Davies5 1 Department of Physics and Chemistry, Sevastopol National Technical University, Crimea, Ukraine;2Department of Biophysical and Medical Physics, Kharkov National University, Ukra
Trang 1Structure–activity relation for synthetic phenoxazone drugs
Evidence for a direct correlation between DNA binding and pro-apoptotic activity Alexei N Veselkov1, Vladimir Ya Maleev2, Evgenie N Glibin3, Leonid Karawajew4and David B Davies5
1
Department of Physics and Chemistry, Sevastopol National Technical University, Crimea, Ukraine;2Department of Biophysical and Medical Physics, Kharkov National University, Ukraine;3Department of Chemistry, St Petersburg State Technological University, Russia;4Department of Haematology, Oncology, and Tumour Immunology, Robert-Ro¨ssle Clinic, Charite´, Humboldt-University
of Berlin, Germany; 5 School of Biological and Chemical Sciences, Birkbeck College, University of London, UK
The structure–activity relations of a series of synthetic
phenoxazone drugs with aminoalkyl side chains of variable
length and different terminal groups were investigated by
examining their biological activity and DNA complexation
affinity Biological activity was determined from their ability
to induce apoptosis and cell cycle perturbations (activation
of cell cycle checkpoints) using the human malignant
MOLT-3 cell line The thermodynamic parameters of drug–
DNA complexation were determined by differential
scan-ning calorimetry By comparing the activities of compounds
with different terminal groups (amino, dimethylamino and
diethylamino), we found that the existence of a terminal
dimethylamino group in the alkylamino side chain is an important factor for anti-tumour activity Minor modifica-tions in the dimethylaminoalkyl side chain (e.g elongation
by one methylene group) led to notable changes in both the anti-tumour activity and DNA-binding properties of the drug, providing unambiguous evidence of a marked struc-ture–activity relation
Keywords: apoptotic activity; differential scanning calori-metry (DSC); drug–DNA binding; phenoxazone drugs; structure–activity relationship
Many anti-tumour drugs are thought to exert their cytotoxic
effect through DNA-specific interactions, resulting in
geno-toxic stress and consequent induction of programmed cell
death (apoptosis) [1–3] Clinically important drugs belong to
structurally different families, reflecting the range of possible
anchoring mechanisms and their different activities with
nucleic acids [4] These drugs include intercalators, groove
binders, and those binding with a combination of the two
mechanisms The antibiotic actinomycin D consists of a
planar phenoxazone chromophore with two identical side
chains consisting of pentapeptide lactone rings It is an
example of an aromatic drug with both intercalative and
groove-binding mechanisms of complexation with DNA
Although the structural significance of the phenoxazone
chromophore is well established, the role of the side chains is
still under discussion One hypothesis suggested [5] that
actinomycin D may be characterized as an
ionophore-antibiotic, because it shows significant complexation of the
side chains with sodium ions but not with potassium ions;
this, in turn, suggested that the activity of actinomycin D
may only be manifested when the pentapeptide rings form
complexes with sodium ions As crown ethers are well
known to exhibit selective binding with metal cations [6], this hypothesis was tested on actinomycin D derivatives with crown-like structures in the side chains [7] None of the derivatives showed significant activity with human leukemia MOLT-3 cell lines, even though the crown side groups had different specificities for metal cation binding, different lengths of spacers in the side chains, etc [7] On the other hand, it was found that the rather simple dimethyl-aminoalkylamidophenoxazone derivative (n¼ 3, Fig 1) chosen as a standard was reasonably active at the 1 lMlevel [7] Interestingly, development of the aminoalkylanthra-quinone family of anti-tumour drugs resulted in a novel synthetic drug, mitoxantrone, with improved characteristics (less cardiac toxicity) compared with natural anthracycline antibiotics such as doxorubicin and daunomycin [8,9] The role of alkylamino side groups in a number of fluorenone derivatives has also been investigated in terms of the structure–antiviral activity of these drugs [10–12]
This work focuses on the role of aminoalkyl side chains in the biological activity and drug–DNA complexation pro-perties of a series of synthetic phenoxazone compounds with aminoalkyl side chains of different length and with different terminal functional groups The biological activity of each drug was investigated in terms of induction of apoptosis and cell cycle perturbations (activation of cell cycle checkpoints) using the human malignant MOLT-3 cell line This cell line shows wild-type status of the tumour suppressor gene p53 [13] Given the well-known role of the p53 protein as a key sensor of DNA damage, this cell line is appropriate for investigating the biological effects of drugs with specific binding to DNA It was found that the series of synthetic phenoxazone compounds with dimethylaminoalkylamido side chains provided the necessary conditions for optimum
Correspondence to D B Davies, School of Biological and Chemical
Sciences, Birkbeck College, University of London, Malet Street,
London WC1E 7HX, UK.
