Protecting-Group-Mediated Diastereoselective Synthesis of C4-Methylated Uridine Analogs and Their Activity against the Human Respiratory Syncytial Virus

Article abstract of DOI:10.1021/acs.joc.9b03425

Adjusting the protecting group strategy, from an alkyl ether to a bidentate ketal at the carbohydrate backbone of uridine, facilitates a switchable diastereoselective α-or -C4/C5-spirocyclopropanation. Using these spirocyclopropanated nucleosides as key intermediates, we synthesized a variety of C4-methylated d-ribose and l-lyxose-configured uridine derivatives by a base-mediated ring-opening of the spirocyclopropanol moiety. Investigations of antiviral activity against the human respiratory syncytial virus were carried out for selected derivatives, showing moderate activity.

Full text of DOI:10.1021/acs.joc.9b03425

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Article
Protecting-Group-Mediated Diastereoselective Synthesis
of C4’-Methylated Uridine Analogs and their Activity
against the Human Respiratory Syncytial Virus
Christoph Köllmann, Svenja M. Sake, Peter G. Jones, Thomas Pietschmann, and Daniel B. Werz
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b03425 • Publication Date (Web): 10 Feb 2020
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Protecting-Group-Mediated Diastereoselective Synthesis of
C4’-Methylated Uridine Analogs and their Activity
against the Human Respiratory Syncytial Virus
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Christoph Köllmann,† Svenja M. Sake,§ Peter G. Jones,‡ Thomas Pietschmann,§ and Daniel B. Werz*,†
†Technische Universität Braunschweig, Institute for Organic Chemistry and ‡Technische Universität
Braunschweig, Institute for Inorganic and Analytical Chemistry, Hagenring 30, 38106 Braunschweig, Germany
§ Institute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research; a
Joint Venture Between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research
(HZI), Hannover, Feodor-Lynen-Str. 7, 30625 Hannover
O
O
O
NH
NH
NH
Ether
Ketal
Protecting
Strategy
Protecting
Strategy
(HO)2(O)PO
HO
N
O
H3C
N
O
N
O
O
O
O
H3C
HO
(HO)2(O)PO
OH
HO
OH
OH
HO
Uridine
ABSTRACT: Adjusting the protecting group strategy, from an alkyl ether to a bidentate ketal at the carbohydrate
backbone of uridine, facilitates a switchable diastereoselective α- or β-C4’/C5’-spirocyclopropanation. Using these
spirocyclopropanated nucleosides as key intermediates, we synthesized a variety of C4’-methylated D-ribose and
L-lyxose configured uridine derivatives by a base-mediated ring-opening of the spirocyclopropanol moiety.
Investigations of antiviral activity against HRSV were carried out for selected derivatives, showing moderate activity.
INTRODUCTION
The observed conformation of the furanose backbone of nucleotides, either in a single molecule or in oligo- and
polynucleotides, is a finely balanced system deeply influenced by its attached substituents. Major electronic
effects, the gauche or anomeric effect, restrict the populated conformations of the pentofuranose to a limited
range among all possible states accessible by theoretical pseudorotation.1 Accordingly, observable characteristics
such as proton coupling constants are determined in the conformational equilibrium state, described by the
Altona-Sundaralingam parameters P and (m).2 Nevertheless, even minor modifications of the substitution
pattern might have a strong and unexpected conformational impact, thereby fundamentally changing the
reactivity of the investigated nucleotides. Guo and coworkers, for example, showed that the half-life of an
oligonucleotide containing C4’-methylated uridine was reduced, by a factor of four compared to its native
unsubstituted counterpart.3 In our previous work we have shown that the Simmons-Smith-Furukawa
spirocyclopropanation proved to be a suitable protocol for the preparation of spirocyclopropane residues in a
variety of monosaccharides and nucleosides, in particular uridine.4 Based on the diastereoselective
cyclopropanation step, feasible synthons for the methylation of a carbohydrate backbone have been
synthesized.5,6
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Previous Work
O
O
5
6
7
NH
NH
HO
HO
N
O
N
O
O
O
CH3
H3C
8
9
OH
OH
HO
HO
I
II
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This Work
O
O
NH
NH
HO
O
N
O
N
O
H3C
O
H3C
HO
HO
OH
HO
OH
III
IV
Figure 1 Previous Work and Our Current Work in the Field of C-Methylation.
In recent literature, various C-methylated nucleosides and nucleotides have been synthesized as proposed
antiviral agents with varying degrees of success.7 In particular, C2’- and C3’-methylated derivatives have been
under the spotlight, as methyl nucleophiles can easily attack ketones that were prepared by prior oxidation at the
hydroxylated carbons such as C2’ and C3’ (I and II, Figure 1). Successful current studies of the antiviral activity
of our spirocyclopropanated nucleoside derivatives prompted us to seek a facile synthetic access to C4’-
methylated uridine derivatives III. Unfortunately, reported procedures suffer from unselective conversions
requiring formaldehyde as a single carbon source; these lead to inseparable product mixtures and an excess of
the unwanted diastereomer.8 Further, we sought to set up a synthesis for a diastereoselective access to the L-lyxo
nucleosides IV, as their previous preparation remained unsuccessful, because of the strong α-selectivity in the
cyclopropanation step. In order to form this motif, we used a modified protecting group strategy, leading finally
to the desired L-configured scaffold as the major diastereomer (for previous preparations of the L-configured
carbohydrate scaffold see References).9
In this work we present a diastereoselective, shortened synthesis for both D- and L-configured C4’-methylated
uridine analogs, depending on the applied protecting group strategy. This further extends the access to the
β-selective spirocyclopropanated uridine derivatives, since these appear as valuable and versatile key
intermediates during synthesis of the L-lyxo series. At this juncture, appropriate ketal cleavage leaves the
spirocyclopropane motif untouched. Finally, we show a detailed structural analysis of all prepared derivatives
and their 5’-monophosphates in aqueous solution and we also submitted selected derivatives to antiviral
investigations against the human respiratory syncytial virus (HRSV). Infection by HRSV, a negative strand RNA
virus, can lead to severe lower respiratory tract disease. Consequently, HRSV is responsible for high
hospitalization rates in vulnerable populations groups such as infants, immunocompromised individuals and the
elderly.10 Although non-nucleoside replication inhibitors such as the benzodiazepine RSV604 reached phase II
clinical trials already 10 years ago, it has not progressed further so far.11 Hence, the guanosine derivative and
broad-spectrum antiviral Ribavirin remains the only licensed, yet debated, therapeutic option for treatment of
HRSV infections.12 For this reason, we prepared a broader rangescope of esterified derivatives, featuring higher
lipophilicity, by exploitation of a prodrug strategy that showed improved results in previous studies.13
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Scheme 1 Synthesis of the L-Lyxose Configured Uridine Series.
4
5
6
7
8
9
O
O
O
N
1) IBX (2.5 equiv)
CH3CN, reflux, 2 h
2) K2CO3 (6 equiv)
(iPrCO)2O (6 equiv)
CH3CN, 50 °C, 3 h
NH
NH
NBOM
O
RO
BOMCl (2.5 equiv)
NiPr2Et (4 equiv)
CH2Cl2, rt
ZnEt2 (5 equiv)
CH2I2 (10 equiv)
ClCH2CH2Cl
N
O
N
O
O
iPrCOO
iPrCOO
O
Y
O
Y
50 °C, 16 h
16 h
O
O
O
O
O
O
Y
10
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pTsOH
MeOH
rt, 4 h
(94%)
1a
1b
1c
2a
2b
3a
3b
R = H, Y = CCy
R = H, Y = CPh2
(54%)
(63%)
Y = CCy (34%, E/Z 1:4 over 2 steps)
Y = CPh2 (39%, E/Z 1:4 over 2 steps)
Y = CCy (72%)
Y = CPh2 (73%)
R = C(OMe)Cy, Y = CCy (14%)
O
O
N
O
1) NaOMe (0.3 equiv)
MeOH, 0 °C, 3 h
NBOM
O
NH
NBOM
2) T1; NaBH4 (1.2 equiv)
MeOH, 0 °C, 10 min
(44%)
Pd/C (0.2 equiv)
H2 (1 atm)
MeOH, rt, 0.5 h
(quant)
N
O
N
O
H3C
H3C
O
O
O
iPrCOO
HO
O
HO
O
O
O
O
O
Y
Y
Y
4a
4b
5
6
Y = CCy (49%,  1:5)
Y = CPh2 (49%,  1:9)
Y = CCy
Y = CCy
1) DDPA (1.2 equiv)
5-MT (3.0 equiv)
CH3CN, rt, 3 h
2) tBuOOH (1.2 equiv)
CH3CN, rt, 0.75 h
DOWEX 50W8
70°C
O
Pd/C (0.2 equiv)
H2 (1 atm)
MeOH, rt, 3 h
(60%)
H2O:MeOH (2:1)
(77%)
NH
N
O
O
(87% over 2 steps)
O
O
iPrCOO
NH
NBOM
O
O
Y
11
N
O
N
O
Y = CPh2
O
H3C
H3C
O
O
Et3SiH (12 equiv)
TFA (13 equiv)
MS 4Å, rt, 2 d
(28%)
NH
HO
(BnO)2(O)PO
O
HO
OH
O
Y
N
O
9
7
Y = CCy
O
(iPrCO)2O (10 equiv)
pyridine, rt
1) Pd/C (0.2 equiv)
H2 (1 atm), rt, 4 h
2) DOWEX 50W8
70°C, 4 h, H2O
(53%)
iPrCOO
OH
O
HO
12
(quant)
(iPrCO)2O (10 equiv)
pyridine, rt, 16 h
(70%)
R = OCiPr
NH
N
O
O
O
O
R = OCiPr
iPrCOO
NH
NH
OR
RO
N
O
N
O
H3C
H3C
O
O
13
(HO)2(O)PO
RO
RO
OH
HO
OR
CCy
=
=
DDPA
5-MT
= Dibenzyl N,N-diisopropyl
phosphoramidite
10
8
CPh2
=
5-Methyl-1H-tetrazole
1 Thermal isomerization of the spirocyclopropanol to the aldehyde.
Ph
Ph
RESULTS AND DISCUSSION
According to our previously reported procedure for a cyclohexylidene ketal, the preparation of the known
compounds 1a and 1b was performed, with minor modifications, from the dimethoxy ketals.14 Crystallographic
information of both compounds is given in the Supporting Information. In the case of cyclohexylidene ketal
(CCy) 1a, the undesired formation of the O5’-protected methoxy ketal 1c was also observed, because of the three
equivalents of protecting group used in this step. Cleaving conditions for ketals at room temperature, applied to
1c, left the spiroketal unaffected and afforded additional 1a in 94% yield (Scheme 1). The primary alcohols were
oxidized by IBX and the crude aldehydes were immediately converted into the isobutyric enol esters 2a and 2b,
respectively.15 Both enol esters needed to be protected with benzyloxymethyl chloride (BOMCl) at the
nucleobase, as a protic nitrogen hampered the spirocyclopropanation by the Simmons-Smith-Furukawa
protocol.16 Interestingly, the cyclopropanation of the nucleoside derivatives 3a and 3b was performed in a
β-diastereoselective fashion, in contrast to our reported procedure for α-selective spirocyclopropanation of the
corresponding ether-protected nucleoside derivatives. The diastereoselective outcome of this reaction might be
explained by the steric bulkiness and reduced flexibility of the cyclohexylidene ketal in compound 4a, which
differs significantly in this respect from to the benzyloxymethyl ether. Clearly, this effect can be further
promoted by increasing the size from the cyclohexylidene ketal residue (α:β 1:5) to a sterically more demanding
benzophenone at the 2’,3’-syn diol motif 4b (α:β 1:9). In case of the cyclohexylidene derivative, previous
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saponification using sodium methoxide (NaOMe) and thermal isomerization of the spirocyclopropanol afforded
the corresponding aldehyde. Subsequent reduction by sodium borohydride gave the C4’-methylated L-lyxose
alcohol 5 in 44% yield over two steps. The L-configured carbohydrate backbone was exemplarily determined by
NOE experiments on compound 5, because a NOE between the methyl group and H6 of the nucleobase was
observed. Branching the synthetic route, hydrogenolytic deprotection of the nucleoside gave 6 and subsequent
acidic cleavage of the cyclohexylidene ketal led to the unprotected C4’-methylated L-lyxose uridine derivative 7.
