DNA sequence-specific ligands: XIII. New dimeric Hoechst 33258 molecules, inhibitors of HIV-1 integrase in vitro

Article abstract of DOI:10.1134/S1068162007060064

Dimeric Hoechst 33258 molecules [dimeric bisbenzimidazoles (DBBIs)] that, upon binding, occupy one turn of the B form of DNA in the narrow groove were constructed by computer simulation. Three fluorescent DBBIs were synthesized; they consist of two bisben

Full text of DOI:10.1134/S1068162007060064

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2007, Vol. 33, No. 6, pp. 569–578. © Pleiades Publishing, Inc., 2007.
Original Russian Text © A.V. Gromyko, V.I. Salyanov, S.A. Strel’tsov, V.A. Oleinikov, S.P. Korolev, M.B. Gottikh, A.L. Zhuze, 2007, published in Bioorganicheskaya Khimiya, 2007,
Vol. 33, No. 6, pp. 613–623.
DNA Sequence-Specific Ligands: XIII. New Dimeric Hoechst
33258 Molecules, Inhibitors of HIV-1 Integrase In Vitro
A. V. Gromykoa, V. I. Salyanova, S. A. Strel’tsova, V. A. Oleinikovb, S. P. Korolevc,
M. B. Gottikhc, and A. L. Zhuzea, 1
a Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow, 119991 Russia
b Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,
ul. Miklukho-Maklaya 16/10, Moscow, 117997 Russia
c Belozersky Institute of Physicochemical Biology and Faculty of Chemistry, Moscow State University,
Vorob’evy gory, Moscow, 119992 Russia
Received December 12, 2006; in final form, January 15, 2007
Abstract—Dimeric Hoechst 33258 molecules [dimeric bisbenzimidazoles (DBBIs)] that, upon binding,
occupy one turn of the B form of DNA in the narrow groove were constructed by computer simulation. Three
fluorescent DBBIs were synthesized; they consist of two bisbenzimidazole units tail-to-tail linked to phenolic
hydroxy groups via penta- or heptamethylene or tri(ethylene glycol) spacers and have terminal positively
charged N,N-dimethylaminopropyl carboxamide groups in the molecule. The absorption spectra of the DBBIs
in the presence of different DNA concentrations showed a hypochromic effect and a small shift of the absorp-
tion band to longer wavelengths, which indicated the formation of a complex with DNA. The presence of an
isobestic point in the spectrum indicates the formation of one type of DBBI–DNA complexes. The interaction
of DBBIs with DNA was studied by CD using a cholesteric liquid-crystalline dispersion (CLD) of DNA. The
appearance of a positive band in the absorption region of ligand chromophores in the CD spectrum of the DNA
CLD indicates the formation of a DBBI–DNA complex in which ligand chromophores are arranged at an angle
close to 54° relative to the helix axis of DNA, which suggests the localization of the DBBI in the narrow groove
of DNA. All the DBBIs were found to be in vitro inhibitors of HIV-1 DNA integrase in the 3'-processing reac-
tion, and, of the three DBBIs, two dimers inhibit HIV-1 integrase even in submicromolar concentrations.
Key words: DNA, HIV-1 DNA integrase, dimeric Hoechst 33258 molecules, inhibitors, molecular modeling,
narrow-groove ligand, optical methods of analysis, synthesis
DOI: 10.1134/S1068162007060064
and penetrating through cellular and nuclear mem-
branes with the effective staining of cell nuclei. How-
ever, a drawback of bis-Hts is poor solubility in water
due to the tendency to form aggregates [2]. Here we
describe the synthesis of three dimeric DBBIs that con-
tain four 2,6-substituted benzimidazole fragments. We
tried to improve the solubility of bis-Ht analogues by
the substitution of more hydrophilic N,N-dimethylami-
nopropyl carboxamide groups for N-methylpiperazine
rings at the ends of Ht molecules. As in the construction
of bis-Hts, linker bridges between two bisbenzimida-
zole fragments of DBBIs were created on the basis of
oligomethylene or oligo(ethylene glycol) chains. The
structure of these linkers should provide such curvature
of the DBBI molecule that it could form a helix isogeo-
metric to the DNA narrow groove.
INTRODUCTION
In the previous communication [1], we have
described the construction and synthesis of three
dimeric molecules of the fluorescent dye Hoechst
33258 (bis-Ht).2 The goal of this study was to create
compounds that noncovalently and site-specifically
bind to DNA in its narrow groove [1]. These com-
pounds may prove convenient tools in molecular biol-
ogy studies and useful therapeutic agents for the control
of gene expression in cells. The studies of bis-Hts
showed that they are new promising fluorescent dyes
capable of both differentially staining chromosomes
1
Corresponding author; phone: +7 (495) 135-9718; fax: +7 (495)
135-1405; e-mail: zhuze@imb.ac.ru.
Abbreviations: CLD, cholesteric liquid-crystalline dispersion;
DBBI, dimeric bisbenzimidazole; Ht, Hoechst 33258 dye.
2
569

570
GROMYKO et al.
