DNA sequence-specific ligands: XV. Synthesis and spectral characteristics of a new series of dimeric bisbenzimidazoles DB(1, 2, 6, 8, 9, 10, 12)

Article abstract of DOI:10.1134/S1068162016020059

Seven fluorescent symmetric dimeric bisbenzimidazoles DB(n) capable of occupying up to one turn of the double-stranded B-form DNA have been designed and synthesized with the aim to develop DNAdependent enzyme inhibitors. The DB(n) compounds contain four 2

Full text of DOI:10.1134/S1068162016020059

ISSN 1068ꢀ1620, Russian Journal of Bioorganic Chemistry, 2016, Vol. 42, No. 2, pp. 183–190. © Pleiades Publishing, Ltd., 2016.
Original Russian Text © A.A. Ivanov, V.I. Salyanov, A.L. Zhuze, 2016, published in Bioorganicheskaya Khimiya, 2016, Vol. 42, No. 2, pp. 205–213.
DNA SequenceꢀSpecific Ligands: XV. Synthesis and Spectral
Characteristics of a New Series of Dimeric Bisbenzimidazoles
DB(1, 2, 6, 8, 9, 10, 12)1
2
A. A. Ivanovb, V. I. Salyanova, and A. L. Zhuzea,
aEngelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow, 119334 Russia
bResearch Institute of Carcinogenesis, Blokhin Russian Oncological Research Center, Russian Academy of Medical Sciences,
Kashirskoe sh. 24, Moscow, 115478 Russia
Received August 9, 2015; in final form, September 1, 2015
Abstract—Seven fluorescent symmetric dimeric bisbenzimidazoles DB(
turn of the doubleꢀstranded Bꢀform DNA have been designed and synthesized with the aim to develop DNAꢀ
dependent enzyme inhibitors. The DB( ) compounds contain four 2,6ꢀsubstituted benzimidazole fragments
and differ by the length of the oligomethylene linker ( = 1, 2, 6, 8, 9, 10, 12) between the bisbenzimidazole
blocks. The formation of DB( )–doubleꢀstranded DNA complexes and the localization of the ligands in the
n) capable of occupying up to one
n
n
n
DNA minor groove have been confirmed by a number of physicochemical methods.
Keywords: dimeric bisbenzimidazole, minor groove ligand, absorption, fluorescence, circular dichroism
DOI: 10.1134/S1068162016020059
2 1
INTRODUCTION
transcription factors to DNA by recognizing and
occupying specific sites in gene promoters [9–15].
Narrow groove ligands form hydrogen bonds with each
of the DNA strands, by binding them in such a way
that results in the formation of a more stable complex
with a higher melting temperature [16]. In addition,
because the narrow DNA groove is important for the
functioning of a great number of DNAꢀdependent
enzymes and the allosteric regulation of transcription
factors [10, 17], it is the most convenient target for
synthetic therapeutic preparations and compounds
capable of specifically interacting with DNA
sequences [7, 10, 17–23].
Doubleꢀstranded DNA is one of the main biomoꢀ
lecular targets in the development of antitumor prepaꢀ
rations. A great number of antibiotics and antitumor
preparations produce the biological effect through the
interaction with DNA, affecting the replication,
repair, and transcription processes [2–4]. Therefore,
the synthesis of compounds capable of binding to
DNA and having a high binding constant and site
specificity is a promising line of research.
Depending on the mechanism of interaction with
DNA, lowꢀmolecularꢀweight siteꢀspecific compounds
capable of forming noncovalent complexes with
dsDNA can be divided into two groups: intercalators
and compounds that bind either in the narrow or the
broad groove of the double helix. Among the latter
compounds are narrow groove ligands, such as the
antibiotics netropsin and distamycin A, fluorescent
dyes Hoechst 33258 (Ht 33258) and DAPI, as well as
berenil and related diamidines [5–9]. There is a direct
relationship between the intensity of biological activity
of narrow groove ligands and the magnitude of their
interaction with AT sequences of the narrow groove,
which determines the capacity of these compounds to
prevent the binding of topoisomerase I (topo I) and
We have earlier synthesized five dimeric bisbenzimꢀ
idazoles DB(n) whose structure was based on the motif
characteristic for such ATꢀspecific narrow groove
ligand as the dye Ht 33258. The DB( compounds
differed from one another by the number of methylene
groups ( ) of the linker connecting bisbenzimidazole
n)
n
residues [1]. The derivatives of aliphatic dicarboxylic
acids whose oligomethylene chains possess a conforꢀ
mational mobility and a low affinity to the sugar phosꢀ
phate backbone and bases of DNA served as linkers
between two bisbenzimidazole fragments of DB(
n).
The structure of the linker had to allow DB( moleꢀ
n
)
cules to acquire the shape isohelical with the narrow
DNA groove owing to which these compounds could
bind to DNA as bidentate ligands (Fig. 1).
Abbreviations:
lamino)phoshonium hexafluorophosphate; DB(
benzimidazoles; DIPEA, ꢀdiisopropylethylamine; MB,
BOP,
(benzotriazolꢀ1ꢀyloxy)tris(dimethyꢀ
n)
, dimeric bisꢀ
N,N
In this case, both bisbenzimidazole residues
responsible for the high affinity of DB(n) for DNA AT
sequences could be simultaneously in the positions
providing the optimal conditions for the formation of
hydrogen bonds between the N1 atoms of four benzꢀ
imidazole fragments acting as donors and O2 atoms of
monomeric bisbenzimidazole; dsDNA, doubleꢀstranded DNA;
PEG, polyethylene glycol; CLCD, cholesteric liquid crystalline
dispersion.
For XIV, see [1].
Corresponding author: phone: +7 (499) 135ꢀ97ꢀ18; fax: (499)
135ꢀ14ꢀ05; eꢀmail: zhuze@eimb.ru.
