Synthesis and studies of new 6-[halo(diphenyl)methyl]- and 6-(thiophen-2-ylmethyl)pyrimidin-4(3H)-ones as possible HIV-1 reverse transcriptase inhibitors

Article abstract of DOI:10.1007/s11172-013-0108-z

Six new 6-[halo(diphenyl)methyl]- and 6-(thiophen-2-ylmethyl)pyrimidin- 4(3H)-one derivatives were synthesized and studied for biological activity. The studies showed that the 6-(thiophen-2-ylmethyl)pyrimidin-4(3H)-one derivatives inhibited activity of the HIV-1 recombinant reverse transcriptase in the micromolar range of concentrations. The 6-[halo-(diphenyl)methyl]pyrimidin-4(3H) -one derivative showed no activity in the concentrations up to 200 μmol mL^-1.

Full text of DOI:10.1007/s11172-013-0108-z

Russian Chemical Bulletin, International Edition, Vol. 62, No. 3, pp. 797—801, March, 2013
797
Synthesis and studies of new 6ꢀ[halo(diphenyl)methyl]ꢀ
and 6ꢀ(thiophenꢀ2ꢀylmethyl)pyrimidinꢀ4(3H)ꢀones as possible
HIVꢀ1 reverse transcriptase inhibitors
V. T. ValuevꢀElliston,a A. V. Ivanov,a B. S. Orlinson,b E. N. Gerasimov,b L. L. Brunilina,b
E. K. Zakharova,b S. N. Kochetkov,a I. A. Novakov,b and M. B. Navrotskiib
aV. A. Engel´gardt Institute of Molecular Biology, Russian Academy of Sciences,
32 ul. Vavilova, 119991 Moscow, Russian Federation.
bVolgograd State Technical University,
28 prosp. Lenina, 400131 Volgograd, Russian Federation.
Fax: +7 (8442) 238 125. Eꢀmail: kholstaedt@yandex.ru
Six new 6ꢀ[halo(diphenyl)methyl]ꢀ and 6ꢀ(thiophenꢀ2ꢀylmethyl)pyrimidinꢀ4(3H)ꢀone deꢀ
rivatives were synthesized and studied for biological activity. The studies showed that the
6ꢀ(thiophenꢀ2ꢀylmethyl)pyrimidinꢀ4(3H)ꢀone derivatives inhibited activity of the HIVꢀ1 recomꢀ
binant reverse transcriptase in the micromolar range of concentrations. The 6ꢀ[haloꢀ
(diphenyl)methyl]pyrimidinꢀ4(3H)ꢀone derivative showed no activity in the concentrations up
to 200 mol mL–1
.
Key words: 6ꢀbenzhydrylpyrimidinꢀ4(3H)ꢀone derivative, 3ꢀoxoester, cyclocondensation,
alkylation, inhibiting activity, HIVꢀ1 reverse transcriptase inhibitors.
At the present time, only limited number of publicaꢀ
tions on the ability of the 6ꢀ(thiophenꢀ2ꢀylmethyl)ꢀ and
6ꢀbenzhydrylpyrimidinꢀ4(3H)ꢀone derivatives to inhibit
activity of HIVꢀ1 reverse transcriptase (RT) is available in
the literature.1,2 At the same time, the activity of the
corresponding 6ꢀbenzylꢀsubstituted analogs was studied
in details. Thus, it was found that the antiviral effect
of 2ꢀ(alkylsulfanyl)ꢀ6ꢀ(thiophenꢀ2ꢀylmethyl)pyrimidinꢀ
4(3H)ꢀone derivatives against HIVꢀ1 is slightly lower than
that of the benzyl analogs.1 However, the activity of the
corresponding derivatives containing a heteroalkyl subꢀ
stituent at the exocyclic sulfur atom has not been studied.
Some 2ꢀsubstituted halogenated derivatives of 6ꢀbenzylꢀ
pyrimidinꢀ4(3H)ꢀone were found to possess a high antiꢀ
HIVꢀ1ꢀactivity.3 At the same time, the antiviral activity of
6ꢀbenzhydrylꢀsubstituted analogs of this series has not been
studied.