The anti-orientation of nucleobase and furanose moiety was confirmed in aqueous media on the basis of a NOE
between H6 of the nucleobase and the C4’-methyl group. For biological investigations, alcohol 7 was
quantitatively converted to the fully esterified derivative 8 by treatment with isobutyric anhydride in pyridine.
Further transformation of the alcohol 5 to the 5’-monophosphate using a phosphoramidite preocedure with
dibenzyl N,N-diisopropylphosphoramidite (DDPA) and 5-methyl-1H-tetrazole (5-MT), led to compound 9 in
87% yield over two steps. Simultaneous deprotection of the monophosphate and the nucleobase under
hydrogenolytic reaction conditions and subsequent conversion by DOWEX at 70 °C in aqueous solution
afforded the nucleotide derivative 10.17
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Alternatively, compound 4b was deprotected at the nucleobase by the application of palladium on charcoal and a
hydrogen atmosphere in 60% yield. The configuration after diastereoselective cyclopropanation of the resulting
compound 11 was confirmed by X-ray analysis (see Supporting Information). As neither acidic deprotection of
the benzophenone ketal nor palladium-mediated hydrogenolysis led to formation of the free diol, ionic
hydrogenolytic deprotection was investigated. Application of comparable reaction conditions reported by Parnes
et al. effected immediate formation of product 12, although full conversion of starting material was not achieved
even after extended reaction times.18 Successive esterification with isobutyric anhydride gave compound 13 in
good yield.
Scheme 2 Synthesis of the D-Ribose Configured Uridine Series.
O
O
NBOM
NBOM
iPrCOO
LiBH4 (3.0 equiv)
HO
N
O
N
O
O
THF, MeOH
0 °C, 0.5 h
(47%)
O
OBOM
BOMO
OBOM
BOMO
14
15
(ref. 6)
1) K2CO3 (0.9 equiv)
EtOH:H2O (20:1), 0 °C to rt, 16 h
2) NaBH4 (1.2 equiv)
EtOH, 0 °C, 2 h, (25% over two steps)
O
O
O
1) DDPA (1.2 equiv)
5-MT (3.0 equiv)
CH3CN, rt, 1.5 h
2) tBuOOH (1.2 equiv)
CH3CN, rt, 0.75 h
(59%)
NH
NR
NR
Pd/C (0.2 equiv)
H2 (1 atm)
MeOH, rt, 1 h
(72%)
HO
(BnO)2(O)PO
HO
N
O
N
O
N
O
O
O
O
H3C
HO
H3C
RO
H3C
OR
OH
OR
RO
17
16
19 R = BOM
R = BOM
O
O
NH
NH
1) Pd/C (0.2 equiv)
H2 (1 atm)
MeOH, rt, 9 h
2) D2O, 50 °C, 2 d
(79%)
(iPrCO)2O (10 equiv)
pyridine, rt, 14 h
(quant)
RO
(HO)2(O)PO
N
O
N
O
O
O
H3C
RO
H3C
HO
OR
OH
i
18
20
R = OC Pr
Compound 14 was prepared according to our reported procedure.6 In contrast to the synthetic route presented in
Scheme 1, the isomerization of the spirocyclopropanol was achieved via the BOM-protected nucleoside
derivative. As alkaline ester cleavage showed undesired side reactions, spirocyclopropanol 15 was prepared in a
separate step from the isobutyric ester by reduction with LiBH4. Successive isomerization to the according
aldehyde and reduction of this by acetonitrile to compounds 5 and 6 of the L-lyxose series, could be provided by
analogous isomerization reactions, further attempts to follow this synthetic route were aborted because yields
dropped significantly in contrast to the L-configured diastereomer (for analytical data of 5a and 6a see
Experimental Section). Hydrogenolysis by palladium on charcoal under a hydrogen atmosphere afforded the
unprotected D-ribose configured C4’-methylated uridine 17 (X-ray analysis, see Supporting Information). The
reported analytical data of Meguro et al. were in accordance with our results.19 Complete esterification by
isobutyric anhydride in pyridine was performed in quantitative yield, giving nucleoside 18. The
5’-monophosphate of compound 16 was afforded by tetrazole-mediated DDPA coupling and successive
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oxidation by tBuOOH affording 19 over two steps. Hydrogenolytic cleavage of all BOM protecting groups led
finally to monophosphate 20 in 79% yield. In case of the D-ribo-configured nucleotide, BOM deprotection
resulted the nucleotide, bearing a hydroxymethylene group at the nucleobase.6 Thermal promoted hydrolysis of
this hemiaminal structure was monitored by NMR; thus the sample was prepared in D2O. Although the direct
transfer of structural information from the solid state to the conformation of molecules in solution should be
regarded with caution, the major part of our current structural knowledge of nucleosides and nucleotides has
doubtless been obtained from X-ray analysis.
10
11
12
13
14
15
16
17
18
19
20
21
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Table 1. Conformational Analysis, based on X-ray analysis, of Compounds 1a, 1b, 11, C4’-methylated 17
and Uridine Monophosphate (UMP).
Compound
P
χ
γ
Type
(m)
164.4(2) 31.7(1) -134.21(12) 48.15(16)
191.0(3) 21.7(1) -147.66(17) 45.8(2)
1a
1b
C2’-endo
C3’-exo
O
NH
11, mol. 1 212.8(2) 29.8(1) 73.35(15) -146.55(13) C3’-exo, C4’-endo
11, mol. 2 262.8(2) 25.2(1) 79.05(15) -144.24(13) envelope O4’-exo
C2
O5'
O
N1
O4'
O

N
O
C5'




C4'
C1'
17
163.5(2) 36.4(1) -130.53(14) 48.99(16)
157.26 33.91 -129.44 49.63
C2’-endo
C2’-endo
C3'
O3'
C2'
O2'
O
UMP20
O
All values are given in degrees °.
P: pseudorotation phase angle
(m): puckering amplitude
γ: torsion angle [O5’-C5’-C4’-C3’]
χ: torsion angle [O4’-C1’-N1-C2]
The entries of Table 1, including the Altona-Sundaralingam pseudorotational parameters P and (m) were
generated from the X-ray data using the PLATON program.21 Interestingly, compound 11 crystallized in two
different conformers (two independent molecules), one of which showed a C3’-exo/ C2’-endo conformation and
the other an O4’-exo envelope (see Table 1). As P values higher than 180 ° are typically observed for α-D-
nucleosides, a phase angle of 212.8 ° is a conspicuous feature of this β-D-nucleoside. The other conformer
presented endocyclic torsion angles ν0 [C4’-O4’-C1’-C2’] of 22.84(14) °, ν2 [C1’-C2’-C3’-C4’] -3.02(14) ° and
ν4 [C3’-C4’-O4’-C1’] of -25.25(14) °. Furthermore, its exocyclic torsion angle [O2’-C2’-C3’-O3’] of -2.27(13) °
revealed a nearly ecliptic orientation of the corresponding substituents. Regarding this conformer to be close to
OE, potential influence of the isobutytric ester substituent, preferring equatorial orientation at the
spirocyclopropane, should be considered. E characterizes the conformer as an envelope (molecule 2), showing
O
O4’ beneath the ring plane. Relief from burdening 1,3-diaxial repulsive effects across the furanose ring is
thereby achieved, as the C4’-oxymethylene unit is inverted (γ = 146.55, 144.24 ° in the two molecules),
compared to the D-ribose configuration. Probably, additional energetic benefit is gained from the anomeric effect
between the axial oriented nucleobase and O4’ of the pentofuranose. As O4’-exo conformations are regularly
observed for 2’,3’-cyclic phosphates, 1,3-diaxial interactions enforce an anti-orientation of the nucleobase.22
Astonishingly, in case of nucleoside 11, the nucleobase showed for both conformers a syn conformation in solid
state, rarely observed for pyrimidine nucleosides. This led to a positive χ value (73.35, 79.05°) in comparison to
the anti-oriented benzophenone protected precursor structure 1b. Additionally, hydrogen bonding between both
conformers via the nucleobase was observed and may represent a stabilizing factor. In contrast to a cyclic
phosphate, a ketal-protection is not sufficient to induce this kind of conformational abnormality, as proved by X-
ray analysis of compound 1a and 1b.
3
3
1
Table 2. Coupling constants JH1’-2’ and JH2’-3’ determined by H NMR and 13C carbon shifts of the C4’-
methyl group and the C5’-oxymethylene group of compounds 7, 10, 17, and 20.
Compound
3JH1’-2’
5.9
3JH2’-3’
5.8
CH3
24.6
24.7
20.2
20.7
C5’
67.0
70.5
69.0
71.9
7
10
17
20
6.5
5.5
6.3
6.1
7.1
5.6
All 3J couplings are given in Hz and 13C shifts in ppm.
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In order to predict the conformation of 17, Meguro et al. performed intensive NOE investigations, the results of
which were confirmed by X-ray analysis. Remarkably, inversion of the configuration at C4’ did not lead to a
major difference between the conformation of the L and D-furanose for either the nucleoside or the
3
3
corresponding 5’-monophosphate. Table 2 shows the JH1’-2’ and JH2’-3’ coupling constants, determined for the
unprotected C4’-methylated derivatives 7 and 17 and their 5’-monophosphates 10 and 20, in D2O as solvent. A
1
stacked view of their respective H NMR spectra versus natural uridine and uridine monophosphate (UMP) is
given in the Supporting Information. From these results one might conclude that L-lyxose nucleoside 7 remains
in a C2’-endo conformation (3JH1’-2’, ΔHz = 0.4) as observed for compound 17 by X-ray analysis. The same can
be assumed for the 5’-monophosphates 10 and 20, which show a maximum discrepancy of ΔHz = 0.6 Hz for
3JH1’-2’ Only a minor difference is observed between the conformation of natural UMP in the solid state and
nucleoside derivative 17. Vicinal oxygen atoms lead to a resonance at higher field of the investigated carbon in
13C-NMR, unlike their diastereomeric counterparts.23 In addition to in-depth NOE studies, this effect has been
taken into account for the purpose of structural elucidation of compound 7 and 17. As the D-ribose configuration
shows a vicinal methyl group (20.2 ppm) related to O3’ and an anti-oriented hydroxymethylene group
(69.0 ppm), these carbons resonate for the L-lyxose scaffold at 24.6 ppm (methyl group) and 67.0 ppm for the
hydroxymethylene group.
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Figure 2 Antiviral activity and cell toxicity of compound 13 compared to Ribavirin and RSV604. Mean
and standard deviations of three independent experiments are shown.
Finally, compounds 7, 8, 13, 17 and 18 were selected for investigation of their antiviral activity against HRSV.
To this end, we quantified infection of cells after inoculation with HRSV and treatment with various compound
concentrations (infection round I). Additionally, infectivity of newly produced virus was analyzed by transfer of
supernatants from treated cells onto naive cells (infection round II). The guanosine analog Ribavirin and the non-
nucleoside viral nucleoprotein inhibitor RSV60411a served as assay controls. All tested uridine derivatives were
non-toxic to the cells in the tested dose range (Figure 2 and Supporting Information Figure S77). No antiviral
activity was observed for the alcohols 7 and 17 and esterification of either compound did not result in
reproducible virus inhibition by compounds 8 or 18 at the tested concentrations (Supporting Information Figure
S77). On the contrary and albeit only minimal effects at high concentrations in infection round I, the
spirocyclopropanated compound 13 reduced infectivity of cellular supernatants to about 30% at 100 µM
compared to the solvent control (Figure 2). Further and more extensive cellular and virologic experiments would
be needed to determine whether differences regarding cell membrane transfer, incorporation into viral RNA or
efficacy in base mispairing account for the distinct antiviral properties of the tested compounds.