N
N
N
H
N
N
H
N
H C
3
Hoechst 33258
OH
N
N
N
N
N
1
1
N
H
R
R
N
H
N
H
H
R
O
O
bis-Ht, DBBI
Compound
R1
R
bis-Ht(5)
bis-Ht(7)
bis-Ht(8)
(CH2)5
(CH2)7
N
N CH3
(CH2)2–O–(CH2)2–O–(CH2)2
DBBI(5)
DBBI(7)
DBBI(8)
(CH2)5
(CH2)7
–CONH–(CH2)3–N(CH3)2
(CH2)2–O–(CH2)2–O–(CH2)2
A preliminary analysis by the computer simulation DNA bases (O2 atoms of thymines and/or N3 atoms of
method showed that this substitution not only should
not impair the binding of these new dimeric bisbenzim-
idazole ligands to DNA, as compared with bis-Ht, but
should increase the affinity of these compounds to the
sequence d(GCTTTTGCAAAAGC)2[N(A/T)n-NN-
(A/T)nN, where n = 3 or 4], which was planned to be
one of probable targets for bis-Ht [1].
adenines, which act as acceptors in hydrogen bond).
The use of computer simulation allowed us to deter-
mine the parameters of interaction of three DBBIs
(which differ in linker structure) with the tetradecanu-
cleotide d(GCTTTTGCAAAAGC)2 and to compare
the corresponding parameters of interaction of three
bis-Hts having the same linkers with the same duplex
(Table 1).
RESULTS AND DISCUSSION
The effect of DNA sequence on the DNA–ligand
binding energy was estimated by the example of three
DBBIs (Table 2). It was shown that the presence ofA or
T bases at the sites of binding of DBBI dibenzimidazole
fragments to the DNA is obligatory. A change in the
sequence of AAAA and TTTT fragments practically
does not affect the binding energy. However, the intro-
duction of G·C pairs into these sites markedly impairs
Molecular modeling was used to construct the
DNA-specific bidentate ligands on the basis of the bis-
benzimidazole motif, which was applied as an AT-spe-
cific unit. The structure of the ligand suggested their
localization in the narrow groove, and the length was
approximately equal to one turn of the double-stranded
DNA. As in the case of bis-Ht [1], the linkers between
two bisbenzimidazole fragments of DBBI were the binding.
designed on the basis of the oligomethylene or
The calculations were carried out using the
oligo(ethylene glycol) chains with taking into account
their conformation mobility and a low affinity for the
sugar phosphate backbone and DNA base pairs. The
structure of the linkers should allow the DBBI molecule
to adopt the form isohelical to the narrow DNA groove,
and their parameters should be such that the DBBI mol-
ecule acts as a bidentate ligand. This means that both
bisbenzimidazole fragments (which are responsible for
the high affinity to A/T sequences of the double-
stranded DNA) should simultaneously be in the posi-
AMBER3 method of molecular mechanics. The effect
of solvent (water molecules) was accounted for by
choosing a distance-dependent value of dielectric per-
meability, an approach that enables to find the condi-
tions of electrostatic interaction close to the conditions
of screening by polar solvent molecules. The model
parameters were chosen so that the conformations of
DNA in the free state and in the complex with ligand
are close to the conformations known from X-ray dif-
tion optimal for hydrogen bonding between them and fraction studies [3, 4].
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 33 No. 6 2007

DNA SEQUENCE-SPECIFIC LIGANDS
571
Table 1. Parameters of interaction of bisbenzimidazole
ligands (energy, kcal/mol)* of different chemical structures
with the duplex d(GCTTTTGCAAAAGC)2
The energy of the ligand interaction with DNA
(Ebound) is determined by the expression:
Ebound = Ecompl – (EDNA + Elig),
Ligand
Ebound
∆EDNA
∆Elig
where EDNA and Elig are the energies of DNA and ligand
in free state, and Ecompl is the energy of the DNA–ligand
complex.
bis-Ht(5)
bis-Ht(7)
bis-Ht(8)
DBBI(5)
DBBI(7)
DBBI(8)
– 93.43
–98.85
–105.17
–110.09
–115.18
–113.57
11.59
16.78
13.14
15.69
20.55
20.85
5.94
8.09
5.62
6.58
5.45
4.18
Interaction of the ligand with DNA leads to changes
in the structures of both components. The distortion in
the ligand structure caused by the interaction with DNA
(∆Elig) was estimated by determining the changes in the
energy of optimal conformation in free state (Elig free
)
* Energy values were calculated by the AMBER-3 method of
molecular mechanics.
and the conformation corresponding to its bound state
but in the absence of DNA (Elig bound):
∆Elig = Elig free – Elig bound
Similarly, the distortion of DNA structure was esti-
mated by the expression
.
Table 2. Energy of ligand binding to different double-strand-
ed DNA sequences
Ligand
DNA sequence 5'
3' Ebound, kcal/mol
∆EDNA = EDNA free – EDNA bound
.
DBBI(5)
GCTTTTGCAAAAGC
GCAAAAGCTTTTGC
GCATGCATGCATGC
GCGCGCGCGCGCGC
GCTTTTGCAAAAGC
GCAAAAGCTTTTGC
GCATGCATGCATGC
GCGCGCGCGCGCGC
GCTTTTGCAAAAGC
GCAAAAGCTTTTGC
GCATGCATGCATGC
GCGCGCGCGCGCGC
–110.09
–109.59
– 99.72
–101.76
–115.18
–116.61
–102.84
–104.07
–113.57
–117.42
–109.29
–109.09
As in the previous case, EDNA free and EDNA bound are
the DNA energies in the free state and in the optimal
conformation and in complex with the ligand but in the
absence of the latter.
