Synthesis of 1,4-triazole linked zanamivir dimers as highly potent inhibitors of influenza A and B

Article abstract of DOI:10.1039/c2md20300f

The copper catalyzed azide alkyne cycloaddition (CuAAC) reaction-the quintessential 'click' reaction-was used to synthesise dimers of the neuraminidase inhibitor zanamivir in high yields. The effect upon anti-viral activity of varying the linker length and the number of triazole units was explored. All dimers were tested for anti-viral activity against influenza A/Sydney/5/97 and B/Harbin/7/94 in a cytopathic effect (CPE) assay. The Royal Society of Chemistry.

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ARTICLE TYPE
Synthesis of 1,4ꢀtriazole linked zanamivir dimers as highly potent
inhibitors of influenza A and B
Benjamin H. Frasera*, Stephanie Hamiltonb, Anwen M. KrauseꢀHeuera, Philip J. Wrightb, Ivan
Gregurica, Simon P. Tuckerb, Alistair G. Draffanb,Valery V. Fokinc, K. Barry Sharplessc
5
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
The successful influenza antiviral drugs Relenza® (zanamivir)
and Tamiflu® (oseltamivir) are established neuraminidase
35 inhibitors and a new long acting compound was launched in
Japan in 2010 as Inavir® (laninamivir octanoate; Figure 1). In
2004 Macdonald et al demonstrated that dimers of zanamivir
showed significantly improved in vitro antiviral activity (>100ꢀ
fold better than zanamivir), more potent in vivo efficacy and
40 extremely long residence times in lung tissue following intranasal
The copper catalyzed azide alkyne cycloaddition (CuAAC)
reaction – the quintessential ‘click’ reaction – was used to
synthesise dimers of the neuraminidase inhibitor zanamivir in
10 high yields. The effect upon antiꢀviral activity of varying the
linker length and the number of triazole units was explored.
All dimers were tested for antiꢀviral activity against influenza
A/Sydney/5/97 and B/Harbin/7/94 in a cytopathic effect (CPE)
assay.
5
doing.4, The ability to modify the dimeric linkers allows the
pharmacokinetic properties of the compounds to be tuned through
modification of the molecular properties (such as increased
lipophilicity). The same group of researchers also demonstrated
45 that trimers and tetramers of zanamivir have increased efficacy
and can be administered infrequently in a mouse model of
influenza infection.6 Although dimeric zanamivir compounds
have significant therapeutic potential, the original published diꢀ
isocyanate coupling method provided only moderate yields of the
50 desired dimers (~50%).4, 5
15 Introduction
Influenza is an acute upper respiratory illness caused by single
stranded negative sense RNA viruses of the family
orthomyxoviridae. Worldwide the disease causes 3ꢀ5 million
cases of severe illness and up to half a million deaths annually.1
20 Although vaccination programs significantly reduce the risk of
epidemic influenza outbreak there is an ongoing need for
therapeutic and prophylactic antiviral drugs. Decades of work on
the influenza virus has provided researchers with several targets
for therapeutic intervention.2 One such target – neuraminidase –
25 is a tetrameric glycoside hydrolase enzyme on the surface of the
virus that cleaves sialic acid units from galactose. This process
allows the virus to move freely through the respiratory tract and
allows ‘budding’ virion progeny to escape from the host cell
surface.3
Fig. 2 1,4ꢀtriazole linked zanamivir dimers
We wanted to explore using the robust, and often high yielding,
copper catalysed azide alkyne cycloaddition (CuAAC)7 reaction
55 to prepare a new class of 1,4ꢀtriazole linked zanamivir dimers. It
was envisaged the CuAAC reaction would improve coupling
yields and allow rapid optimization of antiviral activity as a
30
Fig. 1 Currently available influenza drugs Relenza® (zanamivir),
Tamiflu® (oseltamivir) and Inavir® (laninamivir octanoate).
