Synthesis and biological properties of α-thymidine 5'-aryl phosphonates

Article abstract of DOI:10.1134/S1068162013060058

Diethyl(N-arylaminocarbonyl)methyl phosphonates have been obtained by the reaction of dieth-ylphosphonoacetic acid imidazolides with methyl-4- aminobenzoate or 3,5-bis(trifluoromethyl)phenylamine. Their treatment with Me3SiBr in DMF led to a mixture of the corresponding (N-arylaminocarbonylmethyl) phosphonic acids and their monoethyl esters. After separation, they were condensed with 3'-O-acetyl-α-thymidine, which, after the removal of the acetyl protecting group, gave (α-D-thymidine-5'-yl)-[4-aminocarbonyl, methoxycarbonyl-, or carboxy)phenylaminocarbonyl]methyl phosphonates and (α-D-thymidine-5'-yl)-[3,5-bis(trifluoromethyl)phenylaminocarbonyl]methyl phosphonate and their ethyl esters. It was shown that the compounds are stable under different conditions, low toxic (in Vero and K-562 cell cultures), and capable of penetrating into K-562 cells. Only ethyl (α-D-thymidine-5'-yl)- [4-(methoxycarbonyl)phenylaminocarbonyl]methyl phosphonate at a high concentration (200 μg/mL) inhibited in vitro the growth of the laboratory strain M. tuberculosis H37Rv. Pleiades Publishing, Ltd., 2013.

Full text of DOI:10.1134/S1068162013060058

ISSN 1068ꢀ1620, Russian Journal of Bioorganic Chemistry, 2013, Vol. 39, No. 6, pp. 639–648. © Pleiades Publishing, Ltd., 2013.
Original Russian Text © M.A. Ivanov, I.L. Karpenko, L.N. Chernousova, S.N. Andreevskaya, T.G. Smirnova, L.A. Alexandrova, 2013, published in Bioorganicheskaya Khimiya,
2013, Vol. 39, No. 6, pp. 718–727.
Synthesis and Biological Properties of
5'ꢀAryl Phosphonates
αꢀThymidine
M. A. Ivanova, I. L. Karpenkoa, L. N. Chernousovab, S. N. Andreevskayab,
T. G. Smirnovab, and L. A. Alexandrovaa,
1
a Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow, 119991 Russia
b Central Research Institute of Tuberculosis, Russian Academy of Medical Sciences, Yauzskaya alleya 2, 107564 Russia
Received March 20, 2013; in final form, May 24, 2013
Abstract—Diethyl(
ylphosphonoacetic acid imidazolides with methylꢀ4ꢀaminobenzoate or 3,5ꢀbis(trifluoromethyl)phenylꢀ
amine. Their treatment with Me3SiBr in DMF led to a mixture of the corresponding ( ꢀarylaminocarbonꢀ
ylmethyl)phosphonic acids and their monoethyl esters. After separation, they were condensed with 3'ꢀ
acetylꢀ ꢀthymidine, which, after the removal of the acetyl protecting group, gave ( ꢀDꢀthymidineꢀ5'ꢀyl)ꢀ[4ꢀ
aminocarbonylꢀ, methoxycarbonylꢀ, or carboxy)phenylaminocarbonyl]methyl phosphonates and ( ꢀthyꢀ
Nꢀarylaminocarbonyl)methyl phosphonates have been obtained by the reaction of diethꢀ
N
O
ꢀ
α
α
αꢀD
midineꢀ5'ꢀyl)ꢀ[3,5ꢀbis(trifluoromethyl)phenylaminocarbonyl]methyl phosphonate and their ethyl esters. It
was shown that the compounds are stable under different conditions, low toxic (in Vero and Kꢀ562 cell culꢀ
tures), and capable of penetrating into Kꢀ562 cells. Only ethyl (αꢀDꢀthymidineꢀ5'ꢀyl)ꢀ[4ꢀ(methoxycarboꢀ
nyl)phenylaminocarbonyl]methyl phosphonate at a high concentration (200 μg/mL) inhibited in vitro the
growth of the laboratory strain M. tuberculosis H37Rv.
Keywords: nucleosides, αꢀthymidine, phosphonates, synthesis, cytotoxicity, stability, tuberculosis, mycobacteria,
antimycobacterial activity, Mycobacterium tuberculosis
DOI: 10.1134/S1068162013060058
1
INTRODUCTION
Among modified nucleosides that exhibited a
marked antimycobacterial activity in vitro, the inhibiꢀ
tors of M. tuberculosis thymidine monophosphate
kinase (TMPKmt) showed a pronounced antitubercuꢀ
In the 20th century, effective antibiotics and antiviꢀ
ral preparations were created, which made it possible
to significantly reduce the mortality from infectious
diseases. However, to date, most pathogenic microorꢀ
ganisms and viruses have developed resistance to the
main pool of drugs used for their therapy, which necesꢀ
sitates a search for novel classes of compounds inhibitꢀ
ing the growth of pathogenic bacteria and viruses [1].
losis activity [3, 4]. A comparison of 5'ꢀdeoxyꢀ5'ꢀ(
Nꢀ
arylthiocarbamide) derivatives of ꢀthymidine [3]
α
with its arylaminocarbonyl phosphonates, performed
using the Molecular Operating Environment (MOE)
complex [5], demonstrated a spatial similarity of these
compounds.
The goal of this study was the synthesis of αꢀthymiꢀ
Tuberculosis, which is the cause of the annual
deaths of about two million people, ranks second in
the world among infectious diseases next to AIDS [2].
The simultaneous development of the two diseases in
man substantially aggravates the clinical picture of
each disease. In 1993, WHO declared a critical global
situation with tuberculosis. Of special note is the
emergence of novel Mycobacterium tuberculosis multiꢀ
drugꢀresistant and practically incurable “extensively
drugꢀresistant” strains [2], which remain almost unafꢀ
fected by standard chemotherapy schemes. Therefore,
a search for novel antituberculosis drugs is needed.
dine phosphonate derivatives and the study of their
stability under different conditions, the toxicity in cell
cultures, and their inhibitory effect on the growth of
M. tuberculosis
.
RESULTS AND DISCUSSION
α
ꢀThymidine phosphonate derivatives were synꢀ
thesized by the method similar to [6] (Schemes 1 and 2).
The first stage of the synthesis was the condensation of
diethylphosphonoacetic acid (
ing phenylamine. The activation of acid (
I
) with the correspondꢀ
I) through
Abbreviations: CDI,
forming unit; DCC,
N
,
N
'ꢀcarbonyldiimidazole; CFU, colonyꢀ
the formation of imidazolide (CDI in DMF [7]) and
its subsequent reaction with methylꢀ4ꢀaminobenzoate
N,N'ꢀdicyclohexylcarbodiimide; HFBA,
heptafluorobutyric acid; PBS, phosphateꢀbuffered saline.
Corresponding author: phone: +7 (499) 135ꢀ60ꢀ65; fax:
+7 (499) 135ꢀ14ꢀ05; eꢀmail: ala2004_07@mail.ru.
