A Divergent Approach for the Synthesis of d - And l -4′-Ethynyl Dioxolane Nucleosides with Potent Anti-HIV Activity

Article abstract of DOI:10.1055/s-0035-1561637

Novel 4′-C-ethynyl isomeric dioxolane nucleoside analogues (β-d, α-d, β-l, and α-l, respectively) are successfully synthesized via a divergent strategy from the common starting material, (Z)-but-2-ene-1,4-diol, and are characterized and evaluated for their anti-HIV-1 and anti-HIV-2 activities. The β-d and β-l products display potent in vitro activities against HIV-1 (IIIB) with EC₅₀ values of 0.75 and 0.87 μM, respectively, and against HIV-2 (ROD) with EC₅₀ values of 0.75 and 0.35 μM, respectively, being better in comparison with 3TC [EC₅₀, 5.27 μM (HIV-1) and 1.30 μM (HIV-2)]. The β-d and β-l nucleosides also potently inhibit different drug-resistant strains of the HIV-1 virus (L100I, K103N, Y181C, and V106A). The selectivity indices and cytotoxic profiles of the β-d and β-l nucleosides are much better than those of the standard drugs AZT and d4T.

Full text of DOI:10.1055/s-0035-1561637

SYNTHESIS
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© Georg Thieme Verlag Stuttgart · New York
2016, 48, A–G
paper
A
S. Singh et al.
Paper
Synthesis
A Divergent Approach for the Synthesis of D- and L-4′-Ethynyl
Dioxolane Nucleosides with Potent Anti-HIV Activity
Sarbjit Singha,b, §
Veeraswamy Gajulapatib, §
Minkyoung Kimb
Ja-Il Goob
Jae Kyun Leec
Kyeong Leea
Chong-Kyo Leed
Lak Shin Jeong*e
Yongseok Choi*b
O
N
CH2OBn
NH
HIV-1 (IIIB):
(EC50) = 0.75 mM
O
4 steps
CH2OH
O
O
O
O
HIV-2 (ROD):
(EC50) = 0.75 mM
CH2OH
7 steps
(39%)
+
b-D (31%)
HO
OH
HIV-1 (IIIB):
(EC50) = 0.87 mM
O
4 steps
O
O
O
N
O
HIV-2 (ROD):
(EC50) = 0.35 mM
CH2OH
O
CH2OBn
NH
a College of Pharmacy, Dongguk University-Seoul, Goyang
10326, Republic of Korea
b College of Life Sciences and Biotechnology, Korea University,
Seoul 02842, Republic of Korea
CH2OH
(22%)
b-L (34%)
c Center for Neuromedicine, Korea Institute of Science and
Technology, Seoul 02792, Republic of Korea
d Virus Research and Testing Center, Korea Research Institute of
Chemical Technology, Daejeon, 305-600, Republic of Korea
e The Research Institute of Pharmaceutical Sciences, College of
Pharmacy, Seoul National University, Seoul 151-742, Republic
of Korea
lakjeong@snu.ac.kr
ychoi@korea.ac.kr
§ These authors contributed equally to this work.
Received: 22.02.2016
Accepted after revision: 13.04.2016
Published online: 24.05.2016
tors play a pivotal role along with other protease inhibitors
and integrase inhibitors. However, the effectiveness of cART
is compromised by the rapid development of multidrug-re-
sistant strains of HIV and cumulative cytotoxic effects ob-
served during prolonged uses of this therapy. Thus, there is
a continuous need of better anti-HIV agents having in-
creased potency and improved pharmacokinetic profile.6
It is evident from the literature that acetylene-contain-
ing nucleosides have shown potent anti-HIV activities.7 In
addition, 1,3-dioxalane-8 and 1,3-oxathiolane-based9 nucle-
osides have good potential as shown by their promising an-
tiviral activities. Lamivudine (3TC) and emtricitabine (FTC)
have been approved by the FDA as anti-HIV agents.9 Until
now, there have been no reports of 4′-C-ethynyl dioxolane
nucleosides in the literature. Thus, it is of great interest to
combine both structures to prepare 4′-C-ethynyl dioxolane
DOI: 10.1055/s-0035-1561637; Art ID: ss-2016-f0124-op
Abstract Novel 4′-C-ethynyl isomeric dioxolane nucleoside analogues
(β-D, α-D, β-L, and α-L, respectively) are successfully synthesized via a di-
vergent strategy from the common starting material, (Z)-but-2-ene-
1,4-diol, and are characterized and evaluated for their anti-HIV-1 and
anti-HIV-2 activities. The β-D and β-L products display potent in vitro ac-
tivities against HIV-1 (IIIB) with EC50 values of 0.75 and 0.87 μM, respec-
tively, and against HIV-2 (ROD) with EC50 values of 0.75 and 0.35 μM,
respectively, being better in comparison with 3TC [EC50, 5.27 μM (HIV-
1) and 1.30 μM (HIV-2)]. The β-D and β-L nucleosides also potently in-
hibit different drug-resistant strains of the HIV-1 virus (L100I, K103N,
Y181C, and V106A). The selectivity indices and cytotoxic profiles of the
β-D and β-L nucleosides are much better than those of the standard
drugs AZT and d4T.