Fax: + 44 207 631 6246, Tel.: + 44 207 631 6238,
E-mail: davidbry@ndavies.co.uk
Abbreviations: DSC, differential scanning calorimetry;
FITC, fluorescein isothocyanate; PI, propidium iodide.
(Received 3 January 2003, revised 29 July 2003,
accepted 5 September 2003)
Trang 2biological activity so that meaningful biophysical studies
could be undertaken with a view to understanding the basis
of the anticancer activity The thermodynamic parameters
of complexation of the drugs with DNA were determined by
differential scanning calorimetry (DSC), which is a
con-venient and informative method for obtaining direct data
on the thermal stability of drug–DNA complexes Such
information is crucial to the rational design of drugs and for
determing the molecular basis of hetero association with
other aromatic ligands and their competitive binding with
DNA [14,15]
The investigations show that minor modifications in the
aminoalkyl side chain of synthetic phenoxazone derivatives
(e.g elongation by one methylene group) lead to
consider-able changes in both their anti-tumour activity and
DNA-binding properties, providing unambiguous evidence of a
marked structure–activity relation
Materials and methods
Drugs and DNA
A series of actinomycin derivatives with
dimethyl-aminoalkyl side chains with different numbers of
methylene groups (CH2)n, n¼ 2, 3, 4, and 5 (Fig 1) were
used to investigate the effect of molecular structure on
drug-DNA complexation The phenoxazone derivatives
were synthesized as described previously [16,17] and
characterized by IR, UV and 1H NMR spectroscopy
[16–18] All of the derivatives gave similar experimental
values for absorption coefficients at k¼ 400 nm in the
range (1.596–1.603)· 104
M )1Æcm)1 Therefore ligand con-centrations were determined using the molar absorption
coefficient e400¼ 1.6 · 104
M )1Æcm)1 at the isosbestic point of the absorption spectrum The concentrations of
the freeze-dried aromatic compounds determined by
weighing were the same as those determined
spectrophoto-metrically
For cellular experiments a stock solution of each
compound was prepared in dimethyl sulfoxide at a
concen-tration of 1 mM Subsequent dilutions of the drug stock
solutions were made in RPMI 1640 medium (Biochrom,
Berlin, Germany)
Calf thymus DNA (molecular mass > 107Da,
charac-terized by a nucleotide content of AT/GC¼ 1.36 and a
level of hyperchromicity of 38–39% at k¼ 260 nm) was a
gift from Professor D Lando (Institute of Bioorganic
Chemistry, Minsk, Belarus) Calf thymus DNA from Serva
was also used DNA concentrations were determined
spectrophotometrically using a molar absorption coefficient
e260¼ 6.4 · 103
M )1Æcm)1[19] Solutions of DNA and its complexes with drugs were prepared in 0.1MNaCl with a phosphate/drug ratio of 5.1–5.5 The concentration of DNA
in solution was determined spectrophotometrically at
k¼ 270 nm and k ¼ 290 nm after hydrolysis in 6% HClO4 solution [20] and was equal to 0.04–0.05% The corres-ponding molar concentration of DNA phosphates was
in the range (1.4–1.7)· 10)3M Aqueous salt DNA solu-tions (0.1M NaCl) were used in the DSC experiments,
pH¼ 6.5
Cell culture and drug treatment The human leukemia MOLT-3 cell line [13] was obtained from the DSM Cell Culture Bank (Braunschweig, Ger-many) Cells were maintained in RPMI 1640 standard medium containing 2 mM L-glutamine and supplemented with 10% heat-inactivated fetal calf serum (Gibco BRL, Paisley, Scotland, UK) All cultures were free of myco-plasma contamination To assess drug-induced effects, 0.2· 106cells per well were cultured in 24-well microtiter plates (Nunc, Roskilde, Denmark) in standard medium at
37C in a humidified atmosphere of 5% CO2in air [13] Cells were treated with drugs for 20 h
