DNA sequence-specific ligands: XII. Synthesis and cytological studies of dimeric hoechst 33258 molecules

Article abstract of DOI:10.1007/s11171-005-0047-z

We synthesized dimeric Hoechst dye molecules composed of two moieties of Hoechst 33258 fluorescent dye with the phenolic hydroxy groups tethered via pentamethylene, heptamethylene, or triethylene oxide linkers. A characteristic pattern of differential staining of chromosome preparations from human HL60 premonocytic leukemia cells was observed for all the three fluorescent dyes. The most contrasting pattern was obtained for the bisHoechst analogue with the heptamethylene linker; its quality was comparable with the picture obtained in the case of chromosome staining with 4′,6-diamidino-2-phenylindole. The ability to penetrate into live human fibroblasts was studied for the three bisHoechst compounds. The fluorescence intensity of nuclei of live and fixed cells stained with the penta- and heptamethylene-linked bisHoechst analogues was found to differ only slightly, whereas the fluorescence of the nuclei of live cells stained with triethylene oxide-linked bisHoechst was considerably weaker than that of the fixed cells. The bisHoechst molecules are new promising fluorescent dyes that can both differentially stain chromosome preparations and penetrate through cell and nuclear membranes and effectively stain cell nuclei.

Full text of DOI:10.1007/s11171-005-0047-z

Russian Journal of Bioorganic Chemistry, Vol. 31, No. 4, 2005, pp. 344–351. Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 4, 2005, pp. 385–393.
Original Russian Text Copyright © 2005 by Gromyko, Popov, Mosoleva, Streltsov, Grokhovsky, Oleinikov, Zhuze.
DNA Sequence-Specific Ligands:
XII.1 Synthesis and Cytological Studies
of Dimeric Hoechst 33258 Molecules
A. V. Gromyko*, K. V. Popov*, A. P. Mosoleva*, S. A. Streltsov*, S. L. Grokhovsky**,
, 2
V. A. Oleinikov***, and A. L. Zhuze*
* Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow, 119991 Russia
** Moscow Center of Medical Research of Oslo University, ul. Vavilova 34/5, Moscow, 119991 Russia
*** Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,
ul. Miklukho-Maklaya 16/10, Moscow, 117997 Russia
Received October 7, 2004; in final form, November 27, 2004
Abstract—We synthesized dimeric Hoechst dye molecules composed of two moieties of Hoechst 33258 fluo-
rescent dye with the phenolic hydroxy groups tethered via pentamethylene, heptamethylene, or triethylene
oxide linkers. A characteristic pattern of differential staining of chromosome preparations from human HL60
premonocytic leukemia cells was observed for all the three fluorescent dyes. The most contrasting pattern was
obtained for the bisHoechst analogue with the heptamethylene linker; its quality was comparable with the pic-
ture obtained in the case of chromosome staining with 4',6-diamidino-2-phenylindole. The ability to penetrate
into live human fibroblasts was studied for the three bisHoechst compounds. The fluorescence intensity of
nuclei of live and fixed cells stained with the penta- and heptamethylene-linked bisHoechst analogues was
found to differ only slightly, whereas the fluorescence of the nuclei of live cells stained with triethylene oxide-
linked bisHoechst was considerably weaker than that of the fixed cells. The bisHoechst molecules are new
promising fluorescent dyes that can both differentially stain chromosome preparations and penetrate through
cell and nuclear membranes and effectively stain cell nuclei.
Key words: chromosome, dimeric Hoechst 33258 dye, DNA, fluorescent dye, minor groove ligand, synthesis
21
INTRODUCTION
with theAT-specific antibiotics netropsine and distamy-
cin A and designed and synthesized dimeric ligands,
bis-netropsines [3–6]. They are based on the pyrrole-
carboxamide structural motif and proved to be the first
class of ligands that can noncovalently and site-specif-
ically be bound by DNA. The general principle of the
design of these compounds consists in the linking one
or several pyrrolecarboxamide blocks via various spac-
ers. This structure enables the orientation and fixing
these Ä·í-recognizing pyrrolecarboxamide blocks in
the complex with DNA [5, 6]. The study of physico-
chemical [4, 6] and biochemical [1, 6–10] properties of
these DNA complexes with dimeric ligands showed
that the netropsine fragments are specifically bound by
two Ä·í base pair clusters. Later, this approach was
The design of medicines that can site-specifically
bind to DNA is an important pharmacological task,
since the therapeutic activity of most of the currently
used antitumor drugs depends on the potency and selec-
tivity of their interaction with DNA.
