Nickel(II) 2,6-diacetylpyridine bis(isonicotinoylhydrazonate) and bis(benzoylydrazonate) complexes: Structure and antimycobacterial evaluation. Part XI

Article abstract of DOI:10.1016/S0968-0896(01)00133-X

The reaction of 2,6-diacetylpyridine (dap) and isonicotinoyl- or benzoylhydrazide leads to bishydrazones H₂dapin (1a) and H₂dapb (1b), respectively. The condensation can either take place as a bimolecular kinetic process between the two reactants or as a monomolecular metal-templated synthesis in the presence of nickel(II) ions. In the latter case the reaction products are charged 2,6-diacetylpyridine bis(hydrazone) nickel(II) complexes, which can be easily deprotonated to neutral hydrazonates. Diffractometric analysis of one of these [Ni(dapb)]₂ (8b) has shown a binuclear structure with two octahedral nickel(II) ions bridged by two helicoidal dap (bishydrazonates) in a spheroidal structure of C_2V symmetry. The synthesized complexes 8 are promising as antimycobacterial agents against M. tuberculosis H37Rv. In particular, 8b displays significant activity (MIC = 0.025 μg/mL) 10-fold higher than rifampin and equal to isoniazid, while its ligand is ineffective. Compound 8b is also capable of reducing HIV-induced cytopathogenic effect in human T₄lymphocytes.

Full text of DOI:10.1016/S0968-0896(01)00133-X

Bioorganic & Medicinal Chemistry9 (2001) 2203–2211
Nickel(II) 2,6-Diacetylpyridine Bis(isonicotinoylhydrazonate) and
Bis(benzoylhydrazonate) Complexes: Structure and
Antimycobacterial Evaluation. Part XI
B. Bottari,a R. Maccari,a F. Monforte,a R. Ottana,a M. G. Vigorita,a,* G. Bruno,b
F. Nicolo,b A. Rotondob and E. Rotondob
a
`
`
Dipartimento Farmaco-chimico, Facolta di Farmacia, Universita di Messina, Vl. SS. Annunziata, 98168 Messina, Italy
b
`
`
Dipartimento Ch. Inorg., Anal., Ch.-Fis., Facolta di Scienze MMFFNN, Universita di Messina, Salita Sperone 31,
98166 Messina, Italy
Received 9 January2001; accepted 23 April 2001
Abstract—The reaction of 2,6-diacetylpyridine (dap) and isonicotinoyl- or benzoylhydrazide leads to bishydrazones H2dapin (1a)
and H2dapb (1b), respectively. The condensation can either take place as a bimolecular kinetic process between the two reactants or
as a monomolecular metal-templated synthesis in the presence of nickel(II) ions. In the latter case the reaction products are charged
2,6-diacetylpyridine bis(hydrazone) nickel(II) complexes, which can be easily deprotonated to neutral hydrazonates. Diffractometric
analysis of one of these [Ni(dapb)]2 (8b) has shown a binuclear structure with two octahedral nickel(II) ions bridged bytwo heli-
coidal dap (bishydrazonates) in a spheroidal structure of C2V symmetry. The synthesized complexes 8 are promising as anti-
mycobacterial agents against M. tuberculosis H37Rv. In particular, 8b displays significant activity (MIC=0.025 mg/mL) 10-fold
higher than rifampin and equal to isoniazid, while its ligand is ineffective. Compound 8b is also capable of reducing HIV-induced
cytopathogenic effect in human T4 lymphocytes. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
applications.9,10 In manycases coordination has been
used to confer antibacterial and antiproliferative prop-
erties to different INH derivatives.11ꢀ16 In particular it
was reported that Cu2+ improved the uptake of ISNEs
into both susceptible and resistant mycobacteria,17
whose emergence is greatlyinvolved in the current
recrudescence of TB world-wide, TB in turn being
greatlyinvolved in AIDS pandemic.
