A general approach to the synthesis of 5-S-functionalized pyrimidine nucleosides and their analogues

Article abstract of DOI:10.1039/c4ob00597j

A general and efficient approach was developed for the introduction of S-functionality at the C-5 position of cytosine and uracil nucleosides and their analogues. The key step is a palladium-catalyzed C-S coupling of the corresponding 5-bromo nucleoside derivative and alkyl thiol. The butyl 3-mercaptopropionate coupling products were further converted to the corresponding disulphides, the stable precursors of 5-mercaptopyrimidine nucleosides. the Partner Organisations 2014.

Full text of DOI:10.1039/c4ob00597j

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A general approach to the synthesis of
5-S-functionalized pyrimidine nucleosides and
their analogues†
Cite this: Org. Biomol. Chem., 2014,
12, 5634
Dzmitry G. Kananovich,a Alli Reino,a Kaja Ilmarinen,a Marko Rõõmusoks,a
Mati Karelsonb and Margus Lopp*a
A general and efficient approach was developed for the introduction of S-functionality at the C-5 position
of cytosine and uracil nucleosides and their analogues. The key step is a palladium-catalyzed C–S coup-
ling of the corresponding 5-bromo nucleoside derivative and alkyl thiol. The butyl 3-mercaptopropionate
coupling products were further converted to the corresponding disulphides, the stable precursors of
5-mercaptopyrimidine nucleosides.
Received 19th March 2014,
Accepted 11th June 2014
DOI: 10.1039/c4ob00597j
www.rsc.org/obc
furnishing 5-mercaptopyrimidine derivatives under mild
conditions is required.
Introduction
Nucleosides and their analogues are important substances
owing to their broad medical use. Structural modifications of
these molecules are of great practical importance, making it
possible to expand the number of compounds with anti-
tumour or antiviral activity, and/or to adjust pharmacological
properties of the parent compounds.1 One of the modifi-
cations of substantial interest is the introduction of S-function-
ality into a nucleobase ring. For instance, double-stranded
RNAs modified at the 5-position by selective thiolation of
some cytosine bases have shown high activity against HIV-1 in
human cells and against DNA polymerases of DNA and RNA
tumours.2
5-Mercaptopyrimidine nucleosides have been prepared by a
number of methods, including reactions of 5-halogenated pyri-
midine derivatives with sulphur nucleophiles,3 the addition of
the electrophilic sulphur species (ClSCN) to the double bond
of the pyrimidine base,4 and palladium-mediated substitution
in 5-mercuropyrimidine derivatives.5 The thiolated nucleosides
have also been prepared by the Hilbert–Johnson reaction of
the corresponding pyrimidine bases and sugar derivatives.3,6
However, most of these methods include tedious multi-
step procedures, require harsh conditions and cannot be
universally applied. Therefore, a new and general approach
Results and discussion
During our work on nucleoside analogues, we needed to
modify the structure of the well-known antiviral compound
lamivudine 1a (Scheme 1) and to synthesize its 5-mercapto
substituted derivative 2a for the evaluation of its antiviral
properties.
Several known approaches for the synthesis of compound
2a have been tested by us, mostly unsuccessfully or with
limited success. For example, the Hilbert–Johnson synthesis3,6
from 5-benzylthiouridine and the corresponding sugar ana-
logue led to a mixture of diastereomers, and required harsh
conditions (sodium metal/liquid ammonia) for the removal of
the benzyl protecting group and release of the free thiol func-
tion. As a result, the desired product was obtained in very low
yield. Therefore, the chemical modification of commercially
available lamivudine 1a seemed to be a more attractive
approach. However, a number of attempts to introduce the
thiol group via substitution of the bromine atom into the
aTallinn University of Technology, Department of Chemistry, Akadeemia tee 15,
12618 Tallinn, Estonia
bTartu University, Institute of Chemistry, Ravila 14a, 50411 Tartu, Estonia.
E-mail: margus.lopp@ttu.ee
†Electronic supplementary information (ESI) available: Copies of NMR spectra for
new compounds (.pdf); crystallographic data for compound 1b. CCDC 992209. For
ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/
c4ob00597j
Scheme 1 Lamivudine 1a and its thiolated analogue 2a.
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Scheme 2 Attempted synthesis of 5-mercaptocytosine derivatives.
Scheme 3 Preparation of 4-thiouracil derivative 6.
corresponding 5-bromo derivative 3a by reactions with several
sulphur nucleophiles3 (e.g. Na2S, NaSH and Na2S2) failed. A
complete reductive debromination,3,7 which led to the starting
lamivudine (1a), was observed instead of the desired substi-
tution reaction (Scheme 2).
In order to determine the optimal experimental conditions
and to avoid the undesired debromination process, several
experiments were undertaken with a model 5-bromocytosine
nucleoside analogue 3b. Thus, we tried to achieve the substi-
tution of the bromine atom in some N-acyl protected deriva-
tives of 3b in the hope of increasing activity towards
nucleophile attack at the C-5 position due to the electron-with-
drawing nature of the acyl group. For example, phthaloyl-
protected compound 4 (Scheme 3) smoothly reacted with butyl
3-mercaptopropionate (5) in the presence of DIPEA in an aceto-
nitrile–chloroform solution, giving the product in high (90%)
yield.
However, it was unexpectedly found that, instead of substi-
tution of the C-5 bromine, replacement of the C-4 phthalimide
group occurred, leading to the C-4 substituted sulphur deriva-
tive 6 (Scheme 3). This result may be considered to be evidence
of the deactivation at the C-5 position of the pyrimidine-2(1H)-
one system in compound 4 towards nucleophilic substitution
in favour of substitution at the C-4 position.
These observations, together with the mechanistical con-
siderations made earlier,7,8 allowed us to assume that attempts
to substitute the C-5 halogen of the cytosine ring via reactions
with sulphur nucleophiles are inefficient. So we proposed that
it is possible to achieve the goal by changing the mechanism
of the substitution reaction by using transition-metal catalysis,
which is known to efficiently promote halide substitution at an
sp2-hybridized carbon atom.9 A number of experimental proto-
cols have been developed in recent decades for the copper-,
nickel- and palladium-promoted C–S coupling reactions at
sp2-carbons and S-nucleophiles.10 Among the various possible
options, we selected the procedure described by Itoh and
Mase,11 due to its mild conditions, easily available reagents,
simple experimental protocol and low catalyst load.
To our delight, when the pivaloyl-protected model com-
pound 7b was involved in the palladium-catalyzed reaction
with butyl 3-mercaptopropionate 5, the desired coupling
product 8b was obtained in 77% yield (Scheme 4; Table 1,
entry 2). The use of pivaloyl protection for the amine group
considerably increases the solubility of the cytosine derivative
7b in common organic solvents, which is necessary to carry
out the reaction and makes the isolation of the product con-
venient. This protecting group also seems to facilitate the
Scheme 4 Synthesis of S-functionalized cytosine and uracil derivatives.
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Table 1 Preparation of 5-S-functionalized cytosine and uracil derivatives
Entry
1
Starting bromide
Thiol
Product
Yielda (%)
83
2
77
3
81
4
52
5
88
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Table 1 (Contd.)
Entry
6
Starting bromide
Thiol
Product
Yielda (%)
87
7
8
9
58b
87
c
No reaction
—
a Isolated yields. b Tetrapivaloylcytidine was isolated as a by-product in 20% yield. c Starting material was recovered in 90% yield.
coupling reaction. Thus, the Pd-catalyzed reaction of and 95% yields, respectively), and in moderate yield with
the unprotected 5-bromocytosine derivative 3b with butyl 5-bromocytidine 3c (41% yield). Acylation could be con-
3-mercaptopropionate (5) in DMF at 100 °C resulted in only veniently performed in one flask, followed by bromination
5% conversion of the starting material after 24 h.