1: R1 = Br, R2 = SMe (a); R1 = Br, R2 = NH2 (b); R1 = I, R2 = NH2 (c)
2: R = SMe (a), CH2SMe (b), SPh (c)
The halogenated derivatives of 6ꢀbenzhydrylpyrimidinꢀ
4(3H)ꢀone 1a—c were synthesized from methyl 3ꢀoxoꢀ
4,4ꢀdiphenylbutanoate (3).4 The
latter was converted to 6ꢀbenzhyꢀ
drylꢀ2ꢀ(methylsulfanyl)pyrimidꢀ
inꢀ4(3H)ꢀone (4) according to the
known procedure.5
To prepare 2ꢀaminoꢀ6ꢀbenzꢀ
hydrylpyrimidinꢀ4(3H)ꢀone (5),
In this connection, the present work is devoted to the
synthesis of new 2ꢀsubstituted halogenated derivatives of
6ꢀbenzhydrylpyrimidinꢀ4(3H)ꢀone 1a—c and the studies
of their ability to inhibit the activity of HIVꢀ1 reverse
transcriptase. Besides, we synthesized and studied new
5ꢀmethylꢀ6ꢀ(thiophenꢀ2ꢀylmethyl)pyrimidinꢀ4(3H)ꢀone
derivatives containing a heteroalkylsulfanyl substituent at
position 2 of the pyrimidine heterocycle 2a—c.
the corresponding 3ꢀoxoester 3 was involved in the cycloꢀ
condensation with the obtained in situ guanidine in the
presence of sodium methoxide in methanol (Scheme 1).
The product 5 was obtained as a sesquihydrate, which
can be dehydrated by heating.
The bromination of compounds 4 and 5 with molecuꢀ
lar bromine in anhydrous dimethylformamide and pyridiꢀ
ne (Scheme 2) led to compounds 1a,b.
2ꢀAminoꢀ6ꢀ[diphenyl(iodo)methyl]pyrimidinꢀ4(3H)ꢀ
one (1c) was obtained by treatment of sesquihydrate of
* Dedicated to Academician of the Russian Academy of Sciences
I. P. Beletskaya on the occasion of her anniversary.
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 3, pp. 0796—0800, March, 2013.
1066ꢀ5285/13/6203ꢀ797 © 2013 Springer Science+Business Media, Inc.

798
Russ.Chem.Bull., Int.Ed., Vol. 62, No. 3, March, 2013
ValuevꢀElliston et al.
Scheme 1
Scheme 4
Reagents: 1) (H2N)2CS, EtONa, EtOH; 2) AcOH, H2O.
Reagents and conditions: H2NC(NH)NH2•AcOH, MeONa,
The corresponding 3ꢀoxoester 7 was synthesized from
thiopheneꢀ2ꢀacetonitrile (8) using the Blaise organozinc
synthesis (Scheme 5).
MeOH, reflux, 72 h.
Scheme 2
Scheme 5
Reagents: 1) MeCH(Br)COOEt, Zn, THF; 2) HCl, H2O.
R = SMe (4), NH2 (5)
Hal2 = Br2 (for 1a, b), I2 (for 1c)
The synthesized compounds 1a—c and 2a—c were
studied as potential wild type HIVꢀ1 inhibitors. Nevirapin,
the nonꢀnucleoside HIVꢀ1 RT inhibitor the most frequentꢀ
ly used in medicine, was chosen as a control.
The inhibition constants of the wild type HIVꢀ1 reꢀ
verse transcriptase homodimer (Ki) for the compounds
under study are as follows:
compound 5 with molecular iodine in a mixture of aqueꢀ
ous KOH and dichloromethane.
The target 5ꢀmethylꢀ6ꢀ(thiophenꢀ2ꢀylmethyl)pyrꢀ
imidinꢀ4(3H)ꢀone derivatives 2a—c were obtained by the
Sꢀalkylation of 6ꢀ(thiophenꢀ2ꢀylmethyl)ꢀ2ꢀthiothymine
(6) with (chloromethyl) methyl sulfide, (2ꢀchloroethyl)
methyl sulfide, or (chloromethyl) phenyl sulfide. In the
case of the former alkylating agent, the reaction was carꢀ
ried out in anhydrous dimethylformamide in the presence
of potassium carbonate (Scheme 3).
