Synthesis of conjugates of 2′,3′-dideoxynucleoside-5′- monophosphates with α-amino acids

Article abstract of DOI:10.1007/s10600-006-0080-z

Previously undescribed conjugates of 2′,3′-dideoxyuridine- 5′-monophosphate-(L)-methoxytryptophylphosphoramidate (5) and 2′,3′-dideoxycytidine-5′-monophosphate-(L)- methoxytryptophylphosphoramidate (7) were isolated by a chemical enzymatic method in order to study their pharmacological properties and to prepare new medicinal preparations based on them. 2006 Springer Science+Business Media, Inc.

Full text of DOI:10.1007/s10600-006-0080-z

Chemistry of Natural Compounds, Vol. 42, No. 2, 2006
SYNTHESIS OF CONJUGATES OF 2′,3′-DIDEOXYNUCLEOSIDE-
5
′-MONOPHOSPHATES WITH α-AMINO ACIDS
1
2
2
E. V. Zhernosek, S. V. Kvach, L. A. Eroshevskaya,
UDC 547.92+577.113.3+577.152.271
1
2
E. N. Kalinichenko, and A. I. Zinchenko
Previously undescribed conjugates of 2′,3′-dideoxyuridine-5′-monophosphate-(L)-
methoxytryptophylphosphoramidate (5) and 2′,3′-dideoxycytidine-5′-monophosphate-(L)-
methoxytryptophylphosphoramidate (7)were isolated by a chemical enzymatic method in order to study their
pharmacological properties and to prepare new medicinal preparations based on them.
Key words: nucleotides, nucleosides, 2′,3′-dideoxycytidine, 2′,3′-dideoxyuridine, conjugates, L-tryptophan,
cytidinedeaminase, nucleosidephosphotransferase, Escherichia coli, Erwinia herbicola.
Until now, most synthesized 2′,3′-dideoxyanalogs of natural nucleosides (2′,3′-dideoxynucleosides) have exhibited
distinct antiviral activitytoward human immunodeficiencyvirus (HIV) [1]. Certain representives ofthis group are preparations
such as3′-azidothymidine(AZT), 2′,3′-dideoxycytidine(1, ddC), 2′,3′-dideoxyinosine(ddI), and(-)-3′-thio-2′,3′-dideoxycytidine
(
3TC) have been used clinically to treat HIV-infected patients. The exception is 2′,3′-dideoxyuridine (2, ddU), which has
practically no antiviral activity. On the other hand, it is known that the 5′-triphosphate derivative of this nucleoside,
′,3′-dideoxyuridine-5′-triphosphate (ddU-5′-TP) is a potent (and specific!) inhibitor of HIV reverse transcriptase [2]. This
2
contradiction is explained by the very low rate of the first step in metabolic activation of a nucleoside in the cell, conversion of
it intothe5′-monophosphatederivative, 2′,3′-dideoxyuridine-5′-monophosphate (3, ddU-5′-MP). Thelowpermeabilitythrough
the cell membrane of ddU-5′-MP prevents its use in medical procedures. Furthermore, ddU-5′-MP, like other nucleoside-5′-
monophosphates, is easily dephosphorylated in the intercellular medium and on the surface of cell membranes by
phosphomonoesterase.
Conjugates of nucleoside-5′-monophosphates with certain natural compounds can be used to circumvent partially the
deficiencies of these compounds mentioned above. The resulting "pronucleotides" possess increased lipophilicity and, as a
result, permeate more easilythrough cell membranes. Metabolism ofthe conjugate within the cell liberates active nucleoside-5′-
monophosphate. This avoids the monophosphorylation of the nucleoside. Furthermore, such compounds are rather stable
because they are not substrates for phosphohydrolases and can be viewed as a unique storage form of the preparation. Data on
the activity of certain conjugates of AZT toward viral types resistant to the starting preparation are also known [3].
The use ofnucleotides bonded through a phosphoramide bond tonatural aromaticα-aminoacids as pronucleotides was
proposed earlier [4]. Such conjugates exhibited several advantages, namely stability and good solubility in water owing to the
residual negative charge. Such a conjugate of AZT with L-tryptophan methyl ester turned out to be eight times more active than
starting AZT and less toxic.
We proposed and demonstrated experimentally a rational scheme for chemical enzymatic transformation of ddC (1)
prepared in several steps from cytidine [5] into the two prodrugs 2′,3′-dideoxyuridine-5′-monophosphate-(L)-
methoxytryptophylphosphoramidate (5) [ddU-5′-MP-(L)-TrpOMe] and 2′,3′-dideoxycytidine-5′-monophosphate-(L)-
methoxytryptophylphosphoramidate (7) [ddC-5′-MP-(L)-TrpOMe], which are conjugates of ddU-5′-MP (3) and ddC-5′-MP (6)
with L-tryptophan methyl ester (4).
1
) Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus′, 220141, Belarus′, Minsk, ul.
Kuprevicha, 5/2, e-mail: zhernosek@iboch.bas-net.by; 2) Institute of Microbiology, National Academyof Sciences of Belarus′,
20141, Belarus′, Minsk, ul. Kuprevicha, 2, e-mail: zinch@mbio.bas-net.by. Translated from Khimiya Prirodnykh Soedinenii,
2
No. 2, pp. 173-175, March-April, 2006. Original article submitted December 2, 2005.
©
0
009-3130/06/4202-0208 2006 Springer Science+Business Media, Inc.
2
08