Assessment of drug-induced apoptosis One of the early events of apoptosis is the loss of membrane asymmetry of phospholipids At this early stage, the plasma membrane stays intact, but phosphatidylserine, normally located in the inner leaflet of the membrane, redistributes and appears in the outer leaflet Annexins are a family of proteins that bind to phospholipid membranes in the presence of Ca2+ Annexin V binds specifically to phos-phatidylserine on apoptic cell surfaces and can be used as a marker of apoptosis
To determine the extent of apoptosis, cells were stained with fluorescein isothocyanate (FITC)-conjugated annexin V and propidium iodide (PI) using the annexin V kit (Immunotech, Marseille, France) as recommended by the manufacturer Thereafter, samples were analysed by flow cytometry (FACScan; Becton Dickinson, San Jose,
CA, USA) for the presence of viable (annexin V-negative and PI-negative), early apoptotic (annexin V-positive, PI-negative), and late apoptotic (annexin V-positive and PI-positive) cells The extent of apoptosis was quanti-fied as the percentage of annexin V-positive cells [21] The extent of drug-specific apoptosis (%) was assessed from:
ðdrug-induced apoptosis apoptosis in mediumÞ100
ð100 apoptosis in mediumÞ
ð1Þ where drug-induced apoptosis is the percentage of annexin V-positive cells in the presence of the drugs, and sponta-neous apoptosis in the medium is the percentage of annexin V-positive cells in control samples [22] Cytotoxic activity has been defined using calculated values of drug concentrations at which 50% of lethality (drug-specific apoptosis) is achieved, LC
Fig 1 Chemical structures of the phenoxazone derivatives Act, ActII–
ActV.
Trang 3Assessment of drug-induced cell cycle perturbations
A flow cytometric method developed previously [7] was
used to discriminate cell cycle distribution in subpopulations
of viable and apoptotic cells identified by specific annexin V
staining (annexin V/DNA-staining method) Briefly, cell
samples were first stained with FITC-conjugated annexin V
and consequently fixed by addition of 2 mL ice-cold 70%
ethanol for 1 h at 4C After being washed, the cells were
resuspended in 0.5 mL NaCl/Picontaining 50 lgÆmL)1PI,
pH 7.5 After treatment with 10 lL 10 mgÆmL)1 RNase
(type I-A; Boehringer Mannheim, Mannheim, Germany)
for 30 min at room temperature in the dark, the cells were
analysed by flow cytometry Cell cycle analysis was carried
out usingCELLQUEST(Becton Dickinson) software A total
of 10 000 and 20 000 cells were characterized by flow
cytometry for apoptosis and cell cycle distribution analysis,
respectively All tests were performed in triplicate
DSC
Direct measurement by DSC of heat effects caused by the
melting of DNA and its complexes with drugs results in
determinations of such energy parameters of structural
transition as enthalpy change DH, entropy change DS, free
energy change DG, melting temperature Tmand the interval
of melting DT
The calorimetry experiments were carried using a
differ-ential scanning microcalorimeter (DASM-4, Pushchino,
Moscow Region, Russia) over the working range of
temperatures 40–130C and with a measuring cell volume
of 0.455 mL The constant impulse power in all
measure-ments was 25ÆlW The solution was kept under an excess
pressure of 253 kPa (2.5 atm) to avoid boiling up to 130C
The heating rate of all solutions was 1CÆmin)1 The DSC
baseline was recorded for the aqueous salt solution over the
temperature range studied The heat effect of melting of pure
DNA and ligand–DNA complexes was calculated from the
area under the heat absorption curve with a precision of
± 1% The melting point Tmcorresponds to the value of
temperature at the maximum of the heat absorption curve