Compound
1a
1b
1c
Ki/mmol L–1
>200
>200
>200
198
2a
2b
97.8
2c
191
Nevirapin
4.20
Scheme 3
Based on the data obtained, a conclusion can be drawn
that the halogenated derivatives of 6ꢀbenzhydrylpyrimiꢀ
dinꢀ4(3H)ꢀone, in contrast to the corresponding 6ꢀbenꢀ
zylꢀsubstituted analogs, exhibit no inhibiting activity with
respect to HIVꢀ1 reverse transcriptase. It should be noted
that a similar behavior is also characteristic of the nonꢀ
halogenated derivatives of 6ꢀbenzhydrylpyrimidinꢀ4(3H)ꢀ
one.6 To sum up, the introduction of an additional aroꢀ
matic ring at -position of the benzyl group cancels the
inhibiting activity of these compounds with respect to the
HIVꢀ1 revertase. At the same time, as it has been shown
earlier, the introduction of small alkyl fragments at the
corresponding position of the benzyl group increases the
activity of the analogs obtained.7
R = SMe, CH2SMe, SPh
In the case of (2ꢀchloroethyl) methyl sulfide and (chloꢀ
romethyl) phenyl sulfide, the alkylation was accomplished
in an ethanolic solution of KOH.
The starting 6ꢀ(thiophenꢀ2ꢀylmethyl)ꢀ2ꢀthiothymꢀ
ine (6) was obtained by cyclocondensation of ethyl
2ꢀmethylꢀ3ꢀoxoꢀ4ꢀ(thiophenꢀ2ꢀyl)butanoate (7) with
thiourea in the solution of sodium ethoxide in ethanol
(Scheme 4).
For the compounds described, as in the case of other
2ꢀ(alkylsulfanyl)pyrimidinꢀ4(3H)ꢀone derivatives,1 on goꢀ
ing from 6ꢀbenzylꢀ to 6ꢀ(2ꢀthienylmethyl)ꢀsubstituted anꢀ

Novel pyrimidinones for antiꢀHIVꢀ1 therapy
Russ.Chem.Bull., Int.Ed., Vol. 62, No. 3, March, 2013
799
yield was 61%, m.p. 287—288 C (with decomp., EtOH).
1H NMR (DMSOꢀd6), : 5.76 (s, 1 H, C(5)H); 6.65 (s, 2 H,
NH2); 7.16—7.23 (m, 10 H, (C6H5)2); 11.31 (s, 1 H, NH). Found
(%): C, 56.99; H, 3.90; Br, 22.51; N, 11.47. C17H14BrN3O. Calꢀ
culated (%): C, 57.32; H, 3.96; Br, 22.43; N, 11.80.
alogs low active compounds are obtained. However, it is
important to note that the replacement of the (methylꢀ
sulfanyl)methyl group at the exocyclic sulfur atom with
the different in size (phenylsulfanyl)methyl substituent virꢀ
tually does not change the activity, thus indicating the
different binding pattern of these groups with the correꢀ
sponding site in the enzyme molecule. In this case, on
going from compound 2a to its homoꢀanalog compound 2b,
virtually twofold increase in the activity is achieved. Such
a change in the structure of 6ꢀbenzylꢀ2ꢀ{[(methylsulfanyl)ꢀ
methyl]sulfanyl}pyrimidinꢀ4(3H)ꢀones derivatives, which
earlier has not been described in the literature, can be
considered as an important approach to the targeted conꢀ
struction of new highly active HIVꢀ1 RT inhibitors in the
series of 6ꢀbenzylpyrimidinꢀ4(3H)ꢀone derivatives.
2ꢀAminoꢀ6ꢀ[iodo(diphenyl)methyl]pyrimidinꢀ4(3H)ꢀone (1c).
