DNA sequence-specific ligands: XVI. Series of the DBP(n) fluorescent dimeric bisbenzimidazoles with 1,4-piperazine-containing linkers

Article abstract of DOI:10.1134/S106816201702008X

A novel series of the DBP(n) fluorescent symmetric dimeric bisbenzimidazoles in which the bisbenzimidazole fragments were attached to an oligomeric linker with the 1,4-piperazine residue in its center were prepared. The DBP(n) molecules were distinguished by the number of methylene groups n (where n = 1, 2, 3, 4) in the linker. The DBP(n) synthesis was based on a condensation of the monomeric bisbenzimidazole (MB) with 1,4-piperazinedialkylcarbonic acids. The ability of the DBP(n) dimeric bisbenzimidazoles to form complexes with the double-stranded DNA was demonstrated by a complex of physicochemical methods, including spectroscopy in the visual UV-area, circular dichroism (CD), and fluorescence. The DBP(1–4) molecules were localized in the DNA minor groove by the CD method with the use of cholesteric liquid-crystalline dispersions (CLCD) of the double-stranded DNA. The DBP(n) dimeric bisbenzimidazoles were easily soluble in water, penetrated through cellular and nuclear membranes, and stained DNA in living cells distinct from the previously synthesized DB(n) series.

Full text of DOI:10.1134/S106816201702008X

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2017, Vol. 43, No. 2, pp. 143–149. © Pleiades Publishing, Ltd., 2017.
Original Russian Text © V.S. Koval, A.A. Ivanov, V.I. Salyanov, A.A. Stomakhin, V.A. Oleinikov, A.L. Zhuze, 2017, published in Bioorganicheskaya Khimiya, 2017, Vol. 43, No. 2,
pp. 167–173.
DNA Sequence-Specific Ligands: XVI. Series of the DBP(n)
Fluorescent Dimeric Bisbenzimidazoles
with 1,4-Piperazine-Containing Linkers1
V. S. Kovala, A. A. Ivanovb, V. I. Salyanova, A. A. Stomakhina, V. A. Oleinikovc, and A. L. Zhuzea, 2
aEngelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991 Russia
bBlokhin Russian Cancer Research Center,
Ministry of Health of the Russian Federation, Moscow, 115478 Russia
cShemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997 Russia
Received July 26, 2016; in final form, September 29, 2016
Abstract—A novel series of the DBP(n) fluorescent symmetric dimeric bisbenzimidazoles in which the bis-
benzimidazole fragments were attached to an oligomeric linker with the 1,4-piperazine residue in its center
were prepared. The DBP(n) molecules were distinguished by the number of methylene groups n (where n =
1, 2, 3, 4) in the linker. The DBP(n) synthesis was based on a condensation of the monomeric bisbenzimid-
azole (MB) with 1,4-piperazinedialkylcarbonic acids. The ability of the DBP(n) dimeric bisbenzimidazoles
to form complexes with the double-stranded DNA was demonstrated by a complex of physicochemical meth-
ods, including spectroscopy in the visual UV-area, circular dichroism (CD), and fluorescence. The DBP(1–4)
molecules were localized in the DNA minor groove by the CD method with the use of cholesteric liquid-crys-
talline dispersions (CLCD) of the double-stranded DNA. The DBP(n) dimeric bisbenzimidazoles were eas-
ily soluble in water, penetrated through cellular and nuclear membranes, and stained DNA in living cells dis-
tinct from the previously synthesized DB(n) series.
Keywords: dimeric bisbenzimidazole, DBP(n), complexes with the double-stranded DNA, DNA, minor-
groove binder, fluorescence, circular dichroism
DOI: 10.1134/S106816201702008X
INTRODUCTION
most promising. These minor grove binders are free
from many disadvantages of the traditional biologi-
cally active preparations on the basis of alkylating and
intercalating agents. In particular, they do not damage
DNA, do not significantly distort the DNA spatial
structure, and are almost free from the side mutagenic
effect.
