Synthesis of novel acyclic nucleoside analogues with anti-retroviral activity

Article abstract of DOI:10.1080/15257770.2010.501776

A series of novel acyclic thymine nucleoside analogues were prepared by the Mitsunobu reaction from appropriately protected chiral triols. The enantiomeric triols were obtained from substituted-lactone acids, prepared by asymmetric oxidation of 3-substituted-1,2-cyclopentanediones. The cytotoxic activity of new analogues was evaluated on MCF-7 human breast cancer and HeLa cells, and antiviral activities on human immunodeficiency virus type 1 and hepatitis C virus models. The synthesized compounds revealed specific anti-retroviral activity and no cytotoxic side effects. Taylor and Francis Group, LLC.

Full text of DOI:10.1080/15257770.2010.501776

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Synthesis of Novel Acyclic Nucleoside
Analogues with Anti-Retroviral Activity
A. Paju a , M. Päri a , A. Selyutina b , E. Žusinaite b , A. Merits b , T.
Pehk c , K. Siirde d , A.-M. Müürisepp a , T. Kailas a & M. Lopp a
a Department of Chemistry , Tallinn University of Technology ,
Tallinn, Estonia
b Institute of Technology , Tartu University , Tartu, Estonia
c National Institute of Chemical and Biological Physics , Tallinn,
Estonia
d AS Competence Centre for Cancer Research , Tallinn, Estonia
Published online: 11 Aug 2010.
To cite this article: A. Paju , M. Päri , A. Selyutina , E. Žusinaite , A. Merits , T. Pehk , K. Siirde ,
A.-M. Müürisepp , T. Kailas & M. Lopp (2010) Synthesis of Novel Acyclic Nucleoside Analogues
with Anti-Retroviral Activity, Nucleosides, Nucleotides and Nucleic Acids, 29:9, 707-720, DOI:
10.1080/15257770.2010.501776
To link to this article: http://dx.doi.org/10.1080/15257770.2010.501776
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Nucleosides, Nucleotides and Nucleic Acids, 29:707–720, 2010
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2010.501776
SYNTHESIS OF NOVEL ACYCLIC NUCLEOSIDE ANALOGUES
WITH ANTI-RETROVIRAL ACTIVITY
1
1
2
2
2
3
4
ˇ
A. Paju, M. Pa¨ri, A. Selyutina, E. Zusinaite, A. Merits, T. Pehk, K. Siirde,
A.-M. Mu¨ u¨ risepp,1 T. Kailas,1 and M. Lopp1
1Department of Chemistry, Tallinn University of Technology, Tallinn, Estonia
2Institute of Technology, Tartu University, Tartu, Estonia
3National Institute of Chemical and Biological Physics, Tallinn, Estonia
4AS Competence Centre for Cancer Research, Tallinn, Estonia
A series of novel acyclic thymine nucleoside analogues were prepared by the Mitsunobu reaction
2
from appropriately protected chiral triols. The enantiomeric triols were obtained from substituted
γ -lactone acids, prepared by asymmetric oxidation of 3-substituted-1,2-cyclopentanediones. The cy-
totoxic activity of new analogues was evaluated on MCF-7 human breast cancer and HeLa cells,
and antiviral activities on human immunodeficiency virus type 1 and hepatitis C virus models.
The synthesized compounds revealed specific anti-retroviral activity and no cytotoxic side effects.
Keywords Acyclic nucleosides; anti-retroviral activity; human immunodeficiency virus
type 1; hepatitis C virus
1. INTRODUCTION
Acyclic nucleosides are a part of an important class of nucleosides that
display remarkable biological activity. The discovery of acyclovir 1[1] 20 years
ago made this group of compounds especially attractive for researchers.
This has led to the development of various new acyclic nucleoside structures
(Figure 1; 2–4) and methodologies for their synthesis.
Acyclic nucleoside analogues have shown activity against several viral
infections, such as herpes simplex virus (HSV), human immunodeficiency
virus (HIV), hepatitis B virus (HBV), and other infections.[2–5] Some acyclic
nucleoside analogues have become popular drugs with wide clinical use.
Received 12 May 2010; accepted 27 June 2010.
The authors are grateful to the Estonian Ministry of Education and Research (Grant No:
0142725s06), the Centre of Excellence in Chemical Biology, the EU European Regional Development
Fund 3.2.0101.08-0017, and the AS Competence Centre for Cancer Research for their support.
Address correspondence to M. Lopp, Department of Chemistry, Tallinn University of Technology,
Ehitajate tee 5, 19086 Tallinn, Estonia. E-mail: margus.lopp@ttu.ee
707

