Polymethylene derivatives of nucleic bases with ω-functional groups: VII. Cytotoxicity in the series of N-(2-oxocyclohexyl)-ω-oxoalkyl- substituted purines and pyrimidines

Article abstract of DOI:10.1134/S1068162009010105

New polymethylene derivatives of the nucleic bases with β-diketo function in the ω-position have been synthesized by alkylation of uracil, thymine, cytosine, hypoxanthine, adenine, and N ²-isobutyryl guanine with 2-(ω-chloroalkanoyl)cyclohexano

Full text of DOI:10.1134/S1068162009010105

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2009, Vol. 35, No. 1, pp. 75–85. © Pleiades Publishing, Ltd., 2009.
Original Russian Text © V.V. Komissarov, G.M. Volgareva, Ya.S. Ol’shanskaya, M.E. Chernyshova, L.E. Zavalishina, G.A. Frank, A.A. Shtil’, A.M. Kritzyn, 2009, published in
Bioorganicheskaya Khimiya, 2009, Vol. 35, No. 1, pp. 84–94.
Polymethylene Derivatives of Nucleic Bases with w-Functional
Groups: VII.1 Cytotoxicity in the Series of N-(2-Oxocyclohexyl)-
w-Oxoalkyl-Substituted Purines and Pyrimidines
V. V. Komissarova, G. M. Volgarevab, Ya. S. Ol’shanskayab, M. E. Chernyshovab,
L. E. Zavalishinac, G. A. Frankc, A. A. Shtil’b, A. M. Kritzyna,2
a Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow, 119991 Russia
b Blokhin Russian Cancer Research Center of Oncology, Russian Academy of Medical Sciences,
Kashirskoe sh. 24, Moscow, 115478 Russia
c Moscow Hertzen Institute of Oncology, Russian Academy of Medical Sciences, Moscow, Russia
Received March 20, 2008; in final form, April 15, 2008
Abstract—New polymethylene derivatives of the nucleic bases with β-diketo function in the ω-position have
been synthesized by alkylation of uracil, thymine, cytosine, hypoxanthine, adenine, and N2-isobutyryl guanine
with 2-(ω-chloroalkanoyl)cyclohexanones. The physicochemical characteristics of compounds synthesized and
their effect on tumor K562 and HCT116 cell lines have been studied.
Key words: alkylation, antineoplastic activity, cytotoxicity, β-diketones, nucleosides, polymethylene analogues
DOI: 10.1134/S1068162009010105
INTRODUCTION
This effect depended both on the length of the polyme-
thylene chain and from the ω-functional group.
Versatile studies of biological properties of polyme-
thylene derivatives of nucleic bases with ω-functional
The development of new antineoplastic drugs
groups have shown the availability of their use as con- remains an actual problem despite of the successes
venient tools for research of enzymes involved in reached in the field of chemotherapy of oncological dis-
nucleic exchange.3 For example, compounds bearing eases. One of the main reasons is the ability of a tumor
the polymethylene chain in position-9 of adenine are to become resistant to originally effective medicines.
known to suppress the activity of adenosine-5'-mono- The products of p53 and MDR1 genes play the key role
phosphate deaminase that is the enzyme, which inhibi- in the control of resistance of tumor cell to chemother-
tors can be potential medicines for treating the ischemic apeutic drugs. Protein p53, being the transcription fac-
illness [2]. Polymethylene derivatives of the nucleic tor, regulates the activity of the genes responsible, in
bases, which inhibit lipoprotein-dependent phospholi- particular, for apoptosis and reparation. P-glycoprotein
pase Ä2 playing the significant role in the progress of (Pgp) encoded with MDR1 reduces the accumulation of
atherosclerosis, [3] are known.
medicines in tumor cells [10, 11]. Antimetabolites,
including nucleoside analogues and derivatives of the
nucleic bases, fludarabine, cladribine, phthorafurum,
and cytarabine, are used for the treatment of various
malignant diseases (hemoblastoses, epithelial tumors,
etc.) [12, 13].
In the last few years, 4-aryl-2,4-dioxobutanoic acids
that are effective inhibitors of HIV integrase have been
reported in the series of publications from various lab-
oratories [4–6]. Moreover, such compounds have been
found to reversibly and selectively inhibit RNA-depen-
dent RNA-polymerase of hepatitis C virus [7–9]. In the
previous communication [1] we have described the
synthesis and characteristics of 1-[8-(2-oxocyclo-
hexyl)-8-oxooctyl] derivatives of pyrimidine nucleic
bases. Thymine and uracil derivatives effectively inhib-
ited thymidine and uridine phosphorylase from E. coli.
Polymethylene derivatives of the nucleic bases with
terminal functional groups can be potential antimetab-
olites. Enzymes and nucleic acids can be endocellular
targets of such derivatives of nucleic bases. This class
of compounds showed antimetabolic effect in prokary-
otes, in particular, inhibited thymidine kinase of herpes
virus and reduced the activity of purine nucleoside
phosphorylase and thymidine phosphorylase from
E. coli [1, 14–17]. Polymethylene derivatives of
nucleic bases with antipsychotic and immunostimulat-
ing activities have also been known [18, 19].
1
For communication VI see [1].
Corresponding author; phone/fax: +7 (499) 135-1405; e-mail:
amk@eimb.ru.
Abbreviations: DAPI, 4',6-diamidimo-2-phenylindole dihydro-
chloride; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene.
2
3
75

76
KOMISSAROV et al.
Antineoplastic activity of polymethylene deriva- groups. In the present work we report synthesis, physi-
tives of nucleic bases with terminal functional groups
has not been studied. It seemed to be urgent to investi-
gate, whether such compounds display any toxic effect
towards human tumor cells.
cochemical properties, and cytotoxicity of 9-polymeth-
ylene derivatives of adenine (Ia)–(Ic), hypoxanthine
(IIa)–(IIc), and guanine (IIIa)–(IIIc) and 1-substituted
derivatives of uracil (IVa)–(IVc), thymine (Va)–(Vc),
and cytosine (VIa)–(VIc), containing the β-diketone
fragment in the ω-position of the polymethylene chain.
The polymethylene chain consisted of 6, 7 or 8 methyl-
ene units.4 Synthesis of the compounds afore-men-
tioned was carried out using the developed strategy of
alkylation of the nucleic bases or their sodium salts
RESULTS AND DISCUSSION
In order to study the effect of the length of the poly-
methylene chain on biological activity of the of nucleic
base derivatives with the terminal β-dicarbonyl group,
it is important to have a series of the compounds differ-
ing from one another by one, two, or several methylene with chlorides of (2-oxocyclohexyl)-ω-oxoalkanes.
OH
O
7'
OH
9'
NH
OH
N
2
1'
3'
5'
a) R =
N
N
N
10'
11'
N
N
N
4'
6'
6'
8'
13'
2'
2'
N
R
N
R
N
R
N
N
H N
12' OH
8'
9'
14'
2
O
4'
10'
11'
12'
b) R =
c) R =
(Ia-c)
(IIa-c)
(IIIa-c)
3'
3'
5'
5'
7'
7'
1'
13'
O
OH
NH
O
O
2
1'
9'
11'
12'
13'
N
10'
15'
NH
NH
8'
2'
4'
6'
N
R
O
N
O
N
R
O
14'
R
(IVa-c)
(Va-c)
(VIa-c)
R Cl
(VIIa-c)
7-Chloroheptanoic and 9-chlorononanoic acids between the base and the alkylating agent was carried
were synthesized by hydrolysis of 1,1,1,7-tetrachloro- out in the presence of DBU. Method B included the pre-
liminary preparation of the sodium salt by treating the
base with sodium hydride and the subsequent interac-
tion of the resulting salt with the corresponding alkylat-
ing agent.
heptane and 1,1,1,9-tetrachlorononane according to the
procedures described in [22]. They were treated with
thionyl chloride to give the corresponding acid chlo-
rides. 8-Chlorooctanoic acid chloride was obtained
starting from ε-caprolactone by the method developed
earlier [1]. 1-Morpholincyclohex-1-ene was acylated
with 7-chloroheptanoic, 8-chlorooctanoic or 9-chlo-
rononanoic acid chloride according to the Stork reac-
tion. Acidic hydrolysis of acylated enamines resulted in
the
This approach was used to obtain 2-(7-chlorohep-
tanoyl)cycloheptanone (VIIa), 2-(8-chlorooc-
tanoyl)cycloheptanone (VIIb), and 2-(9-chlo-
rononanoyl)cycloheptanone (VIIc). Their structure
was confirmed by NMR spectroscopy data.