Here, we continued our studies [1] of a creation of
the DBP(n) novel series of the DNA site-specific
ligands based on the Hoechst 33258 (Ht) dye (Fig. 1),
which is widely used in cytology as a DNA-specific
fluorescent label [2]. Ht is known to be noncovalently
and AT-specifically bound to the DNA minor groove
[3, 4]. It inhibits TATA box binding protein [5], effec-
tively inhibits DNA-topoisomerase I [6], topo-II [7],
and DNA-helicases [8], and exhibits the radioprotec-
tive effect [9]. The AT-specificity of Ht is determined
by the backbone of the dye molecule that consists of
the two covalently-bound benzimidazole fragments.
Each of these fragments interacts with DNA, forms
bifurcational (three-center) hydrogen bound with the
O2 atom of thymine and/or with the N3 atom of ade-
nine of the neighboring AT pairs, and covers a region
of approximately one and a half base pairs [3]. The Ht
Preparation of low-molecular-weight compounds
that site-specifically recognize nucleotide sequences
in double-stranded DNAs is one of the urgent prob-
lems in bioorganic chemistry and molecular biology.
In the long term, such molecular instruments which
are specifically bound to definite nucleotide sequences
of the genome could be used for an investigation and a
control of an expression of concrete genes. Moreover,
these target-directed compounds are of special inter-
est for pharmacology, because a chemotherapeutic
activity of the majority of the known antitumor agents
depends on their affinity and selectivity of their inter-
action with a double-stranded DNA. From this point
of view, low-molecular-weight compounds that non-
covalently interact with the DNA minor groove are the
1
2
The XVI series was published in [1].
Corresponding author: phone: +7 (499) 135-97-18; e-mail:
zhuze@eimb.ru.
Abbreviations: dsDNA, the double-stranded DNA; X-ray;
X-ray structural analysis; CD, circular dichroism; CLCD, the
cholesteric liquid-crystalline dispersion; BOP, benzotriazolyl-
1-oxy)tris(dimethylamino)-phosphonium
phate; DIPEA, N,N-diisopropylamine.
hexafluorophos-
143

144
KOVAL et al.
binding is also stabilized by electrostatic and intensive
Van-der-Waals interactions with walls of the DNA
minor groove.
Previously, we have synthesized the DB(n) dimeric
analogues of Ht and studied their biochemical proper-
ties (Fig. 1) in order to create the compounds that
would be noncovalently and site-specifically bound to
the DNA minor groove [1, 11].
The benzimidazole fragments of the DB(n) mole-
cules are attached “tail-to-tail’ to oligomethylene
linkers of different length (n = 1–12; Fig. 1), which
allows them to form the structure that is isogeometri-
cal to the DNA minor groove. Thus, these compounds
act as bidentate ligands that are able to recognize
nucleotide sequences with blocks from two AT-pairs
which are located at a different distance from each
other. DB(n) in low micromolecular concentrations
prove to be inhibitors of a number of DNA-dependent
enzymes: the eukaryotic DNA-topoisomerase I [12],
the mouse DNA-methyltransferase Dnmt 3a [13], and
helicase activity of the NS3 protein of the human hep-
atitis C virus [14]. The studies of DB(n) demonstrate
that these compounds are novel promising fluorescent
dyes that can penetrate through a cellular and nuclear
membranes with an effective staining of the cell nuclei
and differential staining of chromosomes [15]. How-
ever, these compounds are poorly soluble in water due
to the aggregate formation.
The goal of our investigation is a creation and stud-
ies of the DNA interaction with new series of the
DBP(n) dimeric bisbenzimidasoles (n = 1–4) (see Fig. 1)
which do not have this disadvantage. The residue of
1,4-piperazine has been introduced into the structure
of the oligomethylene linker of the DB(n) molecules in
order to increase their solubility in aqueous solutions
and affinity of the DNA complexes. Therefore, the
novel compounds exist as tetracations at neutral pH
values distinct from the dicationic structure of com-
pounds of the DB(n) series, suggesting both the higher
affinity to the double-stranded DNA due to the stron-
ger electrostatic interactions and the better solubility
in water.
