Novel antiviral activity of l-dideoxy bicyclic nucleoside analogues versus vaccinia and measles viruses in vitro

Article abstract of DOI:10.1021/jm301778x

Dideoxy bicyclic pyrimidine nucleoside analogues (ddBCNAs) with d-chirality have previously been described by us to inhibit replication of human cytomegalovirus. We herein report for the first time that activity against vaccinia virus (VACV) was achieved

Full text of DOI:10.1021/jm301778x

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Article
Novel antiviral activity of L-dideoxy bicyclic nucleoside
analogues versus vaccinia and measles viruses in vitro
Christopher McGuigan, Karen Hinsinger, Laura Farleigh, Ranjith N Pathirana, and Joachim J Bugert
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm301778x • Publication Date (Web): 15 Jan 2013
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Novel antiviral activity of Lꢀdideoxy bicyclic nucleoside
analogues versus vaccinia and measles viruses in vitro
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†
†
‡
†,º
Christopher McGuigan, Karen Hinsinger, Laura Farleigh, Ranjith N. Pathirana, and
‡
,*
Joachim J. Bugert
†
. Cardiff School of Pharmacy & Pharmaceutical Sciences, Cardiff University, Redwood Building, King
Edward VII Avenue, Cardiff, CF10 3NB, UK.
. Cardiff Institute of Infection and Immunity/ Medical Microbiology and Infectious Diseases, School of
‡
Medicine, Cardiff University, Heath Park, Cardiff, CF14 4XN, UK.
º. Department of Chemistry, University of Ruhuna, Matara, Sri Lanka.
Abstract
Dideoxy bicyclic pyrimidine nucleoside analogues (ddBCNAs) with [D] chirality have
previously been described by us to inhibit replication of human cytomegalovirus. We
herein report for the first time that activity against vaccinia virus (VACV) was achieved
using novel [L] analogues. A structure activity relationship was established: Antiviral
activity versus VACV was highest with an ether side chain with an optimum of nC H ꢀ
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OꢀnC H . This gave an IC of 190 nM, a 60 fold enhancement over the FDA approved
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antiviral cidofovir. Interestingly, LꢀddBCNAs also inhibit wild type measles virus
syncytia formation with a TCID of 7.5 ꢁM for the lead compound. We propose that Lꢀ
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ddBCNAs represent significant innovative antiviral candidates versus measles and
poxviruses and we suggest a mechanism of action versus one or more cellular targets that
are essential for viral replication.
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Introduction
Small pox – variola was declared eradicated in 1979. However, threats of bioterrorism
have reꢀignited interest in finding suitable treatments that could be used in case of a small
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pox outbreak. At present, the only drug that has been approved for treatment of
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poxviruses is cidofovir. This drug is not ideal as it has severe side effects. Vaccinia
Virus (VACV) is a convenient surrogate poxvirus for the development of new
antipoxviral agents in vitro VACV strains that are resistant to cidofovir have also been
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described. Alternative treatments would also be useful for related infections with
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Molluscum contagiosum virus and human monkey pox virus.
A number of new potential antipoxviral agents have recently been reported. These
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include STꢀ246, a molecule that targets the F13L protein of the virus, although resistance
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to this drug has already been described, and terameprocol, which reduces the spread of
the virus by inhibiting the formation of actin tails at the surface of infected cells (cellular
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target).
Human measles is usually a mild childhood infection but is a known cause of severe
pneumonia in developing countries with over 1 million fatalities worldwide in 2005. The
live measles vaccine is not given to infants under 12 months in age and maternal measles
virus antibodies are lost at 4ꢀ9 months, so individuals are susceptible to the virus before
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the vaccination is given. The ideal antiviral therapy for measles would be a small
molecule inhibitor that is safe, effective, inexpensive to reproduce and stable. Having a
host target may offer the additional benefit of reducing the emergence of rapid resistance.
Ribavirin inhibits measles virus in vitro but only at supraꢀphysiological levels (IC of
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160 ꢂM), but is not effective for the treatment of measles virus infections. At present
there are no measles antiviral drugs on the market.
Previously, we have reported novel potent and selective inhibitors of human
cytomegalovirus based on Dꢀdideoxy bicyclic furanopyrimidine nucleoside analogues
(DꢀddBCNAs) bearing alkyl side chains at the C5ꢀposition, with action suggested early in
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the virus lifecycle. Further potency of DꢀddBCNAs versus VACV suggested a non
virus specific mechanism of action and possibly a cellular target.
In order to further increase potency and explore the structure activity relationships
(
SARs), we were interested in other unusual sugars and particularly in unnatural Lꢀ
configuration nucleosides. Indeed, several Lꢀnucleosides have emerged as potent antiviral
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agents,
which
include
βꢀLꢀ[2ꢀ(hydroxymethyl)ꢀ1,3ꢀoxathiolanꢀ4ꢀyl]cytosine
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(
Lamivudine, 3TC), 5ꢀfluoroꢀβꢀLꢀ2′,3′ꢀdideoxyꢀ3′ꢀthiacytidine (Emtricitabine, FTC),
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ꢀfluoroꢀβꢀLꢀ2′,3′ꢀdideoxycytidine (LꢀFddC), βꢀLꢀ2′ꢀfluoroꢀ5ꢀmethylarabinofuranosyl
1
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uracil (clevudine, LꢀFMAU),
2′,3′ꢀdideoxyꢀ2′,3′ꢀdidehydroꢀβꢀLꢀ5ꢀfluorocytidine
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(
Elvucitabine, βꢀLꢀFd4C) and carbocyclic nucleoside LꢀCd4U. Moreover, it has been
reported that some of them have more potent antiviral activity than their Dꢀenantiomers
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1,22
with reduced toxicity.
Therefore, it was of particular interest to synthesise and study the antiviral activity of Lꢀ
dideoxy bicyclic furanopyrimidine nucleoside analogues (LꢀddBCNAs) with different
side chains.
Results and discussion
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Chemistry. We previously presented the successful synthesis of different DꢀBCNAs.
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For the Dꢀdideoxy family, the bicyclic nucleosides were obtained from the key synthon
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Dꢀ5ꢀiodoꢀ2’,3’ꢀdideoxyuridine (DꢀIddU) via Sonogashira reaction with different
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acetylenes followed by an intramolecular cyclisation. The described formation of the Dꢀ
IddU started from the Dꢀ2’ꢀdeoxyuridine. We wanted to apply our methodology for the
synthesis of the LꢀddBCNAs. However, the Lꢀ2’ꢀdeoxyuridine is not commercially
available. Thus, we sought a new route, illustrated in Scheme 1, starting from Lꢀarabinose
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Scheme 1. Synthetic route to target LꢀddBCNAs.
(
(
(
i) NCꢀNH , NH OH, MeOH, rt, 3 days (70%); (ii) Methyl propiolate, EtOH, reflux, 3 h
2 4
69%); (iii) AcBr, CH CN, reflux, 2 h (75%); (iv) Zn, glacial AcOH, EtOH, reflux, 2 h
3
53%); (v) H , Pd/C, MeOH, rt, 18 h (97%); (vi) 1) I , CAN, CH CN, reflux, 6 h; 2)
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MeONa, MeOH, rt, 18 h (60% over 2 steps); (vii) acetylenes, Pd(PPh ) , CuI, DIPEA,
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o
DMF, rt, 18 h and then addition of Et N, CuI, 80 C, 8 h (7ꢀ53%).
3
First, reaction of the carbohydrate 1 with cyanamide, according to the methodology
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reported by Ladurée et al., afforded the corresponding bicyclic oxazoline 2. Treatment
with methyl propiolate in methanol yielded the 2,2’ꢀanhydroꢀnucleoside 3 which via a
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bromoacetylation reaction with acetyl bromide in acetonitrile gave the nucleoside 4 in
good yield. Reductive βꢀelimination with activated zinc powder in the presence of glacial
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acetic acid afforded the 5’ꢀOꢀacetylꢀ2’,3’ꢀdidehydroꢀ2’,3’ꢀdideoxyuridine 5 which gave
almost quantitatively the dideoxy nucleoside 6 upon catalytic hydrogenation. The Lꢀ5ꢀ
iodoꢀ2’,3’ꢀdideoxyuridine 7 was obtained after first iodination at the C5ꢀposition of the
3
0
base with iodine in the presence of ammonium cerium(IV) nitrate (CAN) and then
deacetylation using sodium methoxide, in 60% yield over two steps. Palladiumꢀcatalysed
Sonogashira coupling of the key synthon 7 with a series of acetylenes gave the
corresponding 5ꢀalkynyl nucleosides. Addition of copper (I) iodide in situ with heating of
the reaction resulted in their intramolecular cyclisation to afford the desired
furanopyrimidine compounds 8aꢀu with alkyl, aryl or ether side chains. Indeed, we have
reported in the case of Dꢀdeoxy BCNAs that aryl and ether groups decrease the
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lipophilicity of nucleosides and improve their activity.
The alkyl and aryl acetylenes needed for the Sonogashira reaction were commercially
available. However, the ether alkynes were synthesised following two different pathways
depending on the target ethers (Scheme 2). The first method (pathway A) started with the
mesylation of alkynꢀ1ꢀols to obtain the intermediates 9aꢀc, which were then coupled with
different alcohols to give the ethers 10aꢀj. In the second method (pathway B), the starting
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materials were alcohols, which were first transformed into the mesylated compounds 11a
and 11b and then reacted with selected alkynols to obtain the ethers 12a and 12b.
