A new approach to the synthesis of optically active alkylated adenine derivatives

Article abstract of DOI:10.1016/j.bmcl.2003.12.107

A new synthesis of chiral acyclic nucleoside and nucleotide analogues starting from D(-)- or L(+)-riboses was proposed. Antiviral properties of the synthesized compounds towards the pox virus family were evaluated.

Full text of DOI:10.1016/j.bmcl.2003.12.107

Bioorganic & Medicinal Chemistry Letters 14 (2004) 3357–3360
A new approach to the synthesis of optically active
alkylated adenine derivatives
N. F. Zakirova,a,* A. V. Shipitsyn,a E. F. Belanovb and M. V. Jaskoa
aEngelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilov St., Moscow 119991, Russian Federation
bSRC Virology and Biotechnology ‘Vector’, Koltsovo 630059, Russian Federation
Received 21 October 2003; revised 29 November 2003; accepted 5 December 2003
Abstract—A new synthesis of chiral acyclic nucleoside and nucleotide analogues starting from
Antiviral properties of the synthesized compounds towards the pox virus family were evaluated.
D
())- or L(+)-riboses was proposed.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
ods suffer serious disadvantages. In particular, they are
multi-step and laborious and are targeted at the syn-
thesis of the analogues of one type.
Acyclic nucleoside and nucleotide analogues are cur-
rently used as antiviral drugs possessing the broad
spectrum of activities.1–3 These compounds are targeted
at inhibition of viral DNA polymerases and require
intracellular phosphorylation to become active. Addi-
tionally, adenine derivatives of this type can inhibit S-
We describe herein the synthesis of optically active (S)-
and (R)-9-(3-hydroxy-2-oxy-((R,S)-1-methoxy-2-hydr-
oxyethyl)propyl)adenines (1a and 1b), which can be used
as key intermediates in the synthesis of HPMPA and its
analogues and (S)-DHPA-like nucleoside analogues.
The proposed uniform scheme allows the preparation of
both acyclic nucleosides and nucleotides.
adenosyl-L-homocysteine hydrolase (SAH hydrolase)
another enzyme essential for the viral life cycle.4;5
The reported syntheses of 3-hydroxy-2-phosphonyl-
methoxypropyl (HPMP) derivatives involve either
alkylation of purine or pyrimidine bases with an acyclic
fragment containing a phosphonomethyl group6 or
phosphonomethylation of the corresponding acyclic
nucleoside analogue.7 Although the preparation of
(R,S)-3-(adenin-9-yl)-2-hydroxypropanoic acid [(R,S)-
AHPA] has been described,8 chiral AHPA isomers have
only been obtained as byproducts in the process of the
synthesis of eritadenine and its derivatives.9 A major
approach to the synthesis of (S)-9-(2,3-dihydroxyprop-
yl)adenine [(S)-DHPA] is based on the adenine coupling
of with (S)-1,2-O-isopropylidene-3-tosyl-sn-glycerol.10
In addition, (S)-DHPA and its analogues can be
obtained by oxidation of the propenyladenine double
bond to the vicinal diol system,11 but this procedure did
not allow an optically active product.10 All these meth-
2. Chemistry
The starting compounds for the synthesis of synthons of
S-series (1a) and R-series (1b), which are commonly
used for the synthesis of various acyclic nucleoside
analogues, were obtained from commercially available
L
(+)-ribose (2a) and
D
())-ribose (2b), respectively (Fig.
1).
Ribose (2a) was methylated with methanol in the pres-
ence of hydrogen chloride obtained in situ by the reac-
tion of acetyl chloride with methanol (Scheme 1). In
A
A
1
3
3
1
2
2
OH
OH
O
4
O
HO
HO
5
5
4
OMe
OMe
Keywords: Acyclic nucleosides and nucleotides; Synthesis.
* Corresponding author. Tel.: +7-095-135-22-55; fax: +7-095-135-14-
05; e-mail: naucik@aport2000.ru
1a
1b
Figure 1. Key intermediates 1a and 1b.
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.12.107