1
2
183

184
IVANOV et al.
thymines and/or N3 adenines of DNA, which serve as on the length of the linker led to the idea to synthesize
acceptors of hydrogen bonds [1]. a complete set of DB( with the number of methylene
groups in the linker from 1 to 12.
n)
The design and synthesis of DB(n) were carried out
with the aim to develop new inhibitors of DNAꢀdependent
enzymes, including the DNA methyltransferase Dnmt3a
and DNA topo I. Excessive expression of Dnmt3a is
observed in various tumor types, which suggests an imporꢀ
tant role of this enzyme in their formation. Because a
decrease in the amount of Dnmt3a significantly suppresses
the growth of tumor cells through the reactivation of epigeꢀ
netically silenced tumor suppressor genes, the inhibition of
this enzyme can serve as a basis for the development of
antineoplastic preparations. We have earlier studied the
inhibition of the catalytic domain of the mouse DNA
methyltransferase Dnmt3a using a series of dimeric bisꢀ
benzimidazoles DB(3, 4, 5, 7, 11) [11, 24].
RESULTS AND DISCUSSION
In the present work, we continued the synthesis of
symmetric DB( in order to develop new DNAꢀ
dependent enzyme inhibitors. The synthesis of new
compounds, in addition to the earlier synthesized
DB(3, 4, 5, 7, 11) [1], enabled us to prepare a complete
n)
set of narrow groove ligands of the DB(
the linker length from 1 to 12 methylene groups (
The chemical synthesis of seven new dimeric
DB( is shown in the Scheme 1. 5ꢀ(4ꢀMethylpiperꢀ
azinꢀ1ꢀyl)ꢀ2ꢀnitrophenylamine obtained by the conꢀ
n)
series with
n).
n)
It was shown that DB(3, 4, 5, 7, 11) bind to DNA densation of 5ꢀchloroꢀ2ꢀnitroaniline with 1ꢀmethꢀ
as bidentate ligands. In this case, the inhibitory activꢀ ylpiperazine [25] was reduced by hydrogenation over
ity of these narrow groove ligands toward Dnmt3a, the Adams’ catalyst to 4ꢀ(4ꢀmethylpiperazinꢀ1ꢀ
which is characterized by the IC50 value, nonlinearly yl)benzeneꢀ1,2ꢀdiamine (
depended on the number of methylene groups in the condensed with imido ether dichlorohydrate (II
linker [1, 24]. Compounds DB(3) and DB(11) inhibꢀ obtained by the Pinner reaction from ꢀ[(6ꢀcyanoꢀ
ited the activity of Dnmt3a to a maximum degree, ꢀbenzimidazolꢀ2ꢀyl)methyl]acetamide [1]. The Nꢀ
I) [25]. Diamine (I) was
)
N
1H
which could be explained by the structural features of acetyl derivative of bisbenzimidazole MB(Ac) resultꢀ
the duplex used in the studies since the mutual posiꢀ ing from the oxidative condensation was deacetylated
tion in the duplex of two regions, each composed of by boiling with concentrated HCl to form MB [1]. The
two successive AT pairs, was most optimal for the condensation of MB in DMF with
α
, ꢀalkandicarꢀ
ω
binding of DB(3) and DB(11) in the immediate vicinꢀ boxylic acids (in the ratio 2 : 1) in the presence of
ity of the methylation site (5'ꢀGCGC) [1, Scheme 4]. DIPEA and the condensing reagent BOP gave DB(1,
The dependence of the degree of Dnmt3a inhibition 2, 6, 8–10, 12) with yields of 22.3–49.9%.
NH2
NH2
N
+
CH3
EtO
N
H
NH
N
O
NH2Cl
N
H3C
(I
)
(II)
5
4
4'
5'
N
2''
N
AcOH/EtOH
N
H
3''
N
95°C, 1 h, N
7
2
7'
NH
CH3
N
N
6''
H
H3C
5''
O
MB(Ac)
O
O
N
HCl
, 105°C
conc
20 min
⋅
4 HCl
+
N
HO
OH
N
H
N
(CH2)n
NH2
N
N
H
(
III),
n
= 1, 2, 6, 8, 9, 10, 12
H3C
MB
4
4'
5
5'
BOP, DIPEA, DMF
N
N
N
20°
C, 14 h
2''
⋅
6HCl
N
N
3''
N
H
N
7
N
(CH2)n
NH
NH
7'
H
N
N
H
N
6''
N
H
H3C
CH3
O
O
5''
DB(1)
n
= 1; DB(2)
n
= 2; DB(6)
n
= 6; DB(8)
n
= 8; DB(9)
n
= 9; DB(10)
n
= 10; DB(12)
n
= 12
Scheme 1. Synthesis of symmetric dimeric bisbenzimidazoles DB(
n).
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 42
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DNA SEQUENCEꢀSPECIFIC LIGANDS
185
DNA
Monodentate binding
Bidentate binding
Fig. 1. Schematic representation of two different types of ligand binding in the DNA narrow groove.
The interaction of compounds DB(1, 2, 6, 8–10, between bisbenzimidazole residues. The structure of
12) with DNA was established by several spectral the linker was chosen so as to provide the conformaꢀ
methods. We measured the absorption spectra of these tional mobility and low affinity for the sugar phosꢀ
compounds in the absence and presence of DNA. All phate backbone and DNA bases as well as to allow
spectra had a similar shape. Figure 2 (a, b) shows, as an DB(n) molecules to acquire the shape isogeometric to
example, the spectra of DB(1) and DB(9) in the presꢀ the DNA narrow groove and thus be capable of bindꢀ
ence of DNA at different concentrations (curves 1–3). ing to DNA as bidentate ligands. Should this be the
Increasing the concentration of DNA in the solution case, then both bisbenzimidazole residues responsible
(in terms of bp) led to a hypochromic effect and a for the high affinity of DB(n) for DNA AT sequences
slight shift of the absorption band to the longꢀwaveꢀ would be simultaneously in the position optimal for the
length region of the spectrum. These changes in the siteꢀspecific binding. The presence of the dimeric bisbenꢀ
absorption spectra of DB(
DB( –DNA complexes.