CONCLUSION
In this work we have shown a feasible access to C4’-methylated uridine derivatives with either a D-ribose or
L-lyxose configured carbohydrate scaffold, and to their corresponding 5’-monophosphates. Further, we have
exploited the steric bulkiness of the 2’,3’-syn diol protecting group as a suitable property for a diastereoselective
control in the spirocyclopropanation step. Appropriate deprotection of the diol opened new opportunities for
access to the L-lyxose configured spirocyclopropanols, of which 13 showed a moderate activity in an antiviral
HRSV assay. Additionally, deeper analysis of NMR and X-ray data revealed only a small conformational impact
on the pentofuranose by inversion of C4’ in general and for C4’-methylation in particular.
EXPERIMENTAL SECTION
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General Information. Solvents were purchased as synthetic grade solvents and stored over suitable molecular
sieves. All reactions were performed in oven-dried glassware, septum-capped and under atmospheric pressure of
argon. Commercially available compounds were used without further purification unless otherwise stated. Proton
(1H) and carbon (13C) NMR spectra were recorded on a Bruker Avance II 300, a Bruker Avance III 400, a Bruker
Avance III HD 500, a Bruker Avance II 600 and a Bruker Ascend 300 spectrometer using the signals from
tetramethylsilane as internal references for 1H and 13C chemical shifts, respectively. Abbreviations for
multiplicities were used as following s = singlet, d = doublet, t = triplet, q = quartet, p = quintet, hept = septet, m
= multiplet. HRMS (ESI) mass spectrometry was carried out on a FTICR instrument and IR spectra were
received from an ATR spectrometer. Optical rotations were determined using a Dr. Kernchen polarimeter at
20 °C. Melting points were received from a Büchi Melting Point M-560. An Agilent 1260 Infinity Series II
system and an Agilent C18 column (Pursuit XRs 5 C18, 250·10.0 mm, flow rate at 4.50 ml·min-1, wavelength
for detection: 254 nm) were used for separations on HPLC. If necessary, the reaction mixtures were heated in an
oil bath.
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Synthetic Procedures and Analytical Data.
1-((3a'R,4'R,6'R,6a'R)-4'-(Hydroxymethyl)tetrahydrospiro[cyclohexane-1,2'-furo[3,4-d][1,3]dioxol]-6'-
yl)pyrimidine-2,4(1H,3H)-dione (1a)
Uridine (8.40 g, 34.4 mmol, 1.00 eq.) was suspended in 1,2-dichloroethane (80.0 mL) and cyclohexanone
dimethyl ketal (15.7 mL, 103 mmol, 3.00 eq.) was added. pTsOH (0.60 g, 3.44 mmol, 0.10 eq.) was added in
three portions and the reaction mixture was heated to 50 °C, stirred for 5 h and finally quenched by the addition
of NEt3 (6.00 mL). SiO2 was added and the mixture evaporated to dryness. Purification by column
chromatography of the powdery residue (SiO2, gradient, pentane/ethyl acetate, 3:1, v/v; 1:1, v/v; 0:1, v/v)
afforded the ketal protected nucleoside 1a (6.00 g, 18.5 mmol, 54%) as a colorless solid and 1c (2.10 g, 4.81
1
mmol, 14%) as a colorless syrup (side product). 1a (spectra shown) mp = 167-169 °C; H NMR (500 MHz,
CDCl3) δ (ppm) 9.48 (s, 1H), 7.40 (d, J = 8.1 Hz, 1H), 5.74 (d, J = 8.0 Hz, 1H), 5.60 (t, J = 2.5 Hz, 1H), 5.04 (dt,
J = 6.2, 2.6 Hz, 1H), 4.95 (ddd, J = 6.3, 3.5, 1.2 Hz, 1H), 4.29 (q, J = 3.6 Hz, 1H), 3.92 (dd, J = 12.1, 2.6 Hz,
1H), 3.86 – 3.76 (m, 1H), 3.35 – 3.08 (m, 1H)1.82 – 1.75 (m, 2H), 1.66 (tt, J = 8.7, 5.1 Hz, 2H), 1.62 – 1.50 (m,
4H), 1.44 – 1.35 (m, 2H). 13C{1H} NMR (126 MHz, CDCl3) δ (ppm) 163.4, 150.4, 143.1, 115.2, 102.6, 95.9,
87.0, 83.1, 79.9, 62.6, 37.1, 34.6, 24.9, 23.9, 23.5.
Preparation of 1a from compound 1c
Nucleoside 1c (1.94 g, 4.44 mmol, 1.00 eq.) was dissolved in methanol (10.0 mL) and pTsOH (75.8 mg,
0.44 mmol, 0.10 eq.) was added. The mixture was stirred for 4 h at room temperature and the reaction was
terminated by the addition of NEt3 (1.00 mL). SiO2 was added to the reaction mixture and the solvent removed
under reduced pressure. Purification of the powdery residue by column chromatography (SiO2, gradient,
pentane/ethyl acetate, 1:1, v/v; 0:1, v/v) afforded the primary alcohol 1a (1.35 g, 4.16 mmol, 94%).
Single crystals of 1a were obtained.
1-((3aR,4R,6R,6aR)-6-(Hydroxymethyl)-2,2-diphenyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)pyrimidine-
2,4(1H,3H)-dione (1b)
To a suspension of uridine (1.00 g, 4.10 mmol, 1.00 eq.) in 1,2-dichloroethane (15.0 mL) was added
benzophenone dimethyl ketal (2.80 g, 12.3 mmol, 3.00 eq.) and pTsOH (68.9 mg, 0.41 mmol, 0.10 eq.) in three
portions. The mixture was stirred for 1 d at 50 °C, the reaction being terminated by the addition of NEt3
(2.00 mL) and SiO2, and finally evaporated to dryness. Purification by column chromatography of the powdery
residue (SiO2, gradient, pentane/ethyl acetate, 4:1, v/v; 1:1, v/v; 0:1, v/v) afforded the ketal protected nucleoside
1b (1.05 g, 2.57 mmol, 63%) as a colorless solid. 1H NMR (500 MHz, CD3OD) δ (ppm) 7.81 (d, J = 8.1 Hz, 1H),
7.54 – 7.48 (m, 4H), 7.39 – 7.29 (m, 6H), 6.02 (d, J = 2.6 Hz, 1H), 5.66 (d, J = 8.1 Hz, 1H), 4.93 (dd, J = 6.6, 2.7
Hz, 1H), 4.82 (dd, J = 6.6, 3.3 Hz, 1H), 4.38 (dt, J = 4.8, 3.6 Hz, 1H), 3.77 (dd, J = 11.9, 3.9 Hz, 1H), 3.72 (dd, J
= 11.9, 4.8 Hz, 1H). 13C{1H} NMR (126 MHz, CD3OD) δ (ppm) 166.2, 152.1, 144.1, 143.1, 142.8, 129.6, 129.5,
-1
푣
129.4, 129.1, 127.4, 127.3, 115.4, 102.6, 94.2, 88.2, 86.5, 83.2, 63.1. IR (ATR): (cm ) = 3061, 2923, 2854,
1690, 1457, 1384, 1271, 1209, 1096. HRMS (ESI) m/z: [M + Na]+ calcd for C22H20N2O6Na+ 431.1214, found:
[α]20
431.1218;
= -21.6 ° (c 0.38, CHCl3). Single crystals of 1b were obtained.
D
1-((3a'R,4'R,6'R,6a'R)-4'-(((1-Methoxycyclohexyl)oxy)methyl)tetrahydrospiro[cyclohexane-1,2'-furo[3,4-
d][1,3]dioxol]-6'-yl)pyrimidine-2,4(1H,3H)-dione (1c)
1
Colorless syrup (2.10 g, 4.81 mmol, 14%), H NMR (500 MHz, CDCl3) δ (ppm) 8.65 (s, 1H), 7.70 (d, J = 8.1
Hz, 1H), 5.99 (d, J = 2.6 Hz, 1H), 5.70 (dd, J = 8.1, 1.3 Hz, 1H), 4.80 (dd, J = 6.1, 3.6 Hz, 1H), 4.75 (dd, J = 6.1,
2.6 Hz, 1H), 4.37 (td, J = 3.8, 2.7 Hz, 1H), 3.70 (dd, J = 10.8, 2.8 Hz, 1H), 3.61 (dd, J = 10.8, 4.0 Hz, 1H), 3.17
(s, 3H), 1.86 – 1.32 (m, 20H). 13C{1H} NMR (126 MHz, CDCl3) δ (ppm) 162.9, 149.9, 141.2, 115.1, 102.1,
푣
100.6, 91.9, 85.7, 84.5, 80.2, 59.7, 47.8, 37.1, 34.8, 33.1, 33.0, 25.4, 24.9, 23.9, 23.6, 22.8, 22.8. IR (ATR):
(cm-1) = 3200, 3063, 2935, 2859, 1693, 1456, 1376, 1269, 1159, 1102, 1048. HRMS (ESI) m/z: [M + Na]+ calcd
[α]20
+
for C22H32N2O7Na 459.2102, found: 459.2107;
= -17.4 ° (c 0.94, CHCl3).
D
(Z)-((3a'R,4'R,6a'S)-4'-(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)dihydrospiro[cyclohexane-1,2'-furo[3,4-
d][1,3]dioxol]-6'(4'H)-ylidene)methyl isobutyrate (2a)
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IBX (2.00 g, 7.14 mmol, 2.47 eq.) was added to a solution of alcohol 1a (0.94 g, 2.89 mmol, 1.00 eq.) in
acetonitrile (28.0 mL). The suspension was refluxed for 2 h and finally filtered through a plug of Celite 545®.
The plug was rinsed with ethyl acetate (3x, 25.0 mL) and the solvent removed under reduced pressure. The
remaining crude aldehyde was immediately dissolved in acetonitrile (28.0 mL). Potassium carbonate (2.40 g,
17.4 mmol, 6.00 eq.) and isobutyric anhydride (2.90 mL, 17.4 mmol, 6.00 eq.) were added and the reaction
mixture heated to 50 °C for 3 h. Finally, the reaction was terminated by the addition of ethyl acetate (100 mL)
and water (100 mL). The aqueous layer was extracted with ethyl acetate (3x, 50 mL,) and the combined organic
phases were dried over Na2SO4, filtered and evaporated to dryness. Purification by column chromatography
(SiO2, gradient, pentane/ethyl acetate, 10:1, v/v; 1:1, v/v) afforded the exocyclic enol ester 2a as an oily mixture
of diastereomers (0.39 g, 0.99 mmol, 34% yield). Because of the excess anhydride, prolonged reaction times are
not recommended as the formation of side product 2c, bearing a further isobuyric amide at the nucleobase, is
then favoured. 2a Major diastereomer (Z) 1H NMR (300 MHz, CDCl3) δ (ppm) 9.64 (s, 1H), 7.20 (d, J = 8.2 Hz,
1H), 6.93 (d, J = 0.8 Hz, 1H), 5.76 (dd, J = 8.1, 1.4 Hz, 1H), 5.73 (d, J = 1.1 Hz, 1H), 5.41 (dd, J = 6.3, 0.8 Hz,
1H), 5.09 (dd, J = 6.2, 1.2 Hz, 1H), 2.67 (hept, J = 7.0 Hz, 1H), 1.72 (p, J = 3.6, 3.0 Hz, 2H), 1.61 (td, J = 10.2,
8.5, 6.0 Hz, 6H), 1.48 – 1.35 (m, 2H), 1.21 (d, J = 7.0 Hz, 3H), 1.20 (d, J = 7.0 Hz, 3H). 13C{1H} NMR (101
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MHz, CDCl3) δ (ppm) 173.3, 163.4, 149.9, 144.3, 142.6, 115.4, 115.0, 102.8, 97.5, 82.5, 78.5, 36.3, 34.8, 33.6,
-1
푣
24.8, 23.8, 23.7, 18.9, 18.8. IR (ATR): (cm ) = 3196, 2938, 2862, 1688, 1455, 1378, 1341, 1259, 1138, 1099,
[α]2D0
1063. HRMS (ESI) m/z: [M + Na]+ calcd for C19H24N2O7Na+ 415.1476, found: 415.1479;
= 116.0 ° (c
0.62, CHCl3).