1
or 3,5ꢀbis(trifluoromethyl)phenylamine led to diꢀ
Oꢀ
ethylꢀ( ꢀ(4ꢀmethoxycarbonyl)phenylaminocarboꢀ
N
639

640
IVANOV et al.
nyl)methyl phosphonate (II) and diꢀ
O
ꢀethylꢀ[3,5ꢀ
(
IV), and (XI) with 3'ꢀ
dine in the presence of DCC [8] led to (3'ꢀ
Dꢀthymidineꢀ5'ꢀyl)ꢀ[4ꢀmethoxycarbonyl)phenylamiꢀ
nocarbonyl]methyl phosphonate ( ) and (3'ꢀ ꢀacetylꢀ
ꢀDꢀthymidineꢀ5'ꢀyl)ꢀ[3,5ꢀbis(trifluoromethyl)phenylaꢀ
O
ꢀacetylꢀ
α
ꢀthymidine in pyriꢀ
bis(trifluoromethyl)phenylaminocarbonyl]methyl phosꢀ
Oꢀacetylꢀ
αꢀ
phonate (IX), respectively.
N
ꢀArylaminocarbonylmethylphosphonic
III) and ( ) and their monoethyl esters (IV) and (XI
were obtained by a gentle treatment of diesters (II) and
IX) with trimethylbromosilane in DMF and isolated
by chromatography on DEAE cellulose and reverseꢀ
phase silica gel. The reaction of derivatives (III), (
acids
V
O
(
X
)
α
minocarbonyl]methyl phosphonate (XII) and the corꢀ
responding ethyl esters (VI) and (XIII) (Schemes 1
(
X), and 2).
O
P
O
O
H
O
N
i, ii
O
O
O
HO
P
O
(II)
O
O
(I)
iii
H
H
HO
O
N
N
+
O
O
OH
OH
P
P
O
O
O
O
O
O
(III)
(IV)
iv; v
iv; v
H
O
N
O
O
O
P
H
N
O
HO
N
O
O
O
O
O
(VI)
P
AcO
O
N
O
N
O
H
O
O
(V)
v
i
vii
AcO
O
N
O
H
(VIIIa) (VIIIb)
vi
v
ii
viii
H
O
N
(VIIa)
(VIIb)
(VIIc)
R
O
O
H
P
HO
P
N
N
O
R
O
O
O
O
HO
O
N
N
O
O
O
O
H
(VIIIa): R = OMe
(VIIIb): R = NH2
HO
O
N
O
(VIIa): R = OMe
(VIIb): R = NH2
H
(VIIc): R = OH
i: CDI, DMF;
ii
:
methylꢀ4ꢀaminobenzoate
С;
ꢀacetylꢀαꢀthymidine, Py;
iii: Me3SiBr, DMF, 5
: DCC, 3'ꢀ
: MeONa/MeOH;
°
iv
v:
O
v
i
vii: NH3/H2O/EtOH; viii: NaOH/H2O.
Scheme 1.
α
ꢀDꢀThymidineꢀ5'ꢀyl)ꢀ[4ꢀ(methoxycarbonylꢀ, amiꢀ
(VIIa)–(VIIc) were isolated by column chromatography
nocarbonylꢀ, and carboxy)phenylaminocarboꢀ on reverseꢀphase silica gel LiChroprep RPꢀ8 with a yield
nyl]methyl phosphonates (VIIa)–(VIIc) and their ethyl of 53 (95%), 40 (75%), and 21 mg (80%), respectively.
esters (VIIIa) and (VIIIb) were obtained by treating comꢀ Compounds (VIIIa) and (VIIIb) were isolated by column
pounds (
V) or (VI) with sodium methylate in methanol, chromatography on silica gel in a linear gradient of ethaꢀ
aqueous ammonia, or an aqueous alkaline solution [in nol concentrations in dichloromethane with a yield of 54
the case of (V), respectively (Scheme 1). Compounds (95%) and 45 mg (82%), respectively.
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SYNTHESIS AND BIOLOGICAL PROPERTIES
641
αꢀDꢀThymidineꢀ5'ꢀylꢀ[3,5ꢀbis(trifluoromethyl)pheꢀ thymidine with a water–alcohol ammonia solution led
nylaminocarbonyl]methyl phosphonate (XIV) and its to target products (XIV) and (XV) (Scheme 2), which
ethyl ester (XV) were obtained without the isolation of were then isolated by column chromatography on silꢀ
intermediate acetylated derivatives (XII) and (XIII). ica gel in a linear gradient of ethanol concentrations in
The treatment of the reaction mixture obtained by dichloromethane with a yield of 144 (81%) and 152 mg
coupling compounds (
X
) and (XI) to 3'ꢀ
O
ꢀacetylꢀ
α
ꢀ
(84%), respectively.
O
O
P
F3C
H
i; ii
O
O
N
O
HO
O
P
O
O
(I)
iii
F3C
(IX)
F3C
F3C
H
H
HO
N
O
N
+
OH
OH
P
P
O
O
O
O
F3C
F3C
iv; v
(XI)
(X)
F3C
H
iv; v
O
N
O
O
P
N
O
O
F3C
F3C
AcO
O
N
H
N
O
H
HO
O
O
(XIII)
P
N
O
O
v
i
F3C
AcO
O
N
H
O
(XII)
F3C
H
v
i
O
N
O
O
P
N
O
O
F3C
F3C
HO
H
O
N
O
HO
N
H
O
O
P
(XV)
N
O
O
F3C
HO
O
N
H
O
(XIV)
i: CDI, DMF;
ii
:
3,5ꢀbis(trifluoromethyl)phenylamine
С;
ꢀacetylꢀ
i: NH3/H2O/EtOH.
;
iii: Me3SiBr, DMF, 5
°
iv
: DCC, 3'ꢀ
v
:
O
α
ꢀthymidine, Py;
v
Scheme 2.
The structure of the compounds synthesized was NMR spectra of all compounds contain a characterisꢀ
confirmed by UV, 1Н, 31P, and 13C NMR spectra. tic signal of the phosphonate methylene group with the
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 39
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642
IVANOV et al.
included in the stage of preclinical tests of compounds
with potential medicamentosus activity. An analysis of
possible products of the conversion of compounds
spin–spin coupling constant: JCH P, P = 18.0–21.0 Hz in
2
1H NMR spectra and JCH P, P = 122.0–130.0 Hz in
2
13C NMR spectra [9].
(
VIIa)–(VIIc), (VIIIa), (VIIIb), (XIV), and (XV) was
carried out by reverseꢀphase HPLC. The compounds
were stable under conditions of chemical hydrolysis
for a period of more than 24 h at three different pH
values: 2.2 (glycineꢀHCl), 7.4 (PBS), and 9.0 (glycineꢀ
NaOH).
Ethyl phosphonates (VI), (VIIIa), (VIIIb), and
(
XV) are mixtures of diastereomers. Because they were
not separated during the synthesis, the NMR spectra
of these compounds contain two groups of signals,
which corresponds to a mixture of two isomers in the
ratio of about 1 : 1. The resolution of the NMR specꢀ
trometer made it possible to correlate chemical shifts
and most of the spin–spin coupling constants of each
isomer using the data of [9] (see the Experimental secꢀ
tion). A difference in chemical shifts was observed for
atoms separated by a distance of 1–3, less common 4–
5 bonds from the chiral center, the phosphorus atom,
and was ~0.01 ppm; the spin–spin coupling constant
was ~0.1 Hz. For instance, the signals of the methylꢀ
ene groups of the phosphonoacetate residue of ethylꢀ
The stability of the compounds to enzymatic
hydrolysis was determined using fetal calf serum. All
the compounds were stable under these conditions
over a period of more than 24 h. The cytotoxicity of the
compounds was determined by the methods described
in [10]. Compounds (VIIa)–(VIIc), (VIIIa), (VIIIb),
(
XIV), and (XV) exhibited no toxicity in Vero and
Kꢀ562 cell cultures at concentrations up to 500 and
250 M, respectively, which is close to, or higher than,
μ
the toxicity of most of M. tuberculosis thymidine
monophosphate kinase inhibitors described in the litꢀ
erature [3, 4].