There is now a tendency in anticancer and antiviral
chemotherapy to replace the standard drugs that disrupt
DNA in a nonselective manner by new agents that non-
covalently interact with DNA and affect its replication
and transcription. Therefore, the design of low-molec-
ular ligands capable of noncovalent and site-specific
binding to the DNA minor groove is of a great interest.
These site-specific compounds would play an impor-
tant role in the direction of them to the certain genes of
genome and for the use in chemotherapy. Such com-
pounds could serve as both the tools for studies and the
therapeutic agents capable of controlling the gene
expression in cells.
N
N
Previously, we had used a molecular model sug-
gested by Zasedatelev et al. [2] for the DNA complexes
N
N
H
N
H
O
1
For communication XI, see [1].
Corresponding author: phone: +7 (095) 135-9718; fax: +7 (095)
135-1405; e-mail: zhuze@imb.ac.ru
N
2
R
Hoechst 33258, R = H
Hoechst 33342, R = CH2CH3
H3C
1068-1620/05/3104-0344 © 2005 Pleiades Publishing, Inc.

DNA SEQUENCE-SPECIFIC LIGANDS
345
successfully used for the design of other dimeric
Three methods were tested for the synthesis of
sequence-specific compounds based on various combi- dimeric bis(benzimidazoles), with bisHoechst (Va)
nations of pyrrolecarboxamide and imidazolecarbox- being an example. The coupling of 2-amino-4-[6-(4-
amide blocks [11–15].
methylpiperazine-1-yl)-1H-benzimidazole-2-yl]phenyl-
amine (IV) [29] with ethyl 4-[(5-{4-
In this work, we chose the dye Hoechst 33258,
which is widely used in cytology as the DNA-specific
fluorescent label [16], as the starting compound for the
design of a new class of dimeric site-specific ligands. It
is known that Hoechst 33258 inhibits the interaction of
the TATA-box-binding protein with DNA [17], is an
efficient inhibitor of DNA topoisomerase I [18] and
DNA helicases [19], and is a radioprotector [20].
Hoechst 33258 is capable of noncovalent binding to
the minor groove of DNA B form. It preferentially
interacts with the clusters composed of three to four
Ä·í pairs (with the stoichiometry of one ligand mole-
cule per binding site) and covers a halfturn of the dou-
ble DNA helix [21–24]. It was found that, among all the
Ä·í triplets, Hoechst 33258 prefers the (5')-AA(A/T)
sequence and, in the case of Ä·í tetraplets, the (5')-
AATT sequence [25].
The Ä·í specificity of Hoechst 33258 is determined
by the backbone of the dye molecule consisting of two
benzimidazole fragments linked to each other accord-
ing to the head-to-tail principle. While interacting with
DNA, each benzimidazole fragment forms a bifurcated
(three-centered) hydrogen bond with the thymine O(2)
atom and/or adenine N(3) atom of two adjacent A·T
pairs and covers a region of about 1.5 bp in length [23,
26]. The Hoechst 33258 binding is also stabilized by
electrostatic and strong van der Waals interactions with
the minor groove walls.
We synthesized three dimeric bis(benzimidazole)
molecules, namely, bisHoechsts (Va)–(Vc) composed
of two dimeric Hoechst 33258 molecules whose phenyl
hydroxyl groups are linked via pentamethylene, hep-
tamethylene, or triethylene glycol bridges, respectively.
Our goal was to design more specific ligands capable of
recognizing two sites comprising three or four Ä·í
pairs, viz., (A/T)n–NN–(A/T)n (where n = 3 or 4), and
covering one turn of the DNA double helix. The choice
of these bridges was based on both speculative models
and the analysis of the oligonucleotide complexes with
known bis(benzimidazole) ligands [27, 28].
[ethoxy(imino)methyl]phenoxy}pentyl)oxy]-1-phenyl-
imidoate (IIIa) (reflux in glacial AcOH for 20 min
under nitrogen) resulted in 29.4% yield of (Va) and
proved to be the most efficient method (Scheme 1).
Diimidate (IIIa) was synthesized by the Pinner reac-
tion from 4-{[5-(4-cyanophenoxy)pentyl]oxy}ben-
zonitrile (IIa), which, in its turn, was prepared by the
alkylation of 4-hydroxybenzonitrile alcoholate with
1,5-dibromopentane in DMF.