Key words anti-HIV, AIDS, dioxolane nucleosides, 4′-acetylene nucle-
osides, divergent synthesis, asymmetric synthesis
HO
HO
O
O
+
Acquired immune deficiency syndrome (AIDS), which is
caused by human immunodeficiency virus 1 (HIV-1) and
human immunodeficiency virus 2 (HIV-2) infection, is a se-
rious worldwide health concern.1,2 Conventional treatment3
of HIV infection is based on combinatorial antiretroviral
therapy (cART), which uses combinations of three or more
antiretroviral agents to control rapidly HIV replication.4,5 In
cART, nucleoside analogues as reverse transcriptase inhibi-
O
R1
R2
R
B
A
HO
O
O
C
Scheme 1 Rational design of 4′-C-ethynyl dioxolane nucleosides
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2016, 48, A–G

B
S. Singh et al.
Paper
Synthesis
nucleosides in order to develop novel nucleoside reverse
transcriptase inhibitors as shown in Scheme 1. Since the
FDA-approved anti-HIV drugs AZT and d4T contain thymine
as an integral part of their structures, we initially planned
to synthesize ethynyl dioxolane nucleosides with thymine
as the base to test the plausibility of this template.
Herein, the syntheses and anti-HIV activity of four iso-
meric 4′-C-ethynyl dioxolane nucleoside analogues 15a–d
are reported (Figure 1).
age of compound 3 was performed with NaIO4 affording al-
dehyde 4 in 81% yield, followed by immediate reaction with
trimethylsilylacetylene in THF using n-BuLi as the base. The
resulting alkyne 5 was then oxidized to give compound 6
using NaOCl as the oxidizing agent. The freshly prepared
compound 6 was then immediately coupled with com-
pound 11 in benzene using a catalytic amount of PTSA un-
der reflux conditions employing a Dean–Stark assembly. Si-
lyl group deprotection of resulting compound 7 with TBAF
in THF afforded diastereomeric compounds 8a and 8b in
39% and 22% yields, respectively.
O
CH2OH
NH
Next, the CH2OH group of compound 8a was trans-
formed into a COOH group using PDC as the oxidizing agent
in DMF to give compound 12a as a diastereomeric mixture.
Compound 12a, without any purification, was directly con-
verted into acetate 13a using Pb(OAc)4 and pyridine. Com-
pound 13a was then coupled with thymine to give product
14a as a mixture of diastereomers. Benzyl group deprotec-
tion in compound 14a with AlCl3 and anisole afforded the
desired products 15a and 15b in 36% and 21% yields, re-
spectively. A similar synthetic strategy was used for the
synthesis of compounds 15c and 15d starting from com-
pound 8b (Scheme 3). At this point, it is important to men-
tion that debenzylation of compounds 14a and 14b was
very challenging. All the available conditions for debenzyla-
tion failed leading to decomposition of the dioxolane ring
due to its instability under acidic conditions, but finally,
deprotection with AlCl3/anisole afforded the desired prod-
ucts. The different reaction conditions employed for the de-
benzylation of compound 14a are given in the Supporting
Information.
O
CH2OH
O
N
O
O
N
O
O
NH
NH
O
15a
15b
O
O
N
O
O
O
N
O
O
CH2OH
NH
CH2OH
O
15c
15d
Figure 1 Structures of compounds 15a–d
Compounds 15a–d were synthesized using the diver-
gent strategies illustrated in Schemes 2 and 3. Initially, the
hydroxy groups of (Z)-but-2-ene-1,4-diol (1) were protect-
ed with benzyl groups using benzyl bromide and NaH in
DMF (Scheme 2). Subsequent cis-hydroxylation with
OsO4/NMO in acetone/H2O gave diol 3 in 76% yield. Cleav-
OH
O
BnO
OBn
(iv)
(ii)
76%
(iii)
(i)
BnO
HO
OH
BnO
OBn
BnO
H
77%
99%
81%
HO
OH
TMS
1
2
3
4
5
TMS
CH2OBn
O
O
(v)
97%
(vi)
(vii)
O
BnO
O
+
BnO
O
11
O
O
CH2OBn
8b (22%)
TMS
CH2OH
CH2OH
OTBDPS
6
7
8a (39%)
O
O
HO
(ix)
(viii)
77%
O
O
HO
(R)
OTBDPS
76%
(S)
OH
(R)
OTBDPS
9
10
11
Scheme 2 Reagents and conditions: (i) BnBr, NaH, DMF, 0 °C to r.t.; (ii) OsO4, NMO, acetone/H2O; (iii) NaIO4, MeOH, 0 °C to r.t.; (iv) n-BuLi, trimethyl-
silylacetylene, THF, –78 °C; (v) 5% NaOCl, NaHCO3, KI, TEMPO, CH2Cl2, r.t.; (vi) 11, PTSA, benzene, 90 °C; (vii) TBAF, THF, 0 °C; (viii) TBDPSCl, DMAP, Et3N,
CH2Cl2, 0 °C; (ix) AcOH, H2O, 90 °C.
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2016, 48, A–G

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S. Singh et al.