The calculations indicate that DBBI molecules are
more promising than the corresponding bis-Ht mole-
cules. The DBBI molecules showed the greatest
decrease in the energy of the system caused by binding.
In this case, the DNA structure is distorted to the great-
est extent. We have previously shown [5, 6] that the
increase in the distortion of DNA upon binding with the
inhibitor operating by the suppressive mechanism [7]
increases its inhibitory effect. This also suggests the
prospect of using these compounds as a basis for the
synthesis of the inhibitors of DNA-dependent enzymes.
DBBI(7)
DBBI(8)
Chemical synthesis of DBBIs is shown in the
scheme. As in the case of bis-Ht [1], the key event in the
synthesis of DBBIs is the condensation of 2-(3,4-
diaminophenyl)-N-[3-(dimethylamino)propyl]-1H-
benzimidazole-6-carboxamide (III) with dihydrochlo-
rides of diimidoesters (IVa)–(IVc), we have synthe-
sized earlier [1]. Diamine (III) was synthesized by
acetylation of the commercial 4-aminobenzonitrile by
acetic anhydride with subsequent nitration of 4-aceta-
midobenzonitrile by a KNO3/H2SO4 mixture [8]. The
resulting 4-acetamido-3-nitrobenzonitrile was deacety-
lated with dilute H2SO4, and then 4-amino-3-nitroben-
zonitrile was converted into the corresponding diimi-
doester dihydrochloride by the Pinner reaction. Boiling
of the latter in methanol with 3,4-diaminobenzoic acid
led to the formation of 2-(4-amino-3-nitrophenyl)-1H-
benzimidazole-6-carboxylic acid (I), which was then
condensed with 3-dimethylaminopropylamine by
means of 1,1'-carbonyldiimidazole to form 2-(4-amino-
3-nitrophenyl)-N-[3-(dimethylamino)propyl]-1H-benz-
imidazole-6-carboxamide (II). After the hydrogenation
of (II) over palladium on carbon, the resulting diamine
(III) was condensed with diimidoester dihydrochlo-
inert atmosphere. Compounds DBBI(5), DBBI(7), and
DBBI(8) synthesized represented hexahydrochlorides;
the yields at the final stage were 9.5, 19.0, and 20.3%,
respectively.
Physicochemical studies of the interaction of
DBBI(5), DBBI(7), and DBBI(8) with double-stranded
DNA were carried out by optical methods. The absorp-
tion spectra of the DBBIs in the presence of calf thymus
DNA were recorded. Figure 1 shows the absorption
spectra of DBBI(8) in the presence of DNA (curves 2,
3). One can see that an increase in the DNA concentra-
tion in solution leads to a hypochromic effect and a
slight shift of the absorption band to longer wave-
lengths. These changes in the absorption spectrum of
ligand indicate that the ligand forms a complex with
DNA. Similar changes were observed in the absorption
spectra of DBBI(5) and DBBI(7). Thus, the changes in
the absorption spectra of DBBIs in the presence of
DNA prove the formation of a complex, and the pres-
ence of the isobestic point indicates that the complexes
rides (IVa)–(IVc) by boiling in glacial AcOH in an are of one type.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 33 No. 6 2007

572
GROMYKO et al.
NO2
NH2
NO2
NH2
HO
O
ClH2N
O
HCl/EtOH
+
NH2
NH2
N
H3C
MeOH
6'
4
7
5'
N
5
(I)
NH2
NO2
O
N
H
2'
OH
CH3
CH3
N
CDI
H2N
N
CH3
H3C
N
(II)
NH2
HN
N
H
NO2
O
CH3
N
H2, Pd/C
H3C
N
NH2
NH2
(III)
HN
N
H
O
O
O
R
H3C
O
O
CH3
AcOH, N , 120°C
2
NH2Cl
NH2Cl
(IV‡) R = (CH2)5
(IV·) R = (CH2)7
(IV‚) R = (CH2)O(CH2)2O(CH2)2
2
O
O
3
R
6
N
N
4'
5
NH
NH
5'
7'
N
N
HN
NH
4"
5"
7"
H3C
CH3
N
NH
O
O
NH
N
CH3
CH3
DBBI(5), R = (CH2)5
DBBI(7), R = (CH2)7
DBBI(8), R = (CH2)2O(CH2)2O(CH2)2
Scheme.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 33 No. 6 2007

DNA SEQUENCE-SPECIFIC LIGANDS
573
Ä
(a)
1
0.6
2
3
0.4
(b)
0.2
0
280
320
360
400
440
λ, nm
Fig. 2. Hypothetical models of (a) an intramolecular
DBBI(8) sandwich and (b) an antiparallel dimer consisting
of two intramolecular DBBI(8) sandwiches. The front sand-
wich in the dimer is shown by bold lines, and hydrogen
bonds between nitrogen atoms that belong to different sand-
wiches and stabilize the dimer are designated by dashed
lines.
Fig. 1. Absorption spectra of DBBI(8) (curve 1) in the
absence and (2) in the presence of 1.35 and (3) 5.40 µg/ml
–5
of DNA. Concentration of DBBI(8) 1 × 10 M; Na-phos-
phate buffer, pH 6.86, containing 0.3 M NaCl.