(Z)-((3a'R,4'R,6a'S)-4'-(3-Isobutyryl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)dihydrospiro[cyclohexane-
1,2'-furo[3,4-d][1,3]dioxol]-6'(4'H)-ylidene)methyl isobutyrate (2c)
Dark yellow oil,1H NMR (300 MHz, CDCl3) δ (ppm) 7.17 (dd, J = 8.1, 0.3 Hz, 1H), 6.93 (d, J = 0.8 Hz, 1H),
5.78 (d, J = 8.1 Hz, 1H), 5.73 (d, J = 1.0 Hz, 1H), 5.35 (ddd, J = 6.2, 0.8, 0.4 Hz, 1H), 5.06 (dd, J = 6.2, 1.2 Hz,
1H), 3.03 (hept, J = 7.0 Hz, 1H), 2.68 (hept, J = 7.0 Hz, 1H), 1.77 – 1.68 (m, 2H), 1.59 (d, J = 2.5 Hz, 6H), 1.45
– 1.36 (m, 2H), 1.26 (d, J = 7.0 Hz, 3H), 1.25 (d, J = 7.0 Hz, 3H), 1.22 (d, J = 7.0 Hz, 3H), 1.21 (d, J = 7.0 Hz,
3H). 13C{1H} NMR (126 MHz, CDCl3) δ (ppm) 179.1, 173.3, 161.6, 148.8, 143.9, 141.7, 115.8, 115.1, 102.8,
-1
푣
97.7, 82.5, 78.4, 39.7, 36.3, 34.7, 33.7, 24.8, 23.9, 23.7, 18.9, 18.8, 17.8, 17.7. IR (ATR): (cm ) = 2935, 2861,
1782, 1749, 1713, 1672, 1441, 1370, 1265, 1140, 1101, 1018. HRMS (ESI) m/z: [M + Na]+ calcd for
[α]20
+
C23H30N2O8Na 485.1894, found: 485.1891;
= 98.1 ° (c 0.93, CHCl3).
D
(Z)-((3aS,6R,6aR)-6-(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2-diphenyldihydrofuro[3,4-d][1,3]dioxol-
4(3aH)-ylidene)methyl isobutyrate (2b)
IBX (4.02 g, 14.4 mmol, 2.50 eq.) was added to a solution of alcohol 1b (2.37 g, 5.81 mmol, 1.00 eq.) in
acetonitrile (58.0 mL). The suspension was refluxed for 2 h and finally filtered through a plug of Celite 545®.
The plug was rinsed with ethyl acetate (3x, 25.0 mL) and the solvent evaporated. The remaining aldehyde was
dissolved in acetonitrile (58.0 mL) and potassium carbonate (4.81 g, 34.9 mmol, 6.00 eq.) and isobutyric
anhydride (5.78 mL, 34.9 mmol, 6.00 eq.) were added. The reaction mixture was heated to 50 °C for 3 h. Finally,
the reaction was terminated by the addition of ethyl acetate (100 mL) and water (100 mL). The aqueous layer
was extracted with ethyl acetate (3 x 50.0 mL,) and the combined organic phases were dried over Na2SO4,
filtered and evaporated to dryness. Purification by column chromatography (SiO2, pentane/ethyl acetate, 10:1 to
1:1) afforded product 2b as an oily mixture of diastereomers (1.07 g, 2.25 mmol, 39% yield). Major
1
Diastereomer (Z) H NMR (500 MHz, CDCl3) δ (ppm) 9.35 (s, 1H), 7.54 – 7.49 (m, 2H), 7.45 – 7.38 (m, 2H),
7.38 – 7.29 (m, 6H), 7.15 (d, J = 8.1 Hz, 1H), 7.01 (d, J = 0.7 Hz, 1H), 5.83 (d, J = 1.0 Hz, 1H), 5.72 (dd, J =
8.1, 2.1 Hz, 1H), 5.34 (dd, J = 6.4, 0.8 Hz, 1H), 5.11 (dd, J = 6.4, 1.2 Hz, 1H), 2.66 (hept, J = 7.0 Hz, 1H), 1.24
– 1.14 (m, 6H). 13C{1H} NMR (126 MHz, CDCl3) δ (ppm) 173.2, 163.3, 149.8, 143.4, 142.8, 140.2, 139.8,
-
푣
128.9, 128.7, 128.5, 128.1, 126.5, 126.3, 115.8, 114.4, 102.7, 97.1, 83.3, 79.6, 33.6, 19.0, 18.7. IR (ATR): (cm
1) = 3174, 3061, 2979, 1682, 1455, 1381, 1341, 1262, 1137, 1067, 1021. HRMS (ESI) m/z: [M + Na]+ calcd for
[α]20
+
C26H24N2O7Na 499.1476, found: 499.1473;
= 69.6.0 ° (c 3.13, CHCl3).
D
(Z)-((3a'R,4'R,6a'S)-4'-(3-((Benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
yl)dihydrospiro[cyclohexane-1,2'-furo[3,4-d][1,3]dioxol]-6'(4'H)-ylidene)methyl isobutyrate (3a)
To a solution of compound 2a (0.39 g, 1.00 mmol, 1.00 eq.) in dichloromethane (10.0 mL) was added NEtiPr2
(0.85 mL, 5.00 mol, 5.00 eq.). Finally, BOMCl (0.28, 2.00 mmol, 2.00 eq.) was added and the reaction mixture
was stirred for 16 h at room temperature. The reaction was quenched by the addition of dichloromethane
(10.0 mL) and water (5.00 mL). The aqueous layer was extracted with dichloromethane (3x, 10.0 mL). The
combined organic layers were dried over Na2SO4, filtered and evaporated to dryness. Purification by column
chromatography (SiO2, gradient, pentane/ethyl acetate, 10:1, v/v; 1:1, v/v) afforded the protected uridine
1
derivative 3a (0.37 g, 0.72 mmol, 72% yield) as a slightly yellow oil. Major diastereomer (Z) H NMR (300
MHz, CDCl3) δ (ppm) 7.39 – 7.26 (m, 5H), 7.08 (d, J = 8.1 Hz, 1H), 6.95 (d, J = 0.8 Hz, 1H), 5.73 (d, J = 8.0
Hz, 1H), 5.67 (d, J = 1.1 Hz, 1H), 5.51 – 5.40 (m, 2H), 5.42 – 5.37 (m, 1H), 5.00 (dd, J = 6.2, 1.2 Hz, 1H), 4.68
(d, J = 1.6 Hz, 2H), 2.66 (hept, J = 7.0 Hz, 1H), 1.73 (m, 2H), 1.60 (dd, J = 11.7, 8.7 Hz, 6H), 1.48 – 1.39 (m,
2H), 1.19 (m, 6H). 13C{1H} NMR (126 MHz, CDCl3) δ (ppm) 173.3, 162.2, 150.6, 144.3, 141.0, 137.8, 128.3,
127.7, 127.6, 115.4, 115.0, 102.4, 98.1, 82.5, 78.5, 72.4, 70.3, 36.3, 34.8, 33.7, 24.9, 23.9, 23.7, 18.9, 18.8. IR
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(ATR): (cm ) = 2938, 2863, 1748, 1722, 1675, 1454, 1358, 1141, 1099. HRMS (ESI) m/z: [M + Na] calcd for
[α]20
+
C27H32N2O8Na 535.2051, found: 535.2051;
= 108.2 ° (c 0.78, CHCl3).
D
(Z)-((3aS,6R,6aR)-6-(3-((Benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2-
diphenyldihydrofuro[3,4-d][1,3]dioxol-4(3aH)-ylidene)methyl isobutyrate (3b)
To a solution of compound 2b (0.41 g, 0.85 mmol, 1.00 eq.) in dichloromethane (9.00 mL) was added NEtiPr2
(0.72 mL, 4.25 mol, 5.00 eq.). Finally, BOMCl (0.23, 1.70 mmol, 2.00 eq.) was added and the reaction mixture
was stirred for 16 h at room temperature. The reaction was quenched by the addition of dichloromethane
(10.0 mL) and water (5.00 mL). The aqueous layer was extracted with dichloromethane (3x, 10.0 mL). The
combined organic layers were dried over Na2SO4, filtered and evaporated to dryness. Purification by column
chromatography (SiO2, gradient, pentane/ethyl acetate, 10:1, v/v; 1:1, v/v) afforded the protected uridine
derivative 3b (0.37 g, 0.72 mmol, 73% yield) as a yellowish oil. (Z) 1H NMR (300 MHz, CDCl3) δ (ppm) 7.58 –
7.51 (m, 2H), 7.47 – 7.21 (m, 13H), 7.06 (d, J = 8.1 Hz, 1H), 7.03 (d, J = 0.7 Hz, 1H), 5.78 (d, J = 1.0 Hz, 1H),
5.71 (d, J = 8.1 Hz, 1H), 5.44 – 5.33 (m, 4H), 5.05 (dd, J = 6.5, 1.1 Hz, 1H), 4.64 (d, J = 2.3 Hz, 2H), 2.65 (hept,
J = 7.0 Hz, 1H), 1.20 (d, J = 7.0 Hz, 3H), 1.17 (d, J = 6.9 Hz, 3H). 13C{1H} NMR (101 MHz, CDCl3) δ (ppm)
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173.2, 162.2, 150.7, 143.5, 141.1, 140.3, 139.9, 137.8, 128.9, 128.7, 128.5, 128.3, 128.1, 127.7, 127.5, 126.6,
-1
푣
126.3, 115.8, 114.4, 102.4, 97.8, 83.3, 79.6, 72.4, 70.3, 33.6, 19.0, 18.7. IR (ATR): (cm ) = 2975, 2936, 1748,
1721, 1671, 1451, 1356, 1267, 1204, 1139, 1073, 1023. HRMS (ESI) m/z: [M + Na]+ calcd for C34H32N2O8Na+
[α]20
619.2051, found: 619.2054;
= 85.0 ° (c 2.17, CHCl3).
D
(2''R,3a'S,4'S,6'R,6a'R)-6'-(3-((Benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)dihydro-6'H-
dispiro[cyclohexane-1,2'-furo[3,4-d][1,3]dioxole-4',1''-cyclopropan]-2''-yl isobutyrate (4a)
Enol ester 3a was azeotropically distilled with toluene (3 x 1.00 mL) and dried for 2 h under high vacuum. To a
solution of 3a (0.37 g, 0.72 mmol, 1.00 eq.) in 1,2-dichloroethane (7.00 mL) was added molecular sieve (4 Å)
and diiodomethane (0.56 mL, 7.03 mmol, 10.0 eq.). The suspension was stirred at room temperature for 0.5 h
and then a solution of diethylzinc (1M in hexane, 3.51 mL, 3.51 mmol, 5.00 eq.) was added. The reaction
mixture was heated to 50°C and stirred for 16 h. Dichloromethane (50.0 mL) was added and the molecular sieve
was filtered off. The reaction was quenched by a saturated, aqueous solution of Na2CO3 (50.0 mL) and the
aqueous phase was extracted with dichloromethane (3 x 25.0 mL). The combined organic phases were dried over
Na2SO4 and evaporated to dryness. Purification by column chromatography (SiO2, gradient, pentane/ethyl
acetate, 10:1, v/v; 1:1, v/v) afforded the spirocyclopropanated uridine derivative 4a as an oily diastereomeric
mixture α:β (1:5) (0.18 g, 0.34 mmol, 49% yield). Major Diastereomer (β) 1H NMR (400 MHz, CDCl3) δ (ppm)
7.43 – 7.19 (m, 5H), 7.13 (d, J = 8.1 Hz, 1H), 5.73 (d, J = 8.1 Hz, 1H), 5.53 (d, J = 1.3 Hz, 1H), 5.51 – 5.41 (m,
2H), 5.17 (dd, J = 6.4, 1.3 Hz, 1H), 4.81 (d, J = 6.4 Hz, 1H), 4.69 (s, 2H), 4.29 (dd, J = 7.8, 4.6 Hz, 1H), 2.60
(hept, J = 7.0 Hz, 1H), 1.84 (q, J = 5.8 Hz, 2H), 1.72 – 1.51 (m, 9H), 1.36 (dd, J = 8.4, 7.7 Hz, 1H), 1.18 (d, J =
3.0 Hz, 3H), 1.16 (dd, J = 6.1, 3.4 Hz, 3H). 13C{1H} NMR (101 MHz, CDCl3) δ (ppm) 177.3, 162.5, 150.7,
141.5, 137.8, 128.3, 127.7, 127.6, 115.3, 102.1, 96.8, 84.8, 82.8, 72.3, 71.5, 70.3, 51.3, 35.8, 35.3, 33.7, 25.0,
24.0, 23.7, 19.1, 18.9, 18.0. IR (ATR): (cm ) = 2935, 2861, 1744, 1720, 1667, 1450, 1353, 1259, 1086. HRMS
(ESI) m/z: [M + Na] calcd for C28H34N2O8Na 549.2207, found: 549.2209;
(1S,3a'S,6'R,6a'R)-6'-(3-((Benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2',2'-
-1
푣
[α]20
+
+
= 68.3 ° (c 1.49, CHCl3).
