Regioselective alkylation of guanine derivatives in the synthesis of peptide nucleic acid monomers

Article abstract of DOI:10.1007/s11172-015-0986-3

A method for the synthesis of 6-O-benzyl- and 6-O-benzyl-2-N-benzyloxycarbonyl-protected guanine derivatives starting from 2-amino-6-chloropurin is described. A regioselective alkylation of these N(9)-protected guanine derivatives gave the corresponding α-monomers of chiral peptide nucleic acids, the L-glutamic acid derivatives. It was shown that these compound do not inhibit (in the concentrations <20 μmol L^-1) the topoisomerase I activity.

Full text of DOI:10.1007/s11172-015-0986-3

1
100
Russian Chemical Bulletin, International Edition, Vol. 64, No. 5, pp. 1100—1106, May, 2015
Regioselective alkylation of guanine derivatives
in the synthesis of peptide nucleic acid monomers*
a
b
a
c
a
a
A. V. Dezhenkov, D. A. Cheshkov, I. A. Prokhorov, L. G. Dezhenkova, V. I. Shvets, and Yu. G. Kirillova
aM. V. Lomonosov Moscow State University of Fine Chemical Technologies,
8
6 prosp. Vernadskogo, 119571 Moscow, Russian Federation.
Eꢀmail: pnaꢀmitht@yandex.ru
b
Research Institute of Chemistry and Technology of Organoelement Compounds,
3
8 sh. Entuziastov, 111123 Moscow, Russian Federation
c
G. F. Gause Research Institute of New Antibiotics
1
1 ul. Pirogovskaya, 119021 Moscow, Russian Federation.
A method for the synthesis of 6ꢀOꢀbenzylꢀ and 6ꢀOꢀbenzylꢀ2ꢀNꢀbenzyloxycarbonylꢀproꢀ
tected guanine derivatives starting from 2ꢀaminoꢀ6ꢀchloropurin is described. A regioselective
alkylation of these N(9)ꢀprotected guanine derivatives gave the corresponding ꢀmonomers of
chiral peptide nucleic acids, the Lꢀglutamic acid derivatives. It was shown that these compound
do not inhibit (in the concentrations <20 mol L– ) the topoisomerase I activity.
1
Key words: peptide nucleic acids, diprotected guanine, topoisomerase I.
Guanineꢀcontaining compounds alkylated at N(9)
noticeably decrease the stability of their complexes with
ON, as well as facilitates the bioavailability of PNA when
using a standard cationic lipofection. The introduction
atom are of special interest. First of all, this is due to the
1
10
fact that acyclic guanine analogs are widely used in the
therapy of viral infections. It is known that 6ꢀOꢀsubstitutꢀ
of a substituent at positions 2() or 5() of the PNA core
leads to the appearance of a chiral center, whose configuꢀ
ration is responsible for the formation of the oligomer
preorganized structure, that, in the end, determines the
hybridizing properties of PNA. Thus, we consider it
a promising direction to develop chiral negatively charged
PNA, which, together with nucleic bases responsible for
molecular recognition, contain a negative charge per each
monomer unit, like in natural ON.
ed guanine in the DNA composition hinders activity of
2
topoisomerase I, whereas 6ꢀCꢀsubstituted purins are inꢀ
3
hibitors of topoisomerase II activity. In this case, the
1
1
enzyme serves as a target for antitumor agents, therefore
the screening of new compounds on the topoisomerase
activity is still important in the present time. Other well
known derivatives are the guanine monomers of funcꢀ
tional polyamide DNA analogs, the peptide nucleic acids
4
(
PNA), which are used in molecular biology and diꢀ
5
agnosis.
At the present time, PNA are considered as a new class
of stable compounds capable of overcoming a number of
disadvantages of natural oligonucleotides (ON) and their
analogs when used as genetic therapeutic agents. An
Nꢀ(2ꢀaminoethyl)glycine (aeg) fragment with nucleic
heterocyclic bases attached through the acetyl linkers
serves as a structural unit of the PNA skeleton. Such
a structure, on the one hand, due to the "restrained flexiꢀ
aegꢀPNA
ꢀPNA
6
bility" secures an efficient recognition of complimentary
sequences by the nucleic bases incorporated in the skeleꢀ
7
ton, on the other hand, due to the unnatural bonds makes
8
the aegꢀPNA stable to nucleases and proteases. Earlier, it
was shown that the introduction of a negative charge in
9
the structure of PNA using the phosphate groups did not
*
On the occasion of the 100th anniversary of the birth of Acadeꢀ
ꢀPNA
mician N. K. Kochetkov (1915—2005).
B is Thy, Cyt, Ade, Gua.
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 5, pp. 1100—1106, May, 2015.
066ꢀ5285/15/6405ꢀ1100 © 2015 Springer Science+Business Media, Inc.
1