We have previously studied the interaction of DNA
with bis-Ht(NMe), which differs from DBBI(7) in that
the central ëH2 group of the pentyl residue in the linker
connecting two Hoechst 33258 molecules is exchanged
by a N(CH3) group [2]. It was shown that the spectral
changes observed for the complex of DBBI(8) with
DNA (Fig. 1) are similar to those for the third-type
complexes of bis-Ht(NMe) with DNA [2]. We believe
that, in this type of complexes, dimers of bis-Ht sand-
wiches are adsorbed on DNA molecules [2]. Note that
an isolated DBBI molecule, in particular a molecule of
DBBI(8), can exist in solution in two conformations:
open and of sandwich type. We attribute the name of
sandwich type to the molecule in which the first benz-
imidazole fragment is in a stacking interaction with the
fourth fragment, and the second fragment interacts with
the third (Fig. 2a). As proposed earlier [9], these sand-
wiches can in turn form dimers stabilized by hydrogen
bonds between benzimidazole fragments belonging to
different sandwiches rather than by stacking interac-
tions (Fig. 2b). It is not excluded also that two bis-Ht
molecules in open conformation can form an intermo-
lecular sandwich stabilized by stacking interactions
between benzimidazole residues belonging to different
bis-Ht molecules. Such sandwiches are also capable of
forming dimers stabilized by hydrogen bonds (hypo-
thetical structures of the intermolecular DBBI(8) sand-
wich and its dimer are not shown).
wich adsorbed on one DNA molecule can interact with
a sandwich dimer adsorbed on another DNA molecule,
probably through hydrogen bonding. As a result,
bridges linking two double-stranded DNA molecules
are formed.
It was shown earlier that polymeric chelate com-
plexes consisting of alternating molecules of the antibi-
otic daunomycin and Cu2+ may play a role of these
bridges [10, 11]. These data agree well with the results
obtained with DBBIs(5,7,8).
The localization of DBBI in the complex with DNA
was determined by the CD spectroscopy using a CLD
of DNA. The appearance of an intense band in the CD
spectrum in the absorption region of ligand chro-
mophores not only indicates the formation of a DNA–
ligand complex, but also enables one to estimate the
geometric parameters of the complex.
The amplitude of the intense band in the CD spec-
trum, which reflects an anomalous optical activity of a
DNA CLD, is determined by equation (1) [12, 13]:
∆εν = kPν3i ∆n(A|| – A⊥)/2(νi2 – ν20),
(1)
i
where ∆ε = εL – εR – CD at frequency νi; ν0 is the fre-
quency of the band of selective reflection of a choles-
teric, which is related to the pitch P of its helical struc-
ture (P = k1nν0), n is the refraction index; ∆n is the opti-
cal anisotropy, which has the negative sign in our case;
A|| – A⊥ is the linear dichroism of DNA, which is
described, according to [14], by equation (2):
The binding to DNA of the dimers of DBBI(8) sand-
wiches of any type leads to the formation of the third
type DNA–ligand complexes. These complexes can
interact with each other [2]: the dimer of bis-Ht sand-
(A|| – A⊥)/A = k2(3cos2α – 1),
(2)
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 33 No. 6 2007

574
GROMYKO et al.
∆ Α
1000
but also the orientation of the ligand relative to the
DNA helical axis.
3
2
1
Figure 3 shows the CD spectra of a CLD of DNA in
the absence (curve 1) and in the presence (curves 2
and 3) DBBI(8) of DBBI(8). The appearance of an
intense negative band in the CD spectrum in the absorp-
tion region of DNA bases (curve 1) indicates the forma-
tion of a CLD of DNA. It is obvious that, as the concen-
tration of the ligand in solution increases, an intense
positive band appears in the CD spectrum in the absorp-
tion region of ligand chromophores (curves 2 and 3) and
the negative band in the region of DNA absorption
changes insignificantly. The calculations show that the
molar CD (∆ε) of the positive band is approximately
300 å–1 cm–1, which is comparable in the order of mag-
nitude with the (∆ε value of DNA CLD. The positive
sign of the band in the absorption region of ligand chro-
mophores indicates that the angle of their inclination
inside the complex with the DNA molecule lies
between 0 and 54°.
This suggests that DBBI(8) is not an intercalator of
DNA but is located upon complex formation in its nar-
row groove. In the CD spectra of DNA CLD in the pres-
ence of DBBI(5) and DBBI(7), no intense bands appear
in the absorption region of these compounds, and the
amplitude of the negative CD band in the absorption
region of DNA chromophores substantially decreases.
Because the absorption spectra of DBBI(5) and
DBBI(7) confirm the interaction of these ligands with
DNA, and the absence of CD in their long-wavelength
absorption band indicates that the chromophores of
these ligands in the complex with DNA are located at
an angle close to 54° relative to its helix axis, they are
the narrow-groove ligands, like DBBI(8). The decrease
in the amplitude of negative band with increasing con-
centration of the DNA–ligand complex probably results
from the distortion of the secondary structure of the
double-stranded DNA during complex formation.