A chemical synthesis of the DBP(n) dimeric bis-
benzimidazoles was presented in the Scheme 1. 4-Pip-
erazinedialkylcarbonic acids (II) were prepared by
alkylation of 1,4-piperazine by nitriles of the corre-
sponding ω-chloroalkylcarbonic acids with the forma-
tion of dinitriles of 1,4-piperazinedialkylcarbonic
acids (I) and the subsequent hydrolysis with hydro-
chloric acid. The DBP(n) dimeric benzimidazoles
were prepared by a condensation of the monomeric
bisbenzimidazole (MB), which we synthesized previ-
ously [1], with 1,4-piperazinedialkylcarbonic acids
(II) with the use of the BOP coupling reagent.
a
NC−(CH2)n Cl
NC−(CH2)n
+
HN
NH
HN
(I)
N
NH (CH2)n CN
n = 1, 2, 3, 4
n = 1, 2, 3, 4
4HCl
·
N
b
N
H
HOOC (CH2)n
(II)
N
4
N (CH2)n COOH
+
N
NH2
N
H
N
n = 1, 2, 3, 4
H3C
c
MB
4'
5
7
5'
N
N
·
8HCl
N
2'''
NH (CH2)n NH
6'''
3'''
2''
5''
N
N
N
H
3''
N
H
N
N
(CH2)n
NH
NH
5'''
7'
N
H
N
H
N
6''
CH3
H3C
O
O
DBP(1) n = 1; DBP(2) n = 2; DBP(3) n = 3; DBP(4) n = 4
Scheme 1. Synthesis of DBP(1,2,3,4). Reagents and conditions: (a), DIPEA, 82°C, 40–45%; (b) concentrated HCl,
100°C, 2 h, 65–88%; (c) BOP, DIPEA, DMF, 0°C—room temperature 12 h, the yields 76–82%.
As in the case of the previous series of the DB(n) DBP(n) with an increase in the concentration of the
double-stranded DNA pointed to the formation of the
dimeric bisbenzimidazoles [1, 16], the formation of
the complexes of these ligands with the double-
stranded DNA and their characteristics were studied
by physicochemical methods. The hypochromic effect
complexes (Fig. 2).
The fluorescent spectra also confirmed the inter-
action between the ligands and the double-stranded
and bathochromic shift in the absorption spectra of DNA, because they demonstrated the multifold
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
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DNA SEQUENCE-SPECIFIC LIGANDS
145
N
N
N
N
H
N
H
N+
H3C
H
OH
Hoechst 33258
N
N
N
N
NH
NH
(CH2)n
N
N
H
N
N
H
O
H
O
H
N
N
N+
N+
H
DBP(n) n = 1−12
H
H3C
CH3
H
N+
N
N
(CH2)n
NH
O
N
N
N
N
H
N+
H
H
O
(CH2)n
N
NH
N
N
H
H
N
N+
H
N+
H
H3C
DBP(n) n = 1
−4
CH3
Fig. 1. Hoechst 333258 and the DB(n) and DBP(n) dimeric bisbenzimidazoles.
(а)
(b)
A
A
1
1
3
0.20
0.25
5
5
0.20
0.15
0.10
0.05
0
2
0.15
0.10
0.05
0
2
4
4
3
300
350
400
450
300
350
400
450
Wave length, nm
Wave length, nm
Fig. 2. The absorption spectra of (a) DBP(2) and (b) DBP(4) (curve 1) in the absence and (curves 2–5) in the presence of the
–5
double-stranded DNA: (a) [DBP(2)] = 5.42 μM; [DNA], 10 M of bp: 0.4.2 (2), 2.08 (3), 15.73 (4), 38.02 (5), (b) [DBP(4)] =
–5
5.47 μM; [DNA], 10 M of bp: 0.42 (2), 2.08 (3), 19.91 (4), 38.02 (5). The buffer was 1 mM sodium cacodylate (pH 6.8). Here-
inafter, the DNA molar concentration is given in M of bp (mol of bp/L).
increase in the DBP(n) fluorescence in the presence of cated of the formation of CLCDs of the DNA mole-
the double-stranded DNA (Fig. 3).
cules.