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Scheme 2. Synthetic routes to alkynyl ethers 10aꢀj and 12aꢀb.
o
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i) MsCl, Et N, THF, rt, 18 h; (ii) Alcohol, NaH, THF, 70 C, 24 h; (iii) Alkynol, NaH,
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THF, 60 C, 36 h.
The structure and the purity of all LꢀddBCNAs were confirmed by spectroscopic,
chromotographic and analytical data before they were tested for inhibition of viral
infections.
Antiviral activity. The LꢀddBCNAs 8aꢀu were evaluated by luciferase assay for their
ability to inhibit the early stages of VACV replication from entry to early gene
transcription in RK 13 cells (Table 1), following the initial notion of an effect early in the
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Table 1. LꢀddBCNA structures, clogP and luciferase reporter assay versus VAVC.
a
b
Cpds
8a
R
clogP
2.60
3.13
3.68
4.19
4.72
5.77
3.64
4.17
3.30
3.83
4.36
4.89
5.42
3.61
4.14
4.23
4.01
4.89
4.89
5.46
5.03
Luciferase reporter (%)
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nC H
70.7
43.2
41.9
38.1
45.7
96.4
43.7
73.6
79.9
46.8
27.5
27.7
35.7
79.9
69.9
64.3
70.7
37.2
26.1
43.9
60.5
6
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b
nC H
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c
nC H
8
8
d
nC H
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8
e
nC H
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8f
nC H
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8
g
h
PhꢀnBu
PhꢀnC H
8
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8
i
nC H ꢀOꢀnEt
9 18
8j
nC H ꢀOꢀnPr
9 18
8
k
nC H ꢀOꢀnBu
9 18
8
l
nC H ꢀOꢀnC H
9 18 5
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8
m
nC H ꢀOꢀnC H
9 18 6
13
8n
8o
nC H ꢀOꢀisoPr
9 18
nC H ꢀOꢀsecBu
9
18
8
p
nC H ꢀOꢀisoBu
9 18
8q
nC H ꢀOꢀterBu
9 18
8
r
nC H ꢀOꢀnC H
8 16 6 13
8s
nC H ꢀOꢀnC H
4 8 10 21
8
t
CH ꢀOꢀnC H
2
13 27
8
u
nC H ꢀOꢀMe
13
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Cidofovir
ꢀ2.39
94.6
a
b
clogP values calculated with Chemdraw ultra 12.0. Luciferase reporter activity as
percent of no drug control (100%) in RK13 cells.
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Luciferase inhibition was observed in compounds 8aꢀh bearing alkyl and aryl chains,
with an optimum at the analogue 8d (nC H chain). The apparent reduction in activity
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for longer alkyl chain derivatives 8e (nC H chain) and 8f (nC H chain) may be
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caused by their low water solubility (calculated logP data of 4.72 and 5.77). This led to
the synthesis of ether compounds with a nonyl chain prior to oxygen (8iꢀq). The best
reduction of luciferase values was then identified among the LꢀddBCNAs 8jꢀm with
linear ether side chains and a SAR regarding the length of the chain emerged; best
activity was found for compounds 8k and 8l with butyl and pentyl termini. Using the
length of side chain in 8l alteration of the oxygen atom position toward the base
(analogues 8rꢀu) did not improve the activity. However the good luciferase values of
derivatives 8e (nC H ꢀOꢀnC H chain) and 8f (nC H ꢀOꢀnC H chain) in spite of high
8
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4
8
10 21
calculated logP data (of 4.89) shows that lipophilicity of compounds is only one
parameter in their mechanism of action.
These results were followed up for the most potent ether compounds by plaque assay and
determination of IC50 in BSCꢀ1 cells with data shown in Table 2. The effect of Lꢀ
ddBCNAs on cell viability (CC ) was tested measuring ATP levels in live BSCꢀ1 cells
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(CellTiterꢀGlo® Luminescent Cell Viability Assay).
Table 2. IC and CC data versus VACV of selected ether LꢀBCNAs.
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50
a
a
b
Cpds
R
IC50 (ꢂM) CC50 (ꢂM) CC50 (ꢂM)
8
8
i
j
nC H ꢀOꢀnEt
12.9
1.9
79.4
89.1
>100
>100
>100
>100
9
18
nC H ꢀOꢀnPr
9
18
8
k
nC H ꢀOꢀnBu
1.8
9
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l
nC H ꢀOꢀnC H
0.19
0.19
1.7
>100
>100
9
18
5
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8
m
nC H ꢀOꢀnC H
74.1
>100
9
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8
r
nC H ꢀOꢀnC H
ꢀ
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s
nC H ꢀOꢀnC H
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2.2
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ꢀ
ꢀ
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8
8t
CH ꢀOꢀnC H
3.4
2
13 27
Cidofovir
11.5
>100
>100
a
b
in BSC-1 cells. in HFFF cells.
The IC values from 4 day plaque assays of ethers with increasing chains (compounds
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8
iꢀm) followed a similar SAR to that identified for the 2 hour luciferase activity data.
Both assays showed the same trend perhaps implying the same mechanism of action. The
hour luciferase assay was used as a predictor of compound efficacy. Moreover, all Lꢀ
2
ddBCNAs with the exception of 8i performed better than the cidofovir control. The IC50
of nucleosides 8l and 8m of 0.19 ꢂM is a 60 fold improvement of activity.
The ether series LꢀddBCNAs have a mild effect on BSCꢀ1 cell viability with a selectivity
index of >500 for the lead compound 8l (IC = 0.19 ꢂM and CC >100 ꢂM). BSCꢀ1
5
0
50
cells did not show visible cytotoxicity in phase contrast microscopy after 2 hours in the
luciferase reporter assays or after 4 days at CC in the plaque reduction assays. The mild
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toxicity observed in BSCꢀ1 cells for some compounds does not follow the SAR of the
ether series LꢀddBCNAs and is not predictive of antiviral activity. No cytotoxic effects of
LꢀddBCNAs 8iꢀm were observed in HFFF cells up to 100 ꢂM.
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The lead bicyclic nucleosides 8l was then evaluated by TCID assay for its ability to
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inhibit formation of cytopathogenic effect of adenovirus, herpesvirus (type 1 and 2) and
vaccine and wild type measles viruses (Table 3).
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Table 3. TCID (ꢂM) of compound 8l versus other viruses.
5
0
HFFF BSCꢀ1 B95a
Adenovirus 5
>50 >50 nd
>50 >50 nd
HSV 1 NCPV 17+
HSV 2 NCPV 132349
>50 >50 nd
a
Measles virus Edmonston (vaccine) nd
>50 >50
a
a
Measles virus wild type mWTFb
nd
nd
7.5
a
cells not susceptible. nd: not determined.
Nucleoside 8l was unable to suppress the cytopathogenic effects of adeno, herpes simplex
and measles vaccine viruses in BSCꢀ1 and HFFF cells. However, it inhibits wild type
measles virus syncytia formation by 50% at 7.5 ꢂM. This is a 150 fold improvement over
1
0
ribavirin in vitro (IC of 1160 ꢂM; Figure 1). When LꢀddBCNAs 8iꢀm of the ether
5
0
series were tested versus wild type measles virus in B95a cells (Figure 1), the SAR was
found to follow closely the one found previously for VACV in RK13 (luciferase assay)
and BSCꢀ1 (plaque assay) cells. Cidofovir, a DNA polymerase inhibitor, active against
VACV, shows as expected, no effect in this assay. The ribavirin IC (1.2 mM, data not
50
shown) is out of the range of this figure. Wortmannin, a furanosteroid inhibitor of PI3
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like kinases, inhibited measles wild type virus in this assay with an IC of 75 ꢂM. (data
5
0
not shown).
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Figure 1. SAR of ether series of LꢀddBCNAs versus wild type measles virus.
The SAR observed versus both vaccinia and wild type measles viruses is highly
suggestive of a drug interaction with a specific target. While measles wild type viruses
are only able to utilize the CD150 receptor found on human and simian B cells, the
vaccine strain of measles virus uses the ubiquitous CD46 receptor. CD150 and CD46
receptors are located in different membrane microdomains, so a differential effect of Lꢀ
ddBCNAs is possible. Membrane microdomains are involved in both measles and
3
2, 33
vaccinia virus replication.
Is it possible that the reduction of VACV plaque size herein observed could be due to an
3
4
effect on the dissemination of VACV in cell culture ‘conveyor belt’.
8
The phenolic antioxidant terameprocol shows a similar activity profile to LꢀddBCNAs,
where the target of the antiviral activity is the actinꢀcytoskeleton, clearly a cellular target.
The main effect is a quantifiable reduction in plaque size, just as observed with the Lꢀ
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ddBCNAs. With a cellular target of this nature, viral infection in the host would not be
completely shut down, but rather the dissemination of the virus would be affected.
Inhibiting specific aspects of a virus lifecycle induces the virus to develop resistance to
the drug by altering the target of the drug. Targeting cellular processes means that there
may be a possibility of increased toxicity, but reduces the opportunity for resistance.
Antivirals versus cellular targets are not subject to viral resistance, which may develop
quickly with all virusꢀtargeting drugs.
0
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Conclusion
In summary we have described the successful synthesis of substituted Lꢀdideoxy
bicyclic furanopyrimidine nucleoside analogues in 8 steps starting from Lꢀarabinose.
