Asymmetric Synthesis of 2′- C-Methyl-4′-selenonucleosides as Anti-Hepatitis C Virus Agents

Article abstract of DOI:10.1021/acs.joc.9b01462

In search of a new template for anti-hepatitis C virus (HCV) agents, we designed and synthesized the 2′-C-methyl-4′-selenopyrimidine and -purine nucleosides and their phosphoramidate prodrugs to replace a furanose oxygen of anti-HCV nucleos(t)ides with a selenium atom on the basis that selenium is a chemical isostere of oxygen. These nucleosides are expected to show different physicochemical properties such as better lipophilicity which might enhance the penetration across cell membranes and the conformational constraint induced by a bulky selenium atom in the sugar ring. The 2′-C-methyl-4′-selenopyrimidine and -purine nucleosides 8 and 9 were synthesized from 2-C-methyl-d-ribono-γ-lactone (14) via construction of 2-C-methyl-d-selenosugar 18 through C-4 epimerization and S_N2 cyclization with Se^2- as key steps. The key 4′-selenosugar was converted to the 2′-C-methyl-4′-selenopyrimidine and -purine nucleosides using Pummerer-type rearrangement and Vorbrüggen glycosylation, respectively. In addition, the ProTide strategy has been applied to synthesize the adenine and uracil phosphoramidate derivatives 10a and 10b to overcome the limitations associated with parent nucleosides such as inefficient conversion to their corresponding 5′-monophosphate form and poor cellular uptake. The regio- and stereochemistry of 4′-selenonucleosides were confirmed by 2D NOESY NMR spectroscopy and X-ray crystallography. None of the final pyrimidine and purine nucleosides and their prodrugs exhibited significant anti-HCV activity up to 100 μM.

Full text of DOI:10.1021/acs.joc.9b01462

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Article
Asymmetric Synthesis of 2#-C-Methyl-4#-
selenonucleosides as Anti-Hepatitis C Virus (HCV) Agents
Hyejin Lee, Dnyandev B Jarhad, Jinha Yu, Choongho Lee, and Lak Shin Jeong
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b01462 • Publication Date (Web): 14 Oct 2019
Downloaded from pubs.acs.org on October 20, 2019
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Asymmetric Synthesis of 2′-C-Methyl-4′-selenonucleosides as
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Anti-Hepatitis C Virus (HCV) Agents
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,#
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Hyejin Lee, Dnyandev B. Jarhad, Jinha Yu, Choongho Lee, Lak Shin
Jeong1,*
1
Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul
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National University, Seoul 08820, Korea and College of Pharmacy, Dongguk
University, Goyang, Gyeonggi-do, 10326, Korea
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In search of a new template for anti-hepatitis C virus (HCV) agents, we designed
and synthesized the 2′-C-methyl-4′-selenopyrimidine and -purine nucleosides and
their phosphoramidate prodrugs to replace a furanose oxygen of anti-HCV
nucleos(t)ides with a selenium atom, on the basis that selenium is a chemical
isostere of oxygen. These nucleosides are expected to show different
physicochemical properties such as better lipophilicity which might enhance the
penetration across cell membranes, and the conformational constraint induced by
bulky selenium atom in the sugar ring. The 2′-C-methyl-4′-selenopyrimidine and -
purine nucleosides were synthesized from 2-C-methyl-D-ribono-γ-lactone via
construction of 2-C-methyl-D-selenosugar through C-4 epimerization and SN2
2
-
cyclization with Se as key steps. The key 4′-selenosugar was converted to the 2′-
C-methyl-4′-selenopyrimidine and -purine nucleosides using Pummerer-type
rearrangement and Vorbrüggen glycosylation, respectively. In addition, the
ProTide strategy has been applied to synthesize the adenine and uracil
phosphoramidate derivatives to overcome the limitations associated with parent
nucleosides such as inefficient conversion to their corresponding 5′-
monophosphate form and poor cellular uptake. The regio- and stereochemistry of
4
′-selenonucleosides were confirmed by 2D NOESY NMR spectroscopy and X-ray
crystallography. None of the final pyrimidine and purine nucleosides and their
prodrugs exhibited significant anti-HCV activity up to 100 μM.
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Keywords: Hepatitis C virus, 2′-C-methyl-4′-selenonucleosides, phosphoramidate
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prodrug, Pummerer-type base condensation, Vorbrüggen condensation
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Introduction
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Hepatitis C is caused by the hepatitis C virus (HCV), which has a 50 nm
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,2
enveloped virion containing a single strand of linear RNA. HCV is a member of
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,2
the Flaviviridae family and is classified into multiple genotypes. Hepatitis C has
reached epidemic proportions worldwide, with more than 1 million new cases
3
reported annually. In most cases, an acute HCV infection leads to a chronic disease
state.4
The treatment of chronic hepatitis C infection was revolutionized with the
approval of direct-acting antiviral agents (DAAs).5-7 DAAs are a relatively new
class of medication that acts to target-specific steps in the life cycle of HCV. There
are four current classes of DAAs, which are defined by their mechanism of action
and therapeutic targets: nonstructural protein (NS) 3/4A protease inhibitors, NS5B
nucleoside polymerase inhibitors, NS5B non-nucleoside polymerase inhibitors, and
7
NS5A inhibitors. The main targets of the DAAs are the HCV-encoded proteins that
are vital to the replication of the virus.5-7
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NH2
O
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HN
O
O
HO
N
O
RO
N
Y
O
O
O
OPh
Me
Me
Me
OH OH
OR OH
OH
F
1
(sofosbuvir)
2 (X = NH2, Y = H, R = H)
(X = OH, Y = NH , R = H)
2
(X = OH, Y = NH , R = A) (IDX-184)
(X = OMe, Y = NH , R = A') (INX-08189)
6 (R = H) (NM-107)
7 (R = val) (NM-283)
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2
2
O
S
O
O
O
P NH
O
O
A' =
A =
P O
HN
HO
Figure 1. Chemical structures of anti-HCV nucleos(t)ides 1-7
The past few years have witnessed an expanding interest in the prodrug approach
8
in the antiviral drug development. Among these, recently approved nucleos(t)ide
9
analogue, sofosbuvir (1) (Figure 1), a phosphoramidate prodrug, has been proven
to be most effective orally available antiviral drug for chronic HCV treatment as a
highly potent NS5B inhibitor. Ribavirin was being used for the treatment of HCV
infection in combination with pegylated interferon (peg-IFN) α before the approval
of sofosbuvir.10 Compound 1 is available in a fixed-dose combination with
1
1
ledipasvir for the treatment of HCV genotypes 1, 4, 5, and 6. In addition, 2′-C-
methylribofuranosylpurine derivatives 2 and 3 were also identified as potent HCV
1
2
polymerase inhibitors. However, the therapeutic potency of adenosine derivative
2
and guanosine derivative 3 was limited due to metabolic deamination and poor
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conversion to the corresponding active triphosphate, respectively. To solve these
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drawbacks, the phosphoramidate prodrugs, IDX-184 (4) and INX-08189 (5)
were developed as potent and selective inhibitors of HCV replication.