Ht is bound to the double-stranded DNA in its
The location of the synthesized compounds in the
DNA groove was shown by the CD method with the
use of the cholesteric liquid-crystalline dispersions
(CD CLCDs) of the double-stranded DNA. The pos-
itive character of the new band in the CD CLCD spec-
trum of the ligands in the presence of DNA gave evi-
dence of the DBP(n) binding in one of the grooves of
the DNA double helix [1, 16] (Fig. 4).
minor groove according to the X-ray data [3, 17]. We
believe that the DBP(n) compounds are also placed in
the minor groove and form a complex with the double-
stranded DNA, because they are the Ht dimers. The
inhibitory activity of DBP(1–4) towards the eukary-
otic DNA of topoisomerase I and the prokaryotic
DNA-methyltransferase M.Sssl [18] in micromolar
concentrations has been demonstrated in the bio-
chemical experiments in vitro. Cytotoxicity of all the
An appearance of the intensive band (λmax = 270 nm,
Δε = 130–140 M–1 cm–1) in the CD spectrum indi- synthesized compounds was investigated in the MTT
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 43 No. 2 2017

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KOVAL et al.
(а)
(b)
4
4
1200
1000
800
600
400
200
0
1600
1400
1200
1000
800
600
400
200
0
3
3
1
2
2
1
480
400
440
480
520
560
600
400
440
520
560
600
Wave length, nm
Wave length, nm
Fig. 3. The fluorescent spectra of (1) the buffer, (а) DBP(2), and (b) DBP(4) (2) in the absence and (3, 4) in the presence of the
–6
–6
–6
double-stranded DNA: [DBP(2)] = 5.42 × 10 M; [DBP(4)] = 5.47 × 10 M; [DNA], 10 M of bp: 20.8 (3); 62.02 (4). The
buffer was 1 mM sodium cacodylate (pH 6,8). The excitation wavelength was 320 nm and the slit width was 5 nm.
(а)
(b)
ΔA × 106
ΔA × 106
4
4
300
200
100
0
100
3
2
1
3
2
0
1
−1000
−1000
−2000
−3000
−4000
−5000
−2000
−3000
−4000
4
4
1
1
260 280 300 320 340 360 380 400
260 280 300 320 340 360 380 400
Wave length, nm
Wave length, nm
Fig. 4. The CD spectra of the cholesteric liquid-crystalline suspension of DNA from the calf thymus (curve 1) in the absence and
–6
(curves 2–4) in the presence of (а) DBP(2) and (b) DBP(4). [DBP(n)], 10 M: 0 (1), 1.1 (2), 2.72 (3), 5.42 (4); [DNA] = 1.5 ×
–5
10 M of bp; the concentration of PEG-4000 was 170 mg/mL; 0.3 М NaCl + 2 mМ sodium phosphate buffer, pH 6.85. The
length of the optical path was 10 mm.
test towards the MCF-7 cellular lines of the breast butironitrile (Acros Organics, Belgium), sodium cac-
cancer and the NKE-hTERT normal fibroblasts of the
epithelium of the human kidney. The DBP(1–4) cyto-
toxicity was low (several dozens of micromoles) in all
the cases [18].
odylate, DMSO (Sigma, United States), PEG 4000
(Fluka, Switzerland), Boc-glycine, dioxane, DMF,
AcOH, AcOEt, Ac2O, EtOH, MeOH, PriOH
(Reakhim, Russia) were used in this study. Solutions
of the substances in organic solvents were dried over
Na2SO4. The solvents were evaporated on a rotary
evaporator in a vacuum of a water-jet pump, usually, at
40–50°С. The substances were dried in vacuum over
P2O5/NaOH. The boiling points were determined on
a Boethius device (Germany) and were not corrected.