(αꢀDꢀthymidineꢀ5'ꢀyl)ꢀ[4ꢀmethoxycarbonyl)phenylꢀ
aminocarbonyl]methyl phosphonate (VIIIa) isomers
are equal to 3.19 and 3.18 ppm, and the constants
One of the factors affecting the activity of comꢀ
pounds is the rate of their accumulation and possible
conversion inside cells. We examined the accumulaꢀ
tion of phosphonates (VIIa) and (VIIb) in Kꢀ562 cells
(human myelogenous leukemia cell line) by HPLC.
After the incubation of cells with phosphonates, these
compounds were identified reliably in cell lysates. As
seen from the figure, the accumulation of methoxycarꢀ
bonyl derivative (VIIa) significantly exceeds the
amount of 4ꢀaminocarbonyl derivative (VIIb) in the
cell. It is seen from the elution profile that cell lysates
contain no products of the conversion of (VIIa) and
JCH P, P are equal to 21.6 and 21.7 Hz, respectively.
The chemical shifts of the atoms of thymine and aroꢀ
matic residues of the two isomers coincide.
2
The condensation of compounds (III) and (
X) with
α
ꢀthymidine also resulted in the formation of bisthyꢀ
midine derivatives (XVI) and (XVII), which were isoꢀ
lated by preparative TLC with a yield of 6 and 8%
(starting from compounds (III) and (X), respectively.
H
O
N
(VIIb).
R'
O
P O
The antimycobacterial effect of the compounds
O
N
synthesized was studied using a Bactec MGIT960
automated system [11] by measuring the bacteriostatic
activity toward the laboratory strain M. tuberculosis
H37Rv by the method developed earlier [12]. Only
AcO
O
N
O
H
phosphonate
(VIIIa) at a high concentration
(XVI), (XVII)
2
(200 g/mL) inhibited the growth of the mycobacteꢀ
μ
rium, whereas the other compounds appeared to be
inactive.
(XVI): R' =
C(O)OMe
CF3
;
Thus, despite the spatial similarity of 2'ꢀdeoxyꢀ
Dꢀthymidine arylaminocarbonyl phosphonates to
α
α
ꢀ
ꢀ
thymidine 5'ꢀdeoxyꢀ5'ꢀ(
tives, they [except for (VIIIa)] did not inhibit in vitro
the growth of M. tuberculosis
Nꢀarylthiocarbamide) derivaꢀ
(XVII)
:
.
CF3
In bisthymidine derivatives (XVI) and (XVII),
owing to a large volume of nucleoside residues at the
phosphorus atom, the latter adopt different conformaꢀ
tions. As a result, some signals of protons (H1', H6) in
1H NMR spectra are nonequivalent and are present as
a double set.
EXPERIMENTAL
The commercial reagents of the companies Fluka
(Germany), AldrichꢀSigma (United States), and
Acrus (Belgium) were used. Solvents were purified by
standard methods. The staring
αꢀDꢀthymidine and 3'ꢀ
The determination of the stability of compounds
O
ꢀacetylꢀ2'ꢀdeoxyꢀ ꢀDꢀthymidine were obtained as
α
during the chemical and enzymatic hydrolysis is described in [3, 13].
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SYNTHESIS AND BIOLOGICAL PROPERTIES
643
100
Lysate of Kꢀ562
80
60
40
20
0
A264
1.2 nmol
104 cells
80
(VIIa)
10 20 30
0.08 nmol
104 cells
(VIIIb)
40
0
0
10
20
30
40
10
20
30
Retention time, min
Accumulation of
Kꢀ562 cell lysate after a 24ꢀh incubation with the compounds.
is the elution profile of the cell lysate without incubation. For the elution conditions, see the Experimental section.
α
ꢀthymidine phosphonates (VIIa) and (VIIb) in Kꢀ562 myeloid leukemia cells (24 h). Elution profile of the
of (VIIa) = 37 min, and of (VIIb) = 26 min. On the insert
T
T
ret
ret
Column chromatography was carried out using
Kieselgel 60 silica gel (40–63 m), LiChroprep RPꢀ8 bonyl]methyl phosphonate (II). CDI (486 mg, 3 mmol)
(25–40 m) (Merck, Germany), and DEAE cellulose
O,O'ꢀDiethylꢀ[4ꢀ(methoxycarbonyl)phenylaminocarꢀ
μ
was added to a solution of 2ꢀ(diethylphosphono)acetic
acid (392 mg, 2 mmol) in dry DMF (5 mL). The soluꢀ
μ
(Whatman, England); TLC was performed on Kieselꢀ
gel 60 F254 plates (Merck, Germany) in systems: chloꢀ
roform–ethanol 20 : 1 (A), chloroform–ethanol 9 : 1 (B),
chloroform–ethanol 4 : 1 (C), isopropanol–25%
ammonia–water 7 : 1 : 3 (D), dioxane–25% ammonia
8 : 2 (E), or dioxane–25% ammonia–water 6 : 1 : 4 (F).
tion was stirred for 1 h at 20
niline (302 mg, 2 mmol) was added, and the mixture
was kept for 12 h at 37 . The reaction was monitored
°С, 4ꢀmethoxycarbonylaꢀ
°
С
by TLC in system A. Then Н2О (1 mL) was added, and
the mixture was evaporated in vacuo and applied to a
The preparative TLC was carried out on plates (20
20 cm) with Kieselgel 60 F254 silica gel; the thickness of
the layer was 1 mm (Merck, Germany).
×
silica gel column (3.0
tion was carried out in a linear gradient of ethanol conꢀ
centrations in dichloromethane ( 5%). Fractions
containing compound (II) were evaporated in vacuo.
Yield of phosphonate (II): 462 mg (70%); f 0.25 (A);
UV (Н2О), λmax, nm (
, М–1 cm–1): 269 (21850). Н
NMR (DMSOꢀd6 : 10.40 (1 H, br s, NH) 7.92 (2 H,
d, Jm ꢀPh 8.7, ꢀPh), 7.70 (2 H, d, ꢀPh), 4.06 (4 H,
m, (СН2СН3)2), 3.82 (3 Н, s, СН3О), 3.12 (2 H, d,
JCH P, P 0.4, CH2ꢀP), 1.24 (6 H, t, 7.0,
(СН2СН3)2); 31P NMR (D2O)
: 24.19 s.
× 35 cm, 40–63 μm). The eluꢀ
0 →
HPLC was performed in the pseudo ionꢀpair
regime on a Nucleosil Cꢀ18 reverseꢀphase column
R
1
ε
(
4
×
150 mm, 5
a gradient of 80% EtOH in 0.1% HFBA (pH 3): 0%, 5
min; 15%, 20 min; 15 40%, 13 min; and 40 →
μm; Dr. Maisch GmbH, Germany) in
)
δ
m
O
ꢀPh,
о
0
→
→
100%, 7 min.