The width of the transition interval DT was determined as a
half-width (i.e width at half height) of the heat absorption
curve All values of thermodynamic parameters were
calculated for 1 mol base pairs, taking an average molecular
mass of a DNA base pair as 660 Da
Results
Dose-dependent apoptosis and cell cycle
in the drug-treated leukemia cells
The biological activity of the series of phenoxazone
deriva-tives Act–ActV (Fig 1) was assessed by the annexin/
PI method [7] Fig 2 shows that the dose-dependent
induction of apoptosis depends on the length of the
dimethylaminoalkyl side chain Although all the
phenoxa-zone derivatives induce apoptosis at very high concentrations
(100 lM), only ActII (containing two methylene groups,
n¼ 2, in the side chain) and ActIII (n ¼ 3) are significantly
effective at lower concentrations (10 lM), and only ActII is
effective at the lowest concentrations tested (£ 1 lM; Fig 2)
The same systems of drug-treated cells were examined for cell cycle distributions by the annexin/DNA method [7] Figure 3 shows that the apoptotic effects of the biologically active compounds Act–ActV are associated with cell cycle perturbations, in which similar cell cycle changes, charac-terized by accumulation of cells preferentially in early S-phase and in G2/M-phase, are shown by compounds II, III and IV However, the concentrations at which the drugs are able to induce cell cycle perturbations depend strongly
on the length of the side chain, with ActII being effective at the lowest concentration (1 lM) whereas ActIII and ActIV are only effective after a 10-fold or 100-fold increase in concentration, respectively
To understand further the molecular basis of the structure–activity relation of this series of phenoxazone drugs, the anti-tumour properties of derivatives with different variations (e.g amino and diethylamino) in the terminal groups of the aminoalkyl side chains were inves-tigated (compounds 1–7, Table 1)
DSC study of thermostability of drug–DNA complexes The results of microcalorimetric measurements of the heat absorption curves q(T) for solutions of pure DNA and complexes with actinocin derivatives ActII–ActV are shown
in Fig 4
The area under the curve of heat capacity dependence on temperature, DCp¼ ƒ(T), and the baseline drawn between the temperatures at the beginning (T1) and the end (T2) of the transition, corresponds to the heat change DQ0 (enthalpy change DH at constant pressure P) induced by the thermal transition of the biopolymers [23]:
DQ0¼ DH ¼
ZT2
T 1
The entropy change (DS) is derived by integration of the following equation:
DS¼
ZT 2
T1
DCp
The change in Gibbs free energy (DG) for the melting of DNA and its complexes with ligands may be calculated from the general thermodynamic relation:
The thermostabilities of DNA and its complexes with ligands were investigated using the melting curvesQ(T), derived from the heat absorption curves DCp(T) using the following relation:
where DQðTÞ ¼RT
T 1DCpðTÞ dT is the heat effect measu-red calorimetrically in the temperature range from T1to the current temperature T The melting curves Q(T) obtained from the heat absorption curves q(T) using eqn
5 are shown in Fig 5
The binding of ligands with natural and model nucleic acids results in an increase in T and DT of complexes
Trang 4compared with free nucleic acids [24,25] The melting
enthalpy, DHmelt, of nucleic acid complexes with either
groove binding or intercalating ligands is higher than DHmelt
of pure nucleic acids, whereas the entropy of ligand binding
(DSbind) can have both positive and negative values, which
mainly results from changes in the environment of the
hydrated structure of the ligand– nucleic acid complex
relative to the free nucleic acid [26] The results of
