The search of novel inhibitors of HIV-1 Integrase among 5-(4-Halogenophenyl)-5-oxopentyl derivatives of nucleic bases

Article abstract of DOI:10.1134/S1068162014050094

New nucleic base derivatives were obtained by alkylation of uracil, thymine, cytosine, adenine, 6-chloropurine, and 2-Amino-6-chloropurine with 5-chloro-1-(4-halogenophenyl)-1-pentanones, and their physical and chemical properties were studied. The influe

Full text of DOI:10.1134/S1068162014050094

ISSN 1068ꢀ1620, Russian Journal of Bioorganic Chemistry, 2014, Vol. 40, No. 5, pp. 532–540. © Pleiades Publishing, Ltd., 2014.
Original Russian Text © V.V. Komissarov, E.S. Knyazhanskaya, A.V. Atrokhova, M.B. Gottikh, A.M. Kritzyn, 2014, published in Bioorganicheskaya Khimiya, 2014, Vol. 40, No. 5,
pp. 578–587.
The Search of Novel Inhibitors of HIVꢀ1 Integrase
among 5ꢀ(4ꢀHalogenophenyl)ꢀ5ꢀoxopentyl
Derivatives of Nucleic Bases
V. V. Komissarova, E. S. Knyazhanskayab, A. V. Atrokhovab,
1
M. B. Gottikhb, and A. M. Kritzyna,
a Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow, 119991 Russia
b Lomonosov Moscow State University, Chemistry Department and Belozersky Institute
of Physical and Chemical Biology, Moscow, 119991 Russia
Received December 19, 2013; in final form, January 24, 2014
Abstract—New nucleic base derivatives were obtained by alkylation of uracil, thymine, cytosine, adenine,
6ꢀchloropurine, and 2ꢀaminoꢀ6ꢀchloropurine with 5ꢀchloroꢀ1ꢀ(4ꢀhalogenophenyl)ꢀ1ꢀpentanones, and
their physical and chemical properties were studied. The influence of the compounds synthesized on the
HIVꢀ1 integrase activity was studied.
Keywords: alkylation, HIVꢀ1integrase, nucleosides, polymethylene analogues
DOI: 10.1134/S1068162014050094
1
INTRODUCTION
enzyme is involved in angiogenesis and apoptosis [5].
Compounds (II), whose synthesis and properties were
described earlier [6], inhibited both E. coli thymidine
and uridine phosphorylases with an efficacy close to
that of the known inhibitors of these enzymes.
A series of drugs used for the therapy of various
human diseases has been developed on the basis of
nonglycoside nucleoside analogues. Nonglycoside
guanine analogues applied for the treatment of herpes
infections can serve an example. Particularly, 2HMꢀ
HBG is effective against Varicella zoster virus, penciꢀ
clovir, against type 1 and 2 Herpes simplex and V. zoster
viruses, EpsteinꢀBarr virus, and cytomegalovirus [1].
Similar adenine and hypoxantine derivatives with a
hydrocarbon chain of nine atoms in length were cytoꢀ
toxic toward
К562 and HCT116 tumor cells [7].
Earlier, we described
idines and purines with a varied length of the polymeꢀ
ω
ꢀoxoꢀ ꢀphenylalkyl pyrimꢀ
ω
Heterocyclic base derivatives are used in the treatꢀ
ment of HIV infection: along with standard nucleoꢀ
sideꢀbased drugs (azidothymidine, dideoxyinosine, thylene chain [8]. It was shown in [9, 10] that polymꢀ
dideoxycytidine, emtricitabine, and tenofovir) bound
in the HIVꢀ1 reverse transcriptase active site, nonnuꢀ
cleoside inhibitors of the HEPT family [2] bound in
the hydrophobic pocket adjacent to the enzyme active
site [3] are intensely studied.
ethylene derivatives of heterocyclic bases bearing
hydroxyl, alkoxycarbonyl, or carboxyl groups in the
position can be successfully used for studies of such
significant enzymes as human topoisomerase I and/or
HIVꢀ1 reverse transcriptase. Another key HIVꢀ1
ωꢀ
In addition, nonglycoside derivatives of heterocyꢀ enzyme is integrase, which provides incorporation of
clic bases are convenient models for studying the
mechanisms of action of various enzymes involved in
the nucleic acid exchange. Among these compounds,
derivatives of both natural and modified heterocyclic
viral DNA into the genome of the infected cell [11].
The first integrase inhibitor approved for the therꢀ
apy of HIV infection was raltegravir [12]. The critical
element of its structure is derived from the pyrimidine
cycle and is responsible for the binding of metal ions in
bases bearing various functional groups in the
ω
ꢀposiꢀ
tion of the hydrocarbon chain attract much attention.
For example, some compounds of a general structural the enzyme active site [13]. The other important strucꢀ
formula ( ) were effective inhibitors of E. coli thymiꢀ
tural element of raltegravir is a pꢀfluorophenyl substitꢀ
I
dine phosphorylase [4]. The analogous human
uent [14]. It is noteworthy that the halogenated aroꢀ
matic residue is a part of the most effective HIVꢀ1
integrase inhibitors [15]. Normally, a fluorine atom is
used as a halogen, but some inhibitors (elvitegravir,
MKꢀ2048) also contain a chlorine atom.
Abbreviations: DBU, 1,8ꢀdiazabicyclo[5.4.0]undecꢀ7ꢀene;
HIVꢀ1, human immunodeficiency virus type 1.
Corresponding author: phone: +7 (499) 135ꢀ14ꢀ05; eꢀmail:
1
amk@eimb.ru.
532

THE SEARCH OF NOVEL INHIBITORS
533
O
O
N
N
N
N
NH
NH2
NH
NH2
HO
N
N
OH
HO
OH
2HM_HBG
Penciclovir
NH2
NH2
N
F
N
N
O
OH
P
N
N
N
O
O
HO
S
O
OH
Tenofovir
(Viread)
Emtricitabin
(Emtriva)
Truvada
F
O
N
O
O
HO
Cl
OH
H
N
O
O
N
O
N
HO
O
N
N
F
Raltegravir
Elvitegravir
Formulas 1. Some inhibitors of HIVꢀ1 reverse transcriptase and integrase
.
ing a
p
ꢀhalogenated phenone fragment in the
ω
ꢀposiꢀ
O
N
O
N
tion can affect the activity of HIVꢀ1 integrase.
R
HN
HN
RESULTS AND DISCUSSION
O
O
OH
O−
n
P
+NH4
In this work we set a task to prepare comꢀ
pounds (IVa–c)–(IXa–c), in which the heterocyclic
base
with a chain of four methylene units (Scheme). We
obtained six series of compounds (IVa–c)–(IXa–c
based on six heterocyclic bases, thymine, uracil,
cytosine, adenine, guanine, and hypoxanthine. Since
the role of the halogen atom for the inhibitory properꢀ
ties toward integrase is not clear, we prepared three
compounds in each series containing atoms of differꢀ
n
O
OH
O
B was linked to a pꢀhalogenophenone fragment
(
I
)
( )
II
R = H, CH3
= 6, 7
n
= 6, 7
)
n
Formulas 2. Some inhibitors of thymidine
and uridine phosphorylases.