2
J
2
NMR spectra (δ, ppm, spin–spin coupling conꢀ
δ
stant, Hz) were recorded on an AMX IIIꢀ400 specꢀ
trometer (Bruker, United States) with an working freꢀ
(4ꢀMethoxycarbonylphenylaminocarbonyl)methyl phosꢀ
phonate (III) and ꢀethylꢀ[4ꢀ(methoxycarbonyl)pheꢀ
nylaminocarbonyl)methyl phosphonate (IV). Me3SiBr
(0.5 mL, 3.75 mmol) was added at to a solution of
1
O
quency of 400 MHz for H NMR [internal standard
Me4Si for DMSOꢀd6 and sodium 3ꢀ(trimethylsilyl)ꢀ1ꢀ
propane sulfonate (DSS) for D2O], 101 MHz for
13C NMR (internal standard methanol), and 162 MHz
for 31P NMR (with the suppression of phosphorus proton
spin–spin coupling; internal standard 85% phosphoric
5°С
compound (II) (247 mg, 0.75 mmol) in dry DMF
(5 mL), the reaction mixture was stirred for 12 h at
5°С, a 25% NH3 solution (10 mL) was added, and the
acid). UV spectra were recorded on a UVꢀ2401P specꢀ mixture was evaporated in vacuo. The resulting residue
trophotometer (Shimadzu, Japan). was dissolved in 10% aqueous ethanol (200 mL) and
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644
IVANOV et al.
applied to a column (7.5
×
25 cm) with DEAE celluꢀ phonate (VI). DCC (500 mg, 1.5 mmol) was added to
a solution of compound (IV) (103 mg, 0.3 mmol) and
lose (HCO–3 ). The column was washed with 10%
3'ꢀ
Oꢀacetylꢀ
α
ꢀDꢀthymidine (129 mg, 0.45 mmol) in
for 12 h.
aqueous ethanol (150 mL) and eluted in a linear gradiꢀ
ent of (NH4)HCO3 concentrations in 10% water ethꢀ
anol (0 → 0.2 M, 800 mL). Fractions containing comꢀ
pounds (III) and (IV) were evaporated in vacuo to dryꢀ
ness, dissolved in Н2О (1 mL), and applied to a
LiChroprep RPꢀ8 column (
elution was with 0.01 M (NH4)HCO3.
dry pyridine. The mixture was kept at 37
°С
The reaction was monitored by TLC in system B.
Then H2O (1 mL) was added and the mixture was
evaporated in vacuo. Compound (VI) was purified on
preparative plates for TLC using ethyl acetate as an
2 × 15 cm, 25–40 μm);
eluent. Yield of (VI): 164 mg (90%);
(CH3OH), λmax, nm (
, М–1 cm–1): 265 (27050).
1Н NMR (DMSOꢀd6
: 11.25 (1 H, br s, ArꢀNH),
11.50 (1 H, s, 3ꢀNH), 7.91 (2 H, d, Jm 8.7
Rf 0.66 (B); UV
ε
Fractions containing target compounds were lyoꢀ
philized. The yield of the ammonium salt of III
99 mg (30%); Rf 0.49 (D); UV (Н2О), λmax, nm (
, М–1
: 11.34
ꢀPh),
ꢀPh), 3.80 (3 Н, s, СН3О), 2.58 (2 H, d,
JCH P, P 18.9, CH2ꢀP); 31P NMR (D2O): 12.55 s. The
)
δ
(
)
, mꢀ
ꢀPh, оꢀPh
ε
Ph), 7.70 (2 H, d
о
ꢀPh), 7.56 (1 H, s, H6), 6.18, 6.16
1
cm–1): 270 (22360). Н NMR (DMSOꢀd6
) δ
(1 H (dd, 2.7, 1.2, isomer A; dd,
J
J
2.7, 1.2, isomer B)
(1 H, br s, NH) 7.82 (2 H, d, Jm 8.7
,
m
H1'), 5.19 (1 H, m, H3'), 4.69 (1 H, m, H4'), 4.15–
ꢀPh, оꢀPh
7.67 (2 H, d,
о
4.07 (4 H, m, СН2CH3, H5'), 3.82 (3 H, s, СН3О),
3.22, 3.21 (2 H (d, JCH P, P 21.6, isomer A; d, JCH P, P
2
2
2
yield of the ammonium salt of
(
IV) 74 mg, (22.5%);
21.6, isomer B) CH2ꢀP), 2.75 (1 H, m, H2'), 2.13, 2.09
(1 H (m, isomer A; m, isomer B) H2''), 1.99 (3 H, s,
СН3С(О)), 1.80 (3 H, s, 5ꢀCH3), 1.258, 1.254 (3 H (t,
R
f 0.57 (D); UV (Н2О), λmax, nm (
ε
, М–1 cm–1): 272
1
(22150). Н NMR (DMSOꢀd6
)
δ: 10.16 (1 H, br s,
J
6.9, isomer A; t,
J
7.1, isomer B) CH2CH3); 31P NMR
NH) 7.84 (2 H, d, Jm ꢀPh 8.7
,
m
ꢀPh), 7.70 (2 H, d,
ꢀPh,
о
(DMSOꢀd6 : 25.14 s, isomer A; 25.01 s, isomer B.
)
δ
о
ꢀPh), 3.80 (3 Н, s, СН3О), 3.73 (2 H, d q, JCH CH ~
2 3
JPOCH , P 7.0 СН2СН3), 2.53 (2 H, d, JCH P, P 18.6,
O α
ꢀ( ꢀDꢀThymidineꢀ5'ꢀyl)ꢀ[4ꢀ(methoxycarbonyl)pheꢀ
2
2
nylaminocarbonyl]methyl phosphonate (VIIa). A 1 M
CH2ꢀP), 1.08 (3 H, t, СН2СН3); 31P NMR (DMSOꢀd6
: 13.10 s.