Unfortunately, both compounds have been removed from clinical trials due to their
cardiotoxicity complications.8,15 2′-C-Methylcytidine (6) and its 3′-O-valine ester
16
prodrug, NM-283 (7) have also been reported as potent and selective inhibitors of
HCV replication in cell culture. These prodrugs have been developed to improve
pharmacokinetic properties such as low oral bioavailability of parent
nucleosides.17,18 These nucleos(t)ide analogues target the catalytic site of NS5B and
are activated within a hepatocyte through phosphorylation by cellular kinases to
nucleoside 5′-triphosphates, which then mimic the natural nucleoside 5′-
triphosphates, and are incorporated into the growing HCV RNA chain by the NS5B
polymerase, resulting in the viral RNA chain termination.17,18 Introduction of 2′-C-
methyl group to the ribofuranosyl nucleosides has been reported to shift the sugar
puckering toward the 3′-endo (North) conformation in solution.12,19 It has been also
suggested that the presence of 2′-C-methyl group induce non-obligate viral RNA
chain termination by restricting the subsequent elongation step through steric
hindrance exerted between the 2′‑C-methyl group and the ribose moiety of the next
incoming natural nucleoside 5′-triphosphate.8,20 Based on the mechanism of action,
it has been identified that 2′-C-methylribofuranosyl nucleosides inhibit the
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replication of other RNA viruses such as yellow fever, bovine diarrhea virus, and
West African Nile viruses which are genetically homologous with HCV.20
On the other hand, it has been reported that the sugar conformation of nucleosides
is closely related to the antiviral activity, which might be decided by the binding
affinity to cellular kinases and/or DNA/RNA polymerases.21-24 Van Roey et. al.22
have reported the correlation between the sugar conformation of nucleosides and
anti-HIV activity, suggesting that anti-HIV nucleosides adopt the C3'-exo (South)
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conformation. Marquez et al. have reported that herpes 1 kinase and cellular DNA
polymerase can discriminate between two isomers of methanocarba thymidine
(MCT), on the basis of specific sugar conformation, wherein the kinases preferred
the substrate that adopt the South sugar puckering (S-MCT), while the DNA
polymerase preferred the substrate that adopt North sugar puckering (N-MCT).
23
Marquez et al. have also demonstrated that HIV-1 reverse transcriptase (RT)
prefers specific sugar conformation of nucleotides using two conformationally
restricted carbocyclic 3′-azidothymidine (AZT)-5′-triphosphate analogues. In this
study, the (N)-methanocarba-AZT-5′-triphosphate (North conformer) exhibited
potent inhibition against RT, while (S)-methanocarba-AZT 5′-triphosphate (South
conformer) was found to be inactive against RT, demonstrating that RT prefers the
2
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northern sugar puckering. Kirby et al. have also suggested that North (2′-exo/3′-
endo) sugar puckering of 4′-ethynyl-2-fluoro-2′-deoxyadenosine is essential for
recognition by RT at both dNTP incorporation step and primer (elongation step)
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binding sites. Thus, the sugar conformation of the nucleos(t)ides is very crucial to
meet the specific conformational demands of the catalytic enzymes.
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Recently, we have reported the 4′-selenonucleosides as next generation
nucleosides, which belong to nonclassical nucleosides in that the furanose ring
oxygen is replaced by selenium.25,26 The bulky selenium atom is expected to change
the profiles of the nucleoside such as the sugar conformation and lipophilicity. For
example, it was identified that the X-ray crystal structure of 4′-selenouridine
showed unusual 2′-endo/3′-exo (South) conformation, which is opposite to uridine,
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adopting a 2′-exo/3′-endo (North) conformation. This conformational difference
might affect the phosphorylation by the cellular kinases or the binding affinity to
cellular or viral DNA/RNA polymerases. The cellular phosphorylation study of 4′-
selenonucleosides and 5′-homo-4′-selenonucleosides demonstrated that 4′-
selenonucleosides were not phosphorylated by cellular kinases probably due to the
steric repulsion between cellular kinase and bulky selenium, possibly explaining
2
5
their poor antiviral activity or anticancer activity. In contrast, the corresponding
5
′-homo-4′-selenonucleosides were phosphorylated by cellular kinases most likely
due to no steric repulsion between cellular kinase and bulky selenium, resulting in
potent antiviral or antitumor activity.25
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B
O P O
O
N
N
Y
Se
HO
HO
Se
Se
NH
Me
Me
Me
OH OH
OH OH
OH OH
O
O
8
9
10
X = OH or NH2
Y = H or CH3
X = Cl, OH or NH2
B = Pyrimidine and Purine
Y = H or NH2
Figure 2. The structures of target nucleos(t)ides 8-10
Thus, based on the bioisosteric relationships between oxygen and selenium, we
designed and synthesized the 4′-seleno derivatives 8 and 9 of 2′-C-
methylribofuranosyl-pyrimidines and purines shown in Figure 2 to discover new
anti-HCV agents. We also synthesized the pyrimidine and purine phosphoramidate
prodrugs 10, which are designed to avoid the rate-determining
monophosphorylation by the cellular kinases as well as the poor cellular uptake.