On the basis of the above facts, we believe it reasonꢀ
able to study whether nucleic base derivatives containꢀ ent halogens in the pꢀposition of the aromatic ring.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 40
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534
KOMISSAROV et al.
O
O
AlCl
→
3
20°C
+
0
Cl
Cl
Cl
O
X
X
(
III)
a
b
c
: X = F, yield 85%
: X = Cl, yield 18%
: X = Br, yield 36%
(
IVа)–(IVc): B = Ura
Vа)–(Vc): B = Try
B
O
(
(
(
(
(
*
VIа)–(VIc): B = Cyt
VIIа)–(VIIc): B = Ade
VIIIа)–(VIIIc): B = Hyp
IXа)–(IXc): B = Gua
BH
Cl
+
(
III)
X
X
a
b
c
: X = F;
: X = Cl;
: X = Br
a
: X = F; b: X = Cl; c: X = Br
Scheme 1. A scheme of synthesis of some heterocyclic base derivatives with a terminal
ꢀhalogenophenyl) oxo group
(
p
.
The compounds were synthesized as shown in the thylene chains, and those of aryl residues (see the
1
13
scheme. Compounds (III) served starting alkylating Experimental section). H and C NMR spectra of
agents. They were obtained by the Friedel–Crafts fluorobenzene derivatives (IVa)–(IXa) are characterꢀ
reaction by acylation of fluoroꢀ, chloroꢀ, or broꢀ ized by the presence of additional resonances of the
mobenzene with ꢀchloropentanoyl chloride similar aromatic radical due to spinꢀspin interactions with the
δ
the procedure described in [16]. Aluminum chloride fluorine atom.
was used as a catalyst. Unlike benzene, acylation of
NMR spectra of the compounds synthesized
halobenzenes proceeded more slowly and was accomꢀ
panied by considerable resinification of the reaction
mixture [17], which resulted in a substantial reduction
of product yields.
Heterocyclic bases were alkylated using the previꢀ
ously developed method of heating the mixture of a
pyrimidine or purine base, compound (III) and DBU
in DMF (method A). For the preparation of cytidine
derivatives we used alkylation of its Na salt in DMF
(method B).
allowed us to identify the substituent positions in the
heterocyclic bases due to significant differences in the
sets of chemical shifts of the base atoms and atoms of
the first methylene unit of the side chain for various
position isomers. Critical differences in the spectra of
3ꢀ and 9ꢀadenine substituents were described by Holy
et al. [18]. For 7ꢀ and 9ꢀsubstituted hypoxanthine and
guanine the differences were also essential. Due to
these differences the position of the purine side chain
once determined by the HMBC method (Heteronuꢀ
clear Multiple Bond Correlation) can be used for
unambiguous assignment of the structure of isomeric
purine derivatives using their oneꢀdimensional NMR
spectra.
For the studies of the capacity of the compounds to
inhibit HIVꢀ1 integrase we took 1ꢀsubstituted pirimiꢀ
dine isomers and 9ꢀsubstituted purine isomers with the
highest solubility. The inhibitory properties of these
compounds were analyzed using the standard protoꢀ
cols [19]. Integrase catalyzes two reactions of the virus
replication: 3'ꢀterminal processing resulting in the
removal of GT nucleotides from both viral DNA
3'ꢀends and strand transfer resulting in the incorporaꢀ
tion of the viral DNA into cell DNA. The capacity of
Final products were isolated by column chromaꢀ
tography on silica gel. Their structures were confirmed
by mass and NMR spectra.
Alkylation of uracil and thymine resulted in 1ꢀsubꢀ
stituted derivatives together with a small amount of
1,3ꢀbisꢀsubstituted pyrimidines. The major product of
adenine alkylation was its 9ꢀsubstituted isomer,
although its 3ꢀsubstituted derivatives were also isolated
in insignificant yields. Alkylation of hypoxanthine
proceeded typically to give the target 9ꢀsubstituted
derivative together with the corresponding 7ꢀderivaꢀ
tive. In the case of compound (VIIIa), the 7ꢀisomer
was separated by multistep crystallization from ethyl
acetate. Since this procedure is rather labor consumꢀ
ing, for the preparation of compounds (VIIIb) and
p
ꢀhalogenated phenone derivatives of heterocyclic
compounds to inhibit the integrase catalytic activity
was studied in both reactions with a recombinant proꢀ
tein and DNA U5B/U5A and U5Bꢀ2/U5A duplexes
corresponding to the terminal region of the U5 fragꢀ
ment of the long DNA repeat before and after the
cleavage of the GT nucleotide. Accordingly, an inteꢀ
grase substrate in the reaction of 3'ꢀterminal processꢀ
(
VIIIc) we used alkylation of 6ꢀchloropurine followed
by hydrolysis (see the Experimental section).
In the NMR spectra of the compounds synthesized
there are three resonance groups clearly observed: resꢀ
onances of heterocyclic bases, resonances of polymeꢀ
* For the conditions, see the Experimental section.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 40
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THE SEARCH OF NOVEL INHIBITORS
535
(а)
(b)
3'ꢀprocessing
21 bp
19 bp
Integrase
21ꢀbp DNA
19ꢀbp product
Mg2+
32Pꢀ19
32P
32P
Substrate of 3'ꢀprocessing (U5)
32P
1 2 3 4 5 6 7 8 9 10
(c)
Strand transfer
(d)
32P
Products of
strand transfer
Mg2+
32P
32P
Substrate of strand transfer (U5ꢀ2)
19ꢀbp DNA
11 12 13 14 15 16 17 18 19 20
Fig. 1. The influence of compound (IXb) on the HIVꢀ1 integrase activity in the 3'ꢀprocessing and strand transfer reactions. (a) A
scheme of the 3'ꢀprocessing catalyzed by HIVꢀ1 integrase in vitro. (b) A representative radioautogram of the inhibition of the 3'ꢀ
processing reaction in the presence of increasing concentrations of compound (IX) in PAG under denaturing conditions (7 M urea).
Lane 1, without inhibitors; lanes 2–10, increasing inhibitor concentrations shown in the table. (c) A scheme of the strand transfer
reaction catalyzed by HIVꢀ1 integrase in vitro. (d) A representative radioautogram of the inhibition of the strand transfer reaction
in the presence of increasing concentrations of compound (IX) in PAG under denaturing conditions (7 M urea). Lane 11, without
inhibitors; lanes 12–20, increasing inhibitor concentrations shown in the table.
ing was the U5B/U5A duplex and in the reaction of
strand transfer, the U5Bꢀ2/U5A duplex. It is noteworꢀ
thy that integrase interacts with cell DNA without
regard to its primary structure. Therefore, in the reacꢀ
tion of strand transfer the enzyme can incorporate the
U5Bꢀ2/U5A substrate into any DNA including the
substrate itself. The schemes of 3'ꢀterminal processing
and strand transfer with these substrates are shown in
Fig. 1a and 1b.