A hydrogenation was performed using 10% Рd/C
(Merck, Germany) at atmospheric pressure and room
EXPERIMENTAL
4-Aminobezonitrile, 4-methylmorpholin, isobutyl
choroformate, DIPEA (N, N-diisopropylethylamine),
BOP
(benzotriazolyl-1-oxy)tris(dimethylamino)-
phosphonium hexafluorophosphate), chloroacetoni-
trile, acrylonitrile, 5-chlorovaleronitrile (Alfa Aesar,
United States), 1-methylpiperazine (Merck, Ger-
many), 5-chlor-2-nitroaniline, piperazine, 4-chloro- temperature to the end of the hydrogen absorption.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
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DNA SEQUENCE-SPECIFIC LIGANDS
147
The homogeneity of the compounds were controlled The cholesteric liquid-crystalline dispersions of
by TLC on Kieselgel 60 F254 plates (Merck, Germany) DNA (CD CLCDs) were prepared by mixing of equal
in the mixture of PriOH and concentrated NH4OH
(3 : 1). The substances were detected on the plates in
UV-light at 254 nm and/or according to their fluores-
cence at 365 nm.
The 1H NMR spectra were recorded on a AMX-400
spectrometer (Bruker, Germany, 400.0 MHz) in
DMSO-d6 (δ, ppm; J, Hz) at 32°С (unless otherwise
indicated). A resonance from the residual protons of
the solvent was used as an internal standard. The
numeration of the hydrogen atoms of piperazine (Pip)
and benzimidazole (Bim) cycles was given in the
Scheme 1 of the DBP(n) synthesis. The resonances in
the 1H NMR spectra of DBP(n) were attributed on the
basis of the published data [19].
volumes of the water-salt (0.3 M NaCl, pH 6.8) solu-
tions of DNA and PEG (4000 Da, Fluka, Germany,
340 mg/mL). The CD spectrum was recorded one
hour after the mixing. We observed an appearance of
the intensive band (λmax 270 nm, Δε 130–140 M–1 cm–1)
that suggested the formation of CLCDs of the DNA
molecules. The ligand-DNA complexes in CLCDs
were formed by the addition of the initial solution of
DBP(2) or DBP(4) in portions from 2 to 5 μL to
CLCD (2 mL) with intensive stirring. Then, the CD
spectrum was recorded again in the area of absorption
of DNA and the ligand (the interval of the wavelengths
from 250 to 380 nm).
The mass spectra were recorded by the MALDI-
TOF method on a 4800 Plus mass spectrometer (AB
Sciex, United States;) in the regime of a registration of
positive ions with the use of a reflectrone (unless oth-
erwise indicated). 2,5-Dihydroxybenzoic acid was
used as a matrix. The Nd:YAG laser (355 nm) was
applied.
The Synthesis of the DBP(1–4) Dimeric
Bisbenzimidazoles
The general method for the synthesis of 1,4-piperas-
inedialkylnitriles. The corresponding ω-chloroalkylni-
trile (0.12 mol) was added dropwise within 40 min to
the suspension of piperazine (5.00 g, 0.058 mmol) and
DIPEA (0.1 mL, 0.57 mol) in anhydrous acetonitrile
(30 mL) on stirring and ice-cooling. The reaction
mixture was heated to 110°С for 2 h, cooled, and
maintained at room temperature for 12 h. The solvent
was evaporated, and the dry residue was dissolved in
chloroform (40 mL) and water (30 mL). The organic
layer was separated, and the aqueous layer was extracted
with two portions of chloroform (10 mL each). The
organic solvent was evaporated, and the dry residue was
recrystallized from ethyl acetate (35 mL).
The IR spectra were recorded on a 3100 FT-IR
Excalibur Series spectrometer (Varian, United States).
Spectral Studies of DBP(n)
The absorption spectra, the fluorescent spectra of
solutions, and the CD spectra were recorded on a
Cary100 spectrophotometer (Varian, United States), a
Cary Exlipse spectrofluorimeter (Varian, United
States), and an SKD-2 portable dichrometer (the
Institute of Spectroscopy of the Russian Academy of
Sciences, Troitsk), respectively. All the spectra were
recorded in quartz cuvettes with the optical path
length of 1 cm at 22°С.