Regioselective alkylation of guanine derivatives
Russ.Chem.Bull., Int.Ed., Vol. 64, No. 5, May, 2015
1101
Lately, both the classic and the modified PNA are used
in the studies of noncanonical structures of nucleic acids
Scheme 1
1
2
in order to determine a regulatory function of these eleꢀ
ments in the process of gene expression, as well as in the
analysis of telomeric repeats. To study such interactions,
it is necessary to obtain PNA oligomers with a large conꢀ
tent of guanine bases, which requires a preparative,
well reproducible procedure for the synthesis of guanine
monomers.
The developed by now strategies for the synthesis of
1
3—15
PNA monomers for their subsequent oligomerization,
suggest either the condensation of a pseudopeptide fragꢀ
1
6
ment with a heterocycle carboxymethylated derivative
or the alkylation of protected heterocycles with a bromoꢀ
acetamide derivative1 of the pseudopeptide fragment. Earꢀ
lier, it was shown that the second strategy is more optimal
for the synthesis of monomers, whose side chain is repreꢀ
7
i. PhCH OH, NaH, 60 C, 62%; ii. CbzCl, NaH, DMF, 44%.
2
1
8
sented by the 3ꢀbenzyloxycarbonyl(ethyl) moiety, since
it gives higher yields. An aegꢀguanine monomer was obꢀ
tained by the condensation of a pseudopeptide fragment
rivative 5 (Scheme 2). Earlier, this synthesis24 was accomꢀ
plished by the addition of bromoacetyl bromide to a mixꢀ
1
6
with 9ꢀNꢀalkylꢀ6ꢀOꢀbenzyl guanine derivative, howevꢀ
er, the unprotected exocyclic amino group of the heteroꢀ
cycle makes it impossible to carry out a qualitative Kaiser
test and capping the amino groups in the growing oligoꢀ
meric chain. Later, guanine 2ꢀNꢀbenzyloxycarbonylꢀ9ꢀNꢀ
ture of pseudopeptide 4 and Et N in dichloromethane.
3
However, when the amount of compound 4 was scaled to
5 g, the yield of bromide 5 was only 43%. Apparently,
2
3
a side cyclic product 6 is formed upon the initial addition
of Et N to the secondary amine 4 (see Scheme 2), and this
3
1
9
carboxymethyl derivative was synthesized, however, in
process becomes very fast upon an increase in the loading
of amine 4. To avoid this side reaction, we changed the
addition order of the reagents. Thus, bromoacetyl bromide
was added to a solution of compound 4 in dichloromethane
all these cases1
the heterocycle was carried out after 9ꢀNꢀalkylation of
ꢀaminoꢀ6ꢀchloropurin 1. For the synthesis of the PNA
monomers through the alkylation of protected heterocycles
6,19
the introduction of protecting groups in
2
at 0 C and only after this Et N was added slowly dropꢀ
3
1
8
with chloro(bromo)acetic acid esters, it is also necessary
to protect O(6) in order to prevent the side alkylation
process at atom N(7). However, it was shown that the
alkylation of 2ꢀNꢀisobutirylꢀ6ꢀOꢀdiphenylcarbamoylꢀ
guanine with methyl bromoacetate led to a mixture of
products of N(9)/N(7)ꢀsubstitution, the content of the
wise. The yield of compound 5 with the changed order of
addition of reagents was 85%.
2
0
The guanineꢀcontaining monomers 7 and 9 protected
at the Cꢀend were obtained by the alkylation of protected
heterocyclic bases 2 and 3 with bromoacetamide derivaꢀ
tive 5 in the presence of 1.1—1.2 equiv. of K CO . Monoꢀ
2
3
7
ꢀNꢀregioisomer being ~15%.
To sum up, to obtain the PNA guanine monomers
mer 7 was obtained in 65% yield, whereas a completely
protected monomer 9 in 41% yield. The moderate yields
can be explained by the incomplete conversion of the startꢀ
ing compounds 2 and 3, which were separated from the
target products by column chromatography.
based on LꢀGlu, the Bocꢀprotocol of the solidꢀphase synꢀ
thesis requires a preliminary protection of the guanine
O(6) and N(2) according to the earlier described tactics
for the introduction of protecting groups in the synthesis
of the thymine monomer.21
The diprotected guanine derivative 2 was obtained in
two steps. In the first step, a benzyl protecting group was
introduced at atom O(6) according to a slightly changed
procedure described earlier:22 sodium hydride was used
instead of sodium for the formation of sodium benzylate.
The yield of compound 3 was 62%. The reaction of 6ꢀOꢀ
benzylguanine 3 with benzyloxycarbonyl chloride (CbzCl)
in the presence of sodium hydride (Scheme 1) led to guaꢀ
nine derivative 2 in 44% yield.
When evaluating the regioselectivity of the alkylation
reaction of purin bases 2 and 3 with bromoacetamide deꢀ
rivative 5, the N(7)/N(9)ꢀsubstitution cannot be inferred
1
3
from the data of C NMR spectroscopy because of
a possibility of the incorrect interpretation of close signals
for the carbon atoms of the allyl protecting group and
atom C(5) of the purin bases. Therefore, the regioselectivꢀ
ity of alkylation was studied using 2D NMR spectroscopy
1
1
1
13
1
13
( H/ H COSY, H/ C HSQC, H/ C HMBC) for
monomer 7, since the interpretation of its spectral data is
simpler than that for monomer 9.
13
1
The starting compound for the synthesis of guanine
monomers was the secondary amine 4 (see Ref. 23), which
was converted to the corresponding bromoacetamide deꢀ
During analysis, we assigned the signals for C and H
nuclei in the NMR spectra of monomer 7, in which the
double sets of signal for all the H and C atoms were observꢀ