EXPERIMENTAL
In this work we used adenine, hypoxantine, guaꢀ
nine, uracil, thymine, cytosine, and 6ꢀchloropurin
purchased from Sigma (United States); DBU, sodium
hydride 60% suspension in mineral oil, from Fluka
(Switzerland); CDCl3, DMSOꢀd6, fluorobenzene,
chlorobenzene, bromobenzene, and aluminum chloꢀ
ride, from Acros Organics (Belgium). 5ꢀChloropenꢀ
tanoyl chloride was obtained as described in [16]. The
solvents were purified using standard procedures [22].
TLC was carried out on Kieselgel 60 F254 plates
(Merck, Germany) with the following systems: 19 : 1
chloroform–ethanol (A), 18 : 2 (B), 17 : 3 (C), and
19.5 : 0.5 (D). The compounds were detected by UV at
254 nm. Column chromatography was performed on
silica gel 60 (0.040–0.063 mm) (Merck, Germany).
None of the 1ꢀsubstituted derivatives of the pyrimꢀ
idine series inhibited the integrase catalytic activity to
a concentration of 1 mM in any of the reactions. Simꢀ
ilar adenine and hypoxanthine derivatives did not disꢀ
play inhibitory properties within the concentration
range tested (10–1500
M) either. At the same time,
µ
the guanine Clꢀderivative (IXb) inhibited both of the
integraseꢀcatalyzed reactions (Fig. 1b and 1d, table),
the inhibitory efficacy for the reaction of strand transꢀ
fer being somewhat higher than that of the 3'ꢀprocessꢀ
ing: the (IXb) concentration, at which the reaction effiꢀ
Mass spectra were registered on an MSꢀ30 massꢀ
spectrometer (Kratos, Japan) using electron impact as
the ionization method. NMR spectra were registered
on an АМХIIIꢀ400 Bruker spectrometer (Germany)
ciency was reduced by 50% (IC50) was 516 107
µ
M for
(
δ
, ppm,
400 MHz for 1H and 100 MHz for 13C spectra at 300 K
in CDCl3 and DMSOꢀd6
K, Hz) with the working frequency of
the 3'ꢀprocessing reaction and 284 59
µ
M for the
strand transfer.
.
Recombinant HIVꢀ1 integrase was isolated from
the E. coli producer strain Rosetta and purified withꢀ
out a detergent as described in [23].
As a reference compound we used raltegravir, an
inhibitor of the strand transfer reaction (IC50 0.5
for the 3'ꢀprocessing reaction and 0.01 M for the
µ
M
µ
strand transfer [20]). Compound (IXb) also inhibited
Oligodeoxyribonucleotides U5B (5'ꢀGTGTGꢀ
GAAAATCTCTAGCAGTꢀ3'), U5Bꢀ2 (5'ꢀGTGTGꢀ
GAAAATCTCTAGCAꢀ3'), and U5A (5'ꢀACTꢀ
GCTAGAGATTTTCCACACꢀ3') were synthesized by
the phosphoamidite procedure on an automatic ABI
3400 DNA synthesizer (Applied Biosystems, United
this reaction more effectively. It is noteworthy that
analogues of this compound containing fluorine (IXa
)
and bromine (IXc) atoms were ineffective as integrase
inhibitors. However, in the mechanism of integrase
inhibition the role of a halogen atom, which is borne
by nearly all inhibitors of the strand transfer [15], is States) using a standard protocol and commercial
still unclear [21]. reagents (Glen Research, United States).
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 40
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536
KOMISSAROV et al.
Yields of the 3'ꢀprocessing and strand transfer reactions at varied concentrations of compound (IXb). The reaction effecꢀ
tiveness is given relative to the reaction without inhibitors
Inhibition of 3'ꢀprocessing
Inhibition of strand transfer
concentration
concentration
of compound
lane number
on figure b
reaction
effectiveness, %
lane number
on figure d
reaction
effectiveness, %
of compound (IXb),
µ
M
(IXb),
µ
M
2
3
4
5
6
7
8
9
200
350
81.27 14.3
70.02 14
52.76 12.1
34.2 6.8
17.76 3.7
9.4 1.2
11
12
13
14
15
16
17
18
10
98.04 16.6
80.88 15.3
60.19 13.3
50.98 10
100
200
300
400
500
750
1000
500
750
900
39.22 8.5
26.08 5.2
1100
1300
1500
3.27 0.5
2.81 0.52
22.94
1
17.65 2.9
1
Preparation of 5ꢀhalogenoꢀ1ꢀ(4ꢀfluorophenyl)penꢀ 275.6 (C11H12ClBrO). H NMR (CDCl3): 1.75–1.95
2C 2CH2Cl); 2.96 (2 H, t, 6.8, COC 2);
6.1, C 2Cl); 7.59 (2 H, d, Jo
ꢀH, PhBr), 7.80 (2 H, d, Jm
tanoneꢀ1 (IIIa)–(IIIc). Anhydrous aluminum chloꢀ (4 H, m,
ride (6.7 g, 50 mmol) was added in portions under stirꢀ 3.57 (2 H, t,
ring to a solution of 5ꢀchloropentanoyl chloride (43.3 g,
C
H
H
J
H
J
H
ꢀH 8.6,
ꢀꢀHH,,mPhBr).
o
8.6,
m
50 mmol) in the proper halobenzene (20 mL) cooled 13C NMR (CDCl3): 21.52 ꢀH(,Co H2CH2CO); 32.06
ꢀH
to
0°C. The mixture was stirred at
0°C
for 30 min and
(CH2CH2Cl); 37.61 (COCH2); 44.68 (CH2Cl);
then at room temperature for 1 h. The solution was [128.32 (C4); 129.62 (2 C, C2, and C6); 132.02 (2 C,
poured onto ice (150 g), the organic layer was sepaꢀ C3, and C5); 135.69 (C1)] (С6H4Br); 198.55 (CO).
rated, and the aqueous layer was extracted with methꢀ
Alkylation of heterocyclic bases using DBU (method A).