3358
N. F. Zakirova et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3357–3360
HO
TsO
positions and shapes. For example, in compound 1a this
O
O
a
L (+) - ribose
2a
b
OMe
OH
OMe
OH
proton resonance was observed as two doublet of dou-
blets at 3.03 and 3.19 ppm with J3;2 4.7 Hz and J3a;3b
11.9 Hz, whereas for 1b it appeared as a doublet at
3.35 ppm with J3;2 5.0 Hz. This may imply the lack of
equivalence of the C3 protons in the S-isomer (1a) and
their equivalence in the R-isomer (1b).12
90%
90%
HO
HO
4a
3a
c
40%
Ade
Ade
Ade
HO
O
OH
O
O
d
e
OMe
OH
O
85%
The resulting chiral 1a and 1b were used for the syn-
thesis of both nucleotide-like HPMPA analogues and
nucleoside-like analogues.
HO
O
OMe
OMe
5a
1a
6a
Scheme 1. Reagents and conditions: (a) AcCl/MeOH, rt, 16 h; (b)
TsCl/Py, 4 °C, 16 h; (c) Adeꢀ/DMF, 80 °C, 5 days; (d) NaIO4, 4 °C, 1 h;
(e) NaBH4, 4 °C, 3 h.
Alkylation of compound 1b with an excess of iodo-
methylphosphonic acid in pyridine in the presence of
DCC resulted in phosphonate 7b, which was isolated in
a 20% yield by successive ion-exchange chromatography
on DEAE-Spheron and reverse-phase chromatography
on LiChroprep RP-18. Compound 7b was hydrolyzed
with 60% formic acid for 24 h at 37 °C to give 30-iodo-
methylphosphonate of (R)-DHPA (8b) in an 80% yield.
It is worth noting that no hydrolysis of 7b was observed
in the presence of Dowex 50 (Hþ) for 12h at room
temperature.
addition to a,b-methylribofuranoside (3a), approxi-
mately 10% of a,b-methylribopyranoside was formed.
The crude reaction mixture was treated with tosyl
chloride in pyridine at 4 °C to give 5-tosyl(methyl)ribo-
side (4a) in a yield of 90%. The distribution of the
reaction mixture between chloroform and a saturated
NaHCO3 solution allowed the isolation of 90% of tos-
ylate (4a). A key stage was the coupling of adenine with
5-tosylriboside (4a) under anhydrous conditions at 80 °C
in the presence of NaH. Unreacted tosylate (4a) was
removed by the distribution of the reaction mixture
between chloroform and water. An excess of adenine
was removed using ion-exchange chromatography on
Dowex 1 (OHꢀ). The subsequent column chromato-
Since the overall yield of 30-iodomethylphosphonate (8b)
was low, we used another method of preparation
(Scheme 3). Synthon 1b was either treated with 60%
formic acid for 24 h at 37 °C or with Dowex 50 (Hþ) for
6 h to give (R)-DHPA (9b) nearly quantitatively. The
target product 9b was eluted from Dowex 50 (Hþ) with
1 M aqueous ammonia with more than 95% purity. It
should be mentioned that the acid-induced cleavage of
the diacetal residue of diphosphonate 7b (Scheme 2)
proceeded much more slowly than that of compound 1b.
The synthesis of (S)-DHPA (9a) was carried out from
graphy on LiChroprep RP-8 afforded the target (L)-9-
(a,b-1-methyl-5-ribofuranosyl)adenine (5a) in a yield of
40%.
The diol system of riboside (5a) was oxidized with an
aqueous sodium periodate solution (4 °C, 1 h). The
inorganic salts were precipitated with ethanol, and the
supernatant containing dialdehyde (6a) was treated with
an aqueous solution of NaBH4 to give the optically
active (S)-9-(3-hydroxy-2-oxy-[(R,S)-1-methoxy-2-hy-
droxyethyl)propyl]adenine (1a). Compound 1a was iso-
lated using ion-exchange chromatography on Dowex 50
(NH4þ) in a good yield (85%). The total yield of chiral
1
compound 1a according to Scheme 3. The H NMR
spectral data for the (S)- and (R)-isomers (9a and 9b)
were identical.13
Chirality of 9a and 9b was confirmed by the data of
circular dichroism. As was expected, the spectral pat-
Ade
Ade
O
P
1a relative to commercially available
achieved 28%.
L-ribose (2a)
Ade
CH2I
OH
O
O
P
OH
a
b
O
O
CH2I
OH
P
O
20%
80%
HO
O
OH
IH2C
OMe
OMe
7b
OH
The synthesis of 1b was performed from
D
())-ribose
1b
8b
O
using the scheme similar to the synthesis of 1a (Scheme
1). The total yield of compound 1b was varied from 15%
to 30% and depended on the nature of the base used in
the reaction of adenine coupling with 5-tosylriboside
(4b). For example, the yields of the product (5b) were
20% in the presence of potassium bis(trimethylsilyl-
amide) (K-HMDS) and 40% in the presence of NaH or a
1:5 mixture of Cs2CO3–K2CO3.
Scheme 2. Reagents and conditions: (a) ICH2P(O)(OH)2/DCC/Py, rt,
28 h; (b) Hþ, 2 4 h.
Ade
Ade
O
P
a
b
1b
OH
O
CH2I
95%
35%
OH
OH
OH
9b
8b
c,d
81%
c
According to the NMR analysis, compounds 1a and 1b
are diastereomers differing in the configuration at the C2
atom. Chemical shifts of the proton resonances at C4
and the methoxy group of 1a were observed at 4.17 and
3.21 ppm, respectively, whereas in the case of 1b they
were 4.43 and 2.64 ppm, respectively. In addition, the
resonances of the C3 protons also differed in their
Ade
Ade
O
P
OH
e
O
P
10b
OH
O
O
90%
O
HO
OH
11b
10b
Scheme 3. Reagents and conditions: (a) Hþ, 6 h; (b) ICH2P(O)(OH)2/
DCC/Py, 16 h; (c) NaH/DMF, 6 h; (d) H2O, 3 h; (e) glacial AcOH, 3 h.