708
A. Paju et al.
FIGURE 1 Some different structures of acyclic nucleosides. (Missing or changed adenosine fragment is
designated with dotted line.)
Research is still intensively being undertaken to achieve new highly ac-
tive compounds with low toxicity and minimal side effects.[6–10] The ab-
solute configuration of the nucleoside analogues often play an important
role in determining their biological activity and selectivity.[11–13] There are
some enantiomeric acyclic nucleoside analogues that have already found
clinical use: (R)-enantiomer of PMPA, tenofovir 4 is about 50 times more
effective against HIV than its (S)-counterpart, whereas for the anti-human
cytomegalovirus drug HPMPC, cidofovir 5, the (R)-enantiomer is much less
potent12[13] (Figure 1).
A number of stereoselective synthetic routes to acyclic nucleoside ana-
logues in enantiomerically pure forms have been reported, including such
starting from optically active natural compounds as carbohydrates,[14,15,] (+)-
diethyl L-tartrate,[16] L-ascorbic and D-isoascorbic acids,[17] D-lactose,[18] or
commercially available purine nucleosides.[19] Also, in some cases the meth-
ods of asymmetric synthesis have been applied: for example, Sharpless asym-
metric dihydroxylation,[20,21] asymmetric Michael addition,[22] etc.
We have recently reported that optically pure 5-oxo-2-substituted
tetrahydrofuran-2-carboxylic acids[23,24] are suitable precursors for the syn-
thesis of 4ꢁ-alkyl− and 4ꢁ-aryl-2ꢁ,3ꢁ-dideoxynucleoside analogues.[25,26] The
dideoxy analogues are known to possess antivirus activity[27–30] and, there-
fore, a further search for new compounds with improved selectivity and
resistance profiles, as well as effectiveness against various drug-resistant re-
verse transcriptase variants, is the subject of extensive interest.

Analogues with Anti-Retroviral Activity
709
In this article, we describe a synthetic route to novel acyclic nucleo-
sides, starting from enantiomeric 2-alkyl-substituted 2-hydroxyglutaric acid-
γ -lactones[31] (Scheme 1).
H
O
OH
R
O
O
O
B
B
OH
O
HO
OH
HO
HO
R
R
R
COOH
7
8
9
6
R
6a R = CH3
6b R = CH2CH3
6c R = Bn
O
6d R = CH2CH2OBn
6e R = CH2CH2OH
SCHEME 1 Retrosynthetic route to acyclic 4ꢁ-alkyl-2ꢁ,3ꢁ-dideoxynucleoside analogues 6 from 2-alkyl-
substituted 2-hydroxyglutaric acid-γ -lactones 9.
These acyclic structures can be considered as 1ꢁ,4ꢁ-seco nucleoside ana-
logues of the corresponding cyclic compounds. Indeed, the oxygen of the ter-
tiary hydroxyl group of the structure 6 may act in a way similarly to the oxygen
atom in a furanose ring. These compounds may benefit from the flexibility
of the acyclic chain, which makes it possible to adopt the active site of the
enzyme and, therefore, may have high potency against some drug-resistant
viruses. It is reported that 4ꢁ-C-methyl and 4ꢁ-C-ethyl -2ꢁ-deoxy- nucleosides
have revealed significant anti-HIV activity.[32,33] Also, the substituent R (sub-
stituted aryl- and hydroxyalkyl groups) in the compound may be used to tune
properties of the compound, such as hydrophobicity, solubility, electronic
properties, specific binding, etc.
2. RESULTS AND DISCUSSION
2.1. Synthesis of the Compounds
According to retrosynthetic analysis, the acyclic nucleoside analogues
6a–e can be prepared directly by a coupling of enantiomeric acetals 7 with
nucleobase B. Acetals 7 can be easily prepared from triols 8 via selective
acetalization of the 1,2-diol moiety; triols 8 can be obtained from γ -lactone
acids 9 (Scheme 1).
According to this general procedure, the starting compounds of the syn-
thesis are γ -lactone acids 9a–d. A general method for the synthesis of differ-
ent enantio-enriched γ -lactone acids has been published by us previously.[31]
The enantiomeric purity of crude compounds 9a and 9c was improved from
94–96% to ≥99%ee by recrystallization from an EtOAc-heptane mixture.

710
A. Paju et al.
Compounds 9b and 9d were used directly as obtained from asymmetric ox-
idation reaction and chromatography on silica gel with 94% and 96%ee,
correspondingly, without additional crystallization.
For the synthesis of triols 8, lactone acids 9 were first converted to their
methyl esters by refluxing in MeOH in the presence of a catalytic amount
of concentrated HCl[34] to give a mixture of diesters 10 and lactone esters
11. The structure of these esters, which were obtained in a ratio of 5:1, was
confirmed by nuclear magnetic resonance (NMR) in the case of benzyl-
substituted compounds 10c and 11c after chromatographical separation. In
all other cases, the obtained crude mixture of esters 10 and 11 was reduced
with LiAlH4,[35] affording the corresponding diols 8a–d, with 71–81% yield
in two steps (Scheme 2).
O
O
OH
R
OH
i
O
O
ii
+
HO
OH
MeOOC
COOMe
COOH
COOMe
R
R
R
11a-d
8a-d
9a-d
10a-d
SCHEME 2 Synthesis of triols 8. Reagents: (i) HCl/MeOH; (ii) LiAlH4, THF.
Selective acetalization of 1,2-hydroxyl groups of triols 8 was carried
out in acetone using the catalytic amount of p-TsOH, to afford acetals 7
in high yield (90–96%). For the direct coupling of acetals 7a–d with N 3-
benzoylthymine,[36] a Mitsunobu[37] reaction, which is widely applied for the
synthesis of carbocyclic,[38–40] and also acyclic nucleosides,[41–43] was used.
Thus, the coupling of acetals 7 with N 3-Bz-thymine, followed by the sequen-
tial treatment of compounds 12 with methanolic ammonia and hydrochloric
acid, produced the target acyclic nucleoside analogues 6a-d, with a 65–80%
yield, in three steps (Scheme 3).
O
O
NBz
NH
O
OH
R
O
HO
iii, iv
O
O
OH
ii
O
HO
N
OH
i
N
O
OH
R
R
R
6a-d
12a-d
8a-d
7a-d
SCHEME 3 Synthesis of acyclic nucleoside analogues 6a–d. Reagents: (i) Acetone, p-TsOH; (ii) N 3-Bz-
thymine, PPh3, DEAD THF; (iii) NH3/MeOH, (iv) HCl/MeOH/H2O.
In the case of compound 6d, the benzyl protecting group was removed by
reduction with H2 on Pd-catalyst, affording the acyclic nucleoside analogue
6e, in 89% yield (Scheme 4).