(Scheme 4).
The coupling products of butyl 3-mercaptopropionate 8a,
To prove the scope of the method, we introduced S-func-
tionality into different natural pyrimidine nucleosides (cyti- 8c and 9d (Table 1, entries 1, 3 and 5) were obtained in high
dine 1c, 2′-deoxycytidine 1d and 2′-deoxyuridine) and into the yields (81–88%), with the only exception being the 2′-deoxycyti-
nucleoside analogue lamivudine 1a. Although an additional dine derivative 8d (entry 4), which was isolated in moderate
step was necessary to introduce the acyl protective group, it yield (52%). In the present work, we mostly used the pivaloyl-
proceeded in good yields with compounds 3a and 3b (80% protected compounds 7a–d and 10d. However, TBS-protected
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Scheme 5 Preparation of disulphides 13a and 13b, and generation of thiol 2a.
5-bromo-2′-deoxyuridine 11d (entry 6) gave the corresponding tion with aqueous ammonium chloride, the insoluble disul-
product 12d in the same high yield as its pivaloyl-protected phide 14a was collected in 78% yield by simple filtration.
analogue 10d. On the other hand, benzoyl-protected 5-bromo- Compound 8d was deprotected in an analogous manner to
cytidine was non-reactive under these conditions, giving only a afford 2′-deoxycytidine disulphide 14d in 80% yield.
trace amount of the desired coupling product.
5-Mercaptopyrimidine nucleosides 2 can be obtained from
A further example of a functionalized alkyl thiol, 4-methoxy- disulphides 14 by their in situ reduction with dithiothreitol
benzyl mercaptan, in reaction with the pivaloyl-protected (DTT) (Scheme 5).3,4b,c Thus, a colourless solution of disul-
5-bromo-2′-deoxyuridine 10d, gave the corresponding coupling phide 14a in DMSO gave a bright yellow solution of the corres-
product 13d in 87% yield (entry 8). The reaction of the same ponding 5-mercapto derivative 2a when treated with an excess
thiol with 5-bromocytidine derivative 7c afforded the product of DTT.
13c in only 58% isolated yield (entry 7), and was accompanied
by a reductive debromination reaction, leading to a tetrapiva-
loylcytidine by-product. Although the coupling reaction pro-
ceeded smoothly with the tested examples of alkyl thiols,
Conclusions
thiophenol failed to give a coupling product and the starting
material 10d was recovered in 90% yield in this case (entry 9).
In conclusion, a new general and short approach for the intro-
duction of sulphur at the C-5 position of cytosine and uracil
The developed reaction sequence may be considered as a from available pyrimidine nucleosides and their analogues as
general approach for the introduction of alkyl S-functionality,
precursors was elaborated. The key step of the proposed reac-
as well as the thiol group, into pyrimidine nucleosides, tion sequence is a palladium-mediated C–S coupling reaction
because the butyl 3-mercaptopropionate group serves as a of the corresponding bromo-derivatives and alkyl thiols, which
source of hidden thiol function.11 It is known that the free
proceeds under mild conditions and gives the coupling pro-
thiol group is released when 3-mercaptopropionate derivatives ducts in good yields. The S–H moiety is easily obtainable from
are treated with a strong base, such as t-BuOK12 or EtONa,11 the intermediate thiopropionates.
and the removal of the acyl groups of compounds 8a–d may
follow in the same flask.
This one-pot protocol was first tested for the thiol gene-
ration/deprotection of compounds 8a and 8b. The procedure
included the treatment of 8a and 8b with an excess (5 equiv.)
Experimental
General methods
of potassium tert-butylate in THF in a temperature range from
−78 °C to −15 °C within 2 h,12 followed by reaction with
methanolic ammonia for the removal of acyl groups
(Scheme 5). Since the product with free thiol functions is sus-
ceptible to air oxidation,3,13 it was isolated as a stable disul-
phide from 14b in ∼60% yield. Following the same procedure,
compound 14a was obtained in 39% isolated yield, which
might have been caused by its low solubility in organic sol-
vents applied during the isolation and chromatographical
purification, as well as due to strong adsorption on silica gel.
That is why a modification of the deprotection procedure was
required. When compound 8a was treated with sodium meth-
oxide (1.3 equiv.) in methanol overnight under an argon
atmosphere and then exposed to air after quenching the reac-
Dioxane was distilled over LiAlH4 under argon. Acetonitrile
and pyridine were distilled over CaH2. Methanol was dried
over 3 Å molecular sieves or sodium and distilled. All other sol-
vents were purified and dried by conventional methods prior
to use. Unless noted otherwise, reagents and starting materials
were used as purchased from commercial suppliers. Silica gel
40–100 μm was used for column chromatography; precoated
1
silica gel 60 F254 plates were used for TLC. H NMR (400 MHz)
and 13C NMR (100.6 MHz) spectra were taken on a Bruker
Avance III spectrometer. Peaks of residual protons of deuter-
ated solvents or TMS signal were used as chemical shift refer-
ences. FT-IR spectra were recorded on a Bruker Tensor 27 FT
spectrometer. RP-HPLC-MS was performed on an Agilent 6540
UHD Accurate-Mass Q-TOF LC/MS spectrometer using AJ-ESI
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ionization. Specific rotation was measured using an Anton (br s, 1H, NH), 5.71 (d, J = 7.4 Hz, 1H), 5.51 (dd, J = 10.3,
Paar MCP 500 polarimeter. Single-crystal X-ray diffraction data 1.7 Hz, 1H), 4.02–3.95 (m, 1H), 3.61–3.49 (m, 1H), 1.91–1.81
were obtained using a Bruker SMART X2S bench top diffracto- (m, 1H), 1.70–1.40 (m, 5H). 13C NMR (DMSO-d6, 100.6 MHz)
meter with Mo-Kα radiation (λ = 0.71070 Å).
δ 165.47, 154.50, 141.21, 93.93, 81.93, 68.03, 30.43, 24.64,
22.56. IR (KBr, cm−1) νmax 3363, 3162, 2942, 1658, 1642, 1604,
1492, 1207. Anal. Calcd for C9H13N3O2: 21.52% N, 55.37% C,
Tetrahydropyran-2-yl acetate14
Acetic acid (5.7 mL, 0.1 mol) was added to the solution of pyri- 6.71% H; found: 21.65% N, 55.07% C, 6.74% H. HRMS (ESI)
dinium p-toluenesulfonate (0.25 g, 1 mmol, 1 mol%) in calcd for C9H14N3O2 [M + H]+ 196.1086, found m/z 196.1081.