0
–1000
–2000
–3000
–4000
–5000
–6000
–7000
240 260 280 300 320 340 360 380
λ, nm
Fig. 3. CD spectra of CLD of DNA in solution (curve 1) in
the absence and (2) in the presence of 0.6 and (3) 2.5 µM
DBBI(8). The concentration of DNA 16.2 µg/ml; the con-
centration of PEG (4000) 170 mg/ml; 2 mM Na-phosphate
buffer, pH 6.86, containing 0.3 M NaCl.
where α is the angle of inclination of DNA bases rela-
tive to the DNA helix axis; A||, A⊥, and A are the absorp-
tion by DNA bases of the light polarized parallel and
perpendicular to the DNA helix axis, and of nonpolar-
ized light, respectively; and k, k1, and k2 are the propor-
tionality factors.
The linear dichroism of nitrogen bases inside DNA
has the negative sign [14] since, in this case, angle α is
close to 90°. The sign of the band in the CD spectrum
in the absorption region of nitrogen bases is determined
by the direction of twisting of the cholesteric helix. A
cholesteric formed of linear DNA molecules has a left-
handed twist and is characterized by an intense negative
CD band in the absorption region of bases. It follows
from equation (2) that, at the inclination angles of
ligand chromophores relative to the DNA helical axis
lying within the range 54–90° (if the ligand is interca-
lated into the DNA molecule, α is 90°), a negative band
should appear in the absorption region of the ligand in
the CD spectrum. If the ligand molecule is located on
DNA so that the angle α lies between 0 and 54°, a pos-
itive band should appear in the CD spectrum. At α
value equal to 54°, the optical activity in the absorption
Biochemical investigation of the DBBIs as the
inhibitors of the 3'-processing reaction was carried out
with HIV-1 integrase (hereinafter referred to as inte-
grase). Integrase is one of three major enzymes of HIV-
1 that are necessary for the virus replication [15]. It is
responsible for the incorporation of the viral DNA into
the host chromosome. Integrase is a rather attractive
target for the antiviral therapy. Up to now, its cellular
homologues in mammals have not been described;
therefore, it can be assumed that the inhibitors of inte-
grase are relatively nontoxic. Integrase specifically rec-
region of ligand chromophores is absent. In this case, ognizes the terminal sequences of regions U3 and U5 of
long terminal repeats of the viral DNA and splits off the
dinucleotide GT from the 3'-termini of both DNA
strands [16]. This is the first function of integrase; it is
called 3'-processing. Then integrase accomplishes the
second of its functions, the incorporation of the viral
DNA into the chromosomal DNA.
the higher the concentration of the ligand complexed
with DNA, the greater the amplitude in the region of its
absorption band in the CD spectrum (provided that the
complex formation does not disturb the structural
parameters of the double-stranded DNA molecule sub-
stantially). Thus, the intense bands in the CD spectrum
of a CLD of DNA in the presence of the ligand allow
Here we studied the inhibitory activity of DBBIs in
one to establish not only the fact of complex formation the 3'-processing reaction using a model 21-nt duplex
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 33 No. 6 2007

DNA SEQUENCE-SPECIFIC LIGANDS
575
Table 3. Inhibition of HIV-1 DNA integrase in the reaction
of 3'-processing of duplex U5B/U5A
U5B/U5A (Table 3), which mimics the terminal
sequence of region U5 of HIV-1 DNA:
U5B 5'-GTGTGGAAAATCTCTAGCA↓GT
U5A 3'-CACACCTTTTAGAGATCGTCA
*
*
IC50, µM
IC50, µM
Compound
Compound
Note that, by itself, Ht did not inhibit the 3'-processing
reaction at concentrations up to 100 µM. So, the dimer-
ization of the Ht molecule into a bis-Ht molecule or
DBBI led to the appearance of a new property in the lat-
ter compounds, namely, the inhibitory activity, which is
absent from Ht.
Thus, we established that, at micro/nanomolar con-
centrations, bis-Hts and DBBIs inhibit in vitro HIV-1
DNA integrase, one of the key enzymes of the replica-
tion of human immunodeficiency virus. Moreover, as
seen from a pairwise comparison of the structurally
homologous compounds (Table 3), the inhibitory activ-
ity of DBBI molecules is by one order of magnitude
higher than that of bis-Ht molecules.
It is noteworthy that the angles formed by chro-
mophores DBBI(5) and DBBI(7) with the DNA helical
axis differ from the angle formed by DBBI(8) with
DNA. This indicates that the conformations of com-
plexes of DBBI(5) and DBBI(7) with DNA are differ-
ent from that of the complex of DBBI(8) with DNA.
The differences in the conformations of these com-
plexes correlate well with their different abilities to
inhibit HIV-1 integrase. Note that the complexes of
DBBI molecules with CLD of DNA are of the third
type [2]; i.e., they have the form of dimers of DBBI
sandwiches adsorbed on DNA.
bis-Ht(5)
bis-Ht(7)
bis-Ht(8)
3.2
2.6
DBBI(5)
DBBI(7)
DBBI(8)
0.5
0.2
5.0
40.0
Note: * IC , the concentration of the inhibitor at which the reac-
50
tion is inhibited by 50%.
hydrogen atoms of benzene and benzimidazole (Bim)
cycles is given in the scheme. Mass spectra were
recorded on a Vision-2000 time-of-flight instrument
(ThermoBioanalysis, UK) in the regime of registration
of positive ions; 2,5-dihydroxybenzoic acid was used as
a matrix; N2 laser, 337 nm.