All compounds of the new series of DB(
n
)
indicate the formation of zimidazole structure should lead to a substantial increase
n)
in the affinity of DB( for DNA, as compared with
n)
MB, which has been demonstrated earlier [31].
n) fluoꢀ
resced with an emission maximum at 470–475 nm at
an excitation of 320 nm. Fluorescence spectra in the
absence and presence of DNA were obtained. Figure 3
(a, b) shows, as an example, the fluorescence spectra
of DB(1) and DB(9). The fluorescence intensity in the
presence of DNA increased many times in the case of
The results of tests of the biological activity of the
new DB(n) compounds, which will be carried out in
much the same way as experiments with DB(3, 4, 5, 7,
11) [1], will be reported in a separate paper.
EXPERIMENTAL
DB(
mately 1.5 times with ligands having long linkers. The
fluorescence firing of DB( in the presence of DNA
n) with short and moderate linkers and approxiꢀ
The following reagents were used: DIPEA, BOP,
and tetradecanedioic acid (Fluka, Switzerland); malꢀ
onic and succinic acids (Reanal, Hungary); sebacic,
suberic, and undecanedioic acids (Aldrich, United
States); dodecanedioic acid (Acros Organics, Belꢀ
gium); and dioxane, DMF, glacial AcOH, AcOEt,
n)
also confirms the above conclusion that these moleꢀ
cules form complexes with DNA.
The localization of DB(n) in the complex with
DNA was determined by the CD method, which is
based on the use of CLCDs of DNA. Figures 4a and 4b
show, as examples, the CD spectra of DNA CLCDs in
i
PrOH, CHCl3, NH4OH, and malonic acid (Reakhim,
Russia). The Adams’ catalyst was prepared as
described in [32]. Solutions of substances in organic
solvents were dried over Na2SO4. Solvents were evapꢀ
orated on a rotor evaporator in a vacuum of a waterꢀjet
the absence (curve
DB(9) (curves and
1
3
) and presence of DB(1) and
).
2
A comparison of curves
the CD spectra of complexes of DB(
1
,
2
, and
3
showed that, in
with DNA
pump as a rule at 40–50°С. The substances were dried
n
)
in vacuo over P2O5 and NaOH. The melting temperaꢀ
tures were determined on a Boethius device (Gerꢀ
many) and were not corrected. The hydrogenation
was carried out over the Adams’ catalyst at atmoꢀ
spheric pressure and at room temperature until hydroꢀ
gen absorption terminated. The purity of the resulting
compounds was determined by TLC on Kieselgel 60
CLCDs, there appears a minor positive band in the
absorption region of the ligands (280–335 nm). The
positive values of CD in the region of the absorption of
DB(n) chromophores indicate that the angle between
the plane of the chromophores of the ligand and the
helical axis of the DNA molecule is less than 54
degrees, which is possible only in the case that these
chromophores are localized in one of the DNA
grooves [26–28]. Because, according to the Xꢀray difꢀ
fraction data, Ht 33258 binds to DNA in the narrow
F254 plates (Merck, Germany) in a system iPrOH–
concentrated NH4OH (3 : 1). Substances on chroꢀ
matograms were detected in UV light from the absorpꢀ
tion at 254 nm and/or fluorescence at 365 nm.
groove [29, 30], and DB(n) molecules are the dimers
1H NMR spectra were recorded on an AMXꢀ400
of the Ht 33258 derivative, we assume that they, too,
are localized in the narrow groove of DNA, i.e., are
narrow groove ligands.
device (Bruker, Germany; 400.0 MHz) in DMSOꢀd6
( , ppm; J, Hz) at 32°C (unless otherwise specified);
δ
DB(
n
)
synthesized in this work differ by the length the signal of residual protons of the solvent was used as
= 1, 2, 6, 8, 9, 10, 12) an internal standard. The numbering of hydrogen
of the oligomethylene linker (
n
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 42
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186
IVANOV et al.
(а)
(b)
A
A
1
0.4
0.3
0.2
0.1
0
1
0.4
2
2
3
0.3
0.2
0.1
0
3
300
350
400
450
300
350
400
450
Wavelength, nm
Wavelength, nm
Fig. 2. Absorption spectra of DB(1) (a) and DB(9) (b) in the absence (1) and presence of calf thymus DNA [curves 2, 3; hereafter
the DNA molar concentration (M) is given in bp]; buffer 0.3 M NaCl + 0.002 M Na PO , pH 6.85. The optical path length is
3
4
–6
–6
–6
–6
10 mm. (a) [DNA]: (
2
)
2.75
×
10
×
М
; (
М.
3
)
38.25
×
10 М; [DB(1)]: 10.40
×
10
М
; (b) [DNA]: (
2)
12.75
×
10
М; (3)
–6
–6
53.13
×
10 М. [DB(9)]: 10.60
10
(а)
(b)
500
400
300
200
100
0
900
3
3
800
700
600
500
400
2
300
2
200
100
0
1
1
400
450
500
550
600
400
450
500
550
600
650
Wavelength, nm
Wavelength, nm
Fig. 3. Fluorescence spectra of buffer (
1), DB(1) (a), and DB(9) (b) in the absence (2) and presence of calf thymus DNA (3);
buffer 0.3 M NaCl + 0.002 M Na PO , pH 6.85. Fluorescence excitation was at 320 nm, with slit widths at excitation and emisꢀ
3
4
–6
–5
–6
sion of 5 nm. The optical path length is 10 mm. (a) [DB(1)] 2.61
×
10
М
, [DNA] 4.25
×
10 M; (b) [DB(9)] 2.61
×
10
М,
–5
[DNA] 4.25
× 10 M.