A series of etherꢀsubstituted compounds has been shown to induce suppression of
orthopoxꢀ and measles virus replication in vitro. Actually, early (2 hours p.i.) luciferase
reporter assays against VACV and plaque/syncytium reduction assays against VACV and
measles virus highlighted a SAR regarding the length of the chain. Notably the lead Lꢀ
ddBCNA 8l with nC H ꢀOꢀnC H side chain possessed an IC50 value of 0.19 ꢁM
9
18
5
11
against VACV, with good selectivity index, and a TCID50 value of 7.5 ꢁM against
measles virus.
We propose that interactions between LꢀddBCNAs and membrane microdomains are
involved in the inhibition of virus replication. This kind of host targeting action would
prevent drug resistance issues. Further studies about the mechanism of action of Lꢀ
ddBCNAs are currently under investigation in our laboratory.
Experimental section
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Chemistry. The numbering of the bicyclic ring follows the recommended IUPAC
nomenclature guidelines. The naming of the compounds follows IUPAC nomenclature
and/or standard accepted nomenclature for nucleoside chemistry.
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All solvents used were anhydrous and used as supplied by Aldrich. All nucleosides and
solid reagents were dried for several hours under high vacuum over potassium hydroxide.
All glassware was oven dried at 130 °C for several hours or overnight and allowed to
cool in a desiccator or under a stream of dry nitrogen. TLC was performed on precoated
aluminumꢀbacked plates (60 Fꢀ54, 0.2 mm thickness; supplied by E. Merck AG,
Darmstadt, Germany) and spots visualized by UV (at 254 or 336 nm depending on the
compound). Column chromatography was performed with silica gel supplied by Fisher
1
13
(60A, 35ꢀ70 ꢁm). H and C NMR spectra were recorded in a Bruker spectrometer (500
MHz and 125 MHz, respectively) and autoꢀcalibrated to the deuterated solvent reference
1
3
peak. All C NMR spectra were protonꢀdecoupled. Coupling constants (J) are measured
in Hertz. The following abbreviations are used in the assignment of NMR signals: br
(broad), s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), dd (doublet of
doublet), ddd (doublet of doublet of doublet), dt (doublet of triplet). Low resolution and
accurate mass analysis were performed by the service at the Department of Chemistry,
University of Wales, Cardiff, UK, with electrospray (ES). The ≥95% purity of all the
final compounds was confirmed using analytical HPLC analysis. HPLC experiments
were done on a Thermo Fisher Scientific Spectra System SCM1000 provided with a
System Controller SN4000, a pump Spectra System P4000 and a Spectra UV2000
detector set (detection at 254 nm) and using a 5 ꢁM Hypersil GOLD (150 × 4.6 mm)
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reverse phase column eluting with the indicated mobile phase with a flow rate of 1
mL/min.
2
ꢀaminoꢀ[1,2ꢀd]arabinosylꢀoxazole (2). A suspension of Lꢀarabinose 1 (17 g, 113
0
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mmol), cyanamide (10 g, 238 mmol, 2.1 eq.) in anhydrous MeOH (30 mL) and 6M
NH OH (5 mL) were stirred at room temperature for 65 h. The solvents were then
4
evaporated from the resulting suspension under reduced pressure. The crude was
1
triturated with MeOH to give a white solid (13.16 g, 66%). H NMR (500 MHz, DMSOꢀ
d ): δ 6.27 (2H, br s, NH ), 5.67 (1H, d, J = 5.5 Hz, Hꢀ1’), 5.39 (1H, br s, OHꢀ3’), 4.68
6
2
(1H, bs, OHꢀ5’), 4.53 (1H, d, J = 5.5 Hz, Hꢀ2’), 4.02ꢀ3.99 (1H, m, Hꢀ3’), 3.67ꢀ3.62 (1H,
1
3
m, Hꢀ4’), 3.31ꢀ3.23 (2H, m, Hꢀ5’); C NMR (125 MHz, DMSOꢀd ): δ 162.18 (Cꢀ2),
6
99.95 (Cꢀ1’), 88.04 (Cꢀ2’), 84.58 (Cꢀ4’), 75.62 (Cꢀ3’), 6.55 (Cꢀ5’).
2
,2’ꢀAnhydrouridine (3). A solution of 2ꢀaminoꢀ[1,2ꢀd]arabinosylꢀoxazole 2 (24.0 g,
1
37.81 mmol) and methyl propiolate (23 mL, 275.62 mmol, 2 eq.) in 50% aqueous
ethanol solution (350 mL) were refluxed for 2 hours. The solvent was removed under
reduced pressure to obtain a residue that was dissolved in acetone (400 mL) and left at 0
°C overnight. The resulting precipitate was filtered and washed with acetone to obtain a
1
white solid (23.06 g, 74%); H NMR (500 MHz, DMSOꢀd ): δ 7.83 (1H, d, J = 7.4 Hz,
6
Hꢀ5), 6.31 (1H, d, J = 5.8 Hz, Hꢀ1’), 5.87 (1H, d, J = 4.3 Hz, OHꢀ3’), 5.84 (1H, d, J = 7.4
Hz, Hꢀ6), 5.20 (1H, d, J = 5.5 Hz, Hꢀ2’), 4.97 (1H, t, J = 5.0 Hz, OHꢀ5’), 4.41ꢀ4.37 (1H,
m, Hꢀ3’), 4.08 (1H, t, J = 4.1 Hz, Hꢀ4’), 3.31ꢀ3.25 (1H, m, Hꢀ5’), 3.23ꢀ3.16 (1H, m, Hꢀ
1
3
5
’); C NMR (125 MHz, DMSOꢀd ): δ 171.10 (Cꢀ4), 159.77 (Cꢀ2), 136.77 (Cꢀ5), 108.59
6
(Cꢀ6), 89.97 (Cꢀ1’), 89.16 (Cꢀ4’), 88.71 (Cꢀ2’), 74.71 (Cꢀ3’), 60.81 (Cꢀ5’).
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’,3’ꢀDiacetylꢀ2’ꢀbromoꢀ2’ꢀdeoxyuridine (4). 2,2’ꢀAnhydrouridine 3 (22.0 g, 97.26
mmol) was suspended in CH CN (400 mL) and heated under reflux. Acetyl bromide
3
(64.7 mL, 875.37 mmol, 9 eq.) was added dropwise. The mixture was stirred at reflux for
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hours. The solvent was removed under reduced pressure and the residue obtained was
dissolved in dichloromethane (400 mL), washed with a solution, dried over MgSO and
4
1
evaporated to obtain the title compound as a brown oil (33.0 g, 87%); H NMR (500
MHz, CDCl ): δ 8.99 (1H, s, NH), 7.45 (1H, d, J = 8.4 Hz, Hꢀ6), 6.22 (1H, d, J = 5.21
3
Hz, Hꢀ1’), 5.82 (1H, dd, J = 7.4 Hz, 1.57 Hz, Hꢀ5), 5.16 (1H, t, J = 5.04 Hz, Hꢀ3’), 4.61
(1H, t, J = 5.6 Hz, Hꢀ2’), 4.46ꢀ4.41 (1H, m, Hꢀ4’), 4.42ꢀ4.36 (1H, m, Hꢀ5’), 3.31ꢀ3.25
1
3
(1H, m, Hꢀ5’), 2.22 (3H, s, CH ), 2.16 (3H, s, CH ); C NMR (125 MHz, CDCl ): δ
3
3
3
171.04 (CO), 169.69 (CO), 162.50 (Cꢀ4), 149.93 (Cꢀ2), 138.92 (Cꢀ5), 103.29 (Cꢀ6), 91.01
Cꢀ1’), 80.11 (Cꢀ4’), 70.76 (Cꢀ3’), 62.55 (Cꢀ5’), 48.41 (Cꢀ2’), 20.78 (CH ), 20.61 (CH );
(
3
3
+
+
MS (ES ) m/z 413ꢀ415 (M+Na ).
5
’ꢀAcetylꢀ2’, 3 ꢀdideoxyꢀ2’,3’ꢀdehydroꢀuridine (5). Glacial acetic acid (1.5 mL, 26.20
0
mmol, 1.6 eq.) was added dropwise at 0 C to a stirred suspension of zinc (4.20 g, 64.23
mmol, 4 eq.) and 5’,3’ꢀdiacetylꢀ2’ꢀbromoꢀ2’ꢀdeoxyuridine 4 (6.2 g, 15.85 mmol) in
ethanol (195 mL). The resulting mixture was stirred at room temperature until the TLC
(3% methanol in ethyl acetate) showed that the starting material had completely reacted.