Herein, we report the synthesis and anti-HCV activity of 2′-C-methyl-4′-
selenonucleosides 8 and 9 and their phosphoramidate prodrugs 10.
Results and Discussion
The retrosynthetic plan towards designed nucleosides 8 and 9 is illustrated in
Scheme 1.
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SN2 cyclization
with Se2-
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Pummerer-type
condensation
Me
OH
Se
N
O
HO
OH
Me
Se
RO
Me
O
O
O
O
HO
OH
13
8
11
Epimerization
&
Reduction
Pummerer
rearrangement
X
N
N
N
RO
Vorbrüggen
Y condensation
Se
HO
OAc
Me
Me
O
O
HO
N
Se
Me
O
O
HO
OH
HO
OH
14
9
12
Scheme 1. Retrosynthetic plan towards the target nucleosides 8 and 9.
The synthetic strategy to the desired pyrimidine nucleosides 8 was to synthesize the
glycosyl donor 11 and then to condense with pyrimidine bases under Pummerer-
type conditions. The targeted purine nucleosides 9 could be derived from the
condensation of another glycosyl donor 12, obtained from Pummerer
rearrangement of 11, with purine bases under Vorbrüggen conditions. The key
compound 11 could be easily obtained from diol 13 via mesylation followed by SN2
2
-
ring cyclization with Se . Diol 13 could be derived from commercially available 2-
C-methyl-D-ribono-γ-lactone (14) via C4-epimerization and subsequent reduction
of lactone.
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Me
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Me
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b
a
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
HO
14
16
15
R = TBDPS
e
79%
from 14
c
RO
OMs Me
OH Me
Se
Me
OMs
d
OH
30%
RO
RO
O
O
from 13
O
O
O
O
17
13
1
8
Reagent and conditions: (a) i. acetone, H SO (cat.), CuSO , rt, 5 h; ii. MsCl, DMAP,
pyridine, 0 °C-rt, 15 h; iii. KOH,dioxane/H O, 0 °C-rt, 4 h; (b) TBDPSCl, imidazole,
CH Cl , rt, 12 h; (c) LiBH , ether,THF/H O, 0 °C to rt, 1.5 h; (d) MsCl, Et N, CH Cl ,
2
4
4
2
2
2
4
2
3
2
2
0 °C- rt, 2 h; (e) Se, NaBH , DMF, EtOH, 140 °C, 5.5 h.
4
Scheme 2. Synthesis of 2-C-methyl-4-selenosugar 18.
Synthesis of the final nucleosides 8 and 9 began with the synthesis of the key
intermediate, 2-C-methyl-4-selenosugar 18, starting from commercially available
2
-C-methyl-D-ribono-γ-lactone (14), as shown in Scheme 2. The starting material
1
4 was converted to 15 in three steps, according to similar procedure used in the
2
7
preparation of L-lyxono-γ-lactone from D-ribono-γ-lactone. Briefly, treatment of
4 with acetone in the presence of a catalytic amount of sulfuric acid followed by
1
the treatment with mesyl chloride afforded 5-mesyl-2-C-methyl-D-ribono-γ-
lactone-2,3-O-isopropylidene. Without purification, the crude mesylate was treated
with aqueous potassium hydroxide, upon which the inversion of the configuration
at the C4 chiral center in 14 occurred to yield 2-C-methyl-L-lyxono-γ-lactone-2,3-
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O-isopropylidene (15) via intramolecular S 2 ring opening of the epoxide
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
intermediate by the carboxylate nucleophile. The primary hydroxyl group of 15 was
protected with TBDPS group to give 16, which was directly subjected to reduction
with LiBH to give diol 13. Mesylation of 13 followed by treatment of resulting
4
dimesylate 17 with selenium powder in the presence of NaBH in DMF-EtOH
4
afforded the desired 2-C-methyl-4-selenosugar 18.
X
Y
RO
Me
O
N
Se
Y
O
RO
N
O
RO
Se
N
X
O
Se
O

b
a
Me
Me
18
N
O
O
O
O
1
1
1
9a (X = OH, Y = H) (37%)
20a (X = OH, Y = H) (14%)
11 (R = TBDPS)
9b (X = OH, Y = CH ) (31%)
20b (X = OH, Y = CH ) (12%)
3
3
9c (X = NHBz, Y = H) (31%)
20c (X = NHBz, Y = H) (13%)
c
c
X
HO
Me
Se
Y
N
N
X
O
HO
N
O
HO HO
Y
Se
Me
N
HO
OH
8a (X = OH, Y = H) (86%)
8aa (X = OH, Y = H) (93%)
8b (X = OH, Y = CH ) (85%)
8bb (X = OH, Y = CH ) (80%)
3
3
21c (X = NHBz, Y = H)
21cc (X = NHBz, Y = H)
d
d
8
c (X = NH , Y = H) (76%)
8cc (X = NH , Y = H) (73%)
2
2
Reagent and conditions: (a) m-CPBA, CH Cl , -78 °C, 45 min; (b) pyrimidine bases, TMSOTf, Et N,
2
2
3
toluene/CH Cl , rt, 15 h; (c) TFA/H O (1/1), MeOH, rt, 4-14 h; (d) NH , MeOH, rt, 15 h.