calculations of the heat stability (melting temperatures,
Tm, and intervals of melting, DT) of DNA and its complexes
are presented in Table 2
A quantitative estimate of the binding parameters was obtained by subtracting the values describing the thermal transition of pure DNA from those derived for the drug– DNA complexes [27]: DHbind¼ DH) DH0, DSbind¼
DS – DS0, DGbind¼ DG) DG0 (the zero index relates to pure DNA) The thermodynamic parameters of the endo-thermic melting of DNA and its complexes with drugs, calculated using eqns 2–4 and the binding parameters,
DHbind, DSbind and DGbind, are summarized in Table 2 Differences in interaction of ActII–ActV with DNA can also be estimated using the binding parameters DH ,
Fig 2 Dose-dependent induction of apoptosis by the drugs Act–ActVin leukemic MOLT-3cells Cells were incubated in the presence of different concentrations of the drugs for 20 h at 37 C After incubation, cells were stained with FITC-conjugated annexin V (FL1-H) and PI (FL3-H) before flow cytometric analysis The extent of apoptosis (normalized with respect to spontaneous apoptosis in the absence of drug) was determined
by flow cytometry as described in Materials and methods.
Trang 5DSbind, DGbind per molecule of drug Spectrophotometric
investigation of actinomine–DNA complexes has shown
[28] that intercalation and external binding of ligand with
DNA, characterized by the parameter r (the number of mol
of ligand per mol of base pairs), depend on the ratio of
DNA and ligand concentrations in solution, and at
phosphate/drug ratio¼ 5.5, the value of the parameter r
is 0.33 The relation of DHbind, DSbind, DGbindto r gives
the changes in enthalpy, entropy and free energy of binding
of ActII–ActV to DNA per mol of ligand (Table 3)
Discussion
Examination of the cytotoxic effects in leukemic cells showed that cytotoxic activity (Figs 2 and 3) was a function
of the number (n) of CH2groups in the side chain (Table 1) The results, expressed in LC50units, exhibit a pronounced maximum in cytotoxic activity for n¼ 2 (Fig 6) Hence, the anti-tumour activity of Act–ActV is found to be very sensitive to minor modifications in the side chain of actinomycin D derivatives, indicating a direct correlation
Fig 3 Flow cytometric analysis of the cell cycle perturbations induced by the drugs Act–ActVin MOLT-3 cells Cells were incubated in the presence
of different concentrations of drugs for 20 h at 37 C and analysed by the annexin V/DNA method [7] Cell cycle distributions in subpopulations of viable (dotted lines) and apoptotic cells (solid lines) are presented as histogram overlays.
Trang 6between structure and activity of the drugs It is of interest
that investigations by stopped-flow spectrophotometry of
the relations between binding mode to DNA and the
anti-tumour activity of mitoxantrone, ametantrone and its
derivatives have shown [9] that variations in the structure
of the aminoalkyl side chains of ametantrone analogs had little effect on the kinetic stability of the complexes
It can be seen from Table 1 that a reduction in the cytotoxic effect of the synthetic phenoxazone drugs results from the presence of short side chains (compounds 1 and 2)
or having diethyl (compounds 3 and 4) or amino (com-pounds 5, 6, and 7) groups at the terminal sites of the alkylamino side chains instead of dimethyl groups It follows that the presence of terminal dimethyl groups in the alkylamino side chains in the series of phenoxazone
Fig 4 Heat absorption curves q (JÆs-1) as a function of temperature
(C) for solutions of pure DNA and its complexes with ActII–ActV(after
baseline correction) The value of calibrating impulse (10)5JÆs)1) is
shown for the case of pure DNA, as an example.