ꢀ(3'ꢀ
)
solution of MeONa (0.3 mL) in MeOH was added to
a solution of compound (V) (58 mg, 0.1 mmol) in anhyꢀ
drous MeOH (50 mL). The solution was stirred for 1 h,
СН3СООН (0.04 mL) was added, and the mixture was
evaporated in vacuo to dryness. The residue was
δ
O
OꢀAcetylꢀαꢀDꢀthymidineꢀ5'ꢀyl)ꢀ[4ꢀmethoxyꢀ
carbonyl)phenylaminocarbonyl]methyl phosphonate
(V). DCC (500 mg, 1.5 mmol) was added to a solution
of compound (III) (100 mg, 0.3 mmol) and 3'ꢀOꢀ
coevaporated with Н2О (2
(1 mL), and applied to a LiChroprep RPꢀ8 column
25 cm, 25–40 m). The reaction was monitored
× 3 mL), dissolved in Н2О
acetylꢀαꢀthymidine (129 mg, 0.45 mmol) in dry pyriꢀ
dine. The mixture was kept at 37°C for 12 h. The reacꢀ
tion was monitored by TLC in system D. Then, Н2О
(3 mL) was added, the mixture was evaporated in
vacuo, and the residue was dissolved in 10% aqueous
ethanol (200 mL) and applied to a with DEAE celluꢀ
(
2
×
μ
by TLC in system (D). Fractions containing comꢀ
pound (VIIa) were evaporated in vacuo. Yield of
VIIa): 53 mg (95%); Rf 0.59 (D); UV (Н2О), λmax, nm
(
1
(
ε
, М–1 cm–1): 272 (27340). Н NMR (D2O)
ꢀPh 8.7 ꢀPh), 7.51 (2 H, d, ꢀPh), 7.32
(1 H, s, H6), 5.86 (1 H, dd, 7.1, 4.0, H1'), 4.49 (2 H,
m, H3', H4'), 4.03 (1 H, ddd, J5a
δ: 7.84
lose (HCO–3 ) (3.5
× 25 cm). The column was
(2 H, d, Jm
,
m
о
ꢀPh,
о
washed with 10% aqueous ethanol (200 mL), target
compounds were eluted in a linear gradient of
(NH4)HCO3 concentrations in 10% aqueous ethanol
J
'
'
'
,
,
5b, 11.2, J5a, P
a ), 3.89 (1 H, m, H5'b), 3.85 (3 H, s,
СН3О), 2.92 (2 H, d, JCH P, P 20.3, CH2ꢀP), 2.62
4.9,
'
'
J5a, 4 , 2.6, Н5
(
0
→
0.15 M, 600 mL). Fractions containing comꢀ
pound ( ) were combined and evaporated in vacuo to
dryness. The yield of ammonium salt ): 154 mg
(88%); f 0.65 (D); UV (CH3OH), λmax, nm (
, М–1
cm–1): 265 (26 950). Н NMR (DMSOꢀd6
: 11.03
(1 H, s, 3ꢀNH), 7.87 (2 H, d, Jm 8.7 ꢀPh),
ꢀPh), 7.47 (1 H, s, H6), 5.86 (1 H, dd,
7.1, 4.0, H1'), 5.19 (1 H, m, H3'), 4.54 (1 H, m, H4'),
V
2
(V
(1 H, m, H2'), 1.93 (1 H, ddd,
J
14.4, 4.0, 3.9, H2''),
: 16.40 s;
R
ε
δ
1.74 (3 H, s, 5ꢀCH3); 31P NMR (D2O)
δ
1
)
13C NMR (D2O): 171.10 (C(O)Ph), 169.86 (d, JС(О), Р
5.3, C(O)CH2), 168.86 (C4), 153.69 (C2), 145.04
,
m
ꢀPh, оꢀPh
7.64 (2 H, d,
о
(
ipsoꢀPh), 140.26 (C6), 133.11 (
Ph), 122.52 ( ꢀPh), 112.91 (C5), 89.54 (C1'), 89.29
(d, JC4', P 7.6 C4'), 73.62 (C3'), 67.41 (d, JC5', P .8,
mꢀPh), 127.52 (pꢀ
J
o
3.84–3.78 (4 H, m, СН3О, H5'a), 3.72 (1 H, m,
5
H5'b), 3.33 (2 H, d, JCH P, P 22.5, CH2ꢀP), 2.74 (1 H,
2
C5'), 55.31 (CH3O), 42.31 (C2'), 40.20 (d, JCH P, P
2
m, H2'), 2.00–1.95 (4 H, m, СН3С(О), H2''), 1.79 (3
122.0, CH2ꢀP), 14.24 (5ꢀСH3).
H, s, 5ꢀCH3); 31P NMR (D2O)
δ
: 13.27 s.
ꢀDꢀthymidineꢀ5'ꢀyl)ꢀ[4ꢀ
(methoxycarbonyl)phenylaminocarbonyl]methyl phosꢀ laminocarbonyl]methyl phosphonate (VIIb). An aqueꢀ
O
ꢀEthylꢀ
O
'ꢀ(3'ꢀ
O
ꢀacetylꢀ
α
Oꢀ(αꢀDꢀThymidineꢀ5'ꢀyl)ꢀ[4ꢀ(aminocarbonyl)phenyꢀ
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 39
No. 6
2013

SYNTHESIS AND BIOLOGICAL PROPERTIES
645
ous NH3 (25%) solution (50 mL) was added to a soluꢀ The reaction was monitored by TLC in system (B).
The residue was applied to a silica gel column (2.0
25 cm, 40–63 m). The elution was carried out in a
linear gradient of ethanol concentrations in dichloꢀ
romethane ( 9%). Fractions containing comꢀ
pound (VIIIa) were evaporated in vacuo. The yield of
VIIIa): 54 mg (95%); Rf 0.36 (B); UV (СН3ОН),
×
tion of compound (V) (58 mg, 0.1 mmol) in ethanol
(100 mL). The solution was kept for 12 h, evaporated
in vacuo to dryness, and the residue was coevaporated
μ
0
→
with Н2О (2
applied to a LiChroprep RPꢀ8 column (
reaction was monitored by TLC in system (D). Fracꢀ
tions containing compound (VIIb) were evaporated in λmax, nm (
vacuo. The yield of ammonium salt (VIIb): 40 mg
(75%); f 0.55 (D); UV (Н2О), λmax, nm (
, М–1 cm–1):
268 (20000). 1Н NMR (D2O)
: 7.67 (2 H, d, Jm
8.7 ꢀPh), 7.48 (2 H, d, ꢀPh), 7.41 (1 H, s, H6), 5.85
(1 H, dd, 7.1, 3.9, H1'), 4.37 (2 H, m, H3', H4'), 4.02
(2 H, ddd, J5a
3.89 (1 H, ddd, H5'b) 2.91 (2 H, d, JCH P, P 20.2, CH2ꢀ
×
3
mL), dissolved in Н2О (1 mL), and
2
×
25 cm). The
(
1
ε
, М–1 cm–1): 270 (22620). Н NMR
: 11.21 (1 H, s, ArꢀNH), 10.40, 10.41
(1 H (s, isomer A; s, isomer B) 3ꢀNH), 7.91 (2 H, d,
Jm 8.5 ꢀPh), 7.72 (1 H, s, H6), 7.69 (2 H, d,
(DMSOꢀd6) δ
R
ε
m
δ
ꢀPh, оꢀPh
ꢀPh, оꢀPh
o
ꢀPh), 6.14, 6.13 (1 H (dd,
7.4, 3.9, isomer B) H1'), 5.46 (1 H, d, J3'OH,3'
J
7.4, 3.6, isomer A; dd,
J
,
m
о
J
3
.3, 3'ꢀ
'
' '
'
,
,
5b, 11.2, J5a, P , 5.1, J5a, 4' 3.1, H5'a,),
OH), 4.32 (1 H, m, H3'), 4.27 (1 H, m, H4'), 4.05 (4
H, m, H5', CH2CH3), 3.82 (3 H, s, CH3O), 3.19, 3.18
(2 H (d, JCH P, P 21.6, isomer A; d, JCH P, P 21.7, isoꢀ
2
2
2
P), 2.62 (1 H, m, H2'), 1.93 (1 H, ddd,
H2''), 1.74 (3 H, s, 5ꢀCH3); 31P NMR (D2O): 16.40 s;
13C NMR (D2O)
: 174.27 (NH2C(O)), 171.00 (d,
JС(О), Р 5.3, C(O)CH2), 169.20 (C4), 153.97 (C2),
143.86 (ipsoꢀPh), 140.47 (C6), 131.19 ( ꢀPh), 130.86
ꢀPh), 123.00 ( ꢀPh), 113.12 (C5), 89.64 (C1'), 89.37
(d, JС4', Р 7.1 C4'), 73.46 (C3'), 67.43 (d, JC5', P .7,
J 14.4, 3.7, 3.7,
mer B) CH2ꢀP), 2.57 (1 H, m, H2'), 1.93, 1.92 (1 H,
m, H2''), 1.77 (3 H, s, 5ꢀCH3), 1.24, 1.25 (3 H (t, 7.0
isomer A; t,
7.0, isomer B) CH2CH3), 31P NMR
(DMSOꢀd6 : 24.92 s, isomer A; 24.79 s, isomer B.