2
2
2
3
Scheme 3. Synthesis of pyrimidine nucleosides 8a-c and 8aa-cc.
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5
6
7
8
9
With the key precursor selenosugar 18 in hand, we first synthesized the desired
pyrimidine nucleosides 8, as illustrated in Scheme 3. The selenosugar 18 was
oxidized to selenoxide 11, which was directly condensed with various pyrimidine
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
bases such as uracil, thymine, and N -benzoylcytosine using Pummerer
rearrangement in the presence of TMSOTf and Et N to afford the β anomers 19a-c
3
and the α anomers 20a-c, respectively. The anomeric configurations of 19a and 20a
were confirmed on the basis of their 2D NOESY NMR spectral data, and also
confirmed by further transformation of 19a into 8a whose structure was finally
decided based on 2D NOESY, NOE, and the X-ray crystallography (vide infra).
The 2D NOESY NMR spectrum of 19a displayed a correlation between the 5′-H
and H-6, indicating the -anomeric configuration. Anomeric configurations of other
pyrimidine nucleosides 19b-c and 20b-c were assigned based on their spectral
characteristics and the comparison with the spectral features of 19a and 20a. For
example, the chemical shifts of the β anomeric protons of 19a-c in the NMR spectra
appeared downfield relative to those of the α anomeric protons of 20a-c. Treatment
of 19a-c with 50% aqueous trifluoroacetic acid afforded the 8a, 8b, and 21c,
respectively. Compound 21c was further treated with methanolic ammonia to
produce 8c. Similarly, the α anomers 20a-c were converted to the final nucleosides
8
aa-cc. The stereochemistry of the desired final nucleosides 8a-c and 8aa-8cc were
confirmed by 2D NOESY NMR spectroscopy and comparison with the NMR
spectral features of 8a whose structure was established by X-ray crystallography
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2
3
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
see the Supporting Information, Figure S1). 2D NOESY spectrum of the -anomer
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8
a showed correlations between H-6 and protons 5′-H, 2′-C-methyl and 3′-H,
confirming the -anomeric configuration, while 2D NOESY NMR spectrum of -
anomer 8aa indicated the correlation between 1′-H and 3′-H and also a correlation
between 1′-H and 2′-C-methyl protons, demonstrating the -anomeric
configuration. Furthermore, due to the deshielding effects by heteroaromatic
2
8
pyrimidine bases, the chemical shifts of the 4′-H’s in the β anomers 8a-c appeared
more upfield than those of the α anomers 8aa-cc, while the 3′-H’s of the β anomers
8
a-c appeared more downfield than those observed for the α anomers 8aa-cc. The
X-ray crystal structure of 8a illustrated that it adopts a 2′-exo/3′-endo (North)
26
conformation, which is opposite to that of 4′-selenouridine, taking a 2′-endo/3′-
exo (South) conformation. This conformational difference might be attributed to
the steric effects between 2′-C-Me group and bulky selenium atom. We have also
investigated the sugar conformation of 8a in the solution state using 2D NOESY
NMR spectroscopy because we could not determine the North or South
conformation from the comparison of 3JH-H coupling constants in H NMR due to
the absence of 2′-H in the structure of 8a (Figure 3).
1
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8
9
O
NOE
O
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H
NH
Me
H
H
H
H
HO
3'
N
HO
5'
H
2'
NH
OH
O
N
HO
1'
Se
5
'
Se
H
H
1'
H
O
H
Me
H
2
'
3'
OH
OH
X
8a
no NOE
8a
2
'-endo/3'-exo (South)
2
'-exo/3'-endo (North)
Figure 3. Confirmation of a North conformation in sugar puckering of 8a in the
solution state using 2D NOESY NMR spectroscopy.
2
3
D NOESY spectrum of 8a in D O displayed a strong NOE between H-6 and the
2
'-H, whereas no correlation between 5'-H and 2'-Me, confirming that the North
sugar conformation of 8a is predominant in the solution state.
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2
3
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
8
a 47%
Se Me
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
RO
OAc
O
12 (R = TBDPS)
b
Cl
N
N
N
N
RO
N
Se
Me
N
RO
N 7
RO
N9
Se

N9

Se
N
O
O
Me Cl
Me
N
N
O
O
O
O
Cl
23 (8%)
24 (16%)
2
2 (32%)
c
Cl
NH2
O
N
N
N
N
N
N
NH
d
 HO
N9
HO
N9
HO
N9
N
N
Se
Se
Se
99%
Me
Me
Me
HO
OH
HO
OH
HO
OH
9
a (19%)
9b (79%)
9c
Reagent and conditions: (a) i, m-CPBA, CH Cl , -78 °C, 45 min, ii, Ac O, 50 °C, 15 h; (b) W,
6-chloropurine, BSA, TMSOTf, toluene, 100 °C, 17 h; (c) TFA/H O (1/1), THF, rt, 12 h; (d)
2
2
2
2
NH , t-BuOH, 88 °C, 20 h.
3
6
Scheme 4. Synthesis of N -substituted purine nucleosides 9a-c
6
For the synthesis of 2′-C-Me-4′-seleno-N -substituted-purine nucleosides 9a-c, the
key intermediate 18 was converted to the glycosyl donor 12, which is suitable for
the Lewis acid-catalyzed Vorbrüggen condensation (Scheme 4). Thus, oxidation of
1
8 with mCPBA followed by heating with acetic anhydride at 50 °C gave 12.