The preparative scheme includes deamination of 1 by Escherichia coli BM-11 cells [6], which exhibit
cytidinedeaminase (KF 3.5.4.5) activity, 5′-monophosphorylation of 1 and the product of its deamination ddU (2) by
nucleosidephosphotransferase(KF2.7.1.77)within Erwiniaherbicola47/3cells[7], and, finally, chemicalsynthesisofthetarget
products 5 and 7 by dicyclohexylcarbodiimide (DCC) mixed with ddU-5′-MP (3) or ddC-5′-MP (6) and 4 in aqueous t-butanol
according to the modified procedure proposed in the literature [8].
NH
O
O
2
N
N
NH
O
N
O
N
O
N
O
HO
HO
O
P
O
O
O
O
O
1
2
3
NH
4, DCC, t-BuOH, H2O
2
N
O
NH
O
N
O
NH
O
P
O
O
O
N
O
O
MeO
NH
P
O
O
O
O
6
NH
2
NH
O
5
4
, DCC, t-BuOH, H2O
N
N
O
MeO
NH
P
O
O
O
O
7
The structures of the prepared compounds were confirmed by spectral data. Thus, PMR spectra of conjugates 5 and
contained signals for the carbohydrate fragment and the pyrimidine base, for the indole group at 6.9-7.8 ppm, and a signal
7
for the O-methyl. The UV spectra of the synthesized compounds exhibited absorption bands near 220 nm, characteristic of
tryptophan.
A study of the enzymatic deamination of 7 by bacterial cytidinedeaminase under the conditions described in the
literature [6] showed that this conjugate is stable to cytidinedeaminase of E. coli because even traces of its deamination were
not found after 24 h whereas starting 1 under these same conditions was transformed into 2 almost quantitatively after 2 h.
Thus, we prepared for the first time new conjugates of ddU and ddC with L-tryptophan methyl ester (5 and 7) and
developed a microbiological method to prepare ddU-5′-MP (3) and ddC-5′-MP (6) starting from 1. The synthesized 5 and 7
were sent for biotesting of their antiviral activity.
EXPERIMENTAL
Melting points were determined on a Kofler block and are uncorrected. UV spectra of solutions were taken on a
Specord M-400 instrument; PMR spectra, on a Bruker AC-200 NMR spectrometer at working frequency 200 M Hz with TMS
internal standard. BiomassofErw. herbicola47/3bacteria cells,which exhibit nucleosidephosphotransferaseactivity(capability
to transfer phosphate from p-nitrophenylphosphate to nucleoside 5′-hydroxyl), were prepared as before [7]. Cells of E. coli
BM-11, which possess high cytidinedeaminase activity, were grown as before [6]. The sorbent for preparative column
chromatographywas silica gel 60H (70-230 mesh, Merck). The course ofreactions was monitored byTLC using Silufol-UV254
plates (Serva).
209