In the last few years a renewed interest in metal-based
therapyhas been raised: in fact, on coordination,
bioactive ligands might improve their bioactivitypro-
files, while inactive ligands mayacquire pharmacologi-
cal properties.1ꢀ6 In addition, metal-coordination is one
of the most efficient strategies in the design of reposi-
tory, slow-release or long-acting drugs.7 Furthermore,
metal complexes have gained importance as enzyme
inhibitors: in fact, theycan either stronglyattach to
enzymes (through covalent and ionic bonds) preventing
substrate interaction or perturbing the active site, or else
disturb metals that are essential for enzymatic action.8
Selectivityand potencyof INH against M. tuberculosis has
so far remained unattainable. However, its efficacyresults
18,19
greatlyreduced against resistant mycobacteria.
In the last years we have tried to extend the spectrum of
action of the drug byfunctinalizations of INH, aimed to
increase its lipophilic character, achieving some
interesting results.20ꢀ22
The metal coordination capabilityof isonicotinoly-
hydrazide (isoniazid, INH), primary antituberculous
drug, and its hydrazones (ISNEs) has been widely
exploited for manybiochemical and pharmacological
More recentlywe have addressed our interest to metal
coordination as a strategyto ameliorate lipophilicity. In
fact, coordination, locking the polar electronegative
atoms in the inner core around the metal and confining
the apolar residues in an external lipophilic envelope,
*Corresponding author. Tel.: +39-090-6766-469; fax: +1-39-090-355-
613; e-mail: vigorita@pharma.unime.it
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00133-X

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B. Bottari et al. / Bioorg. Med. Chem. 9 (2001) 2203–2211
should favour diffusion through biomembranes.23,24 In
this perspective a number of copper(II), nickel(II) and
cobalt(II) complexes of active ISNEs of stoichiometry
[MeL2(H2O)2], endowed with antimycobacterial activity
comparable to rifampin (RMP), have been pre-
pared;25,26 according to our suggestion theymight act as
inside cell repositories for the active ligands.
hydrazonates where the negatively charged oxygens are
27ꢀ30
firmlybonded to the metal.
The reaction of H2dapin and H2dapb with NiCl2 in
methanol (1:1 molar ratio), as well as the NiCl2 tem-
plated reaction of dap with INH or BH (1:2 molar
ratio), leads to soluble charged species (Scheme 1).
Evaporation of the solvent allows the recovering of
mixtures whose composition shows small but significant
differences for each preparation.
In pursuing this search, in the present paper we describe
the preparation and the antimycobacterial activity of
the 2,6-diacetylpyridine bis(isonicotinoylhydrazone)
H2dapin (1a) and its isostere bis(benzoylhydrazone)
H2dapb (1b): moreover, we prepared nickel(II) com-
plexes of these well-known versatile ligands,27,28 with
the objective to extend the lipophilic external envelope
of complexes (Fig. 1).
In fact, the crude products obtained in different experi-
ments, if redissolved in methanol, show equivalent con-
ductances ranging from 145 to 160 ohmꢀ1 molꢀ1 cm2,
values consistent with the existence of both mono- and
di-positivelycharged species in equilibrium (Scheme 1).
Accordingly, the IR spectra of all the samples show
medium NH stretchings centred at 3320–3300 cmꢀ1 and
two verystrong stretching vibrations at 1680 and 1660
cmꢀ1, respectively, attributed to the uncoordinated and
nickel(II) coordinated carbonyl groups, their relative
intensitybeing different in each sample.
The antimycobacterial in vitro activity against
M. tuberculosis H37Rv of the two ligands and their NiII
chelates has been evaluated in comparison with
established anti-TB drugs.
In addition, taking into account the aim of our con-
tinuing search on new INH-analogues, complex 8b was
assayed for anti-HIV-1 activity, which is also reported
in this note.