1
102
Russ.Chem.Bull., Int.Ed., Vol. 64, No. 5, May, 2015
Dezhenkov et al.
Scheme 2
i. Pd(PPh ) , Nꢀethylaniline.
3
4
ed caused by the presence of two conformers of compound
(Fig. 1) due to the hindered rotation around the amide
Monomer 9 was not characterized by 2D NMR specꢀ
1
13
7
tra, however, the regular 1D H and C NMR spectra
showed similar signals for all the H and C atoms with the
additional signals attributed to the Cbz group. The AAL
methylene protons are assigned to  5.16 (a major conꢀ
former) and  5.08 (a minor conformer), whereas the sigꢀ
nals for atoms C(8), C(4), and C(5) of the guanine fragꢀ
ment are found at  140.0, 156.0, and 119.1, respectively.
It is known that in the case of N(7)ꢀsubstitution, the chemꢀ
ical shifts for atoms C(4) and C(5) can be found at
bond between the AAL and the pseudopeptide fragment.
To determine the regioselectivity of alkylation at atom
N(9), we used the HMBC spectra. The analysis of the
spectra showed the presence of a correlation between the
methylene protons of AAL (H 5.13, a major conformer;
H 5.03, a minor conformer) and atoms C(8) (C 142.1)
and C(4) ( 156.6) of the guanine fragment of the bases.
C
At the same time, there were no correlation between the
2
0
linker protons and atom C(5) ( 115.9) of the base, which
 157—164 and 108—112, respectively.
C
indicated the presence of the exclusive N(9)ꢀsubstitution
products only (see Fig. 1, b). A fragment of the 2D HMBC
spectrum of monomer 7 is shown in Fig. 2 with the indicaꢀ
tion of the correlating signals.
To sum up, the alkylation of protected guanine bases 2
and 3 with the pseudopeptide bromoacetamide derivatives
5 proceeds regioselectively and leads to the target products
of N(9)ꢀalkylation.
R = CH CH COOCH Ph
2
2
2
AAL
R = H, Cbz
Fig. 1. (a) Structures of conformers formed owing to the hindered rotation around the amide bond between the N(3)ꢀmonomeric unit
and the C(O)ꢀacetamide linker (AAL); (b) heteronuclear correlation indicating the regioselectivity of the alkylation reaction.

Regioselective alkylation of guanine derivatives
Russ.Chem.Bull., Int.Ed., Vol. 64, No. 5, May, 2015
1103
AAL

Gua C(5)
1
1
1
1
1
1
20
30
40
50
60
70
Gua C(8)
Gua C(4)
5
.5
5.0
4.5

Fig. 2. A fragment of the HMBC spectrum of ꢀguanineꢀcontaining monomer 8.
Earlier, we used to remove an allyl protection in the
presence of morpholine and Pd(PPh ) as a catalyst, which
gave moderate yields of the target products (45—53%).
tal composition by the high resolution mass spectroꢀ
metry data.
3
4
2
5
It is known that guanine derivatives are widely used as
antineoplastic agents in the therapy of cancer diseases.
For example, 6ꢀOꢀbenzylguanine 3 inhibits activity of
alkyltransferase, thus decreasing toxicity of antitumor
An alternative procedure which used polymethylhydrosiꢀ
loxane (PMHS)/ZnCl (see Ref. 26) and a palladium catꢀ
2
alyst did not increase the yields (<40%) of the target prodꢀ
2
8
ucts. The Pd(PPh ) ꢀcatalyzed removal of the Cꢀterminal
agents, whereas 2ꢀNꢀsubstituted purin analogs decrease
3
4
2
9
allyl protecting group in compounds 7 and 9 in the
presence of Nꢀethylaniline2 had proved to be the optimal
and gave monomers 8 and 10 in 77 and 87% yields, reꢀ
spectively. The structure and purity of the syntheꢀ
the activity of proteinekinases. In order to study biologiꢀ
cal activity of guanine derivatives 2 and 8, we examined
their inhibiting activity against enzyme topoisomerase I
(Topo I). 6ꢀOꢀBenzylguanine 3 was used as a comparison
7
1
13
3
sized compounds were confirmed by H and C NMR
spectroscopy (no signals for the H and C atoms of
the allyl group were observed in the spectra), elemenꢀ
sample, which is known to inhibit this enzyme (Fig. 3, a).
Topo I makes a supercoiled DNA (scDNA) to relax,
resulting in the change of its conformation, which reflects
2
8
a
b
scDNA Topo I 0.5
1
5
10 С/mol L–1
scDNA Topo I 0.5
1
5
10
20 0.1 С/mol L–1
Fig. 3. Electrophoretograms of the relaxation reaction products of supercoiled DNA (scDNA) upon treatment with topoisomerase I
(
Topo I) in the presence of compound 3 (a) and compounds 2 and 8 (b).