2-(4-Amino-3-nitrophenyl)-1H-benzimidazole-6-
carboxylic acid (I). A suspension of 3,4-diaminoben-
zoic acid (0.270 g, 1.775 mmol) and 4-amino-3-nitro-
phenylimidic acid hydrochloride ethyl ester (0.436 g,
1.775 mmol), obtained from 4-aminobenzonitrile as
described in [8], was boiled under stirring in MeOH
(5 ml) for 1 h in an nitrogen atmosphere. Then Et3N
(0.25 ml, 1.775 mmol) was added to the reaction mix-
ture, and the mixture was boiled for several minutes.
After addition of water (5 ml), the boiling was contin-
ued for additional several minutes. After cooling, an
abundant precipitate was filtered and washed three
times with water and then three times with acetone.
Drying in a vacuum yielded 0.398 g (75.24%) of orange
(I); mp > 300°C; Rf 0.76; 1H NMR: 7.17 (1 H, d, J 8.1,
H5'), 7.59 (1 H, d, J 7.5, H4), 7.82 (1 H, d, J 7.5, H5),
7.87 (2 H, s, NH2), 8.13 (1 H, s, H7), 8.21 (1 H, d, J 8.1,
H6'), 8.85 (1 H, s, H2'); MS, m/z: 299.0 [M + H]+. Cal-
culated M 298.25 (C14H10N4O4).
EXPERIMENTAL
The following preparations were used: 4-aminoben-
zonitrile (Fluka, Switzerland), 3,4-diaminobenzoic
acid (EGA-CHEMIE, Germany), 3-dimethylaminopro-
pylamine (Aldrich, United States), 1,1'-carbonyldiimi-
dazole (Ferrak, Germany), and 10% Pd/C (Merck, Ger-
many). Solutions of the compounds in organic solvents
were dried with Na2SO4. Solvents were as a rule evap-
2-(4-Amino-3-nitrophenyl)-N-[3-dimethylamino)
orated on a rotor evaporator in a vacuum of a water-jet propyl]-1H-benzimidazole-6-carboxamide (II). Acid
pump at 50°ë. The substances were dried in a vacuum (I) (2.60 g, 8.72 mmol) was dissolved in anhydrous
over P2O5 and NaOH. Mps were determined on a Boet- DMF (15 ml) in a flask equipped with a calcium chlo-
hius device (Germany) and were not corrected. Hydro- ride tube, 1,1'-carbonyldiimidazole (3.73 g, 2.30 mmol)
genation was carried out over 10% Pd/C at the atmo- was at once added, and the mixture was stirred for 45
spheric pressure and room temperature until the hydro- min at 40°ë. Then 3-dimethylaminopropylamine (6.0
gen absorption stopped. The purity of the compounds ml, 47.68 mmol) was added (the solution got warm),
was monitored by TLC on Kieselgel 60 F254 plates and the solution was kept overnight at 20°ë. The sol-
(Merck, Germany) in 5 : 1 MeOH–Et3N system. Sub- vent was evaporated, and the residue was coevaporated
stances on chromatograms were detected in UV light three times with water until the whole compound
from the light absorption at 254 nm or from fluores- became crystalline. It was dissolved in boiling water (5
cence at 365 nm. Absorption spectra were measured in ml), concentrated ammonia was added until the solu-
water; DBBI samples were preliminarily dissolved in tion became turbid, and the solution was kept after
1
DMSO. ç NMR spectra were recorded on an AMX- cooling overnight in a refrigerator. The flocculent pre-
400 instrument (Bruker, Germany; 400.0 MHz) in cipitate was filtered and washed on filter with concen-
DMSO-d6 (δ, ppm; J, Hz) at 23°ë (unless otherwise trated ammonia preliminarily cooled to –15°C. After
specified); the signal from the solvent residual protons drying in a vacuum over P2O5, 2.62 g of gumlike sub-
was used as an internal standard. The numbering of stance was obtained. The recrystallization of the sub-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 33 No. 6 2007

576
GROMYKO et al.
stance from MeOH yielded 2.034 g (61.0%) of orange- for 30 min in nitrogen atmosphere. After evaporation of
1
colored (II); mp 161°C; Rf 0.54; H NMR: 1.62 [2 H, solvent in a vacuum, the residue was dissolved in a mix-
m, (CH3)2NCH2), 2.15 (6 H, s, N(CH3)2), 2.83 [2 H, m, ture of MeOH (4 ml) and water (3 ml), and 1 : 1 PriOH–
NCH2CH2), 3.29 (2 H, m, CONHCH2), 7.17 (1 H, d, J con. ammonia mixture (3 ml) was added. The precipi-
8.7, H5'), 7.16 (1 H, d, J 6.8, H5), 7.70 (1 H, d, J 8.1, tated solid was filtered and reprecipitated twice from
H7), 7.8 (2 H, s, NH2), 8.05 (1 H, s, H4), 8.25 (1 H, d, 50% aqueous MeOH by equal volumes of con. ammo-
J 11.2, H6'), 8.45 (1 H, t, J 5.1, NHCO), 8.85 (1 H, s, nia. The precipitate was purified on a column (2 ×
H2'); EI MS, m/z: 383.2 [M + H]+. Calculated M 382.42 110 cm) of Sephadex LH-20 in DMF; the rate of elu-
(C19H22N6O3).
tion of DMF was 1.5 ml/h. The yield of DBBI(7) in the
form of a free base was 0.101 g (21.3%). For the con-
version to hexahydrochloride, the substance (0.085 g)
was dissolved in methanol (7 ml) containing 0.1 ml of
AcOR, and 2 : 1 MeOR–conc. HCl mixture (1 ml) was
added. Evaporation and drying in a vacuum yielded
0.101 g (89.4%) of DBBI(7) hexahydrochloride; mp
>300°C, Rf 0.21, λma 338 nm (ε338 – 112000 å–1 cm–1).