The mixture was filtered through a sintered funnel packed with celite to remove zinc and
triethylamine (1.4 mL) was added to the filtrate. The solvent was removed under reduced
pressure and the residue obtained was extracted with ethyl acetate (250 mL). Removal of
solvent under reduced pressure gave a brown oil which was purified by column
chromatography (1% methanol in ethyl acetate) to afford the title compound as a yellow
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oil (2.24 g, 53%); H NMR (500 MHz, DMSOꢀd ): δ 11.37 (1H, br s, NH), 7.44 (1H, d, J
6
=
8.1 Hz, Hꢀ6), 6.79 (1H, m, Hꢀ1’), 6.42 (1H, dt, J = 6.1 Hz, 1.7 Hz, Hꢀ3’), 6.00 (1H,
ddd, J = 6.1 Hz, 2.3 Hz, 1.3 Hz, Hꢀ2’), 5.66 (1H, d, J = 8.1 Hz, Hꢀ5), 4.97 (1H, m, Hꢀ4’),
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13
4
.17 (2H, d, J = 3.4 Hz, Hꢀ5’), 2.00 (3H, s, CH ); C NMR (125 MHz, DMSOꢀd ): δ
3
6
170.07 (CO), 163.09 (Cꢀ4), 150.72 (Cꢀ2), 140.52 (Cꢀ6), 133.85 (Cꢀ3’), 126.37 (Cꢀ2’),
+
1
01.90 (Cꢀ5), 89.34 (Cꢀ1’), 83.80 (Cꢀ4’), 64.48 (Cꢀ5’), 20.56 (CH ); MS (ES ) m/z 275
3
+
(
M+Na ).
5’ꢀAcetylꢀ2’,3’ꢀdideoxyuridine (6). To a solution of 5’ꢀAcetylꢀ2’,3ꢀdideoxyꢀ2’,3’ꢀ
dehydroꢀuridine 5 (4.88 g, 19.34 mmol) in anhydrous methanol (80 mL) was added 10%
Pd on carbon (0.23 g, 5%w). The mixture was stirred under a hydrogen atmosphere for 6
h. The reaction was monitored by NMR analysis. The mixture was then filtered through a
Celite pad. The solvent was removed under reduced pressure to afford the title compound
1
as a pale brown oil (5.0 g, 99%); H NMR (500 MHz, DMSOꢀd ): δ 11.27 (1H, br s, NH),
6
7
.66 (1H, d, J = 8.0 Hz, Hꢀ6), 5.98 (1H, dd, J = 7.2 Hz, 3.8 Hz, Hꢀ1’), 5.64 (1H, d, J =
8
.0 Hz, Hꢀ5), 4.21ꢀ4.18 (3H, m, Hꢀ5’, Hꢀ4’), 2.34ꢀ2.22 (1H, m, Hꢀ2’), 2.06ꢀ1.95 (5H, m,
1
3
Hꢀ2’, Hꢀ3’, CH ), 1.81ꢀ1.72 (1H, m, Hꢀ3’); C NMR (125 MHz, DMSOꢀd ): δ 170.21
3
6
(
CO), 163.08 (Cꢀ4), 150.48 (Cꢀ2), 140.43 (Cꢀ6), 101.51 (Cꢀ5), 85.24 (Cꢀ1’), 77.95 (Cꢀ4’),
+
+
6
4.88 (Cꢀ5’), 30.8 (Cꢀ2’), 25.56 (Cꢀ3’), 20.60 (CH ); MS (ES ) m/z 277 (M+Na ).
3
Lꢀ5ꢀiodoꢀ2’,3’ꢀdideoxyuridine (7). To a solution of 5’ꢀAcetylꢀ2’,3’ꢀdideoxyuridine 6
2.00 g, 7.86 mmol) in anhydrous acetonitrile (8 mL) were added I (1.20 g, 4.72 mmol,
(
2
0
.6 eq.) and CAN (2.16 g, 3.94 mmol, 0.5 eq.). The mixture was stirred at reflux for 3 h
(
progress of the reaction was monitored by TLC using 1% methanol in ethyl acetate as
the eluent). The solvent was removed under reduced pressure and the residue was
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dissolved in dichloromethane (300 mL) and washed with a saturated solution of sodium
bicarbonate. The two phases were separated and the organic layer was dried over sodium
sulphate and evaporated under reduced pressure to obtain the 5’ꢀAcetylꢀ2’,3’ꢀdideoxyꢀ5ꢀ
iodoꢀuridine intermediate as a brown solid. This residue was dissolved in MeOH (60 mL)
and MeONa (0.63 g, 11.66 mmol, 1.5 eq.) was added. The solution was stirred for 21 h.
As the reaction was not complete, an additional amount of MeONa (0.21 g, 3.88 mmol,
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.5 eq.) was added and the resulting mixture was stirred for further 1 h. Amberlite IR 120
was then added until the pH of the solution became 6.0 and the mixture was filtered. The
solution was evaporated and the solid was washed with diethyl ether to yield the title
1
compound (offꢀwhite solid, 1.57 g, 60%); H NMR (500 MHz, DMSOꢀd ): δ 11.27 (1H,
6
br s, NH), 8.57 (1H, s, Hꢀ6), 5.87 (1H, dd, J = 6.6 Hz, 2.8 Hz, Hꢀ1’), 5.22 (1H, t, J = 5.0
13
Hz, OH), 4.05 (1H, m, Hꢀ4’), 4.72ꢀ4.53 (2H, m, Hꢀ5’), 2.26ꢀ1.83 (4H, m, Hꢀ2’, H3’); C
NMR (125 MHz, DMSOꢀd ): δ 160.59 (Cꢀ4), 150.05 (Cꢀ2), 145.19 (Cꢀ6), 85.83 (Cꢀ1’),
6
8
2.10 (Cꢀ4’), 68.11 (Cꢀ5), 61.20 (Cꢀ5’), 32.45 (Cꢀ2’), 23.93 (Cꢀ3’); Reverse HPLC
eluting with H O/MeOH (gradient from 90:10 to 0:100 in 30 min), t = 08.96 min.
2
R
General procedure A: formation of mesylates (9aꢀc and 11aꢀb).
To a stirred solution of alcohol in THF (0.7 mL/mmol) was added triethylamine (1.3 eq.)
o
and methanesulphonyl chloride (1.3 eq.) dropwise at 0 C under a nitrogen atmosphere.
The reaction was left stirring for 18 h at room temperature. The solvent was evaporated;
water was added to the reaction mixture and then extracted with dichloromethane. The
organic layer was dried over sodium sulphate, filtered and evaporated under reduced
pressure to obtain the desired product.
1
0ꢀUndecynyl mesylate (9a). The procedure A was carried out using 10ꢀundecynꢀ1ꢀol
1
(
10 g, 59.42 mmol) to give the desired compound as a yellow oil (15.5 g, 100%); H
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NMR (500 MHz, CDCl ): δ 4.26 (2H, t, J = 7.1 Hz, OCH ), 3.05 (3H, s, CH ), 2.20 (2H,
3
2
3
dt, J = 7.1 Hz, 2.6 Hz, αCH ), 1.97 (H, t, J = 2.5 Hz, HC≡C), 1.60ꢀ1.28 (14H, m, 7 x
2
CH2).
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ꢀDecynyl mesylate (9b). The procedure A was carried out using 9ꢀdecynꢀ1ꢀol (2.50 g,
1
16.21 mmol) to give the desired compound as a yellow oil (3.10 g, 82%); H NMR (500
MHz, CDCl ): δ 4.24 (2H, t, J = 6.6 Hz, OCH ), 3.02 (3H, s, CH ), 2.01 (2H, dt, J = 7.1
3
2
3
13
Hz, J = 2.6 Hz, αCH ), 1.96 (1H, t, J = 2.6 Hz, HC≡C), 1.58ꢀ1.31 (12H, m, 6 x CH ); C
2
2
NMR (125 MHz, CDCl ): δ 84.64 (HC≡C), 70.13 (OCH ), 68.19 (HC≡C), 37.39 (CH ),
3
2
3
29.13, 2 x 28.89 (double intensity), 28.57, 28.39, 25.38 (6 x CH ), 18.38 (αCH ).
2
2
1
4ꢀpentadecynyl mesylate (9c). The procedure A was carried out using 14ꢀpentadecynꢀ
3
0
1ꢀol (1.80 g, 8.022 mmol) to give the desired compound as a pale brown oil (2.07 g,
1
8
2
5%); H NMR (500 MHz, CDCl ): δ 4.24 (2H, t, J = 6.1 Hz, OCH ), 3.01 (3H, s, CH ),
3
2
3
.20 (2H, dt, J = 7.1 Hz, 2.7 Hz, HC≡CCH ), 1.95 (1H, t, J = 2.7 Hz, HC≡C), 1.76 (2H,
2
13
m, CH ), 1.56 (2H, m, CH ), 1.43 (2H, m, CH ), 1.40ꢀ1.23 (16H, m, 8 x CH ); C NMR
2
2
2
2
(
125 MHz, CDCl ): δ 84.79 (HC≡C), 70.18 (OCH ), 68.03 (HC≡C), 37.65 (CH ), 29.59,
3
2
3
29.55, 29.47, 29.40, 29.13, 29.09, 28.89, 28.85, 28.74, 28.49, 25.73 (11 x CH ), 18.63
2
(C≡CCH2).
Tridecyl mesylate (11a). The procedure A was carried out using 1ꢀtridecanol (5.00 g,
1
24.95 mmol) to give the desired compound as a pale brown oil (6.39 g, 93%); H NMR
(
500 MHz, CDCl ): δ 4.25 (2H, t, J = 6.6 Hz, OCH ), 3.03 (3H, s, CH ), 1.76 (2H, m,
3
2
3
1
3
OCH CH ), 1.47ꢀ1.23 (20H, m, 10 x CH ), 0.91 (3H, t, J = 7.0 Hz, CH ); C NMR (125
2
2
2
3
MHz, CDCl ): δ 70.19 (OCH ), 37.38 (CH ), 31.91, 29.64, 29.62, 29.60, 29.51, 29.41,
3
2
3
2
9.34, 29.13, 29.03, 25.42 (10 x CH ), 22.68 (CH CH ), 14.10 (CH ).