1
104
Russ.Chem.Bull., Int.Ed., Vol. 64, No. 5, May, 2015
Dezhenkov et al.
in the change of the electrophoretic lability of its forms
topoisomers). The presence of the inhibitor in the studied
ethanol or a mixture of molybdic acid—cerium(IV) sulfate monoꢀ
hydrate—concentrated sulfuric acid—water (2.5 : 1 : 3 : 94) with
subsequent heating.
(
range of concentrations hinders the scDNA relaxation,
which is reflected in the more rapid migration in the gel as
compared to the DNA relaxed with the tested enzyme.
Electrophoresis of the products of the reaction of scDNA
with Topo I in the presence/absence of compounds 2 and
Solvents were evaporated on a vacuum rotary evaporator
(
14 Torr). The drying of compounds was finalized using a high
vacuum of an oil pump (0.5 Torr).
ꢀOꢀBenzylguanine (3). Sodium hydride (1.2 g, 35.4 mmol,
5% suspension in mineral oil) was added to benzyl alcohol
6
5
8
(see Fig. 3, b) showed that, in contrast to heterocyclic
(50 mL) with vigorous stirring under argon. Then, 2ꢀaminoꢀ6ꢀ
chloropurin (2.0 g, 11.8 mmol) was added to the suspension
obtained. The reaction mixture was heated to 60 C and stirred
for 12 h. After cooling, glacial acetic acid (1.4 mL, 24.3 mmol)
was added to the suspension. The resulting mixture was extractꢀ
ed with 2 M solution of sodium hydroxide (5×5 mL). The organꢀ
ic phase was diluted with diethyl ether (100 mL) and again exꢀ
tracted with 2 M solution of sodium hydroxide (10×10 mL). The
aqueous layers were combined, washed with diethyl ether
base 3 (see Fig. 3, a), these compounds do not inhibit (in
the studied range of concentrations) the tested enzyme.
Apparently, this can be due to the fact that the open posiꢀ
tions N(2) and N(9) in guanine are necessary for the stronꢀ
ger binding with DNA, that hinders the Topo I activity.
In conclusion, the ꢀPNA guanine monomers 8 and
1
0 based on LꢀGlu were obtained by regioselective alkylaꢀ
tion. It was also found that the order of addition of reꢀ
agents is important in the acylation of the pseudopeptide
with bromoacetyl bromide. It was shown that the diproꢀ
tected guanine derivative 2 and the monoprotected monoꢀ
mer 8 did not inhibit Topo I activity. The use of diprotectꢀ
ed guanineꢀcontaining monomer 10 seems to be the optiꢀ
mal for further oligomerization on a polymeric support,
since it will be possible to carry out the capping step under
(
50 mL), glacial acetic acid was added to neutralize the solution
(pH 7). Then, the solution was allowed to stand for 12 h at 4 C.
A precipitate formed was filtered and washed with water. After
recrystallization (ethanol—water (1 : 1)), the crystals were dried
in vacuo. The yield was 1.76 g (62%), yellow crystals, m.p.
2
1
Ph); 6.29 (s, 2 H, NH Gua); 5.48 (s, 2 H, CH Ph(Bn)). C NMR
(
128.1, 126.5, 112.8, 66.7. Found (%): C, 56.0; H, 4.8; N, 27.5.
C H N O•H O. Calculated (%): C, 55.6; H, 5.1; N, 27.0. MS
(ESI), found: m/z 243.1033 [M + H] . C H N O. Calculated:
1
01—202 C. H NMR (DMSOꢀd , 300 MHz), : 12.25 (br.s,
6
H, N(9)HGua); 7.82 (s, 1 H, C(8)HGua); 7.56—7.21 (m, 5 H,
13
2
2
DMSOꢀd , 75 MHz), : 159.7, 159.6, 156.1, 138.5, 136.8,
6
the standard conditions (Ac O/TEA) and a qualitative
2
Kaiser test in the course of the solidꢀphase synthesis of
oligomers.
12
11
5
2
+
12
12
5
M = 242.1042.
ꢀOꢀBenzylꢀ2ꢀNꢀ(benzyloxycarbonyl)guanine (2). A suspenꢀ
6
Experimental
sion of NaH (1.74 g, 50.0 mmol, 55% in mineral oil) was washed
with hexane, suspended in anhydrous DMF (50 mL), cooled to
0 C, followed by an in portions addition of compound 3 (3.0 g,
12.45 mmol). The suspension was stirred for 15 min, followed by
a dropwise addition of CbzCl (3.5 mL, 24.9 mmol). The mixture
obtained was stirred for 12 h and neutralized by the addition of
glacial acetic acid (pH 7), DMF was evaporated on a rotary
evaporator. The residue was dissolved in CH Cl (50 mL) and
The following commercially available reactants were used in
the work: [Pd(PPh ) ] (Sigma—Aldrich, USA), isobutyl chloroꢀ
3
4
formate (Sigma—Aldrich, USA), 2ꢀaminoꢀ6ꢀchloropurin (Acros,
Belgium), Nꢀethylaniline (Sigma—Aldrich, USA), bromoacetyl
bromide (Sigma—Aldrich, USA); benzyl alcohol, acetic acid,
triethylamine, THF, DMF, methanol, ethanol, dichloromeꢀ
thane, ethyl acetate, hexane, diethyl ether, P O , KOH, NaOH,
2
2
washed with water (2×20 mL). The organic layer was dried with
Na SO , the solvent was evaporated. The product was isolated
2
5
LiAlH , sodium chloride, sodium sulfate, sodium hydride, calcium
4
2
4
hydride, barium oxide, potassium carbonate — all of reagent or
analytical grade (Russia). The following solvents were purified
before use: benzyl alcohol (distilled in high vacuo), dichloroꢀ
methane (distilled over P O ), DMF (distilled over phthalic anhyꢀ
by column chromatography (eluent methanol—CH Cl gradiꢀ
2 2
ent, 0—10%) and dried in vacuo. The yield was 2.05 g (44%),
white crystals, Rf 0.56 (CH Cl —methanol (8 : 1)), m.p.
2
2
1
207—209 C. H NMR (DMSOꢀd , 300 MHz), : 10.33 (s, 1 H,
2
5
6
dride in vacuo), THF (distilled over КOH and directly before
CbzNH—); 8.18 (s, 1 H, C(8)HGua); 7.77—7.03 (m, 10 H,
reactions over LiAlH ), triethylamine (distilled over KOH
2 Ph(Cbz, Bn); 5.58 (s, 2 H, CH Ph(Cbz)); 5.18 (s, 2 H,
CH Ph(Bn)). C NMR (DMSOꢀd , 75 MHz), : 159.3, 156.2,
2 6
4
2
13
and CaH ).
2
NMR spectra were recorded on Bruker DPXꢀ300 and Bruker
DPXꢀ600 pulse Fourierꢀtransform spectrometers (Germany) at
152.5, 152.1, 142.4, 137.1, 136.8, 129.4, 128.9, 128.7, 128.4,
128.3, 115.8, 67.9, 66.1. Found (%): C, 63.58; H, 4.77; N, 18.55.
C H N O . Calculated (%): C, 63.99; H, 4.56; N, 18.66.
2
5 C relative to the internal standard Me Si.
4
20 17
5
3
+
High resolution mass spectra were recorded on a miꢀ
MS (ESI), found: m/z 376.1388 [M + H] . C H N O . Calcuꢀ
20 18 5 3
crOTOFꢀQ II instrument (Bruker Daltonics GmbH, Germany).
Values of optical angular rotation were determined on an AAꢀ55
polarimeter (Optical Activity Ltd, Great Britain). Melting points
were measured using a Carl Zeiss microscope.
lated: M = 376.1410.
ꢀAllyl ꢀbenzyl N´ꢀbromoacetylꢀNꢀ[2ꢀ(tertꢀbutyloxycarbꢀ
onyl)aminoethyl]ꢀLꢀglutamate (5). Bromoacetyl bromide (1.30 mL,
14.60 mmol) was added to a solution of compound 4 (5.11 g,
12.17 mmol) in CH Cl (50 mL) at 0 C, followed by a dropwise
Column chromatography was carried out on Silica gel 60
2
2
(
0.040—0.063 mm) (Merck, Germany). Reaction progress was
addition of Et N (2.54 mL, 18.25 mmol) at such a rate that to
3
monitored by TLC on Silica gel 60 F254 plates (Merck, Germany).
Compounds on the plates were visualized either under UV light
keep the temperature of the mixture below 10 C, then the mixꢀ
ture was stirred for 1 h. The solvent was evaporated, the residue
was dissolved in ethyl acetate (50 mL) and sequentially washed
(
254 nm) or by spraying with a 0.5% solution of ninhydrin in