Due to the low solubility of guanine in DMF, we
used alkylation of N2-isobutyryl guanine with the sub-
sequent deprotection of the amino group according to
the procedure described earlier [21] (method C, see the
Experimental) to obtain guanine derivatives
(IIIa)−(IIIc).
The structure of the compounds obtained for the first
time, namely, (Ib), (Ic), (IIb), (IIc), (IIIb), (IIIc),
(IVc), (Vc), and (VIc), was corroborated by NMR
spectroscopy and mass spectrometry data.
corresponding-chloroalkanoylcyclohexanones.
4
Synthesis and characteristics of the N-[7-(2-oxocyclohexyl)-7-
Nucleic bases were alkylated with the resulting
chloroalkanoylcyclohexanones (VIIa)–(VIIc) in DMF
by the two ways. According to method A, the reaction
oxoheptyl] derivatives of nucleic bases (Ia)–((VIa) and 1-[8-(2-
oxocyclohexyl)-8-oxooctyl]pyrimidine derivatives (IIIb)–(VIb)
were reported earlier [1, 20, 21].
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POLYMETHYLENE DERIVATIVES OF NUCLEIC BASES
77
1
2
3
1
2
3
120
100
80
120
100
80
(a)
(b)
60
60
40
40
20
20
0
0.1 0.2 0.4 0.8 1.6 3.2 6.3 12.5 25 50
0
0.1 0.2 0.4 0.8 1.6 3.2 6.3 12.5 25 50
C, µM
C, µM
Fig. 1. Curves of survival rate of (a) K562 and (b) K562/4 cell lines (1) after their treatment with compound (Ic) in comparison with
the data for (2) Mitoxantrone and (3) Etoposide. The share of the cells survived at the given concentration of the compound under
study (in percent) is shown at the Y-axis.
Alkylation of adenine also resulted in the corre- sponding
mutant
sublines
(K562/4
and
sponding 3-isomers as by-products isolated in insignif- HCT116p53KO) was observed in the experiments with
icant yields. This fact is in a good agreement with the compound (Ic) for the most of the concentrations
data obtained for adenine upon using other alkylating tested. In the case of Etoposide, the percent of
agents reported by A. Holy et al. [23]. The generation HCT116p53KO cells survived appeared to be reliably
of the corresponding side 7-isomers in the case of alky- higher than that of HCT116 cells in a concentration
lation of N2-isobutyryl guanine and the products of 1,3- range from 0.4 to 3.2 µM. The ability of compound (Ic)
bisalkylation in the case of uracil and thymine was to display the equally effective toxic action on both
observed (see the Experimental).
potentially sensitive cells (the tumor model before the
beginning of chemotherapy) and the cells with drug
resistance acquired can be an argument for design of an
antitumor drug based on compound (Ic).
The derivatives of adenine (Ia)–(Ic), hypoxanthine
(IIa)−(IIc), guanine (IIIa)−(IIIc), uracil (IVa)−(IVc),
thymine (Va)−(Vc), and cytosine (VIa)–(VIc) were
tested on K562 human leukemia cell line in the MTT-
We studied the antitumor effect in more detail using
test (named after the reagent used, 3-(4,5-dimethylthia- the most active compounds (Ia) and (Ic) as an example.
sol-2-yl)-2,5-diphenyl-2H-tetrasolium bromide). Only
To clarify the mechanism of cytotoxicity for com-
pound (Ia), K562 cells were incubated with derivative
adenine derivatives (Ia)−(Ic) and hypoxanthine deriva-
tives (IIb) and (IIc) displayed cytotoxicity. Their toxic-
(Ia); then fluorescent dyes DAPI and propidium iodide
ity toward K562 cells was lower than that of Etoposide,
were added to the culture medium. No cells were found
a topoisomerase II inhibitor used in clinic, and Mitox-
to entrap propidium iodide in a wide concentration
antrone, a DNA-intercalator (used as the positive con-
range up to 50 µM after treating with the compound
trol). The values of cytotoxic parameter IC50 for ade-
under study for 24–72 h.
nine and hypoxanthine derivatives fell in a range of 6–
This result points to the absence of cells with the
50 µM, whereas the IC50 values for Etoposide and
damaged plasmatic membrane (that is an sign of necro-
Mitoxantrone were 0.8 µM, and less than 0.1 µM,
sis). The appearance of chromatin corpuscles in the
respectively. Compound (Ic) appeared to be one of the
cells treated with compound (Ia) and colored with
most active (Fig. 1a).
DAPI differed from those in the control cultures: these
Compounds (Ia)−(Ic) and (IIa)−(IIc) were also
corpuscles were often of the smaller size than nuclei of
toxic toward HCT116 human large intestine carcinoma
intact cells and formed groups.After 72 h of incubation,
cell line (IC50 <= 25 µM) (Fig. 2a).
the morphological specialties of the toxic effect were
K562 cells acquire multiple drug resistance upon also observed in smears colored with hematoxylin and
the selection for survival rate in the presence of an anti- eosin (these dyes allow the obtaining of the contrast
neoplastic medicine doxorubicin due to hyperexpres- image of nuclei on the cytoplasm background). The
sion of glycoprotein Pgp (K562/4 subline). Protein p53 compaction of nuclei was the first specialty: such cells
is not expressed in the HCT116p53KO subline were not observed in the control cultures; their share
obtained from HCT116 line. Pair models, K562 and increased with the concentration of compound (Ia) and
K562/4 (Fig. 1a, b) and HCT116 and HCT116p53KO reached ~50% at 50 µM. This result agreed with the
(Fig. 2a, b) were chosen for the investigation of the role observations made upon coloring cells with DAPI:
of glycoprotein Pgp and protein p53 in cell death chromatin corpuscles of the size smaller than the nuclei
caused by the compounds under study. No difference of cells of the control culture were apparently the com-
within the limits of the experiment deviation between pacted nuclei. The unusually high rate of the cells with
the wild cell lines (K562, HCT116) and the corre- the appearance reminiscent of telophase stage was the
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78
KOMISSAROV et al.
1
2
3
1
2
3
(a)
(b)
160
140
120
100
80
160
140
120
100
80
60
60
40
40
20
20
0
0.1 0.2 0.4 0.8 1.6 3.2 6.3 12.5 25 50
0
0.1 0.2 0.4 0.8 1.6 3.2 6.3 12.5 25 50
C, µM
C, µM
Fig. 2. Curves of survival rate of (a) HCT116 and (b) HCT116p53KO cell lines (1) after their treatment with compound (Ic) in
comparison with the data for (2) Mitoxantrone and (3) Etoposide. The share of the cells survived at the given concentration of the
compound under study (in percent) is shown at the Y-axis.
second specialty. We conventionally marked out such tiple chromosome damages, whose nature and amount
cells as incomplete mitoses. The incomplete mitoses could not be precisely established (Fig. 3b). The pres-
comprised more than 10% of cells after 72 h at 12.5 and ence of the significant share of chromatid type aberra-
50 µM of compound (Ia). As in the cultures of trans- tions in the spectrum of chromosome rearrangements
formed cells 5% of cells in the aggregate are at different testifies that the part of the primary damages induced by
mitosis stages, it is obvious that the share of cells with the compound under study in chromatin is realized to
incomplete mitosis appeared to be significantly the structural rearrangements, when the chromosome is
increased in this experiment. The groups of chromatin represented by two chromatids, i.e. during DNA repli-
corpuscles revealed with DAPI could correspond just to cation or after its completion.
