Synthesis and antiviral properties of spirocyclic [1,2,3]-Triazolooxazine nucleosides

Article abstract of DOI:10.1002/chem.201403560

An efficient synthesis of spirocyclic triazolooxazine nucleosides is described. This was achieved by the conversion of β-D-psicofuranose to the corresponding azido-derivative, followed by alkylation of the primary alcohol with a range of propargyl bromide

Full text of DOI:10.1002/chem.201403560

DOI: 10.1002/chem.201403560
Communication
&
Non-natural Nucleosides
Synthesis and Antiviral Properties of Spirocyclic [1,2,3]-
Triazolooxazine Nucleosides
Antonio Dell’Isola,[a] Matthew M. W. McLachlan,[b] Benjamin W. Neuman,[c] Hawaa M. N. Al-
Mullah,[c] Alexander W. D. Binks,[c] Warren Elvidge,[c] Kenneth Shankland,[a] and
Alexander J. A. Cobb*[a]
alone, but there are also examples whereby the nucleobase is
Abstract: An efficient synthesis of spirocyclic triazolooxa-
zine nucleosides is described. This was achieved by the
conversion of b-d-psicofuranose to the corresponding
azido-derivative, followed by alkylation of the primary al-
cohol with a range of propargyl bromides, obtained by
Sonogashira chemistry. The products of these reactions
underwent 1,3-dipolar addition smoothly to generate the
protected spirocyclic adducts. These were easily depro-
tected to give the corresponding ribose nucleosides. The
library of compounds obtained was investigated for its an-
tiviral activity using MHV (mouse hepatitis virus) as
a model wherein derivative 3 f showed the most promis-
ing activity and tolerability.
directly involved in the conformational restriction of the nu-
cleoside (so-called “cyclonucleosides”).[4] In this respect, we
have an interest in the synthesis and use of anomeric spironu-
cleosides, whereby the anomeric carbon belongs to both the
sugar moiety and the nucleobase (Figure 1). This fixes the nu-
cleobase in a specific orientation around the N-glycosidic
bond, imposing an altered flexibility on the sugar moiety.
Spiro-functionalised nucleosides have gained considerable in-
terest with the discovery of (+)-hydantocidin (1), a natural spi-
ronucleoside with potent herbicidal and plant growth regula-
tory activity.[5] However, to the best of our knowledge, synthet-
ic work in this field is limited, with the majority of anomeric
spirocycles being hydantoine or diketopiperazine analogues, or
simple pseudonucleosides with anchored purinic and pyrimi-
dinic bases.[6]
The design and synthesis of nucleoside analogues has been
a subject of great interest in the discovery of novel anticancer
and antiviral agents owing to the fact that they can be in-
volved in the disruption of nucleic acid biosynthesis and thus
inhibit cellular division and viral replication.[1] Additionally, they
have been utilised for various gene-silencing techniques as
constituents of antisense oligonucleotides, small interfering
RNAs (siRNAs) and microRNA-targeting oligonucleotides (anti-
miRNAs).[2]
In particular, conformationally restricted nucleosides such as
“locked nucleic acids” (LNAs), whereby the sugar moiety of the
nucleoside is locked in the bioactive C3’-endo (North) or C2’-
endo (South) conformations, represent an interesting class of
nucleoside inhibitor as they can show a dramatic improvement
in enzymatic recognition, as well as enhancing base stacking
and backbone pre-organisation.[3] Most of these systems are
locked by virtue of bridging groups on the furanose unit
Figure 1. Representation of a spironucleoside (where the shared carbon is at
the anomeric position), the spironucleoside hydantocidin, and the triazolyl
antiviral ribavirin.
As part of an on-going programme within our laboratories
on the synthesis of non-natural nucleic acids,[7] we aimed to
prepare a library of spiro-fuctionalised nucleosides, containing
a [1,2,3]-triazolyl moiety using a straightforward and highly ste-
reoselective route. It was felt that this class of spironucleoside
would make an interesting alternative to the [1,2,4]-triazolyl
class of nucleoside the biological activity of which is well
known, owing to their resemblance to ribavirin 2.[8] We there-
fore evaluated our resulting [1,2,3]-triazolospironucleosides for
their anti-HMV (mouse hepatitis virus) activity in vitro.