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8
9
Condensation of 12 with silylated 6-chloropurine in the presence of TMSOTf under
9
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
microwave conditions afforded N -β-anomer 22 (32%), N -α-anomer 23 (8%), and
1
7
inseparable anomeric mixture (α:β = 1:3.4, based on H NMR) of N -regioisomer
2
4 (16%). It is interesting to note that Vorbrüggen condensation without microwave
irradiation gave the desired products in low yield. In order to improve the formation
of desired β-anomer 22, the direct condensation of selenoxide 11 with 6-
chloropurine under Pummerer-type base condensation conditions, previously
successful for the synthesis of pyrimidine analogues was also attempted, but no
significant improvement was observed on anomeric ratio, regioisomeric ratio and
9
7
condensation yield. It was extremely difficult to distinguish the N /N -regioisomers,
1
along with α/β-stereoisomers, based on H NMR spectra. First, the structural
9
7
assignment of the N /N -regioisomers was accomplished by the comparison of UV
and H NMR spectra in the literature25d and finally by the X-ray crystallography.
The UV spectra of 22 and 24 showed λmax’s at 264.9 and 259.9 nm, indicating the
1
9
7
N - and N -isomers, respectively. Secondly, anomeric configurations of 22 and 23
were confirmed by 2D NOESY NMR spectroscopy after the transformation of 22
into 9b, which was further confirmed by the X-ray crystallography. Treatment of
2
2 with 50% TFA afforded 9b along with the concomitant formation of the
hydrolyzed product 9a. Treatment of 9b with tert-butanolic ammonia yielded 9c.
The 2D NOESY experiment of 9b demonstrated the correlations between 3′-H, 5′-
H, or 2′-C-methyl protons and H-8, confirming the -anomeric configuration,
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
which was also determined by the X-ray crystallography, as shown in Figure S2
(see the Supporting Information). The 2D NOESY NMR analysis of 9b revealed
that it preferred the North sugar conformation in the solution state because of no
NOE between 5'-H and 2'-Me (see the Supporting Information), which is the same
as that obtained from the X-ray crystal structure of 9b.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Using a similar strategy, 2′-C-Me-4′-seleno-2,6-disubstituted-purine nucleosides
9
d-f were synthesized according to the procedure shown in Scheme 5.
Condensation of 12 with silylated 2-amino-6-chloropurine afforded an inseparable
9
anomeric mixture of N -isomer 25a (57%) with concomitant minor formation of an
inseparable anomeric mixture of N -isomer 25b (14%). The anomeric α and β ratios
7
9
7
of N - and N -isomers were 1:3 and 1:2, respectively. In order to separate the
anomers from mixture 25a, it was protected with tert-butyloxycarbonyl (Boc) group
to yield β-anomer 26 and its α-anomer 27 after separation by silica gel column
chromatography.
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9
1
2
a
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Cl
N
N
NH2
N
N
N

N
TBDPSO
N9
TBDPSO
7N
NH2
Se
Se
Cl
Me
Me
O
O
O
O
2
5a (57%)
25b (14%)
b 85%
X
N
Cl
N
N
N
N
N
N
HO
NH2
Se
TBDPSO
c
N
N(Boc)2
Me
Se
Me
HO
OH
O
O
9d (X = Cl) (37%)
9e (X = OH) (32%)
d
89%
2
6

9f (X = NH2)
-isomer, 27
Reagent and conditions: (a) W, 2-amino-6-chloropurine, BSA, TMSOTf,
toluene, 100 C, 15 h; (b) Boc O, DMAP, THF, rt, 15 h; (c) TFA/H O (1/1),
o
2
2
THF, 28 °C, 12 h; (d) NH , t-BuOH, 95 °C, 68 h.
3
Scheme 5. Synthesis of 2,6-disubstituted purine nucleosides 9d-f
Treatment of 26 with 50% TFA afforded 2-amino-6-chloropurine derivative 9d
along with its hydrolyzed guanine derivative 9e. The anomeric configuration of 9d
was confirmed by 2D NOESY NMR experiment showing the correlation between
3′-H and H-8, and further by X-ray crystallography, illustrated in Figure S3 (see the
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Supporting Information). Amination of 9d with tert-butanolic ammonia yielded
2,6-diamino derivative 9f.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Phosphoramidate prodrugs have stimulated interest in the development of antiviral
drug discovery due to their pharmacokinetics profiles.14,16-18 They were especially
synthesized to overcome the possibility of low cellular uptake and poor conversion
to their corresponding 5′-monophosphate form by cellular kinases. Thus, we
decided to synthesize the phosphoramidate prodrugs 10a-b of 2′-C-methyl-4′-
selenonucleosides 8a and 9c, as shown in Schemes 6 and 7.
O
N
O
N
O
NH
O
O
NH
O
P
a
b
O
O
NH
19a
O
O
c
HO
HN
O
O
P
Se
Se
8
1%
37%
N
O
68%
HN
O
Me
OPh
Me
Se
OPh
Me
O
O
O
O
HO
0a
OH
28
29
1
O
F
F
O
P
C =
O
HN
O
F
OPh
F
F
Reagent and conditions: (a) TBAF, THF, rt, 1.5 h; (b) C, t-BuMgCl, molecular sieves, THF, 0 °C-rt, 48 h;
c) 50% HCO2H, rt, 12 h.
Scheme 6. Synthesis of uracil phosphoramidate prodrug 10a
The removal of the TBDPS group of 19a with TBAF afforded the 5′-hydroxyl
2
9
derivative 28, which was treated with the phosphoramidate reagent (C) in the
presence of tert-butylmagnesium chloride to afford the phosphoramidate derivative
2
9 (Scheme 6). Treatment of 29 with 50% HCOOH yielded the final uracil
phosphoramidate derivative 10a.