The second pathway involved the synthesis of dial-
dehydes (VIa)–(VId) by the alkylation of p-hydroxy-
benzaldehyde alcoholate with α,ω-dibromoalkanes in
order to couple them further with diamine (IV) and
obtain the corresponding bis(benzimidazoles). How-
ever, it was found that a prolonged heating of diamine
(IV) in ethanol with 4-[5-(4-formylphenoxy)pentyl-
oxy]benzaldehyde (VI‡) in the presence of p-benzo-
quinone in inert atmosphere did not result in the target
(V‡) because of considerable resinification of the reac-
tion mixture (Scheme 2). Therefore, (VIb)–(VId) were
not used further for the preparation of the correspond-
ing bisHoechsts.
In the third pathway, the coupling of diamine (IV)
with methyl 4-{5-[4-(methoxycarbonyl)phenoxy]pen-
tyloxy}benzoate (VII) in the presence of P2O5 in meth-
anesulfonic acid [30] began to form (V) after the first
15 min. However, this later degraded in the course of
reaction to give the Hoechst 33258 mesyl derivative
(according to MALDI TOF mass spectrometry). There-
fore, this approach also has not been developed.
These results made us to synthesize (Vb) and (Vc) in
24.8 and 23.3% yields by coupling diamine (IV) with
diimidates (IIIb) and (IIIc), respectively (Scheme 1).
An alteration of absorption spectrum of (Va) in the
presence of calf thymus DNA indicated the formation
of their complex [31]. Chromosomes are identified in
most of cytogenetic studies by the pattern of differential
staining. A large number of procedures have been
developed for the specific staining of pairs of homolo-
gous chromosomes [32]. For the procedures based on
the fluorescence in situ hybridization (FISH) of DNA
sequences, various types of the so-called Q-staining are
RESULTS AND DISCUSSION
The choice of Hoechst 33258 as the basic compound used. Its basis is the staining with the AT-specific fluo-
for the design of new site-specific ligands was due to rochromes due to uneven distribution of AT/GC pairs
both its AT-specificity and the ability of its analogue, along chromosomes. DAPI (4',6-diamidino-2-phe-
Hoechst 33342, to penetrate through cell membranes nylindol) is the dye that is most often used for these
while retaining its fluorescent properties. We believed purposes, since its spectral characteristics are compati-
that the preservation of fluorescent properties would ble with those of the dye used for DNA labeling [33].
permit a reliable detection of even small amounts of the In addition to DAPI, Hoechst 33258 is also used for the
new ligands, which would make the designed bisHoe- differential chromosome staining in a number of cases
chsts (Va)–(Vc) superior to the bisnetropsines, we pre- [34]. We tested each of the synthesized fluorochromic
viously synthesized, by enabling their localization in dyes (Va)–(Vc) and found that all of them provide the
cells.
differential staining. The most contrast pattern was
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 4 2005

346
GROMYKO et al.
OH
‡: X = Br, R = (CH2)5
b: X = Br, R = (CH2)7
R
+
X
X
c: X = Cl, R = (CH2)2O(CH2)2O(CH2)2
(I‡–Ic)
N
NaH, DMF, t°
2
O
O
3
R
6
(II‡–IIc)
5
N
N
HCl, EtOH
O
O
R
ClH2N
NH2Cl
OEt
(III‡–IIIc)
OEt
N
H2N
AcOH, N2, 120°C
N
H
N
H2N
N
(IV)
CH3
2
O
O
3
R
6
N
N
4'
5
NH
HN
5'
(V‡–Vc)
7'
N
N
NH
HN
4''
5''
7''
bisHoechst (V‡), R = (CH2)5
bisHoechst (Vb), R = (CH2)7
bisHoechst (Vc), R = (CH2)2O(CH2)2O(CH2)2
N
N
2'''
3'''
6'''
5'''
N
N
H3C
CH3
Scheme 1.The scheme of synthesis of bisHoechsts (Va)–(Vc).
obtained with Hoechst (Vb) (see the figure); its pattern dyes capable of penetration through the cell and nuclear
was comparable with that obtained with DAPI.
membranes and effectively staining the cell nuclei.
It is known that Hoechst 33258 can penetrate
through the intact cell membranes [35]. The study of
the ability of bisHoechsts (Va)–(Vc) to pass into live
human fibroblast cells demonstrated that the fluores-
cence brightness of live and fixed cells stained with
bisHoechsts (Va) and (Vb) only slightly differed from
one another. The fluorescence of nuclei of live cells
stained with bisHoechst (Vc) was considerably lower
than that of fixed cells. However, we did not study the
time-dependent dynamics of dye penetration into cells.