Analogues with Anti-Retroviral Activity
711
O
O
N
NH
NH
OH
OH
i
N
O
O
HO
HO
6d
6e
BnO
HO
SCHEME 4 Synthesis of acyclic nucleoside analogue 6e. Reagents: (i) H2, 10%Pd/C, MeOH.
2.2. Biological Testing: Cytotoxicity of the Compounds
Two different assays were used to detect the cytotoxic activity of the
synthesized nucleoside analogues. First, a cell proliferation assay using the
MCF-7 cells was carried out. (The results are presented in Table 1). As is
evident from the obtained data, this analysis failed to detect any cytotoxic
or cytostatic effect by day 9, while cell viability in the presence of 2 µM
Camptothecin or 1 µM Gemcitabine was estimated as 2% or 5%, respectively.
Next, the cytotoxicity of the compounds was determined using HeLa cells
and an MTT cell viability assay. The normalized results of this assay are shown
in Table 1.
As is evident from this data, none of the compounds caused cell death
or inhibited cell proliferation; the results of the MTT assay also confirmed
that there was no reduction of the energy production (judged by the activity
of mitochondrial dehydrogenases) in response to the treatment of the cells
with novel nucleoside analogues. Thus, these compounds did not possess
TABLE 1 Results of the analysis of cytotoxic properties of compounds
Cell viability
No.
Compounds 6
MCF-7∗
HeLa∗∗
1
2
3
4
5
6
a
b
c
d
119
119
129
102
117
100
97
104
110
108
103
100
e
DMSO (control)
∗The number of viable cells in negative control samples (treated with DMSO alone)
was taken as 100%. Cell viability is presented as a ratio (in percentage) of number of
viable cells in experimental (treated with tested compound) to that in DMSO treated
control cells.
∗∗The OD540 nm of DMSO-treated control cells was taken as 100%. Cell viability is
presented as a ratio (in percentage) of the OD540 nm of cells treated with the tested
compounds to the OD540 nm of DMSO-treated cells.

712
A. Paju et al.
cytotoxic or cytostatic properties; if anything, the treatment of cells with
these compounds slightly activated cell division and energy metabolism.
2.3. Biological Testing: Anti-Retroviral Activities
of the Compounds
The anti-retroviral activity of synthesized nucleoside compounds was
tested using human immunodeficiency virus type 1 (HIV-1)-based virus-like
particles (VLPs), prepared by use of the Invitrogen ViraPower Lentiviral Ex-
pression System. These particles contained, along with HIV-1 reverse tran-
scriptase, a recombinant RNA genome which, if reverse transcribed and
integrated by viral enzymes, expresses resistance to the antibiotic blasticidin.
Thus, the formation of colonies from VLP-infected cells in the presence of
blasticidin depends on reverse transcription taking place in early stages of
infection and can be used as a measure for the efficiency of reverse transcrip-
tase inhibitors. Two well-characterized nucleoside inhibitors of HIV reverse
transcriptase, Lam (Lamivudine, 2ꢁ,3ꢁ-dideoxy-3ꢁ-thiacytidine) and AZT (azi-
dothymidine), were used as positive controls. The results of such analysis
are shown in Table 2. These results are in agreement with known activities
of these compounds. Importantly, all five tested acyclic compounds showed
some inhibitory effect, although the effect was not as prominent as in the
case of the positive controls (Table 2). Since the compounds lacked any
cytotoxic side effects, the inhibition must have originated from the direct
antiviral action of the compounds. The mechanisms of their action were not
studied in detail. However, action as chain-terminators, typical for acyclic
compounds, represents the most likely option.
No acyclic compound synthesized in this study bore an -OH group which
could be recognized as a 2ꢁOH group of the ribose ring by RNA dependent
RNA polymerases of viruses with RNA genomes. However, it was recently
demonstrated that compounds without a 2ꢁOH group,[42] or even acyclic
TABLE 2 Inhibition of activity of HIV reverse transcription by acyclic nucleosides and
control substances (colony formation assay)
Anti-retroviral viral activity
Number of
colonies
Efficiency of colony
formation (%)
No.
Compounds 6
DMSO (control)
75
45
36
39
34
47
7
100
60
48
52
45
63
9
1
2
3
4
5
6
7
a
b
c
d
e
Lam
AZT
2
3