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8
9
NH2
N
N(Boc)2
N(Boc)2
N
N
N
N
N
N
N
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
N
b
N
c
N
RO
RO
2
2
Se
HO
Se
Se
97%
Me
95%
Me
97%
Me
O
O
O
O
O
O
3
0 (R = TBDPS)
31
32
O
F
F
F
F
O
P
d
42%
C =
O
HN
O
F
OPh
NHBoc
NH2
N
N
N
O
O
N
N
O
O
O
P
N
N
N
e
HN
P
O
O
HN
O
Se
Se
OPh
Me
OPh
Me
61%
O
O
HO
OH
0b
1
3
3
Reagent and conditions: (a) NH , t-BuOH, 88 °C, 25 h; (b) Boc O, DMAP, THF, rt, 12 h; (c) TBAF, THF, rt, 30 min;
3
2
(
d) C, t-BuMgCl, Molecular sieves, THF, 0 °C-rt, 24 h; (e) 50% HCO H, rt, 12 h.
2
Scheme 7. Synthesis of adenine phosphoramidate prodrug 10b
In order to synthesize the adenine phosphoramidate derivative 10b, the key
intermediate 22 was converted into 32 in three steps as shown in Scheme 7. N6-
Amination of 22 with tert-butanolic ammonia gave 30, which was protected with
Boc group to yield N,N-di-Boc derivative 31. The removal of the TBDPS group of
2
9
31 produced 32, which was treated with phosphoramidate reagent (C) in the
presence of tert-butylmagnesium chloride to afford 33 in 42% yield. Final removal
of acetonide and Boc groups of 33 with 50% formic acid yielded the desired adenine
phosphoramidate derivative 10b in 61% yield.
Conclusion
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In summary, the key intermediate, 2-C-methyl-D-4-selenosugar 18 was synthesized
0
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0
from 2-C-methyl-D-ribono-γ-lactone (14) via C4-epimerization and S 2 cyclization
N
2-
with Se as key steps. 2′-C-Methyl-4′-selenopyrimidine nucleosides 8a-c were
synthesized from condensation of selenoxide 11 with pyrimidine bases under a
Pummerer-type rearrangement conditions, while 2′-C-methyl-4′-selenopurine
nucleosides 9a-f were synthesized from condensation of 4-selenoacetate 12 with
silylated purine bases under Vorbrüggen conditions. In addition, 2′-C-Methyl-4′-
selenouridine 19a and 2′-C-methyl-4′-selenoadenosine 30 were converted to their
corresponding phosphoramidate prodrugs 10a and 10b in order to circumvent the
rate-determining 5′-monophosphorylation step of parent compounds by the cellular
kinases. The structures of pyrimidine and purine nucleosides were confirmed by
UV, NOE correlation, the NOESY NMR spectroscopy and X-ray crystallography.
The X-ray crystallographic analyses of final nucleosides 8a, 9b, and 9d indicated
that they adopted the C2′-exo/C3′-endo (North) conformations, which are same as
the corresponding 4′-oxonucleosides, which is opposite to that of 4′-
selenoribofuranosyl nucleosides,25d,26 taking a 2′-endo/3′-exo (South) conformation.
This result might indicate that the effect of the 2′-methylation on the sugar
puckering of 4′- selenoribofuranosyl nucleosides cancels the one induced by the 4′-
selenium replacement. All the final nucleosides including prodrugs were assayed
for anti-HCV activity, but they did not show significant anti-HCV activity up to
1
00 μM probably due to no phosphorylation by cellular kinases or slow hydrolysis
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of phosphoramidate by steric effects induced by bulky selenium atom. We believe
that the results described here may find applications in nucleic acid structural
biology or enzyme mechanistic studies in determining the conformational
preferences of kinases and nucleases, and will also contribute to the conformational
studies of oligonucleotide containing 4′-selenonucleoside.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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Experimental Section
General Methods
1
13
1
Proton ( H) and carbon C{ H} NMR spectra were obtained on a Bruker AV 400
(400/100 MHz), AMX 500 (500/125 MHz), Jeol JNM-ECA 600 (600/150 MHz) or
1
Bruker AVANCE III 800 (800/200 MHz) spectrometer. The H NMR data were
reported as peak multiplicities: s for singlet; d for doublet; dd for doublet of doublets;
t for triplet; td for triplet of doublet; q for quartet; quin for quintet; bs for broad
singlet and m for multiplet. Coupling constants were reported in Hertz. The
chemical shifts were reported as ppm (δ) relative to the solvent peak. All reactions
were routinely carried out under an inert atmosphere of dry nitrogen. IKA RCT
basic type heating mantle was used to provide a constant heat source.
Microwave-assisted reactions were carried out in sealed vessels using a Biotage
Initiator+ US/JPN (part no. 356007) microwave reactor and the reaction
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
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3
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5
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5
6
temperatures were monitored by an external surface IR sensor. High resolution
mass spectra were measured with electrospray-ionization quadrupole time-of-flight
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9
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8
9
0
(ESI-Q-TOF) techniques. Melting points were recorded on Barnstead
electrothermal 9100 instrument and are uncorrected. Reactions were checked by
thin layer chromatography (Kieselgel 60 F254, Merck). Spots were detected by
viewing under a UV light, and by colorizing with charring after dipping in a p-
anisaldehyde solution. The crude compounds were purified by column
chromatography on a silica gel (Kieselgel 60, 70-230 mesh, Merck). All the
anhydrous solvents were redistilled over CaH , or P O , or sodium/benzophenone
2
2
5
prior to the reaction.
S)-2-((tert-Butyldiphenylsilyl)oxy)-1-((4R,5S)-5-(hydroxymethyl)-2,2,5-
(
trimethyl-1,3-dioxolan-4-yl)ethan-1-ol (13).
To a suspension of 2-C-Methyl-D-ribono-1,4-lactone (14) (25 g, 0.15 mol) in
acetone (500 mL) were added anhydrous copper(II) sulfate (10 g) and catalytic
concentrated sulfuric acid (2.5 mL) at room temperature under nitrogen atmosphere.