An alkylating agent (5.5 mmol) and DBU (0.78 mL,
5.5 mmol) were added to a suspension of a heterocyꢀ
clic base or its protected derivative (5 mmol) in anhyꢀ
drous DMF (10 mL). The mixture was heated at 80–
ylene chloride (
3
×
15 mL).The extracts were united,
washed with saturated sodium hydrocarbonate (20 mL),
dried with anhydrous sodium sulfate, and the solvent
was evaporated. The residue was chromatographed on
a silica gel column (
5 10 cm, 70 g) eluting with methꢀ
×
100°C for 20 h (TLC control). The reaction mixture
ylene chloride.
was cooled and evaporated in vacuum. The residue was
5ꢀChloroꢀ1ꢀ(4ꢀfluorophenyl)pentanoneꢀ1 (IIIa). suspended in a minimal volume of methylene chloride
The yield 85%; bp 135–137 (1 mm Hg). Mass: m/z and chromatographed on a silica gel column (70 g,
214.7 [M+
. Calculated 214.7 (C11H12ClFO). 10 cm) eluting in a gradient of ethanol in methylene
1H NMR (CDCl3): 1.65–1.85 (4 H, m,
2C 2CH2Cl); chloride ( 20%). The target fractions were evapoꢀ
2.86 (2 H, t, 6.8, COC 2); 3.45 (2 H, t, 6.2, C 2Cl); rated and the residue was recrystallized.
6.99 (2 H, dd, Jm ꢀH 8.7, JmꢀH, F 8.7, ꢀH, PhF); 7.86
ꢀH, PhF). 13C NMR
H2CH2CO); 31.95 ( H2CH2Cl);
H2); 44.65 ( H2Cl); [115.50 (2 C, d, J3ꢀC, F
°
M
C
5
×
]
C
H
J
H
0
→
J
H
H
m
ꢀH,
o
Alkylation of cytosine sodium salt (method B).
A
(2 H, dd, Jo
ꢀH 8.7, JoꢀH, F 5.3, o
ꢀH,
m
suspension of sodium hydride (0.21 g, 5.2 mmol) in
mineral oil was added to a suspension of cytosine (0.55 g,
5 mmol) in anhydrous DMF (10 mL). The reaction
(CDCl3): 21.38 (
37.34 (CO
C
C
C
C
22.1, C3 and C5); 130.54 (2 C, d, J2 C, F 9.1, C2 and
C6); 133.30 (C1); 165.56 (1 C, d, J4ꢀ C, F 254.1, C4)]
mixture was heated at 80–100°C for 20 h (TLC conꢀ
trol). The reaction mixture was cooled and evaporated
in vacuum. The residue was suspended in a minimal
ꢀ
(С6H4Cl); 198.25 (CO).
5ꢀChloroꢀ1ꢀ(4ꢀchlorophenyl)pentanoneꢀ1 (IIIb). volume of methylene chloride and chromatographed
The yield 49%. Mass: . Calculated on a silica gel column (70 g, 10 cm) eluting in a
275.6 [M+
231.1 (C11H12Cl2O). 1H NMR (CDCl3): 1.80–1.95 (4 gradient of ethanol in methylene chloride (
20%).
H, m, 2C 2CH2Cl); 2.97 (2 H, t, 6.8, COC 2); The target fractions were evaporated and the residue
3.57 (2 H, t, 6.2, C 2Cl); 7.42 (2 H, d, Jm ꢀH 8.7, was recrystallized.
ꢀH, PhCl); 7.88 (2 H, d, Jo
13C NMR (CDCl3): 21.43ꢀH(,CmHꢀH2CH2CO); 31.97
H2CH2Cl); 37.52 (CO H2); 44.59 ( H2Cl)
[128.19 (2 C, C3, and C5); 129.40 (2 C, C2, and C6);
135.19 (C1); 139.50 (C4)] ( 6H4Cl); 198.55 ( ).
5ꢀChloroꢀ1ꢀ(4ꢀbromophenyl)pentanoneꢀ1 (IIIc). m, H2' and H3'); 2.99 (2 H, t,
The yield 39%. Mass: 275.6 [ 6.7, H 1'); 5.68 (1 H, d, 5, 6 7.8, H5); 7.10 (2 H, dd,
+]. Calculated
m/z
]
M
5
×
0
→
CH
H
J
J
H
H
ꢀH,
m
8.7, o
ꢀH, oPhCl).
1ꢀ[5ꢀ(4ꢀFluorophenyl)ꢀ5ꢀoxopentyl] uracil (IVa)
was obtained by method
A
in a yield of 28%;
(ethyl acetate). Mass:
+ H+]. Calculated
290.3
(C15H15FN2O3). H NMR (CDCl3): 1.68–1.83 (4 H,
6.4, H4'); 3.76 (2 H, t,
Rf 0.25
(C
C
C
;
(19.5 : 0.5); mp 101–102
°
C
m/z
290.3 [M
+], 291.3 [
M
M
С
CO
1
J
m/z
M
M
J
J
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 40
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THE SEARCH OF NOVEL INHIBITORS
537
Jmꢀ ꢀH 8.6, JmꢀH, F 8.6, mꢀH, PhF); 7.18 (1 H, d, J6, 5 (C5); 140.71 (C8); 149.51 (C4); 152.23 (C2); 155.85
7.8,H,H6); 7.95 (2 H, dd, Jo
ꢀH 8.9, JoꢀH, F 5.4, oꢀH, (C6); 198.15 (C5').
o
ꢀH,
m
PhF), 9.48 (1 H, br s, H3). 13C NMR (CDCl3): 20.74
(C3'); 28.54 (C2'); 37.54 (C4'); 48.55 (C1'); 102.33
In this reaction3ꢀ[5ꢀ(4ꢀfluorophenyl)ꢀ5ꢀoxopenꢀ
tyl]adenine, Rf 0.09 (18 : 2), was obtained in a yield of
6% as a side product.
(C5); [115.79 (2 C, d, JC, F 22.0); 130.69 (2 C, d, JC, F
8.9); 133.27 (1 C, d, JC, F 3.1); 165.84 (1 C, d, J4ꢀC, F
254.9)] (C6H4F); 144.39 (C6); 151.06 (C2); 163.87
(C4); 197.86 (C5').
9ꢀ[5ꢀ(4ꢀFluorophenyl)ꢀ5ꢀoxopentyl] hypoxantine
(VIIIa) was obtained by method
0.40 (18 : 2); mp 202–203
+], 315.3 [ + H+]. Calculated
pentyl]uracil, Rf 0.61 (19.5 : 0.5), oil, was obtained in (C16H15FN4O2). 1H NMR (DMSOꢀ
6): 1.56 (2 H, m,
H2'); 1.86 (2 H, m, H3'); 3.05 (2 H, t, 7.0, H4'); 4.18
(2 H, t, 6.8, H1'); 7.32 (2 H, dd, Jm ꢀH 8.6, JmꢀH, F 8.6,
ꢀH, PhF); 8.01 (3 H, m,
(1 H, s, H2); 12.22 (1 H, br s, H1). 13C NMR
(DMSOꢀ 6): 20.52 (C3'); 28.96 (C2'); 37.01 (C4');
43.06 (C1'); [115.56 (2 C, d, C, F 22.0); 130.74 (2 C, d,
C, F 9.4); 133.32 (1 C, d, C, F 2.7); 164.89 (1 C, d, J4ꢀC, F
A
in a yield of 21%; Rf
(ethyl acetate). Mass:
314.3
°
C
In this reaction 1,3ꢀbis[5ꢀ(4ꢀfluorophenyl)ꢀ5ꢀoxoꢀ m/z 314.3 [
M
M
M
d
a yield of 15% as a side product.