The preparation of the calf thymus DNA (Sigma,
United States) was dissolved in a water-salt solution of
0.3 M NaCl + 2 mM Na3PO4 (buffer A, pH 6.85) and
depolymerized on an UZDN-2T ultrasound dis-
perser. The DNA molecular mass after the ultrasound
depolymerization was determined by an electrophore-
sis in the 1% agarose gel and proved to be (0.5–0.8) ×
106 Da. The DNA concentration in solutions was
determined spectrophotometrically using the molar
extinction coefficient of ꢀ260 = 13200 М–1 сm–1 (rela-
tive to one base pair).
The initial solutions of DBP(2) and DBP(4) with a
concentration of ~1 mM were prepared by an addition
of the ligand sample to DMSO. The DNA complexes
with DBP(2) and DBP(4) were formed by the addition
of the concentrated DNA solution (~4 mM of bp) to
the ligand solution in 1 mM sodium cacodylate. The
solutions were carefully stirred at room temperature
and the absorption or fluorescent spectra were
recorded.
2,2'-(Piperazine-1,4-diyl)diacetonitrile: the yield
45.5%; mp 170–171°С; 1H NMR: 2.56 (8 Н, m, Pip),
3.67 (4 Н, s, CH2–CN).
3,3'-(Piperazine-1,4-diyl)dipropanonitrile:
the
yield 45.4%; mp 173–175°С; 1H NMR: 2.50 (8 Н, m,
Pip), 2.55 (4 Н, t, J 6.6, CH2–CN), 2.65 (4 Н, t, J 6.6,
N–CH2).
4,4'-(Piperazine-1,4-diyl)dibutanonitrile: the yield
45.7%; mp 101–102°С; 1H NMR: 1.70 (4H, q, J 6.8,
N-CH2-CH2), 2.33 (4H, t, J 7.0, CH2–CN), 2.36
(8H, broadened s, Pip), 2.46 (4H, t, J 7.0, N–CH2).
5,5'-(Piperazine-1,4-diyl)dipentanonitrile:
the
yield 36.8%; mp 183–185°С; 1H NMR: 1.64 (4H, q, J
7.6, CH2–CH2–CN), 1.80 (4H, q, J 7.6, N–CH2–
CH2), 2.56 (4H, t, J 7.6, CH2–CN), 3.13 (4H, broad-
ened s, N–CH2), 3.33–3.80 (8H, m, Pip).
General method for the synthesis of 1,4-piperazine-
dialkylcarbonic acids. 1,4-Piperazinedialkylnitriles
(5 mmol) were boiled in concentrated HCl (5 mL) for
2 h and remained to stay for a night at 4°С. The pre-
cipitate was filtered, washed with cool water, and dried
in vacuum over P2O5/NaOH.
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KOVAL et al.
(KBr), cm–1: 845, 963, 1149, 1210, 1253, 1402, 1462,
Dihydrochloride of 1,4-piperazinediacetic acid: the
1
yield 65.3%; mp 250–252°С; H NMR: 2.51 (8Н, 1639, 2931, 2961.
broadened s, Pip), 3.35 (4Н, s, СН2).