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afforded the alkyne precursors (14ꢀ16) in very good yields (79ꢀ
may provide a handle to influence the physicochemical properties 25 85%). The corresponding azide precursors (23ꢀ25) were
function of linker length. It was also envisaged the triazole unit
and pharmacokinetic behaviour of the compounds. As previous
research had shown that 14ꢀ18 atom linkers were preferred for
antiviral activity4,5 we aimed to prepare dimers with linkers
spanning 12 to 22 atoms with one triazole unit (compounds 1ꢀ5)
synthesised from commercially available bromoꢀfunctionalised
carboxylic acids (17ꢀ19). Treatment of the acids (17ꢀ19) with
sodium azide in acetone/water gave the corresponding acid azides
which were treated with thionyl chloride and trimethylsilyl azide
5
(Figure 2) and linkers with 19 to 25 atoms bearing two triazole 30 to yield the corresponding acyl azides. Due to the potential
units (compounds 6ꢀ7) (Figure 2). The linker length is defined as
the number of atoms from the carbon atom of one carbamate
instability of these compounds they were not isolated but were
immediately converted via Curtius rearrangement to the
10 group – through the chain including the two carbons of the
triazole unit – to the carbon atom on the other carbamate group.
corresponding isocyanates (20ꢀ22). To a solution of the
isocyanates (20ꢀ22) in DCM was then added a protected
35 zanamivir derivative which yielded the azide precursors (23ꢀ25)
in very good yield (82ꢀ86%). With azide and alkyne precursors in
hand optimization of the CuAAC coupling reaction was
undertaken. Experiments were performed with the alkyne
precursor 15 and the azide precursor 24 (Scheme 2 and Table 1).
Chemistry
The alkyne and azide precursors for the CuAAC reaction were
synthesized from commercially available carboxylic acids using
15 an adaptation of the procedure of Carboni (Scheme 1).8
40 Table 1 Conditions for optimization of CuAAC reaction of alkyne 15 and
azide 24 to form the dimer 26
sodium solv.
ascorbate (H2O/
(mol%) tBuOH)
temp.
C)
CuSO4
(mol%)
conc. time
yield
(%)
entry
(M)
(hr)
(
◦
1
2
1
1
5
5
2:1
1:1
1:2
1:2
1:2
1:2
1:2
1:2
1:2
1:2
1:2
1:2
0.05
0.05
0.05
0.05
0.5
18
18
18
48
18
18
18
18
18
18
18
18
rta
39
45
56
54
42
54
33
59
63
85
82
58
rta
rta
rta
rta
40
60
rta
rta
rta
rta
rta
3
1
5
4
1
5
5
1
5
6
1
5
0.05
0.05
0.05
0.05
0.05
0.05
0.05
7
1
5
8
5
5
9
10
30
50
100
50
150
250
500
10
11
12
aroom temperature
Firstly, to a small RBF (5 ml) containing a 1:1 suspension of
azide (24) (0.05 mmol) and alkyne (15) (0.05 mmol) in 2:1
45 water/tertꢀbutanol (1 ml) was added copper (II) sulfate (1 mol%)
and sodium ascorbate (5 mol%). The reaction was stirred at room
temperature for 18 hours after which the crude reaction mixture
was purified directly on C18 reverse phase silica. The 1,4ꢀlinked
triazole dimer 26 was isolated in 39% yield (Entry 1, Table 1).
50 Changing the solvent to 1:1 water/tertꢀbutanol aided dissolution
of the starting materials and the yield increased to 45% (entry 2).
Changing the solvent to 1:2 water/tBuOH completely dissolved
the azide/alkyne starting materials and the yield further increased
to 56% (entry 3). Increasing the reaction time to 48 hrs gave no
55 improvement in yield (54%) (entry 4). Increasing the
concentration of the reaction (to 0.5 M) reduced the yield to 42%
which may have been due to starting material precipitation
Scheme 1 Synthesis of azide and alkyne precursors
The alkyne functionalized acids (8ꢀ10) were treated with thionyl
chloride and trimethylsilyl azide to give the corresponding acyl
20 azides which were not isolated but immediately heated to reflux
in toluene to induce Curtius rearrangement to the corresponding
isocyanates (11ꢀ13). To a solution of the isocyanates (11ꢀ13) in
DCM was then added a protected zanamivir derivative which
◦
(entry 5). Increasing the reaction temperature to 40 C did not
◦
affect the yield (entry 6) whilst increasing to 60 C decreased the
60 yield to 33% (entry 7). Increasing the catalyst loading (entries
8ꢀ11) showed that 30mol% of CuSO4 gave the highest yield
(85%) (entry 10). Stoichiometric loading (100mol%) of CuSO4
gave a diminished isolated yield due to difficulty in purifying the
2
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Table 2 Yields of zanamivir dimers (1ꢀ7) from the CuAAC coupling
reaction
product (entry 12). An alternative method for isolating the
product by quenching the reaction with water (5 ml) and
collecting the resulting precipitate gave only contaminated
product (~60% by 1H NMR). The final removal of the acid labile
protecting groups was completed by treatment with TFA in
water/acetonitrile (91%). Using the optimized conditions for the
CuAAC reaction (entry 10) we prepared a series of 1,4ꢀtriazole
linked zanamivir dimers (1ꢀ7) in very good yields (65ꢀ78%) over
two steps (Table 2).