J
J
m
o
ꢀH, PhoF and H8); 8.10
ꢀH,
1ꢀ[5ꢀ(4ꢀFluorophenyl)ꢀ5ꢀoxopentyl] thymine (Va)
was obtained by method
A
in a yield of 32%;
(ethyl acetate). Mass:
+ H+]. Calculated
304.3
(C16H17FN2O3). H NMR (CDCl3): 1.68–1.82 (4 H,
m, H2' and H3'); 1.89 (3 H, s, 5ꢀCH3 ; 2.98 (2 H, t,
6.4, H4'); 3.72 (2 H, t, 6.7, H1'); 6.99 (1 H, s, H6);
7.09 (2 H, dd, Jm ꢀH 8.7, JmꢀH, F 8.7, ꢀH, PhF); 7.95
ꢀH, PhF), 9.45 (1 H,
br s, H3). C NMR (CDCl3): 12.21 (5ꢀ H3), 20.69
(C3'); 28.45 (C2'); 37.46 (C4'); 48.09 (C1'); 110.72
(C5); [115.66 (2 C, d, C, F 22.0); 130.58 (2 C, d, JC, F
9.4); 133.20 (1 C, d, C, F 2.7); 165.71 (1 C, d, J4
Rf 0.33
(19.5 : 0.5); mp 121–122
°
C
m/z
d
304.3 [
M
+], 305.3 [
M
M
J
1
J
J
)
251.3)] (C6H4F); 123.91 (C5); 140.21 (C8); 145.31
(C2); 148.33 (C4); 156.60 (C6); 198.18 (C5').
J
J
m
ꢀH,
o
In this reaction 20% 7ꢀ[5ꢀ(4ꢀfluorophenyl)ꢀ5ꢀoxoꢀ
(2 H, dd, Jo
ꢀH 8.7, JoꢀH, F 5.3, o
ꢀH,
13
m
pentyl]hypoxantine, Rf 0.40 (18 : 2), was obtained as a
side product. The target product (VIIIa) was separated
from it by multiple crystallization from ethyl acetate.
C
J
J
9ꢀ[5ꢀ(4ꢀFluorophenyl)ꢀ5ꢀoxopentyl] guanine (IXa)
254.9)] (С6H4F); 140.22 (C6); 151.05 (C2); 164C.9, 7F was obtained by method
A by alkylation of 2ꢀaminoꢀ6ꢀ
ꢀ
(C4); 197.85 (C5').
chloropurine followed by hydrolysis of the resulting
raw 2ꢀaminoꢀ6ꢀchloropurine derivative (reflux in 0.33 M
NaOH, 3.5 h) in a total yield of 26%; Rf 0.40 (18 : 2);
In this reaction 1,3ꢀbis[5ꢀ(4ꢀfluorophenyl)ꢀ5ꢀoxoꢀ
pentyl]thymine, Rf 0.64 (19.5 : 0.5), oil, was obtained
in a yield of 14% as a side product.
mp 128–129
330.3 [
+ H+]. Calculated
cytosine 1H NMR (CDCl3): 1.75 (2 H, m, H2'); 1.89 (2 H, m,
in a yield of 41%; Rf H3'); 2.94 (2 H, t, 7.2, H4'); 4.04 (2 H, t, 7.0, H1');
(ethanol). Mass: m/z 4.84 (2 H, br s, 2ꢀN
2); 7.02 (2 H, dd, Jm ꢀH8.6,
+ H+]. Calculated
289.3 Jm 8.6, ꢀH, PhF); 7.45 (1 H, s, H8); 7.91 (2 H,
(C15H16FN3O2). H NMR (DMSOꢀ 6): 1.50–1.70 dd, Jo ꢀH8.7, Jo 5.5,
ꢀH, PhF). 13C NMR
(4 H, m, H2' and H3'); 3.04 (2 H, t,
6.7, H4'); 3.66 (CDCꢀlH3),:m21.16 (C3'); 29.44 (C2'); 37.62 (C4'); 43.05
(2 H, t, 6.7, H1'); 5.63 (1 H, d, 5, 6 7.2, H5); 6.93 (C1'); 115.24 (C5) [115.70 (2 C, d,
C, F 22.0); 130.65
(2 H, br s, 4ꢀN 2); 7.33 (2 H, dd, Jm ꢀH 8.9, JmꢀH, F 8.9, (2 C, d, C, F 9.4); 133.34 (1 C, d, C, F 2.7); 167.7 (1 C,
6, 5 7.2, ꢀHH,6); 8.03 (2 H, dd, d, J4ꢀC, F 254.5)] (C6H4F); 135.98 (C8); 152.43 (C4);
ꢀH, PhF). 13C NMR (DMSOꢀ 155.43 (C2); 159.25 (C6); 198.18 (C5').
°C
(ethyl acetate). Mass:
m/z 329.3 [M
+],
M
M
329.3 (C16H16FN5O2).
1ꢀ[5ꢀ(4ꢀFluorophenyl)ꢀ5ꢀoxopentyl]
(VIa) was obtained by method
0.47 (17 : 3); mp 197–198
289.3 [
+], 290.3 [
B
C
J
J
°
H
ꢀH,
o
M
M
M
m
ꢀH, F
1
d
o
ꢀH, F
J
J
J
J
H
J
J
o
m
ꢀH, PhF); 7.57 (1 H, d,
ꢀH 8.9, JoꢀH 5.6,
J
Jo
о
d6ꢀ)H:,20.54 (C3'); 28.18 (C2'); 37.29 (C4'); 48.18 (C1');
m
1ꢀ[5ꢀ(4ꢀChlorophenyl)ꢀ5ꢀoxopentyl] uracil (IVb)
was obtained by method in a yield of 30%; Rf 0.4 (19 : 1);
mp 171–172 (ethyl acetate). Mass: 306.7 [
+],
307.7 [ 306.7 (C15H15ClN2O3).
+ H+]. Calculated
1H NMR (CDCl3): 1.19–1.41 (4 H, m, H2' and H3');
2.66 (2 H, t, 6.5, H4'); 3.29 (2 H, t, 6.5, H1'); 5.11
(1 H, d, 5, 6 7.8, H5); 6.89 (1 H, d, 6, 5 7.8, H6); 6.89
(2 H, d, Jm ꢀH 8.6, ꢀH, PhCl); 7.44 (2 H, d, Jmꢀ
92.97 (C5); [115.56 (2 C, d,
C, F 9.4); 133.38 (1 C, d,
J4 251.8)] (С6H4F); 145.88 (C6); 155.74 (C2);
J
C, F 22.0); 130.75 (2 C, d,
JC, F 2.7); 164.87 (1 C, d,
A
J
°
C
m/z
M
ꢀC, F
M
M
165.81 (C4); 198.33 (C5').