incomplete mitosis. The cells with sharply increased
Besides metaphases with chromosome aberrations
nuclei, which were much less colored than the nuclei of
on cytogenetic preparations, we observed the morpho-
control cells, were the third specialty. We convention-
logical form of nuclei that was not found in the control
ally marked out such cells as the cells with friable
material. These were pair nuclei (Fig. 3c), which were
nuclei. Such cells comprised no more than 3% of the
kept together in spite of the fact that the method of
control cells. Treating with compound (Ia) in a concen-
preparation of cytogenetic specimens from cell suspen-
sions, including application of the drop of the fixed
tration of 50 µM resulted in a 10-fold increase of the
amount of these cells.
material from a height of 30–40 cm on the cooled wet
Genotoxicity of compound (Ic) was studied by the glass, favors the division of all structures occasionally
method of counting the aberrations of metaphase chro- appeared to be in close proximity or weakly bound to
mosomes in K562 cell line (see the table). The com- each other. The rate of such pair nuclei was 5–7% from
pound under study had a reliable damaging effect on the total amount of cells. The rate of incomplete mito-
chromosomes at both tested dozes, 3.2 and 25.0 µM ses was ~10% (see above) that is comparable to the rate
(P < 0.01). Alongside with single identified chromo- of occurrence of the pair nuclei. Mitosis in the cells
some rearrangements, chromatid breaks and chromatid treated with cytotoxic polymethylene derivatives of the
exchanges being the most frequent (Fig. 3a), compound nucleic bases is apparently disrupted at one of terminal
(Ic) in the higher of the tested dozes also induced mul- stages, namely, the stage of nucleus reconstruction. It is
Chromosome damaging action of compound (Ic) on cells of the K562 line
Doze,
µM
Metaphases with chromosomal
aberrations, %
Metaphases with plural damages
of chromosomes, %
Number of metaphase plates analyzed
0
150
100
200
2.0 0.0
20.0 0.0
13.5 3.9
0
3.2
25.0
0
4.0 1.0
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POLYMETHYLENE DERIVATIVES OF NUCLEIC BASES
79
a
b
c
d
Fig. 3. (a, b) Metaphase plates (magnification ×1000) and (c, d) nuclei (magnification ×1000) in the culture of K562 cell line after
treatment with (Ic) (25 µM, 24 h): the view on cytogenetic preparations. (a) The metaphase plate with chromatid exchange, (b) the
metaphase plate with multiple unidentified chromosome damages, (c) the pair nuclei, (d) the group of chromatin corpuscles encap-
sulated in the nucleus membrane corresponding to the apoptotic cells.
not improbable that this is the consequence of raising encapsulated in the nuclear membrane obviously corre-
the exchange rearrangements of the chromosomes sponding to apoptotic cells with a rate no more than 1%
causing the disturbance of chromosome divergence in (Fig. 3d). As the period of exposition with derivative
the anaphase–telophase in the form of bridges between (Ic) in this experiment was only 24 h, we can assume
the poles of terminating mitosis. Just retaining such that the share of apoptotic cells is probably increased
bridges till the end of telophase can prevent the comple- upon the longer treatment.
tion of nucleus formation in the daughter cells.
The character of chromosome damages induced by
As for the cells with large poorly colored nuclei (the
cells with friable nuclei) noted upon the analysis of
smears, it is not improbable that they correspond to
metaphases with multiple chromosome damages of
cytogenetic preparations.
compound (Ic) can specify that genotoxicity contrib-
utes to the cytotoxic affect of this compound. These
damages include the loss of the significant part of
genome due to chromatid break; disturbances of the
next mitosis in the cells after chromatid exchange, the
The cytogenetic preparations of the investigated so-called bridges in anaphase entailing with the inevi-
material contained the groups of chromatin corpuscles table loss of genetic material; and disturbance of
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80
KOMISSAROV et al.
genome functions upon its multiple damages. The fact chlorooctanoic acid chloride was obtained by the
that the rate of the cells with chromosome damages in method [1].
our experiences did not exceed 20% (the table) does not
TLC was performed on Silica gel 60 F254 (Merck,
indicate the insignificance of the contribution of com-
Germany) precoated plates, using chloroform–ethanol
pound (Ic) genotoxicity to its cytotoxic activity. On the
(A) 19 : 1, (B) 18 : 2, (C) 17 : 3, and (D) 19.5 : 0.5 as
contrary, the cells with chromosome rearrangements
developing systems. The spots on chromatograms were
most likely die or slow down the progress from one to
visualized by UV absorption at 254 nm; β-diketones
another stage of the cell cycle selectively. Many of the
were visualized by treating with FeCl3 solution. Col-
cells aforementioned did not probably reach the first
umn chromatography was performed on Silica gel L
metaphase after the chromosome rearrangement, when
40/100 (Chemapol, Czech Republic).
the chromosome damaging effect of (Ic) was
Mass spectra were registered on a MS-30 (Kratos,
Japan) mass spectrometer using the electron impact as
accounted. The cytostatic effect of derivative (Ic)
observed also corroborates this assumption. We have
ionization method. NMR spectra (δ, ppm; J, Hz) were
estimated the effect of compound (Ic) on the prolifera-
registered on a Bruker AMXIII-400 (Germany) spec-
tive activity of K562 cells using the metaphase index.
1
trometer at a working frequency of 400 MHz for H
This parameter was 62 4% for the control cultures; in
spectra and 100 MHz for 13C spectra at 300 K in CDCl3
the case of cultures treated with compound (Ic) in a
and DMSO-d6.
doze of 0.8, 3.2, or 25 µM this parameter was 60 3,
2-(9-Chlorononanoyl) cyclohexanone (VIIc). A
solution of 9-chlorononanoic acid chloride (54.4 g,
0.26 mol) in chloroform (120 ml) was added dropwise
during 1 h to a solution of 4-(1-cyclohexen-1-yl)mor-
pholine (48 g, 0.29 mol) and triethylamine (46 ml,
0.33 mol) in anhydrous chloroform (320 ml) at stirring.
The reaction mixture was kept for 14 h at 20°ë, then
55% H2SO4 (170 ml) was added dropwise at vigorous
stirring during 40 min. The mixture was refluxed at vig-
orous stirring for 4 h and cooled. The organic layer was
separated, the aqueous layer was extracted with chloro-
form (2 × 100 ml), the combined extracts were washed
with the saturated solution of sodium bicarbonate
(150 ml) and dried over anhydrous sodium sulfate, and
the solvent was evaporated. The residue was distilled in
a vacuum.Yield 46.8 g (57%); bp 182–184°ë/1 mm Hg;
MS: m/z 272.8 [M]+, calculated 272.8 (C15H25ClO2)
1H NMR (CDCl3): 1.15–1.70 (16 H, m, H2–H7, H13,
H14), 2.23 (4 H, m, H12, H15), 2.30 (2 H, t, J = 7.5,
H8), 3.43 (2 H, t, J = 6.5, H1), 15.93 (1 H, s, 11-OH);
13C NMR (CDCl3): 21.45 (C14), 22.65 (C15), 23.58
(C13), 23.96 (C7), 26.57 (C3), 28.47 (C4), 29.02 (2 C,
C5, C6), 30.89 (C2), 32.37 (C12), 36.59 (C8), 44.79
(C1), 106.43 (C10), 181.38 (C11), 201.22 (C9).
Alkylation the nucleic bases with use as bases
DBU (method A). The corresponding alkylating
reagent (15 mmol) and DBU (2.2 ml, 15 mmol) were
added to a suspension of a nucleic base (10 mmol) or its
protected derivative in absolute DMF (25 ml). The mix-
ture was heated for 20 h at 80–100°ë; the reaction was
monitored by TLC in suitable chloroform–ethanol
developing system. The reaction mixture was cooled
and evaporated in a vacuum. The residue was dissolved
in the minimum volume of chloroform and chromato-
graphed on a column (5 × 28 cm) with Silica gel
(200 g), elution with a gradient of ethanol in chloro-
34 5, and 36 3%, respectively.
Thus, compound (Ic) in the concentration giving
rise to the chromosome damages also displayed cyto-
static activity resulting in an almost twice decrease of
the amount of cells entered mitosis. These results give
grounds to consider the cytotoxicity of the compounds
under study to be due to several mechanisms including
the disturbance of the cell cycle.