atoms of the piperazine (Pip) and bisbenzimidazole continued for 3.5 h until the termination of hydrogen
(Bim) cycles is given in the Scheme 1. The assignment absorption; in this case, the solution became almost
of signals in 1H NMR spectra of DB(
using the data reported in [33].
n)
was carried out completely bleached. The catalyst was filtered and
washed with glacial AcOH and absolute EtOH. The
filtrate was evaporated in vacuo and reevaporated
MALDIꢀTOF mass spectra were recorded on a
Visionꢀ2000 timeꢀofꢀflight device (ThermoBioanalyꢀ
sis, Great Britain) in the positive ion mode; the matrix
was 2,5ꢀdihydroxybenzoic acid; N2 laser, 337 nm.
twice with absolute EtOH. 4ꢀ(4ꢀMethylpiperazinꢀ1ꢀ
yl)benzeneꢀ1,2ꢀdiamine ( ) was obtained as a solid
I
easily oxidizable product in a quantitative yield and
was immediately used in the synthesis of compound
4ꢀ(4ꢀMethylpiperazinꢀ1ꢀyl)benzeneꢀ1,2ꢀdiamine (I).
5ꢀ(4ꢀMethylpiperazinꢀ1ꢀyl)ꢀ2ꢀnitrophenylamine
(5.61 g, 23.76 mmol) [25] in glacial AcOH (30 mL)
was added to the Adams’ catalyst (0.95 g) preliminarꢀ benz[
MB
(
Ac
).
Dichlorohydrate of 2ꢀ(acetamidomethyl)ꢀ1
Hꢀ
d]imidazolꢀ6ꢀimide acid ethyl ether (II). An
ily reduced in 15 mL of glacial AcOH (until the hydroꢀ intensive flow of HCl was passed through a suspension
gen absorption terminated). The hydrogenation was of ꢀ[(6ꢀcyanoꢀ ꢀbenzo[ ]imidazolꢀ2ꢀyl)methyl]aceꢀ
N
1H
d
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 42
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DNA SEQUENCEꢀSPECIFIC LIGANDS
187
(а)
(b)
Δ
A
×
103
Δ
A
×
103
1
1
3
2
1
3
2
1
0
0
–1
–2
–3
–4
–5
–1
–2
–3
–4
3
3
2
2
1
1
250
275
300
325
350
375
250
275
300
325
350
375
Wavelength, nm
Wavelength, nm
Fig. 4. CD spectra of a CLCD of calf thymus DNA in solution in the absence (curve 1) and presence of DB(1) (a), and DB(9)
(b) (curves
path length is 10 mm. (a)
2 and 3). The concentration of PAG (4000) 170 mg/mL; buffer 0.3 M NaCl + 0.002 M Na PO , pH 6.85. The optical
3 4
–6
–6
–5
–6
1
, 0;
2,
7.80
×
10 M; 3, 15.60 × 10 M DB(1). [DNA]: 1.55 × 10 M; (b) 1, 0; 2, 10.60 × 10 M;
–6
–5
3, 15.90 × 10 M DB(9). [DNA]: 1.55 × 10 M.
tamide [1] (1.12 g, 5.22 mmol) in 45 mL of absolute H4), 8.36 (1 H, d,
dioxane/absolute EtOH (2 : 1) at until complete (1 H, m, СH2N ), and 11.19 [1 H, m, NH(Bim)];
saturation of the solution. The reaction flask was herꢀ EIMS, : 404.9 [ 403.44
+ H]+. Calculated:
metically closed, and the homogenous solution of (C22H25N7O1)
J 7.5, H5), 8.75 (1 H, s, H7), 8.87
0°C
H
m/z
M
M
.
dark color was held for 4 h at 0°C and then allowed to
(6ꢀ(4ꢀMethylpiperazinꢀ1ꢀyl)ꢀ
1
H
,3'
Hꢀ2,5'ꢀdibenzꢀ
stand overnight at room temperature. After a 4ꢀday
standing at room temperature, the solvent was evapoꢀ
rated, and the residue was reevaporated twice with
absolute EtOH. Dichlorohydrate (II) was obtained as
a solid light yellow substance with a quantitative yield
imidazolꢀ2'ꢀyl)methanamine
tetrachlorohydrate
(MB). Concentrated HCl (5 mL) was added to acetaꢀ
mide trichlorohydrate (MB(Ac)) (1.54 g, 3.00 mmol),
and the resulting suspension was refluxed for 20 min.
After the evaporation of the solvent, the residue was
dried in vacuo over СаCl2/NaOH, dissolved in 20 mL
of a MeOH/H2O mixture (3 : 1), and boiled for 5 min
with activated carbon (0.5 g). After the removal of carꢀ
bon, the dark claret solution was evaporated, the resiꢀ
due was heated in 20 mL of an EtOH/H2O mixture
(9 : 1) until boiling, and the resulting bulky suspension
was allowed to stand overnight at room temperature.
The precipitate was filtered and washed with EtOH
and an EtOH/H2O mixture (9 : 1). After drying, comꢀ
pound MB was obtained as a tetrachlorohydrate of
yellowish green color. Yield: 1.35 g (88.8%); mp
and used immediately for the synthesis of
methylpiperazinꢀ1ꢀyl)ꢀ 3' ꢀ2,5'ꢀdibenzo[
dazolꢀ2'ꢀyl)methyl)acetamide trichlorohydrate
MB(Ac)).
ꢀ((6ꢀ(4ꢀMethylpiperazinꢀ1ꢀyl)ꢀ
dibenzo[ ]imidazolꢀ2'ꢀyl)methyl)acetamide trichloroꢀ
hydrate (MB(Ac)). A solution of 4ꢀ(4ꢀmethylpiperꢀ
azinꢀ1ꢀyl)benzeneꢀ1,2ꢀdiamine ( ) (1.07 g, 5.2 mmol)
in 26 mL of a glacial АсОН–absolute ЕtОН mixture
(1 : 1) was added to imidate dichlorohydrate (II
(1.74 g, 5.2 mmol). The mixture was heated on an oil
bath in an atmosphere of nitrogen for 30 min at 95
N
ꢀ((6ꢀ(4
ꢀ
1H,
H
d]imiꢀ
(
N
1H,3'Hꢀ2,5'ꢀ
d
I
)
°C.
