements
A. J. and A. K. D. K. thank Dr Adrian Whitwood and Dr Robert
Thatcher for help with the X-ray crystal structure determinations,
Karl Heaton for recording ESI mass spectra and the Medical
Research Council and the Holbeck Trust for supporting a
Dorothy Hodgkin Post Graduate Award. P. D. and S. P. are thank-
ful to Mr P. A. Inamdar and ISTRA for providing funds for this
project. K. V. acknowledges the Department of Biotechnology
(DBT) for a fellowship and M. S. thanks the Department of
Science and Technology for financial assistance for the project
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