N. F. Zakirova et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3357–3360
3359
Ade
4
3
O
Ade
Ade
O
OH
9b
9a
O
b
2
c
a
O
OH
5a
O
60%
1
95%
HO
OH
13a
O
OMe
12a
OMe
0
O
6a
-1
-2
-3
-4
-5
Scheme 4. Reagents and conditions: (a) NaIO4, 4 °C, 1 h; (b) KMnO4
or RuCl3 (pH 13), 12h; (c) Dowex 50 (H þ), 12h.
190
210
230
250
270
290
310
Wavelength,nm
The NMR spectral patterns for 12a and 12b were vir-
tually identical, with the exception of the resonances of
methoxy groups. In particular, for 12a the chemical shift
of these protons was observed at 3.08 ppm, whereas in
the case of 12b it was at 2.64 ppm.15 After acid hydro-
lysis of 12a or 12b, optically active (S)- or (R)-3-(adenin-
9-yl)-2-hydroxypropanoic acid (13a or 13b, respectively)
were obtained. In particular, in the presence of 60%
HCOOH (24 h, 20 °C) compounds 12a and 12b were
hydrolyzed to give about 70% of 13a or 13b, respec-
tively, whereas treatment of 12a or 12b with Dowex 50
(Hþ) (12h, 20 °C) yielded these products in nearly
quantitative yields. The products were eluted with a 1 M
ammonia solution from a Dowex 50 (Hþ) column, and
the purity exceeded 95%. The NMR spectra for the
isomers agreed well with that described for (R)-AHPA.9
Figure 2. CD spectra of (S)-DHPA (9a) and (R)-DHPA (9b) at 2 0°C
in water.
terns of these compounds were observed as curves with
negative and positive elipticity, respectively (Fig. 2).
The reaction of (R)-DHPA (9b) with 1.5 equiv of
iodomethylphosphonic acid in the presence of DCC in
pyridine resulted in iodomethylphosphonate 8b (35%).
When treated with 5 equiv NaH in DMF for 6 h, com-
pound 8b underwent an intermolecular cyclization to
give derivative 10b. The treatment of the reaction mix-
ture with glacial acetic acid allowed the isolation of
cyclo-HPMPA in a yield of about 90%. If the reaction
mixture was treated with water, the isolated product was
(R)-HPMPA. Its yield after chromatographic purifica-
tion exceeded 90%. The corresponding (S)-cyclo-
HPMPA (10a) and (S)-HPMPA (11a) were obtained in
a similar fashion from (S)-DHPA (9a).
3. Biology
All of the synthesized compounds were evaluated as
potential antiviral agents and were tested in Vero cell
cultures against vaccinia virus (VV), monkeypox (MPV)
and cowpox (CPV) viruses. The antiviral activity of the
compounds under study is shown in Table 1. It can be
mentioned that the activities against VV and CPV of 9a
and 11a synthesized by us were very close to those de-
scribed earlier for these analogues.2;16
The structures of the synthesized compounds were
confirmed by NMR spectroscopy data and coincided
with the published data.14 Figure 3 presents CD spectral
patterns of compounds 11a and 11b.
We also synthesized some nucleoside analogues starting
from chiral synthons 1a and 1b. First, we tried oxidation
of compound 1b with potassium permanganate (pH 13).
However, since the yield of the target (R)-3-(adenin-9-
yl)-2-oxy-((R,S)-1-methoxy-1-carboxy)propanoic acid
(12b) only was 10%, we used another approach (Scheme
In addition, compounds 1b, 9a and 13a inhibited the
replication of small pox virus (strain India 3a) in Vero
cells with IC50 values of 62, 41 and 91 lM, respectively.
They did not exhibit toxicity up to 500 lM.
4). Oxidation of
(L)-9-(a,b-1-methyl-5-ribofurano-
syl)adenine (5a) with aqueous solution of NaIO4 re-
sulted in dialdehyde 6a, which was further oxidized
under alkaline conditions (pH 13) with either RuCl3 or
KMnO4. The yield of acid 12a was 60% in both cases,
and isomeric 12b was obtained in a similar yield.
To summarize, we have developed a new approach to
the synthesis of chiral (both S- and R-) 9-(3-hydroxy-2-
oxy-((R,S)-1-methoxy-2-hydroxyethyl)-propyl)adenine
(1a and 1b) starting from commercially available
D
())-
4
3
Table 1
11b
2
Compound
IC50, lM
CC50, lM
1
0
VV
34
MPV
CPV
9a
9b
54
>500
48
49
>500
61
>500
>500
>350
>350
>300
>300
-1
>500
41
>350
11a
-2
-3
-4
10a
10b
11a
11b
>350
22
>300
>350
13
>300
190
210
230
250
270
290
310
12.5
>300
Wavelength, nm
Figure 3. CD spectra of (S)-HPMPA (11a) and (R)-HPMPA (11b) at
20 °C in water.
IC50––concentration, at which the virus replication was inhibited by
50%; CC50––concentration, at which cell viability was reduced by 50%.