As a whole, the results of this study testify that 9-
polymethylene derivatives of adenine (Ia)−(Ic) and
hypoxanthine (IIb) and (IIc) with the β-diketone frag-
ment in the ω-position of the polymethylene chain offer
promise as antineoplastic agents. Cytotoxicity of these
compounds observed in in vitro experiments on human
leukemia and carcinoma cells is the main argument
favoring such conclusion. This activity is due to the
sum of several effects of the compounds under study,
namely, the induction of apoptosis and chromosome
damages and also inhibition of the mitotic activity of
cells. The ability of compound (Ic) to possess the
equally high toxic effect both on potentially sensitive
K562 and HCT116 (model of the tumor before the
beginning of chemotherapy) cells and on the cells that
have acquired drug resistance (K562/4 and
HCT116p53KO cells) also indicates the promises of
the compounds aforementioned.
EXPERIMENTAL
Adenine, hypoxanthine, guanine, uracil, thymine,
and cytosine were from Sigma (USA); DBU and
sodium hydride (80% suspension in mineral oil) were
from Fluka (Switzerland); DMF, chloroform (reagent
grade), triethylamine (pure), and ethanol (95% recti-
fied) were of domestic manufacture; CDCl3 and
DMSO-d6 were from Acros Organics (Belgium). Sol-
vents were purified and dried by standard procedures
[24].
form (0
20%). The fractions containing the target
product were evaporated; the residue was crystallized.
Alkylation of sodium salts of adenine and
7-Chloroheptanoic and 9-chlorononanoic acid chlo- cytosine (method B). Sodium hydride (80% suspen-
rides were obtained according to the procedure [22]; 8- sion in mineral oil) (0.33 g, 11 mmol) was added at stir-
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POLYMETHYLENE DERIVATIVES OF NUCLEIC BASES
81
ring to a suspension of adenine or cytosine (10 mmol) (C5), 140.77 (C8), 149.53 (C4), 152.32 (C2), 155.92
in absolute DMF (25 ml). The mixture was stirred for
30 min at 20°ë; then the corresponding alkylating
reagent (12 mmol) was added. The reaction mixture
was heated to 80–100°ë for 20 h upon TLC monitoring
in the suitable chloroform–ethanol developing system.
Dimethyl formamide was evaporated in a vacuum; the
residue was dissolved in chloroform (50 ml); water
(20 ml) was added; and the resulting mixture was
placed in the separating funnel. The organic phase was
separated; the aqueous layer was extracted with chloro-
form (5 × 30 ml); the combined extracts were dried with
sodium sulfate; the solvent was evaporated; and the res-
idue was chromatographed (for conditions see method
A). The fractions containing the target products were
evaporated; the residue was crystallized from the suit-
able solvent.
(C6), 180.71 (C11'), 201.82 (C9').
3-[9-(2-Oxocyclohexyl)-9-oxononyl] adenine was
obtained in 8% yield as a by-product upon the synthesis
of derivative (Ic) by method B; Rf = 0.38 (B), mp 143°ë
(decomp.) (EtOH); MS: m/z 371.5 [M]+, m/z 372.5 [M +
H]+, calculated [M]+ 371.5 (C20H29N5O2); H NMR
1
(DMSO-d6): 1.15–1.90 (16 H, m, H2'–H7', H13', H14'),
2.24 (4 H, m, H12', H15'), 2.38 (2 H, t, J = 7.2, H8'),
4.27 (2 H, t, J = 6.9, H1'), 7.75 (1 H, s, H2), 7.99 (2 H,
s, 6-NH2), 8.34 (1 H, s, H8), 15.89 (1 H, s, 11'-OH); 13C
NMR (DMSO-d6): 21.11 (C13'), 22.24 (C14'), 22.95
(C15'), 23.55 (C7'), 25.75 (C3'), 28.29 (C4'), 28.52
(C5'), 28.57 (C6'), 28.60 (C2'), 30.53 (C12'), 36.22
(C8'), 49.28 (C1'), 106.54 (C10'), 120.44 (C5), 143.23
(C2), 149.65 (C4), 152.45 (C8), 154.91 (C6), 180.80
(C11'), 201.55 (C9').
9-[8-(2-Oxocyclohexyl)-8-oxooctyl] adenine (Ib)
was obtained by method B in 40% yield; Rf = 0.25 (A),
mp 100–101°ë (1 : 1 EtOAc–heptane); MS: m/z 357.4
[M]+, m/z 358.4 [M + H]+, calculated [M]+ 357.4
(C19H27N5O2); 1H NMR (DMSO-d6): 1.13–1.80 (14 H, m,
H2'–H6', H12', H13'), 2.22 (4 H, m, H11', H14'), 2.36
(2 H, t, J = 7.2, H7'), 4.11 (2 H, t, J = 7.2, H1'), 7.15
(2 H, s, 6-NH2); 8.11 (1 H, s, H8), 8.13 (1 H, s, H2),
15.95 (1 H, s, 10'-OH); 13C NMR (DMSO-d6): 21.06
(C13'), 22.19 (C14'), 22.89 (C6'), 23.42 (C12'), 25.79
(C4'), 28.16 (C5'), 28.43 (C3'), 29.25 (C2'), 30.45
(C11'), 36.11 (C7'), 42.79 (C1'), 106.49 (C9'), 118.72
(C5), 140.71 (C8), 149.51 (C4), 152.26 (C2), 155.88
(C6), 180.74 (C10'), 201.53 (C8').
9-[8-(2-Oxocyclohexyl)-8-oxooctyl]
hypoxan-
thine (IIb) was obtained in 57% yield by method A;
Rf = 0.45 (A), mp 142–143°ë (1 : 1 EtOAc–heptane);
MS: m/z 358.4 [M]+, m/z 359.4 [M + H]+, calculated [M]+
1
358.4 (C19H26N4O3); H NMR (DMSO-d6): 1.14–1.88
(14 H, m, H2'–H6', H12', H13'), 2.24 (4 H, m, H11',
H14'), 2.37 (2 H, t, J = 7.2, H7'), 4.11 (2 H, t, J = 7.2,
H1'), 8.00 (1 H, s, H8), 8.06 (1 H, s, H2), 12.21 (1 H, s,
13
6-OH), 15.94 (1H, s, 10'-OH); C NMR (DMSO-d6):
21.07 (C13'), 22.20 (C14'), 22.89 (C6'), 23.40 (C12'),
25.71 (C4'), 28.12 (C5'), 28.41 (C3'), 29.40 (C2'), 30.44
(C11'), 36.12 (C7'), 43.17 (C1'), 106.50 (C9'), 123.89
(C5), 140.14 (C8), 145.24 (C2), 153.22 (C4), 156.60
(C6), 180.74 (C10'), 201.56 (C8').
3-[8-(2-Oxocyclohexyl)-8-oxooctyl] adenine was
obtained as a by-product in 0.9% yield upon the synthe-
sis of derivative (Ib) by method B; Rf = 0.15 (A),
mp 184–185°ë (EtOH); MS: m/z 357.4 [M]+, m/z 358.4
[M + H]+, calculated [M]+ 357.4 (C19H27N5O2); 1H NMR
(DMSO-d6): 1.16–1.95 (14 H, m, H2'–H6', H12', H13'),
2.26 (4 H, m, H11', H14'), 2.38 (2 H, t, J = 7.5, H7'),
4.30 (2 H, t, J = 7.2, H1'), 7.70 (2 H, s, 6-NH2), 7.76
(1 H, s, H2), 8.30 (1 H, s, H8), 15.92 (1H, s, 10'-OH);
13C NMR (DMSO-d6): 20.91 (C13'), 22.07 (C14'), 22.79
(C6'), 23.33 (C12'), 25.45 (C4'), 27.95 (C5'), 28.21
(C3'), 28.35 (C2'), 30.44 (C11'), 35.82 (C7'), 49.09
(C1'), 106.37 (C9'), 120.35 (C5), 142.88 (C2), 149.81
(C4), 152.32 (C8), 154.67 (C6), 181.09 (C10'), 200.86
(C8').