These data are consistent with the existence of competi-
tion for NiII coordination sites between the bishy-
drazone carbonyl oxygens, chloride ions and eventually
solvent molecules (Scheme 1). During each preparation,
equilibria can be driven towards mono- (3, 5, 6) or di-
positively( 2) charged species bysubtle unwanted chan-
ges of the experimental conditions, occurring probably
during the evaporation of the solvent.
Results and Discussion
Chemistry
The reaction of 2,6-diacetylpyridine (dap) with INH or
benzoylhydrazide (BH) in 1:2 molar ratio in methanol
at room temperature quantitativelyleads to the forma-
tion of bishydrazones H2dapin (1a) and H2dapb (1b).
When the Ni2+ templated synthesis is carried out with
an excess of hydrazide (Ni2+/dap/hydrazide 1:1:4), only
neutral enolate complexes (7, 8) are obtained, since the
coordinated hydrazones are quantitatively deprotonated
bythe unreacted hydrazide. It is known, in fact, that
coordination to transition metals mayincrease the acid
dissociation constant of some hydrazones by 105 to 108
factor.31,32
When the condensation between dap and hydrazides is
carried out in the presence of Ni2+, the reaction takes
place within the coordination sphere bya metal-tem-
plated first-order kinetic process (see Kinetics) and neu-
tral or charged NiII complexes of 1a and 1b are formed
(Scheme 1).
With deprotonated ligands, besides pentagonal bipyr-
amidal, other coordination geometries have been
observed where the hydrazonates lack the planar array
to give rise to dinuclear or sometimes tetranuclear
highlysymmetric structures, byhelicoidal wrapping. A
delicate balance of electronic and steric effects can drive
the mononuclear pentacoordinate towards polynuclear
species or viceversa.27,28,33,34
2,6-Diacetylpyridine has often been used to synthesize
hydrazonic ligands.27 The reaction of these ligands with
chlorides of the first row transition metals usuallygives
pentagonal bipyramidal complexes, in which the five
equatorial coordination sites are occupied byhdyra-
zones in a planar arrangement of four adjacent penta-
dentate rings. Two molecules of solvents or two chloride
ions usuallyoccupythe two residual apical sites of the
bipyramid.28ꢀ30
Even though the pentacoordination has often been
observed in these complexes, it must be reminded that
the two oxygen atoms of the carbonyl groups are rather
weak donors whose coordination is favoured bythe
planar arrangement of the hydrazone structure and by
the chelate effect. Thus, in solution carbonyl displace-
ment equilibria driven bythe metal and/or bythe nature
of hydrazone acyl group, must be expected. Alternatively
these structures can be stabilised bydeprotonation to
Figure 1.

B. Bottari et al. / Bioorg. Med. Chem. 9 (2001) 2203–2211
2205
On the other hand, the reaction of 1a and 1b with
Ni(CH3COO)2 in methanol (Ni2+/dap/hydrazide 1:1:2),
leads to quantitative formation of enolates 7 and 8,
since the acetate ion acts as a base strong enough to
quantitativelydeprotonate the bishydrazones.
bythe plots of kobs versus hydrazide (Fig. 2) are
reported in Table 1.
The reaction of dap/hydrazide/nickel(II) in 1:1:1 ratio
takes place in two different kinetic stages. We attri-
bute the first one, too fast to be measured, to the
formation of a ternarycomplex in accordance with
the equation:
The dinuclear structure of 8b was unambiguously
8a could
shown bythe X-raydiffractometric analysis;
not be obtained as crystals suitable for the diffracto-
metric analysis. The poor solubility of the latter com-
plex prevented its spectroscopic characterization;
however, the lack of NH and C¼O stretchings in the IR
spectrum proved that 8a, similar to 8b, is an enolate.