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Synthesis
O
N
CH2OH
NH
O
O
CH2OBn
O
CH2OBn
O
CH2OBn
CH2OH
CH2OBn
O
O
OAc
COOH
O
O
(i)
(ii)
(iii)
NH
(iv)
O
N
N
O
O
O
O
O
58%
+
38%
74%
O
O
CH2OH
NH
β-D
α-D
O
8a
12a
13a
14a
15a (36%)
15b (21%)
O
NH
O
O
O
N
O
O
(ii)
60%
(i)
(iii)
(iv)
O
COOH
OAc
O
O
NH
+
N
O
O
CH2OH
N
O
O
77%
O
43%
O
O
CH2OH
O
CH2OBn
CH2OBn
CH2OH
CH2OBn
NH
OCH2OBn
β-L
15c (34%)
α-L
15d (18%)
O
8b
12b
13b
14b
Scheme 3 Reagents and conditions: (i) PDC, DMF, r.t.; (ii) Pb(OAc)4, py, THF, r.t.; (iii) thymine, 2,4,6-collidine, TMSI, TBDMSOTf, CH2Cl2, 40 °C; (iv) AlCl3,
anisole, CH2Cl2, 0 °C.
Attempts were also made to assign the absolute config-
urations of nucleosides 15a–d. Compound 15d was able to
be recrystallized, and X-ray crystal structure analysis re-
vealed that it had α-L configuration (Figure 2).
The NMR spectra, HPLC retention times, Rf values (TLC)
and melting points of 15a,c and 15b,d were the same, indi-
cating their enantiomeric relationships with each other. On
the basis of these experiments, the absolute configurations
of compounds 15a, 15b, 15c and 15d were assigned as β-D,
α-D, β-L, and α-L, respectively.
Compounds 15a–d were then evaluated for their activi-
ties against HIV-1 and HIV-2 using the IIIB and ROD strains,
respectively. The results are shown in Table 1, which also
includes the activities of the standard drugs AZT, 3TC and
d4T against these viral strains. As is evident from Table 1,
nucleosides 15a and 15c with β-configurations displayed
potent anti-HIV-1 and anti-HIV-2 activities, whereas com-
pounds 15b and 15d having α-configurations were found to
be inactive. The activities of both compounds 15a and 15c
were better than the standard drug 3TC. All the compounds
also showed excellent cytotoxic and selectivity index pro-
files suggesting their scope for further development.
Figure 2 X-ray crystal structure of compound 15d (CCDC 1479802)
Table 1 Anti-HIV Activities of Compounds 15a–d
Compound
Toxicity CC50
Anti-viral activity (EC50) (μM)
Selectivity index
HIV-1 strain (IIIB)
HIV-2 strain (ROD)
IIIB
>562.32
ROD
>562.32
15a
15b
15c
15d
AZT
3TC
d4T
>100
>100
>100
>100
3.522
>100
7.55
0.75
0.75
14.78
0.87
16.18
0.35
>26.82
>447.16
>3.54
864
>24.48
>1132.43
> 9.96
112.0
0.015
5.27
39.80
0.0015
1.30
8427
>82.64
127
>333.33
1373
0.27
0.024
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2016, 48, A–G

D
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Table 2 Anti-HIV Activities of Compounds 15a and 15c against Drug-Resistant Strains of the HIV-1 Virus
Compound
Anti-viral activity (EC50) (HIV-1) (μM)
RTMDR1
L100I
K103N
V106A
Y181C
Y188H
Y188L
G190A
15a
5.94
5.590
1.65
0.13
0.24
0.24
nda
0.47
nda
0.25
0.28
0.30
0.01
0.011
0.31
0.30
0.09
0.011
0.194
0.225
0.014
0.0063
15c
0.21
nda
ddC
0.33
0.33
0.0067
0.57
0.012
0.331
0.0052
AZT
0.0026
a Not determined.
As the emergence of resistant viral strains is a major
challenge and a matter of concern, we evaluated the activi-
ties of compounds 15a and 15c against different drug-resis-
tant strains of the HIV-1 virus. The results are shown in Ta-
ble 2. Compounds 15a and 15c were able to potently inhibit
different drug-resistant strains of the HIV-1 virus. The ac-
tivity of 15a was better than ddC against the L100I, K103N,
V106A and Y181C strains of virus. Similarly, compound 15c
also displayed better activity than ddC against the L100I vi-
rus strain.
In summary, all four isomers of novel 4′-C-ethynyl diox-
olane nucleosides 15a–d have been successfully synthe-
sized (and characterized) via a divergent strategy from the
same starting material, (Z)-but-2-ene-1,4-diol (1). Com-
pounds 15a and 15c displayed potent in vitro activities
against HIV-1 and HIV-2 viruses with no cytotoxic effects.
Compounds 15a and 15c also displayed potent activities
against different drug-resistant strains of the HIV-1 virus.
These results warrant further study on the mode of action
and the derivatization of these analogues to search for more
potent anti-HIV agents.
All of the HIV strains were amplified in MT-4 (HTLV-1-infected hu-
man T lymphocytes) cells, which were kindly provided by N. Yama-
moto of the Tokyo Medical and Dental University, Japan, and grown in
RPMI 1640 medium with 10% FBS and 4 μg/mL gentamycin. The
aforementioned medium was used for making dilutions of the drugs
and maintenance of the cultures during the assays. Aliquots of the vi-
rus stocks were stored as culture supernatants at –70 °C until used.
