Stereoselective Synthesis of Highly Functionalized Arabinosyl Nucleosides through Application of an N-Nitro Protecting Group

Article abstract of DOI:10.1021/acs.joc.8b01834

2′-Deoxy-2′,5-disubstituted arabinosyl uridine derivatives bearing a halogen (Cl, Br or I) at C2′ and an ethynyl group at C5 have been synthesized in 6 steps from 2′,3′,5′-tri-O-acetyl-5-iodo-uridine in overall yields of 61% (compound 3, Cl), 47% (compound 4, Br), and 19% (compound 5, I). Stabilization of a 2′-O-triflyl leaving group intermediate to overcome spontaneous intramolecular 2,2′-anhydro uridine formation was pivotal to the synthesis. Specifically, to favor S_N2 reaction with a halogen nucleophile over intramolecular cyclization, the nucleophilicity of O-2 oxygen was reduced by incorporation of an adjacent electron withdrawing nitro substituent at N-3. The introduction of the 3-N-nitro group proceeded rapidly (nitronium trifluoroacetate, 1 min) and in quantitative yield. A one-pot method to remove the 3-N-nitro group by reductive nitration (zinc metal in acetic acid, 5 min) and the silyl protecting groups of the alkyne and 3′,5′ hydroxyls (fluoride reagent, 16 h) was established as the final synthetic step. This application of the 3-N-nitro protecting group addresses the significant shortfalls of the conventional approach to synthesis of 2′ modified nucleosides, wherein condensation of a 2′ modified sugar fragment with a pyrimidine base provides poor stereocontrol of N-glycosylation, low yields and incompatibility with 2′ iodo sugars.

Full text of DOI:10.1021/acs.joc.8b01834

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Article
Stereoselective synthesis of highly functionalised arabinosyl
nucleosides through application of a N-nitro protecting group
David H. Hilko, Laurent F. Bornaghi, and Sally-Ann Poulsen
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b01834 • Publication Date (Web): 29 Aug 2018
Downloaded from http://pubs.acs.org on August 30, 2018
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Stereoselective synthesis of highly functionalised arabinosyl nucleosides through
application of a Nꢀnitro protecting group
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David H. Hilko, Laurent F. Bornaghi and SallyꢀAnn Poulsen
Griffith Institute for Drug Discovery, Griffith University, Don Young Road, Nathan,
Brisbane, Queensland 4111, Australia
*
Corresponding author. Tel.: +61 7 3735 7825; e-mail: s.poulsen@griffith.edu.au
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Abstract
ʹꢀDeoxyꢀ2ʹ,5ꢀdisubstituted arabinosyl uridine derivatives bearing a halogen (Cl, Br or I) at
2
C2′ and an ethynyl group at C5 have been synthesized in 6 steps from 2ʹ,3ʹ,5ʹꢀtriꢀOꢀacetylꢀ5ꢀ
iodoꢀuridine in overall yields 61% (compound 3, Cl), 47% (compound 4, Br), 19%
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(
compound 5, I). Stabilisation of a 2ʹꢀOꢀtriflyl leaving group intermediate to overcome
spontaneous intramolecular 2,2ʹꢀanhydro uridine formation was pivotal to the synthesis.
Specifically, to favor S 2 reaction with a halogen nucleophile over intramolecular
N
cyclisation, the nucleophilicity of Oꢀ2 oxygen was reduced by incorporation of an adjacent
electron withdrawing nitro substituent at Nꢀ3. The introduction of the 3ꢀNꢀNitro group
proceeded rapidly (nitronium trifluoroacetate, 1 min) and in quantitative yield. A oneꢀpot
method to remove the 3ꢀNꢀnitro group by reductive nitration (zinc metal in acetic acid, 5 min)
and the silyl protecting groups of the alkyne and 3ʹ,5ʹ hydroxyls (fluoride reagent, 16 h) was
established as the final synthetic step. This application of the 3ꢀN-nitro protecting group
addresses the significant shortfalls of the conventional approach to synthesis of 2ʹ modified
nucleosides, wherein condensation of a 2ʹ modified sugar fragment with a pyrimidine base
provides poor stereocontrol of Nꢀglycosylation, low yields and incompatibility with 2ʹ iodo
sugars.
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Introduction
Nucleoside derived chemical probes are small molecule reagents that have had a profound
impact on our ability to answer fundamental questions about DNA synthesis, a process
central to all proliferating cells. An exemplar nucleoside chemical probe is 5ꢀethynylꢀ2′ꢀ
deoxyuridine (EdU, 1), Figure 1(a), wherein the 5ꢀethynyl group of 1 replaces the 5ꢀmethyl
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group of thymidine. When proliferating mammalian cells are treated with 1 this probe
molecule is incorporated into DNA. Subsequent conjugation of the alkyne moiety in the
modified DNA to a fluorescent azide molecule enables detection of cellular DNA using
1
fluorescence microscopy. The cytotoxicity of 1 does however restrict its applications to short
2
ꢀ6
exposure and low dose experiments such that next generation chemical probes with
improved properties are desirable to extend our current capabilities for studying DNA
synthesis. Luedtke and colleagues reported that the mammalian cell toxicity of 1 is mitigated
(i.e. little/no cellular arrest for up to 60 days treatment) by modification of the ribose of 1 to
the fluorinated arabinosyl derivative, (2ʹS)ꢀ2ʹꢀdeoxyꢀ2ʹꢀfluoroꢀ5ꢀethynyluridine (FꢀaraꢀEdU,
7
2
) Figure 1(a). Probe compound 2 retains DNA labelling capability in proliferating cells
similarly to 1. The 2ʹꢀhaloꢀaraꢀEdU analogues ClꢀaraꢀEdU (3), BrꢀaraꢀEdU (4) and IꢀaraꢀEdU
5) are not known, but may also have potential as next generation chemical probes or as
(
therapeutics, Figure 1(b). Herein we describe the stepwise development of a stereoselective
synthesis of 2ʹꢀhaloꢀaraꢀEdU analogues 3ꢀ5.
2ʹꢀDeoxyꢀ2ʹꢀsubstituted arabinosyl nucleosides are not found in nature. The conventional
synthesis of 2ʹ modified nucleosides proceeds by condensation of the sugar fragment with a
purine or pyrimidine base. Poor stereocontrol of this Nꢀglycosylation reaction, irrespective of
the anomeric configuration of the sugar precursor, is a wellꢀknown flaw of this synthetic
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method. For example, the reported synthesis of 2ʹꢀdeoxyꢀ2ʹꢀfluoroꢀarabinosyl pyrimidine
nucleosides from 2ʹꢀdeoxyꢀ2ʹꢀfluoroꢀ3ʹ,5ʹꢀbisꢀacylꢀαꢀ
Dꢀarabinofuranosyl bromide proceeds to
8
,9
form the nucleoside product as a mixture of α and β anomers. Moreover, reaction yields
with 2ʹꢀchloroꢀ2ʹꢀdeoxyꢀarabinosyl or 2ʹꢀbromoꢀ2ʹꢀdeoxyꢀarabinosyl sugars are considerably
reduced compared to the 2ʹꢀdeoxyꢀ2ʹꢀfluoroꢀarabinosyl sugar, while there are no reports of
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this reaction with a 2ʹꢀdeoxyꢀ2ʹꢀiodoꢀarabinosyl sugar.
Reported syntheses of 5ꢀ
substitutedꢀ2ʹꢀdeoxyꢀ2ʹꢀhaloꢀarabinosyl pyrimidine nucleosides are even fewer than that for
2ʹꢀdeoxyꢀ2ʹꢀhaloꢀarabinosyl pyrimidine nucleosides but are approached similarly.
Stereocontrol of the Nꢀglycosylation reaction of the 2ʹ sugar fragment with the 5ꢀsubstituted
pyrimidine remains a problem, while the reactions are slower than with the corresponding
7,11,12
unsubstituted pyrimidines and typically require many days to reach completion.
Thus it
was anticipated that employing this sugarꢀbase condensation approach to the synthesis of the
target 2ʹꢀhaloꢀaraꢀEdU compounds 3ꢀ5, even if successful, would suffer from poor
stereocontrol, extended reaction times and low yields. The motivation for the current study
was to overcome the existing obstacles inherent in approaches to 2ʹ modified nucleosides to
provide a reliable synthetic methodology to the synthesis of 2ʹ,5ꢀbisfunctionalised
nucleosides, target compounds 3ꢀ5.