2þ
Ni2þ þ dap þ hydrazide ! ½NiðdapÞðhydrazideފ
The second stage, which we have measured and repor-
ted in Tables 1 and 2 as k1, is a rather slow first-order
kinetic process which can be conceivablyattributed to
an intramolecular condensation. According to this
interpretation, the reactants, held in close proximityby
the metal, are forced to react within the coordination
sphere of nickel(II) in a single first-order kinetic stage by
a metal-templated synthesis. The ‘matchmaking’ role
Kinetics
The reaction between dap and INH or BH takes place
in methanol as a second-order single kinetic stage. The
s
second-order rate constants, k2 (molꢀ1 ꢀ1), determined
Scheme 1. For simplicitythe coordinated molecules of solvent have not been reported in the Scheme.

2206
B. Bottari et al. / Bioorg. Med. Chem. 9 (2001) 2203–2211
2+
played by the metal in these reactions recalls that of a
rudimentaryenzyme, but, of course, the strong metal–
ligand interaction does not allow any catalytic cycle.35
intermediate ternarycomplexes [Ni(dap)(hydrazide)]
above described.
Crystal structure of [Ni(dapb)]2 (8b)
If the same condensations are performed byadding an
excess of hydrazide to the 1:1 molar ratio solution of
nickel(II)/ dap, a two terms kinetic law is observed:
The crystal structure of compound 8b consists of dis-
crete binuclear molecules of [Ni(dapb)]2 in an I-non-
standard monoclinic packing. The binuclear complex
possesses a C2 axis of symmetry which coincides with a
binarycrsytallographic axis; consequentlythe two
ligands are coordinated to nickel atoms in the same
way. A view of the molecular structure is shown in Fig-
ure 3a and b together with the atomic numbering
scheme. Each ligand, that in principle could act as pen-
tadentate bymeans of Z2-coordination of pyridine
nitrogen atom (N1) is able to exhibit an esa coordina-
tion mode.
kobs ¼ k1 þ k2 ½hydrazideŠ
The first-order term k1 (sꢀ1) accounts for the metal-
templated synthesis, the second-order term k2 (sꢀ1
molꢀ1) for the bimolecular nucleophilic attack of the
‘free’ hydrazide onto co-ordinated dap. The two differ-
ent contributions are clearlyevidenced bythe intercept
and bythe slope of the plots of kobs versus hydrazide,
respectively(Tables 1 and 2, Figure 2).
The k1 values calculated bymeans of the intercept of the
plots agree, within the range of experimental error, with
those calculated from the pure first-order reaction of the
The two metal centres show a distorted octahedral geo-
metry; the distortions are evidenced by the N2–Ni–O1
and N1–Ni–N2 angles which are 78.62(7)ꢁ and 76.22(7)ꢁ
Figure 2. Plots of kobs (sꢀ1) versus hydrazide concentration for the condensation of dap and hydrazide in the presence and in the absence of Ni2+
.

B. Bottari et al. / Bioorg. Med. Chem. 9 (2001) 2203–2211
2207
ꢁ
significantlydifferent from 90 and bythe deviations of
culosis H37Rv (ATCC 27294, susceptible both to RMP
and INH) according to the Tuberculosis Antimicrobial
the O2–Ni–N1 [153.42(6)] and O1–Ni–N1 154.77(7)ꢁ
from linearity.
Acquisition Coordinating Facility(TAACF) anti-
39ꢀ41
tuberculosis drug discoveryprogram.
are collected in Table 3.
The results
The Ni. . .Ni separation of 3.0933(8) A is comparable
with those found in other related binuclear complexes
reported in the April 2000, 5.19 release of the CSD;36 it
is also comparable with the value reported for the
[Ni(dapz)]2 complex37 where the two 2,6-diacetylpyr-
idine-bis(10-phthalazinylhydrazone) (H2dapz) ligands
show the same coordination type toward metal centres.
On the contrary, the very similar 2,6-diacetylpyridine-
bis{[dl-hydroxy(phenyl)acetic] hydrazone} and the 2,6-
diacetylpyridine-bis(octanoylhydrazone) ligands chelate
a single metal centre.29,38
As it was to be expected because of the presence of the
isoniazid moiety, 1a and 8a reach 99% inhibition of M.
tuberculosis H37Rv growth with MIC values 0.39 and
0.78 mg/mL, respectively.