2
′,3′-Dideoxyuridine (ddU) (2). A reaction mixture (11.0 mL) containing 1 (70.4 mg, 0.33 mol), Tris-HCl-buffer
30 mM, pH 7.25), and E. coli BM-11 cells (0.2% dry mass) was incubated and stirred on a magnetic stirrer for 3.5 h at 50°C.
The course of the deamination was monitored using TLC of aliquots of the reaction mixture and CHCl :CH OH (4:1). Based
(
3
3
on UV spectroscopy, the transformation of ddC into ddU was quantitative. After the reaction was complete, the mixture was
evaporated, placed on a silica-gel column, and eluted by CHCl :CH OH (5:1, 200 mL). Fractions containing product were
3
3
evaporated to afford 2 (66.1 mg, 94%), mp 113-115°C (acetone:diethylether), lit. [9] mp 116-117°C (acetone:diethylether).
TLC, R 0.75 (CHCl :CH OH, 4:1). UV spectrum (dist. H O, λ, nm, ε): 262 max (9600), 232 min (3500).
f
3
3
2
PMR spectrum (CD OD, δ, ppm, J/Hz): 8.08 (1H, d, J = 8, H-6), 6.04 (1H, m, H-1′), 5.70 (1H, d, J5,6 = 8, H-5), 4.16
3
5,6
(
(
1H, m, H-4′), 3.88 (1H, dd, J4′,5′ = 4, J5′,5′′ = 11, H-5′), 3.68 (1H, dd, J4′,5′′ = 4, J5′,5′′ = 11, H-5″), 2.40 (1H, m, H-2′), 2.16-1.9
3H, m, H-2″, H-3′, H-3″). The reaction mixture was used in the next step without work up or product isolation.
2
′,3′-Dideoxyuridine-5′-monophosphate (3). A reaction mixture (11.0 mL) containing 2 (70.7 mg, 0.33 mM),
p-nitrophenylphosphate (0.15 M), sodium-acetate buffer (0.2 M, pH 4.2), and Erw. herbicola 47/3 cells (0.5% dry mass) was
incubated and stirred on a magnetic stirrer for 19 h at 35°C. The course of the reaction was monitored using TLC and
CHCl :C H OH (4:1) and then i-propanol:conc. NH OH:H O (7:1:2). Based on UV data, the transformation of 2 into its
3
2
5
4
2
5
′-monophosphate 3 was 49%. After the reaction was finished, cells were removed from the mixture by centrifugation
-
(
10,000 g, 5 min). The supernatant was placed on a column (10 mL) with Dowex 2×10 resin in the Cl form. Washing the
column with NaCl solution (0.1 M) and HCl gradient (60 mL, 0→40 mM) produced 3, which was then desalted over a column
(
2 mL) of activated carbon and eluted with ethanol (30%) containing ammonia (1 M). The effluent was evaporated. The
+
resulting monophosphate 3 was converted to the H -form by a standard procedure (over Dowex 50W × 2 resin, 200-400 mesh
in the H -form) to produce 3 (30 mg, 31%). TLC, R 0.18 (isopropanol:NH OH:H O, 7:1:2). UV spectrum (H O, λ, nm, ε):
+
f
4
2
2
2
62 max (9700), 230 min (3800).
′,3′-Dideoxyuridine-5′-monophosphate-(L)-methoxytryptophylphosphoramidate (5). Asolution ofL-tryptophan
2
methyl ester (4, 25 mg, 0.115 mmol) and 3 (16 mg, 0.05 mmol) in a mixture of t-butanol and water (4:1, 1 mL) was treated with
DCC (30 mg, 0.135 mmol). The reaction mixture was heated at 65-70°C for 7 h with stirring on a magnetic stirrer. The course