The compound 5•1.5H2O (1.5 g, 4.9 mmol) was added to
a solution of KOH (0.4 g, 6.0 mmol) in water (20 mL), the
mixture obtained was heated until a clear solution was formed.
After cooling to room temperature, CH2Cl2 (40 mL) and I2 (1.4 g,
5.5 mmol) were added to the solution, then the reaction mixture
was vigorously stirred for 5 h at room temperature. Then,
a saturated aq. Na2S2O5 (10 mL) was added and the stirring was
continued for another 5 min. A precipitate was filtered off and
crystallized. The yield was 0.7 g (35%), m.p. 273—274 C (with
decomp., EtOH—DMF). 1H NMR (DMSOꢀd6), : 5.76 (s, 1 H,
C(5)H); 6.60 (s, 2 H, NH2); 7.16—7.23 (m, 10 H, (C6H5)2);
11.14 (s, 1 H, NH). Found (%): C, 50.50; H, 3.50; I, 30.99;
N, 10.33. C17H14IN3O. Calculated (%): C, 50.64; H, 3.50;
I, 31.74; N, 10.42.
Experimental
5ꢀMethylꢀ2ꢀ{[(methylsulfanyl)methyl]sulfanyl}ꢀ6ꢀ(thiophenꢀ
2ꢀylmethyl)pyrimidinꢀ4(3H)ꢀone (2a). A mixture of compound 6
(1 g, 4.2 mmol), methyl (chloromethyl) sulfide (0.4 mL, 0.46 g,
4.8 mmol), anhydrous potassium carbonate (0.61 g, 4.4 mmol),
and anhydrous DMF (10 mL) was stirred at room temperature
until the complete conversion of the starting compound (TLC
monitoring, eluent system B), diluted with water, and extracted
with ethyl acetate. The organic phases were combined, washed
with brine, dried with anhydrous magnesium sulfate, and filtered
through a short layer of silica gel. The filtrate was concentrated
at reduced pressure, the residue was crystallized from cyclohexꢀ
ane. The yield was 0.87 g (68%), m.p. 155—156 C (cyclohexꢀ
ane). 1H NMR (CDCl3), : 2.06 (s, 3 H, CH3 (pyrimidine));
2.14 (s, 3 H, SCH3); 4.02 (s, 2 H, CCH2); 4.31 (s, 2 H, SCH2S);
6.80—6.90 (m, 2 H, C(4)H, C(5)H (thienyl)); 7.08—7.13 (m, 1 H,
m, C(3)H (thienyl)). Found (%): C, 48.11; H, 4.70; N, 9.51;
S, 32.23. C12H14N2OS3. Calculated (%): C, 48.29; H, 4.73;
N, 9.39; S, 32.23.
5ꢀMethylꢀ2ꢀ{[2ꢀ(methylsulfanyl)ethyl]sulfanyl}ꢀ6ꢀ(thiophenꢀ
2ꢀylmethyl)pyrimidinꢀ4(3H)ꢀone (2b). A mixture of compound 6
(1 g, 4.2 mmol) and a solution of potassium hydroxide (277 mg,
4.2 mmol, the content of the main compound 84.5%) in 95% aq.
ethanol (20 mL) was stirred for 1 h at room temperature, folꢀ
lowed by the addition of (2ꢀchloroethyl) methyl sulfide (0.5 mL,
0.56 g, 5.0 mmol) and stirring for 1 h, then it was kept for 16 h.