dichloromethane (40 mL) followed by addition of 3,4-dihydro-
5-Bromo-1-(tetrahydro-2H-pyran-2-yl)cytosine (3b)
4H-pyran (10 mL, 0.11 mol) at 0 °C. The reaction mixture was
allowed to stand overnight at room temperature, volatiles were The title compound was prepared by an adaptation of the
removed under reduced pressure, and the residue was purified already described procedures.8,19 A suspension of 1-(tetra-
by short-column flash chromatography on silica gel (hexane– hydro-2H-pyran-2-yl)cytosine (1.20 g, 6.15 mmol) in pyridine
ethyl acetate, 5 : 1). The obtained product (12.2 g, 85%) was (20 mL) was cooled in an ice bath, and then a solution of
additionally purified by distillation collecting the fraction bromine (1.08 g, 0.35 mL, 6.75 mmol) in tetrachloromethane
boiling at 67–69 °C (6–7 Torr) [lit.15 55–56 °C (2 Torr)]. Yield (13 mL, approx. 5% w/w) was added under stirring. The reac-
9.8 g (68%). Rf 0.4 (hexane–ethyl acetate, 5 : 1). 1H NMR tion mixture was allowed to warm to room temperature and
(CDCl3, 400 MHz) δ 5.95 (m, 1H), 3.91 (ddd, J = 11.9, 9.1, stirred for approx. 1–2 hours until the reaction was complete
3.1 Hz, 1H), 3.73–3.64 (m, 1H), 2.10 (s, 3H), 1.90–1.75 (m, 2H), (TLC monitoring). Then
a solution of Na2CO3 (0.50 g,
1.73–1.47 (m, 4H). 13C NMR (CDCl3, 100.6 MHz) δ 170.02, 3.1 mmol) in water (2 mL) was added, volatiles were removed
92.82, 63.53, 29.33, 25.06, 21.35, 18.86. Spectral data are in under reduced pressure (bath temperature 30–35 °C), and the
accordance with those reported.16,17
residue was coevaporated with toluene (3 × 15 mL) to remove
pyridine. Column chromatography on silica gel (dichloro-
methane–methanol, 20 : 1 to 5 : 1) afforded the title compound
1-(Tetrahydro-2H-pyran-2-yl)cytosine (1b)18
A two-necked round-bottom flask (250 mL), carrying a reflux as a yellowish solid (1.61 g, 95% yield). An analytically pure
condenser with a calcium chloride drying tube in one neck sample was prepared by recrystallization from ethanol. Rf 0.50
and a septum cap in another neck, was charged with cytosine (dichloromethane–methanol, 10 : 1). 1H NMR (CDCl3,
(2.2 g, 20 mmol) and dry acetonitrile (70 mL). Then bis(tri- 400 MHz) δ 8.37 (br s, 1H, NH), 7.75 (s, 1H), 5.71 (br s, 1H,
methylsilyl)acetamide (17 mL, 70 mmol) was added via a NH), 5.61 (dd, J = 10.6, 2.2 Hz, 1H), 4.14 (m, 1H), 3.69 (m, 1H),
syringe and the obtained suspension was heated to 60 °C (oil 2.06 (m, 1H), 1.95 (m, 1H), 1.78–1.53 (m, 3H), 1.35 (m, 1H).
bath) under stirring. Within 30–40 min at this temperature a 13C NMR (CDCl3, 100.6 MHz) δ 162.25, 154.06, 141.58, 87.50,
transparent solution was obtained, and the reaction flask was 83.63, 69.22, 32.05, 25.13, 22.69. IR (KBr, cm−1) νmax 3438,
cooled to room temperature. Then a solution of tetrahydro- 3047, 2950, 1659, 1612, 1503, 1458, 1086, 775, 501. Anal. Calcd
pyran-2-yl acetate (3.2 g, 22 mmol) in dry acetonitrile (20 mL) for C9H12BrN3O2: 15.33% N, 39.43% C, 4.41% H; found:
was added via a syringe followed by trimethylsilyl trifluoro- 15.56% N, 39.83% C, 4.61% H. HRMS (ESI) calcd for
methanesulfonate (4.0 mL, 22 mmol), and the reaction C9H13BrN3O2 [M + H]+ 274.0191, found m/z 274.0196.
mixture was stirred at room temperature for 24 h. The reaction
Following the same procedure, 5-bromo-2′,3′-dideoxy-3′-
was stopped by the addition of 25% aq. ammonia (7 mL) in thiacytidine (3a) was obtained from 2′,3′-dideoxy-3′-thiacyti-
methanol (10 mL) and the reaction mixture was stirred for dine (lamivudine, 1a) in 55% yield as a colourless solid. Rf
another 24 h. A colorless crystalline precipitate formed was col- 0.35 (dichloromethane–methanol, 10 : 1). [α]2D5 −97.4 (c 0.34 in
1
lected by filtration, washed with acetonitrile and dried. The methanol). H NMR (DMSO-d6, 400 MHz) δ 8.38 (s, 1H), 7.90
obtained product (1.35–1.50 g), according to 1H NMR (br s, 1H, NH), 7.04 (br s, 1H, NH), 6.15 (dd, J = 5.3, 3.7 Hz,
spectroscopy, contains 1-(tetrahydro-2H-pyran-2-yl)cytosine 1H), 5.46 (br t, J = 4.7 Hz, 1H, OH), 5.20 (t, J = 3.7 Hz, 1H), 3.82
(1b) as a major component, contaminated with 15–30 mol% of (dt, J = 12.3, 3.7 Hz, 1H), 3.73 (dt, J = 12.3, 3.7 Hz, 1H), 3.45
unreacted cytosine. The filtrate was evaporated under a (dd, J = 12.0, 5.3 Hz, 1H), 3.17 (dd, J = 12.0, 3.7 Hz, 1H).
reduced pressure to give the oily residue, from which an 13C NMR (DMSO-d6, 100.6 MHz) δ 162.01, 153.35, 142.15,
additional portion of pure 1-(tetrahydro-2H-pyran-2-yl)cytosine 87.19, 86.60, 86.00, 61.72, 37.38. IR (KBr, cm−1) νmax 3332,
(1b) (0.40–0.85 g) was obtained after standing at room temp- 2927, 1638, 1593, 1496, 1285, 1169, 1057, 779. Anal. Calcd for
erature for a few days (the prismatic colorless single crystal C8H10BrN3O3S: 13.64% N, 31.18% C, 3.27% H; found: 13.78%
grown from the reaction mixture was used for X-ray analysis). N, 31.51% C, 3.54% H. HRMS (ESI) calcd for C8H11BrN3O3S
Total yield of 1-(tetrahydro-2H-pyran-2-yl)cytosine (1b) was [M + H]+ 307.9704, found m/z 307.9715.
1.6–2.1 g (41–54%), and the obtained material was used in the
Following the same procedure, 5-bromocytidine (3c) was
next step without further purification. An analytically pure obtained from cytidine 1c (Sigma-Aldrich, 0.751 g, 3.1 mmol)
sample was prepared by recrystallization from water. Rf and bromine (0.853 g, 5.3 mmol) in 0.916 g (92%) yield as a
0.38 (dichloromethane–methanol, 10 : 1). 1H NMR (DMSO-d6, pale-yellow solid. Spectral data are in accordance with those
400 MHz) δ 7.58 (d, J = 7.4 Hz, 1H), 7.19 (br s, 1H, NH), 7.13 reported.20
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5-Bromo-4-phthalimido-1-(tetrahydro-2H-pyran-2-yl)pyrimidin- overnight, and then methanol (4 mL) was added and the reac-
2(1H)-one (4)
tion mixture was stirred for an additional hour. The volatiles
were removed under reduced pressure, and the residue was
coevaporated with toluene (3 × 3 mL) to remove pyridine. The
residue was dissolved in CH2Cl2 (8 mL), washed with water
(5 mL), 5% aq. HCl (5 mL), std. aq. NaHCO3 (5 mL), and brine
(5 mL) and dried (MgSO4). The solvent was removed under
reduced pressure, and the residue was purified by short-
column chromatography on silica gel (dichloromethane–
methanol 100 : 1 to 40 : 1) to afford the title compound after
solvent evaporation as a foamy solid (0.578 g, 95% yield). Rf
0.60 (dichloromethane–methanol 10 : 1). 1H NMR (CDCl3,
400 MHz) δ 12.1 (br s, 1H, NH), 7.88 (s, 1H), 5.56 (dd, J = 10.2,
2.2 Hz, 1H), 4.22–4.11 (m, 1H), 3.72–3.64 (m, 1H), 2.08–1.92
To a stirred suspension of 5-bromo-1-(tetrahydro-2H-pyran-
2-yl)cytosine (3b) (0.247 g, 0.90 mmol) in pyridine (6 mL) was
added DMAP (12 mg, 0.1 mmol) followed by phthaloyl chloride
(0.274 g, 1.35 mmol). The resulting dark-red reaction mixture
was stirred at room temperature for 3 h and then pyridine was
removed under reduced pressure. The residue was dissolved in
CH2Cl2 (10 mL), washed with water (5 mL), 5% aq. HCl (5 mL),
std. aq. NaHCO3 (5 mL), and brine (5 mL) and dried (MgSO4).