9-[9-(2-Oxocyclohexyl)-9-oxononyl]
hypoxan-
thine (IIc) was obtained in 51% yield by method A;
Rf = 0.80 (A), mp 165–166°ë (1 : 1 EtOAc–heptane);
MS: m/z 372.4 [M]+, m/z 373.4 [M + H]+, calculated [M]+
1
372.4 (C20H28N4O3); H NMR (DMSO-d6): 1.12–1.80
(16 H, m, H2'–H7', H13', H14'), 2.25 (4 H, m, H12',
H15'), 2.38 (2 H, t, J = 7.2, H8'), 4.10 (2 H, t, J = 7.2,
H1'), 8.01 (1 H, s, H8), 8.08 (1 H, s, H2), 12.26 (1 H, s,
6-OH), 15.98 (1 H, s, 11'-OH); 13C NMR (DMSO-d6):
21.06 (C13'), 22.19 (C14'), 22.89 (C15'), 23.50 (C7'),
25.77 (C3'), 28.16 (C4'), 28.45 (C5'), 28.56 (C6'), 29.39
(C2'), 30.45 (C12'), 36.12 (C8'), 43.16 (C1'), 106.52
(C10'), 123.87 (C5), 140.13 (C8), 145.24 (C2), 154.20
(C4), 156.57 (C6), 180.79 (C11'), 201.58 (C9').
9-[9-(2-Oxocyclohexyl)-9-oxononyl] adenine (Ic)
was obtained in 39% yield by method B; Rf = 0.80 (B),
Removal of isobutyryl groups in derivatives N2-
mp 73–74°ë (1 : 1 EtOAc–heptane); MS: m/z371.5 [M]+, isobutyryl guanine (method B). Triethylamine
m/z 372.5 [M + H]+, calculated [M]+ 371.5 (C20H29N5O2); (0.23 ml, 1.6 mmol) was added to a solution of the pro-
1H NMR (DMSO-d6): 1.11–1.82 (16 H, m, H2'–H7', tected guanine derivative (0.8 mmol) in ethanol (9 ml)
H13', H14'), 2.26 (4 H, m, H12', H15'), 2.38 (2 H, t, J = and water (1.5 ml); the resulting solution was refluxed;
7.2, H8'), 4.10 (2 H, t, J = 7.2, H1'), 7.20 (2 H, s, 6- the course of the reaction was monitored by TLC (sys-
NH2), 8.11 (1 H, s, H8), 8.12 (1 H, s, H2), 15.99 (1 H, tem A). After 16 h, the solvents and triethylamine were
13
s, 10'-OH); C NMR (DMSO-d6): 21.12 (C13'), 22.24 evaporated in a vacuum. The residue was dissolved in
(C14'), 22.93 (C15'), 23.54 (C7'), 25.91 (C3'), 28.29 the minimum quantity of boiling ethanol; the solution
(C4'), 28.54 (C5'), 28.66 (C6'), 29.31 (C2'), 30.46 was cooled to room temperature and then placed in the
(C12'), 36.23 (C8'), 42.82 (C1'), 106.59 (C10'), 118.73 refrigerator (0°ë) to finish crystallization.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 1 2009

82
KOMISSAROV et al.
N2-(2-methylpropionyl)-9-[8-(2-oxocyclohexyl)-
(14 H, m, H2'–H6', H12', H13'), 2.25 (4 H, m, H11',
8-oxooctyl] guanine was obtained in 17% yield upon H14'), 2.38 (2 H, t, J = 7.2, H7'), 4.14 (2 H, t, J = 6.7,
alkylation of N2-isobutyryl guanine with (VIIb) by H1'), 6.31 (2 H, s, 2-NH2), 7.88 (1 H, s, H8), 11.10 (1
13
method A; Rf = 0.27 (A); mp 115–116°ë (1 : 2 EtOAc– H, s, H1), 15.91 (1 H, s, 10'-OH); C NMR (DMSO-
heptane); MS: m/z 443.5 [M]+, m/z 444.5 [M + H]+, calcu- d6): 21.07 (C13'), 22.20 (C14'), 22.90 (C6'), 23.46
lated [M]+ 443.5 (C23H33N5O4); 1H NMR (CDCl3): 1.19 (C12'), 25.48 (C4'), 28.18 (C5'), 28.46 (C3'), 30.32
(6 H, a, J = 6.2, CH2(CH3)2); 1.12–1.70 (14 H, m, H2'– (C2'), 30.48 (C11'), 36.13 (C7'), 45.95 (C1'), 106.54
H6', H12', H13'), 2.25 (4 H, m, H11', H14'), 2.30 (2 H, (C9'), 107.97 (C5), 142.84 (C8), 153.02 (C4), 154.96
t, J = 7.2, H7'), 2.86 (1 H, m, CH(CH3)2), 3.92 (2 H, t, (C2), 159.98 (C6), 180.83 (C10'), 201.55 (C8').
J = 5.9, H1'), 7.62 (1 H, s, H8), 10.27 [1 H, s,
N2-(2-methylpropionyl)-9-[9-(2-oxocyclohexyl)-
2-NHCOCH(CH3)2], 12.20 (1 H, s, H1), 15.93 (1H, s,
9-oxononyl] guanine was obtained in 31% yield by
13
10'-OH). C NMR (CDCl3): 18.92 (2 C, CH(CH3)2);
alkylation of N2-isobutyryl guanine with (VIIc) by
21.48 (C13'), 22.67 (C14'), 23.86 (C6'), 23.65 (C12'),
method A; Rf = 0.41 (A), mp 116–117°ë (1 : 2 EtOAc–
26.15 (C4'), 28.62 (C5'), 28.86 (C3'), 29.69 (C2'), 31.01
heptane); MS: m/z 457.5 [M]+, m/z 458.5 [M + H]+, calcu-
(C11'), 35.95 (CH(CH3)2); 36.58 (C7'), 43.71 (C1'),
1
lated [M]+ 457.5 (C24H35N5O4); H NMR (CDCl3): 1.20
106.69 (C9'), 120.77 (C5), 138.76 (C8), 147.67 (C2),
(6 H, a, J = 6.8, CH2(CH3)2); 1.10–1.79 (16 H, m, H2'–
148.74 (C4), 155.89 (C6), 179.47 (COCH(CH3)2);
H7', H13', H14'), 2.28 (4 H, m, H12', H15'), 2.35 (2 H,
181.73 (C10'), 201.27 (C8').
t, J = 7.2, H8'), 2.81 (1 H, m, CH(CH3)2); 3.97 (2 H, t,
N2-(2-methylpropionyl)-7-[8-(2-oxocyclohexyl)-
J = 7.2, H1'), 7.63 (1 H, s, H8), 9.75 (1 H, s,
8-oxooctyl] guanine was obtained in 17% yield as a 2-NHCOCH(CH3)2); 12.27 (1 H, s, H1), 15.93 (1 H, s,
13
by-product upon alkylation of N2-isobutyryl guanine 11'-OH); C NMR (CDCl3): 18.99 (2 C, CH(CH3)2);
with (VIIb) by method A; Rf = 0.36 (A), oil; MS: m/z 21.58 (C13'), 22.78 (C14'), 23.75 (C15'), 24.01 (C7'),
443.5 [M]+, m/z 444.5 [M + H]+, calculated [M]+ 443.5 26.33 (C3'), 28.61 (C4'), 29.02 (2 C, C5', C6'), 29.83
(C23H33N5O4); 1H NMR (CDCl3): 1.15 (6 H, a, J = 6.8, (C2'), 31.09 (C12'), 36.16 (CH(CH3)2); 36.72 (C7'),
CH2(CH3)2); 1.12–1.87 (14 H, m, H2'–H6', H12', H13'), 43.79 (C1'), 106.75 (C10'), 120.97 (C5), 138.87 (C8),
2.22 (4 H, m, H11', H14'), 2.28 (2 H, t, J = 7.5, H7'), 147.32 (C2), 148.64 (C4), 156.10 (C6), 178.66
2.85 (1 H, m, CH(CH3)2); 4.25 (2 H, t, J = 7.0, H1'), (COCH(CH3)2); 181.83 (C11'), 201.27 (C9').