In our future work, we plan to study the site speci-
ficity of bisHoechst analogues using the footprinting
with DNase I and evaluate the ability of these com-
pounds to inhibit the catalytic activity of human DNA
topoisomerase I.
EXPERIMENTAL
4-Hydroxybenzaldehyde, 1,5-dibromopentane, 1,7-
dibromoheptane, and sodium hydride (80% suspension
To summarize, the synthesized compounds, in Vaseline oil) were from Fluka (Switzerland); 4-
bisHoechsts (Va)–(Vc) are new promising fluorescent hydroxybenzonitrile was from Aldrich (United States);
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 4 2005

DNA SEQUENCE-SPECIFIC LIGANDS
347
N
2
O
(CH2)n
O
NH2
3
N
H
2
+
N
6
NH2
R
R
N
5
H3C
(IV)
(VI‡) R = CHO; n = 5
(VIb) R = CHO; n = 2
(VIc) R = CHO; n = 3
(VId) R = CHO; n = 4
(VII) R = COOMe; n = 5
O
O
N
N
NH
HN
N
N
NH
HN
N
N
N
N
H3C
CH3
Scheme 2. The alternative scheme of synthesis of bisHoechst (Va).
and 1,2-bis(2-chloroethoxy)ethane was from Merck was an internal standard. The numerations of hydrogen
atoms in benzimidazole and piperazine cycles are given
in Scheme 1. The assignment of H NMR signals of
(Va)–(Vc) was done using the information in [37]. The
bisHoechst mass spectra were registered on a time-of-
flight mass spectrometerVision-2000 (ThermoBioanal-
ysis, UK) using a linear mode of positive ion registra-
tion; 2,5-dihydroxybenzoic acid as a matrix ionization
(Germany).
1
Solutions of compounds in organic solvents were
dried with Na2SO4. As a rule, the solvents were evapo-
rated on a rotor evaporator at 50oë in a vacuum of a
water-jet pump. The compounds were dried in a vacuum
over P2O5 and NaOH. Melting points were determined
on a Boethius hot plate (Germany) and were uncor- by N2 laser (337 nm) were used. Mass spectra of dini-
rected. Hydrogenation was carried our in the presence
of the Adams catalyst [36] at atmospheric pressure and
room temperature until the hydrogen absorption was
ceased. The homogeneity of the compounds synthe-
triles (IIa)–(IIc), dialdehydes (VIa)–(VId), and (VII)
were obtained by electron impact ionization on a
Polaris CQ 230 (Finnigan, United States) and a MS-
890 (Kratos, UK) at 160–200°C using the direct inlet
sized was monitored by TLC on the Kieselgel 60 F254 and the electron energy of 70 eV; the temperature of
precoated plates (Merck, Germany) in system A, 50 : 1 ionization chamber was 250oë.
methanol–Et3N, and system B, 100 : 1 CHCl3–isopro-
A general procedure for the synthesis of dini-
panol. The compounds on chromatograms were
triles (IIa)–(IIc). 4-Hydroxybenzonitrile (2.4 mmol)
detected in UV light according to the absorption at
was added to a suspension of NaH (2.4 mmol) in DMF
254 nm or fluorescence excited at 365 nm. The absorp-
(3.0 ml) at 0°C, the mixture was heated at 80°C under
tion spectra of bisHoechsts (Va)–(Vc) were registered
stirring until the complete dissolution of NaH, and α,ω-
in 1 mM cacodylate buffer (pH 6.8) in the presence of
dihalogenoalkane (Ia)–(Ic) (1.0 mmol) was added. The
1
10 vol % DMSO. The H NMR spectrum was regis-
tered on an AMX-400 spectrometer (Bruker, Germany)
(working frequency of 400 MHz) in DMSO-d6 at 23°C
solution was heated at 80°ë for 1 h [for (Ia) and (Ib)]
or at 110–120°C for 1 h in the presence of NaI
(0.1 mmol) [in the case of 1,2-bis(2-chloroeth-
(if not stated otherwise); the residual DMSO resonance oxy)ethane (Ic)], cooled, and water (10 ml) was added.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 4 2005

348
GROMYKO et al.
rated in a vacuum to dryness, and the solid residues of
(IIIa)–(IIIc) were dried over NaOH and P2O5 in a vac-
uum and used in further reactions without purification.