Regioselective alkylation of guanine derivatives
Russ.Chem.Bull., Int.Ed., Vol. 64, No. 5, May, 2015
1105
with water (2×40 mL) and saturated aqueous NaCl (2×30 mL).
followed by a dropwise addition of a solution of compound 5
(333 mg, 0.62 mmol) in DMF (5 mL). After 12 h, the solvent was
evaporated, the mixture obtained was dissolved in ethyl acetate
(50 mL), filtered through a layer of silica gel (10×10 mm), and
washed with water (2×20 mL) and a saturated aqueous NaCl
(2×20 mL). The organic phase was dried with Na SO , the solꢀ
The organic phase was dried with Na SO , the solvent was evapꢀ
2
4
orated. The product was isolated by column chromatography
eluent hexane—ethyl acetate (2 : 3)) and dried in vacuo. The
yield was 4.70 g (85%), an yellow oil, R 0.65 (hexane—ethyl
(
f
acetate (1 : 1)), []D2 –10.0 (c 1.0, MeOH). H NMR (CDCl ,
0
1
3
2
4
3
00 MHz), : 7.35—7.28 (m, 5 H, Ph(Bn)); 5.91—5.85 (m, 1 H,
vent was evaporated. The product was subjected to chromatoꢀ
graphy, using ethyl acetate as the eluent. The solvent was evapoꢀ
rated, the compound was dried in vacuo. The yield was 200 mg
CH —CH=CH ); 5.36—5.24 (m, 2 H, —CH —CH=CH );
5
2
2
2
2
.12, 5.05 (both d, 1 H each, CH Ph(Bn), J = 7.5 Hz); 4.62—4.48
2
2
0
(
(
(
3
m, 2 H, CH —CH=CH ); 3.91—3.82 (m, 2 H, N(C=O)CH —
AAL)); 3.75—3.71 (d, 1 H, CH(Glu), J = 10.8 Hz); 3.62—3.43
m, 1 H, BocHNCH CH ); 3.32—3.21 (m, 1 H, BocHNCH CH );
(41%), white crystals. R 0.71 (ethyl acetate), []
–1.7 (c 1.0,
2
2
2
f
D
1
MeOH), m.p. 47—49 C. H NMR (CDCl , 300 MHz), : 7.70
3
(s, 1 H, C(8)HGua); 7.50—7.30 (m, 15 H, 3 Ph(Cbz, Bn));
2
2
2
2
.07—2.94 (m, 2 H, BocHNCH CH ); 2.55—2.25 (m, 4 H,
5.82—5.75 (m, 1 H, CH CH=CH ); 5.61 (s, 1 H, BocNH);
2
2
2
2
t
13
CH (Glu), CH (Glu)); 1.39 (s, 9 H, Bu ). C NMR (CDCl3,
5.40—4.29 (m, 12 H, CH CH=CH , 3 CH Ph, N(C=O)CH —
2 2 2 2
2
2
7
1
2
5 MHz), : 172.1, 169.5, 167.0, 155.2, 134.9, 130.6, 127.8,
(AAL), CH CH=CH ); 3.99—3.82 (m, 1 H, CH(Glu));
3.70—3.62 (m, 1 H, BocHNCH CH ); 3.45—3.33 (m, 1 H,
2 2
2 2
27.6, 127.4, 118.5, 79.0, 65.9, 65.7, 65.4, 58.6, 49.2, 37.7, 29.2,
+
7.9, 25.5, 22.7. MS (ESI), found: m/z 563.1390 [M + Na] .
BocHNCH CH ); 3.30—3.18 (m, 2 H, BocNHCH CH );
2 2 2 2
t
C H N O BrNa. Calculated: M = 563.1369.
2.50—2.30 (m, 4 H, CH (Glu), CH (Glu)); 1.39 (s, 9 H, Bu ).
2
4
33
2
7
2 2
13