After stirring for 5 h, the reaction mixture was neutralized with anhydrous sodium
carbonate (63 g), filtered through a Celite bed, and washed with ethyl acetate. The
filtrate was concentrated in vacuo to afford the acetonide, which was used directly
for next step without any further purification,
To a solution of above obtained crude acetonide in pyridine (230 mL) were added
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methanesulfonyl chloride (24 mL, 0.31 mol) and N,N-dimethylaminopyridine (1.9
g, 0.016 mol) under nitrogen atmosphere at 0 °C. After which the reaction mixture
was stirred for 15 h at room temperature, concentrated in vacuo, and co-evaporated
with toluene (70 mL x 2). The residue was dissolved in CH Cl (250 mL) and
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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0
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0
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0
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9
0
1
2
3
4
5
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8
9
0
2
2
washed with water (100 mL) and brine (100 mL). The aqueous layer was extracted
with CH Cl (150 mL x 3). The combined organic layers were dried over anhydrous
2
2
magnesium sulfate and filtered. The filtrate was evaporated to give crude mesylate.
To a solution of crude mesylate in dioxane (560 mL) was added a solution of
potassium hydroxide (26.2 g, 0.467 mol) in water (440 mL) at 0 °C and the reaction
mixture was stirred vigorously for 4 h at room temperature. After which it was
acidified to pH 2 with 2 M hydrochloric acid. The reaction mixture was extracted
with CH Cl (150 mL x 3) and the combined organic layer was dried over
2
2
anhydrous magnesium sulfate and filtered. The filtrate was evaporated to give crude
2
0
1
5 (19 g), which was directly subjected to TBDPS protection reaction as follows.
To a stirred solution of 15 (19 g) in CH Cl (300 mL) were added imidazole (12.8
2
2
g, 0.188 mol) and tert-butyldiphenylsilyl chloride (25.8 g, 24.4 mL, 0.094 mol) at
room temperature and the reaction mixture was stirred for 12 h at the same
temperature. After which it was partitioned between dichloromethane (150 mL) and
water (100 mL) and the organic layer was washed with brine (125 mL), dried over
anhydrous MgSO , and evaporated to give crude 16 (35 g), which was directly used
4
for next reduction step without any further purification as follows.
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5
6
To a suspension of lithium borohydride (4.07 g, 0.187 mol) in ether (125 mL) was
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9
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0
added a solution of 16 (35 g) in THF/H O (125 mL/2.5 mL) at 0 °C and the reaction
2
mixture was stirred at room temperature for 1.5 h. After which it was neutralized
with glacial acetic acid and brine (100 mL) was added to the mixture. The reaction
mixture was extracted with ethyl acetate (200 mL x 3), dried over anhydrous
magnesium sulfate, and evaporated. The residue was purified by silica gel column
chromatography (hexane/ethyl acetate = 4/1) to give 13 (54 g, 79% from 14) as a
colorless syrup: [α]D25 = -3.55 (c 0.45, CH OH); H NMR (400 MHz, CDCl ) δ
1
3
3
7
.66-7.70 (m, 4 H), 7.34-7.48 (m, 6 H), 3.97 (s, 1 H), 3.89-3.92 (m, 1 H), 3.82 (dd,
J = 9.9, 6.2 Hz, 1 H), 3.72 (dd, J = 9.7, 6.9 Hz, 1 H), 3.62 (d, J = 11.9 Hz, 1 H),
.42 (d, J = 11.9 Hz, 1 H), 3.15 (bs, 2 H), 1.48 (s, 3 H), 1.38 (s, 3 H), 1.29 (s, 3 H),
3
1
3
1
1.07 (s, 9 H); C{ H} NMR (100 MHz, CDCl ) δ 135.7, 133.2, 130.1, 130.0, 128.0,
3
1
27.9, 107.8, 81.8, 80.9, 68.23, 68.17, 65.7, 65.4, 28.1, 27.0, 26.8, 22.1, 19.3;
+
+
HRMS (ESI-Q-TOF) m/z: [M + Na] Calcd for C H NaO Si 467.2224; Found
2
5
36
5
467.2235.
(S)-2-((tert-Butyldiphenylsilyl)oxy)-1-((4R,5S)-2,2,5-trimethyl-5-
(((methylsulfonyl)oxy)methyl)-1,3-dioxolan-4-yl)ethyl methanesulfonate (17)
To a solution of 13 (25 g, 0.056 mmol) in CH Cl (500 mL) were added
2
2
triethylamine (47 mL, 0.337 mol) and methanesulfonyl chloride (17.4 mL, 0.225
mol) at 0 °C and the reaction mixture was stirred at room temperature for 2 h. After
which it was diluted with saturated aqueous NH Cl solution (100 mL) and extracted
4
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with dichloromethane (170 mL x 3). The combine organic layer was washed with
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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brine (150 mL), dried over anhydrous MgSO , filtered, and evaporated. The residue
4
was dissolved in ethyl acetate (300 mL), filtered through a pad of silica gel, and
washed with ethyl acetate (100 mL x 2). After the combined filtrate was evaporated
to give crude mesylate 17, which was used for next reaction without further
purification.