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Decyl mesylate (11b). The procedure A was carried out using 1ꢀdecanol (8.6 mL, 45
1
mmol) to give the desired compound as a pale brown oil (11.00 g, 100%); H NMR (500
MHz, CDCl ): δ 4.24 (2H, t, J = 6.6 Hz, OCH ), 3.03 (3H, s, CH ), 1.78 (2H, m,
3
2
3
10
11
12
13
14
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16
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60
1
3
OCH CH ), 1.46ꢀ1.23 (14H, m, 7 x CH ), 0.91 (3H, t, J = 7.0 Hz, CH ); C NMR (125
2
2
2
3
MHz, CDCl ): δ 70.19 (OCH ), 37.38 (CH ), 31.85, 29.46, 29.41, 29.25, 29.13, 29.02,
3
2
3
2
5.42 (7 x CH ), 22.66 (CH CH ), 14.08 (CH ).
2 2 3 3
General procedure B: synthesis of alkyloxyꢀalkyne (10aꢀj and 12aꢀb).
o
To a stirred solution of alcohol (1.2 eq.) in THF (2.5 mL/mmol) at 0 C under a nitrogen
atmosphere was added slowly NaH (dispersion, 60%, 1.25 eq). After 5 min, the mesylate
o
was added and the reaction was heated at 70 C for 19 h. Then the solvent was removed
under reduced pressure and the residue was dissolved in dichloromethane (100 mL) and
washed with water (2 x 100 mL). The organic layer was dried over sodium sulphate,
evaporated to dryness and purified by column chromatography to obtain the desired
compound.
1
1ꢀethyloxyꢀ1ꢀundecyne (10a). The procedure B was carried out using ethanol (0.448 g,
.735 mmol) and 10ꢀundecynyl mesylate 9a (2.04 g, 8.28 mmol). The crude was purified
9
by column chromatography (5% ethyl acetate in hexane) to give the product 10a as a
1
colourless oil (0.87 g, 69%); H NMR (500 MHz, CDCl ): δ 3.54ꢀ3.36 (4H, m, OCH ),
3
2
2.20 (2H, m, αCH ), 1.95 (1H, ddd, J = 3.1 Hz, 2.2 Hz, 1.0 Hz, HC≡C), 1.64ꢀ1.27 (8H,
2
13
m, 4 x CH ), 1.21 (3H, dt, J = 7.4 Hz, 1.1 Hz, CH ); C NMR (125 MHz, CDCl ): δ
2
3
3
84.76 (CHꢀC), 70.76 (OCH ), 68.03 (CHꢀC), 66.04 (OCH CH ), 29.81, 29.77, 29.43,
2
2
3
2
9.03, 28.72, 28.48, 28.25 (7 x CH ), 18.39 (αCH ), 15.24 (CH ).
2 2 3
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1ꢀpropyloxyꢀ1ꢀundecyne (10b). The procedure B was carried out using propanol (0.75
mL, 9.936 mmol) and 10ꢀundecynyl mesylate 9a (2.04 g, 8.28 mmol). The crude was
purified by column chromatography (5% ethyl acetate in hexane) to give the product 10b
0
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as a colourless oil (1.06 g, 61%); H NMR (500 MHz, CDCl ): δ 3.50ꢀ3.36 (4H, m, 2 x
3
OCH ), 2.21 (2H, m, αCH ), 1.95 (1H, ddd, J = 3.1 Hz, 2.2 Hz, 1.0 Hz, HC≡C), 1.65ꢀ
2
2
1
.48 (8H, m, 4 x CH ), 1.46ꢀ1.20 (8H, m, 4 x CH ), 0.94 (3H, dt, J = 7.4 Hz, 1.1 Hz,
2 2
13
CH3); C NMR (125 MHz, CDCl ): δ 84.75 (CHꢀC), 72.55 (OCH ), 70.94 (OCH ),
3
2
2
6
8.01 (CHꢀC), 29.78, 29.69, 29.42, 29.03, 28.72, 28.48, 26.17, 22.94 (8 x CH ), 18.38
2
(αCH ), 10.57 (CH ).
2 3
11ꢀbutyloxyꢀ1ꢀundecyne (10c). The procedure (B) was carried out using butanol
0.77mL, 8.44 mmol) and 10ꢀundecynyl mesylate 9a (1.60g, 6.49 mmol). The crude was
(
purified by column chromatography (5% ethyl acetate in hexane) to give the product 10c
1
as a colourless oil (0.83 g, 57%); H NMR (500 MHz, CDCl ): δ 3.47ꢀ3.38 (4H, m, 2 x
3
OCH ), 2.20 (2H, m, αCH ), 1.95 (1H, t, J = 2.6 Hz, HC≡C), 1.65ꢀ1.26 (18H, m, 9 x
2
2
13
CH ), 0.94 (3H, t, J = 6.5 Hz, CH ); C NMR (125 MHz, CDCl ): δ 84.75 (HC≡C),
2
3
3
7
0.93 (OCH ), 70.63 (OCH ), 68.01 (HC≡C), 31.88, 29.77, 2 x 29.42 (double intensity),
2 2
29.03, 28.72, 28.48, 26.17, 19.37 (9 x CH ), 18.38 (αCH ), 13.91 (CH ).
2
2
3
1
1ꢀpentyloxyꢀ1ꢀundecyne (10d). The procedure B was carried out using pentanol (0.84
mL, 7.79 mmol) and 10ꢀundecynyl mesylate 9a (1.60 g, 6.49 mmol). The crude was
purified by column chromatography (5% ethyl acetate in hexane) to give the product 10d
1
as a colourless oil (1.00 g, 66%); H NMR (500 MHz, CDCl ): δ 3.48 (4H, m, 2 x OCH ),
3
2
2.20 (2H, dt, J = 7.1 Hz, 2.7 Hz, αCH ), 1.95 (1H, t, J = 2.7 Hz, HC≡C), 1.67ꢀ1.22 (20H,
2
13
m, 10 x CH ), 0.92 (3H, t, J = 7.3 Hz, CH ); C NMR (125 MHz, CDCl ): δ 84.75
2
3
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HC≡C), 70.96 (OCH ), 70.94 (OCH ), 68.01 (HC≡C), 31.59, 29.78, 29.49, 29.36, 29.04,
2
2
2
8.73, 28.49, 28.39, 26.17, 22.65 (10 x CH ) 18.39 (αCH ), 14.03 (CH ).
2 2 3
1
1ꢀhexyloxyꢀ1ꢀundecyne (10e). The procedure B was carried out using hexanol (0.98
10
11
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14
15
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18
19
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mL, 7.79 mmol) and 10ꢀundecynyl mesylate 9a (1.60 g, 6.49 mmol). The crude was
purified by column chromatography (5% ethyl acetate in hexane) to give the product 10e
1
as a colourless oil (0.98 g, 60%); H NMR (500 MHz, CDCl ): δ 3.41 (4H, t, J = 6.7 Hz,
3
2
1
x OCH ), 2.19 (2H, dt, J = 7.1 Hz, 2.6 Hz, αCH ), 1.95 (1H, t, J = 2.7 Hz, HC≡C),
2 2
1
3
.65ꢀ1.24 (22H, m, 11 x CH ), 0.92 (3H, t, J = 7.2 Hz, CH ); C NMR (125 MHz,
2
3
CDCl ): δ 84.75 (HC≡C), 70.97 (OCH ), 70.93 (OCH ), 68.02 (HC≡C), 31.73, 29.78, 2 x
3
2
2
29.76 (double intensity), 29.43, 29.03, 28.73, 28.48, 26.18, 25.88, 22.63 (11 x CH2),
1
8.39 (αCH ), 14.03 (CH ).
2 3
1
1ꢀ(isoꢀpropyloxy)ꢀ1ꢀundecyne (10f). The procedure B was carried out using iso-
propanol (0.372 mL, 4.86 mmol) and 10ꢀundecynyl mesylate 9a (1.00 g, 4.06 mmol). The
mixture was heated for total of 24 h at 70 °C, with further 0.75 eq. of NaH added after the
first 7 h. After treatment, the crude was purified by column chromatography (5% ethyl
1
acetate in hexane) to give the product 10f as a colourless oil (0.484 g, 56%); H NMR
(500 MHz, CDCl ): δ 3.56 (1H, m, J = 6.1 Hz, CH), 3.41 (2H, t, J = 6.8 Hz, OCH ), 2.20
3 2
(
2H, dt, J = 7.1 Hz, 2.7 Hz, αCH ), 1.96 (1H, t, J = 2.7 Hz, HC≡C), 1.58ꢀ1.52 (4H, m, 2 x
2
13
CH ), 1.44ꢀ1.29 (10H, m, 5 x CH ), 1.18 (3H, s, CH ), 1.17 (3H, s, CH ); C NMR (125
2
2
3
3
MHz, CDCl ): δ 84.81 (HC≡C), 71.23 (OCH), 68.23 (OCH ), 68.02 (HC≡C), 30.18, 2 x
3
2
2
9.44 (double intensity), 29.04, 28.73, 28.48, 26.18 (7 x CH ), 22.16 (double intensity, 2
2
x CH ), 18.39 (αCH ).