ꢀAllyl ꢀbenzyl Nꢀ[(6ꢀOꢀbenzyl)guaninꢀ9ꢀylacetyl]ꢀNꢀ[2ꢀ
C NMR (CDCl , 75 MHz), : 173.0, 170.2, 167.0, 161.1, 159.3,
3
(
tertꢀbutyloxycarbonyl)aminoethyl]ꢀLꢀglutamate (7). A predried
156.0, 154.1, 140.0, 136.5, 135.4, 131.4, 128.6, 119.3, 119.1, 80.1,
potassium carbonate (232 mg, 1.682 mmol) was added to a susꢀ
pension of compound 3 (406 mg, 1.682 mmol) in freshly distilled
DMF (5 mL) with mechanical stirring. The reaction mixture
was stirred for 15 min at ~20 C, followed by a dropwise addition
of a solution of compound 5 (455 mg, 0.841 mmol) in DMF
68.1, 66.9, 66.7, 66.3, 60.09, 48.9, 44.2, 38.8, 30.3, 28.7, 24.0.
+
MS (ESI), found: m/z 858.3411 [M + Na] . C H N O Na.
4
4
49
7
10
Calculated: M = 858.3439.
ꢀBenzyl Nꢀ[(6ꢀOꢀbenzyl)guaninꢀ9ꢀylacetyl]ꢀNꢀ[2ꢀ(tertꢀ
butyloxycarbonyl)aminoethyl]ꢀLꢀglutamate (8). NꢀEthylaniline
(0.32 mL, 2.06 mmol) and Pd(PPh ) (59 mg, 0.05 mmol) were
(
5 mL). After 12 h, the solvent was evaporated, the mixture
3
4
obtained was dissolved in ethyl acetate (50 mL), filtered through
a layer of silica gel (10×10 mm), and washed with water
sequentially added to a suspension of compound 7 (360 mg,
0.51 mmol) in THF (4 mL) under argon. After 1 h, the reaction
mixture was added dropwise to hexane (100 mL), this resulted in
the formation of a precipitate, which was filtered, washed with
hexane, and subjected to chromatography, using ethyl acetate as
the eluent. The solvent was evaporated, the compound was dried
(
2×20 mL) and saturated aqueous NaCl (2×20 mL). The organic
phase was dried with Na SO , the solvent was evaporated on
2
4
a rotary evaporator. The product was subjected to chromatoꢀ
graphy, using ethyl acetate as the eluent. The solvent was evapoꢀ
rated, the compound was dried in vacuo. The yield was 360 mg
in vacuo. The yield was 260 mg (77%), yellow crystals. R 0.50
f
2
0
(
[
61%), white crystals, R 0.63 (ethyl acetate), m.p. 51—53 C,
(ethyl acetate), m.p. 120—122 C, []
–1.7 (c 1.0, MeOH).
f
D
2
0
1
1
]D –1.7 (c 1.0, MeOH). H NMR (acetoneꢀd , 600 MHz),
H NMR (DMSOꢀd , 300 MHz), : 7.72 (d, 1 H, J = 6.5 Hz);
6
6

6
5
: 7.69 (s, 1 H, C(8)HGua); 7.58—7.24 (m, 10 H, 2 Ph(Bn));
7.55—7.25 (m, 10 H); 7.13—6.95 (m, 1 H); 6.39 (s, 2 H); 5.50
(s, 2 H); 5.16—4.89 (m, 4 H); 4.31—4.00 (m, 1 H); 3.52—2.96
(m, 4 H); 2.69—2.53 (m, 1 H); 2.42—2.19 (m, 2 H), 2.04—1.86
.23 (s, 1 H, BocNH); 5.95—5.84 (m, 1 H, CH —CH=CH );
2
2
.74 (s, 2 H, NH Gua), 5.56—5.52 (d, 2 H, CH Ph(Bn)Gua,
2
2
13
J = 7.2 Hz); 5.34, 5.17 (both d, 1 H each, CH —CH=CH ,
(m, 1 H), 1.41 (s, 9 H). C NMR (DMSOꢀd , 75 MHz), :
2
2
6
J = 17.2 Hz); 5.15 (d, 2 H, CH Ph(Bn), J = 3.2 Hz); 5.13 (d, 2 H,
172.8, 172.6, 167.7, 166.9, 160.1, 159.8, 155.7, 141.1, 137.0,
136.3, 131.7, 128.9, 128.8, 128.4, 128.1, 121.3, 113.3, 78.2, 77.6,
67.0, 65.6, 61.1, 59.8, 46.2, 44.6, 39.7, 31.4, 28.4, 25.0. MS
2
—
N(C=O)CH —(AAL), J = 14.8 Hz); 4.59—4.55 (m, 2 H,
2
CH —CH=CH ); 4.35—4.25 (t, 1 H, CH(Glu), J = 5.9 Hz);
2
2
–
3
.91—3.43 (m, 4 H, BocNHCH CH —); 2.59—2.54 (t, 2 H,
(ESI), found: m/z 660.2790 [M – H] . C H N O . Calculatꢀ
2
2
33 38
7
8
CH (Glu), J = 7.4 Hz); 2.45—2.37 (m, 2 H, CH (Glu)); 1.37
ed: M = 660.2782.
2
2
t
13
(
s, 9 H, Bu ). C NMR (acetoneꢀd , 150 MHz), : 174.0
ꢀBenzyl Nꢀ[(2ꢀNꢀbenzyloxycarbonylꢀ6ꢀOꢀbenzyl)guaninꢀ9ꢀ
ylacetyl]ꢀNꢀ[2ꢀ(tertꢀbutyloxycarbonyl)aminoethyl]ꢀLꢀglutamate
(10). NꢀEthylaniline (0.12 mL, 0.78 mmol) and Pd(PPh3)4
(23 mg, 0.02 mmol) were sequentially added to a suspension of
compound 9 (164 mg, 0.20 mmol) in THF (4 mL) under argon.
After 1 h, the reaction mixture was added dropwise to hexane
(100 mL), this resulted in the formation of a precipitate, which
was filtered, washed with hexane, and subjected to chromatogꢀ
raphy, using ethyl acetate as the eluent. The solvent was evapoꢀ
rated, the compound was dried in vacuo. The yield was 154 mg
(87%), yellow crystals, Rf 0.61 (CH Cl —MeOH—AcOH
6
(
C=O(GluOBn)); 171.5 (C=O(COOAll); 168.7 (N(C=O)ꢀ
CH —(AAL)); 162.3 (CH Ph(Bn)Gua); 161.4 (C(2)HGua);
1
(
1
6
6
2
2
57.6 (C=O(Boc)); 156.6 (C(4)Gua); 142.1 (C(8)HGua); 138.8
C(Ph)); 134.0 (CH —CH=CH ); 130.2—129.4 (CH(Ph));
2
2
19.0 (CH —CH=CH ); 115.9 (C(5)Gua); 80.1 (C—O(Boc));
2 2
8.8 (CH (Bn)Gua); 67.4 (CH (Bn)); 66.9 (CH —CH=CH );
2
2
2
2
1.5 (CH (Glu)); 49.7 (BocNHCH ); 45.1 (N(C=O)CH —
2
2
2
(
(
[
AAL)); 40.8 (BocNHCH CH ); 31.9 (CH (Glu)); 29.3
2 2 2
CH (Boc)); 25.8 (CH (Glu)). MS (ESI), found: m/z 702.3231
3
2
+
M + H] . C H N O . Calculated: M = 702.3251.
3
6
44
7
8
2
2
2
0