D
diphenyldihydro-6'H-spiro[cyclopropane-1,4'-furo[3,4-d][1,3]dioxol]-2-yl isobutyrate (4b)
Enol ester 3b was azeotropically distilled with toluene (3 x 1.00 mL) and dried for 2 h under high vacuum. To a
solution of 3b (0.25 g, 0.41 mmol, 1.00 eq.) in 1,2-dichloroethane (4.00 mL) was added molecular sieve (4 Å)
and diiodomethane (0.33 mL, 4.10 mmol, 10.0 eq.). The suspension was stirred at room temperature for 0.5 h
and then a solution of diethylzinc (1M in hexane, 2.05 mL, 2.05 mmol, 5.00 eq.) was added. The reaction
mixture was heated to 50°C and stirred for 16 h. Dichloromethane (50.0 mL) was added and the molecular sieve
was filtered off. The reaction was quenched by a saturated, aqueous solution of Na2CO3 (50.0 mL) and the
aqueous phase was extracted with dichloromethane (3 x 25.0 mL). The combined organic phases were dried over
Na2SO4 and evaporated to dryness. Purification by column chromatography (SiO2, pentane/ethyl acetate, 10:1 to
1:1) afforded the spirocyclopropanated uridine derivative 4b as an oily diastereomeric mixture (0.12 g,
1
0.20 mmol, 49% yield). H NMR (300 MHz, CDCl3) δ (ppm) 7.59 – 7.23 (m, 15H), 7.11 (d, J = 7.9 Hz, 1H),
5.72 (d, J = 8.1 Hz, 1H), 5.68 (d, J = 1.1 Hz, 1H), 5.42 (d, J = 3.4 Hz, 2H), 5.23 (dd, J = 6.4, 1.1 Hz, 1H), 4.72 –
4.63 (m, 3H), 4.42 (dd, J = 7.8, 4.8 Hz, 1H), 2.62 (hept, J = 7.0 Hz, 1H), 1.38 (dd, J = 8.4, 7.7 Hz, 1H), 1.21 (d, J
= 7.0 Hz, 3H), 1.18 (d, J = 7.0 Hz, 3H), 1.09 (dd, J = 8.4, 4.8 Hz, 1H). 13C{1H} NMR (126 MHz, CDCl3) δ
(ppm) 177.2, 162.5, 150.8, 142.0, 140.4, 140.0, 137.8, 128.9, 128.6, 128.4, 128.3, 128.0, 127.7, 127.6, 127.0,
-1
푣
126.5, 114.1, 102.2, 96.9, 85.5, 84.6, 72.3, 71.8, 70.2, 51.5, 33.7, 19.2, 19.1, 19.0. IR (ATR): (cm ) = 2973,
2933, 1723, 1674, 1451, 1355, 1257, 1207, 1152, 1081, 1022. HRMS (ESI) m/z: calcd for C35H34N2O8Na+
[α]20
633.2207, found: 633.2208;
= 57.5 ° (c 0.49, CHCl3).
D
3-((Benzyloxy)methyl)-1-((3a'S,4'S,6'R,6a'R)-4'-(hydroxymethyl)-4'-methyltetrahydrospiro[cyclohexane-
1,2'-furo[3,4-d][1,3]dioxol]-6'-yl)pyrimidine-2,4(1H,3H)-dione (5)
Nucleoside 4a (0.28 g, 0.53 mmol, 1.00 eq.) was dissolved in methanol (5.00 mL) and the reaction mixture was
cooled to 0 °C. NaOMe (30.0 µL, 0.16 mmol, 0.30 eq., 5.4 mol/L in methanol) was added and the reaction was
stirred at 0 °C for 3 h. After neutralization by the addition of Amberlite IR 120, the resin was filtered off and the
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crude spirocyclopropanol was evaporated to dryness. The product was dissolved in dichloromethane (5.00 mL)
and evaporated to dryness at 45 °C (rotary evaporator). The procedure was repeated three times until thermal
isomerization of the cyclopropanol to the desired aldehyde was achieved. The crude aldehyde was dissolved in
methanol (1.00 mL) and NaBH4 (24.2 mg, 0.64 mmol, 1.20 eq.) was added at 0 °C. After 10 min SiO2 was added
to the reaction mixture and the solvent was evaporated to dryness. Purification by column chromatography of the
powdery residue (SiO2, gradient, pentane/ethyl acetate, 3:1, v/v; 1:1, v/v; 0:1, v/v) yielded the L-lyxo nucleoside
5 (0.11 g, 0.23 mmol, 44%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ (ppm) 7.40 – 7.24 (m, 5H), 7.22 (d,
J = 8.2 Hz, 1H), 5.73 (d, J = 8.1 Hz, 1H), 5.58 (d, J = 2.4 Hz, 1H), 5.50 (d, J = 9.8 Hz, 1H), 5.46 (d, J = 9.8 Hz,
1H), 5.09 (dd, J = 6.6, 2.4 Hz, 1H), 4.74 (d, J = 6.6 Hz, 1H), 4.71 (s, 2H), 3.79 (dd, J = 11.7, 6.4 Hz, 1H), 3.62
(dd, J = 11.5, 7.8 Hz, 1H), 2.40 (dd, J = 7.8, 6.5 Hz, 1H), 1.82 (dd, J = 8.1, 3.9 Hz, 2H), 1.56 (s, 6H), 1.45 – 1.41
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(m, 5H). 13C{1H} NMR (101 MHz, CDCl3) δ (ppm) 162.6, 150.7, 140.4, 138.0, 128.3, 127.7, 127.6, 115.4,
-1
푣
101.9, 95.2, 88.0, 85.5, 85.3, 72.4, 70.4, 66.9, 35.8, 33.8, 24.9, 24.0, 23.5, 22.8. IR (ATR): (cm ) = 3460,
2934, 2860, 1717, 1669, 1453, 1363, 1157, 1099. HRMS (ESI) m/z: [M + Na]+ calcd for C24H30N2O7Na+
481.1945, found: 481.1951; [α]2D0 = 13.2 ° (c 0.39, CHCl3).
3-((Benzyloxy)methyl)-1-((3a'S,4'R,6'R,6a'R)-4'-(hydroxymethyl)-4'-methyltetrahydrospiro[cyclohexane-
1,2'-furo[3,4-d][1,3]dioxol]-6'-yl)pyrimidine-2,4(1H,3H)-dione (5a)
1
Colorless oil (15.7 mg, 34.2 µmol, 10% yield), H NMR (500 MHz, CDCl3) δ (ppm) 7.42 – 7.29 (m, 6H), 5.74
(d, J = 8.1 Hz, 1H), 5.51 – 5.44 (m, 3H), 5.11 (dd, J = 6.5, 4.1 Hz, 1H), 4.89 (d, J = 6.5 Hz, 1H), 4.70 (s, 2H),
3.69 (dd, J = 11.6, 2.8 Hz, 1H), 3.61 (dd, J = 11.6, 8.3 Hz, 1H), 2.96 (dd, J = 8.3, 2.8 Hz, 1H), 1.81 (dd, J = 7.0,
5.3 Hz, 2H), 1.69 – 1.55 (m, 6H), 1.42 (d, J = 5.7 Hz, 2H), 1.29 (s, 3H). 13C{1H} NMR (126 MHz, CDCl3) δ
(ppm) 162.4, 151.2, 141.6, 137.8, 128.3, 127.7, 127.6, 115.0, 102.1, 96.1, 88.3, 83.1, 81.1, 72.4, 70.4, 68.3, 36.6,
-1
푣
34.5, 25.0, 24.0, 23.5, 18.6. IR (ATR): (cm ) = 3474, 2933, 2859, 1714, 1659, 1453, 1362, 1280, 1251, 1096,
1072. HRMS (ESI) m/z: [M + Na]+ calcd for C24H30N2O7Na+: 481.1945, found: 481.1950; [α]2D0 = 1.0 ° (c 0.27,
CHCl3).
1-((3a'S,4'S,6'R,6a'R)-4'-(Hydroxymethyl)-4'-methyltetrahydrospiro[cyclohexane-1,2'-furo[3,4-
d][1,3]dioxol]-6'-yl)pyrimidine-2,4(1H,3H)-dione (6)
Compound 5 (43.8 mg, 96.0 µmol, 1.00 eq.) was dissolved in methanol (2.00 mL) under an argon atmosphere
and palladium on charcoal (10 wt.% on activated carbon, 20.3 mg, 19.2 µmol, 0.20 eq.) was added. The
atmosphere was replaced by hydrogen (balloon, 1 atm) and the reaction was stirred at room temperature for 1 h.
After consumption of all starting material, the reaction mixture was filtered through Celite® 545 and the plug
was rinsed with methanol (3x, 2.00 mL). After evaporation of the solvent, column purification (SiO2, isocratic,
dichloromethane/methanol, 20:1, v/v) yielded nucleoside derivative 6 (34.6 mg, 96.0 µmol, quant) as a sticky
foam. 1H NMR (300 MHz, CD3CN) δ (ppm) 9.02 (s, 1H), 7.43 (d, J = 8.1 Hz, 1H), 5.74 (d, J = 3.1 Hz, 1H), 5.61
(d, J = 8.1 Hz, 1H), 5.00 (dd, J = 6.5, 3.1 Hz, 1H), 4.63 (dd, J = 6.5, 0.5 Hz, 1H), 3.65 (dd, J = 11.6, 6.8 Hz, 1H),
3.58 (dd, J = 11.6, 6.4 Hz, 1H), 2.79 (t, J = 6.6 Hz, 1H), 1.70 – 1.48 (m, 2H), 1.68 – 1.36 (m, 6H), 1.48 – 1.36
(m, 2H), 1.34 (s, 3H). 13C{1H} NMR (126 MHz, CD3CN) δ (ppm) 164.0, 151.4, 142.2, 115.8, 102.6, 92.9, 88.2,
-1
푣
85.8, 85.7, 65.9, 36.8, 34.8, 25.7, 24.9, 24.4, 22.9. IR (ATR): (cm ) = 3477, 3203, 3061, 2939, 1687, 1456,
1378, 1266, 1103, 1054. HRMS (ESI) m/z: [M + Na]+ calcd for C16H22N2O6Na+ 361.1370, found: 361.1375;
[ ]20
α
= -2.1 ° (c 0.53, CHCl3).