DBBI(7) hexahydrochloride in solution produced a
2-(3,4-Diaminophenyl)-N-[3-(dimethylamino)
propyl]-1H-benzimidazole-6-carboxamide (III). A
suspension of nitroamine (II) (0.782 g, 2.05 mmol) in
methanol was hydrogenated at atmospheric pressure
for 1 h over 10% Pd/C (150 mg); the solution became a
light yellow. The catalyst was filtered off, and the sol-
vent was evaporated in a vacuum, following which the
residue was twice coevaporated with abs. EtOH. After
drying, diamine (III) was obtained as a solid substance
in a quantitative yield. The substance was homoge-
neous, as indicated by TLC (Rf 0.41) and was immedi-
ately used in the synthesis of DBBIs(5,7,8).
1
blue fluorescence; H NMR (DMSO-d6 + 1 drop of
CF3COOH): 1.46 (6 H, m, é(ëç2)3ëç2 + 2 ×
NHëç2ëç2), 1.77 (4 H, m, Oëç2ëç2), 1.94 (4 H,
quintet, J 7.9, é(ëç2)2ëç2), 2.79 (12 H, s, N(CH3)2),
3.13 (4 H, m, (CH3)2NCH2), 3.39 (4 H, m, NHëç2),
DBBI(5). A solution of diamine (III) (0.720 g, 4.10 (4 H, t, J 6.2, OCH2), 7.22 (4 H, d, J 8.7, H2, H6),
2.044 mmol) in glacial AcOH (13 ml) was added to 7.85 (2 H, d, J 8.7, H5'), 8.00 (2 H, d, J 8.7, H4'), 8.04
dihydrochloride of ethyl ester of 4-[(5-{4- (2 H, d, J 8.7, H4''), 8.17 (4 H, d, J 8.7, H3, H5), 8.25
[ethoxy(imino)methyl]phenoxy}pentylphenyl)oxy]-1-
(4 H, d, J 8.7, H5''), 8.28 (4 H, s, H7'), 8.55 (2 H, s,
phenylimidic acid (IVa) [1] (0.482 g, 1.022 mmol), and H7''), 8.82 (2 H, t, J 4.9, CONH), 9.51 (4 H, m, NH,
the mixture was refluxed for 20 min in nitrogen atmo- Bim); MS, m/z: 1005.6 [M]+, 1027.9 [M + Na]+. Calcu-
sphere. The mixture was cooled to room temperature, lated M 1005.2 (C59H64N12O4).
and the precipitated solid was filtered and washed twice
DBBI(8). A solution of diamine (III) (0.532 g,
with glacial AcOH and benzene. The resulting hygro-
1.51 mmol) in AcOR (15 ml) was added to 4-{2-[2-(2-
scopic light yellow powder was dried and dissolved
{4-[ethoxy(imino)methyl]phe-
under heating in methanol (11 ml). After the addition of
noxy}ethoxy)ethoxy]ethoxy}- 1-phenylimidic acid ethyl
concentrated ammonia (~10 ml), the resulting suspen-
ester dihydrochloride (IVc) [1] (0.391 g, 0.755 mmol),
sion was cooled and left in a refrigerator for several
and the mixture was refluxed for 30 min in nitrogen
hours. The precipitate formed was filtered, washed
atmosphere. The solvent was evaporated, the residue
twice with water, and dried in a vacuum. Then the resi-
was dissolved in hot water (5 ml), and concentrated
due was dissolved under heating in AcOH (4 ml), and
ammonia (1 ml) was added. The precipitate was fil-
AcOH saturated at 20°C with dry hydrogen chloride
tered, dried in a vacuum, dissolved in EtOH (10 ml),
was added to the hot solution until the precipitation
and treated under boiling with active charcoal
ceased. The precipitate was filtered, washed twice with
(200 mg). After the removal of charcoal, 1 ml of
AcOH and one time with benzene, and dried in a vac-
1 : 3 con. HCl–EtOH mixture was added to the filtrate.
uum. The yield of DBBI(5) hexahydrochloride, exhib-
The precipitated solid was filtered, washed with EtOH,
iting a blue fluorescence in solution, was 0.116 g
and dried in a vacuum. The resulting product was
(9.5%); mp >300°C, Rf 0.18, λmax 338 (ε338 – 90000 å−1
extracted with EtOH (30 ml) under boiling; the still
cm–1); 1H NMR: 1.64 (2 H, m, éëç2ëç2ëç2), 1.86 (4
warm EtOH was filtered off, and the residue was
H, m, NHëç2ëç2), 1.99 (4 H, m, éëç2ëç2), 2.7 (12
washed with EtOH. The solid product was dissolved in
H, d, Nëç3), 3.14 (4 H, m, CH2N(CH3)2), 3.4 (4 H, m,
MeOH (4 ml), and PriOH (3 ml) was added. The precip-
NHCH2), 4.17 (4 H, t J 6.2, éëç2), 7.22 (4 H, d, J 8.7,
itate was filtered and dissolved in EtOH (10 ml) with
H2, H6), 7.78 (2 H, d, J 8.7, H4''), 7.90 (2 H, d, J 8.7,
the addition of water (2.2 ml). Concentrate HCl
H5', H4'), 8.28 (2 H, s, H7'), 8.38 (6 H, m, H3, H5,
(0.05 ml) and then PriOH (2 ml) were added to the solu-
H5''), 8.73 (2 H, s, H7''), 8.90 (2 H, t, J 4.7, CONH),
tion. After cooling, the precipitate was filtered and
10.44 (2 H, m, NH, Bim); EI MS, m/z: 978.2 [M + H]+,
dried in a vacuum. Yield of DBBI(8) hexahydrochlo-
1000.6 [M + Na]+. Calculated M 977.2 (C57H60N12O4).