ꢀAllyl ꢀbenzyl Nꢀ[(2ꢀNꢀbenzyloxycarbonylꢀ6ꢀOꢀbenzyl)ꢀ
(9 : 1 : 0.1)), m.p. 116—118 C, []
–5.0 (c 1.0, CH OH).
3
D
1
guaninꢀ9ꢀylacetyl]ꢀNꢀ[2ꢀ(tertꢀbutyloxycarbonyl)aminoethyl]ꢀLꢀ
H NMR (DMSOꢀd , 300 MHz), : 10.35 (d, 1 H, J = 7.1 Hz);
6
glutamate (9). A predried potassium carbonate (111 mg,
7.72 (d, 1 H, J = 5.2 Hz); 7.65—7.12 (m, 15 H); 6.98—6.73 (m, 1 H);
5.59 (s, 2 H); 5.29—4.89 (m, 6 H); 4.31—3.99 (m, 1 H);
3.21—2.83 (m, 3 H); 2.71—2.51 (m, 1 H); 2.43—2.15 (m, 2 H);
0
0
.8 mmol) was added to a suspension of compound 2 (300 mg,
.8 mmol) in freshly distilled DMF (5 mL) with mechanical
13
stirring. The reaction mixture was stirred for 15 min at ~20 C,
2.04—1.80 (m, 2 H); 1.35 (s, 9 H). C NMR (DMSOꢀd6,