On the contrary, H2dapb (1b) is ineffective (MIC >12.5
mg/mL), in line with our previous finding about hydra-
zone analogues in which isonicotinoyl moiety was
25
replaced byisostere acyl groups.
The molecular packing is essentiallydue to van der
Waals interactions. There are also several inter and
intramolecular contacts involving heteroatoms N and O
less than the sums of the van der Waals radii. Some of
these can be properlydescribed as weak hdyrogen
bonds.
Surprisinglyits nickel(II) binuclear complex 8b displays
verysignificant activity. In fact, its MIC=0.025 mg/mL
is 10-fold lower than that of RMP and reaches INH, so
far the sole established anti-TB drug which possesses
such a MIC value against M. tuberculosis. Instead,
charged mononuclear complexes from the 2,6-diace-
tylpyridine bisbenzoylhydrazone in different mixtures
showed MICs >12.5 mg/mL.
Microbiology
It is worth noting that, for both binuclear complexes 8a
and 8b, no breakdown was evidenced in conditions
mimicking those employed for antimycobacterial assays
(phosphate buffer: pH 6.5/6.8; 37 ꢁC; 15 days).
The antimycobacterial in vitro activity of nickel(II)
chelates 8a and 8b, in comparison with their ligands
H2dapin and H2dapb, has been tested against M. tuber-
A precise estimation of the stabilityconstants of the
complexes in water is precluded bytheir poor solubility.
However, spectrophotometric titrations have allowed us
to establish that the concentration of Ni2+ ion in the
condition of the assays is less than 10ꢀ10 M. It is known
that nickel is generallyan essential nutrient for bacteria,
required as trace elements at nanomolar concentrations,
but at micro- or millimolar concentrations it is
toxic.28,41 However, taken together, our experimental
data rule out the possibilitythat the observed anti-
mycobacterial activity rises from a free metal ion toxic
effect.
Table 1. Pseudo-first-order and first-order rate constants (kobs, sꢀ1
dependence on the concentration of isonicotinoylhydrazide (methanol,
)
25 ꢁC, m=0.1)
104
[dap]
104
[inh]
104
[NiCl2]
104 kobs
k1
(sꢀ1
k2
(sꢀ1
)
)
(molꢀ1 sꢀ1
)
9
2.50
2.50
2.50
2.50
90
180
360
720
—
—
—
—
12.0
23.5
49.2
104
78.0a
>
>
=
—
1.3
2.2
>
>
;
2.50
2.50
2.6
9
2.50
2.50
2.50
2.50
90
180
360
720
2.6
2.6
2.6
2.6
92.0
110
160
240
>
>
=
76.0
>
In addition [Ni(dapb)]2 8b has been submitted to the
anti-HIV-1 in vitro screening in the context of AIDS
Antiviral Screening Program of the US-NCI.42 In the
>
;
aFirst-order rate constant.
preliminarycytotoxicityassayon uninfected human T
lymphocytes it displayed low cytotoxicity (IC50>100
4
Table 2. Pseudo-first-order and first-order rate constants (kobs, sꢀ1
dependence on the concentration of benzoylhydrazide (methanol,
)
Table 3. Antimycobacterial in vitro activity expressed as MIC (mg/
mL)a against M. tuberculosis H37Rv
25 ꢁC, m=0.1)
104
[dap]
104
[bh]
104
[NiCl2]
104 kobs
k1
(sꢀ1
k2
Compdb
% Inhibition
MIC (mg/mL)
(sꢀ1
)
)
(molꢀ1 sꢀ1
)
1a
1b
8a
8b
RMP
INHc
99
65
99
99
0.39
>12.5
0.78
0.025
0.25
0.025–0.05
9
2.50
2.50
2.50
2.50
72
145
290
580
—
—
—
—
13.0
24.1
49.5
>
>
=
—
52
1.0
1.6
>
>
;
106
2.50
2.50
2.50
2.50
2.50
2.50
2.6
2.6
2.6
2.6
2.6
50.0a
63.0
72.2
98.6
9
72
145
290
580
>
>
aMIC is defined as the lowest concentration of compound inhibiting
90% of the inoculum relative to controls.
bCharged mononuclear complexes from 2,6-diacetylpytidine bis(ben-
zoylhydrazone) in different mixtures showed MIC >12.5 mg/mL.
cRef 39.