>
350
1H NMR (77
H(3'', 5'')], 3.55 [4 H, m, H(2'',6'')], 4.38 (2 H, s,
°C, Rf 0.57, λmax 334 nm (
ε
37400 М–1 cm–1).
The resulting solution was evaporated in vacuo, and
the residue was reevaporated three times with absolute
EtOH. The dry substance was dissolved in 25 mL of
EtOH, 0.8 mL of concentrated HCl was added, and
the precipitated suspension was boiled for a few minꢀ
utes and left to stand overnight at room temperature.
The dark claret precipitate was filtered and washed
three times on a filter with cold EtOH. After drying,
compound (MB(Ac)) of dark violet color was
obtained with a yield of 2.08 g (78.0%); mp 239–
°C): 2.83 (3 H, s, NCH3); 3.36 [4 H, m,
+
С
Н
); 7.18 (1 H, d,
J
8.7, H5'), 7.21 (1 H, s, H7'),
8.7, H4'), 7.79 (1 H, d, 8.7, H4), 8.21
8.7, Í5), and 8.64 (1 H, s, H7); EIMS,
+ H)+. Calculated:
361.44 (C20H23N7)
2NH3
7.63 (1 H, d,
(1 H, d,
361.4
J
J
J
m/z:
.
(
M
M
The General Method for the Synthesis of Compounds
DB(1, 2, 6, 8, 9, 10, 12)
240
2.84 (3 H, s, NCH3), 3.1–4.0 [8 H, m, H(Pip)], 4.67
(2 H, m, 2NH), 7.21 (1 H, s, H7'), 7.33 (1 H, d,
8.1, H5'), 7.71 (1 H, d, 8.7, H4'), 7.93 (1 H, d, 8.1, chlorohydrate MB and 0.2 mmol of the corresponding
°C, R °C): 1.98 (3 H, s, COCH3);
f 0.66. 1H NMR (23
СН
J
A weighed sample (202.9 mg, 0.4 mmol) of tetraꢀ
J
J
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188
IVANOV et al.
α
,
ω
ꢀalkandicarboxylic acid (III) was suspended in [1 H, s, NH(Bim)]; EIMS,
m
/
z
: 862.8 [
M
+ H]+. Calꢀ
+ Na +
923.12 (C48H56N14O2NaK), 986.8
+ 2Na + 2K]+; calculated:
985.20 (C48H56N14O2
(195 mg, 0.44 mmol) was added. The mixture was held Na2K2)
for another 15 min at , the cooling was eliminated,
absolute DMF (5 mL) and cooled to
which DIPEA (0.63 mL, 3.6 mmol) was added. The K]+; calculated:
suspension was stirred for 10 min at , and BOP
0°С following culated: М 861.05 (C48H56N14O2), 924.8 [M
М
0°
С
[M
М
.
0°С
DB
107 mg (48.3%); mp: at 245–250°C the compound
melts to the state of resin and further does not melt
up to temperatures of >330°C; f 0.71, λmax 325 nm
47700 М–1 cm–1). 1H NMR: 1.27 CO(CH2)2(C
2)4);
1.55 (4 H, quin, 6.9, COCH2C 2); 2.25 (4 H, t,
7.5, COCH2); 2.84 (6 H, s,
3ꢀNH+); 3.23 [8 H, m,
H(3'', 5'')]; 3.54 [4 H, m, H(2'', 6'')]; 3.88 [4 H, m,
H(2'', 6'')]; 4.68 (4 H, d, 5.6, C 2NH); 7.20 (2 H, s,
H7'); 7.33 (2 H, dd, 1.9, 8.7, H5'); 7.71 (2 H, d,
9.3, H4'); 7.94 (2 H, d, 8.7, H4); 8.36 (2 H, d, 8.7
H5); 8.75 (4 H, m, CH2N , H7); 11.19 [1 H, s,
NH(Bim)]; EIMS, : 890.7 [
+ H]+. Calculated:
889.10 (C50H60N14O2), 912.7 [
+ Na]+; calcuꢀ
lated: 912.10 (C50H60N14O2Na), 952.7 [ + Na +
K]+; calculated:
951.17 (C50H60N14O2NaK)
(8) · 6HCl. The yield after the drying was
and the solution was allowed to stand at room temperꢀ
ature for 12 h. The solvent was evaporated, and the
residue was ground with 5 mL of CHCl3. The solid
sediment was filtered off, dissolved by boiling in 5–
15 mL of MeOH–H2O (5.5 : 1), cooled, and the solid
product was precipitated with 5–10 mL of AcOEt.
The precipitate was filtered off and dissolved again in
the same amount of the MeOH–H2O mixture. Then,
5–10 drops of concentrated HCl were added after
which the substance was precipitated with AcOEt (5–
15 mL). If the product precipitated as an oil, the solꢀ
vent was decanted, and the oil was ground with 5 mL
of AcOEt until the solid substance was obtained. The
product of dark brown color was filtered off and dried.
R
ε
H
J
H
J
CH
J
H
J
J
J
J
J
,
H
m/z
M
М
M
М
M
.
1
If the H NMR spectrum of the product contained
М
foreign signals, the substance was further treated by
boiling in 5 mL of absolute EtOH after which the
undissolved precipitate was isolated.