9ꢀ[5ꢀ(4ꢀFluorophenyl)ꢀ5ꢀoxopentyl] adenine (VIIa)
was obtained by method in a yield of 35%; Rf 0.33
(18 : 2); mp 197–198 (ethyl acetate). Mass: m/z 313.3
+], + H+], 314.3 [ + H+]. Calculated
313.3
(C16H16FN5O). 1H NMR (DMSOꢀ
6): 1.58 (2 H, m,
H2'); 1.88 (2 H, m, H3'); 3.05 (2 H, t, 7.2, H4'); 4.19
(2 H, t, 7.0, H1'); 7.11 (2 H, br s, 6ꢀN 2); 7.30 (2 H,
dd, Jm ꢀH 8.9, JmꢀH, F 8.9, ꢀH, PhF); 8.00 (2 H, dd,
ꢀH, PhF); 8.14 (2 H, br s, H2
and H8). C NMR (DMSOꢀ
(C2'); 37.00 (C4'); 42.57 (C1'); [115.46 (2 C, d, JC, F (19 : 1); mp 172–173
22.0); 130.65 (2 C, d, JC, F 9.4); 133.32 (1 C, d, JC, F 320.7 [
+], 321.7 [
2.7); 164.82 (1 C, d, J4ꢀC, F 251.3)] (C6H4F); 118.70 (C16H17ClN2O3). 1H NMR (CDCl3): 1.68–1.82 (4 H,
J
J
J
J
A
m
ꢀH,
o
H, оꢀH
°C
13
8.6, oꢀH, PhCl); 10.54 (1 H, br s, H3). C NMR
[M
[
M
M
M
(CDCl3): 19.54 (C3'); 27.33 (C2'); 36.54 (C4'); 46.81
(C1'); 100.72 (C5); [127.76 (2 C); 128.41 (2 C);
134.15; 137.99] (С6H4Cl); 143.53 (C6); 150.16 (C2);
163.08 (C4); 197.10 (C5').
d
J
J
H
m
ꢀH,
o
Jo
ꢀH,
ꢀH 8.7, JoꢀH, F 5.6,
m
o
1ꢀ[5ꢀ(4ꢀChlorophenyl)ꢀ5ꢀoxopentyl] thymine (Vb)
6): 20.57 (C3'); 28.76 was obtained by method
13
d
A
C
in a yield of 28%; Rf 0.54
(ethyl acetate). Mass:
+ H+]. Calculated
320.7
°
M
m/z
M
M
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 40
No. 5
2014

538
KOMISSAROV et al.
m, H2' and H3'); 1.89 (3 H, s, 5ꢀC
H
3); 2.98 (2 H, t, mp >250
°
C
(ethanol). Mass:
345.7 (C16H16ClN5O2). 1H NMR
6): 1.54 (2 H, m, H2'); 1.78 (2 H, m, H3');
8.6, oꢀꢀHH, o, PhCl); 9.37 (1 H, br s, H3). 3.04 (2 H, t,
7.2, H4'); 3.96 (2 H, t, 7.0, H1'); 6.40
13C NMR (CDCl3): 12.23 (5ꢀ
H3), 20.63 (C3'); 28.44 (2 H, br s, 2ꢀN 2); 7.56 (2 H, d, Jm ꢀH8.7,
(C2'); 37.53 (C4'); 48.08 (C1'); 110.74 (C5); [128.90 PhCl); 7.68 (1 H, s, H8); 7.93 (2 H,ꢀdH,, oJo
m/z 345.7 [M M
+], 346.7 [
J
6.4, H4'); 3.72 (2 H, t,
7.40 (2 H, d, Jm ꢀH 8.6,
Jo
J M
6.4, H1'); 6.98 (1 H, s, H6); + H+]. Calculated
mꢀH, PhCl); 7.86 (2 H, d, (DMSOꢀd
J
J
ꢀH,
mꢀH
C
H
mꢀH,
8.7,
ꢀH, mꢀH
(2 C); 129.38 (2 C); 135.07; 139.55] (C6H4Cl); 140.19
(C6); 151.03 (C2); 164.26 (C4); 198.22 (C5').
o
ꢀH, PhCl); 10.53 (1 H, brs, H2). 13C NMR (DMSOꢀ
d6):
20.49 (C3'); 28.83 (C2'); 37.14 (C4'); 42.33 (C1');
116.55 (C5); [128.70 (2 C); 129.70 (2 C); 135.26;
137.91] (С6H4Cl); 137.37 (C8); 151.10 (C4); 153.41
(C2); 156.77 (C6); 198.65 (C5').
In this reaction 1,3ꢀbis[5ꢀ(4ꢀchlorophenyl)ꢀ5ꢀ
oxopentyl]thymine, Rf 0.64 (19.5 : 0.5), oil, was
obtained as a side product in a yield of 14%.
1ꢀ[5ꢀ(4ꢀBromophenyl)ꢀ5ꢀoxopentyl] uracil (IVc)
1ꢀ[5ꢀ(4ꢀChlorophenyl)ꢀ5ꢀoxopentyl]
(VIb) was obtained by method in a yield of 3.6%; Rf
0.47 (17 : 3); mp 213–214 (ethanol). Mass: m/z
+], 306.7 [ + H+]. Calculated
305.7 [ 305.7
(C15H16ClN3O2). H NMR (DMSOꢀ 6): 1.50–1.67
(4 H, m, H2' and H3'); 3.03 (2 H, t, 6.7, H4'); 3.65
(2 H, t, 6.7, H1'); 5.64 (1 H, d, 5, 6 7.2, H5); 6.95
(2 H, br s, 4ꢀN 2); 7.56 (1 H, d, 6, 5 7.2, H6); 7.70
(2 H, d, Jm ꢀH 8.6, ꢀH, PhCl); 7.87 (2 H, d, JoꢀH,
mꢀH
cytosine
was obtained by method
(18 : 2); mp 174–175
351.2 [
+], 352.2 [
(C15H15BrN2O3). H NMR (DMSOꢀ
(4 H, m, H2' and H3'); 3.04 (2 H, t,
A
in a yield of 27%; Rf 0.65
(ethyl acetate). Mass:
+ H+]. Calculated
351.2
6): 1.53–1.70
6.8, H4'); 3.68
5, 6 7.8, H5); 7.64
6, 5 7.8, H6); 7.71 (2 H, d, Jo ꢀH 8.6, ꢀH,
ꢀH, PhBr); 11.16
(1 H, br s, H3). C NMRo (DMSOꢀ
6): 20.25 (C3');
B
C
°
M
C
m/z
°
M
M
M
M
M
1
1
d
d
J
J
(2 H, t,
(1 H, d,
J 6.8, H1'); 5.53 (1 H, d, J
J
J
J
J
o
ꢀH,
m
H
PhBr); 7.87 (2 H, d, Jm ꢀH 8.6,
m
ꢀH,
m
ꢀH,
o
13
8.6, o d6): 20.47 (C3');
ꢀH, PhCl). 13C NMR (DMSOꢀ
d
27.86 (C2'); 37.25 (C4'); 47.10 (C1'); 100.74 (C5);
[127.05; 129.82(2 C); 131.66 (2 C); 135.60] (С6H4Br);
145.57 (C6); 150.87 (C2); 163.62(C4); 198.86 (C5').