3
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1ꢀ(sec-butyloxy)ꢀ1ꢀundecyne (10g). The procedure B was carried out using secꢀbutanol
(0.447 mL, 4.87 mmol) and 10ꢀundecynyl mesylate 9a (1.00 g, 4.06 mmol). The crude
was purified by column chromatography (5% ethyl acetate in hexane) to give the product
0
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0g as a colourless oil (0.372 g, 38%); H NMR (500 MHz, CDCl ): δ 3.50ꢀ3.29 (3H, m,
3
CH OCH), 2.20 (2H, dt, J = 7.1 Hz, 2.6 Hz, αCH ), 1.96 (1H, t, J = 2.7 Hz, HC≡C), 1.62ꢀ
2
2
1
.27 (16H, m, 8 x CH ), 1.14 (3H, d, J = 6.1 Hz, CHCH ), 0.92 (3H, t, J = 7.4 Hz,
2 3
1
3
CH CH ); C NMR (125 MHz, CDCl ): δ 84.79 (HC≡C), 76.59 (OCH), 68.47 (OCH ),
2
3
3
2
6
8.01 (HC≡C), 30.21, 29.44 (double intensity), 29.26, 29.04, 28.74, 28.49, 26.18 (8 x
CH ), 19.30 (CHCH ), 18.39 (αCH ), 9.86 (CH CH ).
2
3
2
2
3
11ꢀ(iso-butyloxy)ꢀ1ꢀundecyne (10h). The procedure B was carried out using isoꢀbutanol
0.450 mL, 4.87 mmol) and 10ꢀundecynyl mesylate 9a (1.00 g, 4.06 mmol). The mixture
(
was heated for total of 28 h at 70 °C, with further 0.75 eq. of NaH and 0.6 eq. of isoꢀ
butanol added after the first 18 h. After the treatment, the crude was purified by column
chromatography (5% ethyl acetate in hexane) to give the product 10h as a light yellow oil
1
(0.480 g, 52%); H NMR (500 MHz; CDCl ): δ 3.41 (2H, t, J = 6.7 Hz, CH CH O), 3.19
3
2
2
(2H, d, J = 6.7 Hz, OCH CH), 2.20 (2H, dt, J = 7.1 Hz, 2.6 Hz, αCH ), 1.96 (1H, t, J =
2
2
2
.7 Hz, HC≡C), 1.92ꢀ1.85 (1H, m, CH), 1.59ꢀ1.53 (4H, m, 2 x CH ), 1.36ꢀ1.28 (13H, m,
2
13
2 x CH ), 0.93 (3H, s, CH ), 0.91 (3H, s, CH ); C NMR (125 MHz, CDCl ): δ 84.80
2
3
3
3
(HC≡C), 77.86 (OCH CH), 71.07 (OCH CH ), 68.03 (HC≡C), 29.76, 2 x 29.44 (double
2 2 2
intensity), 29.05, 28.75, 28.50, 28.45, 26.15, 25.89, 22.63 (10 x CH ), 18.39 (αCH ),
2
2
1
4.04 (CH3).
1
1ꢀ(tert-butyloxy)ꢀ1ꢀundecyne (10i). The procedure B was carried out using tertꢀbutanol
(0.925 mL, 9.74 mmol) and 10ꢀundecynyl mesylate 9a (2.00 g, 8.12 mmol). The mixture
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was heated for total of 28 h at 70 °C, with further 0.75 eq. of NaH added after the first 18
h. After treatment, the crude was purified by column chromatography (5% ethyl acetate
1
in hexane) to give the product 10i as a colourless oil (0.478 g, 26%); H NMR (500 MHz;
10
11
12
13
14
15
16
17
18
19
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CDCl ): δ 3.41 (2H, t, J = 6.7 Hz, OCH ), 2.21 (2H, dt, J = 7.1 Hz, 2.7 Hz, αCH ), 1.96
3
2
2
(1H, t, J = 2.7 Hz, HC≡C), 1.58ꢀ1.52 (4H, m, 2 x CH ), 1.41ꢀ1.28 (10H, m, 5 x CH ),
2
2
13
1
.21 (9H, s, 3 x CH ); C NMR (125 MHz, CDCl ): δ 84.81 (HC≡C), 72.97 (OC(CH ) ),
3
3
3 3
70.97 (OCH ), 68.05 (HC≡C), 29.79, 2 x 29.45 (double intensity), 29.06, 28.76, 28.50, (6
2
x CH ), 27.61 (3 x CH ), 26.20, 18.42 (2 x CH ).
2
3
2
1
0ꢀhexyloxyꢀ1ꢀdecyne (10j). The procedure B was carried out using hexanol (1.2mL,
9.29 mmol) and 9ꢀdecynyl mesylate 9b (1.8 g, 7.74 mmol). The mixture was heated for a
total of 55 h at 70 °C, with further 1.2 eq. of NaH added after the first 19 h and again 0.5
eq. of NaH and 0.5 eq. of 9ꢀdecynyl mesylate 9b added after 40 h. After treatment, the
crude was purified by column chromatography (5% ethyl acetate in hexane) to obtain the
1
product 10j as a pale brown oil (0.572 g, 35%); H NMR (500 MHz, CDCl ): δ 3.41 (4H,
3
t, J = 6.6 Hz, 2 x OCH ), 2.20 (2H, dt, J = 7.1 Hz, 2.6 Hz, αCH ), 1.96 (1H, t, J = 2.7 Hz,
2
2
13
HC≡C), 1.70ꢀ1.26 (20H, m, 10 x CH ), 0.92 (3H, t, J = 7.2 Hz, CH ); C NMR (125
2
3
MHz, CDCl ): δ 84.75 (HC≡C), 70.98 (OCH ), 70.91 (OCH ), 68.04 (HC≡C), 31.73, 2 x
3
2
2
2
1
1
9.76 (double intensity), 29.34, 29.05, 28.70, 28.47, 26.15, 25.89, 22.63 (10 x CH2),
8.39 (αCH ), 14.04 (CH ).
2
3
5ꢀmethoxyꢀ1ꢀpentadecyne (10k). The procedure B was carried out using methanol
o
(
0.36mL, 8.76 mmol) and 14ꢀpentadecynyl mesylate 9c (2.04 g, 6.74 mmol) at 60 C for
4 h. Some methanol (1.3 eq.) and NaH (1.35 eq.) were then added and the mixture was
o
heated further 3 h at 60 C. After treatment, the crude was purified by column
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chromatography (5% ethyl acetate in hexane) to obtain the product 10k as a colourless oil
1
(1.11 g, 69%); H NMR (500 MHz, CDCl ): δ 3.40 (2H, J = 6.7 Hz, OCH ), 3.35 (3H, s,
3
2
CH ), 2.20 (2H, dt, J = 7.1 Hz, 2.7 Hz, CH C≡C), 1.95 (1H, t, J = 2.7 Hz, HC≡C), 1.64ꢀ
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3
4
5
6
7
8
9
0
13
1
.50 (6H, m, 3 x CH ), 1.46ꢀ1.23 (16H, m, 8 x CH ); C NMR (125 MHz, CDCl ): δ
2
2
3
8
4.79 (HC≡C), 72.98 (OCH ), 68.01 (HC≡C), 58.51 (CH ), 31.58, 2 x 29.66 (double
2
3
intensity), 29.61, 29.58, 29.50, 29.11, 28.76, 28.50, 26.14, 22.65 (11 x CH ), 18.40
2
(CH C≡C).
2
3
ꢀtridecyloxyꢀ1ꢀpropyne (12a). The procedure B was carried out using 2ꢀpropynꢀ1ꢀol
(0.56 g, 10.00 mmol) and tridecyl mesylate 11a (4.2 g, 15.0 mmol). The mixture was
heated for a total of 36 h at 60 °C with a further 1.0 eq. of NaH added after the first 24 h.
After treatment, the crude was purified by column chromatography (5% ethyl acetate in
1
hexane) to give the desired compound as a pale brown oil (0.90 g, 22%); H NMR (500
MHz, CDCl ): δ 4.17 (2H, d, J = 2.6 Hz, OCH C≡C), 3.54 (2H, t, J = 7.1 Hz, OCH CH ),
3
2
2
2
2
.43 (1H, t, J = 2.6 Hz, HC≡C), 1.62 (2H, m, OCH CH ), 1.62ꢀ1.22 (20H, m, 10 x CH ),
2 2 2
13
0.91 (3H, t, J = 7.1 Hz, CH ); C NMR (125 MHz, CDCl ): δ 80.09 (HC≡C), 73.97
3
3
(
HC≡C), 70.32 (OCH CH ), 57.99 (OCH C≡C), 31.92, 29.67, 29.65, 29.60, 29.57, 29.52,
2 2 2
2
6
9.44, 29.35, 26.21, 26 09 (10 × CH ), 22.68 (αCH ), 14.10 (CH ).