(a)
(b)
O
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OH
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Cl-ara-EdU,
OH
Br-ara-EdU,
OH
I-ara-EdU, 5
EdU, 1
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Figure 1. (a) Nucleoside chemical probes in current use: 5ꢀethynylꢀ2′ꢀdeoxyuridine (EdU, 1)
and (2ʹS)ꢀ2ʹꢀdeoxyꢀ2ʹꢀfluoroꢀ5ꢀethynyluridine (FꢀaraꢀEdU, 2). (b) Target compounds: 2ʹꢀhalo
modified EdU nucleosides: ClꢀaraꢀEdU, 3; BrꢀaraꢀEdU, 4; and IꢀaraꢀEdU, 5.
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Results and Discussion
In principle, an alternative synthesis of 2ʹꢀdeoxyꢀ2ʹꢀhaloꢀarabinosyl pyrimidines is S
N
2
displacement of an activated 2ʹꢀhydroxyl group on the native pyrimidine scaffold with a
halide ion nucleophile. Here the Nꢀglycosidic bond configuration is both predefined and
maintained, while an inversion of configuration at 2ʹ is expected. In practice however this
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S
N
2 product does not form and instead an intramolecular cyclisation proceeds to the 2,2ʹꢀ
anhydro nucleoside 6, Scheme 1.
Scheme 1. Formation of 2,2ʹꢀanhydrouridine 6 by intramolecular cyclisation of a 2ʹ leaving
group (LG) modified uridine is favoured over S
halide anion).
N
2 displacement. Nu = nucleophile (e.g.
N
An approach to favour S 2 over intramolecular cyclisation is to decrease the nucleophilicity
of the precursor pyrimidine nucleoside Oꢀ2 oxygen by incorporating an electron withdrawing
substituent on Nꢀ3. The first successful application of this approach was accomplished by
utilising a benzoyl group at Nꢀ3 with azide anion as the nucleophile. Displacement of the 2ʹ
hydroxyl of 3ꢀN-benzyolꢀ3ʹ,5ʹꢀOꢀtetraisopropyldisiloxaneꢀ1,3ꢀdiyl)ꢀuridine 7 with the azide
13
ion and inversion of configuration was achieved under Mitsunobu conditions, Scheme 2.
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The desired S
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2 product, arabinosyl derivative 8 was formed as product. To the best of our
knowledge no attempt to perform a S
N
2 displacement on 7 with other nucleophiles under
these conditions has since been reported. Furthermore, attempts to isolate the 2ʹꢀtriflate
derivative of 7 were also unsuccessful, this derivative is not stable and converts to the 2,2ʹꢀ
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intramolecular cyclisation product.
Scheme 2. Synthesis of 2ʹꢀdeoxyꢀ2ʹꢀazidoꢀarabinosyl uridine derivative 8 by displacement of
the 2ʹ hydroxyl of 3ꢀN-benzoyl protected uridine precursor 7 with azide ion under Mitsunobu
conditions. TPP: triphenylphosphine, TIPDS: tetraisopropyldisilyloxane, DPPA: diphenyl
phosphoryl azide.
A more detailed exploration of this concept found that a nitro group at Nꢀ3 in combination
with a triflate moiety as the 2ʹ leaving group overcame competing intramolecular cyclisation.
The introduction of the 3ꢀNꢀnitro protecting group on to the native pyrimidine bases, uracil
1
4,15
and thymine uses nitronium trifluoroacetate (TFAN)
generated from TFAA and a nitrate salt such as NH
mechanism of nitration in pyrimidine systems, while the mechanism of nitration of purines
as the nitrating reagent. TFAN is
4
NO . There is little known about the
3
16
17,18
using TFAN is disputed; with both ionic and radical
processes proposed. The 3ꢀNꢀnitro,
2
ʹꢀtriflate nucleoside 9 is a stable compound and is reported to undergo reaction with
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chloride, bromide or iodide ions via a S
N
2 displacement mechanism to furnish the
corresponding 3ꢀNꢀnitroꢀ2ʹꢀdeoxyꢀ2ʹꢀhaloꢀarabinosyl uridines 12aꢀ12c in high yield, Scheme
14
3.
However subsequent denitration of 12aꢀ12c is not reported. To synthesise 9, the
precursor nucleoside 10 is first treated with pyridine and Tf O to briefly form the 2ʹ triflate
2
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intermediate 11, which is then trapped in situ by 3ꢀNꢀnitration with added TFAN. In our
hands attempts to reproduce the synthesis of 9 gave inconsistent results and were not
successful. We note that the literature specified significant care with addition of TFAN (time,
rate of addition and temperature) to successfully trap intermediate 11 without degradation
1
4
first occuring.
Scheme 3. Synthesis of 3ꢀNꢀnitroꢀ3ʹ,5ʹꢀOꢀ(tetraisopropyldisiloxaneꢀ1,3ꢀdiyl)ꢀ2ʹꢀOꢀtriflylꢀ
uridine 9 from 3ʹ,5ʹꢀOꢀ(tetraisopropyldisiloxaneꢀ1,3ꢀdiyl)ꢀuridine 10 and subsequent S
displacement of the 2ʹꢀtriflate with halide ions to yield 2ʹꢀdeoxyꢀ2ʹꢀhaloꢀ3ꢀNꢀnitroꢀ3ʹ,5ʹꢀOꢀ
N
2
1
4
tetraisopropyldisiloxaneꢀ1,3ꢀdiyl)ꢀarabinofuranosyl uridines 12aꢀ12c. Reproduced with
permission from Tetrahedron Lett. Vol. 39. Copyright 1998 Elsevier.
The application of a 3ꢀN-nitro group to our target nucleoside derivatives 3ꢀ5 necessitates that
conditions for the introduction and removal of this group are compatible with either a 5ꢀiodo
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or 5ꢀethynyl group in the nucleoside precursor. To evaluate if introduction of a 3ꢀNꢀnitro
group is feasible with these groups in place 2ʹ,3ʹ,5ʹꢀtri-O-acetylꢀ5ꢀiodoꢀuridine 15 and 2ʹ,3ʹ,5ʹꢀ
tri-O-acetylꢀ5ꢀ(ethynyl(2ꢀtrimethylsilyl))ꢀuridine 17 were treated with the TFAN reagent. As
a positive control for the reaction we employed nitration conditions known for 2ʹ,3ʹ,5ʹꢀtriꢀOꢀ
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acetylꢀuridine 13 (8 equiv. KNO /TFAA, 20 min, 0 C, CH Cl ). The reaction with 13 gave
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2
1
5
the expected 3ꢀNꢀnitro compound 14 in quantitative yield, consistent with the literature. The
desired 3ꢀNꢀnitro product 16 was obtained from 5ꢀiodo derivative 15, however deiodination
of 15 was also observed, leading to 14 as a significant side product. Reaction of 5ꢀethynyl
derivative 17 produced a complex mixture of products with no analytical evidence to indicate
formation of the desired product 18. As the TFAN reagent is both an oxidant and is acidic,
17
we speculate that these properties and/or the presence of a radical species may have
attributed to the observed deiodination of 15 and the competing side reactions with alkyne
16
1
7. TFAN prepared from TFAA and inorganic nitrate salts requires a 4ꢀ8 fold excess of
1
9
reagent for nitration of pyrimidines to reach completion (~20 min). In contrast, TFAN
prepared from TFAA and tetrabutylammonium nitrate (TBAN) is reported as an efficient Cꢀ
nitrating system in purine nucleosides, requiring less than twoꢀfold TFAN excess for rapid
17,18
and complete reaction.
When we applied 2 equivalents of TBAN/TFAA to the 5ꢀiodo
derivative 15 nitration proceeded to completion within 1 min as evidenced by TLC. This is a
remarkable 20ꢀfold faster than with either KNO3 or NH NO3 as the nitrate source.
3
Furthermore no deiodination product 14 was observed. Similarly, the reaction of 17 with
o
TBAN/TFAA (2 equiv. TBAN/TFAA, 1 min, 0 C, CH
Cl
2 2
) also proceeded rapidly, with full
conversion to 18, as evidenced by TLC. The reactions were quenched with MeOH and
purified directly by silica gel flash chromatography without further workup. The products 16
and 18 were obtained in quantitative yield, Scheme 4. Next, to establish if conversion of 5ꢀ
iodo derivative 16 to the 5ꢀethynyl derivative 18 could be achieved, compound 16 was
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subjected to the Sonagashira reaction, however all attempted conditions failed to give 18 and
instead resulted in formation of a complex mixture, Scheme 4. These findings indicated that
the 5ꢀethynyl group of the target compounds 3ꢀ5 would need to be in place prior to
installation of the 3ꢀNꢀnitro group. Compound 18 was thus the focus for further synthetic
development, specifically to ready the ribose moiety for installation of a 2′ leaving group.