Standard Compounds
The following standard compounds: 2′,3′-dideoxycytidine (ddC) (Sig-
ma), 3TC (Sigma), d4T (Sigma) and azidothymidine (AZT) (Sigma)
were used as references in the anti-HIV tests. All of the compounds
were dissolved in 100% dimethyl sulfoxide (DMSO) at a stock concen-
tration of 20 mg/mL.
Antiviral Assays
The virus-induced-CPE inhibition assay10 was used to measure the
anti-HIV activities. Log-phase MT-4 cells were plated and infected
with the virus at a multiplicity of infection of 20~100 CCID50 (50% cell
culture inhibitory dose) per well. The cells were immediately resus-
pended in RPMI 1640 plus 10% FBS at a concentration of 105 cells/mL.
Aliquots of 100 μL of the resuspended cells were placed in the wells of
a 96-well plate that contained 100 μL of twofold-concentrated test
samples. After 5 d of incubation at 37 °C, the cells were observed mi-
croscopically, and cell viability was quantified using the MTT assay,
which is based on the mitochondrial reduction of 3-(4,5-dimethylthi-
azol-2-yl)-2,5-diphenyltetrazolium bromide.11 The effective antiviral
concentration was expressed as the EC50, which is the concentration
of compound required to inhibit virus-induced CPE by 50%. The cyto-
toxic concentration is expressed as the CC50, which is the concentra-
tion of the compound that killed 50% of the mock-infected cells.
TLC was performed on Merck silica gel 60 F254 plates. Column chro-
matography was conducted using silica gel 60 N (Kanto, 100e210
mm) or silica gel 60 (Kanto, 40e50 mm). All melting points were de-
termined using a Yamato melting point apparatus (model MP-J3). The
NMR spectra were recorded on a JEOL JNM-ECA-500 spectrometer.
The chemical shifts (δ) are reported in parts per million relative to
TMS (0.0 ppm) as the internal standard, and the signals are expressed
as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), or br
(broad). The values of the coupling constants (J) are given in hertz.
The HRMS spectra were obtained using JEOL JMS-HX110, JEOL JMS-
700TZ, and JEOL AccuTOF LC-plus systems with electrospray ioniza-
tion (ESI) operating in positive modes.
(Z)-1,4-Bis(benzyloxy)but-2-ene (2)
To a mixture of 55% NaH (13.0 g, 298 mmol) and DMF (250 mL) was
slowly added a solution of 1 (10.0 g, 114 mmol) in DMF (50 mL) at
0 °C. After stirring the mixture for 1 h at r.t., benzyl bromide (35.3 mL,
398 mmol) was added dropwise. The resulting mixture was stirred
for 4 h at r.t. The reaction was quenched by the addition of sat. aq
NH4Cl solution, followed by extraction with EtOAc. The organic layer
was washed with H2O, dried over MgSO4 and concentrated to afford
crude compound 2. The crude was purified by silica gel column chro-
matography (hexane–EtOAc, 8:1) to give 2 (28.4 g, 99%) as a colorless
oil.
1H NMR (500 MHz, CDCl3): δ = 4.15–4.16 (d, J = 2.5 Hz, 4 H), 4.58 (s, 4
H), 5.89 (br s, 2 H), 7.36–7.42 (m, 10 H).
13C NMR (125 MHz, CDCl3): δ = 66.0, 72.3, 127.8, 127.9, 128.6, 129.7,
138.6.
Viruses and Cell Lines
The virus strains used in these anti-HIV tests are as follows: (1) hu-
man immunodeficiency virus type 1 (HIV-1), strain IIIb and the type
2 (HIV-2), strain ROD were kindly provided by the National Institute
for Biological Standards and Control (NIBSC), U.K., as part of the MRC
AIDS Reagents Program. (2) Drug-resistant strains of HIV-1 having
mutation(s) on the reverse transcriptase gene were obtained from the
Centralized Facility for AIDS Reagents, NIBSC according to the EU pro-
gram EVA (Framework V), U.K. Medical Research Council.
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HRMS: m/z [M + H]+ calcd for C18H21O2: 269.1542; found: 269.1536.
meso-1,4-Bis(benzyloxy)butane-2,3-diol (3)
night with continuous removal of in situ formed H2O using a Dean–
Stark apparatus. The reaction was worked up by extracting the mix-
ture with EtOAc. The organic layer was washed with H2O and dried
over MgSO4, the solvent evaporated and the residue was purified
through a silica column to give crude compound 7 (4 g, 40%).
To a solution of 2 (25.0 g, 93 mmol) in acetone (200 mL) and H2O (200
mL), NMO (12 g, 102 mmol) and a 0.02 M solution of OsO4 in t-BuOH
(5 mL) were added at r.t. The mixture was stirred at r.t. for 12–14 h.
The work-up was performed by diluting the mixture with EtOAc and
washing with sat. aq Na2S2O4 solution, H2O, and brine, respectively.
The organic layer was dried over MgSO4 and concentrated. The resi-
due was purified by silica gel column chromatography (hexane–
EtOAc, 1:1) to give 3 (21.3 g, 76%) as a white solid; mp 58–59 °C.