Analogues with Anti-Retroviral Activity
713
nucleosides,[43] can act as inhibitors of hepatitis C virus (HCV) RNA poly-
merase. To find out whether this was also the case for compounds described
in this study, the anti-HCV activity was tested using an Huh7-Luc-neo/ET cell
line, which carries a stably replicating HCV RNA replicon with an inserted
firefly luciferase gene as a reporter.[44] In most cases, the compounds did
not cause any reduction of HCV replication; in contrast, a slight activation
was observed for compounds 6b–e (data not shown). Interestingly, all these
compounds also showed positive effects on cellular activities (Table 1). Thus,
it is likely that the observed increase of HCV replication was mediated by the
activation of cellular processes. The compound 6a did not stimulate HCV
replication, but its inhibitory effect on the HCV replication was very mild (in-
hibitory concentration 50 >100 µM). Thus, none of the acyclic compounds
described in this study acted as inhibitors of HCV replication.
3. EXPERIMENTAL
3.1. General
1H and 13C spectra were determined in deuterated solvents on a Bruker
AMX-500 spectrometer. Deuterated solvent peaks were used as references.
1
Two-dimensional FT methods were used for the full assignment of H and
13C chemical shifts. Mass spectra were measured on a Hitachi M80B spec-
trometer, using an EI (70 eV) or Shimadzu GCMS–QP 2010 spectrometer,
with EI (70 eV). Infrared (IR) spectra were recorded on a Perkin-Elmer
Spectrum BX FTIR spectrometer. Elemental analyses were performed on
a Perkin-Elmer C,H,N,S-Analyzer 2400 ga. Optical rotations were obtained
using a Kru¨ss Optronic GmbH polarimeter P 3002 and Anton Paar GWB
polarimeter MCP 500. Thin layer chromatography (TLC) was performed
using Merck DC-Alufolien Kieselgel 60 F254 silica gel plates. For column
chromatography, silica gel KSK 40–100 µm was used. All reactions sensi-
tive to oxygen or moisture were conducted under an argon atmosphere in
oven-dried glassware. Commercial reagents were generally used as received.
Tetrahydrofuran (THF) was distilled from LiAlH4 before use.
General procedure for synthesis of triols 8a–d. A solution of lactone acid
9a (720 mg, 5 mmol) and 1 drop of concentrated HCl in dry MeOH (15 mL)
was heated to reflux overnight. After cooling, EtOAc (150 mL) was added
and the mixture was washed with 5% NaHCO3 (50 mL), brine (50 mL),
and dried (MgSO4). The solvents were removed under reduced pressure to
yield 0.848 g of a crude mixture of esters. These esters were dissolved in dry
THF (12 mL) and added a drop at a time at 0◦C to a suspension of LiAlH4
(339 mg, 8.9 mmol) in THF (12 mL). The mixture was heated to reflux
for 3 hours. After cooling to 0◦C, water (0.34 mL) was carefully added. The
mixture was stirred at room temperature for 20 minutes, and 10% aqueous