BisHoechst (Va). A solution of ethyl 4-[(5-{4-
[ethoxy(imino)methyl]phenoxy}pentyloxy]-1-phenyl-
imidate dihydrochloride (IIIa) (0.313 g, 0.665 mmol)
and 2-amino-4-[6-(4-methylpiperazin-1-yl)-1H-benz-
imidazol-2-yl]phenylamine (IV) [29] (0.429 g,
1.330 mmol) in AcOH (5 ml) was refluxed for 20 min
under nitrogen, cooled, the precipitate of bright yellow
crystals was filtered, washed with AcOH and benzene,
the filtrate was evaporated in a vacuum, and the residue
was dissolved in AcOH (3 ml). A thick suspension
formed after the addition of three drops of concentrate
HCl was refluxed until the complete dissolution of all
crystals. Bright yellow crystals precipitated after cool-
ing the mixture were filtered and washed with AcOH
and benzene. Both portions of crystals were combined
and recrystallized from a mixture of methanol (15 ml)
and Et3N (1 ml). The precipitate was washed on filter
with methanol, dissolved in a 10 : 2 methanol–AcOH
mixture (12 ml), and refluxed with activated charcoal.
The charcoal was removed, the solution was evaporated
in a vacuum, and the residue was recrystallized from a
10 : 1 methanol–Et3N mixture (11 ml). The formed pre-
cipitate was washed on filter with methanol, dissolved
in AcOH (3 ml), and two drops of concentrated HCl
were added. The solution was evaporated in a vacuum,
the residue was suspended in boiling methanol (4 ml),
and a solution of conc. HCl (five drops) in methanol
(3 ml) was added. The resulting suspension was kept
overnight in a refrigerator, the yellow crystalline prod-
uct was separated, washed twice with methanol, and
dried over NaOH and P2O5 in a vacuum to give 0.222 g
(29.4%) of (Va) hexahydrochloride; mp > 260°C; Rf
A negative image of metaphase chromosomes of the HL-60
cells stained with bisHoechst (Vb). A scale is 10 µm.
The precipitate was filtered, washed with water, dried,
and the resulting dinitrile (II) was twice recrystallized
from ethanol.
4-{[5-(4-Cyanophenoxy)pentyl]oxy}benzonitrile
1
(IIa); yield 95.7%; Rf 0.34 (B); mp 109–110°C; H
NMR: 1.56 (2 H, m, OCH2CH2CH2), 1.79 (4 H, m,
OCH2CH2), 4.07 (4 H, t, J 6.23, OCH2), 7.09 (4 H, d, J
9.03, H2 + H6), and 7.75 (4 H, d, J 9.03, H3 + H5); MS
(Polaris CQ 230), m/z: 307.1 [M + H]+., calc. for
C19H18N2O2: M 306.3.
0.14 (A); λmax 341 nm (ε341 70000 å–1 cm–1). BisHoe-
chst (Va) exhibits a yellow–green fluorescence in solu-
tion. For more accurate assignment of resonances
4-{[7-(4-Cyanophenoxy)heptyl]oxy}benzonitrile
1
(IIb); yield 85.7%; Rf 0.38 (B); mp 112°C; H NMR:
1.39 [6 H, m, OCH2CH2(CH2)3], 1.72 (4 H, m,
OCH2CH2), 4.04 (4 H, t, J 6.54, OCH2), 7.08 (4 H, d, J
8.72, H2 + H6), 7.74 (4 H, d, J 8.72, H3 + H5); MS
(Polaris CQ 230), m/z: 333.9 [M + H]+, calc. for
C21H22N2O2: M 334.4.
1
within the ranges of 3.0–4.0 and 7.0–8.5 ppm, its H
NMR spectrum was registered at 380 K: 1.69 (2 H, m,
OCH2CH2CH2), 1.91 (4 H, m, OCH2CH2), 2.85 (6 H, s,
NCH3), 3.37 (8 H, m, H3''' + H5'''), 3.57 (8 H, m, H2''' +
H6'''), 4.19 (4 H, t, J 6.30, OCH2), 7.14 (4H, d, J 8.72,
H2 + H6), 7.15 (2 H, dd, J 9.03, 1.87, H5''), 7.23 (2 H,
d, J 1.87, 2 x H7''), 7.62 (2 H, d, J 9.03, H4''), 7.76 (2
H, d, J 8.41, H4'), 8.12 (2 H, dd, J 8.41, 1.56, H5'), 8.22
(4 H, d, J 8.72, H3 +,H5), 8.51 (2 H, d, J 1.56, H7'), MS
(Vision-2000), m/z: 918.6 [M + H]+, calc. for
C55H56N12O2: M 917.1.