28.16 (C2'); 37.38 (C4'); 48.18 (C1'); 93.03 (C5);
[128.71 (2 C); 129.72 (2 C); 135.30; 137.90] (C6H4Cl);
145.89 (C6); 155.78 (C2); 165.82 (C4); 198.76 (C5').
1ꢀ[5ꢀ(4ꢀBromophenyl)ꢀ5ꢀoxopentyl] thymine (Vc)
9ꢀ[5ꢀ(4ꢀChlorophenyl)ꢀ5ꢀoxopentyl]
(VIIb) was obtained by method in a yield of 36%; Rf
0.31 (18 : 2); mp 209–210 (ethyl acetate). Mass:
329.7 [ 329.7
+], 330.7 [ + H+]. Calculated
(C16H16ClN5O). 1H NMR (DMSOꢀ
6): 1.55 (2 H, m,
H2'); 1.86 (2 H, m, H3'); 3.05 (2 H, t, 7.2, H4'); 4.17
(2 H, t, 7.0, H1'); 7.13 (2 H, br s, 6ꢀN 2); 7.55 (2 H,
d, Joꢀ ꢀH 8.4, ꢀH, PhCl); 7.92 (2 H, d, Jm ꢀH 8.4,
adenine
was obtained by method
(19 : 1); mp 187–190
A
in a yield of 22%; Rf 0.48
A
°C
(ethyl acetate). Mass: m/z
°C
365.2 [M M M 365.2
+], 366.2 [ + H+]. Calculated
m/
z
M
M
M
(C16H17BrN2O3). 1H NMR (CDCl3): 1.70–1.80 (4 H,
m, H2' and H3'); 1.90 (3 H, s, 5ꢀCH3); 2.98 (2 H, t,
d
J
H
J
6.4, H4'); 3.73 (2 H, t,
J
7.0, H1'); 6.98 (1 H, s, H6);
ꢀH, PhBr); 7.79 (2 H, d,
ꢀH, PhBr); 9.09 (1 H, br s, H3). 13C NMR
(CDCl3): 12.36 (5ꢀC 3), 20.74 (C3'); 28.56 (C2');
J
m
7.68 (2 H, d, Jo ꢀH 8.6,
o
m
ꢀH,
m
ꢀH, o
o
ꢀH,HP, hCl); 8.12 (1 H, s, H8); 8.14 (1 H, s, H2). 13C
Jm ꢀH 8.6,
m
ꢀH,
o
H
NMR (DMSOꢀ 6): 20.50 (C3'); 28.77 (C2'); 37.11
d
37.63 (C4'); 48.22 (C1'); 110.88 (C5); [128.40; 129.60
(2 C); 132.04 (2 C); 135.58] (С6H4Br); 140.28 (C6);
151.04 (C2); 164.23 (C4); 198.50 (C5').
(C4'); 42.59 (C1'); 118.71 (C5); [128.68 (2 C); 129.68
(2 C); 135.26; 137.89] (С6H4Cl); 140.76 (C8); 149.52
(C4); 152.28 (C2); 155.89 (C6); 198.63 (C5').
1ꢀ[5ꢀ(4ꢀBromophenyl)ꢀ5ꢀoxopentyl] cytosine (VIc)
9ꢀ[5ꢀ(4ꢀChlorophenyl)ꢀ5ꢀoxopentyl] hypoxantine
was obtained by method
(17 : 3); mp 217–218
+], 351.2 [ + H+]. Calculated
(C15H16BrN3O2). 1H NMR (DMSOꢀ
6): 1.46–1.77 (4
H, m, H2' and H3'); 3.02 (2 H, t, 6.8, H4'); 3.65 (2
H, t, 6.6, H1'); 5.62 (1 H, d, 5, 6 7.2, H5); 6.92 (2 H,
br s, 4ꢀN 2); 7.57 (1 H, d, 6, 5 7.2, H6); 7.71 (2 H, d,
Jo ꢀH 8.6, ꢀH, PhBr); 7.87 (2 H, d, Jm ꢀH 8.6,
Hꢀ,HP, mhBr);. 13C NMR (DMSOꢀ
B
in a yield of 8.2%; Rf 0.47
(ethanol). Mass: 350.2
350.2
(VIIIb) was obtained by method
A by alkylation of
°C
m/z
M
6ꢀchloropurine followed by hydrolysis of the resulting
raw 6ꢀchloropurine derivative (reflux in 4% HCl, 3 h)
[M
M
d
in a total yield of 27%; Rf 0.28 (18 : 2); mp 231–232
(ethyl acetate). Mass: 330.7 [
+], 331.7 [
H+]. Calculated
330.7 (C16H15ClN4O2). H NMR
(DMSOꢀ 6): 1.55 (2 H, m, H2'); 1.85 (2 H, m, H3');
°C
M +
J
m/z
M
1
J
J
M
H
J
d
o
mꢀ
3.04 (2 H, t, J 7.2, H4'); 4.17 (2 H, t, J 6.9, H1'); 7.55
ꢀH,
o
d
6): 20.44 (C3'); 28.14
(2 H, d, Jm
H 8.7, mꢀH, PhCl); 7.93 (2 H, d, Jo
ꢀH, oꢀ
ꢀH8.7,
o
ꢀH, PhCl); 8.00 (1 H, s, H8); 8.09 (1 H,ꢀHs,,
(C2'); 37.35 (C4'); 48.16 (C1'); 92.97 (C5); [127.02;
129.83 (2 C); 131.66 (2 C); 135.62] (С6H4Br); 145.88
(C6); 155.72 (C2); 165.79 (C4); 198.95 (C5').
m
13
H2); 12.22 (1 H, br s, H1). C NMR (DMSOꢀ
d
6):
20.42 (C3'); 28.93 (C2'); 37.07 (C4'); 43.03 (C1');
123.91 (C5); [128.68 (2 C); 129.68 (2 C); 135.24;
137.91] (С6H4Cl); 140.20 (C8); 145.29 (C2); 148.32
(C4); 156.59 (C6); 198.58 (C5').