7.73 (1 H, s, H8), 11.00 (1 H, s, 2-NHCOCH(CH3)2);
N2-(2-methylpropionyl)-7-[9-(2-oxocyclohexyl)-
13
12.29 (1 H, s, H1), 15.91 (1 H, s, 10'-OH); C NMR
9-oxononyl] guanine was obtained in 22% yield as a
(CDCl3): 18.81 (2 C, CH(CH3)2); 21.43 (C13'), 22.63
by-product upon alkylation of N2-isobutyryl guanine
(C14'), 23.58 (C6'), 23.85 (C12'), 25.92 (C4'), 28.64
with (VIIc) by method A; Rf = 0.70 (A), oil; MS: m/z
(C5'), 28.89 (C3'), 30.81 (C2'), 30.93 (C11'), 35.75
457.5 [M]+, m/z 458.5 [M + H]+, calculated [M]+ 457.5
(CH(CH3)2); 36.52 (C7'), 47.25 (C1'), 106.54 (C9'),
1
(C24H35N5O4); H NMR (CDCl3): 1.18 (6 H, a, J = 6.8,
111.79 (C5), 142.84 (C8), 147.86 (C4), 153.00 (C2),
CH2(CH3)2); 1.20–1.90 (16 H, m, H2'–H7', H13', H14'),
156.63 (C6), 179.86 (COCH(CH3)2); 181.62 (C10'),
2.27 (4 H, m, H12', H15'), 2.33 (2 H, t, J = 7.5, H8'),
201.14 (C8').
2.97 (1 H, m, CH(CH3)2); 4.29 (2 H, t, J = 7.2, H1'),
9-[8-(2-Oxocyclohexyl)-8-oxooctyl]guanine (IIIb) 7.72 (1 H, s, H8), 10.86 (1 H, s, 2-NHCOCH(CH3)2);
13
was obtained from the corresponding 9-derivative of 12.34 (1 H, s, H1), 15.97 (1 H, s, 11'-OH); C NMR
N2-isobutyryl guanine in 87% yield by method C; Rf = (CDCl3): 18.98 (2 C, CH(CH3)2); 21.57 (C13'), 22.77
0.18 (C), mp 181–182°ë (H2O–EtOH, 1 : 1); MS: m/z (C14'), 23.73 (C15'), 24.09 (C7'), 26.17 (C3'), 28.76
373.4 [M]+, m/z 374.4 [M + H]+, calculated [M]+ 373.4 (C4'), 29.11 (2 C, C5', C6'), 30.98 (C2'), 31.06 (C12'),
1
(C19H27N5O3); H NMR (DMSO-d6): 1.12–1.90 (14 H, 35.80 (CH(CH3)2); 36.70 (C7'), 47.36 (C1'), 106.63
m, H2'–H6', H12', H13'), 2.20 (4 H, m, H11', H14'), (C9'), 112.02 (C5), 142.84 (C8), 147.80 (C4), 153.21
2.25 (2 H, t, J = 7.2, H7'), 3.90 (2 H, t, J = 6.2, H1'), 6.57 (C2), 156.97 (C6), 179.84 (COCH(CH3)2); 181.70
(2 H, s, 2-NH2), 7.68 (1 H, s, H8), 10.83 (1 H, s, H1), (C10'), 201.30 (C8').
15.93 (1 H, s, 10'-OH); 13C NMR (DMSO-d6): 21.11
9-[9-(2-Oxocyclohexyl)-9-oxononyl]
guanine
(C13'), 22.24 (C14'), 22.94 (C6'), 23.48 (C12'), 25.85
(C4'), 28.31 (C5'), 28.52 (C3'), 29.35 (C2'), 30.49
(C11'), 36.18 (C7'), 42.60 (C1'), 106.53 (C9'), 116.55
(C5), 137.34 (C8), 151.21 (C4), 153.72 (C2), 157.31
(C6), 180.78 (C10'), 201.58 (C8').
(IIIc) was obtained in 87% yield from the correspond-
ing 9-derivative of N2-isobutyryl guanine by method C;
Rf = 0.29 (B), mp 166–167°ë (1 : 1 H2O–EtOH); MS:
m/z 387.5 [M]+, m/z 388.5 [M + H]+, calculated [M]+
1
387.5 (C20H29N5O3); H NMR (DMSO-d6): 1.10–1.75
7-[8-(2-Oxocyclohexyl)-8-oxooctyl] guanine was (16 H, m, H2'–H7', H13', H14'), 2.26 (4 H, m, H12',
obtained from the corresponding 7-derivative of N2- H15'), 2.38 (2 H, t, J = 7.2, H8'), 3.89 (2 H, t, J = 6.8,
isobutyryl guanine in 44% yield by method C; Rf = 0.18 H1'), 6.53 (2 H, s, 2-NH2), 7.67 (1 H, s, H8), 10.83 (1 H,
13
(C), mp 218–219°ë (decomp.) (1 : 1 H2O–EtOH); MS: s, H1), 15.93 (1 H, s, 11'-OH); C NMR (DMSO-d6):
m/z 373.4 [M]+, m/z 374.4 [M + H]+, calculated [M]+ 21.17 (C13'), 22.29 (C14'), 22.99 (C15'), 23.62 (C7'),
1
373.4 (C19H27N5O3); H NMR (DMSO-d6): 1.11–1.90 25.98 (C3'), 28.43 (C4'), 28.61 (C5'), 28.78 (C6'), 29.44
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 1 2009

POLYMETHYLENE DERIVATIVES OF NUCLEIC BASES
83
(C2'), 30.55 (C12'), 36.32 (C8'), 42.56 (C1'), 106.57 H12', H15'), 2.30 (2 H, t, J = 7.5, H8'), 3.60 (2 H, t, J =
(C10'), 116.58 (C5), 137.01 (C8), 151.40 (C4), 155.03 7.5, H1'), 6.95 (1 H, s, H6), 10.07 (1 H, s, H3), 15.94
(C2), 159.06 (C6), 180.74 (C11'), 201.58 (C9').
(1 H, s, 11'-OH); 13C NMR (CDCl3): 12.03 (5-CH3),
21.40 (C13'), 22.60 (C14'), 23.54 (C15'), 23.93 (C7'),
26.09 (C3'), 28.76 (C4'), 28.79 (C5'), 28.97 (2 C, C2',
C6'), 30.89 (C12'), 36.57 (C8'), 48.19 (C1'), 106.49
(C10'), 110.21 (C5), 140.31 (C6), 150.97 (C2), 164.50
(C4), 181.47 (C11'), 201.25 (C9').
7-[9-(2-Oxocyclohexyl)-9-oxononyl] guanine was
obtained in 53% yield from the corresponding 7-deriv-
ative of N2-isobutyryl guanine by method C; Rf = 0.29
(B), mp 204–205°ë (decomp.) (1 : 1 H2O–EtOH); MS:
m/z 387.5 [M]+, m/z 388.5 [M + H]+, calculated [M]+
1
387.5 (C20H29N5O3); H NMR (DMSO-d6): 1.10–1.80
1,3-Bis-[9-(2-Oxocyclohexyl)-9-oxononyl] thym-
(16 H, m, H2'–H7', H13', H14'), 2.25 (4 H, m, H12', ine was obtained in 10% yield as a by-product upon the
H15'), 2.40 (2 H, t, J = 7.2, H8'), 4.14 (2 H, t, J = 6.8, synthesis of (Vc) by method A, Rf = 0.86 (D); MS: m/z
H1'), 6.38 (2 H, s, 2-NH2), 7.86 (1 H, s, H8), 11.37 (1 H, 598.8 [M]+, 599.8 [M + H]+, calculated M 598.8
13
1
s, H1), 15.88 (1 H, s, 11'-OH); C NMR (DMSO-d6): (C35H54N2O6). H NMR(CDCl3): 1.20–1.70 (32 H, m,
21.07 (C13'), 22.19 (C14'), 22.90 (C15'), 23.54 (C7'), H2'–H7', H2''–H7'', H13', H13'', H14', H14''), 1.87 (3 H,
25.52 (C3'), 28.23 (C4'), 28.48 (C5'), 28.59 (C6'), 30.44 s, 5-CH3), 2.26 (8 H, m, H12', H12'', H15', H15''), 2.33
(C2'), 30.50 (C12'), 36.15 (C8'), 45.89 (C1'), 106.53 (4 H, m, H8', H8''), 3.64 (2 H, t, J = 6.8, H1''), 3.87 (2
(C10'), 108.00 (C5), 142.66 (C8), 153.35 (C4), 155.31 H, t, J = 6.5, H1'), 6.92 (1 H, s, H6), 15.97 (1 H, s, 11''-
13
(C2), 159.94 (C6), 180.85 (C11'), 201.51 (C9').