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N. F. Zakirova et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3357–3360
7. Holy, A.; Rosenberg, I. Collect. Czech. Chem. Commun.
L(+)-riboses, which allowed the preparation of a
or
1987, 52, 2775.
number of acyclic nucleoside and nucleotide analogues.
Some of these analogues showed antiviral properties in
cell cultures infected with the viruses of the pox family.
8. Holy, A. Collect. Czech. Chem. Commun. 1984, 49, 2148.
9. Kawazu, M.; Kanno, T.; Yamamura, S.; Mizaguchi, T.;
Saito, S. J. Org. Chem. 1973, 38, 2887.
10. Holy, A.; Ivanova, G. S. Nucl. Acids Res. 1974, 1, 19.
11. Wang, G. X.; Wang, L.; Zhao, Z. Z.; Zhang, T.; Tao, P. Z.
Yao Xue Xue Bao 1997, 32, 188.
Acknowledgements
12. 1a: (D2O) d 8.11 (s, 1H, H-8 Ade), 8.02(s, 1H, H-2Ade),
4.23–4.36 (m, 2H, H-1), 4.17 (t, 1H, H-4), 4.01–4.06 (m,
1H, H-2), 3.64 (dd, 1H, H-5a, J5a;5b ¼ 12:2Hz and
J5a;4 ¼ 4:5 Hz), 3.58 (dd, 1H, H-5b, J5b;4 ¼ 4:5 Hz), 3.21
(s, 3H, OMe), 3.19 (dd, 1H, H-3a, J3a;3b ¼ 11:9 Hz and
J3a;2 ¼ 4:7 Hz), 3.03 (dd, 1H, H-3b, J3b;2 ¼ 4:7 Hz); 1b:
(D2O) d 8.02(s, 1H, H-8 Ado), 7.97 (s, 1H, H-2Ado),
4.17–4.28 (m, 2H, H-1), 4.43 (t, 1H, H-4), 4.00–4.05 (m,
1H, H-2), 3.71 (dd, 1H, H-5a, J5a;5b ¼ 12:5 Hz and
J5a;4 ¼ 4:5 Hz), 3.58 (dd, 1H, H-5bJ5a;4 ¼ 4:5 Hz), 3.35
(dd, 2H, H-3, J3;2 ¼ 5:0 Hz), 2.64 (s, 3H, OMe).
13. 9a and 9b: (D2O) d 7.87 (s, 1H, H-8 Ade), 7.84 (s, 1H, H-2
Ade), 4.13 (dd, 1H, H-1a, J1a;1b ¼ 14:2Hz and
J1a;2 ¼ 1:6 Hz), 3.97 (dd, 1H, H-1b, J1b;2 ¼ 8:1 Hz), 3.89–
3.94 (m, 1H, H-2), 3.42 (dd, 1H, H-3a, J3a;3b ¼ 11:8 Hz and
J3a;2 ¼ 4:6 Hz), 3.53 (dd, 1H, H-3b, J3b;2 ¼ 4:6 Hz).
14. Rosenberg, I.; Holy, A. Collect. Czech. Chem. Commun.
1987, 52, 2792.
The work was supported by International Science and
Technology Center (project 1989) and Russian Foun-
dation for Basic Research (project 02-04-49009).
The authors are grateful to Dr. A. N. Surovaya
(Engelhardt Institute of Molecular Biology, Russian
Academy of Sciences) for the registration of CD spectra.
References and notes
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15. 12a: (D2O) d 8.06 (s, 1H, H-8 Ade), 7.98 (s, 1H, H-2Ade),
4.21–4.45 (m, 3H, H-1, H-2 and H-4), 3.08 (s, 3H, OMe);
12b: (D2O) d 7.94 (s, 1H, H-8 Ade), 7.91 (s, 1H, H-2Ade),
4.54 (s, 1H, H-4), 4.11–4.34 (m, 2H, H-1 and H-2), 2.64 (s,
3H, OMe).
6. Bronson, J. J.; Ghazzouli, I.; Hitchcock, M. J.; Webb,
R. R., II; Martin, J. C. J. Med. Chem. 1989, 32, 1457.
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