The reaction mixture was refluxed with stirring until the comꢀ
plete consumption of the starting compound (TLC monitoring
of the acidified sample in system B), diluted with water, and
extracted with ethyl acetate. The organic phases were combined,
washed with brine, dried with anhydrous magnesium sulfate,
and filtered through a short layer of silica gel. The filtrate was
concentrated at reduced pressure, the residue was crystallized
from cyclohexane. The yield was 0.76 g (58%), m.p. 147—148.5 C
(cyclohexane). 1H NMR (CDCl3), : 2.06 (s, 3 H, CH3 (pyrimꢀ
idine)); 2.09 (s, 3 H, SCH3); 2.71—2.76 (m, 2 H, CH2CH2SMe);
3.29—3.34 (m, 2 H, SCH2CH2); 4.00 (s, 2 H, CCH2); 6.81
(d, 1 H, C(5)H (thienyl), J = 3.4 Hz); 6.87 (dd, 1 H, C(4)H
(thienyl), J1 = 3.4 Hz, J2 = 5.1 Hz); 7.10 (d, 1 H, C(3)H
(thienyl), J = 5.1 Hz). Found (%): C, 50.01; H, 5.14; N, 9.05;
S, 30.80. C13H16N2OS3. Calculated (%): C, 49.97; H, 5.16;
N, 8.97; S, 30.78.
1
H NMR spectra of compounds were recorded on a Bruker
AVANCE spectrometer (600 MHz), using hexamethyldisiloxane
as an internal standard. The individuality of the compounds obꢀ
tained were confirmed by TLC on the ALUGRAM NanoꢀSIL
G/UV254 plates for HPLC in the systems A (chloroform—methꢀ
anol (19 : 1)) and B (light petroleum (b.p. 40—70 C)—ethyl
acetate—methanol (12 : 3 : 1)). The plates were visualized under
UV light. Melting points were determined on a Fisher—Jones
(Cole—Palmer) heating stage at the rate of heating 10 deg min–1
(reported are corrected values). The reagents and the solvents
were prepared according to the standard procedures.8
The following materials were used for the biological studies:
[ꢀ32P]dATP (5000 Ci mol–1) from Isotop (Russia), 2´ꢀdeoxyriꢀ
bonucleosideꢀ5´ꢀtriphosphate (Promega, USA), the Whatman
3MM cellulose filters (Whatman, Great Britain). All the other
reactants of the maximal purity were purchased from Sigꢀ
ma—Aldrich or Fluka. Activated DNA was obtained from
the salmon sperm DNA (Pharmacia Biotech, USA) by
treatment with the bovine pancreas DNAase (Fermentas, Liꢀ
thuania) as described in the work.9 The HIVꢀ1 reverse tranꢀ
scriptase was expressed in the E.coli and isolated as described
earlier.10
6ꢀ[Bromo(diphenyl)methyl]ꢀ2ꢀ(methylsulfanyl)pyrimidinꢀ4(3H)ꢀ
one (1a). Bromine (0.35 mL, 1.09 g, 6.8 mmol) was added to
a suspension of 4 (2 g, 6.8 mmol) in a mixture of anhydrous
pyridine (6 mL, 5.87 g, 74.3 mmol) and anhydrous DMF (10 mL).
The reaction mixture was stirred for 5 h at room temperaꢀ
ture, diluted with water (150 mL), and extracted with CHCl3
(4×50 mL). The organic phases were combined, sequentially
washed with water (2×50 mL), saturated aqueous Na2S2O5
(25 mL), and brine (2×25 mL) and dried with Na2SO4. The
filtrate was concentrated in vacuo, the residue was coconcenꢀ
trated in vacuo with PhMe (25 mL) and recrystallized. The yield
was 1.9 g (76%), m.p. 228—230 C (from PhMe). 1H NMR
(DMSOꢀd6), : 2.23 (s, 3 H, CH3); 5.76 (s, 1 H, C(5)H); 7.18—7.26
(s, 10 H, (C6H5)2); 13.21 (br.s, 1 H, NH). Found (%): C, 55.42;
H, 3.94; Br, 20.60; N, 7.03; S, 8.07. C18H15BrN2OS. Calculatꢀ
ed (%): C, 55.82; H, 3.90; Br, 20.63; N, 7.23; S, 8.28.
2ꢀAminoꢀ6ꢀ[bromo(diphenyl)methyl]pyrimidinꢀ4(3H)ꢀone
(1b) was obtained similarly from compound 5•1.5 H2O. The

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ValuevꢀElliston et al.
5ꢀMethylꢀ6ꢀ(thiophenꢀ2ꢀylmethyl)ꢀ2ꢀ{[(phenylsulfanyl)meꢀ
washed with several portions of anhydrous THF (50 mL each).