Fig 5 Melting curves of calf thymus DNA and its complexes with ActII–ActVin 0.1 M NaCl at pH 6–6.5 DNA concentration is 0.04– 0.05%; DNA phosphate/drug (P/D), 5.1–5.5.
Table 1 Anticancer activity (% drug-specific apoptosis in human
leukemia MOLT-3 cell lines) of symmetrically substituted synthetic
phenoxazone derivatives.
% Apoptosis
1 l M 10 l M 100 l M
Table 2 Thermodynamic data of helix to coil transition of calf thymus DNA and its complexes with ActII/ActVdetermined from DSC meas-urements All thermodynamic parameters are calculated per mol of DNA base pairs DH and DS, as well as DH bind and DS bind values were determined at T ¼ T m Temperatures are given in C, and changes in enthalpy as kcalÆmol)1, entropy as calÆmol)1ÆK)1, and free energy as kcalÆmol)1.
Sample
Helix–coil transition
Drug–DNA complexation
T m DT DH DS DG 293 –DH bind –DS bind –DG bind293
Table 3 Binding parameters for ActII/ActVdrug–DNA complexation, calculated per mol of ligand at r = 0.33 (ratio of moles of bound ligand to moles of base pairs) Values are mean ± average deviation.
Sample
–DH bind
(kcalÆmol)1)
–DS bind
(calÆmol)1ÆK)1)
– DG bind
(kcalÆmol)1)
Trang 7derivatives is an important factor in their anti-tumour
activity
The thermal studies of drug–DNA complexation also
show that different lengths of the aminoalkyl side chains in
the series of Act–ActV phenoxazone drugs results in
different stabilizing effects on the structure of DNA It
can be seen from Table 2 that the stability of all the drug–
DNA complexes is higher than that of pure DNA For
example, as shown in Fig 7, both the melting temperature
Tmand free energy changes due to melting of the complexes,
DGbind, increase nonlinearly with a decrease in the number
of methylene groups in the side chains of the drugs, reaching
maximum at n¼ 2 Thus, the DNA-binding affinity for
ActII (which contains two CH2groups in the side chain and
has maximum biological activity, Fig 6) is much higher
than that of ActIII–ActV (containing more than two CH2
groups in the side chain), indicating that the degree of drug–
DNA complexation and the activity of the drug are related
processes
NMR studies of the self-association of ActII–ActV have
also shown different behavior for ActII compared with the
other phenoxazone drugs [18]; namely, the entropy change during self-association of ActII was appreciably smaller than that of ActIII–ActV, which have longer dimethyl-aminoalkyl side chains This effect is probably due to the differences in electrostatic and hydrophobic interactions in the ActII molecule with short side chains (n¼ 2) compared with ActIII–ActV, which have longer dimethylaminoalkyl side chains (n > 2) but the same charge
Although there are small, systematic changes in the binding parameters of ActIII–ActV with DNA, it is seen that their characteristic energies of complexation are quite similar (in comparison with ActII), and the average binding free energy change DGbindis 0.74 kcal per mol base pairs (Table 2) or 2.25 kcal per mol ligand (Table 3) It appears that the binding enthalpy, DHbind, is mainly responsible for the intercalation type of molecular complexation, whereas hydrogen bonds (as a result of direct contact between the chromophore and GC base pairs) and water bridges may also make a significant contribution The values of the melting entropy, DS, of the complexes are larger than those for pure DNA (Table 2), which is probably due to the more ordered structure of the hydration environment of drug– DNA complexes compared with pure DNA The effect for ActII–DNA complexation is significantly greater than for complexation of DNA with ActIII–ActV
Table 3 shows that DHbind for ActII–DNA complexa-tion per mole of ligand, 14.5 kcalÆmol)1, is larger by
7 kcalÆmol)1than the mean value for DHbindfor complex formation for ActIII–ActV with DNA Assuming that the nature of intercalation with DNA is similar for all the drugs investigated, then the additional enthalpy of com-plexation found for ActII–DNA may be due to other types
of interactions in this system, e.g the direct contact between cationic groups of the drug and the sugar– phosphate backbone of DNA This is currently being investigated
In summary, both the biological activity of synthetic phenoxazone derivatives and the thermodynamic properties
of drug–DNA complexation revealed a direct and quite marked structure–activity relation, in which significant changes occur with variation of only one methylene group
in the dimethylaminoalkyl side chains Synthetic phenoxa-zone drugs provide an important series of molecules for investigating structure–activity relations They also provide some of the basic molecular requirements for the search for compounds of greater biological potency and efficacy
Acknowledgements
This work was supported, in part, by INTAS (grant No
INTAS-97 31753).