The solvent was removed under reduced pressure, and the
residue was purified by column chromatography on silica gel
(dichloromethane–methanol, 100 : 1) to give the title com-
pound 4 after solvent evaporation as a foamy yellowish solid
(0.281 g, 77% yield). Rf 0.45 (hexane–ethyl acetate, 1 : 2). 1H
NMR (CDCl3, 400 MHz) δ 8.30 (s, 1H), 8.03–7.92 (m, 2H),
7.87–7.77 (m, 2H), 5.62 (dd, J = 10.3, 2.3 Hz, 1H), 4.25 (m, 1H),
3.74 (dt, J = 11.4, 3.2 Hz, 1H), 2.40–2.29 (m, 1H), 2.07–1.95 (m,
1H), 1.84–1.58 (m, 3H), 1.35 (tdd, J = 12.5, 10.3, 4.2 Hz, 1H).
13C NMR (CDCl3, 100.6 MHz) δ 164.81, 164.62, 158.55, 152.75,
147.06, 135.10, 131.83, 124.58, 96.21, 85.43, 62.58, 32.17,
25.05, 22.47. IR (KBr, cm−1) νmax 3072, 2948, 1789, 1734, 1672,
1599, 1493, 1429, 1329, 1239, 1096, 717. Anal. Calcd for
C17H14BrN3O4: 10.40% N, 50.51% C, 3.49% H; found: 10.10%
N, 49.94% C, 3.55% H. HRMS (ESI) calcd for C17H15BrN3O4
[M + H]+ 404.0246, found m/z 404.0236.
(m, 2H), 1.77–1.53 (m, 3H), 1.50–1.33 (m, 1H), 1.29 (s, 9H). 13
C
NMR (CDCl3, 100.6 MHz) δ 174.12 (broad),21 155.81 (broad),
148.93 (very broad), 141.33 (broad), 94.65 (very broad), 83.54,
69.33, 42.26, 31.72, 27.37, 24.95, 27.58. IR (KBr, cm−1) νmax
2953, 1713, 1634, 1573, 1455, 1241, 1158, 1088, 1046, 998, 780,
661. HRMS (ESI) calcd for C14H21BrN3O3 [M + H]+ 358.0766,
found m/z 358.0759.
Following the same procedure, 5-bromo-N,O-dipivaloyl-2′,3′-
dideoxy-3′-thiacytidine (7a) was prepared from 5-bromo-2′,3′-
dideoxy-3′-thiacytidine (3a) (0.783 g, 2.54 mmol) and pivaloyl
chloride (0.766 g, 6.36 mmol, 2.5 equiv.). Yield 0.964 g (80%),
foamy colourless solid. Rf 0.60 (dichloromethane–methanol
10 : 1). [α]2D5 −99.8 (c 0.48 in methanol). 1H NMR (CDCl3,
400 MHz) δ 12.7 (br s, 1H, NH), 8.03 (s, 1H), 6.26 (dd, J = 5.4,
4.3 Hz, 1H), 5.38 (dd, J = 5.4, 3.3 Hz, 1H), 4.60 (dd, J = 12.4,
5.4 Hz, 1H), 4.38 (dd, J = 12.4, 3.3 Hz, 1H), 3.58 (dd, J = 12.4,
5.4 Hz, 1H), 3.14 (dd, J = 12.4, 4.3 Hz, 1H), 1.28 (s, 9H), 1.27 (s,
9H). 13C NMR (CDCl3, 100.6 MHz) δ 178.11, 156.04 (broad),
140.77 (broad), 87.57, 84.21, 64.03, 42.09, 39.05, 38.18, 27.37,
27.31 (signals from quaternary carbons C-4 and C-5 of the cyto-
sine ring and quaternary carbon of the pivaloylamide carbonyl
group were not detected due to strong line broadening and low
intensity).21 IR (KBr, cm−1) νmax 3413, 2972, 1732, 1715, 1575,
1478, 1460, 1282, 1255, 1154. Anal. Calcd for C18H26BrN3O5S:
8.82% N, 45.38% C, 5.50% H; found: 8.65% N, 45.34% C,
5.56% H. HRMS (ESI) calcd for C18H26BrN3O5S [M + H]+
476.0855, found m/z 476.0856.
5-Bromo-4-[2′-(butoxycarbonyl)ethylsulfanyl]-1-(tetrahydro-2H-
pyran-2-yl)pyrimidin-2(1H)-one (6)
Starting bromide 4 (0.103 g, 0.26 mmol), butyl 3-mercaptopro-
pionate (5) (0.054 g, 0.33 mmol) and DIPEA (0.082 g,
0.64 mmol) were combined together in a mixture of aceto-
nitrile (2.5 mL) and chloroform (1 mL) and refluxed until the
full conversion of the starting material (TLC monitoring).
Then solvents were removed under reduced pressure, and the
product was isolated by column chromatography on silica gel
(hexane–ethyl acetate, 2 : 1). Yield 0.098 g (90%), colourless oil.
1
Rf 0.61 (hexane–ethyl acetate, 1 : 2). H NMR (CDCl3, 400 MHz)
δ 7.80 (s, 1H), 5.59 (dd, J = 2.3, 10.4 Hz, 1H), 4.17 (m, 1H), 4.11
(t, J = 6.7 Hz, 2H), 3.69 (m, 1H), 3.43 (t, J = 6.7 Hz, 2H), 2.78 (t,
J = 6.7 Hz, 2H), 2.16 (m, 1H), 2.01–1.91 (m, 1H), 1.80–1.54 (m,
5H), 1.44–1.28 (m, 3H), 0.93 (t, J = 7.4 Hz, 3H). 13C NMR
(CDCl3, 101 MHz) δ 176.26, 171.94, 151.74, 140.67, 97.22,
84.28, 69.33, 64.89, 33.69, 32.11, 30.74, 26.19, 25.10, 22.56,
19.27, 13.84. IR (neat, cm−1) νmax 3072, 2957, 1734, 1672, 1603,
1478, 1257, 1088. HRMS (ESI) calcd for C16H24BrN2O4S
[M + H]+ 419.0640, found m/z 419.0646.
Following the same procedure, 5-bromo-O2′,O3′,O5′,N4-tetra-
pivaloylcytidine (7c) was prepared from 5-bromocytidine (3c)
(1.12 g, 3.47 mmol) and pivaloyl chloride (2.31 g, 19.1 mmol,
5.5 equiv.). Yield 0.94 g (41%), foamy colorless solid. Rf 0.80
(dichloromethane–methanol 15 : 1). [α]2D5 +29.1 (c 0.56 in
1
methanol). H NMR (400 MHz, CDCl3) δ 12.7 (br s, 1H, NH),
7.80 (s, 1H), 6.20 (d, J = 5.8 Hz, 1H), 5.31–5.23 (m, 2H), 4.49
(dd, J = 12.5, 3.2 Hz, 1H), 4.38–4.31 (m, 1H), 4.30 (dd, J = 12.5,
2.3 Hz, 1H), 1.30 (s, 9H), 1.27 (s, 9H), 1.25 (s, 9H), 1.20 (s, 9H).
5-Bromo-N-pivaloyl-1-(tetrahydro-2H-pyran-2-yl)cytosine (7b)
Pivaloyl chloride (0.308 g, 2.56 mmol) was added to a stirred 13C NMR (100.6 MHz, CDCl3) δ 178.07, 177.44, 177.39, 139.1
and cooled (ice bath, 0 °C) suspension of 5-bromo-1-(tetra- (very broad; extracted from HSQC data), 86.51, 81.67, 73.39,
hydro-2H-pyran-2-yl)cytosine (3b) (0.467 g, 1.70 mmol) and 70.90, 63.65, 42.62 (broad), 39.09, 39.01 (2×), 27.51, 27.34,
DMAP (20 mg, 0.17 mmol) in a mixture of pyridine (4 mL) and 27.22, 27.16 (signals from quaternary carbons of the cytosine
dichloromethane (4 mL). A clear solution was obtained within ring were not detected due to strong line broadening and low
3–5 min. The reaction mixture was stirred at room temperature intensity).21 IR (KBr, cm−1) νmax 2975, 1742, 1638, 1579, 1151.