1
1
1
1
1
1
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1
2
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0
Scheme 4. 3ꢀNꢀNitration of 2ʹ,3ʹ,5ʹꢀtri-O-acetylꢀuridine 13 and 5ꢀsubsitutedꢀ2ʹ,3ʹ,5ʹꢀtri-O-
acetylꢀuridines 15 and 17.
The Nꢀnitro group is known to be base labile and is incompatible with the more usual basic
15,20
deacetylation conditions of NaOMe in MeOH or ammonolysis.
The reported
19,21
deacetylation conditions for 14 are acidic (HCl catalysed methanolysis
), however acid
catalysed hydration and electrophilic addition of HCl to the 5ꢀethynyl group of 5ꢀethynyl
2
2ꢀ25
uridine derivatives is known
and this presents a potential hurdle to deacetylation of the
TMS protected 5ꢀethynyl derivative 18. When compound 18 was subjected to HCl in
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methanol the desired free sugar product 19 was observed by LCꢀMS only as a minor product.
The HCl electrophilic addition product, vinyl chloride 20, was also detected as a minor
product, while the major product is compound 21, consistent with loss of the TMS group and
hydration of the alkyne, Scheme 5. In an endeavour to limit electrophilic addition and/or
hydration we investigated deacetylation of 18 using a panel of five sulfonic acids:
0
1
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4
5
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2 4
trifluoromethanesulfonic acid (TfOH); H SO ; methanesulfonic acid (MsOH); paraꢀ
toluenesulfonic acid (TsOH); and polymeric vinylbenzenesulfonic acid (amberlite IRAꢀ120
+
H ). The reactions using homogeneous acids produced as the major product, the desired
product 19 as evidenced by TLC and LCꢀMS. The relative reaction rate order as estimated by
+
TLC was H SO > TfOH > MsOH > TsOH >> IRAꢀ120 H . Side reactions, including loss of
2
4
the 5ꢀethynyl TMS group, were comparatively slower than removal of the acetate groups,
+
with all except IRAꢀ120 H providing kinetic control. Reaction workup proved problematic
3
however as neutralisation using either CaCO or weakly basic resins (Amberlyst Aꢀ21 and
Amberlite IRAꢀ67) led to substantial decomposition of 19. Direct adsorption of the crude
reaction mixture onto silica gel also caused substantial product degradation (recovered yield
of 19 <20% yield) with the exception of the TsOH reaction, which provided 19 in 65 – 80%
yield after purification. We speculate that removal of the solvent under reduced pressure at
room temperature during adsorption onto silica concentrated the sulfonic acids and initiated
decomposition of 19, including loss of the ethynyl TMS group. This effect may have been
less pronounced with TsOH, which in combination with being the weakest of the acids, gave
a solid when concentrated (mp ~105.3 ˚C). The latter heterogeneous solution may have
contributed to less degradation of 19 during adsorption onto silica in the presence of TsOH,
Scheme 5.
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Scheme 5. Deacetylation of nucleoside 18 under acidic conditions to give the target product
19. Hydration, loss of the TMS group and electrophilic addition of HCl to the 5ꢀethynyl
group gives byꢀproducts 20 and 21 using AcCl.
In addition to the acetate group, the TBDMS group has successfully been utilised as a
1
4,19
hydroxyl protecting group for 3ꢀNꢀnitration of uridines.
The TBDMS protected derivative
26
2
3 was prepared from 5ꢀiodo uridine 22 in quantitative yield, Scheme 6. The Sonogashira
reaction of 23 with ethynylꢀ2ꢀtrimethylsilane to give 24 followed by 3ꢀNꢀnitration (2 equiv
o
TBAN/TFAA, 1 min, 0 C, CH
2
Cl
2
) to give 25 both proceeded in excellent yield, Scheme 6.
Triethylamine trihydrofluoride (Et
3
N·(HF)
3
) was initially chosen for desilylation of 25 as it is
27,28
considered a mild acidic source of fluoride.
3 3
Indeed, the reaction of 25 with Et N·(HF)
yielded the fully desilylated product 26, although upon purification by solid addition silica
gel chromatography complete degradation of 26 was observed. Olah’s reagent (~70% HF,
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5
5
6
5
~30% pyridine w/w or py·(HF) ) was employed as an alternative mild fluoride source for
desilylation of 25. The HF in Olah’s reagent forms immobile fluorosilicates on addition of
the reaction residue to silica gel, enabling the HF to be quenched on workup with silica gel
chromatography. This avoids the product degradation found with Et N·(HF) . Unexpectedly
3
3
0
1
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4
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0
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9
0
the 5ꢀethynyl TMS group of 25 proved stable towards Olah’s reagent and only the TBDMS
19
groups were removed to give 19 as the product, Scheme 6. F NMR confirmed no fluorine
contamination in 19. The deprotection of the acetyl groups of 18 and the silyl groups of 25
each gave the free ribose 19 as product. The acetylation pathway was selected in the synthetic
route toward the target final compounds 3ꢀ5 as the reagents required are less hazardous than
handling and disposal of HF reagents.
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Scheme 6. Application of the TBDMS group as a 2ʹ, 3ʹ,5ʹ hydroxyl protecting group for 3ꢀNꢀ
nitration of uridines.
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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9
0
With the deprotected ribose nucleoside 19 in hand, the 3ʹ and 5ʹ hydroxyl groups of 19 were
selectively protected with the tetraisopropyldisiloxane (TIPDS) protecting group and the 2ʹꢀ
hydroxyl converted to the 2ʹꢀtriflate with trifluoromethanesulfonic anhydride (triflylic
2
anhydride, Tf O). This hydroxyl group protection was performed as a one pot procedure and
1
4
gave 27 in 80% yield, Scheme 7. Similarly to 3ꢀN-nitroꢀ2ʹꢀtriflate 9, purified 27 was stable
in solution at room temperature for >12 h and stable as a solid at 4 ˚C for several weeks. It
ꢀ
ꢀ
ꢀ
was expected that 27 would undergo efficient nucleophilic substitution with Cl , Br , and I
similarly to triflate 9 (Scheme 3) as they differ only by a TMS protected ethynyl group in
position 5 of the pyrimidine ring. Nucleophilic displacement of the 2ʹꢀtriflate of 27 with
tetrabutylammonium chloride (TBAC) or bromide (TBAB) proceeded readily at room
temperature in both toluene and CH Cl as solvent to give 28 and 29, respectively, in
2
2
quantitative yield, Scheme 7. Displacement of the 2ʹꢀtriflate of 27 with tetrabutylammonium
iodide (TBAI) to form the 2ʹꢀiodo species 30 gave variable yields. The maximum yield of 30
was 60%, with yields as low as 16% obtained. Decomposition to multiple products (including
the denitrated compound 31) occurred with longer reaction times, higher temperatures or
increased equivalents of TBAI, Scheme 7. In an attempt to eliminate the variability in
iodination of 27 we assessed alternative iodide sources (Bu
toluene, CH CN, acetone, CH Cl , EtOAc, and THF). Only one combination, TBAI with
acetone, provided any rate enhancement. No combinations prevented formation of I and
4
NI, LiI, NaI, and KI) and solvents
(
3
2
2
2
denitration to give 31 as evident by TLC. Consequently, we continued with TBAI in toluene
and kept reaction times to a minimum to favour the production of 30 over 31.
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Scheme 7. Oneꢀpot conversion of deprotected ribose nucleoside 19 to 2ʹꢀtriflate precursor 27
followed by nucleophilic substitution and inversion of configuration of the 2ʹꢀtriflate with
tetrabutylammonium halide salts to give target 2ʹꢀhalo compounds 28ꢀ30.
There are three reported methods for denitration of 3ꢀNꢀnitroꢀuridines: catalytic
14
20
29
3
hydrogenation over palladium, NaI in acetone, and Bu SnH with AIBN. Each method
has drawbacks for application to our compounds including (i) potential for reduction of the
alkyne and halides of our compounds using hydrogenation, (ii) NaI in acetone is a slow
reaction (c.a. 24 hours) where side reactions are prevalent with time causing a reported lack
of reproducibility, and (iii) use of Bu SnH and AIBN is described for radical reductive
3
30
elimination of 2ʹꢀdeoxyꢀ2ʹꢀhaloꢀuridines, this would be an unwanted competing reaction.
