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A.N. Veselkov et al. / Journal of Molecular Structure 701 (2004) 31–37
Different mathematical models have been proposed for
interpretation of experimental data of hetero-association of
aromatic drug molecules in aqueous solution [2–5,7–10].
Dimer models are mostly used to interprete spectrophoto-
metric data, i.e. they take into account the formation of self-
aggregates and hetero-association complexes with not more
than two molecules in the stack [3,7]. When comparatively
large concentrations of molecules are used in the NMR
experiment, more general models are considered as they
take into consideration indefinite self- and hetero-associ-
ation of aromatic molecules in solution [8–10]. Such
models consider formation of hetero-complexes with not
more than two hetero-stacks in the dynamic equilibrium in
solution. At the same time investigation of different models
of hetero-association of aromatic molecules based on NMR
data enables to conclude [10], that detailed calculation of
such hetero-complexes results in essential complication of
the procedure of transformation of the initial equations to
the final analytical form for the observed proton chemical
shifts of the molecules in the NMR experiment. The analysis
has shown that introduction of new reactions to the
generalized model [10] to describe the dynamic equilibrium
in solution considerably complicates the form of the initial
equations and, in general, it is impossible to get finite
analytical expressions for the obtained experimental
parameter. As a result, the probability (stochastic) model
of hetero-association of aromatic compounds has been
developed [11,12], which is not associated with functional-
analytical modelling of dynamic equilibrium in solution
and, hence, may be used for analysis of multicomponent
systems of any dimension.
Fig. 1. Structures of novatrone (a) and daunomycin (b).
from D
99.95% D
O and re-dissolved in 0.1 M phosphate buffer in
2
2
4
O, pD ¼ 7.1, containing 10 M EDTA. The
2
concentrations of the stock solutions of the aromatic
molecules were measured spectrophotometrically on
appropriate dilution using the following molar absorption
2
1
21
coefficients: 1 ¼ 8360 M cm (l ¼ 682 nm) for NOV
2
1
21
[15] and 1 ¼ 11; 500 M cm (l ¼ 477 nm) for DAU
1
[16,17]. 1D and 2D H NMR spectra were recorded on a
Bruker DRX spectrometer (500 MHz). NOV showed
limited solubility in aqueous buffered solution and, there-
fore, chemical shifts measurements of non-exchangeable
protons of the aromatic molecules were made as a function
of concentration of DAU at relatively high temperature
T ¼ 318 K (from 3.33 to 0.25 mM), keeping NOV
concentration constant (C
¼ p ¼ 0:68 mM). Tempera-
NOV
0
ture dependences of proton chemical shifts were measured
at constant concentrations of drug molecules in the
temperature range 312–351 K. All NMR measurements
were made in the fast-exchange condition on the NMR
time-scale. Chemical shifts were measured relative to an
internal reference tetramethylammonium bromide (TMA)
and recalculated with respect to sodium 2.2 dimethyl
In this work, the analytical statistical-thermodynamic
model of hetero-association of molecules, taking into
account formation of associated complexes of different
dimensions for reactions of self- and hetero-association
of aromatic molecules in two-component system [8,9],
has been used for analysis of reactions of complexation
of antitumour antibiotics novatrone (NOV) and dauno-
mycin (DAU) in aqueous salt solution. Hetero-association
of antibiotics DAU and NOV have been investigated
2-silapentane-5-sulphonate (DSS), i.e. dDSS ¼ dTMA
þ
3:178 (ppm).
Signal assignments of the non-exchangeable protons of
the drugs were obtained using two-dimensional homo-
nuclear TOCSY, NOESY and ROESY experiments. The
methods of preparation of samples and performance of
the NMR experiments are described in detail elsewhere
[5,14].
1
by 1D and 2D H NMR-spectroscopy (500 MHz);
parameters of complexation of aromatic molecules have
been determined from concentration and temperature
dependences of proton chemical shifts of the interacting
molecules [5,8,9]. The self-association of NOV and DAU
3. Results
The 2D-ROESY spectrum of DAU–NOV mixed sol-
ution (Fig. 2) obtained at initial drugs concentrations
exhibits intermolecular ROE contacts between protons of
novatrone and DAU: H6/H7 NOV-4CH3 DAU and H6/H7
NOV-H1/3 DAU (shown by arrows), which provide
unambiguous evidence of the mutual arrangements of
DAU and NOV chromophores in 1:1 hetero-complex, i.e.
aromatic ring D of DAU (Fig. 1) is situated above an
aromatic ring of novatrone chromophore, containing H6/H7
protons.
molecules has been investigated earlier by 1D- and
1
D- H NMR-spectroscopy under the same experimental
2
conditions [13,14].
2
. Experimental
Antibiotics novatrone (1,4-dehydroxy-5,8-bis [[[2-(2-
hydroxyethyl) amino] ethyl] amino]-9,10-antracenedion)
and DAU (Fig. 1) were purchased from ‘Sigma’ and used
without further purification. The samples were lyophilized
Structural and thermodynamical parameters of hetero-
association between DAU and NOV have been determined,