[a] A. Dell’Isola, Dr. K. Shankland, Dr. A. J. A. Cobb
School of Chemistry, Food and Pharmacy (SCFP)
University of Reading, Whiteknights, Reading, Berks RG6 6AD (UK)
E-mail: a.j.a.cobb@reading.ac.uk
[b] Dr. M. M. W. McLachlan
Syngenta, Jealott’s Hill International Research Centre
Bracknell, Berks RG42 6EY (UK)
As depicted in the retrosynthetic path (Scheme 1), the versa-
tility of the synthetic strategy towards novel anomeric spironu-
cleosides 3 lies in the strategic installation of azide and alkyne
moieties on the d-psicofuranose derivative 4, followed by an
intramolecular Huisgen 1,3-dipolar cycloaddition to generate
the spirocyclic [1,2,3]-triazolooxazine ring.[9]
[c] Dr. B. W. Neuman, H. M. N. Al-Mullah, A. W. D. Binks, W. Elvidge
School of Biological Sciences, University of Reading
Whiteknights, Reading, Berks RG6 6AJ (UK)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201403560.
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Table 1. Alkylation and 1,3-dipolar cycloaddition to access the spirocyclic
nucleoside system.
Scheme 1. Retrosynthetic access to [1,2,3]-spirotriazolooxazines.
The first part of the synthesis is shown in Scheme 2 and fol-
lowed the general procedure described by Fuentes and co-
workers.[10] This involved isomerisation of 1,2:4,5-di-O-isopropy-
lidene-d-psicopyranose (7; easily prepared in a multigram scale
using a straightforward three-step procedure from b-d-fructo-
pyranose)[11] to its furanose form 6a using amberlyst acid resin
in acetone. The remaining alcohol was then converted to the
benzoate ester 6b with a satisfactory overall yield of 70%.
Entry
Product
R
Overall yield [%][a]
1
2
3
4
5
6
7
8
10a
10b
10c
10d
10e
10 f
10g
10h
10i
H
Me
Et
2-naphthyl
Ph
4-Cl-C6H4
4-MeO-C6H4
4-F-C6H4
3-F-C6H4
2-F-C6H4
n-pentyl
51
53
43
59
44
45
43
43
39
45
36
9
10
11
10j
10k
[a] Overall isolated yield for alkylation and cycloaddition.
cycloaddition upon heating in toluene for 24 h to give the
novel protected anomeric spironucleoside library 10
(Table 1).[13]
Finally, deacylation of the spiroderivative 10, using a 7N so-
lution of ammonia in methanol, followed by hydrolysis of the
isopropylidene group with acidic resin (Dowexꢁ 50W) gave
straightforward access to anomeric spironucleosides 3 in good
yield (Scheme 3).
As proof of final structure and to gain an understanding of
the conformation of these systems, an X-ray crystal structure
of 3g was obtained from a thin (0.02ꢂ0.03ꢂ0.31 mm) single
crystal (Figure 2). The structure in space group P21 has two in-
dependent molecules in the asymmetric unit, each having
a disordered benzene ring occupying two distinct conforma-
tions (A and B) at about 608 different rotations about the aryl
bond. In the upper molecule, conformation A is 92% occupied
Scheme 2. Synthesis of the azido-ribose system.
The benzoate ester was then treated with azidotrimethylsi-
lane in the presence of trimethylsilyl triflate in acetonitrile
under stringently anhydrous conditions at 08C for 5 min to
provide the b-azido-1-trimethylsilyl ether (8) as the sole
anomer. The silyl group was then removed smoothly with
a mixture of acetone, acetic acid and methanol, giving alcohol
5a in 98% yield.