714
A. Paju et al.
NaOH (0.34 mL) was added a drop at a time, and stirred for another 20
minutes. Then water (1.02 mL) was added and the mixture was stirred for
30 minutes. After filtration with EtOAc (6 × 10 mL), the combined filtrate
was dried (Na2SO4) and concentrated. The residue was purified by flash
chromatography (silica gel, CH2Cl2/MeOH 30:1 to 10:1).
(S)-2-methylpentane-1,2,5-triol 8a. Obtained as a colorless oil (543 mg,
81%); [α]2D3−1.7 (c 5.95, MeOH); 1H NMR (500 MHz, CDCl3 + ꢀCD3OD):
δ 4.55 (bs, 1H, 5-OH), 4.41 (bs, 1H, 1-OH), 4.17 (s, 1H, 2-OH), 3.47–3.52
(m, 2H, H-5), 3.26–3.33 (m, 2H, H-1), 1.40–1.56 (m, 4H, H-3,4), 1.04 (s, 3H,
CH3); 13C NMR (125 MHz, CDCl3 + ꢀCD3OD): δ 72.37 (C-2), 69.23 (C-1),
62.389 (C-5), 34.55 (C-3), 26.32 (C-4), 22.68 (CH3); IR (neat, cm−1): 3351,
2946, 2875, 1454, 1416, 1378, 1057, 635; MS (m/z): 103, 101, 85, 75, 57, 55,
43 (base). Calculated for C6H14O3: C, 53.71; H, 10.52. Found: C, 53.58; H,
10.62.
(S)-2-ethylpentane-1,2,5-triol 8b. Obtained from 9b (730 mg, 4.62 mmol)
as a colorless oil (485 mg, 71%); [α]2D5 − 0.3 (c 13.0, MeOH); 1H NMR (500
MHz, CDCl3 + ꢀCD3OD): δ 4.62 (bs, 1H, 5-OH), 4.35 (bs, 1H, 1-OH), 3.95
(s, 1H, 2-OH), 3.42 (J = 5.4 Hz, 2H, H-5), 1.29–1.43 (m, 6H, H-3,4,6), 0.71
(t, J = 7.5 Hz, 3H, H-7); 13C NMR (125 MHz, CDCl3 + ꢀCD3OD): δ 74.11
(C-2), 66.60 (C-1), 62.24 (C-5), 31.23 (C-3), 27.85 (C-6), 25.81 (C-4), 7.19
(C-7); IR (neat, cm−1): 3351, 2944, 2880, 1462, 1337, 1137, 1057, 633; MS
(m/z): 117, 101, 99, 89, 83, 71, 57 (base), 43. Calculated for C7H16O3: C,
56.73; H, 10.88. Found: C, 56.71; H, 10.94.
(R)-2-benzylpentane-1,2,5-triol 8c. Obtained from 9c (450 mg, 2.05
mmol) as a colorless oil, which solidified on standing (304 mg, 71%); [α]D25
+ 0.4 (c 8.12, MeOH); 1H NMR (500 MHz, CD3OD): δ 7.27 (m, 2H, m-Ph),
7.22 (m, 2H, o-Ph), 7.21 (m, 1H, p-Ph), 3.57 (t, J = 5.8 Hz, 2H, H-5), 3.41 (s,
2H, H-1), 2.82 and 2.76 (2d, J = 13.5 Hz, 2H, Bn-CH2), 1.67 (m, 2H, H-4),
1.51 (m, 2H, H-3); 13C NMR (125 MHz, CD3OD): δ 136.94 (s), 130.40 (o),
128.16 (m), 126.41 (p), 74.57 (C-2), 66.73 (C-1), 62.72 (C-6), 42.42 (Bn),
32.47 (C-3), 26.23 (C-4); IR (neat, cm−1): 3368, 3086, 3062, 3029, 2947, 1603,
1495, 1454, 1055, 735, 703; MS (m/z): 179, 161, 151, 133, 119, 101 (base),
92, 91, 83. Calculated for C12H18 O3: C, 68.55; H, 8.63. Found: C, 68.46; H,
8.67.
(R)-2-(2-benzyloxyethyl)-pentane-1,2,5-triol 8d. Obtained from 9d
(1.87 g, 7.08 mmol) as a colorless oil (1.32 g, 73%); [α]2D3 − 0.8 (c 43.1,
MeOH/CHCl3 1:1); 1H NMR (500 MHz, CDCl3): δ 7.28–7.36 (m, 5H, Ph),
4.50 (s, 2H, CH2Ph), 3.95 (bs, OH), 3.53–3.70 (m, 6H, 2xOH, H-1, CH2OBn),
3.45 (t, J = 5.0 Hz, 2H, H-5), 1.77–1.87 (m, 2H, CH2CH2OBn), 1.55–1.62
(m, 4H, H-3,4); 13C NMR (125 MHz, CDCl3): δ 137.39 (s-Ph), 128.43 (m-Ph),
127.84 (p-Ph), 127.74 (o-Ph), 73.79 (C-2), 73.33 (CH2Ph), 67.54 (C-1), 66.57
(CH2OBn), 62.78 (C-5), 35.96 (CH2CH2OBn), 33.82 (C-3), 26.25 (C-4); IR
(neat, cm−1): 3371, 3089, 3031, 296, 2872, 1954, 1586, 1496, 1454, 1058, 747,
699, 608; MS (m/z): 205, 195, 169, 159, 143, 129, 115, 107, 101, 99, 92, 91