DB · 6HCl. The yield after the drying was
112 mg (49.9 %); mp: at 245–247 the compound
melts to the state of resin and further does not melt up
to temperatures of >330 Rf 0.72, λmax 326 nm (
44800 М–1 cm–1). 1H NMR: 1.25 CO(CH2)2(C
2)5);
1.54 (4 H, quin, 6.5, COCH2CH2 , 2.25 (4 H, t,
7.5, COCH2), 2.84 (6 H, s, C (6 H, s, C
3ꢀNH+), 3.23
[8 H, m, H(3'', 5'')], 3.54 [4 H, m, H(2'', 6'')]; 3.88 [4
H, m, H(2'', 6'')], 4.68 (4 H, d, 5.6, C 2NH), 7.20
(2 H, s, H7'), 7.34 (2 H, dd, 2.2, 8.7, H5'), 7.72
(2 H, d, 8.7, H4'), 7.95 (2 H, d, 8.7, H4), 8.37 (2 H,
d, 8.7, H5), 8.76 (4 H, m, CH2N , H7), and 11.18
[1 H, s, NH(Bim)]; EIMS, : 903.4 [
+ H]+. Calꢀ
culated: 903.13 (C51H62N14O2)
DB 10 · 6HCl. The yield after the drying was
98 mg (43.1%); mp: at 241–243 the compound
melts to the state of resin and further does not melt up
to temperatures of 330 Rf 0.73 λmax 327 nm (
39100 М–1 cm–1). 1H NMR: 1.24 CO(CH2)2(C
2)6);
1.54 (4 H, quin, 6.9, COCH2CH2 , 2.25 (4 H, t,
7.5, COCH2), 2.84 (6 H, s,
3ꢀNH+), 3.23 [8 H, m,
H(3'', 5'')], 3.54 [4 H, m, H(2'', 6'')], 3.88 [4 H, m,
H(2'', 6'')], 4.69 (4 H, d, 5.6, C 2NH), 7.20 (2 H, s,
H7'), 7.34 (2 H, dd, 1.9, 9.3, H5'), 7.71 (2 H, d,
8.7, H4'), 7.95 (2 H, d, 8.1, H4), 8.39 (2 H, d, 8.7
H5), 8.77 (4 H, m, CH2N
NH(Bim)]; EIMS, : 918.3 [
917.16 (C52H64N14O2), 980.2 [
lated:
2Na + 2K]+; calculated:
Na2K2)
DB
3.23 [8 H, m, H(3'', 5'')], 3.54 [4 H, m, H(2'', 6'')], 3.88 115 mg (49.4%); mp: at 237–239
[4 H, m, H(2'', 6'')], 4.69 (4 H, d, 5.0, C 2NH), 7.20 melts to the state of resin and then does not melt up to
(2 H, s, H7'), 7.33 (2 H, dd, 2.5, 8.7, H5'), 7.71 (2 temperatures of >330 Rf 0.74 λmax 328 nm (
H, d, 9.3, H4'), 7.94 (2 H, d,
8.7, H4), 8.36 (2 H, 37300 М–1 cm–1). 1H NMR: 1.22 [16 H, m,
d, 8.7, H5), 8.77 (4 H, m, CH2N , H7), and 11.20 CO(CH2)2(C 2)8); 1.53 (4 H, quin, 6.9,
(9)
°C
DB
(1)
· 6HCl. The yield after the drying was 45 mg
Rf 0.60, λmax 323 nm (
°
C
;
ε
(22.3%); mp >330
°
C;
ε
H
37300 М–1 cm–1). H NMR: 2.84 (6 H, br s,
CH3ꢀ
1
J
)
J
NH+), 3.23 [8 H, m, H(3'', 5'')], 3.48 (2 H, s,
COCH2); 3.54 [4 H, m, H(2'', 6'')], 3.88 [4 H, m,
H
H(2'', 6'')], 4.79 (4 H, d,
s, H7'), 7.34 (2 H, d, 9.3, H5'), 7.69 (2 H, d,
H4'), 7.92 (2 H, d, 8.7, H4), 8.34 (2 H, d, 8.7, H5),
, and 11.23
+ H]+. Calꢀ
+ Na + K)+
J
5.0, C
H
2NH), 7.19 (2 H, br
J
H
J
J
8.7
,
J
J
J
J
H)
J
J
8.75 (2 H, s, H7), 9.19 (2 H, t,
J
m
5.3, CH2N
J
H
[2 H, br s, NH(Bim)]; EIMS,
/
z
: 793.4 [M
m/
z
M
culated:
calculated:
M
790.92 (C43H46N14O2); 855.7 (
М
.
;
М
.
M
853.00 (C43H46N14O2NaK)
(
)
DB · 6HCl. The yield as hexachlorohydrate after
the drying was 49 mg (23.9%); mp >330 Rf 0.59,
λmax 323 nm (
46300 М–1 cm–1). 1H NMR: 2.63 (4 H,
s, COCH2), 2.84 (6 H, br s,
3–NH+), 3.23 [8 H, m,
H(3'', 5'')], 3.54 [4 H, m, H(2'', 6'')], 3.87 [4 H, m,
H(2'', 6'')], 4.74 (4 H, d, 5.6, C 2NH), 7.17 (2 H, br
s, H7'), 7.32 (2 H, d, 8.7, H5'), 7.68 (2 H, d, 9.3
H4'), 7.87 (2 H, d, 8.7, H4), 8.32 (2 H, d, 8.7, H5),
8.72 (2 H, s, H7), 8.99 (2 H, t, 5.6, CH2N , and
11.24 [1 H, br s, NH(Bim)]; EIMS, : 806.3 [
H]+. Calculated:
804.94 (C44H48N14O2), 866.8 [
Na + K]+; calculated:
867.01 (C44H48N14O2NaK)
DB · 6HCl. The yield after the drying was
101 mg (49.8%); mp 2264–266 C, Rf 0.69, λmax
325 nm (
45000 М–1 cm–1). 1H NMR: 1.30 [4 H, m,
CO(CH2)2C 2); 1.56 (4 H, quin, 6.5, COCH2CH2
2.26 (4 H, t, 7.5, COCH2), 2.84 (6 H, s, C
3–NH+);
(2)
°C
°C;
ε
°
C
;
;
ε
CH
H
J
)
J
J
H
CH
J
J
,
J
J
J
H
J
H)
J
J
m/z
M +
M +
J
J
J
,
М
H
, H7), 11.23 (1 H, s,
m/z
M
M
+ H]+. Calculated:
М
.