OH), 16.00 (1 H, s, 11'-OH); C NMR(CDCl3): 12.88
(5-CH3), 21.56 (2 C, C13', C13''), 22.76 (2 C, C14',
C14''), 23.71 (2 C, C15', C15''), 24.07 (C7''), 24.19
(C7'), 26.31 (C3''), 26.80 (C3'), 27.44 (C4''), 28.88
(C4'), 28.95 (C5''), 29.02 (C5'), 29.12 (2 C, C2', C2''),
29.19 (2 C, C6', C6''), 31.03 (C12''), 31.07 (C12'), 36.70
(C8''), 36.73 (C8'), 41.32 (C1''), 49.32 (C1'), 106.60 (2
C, C10', C10''), 109.49 (C5), 138.21 (C6), 151.26 (C2),
163.65 (C4), 181.66 (2 C, C11', C11''), 201.34 (2 C,
C9', C9'').
1-[9-(2-Oxocyclohexyl)-9-oxononyl] uracil (IVc)
was obtained in 34% yield by method A; Rf = 0.33 (D),
mp 71–72°ë (1 : 2 EtOAc–heptane); MS: m/z 348.4
[M]+, m/z 349.4 [M + H]+, calculated [M]+ 348.4
1
(C19H28N2O4); H NMR (CDCl3): 1.20–1.70 (16 H, m,
H2'–H7', H13', H14'), 2.28 (4 H, m, H12', H15'), 2.35
(2 H, t, J = 7.2, H8'), 3.68 (2 H, t, J = 7.2, H1'), 5.66 (1
H, d, J = 7.8, H5), 7.13 (1 H, d, J = 7.8, H6), 9.59 (1 H,
s, H3), 15.99 (1 H, s, 11'-OH); 13C NMR (CDCl3): 21.61
(C13'), 22.80 (C14'), 23.76 (C15'), 24.11 (C7'), 26.26
1-[9-(2-Oxocyclohexyl)-9-oxononyl]
cytosine
(C3'), 28.89 (C4'), 28.93 (C5'), 29.14 (2 C, C2', C6'), (VIc) was obtained in 31% yield by method B; Rf =
31.10 (C12'), 36.74 (C8'), 48.76 (C1'), 102.00 (C5), 0.44 (C), mp 156–157°ë (ethanol); MS: m/z 347.5 [M]+,
106.67 (C10'), 144.35 (C6), 150.84 (C2), 163.79 (C4), 348.5 [M + H]+, calculated [M]+ 347.5 (C19H29N3O3); H
1
181.75 (C11'), 201.36 (C9').
NMR (DMSO-d6): 1.15–1.70 (16 H, m, H2'–H7', H13',
H14'), 2.26 (4 H, m, H12', H15'), 2.41 (2 H, t, J = 7.5,
H8'), 3.58 (2 H, t, J = 7.2, H1'), 5.60 (1 H, d, J = 7.2,
H5), 6.97 (2 H, s, 4-NH2), 7.54 (1 H, d, J = 7.2, H6),
15.99 (1 H, s, 11'-OH); 13C NMR (DMSO-d6): 21.13
(C13'), 22.25 (C14'), 22.94 (C15'), 23.58 (C7'), 25.91
(C3'), 28.57 (C4'), 28.61 (C5'), 28.66 (C2'), 28.76 (C6'),
30.47 (C12'), 36.25 (C8'), 48.54 (C1'), 92.94 (C5),
106.60 (C10'), 145.92 (C6), 155.75 (C2), 165.84 (C4),
180.71 (C11'), 201.82 (C9').
1,3-Bis-[9-(2-Oxocyclohexyl)-9-oxononyl] uracil
was obtained in 23.6% yield as a by-product upon the
synthesis of (IVc) by method A, oil; Rf = 0.85 (D); MS:
m/z 584.8 [M]+, 585.8 [M + H]+, calculated M 584.8
1
(C34H52N2O6); HNMR (CDCl3): 1.20–1.70 (32 H, m,
H2'–H7', H2''–H7'', H13', H13'', H14', H14''), 2.26 (8 H,
m, H12', H12'', H15', H15''), 2.32 (4 H, m, H8', H8''),
3.66 (2 H, t, J = 7.2, H1''), 3.85 (2 H, t, J = 7.2, H1'),
5.66 (1 H, d, J = 7.8, H5), 7.06 (1 H, d, J = 7.8, H6),
15.97 (1 H, s, 11''-OH), 15.99 (1 H, s, 11'-OH); 13C
All reagents used in the biological part of this study,
NMR (CDCl3): 21.60 (2 C, C13', C13''), 22.80 (2 C, except for those specified separately, were from Sigma
C14', C14''), 23.75 (2 C, C15', C15''), 24.09 (C7''), (USA).
24.22 (C7'), 26.32 (C3''), 26.80 (C3'), 27.43 (C4''),
Cell cultures. K562 and HCT116 cells lines were
28.89 (C4'), 28.92 (C5''), 29.05 (C5'), 29.14 (C2''),
purchased from American Type Culture Collection
29.16 (C2'), 29.23 (2 C, C6', C6''), 31.07 (C12''), 31.11
(USA). K562/4 subline was obtained by A.N. Saprin
(C12'), 36.73 (C8''), 36.76 (C8'), 41.14 (C1''), 49.69
(Center for Theoretical Problems in Physico-Chemical
(C1'), 101.44 (C5), 106.63 (2 C, C10', C10''), 142.02
Pharmacology, Russian Academy of Sciences),
(C6), 151.33 (C2), 162.99 (C4), 181.66 (2 C, C11',
HCT116p53KO subline was obtained by B. Vogelstein
C11''), 201.37 (2 C, C9', C9'').
(John Hopkins Cancer Center, USA) and purchased by
thymine B.P. Kopnin. The cells were cultured in Dulbecco mod-
1-[9-(2-Oxocyclohexyl)-9-oxononyl]
(Vc) was obtained in 58% yield by method A; Rf = 0.46 ified Eagle’s medium with addition of 10% embrional
(D), mp 85–86°ë (1 : 2 EtOAc–heptane); MS: m/z 362.4 calf serum (Invitrogen, USA) and 2 mM L-glutamine at
[M]+, m/z 363.4 [M + H]+, calculated [M]+ 362.4 36°ë, 5% ëé2. To estimate the cytotoxicity of the prep-
1
(C20H30N2O4); H NMR (CDCl3): 1.15–1.70 (16 H, m, arations, the cells in exponential growth phase were
H2'–H7', H13', H14'), 1.82 (3 H, s, 5-CH3), 2.23 (4 H, m, inoculated in 96-well plates or in the Petri dishes. The
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 1 2009

84
KOMISSAROV et al.
and Whittaker, C.M., Bioorg. Med. Chem. Lett., 2000,
compounds under study were introduced on 24–72 h.
The cytological smears were prepared by the standard
method after 72 h incubation with the compound under
study and were fixed for 10 min in methanol. The
smears were colored with hematoxylin and eosin and
analyzed at magnification ×900.
Cytotoxicity of the compounds under study was
estimated in the MTT test [25]. Medicines Etoposide
and Mitoxantron were used as positive controls.
vol. 10, pp. 2557–2561.