We thus sought to develop alternative denitration conditions that would be compatible with
both 5ꢀethynyl and 2ʹꢀhalo nucleosides.
The only reported reduction of the Nꢀnitro functionality other than in pyrimidines is the
3
1ꢀ33
reduction of N-nitrourethane and N-nitroguanidine using Zn metal in acid.
Although the
34
reported conditions are compatible with alkyne functionality we proceeded cautiously with
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reduction of 28ꢀ30 as alkyne 18 was sensitive to HCl. Additionally, reductive elimination at
higher temperatures is known to occur with these reagents, posing a potential competing
3
5,36
reaction to nitro group reduction.
3
We first assessed Zn and Fe with acetic acid in CH CN
to reduce the nitro group of 14. Both reactions were successful and gave 13 as product,
however with Zn the reaction was rapid (completion within 5 min at 0 ˚C) while with Fe the
reaction was incomplete after more than 2 h at room temperature. The conditions of Zn and
1
1
1
1
1
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1
1
2
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2
2
2
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0
3
acetic acid in CH CN were next explored for reductive denitration of perꢀOꢀacetylated TMSꢀ
alkyne 18. This gave the expected denitrated product 17 in quantitative yield. The now
optimal reductive denitration conditions were applied to 2ʹꢀhalo derivatives 28ꢀ30 and
afforded the expected 2ʹꢀhalo denitrated compounds 31ꢀ33 in good to excellent yields,
Scheme 8. The discrepancy between the yield of 32 and 33 was attributed to product loss
during the physical manipulation of 32 on workup as both reactions were complete and clean
as evidenced by TLC. Some degradation occurred with the iodo compound 30, which
worsened if reaction times were extended or the reaction temperature raised to room
temperature. The final step in the synthesis of the target 2ʹꢀhaloꢀaraꢀEdU compounds was
removal of the silyl protecting groups of 31ꢀ33. To accomplish this, compounds were treated
with Et
give the corresponding target fully deprotected 2ʹꢀhaloꢀaraꢀEdU derivatives 3, 4 and 5 in
2%, 46%, and 82% yields respectively, Scheme 8. Et N·(HF) is a more basic fluoride
3 3 3
N·(HF) in CH CN followed by aqueous workup and silica gel chromatography to
9
3
3
source than Olah’s reagent and is able to remove the TMS group. NOESY confirmed correct
stereochemistry at the 2ʹ position, with Hꢀ2ʹ/Hꢀ4ʹ and Hꢀ3ʹ/Hꢀ6 correlations observed,
consistent with the desired 2ʹS configuration. Furthermore, absence of correlation between Hꢀ
2ʹ/Hꢀ6 confirms arabinosyl configuration.
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Scheme 8. Reductive denitration of 28ꢀ30 followed by desilylation of 31ꢀ32 gave the final
target compounds 3ꢀ5.
To further improve the overall yield of 3ꢀ5 we investigated combining the proceeding two
steps of desilylation and reductive denitration in a oneꢀpot reaction, Scheme 9. Desilylation
was carried out first using Et N·(HF) in CH CN to give the desilylated Nꢀnitro intermediates
3
3
3
34ꢀ36. The subsequent addition of excess Zn reduced the nitro group and quenched any
remaining fluoride by formation of insoluble ZnF . The final products 3, 4 and 5 were
2
purified by solid addition silica gel flash chromatography without further workup and
obtained in 50ꢀ95% yield in oneꢀpot, Scheme 9.
Scheme 9. Oneꢀpot desilyation and reductive denitration of compounds 28ꢀ30 to give the
final target compounds 3ꢀ5.
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9
The overall 8ꢀstep synthetic approach developed to access the target 2ʹꢀhaloꢀaraꢀEdU
analogues, compounds 3, 4 and 5 from uridine 37, is shown in Scheme 10. This synthesis
required the development of a 3ꢀNꢀnitro protecting group strategy to overcome 2,2ʹꢀanhydro
nucleoside formation and that was compatible with a C5ꢀalkyne and lateꢀstage 2ʹ nucleophilic
displacement. The 3ꢀNꢀnitro protecting group in combination with the 2ʹꢀtriflate leaving
group are required to install a halogen at 2ʹ with inversion of configuration. This has not been
established previously with a substituent at the 5ꢀposition of uridine or with a 2ʹ iodo group.
To successfully utilise the 3ꢀNꢀnitro protecting group strategy we developed new synthetic
methodology for its installation and removal that was both rapid (minutes) and compatible
with the multiꢀfunctionalised uridine scaffold. The final synthesis proceeds in 6 steps from
the commercially available 15 to give arabinosyl EdU analogues in overall yields 61%, 47%,
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
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2
3
4
5
6
7
8
9
0
19% for 3, 4 and 5, respectively. Yields for the 8 step synthesis from the natural product
uridine 37 are similar as acetylation and iodination proceed with high efficiency to give 13
and 15, respectively. In summary, in the course of this synthesis we have developed several
synthetic methodologies and protecting group strategies that we anticipate will be a welcome
37
addition to a synthetic chemist’s repertoire when tackling functionalised nucleosides or
other important nitrogen containing compounds.
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4
5
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8
9
0
Scheme 10. Overall synthesis developed for target 2ʹꢀhaloꢀaraꢀEdU 3, 4 and 5 compounds
commencing from uridine 37.
Experimental Section
General Chemistry
All reactions were carried out in dry solvents under anhydrous conditions unless otherwise stated. All
chemicals were purchased from commercial suppliers and used without further purification. All
reactions were monitored by TLC using silica plates with visualisation of eluted bands by UV
fluorescence (λ = 254 nm) and charring with vanillin stain (6 g vanillin in 100 mL of EtOH
containing 1% v/v 98% H
2
SO
4
). Silica gel flash chromatography was performed using silica gel 60 Å
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13
19
(
230ꢀ400 mesh). NMR ( H, C, F, COSY, NOESY, HSQC and HMBC) spectra were recorded on a
Bruker AVANCE III HD 500 MHz NMR spectrometer equipped with a BBO probe at 25 ˚C.
1
13
Chemical Shifts for H and C NMR obtained in DMSOꢀd
6
are reported in ppm relative to residual
1
solvent proton (δ = 2.50 ppm) and carbon (δ = 39.5 ppm) signals, respectively. Chemical Shifts for H
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
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2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
and C NMR obtained in CDCl are reported in ppm relative to residual solvent proton (δ = 7.26
3
ppm) and carbon (δ = 77.2 ppm) signals, respectively. Signal splitting multiplicity is indicated as
follows: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), dd (doublet of doublets), br
(broad signal). Coupling constants are reported in hertz (Hz). LRMS (ESI) data were acquired on a
waters ZQ or Thermo Fisher MSQ Plus single quadruple ESI mass spectrometers using electrospray
as the ionisation technique in positive and/or negative mode as stated. HRMS data were acquired on a
12 T SolariX XR FTꢀICRꢀMS using electrospray as the ionisation technique in positiveꢀion and/or
negative mode as stated. All MS analysis samples were prepared as solutions in either methanol or
acetonitrile. Purity of the compounds were >95% as determined by Thermo Fisher Dionex Ulitmate
3000 series HPLC via UV detection at 254 nm. The melting points are uncorrected. Proton and carbon
atom numbering for NMR chemical shifts are designated as shown below for uridine (37).
General procedure 1. Nꢀnitration of uridine nucleosides with TBAN/TFAA
Uridine derivative and TBAN (2 equiv) were combined dry and placed under an atm of Ar. The
2 2
mixture was dissolved in anhydrous CH Cl (approx. 3.4 mL/mmol) with vigorous stirring. The
resulting mixture was cooled in an ice bath to 0 ˚C then TFAA (2 equiv) added. After approximately 1
min a few drops of MeOH was added to quench the fuming mixture. The resulting pale yellow
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6
mixture was allowed to warm to rt then applied directly to a preconditioned silica gel flash column
chromatography column for purification as described for each compound.
3
General procedure 2. NꢀNitration of uridine nucleosides with KNO /TFAA
0
1
2
3
4
5
6
7
8
9
0
1
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4
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9
0
1
2
3
4
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6
7
8
9
0
To prepare the nitrating mixture TFAA (8 equiv) was added to powdered KNO (8 equiv) previously
3
cooled to 0 ˚C under an atm of argon and the mixture stirred until a white paste formed (~10 min).