Analogues with Anti-Retroviral Activity
715
(base), 79. Calculated for C14H22O4: C, 66.12; H, 8.72. Found: C, 65.92; H,
8.78.
1
2-Benzyl-2-hydroxy-pentanedioic acid dimethyl ester 10c. H NMR (500
MHz, CDCl3): δ 7.14–7.29 (m, 5H, Ph), 3.72 (s, 3H, 1-OCH3), 3.67 (s, 3H,
5-OCH3), 3.17 (d, J = 0.9 Hz, 1H, 2-OH), 3.05 and 2.94 (2d, J = 13.5 Hz,
2H, CH2Ph), 2.50–2.57 (m, 1H, H-4), 2.20–2.28 (m, 2H, H3, H4), 2.09–2.15
(m, 1H, H-3); 13C NMR (125 MHz, CDCl3): δ 175.41 (C-1), 173.48 (C-5),
135.40 (s-Ph), 129.91 (o-Ph), 128.15 (m-Ph), 126.95 (p-Ph), 77.40 (C-2),
52.56 (1-OCH3), 51.59 (5-OCH3), 45.61 (CH2Ph), 62.78 (C-5), 33.72 (C-3),
28.68 (C-4).
1
2-Benzyl-5-oxo-tetrahydrofuran-2-carboxylic acid methyl ester 11c. H
NMR (500 MHz, CDCl3): δ 7.24–7.33 (m, 5H, Ph), 3.77 (s, 3H, OCH3),
3.37 and 3.14 (2d, J = 14.4 Hz, 2H, CH2Ph), 2.43–2.51 (m, 2H, H3, H-4),
2.22–2.30 (m, 1H, H3), 2.10–2.19 (m, 1H, H-4); 13C NMR (125 MHz, CDCl3):
δ 175.48 (C-5), 171.74 (2-COO), 133.83 (s-Ph), 130.48 (o-Ph), 128.54 (m-
Ph), 127.45 (p-Ph), 86.26 (C-2), 52.92 (OCH3), 42.46 (CH2Ph), 29.93 (C-3),
27.96 (C-4).
General procedure for synthesis of acyclic nucleoside analogues 6a–d.
A solution of triol 8a (264 mg, 1.97 mmol) in acetone (10 mL) p-TsOH (3.8
mg, 0.02 mmol) was added. After stirring at room temperature for 4 hours,
Et3N (20 µL) was added, and the mixture was stirred for 5 minutes and
concentrated. The residue was purified by flash chromatography (silica gel,
petroleum ether/acetone 10:2), producing acetal 7a (307 mg, 90%). Acetals
7b (335 mg, 94%), 7c (264 mg, 96%) and 7d (730 mg, 94%) were obtained
from triols 8b.(280 mg, 1.89 mmol), 8c (230 mg, 1.095 mmol), and 8d (673
mg, 2.65 mmol), respectively.
3
To a solution of acetal 7a (161 mg, 0.925 mmol), N -benzoylthymine
(253 mg, 1.1 mmol) and Ph3P (288 mg, 1.1 mmol) in THF (19 mL) DEAD
(173 µL, 1.1 mmol) were added dropwise at a time at 0◦C. The mixture was
stirred at room temperature overnight, and the solvent was removed under
reduced pressure. The residue was purified by flash chromatography (silica
gel and petroleum ether/acetone 10:2) to give 12a (469 mg) as a crude white
solid. This solid was dissolved in MeOH (2.8 mL), and saturated methanolic
ammonia solution (2.8 mL) was added at 0◦C. The mixture was stirred at
room temperature overnight and the solvents were evaporated. The residue
was dissolved in MeOH (2.8 mL), and water (0.93 mL) and 6N HCl solution
(1.85 mL) were added. The mixture was stirred at room temperature for
4 hours and the solvents were evaporated. Flash chromatography on silica
gel (CH2Cl2/MeOH 15:1 for 6a-b and 30:1 to 20:1 for 6c-d) gave the target
compounds.
1-(4,5-Dihydroxy-4-methylpentyl)-5-methyl-1H-pyrimidine-2,4-dione 6a.
Obtained as a white solid (177 mg, 79%); m.p. 169–172◦C; [α]D22 −1.2 (c
16.5, DMSO); 1H NMR (500 MHz, CD3OD): δ 7.43 (q, J = 1.0 Hz, 1H, H-6),
3.72 (t, J = 7.2 Hz, 2H, NCH2), 3.34 (s, 2H, >C < CH2OH), 1.86 (d, J = 1.0