References
1 Au, J.L., Panchal, N., Li, D & Gan, Y (1997) Apoptosis: a new pharmacodynamic endpoint Pharm Res 14, 1659–1671.
2 Chresta, C.M., Arriola, E.L & Hickman, J.A (1996) Apoptosis and cancer chemotherapy Behring Inst Mitt 232–240.
3 Lowndes, N.F & Murguia, J.R (2000) Sensing and responding to DNA damage Curr Opin Genet Dev 10, 17–25.
4 Graves, D.E & Velea, L.M (2000) Intercalative binding of small molecules to nucleic acids Curr Org Chem 4, 915–929.
Fig 6 Cytotoxic activity, expressed in LC 50 units, of Act–ActVwith
different numbers (n) of CH 2 groups in the side chains LC 50 units are the
calculated drug concentrations at which 50% of lethality (drug-specific
apoptosis) is achieved in the human leukemia MOLT-3 cell line.
Fig 7 Dependence of both the melting temperature, T m , and binding
free energy, DG bind , of helix to coil transition of DNA–ActII/ActV
complexes on the number of methylene groups (n) in the ligand side
chains.
Trang 85 Horti, A., Glibin, E & Nesterov, V (1992) Retention behavior of
crown ethers and actinomycin D in reversed-phase HPLC
Chro-matographia 34, 155–158.
6 Lindoy, L.F (1989) The Chemistry of Macrocyclic Ligand
Com-plexes Cambridge University Press, Cambridge.
7 Karawajew, L., Glibin, E.N., Maleev, V., Ya, Czerwony, G.,
Dorken, B., Davies, D.B & Veselkov, A.N (2000) Role of
crown-like side chains in the biological activity of
substituted-phenox-azone drugs Anti-Cancer Drug Design 15, 331–338.
8 Zee-Cheng, R.K.-Y & Cheng, C.C (1978) Antineoplastic agents.
Structure-activity relationship of bis (substituted
aminoalk-ylamino)-anthraquinones J Med Chem 21, 291–298.
9 Denny, W.A & Wakelin, L.P.G (1990) Kinetics of the binding of
mitoxantrone, ametantrone and analogues to DNA: relationship
with binding mode and anti-tumour activity Anti-Cancer Drug
Des 5, 189–200.
10 Witkowski, W., Hoffman, S., Veckenstedi, A., Skolziger, R.,
Luck, G & Zimmer, Ch (1979) Structure-activity relationships of
antiviral bis-basic fluorenone base-pair analogs Acta Biol Med.
Germ 38, 733–737.
11 Bischoff, G., Gromann, U., Lindau, S., Skolziger, R., Witkowsky,
W., Bohley, Ch, Naumann, S., Sagi, J., Meister, W.-V &
Hoff-mann, S (2000) A structure-function study of nucleic
acid-fluor-enone complexes J Biomol Struct Dyn 18, 199–208.
12 Bischoff , G & Hoff mann, S (2002) DNA-binding of drugs used in
medical therapies Curr Med Chem 9, 321–348.
13 Cai, Z., Lin, M., Wuchter, C., Ruppert, V., Dorken, B., Ludwig,
W.D & Karawajew, L (2000) Apoptotic response to
homo-harringtonine in human wt p53 leukemic cells is independent of
reactive oxygen species generation and implicates Bax
transloca-tion, mitochondrial cytochrome c release and caspase activation.