2
2
3
ꢀdecyloxyꢀ1ꢀhexyne (12b). The procedure B was carried out using hexynꢀ1ꢀol (0.66
mL, 6.11 mmol) and decyl mesylate 11b (2.17 g, 9.17 mmol, 1.5 eq.). The mixture was
heated for a total of 90 h at 60 °C, with a further 1 eq. of NaH added after the first 24 h
and another 1 eq. of NaH and 1 eq. of decyl mesylate added after 48 h. After treatment,
the crude was purified by column chromatography (5% ethyl acetate in hexane) to obtain
1
the desired compound as a brown oil (1.2 g, 82%); H NMR (500 MHz, CDCl ): δ 3.47ꢀ
3
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.36 (4H, m, 2 x OCH ), 2.24 (2H, dt, J = 7.0 Hz, J = 2.6 Hz, CH ), 1.96 (1H, t, J = 2.6
2
2
Hz, HC≡C), 1.76ꢀ1.53 (6H, m, 3 x CH ), 1.38ꢀ1.21 (14H, m, 7 x CH ), 0.89 (3H, t, J =
2
2
1
3
7
.0 Hz, CH3); C NMR (125 MHz, CDCl ): δ 84.36 (HC≡C), 71.01 (OCH ), 70.17
3 2
10
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(
OCH ), 68.29 (HC≡C), 31.90, 29.80, 29.32, 29.11, 28.81, 26.20, 25.28, 24.85, 22.67,
2
22.64 (10 × CH ), 18.23 (αCH ), 14.08 (CH ).
2
2
3
General procedure C: synthesis of 3ꢀ(2’,3’ꢀdideoxyꢀriboꢀβꢀLꢀfuranosyl)ꢀ6ꢀ
substitutedꢀfuro[2,3ꢀd]pyrimidinꢀ2(3H)ꢀone analogues (8aꢀu).
To a solution of Lꢀ5ꢀiodoꢀ2’,3’ꢀdideoxyuridine 7 in DMF (5.5 mL/mmol) was added the
alkyne (3 eq.), Tetrakis (triphenylphosphine) Pd(0) (0.1 eq.), CuI (0.2 eq.) and DIPEA (2
eq.). The mixture was stirred at room temperature for 16 h and further CuI (0.2 eq.) and
triethylamine (5.5 mL/mmol) were added. The mixture was stirred at 80 °C for 8 h. The
solvent was removed under high vacuum and the residue was purified by flash column
chromatography.
3
ꢀ(2’,3’ꢀdideoxyꢀriboꢀβꢀ
Lꢀfuranosyl)ꢀ6ꢀhexylfuro[2,3ꢀd]pyrimidinꢀ2(3H)ꢀone
(8a).
The procedure C was carried out using 1ꢀoctyne (0.29 g, 2.66 mmol), which gave the
desired alkyne intermediate (0.130 g, 0.374 mmol, 34%). This compound was dissolved
in DMF (5 mL) and to this solution Et N (5 mL) and CuI (0.014 g, 0.075 mmol, 0.2 eq)
3
0
were added. The resulting solution was heated at 80 C for 8h. Solvent was removed
under high vacuum. The product 8a was then obtained as an offꢀwhite solid (0.026 g,
2
2%) after purification by preparative thin layer chromatography using 20% chloroform
1
in ethyl acetate as the eluent; H NMR (500 MHz, CDCl ): δ 876 (1H, s, Hꢀ4), 6.20 (1H,
3
m, Hꢀ1’), 6.15 (1H, s, Hꢀ5), 4.31 (1H, m, Hꢀ4’), 4.16 (1H, ddd, J = 11.9 Hz, 4.2 Hz, 2.9
Hz, Hꢀ5’), 3.89 (1H, ddd, J = 11.9 Hz, 3.0 Hz, Hꢀ5’), 2.69ꢀ2.54 (3H, m, CH , Hꢀ2’), 2.25ꢀ
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.16 (1H, m, Hꢀ2’), 1.99ꢀ1.90 (2H, m, Hꢀ3’), 1.72 (2H, m, J = 7.1 Hz, CH ), 1.41ꢀ1.22
2
1
3
(6H, m, 3 x CH ), 0.90 (3H, t, J = 6.3 Hz, CH ); C NMR (125 MHz, CDCl ): δ 171.74
2 3 3
(
Cꢀ7a), 159.47 (Cꢀ6), 155.05 (Cꢀ2), 135.98 (Cꢀ4), 107.25 (Cꢀ4a), 99.10 (Cꢀ5), 89.00 (Cꢀ
’), 82.93 (Cꢀ4’), 62.81 (Cꢀ5’), 33.79 (Cꢀ2’), 31.43, 28.69, 28.25 (3 x CH ), 26.78 (Cꢀ3’),
0
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8
9
0
1
2
+
+
23.89, 22.50 (2 x CH ), 14.01 (CH ); MS (ES ) m/z 321 (M+H ). Accurate mass:
2
3
C H N O requires 321.1814; found 321.1824. Reverse HPLC eluting with H O/MeOH
17
25
2
4
2
(gradient from 100:0 to 0:100 in 25 min), t = 21.81 min.
R
3ꢀ(2’,3’ꢀdideoxyꢀriboꢀβꢀ
Lꢀfuranosyl)ꢀ6ꢀseptylfuro[2,3ꢀd]pyrimidinꢀ2(3H)ꢀone
(8b).
The procedure C was carried out using 1ꢀnonyne (0.275 g, 2.22 mmol), which gave, after
purification by flash column chromatography (5% methanol in ethyl acetate), the product
1
8b as an offꢀwhite solid (0.128 g, 53%); H NMR (500 MHz, CDCl ): δ 8.84 (1H, s, Hꢀ
3
4
), 6.20 (1H, dd, J = 6.7 Hz, 2.5 Hz, Hꢀ1’), 6.16 (1H, s, Hꢀ5), 4.30 (1H, m, Hꢀ4’), 4.18
(
1H, m, Hꢀ5’), 3.91 (1H, m, Hꢀ5’), 3.16 (1H, t, J = 5.4 Hz, OH), 2.70ꢀ2.54 (3H, m, αCH2,
Hꢀ2’), 2.21 (1H, m, Hꢀ2’), 1.97 (2H, m, Hꢀ3’), 1.69 (2H, m, J = 7.1 Hz, CH ), 1.44ꢀ1.22
2
13
(8H, m, 4 x CH ) 0.90 (3H, t, J = 6.5 Hz, CH ); C NMR (125 MHz, CDCl ): δ 171.72
2
,
3
3
(Cꢀ7a), 159.43 (Cꢀ6), 155.06 (Cꢀ2), 136.12 (Cꢀ4), 107.25 (Cꢀ4a), 99.15 (Cꢀ5), 89.00 (Cꢀ
’), 83.01(Cꢀ4’), 62.74 (Cꢀ5’), 33.81 (Cꢀ2’), 31.68, 28.98, 28.91, 28.25 (4 x CH ), 26.83
1
2
+
+
(Cꢀ3’), 23.87, 22.59 (2 x CH ), 14.04 (CH ); MS (ES ) m/z 335 (100%, M+H ), 373 (9%,
2 3
+
+
M+K ), 398 (81%, M+CH CN+Na ). Accurate mass: C H N O requires 335.1971;
3
18 27
2
4
found 335.1958. Reverse HPLC eluting with H O/MeOH (gradient from 100:0 to 10:90
2
in 15 min and with 10:90 for 10 min), t = 17.91 min.
R
3
ꢀ(2’,3’ꢀdideoxyꢀriboꢀβꢀLꢀfuranosyl)ꢀ6ꢀoctylfuro[2,3ꢀd]pyrimidinꢀ2(3H)ꢀone (8c). The
procedure C was carried out using 1ꢀdecyne (0.31 g, 2.22 mmol). After purification by
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flash column chromatography (2% methanol in ethyl acetate) to obtain a pale brown oil
and trituration with ethyl acetate, the product 8c was obtained as an offꢀwhite solid (0.060
1
g; 23%); H NMR (500 MHz, DMSOꢀd ): δ 8.83 (1H, s, Hꢀ4), 6.42 (1H, s, Hꢀ5), 5.97
6
10
11
12
13
14
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(
1H, m, Hꢀ1’), 5.15 (1H, t, J = 5.1 Hz, OH), 4.14 (1H, m, Hꢀ4’), 3.80 (1H, m, Hꢀ5’), 3.60
(1H, m, Hꢀ5’), 2.61 (2H, t, J = 7.2 Hz, αCH ), 2.42 (1H, m, Hꢀ2’), 1.97 (1H, m, Hꢀ2’),
2
1
3
1
.88ꢀ1.56 (2H, m, Hꢀ3’), 1.6 (2H, m, J = 7.5 Hz, CH ), 0.84 (3H, t, J = 6.7 Hz, CH ) C
2 3 ;
NMR (125 MHz, DMSOꢀd ): δ 170.99 (Cꢀ7a), 158.01 (Cꢀ6), 153.76 (Cꢀ2), 137.00 (Cꢀ4),
6
107.33 (Cꢀ4a), 99.72 (Cꢀ5), 88.02 (Cꢀ1’), 83.00 (Cꢀ4’), 61.33 (Cꢀ5’), 33.03 (Cꢀ2’), 31.16,
2
8.55, 28.50, 28.29, 27.29 (5 x CH ), 26.35 (Cꢀ3’), 23.61, 22.00 (2 x CH ), 13.88 (CH );
2 2 3
+
+
MS (ES ) m/z 349 (M+H ). Accurate mass: C H N O requires 349.2127; found
19 29
2
4
349.2141. Reverse HPLC eluting with H O/MeOH (gradient from 100:0 to 0:100 in 25
2
min and then at 0:100 for 5 min), t = 26.04 min.
R
3
ꢀ(2’,3’ꢀdideoxyꢀriboꢀβꢀ
Lꢀfuranosyl)ꢀ6ꢀnonylfuro[2,3ꢀd]pyrimidinꢀ2(3H)ꢀone
(8d).