Next, the uridine derivative, dissolved in a minimum amount of CH
mixture. The reaction was stirred for 20 min at 0 ˚C or until completion by as evidenced TLC. The
reaction mixture was poured into 1.0 M phosphate buffer solution (20 mL, pH ≈7) then CH Cl (20
2 2
Cl , was added to the nitrating
2
2
mL) added. The organic and aqueous phases were separated and the aqueous phase extracted with
CH Cl (3 × 20 mL). The organic extracts were combined, dried over MgSO , and concentrated in
2
2
4
vacuo. The crude product was purified by silica gel flash column chromatography as described for
each compound.
General procedure 3. Sonogashira coupling of 5ꢀiodoꢀuridine derivatives with ethynylꢀ2ꢀ
trimethysilane
3 4
5ꢀIodoꢀuridine derivative, CuI (0.1 equiv) and Pd(PPh ) (0.05 equiv) were combined and placed
under an atm of argon. The mixture was dissolved/suspended in anhydrous CH Cl followed by
2
2
immediate addition of ethynylꢀ2ꢀtrimethysilane followed by Et
3
3
N. Upon addition of Et N the reaction
mixture clarifies and develops a yellowꢀorange in colour. The mixture was stirred at rt until complete
consumption of the 5ꢀiodoꢀuridine derivative, as evidenced by TLC (2 – 3 h). The solvent was
removed in vacuo and the residue dissolved in EtOAc. The organic phase was washed with 5% HCl(aq)
(
4
20 mL), NaHCO3(sat. aq) (20 mL), 1.0 M EDTAꢀNa (3 × 20 mL) and brine, then dried over MgSO and
concentrated in vacuo. The crude product was purified as described for each compound.
General procedure 4. NꢀDenitration of uridine derivatives
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The Nꢀnitro uridine analogue was dissolved in CH
3
CN and cooled to 0 ˚C. Acetic acid (10 equiv),
silica gel (∼0.8 g/mol) and Zn (5 equiv) were added sequentially. The mixture was stirred vigorously
at 0 ˚C until complete conversion of the starting material as evidenced by TLC. The mixture was
filtered through cotton wool and a silica plug. The eluant was dissolved in EtOAc (20 mL) and
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
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5
5
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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
washed with NaHCO3(aq. sat.)(20 mL) and brine (20 mL) then dried over MgSO
was purified by silica gel flash chromatography as described for each compound.
4
. The crude product
General procedure 5. Desilylation with Et
3
N·(HF)
3
The silylated substrate was dissolved in CH
3
CN and Et N·(HF) (10 equiv) added. The reaction was
3 3
stirred at rt until desilylation was complete as evidenced by TLC. The crude product was purified by
silica gel flash chromatography as described for each compound.
General procedure 6. Oneꢀpot desilylation and Nꢀdenitration
3 3 3
The silyl protected Nꢀnitroꢀuridine analogue was dissolved in CH CN then Et N·(HF) (10 equiv)
added and the mixture stirred at rt. After complete desilylation, as evidenced by TLC, the mixture was
cooled to 0 ˚C then acetic acid (20 equiv), silica gel (∼0.8 g/mol), and Zn (5 equiv) was added
sequentially. After approximately 5 min the mixture was filtered through a plug of cotton wool and
silica gel. The crude product was purified by solid addition silica gel flash chromatography as
described for each compound.
ʹ,3ʹ,5ʹꢀTriꢀOꢀacetylꢀ3ꢀNꢀnitroꢀuridine (14
2 )
Compound 14 was synthesised from 13 employing both general procedure 1 and general procedure 2
to give spectroscopically identical compounds (quantitative yields from both methods). The NMR
1
5 1
spectra for compounds obtained by both methods were consistent with literature values. H NMR
(500 MHz, CDCl ) δ = 7.44 (d, J = 8.4 Hz, 1H), 5.97 (d, J = 5.0 Hz, 1H), 5.92 (d, J = 8.4 Hz, 1H),
.39 – 5.28 (m, 2H), 4.42 – 4.30 (m, 3H), 2.14 (s, 4H), 2.14 (s, 3H), 2.12 (s, 3H). LRMS (ESI): m/z =
3
H
5
4
ꢀ
60 [M –H + HCOOH] .
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5
6
2ʹ,3ʹ,5ʹꢀTriꢀOꢀacetylꢀ5ꢀiodoꢀ3ꢀNꢀnitrouridine (16
)
Compound 16 was synthesised from 15 (0.200 g, 0.403 mmol) by two methods: Method A: according
to general procedure 1. Method B: according general procedure 2. The crude product was purified by
silica gel flash chromatography (Method A: 20% ꢀ 60 EtOAc in nꢀhexane, Method B: 0ꢀ20% EtOAc
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
in CH
2
2
Cl ) to afford the title compound as a colourless solid (general procedure 1: quantitative,
1
general procedure 2: 50 %). Mp = 145ꢀ150 ˚C R
CDCl ) δ
.39 (m, 2H, Hꢀ4ʹ and Hꢀ5ʹ(α or β)), 4.35 (dd, J = 13.4, 3.3 Hz, 1H, Hꢀ5ʹ(α or β)), 2.24 (s, 3H, AcO), 2.14
f
= 0.64 (5% MeOH in CH
2
Cl
2
). H NMR (500 MHz,
= 7.92 (s, 1H, Hꢀ6), 6.05 – 6.01 (m, 1H, Hꢀ1ʹ), 5.36 – 5.28 (m, 2H, Hꢀ2ʹ and Hꢀ3ʹ), 4.45 –
H
3
4
1
3
(
(
s, 3H, AcO), 2.12 (s, 3H, AcO). C NMR (125 MHz, CDCl
3
) δ
C 3
= 170.1 (H CCOO), 169.8
H
3
CCOO), 169.7 (H
3
CCOO), 152.5 (Cꢀ4), 145.3 (Cꢀ2), 143.2 (Cꢀ6), 88.4 (Cꢀ1ʹ), 81.1 (Cꢀ4ʹ), 73.3
(
Cꢀ2), 70.2 (Cꢀ3ʹ), 66.8 (Cꢀ5), 62.9 (Cꢀ5ʹ), 21.2 (H
3
CCOO), 20.6 (H
3
3
CCOO), 20.5 (H CCOO). LRMS
ꢀ
+
(
ESI): m/z = 586 [M – H + HCOOH] . HRMS (ESI+) m/z [M + Na] calcd for C H IN O Na
15
16
3
11
563.9721, found 563.9719.
ʹ,3ʹ,5ʹꢀTriꢀOꢀacetylꢀ5ꢀ(ethynyl(2ꢀtrimethylsilyl))ꢀuridine (17
2 )
Compound 17 was synthesised from 15 (1.700 g, 3.426 mmol) according to general procedure 3. The
crude product was purified by silica gel flash chromatography to give the title compound as an offꢀ
white foam (1.520 g, 95%). R
f
= 0.37 (20% EtOAc in CH
2
Cl
H NMR (500 MHz, CDCl
H, Hꢀ6), 6.10 (d, J = 4.9 Hz, 1H, Hꢀ1ʹ), 5.36 – 5.28 (m, 2H, Hꢀ2ʹ and Hꢀ3ʹ ), 4.42 – 4.32 (m, 3H, Hꢀ4ʹ
2
). The NMR spectroscopic data was
3
9, 41 1
consistent with literature values.
3
) δ = 8.93 (br s, 1H, NH), 7.76 (s,
H
1
13
and Hꢀ5ʹ(α and β)), 2.21 (s, 3H, AcO), 2.12 (s, 3H, AcO), 2.10 (s, 3H, AcO), 0.21 (s, 9H, (Si(CH ) ). C
3 3
NMR (125 MHz, CDCl
3
) δ
C
= 170.2 (H
3
CCOO), 169.9 (H
3
CCOO), 169.7 (H
3
CCOO), 160.8 (Cꢀ4),
149.3 (Cꢀ2), 142.2 (Cꢀ6), 101.6 (Cꢀ5), 100.5 (C≡C
−
Si), 95.0 (C≡C−
Si), 87.3 (Cꢀ1ʹ), 80.4 (Cꢀ4ʹ), 73.4
(
Cꢀ2ʹ), 70.3 (Cꢀ3ʹ), 63.2 (Cꢀ5ʹ), 21.2 (H
3
CCOO), 20.7 (H
3
3 3 3
CCOO), 20.6 (H CCOO), 0.0 (Si(CH ) ).
ꢀ
+
+
LRMS (ESI): m/z = 465 [M ꢀ H] , 467 [M + H] , 489 [M + Na] .
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2ʹ,3ʹ,5ʹꢀTriꢀOꢀacetylꢀ5ꢀ(ethynyl(2ꢀtrimethylsilyl))ꢀ3ꢀNꢀnitroꢀuridine (18
)
Compound 18 was synthesised from 17 (1.5200 g, 3.2582 mmol) according to general procedure 1.