The alkylation of alcohol 5a with a range of propargyl bro-
mides was then undertaken using BEMP (2-tert-butylimino-2-di-
ethylamino-1,3-dimethylperhydro-1,3,2-diazaphosphorine)
as
base to give the crude propargylic ether intermediates. The 3-
arylprop-2-ynyl partners for the O-alkylation were prepared
from commercially available aryl iodides and propargyl alcohol
using a two-step process involving Sonogashira coupling[12] fol-
lowed by conversion of the resulting 3-arylprop-2-ynyl alcohols
to their corresponding bromides under Appel conditions (see
the Supporting Information). The crude propargyl ether inter-
mediates 9 then underwent efficient intramolecular 1,3-dipolar
Scheme 3. Final deprotection steps to obtain novel anomeric spirocyclic
system 3.
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Figure 2. Crystal structure of spirocyclic nucleoside 3g. CCDC-1003449 (3g)
contains the supplementary crystallographic data for this paper. These data
can be obtained free of charge from The Cambridge Crystallographic Data
Centre via www.ccdc.cam.ac.uk/data_request/cif.
Figure 3. Overlay of 3g (green) with ribavirin (red), showing the similarities
in conformation.
and B is 8% occupied, whilst in
the lower molecule, the occu-
pancies are reversed.
Encouragingly, and consider-
ing the “locked” nature of our
nucleoside system, the crystal
structure when overlaid with the
[1,2,4]-triazolyl drug ribavirin
showed remarkable similarities
in conformation, particularly
with respect to the ribose ring
system (Figure 3). This prompted
us to evaluate the antiviral activi-
ty of our nucleoside analogues
and these studies are described
below.
Coronaviruses are the largest
and most complex RNA viruses
known, encoding an unusually
wide array of proteins that inter-
act with or modify viral RNA.[14]
Examples include severe acute
respiratory syndrome (SARS),
and middle eastern respiratory
syndrome (MERS), which are
amongst the most lethal viruses
currently known. Coronaviruses
are predicted to be sensitive to
Figure 4. Antiviral effects of novel nucleosides. A) Cells were pre-treated with 1 mm compounds, DMSO-containing
vehicle or mock treated 3 h before infection. Virus growth is shown relative to untreated controls. Compounds
that reduced virus growth significantly (P<0.5 after unpaired t-test with Bonferroni correction) are indicated with
RNA-like drugs,[15] and some nu-
cleosides, such as ribavirin, have
anti-coronaviral activity.[16,17] We stars. B) Reduction of cytopathic effects by 3 f. Infected cells were fixed, stained with crystal violet and adherent
cells were imaged by light microscopy. The number of nuclei in single cell bodies and in virus-induced multinu-
cleate syncytia was normalised to the number of nuclei present in uninfected, untreated controls (Uninfected).
C) Experimental compounds were applied 3 h before addition of the virus, and were maintained throughout the
experiment. Data points show the average virus titre Æstandard deviation based on 5–8 replicates. Virus growth
was measured by plaque assay 14 h after inoculation.
therefore chose the model coro-
navirus MHV as
a
proving
ground for the novel nucleoside
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analogues described in this study for antiviral activity.
In order to test for antiviral effects, MHV was grown on cells
that had been pre-treated with the experimental compounds
at a concentration of 1 mm. The amount of MHV released from
infected cells usually peaks at about 14 h after infection. Two
of the treatments, 3b and 3 f reduced the amount of MHV
that was released by about tenfold (Figure 4A).
MHV infection in 17Cl-1 cells normally results in formation of
large multinucleate syncytia followed by detachment of cells
from the culture flask.[18] The most effective experimental com-
pound from the previous assay was screened for the ability to
protect cells from MHV-induced cytopathology. The 17Cl-
1 cells were pre-treated with 3 f 3 h before infection, and sur-
viving cells were photographed 24 h after infection. Treatment
with 3 f resulted in a dose-dependent reduction in both syncy-
tium formation and detachment (Figure 4B). From these data
it was concluded that 3 f exerted a protective effect on treated
cells at concentrations up to 2 mm. This also demonstrated
that the apparent antiviral activity of 3 f was not simply an ar-
tefact of cytotoxicity.
Figure 5. Dose-response effect of experimental compounds on cell viability.