of the reaction was monitored by TLC. After the reaction was finished, the mixture was cooled, evaporated, and
chromatographed over silica gel with elution byCHCl :CH OH:H O(40:10:2, 100mL)andthen CHCl :CH OH:H O (30:15:3,
3
3
2
3
3
2
2
50 mL) to produce 5 (20 mg, 71%, oil), R 0.39 (CHCl :CH OH:H O, 15:5:1). UV spectrum (H O, λ, nm, ε): 220 max
f
3 3 2 2
(31,000), 266 max (9700), 205 min (22,000), 243 min (5300).
PMR spectrum (CD OD, δ, ppm, J/Hz): 7.92 (1H, d, J = 8, H-6), 7.52 (1H, d, J = 8, H-4, indole), 7.30 (1H, d,
3
5,6
J = 8, H-7, indole), 7.10 (1H, s, H-2, indole), 7.06 (1H, t, J = 8, H-6, indole), 6.98 (1H, t, J = 8, H-5, indole), 5.96 (1H, m, H-1′),
.72 (1H, d, J5,6 = 8, H-5), 4.12 (1H, m, CHCO CH ), 3.98 (1H, m, H-4′), 3.82 (1H, m, H-5′), 3.70 (1H, m, H-5″), 3.60 (3H,
5
2
3
s, OCH ), 3.16 (2H, d, J = 7, CH -indole), 2.26 (1H, m, H-2′), 2.04-1.74 (3H, m, H-2″, H-3′, H-3″).
3
2
2
′,3′-Dideoxycytidine-5′-monophosphate (6). A reaction mixture (20 mL) containing ddC (1, 84 mg, 0.4 mmol),
p-nitrophenylphosphate (0.15 M), sodium-acetate buffer (0.2 M, pH 4.2), and Erw. herbicola 47/3 cells (0.5% dry mass) was
incubated for 12 h at 35°C. After the reaction was finished (TLC), the target product 6 was isolated as described above for the
synthesis of 3 to produce 6 (32 mg, 28%) Yield 43% according to UV spectroscopy, R 0.27 (isopropanol:NH OH:H O, 7:1:2).
f
4
2
UV spectrum (H O, λ, nm, ε): 278 max (10,400), 243 min (2500).
2
2
′,3′-Dideoxycytidine-5′-monophosphate-(L)-methoxytryptophylphosphoramidate (7). A solution of 4 (50 mg,
0
0
.23 mmol) and 6 (50 mg, 0.15 mmol) in a mixture of t-butanol and water (4 mL, 4:1) was treated with DCC (30 mg,
.135 mmol). The reaction mixture was heated at 65-70°C for 3 h with stirring on a magnetic stirrer and treated with another
portion of DCC (30 mg, 0.135 mmol). The course of the reaction was monitored by TLC. After the reaction was finished, the
reaction mixture was worked up analogouslyto 5 to produce 7 (62 mg, 70%, oil), R 0.23 (CHCl :CH OH:H O, 10:6:1), R 0.75
f
3
3
2
f
[
(CH ) CHOH:NH OH:H O, 7:1:2]. UV spectrum (H O, λ, nm, ε): 220 max (30,600), 248 min (5400), 272 max (10,000).
3 2 4 2 2
PMR spectrum (CD OD, δ, ppm, J/Hz): 8.06 (1H, d, J = 8, H-6), 7.52 (1H, d, J = 8, H-4, indole), 7.30 (1H, d,
3
5,6
J = 8, H-7, indole), 7.10 (1H, s, H-2, indole), 7.06 (1H, t, J = 8, H-6, indole), 6.96 (1H, t, J = 8, H-5, indole), 5.98 (1H, m, H-1′),
.94 (1H, d, J5,6 = 8, H-5), 4.12 (1H, m, CHCO CH ), 4.02 (1H, m, H-4′), 3.92 (1H, m, H-5′), 3.74 (1H, m, H-5″), 3.56 (3H,
5
2
3
s, OCH ), 3.16 (2H, d, J = 7, CH -indole), 2.30 (1H, m, H-2′), 2.04-1.76 (3H, m, H-2″, H-3′, H-3″).
3
2
2
10

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