The crude product was purified by silica gel flash chromatography (5% EtOAc in CH Cl ) to afford
2
2
1
title compound as a white solid (quantitative). Mp = 135–137 ˚C. R
500 MHz, CDCl ) δ
= 7.78 (s, 1H, Hꢀ6), 6.08 – 6.05 (m, 1H, Hꢀ1ʹ), 5.36 – 5.32 (m, 2H, Hꢀ2ʹ and Hꢀ
ʹ), 4.42 (ddd, J = 2.6, 0.8 Hz, 1H, Hꢀ4ʹ), 4.38 (d, J = 2.6 Hz, 2H, Hꢀ5ʹ(α and β)), 2.21 (s, 3H, AcO), 2.13
f 2 2
= 0.36 (100% CH Cl ). H NMR
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
(
3
H
3
13
(
(
(
s, 3H, AcO), 2.12 (s, 3H, AcO), 0.22 (s, 9H, Si(CH
CCOO), 169.7 (H CCOO), 169.7 (H CCOO), 153.8 (Cꢀ4), 144.7 (Cꢀ2), 140.9 (Cꢀ6), 102.4
C≡C Si), 101.3 (Cꢀ5), 93.2 (C≡C
Si), 88.3 (Cꢀ1ʹ), 80.9 (Cꢀ4ʹ), 73.4 (Cꢀ2ʹ), 70.1 (Cꢀ3ʹ), 62.9 (Cꢀ5ʹ),
1.0 (H CCOO), 20.6 (H CCOO), 20.5 (H CCOO), ꢀ0.2(Si(CH ) ). LRMS (ESI): m/z = 556 [M – H +
3
)
3
). C NMR (125 MHz, CDCl
3
) δ
C
= 169.9
H
3
3
3
−
−
2
3
3
3
3 3
ꢀ
+
+
25 3
HCOOH] , 534 [M + Na] . HRMS (ESI+) m/z [M + Na] calcd for C20H N O11SiNa 534.1150, found
534.1133.
2ʹ,3ʹ,5ʹꢀTriꢀOꢀ(tertꢀbutyldimethylsilyl)ꢀ5ꢀiodoꢀuridine (23)
5ꢀIodoꢀuridine (22) (1.10 g, 2.97 mmol), tertꢀbutylꢀdimethylsilyl chloride (TBDMSCl) (1.79 g, 11.88
mmol, 4 equiv), and imidazole were combined and placed under an atm of argon. The mixture was
treated with a minimum amount of pyridine (~1 mL) to solubilise the starting materials. The resulting
mixture was heated to 50 ˚C with stirring for 2 h at which point a single product was evident by TLC.
To quench the excess TBDMSCl the mixture was allowed to cool to rt then MeOH (0.5 mL) added
with stirring. After 5 min the mixture was dissolved in CH
then extracted with CH Cl (3 × 20 mL). The CH Cl extracts were combined and washed with 5%
HCl(aq) (20 mL). The 5% HCl(aq) (20 mL) phase was extracted with CH Cl (20 mL). The combined
CH Cl extracts were washed with sat. NaHCO3(aq) and brine, then dried over MgSO and
concentrated in vacuo. The crude product was purified by silica gel flash chromatography (1ꢀ5 %
2 2
Cl and poured into 5% HCl(aq) (20 mL)
2
2
2
2
2
2
2
2
4
MeOH in CH
CH Cl
CDCl ) δ
2
Cl
2
f
) to give the title compound as a colourless foam (quantitative). R = 0.17 (100%
1
2
2
), 0.42 (5% EtOAc in CH
2
Cl
2
), 0.81 (40% EtOAc in n-hexane). H NMR (500 MHz,
= 8.17 (s, 1H, NH), 8.05 (s, 1H, Hꢀ6), 6.03 (d, J = 6.7 Hz, 1H, Hꢀ1ʹ), 4.11 (dd, J = 6.7, 4.4
3
H
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2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
4
4
4
4
4
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5
5
5
5
5
5
5
5
5
6
Hz, 1H, Hꢀ2ʹ), 4.07 – 4.03 (m, 2H, Hꢀ3ʹ and Hꢀ4ʹ), 3.90 (dd, J = 11.6, 2.0 Hz, 1H, Hꢀ5ʹ(α or β)), 3.74 (dd,
J = 11.7, 1.9 Hz, 1H, Hꢀ5ʹ(α or β)), 0.99 (s, 9H, (CH ) SiC(CH ) ), 0.91 (S, 9H, (CH ) SiC(CH ) ), 0.86
3 2
3 3
3 2
3 3
(
(
s, 9H, (CH
CH SiC(CH
) δ
3
)
2
SiC(CH
3
)
3
), 0.20 (s, 6H, (CH
3
)
2
SiC(CH
3
)
3
), 0.10 (s, 3H, (CH
3
)
2
SiC(CH
3
)
3
), 0.07 (s, 3H,
13
3
)
2
3
)
3
), 0.03 (s, 3H, (CH
3
)
2
SiC(CH
3
3
) ), ꢀ0.05 (s, 3H, (CH
3
)
2
SiC(CH
3
)
3
). C NMR (125
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
MHz, CDCl
3
C
= 159.7 (Cꢀ4), 150.0 (Cꢀ2), 144.7 (Cꢀ6), 87.6 (Cꢀ1ʹ), 86.7 (Cꢀ4ʹ), 76.0 (Cꢀ2ʹ), 72.8
SiC(CH ), 25.9 ((CH SiC(CH ), 25.8
(Cꢀ3ʹ), 68.7 (Cꢀ5), 63.3 (Cꢀ5ʹ), 26.5 ((CH
)
2
)
3
3
)
3 3
)
3
3
2
((CH ) SiC(CH ) ), 18.8 ((CH ) SiC(CH ) ), 18.2 ((CH ) SiC(CH ) ), 18.0 ((CH ) SiC(CH ) ), ꢀ4.31
3
3
3
2
2
2
3 3
3 2
3 3
3 2
3 3
3 2
3 3
(
(
(
(CH
(CH
)
)
SiC(CH
3
)
)
3
)), ꢀ4.33 ((CH
3
)
2
SiC(CH
3
)
3
)), ꢀ4.5 ((CH ), ꢀ4.8
3
)
2
SiC(CH
3
)
3
), ꢀ4.7 ((CH
3
)
2
SiC(CH
3
)
3
+
ꢀ
SiC(CH
3
3
), ꢀ4.9 ((CH
3
)
2
SiC(CH
3
)
3
). LRMS (ESI): m/z = 712 [M + H] 710 [M ꢀ H] . HRMS
,
+
ESI+) m/z [M + Na] calcd for C H IN O Si Na 735.2148, found 735.2178.