Effect of short (24 h) and long (72 h) exposure to experimental compounds
on cell viability. Experiments were performed in a mouse lung 17Cl-1 fibro-
blast cell line that is highly permissive for MHV. Relative viability as mea-
sured by conversion of formazan to MTT is plotted against treatment dose
More detailed dose-response experiments were performed
for four of the experimental compounds in order to better
gauge their antiviral potential. Pre-treatment with 2 mm of 3 f
produced the strongest antiviral effects, resulting in approxi-
mately one million-fold reduction of MHV growth (Figure 4C).
Together, these results demonstrate that 3 f had antiviral activi-
ty against the model coronavirus MHV.
~
~
*
;
for four representative compounds. Untreated: c; 3b: ; 3d: ; 3 f:
*
.
3k:
coronavirus within about five passages.[20] However, MHV
grown in the presence of 3 f consistently produced about 10%
of the virus produced in untreated control cells, and did not
develop resistance (data not shown). These results suggest
that the mechanism of action of 3 f is unclear, and that effects
of 3 f on the cell cannot be ruled out as a potential explana-
tion of the antiviral effects.
The 17Cl-1 mouse lung fibroblast line supports high-titre
MHV growth, and was therefore chosen for both toxicity and
antiviral testing. The effects of treatment on cell viability were
assessed by MTT assay.[19] Cell viability was assessed after one
day or three days. Of the compounds studied, the most prom-
ising were tested in this assay. Compound 3d was the most cy-
totoxic, while 3b, 3 f and 3k (included as a control) were
better tolerated (Figure 5).
In conclusion, a novel triazolospirocylcic nucleoside array
was assembled efficiently through intramolecular 1,3-dipolar
cycloaddition methodology, and allowed the identification of
agents that showed promising antiviral activity towards
MHV—the most promising of these being the 4-chlorophenyl
derivative 3 f. Further work is underway to establish the mech-
anism of action of this inhibitor.
The concentration which produced a 50% reduction in cell
viability in these assays was greater than 1 mm for each of the
experimental compounds tested (Table 2), demonstrating that
the compounds are relatively non-toxic.
Table 2. Relative activity of spirocyclic nucleosides 3b, 3d, 3 f and 3k.
Acknowledgements
Entry
Nucleoside
EC50
CC50
Therapeutic
index
[mm]
[mm]
The authors wish to acknowledge financial contributions from
the University of Reading (to A.D.I. and H.M.N.A.-M.) and Syn-
genta (to A.D.I.), as well as Fraser White of Agilent Technolo-
gies for X-ray data collection and structure determination of
3g.
1
2
3
4
3b
3d
3 f
410Æ50
>2000
36Æ13
1290Æ110
1510Æ90
1170Æ180
>2000
>2000
3.7
<0.6
>56
3k
>1.6
Keywords: alkynes
nucleosides · spiro compounds
·
antiviral agents
·
cycloaddition
·
A further experiment was performed in order to learn more
about the mechanism of 3 f antiviral activity by evolving drug
resistance. MHV was serially passaged eight times on 17Cl-
1 cells, which had been pre-treated with 1 mm 3 f, a concentra-
tion that reproducibly reduced viral growth by about 90%.
Previous work on antiviral compounds suggested that these
conditions were appropriate for the selection of drug-resistant
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Published online on && &&, 0000
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Communication
COMMUNICATION
&
Non-natural Nucleosides
A. Dell’Isola, M. M. W. McLachlan,
B. W. Neuman, H. M. N. Al-Mullah,
A. W. D. Binks, W. Elvidge, K. Shankland,
A. J. A. Cobb*
&& – &&
Taming the ring: Reported herein is the
synthesis of a novel class of conforma-
tionally restricted nucleoside (see
have conformational similarities to Riba-
viran. Consequently, these systems were
tested for their antiviral properties and
several were shown to have promising
activity against mouse hepatitis virus.
Synthesis and Antiviral Properties of
Spirocyclic [1,2,3]-Triazolooxazine
Nucleosides
scheme). The synthesis relies on an in-
tramolecular 1,3-dipolar cycloaddition
to generate a class of compound which
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Chem. Eur. J. 2014, 20, 1 – 6
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ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!