9ꢀ[5ꢀ(4ꢀBromophenyl)ꢀ5ꢀoxopentyl] adenine (VIIc)
was obtained by method
(18 : 2); mp 206–207
+], 375.2 [
by alkylation of 2ꢀaminoꢀ6ꢀ (C16H16BrN5O). 1H NMR (DMSOꢀ
chloropurine followed by hydrolysis of the resulting H2'); 1.86 (2 H, m, H3'); 3.04 (2 H, t,
A
in a yield of 34%; Rf 0.39
(ethyl acetate). Mass:
+ H+]. Calculated
374.2
6): 1.55 (2 H, m,
7.2, H4'); 4.17
2); 7.70 (2 H,
°
M
C
m/z
9ꢀ[5ꢀ(4ꢀChlorophenyl)ꢀ5ꢀoxopentyl] guanine (IXb) 374.2 [
was obtained by method
M
M
A
d
J
raw 2ꢀaminoꢀ6ꢀchloropurine derivative (reflux in 0.33 N (2 H, t,
J
7.0, H 1'); 7.14 (2 H, br s, 6ꢀN
H
NaOH, 3.5 h) in a total yield of 11%; Rf 0.11 (18 : 2); d, Jo
ꢀH 8.6,
o
ꢀH, PhBr); 7.85 (2 H, d, Jm ꢀH 8.6,
ꢀH,
m
ꢀH,
o
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 40
No. 5
2014

THE SEARCH OF NOVEL INHIBITORS
539
m
ꢀH, PhBr); 8.12 (1 H, s, H8); 8.14 (1 H, s, H2). MgCl2, 1 mM DTT, and 10% DMSO) in the presence
13C NMR (DMSOꢀ
6): 20.48 (C3'); 28.77 (C2'); of increasing concentrations of the inhibitor (1–
37.08 (C4'); 42.58 (C1'); 118.71 (C5); [127.03; 129.79 1500 M) for 2 h at 37 . The reaction was stopped
(2 C); 131.63 (2 C); 135.57] (C6H4Br); 140.75 (C8); the addition of the stop solution (80 L) (7 mM
d
µ
°C
µ
149.51 (C4); 152.27 (C2); 155.88 (C6); 198.82 (C5').
EDTA, 0.3 M sodium acetate, 10 mM TrisꢀHCl,
pH 8.0, and 0.125 mg/mL glycogen). Integrase was
extracted with a phenol/chloroform/isoamyl alcohol
mixture (25 : 24 : 1), and the DNA duplex was precipꢀ
itated with ethanol (250 L) and analyzed by electroꢀ
phoresis in 20% PAG with 7 M urea.
Radioautograms were registered on a GE Typhoon
FLA 9500 scanner; densitometry was performed using
the ImageQuant 5.0 software. For the calculation of
the IC50 the data on the reaction effectiveness were
approximated using the function of exponential decay
followed by the calculation at the point corresponding
to 50%.
9ꢀ[5ꢀ(4ꢀBromophenyl)ꢀ5ꢀoxopentyl] hypoxantine
(VIIIc) was obtained by method
A by alkylation of
6ꢀchloropurine followed by hydrolysis of the resulting
raw 6ꢀchloropurine derivative (reflux in 4% HCl, 3 h).
µ
The total yield 24%; Rf 0.27 (18 : 2); mp 242
ethanol). Mass: 375.2 [
+], 376.2 [
Calculated 375.2 (C16H15BrN4O2). H NMR
(DMSOꢀ 6): 1.54 (2 H, m, H2'); 1.84 (2 H, m, H3');
°
C
(degr.,
m
/
z
M
M
+ H+]
.
1
M
d
3.04 (2 H, t,
J
m
7.2, H4'); 4.17 (2 H, t,
ꢀH 8.5, oꢀH, PhBr); 7.85 (2 H, d, Jm
J
6.9, H 1'); 7.70
(2 H, d, Jo
oꢀH
8.5,
m
ꢀH,ꢀPHh, Br); 8.01 (1 H, s, H8); 8.10 (1 H, s,ꢀHH,2);
13
12.25 (1 H, br s, H1). C NMR (DMSOꢀ
d6): 20.42
(C3'); 28.97 (C2'); 37.09 (C4'); 43.07 (C1'); 123.93
(C5); [127.13; 129.86 (2 C); 131.71 (2 C); 135.58]
(C6H4Br); 140.29 (C8); 145.38 (C2); 148.36 (C4);
156.65 (C6); 198.84 (C5').
Inhibition of the strand transfer reaction. The
32P labeled U5Bꢀ2/U5A duplex was obtained as
32
described above for U5B/U5A starting from the P
labeled oligonucleotide U5Bꢀ2. The reaction was carꢀ
ried out in the same buffer as that for the 3'ꢀprocessing
using the 32P labeled U5B/U5A duplex (0.2 pmol) and
integrase (2 pmol). The reaction was performed in the
presence of increasing inhibitor concentrations (1–
9ꢀ[5ꢀ(4ꢀBromophenyl)ꢀ5ꢀoxopentyl] guanine (IXc)
was obtained by method A by alkylation of 2ꢀaminoꢀ6ꢀ
chloropurine followed by hydrolysis of the resulting raw
2ꢀaminoꢀ6ꢀchloropurine derivative (reflux in 0.33 N
NaOH, 3.5 h). The total yield 13%; Rf 0.34 (A); mp
1500 M) at 37°C for 2 h. The isolation and analysis
µ
m/z 390.2 [M
+], 391.2
of the products were performed as described above.
For the calculation of the IC50 the data on the reaction
effectiveness were approximated using the function of
exponential decay followed by the calculation at the
point corresponding to 50%.
126–127
+ H+]. Calculated
1H NMR (DMSOꢀ
6): 1.71 (2 H, m, H2'); 1.87 (2 H,
H3'); 2.91 (2 H, t, 7.2, H4'); 4.01 (2 H, t, 7.0, H1');
4.79 (2 H, br s, 2ꢀN 2); 7.43 (1 H, s, H8); 7.52 (2 H,
d, Jo ꢀH 8.6, ꢀH, PhBr); 7.71 (2 H, d, Jm ꢀH 8.6,
°C
(ethanol). Mass:
[M
M
390.2 (C16H16BrN5O2).
d
J
J
H
o
ꢀH,
m
ꢀH, o
13
m
ꢀH, PhBr). C NMR (CDCl3): 20.94 (C3'); 29.25
ACKNOWLEDGMENTS
(C2'); 37.50 (C4'); 42.81 (C1'); 115.10 (C5); [120.06;
129.42 (2 C); 131.78 (2 C); 135.79] (C6H4Br);
135.44 (C8); 152.48 (C4); 155.27 (C2); 159.16
(C6); 198.35 (C5').
The work was supported by the Russian Foundaꢀ
tion for Basic Research, project nos. 12ꢀ04ꢀ00237ꢀa
and 12ꢀ04ꢀ32085ꢀmol_a) and the Grant of the Presiꢀ
dent of Russian Federation for Young Scientists and
Postgraduate Students SPꢀ6530.2013.4.
Inhibition of the 3'ꢀprocessing reaction. For the
preparation of a radioactive duplex U5B/U5A, oligoꢀ
nucleotide U5B/U5A (10 pmol) was incubated with
T4 polynucleotide kinase (10 U, Fermentas, Lithuaꢀ
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No. 5
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