entry
azide
alkyne 1,4ꢀlinked
no. of
yield (%)
zanamivir atoms in
dimer
the linker
5
1
2
3
4
5
6
7
23
23
24
25
25
23
24
14
15
15
15
16
a
1
2
3
4
5
6
7
12
14
16
18
22
19
25
75
71
78
76
67
72
65
b
a 1,6ꢀheptadiyne
15 b 1,8ꢀnonadiyne
Antiꢀviral activity assay
The 1,4ꢀtriazole linked dimers (1ꢀ7) were tested for antiviral
activity against representative influenza and viruses
A
B
(A/Sydney/5/97 and B/Harbin/7/94) in a standard cytopathic
20 effect (CPE) assay.4 Zanamivir and oseltamivir carboxylate were
used as controls (Table 3).
Table 3 In vitro antiviral activity for zanamivir dimers (1ꢀ7)
compound
no. of atoms
in linker
A/Sydney/5/97
EC50 (nM)
B/Harbin/7/94
EC50 (nM)
zanamivir
786 ± 610 (n=6)
149 ± 89 (n=6)
oseltamivir
carboxylate
98 ± 98 (n=6)
583 ± 296 (n=6)
1
2
3
4
5
6
7
12
14
16
18
22
19
25
3005 ± 685 (n=3) 0.51 ± 0.30 (n=3)
1285 ± 960 (n=3) 0.43 ± 0.20 (n=3)
1820 ± 820 (n=3) 0.79 ± 0.07 (n=3)
0.73 ± 0.22 (n=3) 0.32 ± 0.07 (n=3)
0.61 ± 0.22 (n=3) 0.39 ± 0.09 (n=3)
1.36 ± 0.66 (n=3) 0.65 ± 0.39 (n=3)
0.80 ± 0.20 (n=3) 0.88 ± 0.61 (n=3)
a Footnote text.
The data show that all 1,4ꢀtriazole linked zanamivir dimers are
25 significantly more potent than zanamivir and oseltamivir
carboxylate against influenza B/Harbin/7/94 with most also
having increased efficacy when tested against A/Sydney/5/97.
Dimers 4 to 7 are 100 times more potent than oseltamivir
carboxylate and 1000ꢀ3000 fold more potent than zanamivir
30 against influenza A/Sydney/5/97. For the influenza
B/Harbin/7/94 virus all dimers were also at least 100 fold more
potent than zanamivir and generally 1000 fold more potent than
oseltamivir carboxylate. Interestingly, the dimers with the
shortest linker lengths (1, 2 and 3) had EC50 values of around 1 to
35 4 ꢁM against influenza A/Sydney/5/97 which could be explained
by the linkers being too short to allow effective biꢀvalent
interactions between binding sites on the same virus particle or
adjacent virions for this particular virus isolate. As the size of the
linker increases from 18 to 25 atoms (4ꢀ7) there is little variation
40 in inhibition potency. It was also observed that as the number of
triazole units in the chain increases (6 and 7) there is little effect
10
Scheme 2 CuAAC reaction of alkyne 15 and azide 24
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on potency. These results are consistent with our previous
research4 and suggest that beyond a minimum value the number
of atoms in the 1,4ꢀtriazole linker up to 25 atoms does not
significantly affect potency. We have previously proposed that
intervirion binding leading to virus aggregation is the most
significant factor contributing to the increased potency of
5. S. J. F. Macdonald, R. Cameron, D. A. Demaine, R. J.
Fenton, G. Foster, D. Gower, J. N. Hamblin, S.