J
,
δ
J
)
δ
m
(
p
o
O
ꢀEthylꢀO' ꢀ(αꢀDꢀthymidineꢀ5' ꢀyl)ꢀ[4ꢀ(aminocarꢀ
bonyl)phenylaminocarbonyl]methyl phosphonate
5
(VIIIb). An aqueous solution (50 mL) of NH3 (25%)
was added to a solution of compound (VI) (61 mg,
0.1 mmol) in EtOH (100 mL). The solution was kept
for 12 h, evaporated in vacuo to dryness, and coevapꢀ
C5'), 42.17 (C2'), 40.10 (d, JCH P, P 122.5 CH2ꢀP),
2
14.30 (5ꢀСH3).
Oꢀ(αꢀDꢀThymidineꢀ5'ꢀyl)ꢀ[4ꢀ(carboxyphenylamiꢀ
nocarbonyl]methyl phosphonate (VIIc). A 1 M soluꢀ
tion of NaOH in water (0.2 mL) was added to a soluꢀ
orated with Н2О (2
tored by TLC in system (C). The residue was applied
to a silica gel column (2.0 25 cm, 40–63 m).
The elution was carried out in a linear gradient of ethꢀ
anol concentrations in dichloromethane ( 15%).
× 3 mL). The reaction was moniꢀ
tion of compound (V) (29 mg, 0.05 mmol) in Н2О
×
μ
(100 mL). The solution was stirred for 1 h, evaporated
in vacuo to dryness, after which 0.04 mL of
СН3СООН was added. The solution was evaporated
in vacuo to dryness, and the residue was coevaporated
0
→
Fractions containing compound (VIIIb) were evapoꢀ
rated in vacuo. The yield of (VIIIb): 45 mg (82%);
ε
, М–1 cm–1):
with Н2О (2
applied to a LiChroprep RPꢀ8 column (
25–40 m). The reaction was monitored by TLC in
system (D). Fractions containing compound (VIIc
were evaporated in vacuo. The yield of product (VIIc):
21 mg (80%); Rf 0.48 (D); UV (Н2О), λmax, nm (
, М–1
cm–1): 269.1 (23000). 1Н NMR (DMSOꢀd6
: 10.70 (1
H, s, 3ꢀNH), 7.81 (2 H, d, 8.7 ꢀPh), 7.73 (1 H, s,
H6), 7.60 (1 H, d, ꢀPh), 6.08 (1 H, dd, 7.6, 3.1,
×
3
mL), dissolved in Н2О (1 mL), and
Rf 0.25 (C); UV (СН3ОН), λmax, nm (
272 (28050). 1Н NMR (DMSOꢀd6
: 11.20 (1 H, br
s, ArꢀNH), 10.29, 10,28 (1 H, s, 3ꢀNH), 7.82 (3 H, d,
Jm ꢀPh 8.7 ꢀPh, s, Hа NH2), 7.74, 7.73 (1 H (s,
isomer A; s, isomer B) H6), 7.61 (2 H, d, ꢀPh), 7.19
(1 H, s, Hb, NH2) 6.16, 6.15 (1 H (dd, 7.6, 3.6, isoꢀ
mer A; dd, 7.4, 3.9, isomer B) H1'), 5.48 (1 H, d,
J3'OH,3' .4, 3'ꢀOH), 4.27 (1 H, m, H3'), 4.32 (1 H, m,
2
×
25 cm,
)
δ
μ
)
,
m
,
ꢀPh,
о
о
ε
J
)
δ
J
J
m
3
о
J
H4'), 4.05 (4 H, m, H5', OCH2CH3), 3.17, 3.16 (2 H
(d, JCH P, P 21.6, isomer A; d, JCH P, P 21.6, isomer B)
H1'), 4.28 (1 H, m, H4'), 4.23 (1 H, m, H3'), 3.71 (2
2
2
H, m, H5'), 3.18 (2 H, d, JCH P, P 20.6, CH2ꢀP), 2.59
2
CH2ꢀP), 2.58 (1 H, m, H2'), 1.94, 1.93 (1 H (ddd,
14.3, 3.8, 3.6, isomer A; ddd, 14.1, 3.6, 3.4, isomer
B) H2''), 1.78 (3 Н, s, 5ꢀСН3), 1.25, 1.24 (3 H (t, 7.0
isomer A; t, 7.0, isomer B) CH2CH3)
31P NMR
(DMSOꢀd6 : 25.15 s, isomer A; 25.02 s, isomer B;
13C NMR (DMSOꢀd6
: 167.29 (2 H, s, NH2C(O)),
163.76 (C4), 163.16 (d, JC4', P 5.8, C(O)CH2), 150.44
(C2), 141.30 (ipsoꢀPh) 136.68 (C6), 129.11 ( ꢀPh),
128.37 ( ꢀPh), 118.17 ( ꢀPh), 108.85 (C5), 85.95,
85.80 ((d, JC4', P 6.7, isomer A; d, JC4', P 7.1, isomer B)
(1 H, m, H2'), 1.83 (1 H, m, H2''), 1.76 (3 H, s, 5ꢀ
J
J
CH3); 31P NMR (DMSOꢀd6
)
δ: 13.60 s.
J
,
O
ꢀEthylꢀO'ꢀ(αꢀDꢀthymidineꢀ5'ꢀyl)ꢀ[4ꢀ(methoxyꢀ
J
;
carbonyl)phenylaminocarbonyl]methyl phosphonate
)
δ
(VIIIa). A 1 M solution of MeONa (0.3 mL) in MeOH
was added to a solution of compound (V) (61 mg,
)
δ
0.1 mmol) in anhydrous MeOH (50 mL). The solution
was stirred for 1 h, CH3COOH (0.04 mL) was added,
and the mixture was evaporated in vacuo to dryness.
p
m
o
The residue was coevaporated with Н2О (2
× 3 mL).
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 39
No. 6
2013

646
IVANOV et al.
1
, М–1 cm–1): 244 (12460). Н NMR
: 12.18 (1 H, br s, ArꢀNH), 8.08 (2 H, s,
ꢀPh), 3.92 (2 H, d q, JCH CH ~
3
C4'), 85.17, 85.12 [(s, isomer A; s, isomer B) C1'],
70.78, 70.05 [(s, isomer A; s, isomer B) C3'], 65.48 (m,
λmax, nm (
(DMSOꢀd6
ꢀPh), 7.22 (1 H, s,
ε
δ
)
C5'), 62.12, 62.09 ((d, JPOCH , P 6.2, isomer A; d,
O
p
2
2
JPOCH , P
5.8, isomer B) CH2CH3), 39.70 (C2'), 35.79
JPOCH , P 7.0 СН2СН3), 2.78 (2 H, d, JPOCH , P 20.5,
2
2
2
CH2ꢀP), 1.18 (3 H, t, СН2СН3);
31P NMR (DMSOꢀ
(d,
J
CH2P, P
131.9 CH2ꢀP), 16.14, 16.07 (1 C (s, isomer
d6)
δ: 13.57 s.
A; s, isomer B) CH2CH3), 12.23 (5ꢀСH3).
O,O'ꢀDiethylꢀ[3,5ꢀbis(trifluoromethyl)phenylamiꢀ
O
ꢀ( ꢀDꢀThymidineꢀ5'ꢀyl)ꢀ[3,5ꢀbis(trifluoromeꢀ
α
nocarbonyl]methyl phosphonate (IX). CDI (486 mg,
thyl)phenylaminocarbonyl]methyl phosphonate (XIV).