4-(2-{2-[2-(4-Cyanophenoxy)ethoxy]ethoxy}etho-
xy)benzonitrile (IIc); yield 71.0%; Rf 0.06 (B); mp
1
77–80°C; H NMR: 3.60 (4 H, s, EtOCH2CH2OEt),
3.75 (4 H, t, J 4.05, PhOCH2CH2), 4.17 (4 H, t, J 4.05,
PhOCH2), 7.10 (4 H, d, J 8.40, H2 + H6), 7.75 (4 H, d,
J 8.40, H3 + H5); MS (Polaris CQ 230), m/z: 352.9
[M + H]+, calc. for C20H20N2O4: M 352.4.
BisHoechst (Vb). A solution of ethyl 4-[(5-{4-
A general procedure for the synthesis of diimido [ethoxy(imino)methyl]phenoxy}heptyloxy]-1-phenyl-
esters dihydrochlorides (IIIa)–(IIIc).A suspension of imidate dihydrochloride (IIIb) (0.323 g, 0.65 mmol)
dinitriles (IIa)–(IIc) (1 mmol) in absolute ethanol and 2-amino-4-[6-(4-methylpiperazin-1-yl)-1H-benz-
(5 ml) was saturated with dry hydrogen chloride at 0°C imidazol-2-yl]phenylamine (IV) (0.425 g, 1.32 mmol)
for 15 min, until the starting compounds were com- inAcOH (4 ml) was refluxed for 30 min under nitrogen,
pletely dissolved. The reaction mixture was kept at cooled to room temperature, the resulting gel-like mass
room temperature for 4 days in a sealed flask, evapo- was separated, washed with benzene, dissolved in
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 4 2005

DNA SEQUENCE-SPECIFIC LIGANDS
349
methanol (10 ml), reprecipitated with a 1 : 1 mixture of crystallized from methanol (6 ml) and suspended in a
isopropanol–concentrated ammonia, and the precipi- mixture of DMF (0.6 ml) and isopropanol (3 ml) under
tate was recrystallized from methanol (5 ml). The meth- heating. The addition of water (2 ml) first resulted in the
anolic filtrate was evaporated in a vacuum, and the res- dissolution of suspension, while the addition of another
idue was crystallized from methanol (10 ml). The 4 ml of water led to crystallization. The precipitate was
mother liquids were combined, evaporated, and recrys- separated, washed with isopropanol and water, and
tallized several times from a 2 : 3 mixture of methanol– dried in a vacuum to give 0.229 g (25.4%) of (Vc). The
isopropanol. All the crystals were combined and dis- base (Vc) was converted to the protonated form by dis-
solved in a methanol–AcOH mixture, the solvents were solution in a methanol–AcOH mixture, evaporation of
removed in a vacuum, and the residue was recrystal- the solvents, dissolution of the residue in methanol
lized from a 10 : 1 methanol–Et3N mixture (11 ml). The (4 ml), and treatment of the solution with a mixture of
methanol (3 ml) and concentrate HCl (1 ml). The yel-
low hexahydrochloride of (Vc) was precipitated in ace-
tone (12 ml), separated, washed with acetone, and dried
in a vacuum to give 0.258 g (23.34%) of (Vc); mp
resulting precipitate was dissolved in a 5 : 1 methanol–
AcOH mixture (12 ml) and treated three times with
activated charcoal, until the solution became light yel-
low. After the solvent removal in a vacuum, the residue
was recrystallized from a 10 : 1 methanol–Et3N mixture >260°C; Rf 0.12 (A); λmax 340 nm (ε340 70 800 M–1 cm–1).