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A. Paju et al.
Hz, 3H, 5-CH3), 1.71–1.77 (m, 2H, CH2CH2CH2), 1.43–1.52 (m, 2H, CH2 >
C<), 1.12 (s, 3H, CH3 > C<); 13C NMR (125 MHz, CD3OD): δ 166.91 (C-4),
152.99 (C-2), 143.22 (C-6), 111.09 (C-5), 73.28 (COH), 70.24 (CH2OH),
49.80 (NCH2), 35.84 (NCH2CH2CH2), 24.35 (CH2CH2CH2), 23.78 (CH3 >
C<), 12.17 (5-CH3); IR (KBr, cm−1): 3354, 3260, 2988, 1674, 1466, 1424,
1350, 1285, 1218, 1104, 1054, 927, 762, 707; MS (m/z): 243, 227, 211, 206,
167, 152, 127, 109, 85 (base), 75, 57, 43. Calculated for C11H18O4N2: C,
54.53; H, 7.49; N, 11.56. Found: C, 54.03; H, 7.51; N, 11.59.
1-[4-Hydroxy-4-(hydroxymethyl)hexyl]-5-methyl-1H-pyrimidine-2,4-dione
6b. Obtained from 7b (169 mg, 0.9 mmol) as a white solid (150 mg, 65%);
22
1
m.p. 41–44◦C; α]D − 2.2 (c 12.9, MeOH); H NMR (500 MHz, CD3OD):
δ 7.43 (q, J = 1.0 Hz, 1H, H-6), 3.71 (t, J = 7.1 Hz, 2H, NCH2), 3.37 (s,
2H, > C < CH2OH), 1.85 (d, J = 1.0 Hz, 3H, 5-CH3), 1.67–1.73 (m, 2H,
CH2CH2CH2), 1.43–1.51 (m, 4H, CH2 > C < CH2), 0.85 (t, J = 7.4 Hz, 3H,
CH3CH2); 13C NMR (125 MHz, CD3OD): δ 166. 87 (C-4), 152.95 (C-2), 143.
26 (C-6), 111.04 (C-5), 75.22 (COH), 67.36 (> C < CH2OH), 49.81 (NCH2),
33.14 (NCH2CH2CH2), 29.52 (> C < CH2CH3), 24.00 (CH2CH2CH2),
12.18 (5-CH3), 7.86 (> C < CH2CH3); IR (KBr, cm−1): 3419, 2942, 1680,
1472, 1358, 1218, 1050, 904, 783, 766; MS (m/z): 256, 238, 225, 197, 167,
152, 127, 112, 99 (base), 96, 83, 57, 41. Calculated for C12H20O4N2: C, 56.24;
H, 7.87; N, 10.93. Found: C, 55.89; H, 7.88; N, 10.84.
1-(4-Benzyl-4,5-dihydroxypentyl)-5-methyl-1H-pyrimidine-2,4-dione 6c.
Obtained f◦rom 7c (225 mg, 0.9 mmol) as a white solid (230 mg, 80%);
m.p. 94–96 C; [α]2D3 + 3.1 (c 23.5, MeOH/CHCl3 1:1); 1H NMR (500 MHz,
CD3OD): δ 7.34 (q, J = 1.0 Hz, 1H, H-6), 7.19–7.23 (m, 4H, o-,m-Ph),
7.13–7.18 (m, 1H, p-Ph), 3.68 (t, J = 7.0 Hz, 2H, NCH2), 3.36 ja 3.33 (2d, J =
11.0 Hz, 2H, CH2OH), 2.78 and 2.74 (2d, J = 13.5 Hz, 2H, CH2Ph), 1.83 (d,
J = 1.0 Hz, 3H, 5-CH3), 1.74–1.82 (m, 2H, CH2CH2CH2), 1.40 (ddd, J = 5.7,
10.8 and 13.7 Hz, 1H, NCH2CH2CH2), 1.35 (ddd, J = 6.2, 11.3 and 13.7 Hz,
1H, NCH2CH2CH2); 13C NMR (125 MHz, CD3OD): δ 166.89 (C-4), 152.94
(C-2), 143.31 (C-6), 138.69 (s-Ph), 131.62 (o-Ph), 128.92 (m-Ph), 127.19
(p-Ph), 110.93 (C-5), 75.45 (COH), 67.28 (CH2OH), 49.75 (NCH2), 43.30
(CH2Ph), 33.21 (NCH2CH2CH2), 24.01 (CH2CH2CH2), 12.22 (5-CH3); IR
(KBr, cm−1): 3418, 3030, 2949, 1674, 1473, 1357, 1218, 1100, 1056, 704; MS
(m/z): 300, 287, 272, 227 (base), 209, 195, 168, 161, 140, 127, 115, 101, 91,
83, 65, 55, 41. Calculated for C17H22O4N2: C, 64.13; H, 6.97; N, 8.80. Found:
C, 63.92; H, 6.93; N, 8.76.
1-[6-Benzyloxy)-4-hydroxy-4-(hydroxymethyl)hexyl]-5-methyl-1H-pyrimi
dine-2,4-dione 6d. Obtained from 7d (368 mg, 1.25 mmol) as a white
solid (343 mg, 76%); m.p. 45–48◦C; [α]D−2.2 (c 10.1, MeOH/CHCl3 1:1);
1H NMR (500 MHz, CDCl3): δ 9.87 (s, 1H, NH), 7.26–7.35 (m, 5H, Ph),
6.96 (q, J = 1.1 Hz, 1H, H-6), 4.49 (s, 2H, BnCH2), 3.62–3.68 (m, 5H,
NCH2, BnOCH2 and OH), 3.41–3.49 (m, 3H, > C < CH2O and OH), 1.87
(d, J = 1.1 Hz, 3H, 5-CH3), 1.77–1.85 (m, 2H, BnOCH2CH2), 1.68–1.79