М
+ Na + K]+; calcuꢀ
(6)
М
979.23 (C52H64N14O2 NaK), 1042.2 [
M +
°
М
1041.30 (C52H64N14O2
ε
.
H
J
),
J
H
(12)
· 6HCl. The yield after the drying was
the compound
°C
J
H
J
J
°C;
;
ε
J
J
J
H
H
J
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 42
No. 2
2016

DNA SEQUENCEꢀSPECIFIC LIGANDS
189
COCH2CH2
s,
3ꢀNH+), 3.23 [8 H, m, H(3'', 5'')], 3.54 [4 H, m, centration of 340 mg/mL. One hour after the mixing,
H(2'', 6'')], 3.88 [4 H, m, H(2'', 6'')], 4.68 (4 H, d, a CD spectrum was recorded; the appearance of an
5.0, C 2NH), 7.21 (2 H, s, H7'), 7.34 (2 H, dd,
2.5, intense band (λmax 270 nm, Δε 130–140 M–1 cm–1) in
8.7, H5'), 7.72 (2 H, d, 9.3, H4'), 7.94 (2 H, d, the CD spectrum indicated the formation of a CLCD
8.7, H4), 8.36 (2 H, d, 8.7, H5), 8.73 (4 H, m, of DNA molecules. At the second stage, an initial
CH2N , H7), and 11.15 (1 H, s, NH(Bim)]; EIMS, DB( solution was added in small portions (from 2 to
: 946.4 [ 945.21 L) to 2 mL of the CLCD, with intensive stirring,
+ H]+. Calculated:
(C54H68N14O2), 1008.4 [
+ Na + K]+; calculated:
1007.28 (C54H68N14O2NaK)
), 2.24 (4 H, t, J 7.5, COCH2), 2.84 (6 H, pH 6.8) solutions of DNA and PAG (4000) at a conꢀ
CH
J
H
J
J
J
J
J
H
n)
m/
z
M
М
5
µ
M
М
after which a CD spectrum was recorded again in the
absorption region of the DNA and the ligand (the
wavelength range 250–380 nm).
.
Physicochemical Studies
ACKNOWLEDGMENTS
Absorption spectra were recorded on a Cary100
spectrophotometer (Varian, United States). Fluoresꢀ
cence spectra were measured on a PTI spectrofluoꢀ
rimeter (Photo Technology Intern., Canada). CD
spectra were recorded using a portable dichrometer
SKDꢀ2 (manufactured at the Institute of Spectrosꢀ
copy, Russian Academy of Sciences, Troitsk). All
This work was supported by the Russian Foundaꢀ
tion for Basic Research (project no. 14ꢀ04ꢀ00388) and
the Program of the Presidium of the Russian Academy
of Sciences “Molecular and Cellular Biology.”
REFERENCES
spectra were measured at 20°С. The DNA molar conꢀ
centration (M in bp) in terms of the number of moles
of base pairs (M = mole of bp/L) was used.
1. Ivanov, A.A., Salyanov, V.I., Streltsov, S.A.,
Cherepanova, N.A., Gromova, E S., and Zhuze, A.L.,
Russ. J. Bioorg. Chem., 2011, vol. 37, pp. 472–482.
A sample of calf thymus DNA (Sigma, United
States) was dissolved in a water–salt buffer solution
(0.3 M NaCl + 0.002 M Na3PO4, pH 6.85) and diaꢀ
2. Baraldi, P.G., Bovero, A., Fruttarolo, F., Preti, D.,
Tabrizi, M.A., Pavani, M.G., and Romagnoli, R.,
Med. Res. Rev., 2004, vol. 32, pp. 475–528.
lyzed at 4°C against three changes of the same buffer.
3. ZewailꢀFoote, M. and Hurley, L.H., AntiꢀCancer Drug
Des., 1999, vol. 14, pp. 1–9.
The duration of each change of dialysis was no less
than 8 h, with the DNA/buffer ratio being 1 : 15. The
DNA concentration was determined spectrophotoꢀ
metrically using the molar absorption coefficient ε260
= 13200 М–1 cm–1 (M in bp, see above) [34].
4. Chaires, J.B., Curr. Opin. Struct. Biol., 1998, vol. 8,
pp. 314–320.
5. Strekowski, L. and Wilson, B., Mutat. Res., 2007,
vol. 623, pp. 3–13.
The starting solutions of DB(n), MB, and
6. Wilson, W.D., Nguyen, B., Tanious, F.A., Mathis, A.,
Hall, J.E., Stephens, C.E., and Boykin, D.W., Curr.
Med. Chem. AntiꢀCancer Agents, 2005, vol. 5, pp. 389–
408.
[
MB(Ac)] at a concentration of about 1 mM were preꢀ
pared in DMSO using a weighed sample. DNA–
ligand complexes were formed by two methods. In the
first method, a small (2–5 L) volume of a solution of
the corresponding substance in DMSO was added in
small portions to an aqueous salt DNA solution. In the
second method, a concentrated DNA solution
µ
7. Neidle, S., Nat. Prod. Rep., 2001, vol. 18, pp. 291–309.
8. Wiederholt, K., Rajur, S.B., Giuliano, J.,
O’Donnell, M., and McLaughlin, L., J. Am. Chem.
Soc., 1996, vol. 118, pp. 7055–7062.