Hamilton, G. J. Hart, A. P. Hill, G. G. A. Inglis, B. Jin,
H. T. Jones, D. B. McConnell, J. McKimmꢀBreschkin,
G. Mills, V. Nguyen, I. J. Owens, N. Parry, S. E.
Shanahan, D. Smith, K. G. Watson, W.ꢀY. Wu and S.
P. Tucker, J. Med. Chem., 2005, 48, 2964ꢀ2971.
6. K. G. Watson, R. Cameron, R. J. Fenton, D. Gower, S.
Hamilton, B. Jin, G. Y. Krippner, A. Luttick, D.
McConnell, S. J. F. MacDonald, A. M. Mason, V.
Nguyen, S. P. Tucker and W.ꢀY. Wu, Bioorg. Med.
Chem. Lett., 2004, 14, 1589ꢀ1592.
60
5
5
zanamivir dimers4, but a contribution from bivalent ligand
binding between sites on the same viral particle cannot be
discounted. Thus the dimers may have a dual mechanism of
10 action: neuraminidase inhibition and enhanced aggregation.
Further results on the dual mechanism of dimeric zanamivir
compounds will be published shortly.
65
7. V. V. Rostovtsev, L. G. Green, V. V. Fokin and K. B.
Sharpless, Angew. Chem., Int. Ed., 2002, 41, 2596ꢀ
2599.
8. M. Khoukhi, M. Vaultier, A. Benalil and B. Carboni,
Synthesis, 1996, 4, 483ꢀ487.
Conclusions
70
A high yielding copper catalyzed azide alkyne cycloaddition
15 (CuAAC) reaction was used to prepare new dimeric zanamivir
conjugates as influenza antiviral compounds in very good yields.
Not only does the CuAAC method improve coupling yields
compared to previously published methods but the 1,4ꢀtriazole
linked dimers are among the most potent inhibitors of influenza
20 reported to date – being up to 3000 times more potent than
zanamivir. Given the very high inhibitory activity of these dimers
further investigation of the synthesis, SAR and properties of the
1,4ꢀtriazole class of zanamivir dimers is warranted and future
progress will be reported.
75
25 Acknowledgements
BF gratefully acknowledges Biota Holdings Limited for the
award of a Biota Fellowship in 2008. BF gratefully acknowledges
the American Australian Association for the award of the Dow
Chemical Company Foundation Fellowship of 2008. AKH
30 gratefully acknowledges the support of the Australian Nuclear
Science and Technology Organisation (ANSTO) through the
Graduate Development Program.
Notes and references
aAustralian Nuclear Science and Technology Organisation, Locked Bag
35 2001, Kirrawee DC, New South Wales, 2232, Australia. Fax:+61 2 9717
9262 Tel:+61 2 9717 3887 E-mail: benjamin.fraser@ansto.gov.au
bBiota Holdings Limited, Unit 10, 585 Blackburn Road, Notting Hill,
Victoria, 3168, Australia.
cThe Scripps Research Institute, 10550 North Torrey Pines Road, La
40 Jolla, California, 92037, The United States of America.
†Electronic Supplementary Information (ESI) is available which includes
full experimental synthetic methods and spectroscopic data for all
compounds and intermediates. Full experimental details of the
cytopathetic effect assay are also given. See DOI: 10.1039/b000000x/
45
1. H. Brody, Nature, 2011, 480, 1.
2. B. N. Fields, in Fields Virology: Volume 1, Lippincott1995,
p. 1504.
3. P. M. Colman, Protein Sci., 1994, 3, 1687ꢀ1696.
50 4. S. J. F. Macdonald, K. G. Watson, R. Cameron, D. K.
Chalmers, D. A. Demaine, R. J. Fenton, D. Gower, J.
N. Hamblin, S. Hamilton, G. J. Hart, G. G. A. Inglis, B.
Jin, H. T. Jones, D. B. McConnell, A. M. Mason, V.
Nguyen, I. J. Owens, N. Parry, P. A. Reece, S. E.
55
Shanahan, D. Smith, W.ꢀY. Wu and S. P. Tucker,
Antimicrob. Agents Chemother., 2004, 48, 4542ꢀ4549.
4
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