1
106
Russ.Chem.Bull., Int.Ed., Vol. 64, No. 5, May, 2015
Dezhenkov et al.
7
1
7
5 MHz), : 173.4, 173.2, 160.2, 160.0, 156.0, 152.3, 137.1,
36.6, 134.9, 131.9, 129.3, 128.7, 128.3, 128.2, 116.9, 110.0,
9. V. A. Efimov, O. G. Chakhmakhcheva, E. Wickstrom,
Nucleosides, Nucleotides and Nucleic Acids, 2005, 24, 1853.
10. V. A. Efimov, K. R. Birikh, D. B. Staroverov, S. A. Lukyaꢀ
nov, M. B. Tereshina, A. G. Zaraisky, O. G. Chakhmakhꢀ
cheva, Nucl. Acids Res., 2006, 34, 2247.
8.4, 78.0, 68.1, 66.1, 65.9, 65.8, 45.0, 38.7, 31.7, 28.9, 25.5. MS
–
(
ESI), found: m/z 794.3155 [M – H] . C H N O . Calculatꢀ
41
44
7
10
ed: M = 794.3150.
Biological trials. The ability of compounds 2, 3, and 8 to
inhibit activity of Topo I in vitro was determined in the relaxꢀ
ation reaction of scDNA. The reaction mixture (20 L) containꢀ
ing Topo I (1—2 activity units) (Promega, USA) was incubated
with supercoiled plasmid DNA pBR322 (205 ng) (Fermentas,
Lithuania) in the reaction buffer (35 mM TrisꢀHCl, pH 8.0,
11. R. Corradini, S. Sforza, T. Tedeschi, F. Totsingan, A. Maniꢀ
cardi, R. Marchelli, Curr. Top. Med. Chem., 2011, 11, 1535.
12. K. Usui, A. Okada, K. Kobayashi, N. Sugimoto, Org. Bioꢀ
mol. Chem., 2015, 13, 2022.
13. L. Christensen, R. Fitzpatrick, B. Gildea, K. H. Petersen,
H. F. Hansen, T. Koch, M. Egholm, O. Buchardt, P. E.
Nielsen, J. Coull, R. H. Berg, J. Pept. Sci., 1995, 3, 175.
14. A. Porcheddu, G. Giacomelli, I. Piredda, M. Carta, G. Niedꢀ
du, Eur. J. Org. Chem., 2008, 5786.
7
2 mM KCl; 5 mM MgCl containing 5 mM dithiothreitol; 2 mM
2
–
1
spermidine, 100 mg mL of bovine serum albumine) (all the
reagent from Sigma, USA) in the presence of 0.1% DMSO (conꢀ
trol of the solvent, the data are not shown) and compounds
under study at 37 C for 30 min. The reaction was stopped by the
addition of sodium dodecylsulfate (to 1%). After addition of proꢀ
teinase K (the final concentration 50 mg mL ), the reaction
mixture was incubated for 30—40 min at 37 C. DNAꢀtopoisoꢀ
mers were separated using electrophoresis in 1% agarose body
15. S. Pothukanuri, Z. Pianowski, N. Winssinger, Eur. J. Org.
Chem., 2008, 3141.
16. K. L. Dueholm, M. Egholm, C. Behrens, L. Christensen,
H. F. Hansen, T. Vulpius, K. H. Petersen, R. H. Berg, P. E.
Nielsen, O. Buchardt, J. Org. Chem., 1994, 59, 5767.
17. P. C. Meltzer, A. Y. Liang, P. Matsudaira, J. Org. Chem.,
1994, 60, 4305.
18. A. V. Dezhenkov, M. V. Tankevich, E. D. Nikolskaya, I. P.
Smirnov, G. E. Pozmogova, V. I. Shvets, Yu. G. Kirillova,
Mendeleev Commun., 2015, 25, 47.
19. S. A. Thomson, J. A. Josey, R. Cadilla, M. D. Gaul, C. F.
Hassman, M. J. Luzzio, A. J. Pipe, K. L. Reed, D. J. Ricca,
R. W. Wiethe, S. A. Noble, Tetrahedron, 1995, 51, 6179.
20. Z. Timár, L. Kovács, G. Kovács, Z. Schmél, J. Chem. Soc.,
Perkin Trans., 2000, 1, 19.
–
1
(
3
3 h, 60 V) in a buffer containing 40 mM Trisꢀbase, 1 mM EDTA,
0 mM glacial acetic acid, after the addition (1 : 10) of the buffer
for the deposition (0.25% bromophenol blue, 50% glycerol). The
gels after electrophoresis were colored with ethidium bromide
and visualized under UV light.
This work was financially supported by the Ministry
of Education and Science of the Russian Federation
(
Grant 4.128.2014/K) and the Russian Science Foundaꢀ
2
1. Haaima, A. Lohse, O. Buchardt, P. E. Nielsen, Angew.
Chem., Int. Ed., 1996, 98, 1939.
tion (Grant 14ꢀ25ꢀ00013).
2
2
2. C. Barth, O. Seitz, H. Kunz, Z. Naturforsch., 2004, 59b, 802.
3. N. P. Boyarskaya, D. I. Prokhorov, Y. G. Kirillova, E. N.
Zvonkova, V. I. Shvets, Tetrahedron Lett., 2005, 46, 7359.
References
1
2
. G. Ferenc, P. Pádár, J. Szolomájer, L. Kovács, Curr. Org.
Chem., 2009, 13, 1085.
. P. Pourquier, J. L. Waltman, Y. Urasaki, N. A. Loktionova,
A. E. Pegg, J. L. Nitiss, Y. Pommier, Cancer Res., 2001,
24. A. V. Baranov, N. S. Tsvid, V. I. Luk´yanchenko, D. I.
Prokhorov, Yu. G. Kirillova, V. I. Shvets, Vestn. MITKhT
[Bull. Moscow Inst. Fine Chem. Technol.], 2007, 2, No. 5, 28
(in Russian).
6
1, 53.
25. S. FriedrichꢀBochnitschek, H. Waldmann, H. Kunz, J. Org.
Chem., 1989, 54, 751.
26. S. Chandrasekhar, Ch. R. Reddy, R. J. Rao, Tetrahedron,
2001, 57, 3435.
3
. L. H. Jensen, A. V. Thougaard, M. Grauslund, B. Søkilde,
E. V. Carstensen, H. K. Dvinge, D. A. Scudiero, P. B. Jensꢀ
en, R. H. Shoemaker, M. Sehested, Cancer Res., 2005,
6
5, 7470.
27. T. Shiraishi, R. Hamzavi, P. E. Nielsen, Nucl. Acids Res.,
2008, 36, 4424.
4
. P. E. Nielsen, M. Egholm, R. H. Berg, O. Buchardt, Sciꢀ
ence, 1991, 254, 1497.
28. M. E. Dolan, A. E. Pegg, Clinical Cancer Res., 1997, 3, 837.
29. N. S. Gray, L. Wodicka, A.ꢀM. W. H. Thunnissen, T. C.
Norman, S. Kwon, F. H. Espinoza, D. O. Morgan, G. Barꢀ
nes, S. LeClerc, L. Meijer, S.ꢀH. Kim, D. J. Lockhart, P. G.
Schultz, Science, 1998, 281, 533.
5
6
. P. E. Nielsen, Chem. Biodiversity, 2010, 7, 786.
. B. Hyrup, M. Egholm, P. E. Nielsen, P. Wittung, B. Norde´n,
O. Buchardt, J. Am. Chem. Soc., 1994, 116, 7964.
. P. Wittung, P. E. Nielsen, O. Buchardt, M. Egholm,
B. Norde´n, Nature, 1994, 368, 561.
7
8
. V. Demidov, V. N. Potaman, M. D. FrankꢀKamenetskii,
O. Buchardt, M. Egholm, P. E. Nielsen, Biochem. Pharmacol.,
Received March 12, 2015;
1
994, 48, 1309.
in revised form April 10, 2015