=
>
>
;
130.0
aFirst-order rate constant.

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B. Bottari et al. / Bioorg. Med. Chem. 9 (2001) 2203–2211
Figure 3. (a) View of the coordination polyhedron around the two equivalent nickel atoms related by the symmetry operation 1=2-x, y,-z. Thermal
ellipsoids are drawn at 30% level. (b) View of the dimer along the crystallographic two-fold axis showing the numbering Scheme of the asymmetric
unit. The bridging N-bonds are represented byshaded lines. Thermal ellipsoids are drawn at 30% level while hydrogen and equivalent atom size is
arbitrary.

B. Bottari et al. / Bioorg. Med. Chem. 9 (2001) 2203–2211
2209
mM). Its capability of protecting infected lymphocytes
from HIV-induced cytopathogenicity reaches 39.07% at
1 mM dose (the activitycriterion for this assayis 50%
protection).
defined as the lowest concentration effecting a reduction
in fluorescence of 90% relative to controls. Rifampin
(RMP) was used as reference drug. GenerallyMIC ꢂ1
mg/mL in a novel compound class is considered an
excellent lead.
Conclusions
Anti-HIV-1 activity
2,6-diacetylpyridine bishydrazones act with respect to
nickel(II) as pentadentate ligands which can be easily
deprotonated leading to enolate complexes. X-raydif-
fractometric analysis shows that nickel(II) 2,6-diace-
tylpyridine bis(benzoylhydrazonate) 8b is a dimer
endowed with an external spheroidal lipophilic structure
which could account for the observed bioactivityof
both complexes 8a and 8b.
The in vitro anti-HIV drug testing system was performed
in the National Cancer Institute Laboratories of
Bethesda (USA) according to reported procedures.42 The
assayinvolves the killing of T 4 lymphocytes by HIV. T4
lymphocytes (CEM cell lines) are exposed to HIV at a
virus to cell ratio (MOI) of approximately0.05. Each
candidate agent, dissolved in DMSO, was added at
varying concentrations ranging from 10ꢀ8 to 10ꢀ4 M.
Uninfected cells treated with the test compound serve as
toxicitycontrol, and infected and uninfected cells with-
out the compound as basic controls. Cultures were incu-
bated at 37 ꢁC in a 5% CO2 atmosphere for 6 days; the
tetrazolium salt, XTT, was added to all wells, and cul-
tures were again incubated to allow formazan colour
development byviable cells. Individual wells were ana-
lysed spectrophotometrically to quantify formazan pro-
duction and in addition were viewed microscopicallyfor
detection of viable cells and confirmation of protective
activity. All tests were compared with positive AZT and
20,30-dideoxycytidine (ddC)- treated controls carried out
at the same time under identical conditions.
In fact, 8a has MIC inferior to 1 mg/mL; its isostere 8b,
obtained from the pharmacologicallyinactive ligand 1b,
proves to be veryeffective against M. tuberculosis
H37Rv reaching the MIC level reported for INH.
Compound 8b is also capable of reducing HIV-induced
cytopathogenic effects in human T4 lymphocytes, with a
favourable therapeutical index. It is known that HIV-
sieropositivityis the strongest risk factor for TB and
other mycobacterial diseases; thus, although the protec-
tion percentage is moderate, this result steps up the
interest in this metalchelate. It is worth designing new
structural modifications to enhance its anti-HIV potency.