Analogues with Anti-Retroviral Activity
717
(m, 2H, NCH2CH2), 1.47–1.54 (m, 2H, NCH2CH2CH2); 13C NMR (125
MHz, CDCl3): δ 164.54 (C-4), 151.18 (C-2), 140.53 (C-6), 137.46 (s-Ph),
128.44 (m-Ph), 127.85 (p-Ph), 127.76 (o-Ph), 110.55 (5-CH3), 73.80 (>C<),
73.36 (BnCH2), 67.53 (> C < CH2OH), 66.55 (BnOCH2), 48.80 (NCH2),
35.93 (BnOCH2CH2), 33.54 (NCH2CH2CH2), 23.12 (CH2CH2CH2), 12.15
(5-CH3); IR (KBr, cm−1): 3386, 3032, 2950, 1674, 1474, 1365, 1213, 1104,
1051, 741, 700; MS (m/z): 362, 331, 313, 271, 256, 238, 223, 207, 182, 167,
152, 127, 109, 91 (base), 79, 65, 45, 41. Calculated for C19H26O5N2: C, 62.97;
H, 7.23; N, 7.73. Found: C, 62.74; H, 7.44; N, 7.68.
1-[4,6-dihydroxy-4-(hydroxymethyl)hexyl]-5-methyl-1H-pyrimidine-2,4-
dione 6e. To a solution of nucleoside analogue 6d (240 mg, 0.66 mmol)
in MeOH (20 mL), 10% Pd/C (93 mg) was added. Through the reaction
mixture, H2 was bubbled at room temperature for an hour and a half.
The catalyst was removed by filtration and the filtrate was evaporated. The
residue was purified by flash chromatography (silica gel, CH2Cl2/MeOH
15:1 to 10:1) affording 3e as a white solid (180 mg, 89%); m.p. 119–121◦C;
[α]2D3 − 1.9 (c 12.2, MeOH); H NMR (500 MHz, CD3OD): δ 7.43 (q, J =
1
1.0 Hz, 1H, H-6), 3.72 (t, J = 7.4 Hz, 2H, NCH2), 3.70 (t, J = 6.8 Hz, 2H,
CH2CH2OH), 3.40 and 3.39 (2d, J = 11.0 Hz, 2H, CH2OH), 1.86 (d, J =
1.0 Hz, 3H, 5-CH3), 1.71–1.77 (m, 4H, CH2CH2CH2 and CH2CH2OH),
1.47–1.55 (m, 2H, NCH2CH2CH2); 13C NMR (125 MHz, CD3OD): δ
166.90 (C-4), 152.98 (C-2), 143.23 (C-6), 111.08 (C-5), 74.81 (COH), 68.05
(CH2OH), 58.90 (CH2CH2OH), 49.75 (NCH2), 39.62 (CH2CH2OH), 34.43
(NCH2CH2CH2), 24.03 (CH2CH2CH2), 12.20 (5-CH3); IR (KBr, cm−1):
3414, 3168, 3047, 2921, 1690, 1474, 1355, 1218, 1047, 873, 757; MS (m/z):
272, 241, 236, 223, 206, 167, 152, 127, 115, 110, 97 (base), 83, 69, 55, 41.
Calculated for C12H20O5N2: C, 52.93; H, 7.40; N, 10.29. Found: C, 52.81;,
H, 7.37; N 10.22.
Procedure for testing cytotoxic activity. A cell proliferation assay was
carried out, using the MCF-7 cells. Compounds were dissolved in DMSO and
added to the cells at a final concentration of 100 µM (the concentration of
DMSO was 0.1% in all cases). Cells were incubated in the presence of the
compound and the number of living cells was counted after incubation for
3, 6, and 9 days. Camptothecin 2 µM and Gemcitabine 1 µM and 10 µM were
used as a positive (cytotoxic) control and PBS + 0.1% DMSO as a negative
control. The analysis was carried out at AS InBio (Tallinn, Estonia).
The cytotoxicity of the compounds was determined using HeLa cells and
an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell
viability assay. Cells were seeded on 96-well plates and incubated with 50 µM
concentrations of the tested substances for 9 days. Cells treated with 0.5%
of DMSO (the same amount of DMSO was used in experimental samples)
were used as controls. Every 3 days, the media were replaced with fresh ones
containing the same concentration of the compounds. After 9 days, an MTT
reagent was added to the media at 10% concentration, cells were incubated

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A. Paju et al.
at 37◦C for 2 hours, lysed in DMSO, and the optical density at 540 nm was
measured.
Procedure for testing anti-retroviral activity. The anti-retroviral activity
of synthesized nucleoside compounds was tested using HIV-1-based virus-
like particles (VLPs) (ViraPower Lentiviral Expression System, Invitrogen).
Nucleoside inhibitors of HIV reverse transcriptase, lamivudine, 2ꢁ,3ꢁ-dideoxy-
3ꢁ-thiacytidine (Lam) and azidothymidine (AZT) were used as positive con-
trols. Briefly, U2OS cells seeded on 60 mm plates at ∼95% of confluency
were infected with 75 cfu (colony forming units) of HIV-1-based VLPs, in
the presence of 50 µM of tested/control substances and 6 µg/µL polybrene.
Cells used as negative control were treated with DMSO (the same amount as
was used in experimental cells). Twenty-four hour postinfection blasticidin
selection (5 µg/mL) was applied. Ten days after infection, the cells were
stained with crystal violet and the antibiotic resistant colonies were counted.
Experiment was repeated three times with similar results. The number of
colonies on the plates treated with DMSO alone (negative control) was taken
as 100%. In the case of the positive controls AZT and Lam, the number of
the formed colonies was reduced 35 and 10 times, respectively.
Procedure for testing anti-HCV-activity. The Huh-luc/neo-ET cells (de-
rived from a human hepatocarcinoma cell line) seeded on 60 mm diameter
dishes were incubated with 100 µM concentrations of the substances for 56
hours (control cells were incubated with the appropriate amount of DMSO),
then lysed, and firefly luciferase activity in the lysates was measured according
to the protocol of the Promega Luciferase Assay Systems. The total protein
concentration in the lysates was determined using a Bio-rad Bradford as-
say and luminescence was normalized to the total protein concentration.
This analysis was carried out at Baltic Technology Development (Tallinn,
Estonia).
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