The combined organic solutions were concentrated at reduced
pressure, the residue was mixed with toluene (500 mL) and diꢀ
luted with 12% aq. hydrochloric acid (600 mL). The mixture
obtained was stirred for 2.5 h, the organic phase was separated
and washed with water until neutral, filtered through a short
layer of silica gel, and dried with anhydrous magnesium sulfate.
The solvent was evaporated at reduced pressure, the residue was
distilled in vacuo. The distilled product was fractionally distilled
in vacuo on a short Vigreux column. The lower boiling fraction
predominantly containing ethyl 2ꢀmethylꢀ3ꢀoxopentanoate
was removed and the fraction of the target product with b.p.
128—131 C (2 Torr) was collected. The yield was 18.5 g (34%).
Found (%): C, 58.30; H, 6.24; S, 13.99. C11H14O3S. Calculatꢀ
ed (%): C, 58.38; H, 6.24; S, 14.17.
thyl]sulfanyl}pyrimidinꢀ4(3H)ꢀone (2c). A mixture of compound
6 (1 g, 4.2 mmol) and a solution of potassium hydroxide (277 mg,
4.2 mmol, the content of the main compound 84.5%) in 95% aq.
ethanol (20 mL) was stirred for 1 h at room temperature, folꢀ
lowed by the addition of (chloromethyl) phenyl sulfide and stirꢀ
ring until the complete consumption of the starting compound
(TLC monitoring of the acidified sample in system B), diluted
with water, and extracted with ethyl acetate. The organic phases
were combined, washed with brine, dried with anhydrous magꢀ
nesium sulfate, and filtered through a short layer of silica gel.
The filtrate was concentrated at reduced pressure, the residue
was crystallized from acetonitrile. The yield was 0.77 g (51%),
m.p. 161—163 C (MeCN). 1H NMR (DMSOꢀd6) of a mixture
of tautomers, : 1.91 (s, 3 H, CH3 (pyrimidine)); 3.99 (s, 2 H,
CCH2); 4.74, 4.76 (both s, 2 H, SCH2S); 6.86—6.90 (m, 2 H,
C(4)H, C(5)H (thienyl)); 7.15—7.21 (m, 1 H, C(3)H (thienyl));
7.23—7.35 (m, 5 H, (phenyl)); 12.59 (br.s, 1 H, NH). Found (%):
C, 56.70; H, 4.49; N, 7.81; S, 26.58. C17H16N2OS3. Calculatꢀ
ed (%): C, 56.64; H, 4.47; N, 7.77; S, 26.68.
2ꢀAminoꢀ6ꢀbenzhydrylpyrimidinꢀ4(3H)ꢀone sesquihydrate
(5•1.5H2O). A solution of compound 3 (5 g, 18.6 mmol),
(H2N)2CNH2•AcOH (4.5 g, 37.8 mmol) and NaOMe (3 g,
55.5 mmol) in anhydrous MeOH (100 mL) was refluxed for 72 h
with protection from moisture and CO2. The solvent was evapoꢀ
rated in vacuo, the residue was diluted with 1 M AcOH (100 mL),
the product 5 was filtered off, dried, and crystallized. The yield
was 3.8 g (67%), m.p. 167 C (H2O), 237 C (without crystal
water, from EtOH). 1H NMR (DMSOꢀd6) of a mixture of tautoꢀ
mers, : 5.06, 5.08 (both s, 1 H, C(5)H); 5.16, 5.18 (both s,
1 H, CH); 6.51 (s, 2 H, NH2); 7.14—7.27 (m, 10 H,
(C6H5)2); 10.70 (s, 1 H, NH). Found (%): C, 66.50; H, 6.01;
N, 13.73. C17H15N3O•1.5H2O. Calculated (%): C, 67.09;
H, 5.96; N, 13.81.