(5.5 ml) and kept overnight in a refrigerator. The result- BisHoechst (Vc) exhibited a yellow–green fluores-
ing precipitate was separated, washed with methanol, cence in solution. For more accurate assignment of res-
and dissolved in a 5 : 1 methanol–AcOH mixture onances within the ranges of 3.0–4.0 and 7.0–8.5 ppm,
1
(12 ml). The organic solvents were removed in a vac-
uum, and the residue was recrystallized from a 8 : 1
methanol–AcOH mixture (9 ml). Concentrate HCl
(0.5 ml) was added to the solution obtained after the
treatment of the crude product with a 10 : 1 methanol–
AcOH mixture (11 ml), and the resulting suspension
was kept overnight in a refrigerator to give a yellow
compound, which was separated, washed twice with
methanol (5 ml) on filter, and dried in a vacuum over
NaOH and P2O5 to give 0.188 g (24.85%) of (Vb)
hexahydrochloride, mp >260°C; Rf 0.16 (A); λmax
the H NMR spectrum was registered at 380 K: 2.85
(6 H, s, NCH3), 3.37 (8 H, m, H3''' + H5'''), 3.59 (8 H,
m, H2''' + H6'''), 3.71 (4 H, s, EtOCH2CH2OEt), 3.85
(4 H, t, J 4.83, PhOCH2CH2O), 4.26 (4 H, t, J 4.83,
PhOCH2), 7.14 (4 H, d, J 8.72, H2 + H6), 7.18 (2 H, dd,
J 9.03 and 1.87, H5''), 7.24 (2 H, d, J 1.87, H7''), 7.64
(2 H, d, J 9.03, H4''), 7.77 (2 H, d, J 8.72, H4'), 8.13
(2 H, dd, J 8.72 and 1.25, H5'), 8.22 (4 H, d, J 8.72,
H3 + H5), and 8.54 (2 H, s, H7'); MS (Vision-2000),
m/z: 963.7 [M + H]+, calc. for C56H58N12O4: M 963.0.
342 nm (ε342 82 700 M–1 cm–1). BisHoechst (Vb) exhib-
A general procedure for the synthesis of dialde-
hydes (VIa)–(VId). Dialdehydes were obtained by the
procedure of the synthesis of dinitriles (II) using the
corresponding α,ω-dibromoalkanes: namely, 1,5-
dibromopentane for (VIa); 1,2-dibromoethane for
(VIb); 1,3-dibromopropane for (VIc), and 1,4-dibro-
mobutane for (VId); 4-hydroxybenzaldehyde was used
instead of 4-hydroxybenzonitrile. The reaction pro-
ceeded for 1 h at 80°C. After the reaction was over, the
target products were precipitated with water, separated,
washed with water, dried, and used in further reactions
without additional purification.
1
its a yellow–green fluorescence in solution; H NMR:
1.46 (6 H, m, OCH2CH2(CH2)3), 1.78 (4 H, m,
OCH2CH2), 2.85 (6 H, s, NCH3), 3.21 (8 H, m, H3''' +
H5'''), 3.54 (4 H, m, H2''' + H6'''), 3.89 (4 H, m, H2''' +
H6'''), 4.10 (4 H, t, J 6.23, OCH2), 7.16 (4 H, d, J 8.72,
H2 + H6), 7.17 (2 H, s, H7''), 7.30 (2 H, dd, J 9.03,
J 1.25, H5''), 7.68 (2 H, d, J 9.03, H4''), 7.87 (2 H, d,
J 8.72, H4'), 8.19 (2 H, d, J 8.72 + 1.25, H5'), 8.25 (4 H,
d, J 8.72, H3 + H5), 8.59 (2 H, s, H7'), and 10.95 (4 H,
s, NH); MS (Vision-2000), m/z: 946.4 [M + H]+, calc.
for C57H60N12O2: M 945.1.
Analytical samples of the dialdehydes were
BisHoechst (Vc). A solution of diamine (IV) (0.603 g, obtained after recrystallization from benzene–hexane.
1.87 mmol) and diimidoester dihydrochloride (IIIc)
4-[5-(4-Formylphenoxy)pentyloxy]benzaldehyde
((0.485 g, 0.935 mmol) was refluxed in AcOH (5 ml)
1
(VIa); yield 94.6%; Rf 0.15 (A); mp 80–82°C; H
for 30 min under nitrogen, and evaporated. The residue
NMR: 1.58 (2 H, m, OCH2CH2CH2), 1.82 (4 H, m,
was dissolved in hot water and treated with concentrate
OCH2CH2), 4.11 (4 H, t, J 6.54, OCH2), 7.12 (4 H, d, J
ammonia to precipitate the product. After the solution
8.72, H2 + H6), 7.85 (4 H, d, J 8.72, H3 + H5), and 9.86
was decanted, the oil-like product was washed with
(2 H, s, CHO); MS (MS-890), m/z: 312.0 [M]+., calc.
water, dissolved in methanol, and refluxed with acti-
for C19H20O4: M 312.36.