In such a test system, activity is expressed as the 50%
effective concentration (EC50) which represents the
concentration of test compound resulting in 50%
reduction of the viral cytopathic effect, whereas the
cytotoxic dose (IC50) represents the concentration of
agent resulting in 50% inhibition of growth of normal
uninfected cells.
In conclusion, 8b could be considered as a novel anti-
mycobacterial lead compound, not related to INH. In
fact, according to TAACF criteria, a novel compound
showing MIC ꢂ 1 mg/mL is considered an excellent
lead. This result is noteworthy, taking into account that
todaydramatic health problems, such as AIDS pan-
demic, multi-drug resistance, etc., have made the search
for new anti-TB drugs even more urgent. It is hopeful
that positive results will be found in the further assays
against single-drug resistant M. tuberculosis and
M. avium complex that are ongoing.
Experimental
Chemistry
Elemental analyses (C, H, N) were performed at Redox
Labs, Cologno Monzese, Milano (Italy).
Microbiology
Antimycobacterial activity. The antimycobacterial in vitro
activitywas assayed according the Tuberculosis Anti-
microbial Acquisition Coordinating Facility(TAACF)
antituberculosis drug discoveryprogram, coordinated
bythe Southern Research Institute (Birmingham, Ala-
bama, USA) under the direction of the National Insti-
tute of Allergyand Infectious Diseases (NIAID).
Physical measurements. IR spectra were recorded as
Nujol mulls in the range 4000–300 cmꢀ1 using CsI disks
on a Perkin-Elmer FT-IR model 1730 spectro-
photometer.
UV–visible spectra were scanned in the thermostated
cell compartment of a Cary219 or a Perkin-Elmer
Lambda 3 spectrophotometer.
All compounds were initiallyscreened against Myco-
bacterium tuberculosis H37Rv (ATCC 27294) at the sin-
gle concentration 12.5 mg/mL in BACTEC 12B medium
A radiometer 4 conductivitybridge provided with a
thermostatated cell was used for the measures of molar
conductivity.
39
using the Microplate Alamar Blue Assay(MABA).
Compounds exhibiting fluorescence are then tested in
the BACTEC 460 radiometric system too.40 Com-
pounds 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
Kinetic experiments
1.5 mL of a methanol solution of dap (5Â10ꢀ4 M)
was thermostated at 25 ꢁC. 1.5 mL of the hydrazide

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B. Bottari et al. / Bioorg. Med. Chem. 9 (2001) 2203–2211
dissolved in methanol at the desired concentration were
added to the dap solution. A similar procedure was
followed for the template reactions; in the latter case
anhydrous NiCl2 (5.5Â10ꢀ4 M) was added to the dap
solution. No significative rate change was observed if
NiCl2 was added to the hydrazide rather than to the dap
solutions. When required pseudo-first-order conditions
were ensured byan at least 20 times excess of hydrazide
with respect to dap. The kinetic calculations were per-
formed using optical densitychanges at 380 nm bya
home-made best fitting procedure. The ionic strength
was maintained constant at the value of 0.1 M byaddi-
tion of anhydrous LiClO4 to the methanol solution. No
significative change in the reaction rate was observed by
addition of 5% of water.
then corrected for Lorentz-polarization effects. Absorp-
tion correction was applied byfitting a pseudo-ellipsoid
to the azimutal scan data of 15 suitable reflections with
high w angles.44
Structure determination. The structure was solved by
standard direct methods and subsequentlycompleted by
a combination of least squares technique and Fourier
Synthesis. All non-hydrogen atoms were refined aniso-
tropically.