Crude compound 7 (3.5 g, 9.65 mmol) was dissolved in dry THF (100
mL) and stirred for 5 min at 0 °C. To this solution was added TBAF
(14.45 mL, 1 M solution in THF, 14.45 mmol). The mixture was stirred
for 1 h at 0 °C and worked up by extraction with EtOAc. The organic
layer was washed with H2O, dried over MgSO4 and evaporated to give
a crude mixture of diastereomers 8a and 8b which were separated by
column chromatography (EtOAc–hexane, 3:1).
1H NMR (500 MHz, CDCl3): δ = 2.69–2.71 (m, 2 H), 3.60–3.67 (m, 4 H),
3.81–3.82 (br s, 2 H), 4.53 (s, 4 H), 7.24–7.35 (m, 10 H).
13C NMR (125 MHz, CDCl3): δ = 71.2, 71.6, 73.5, 127.8, 128.0, 128.5,
137.9.
HRMS: m/z [M + Na]+ calcd for C18H22O4Na: 325.1416; found:
325.1417.
Compound 8a
Compound 8a (0.60 g, 39%) was obtained as a pale yellow solid; mp
36–37 °C.
Purity = 99% [determined by HPLC on a 5 μM Fortis C18 column
(150 × 4.6 mm), MeCN–H2O, 20:80; flow rate = 0.6 mL/min, λ = 254
nm]; tR = 12.07 min.
1H NMR (500 MHz, CDCl3): δ = 2.60 (s, 1 H), 3.32 (br s, 1 H), 3.48 (dd, J
= 15, 5 Hz, 1 H), 3.68–3.76 (m, 3 H), 4.01–4.04 (m, 1 H), 4.12–4.15 (m,
1 H), 4.35–4.37 (m, 1 H), 4.60–4.67 (m, 2 H), 7.31–7.34 (m, 5 H).
13C NMR (125 MHz, CDCl3): δ = 63.1, 66.8, 72.5, 73.7, 74.0, 77.0, 80.0,
102.0, 126.4, 127.85, 127.91, 128.3, 128.5, 137.2.
HRMS: m/z [M + Na]+ calcd for C14H16O4Na: 271.0946; found:
271.0949.
1-(Benzyloxy)-4-(trimethylsilyl)but-3-yn-2-ol (5)
To a solution of 3 (12 g, 39.6 mmol) in MeOH (90 mL), NaIO4 (12.76 g,
59.52 mmol) was added at 0 °C. The resulting mixture was stirred for
8 h at r.t., followed by removal of precipitates by filtration. Most of the
solvent was removed using a rotary evaporator and the residual oil
(4) (9.62 g, 81%) was used as such for further transformation. In an-
other round-bottom flask, a solution of trimethylsilylacetylene (8.4
mL, 59.35 mmol) in THF (50 mL) was stirred at –78 °C. To this solution
was added n-BuLi (37.5 mL, 2.5 M solution in hexanes, 87.88 mmol)
slowly, and the resulting mixture was stirred at –78 °C for 30 min. To
this solution was added compound 4 (9 g, 59.92 mmol) in THF (30
mL) dropwise, and the resulting mixture was further stirred for 5 min
at –78 °C and then at 0 °C for 2 h. Sat. NH4Cl (50 mL) was added to the
mixture at 0 °C, followed by extraction with EtOAc (3 × 100 mL). The
combined organic layers were washed with brine (100 mL) and dried
over MgSO4. The solvent was removed by evaporation and the result-
ing residue was purified by column chromatography (EtOAc–hexane,
1:3) to afford racemic propargylic alcohol (±)-5 as a pale yellow oil
(11.5 g, 77% yield).
Compound 8b
Compound 8b (0.34 g, 22%) was obtained as a pale yellow solid; mp
34–35 °C.
Purity = 99% [determined by HPLC on a 5 μM Fortis C18 column
(150 × 4.6 mm), MeCN–H2O, 20:80; flow rate = 0.6 mL/min, λ = 254
nm]; tR = 11.68 min.
1H NMR (500 MHz, CDCl3): δ = 2.47 (br s, 1 H), 2.62 (s, 1 H), 3.67–3.80
(m, 4 H), 3.99–4.02 (m, 1 H), 4.17–4.20 (m, 1 H), 4.26–4.28 (m, 1 H),
4.68 (s, 2 H), 7.33–7.34 (m, 5 H).
13C NMR (125 MHz, CDCl3): δ = 62.2, 67.3, 73.4, 73.92, 73.93, 78.2,
81.0, 102.2, 127.7, 128.4, 137.8.
HRMS: m/z [M + Na]+ calcd for C14H16O4Na: 271.0946; found:
271.0941.
1H NMR (500 MHz, CDCl3): δ = 0.00 (9 H, s), 3.35–3.44 (m, 3 H), 4.36–
4.40 (m, 3 H), 7.09–7.15 (m, 5 H).
13C NMR (125 MHz, CDCl3): δ = 0.0, 62.1, 73.4, 73.8, 90.2, 104.1, 127.9,
128.0, 128.4, 128.6, 137.9.
HRMS: m/z [M + Na]+ calcd for C14H20O2SiNa: 271.1130; found:
271.1125.