27 53
2
6
3
2ʹ,3ʹ,5ʹꢀTriꢀOꢀ(tertꢀbutyldimethylsilyl)ꢀ5ꢀ(ethynyl(2ꢀtrimethylsilyl))ꢀuridine (24
)
Compound 24 was synthesised from 23 (1.90 g, 2.66 mmol) according to general procedure 3. The
crude product was purified by silica gel flash chromatography (Gradient: 10ꢀ30% EtOAc in n-hexane
to afford the title compound as faint yellow foam (1.675 g, 92%). R
f
= 0.09 (100% CH
2
2
Cl ), 0.42 (5%
1
EtOAc in CH
2
Cl
2
), 0.82 (40% EtOAc in n-hexane). H NMR (500 MHz, CDCl
3
) δ
H
= 8.06 (s, 1H,
NH), 7.95 (s, 1H, Hꢀ6), 6.02 (d, J = 6.3 Hz, 1H, Hꢀ1ʹ), 4.13 (dd, J = 6.3, 4.4 Hz, 1H, Hꢀ2ʹ), 4.06 (dt, J
7.1, 2.2 Hz, 2H, Hꢀ3ʹ and Hꢀ4ʹ), 3.91 (dd, J = 11.6, 2.0 Hz, 1H, Hꢀ5ʹ(α or β)), 3.74 (dd, J = 11.6, 1.8
=
Hz, 1H, Hꢀ5ʹ(α or β)), 0.98 (s, 9H, (CH ) SiC(CH ) ), 0.91 (s, 9H, (CH ) SiC(CH ) ), 0.86 (s, 9H,
3 2
3 3
3 2
3 3
(
CH
3
3
3
)
)
)
2
2
2
SiC(CH
SiC(CH
SiC(CH
3
3
3
)
)
)
3
3
3
), 0.22 (s, 9H, Si(CH
3
)
3
), 0.19 (s, 3H, (CH
3
)
2
SiC(CH
SiC(CH
). C NMR (125 MHz, CDCl ) δ
Si), 95.3 (C≡C
Si), 88.0 (Cꢀ1ʹ), 86.4 (Cꢀ4ʹ), 76.2
SiC(CH ), 25.9 ((CH SiC(CH ), 25.8
), 18.2 ((CH SiC(CH ), 18.0 ((CH SiC(CH ), 0.0
), ꢀ4.4 ((CH SiC(CH ), ꢀ4.6 ((CH SiC(CH ), ꢀ4.6
3
)
3
), 0.18 (s, 3H,
(CH
), 0.09 (s, 3H, (CH
), ꢀ0.04 (s, 3H, (CH
3
)
2
SiC(CH
3
)
3
), 0.07 (s, 3H, (CH
3
)
2
3
)
3
), 0.03 (s, 3H,
13
(
CH
3
)
2
SiC(CH
3
)
3
3
C
= 161.0 (Cꢀ4),
1
49.3 (Cꢀ2), 142.8 (Cꢀ6), 100.7 (Cꢀ5), 100.0 (C≡C
−
−
(
(
(
Cꢀ2ʹ), 72.6 (Cꢀ3ʹ), 63.1 (Cꢀ5ʹ), 26.4 ((CH
(CH SiC(CH ), 18.7 ((CH SiC(CH
Si(CH ), ꢀ4.3 ((CH SiC(CH
3
)
2
3
)
3
3
)
2
3 3
)
3
)
2
3
)
3
3
)
2
3
)
3
3
)
2
3
)
3
3
)
2
3 3
)
3
)
3
3
)
2
)
3 3
3
)
2
3
)
3
3
)
2
3 3
)
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8
9
ꢀ
(
,
(CH
3
)
2
SiC(CH
3
)
3
), ꢀ4.9 ((CH
3
)
2
SiC(CH
3
)
3
3 2 3 3
), ꢀ5.2 ((CH ) SiC(CH ) ). LRMS (ESI): m/z = 682 [M ꢀ H]
+
+
684 [M + H] . HRMS (ESI+) m/z [M + Na] calcd for C H N O Si Na705.3577, found 705.3553.
32 62
2
6
4
2ʹ,3ʹ,5ʹꢀTriꢀOꢀ(tertꢀbutyldimethylsilyl)ꢀ5ꢀ(ethynyl(2ꢀtrimethylsilyl)ꢀ3ꢀNꢀnitroꢀuridine (25
Compound 25 was synthesised from 24 (0.3200 g, 0.4684 mmol) according to general procedure 1.
The crude product was purified by silica gel flash chromatography (100% CH Cl ) to afford the title
= 0.91 (40% EtOAc in n-hexane), 0.88 (100%
)
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
2
2
compound as a colourless foam (quantitative). R
f
1
CH
2
Cl
2
). H NMR (500 MHz, CDCl
3
) δ
H
= 7.99 (s, 1H, Hꢀ6), 6.02 (d, J = 6.3 Hz, 1H, Hꢀ1ʹ), 4.14 (dd,
J = 6.4, 4.4 Hz, 1H, Hꢀ1ʹ), 4.09 (q, J = 1.9 Hz, 1H, Hꢀ4ʹ), 4.06 (dd, J = 4.5, 2.0 Hz, 1H, Hꢀ3ʹ), 3.93
(
(
dd, J = 11.6, 2.1 Hz, 1H, Hꢀ5ʹ(α or β)), 3.75 (dd, J = 11.7, 1.8 Hz, 1H, Hꢀ5ʹ(α or β)), 0.98 (s, 9H,
CH ) SiC(CH ) ), 0.91 (s, 9H, (CH ) SiC(CH ) ), 0.87 (s, 9H, (CH ) SiC(CH ) ), 0.23 (s, 9H,
3
2
3 3
3 2
3 3
3 2
3 3
Si(CH
CH
MHz, CDCl
3
)
3
), 0.19 (d, 6H, (CH
3
)
2
SiC(CH
3
)
3
), 0.10 (s, 3H, (CH
3
)
2
SiC(CH
SiC(CH
= 154.4 (Cꢀ4), 144.8 (Cꢀ2), 141.5 (Cꢀ6), 102.1 (C≡C Si), 100.5 (Cꢀ5) , 93.6
SiC(CH ), 25.9
SiC(CH ), 18.0
SiC(CH ), ꢀ4.6
). LRMS (ESI): m/z = 746 [M
Na750.3427, found 750.3433.
3 3
) ), 0.08 (s, 3H,
1
3
(
3
)
2
SiC(CH
3
)
3
), 0.03 (s, 3H, (CH
3
)
2
SiC(CH
)
3 3
), ꢀ0.05 (s, 3H, (CH
)
3 2
3 3
) )). C NMR (125
3
) δ
C
−
(
(
(
(
C≡C
−
Si), 89.1 (Cꢀ1ʹ), 87.2 (Cꢀ4ʹ), 76.5 (Cꢀ2ʹ), 72.7 (Cꢀ3ʹ), 63.1 (Cꢀ5ʹ), 26.4 ((CH
3
)
2
3 3
)
(CH
(CH
(CH
3
)
)
)
2
SiC(CH
SiC(CH
SiC(CH
3
3
3
)
)
)
3
3
3
), 25.8 ((CH
3
)
2
SiC(CH
3
)
3
), 18.7 ((CH
SiC(CH
), ꢀ5.0, ꢀ5.2 ((CH SiC(CH
Si
3
)
2
SiC(CH
3
)
3
), 18.2 ((CH
3
)
2
3 3
)
3
3
2
2
), ꢀ0.2 (Si(CH
)
3 3
), ꢀ4.3 ((CH
3
)
2
3
)
3
), ꢀ4.4 ((CH
3
)
2
3 3
)
), ꢀ4.7 ((CH
3
)
2
SiC(CH
3
)
3
3
)
2
3 3
)
+
+
+
H
3
O] , HRMS (ESI+) m/z [M + Na] calcd for C32
H
61
N
3
O
8
4
5
ꢀ(Ethynyl(2ꢀtrimethylsilyl))ꢀ3ꢀNꢀnitroꢀuridine (19)
Compound 19 was synthesised by two methods. Method A: desilylation of 25. Method B:
deacetylation of 18. Method A: Within a polypropylene tube 25 (0.3000 g, 0.412 mmol) was
3 3 5
dissolved in CH CN (1.5 mL) and cooled to ꢀ40 ˚C in a dry ice/CH CN bath. Then py·(HF) (0.1606
mL, 6.179 mmol, 15 equiv) previously cooled to ꢀ20 ˚C was added to the mixture and allowed to
warm to rt. After 18 h at rt the mixture was cooled to ꢀ40 ˚C then added directly to a silica gel flash
2 2
chromatography column preconditioned with 5% MeOH in CH Cl . The pcolumn was eluted using a
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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
2 2
gradient of 5ꢀ10% MeOH in CH Cl to afford the title compound as an orange/yellow foam (0.1420 g,
90%). Method B: 18 (0.6000 g, 1.173 mmol) and pꢀtoluenesulfonic acid (0.127 g, 1.1730 mmol, 1
equiv) were placed under an atm of argon and dissolved in MeOH (3 mL) and a minimum amount of
2 2
CH Cl . The mixture was stirred at rt for 48 h then purified by solid addition silica gel flash
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
chromatography (Gradient: 5ꢀ8% MeOH in CH Cl ) to give the title compound as a colourless
2
2
1
foam/gum (80 %, 0.3602 mg). R
f
= 0.59 (10% MeOH in CH
2
Cl
2
). H NMR (500 MHz, CDCl
3
H
) δ =
8.66 (s, 1H, Hꢀ6), 5.66 (d, J = 2.9 Hz, 1H, Hꢀ1ʹ), 5.60 (d, J = 5.0 Hz, 1H, OHꢀ2ʹ), 5.37 (t, J = 4.4 Hz,
1H, OHꢀ5ʹ), 5.11 (d, J = 6.3 Hz, 1H, OHꢀ3ʹ), 4.14 (td, J = 4.8, 2.7 Hz, 1H, Hꢀ2ʹ), 4.01 (q, J = 6.2 Hz,
1H, Hꢀ3ʹ), 3.92 (dt, J = 6.6, 2.4 Hz, 1H, Hꢀ4ʹ), 3.78 (ddd, J = 12.1, 4.5, 2.6 Hz, 1H, Hꢀ5ʹ(α or β)), 3.61
13
(ddd, J = 12.2, 2.2 Hz, 1H, Hꢀ5ʹ(α or β)), 0.20 (s, 9H, Si(CH
)
3
). C NMR (125 MHz, CDCl
C
) δ = 154.8
3
3
(
Cꢀ4), 145.0 (Cꢀ6), 144.5 (Cꢀ2), 98.9 (C≡C Si), 97.3 (Cꢀ5), 95.5 (C≡C
−
−Si), 90.9 (Cꢀ1ʹ), 84.6 (Cꢀ4ʹ),
ꢀ
7
4.0 (Cꢀ2ʹ), 68.1 (Cꢀ3ʹ), 59.1 (Cꢀ5ʹ), ꢀ0.2 (Si(CH ) ). LRMS (ESI): m/z = 384 [M ꢀ H] , 430 [M – H +
3
3
ꢀ
+
19 3 8
HCOOH] . HRMS (ESI+) m/z [M + Na] calcd for C14H N O SiNa 408.0834, found 408.0818.