Hydrogen atoms were added at calculated positions and
included in the structure factor calculations with a
common thermal parameter (U=0.06 A2), and during
the refinement theywere allowed to ride on their
respective parent carbons. The structure model was
refined byfull-matrix least squares technique, minimising
the function w(Fo2ꢀFc2)2, converging to R ¼
2,6-diacetylpyridine bis(isonicotinoylhydrazone) and
2,6-diacetylpyridine bis(benzoylhydrazone) were pre-
pared according to reported procedures.27,28
2
SjFo ꢀ Fcj=SFo ¼ 0:0311 and R0 ¼ ½SwðFo2 ꢀ Fc2Þ =
2 1=2
SwꢀðFo2Þ Š ¼ 0:061 with the final weighting
2
Preparation of nickel(II) 2,6-diacetylpyridine bis(isonico-
tinoylhydrazonate) 8a and nickel(II) 2,6-diacetylpyridine
bis(benzoylhydrazonate) 8b. A mixture of Ni(CH3-
COO)2 (1 mmol; 0.176 g), 2,6-diacetylpyridine (1mmol;
0.163 g) and isoniazid (2 mmol; 0.274 g) or benzoylhy-
drazine (2 mmol, 0.272 g), each dissolved in 10 mL of
CH3OH, was refluxed under stirring for 10 h. After
cooling the reaction mixture, the product was filtered off
and obtained as pure solid.
Scheme=1=½ꢀ2ðFo2Þ þ ð0:0309ꢃPÞ þ 0:00ꢃPŠ
where
P ¼ ðMaxðFo2Þ þ 2ꢃFc2Þ=3.
The last difference map showed the largest electron
densityresiduals (maximum and minumum range=
ꢄ0.26 eAꢀ3). Neutral-atom scattering factors and
anomalous dispersion corrections were taken into
account.45
Data reduction and structure solutions and drawings
were performed with the SHELXTL-PLUS package,46
while structure refinement and final geometrical calcu-
lations were carried out with SHELXL-9747 and
PARST program48 respectively, on DEC MicroVax/
3400 computer.
Nickel(II) 2,6-diacetylpyridine bis(isonicotinoylhydrazo-
nate) 8a. Yield: 95%. IR (nujol), cmꢀ1: 1600 (C¼N),
1145 (C–O). Anal: C42H34N14O4Ni2. Found: C, 55.12;
H, 3.14; N, 21.31.
Nickel(II) 2,6-diacetylpyridine bis(benzoylhydrazonate)
8b. Yield: 98%. IR (nujol), cmꢀ1: 1590 (C¼N), 1156
(C–O). Anal: C46H38N10O4Ni2. Found: C, 60.19; H,
4.37; N, 15.09.
Acknowledgements
The authors gratefullyacknowledge the Staff of the
TAACF organization for the antimycobacterial assays
and the Staff of US-NCI for the anti-HIV assays. This
work was supported byfinancial assistance from Min-
istero Universita Ricerca Scientifica e Tecnologica
(MURST, Italy) and from Consiglio Nazionale
Ricerche (CNR, Italy).
Crystal structure analysis. Crystal data. C46H38N10O4Ni2.
Mr=3649.14, monoclinic, I2/a, a=15.496(3), b=
16.220(3), c=16.416(3)A, ß=90.21(2)ꢁ, V=4126.05(2)
A3, Z=8, Dc=1.4686 g cmꢀ3, l (MoKa)=0.71073 A,
m=0.797 cmꢀ1, F(000)=1888, T=296 K, R=0.0311
for 3663 independent reflections.
Crystals suitable for X-ray analysis were obtained by
recrystallization from methanol solutions. A crystal of
dimensions 0.20Â0.20Â0.30 mm was used for intensity-
data collection at 296 K with a Siemens R3m/V four-circle
diffractometer using graphite-monochromated MoKa(l
= 0.71073 A) radiation. Accurate unit-cell dimensions
and crystal orientation matrices were obtained from
least-squares refinement of 2y, o, w and jf values of 30
strong reflections in the range 12<2y<30ꢁ. Crystal and
electronic stabilitywas confirmed bythe constancyof
three check reflections measured every100 min of X-ray
exposure. Of 7541 reflections measured bythe o/2y scan
technique, in the 2y range 3.64–5y.12; 3663
(Rint=0.037) having net intensityF >4.0 s(F) were used
in the solution and refinement. The diffraction data
were processed with the learnt-profile procedure43 and
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