5640 | Org. Biomol. Chem., 2014, 12, 5634–5644
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HRMS (ESI) calcd for C29H45BrN3O9 [M + H]+ 658.2334, found 2.74 (dd, J = 14.3, 5.3 Hz, 1H), 2.04 (ddd, J = 14.3, 8.6, 6.5 Hz,
m/z 658.2329. 1H), 1.28 (s, 9H), 1.24 (s, 9H), 1.22 (s, 9H) (signal from the
Following the same procedure, 5-bromo-O3′,O5′-dipivaloyl- amide NH group was not detected).21 13C NMR (100.6 MHz,
2′-deoxyuridine (10d) was prepared from 5-bromo-2′-deoxyuri- CDCl3) δ 178.23, 178.08, 156.10 (broad), 140.21 (broad), 86.78,
dine (Sigma-Aldrich, 0.250 g, 0.81 mmol) and pivaloyl chloride 81.74, 74.34, 64.05, 42.15, 39.02, 38.86, 38.79, 27.45, 27.33,
(0.289 g, 2.4 mmol, 3.0 equiv.). Yield 0.184 g (48%), colourless 27.11 (signals from two carbons of the cytosine ring and qua-
solid. Rf 0.25 (hexane–ethyl acetate 2 : 1). [α]2D5 +1.0 (c 0.19 in ternary carbon of the pivaloylamide carbonyl group were not
1
methanol). H NMR (CDCl3, 400 MHz) δ 8.89 (s, 1H, NH), 7.82 detected due to strong line broadening and low intensity).21 IR
(s, 1H), 6.23 (dd, J = 8.8, 5.3 Hz, 1H), 5.20 (dt, J = 6.5, 1.5 Hz, (KBr, cm−1) νmax 3092, 2975, 1734, 1637, 1576, 1480, 1282,
1H), 4.46 (dd, J = 12.4, 3.8 Hz, 1H), 4.32 (dd, J = 12.4, 2.7 Hz, 1152, 1100. HRMS (ESI) calcd for C24H37BrN3O7 [M + H]+
1H), 4.26 (m, 1H), 2.59 (ddd, J = 14.2, 5.3, 1.5 Hz, 1H), 2.11 558.1809, found m/z 558.1798.
(ddd, J = 14.2, 8.8, 6.5 Hz, 1H), 1.26 (s, 9H), 1.23 (s, 9H). 13C
3′,5′-Di-O-tert-butyldimethylsilyl-5-bromo-2′-deoxyuridine
NMR (CDCl3, 100.6 MHz) δ 178.17, 178.06, 158.75, 149.38, (11d) was prepared from 5-bromo-2′-deoxyuridine (Sigma-
138.41, 97.58, 85.78, 83.38, 74.11, 64.05, 39.05, 38.81, 38.54, Aldrich) in 96% yield following the published procedure.22
27.48, 27.13. IR (KBr, cm−1) νmax 3196, 2975, 1734, 1690, 1454, Spectral data are in accordance with those reported.22
1273, 1146. HRMS (ESI) calcd for C19H27BrN2O7Na [M + Na]+
497.0894, found m/z 497.0893. 5-Bromo-O3′,O5′,O4-tripivaloyl-
2′-deoxyuridine was isolated as a by-product, yield 0.102 g
(23%), colourless solid. Rf 0.62 (hexane–ethyl acetate 2 : 1).
1H NMR (CDCl3, 400 MHz) δ 7.84 (s, 1H), 6.22 (br m, 1H), 5.20
(m, 1H), 4.46 (dd, J = 12.3, 3.8 Hz, 1H), 4.32 (dd, J = 12.3,
2.8 Hz, 1H), 4.27 (m, 1H), 2.61 (br m, 1H), 2.11 (br m, 1H),
1.34 (s, 9H), 1.26 (s, 9H), 1.22 (s, 9H). 13C NMR (CDCl3,
100.6 MHz) δ 181.98, 177.99, 177.93, 157.86, 148.38, 137.81,
97.17, 85.94, 83.45, 74.06, 63.95, 44.05, 38.96, 38.71, 38.53,
27.41, 27.32, 27.03. HRMS (ESI) calcd for C24H36BrN2O8
[M + H]+ 559.1655, found m/z 559.1665.
General procedure for the palladium-catalysed coupling of
5-bromopyrimidine nucleoside derivatives 7a–d, 10d, 11d and
alkyl thiols11
Starting bromide 7a–d, 10d, 11d (0.54 mmol), alkyl thiol
(0.65 mmol, 1.2 equiv.), DIPEA (0.160 g, 1.24 mmol,
2.3 equiv.), Pd2(dba)3 (12.4 mg, 0.0135 mmol, 2.5 mol%) and
the Xantphos ligand (15.6 mg, 0.027 mmol, 5 mol%) were com-
bined together in 2 mL of dry dioxane, and the reaction
mixture was heated to reflux under argon for 5 h. The reaction
mixture was allowed to cool to room temperature, and TLC
analysis showed total conversion of the starting material.
Dichloromethane (3 mL) and water (3 mL) were added, the
organic layer was separated and the aqueous layer was
extracted with dichloromethane (3 × 4 mL). The combined
organic phase was washed with brine and dried (MgSO4). After
the solvent removal, the products were isolated by column
chromatography on silica gel (dichloromethane–methanol
50 : 1 or hexane–ethyl acetate 2 : 1).
5-Bromo-O2′,O3′,O5′,N4-tetrapivaloylcytidine (7c), one-pot
preparation from cytidine (1c)
A suspension of cytidine (1c) (0.31 g, 1.3 mmol) in pyridine
(4.1 mL) was cooled in an ice bath, and then a solution of
bromine (0.22 g, 1.4 mmol) in tetrachloromethane (2.4 mL,
approx. 5% w/w) was added under stirring. The reaction
mixture was allowed to warm to room temperature and stirred
for approx. 1–2 hours until the reaction was complete (TLC
monitoring). Then DMAP (0.031 g, 0.25 mmol) and pivaloyl
chloride (0.762 g, 6.3 mmol) were added and the obtained
clear solution was stirred for an additional 60 h. The reaction
was stopped with the addition of saturated Na2CO3 (500 μL)
5-[2′-(Butoxycarbonyl)ethylsulfanyl]-N-pivaloyl-1-(tetrahydro-
2H-pyran-2-yl)cytosine (8b)
and saturated Na2SO3 (500 μL). The volatiles were removed Yield 77%, obtained as a yellow oil. Rf 0.57 (dichloromethane–
1
under reduced pressure, and the residue was coevaporated methanol, 10 : 1). H NMR (CDCl3, 400 MHz) δ 12.9 (very br s,
with toluene (3 × 3 mL) to remove pyridine. The residue was ca. 0.5H, NH), 9.10 (very br s, ca. 0.5H, NH), 7.92 (br s, 1H),
purified by column chromatography on silica gel (hexane– 5.57 (d, J = 9.5 Hz, 1H), 4.15 (d, J = 11.5 Hz, 1H), 4.12–4.03 (m,
ethyl acetate 2 : 1) to afford the title compound after solvent 2H), 3.67 (dt, J = 11.5, 3.3 Hz, 1H), 2.99 (br s, 2H), 2.64–2.49
evaporation as a foamy yellowish solid (0.27 g, 31% yield).