4. Wai, J.S., Egbertson, M.S., Payne, L.S., Fisher, T.E.,
Embrey, M.W., Tran, L.O., Melamed, J.Y.,
Langford, H.M., Guare, J.P., Zhuang, L., Grey, V.E.,
Vacca, J.P., Holloway, M.K., Naylor-Olsen, A.M.,
Hazuda, D.J., Felock, P.J., Wolfe, A.L., Stillmock, K.A.,
Schleif, W.A., Gabryelski, L.J., and Young, S.D., J. Med.
Chem., 2000, vol. 43, pp. 4923–4926.
5. Grobler, J.A., Stillmock, K., Hu, B., Witmer, M.,
Felock, P., Espeseth, A.S., Wolfe, A., Egbertson, M.,
Bourgeois, M., Melamed, J., Wai, J.S., Young, S., Vacca,
J., and Hazuda, D.J., Proc. Natl. Acad. Sci. USA, 2002,
vol. 99, pp. 6661–6666.
Obtaining and analysis of the preparations of
metaphase chromosomes. The preparations were
obtained by the standard method and were colored with
azure and eosin; the analysis was performed at magni-
fication ×1000.
The proliferative activity of cultures was esti-
mated by the metaphase index. We counted 1000 cells
in random vision fields of the cytogenetic preparation
used for the count of chromosome aberrations, marking
all dividing cells. As the cultures were treated with
mitostatic agent Colchicine for 2 h before fixation, the
overwhelming majority of dividing cells were in the
metaphase. The results of calculation were expressed in
per mille (‰).
6. Long,Y.-Q., Jiang, X.-H., Dayam, R., Sanchez, T., Shoe-
maker, R., Sei, S., and Neamati, N., J. Med. Chem.,
2004, vol. 47, pp. 2561–2573.
7. Summa, V., Petrocchi, A., Matassa, V.G., Taliani, M.,
Laufer, R., De Francesco, R., Altamura, S., and Pace, P.,
J. Med. Chem., 2004, vol. 47, pp. 5336–5339.
8. Biswal, B.K., Cherney, M.M., Wang, M., Chan, L., Yan-
nopoulos, C.G., Bilimoria, D., Nicolas, O., Bedard, J.,
and James, M.N.G., J. Biol. Chem., 2005, vol. 280,
pp. 18 202–18 210.
9. Summa, V., Petrocchi, A., Pace, P., Matassa, V.G., De
Francesco, R., Altamura, S., Tomei, L., Koch, U., and
Neuner, P., J. Med. Chem., 2004, vol. 47, pp. 14–17.
ACKNOWLEDGMENTS
10. Kopnin, B.P., in Kantserogenez (Cancerogenesis),
Zaridze, D.G., Ed., Moscow: Meditsina, 2004, pp. 125–
158.
We are grateful to A.R. Khomutov (Engelhardt
Institute of Molecular Biology, Russian Academy of
Sciences) and to T.P. Stromskaya (Blokhin Russian
Cancer Research Center of Oncology, Russian Acad-
emy of Medical Sciences) for effective discussion of
the results and also to B.P. Kopnin (Blokhin Russian
Cancer Research Center of Oncology, Russian Acad-
emy of Medical Sciences) and A.N. Saprin (Center for
Theoretical Problems in Physico-Chemical Pharmacol-
ogy, RussianAcademy of Sciences) for purchase of cell
sublines.
The work was supported by the Federal Target Pro-
gram for Research and Development in Priority Direc-
tions of Development of Russian Scientific and Techni-
cal Complex in 2007–2012 (State Contract
no. 02.512.11.2237) and by the Russian Foundation for
Basic Research (project nos. 06-04-48716-a and 07-04-
00800-a).
11. Stavrovskaya, A.A., in Kantserogenez (Cancerogenesis),
Zaridze, D.G, Ed., Moscow: Meditsina, 2004, pp. 558–
574.
12. Galmarini, C.M., Mackey, J.R., and Dumontet, C., Lan-
cet Oncol., 2002, vol. 3, pp. 415–424.
13. Konstantinova, I.D., Leont’eva, N.A., Galegov, G.A.,
Ryzhova, O.I., Chuvikovsky, D.V., Antonov, K.V., Esi-
pov, R.S., Taran, S.A., Verevkina, K.N., Feofanov, S.A.,
and Miroshnikov, A.I., Rus. J. Bioorg. Chem., 2004,
vol. 30, pp. 553–560; Bioorg. Khim., 2004, vol. 30,
pp. 613–620.
14. Manikowski, A., Verri, A., Lossani, A., Gebhardt, B.M.,
Gambino, J., Focher, F., Spadari, S., and Wright, G.E.,
J. Med. Chem., 2005, vol. 48, pp. 3919–3929.
15. Esteban-Gamboa, A., Balzarini, J., Esnouf, R., and De
Clercq, E., Camarasa M.-J., and Perez-Perez M.-J,
J. Med. Chem., 2000, vol. 43, pp. 971–983.
REFERENCES
16. Halazy, S., Ehrhard, A., and Danzin, C., J. Am. Chem.
Soc., 1991, vol. 113, pp. 315–317.
1. Komissarov, V.V., Panova, N.G., and Kritzyn, A.M., Rus.
J. Bioorg. Chem., 2008, vol. 34, pp. 67–73; Bioorg.
Khim., 2008, vol.34, pp. 75–82.
17. Beauchamp, L.M., Tuttle, J.V., Rodriguez, M.E., and
Sznaidman, M.L., J. Med. Chem., 1996, vol. 39,
pp. 949–956.
2. Pragnacharyulu, P.V.P., Varkhedkar, V., Curtis, M.A.,
Chang, I.F., and Abushanab, E., J. Med. Chem., 2000,
vol. 43, pp. 4694–4700.
18. Kelley, J.L., Bullock, R.M., Krochmal, M.P., McLean,
E.W., Linn, J.A., Durcan, M.J., and Cooper, B.R., J.
Med. Chem., 1997, vol. 40, pp. 3207–3216.
3. Boyd, H.F., Fell, S.C.M., Flynn, S.T., Hickey, D.M.B.,
Ife, R.J., Leach, C.A., Macphee, C.H., Milliner, K.J., 19. Zacharie, B., Gagnon, L., Attrado, G., Connolly, T.P., St-
Moores, K.E., Pinto, I.L., Porter, R.A., Rawlings, D.A.,
Smith, S.A., Stansfield, I.G., Tew, D.G., Theobald, C.J.,
Dennis, Y., and Penney, C.L., J. Med. Chem., 1997,
vol. 40, pp. 2883–2894.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 1 2009

POLYMETHYLENE DERIVATIVES OF NUCLEIC BASES
85
20. Kritzyn, A.M. and Komissarov, V.V., Rus. J. Bioorg. 24. Tietze, L. and Eicher, T., Reaktionen und Synthesen im
Chem., 2004, vol. 30, pp. 436–440; Bioorg. Khim., 2004,
organisch-chemischen Praktikum und Forschungslabo-
ratorium, New York: Georg Thieme Verlag Stuttgart,
1991. Preparativnaya organicheskaya khimiya: Per. s
nem (Preparative Organic Chemistry), Moscow: Mir,
1999.
vol. 30, pp. 487–492.
21. Kritzyn, A.M., Vepsalainen, J., and Komissarov, V.V.,
Rus. J. Bioorg. Chem., 2005, vol. 31, pp. 256–262;
Bioorg. Khim., 2005, vol. 31, pp. 288–294.
22. Nesmeyanov, A.N. and Zakharkin, L.I., in: A.N. Nesm-
eyanov, Izbrannye trudy (Selected Works), Moscow,
1959, vol. 3, pp. 276–286.
25. Sidorova, T.A., Nigmatov, A., Kakpakova, E.S.,
Stavrovskaya, A.A., Gerassimova, G.K., Shtil, A.A., and
Serebryakov, E.P., J. Med. Chem., 2002, vol. 45,
pp. 5330–5339.
23. Meszarosova, K., Holy, A., and Masojidkova, M., Coll.
Czech. Chem. Communs., 2000, vol. 65, pp. 1109–1125.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 1 2009