D
1-((3a'S,4'R,6'R,6a'R)-4'-(Hydroxymethyl)-4'-methyltetrahydrospiro[cyclohexane-1,2'-furo[3,4-
d][1,3]dioxol]-6'-yl)pyrimidine-2,4(1H,3H)-dione (6a)
Colorless syrup (4.13 mg, 12.2 µmol, 55% yield), 1H NMR (300 MHz, CDCl3) δ (ppm) 7.97 (s, 1H), 7.40 (d, J =
8.1 Hz, 1H), 5.73 (dd, J = 8.1, 2.3 Hz, 1H), 5.51 (d, J = 4.4 Hz, 1H), 5.13 (dd, J = 6.4, 4.4 Hz, 1H), 4.89 (d, J =
6.4 Hz, 1H), 3.70 (dd, J = 11.6, 2.9 Hz, 1H), 3.61 (dd, J = 11.5, 8.3 Hz, 1H), 2.97 (dd, J = 8.2, 3.0 Hz, 1H), 1.82
(m, 2H), 1.63 (m, 6H), 1.42 (m, 2H), 1.29 (s, 3H). 13C{1H} NMR (126 MHz, CDCl3) δ (ppm) 162.2, 150.1,
-1
푣
143.1, 115.2, 102.6, 95.5, 88.1, 82.8, 81.2, 68.4, 36.7, 34.5, 24.9, 24.0, 23.5, 18.5. IR (ATR): (cm ) = 3393,
3203, 2927, 2856, 1689, 1460, 1378, 1263, 1165, 1106, 1071. HRMS (ESI) m/z: [M + Na]+ calcd for
[α]20
+
C16H22N2O6Na 361.1370, found: 361.1371;
= 1.0 ° (c 0.27, CHCl3).
D
1-((2R,3R,4S,5S)-3,4-Dihydroxy-5-(hydroxymethyl)-5-methyltetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-
dione (7)
Nucleoside derivative 6 (16.0 mg, 47.0 µmol, 1.00 eq.) was dissolved in a mixture of water and methanol
(1.50 mL, 2:1) and DOWEX 50W8 50-100 mesh (hydrogen form) (34.0 mg) was added. The reaction mixture
was heated to 70 °C, stirred for 2 h and finally the resin was removed by inverse filtration through a syringe
needle covered with cotton. After evaporation of the solvent, RP-column chromatography of the residue (RP-
C18, water/methanol, 1:0, v/v; 1:1, v/v) yielded the L-lyxo nucleoside 7 (9.30 mg, 36.0 µmol, 77%) as a
hygroscopic syrup. 1H NMR (300 MHz, D2O) δ (ppm) 7.68 (d, J = 8.0 Hz, 1H), 5.97 (d, J = 5.9 Hz, 1H), 5.91 (d,
J = 8.0 Hz, 1H), 4.52 (t, J = 5.8 Hz, 1H), 4.23 (d, J = 5.7 Hz, 1H), 3.80 (d, J = 12.0 Hz, 1H), 3.68 (d, J = 12.1
Hz, 1H), 1.41 (s, 3H). 13C{1H} NMR (126 MHz, D2O) δ (ppm) 170.8, 156.0, 144.6, 105.5, 92.4, 89.2, 77.7, 76.3,
-1
푣
67.0, 24.6. IR (ATR): (cm ) = 3359, 3314, 2924, 2854, 1670, 1540, 1465, 1416, 1387, 1259, 1113, 1043.
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[α]2D0
HRMS (ESI) m/z: [M + Na]+ calcd for C10H14N2O6Na+ 281.0744, found: 281.0747;
CHCl3).
= -23.5 ° (c 0.38,
(2S,3S,4R,5R)-5-(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-((isobutyryloxy)methyl)-2-
6
methyltetrahydrofuran-3,4-diyl bis(2-methylpropanoate) (8)
7
8
9
Nucleoside derivative 7 (2.20 mg, 8.52 µmol, 1.00 eq.) was dissolved in pyridine (0.20 mL), isobutyric
anhydride (14.2 µL, 85.2 µmol, 10.0 eq.) was added and the reaction mixture was stirred for 1 d at room
temperature. Finally, the solvent was removed under reduced pressure, after which purification by column
chromatography (SiO2, isocratic, pentane/ethyl acetate, 1:1, v/v) afforded product 8 (4.00 mg, 8.52 µmol, quant)
as a colorless oil. 1H NMR (300 MHz, CD3CN) δ (ppm) 9.00 (s, 1H), 7.40 (d, J = 8.1 Hz, 1H), 5.91 (dd, J = 5.6,
0.4 Hz, 1H), 5.65 (dd, J = 8.1, 0.4 Hz, 1H), 5.53 (dd, J = 6.2, 5.6 Hz, 1H), 5.40 (dd, J = 6.2, 0.4 Hz, 1H), 4.32 (d,
J = 11.5 Hz, 1H), 4.10 (d, J = 11.5 Hz, 1H), 2.70 – 2.46 (m, 3H), 1.46 (s, 3H), 1.18 – 1.07 (m, 18H). 13C{1H}
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13
14
15
16
17
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21
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33
34
35
36
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
NMR (126 MHz, CD3CN) δ (ppm) 177.1, 176.3, 176.0, 163.6, 151.1, 142.2, 103.3, 89.6, 84.1, 74.9, 73.5, 65.7,
-1
푣
34.7, 34.6, 34.4, 22.9, 19.2, 19.2, 19.1, 19.0, 19.0, 18.9. IR (ATR): (cm ) = 3183, 2975, 1740, 1695, 1490,
1457, 1252, 1196, 1149, 1106. HRMS (ESI) m/z: [M + Na]+ calcd for C22H32N2O9Na+ 491.2000, found:
491.1999; [α 20 = 63.4 ° (c 0.35, CHCl3). HPLC (C18, isocratic, CH3CN:H2O, 70:30, tR = 5.81 min).
]
D
Dibenzyl
(((3a'R,4'R,6'S,6a'S)-4'-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-6'-
methyltetrahydrospiro[cyclohexane-1,2'-furo[3,4-d][1,3]dioxol]-6'-yl)methyl) phosphate (9)
Nucleoside derivative 5 (71.2 mg, 155 µmol, 1.00 eq.) was twice azeotropically distilled with acetonitrile
(0.5 mL) and then dissolved in acetonitrile (1.60 mL). After the addition of dibenzyl N,N-
diisopropylphosphoramidite (57.0 µL, 0.17 mmol, 1.10 eq.) and 5-methyl-1H-tetrazole (39.1 mg, 0.47 mmol,
3.00 eq.) the reaction mixture was stirred for 3 h and finally tBuOOH (71.0 µL, 0.39 mmol, 2.50 eq.) added. The
reaction mixture was further stirred for 0.75 h and the solvent removed under reduced pressure. Column
chromatography (SiO2, gradient, pentane/ethyl acetate, 3:1, v/v; 0:1, v/v) yielded the dibenzyl phosphate 9
(96.8 mg, 135 µmol, 87% yield) as a colorless oil. 1H NMR (500 MHz, CD3CN) δ (ppm) 7.39 – 7.31 (m, 16H),
5.70 (d, J = 2.9 Hz, 1H), 5.66 (d, J = 8.2 Hz, 1H), 5.37 (s, 2H), 5.08 – 5.03 (m, 4H), 4.98 (dd, J = 6.4, 2.9 Hz,
1H), 4.63 (d, J = 6.4 Hz, 1H), 4.63 (d, J = 0.5 Hz, 2H), 4.12 (dd, J = 10.7, 5.6 Hz, 1H), 4.04 (dd, J = 10.7, 6.5
Hz, 1H), 1.70 (dd, J = 8.4, 5.3 Hz, 2H), 1.60 – 1.45 (m, 6H), 1.38 (q, J = 6.0 Hz, 2H), 1.33 (s, 3H). 13C{1H}
NMR (126 MHz, CD3CN) δ (ppm) 163.6, 152.1, 141.1, 139.5, 137.4, 137.3, 129.6, 129.6, 129.5, 129.5, 129.3,
129.0, 128.9, 128.5, 128.5, 115.9, 102.2, 94.1, 87.1 (d, J = 8.2 Hz), 85.8, 85.4, 72.6, 71.3, 70.4 (d, J = 5.5 Hz),
70.1 (d, J = 5.5 Hz), 70.0 (d, J = 5.3 Hz), 36.8, 34.8, 25.6, 24.9, 24.4, 22.5. 31P NMR (122 MHz, CD3CN) δ
-1
푣
(ppm) 0.43 (tt, J = 8.3, 6.1 Hz). IR (ATR): (cm ) = 2938, 2860, 1719, 1673, 1453, 1362, 1273, 1097, 1015.
[α]2D0
HRMS (ESI) m/z: [M + Na]+ calcd for C38H43N2O10PNa+ 741.2548, found: 741.2550;
= 5.0 ° (c 0.41,
CHCl3).
((2S,3S,4R,5R)-5-(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3,4-dihydroxy-2-methyltetrahydrofuran-2-
yl)methyl dihydrogen phosphate (10)
Nucleoside derivative 9 (54.1 mg, 75.3 µmol, 1.00 eq.) was dissolved in methanol (2.00 mL) and palladium on
charcoal (10 wt.% on activated carbon, 16.0 mg, 15.1 µmol, 0.20 eq.) was added. The atmosphere was replaced
by hydrogen (balloon, 1 atm) and the reaction was stirred at room temperature for 4 h. After consumption of all
starting material, the reaction mixture was filtered through Celite® 545 and the plug was rinsed with methanol (3
x 2.00 mL). After evaporation of the solvent, the residue was subjected to ketal deprotection without further
purification.
The residue from the previous step was dissolved in water (2.00 mL) and DOWEX 50W8 50-100 mesh
(hydrogen form) (63.0 mg) was added. The reaction mixture was heated to 70 °C, stirred for 4 h and finally the
resin was removed by inverse filtration through a syringe needle covered with cotton. After evaporation of the
solvent, RP-column chromatography of the residue (RP-C18, H2O) yielded the L-lyxo nucleoside
1
monophosphate 10 (13.6 mg, 40.2 µmol, 53%) over two steps as a colorless hygroscopic solid. H NMR (500
MHz, D2O) δ (ppm) 7.69 (d, J = 8.1 Hz, 1H), 5.98 (d, J = 6.5 Hz, 1H), 5.91 (d, J = 8.1 Hz, 1H), 4.58 (dd, J = 6.6,
5.5 Hz, 1H), 4.23 (d, J = 5.5 Hz, 1H), 4.02 (d, J = 5.4 Hz, 2H), 1.45 (s, 3H). 13C{1H} NMR (126 MHz, D2O) δ
(ppm) 169.1, 154.7, 145.1, 105.7, 92.2, 88.7 (d, J = 8.9 Hz), 77.5, 76.0, 70.5 (d, J = 5.1 Hz), 24.7. 31P NMR (203
-1
푣
MHz, D2O) δ (ppm) 0.8. IR (ATR): (cm ) = 3173, 2987, 1685, 1464, 1387, 1250, 1135, 1044. HRMS (ESI)
[α]20
m/z: [M – H + 2Na]+ calcd for C10H14N2O9PNa2 383.0227, found: 383.0226;
= -17.6 ° (c 0.55, CH3OH).
+
D
(1S,3a'S,6'R,6a'R)-6'-(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2',2'-diphenyldihydro-6'H-
spiro[cyclopropane-1,4'-furo[3,4-d][1,3]dioxol]-2-yl isobutyrate (11)
Nucleoside derivative 4b (88.0 mg, 144 µmol, 1.00 eq.) was dissolved in methanol (4.00 mL) and palladium on
charcoal (10 wt.% on activated carbon, 29.7 mg, 27.9 µmol, 0.20 eq.) was added. The atmosphere was replaced
by hydrogen (balloon, 1 atm) and the reaction was stirred at room temperature for 3 h. After consumption of all
starting material, the reaction mixture was filtered through Celite® 545 and the plug was rinsed with methanol (3
x 2.00 mL). After evaporation of the solvent, column purification (SiO2, gradient, pentane/ethyl acetate, 3:1, v/v;
1
1:1, v/v) yielded nucleoside derivative 11 (41.3 mg, 84.0 µmol, 60%). mp 183-185 °C; H NMR (500 MHz,
CDCl3) δ (ppm) 8.34 (s, 1H), 7.51 (d, J = 1.8 Hz, 1H), 7.49 – 7.28 (m, 9H), 7.18 (d, J = 8.1 Hz, 1H), 5.81 – 5.66
(m, 2H), 5.27 (dd, J = 6.4, 1.2 Hz, 1H), 4.64 (d, J = 6.4 Hz, 1H), 4.43 (dd, J = 7.8, 4.7 Hz, 1H), 2.62 (hept, J =
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7.0 Hz, 1H), 1.37 (dd, J = 8.4, 7.8 Hz, 1H), 1.21 (d, J = 7.0 Hz, 3H), 1.19 (d, J = 6.9 Hz, 3H), 1.11 (dd, J = 8.5,
4.7 Hz, 1H). 13C{1H} NMR (126 MHz, CDCl3) δ (ppm) 177.2, 162.6, 149.8, 143.5, 140.4, 140.0, 128.9, 128.6,
-1
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128.4, 128.0, 127.0, 126.5, 114.3, 102.7, 96.1, 85.4, 84.5, 71.6, 51.4, 33.7, 19.1, 19.1, 19.0. IR (ATR): (cm ) =
3219, 3063, 2971, 1691, 1454, 1378, 1257, 1210, 1156, 1078. HRMS (ESI) m/z: [M + Na]+ calcd for
[α]20
+
7
C27H26N2O7Na 513.1632, found: 513.1630;
= 63.4 ° (c 0.35, CHCl3).