(R)-3-(tert-Butyldiphenylsilyloxy)propane-1,2-diol (11)12
In a round-bottom flask containing a solution of compound 9 (24 g,
181.5 mmol) and dry CH2Cl2 (150 mL) was added Et3N (30.39 mL,
217.8 mmol) and DMAP (1.10 g, 9 mmol). The mixture was stirred at
0 °C for 10 min and then TBDPSCl (50 g, 181.5 mmol) was added to
the mixture dropwise. The mixture was slowly allowed to warm to r.t.
and was further stirred for 24 h. The mixture was worked up by ex-
traction with EtOAc. The organic layer was washed with H2O and
brine, dried over MgSO4, evaporated under vacuum and purified
through a silica column to afford compound 10 (51.81 g, 77%).
[(2S,4S)-2-(Benzyloxymethyl)-2-ethynyl-1,3-dioxolan-4-yl]metha-
nol (8a) and [(2R,4S)-2-(Benzyloxymethyl)-2-ethynyl-1,3-dioxol-
an-4-yl]methanol (8b)
In a round-bottom flask containing 5 (9.4 g, 37.8 mmol) and CH2Cl2
(142 mL) was added 5% aq NaHCO3 solution (75.2 mL). After stirring
the mixture for 10 min, TEMPO (58.28 mg, 0.372 mmol), KI (628 mg,
3.78 mmol), and NaOCl (65 mL, 5% solution) were added to the bipha-
sic mixture. The mixture was further stirred for 1.5 h and then ex-
tracted with CH2Cl2. The organic layer was dried over MgSO4, filtered,
and evaporated to yield 6 as a light yellow oil (9.02 g, 97%), which was
immediately used for further transformation.
Compound 10 (10 g, 26.98 mmol) was added to a round-bottom flask
containing 20% aq AcOH (50 mL) and the mixture was heated at reflux
temperature (90 °C) overnight. Excess AcOH was evaporated under
vacuum and the residue was purified by column chromatography
(EtOAc–hexane, 1:9) to afford product 11 as a white solid (7.36 g,
76%); mp 58–60 °C.
In a two-neck round-bottom flask containing a solution of compound
11 (7 g, 21.21 mmol) and dry benzene (120 mL) was added freshly
prepared compound 6 (4.45 g, 18.06 mmol) and PTSA (343.2 mg, 1.80
mmol). The mixture was heated at reflux temperature (90 °C) over-
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2016, 48, A–G

F
S. Singh et al.
Paper
Synthesis
1H NMR (500 MHz, CDCl3): δ = 1.10 (s, 9 H), 2.23 (s, 1 H), 2.72 (s, 1 H),
3.60–3.79 (m, 5 H), 7.37–7.56 (m, 6 H), 7.64–7.63 (m, 4 H).
13C NMR (125 MHz, CDCl3): δ = 19.2, 26.9, 63.9, 65.2, 71.9, 127.9,
crude product was purified by column chromatography (EtOAc–hex-
ane, 3:7) to afford pure 14a (102 mg, 38%) as a white solid (~2:1 mix-
ture of diastereomers).
130.0, 132.9, 135.5.
1H NMR (500 MHz, CDCl3): [mixture of diastereomers (~2:1 ratio)]:
δ = 1.56 (s , 1 H), 1.94 (s, 2 H), 2.62 (s, 0.35 H), 2.77 (s, 0.65 H), 3.74 (s,
1.30 H), 3.93 (s, 0.70 H), 4.08-4.18 (m, 2 H), 4.49–4.52 (m, 0.35 H),
4.57–4.60 (m, 0.65 H), 4.63–4.69 (s, 2 H), 6.32–6.34 (m, 0.65 H), 6.53–
6.54 (d, J = 5 Hz, 0.35 H), 7.30–7.37 (m, 4 H), 7.59–7.60 (d, J = 7 Hz, 1
H), 9.10 (s, 0.35 H), 9.19 (s, 0.65 H).
13C NMR (125 MHz, MeOD): δ = 12.0, 12.6, 70.4, 71.1, 71.6, 73.5, 74.1,
74.4, 74.8, 75.9, 78.1, 78.9, 80.3, 83.2, 103.4, 104.3, 111.3, 111.7,
127.7, 127.9, 128.1, 128.5, 128.6, 135.3, 135.7, 137.0, 137.3, 150.7,
150.8, 163.8, 163.9.
(R)-1-{2-[(Benzyloxy)methyl]-2-ethynyl-1,3-dioxolan-4-yl}-ace-
tate (13a)
To a two-neck round-bottom flask were added compound 8a (800
mg, 3.22 mmol), PDC (18.18 g, 48.33 mmol) and DMF (50 mL). The
mixture was stirred at r.t. for 24 h and worked up by washing with
H2O (40 mL) and extraction with Et2O (4 × 40 mL). The organic layer
was dried over MgSO4 and evaporated to afford 12a (622.3 mg, 74%).
Compound 12a was used directly for the next transformation without
any purification.
HRMS: m/z [M + Na]+ calcd for C18H18N2O5Na: 365.1113; found:
365.1123.