The procedure C was carried out using 1ꢀundecyne (0.405 g, 2.66 mmol), which gave,
after purification by flash column chromatography (40% dichloromethane in ethyl
1
acetate), the product 8d as an offꢀwhite solid (0.150 g, 47%); H NMR (500 MHz,
CDCl ): δ 8.76 (1H, s, Hꢀ4), 6.20 (1H, dd, J = 6.8 Hz, J = 2.6 Hz, Hꢀ1’), 6.15 (1H, s, Hꢀ
3
5
), 4.31 (1H, m, Hꢀ4’), 4.17 (1H, m, Hꢀ5’), 3.91 (1H, m, Hꢀ5’), 2.83 (1H, t, J = 5.5 Hz,
OH), 2.70ꢀ2.55 (3H, m, αCH , Hꢀ2’), 2.24 (1H, m, Hꢀ2’), 1.95 (2H, m, Hꢀ3’), 1.70 (2H,
2
13
m, J = 7.2 Hz, CH ), 1.45ꢀ1.21 (12H, m, 6 x CH ) 0.90 (3H, t , J = 6.7 Hz, CH ); C
2
2 ,
3
NMR (125 MHz, CDCl ): δ 171.75 (Cꢀ7a), 159.49 (Cꢀ6), 155.02 (Cꢀ2), 135.89 (Cꢀ4),
3
1
07.22 (Cꢀ4a), 99.07(Cꢀ5), 88.99 (Cꢀ1’), 82.88 (Cꢀ4’), 62.87 (Cꢀ5’), 33.78 (Cꢀ2’), 31.84,
9.45, 29.26, 29.04, 28.26 (5 x CH ), 26.83 (Cꢀ3’), 23.91, 22.64 (2 x CH ), 14.08 (CH );
2
2
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MS (ES ) m/z 363 (53%, M+H ), 426 (80%, M+CH CN+Na ). Accurate mass:
3
C H N O requires 363.2284; found 363.2289. Reverse HPLC eluting with H O/MeOH
20 31
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4
2
(
gradient from 100:0 to 10:90 in 15 min and then at 10:90 for 10 min), t = 21.57 min.
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ꢀ(2’,3’ꢀdideoxyꢀriboꢀβꢀ
L
ꢀfuranosyl)ꢀ6ꢀdecylfuro[2,3ꢀd]pyrimidinꢀ2(3H)ꢀone
(8e).
The procedure C was carried out using 1ꢀdodecyne (0.516 g, 3.11 mmol), which gave,
after purification by flash column chromatography (40% dichloromethane in ethyl
1
acetate), the product 8e as an offꢀwhite solid (0.100 g, 33%); H NMR (500 MHz,
CDCl ): δ 8.75 (1H, s, Hꢀ4), 6.21 (1H, m, Hꢀ1’), 6.15 (1H, s, Hꢀ5), 4.32 (1H, m, Hꢀ4’),
3
4
.17 (1H, m, Hꢀ5’), 3.90 (1H, m, Hꢀ5’), 2.86 (1H, t, J = 5.4 Hz, OH), 2.68ꢀ2.57 (3H, m,
αCH , Hꢀ2’), 2.21 (1H, m, Hꢀ2’), 1.96 (2H, m, Hꢀ3’), 1.72 (2H, m, J = 7.0 Hz, CH ),
2
2
1
3
1.44ꢀ1.21 (14H, m, 7 x CH ) 9.00 (3H, t, J = 6.4 Hz, CH ); C NMR (125 MHz,
2
,
3
CDCl ): δ 171.75 (Cꢀ7a), 159.49 (Cꢀ6), 155.03 (Cꢀ2), 135.90 (Cꢀ4), 107.23 (Cꢀ4a), 99.07
3
(
Cꢀ5), 88.99 (Cꢀ1’), 82.88 (Cꢀ4’), 62.86 (Cꢀ5’), 33.78 (Cꢀ2’), 31.87, 29.56, 29.50, 29.29,
2
9.27, 29.05, 28.26 (7 x CH ), 26.83 (Cꢀ3’), 23.91, 22.66 (2 x CH ), 14.09 (CH ); MS
2
2
3
+
+
+
+
(
ES ) m/z 377 (16%, M+H ), 399 (6%, M+Na ), 415 (8%, M+K ), 440 (100%,
+
M+CH CN+Na ). Accurate mass: C H N O requires 377.2440; found 377.2426.
3
21 33
2
4
Reverse HPLC eluting with H O/MeOH (gradient from 100:0 to 0:100 in 30 min and
2
then at 0:100 for 5 min), t = 33.00 min.
R
3ꢀ(2’,3’ꢀdideoxyꢀriboꢀβꢀLꢀfuranosyl)ꢀ6ꢀdodecylfuro[2,3ꢀd]pyrimidinꢀ2(3H)ꢀone (8f).
The procedure C was carried out using 1ꢀtetradecyne (0.345 g, 1.77 mmol), which gave,
after purification by flash column chromatography (40% dichloromethane in ethyl
1
acetate), the product 8f as an offꢀwhite solid (0.100 g, 42%); H NMR (500 MHz,
CDCl ): δ 8.77 (1H, s, Hꢀ4), 6.23 (1H, dd, J = 6.6 Hz, 2.5 Hz, Hꢀ1’), 6.16 (1H, s, Hꢀ5),
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.32 (1H, m, Hꢀ4’), 4.18 (1H, m, Hꢀ5’), 3.90 (1H, m, Hꢀ5’), 2.90 (1H, br s, OH), 2.67ꢀ
.56 (3H, m, αCH , Hꢀ2’), 2.21 (1H, m, Hꢀ2’), 1.94 (2H, m, Hꢀ3’), 1.69 (2H, m, J = 7.5
2
1
3
Hz, CH ), 1.43ꢀ1.20 (18H, m, 9 x CH ) 9.00 (3H, t, J = 6.5 Hz, CH ); C NMR (125
2
2 ,
3
10
11
12
13
14
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17
18
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MHz, CDCl ): δ 171.74 (Cꢀ7a), 159.48 (Cꢀ6), 155.03 (Cꢀ2), 135.95 (Cꢀ4), 107.24 (Cꢀ4a),
3
99.08 (Cꢀ5), 88.99 (Cꢀ1’), 82.91 (Cꢀ4’), 62.83 (Cꢀ5’), 33.79 (Cꢀ2’), 31.90, 29.64, 29.61,
2
9.51, 29.33, 29.28, 29.06, 28.26 (9 x CH ), 26.84 (Cꢀ3’), 23.90, 22.67 (2 x CH ), 14.10
2 2
+
+
(
CH ); MS (ES ) m/z 405 (M+H ). Accurate mass: C H N O requires 405.2753; found
3 23 37 2 4
405.2739. Reverse HPLC eluting with H O/MeOH (gradient from 100:0 to 10:90 in 15
2
min, then at 10:90 for 10 min and another gradient from 10:90 to 0:100 in 5 min), t =
R
2
3
2
8.99 min.
ꢀ(2’,3’ꢀdideoxyꢀriboꢀβꢀLꢀfuranosyl)ꢀ6ꢀ(4ꢀnꢀbutylphenyl)ꢀfuro[2,3ꢀd]pyrimidinꢀ
(3H)ꢀone (8g). The procedure C was carried out using 4ꢀnꢀbutylphenylacetylene (0.351
g, 0.39 mL, 2.22 mmol). The obtained residue was purified by column chromatography
(5% methanol in dichloromethane) to give the product 8g as an offꢀwhite solid (0.110 g,
1
4
0%); H NMR (500 MHz, CDCl ): δ 8.90 (1H, s, Hꢀ4), 7.63 (2H, d, J = 8.2 Hz, Ph),
3
7.22 (2H, d, J = 8.3 Hz, Ph), 6.71 (1H, s, Hꢀ5), 6.25 (1H, dd, J = 6.7 Hz, 2.3 Hz, Hꢀ1’),
4
2
.32 (1H, m, Hꢀ4’), 4.2 (1H, m, Hꢀ5’), 3.92 (1H, m, Hꢀ5’), 2.92 (1H, t, J = 5.0 Hz, OH),
.70ꢀ2.59 (3H, m, αCH , Hꢀ2’), 2.25 (1H, m, Hꢀ2’), 2.00 (2H, m, Hꢀ3’), 1.65 (2H, m,
2
13
CH ), 1.39 ( 2H, m, CH ) 0.98 (3H, t, J = 6.6 Hz, CH ); C NMR (125 MHz, CDCl ): δ
2
2 ,
3
3
1
71.58 (Cꢀ7a), 155.58 (Cꢀ6), 155.00 (Cꢀ2), 144.80 (Cꢀpara Ph), 136.79 (Cꢀ4), 128.96
(Ph), 125.98 (Cꢀipso Ph), 124.82 (Ph), 107.81 (Cꢀ4a), 97.22 (Cꢀ5), 89.11 (Cꢀ1’), 83.03
(
Cꢀ4’), 62.79 (Cꢀ5’), 35.53 (CH ), 33.85 (Cꢀ2’), 33.36 (CH ), 23.83 (Cꢀ3’), 22.32 (CH ),
2
2
2
+
+
+
1
3.91 (CH ); MS (ES ) m/z 369 (44%, M+H ), 407 (70%, M+K ), 432 (50%,
3
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