(m, 2H), 2.04 (br s, 1H), 2.00–1.90 (m, 1H), 1.76–1.53 (m, 5H),
Following the same procedure, 5-Bromo-O3′,O5′,N4-tripiva- 1.45–1.31 (m, 3H), 1.28 (s, 9H), 0.93 (t, J = 7.4 Hz, 3H). 13C
loyl-2′-deoxycytidine (7d) was obtained from 2′-deoxycytidine NMR (CDCl3, 100.6 MHz) δ 171.45, 83.66 (broad), 69.29, 64.95,
(1d, Sigma-Aldrich) (0.34 g, 1.5 mmol), bromine (0.22 g, 41.90 (very broad), 34.37 (broad), 31.72 (broad), 30.72, 30.50
1.4 mmol) and pivaloyl chloride (0.72 g, 6.0 mmol). Foamy col- (very broad; extracted from HSQC data), 27.39, 25.02, 22.62,
ourless solid (0.53 g, 63% yield). O3′,O5′,N4-Tripivaloyl-2′-deoxy- 19.23, 13.82 (signals from four carbons of the cytosine ring
cytidine was isolated as a by-product in 0.086 g (10%) yield. Rf and quaternary carbon of the pivaloylamide carbonyl group
0.54 (hexane–ethyl acetate 2 : 1). [α]2D5 +35.8 (c 0.63 in metha- were not detected due to strong line broadening and low inten-
nol). 1H NMR (400 MHz, CDCl3) δ 7.95 (s, 1H), 6.21 (dd, J = sity).21 IR (KBr, cm−1) νmax 3353, 2958, 1732, 1714, 1623, 1565,
8.6, 5.3 Hz, 1H), 5.19 (dt, J = 6.5, 1.6 Hz, 1H), 4.44 (dd, J = 12.3, 1243, 1159, 1045. HRMS (ESI) calcd for C21H34N3O5S [M + H]+
4.0 Hz, 1H), 4.35 (dd, J = 12.3, 2.7 Hz, 1H), 4.30–4.26 (m, 1H), 440.2214, found m/z 440.2195.
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Organic & Biomolecular Chemistry
5-[2′-(Butoxycarbonyl)ethylsulfanyl]-N,O-dipivaloyl-2′,3′-
dideoxy-3′-thiacytidine (8a)
5-[2′-(Butoxycarbonyl)ethylsulfanyl]-O3′,O5′-dipivaloyl-2′-
deoxyuridine (9d)
Yield 83%, obtained as a yellow oil. Rf 0.53 (dichloromethane– Yield 88%, obtained as a yellow oil. Rf 0.18 (hexane–ethyl
methanol, 10 : 1). [α]2D5 −101.2 (c 0.57 in methanol). 1H NMR acetate, 2 : 1). [α]2D5 +6.9 (c 3.1 in methanol). 1H NMR
(CDCl3, 400 MHz) δ 9.23 (br s, 1H, NH), 8.10 (br s, 1H), 6.27 (400 MHz, CDCl3) δ 8.32 (s, 1H, NH), 7.82 (s, 1H), 6.23 (dd, J =
(dd, J = 5.3, 4.0 Hz, 1H), 5.38 (dd, J = 6.1, 3.7 Hz, 1H). 4.63 (dd, 8.9, 5.3 Hz, 1H), 5.21 (dt, J = 6.5, 1.5 Hz, 1H), 4.44 (dd, J = 12.3,
J = 12.3, 6.1 Hz, 1H), 4.38 (dd, J = 12.3, 3.7 Hz, 1H), 4.15–4.03 4.1 Hz, 1H), 4.31 (dd, J = 12.3, 3.0 Hz, 1H), 4.26–4.22 (m, 1H),
(m, 2H), 3.60 (dd, J = 12.3, 5.3 Hz, 1H), 3.18 (dd, J = 12.3, 4.07 (t, J = 6.7 Hz, 2H), 3.10–2.95 (m, 2H), 2.60 (t, J = 7.3 Hz,
4.0 Hz, 1H), 2.97 (br s, 2H), 2.59 (t, J = 7.3 Hz, 2H), 1.64–1.57 2H), 2.54 (ddd, J = 14.2, 5.3, 1.2 Hz, 1H), 2.15 (ddd, J = 14.2,
(m, 2H), 1.42–1.32 (m, 2H), 1.31 (s, 9H), 1.25 (s, 9H), 0.93 (t, J = 8.9, 6.5 Hz, 1H), 1.64–1.55 (m, 2H), 1.42–1.32 (m, 2H), 1.25 (s,
7.4 Hz, 3H). 13C NMR (CDCl3, 100.6 MHz) δ 178.04, 171.22, 9H), 1.23 (s, 9H), 0.93 (t, J = 7.4 Hz, 3H). 13C NMR (CDCl3,
88.10, 84.30, 65.05, 64.30, 41.61, 39.01, 38.41, 34.28, 31.20, 100.6 MHz) δ 178.13, 178.03, 171.66, 161.19, 149.73, 142.64,
30.70, 27.32, 19.22, 13.81 (signals from four carbons of the 108.10, 85.52, 83.17, 74.17, 64.85, 64.07, 39.03, 38.81, 38.28,
cytosine ring were not detected due to strong line broadening 35.03, 30.74, 28.91, 27.49, 27.14, 19.25, 13.85. IR (KBr, cm−1
)
and low intensity).21 IR (KBr, cm−1) νmax 3349, 2961, 2873, νmax 3197, 2964, 1731, 1693, 1451, 1274, 1147. HRMS (ESI)
1733, 1714, 1674, 1624, 1566, 1461, 1282, 1248, 1155. HRMS calcd for C26H40N2O9SNa [M + Na]+ 579.2347, found m/z
(ESI) calcd for C25H40N3O7S2 [M + H]+ 558.2302, found m/z 579.2339.
558.2291.