5
ꢀ(Ethynyl(2ꢀtrimethylsilyl))-3-Nꢀnitroꢀ3ʹ,5ʹꢀOꢀ(tetraisopropyldisiloxaneꢀ1,3ꢀdiyl)ꢀ2ʹꢀOꢀtriflylꢀ
uridine (27)
Compound 19 (0.300 g, 0.778 mmol) was dissolved in pyridine (2 mL) and cooled to 0 ˚C then 1,3ꢀ
dichloroꢀ1,1,3,3ꢀtetraisopropyldisiloxane (TIPDSiCl ) (0.28 mL, 1.1 equiv) added slowly. The
reaction was allowed to warm to rt spontaneously overnight, then cooled again to 0 ˚C and Tf O (1.0
2
2
M CH
was dissolved in EtOAc (20 mL) and washed with 5% HCl(aq) solution (20 mL) and phosphate buffer
20 mL, pH ≈ 7, 1.0 M), with the aqueous phases back extracted with EtOAc (2 × 20 mL). The
organic extracts were combined, dried over MgSO and concentrated in vacuo. The crude product was
purified by silica gel flash chromatography (100% CH Cl ) to afford the title compound as a waxy
2 2
Cl solution) (1.2 mL, 1.5 equiv) added dropwise and stirred for 5 min at 0 ˚C. The mixture
(
4
2
2
f
solid (0.4726 g, 80%). Mp = 98 – 100 ˚C (decomp, darkens at 70 ˚C). R = 0.88 (20% EtOAc in n-
1
hexane). H NMR (500 MHz, CDCl
3
) δ
H
= 7.92 (s, 1H, Hꢀ6), 5.89 (s, 1H, Hꢀ1ʹ), 5.23 (d, J = 4.2 Hz,
1
H, Hꢀ2ʹ), 4.45 (dd, J = 9.5, 4.3 Hz, 1H, Hꢀ3ʹ), 4.33 (d, J = 13.9 Hz, 1H, Hꢀ5ʹ(α or β)), 4.17 (dd, J = 9.5,
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2 2
.5 Hz, 1H, Hꢀ4ʹ), 4.03 (dd, J = 14.0, 2.7 Hz, 1H, Hꢀ5ʹ(α or β)), 1.18 – 0.97 (m, 28H, (Si(iꢀPr) ) ), 0.22
13
(s, 9H, Si(CH
3
)
3
). C NMR (125 MHz, CDCl
3
C
) δ = 153.9 (Cꢀ4), 144.2 (Cꢀ2), 140.1 (Cꢀ6), 102.8
(C≡C−Si), 100.9 (Cꢀ5), 92.9 (C≡C−
Si), 88.8 (Cꢀ1ʹ), 87.3 (Cꢀ2ʹ), 82.7 (Cꢀ4ʹ), 66.9 (Cꢀ3ʹ), 58.6 (Cꢀ5ʹ),
17.6 SiCH(CH ) , 17.6 SiCH(CH ) , 17.4 SiCH(CH ) , 17.3 SiCH(CH ) , 17.0 SiCH(CH ) , 16.9
3
2
3 2
3 2
3 2
3 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
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
SiCH(CH
3
)
)
2
, 16.9 SiCH(CH
3
)
2
, 16.8 SiCH(CH
3
)
2
, 13.7 SiCH(CH
3
)
2
3 2
, 13.0 SiCH(CH ) , 12.9
+
SiCH(CH
3
2
, 12.7 SiCH(CH
3
)
2
, ꢀ0.2 (Si(CH
3
)
3
). LRMS (ESI): m/z = 688 [MꢀTMS+H] , 564 [Mꢀ
+
+
TfO+H] . HRMS (ESI+) m/z [M + Na] calcd for C27
H
44
F
3
N
3
O11SSi
3
Na 782.1848, found 782.1829.
(2ʹS)ꢀ2ʹꢀChloroꢀ2ʹꢀdeoxyꢀ5ꢀ(ethynyl(2ꢀtrimethylsilyl))ꢀ3ꢀNꢀnitroꢀ3ʹ,5ʹꢀOꢀ
(tetraisopropyldisiloxaneꢀ1,3ꢀdiyl)ꢀuridine (28
Compound 27 (0.3000 mg, 0.3950 mmol) and TBAC (0.1317 g, 0.474 mmol, 1.2 equiv) were
combined and placed under an atm of argon. The mixture was dissolved in CH Cl (2 mL) and stirred
at rt for 30 min. The crude product was purified by silica gel flash chromatography (100% CH Cl ) to
)
2
2
2
2
f
afford the title compound as a white solid (quantitative). Mp = 145 – 150 ˚C. R = 0.68 (20% EtOAc
1
in n-hexane). H NMR (500 MHz, CDCl
3
) δ
H
= 7.77 (s, 1H, Hꢀ6), 6.27 (d, J = 6.4 Hz, 1H, Hꢀ1ʹ), 4.57
(
dd, J = 8.1, 6.4 Hz, 1H, Hꢀ2ʹ), 4.39 (t, J = 8.2 Hz, 1H, Hꢀ3ʹ), 4.15 (dd, J = 13.2, 2.5 Hz, 1H, Hꢀ5ʹ(α or
β)), 4.06 (dd, J = 13.3, 3.1 Hz, 1H, Hꢀ5
ʹ(α or β)), 3.84 (dt, J = 8.2, 2.7 Hz, 1H, Hꢀ4ʹ), 1.17 – 0.95 (m,
13
28H, (Si(iꢀPr)
2
)
2
), 0.23 (s, 9H, Si(CH
3
)
3
). C NMR (125 MHz, CDCl
3
) δ
Si), 84.6 (Cꢀ1ʹ), 83.0 (Cꢀ4ʹ), 75.4 (Cꢀ3ʹ), 62.7
), 17.75 (SiCH(CH ), 17.6 (SiCH(CH ), 17.5 (SiCH(CH ),
), 17.24 (SiCH(CH ), 17.22 (SiCH(CH ), 14.1 (SiCH(CH ), 13.3
), 12.75 (SiCH(CH ), 12.67 (SiCH(CH ), 0.0 (Si(CH ). LRMS (ESI): m/z = 690,
C
= 154.3 (Cꢀ4), 144.8 (Cꢀ2),
141.3 (Cꢀ6), 102.4 (C≡C
−
Si), 100.5 (Cꢀ5), 93.5 (C≡C−
(
Cꢀ2ʹ), 60.2 (Cꢀ5ʹ), 17.84 (SiCH(CH
7.3 (SiCH(CH
SiCH(CH
3
)
2
3
)
2
3
)
2
3 2
)
1
3
)
2
3
)
2
)
3 2
3 2
)
(
3
)
2
3
)
2
3
)
2
3 3
)
35
37
ꢀ
+
35
37
692 [M – H + HCOOH, Cl, Cl] , 468 [M + H] , 668, 670 [M + Na, Cl, Cl]. HRMS (ESI+) m/z
+
35
[
3 8 3
(M + Na) , Cl] calcd for C26H44ClN O Si Na 668.2016, found 668.2009.
(
(
2ʹS)ꢀ2ʹꢀBromoꢀ2ʹꢀdeoxyꢀ5ꢀ(ethynyl(2ꢀtrimethylsilyl))ꢀ3ꢀNꢀnitroꢀ3ʹ,5ʹꢀOꢀ
tetraisopropyldisiloxaneꢀ1,3ꢀdiyl)ꢀuridine (29
)
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