tert-Butyldiphenyl(((3aS,6aR)-2,2,6a-trimethyltetrahydroselenopheno[3,4-
d][1,3]dioxol-4-yl)methoxy)silane (18)
To a cooled (0 °C) suspension of selenium powder (11.2 g, 0.141 mol) in anhydrous
ethanol (150 mL) was slowly added sodium borohydride (10 g, 0.264 mol) until the
color of the reaction mixture changed from black to colorless. To this mixture was
added a solution of crude mesylate 17 (33.95 g) in anhydrous DMF (500 mL) and
the mixture was heated at 140 °C for 5.5 h and evaporated. The residue was diluted
with water and extracted with n-hexane (300 mL x 3). The organic layer was
washed with water (50 mL), brine (50 mL), dried over anhydrous MgSO , filtered,
4
and evaporated. The residue was purified by silica gel column chromatography
(hexane/ethyl acetate = 133/1) to give 18 (8.31 g, 30% in 2 steps) as pale yellow
syrup: [α]D25 = 68.49 (c 0.63, CH OH); H NMR (400 MHz, CDCl ) δ 7.66-7.72
1
3
3
(m, 4 H), 7.37-7.45 (m, 6 H), 4.44 (s, 1 H), 3.74-3.80 (m, 3 H), 3.12 (d, J = 11.2
Hz, 1 H), 2.82 (d, J = 10.4 Hz, 1 H), 1.53 (s, 3 H), 1.42 (s, 3 H), 1.35 (s, 3 H), 1.08
1
3
1
(
s, 9 H); C{ H} NMR (125 MHz, CDCl ) δ 135.65, 135.63, 133.21, 133.17,
3
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
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3
3
3
3
4
4
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4
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5
5
5
5
5
5
6
1
29.83, 129.80, 127.8, 127.7, 110.6, 93.7, 90.7, 65.9, 46.9, 34.6, 28.3, 26.82, 26.75,
+
+
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24.9, 19.3; HRMS (ESI-Q-TOF) m/z: [M + H] Calcd for C H O SeSi 491.1517;
25 35 3
Found 491.1545.
General procedure for the synthesis of 19a-c and 20a-c (Pummerer-type
pyrimidine base condensation)
[Oxidation] To a stirred solution of 18 (0.66 g, 1.348 mmol) in anhydrous CH Cl
2 2
(20 mL) was dropwise added a solution of 3-chloroperbenzoic acid (1.1 equiv, 70%)
in CH Cl (5 mL) at –78 °C under nitrogen atmosphere and the reaction mixture
2
2
was stirred at the same temperature for 45 min. After which it was quenched with
saturated aqueous NaHCO solution, and diluted with CH Cl . The organic layer
3
2
2
was washed with water (20 mL) and brine (20 mL), dried over anhydrous MgSO4,
filtered, and evaporated below 10 °C to give 11 as colorless syrup. The residue 11
was used immediately for next step without further purification.
[
Pummerer-type condensation] To a suspension of uracil or thymine or N4-
benzoylcytosine (2 equiv) in anhydrous toluene (7.0 mL) were dropwise added
triethylamine (4 equiv) and trimethylsilyl trifluoromethanesulfonate (6 equiv) at
room temperature under nitrogen atmosphere. The reaction mixture was stirred at
the same temperature for 45 min and diluted with anhydrous CH Cl (6.0 mL). To
2
2
this mixture were dropwise added a solution of 11 (1 equiv) in CH Cl (6.0 mL)
2
2
and additional triethylamine (2 equiv) in toluene (0.5 mL) at 0 °C and the mixture
was stirred at the room temperature for 15 h. The reaction mixture was quenched
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with saturated aqueous NaHCO solution, and diluted with CH Cl . The organic
3
2
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
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6
7
8
9
0
1
2
3
4
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8
9
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1
2
3
4
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6
7
8
9
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2
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
layer was washed with water and brine, dried over anhydrous MgSO , filtered, and
4
evaporated. The residue was purified by repeated silica gel column chromatography
(
hexanes/ethyl acetate = 100/0 to 4/1) to give the β-anomers 19a-c and the α-
anomers 20a-c, respectively.
-((3aR,4R,6R,6aS)-6-(((tert-Butyldiphenylsilyl)oxy)methyl)-2,2,3a-
1
trimethyltetrahydroselenopheno[3,4-d][1,3]dioxol-4-yl)pyrimidine-2,4(1H,3H)-
dione (19a) and 1-((3aR,4S,6R,6aS)-6-(((tert-Butyldiphenylsilyl)oxy)methyl)-
2
2
1
,2,3a-trimethyltetrahydroselenopheno[3,4-d][1,3]dioxol-4-yl)pyrimidine-
,4(1H,3H)-dione (20a)
9a: Yield (299 mg, 37%, 2 steps), pale yellow foam; [α]D25 = 38.20 (c 0.26,
1
CH OH); UV (CH OH) λ 266.7 nm; H NMR (500 MHz, CDCl ) δ 8.86 (bs, 1
3
3
max
3
H, N-H), 7.64-7.67 (m, 4 H, phenyl), 7.54 (d, J = 8.1 Hz, 1 H, C6-H), 7.38-7.46 (m,
6 H, phenyl), 6.57 (s, 1 H, C1′-H), 5.59 (d, J = 8.2 Hz, 1 H, C5-H), 4.28 (s, 1 H,
C3′-H), 3.95-4.00 (m, 2 H, C4′-H, C5′-Ha), 3.83 (dd, J = 13.4, 10.8 Hz, 1 H, C5′-
Hb), 1.62 (s, 3 H, O-C(CH ) -O), 1.31 (s, 3 H, O-C(CH ) -O), 1.18 (s, 3 H, C2′-
3
2
3 2
1
3
1
CH ), 1.06 (s, 9 H, C(CH ) ); C{ H} NMR (150 MHz, CDCl ) δ 162.6 (C4), 150.3
3
3 3
3
(C2), 143.3 (C6), 135.7 (phenyl), 135.6 (phenyl), 132.9 (phenyl), 132.8 (phenyl),
1
30.1 (phenyl), 130.0 (phenyl), 127.9 (phenyl), 127.8 (phenyl), 111.7 (O-C(CH ) -
3
2
２
６
ACS Paragon Plus Environment