DBP(2) · 8 HCl: the yield 200 mg (41.4%); mp
287–288°С; Rf 0.68; λmax 324 nm (ε324 46000 М–1 cm–1);
Dihydrochloride of 1,4-piperazinedipropanic acid:
1
1H NMR: 2.84 (6H, s, N–CH3), 2.94 (4H, t, J 7.5,
CO–CH2), 3.23 (8H, d, J 8.2, H (3'', 5'')), 3.49 (4H, t,
J 7.5, CH2–N), 3.54 (4H, d, J 8.2, H(2'', 6'')), 3.65
(8H, broadened s, H(2''', 3''', 5''', 6''')), 3.86 (4H, d, J
8.2, H(2'', 6'')), 4.76 (4H, d, J 5.1, CH2–NH), 7.20
(2H, d, J 1.9, H7'), 7.34 (2H, dd, J1 8.9, J2 1.9, H5'),
7.71 (2H, d, J 8.9, H4'), 7.97 (2H, d, J 8.3, H4), 8.39
(2H, d, J 8.3, H5), 8.79 (2H, s, H7), 9.18 (2H, t, J 5.1,
the yield 84.5%; mp 266–268°С; H NMR (90°C):
2.79 (4Н, t, J 7.4, CH2–COOH), 3.25 (4Н, t, J 7.4,
N–CH2), 3.40 (8Н, broadened s, Pip).
Dihydrochloride of 1,4-piperazinedibutanic acid:
1
the yield 78.5%; mp 234–235°С; H NMR (90°C):
1.94 (4Н, q, J 7.5, СО–СН2–СН2), 2.36 (4Н, t, J 7.4,
СO–СН2), 3.05 (4Н, t, J 7.4, N–СН2), 3.44 (8Н,
broadened s, Pip).
13
CH2–NH), 11.20 (2H, broadened s, NH(Bim)); C
Dihydrochloride of 1,4-piperazinedivaleric acid: the
1
NMR (100 MHz, DMSO-d6, 32°C): 169.88; 155.37;
148.67; 147.96; 136.87; 134.86; 133.20; 126.27; 123.49;
118.81; 117.17; 115.30; 114.42; 98.93; 52.12; 51.07;
47.66; 46.21; 41.92; 36.49; 29.44; Mass spectrum m/z:
917.58 [M]+, calculated for C50H60N16O2 917.11; IR,
yield 88.3%; mp 249–251°С; H NMR (90°C): 1.59
(4H, q, J 7.4, CH2–CH2–COOH), 1.74 (4H, q, J 7.5,
N–CH2–CH2), 2.27 (4H, t, J 6.8, CH2–COOH),
3.02 (4H, t, J 7.6, N–CH2), 3.43 (8H, broadened s,
Pip).
νmax (KBr), cm–1: 846, 962, 1149, 1211, 1253, 1400,
1459, 1638, 2932, 2961.
The general method for the synthesis of the DBP(1–4)
dimeric bisbenzimidazoles. DIPEA (1.26 mL,
7.2 mmol) was added to the suspension of the mono-
meric bisbenzimidazole (MB, 405 mg, 0.8 mmol) [1]
and the corresponding 1,4-piperazinedialkylcarbonic
acid (0.4 mmol) in an anhydrous DMF (10 mL) with
cooling on an ice-bath and stirring. The reaction mix-
ture was stirred with cooling for 20 min, and BOP
(400 mg, 0.9 mmol) was added. The reaction mixture
was stirred for 15 min at 0°C and allowed to stay at
room temperature for a night. The solvent was evapo-
rated, and the oily dark precipitate was triturated with
chloroform (10 mL) to the formation of beige crystals.
The crystals were filtered, washed with chloroform
(1 × 5 mL) and ethyl acetate (2 × 5 mL), and dried in
air. After 30 min, the precipitate was solved in ethanol
(10 mL), concentrated HCl (1 mL) was added, and the
solution was evaporated. The solid residue was tritu-
rated with an anhydrous ethanol and filtered. The
formed greenish yellow powder was dried in vacuum
over Р2О5/NaOH. The substances were homogenous
according to TLC.