3 mmol) was added to a solution of 2ꢀ(diethꢀ
DCC (500 mg, 1.5 mmol) was added to a solution of
ylphosphono)acetic acid (
DMF (5 mL). The solution was stirred at 20
3,5ꢀbis(trifluoromethyl)phenylamine (458 mg, 2 mmol)
was added, and the mixture was kept at 37 for 5 h.
I
) (392 mg, 2 mmol) in dry
compound (
ꢀthymidine (129 mg, 0.45 mmol) in dry pyridine.
The mixture was kept at 37 for 12 h, Н2О (3 mL)
was added, and the solution was evaporated in vacuo.
The residue was dissolved in ethanol (150 mL), a 25%
aqueous NH3 solution (50 mL) was added, and the
X) (116 mg, 0.3 mmol) and 3'ꢀOꢀacetylꢀ
°С
for 1 h,
α
°С
°С
The reaction was monitored by TLC in system (A).
Then Н2О (1 mL) was added, and the mixture was
evaporated in vacuo and applied to a silica gel column
reaction mixture was kept at 5°С for 12 h. The reacꢀ
(
3.0
romethane. Fractions containing compound (IX
were evaporated in vacuo. The yield of diester IX):
612 mg (75%); Rf 0.57 (A); UV (EtOH), λmax, nm
, М–1 cm–1): 242 (12650). 1Н NMR (DMSOꢀd6
12.42 (1 H, br s, ArꢀNH), 8.16 (2 H, s, ꢀPh), 7.22 (1
H, s, ꢀPh), 4.25 (2 H, m, СН2СН3), 3.37 (2 H, d,
JCH P, P 20.9, CH2ꢀP), 1.34 (3 H, t,
7.1 СН2СН3); 31P
× 35 cm). The elution was carried out in dichloꢀ
tion was monitored by TLC in system (E). Then the
mixture was evaporated in vacuo, the residue was disꢀ
solved in 15% aqueous ethanol (100 mL), and the
solution was applied to a column with DEAE cellulose
HCO–3 ) (3.5
× 25 cm). The column was washed with
15% aqueous ethanol (200 mL), and the target comꢀ
pound was eluted in a linear gradient of (NH4)HCO3
concentrations in 15% aqueous ethanol (0 → 0.25 M,
600 mL). Fractions containing compound (XIV) were
combined and evaporated in vacuo to dryness. The
)
(
(
ε
) δ:
(
o
p
J
2
NMR (DMSOꢀd6
[3,5ꢀBis(trifluoromethyl)phenylaminocarbonyl]methyl
phosphonate (X) and ꢀethylꢀ[3,5ꢀbis(trifluoromeꢀ
) δ: 24.66 s.
yield of the ammonium salt of
(
XIV): 144 mg (81%); Rf
, М–1 cm–1): 248
(14185). Н NMR (D2O): 7.99 (2 H, s, ꢀPh), 7.75
(1 H, s, ꢀPh), 7.46 (1 H, s, H6), 5.86 (1 H, dd, 6.8
O
0.65 (E); UV (CН3ОH), λmax, nm (
ε
thyl)phenylaminocarbonyl]methyl phosphonate (XI).
To a solution of compound (IX) (408 mg, 1.0 mmol) in
dry DMF (7 mL), Me3SiBr (0.66 mL, 5.0 mmol) was
1
o
p
J
,
added at 5°С, the reaction mixture was stirred at 5°С
3.2, H1'), 4.41 (2 H, m, H3', H4'), 4.05 (1 H, m,
for 12 h, a 25% NH3 solution (15 mL) was added, and
the mixture was evaporated in vacuo. The reaction was
monitored by TLC in system (D). The resulting resiꢀ
due was dissolved in 10% alcohol (300 mL) and
H5'a), 3.92 (1 H, m, H5'b), 2.96 (2 H, d, JPOCH , P
2
20.5, CH2ꢀP), 2.66 (1 H, m, H2'), 1.97 (1 H, m, H2''),
1.76 (3 Н, s, 5ꢀСН3), 31P NMR (D2O): 16.10 s.
applied to a column with DEAE cellulose (HCO3–
)
O
ꢀEthylꢀ
romethyl)phenylaminocarbonyl]methyl
(XV). DCC (500 mg, 1.5 mmol) was added to a soluꢀ
tion of compound ( ) (116 mg, 0.3 mmol) and 3'ꢀ
acetylꢀ ꢀthymidine (129 mg, 0.45 mmol) in dry pyriꢀ
dine. The mixture was kept at 37 for 12 h. Then Н2О
O
'ꢀ(
α
ꢀDꢀthymidineꢀ5' ꢀyl)ꢀ[3,5ꢀbis(trifluoꢀ
phosphonate
(7.5 25 cm). The column was washed with 10%
×
aqueous ethanol (200 mL) and then in a linear gradiꢀ
ent of (NH4)HCO3 concentrations in 10% aqueous
X
Oꢀ
α
ethanol (0.05
ing compound (IX) or (
dryness, dissolved in Н2О (1 mL), and applied to a
LiChroprep RPꢀ8 column ( 15 cm). The elution
→
0.2 M, 1000 mL). Fractions containꢀ
°С
X) were evaporated in vacuo to
(3 mL) was added, and the reaction mixture was evapꢀ
orated in vacuo. The residue was dissolved in ethanol
(150 mL), a 25% aqueous NH3 solution (50 mL) was
added, and the reaction mixture was kept for 12 h at
2
×
was carried out with 0.01 M (NH4)HCO3. Fractions
containing target compounds were lyophilized, and
5°С. The reaction was monitored by TLC in system
the ammonium salts of compounds (
obtained. The yield of the ammonium salt of
(32%); f 0.62 (D); UV (EtOH), λmax, nm (
, М–1 cm–1):
242.4 (11890). 1Н NMR (DMSOꢀd6
: 12.56 (1 H, br
s, ArꢀNH), 8.14 (2 H, s, ꢀPh), 7.19 (1 H, s, ꢀPh),
2.83 (2 H, d, JCH P, P 20.7, CH2ꢀP), 31P NMR
X
) or (XI) were
(B). Compound (XV) was purified on preparative
plates for TLC, using ethyl acetate saturated with
water as an eluent. The yield of methyl phosphonate
(X) 123 mg
R
ε
)
δ
(
XV): 152 mg (84%);
nm (
, М–1 cm–1): 248.6 (14185). 1Н NMR (DMSOꢀ
d6 : 11.20 (1 H, s, ArꢀNH), 10.80 (1 H, s, 3ꢀNH),
8.20 (2 H, s, ꢀPh), 7.75 (1 H, s, ꢀPh), 7.71 (1 H, s,
H6), 6.13, 6.12 (1 H (dd, 7.6, 3.8, isomer A; dd,
Rf 0.66 (B); UV (CН3ОH), λmax,
o
p
ε
δ
2
)
(DMSOꢀd6
)
δ: 13.02 s. The yield of the ammonium
о
p
salt of XI) 74 mg (28.0%),
(
Rf 0.77 (D). UV (EtOH),
J
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 39
No. 6
2013

SYNTHESIS AND BIOLOGICAL PROPERTIES
647
Penetration and accumulation of compounds in the
J
4.3, 8.2, isomer B) H1'), 5.48 (1 H, d, J3'OH,3' 3.5, 3'ꢀ
Kꢀ562 cell culture. Suspension of Kꢀ562 cells (2.14
×
OH), 4.33 (1 H, m, H4'), 4.26 (1 H, m, H3'), 4.06
(4 H, m, H5', CH2CH3), 3.20 (2 H, d, JCH P, P 21.6,
106 cells/mL, 5 mL) grown in flasks (25 cm2) was incuꢀ
2
bated with test compounds at a concentration of 1 mM
(5% СO2, humidity 90%, 37°C) for 1, 5, and 24 h.