5-[2′-(Butoxycarbonyl)ethylsulfanyl]-3′,5′-di-O-tert-
5-[2′-(Butoxycarbonyl)ethylsulfanyl]-O2′,O3′,O5′,N4-tetrapivaloyl- butyldimethylsilyl-2′-deoxyuridine (12d)
cytidine (8c)
Yield 87%, obtained as a yellow oil. Rf 0.38 (hexane–ethyl
Yield 81%, obtained as a yellow oil. Rf 0.46 (hexane–ethyl acetate, 2 : 1). [α]2D5 +6.9 (c 1.9 in methanol). 1H NMR
acetate, 2 : 1). [α]D25 +18.6 (c 0.86 in methanol). 1H NMR (CDCl3, (400 MHz, CDCl3) δ 8.20 (s, 1H), 8.00 (s, 1H), 6.28 (dd, J = 8.0,
400 MHz) δ 12.9 (br s, 0.5H, NH), 9.20 (br s, 0.5H, NH), 7.88 5.7 Hz, 1H), 4.46–4.36 (m, 1H), 4.07 (t, J = 6.7 Hz, 2H), 3.97 (q,
(br s, 1H), 6.19 (br s, 1H), 5.30 (m, 2H), 4.48 (dd, J = 13.3, J = 2.6 Hz, 1H), 3.86 (dd, J = 11.4, 2.9 Hz, 1H), 3.77 (dd, J =
4.6 Hz, 1H), 4.36–4.29 (m, 2H), 4.08 (t, J = 6.7 Hz, 2H), 3.00 (br 11.4, 2.6 Hz, 1H), 3.08–2.93 (m, 2H), 2.60 (t, J = 7.4 Hz, 2H),
s, 2H), 2.58 (t, J = 7.2 Hz, 1H), 1.60 (m, 2H), 1.37 (m, 2H), 1.29 2.29 (ddd, J = 13.1, 5.7, 2.4 Hz, 1H), 2.03(ddd, J = 13.1, 8.0,
(s, 18H), 1.25 (s, 9H), 1.19 (s, 9H), 0.93 (t, J = 7.4 Hz, 3H). 5.7 Hz, 1H), 1.65–1.54 (m, 2H), 1.43–1.31 (m, 2H), 0.93 (s, 9H),
13C NMR (CDCl3, 100.6 MHz) δ 178.05, 177.38, 86.96 (broad), 0.93 (t, J = 7.4 Hz, 3H), 0.90 (s, 9H), 0.14 (s, 3H), 0.13 (s, 3H),
81.45, 73.33 (broad), 70.90, 64.93, 63.68, 39.05, 38.98, 34.4 0.09 (s, 3H), 0.08 (s, 3H). 13C NMR (CDCl3, 100.6 MHz)
(broad), 30.71, 27.51, 27.36, 27.23, 27.15, 19.22, 13.81 (signals δ 171.74, 161.50, 149.87, 143.90, 107.35, 88.44, 85.71, 72.62,
from several carbons were not detected due to strong line 64.78, 63.21, 41.82, 35.07, 30.75, 29.01, 26.22, 25.89, 19.26,
broadening and low intensity).21 IR (film, cm−1) νmax 3350, 18.61, 18.15, 13.85, −4.52, −4.69, −5.10, −5.25. IR (KBr, cm−1
)
2967, 1738, 1597, 1281, 1151. HRMS (ESI) calcd for νmax 3191, 2956, 2931, 1722, 1694, 1446, 1255, 1128, 1107, 837,
C36H58N3O11S [M + H]+ 740.3787, found m/z 740.3785.
779. HRMS (ESI) calcd for C28H52N2O7SSi2Na [M + Na]+
639.2926, found m/z 639.2917.
5-[2′-(Butoxycarbonyl)ethylsulfanyl]-O3′,O5′,N4-tripivaloyl-2′-
deoxycytidine (8d)
5-[4′-Methoxybenzylsulfanyl]-O2′,O3′,O5′,N4-tetrapivaloylcytidine
(13c)
Yield 52%, obtained as a yellow oil. Rf 0.28 (hexane–ethyl
acetate, 2 : 1). [α]D25 +42.2 (c 0.55 in methanol). 1H NMR (CDCl3, Yield 58%, obtained as a pale-yellow oil. Rf 0.57 (hexane–ethyl
400 MHz) δ 13.0 (br s, 0.5H, NH), 9.17 (br s, 0.5H, NH), 8.08 acetate, 1 : 1). [α]2D5 +27.6 (c 0.87 in ethyl acetate). 1H NMR
(br s, 1H), 6.21 (dd, J = 8.5, 5.4 Hz, 1H), 5.18 (m, 1H), 4.43 (dd, (CDCl3, 400 MHz) δ 9.20 (br s, ca. 0.5H, NH), 7.47 (s, 1H), 7.02
J = 12.2, 4.5 Hz, 1H), 4.34 (dd, J = 12.2, 2.9 Hz, 1H), 4.29 (m, (d, J = 8.5 Hz, 2H), 6.80 (d, J = 8.5 Hz, 2H), 6.23 (d, J = 6.8 Hz,
1H), 4.07 (t, J = 6.6 Hz, 2H), 2.96 (br s, 3H), 2.57 (t, J = 7.1 Hz, 1H), 5.20 (dd, J = 5.9, 2.8 Hz, 1H), 5.13 (dd, J = 6.8, 5.9 Hz, 1H),
2H), 2.06 (m, 1H), 1.65–1.54 (m, 2H), 1.42–1.30 (m, 2H), 1.31 4.35 (dd, J = 12.4, 3.5 Hz, 1H), 4.25 (pseudo q, J = 2.9 Hz, 1H),
(br s, 9H), 1.23 (s, 9H), 1.22 (s, 9H), 0.93 (t, J = 7.4 Hz, 3H). 4.15 (dd, J = 12.4, 2.5 Hz, 1H), 3.90–3.75 (br m, 2H), 3.78 (s,
13C NMR (CDCl3, 100.6 MHz) δ 178.22, 178.02, 87.2 (very 3H), 1.27 (s, 9H), 1.27 (s, 9H), 1.24 (s, 9H), 1.18 (s, 9H).
broad; extracted from HSQC data), 83.75 (broad), 74.41, 65.05, 13C NMR (CDCl3, 100.6 MHz) δ 177.96, 177.41, 177.34, 159.20,
64.05, 38.98, 38.78, 34.4 (broad), 31.2 (very broad; extracted 130.23, 128.58, 114.43, 86.28, 81.35, 73.50, 70.93, 63.75, 55.34,
from HSQC data), 30.69, 27.42, 27.35, 27.12, 19.22, 13.81 41.69 (broad), 40.0 (very broad), 38.97, 27.54, 27.36, 27.22,
(signals from several quaternary carbons were not detected 27.16 (signals from several carbons were not detected due to
due to strong line broadening and low intensity).21 IR (film, strong line broadening and low intensity).21 IR (film, cm−1
)
cm−1) νmax 3351, 2964, 1734, 1624, 1565, 1460, 1282, 1151. νmax 3347, 2973, 1738, 1684, 1540, 1513, 1479, 1461, 1280,
HRMS (ESI) calcd for C31H50N3O9S [M + H]+ 640.3262, found 1251, 1149. HRMS (ESI) calcd for C37H53N3O10S [M + H]+
m/z 640.3260.
732.3530, found m/z 732.3515. O2′,O3′,O5′,N4-Tetrapivaloylcyti-
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dine was isolated as a major by-product in 20% yield. Rf 0.27 82.43, 68.54, 68.20, 31.04, 30.61, 24.44, 22.29, 22.21. IR (KBr,
1
(hexane–ethyl acetate, 1 : 1). H NMR (CDCl3, 400 MHz) δ 8.18 cm−1) νmax 3453, 2943, 1670, 1630, 1489, 1087, 1041, 782.
(br s, 1H, NH), 7.91 (d, J = 7.6 Hz, 1H), 7.47 (d, J = 7.6 Hz, 1H), HRMS (ESI) calcd for C18H25N6O4S2 [M + H]+ 453.1373, found
6.29 (d, J = 5.5 Hz, 1H), 5.35 (t, J = 5.5 Hz, 1H), 5.30 (dd, J = 5.5, m/z 453.1355.