DBP(3) · 8 HCl: the yield 410 mg (82.8%); mp
274–275°С; Rf 0.69; λmax 324 nm (ε324 50400 М–1 cm–1);
1H NMR (120°C): 2.02 (4H, q, J 7.6, CO–CH2–
CH2), 2.43 (4H, t, J 7, CO–CH2), 2.87 (6H, s, N–
CH3), 3.07 (4H, t, J 7.6, CH2–N), 3.39 (16H, m,
H(2'', 3'', 5'', 6'')), 3.59 (8H, broadened s, H(2''', 3''',
5''', 6''')), 4.66 (4H, broadened s, CH2–NH), 7.23
(2H, dd, J1 8.9, J2 1.9, H5'), 7.25 (2H, s, H7'), 7.68
(2H, d, J 8.9, H4'), 7.81 (2H, d, J 8.9, H4), 8.22 (2H,
d, J 8.2, H5), 8.40 (2H, broadened s, CH2–NH); 8.64
(2H, s, H7); Mass spectrum, m/z: 945.43 [M]+, calcu-
lated for C52H64N16O2 945.17; IR, νmax (KBr), cm–1:
846, 962, 1149, 1211, 1253, 1400, 1459, 1638, 2932,
2961.
DBP(4) · 8 HCl: the yield 205 mg (81.0%); mp 271–
272°C; Rf 0.72; λmax 324 nm (ε324 54800 М–1 cm–1;
1H NMR (90°C): 1.67 (4H, q, J 7, CO–CH2–CH2),
1.78 (4H, q, J 7.5, CH2–CH2–N), 2.33 (4H, t, J 7,
CO–CH2), 2.84 (6H, s, N–CH3), 3.12 (4H, t, J 7.5,
CH2–N), 3.39 (8H, broadened s, H (2''', 3''', 5''', 6''')),
3.54 (16H, s, H (2'', 3'', 5'', 6'')), 4.66 (4H, d, J 5,
CH2–NH), 7.24 (2H, d, J 2.1, H7'), 7.28 (2H, dd, J1 9,
J2 2.1, H5'), 7.70 (2H, d, J 9, H4'), 7.86 (2H, d, J 8.3,
H4), 8.27 (2H, dd, J1 8.3, J2 1.2, H5), 8.49 (2H, t, J
5.1, NH–CH2), 8.69 (2H, d, J 1.2, H7); Mass spec-
DBP(1) · 8 HCl: the yield 360 mg (76.2%); mp
294–295°С; Rf 0.70; λmax 324 nm (ε324 43500 М–1 cm–1);
1H NMR: 2.84 (6H, d, J 2.6, N–CH3), 3.23 (8H, d,
J 8.2, H(3'', 5'')), 3.45 (8H, broadened s, H(2''', 3''',
5''', 6''')), 3.54 (4H, d, J 8.2, H (2'', 6'')), 3.88 (4H, d,
J 8.2, H (2'', 6'')), 3.97 (4 H, s, CO–CH2). 4.81 (4H,
d, J 5.1, CH2–NH), 7.21 (2H, d, J 1.9, H7'), 7.35 (2H,
dd, J1 8.9, J2 1.9, H5'), 7.72 (2H, d, J 8.9, H4'), 7.96
(2H, d, J 8.3, H4), 8.38 (2H, d, J 8.3, H5), 8.79 (2H,
broadened s, H7), 9.43 (2H, broadened s, CH2–NH),
trum, m/z: 973.64 [M]+, calculated for C54H68N16O2
973.22; IR, νmax (KBr), cm–1: 846, 964, 1155, 1213,
1258, 1490, 1460, 1638, 2932, 2962.
11.21 (1H, broadened s, NH(Bim)); 13C NMR
(100 МHz, DMSO-d6, 32°C, δ): 170.00; 155.08;
148,62; 133.22; 122.77; 117.07; 115.06; 114.38; 99.01;
57.04; 52.17; 49.89; 46.27; 41.95; 36.52; Mass spec-
trum, m/z: 889.57 [M]+ , calculated for C48H56N16O2
889.06; IR (Varian 3100 FT-IR Excalibur Series), νmax
ACKNOWLEDGMENTS
This study was supported by the Russian Founda-
tion for Basic Research, project no. 14-04-00388 and
by the Program of the Presidium of the Russian Acad-
emy of Sciences on the molecular and cellular biology.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
Vol. 43
No. 2
2017

DNA SEQUENCE-SPECIFIC LIGANDS
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Translated by L. Onoprienko
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 43 No. 2 2017