Compound 12a (300 mg, 1.14 mmol) was taken up in a two-neck
round-bottom flask containing dry THF (12 mL). To this mixture was
added Pb(OAc)4 (760 mg, 1.71 mmol) and pyridine (0.15 mL, 1.86
mmol) under inert conditions. The mixture was stirred for 40 min un-
der nitrogen and worked up by adding EtOAc (50 mL) to the mixture.
The mixture was filtered and purified to give acetate 13a (184.3 mg,
58%) as a pale yellow liquid (~1:1 mixture of diastereomers).
1H NMR (500 MHz, CDCl3): δ = 1.96 (s, 1.5 H), 2.11 (s, 1.5 H), 2.60 (s,
0.5 H), 2.63 (s, 0.5 H), 3.70–3.76 (m, 2 H), 4.15–4.31 (m, 2 H), 4.67 (s, 1
H), 4.71 (s, 1 H), 6.39–6.41 (br s, 1 H), 7.25–7.37 (m, 5 H).
(S)-1-{2-[(Benzyloxy)methyl]-2-ethynyl-1,3-dioxolan-4-yl}-thy-
mine (14b)
Compound 14b (115 mg, 43%) was obtained as a white solid (~2:1
mixture of diastereomers) using the same sequence of steps as de-
scribed above starting from compound 13b (148 mg scale).
1H NMR (500 MHz, CDCl3): [mixture of diastereomers (~2:1 ratio)]:
δ = 1.56 (s, 1 H), 1.95 (s, 2 H), 2.62 (s, 0.3 5H), 2.77 (s, 0.65 H), 3.74 (s,
1.30 H), 3.94 (s, 0.70 H), 4.10–4.18 (m, 2 H), 4.49-4.53 (m, 0.35 H),
4.57–4.60 (m, 0.65 H), 4.63–4.69 (s, 2 H), 6.32–6.34 (m, 0.65 H), 6.52–
6.54 (dd, J = 5.5, 1.5 Hz, 0.35 H), 7.29–7.38 (m, 4 H), 7.58–7.60 (m, 1
H), 8.68 (s, 0.35 H), 8.83 (s, 0.65 H).
13C NMR (125 MHz, MeOD): δ = 20.9, 21.0, 71.0, 71.3, 73.0, 73.1, 73.8,
74.0, 74.2, 79.1, 79.5, 94.2, 94.9, 103.4, 104.2, 127.7, 128.3, 128.4,
137.7, 137.8, 170.0, 170.1.
HRMS: m/z [M + Na]+ calcd for C15H16O5Na: 299.0895; found:
299.0893.
13C NMR (125 MHz, MeOD): δ = 12.0, 12.6, 14.2, 21.0, 60.3, 70.4, 71.6,
73.5, 74.1, 74.4, 75.9, 83.2, 103.4, 104.3, 111.3, 127.7, 127.9, 128.0,
128.1, 128.5, 135.3, 137.3, 150.7, 163.8.
HRMS: m/z [M + Na]+ calcd for C18H18N2O5Na: 365.1113; found:
(S)-1-{2-[(Benzyloxy)methyl]-2-ethynyl-1,3-dioxolan-4-yl}-ace-
tate (13b)
365.1121.
Compound 13b (189 mg; 60%) was obtained as a pale yellow liquid
using a similar strategy to that described above starting from com-
pound 8b (~1:1 mixture of diastereomers).
1H NMR (500 MHz, CDCl3): δ = 1.99 (s, 1.5 H), 2.11 (s, 1.5 H), 2.60 (s,
0.5 H), 2.63 (s, 0.5 H), 3.71–3.77 (m, 2 H), 4.16–4.32 (m, 2 H), 4.67 (s, 1
H), 4.71 (s, 1 H), 6.40 (br s, 1 H), 7.27–7.37 (m, 5 H).
13C NMR (125 MHz, MeOD): δ = 20.9, 21.0, 71.0, 71.4, 73.0, 73.2, 73.9,
74.1, 74.2, 79.1, 79.5, 94.2, 94.9, 103.5, 104.3, 127.7, 127.8, 128.3,
128.4, 137.7, 137.8, 170.0, 170.2.
Compounds 15a–d
To a perfectly dry two neck round-bottom flask was added compound
14a (200 mg, 0.554 mmol) dissolved in dry CH2Cl2 (8 mL). The mix-
ture was allowed to stir for 5 min at 0 °C, followed by the addition of
anisole (0.23 mL, 2.33 mmol) and AlCl3 (233.6 mg, 1.75 mmol) at the
same temperature. The mixture was allowed to stir overnight at 0 °C.
The reaction was worked up by adding H2O (20 mL) to the mixture,
followed by the addition of 1 M HCl at 0 °C until the pH was 2–3. The
mixture was further stirred at 0 °C for 5 min and then extracted with
EtOAc. The organic layer was washed with H2O, dried over MgSO4 and
evaporated to give the crude product as a diastereomeric mixture of
15a and 15b. Both diastereomers were separated/purified by column
chromatography (MeOH and CH2Cl2). Compounds 15c and 15d were
synthesized by following the same sequence of steps starting from
compound 14b (on 200 mg scale).
HRMS: m/z [M + Na]+ calcd for C15H16O5Na: 299.0895; found:
299.0892.