D
8
Single crystals of 11 were obtained.
9
(3R,5R,6R,7S)-5-(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)-6,7-dihydroxy-4-oxaspiro[2.4]heptan-1-yl
isobutyrate (12)
10
11
12
13
14
15
16
17
18
19
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Compound 11 (22.4 mg, 45.7 µmol, 1.00 eq.) was dissolved in Et3SiH (87.6 µL, 0.55 mmol, 12.0 eq.) and
molecular sieve (4 Å) was added. The mixture was stirred for 20 min at room temperature and then
trifluoroacetic acid (45.0 µL, 0.59 mmol, 13.0 eq.) was added. The reaction mixture was stirred for 2 d and
finally diluted with dichloromethane (5.00 ml) and a saturated, aqueous solution of NaHCO3 (2.00 mL). The
aqueous layer was extracted with dichloromethane (3 x 5.00 mL) and the organic phases were combined, dried
over Na2SO4 and evaporated to dryness. Column purification (SiO2, isocratic, dichloromethane/methanol, 20:1)
afforded diol 12 (4.20 mg, 12.9 µmol, 28% yield) as a colorless and hygroscopic foam. Additional remark: The
reaction has to be performed neat because dilution by a solvent (dichloromethane) lead to insufficient product
formation. Otherwise, the product is formed gradually as the solvent slowly evaporates from the reaction
1
mixture. H NMR (300 MHz, CDCl3) δ (ppm) 8.26 (s, 1H), 7.24 (d, J = 8.2 Hz, 1H), 5.78 (dd, J = 8.2, 2.2 Hz,
1H), 5.68 (d, J = 2.2 Hz, 1H), 4.55 – 4.44 (m, 1H), 4.34 – 4.25 (m, 2H), 3.90 (dd, J = 7.8, 4.7 Hz, 1H), 3.33 (d, J
= 7.6 Hz, 1H), 2.64 (hept, J = 6.9 Hz, 1H), 1.44 (dd, J = 8.8, 4.6 Hz, 1H), 1.38 (dd, J = 8.8, 7.8 Hz, 1H), 1.20 (d,
J = 7.0 Hz, 4H), 1.19 (d, J = 7.0 Hz, 3H). 13C{1H} NMR (126 MHz, CDCl3) δ (ppm) 180.8, 162.3, 149.6, 139.5,
-1
푣
102.9, 91.3, 75.2, 70.2, 66.7, 50.1, 33.7, 19.0, 18.7, 12.4. IR (ATR): (cm ) = 3364, 3194, 2923, 2855, 1693,
1461, 1383, 1258, 1198, 1112, 1075, 1031. HRMS (ESI) m/z: [M + Na]+ calcd for C14H18N2O7Na+ 349.1006,
[α]20
found: 349.1010;
= 89.8 ° (c 0.15, CHCl3).
D
(3S,5R,6R,7S)-5-(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-oxaspiro[2.4]heptane-1,6,7-triyl
tris(2-
methylpropanoate) (13)
Compound 12 (2.70 mg, 8.3 µmol, 1.00 eq.) was dissolved in pyridine (0.10 ml) and isobutyric anhydride
(13.7 µL, 83.0 µmol, 10.0 eq.) was added. The reaction mixture was stirred for 16 h at room temperature and
finally the solvent was removed under reduced pressure. Purification by column chromatography (SiO2, gradient,
pentane/ethyl acetate, 2:1, v/v; 1:1, v/v) afforded the triply esterified nucleoside 13 (2.70 mg, 5.78 µmol, 70%)
as a colorless oil. 1H NMR (300 MHz, CD3CN) δ (ppm) 8.96 (s, 1H), 7.45 (d, J = 8.2 Hz, 1H), 5.88 (dq, J = 6.2,
0.3 Hz, 1H), 5.68 (dd, J = 8.1, 0.4 Hz, 1H), 5.61 – 5.51 (m, 1H), 5.34 (d, J = 6.1 Hz, 1H), 4.05 (dd, J = 8.0, 4.7
Hz, 1H), 2.72 – 2.47 (m, 3H), 1.41 (dd, J = 8.6, 7.9 Hz, 1H), 1.28 (dd, J = 8.6, 4.7 Hz, 1H), 1.19 (d, J = 4.0 Hz,
3H), 1.17 (d, J = 4.0 Hz, 3H), 1.13 – 1.10 (m, 6H), 1.10 – 1.08 (m, 6H). 13C{1H} NMR (126 MHz, CD3CN) δ
(ppm) 178.2, 176.8, 176.5, 163.5, 151.2, 141.6, 103.8, 88.6, 73.7, 72.5, 67.1, 51.4, 34.7, 34.5, 34.4, 19.4, 19.2,
-1
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19.2, 19.1, 19.0, 19.0, 16.0. IR (ATR): (cm ) = 3198, 3065, 2924, 2857, 1742, 1696, 1460, 1382, 1254, 1194,
[α]20
+
+
1152. HRMS (ESI) m/z: [M + Na] calcd for C22H30N2O9Na 489.1844, found: 489.1838;
CHCl3). HPLC (C18, isocratic, CH3CN:H2O, 95:5, tR = 3.44 min).
= -73.5 ° (c 0.17,
D
(3R,5R,6R,7S)-6,7-Bis((benzyloxy)methoxy)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-
1(2H)-yl)-4-oxaspiro[2.4]heptan-1-yl isobutyrate (14)
Compound 14 was prepared according to our reported procedure.6
3-((Benzyloxy)methyl)-1-((3R,5R,6R,7S)-6,7-bis((benzyloxy)methoxy)-1-hydroxy-4-oxaspiro[2.4]heptan-5-
yl)pyrimidine-2,4(1H,3H)-dione (15)
Ester 14 (23.8 mg, 34.7 µmol, 1.00 eq.) was dissolved in a mixture of tetrahydrofuran and methanol (0.63 mL,
20:1). LiBH4 in tetrahydrofuran (26.0 µL, 104 µmol, 3.00 eq., 4 mol/L) was added and the reaction mixture was
stirred at room temperature for 0.5 h until all starting material was consumed. The reaction was terminated by
the addition of SiO2 and the solvent was removed under reduced pressure. Column purification of the powdery
material (SiO2, isocratic, pentane/ethyl acetate, 1:1, v/v) afforded the spirocyclopropanol 15 (10.2 mg,
16.5 µmol, 47% yield) as a colorless oil. 1H NMR (300 MHz, CDCl3) δ (ppm) 7.43 – 7.14 (m, 15H), 7.09 (d, J =
8.2 Hz, 1H), 5.68 (d, J = 8.1 Hz, 1H), 5.56 (d, J = 7.3 Hz, 1H), 5.45 – 5.30 (m, 3H), 4.96 (d, J = 7.2 Hz, 1H),
4.88 (d, J = 7.1 Hz, 1H), 4.77 (s, 2H), 4.71 (d, J = 11.9 Hz, 1H), 4.62 (d, J = 7.2 Hz, 3H), 4.52 (d, J = 2.7 Hz,
2H), 3.92 – 3.76 (m, 2H), 3.35 (ddd, J = 7.4, 4.4, 0.9 Hz, 1H), 1.23 – 1.17 (m, 1H), 1.06 (dd, J = 8.5, 4.4 Hz,
1H). 13C{1H} NMR (126 MHz, CDCl3) δ (ppm) 162.0, 151.7, 143.0, 137.5, 137.3, 136.7, 128.6, 128.5, 128.4,
128.0, 127.9, 127.7, 127.7, 127.3, 102.6, 96.5, 94.7, 94.3, 78.8, 77.5, 72.2, 70.2, 70.0, 69.9, 68.8, 50.9, 14.8. IR
-1
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(ATR): (cm ) = 3450, 2955, 2897, 1715, 1666, 1455, 1355, 1205, 1173, 1067, 1035. HRMS (ESI) m/z: [M +
[α]20
+
+
Na] calcd for C34H36N2O9Na 639.2313, found: 639.2317;
= 8.0 ° (c 0.2, CHCl3).
D
3-((Benzyloxy)methyl)-1-((2R,3R,4S,5R)-3,4-bis((benzyloxy)methoxy)-5-(hydroxymethyl)-5-
methyltetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (16)
Spirocyclopropanol 15 (7.1 mg, 11.5 µmol, 1.00 eq.) was dissolved in a mixture of ethanol and water (0.22 mL,
10:1), and potassium carbonate (1.5 mg, 10.9 µmol, 0.95 eq.) was added at 0 °C. After 5 h at 0 °C, the reaction
mixture was warmed to room temperature and stirred for a further 11 h. The progress of isomerisation was
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followed by TLC and LC-MS, as the ethoxy hemiacetal can easily be monitored by mass analysis. The reaction
mixture was diluted with ethanol (0.10 mL) and cooled to 0 °C. NaBH4 (0.5 mg, 13.2 µmol, 1.10 eq.) was added
and the reaction mixture was stirred for 2 h. Finally, SiO2 was added and the solvent was evaporated to dryness.
Column purification of the powdery material (SiO2, isocratic, pentane/ethyl acetate, 1:1, v/v) afforded the
1
primary alcohol 16 (1.80 mg, 2.90 µmol, 25% yield over two steps) as a colorless syrup. H NMR (500 MHz,
7
CDCl3) δ (ppm) 7.40 (d, J = 8.2 Hz, 1H), 7.39 – 7.21 (m, 15H), 5.72 (d, J = 3.8 Hz, 1H), 5.62 (d, J = 8.1 Hz,
1H), 5.41 (d, J = 9.7 Hz, 1H), 5.37 (d, J = 9.7 Hz, 1H), 4.93 (d, J = 6.9 Hz, 1H), 4.87 (d, J = 6.9 Hz, 1H), 4.85
(d, J = 6.9 Hz, 1H), 4.80 (d, J = 6.8 Hz, 1H), 4.70 (d, J = 11.9 Hz, 1H), 4.66 (s, 2H), 4.63 (d, J = 11.9 Hz, 1H),
4.62 (d, J = 11.8 Hz, 1H), 4.59 – 4.54 (m, 2H), 4.51 (d, J = 6.1 Hz, 1H), 3.70 (dd, J = 11.9, 3.8 Hz, 1H), 3.57
(dd, J = 12.0, 7.5 Hz, 1H), 2.49 (dd, J = 7.5, 3.9 Hz, 1H), 1.31 (s, 3H). 13C{1H} NMR (126 MHz, CDCl3) δ
8
9
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(ppm) 162.4, 151.0, 140.7, 137.8, 137.3, 137.1, 128.5, 128.5, 128.5, 128.3, 127.9, 127.9, 127.7, 127.6, 127.6,
-1
푣
127.5, 101.9, 95.1, 95.0, 92.5, 86.7, 79.3, 74.5, 72.2, 70.2, 70.2, 70.0, 66.7, 18.6. IR (ATR): (cm ) = 3360,
3316, 2953, 2921, 2853, 1715, 1664, 1459, 1375, 1076, 1032. HRMS (ESI) m/z: [M + Na]+ calcd for
[α]20
+
C34H38N2O9Na 641.2470, found: 641.2471;
= 31.0 ° (c 0.57, CHCl3).