(~100 mM in bp) was added in small portions (2–5 L)
µ
to an aqueous salt solution of the corresponding ligand
obtained by the 100–500ꢀfold dilution of the initial
solution in DMSO with the buffer (0.3 M NaCl +
0.002 M Na3PO4). The solutions were thoroughly
stirred at room temperature, and absorption and/or
fluorescence spectra were recorded.
9. Geierstanger, B.H. and Wemmer, D.E., Annu. Rev.
Biophys. Biomol. Struct., 1995, vol. 24, pp. 463–493.
10. Chenoweth, D.M. and Dervan, P.B., J. Am. Chem.
Soc., 2010, vol. 132, pp. 14521–14529.
11. Meng, L.ꢀH., Liao, Z.ꢀY., and Pommier, Y., Curr. Top.
Med. Chem., 2003, vol. 3, pp. 305–320.
The complexes of DB(n) with CLCD of DNA were
12. Bailly, C., Curr. Med. Chem., 2000, vol. 7, pp. 39–58.
studied using a sample of calf thymus DNA (Sigma,
United States) depolymerized on a UZDNꢀ2T ultraꢀ
sound disperser. The molecular weight of DNA after the
ultrasonic depolymerization was (0.5–0.8) × 106 Da, as
determined by electrophoresis in 1% agarose gel.
Quartz cuvettes with an optical path length of 1 cm
were used. The initial concentration of solutions of
13. Turner, P.R. and Denny, W.A., Curr. Drug Targets
2000, vol. 1, pp. 1–14.
,
14. Chiang, S.Y., Welch, J.J., Rauscher, F.J., 3rd, and
Beerman, T.A., J. Biol. Chem., 1996, vol. 271,
pp. 23999–24004.
15. Welch, J.J., Rauscher, F.J., 3rd, and Beerman, T.A.,
J. Biol. Chem., 1994, vol. 269, pp. 31051–31058.
DB(n) in DMSO was ~1 mM.
16. Gabelica, V., Rosu, F., Houssier, C., and De Pauw, E.,
Rapid Commun. Mass Spectrom., 2000, vol. 14, pp. 464–
467.
At the first stage, a CLCD of DNA was prepared by
mixing equal volumes of aqueous salt (0.3 M NaCl;
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 42
No. 2
2016

190
IVANOV et al.
17. White, C.M., Satz, A.L., Gawron, L.S., Bruice, T.C., 27. Yevdokimov, Yu.M., Skuridin, S.G., Nechipurenko, Y.D.,
and Beerman, T.A., Biochim. Biophys. Acta, 2002,
vol. 1574, pp. 100–108.
18. Reddy, B.S.P., Sondhi, S.M., and Lown, J.W., Pharꢀ
macol. Ther., 1999, vol. 84, pp. 1–111.
19. Nelson, S.M., Ferguson, L.R., and Denny, W.A.,
Zakharov, M.A., Salyanov, V.I., Kurnosov, A.A., Kuzꢀ
netsov, V.D., and Nikiforov, V.N., Int. J. Biol. Macroꢀ
mol., 2005, vol. 36, pp. 103–115.
28. Gromyko, A.V., Salyanov, V.I., Strel’tsov, S.A., Oleiniꢀ
kov, V.A., Korolev, S.P., Gottikh, M.B., and Zhuze,
A.L, Russ. J. Bioorg. Chem., 2007, vol. 33, pp. 569–578.
Mutat. Res., 2007, vol. 623, pp. 24–40.
20. Dervan, P.B. and Edelson, B.S., Curr. Opin. Struct.
Biol., 2003, vol. 13, pp. 284–299.
29. Pjura, P.E., Grzeskowiak, K., and Dickerson, R.E.,
J. Mol. Biol., 1987, vol. 197, pp. 257–271.
21. Reddy, B.S., Sharma, S.K., and Lown, J.W., Curr.
Med. Chem., 2001, vol. 8, pp. 475–508.
30. Teng, M.ꢀK., Usman, N., Frederick, C.A., and
Wang, A.H., Nucleic Acids Res., 1988, vol. 16 P,
pp. 2671–2690.
22. Wemmer, D.E., Annu. Rev. Biophys. Biomol. Struct.
,
2000, vol. 29, pp. 439–461.
23. Bailly, C. and Chaires, J.B., Bioconjug. Chem., 1998,
31. Lisitsyna, E.S., Durandin, N.A., Ivanov, A.A.,
Streltsov, S.A., Susova, O.Yu., Shtil, A.A., Zhuze, A.L.,
and Kuzmin, V.A., Mol. Biol. (Moscow), 2012, vol. 46,
pp. 823–827.
vol. 9, pp. 513–538.
24. Cherepanova, N.A., Ivanov, A.A., Maltseva, D.V.,
Minero, A.S., Gromyko, A.V., Streltsov, S.A.,
Zhuze, A.L., and Gromova, E.S., J. Enzyme Inhib.
Med. Chem., 2011, vol. 26, pp. 295–300.
25. Loewe, H. and Urbanietz, J., Arzneim.ꢀForsch. (Drug
Res.), 1974, vol. 24, pp. 1927–1933.
26. Evdokimov, Yu.M., Salyanov, V.I., Nechipurenko, Y.D.,
Skuridin, S.G., Zakharov, M.A., Spener, F., and
Palunbo, M., Mol. Biol., (Moscow), 2003, vol. 37,
pp. 293–306.
32. Sintezy organicheskikh preparatov. (Organic Synthesis),
Moscow: IL, 1953, vol. 1, pp. 357–358.
33. Martin, R.F., Pardee, M., Kelly, D.P., and Mack, P.O.ꢀL.,
Aust. J. Chem., 1986, vol. 39, pp. 373–381.
34. Wells, R.D., Larson, J.E., Grant, R.C., Shortle, B.E.,
and Cantor, C.R., J. Mol. Biol., 1970, vol. 54, pp. 465–
497.
Translated by S. Sidorova
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 42
No. 2
2016