Cells were separated from the culture medium by sedꢀ
CH2ꢀP), 2.57 (1 H, m, H2'), 1.93 (1 H, m, H2''), 1.76
(3 Н, s, 5ꢀСН3), 1.26, 1.25 (3 H (t,
6.9, isomer B) CH2CH3)
31P NMR (DMSOꢀ
24.39 s, isomer A; 24.27 s, isomer B; 13C NMR
(DMSOꢀd6 : 164.09 (1 C, d, JС(О), Р 5.8, C(O)CH2),
163.81 (C4), 150.44 (C2), 140.56 (ipsoꢀPh), 136.68
(C6), 130.88 (q, Jm 33.3, ꢀPh), 123.11 (q, J 272.8
ꢀPh), 116.42 (m, ꢀPh), 108.87 (C5),
J 6.9, isomer A; t,
J
;
d
6): imentation on a Kꢀ23 centrifuge (1000 rpm, 10 min,
4°C), washed twice from the preparation by resuspenꢀ
sion in PBS, and centrifuged (3500 rpm, 5 min). Then
cells were resuspended in an equal volume of PBS
diluted in the ratio 1 : 10, destructed by adding 6%
trichloroacetic acid to a final concentration of 3%
(v/v), and left at −20°C for 10 min. After centrifugaꢀ
tion (13400 rpm, 7 min), the acidꢀinsoluble fraction
)
δ
m
ꢀC, F
CF3), 118.74 (
o
p
85.91 (1 H, m, C4'), 85.20, 85.17 (C1'), 70.71, 70.76
(C3'), 65.69, 65.62 ((d, JC5', P 6.7, isomer A; d, JC5', P
was removed, and the supernatant was neutralized by
adding a saturated Na2CO3 solution (4.3 μL per 100 μL
8
.0, isomer B) C5'), 62.32, 62.30 ((d, JPOCH , P
6.2,
2
of a sample). The resulting samples of the Kꢀ562 cell
lysate were analyzed by HPLC under conditions indiꢀ
cated above. Compounds were identified by the retenꢀ
tion time specified for authentic controls. The amount
of tested compound in the cell lysate was estimated
from the calibration curve (mV/nmol).
isomer A; d, JPOCH , P
6.2, isomer B) CH2CH3), 39.70
2
(C2'), 35.94 (d, JCH P, P 131.9 CH2ꢀP), 16.14, 16.08
2
((s, isomer A; s, isomer B) CH2CH3), 12.22 (5ꢀСH3).
O,O' ꢀDiꢀ(3' ꢀOꢀacetylꢀαꢀDꢀthymidineꢀ5' ꢀyl)ꢀ[4ꢀ
(methoxycarbonyl)phenylaminocarbonyl]methyl phosꢀ
phonate (XVI). Rf 0.72 (D); UV (CH3OH), λmax, nm
Mycobacterium strain. The trials of preparations
were carried out using the laboratory strain M. tuberꢀ
culosis H37Rv, which is sensitive to antituberculosis
drugs. Mycobacteria were transformed into a suspenꢀ
sion of single cells in the same growth phase and norꢀ
malized by CFU [14]. The enriched Dubois medium
(Difco) was used.
(
ε
, М–1 cm–1): 265 (36480). 1Н NMR (DMSOꢀd6
)
ꢀPh 8.9
s, H6),
δ
,
:
10.61 (1 H, br s, ArꢀNH), 7.89 (2 H, d, Jm
ꢀPh,
2
о
m
ꢀPh), 7.71 (2 H, d,
o
ꢀPh), 7.49, 7.48 (2 H,
×
6.17, 6.15 (1 H,
2
×
dd,
J
2.5, 1.7, H1'), 5.18 (2 H, m,
H3'), 4.69 (2 H, m, H4'), 4.20 – 4.12 (4 H, m, H5'),
3.37 (2 H, d, JCH P, P 22.5, CH2ꢀP), 3.81 (3 Н, s,
2
СН3О), 2.75 (2 H, m, H2'), 2.13—2.08 (2 H, m,
Estimation of the efficiency of compounds. The
H2''), 1.98, 1.97 (6 Н, 2
× s, СН3С(О)), 1.79 (6 H, s, effect of compounds on the growth of the mycobacteꢀ
5ꢀCH3); 31P NMR (DMSOꢀd6
) δ: 25.95 s.
rial strain was examined for 42 days on a Bactec MGIT
960 automated system for the detection of growth
(BD, United States) by the standard method [11]. A
O,O ꢀDiꢀ(3 ꢀ ꢀacetylꢀ( ꢀDꢀthymidineꢀ5 ꢀyl)ꢀ[3,5ꢀ
O
α
'
'
'
bis(trifluoromethyl)phenylaminocarbonyl]methyl phosꢀ
phonate (XVII). f 0.81 (D); UV (EtOH), λmax, nm (
М–1 cm–1): 244 (21480). 1Н NMR (DMSOꢀd6
: 11.23
(2 H, s, 3ꢀNH), 10.95 (1 H, br s, ArꢀNH), 8.22 (2 H,
s, ꢀPh), 7.74 (1 H, s, ꢀPh), 7.50, 7.49 (2 H, s,
H6), 6.15 (2 H, dd, 7.1, 2.5, H1'), 5.18 (2 H, m,
mycobacterial suspension (500 μL) was inoculated in
R
ε,
7.9 mL of nutrient medium. The final concentration of
M. tuberculosis in the sample was 105–106 CFU/mL.
Each sample including the control samples lacking the
tested compound were studied in triplicate. The antiꢀ
mycobacterial effect of a compound was estimated
from the dynamics of growth of M. tuberculosis H37Rv
in the presence of different concentrations of the prepꢀ
aration as compared with the growth of the strain in a
medium containing no compound. The detection of
growth was carried out every hour automatically and
was recorded using the software program Epicenter
(BD, United States).
)
δ
о
p
2 ×
J
H3'), 4.71 (2 H, m, H4'), 4.18 (4 H, m, H5'), 3.35
(2 H, d, JPOCH , P 20.2, CH2ꢀP), 2.74 (2 H, m, H2'),
2
2.11 (2 H, m, H2''), 1.97, 1.96 (6 Н, 2
×
s, СН3С(О)),
: 25.26 s.
1.79 (6 Н, s, 5ꢀСН3); 31P NMR (DMSOꢀd6
) δ
Experiments on Cell Cultures
The toxicity of compounds was determined in Vero
cell culture (epithelial kidney cells of African green
monkey) and Kꢀ562 cell culture (human myelogenous
leukemia cells) (both from the collection of cell culꢀ
tures of Ivanovskii Research Institute of Virology,
Ministry of Public Health and Social Development of
Russia). The cytopathic effect of compounds was
tested by MTT assay after 72 h of incubation [10].
ACKNOWLEDGMENTS
This work was supported by the Russian Foundaꢀ
tion for Basic Research (project nos. 11ꢀ04ꢀ00603 and
13ꢀ04ꢀ91441) and the basic research program of the
Presidium of the Russian Academy of Sciences
“Molecular and Cellular Biology.”
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 39
No. 6
2013

648
IVANOV et al.
8. Hauptmann, Z., Graefe, Yu, and Remane, H., Organꢀ
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Translated by S. Sidorova
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 39
No. 6
2013