vated charcoal. The filtrate was evaporated; the residue
was dissolved in ethanol (3 ml) and reprecipitated with
a mixture of water (3 ml) and concentrate ammonia
(0.4 ml). The product was repeatedly reprecipitated
from ethanol (3 ml) with water (1 ml) followed by a
thrice reprecipitation from ethanol (3 ml) with acetone
(7 ml). The resulting compound was refluxed with eth-
4-[2-(4-Formylphenoxy)ethoxy]benzaldehyde (VIb);
1
yield 23.0%; Rf 0.14 (A); mp 118–120°C; H NMR:
4.49 (4 H, s, CH2CH2), 7.19 (4 H, d, J 8.50, H2 + H6),
7.88 (4 H, d, J 8.5, H3 + H5), 9.88 (2 H, s, CHO). MS
.
(MS-890), m/z: 270.0 [M]+ ; calc. for C16H14O4 M
anol (3 ml), cooled, and filtered. The precipitate was 270.28.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 4 2005

350
GROMYKO et al.
4-[3-(4-Formylphenoxy)propoxy]benzaldehyde etrate into live cells. The cells were cultured in the
1
DMEM medium (PanEco, Russia) containing 40 µg/ml
gentamycin (Akrikhin, Russia) and 10% embryonic
calf serum (Hyclone, United States) at 37°C, 5% CO2,
and 95% air humidity. The cells were transferred onto
cover-glasses placed in separate Petri dishes and ana-
lyzed after two days. Two glasses were used for each
dye. One cover-glass of each pair of Petri dishes was
fixed in 70% ethanol, stained with 1 µM dye solution in
the phosphate buffered saline (PBS) for 30 min, washed
with PBS three times, and kept in PBS. The dyes were
added in the cultural medium of the second dish up to
the concentration of 1 µM, the cells were washed out
from the dye after 2 h, kept in PBS, and were immedi-
ately analyzed using the same microscope and the same
combination of filters as used for the study of differen-
tial staining of chromosome preparations. The fluores-
cence brightness of fixed and live cell nuclei was com-
pared visually.
(VIc); yield 88.1%; Rf 0.16 (A); mp 125–126°C; H
NMR: 2.25 (2 H, m, CH2CH2CH2), 4.27(4 H, t, J 6.23,
OCH2), 7.15 (4 H, d, J 8.50, H2 + H6), 7.86 (4 H, d, J
8.50, H3 + H5), 9.84 (2 H, s, CHO); MS (MS-890), m/z:
.
284.0 [M]+ , calc. for C17H16O4: M 284.30.
4-[4-(4-Formylphenoxy)butoxy]benzaldehyde
1
(VId); yield 90.18%; Rf 0.14 (A); mp 100–102°C; H
NMR: 1.91 (4 H, m, OCH2CH2), 4.17 (4 H, m, OCH2),
7.13 (4 H, d, J 8.72, H2 + H6), 7.86 (4 H, d, J 8.72,
H3 + H5), 9.86 (2 H, s, CHO); MS (MS-890), m/z:
.
298.0 [M]+ , calc. for C18H18O4: M 298.33.
Methyl 4-{5-[4-(methoxycarbonyl)phenoxy]pen-
tyloxy}benzoate (VII). Dimethyl ester (VII) was
obtained as described for the preparation of dinitriles
(IIa)–(IIc) using 1,5-dibromopentane and methyl 4-
hydroxybenzoate instead of 4-hydroxybenzonitrile.
The reaction proceeded at 80°C for 2 h. After the reac-
tion was over, the target product was precipitated with
water, separated, washed with water, dried, and used in
further reactions without additional purification. Its
yield was 81.8%; Rf 0.26 (A); mp 91–93°C; 1H NMR:
1.57 (2 H, m, OCH2CH2CH2), 1.80 (4 H, m,
OCH2CH2), 3.80 (6 H, s, CH3), 4.07 (4 H, t, J 6.54,
OCH2), 7.02 (4 H, d, J 8.72, H2 + H6), 7.89 (4 H, d, J
ACKNOWLEDGMENTS
The work was partially supported by the Russian
Foundation for Basic Research, project nos. 04-03-
33144, 04-04-49364, and 03-04-48602, and the Pro-
gram on the Molecular and Cell Biology of the Presid-
ium of Russian Academy of Sciences.
.
8.72, H3 + H5); MS (Polaris CQ 230), m/z: 372.0 [M]+ ,
calc. for C21H24O6: M 372.41.
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