6ꢀ(Thiophenꢀ2ꢀylmethyl)ꢀ2ꢀthiothymine (6). A mixture of
20% aq. sodium ethoxide (~54.5 g, ~801 mmol) in ethanol
(300 mL), compound 5 (8.4 g, ~37 mmol), and thiourea (28 g,
~368 mmol) was refluxed for 38 h with stirring and protection
from the air moisture and carbon dioxide, the solvent was evapoꢀ
rated at reduced pressure, the residue was dissolved in water
(150 mL) and neutralized with glacial acetic acid (50 mL). The
precipitate was filtered off, washed with water (3×50 mL), diꢀ
ethyl ether (2×50 mL), and dried until the weight was constant.
The yield was 5.16 g (58%), m.p. 236—237.5 C (aq. AcOH).
1H NMR (DMSOꢀd6), : 1.79 (s, 3 H, CH3 (pyrimidine)); 3.95
(s, 2 H, CH2); 6.90—6.93 (m, 2 H, C(4)H, C(5)H (thienyl));
7.33—7.36 (m, 1 H, C(3)H (thienyl)); 12.28 (s, 1 H, N(1)H);
12.39 (s, 1 H, N(3)H). Found (%): C, 50.47; H, 4.30; N, 11.62;
The physicochemical spectral parameters agree with those
given in the literature.11
Determination of HIVꢀ1 RT activity in the system of activated
DNA. The standard reaction mixture (10 L) contained the actiꢀ
vated DNA (168 g mL–1), HIVꢀ1 RT (0.1 g), ATP (3 mol L–1),
other nucleosideꢀ5´ꢀtriphosphates (30 mol L–1 each),
[ꢀ32P]dATP (0.04 MBq) in the buffer for the measurement of
the HIVꢀ1 RT activity (50 mmol L–1 Tris•HCl, pH 8.1, MgCl2
(10 mmol L–1), and KCl (200 mmol L–1)). In the experiments
on the studies of inhibiting properties, compounds 1a—c and
2a—c were added into the reaction mixtures as solutions in
DMSO to the final concentration equal to 10%, to the control
samples a similar amount of pure DMSO was added. The reꢀ
action was initiated by the addition of the reverse transcriptase
and incubated for 20 min at 37 C, then the samples were placed
on the Whatman 3MM filters (1×1 cm) saturated with a 0.5 M
solution of EDTA (1 L). Each filter was washed off from
the labeled nucleotide unincorporated in the DNA with 10% aq.
trichloroacetic acid (5×25 mL) during 5 min, then with
ethyl alcohol (25 mL), and dried in air. The radioactivity
absorbed on the filters was measured by the Cherenkov methꢀ
od using an Intertechnique Liquid Scintillation Counter
SLꢀ4000. The inhibition constants were calculated by the Dixon
method.12
This work was financially supported by the Ministry of
Education and Science of the Russian Federation (Federꢀ
al Target Program "Scientific and ScientificꢀPedagogical
Personel of the Innovative Russia" in 2009—2013, the topic
"Development of Highly Efficient antiꢀHIVꢀ1 Agents of
Nonnucleoside Nature Based on Functional Derivatives
of Pyrimidinꢀ4(3H)ꢀone", Grant 14.B37.21.0826).
S, 27.00
N, 11.75; S, 26.91
. C10H10N2OS2. Calculated (%): C, 50.40; H, 4.23;
References
.
Ethyl 2ꢀmethylꢀ3ꢀoxoꢀ4ꢀ(thiophenꢀ2ꢀyl)butanoate (7). Merꢀ
curic chloride (250 mg) and ethyl 2ꢀbromopropionate (0.5 mL)
were added to a mixture of anhydrous THF (500 mL) and freshly
prepared zinc filings (80 g, 1.22 mol). The mixture obtained was
refluxed with stirring and protection from the air moisture and
carbon dioxide until a green color appeared. Then, a solution of
compound 8 (25 mL, 29 g, 0.24 mol) in anhydrous THF (250 mL)
was added to the reaction mixture in one portion, followed by
a dropwise addition of ethyl 2ꢀbromopropionate (120 mL, ~167 g,
0.92 mol) over 2 h and reflux of the reaction mixture for another
30 min. The organic solution was decanted, the residual zinc was
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Received December 26, 2012;
in revised form March 19, 2013