D
1-((2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)-5-methyltetrahydrofuran-2-yl)pyrimidine-
2,4(1H,3H)-dione (17)
Compound 16 (29.5 mg, 47.0 µmol, 1.00 eq.) was dissolved in methanol (2.00 mL) under an argon atmosphere,
and palladium on charcoal (10 wt.% on activated carbon, 10.2 mg, 9.50 µmol, 0.20 eq.) was added. The
atmosphere was replaced by hydrogen (balloon, 1 atm) and the reaction was stirred at room temperature for 1 h.
After consumption of all starting material, the reaction mixture was filtered through Celite® 545 and the plug
was rinsed with methanol (3 x 2.00 mL). After evaporation of the solvent, RP-column chromatography of the
residue (RP-C18, gradient, water/methanol, 0:1, v/v; 5:1, v/v) yielded the D-configured nucleoside derivative 17
(8.7 mg, 34.0 µmol, 72% yield). 213-215 °C (decomposition) 1H NMR (500 MHz, D2O) δ (ppm) 7.88 (d, J = 8.1
Hz, 1H), 5.99 (d, J = 6.3 Hz, 1H), 5.92 (d, J = 8.1 Hz, 1H), 4.52 (t, J = 6.1 Hz, 1H), 4.22 (d, J = 5.8 Hz, 1H),
3.63 (s, 2H), 1.29 (s, 3H). 13C{1H} NMR (126 MHz, D2O) δ (ppm) 169.1, 154.8, 144.8, 105.5, 90.8, 90.5, 76.5,
-1
푣
74.3, 69.0, 20.2. IR (ATR): (cm ) = 3400, 3102, 3061, 1681, 1467, 1391, 1275, 1186, 1113, 1062. HRMS
[α]20
+
+
(ESI) m/z: [M + Na] calcd for C10H14N2O6Na 281.0744, found: 281.0746;
HPLC (C18, isocratic, CH3CN:H2O, 70:30, tR = 2.08 min).
Single crystals of 17 were obtained.
= -22.9 ° (c 0.49, CH3OH).
D
(2R,3S,4R,5R)-5-(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-((isobutyryloxy)methyl)-2-
methyltetrahydrofuran-3,4-diyl bis(2-methylpropanoate) (18)
The unprotected nucleoside derivative 17 (4.20 mg, 16.3 µmol, 1.00 eq.) was dissolved in pyridine (0.20 ml) and
isobutyric anhydride (27.0 µL, 16.0 µmol, 10.0 eq.) was added. The reaction mixture was stirred for 14 h at
room temperature and finally the solvent was removed under reduced pressure. Purification by column
chromatography (SiO2, gradient, pentane/ethyl acetate, 2:1, v/v; 1:1, v/v) afforded the triply esterified nucleoside
18 (7.70 mg, 16.3 µmol, quant) as a colorless solid. mp 133-135 °C;1H NMR (300 MHz, CDCl3) δ (ppm) 8.30
(s, 1H), 7.47 (d, J = 8.2 Hz, 1H), 6.12 (dd, J = 4.0, 1.9 Hz, 1H), 5.77 (dd, J = 8.1, 2.2 Hz, 1H), 5.61 – 5.25 (m,
2H), 4.24 (d, J = 12.1 Hz, 1H), 4.10 (d, J = 12.1 Hz, 1H), 2.79 – 2.42 (m, 3H), 1.35 (s, 3H), 1.27 – 1.20 (m,
12H), 1.19 – 1.13 (m, 6H). 13C{1H} NMR (126 MHz, CDCl3) δ 176.2, 175.5, 175.3, 162.2, 149.9, 139.1, 103.2,
-1
푣
85.9, 84.0, 73.1, 70.9, 67.9, 34.1, 33.9, 33.7, 19.0, 19.0, 18.9, 18.9, 18.7, 18.7, 18.6. IR (ATR): (cm ) = 3208,
2978, 2939, 2880, 1744, 1695, 1462, 1385, 1251, 1191, 1150, 1110. HRMS (ESI) m/z: [M + Na]+ calcd for
[α]20
+
C22H32N2O9Na 491.2000, found: 491.1998;
= 27.7 ° (c 0.34, CHCl3). HPLC (C18, isocratic, CH3CN:H2O,
D
70:30, tR = 5.85 min).
Dibenzyl
(((2R,3S,4R,5R)-3,4-bis((benzyloxy)methoxy)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)-2-methyltetrahydrofuran-2-yl)methyl) phosphate (19)
Compound 18 (32.0 mg, 52.0 µmol, 1.00 eq.) was twice azeotropically distilled with acetonitrile (0.50 mL) and
then dissolved in acetonitrile (1.00 mL). After the addition of dibenzyl N,N-diisopropylphosphoramidite
(19.0 µL, 57.0 µmol, 1.10 eq.) and 5-methyl-1H-tetrazole (13.1 mg, 156 µmol, 3.00 eq.) the reaction mixture
was stirred for 1.5 h and finally the solvent was evaporated to dryness. The crude phosphite was separated from
remaining starting material by a short isocratic column (SiO2, isocratic, pentane/ethyl acetate, 1:1, v/v). After
evaporation of the solvent, the afforded dibenzyl phosphite was immediately submitted to oxidation by tBuOOH
(23.0 µL, 130 µmol, 2.50 eq.) in acetonitrile (1.00 mL) at room temperature. The reaction mixture was stirred for
0.75 h and the solvent removed under reduced pressure. Column chromatography (SiO2, gradient, pentane/ethyl
1
acetate, 1:1, v/v; 0:1, v/v) afforded phosphate 19 (27.0 mg, 31.0 µmol, 59% yield) as a colorless oil. H NMR
(300 MHz, CDCl3) δ (ppm) 7.43 (d, J = 8.2 Hz, 1H), 7.39 – 7.22 (m, 25H), 5.98 (d, J = 2.1 Hz, 1H), 5.45 (d, J =
8.2 Hz, 1H), 5.39 (d, J = 9.7 Hz, 1H), 5.32 (d, J = 9.8 Hz, 1H), 5.02 (dd, J = 4.2, 2.3 Hz, 2H), 4.99 (dd, J = 4.1,
2.2 Hz, 2H), 4.92 (d, J = 6.8 Hz, 1H), 4.83 – 4.76 (m, 3H), 4.68 – 4.54 (m, 6H), 4.29 – 4.23 (m, 2H), 4.05 – 3.93
(m, 2H), 1.33 (s, 3H). 13C{1H} NMR (126 MHz, CDCl3) δ (ppm) 162.4, 150.9, 138.5, 137.9, 137.2, 135.4 (d, J =
1.4 Hz), 135.4 (d, J = 1.4 Hz), 128.8, 128.8, 128.7, 128.7, 128.5, 128.4, 128.3, 128.1, 128.1, 127.9, 127.8, 127.7,
127.6, 127.5, 101.9, 94.7 (d, J = 12.4 Hz), 89.3, 84.6 (d, J = 8.2 Hz), 79.2, 74.2, 72.1, 70.2, 69.9, 69.7 (d, J = 4.4
-
Hz), 69.7 (d, J = 4.3 Hz), 18.4. 31P NMR (122 MHz, CDCl3) δ (ppm) -0.2 (tt, J = 8.9, 4.8 Hz). IR (ATR): (cm
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1) = 2952, 2895, 1718, 1672, 1455, 1278, 1074, 1017. HRMS (ESI) m/z: [M + Na]+ calcd for C48H51N2O12PNa+
[α]20
901.3072, found: 901.3082;
= 46.0 ° (c 0.16, CHCl3).
D
((2R,3S,4R,5R)-5-(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3,4-dihydroxy-2-methyltetrahydrofuran-2-
yl)methyl dihydrogen phosphate (20)
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7
8
9
Compound 19 (2.30 mg, 2.62 µmol, 1.00 eq.) was dissolved in methanol (0.50 mL) under an argon atmosphere
and palladium on charcoal (10 wt.% on activated carbon, 0.60 mg, 0.52 µmol, 0.20 eq.) was added. The
atmosphere was replaced by hydrogen (balloon, 1 atm) and the reaction was stirred at room temperature for 9 h.
After consumption of all starting material, the reaction mixture was filtered through Celite® 545 and the plug
was rinsed with methanol (3 x 2.00 mL). The solvent was evaporated to dryness, the residue was dissolved in
D2O (0.60 mL) and the solution heated to 50 °C for 2 d. The solvent was removed and the residue was dissolved
in a few drops of methanol. Finally, dichloromethane was added dropwise leading to precipitation of the
nucleotide. The precipitation procedure was repeated twice and the residue dried under high vacuum, giving
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compound 20 (0.70 mg, 2.17 µmol, 79% yield) as a colorless, hygroscopic syrup. H NMR (500 MHz, D2O) δ
(ppm) 8.09 (d, J = 8.1 Hz, 1H), 6.07 (d, J = 7.1 Hz, 1H), 6.00 (d, J = 8.0 Hz, 1H), 4.58 (dd, J = 7.1, 5.6 Hz, 1H),
4.31 (d, J = 5.6 Hz, 1H), 3.88 – 3.79 (m, 2H), 1.31 (s, 3H). 13C{1H} NMR (126 MHz, D2O) δ (ppm) 169.1,
155.0, 144.8, 105.9, 90.2 (d, J = 8.8 Hz), 89.7, 76.8, 74.9, 71.9 (d, J = 4.7 Hz), 20.7. 31P NMR (122 MHz, D2O)
-1
+
푣
δ (ppm) 4.1. IR (ATR): (cm ) = 3101, 1679, 1430, 1393, 1282, 1079. HRMS (ESI) m/z: [M - H + 2Na] calcd
[α]20
+
for C10H14N2O9PNa2 383.0227, found: 383.0228;
= -33.6 ° (c 0.11, H2O).
D
MTT Cell Viability Assay.
Cell viability assays were performed as reported previously.6 In brief, HEp2 cells (ATCC; CCL-23) were seeded
in 96 well plates. One day later, supernatant was changed to fresh growth medium containing 10 µM Puromycin,
the indicated compound concentration or 1 % DMSO only. 24 hours after treatment, cells were incubated with
0.5 mg/ml Thiazolyl Blue Tetrazolium Bromide (MTT) for 30 minutes and lysed in Isopropanol. Color change
as indirect measure of cell metabolism was quantified using a microplate reader and normalized to the
Puromycin (0 % cell viability) and DMSO (100 % cell viability) control.
HRSV Infection Assay.
The antiviral effect of the generated nucleoside derivatives was investigated as described previously.6 In brief,
HEp2 cells were seeded in 96 well plates and infected with a recombinant RSV A firefly luciferase reporter
virus24 in presence of the indicated compound concentration or 1 % DMSO only. 24 hours later, cells were lysed
and stored at -20°C until reporter activity was quantified on a microplate luminometer (infection round I).
Additionally, supernatants containing newly produced virus were collected and transferred onto naive cells
(infection round II). Lysis and analysis were performed as described for infection round I. After subtraction of
background levels (0 %) luciferase activity as indirect measure of viral infection was normalized to the DMSO
control (100 %). Assay controls Ribavirin and RSV604 were purchased from Sigma-Aldrich (R9644) and
MedChemExpress (HY-12993), respectively.
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ASSOCIATED CONTENT
Supporting Information.1H NMR, 13C NMR and 31P NMR spectra, graphics concerning antiviral activity and
cell toxicity of selected compounds.
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AUTHOR INFORMATION
*E-mail: d.werz@tu-braunschweig.de (D.B.W.).
ORCID
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Daniel B. Werz: 0000-0002-3973-2212
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank the Studienstiftung des deutschen Volkes (Ph.D. Fellowship to C.K.) and the Fonds der Chemischen
Industrie (Ph.D. Fellowship to C.K.) for funding. Recombinant RSVA luciferase reporter virus was a kind gift
from Marie-Anne Rameix-Welti (Université de Versailles St. Quentin) and Jean-François Éléöuet (Université
Paris Saclay). We thank Christina Grethe (TWINCORE) for her technical assistance and the Helmholtz-Alberta
initiative for infectious disease research (HAI-IDR) for funding of the work in the T.P. laboratory.
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