4.1 Hz, 1H), 4.46 (dd, J = 12.4, 3.3 Hz, 1H), 4.38 (m, 1H), 4.33
Following the same procedure, disulfide 14a was obtained
(dd, J = 12.4, 2.4 Hz, 1H), 1.29 (s, 9H), 1.27 (s, 9H), 1.24 (s, 9H), from compound 8a in 39% yield as a colourless solid. Rf 0.18
1.22 (s, 9H). 13C NMR (CDCl3, 100.6 MHz) δ 178.21, 177.96, (dichloromethane–methanol, 10 : 1). 1H NMR (DMSO-d6,
177.23, 177.16, 162.57, 154.92, 143.58, 96.93, 87.75, 81.04, 400 MHz) δ 7.88 (br s, 1H, NH), 7.54 (s, 1H), 7.29 (br s, 1H,
74.07, 70.43, 63.28, 40.46, 39.00, 38.94, 27.39, 27.19, 27.16. NH), 6.05 (dd, J = 5.3, 3.1 Hz, 1H), 5.15 (dd, J = 6.7, 5.3 Hz,
Spectral data are in accordance with those reported.23
1H), 5.11 (dd, J = 5.4, 4.0 Hz, 1H), 3.76 (ddd, J = 12.0, 5.3,
4.0 Hz, 1H), 3.55 (ddd, J = 12.0, 6.7, 5.4 Hz, 1H), 3.43 (dd, J =
12.1, 5.3 Hz, 1H), 3.05 (dd, J = 12.1, 3.1 Hz, 1H). 13C NMR
(DMSO-d6, 100.6 MHz) δ 164.38, 154.01, 149.48, 95.26, 87.96,
O3′,O5′-Dipivaloyl-5-[4′-methoxybenzylsulfanyl]-2′-deoxyuridine
(13d)
Yield 87%, yellow solid. Rf 0.20 (hexane–ethyl acetate, 2 : 1). 86.90, 61.97, 37.76. IR (KBr, cm−1) νmax 3355, 3045, 1655, 1632,
[α]2D5 −6.3 (c 3.1 in ethyl acetate). 1H NMR (CDCl3, 400 MHz) 1488, 1287, 1058. HRMS (ESI) calcd for C16H21N6O6S4 [M + H]+
δ 8.67 (s, 1H, NH), 7.33 (s, 1H), 7.07 (d, J = 8.7 Hz, 2H), 6.81 (d, 521.0405, found m/z 521.0405.
J = 8.7 Hz, 2H), 6.16 (dd, J = 9.0, 5.3 Hz, 1H), 5.11 (dt, J = 6.6,
Preparation of disulfide 14a by deprotection of the compound
8a with sodium methoxide
1.7 Hz, 1H), 4.27 (dd, J = 12.8, 4.8 Hz, 1H), 4.17–4.14 (m, 2H),
3.92 (d, J = 12.8 Hz, 1H), 3.88 (d, J = 12.8 Hz, 1H), 3.79 (s, 3H),
2.40 (ddd, J = 14.1, 5.3, 1.4 Hz, 1H), 1.86 (ddd, J = 14.1, 9.0, 6.6
Hz, 1H), 1.23 (s, 9H), 1.21 (s, 9H). 13C NMR (CDCl3,
100.6 MHz) δ 178.08, 178.02, 161.42, 158.89, 149.88, 143.16,
130.44, 129.39, 114.06, 107.99, 84.98, 82.82, 74.01, 63.98,
To a solution of compound 8a (0.19 g, 0.34 mmol) in anhy-
drous methanol (1 mL) was added sodium methoxide
(0.42 mmol, 0.42 mL of 1.0 M solution in methanol) and the
bright-yellow reaction mixture was stirred overnight (ca. 18 h)
under an argon atmosphere. Then, the reaction was stopped
by the addition of an ammonium chloride (0.023 g,
0.43 mmol) solution in water (0.3 mL) and the reaction
mixture was exposed to air under vigorous stirring. Within
30 min, a fine precipitate started to form. The reaction mixture
was stirred for 8 h, the precipitate was filtered out and washed
with methanol to afford disulfide 14a (0.065 g, 78% yield).
Disulfide 14d was prepared according to the same pro-
cedure starting from compound 8d (0.096 g, 0.15 mmol) and
sodium methoxide (0.21 mmol, 0.21 mL of 1.0 M solution).
When the reaction mixture was quenched with an ammonium
chloride (0.012 g, 0.22 mmol) solution in water (0.2 mL) and
then exposed to air, a colourless precipitate of disulfide 14d
started to form after 4 h of vigorous stirring. Stirring was con-
tinued for an additional 4 h, and the reaction mixture was
allowed to stand overnight. The precipitate was filtered out
and washed with methanol (2 × 0.5 mL) to afford disulfide 14d
(0.031 g, 80% yield). 1H NMR (DMSO-d6, 400 MHz) δ 7.72 (br s,
1H, NH), 7.70 (s, 1H), 7.10 (br s, 1H, NH), 5.97 (t, J = 6.5 Hz,
1H), 5.16 (d, J = 4.1 Hz, 1H, OH), 4.77 (t, J = 5.2 Hz, 1H, OH),
4.12 (m, 1H), 3.73 (q, J = 3.5 Hz, 1H), 3.44 (pseudo t, J =
4.5 Hz, 2H), 2.14 (ddd, J = 13.1, 6.0, 3.2 Hz, 1H), 1.85 (dt, J =
13.1, 6.5 Hz, 1H). 13C NMR (DMSO-d6, 100.6 MHz) δ 164.25,
154.19, 149.36, 95.87, 87.67, 86.06, 70.47, 61.12, 40.87. IR (KBr,
cm−1) νmax 3324, 1647, 1629, 1488, 1092. HRMS (ESI) calcd for
C18H24N6O8S2 [M + H]+ 517.1175, found m/z 517.1174.
55.39, 38.98, 38.77, 38.00, 37.69, 27.50, 27.11. IR (film, cm−1
)
νmax 3194, 2973, 1724, 1511, 1274, 1250, 1146. HRMS (ESI)
calcd for C27H37N2O8S [M + H]+ 549.2271, found m/z 549.2273.
Bis-[1-(tetrahydro-2H-pyran-2-yl)-cytosine-5-yl]-disulfide (14b)
Compound 8b (66 mg, 0.15 mmol) was dissolved in THF
(1 mL), the reaction flask was flushed with argon and cooled
to −78 °C. At that temperature, a solution of potassium tert-
butoxide (84 mg, 0.75 mmol) in THF (1 mL) was added via a
syringe, and the reaction mixture turned deep yellow colour.
After 1 h of stirring at −78 °C, the reaction mixture was
allowed to warm slowly (within 2 h) to −15 °C, and TLC analy-
sis showed disappearance of the starting material. The reac-
tion was quenched by the addition of NH4Cl (54 mg, 1 mmol)
solution in 50% aq. methanol (ca. 0.5 mL) and allowed to
warm to room temperature. Then saturated methanolic
ammonia solution (2 mL) was added, and the reaction mixture
was stirred overnight while being exposed to air. After evapor-
ation of the volatiles, the residue was chromatographed on
silica gel (dichloromethane–methanol, 15 : 1 to 5 : 1) to afford
disulfide 14b (20 mg, 59% yield) as a colourless solid. Accord-
ing to NMR analysis, compound 14b was obtained as an
equimolecular mixture of DL/meso-diastereomers. Single recrys-
tallization from chloroform gave an analytically pure sample
with the ratio of diastereomers 60 : 40. Rf 0.30 (dichloro-
methane–methanol, 10 : 1). 1H NMR (DMSO-d6, 400 MHz)
δ 7.88 (br s, 0.6H, NH), 7.82 (br s, 0.4H, NH), 7.31 (br s, 0.6H,
NH), 7.31 (s, 0.6H), 7.29 (s, 0.4H), 7.21 (br s, 0.4H, NH), 5.36
(dd, J = 10.6, 1.8 Hz, 0.6H), 5.28 (dd, J = 10.6, 1.4 Hz, 0.4H),
4.02–3.90 (m, 1H), 3.59–3.46 (m, 1H), 1.84–1.73 (m, 1H),
Acknowledgements
1.73–1.65 (m, 1H), 1.65–1.50 (m, 1H), 1.50–1.35 (m, 2H), Support from the Ministry of Education and Research of
1.35–1.20 (m, 1H). 13C NMR (DMSO-d6, 100.6 MHz) δ 164.02, Estonia (grant IUT 19-32) and the EU Regional Development
163.92, 153.34, 153.32, 149.07, 148.56, 96.17, 95.74, 82.81, Fund Grant (3.2.0101.08-0017) are acknowledged.
This journal is © The Royal Society of Chemistry 2014
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Organic & Biomolecular Chemistry
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5644 | Org. Biomol. Chem., 2014, 12, 5634–5644
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