(R)-1-{2-[(Benzyloxy)methyl]-2-ethynyl-1,3-dioxolan-4-yl}-thy-
mine (14a)
To a perfectly dry round-bottom flask were added dry CH2Cl2 (20 mL)
and thymine (102.2 mg, 0.81 mmol). After 2 min of stirring, TBDM-
SOTf (0.37 mL, 1.61 mmol) was added slowly to the mixture which
was allowed to stir for 2 min. Next, 2,4,6-collidine (0.21 mL, 1.62
mmol) was added and the mixture was allowed to stir for 30 min at
40 °C. To this clear solution was added a solution of compound 13a
(148 mg, 0.53 mmol) and dry CH2Cl2 (10 mL) slowly, followed by TMSI
(0.21 mL). The mixture was allowed to stir for 3–4 h and then
quenched with sat. Na2S2O3 solution, followed by extraction with
CH2Cl2. The organic layer was washed with H2O, dried over MgSO4
and evaporated to give crude 14a as a diastereomeric mixture. The
(–)-(2R,4R)-1-[2-Ethynyl-2-(hydroxymethyl)-1,3-dioxolan-4-yl]-
thymine (15a)
Yield: 53 mg (36%); white solid; mp 247–248 °C; Rf = 0.57 (EtOAc–
hexane, 20:80); [α]D20 –16.95 (c 1.0, MeOD); purity >99% [determined
by HPLC on a 5 μM Fortis C18 column (150 × 4.6 mm), MeCN–H2O,
20:80; flow rate = 0.6 mL/min, λ = 254 nm]; tR = 6.12 min.
1H NMR (500 MHz, MeOD): δ = 1.83 (s, 3 H), 3.17 (s, 1 H), 3.88 (dd, J =
13.5, 7.5 Hz, 2 H), 4.27 (d, J = 10.5 Hz, 1 H), 4.42–4.45 (m, 1 H), 6.42 (d,
J = 6 Hz, 1 H), 7.79 (s, 1 H).
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2016, 48, A–G

G
S. Singh et al.
Paper
Synthesis
13C NMR (125 MHz, MeOD): δ = 10.9, 63.7, 69.4, 74.8, 78.2, 80.9,
103.9, 110.5, 136.8, 151.2, 164.9.
Supporting Information
Supporting information for this article is available online at
HRMS (Q-TOF): m/z [M + Na]+ calcd for C11H12N2O5Na: 275.0644;
found: 275.0646.
http://dx.doi.org/10.1055/s-0035-1561637.
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hexane, 20:80); [α]D +9.67 (c 1.0, MeOD); purity >99% [determined
by HPLC on a 5 μM Fortis C18 column (150 × 4.6 mm), MeCN–H2O,
20:80; flow rate = 0.6 mL/min, λ = 254 nm]; tR = 9.93 min.
20
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thymine (15c)
Yield: 50 mg (34%); white solid; mp 247–248 °C; Rf = 0.57 (EtOAc–
hexane, 20:80); [α]D20 +16.21 (c 1.0, MeOD); purity >99% (determined
by HPLC on a 5 μM Fortis C18 column (150 × 4.6 mm), MeCN–H2O,
20:80; flow rate = 0.6 mL/min, λ = 254 nm]; tR = 5.89 min.
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13.0, 8.5 Hz, 2 H), 4.18 (d, J = 10 Hz, 1 H), 4.32–4.35 (m, 1 H), 6.33 (d, J
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103.6, 110.2, 136.5, 150.9, 164.6.
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HRMS (Q-TOF): m/z [M + Na]+ calcd for C11H12N2O5Na: 275.0644;
found: 275.0647.
(–)-(2S,4R)-1-[2-Ethynyl-2-(hydroxymethyl)-1,3-dioxolan-4-yl]-
thymine (15d)
Yield: 26.6 mg (18%); white solid; mp 205–206 °C; Rf = 0.42 (EtOAc–
hexane, 20:80); [α]D20 –9.33 (c 1.0, MeOD); purity >99% [determined
by HPLC on a 5 μM Fortis C18 column (150 × 4.6 mm), MeCN–H2O,
20:80; flow rate = 0.6 mL/min, λ = 254 nm]; tR = 9.87 min.
1H NMR (500 MHz, MeOD): δ = 1.77 (s, 3 H), 3.18 (s, 1 H), 3.59 (dd, J =
13.0, 7.5 Hz, 2 H), 4.07 (d, J = 9.5 Hz, 1 H), 4.42 (t, J = 8 Hz, 1 H), 6.21 (s,
1 H), 7.59 (s, 1 H).
13C NMR (125 MHz, MeOD): δ = 11.2, 65.8, 70.7, 76.4, 79.0, 83.1,
104.7, 110.3, 136.0, 151.1, 164.9.
HRMS (Q-TOF): m/z [M + Na]+ calcd for C11H12N2O5Na: 275.0644;
found: 275.0645.
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Acknowledgment
This work was supported by a Korea University Grant (K1505491) and
a grant of the Basic Science Research Program through the National
Research Foundation of Korea (NRF) funded by the Korean govern-
ment (MSIP) (No. 2014R1A2A2A01005455, 2012M3A9C1053532, and
NRF-2015M3A6A4065734), Republic of Korea.
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2016, 48, A–G