Leukemia 15, 567–574.
14 Davies, D.B., Veselkov, D.A., Djimant, L.N & Veselkov, A.N.
(2001) Hetero-association of caffeine and aromatic drugs and their
competitive binding with a DNA oligomer Eur Biophys J 30,
354–366.
15 Davies, D.B., Veselkov, D.A & Veselkov, A.N (2002) NMR
determination of the hetero association of phenanthridines with
daunomycin and their competitive binding to a DNA oligomer.
Eur Biophys J 31, 153–162.
16 Glibin, E.N., Plekhanova, N.G., Ovchinnikov, D.V &
Kor-shunova, Z.I (1996) Synthesis of analogues of actinomycin XX.
Amides of actinocin containing crown ethers Zhurnal
Orga-nicheskoi Khimii 32, 406–408.
17 Glibin, E.N., Ovchinnikov, D.V & Plekhanova, N.G (1997) Synthesis of analogues of actinomycin XXI Benzo-crown-4-car-boalkyl amides of actinocin Zhurnal Organicheskoi Khimii 33, 1573–1576.
18 Veselkov, D.A., Lantushenko, A.O., Davies, D.B & Veselkov, A.N (2002) The self-association of the antibiotic actinocil-bis (3-dimethylaminopropylamine) in aqueous solution: a 1 H NMR analysis Rus J Bioorg Chem 28, 342–347.
19 Muller, W & Crothers, D.M (1975) Interaction of heteroaro-matic compounds with nucleic acids 1 The influence of hetero-atoms and polarizability on the base specificity of intercalating ligands Eur J Biochem 54, 267–277.
20 Spirin, A.S (1958) Spectrophotometric determination of total quantity of nucleic acids Biochemistry (Rus.) 23, 656–662.
21 Karawajew, L., Ruppert, V., Wuchter, C., Kosser, A., Schrappe, M., Dorken, B & Ludwig, W.D (2000) Inhibition of in vitro spontaneous apoptosis by IL-7 correlates with upregulation of Bcl-2, cortical/mature immunophenotype, and better cyto-reduc-tion in childhood T-ALL Blood 98, 297–306.
22 Karawajew, L., Wuchter, C., Ruppert, V., Drexler, H., Gruss, H.J., Dorken, B & Ludwig, W.D (1997) Differential CD95 expression and function in T and B lineage acute lymphoblastic leukemia cells Leukemia 11, 1245–1252.
23 Marky, L.A & Breslauer, K.J (1987) Calculating thermodynamic data for transition of any molecularity from equilibrium melting curves Biopolymers 26, 1601–1620.
24 Karapetian, A.T., Permogorov, V.I., Frank-Kamenetskii, M.D & Lasurkin, Y.S (1972) Thermodynamic investigation of the DNA complexes with dyes Mol Biol 6, 867–874.
25 Bischoff, G., Gromann, U., Lindau, S., Meister, W.-V & Hoff-mann, S (1999) Spectroscopic and thermodynamic study of taxol nucleic acid complexes Nucleosides Nucleotides 18, 2201–2217.
26 Lane, A.N & Jenkins, T.C (2000) Thermodynamics of nucleic acids and their interactions with ligands Q Rev Biophys 33, 255–306.
27 Schmitz, H.-U., Hu¨bner, W & Ackermann, T (1995) Thermo-dynamic and spectroscopic characterization of the berenil and pentamidine complexes with the dodecanucleotide d(CGCGA TATCGCG) 2 Z Naturforsch 50c, 263–274.
28 Krivtsova, M.A., Moroshkina, E.B., Glibin, E.N & Frisman, E.V (1982) DNA interaction with low molecular ligands of dif-ferent structure II Complexes of DNA with actinomine and its analogues Mol Biol 16, 149–155.