ride was 0.191 g (20.3%); mp >300°C, Rf 0.15, λmax
DBBI(7). A solution of diamine (III) (0.333 g, 338 nm (ε338 – 113000 å–1 cm–1). DBBI(8) hexahydro-
1
0.944 mmol) in glacial AcOH (16 ml) was added to 4- chloride in solution produces a blue fluorescence; H
[(7-{4-[ethoxy(imino)methyl]phenoxy}heptyl)oxy]-1- NMR: 1.98 (4 H, m, ëç2ëç2ëç2), 2.77 (12 H, d, J
phenylimidic acid ethyl ester dihydrochloride (IVb) [1] 3.7, N(CH3)2), 3.14 (4 H, m, ëç2N(CH3)2), 3.38 (4 H,
(0.236 g, 0.472 mmol), and the mixture was refluxed m, NHCH2), 3.66 (4 H, s, Phé(ëç2)2é(ëç2)2), 3.8 (4
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 33 No. 6 2007

DNA SEQUENCE-SPECIFIC LIGANDS
577
H, m, PhOCH2CH2), 4.21 (4 H, m, Phéëç2), 7.17 (4 and 10% DMSO at 37°C for 2 h. The reaction was ter-
H, d, J 8.1, H2, H6), 7.71 (2 H, d, J 8.7, H4"), 7.85 (2 minated by adding 80 µl of a solution containing
H, d, J 8.1, H4', H5'), 8.25 (6 H, m, H3, H5, H5", H7'), 10 mM Tris-HCl (pH 7.5), 0.3 I sodium acetate, 1 mM
8.58 (2 H, s, H7"), 8.89 (2 H, t (br), CONH), 10.32 (2 EDTA, and 0.125 µg/µl of glycogen. The integrase was
H, m, NH, Bim); MS, m/z: 1023.3 [M]+. Calculated M extracted by phenol, and the reaction products were
1023.2 (C58H62N12O6).
precipitated by ethanol and resuspended in an aqueous
80% formamide solution. The products were separated
by 20% PAGE under denaturing conditions (7 M urea),
and the gels were then analyzed on a PhosphoImager
device (Molecular Dynamics, United States). The
inhibitory action of the compounds on 3'-terminal pro-
cessing was estimated from changes in the intensity of
the band corresponding to the duplex chain being pro-
cessed, which was truncated by two nucleotides (19-nt
product). From the results, the curves of the depen-
dence of the reaction efficiency on the inhibitor concen-
tration were plotted. From the curves, the IC50 value as
an inhibitor concentration at which the reaction is
inhibited by 50% was determined.
Physicochemical studies. A preparation of calf thy-
mus double-stranded DNA (Sigma, United States) was
depolymerized by an UZDN-2T ultrasound disintegra-
tor (Russia). The molecular mass of DNA after sonica-
tion was (0.5–0.8) × 106 Da, as determined by 1% aga-
rose gel electrophoresis. Absorption spectra were
recorded on a Specord M40 spectrophotometer (Ger-
many), and CD spectra were measured on an SKD-2
portable dichrometer (manufactured at the Institute of
Spectroscopy, Russian Academy of Sciences; Troitsk,
Russia). Quartz cuvettes with an optical path length of
1 cm were used. The initial concentration of a solution
of DBBI in 80% DMSO was 2.15 mg/ml.
A ligand complex with DNA as part of a CLD was
formed as follows. At the first stage, a CLD of DNA
was prepared by mixing equal volumes of a water salt
solution (0.3 M NaCl; pH 6.8) of DNA and PEG (M
4000 Da, Fluka) at a concentration of 340 mg/ml. A CD
spectrum was recorded 1 h after mixing; the appearance of
an intense band (λmax = 270 nm, ∆ε = 130–140 M–1 cm–1)
in the CD spectrum indicated the formation of a CLD
of DNA molecules. At the second stage, an initial solu-
tion of DBBI was added to CLD (2 ml) in small por-
tions (from 2 to 5 µl) under vigorous stirring, and the
CD spectrum in the absorption region of DNA and the
ligand (wavelength range 250–450 nm) was recorded
again.
ACKNOWLEDGMENTS
The study was supported by the Russian Foundation
for Basic Research (project nos. 07-03-00492, 07-04-
92164, 05-04-48743), the program of the Presidium of
the Russian Academy of Sciences on molecular and
cellular biology, and the State Program Creation of
Drugs for the Treatment and Prophylaxis of Viral Dis-
eases by the Methods of Chemical Synthesis.
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