Toxic on purpose: Ionic liquid fungicides as combinatorial crop protecting agents

Article abstract of DOI:10.1039/c1gc15170c

We present an ionic liquid strategy for novel hydrophobic fungicide forms of the actives thiabendazole and imazalil with increased rain persistence and activity against potato tuber diseases.

Full text of DOI:10.1039/c1gc15170c

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Green Chemistry
Cite this: Green Chem., 2011, 13, 2344
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COMMUNICATION
Toxic on purpose: ionic liquid fungicides as combinatorial crop protecting
agents†
Katharina Bica,a,b Louise R. Cooke,c Patrick Nugent,c Christiaan Rijksenb and Robin D. Rogers*b,d
Received 14th February 2011, Accepted 31st May 2011
DOI: 10.1039/c1gc15170c
We present an ionic liquid strategy for novel hydrophobic
fungicide forms of the actives thiabendazole and imazalil
with increased rain persistence and activity against potato
tuber diseases.
against MRSA or other pathogens, antiseptics or antifouling
agents, or anti-cancer agents.7–9
While these examples mainly deal with traditionally stud-
ied IL ions such as 1-alkyl-3-methyl-imidazolium salts, ILs
can also be directly prepared from pharmaceutically active
ingredients.10,11 We have recently shown that an IL form of a
formerly solid drug cannot only preclude polymorphism, but
also influences important properties such as solubility, bioavail-
ablity, and stability.12 Furthermore, IL forms may provide
novel delivery options with improved membrane penetration or
iontophoresis.13
Another field where the combination of biological activity and
IL form might bring operational benefits is in agrochemistry.14,15
For example, a hydrophobic IL form of an agrochemical could
improve rain-fastness, reduce drift and the amount of chemicals
which need to be applied, and also enhance movement within
the plant to achieve greater uniformity of protection. Improved
persistence might be achieved by movement into plant tissue
or into the cuticle/epicuticular wax followed by gradual re-
distribution.
Enhanced curative activity against plant pathogens might
result from re-distribution into plant tissue, since in the absence
of translaminar or systemic movement, a fungicide can only act
on the target pathogen before it penetrates plant tissue. The
downside of increasing penetration would be the impact on
residues; however, this would not matter if a pre-planting seed
tuber treatment was used for seed-borne diseases. Additionally,
a dual functional approach combining active cations and anion
in one IL formulation might lead to synergistic or antagonistic
effects (as observed for pharmaceutically active ILs) which might
even be used to overcome resistance.16
Only two fungicides are currently approved in the UK for use
against potato tuber diseases such as dry rot (Fusarium spp.),
gangrene (Phoma spp.), silver scurf (Helminthosporium solani),
and skin spot (Polyscytalum pustulans): the benzimidazole
derivative thiabendazole 1 and the imidazole-derivative imazalil
2.1 Thiabendazole has been widely used on potatoes since
the mid-1970s both pre-planting and post-harvest.2 However,
problems with its use include resistance conferred by specific
mutations (single base changes) in the b-tubulin gene, which
have affected its activity in controlling silver scurf, skin spot,
and dry rot caused by F. sambucinum, and its limited penetration
into the potato tissue, since most of the active stays on the skin.3
Imazalil was introduced for potato tuber disease control in the
early 1980s4 and is now available both alone and formulated with
thiabendazole, but, like thiabendazole, it suffers from limited
penetration into the tuber.
Within the last decade, ionic liquids (ILs, currently defined as
salts melting below 100 ◦C) have been studied for an increasing
number of applications where low or no toxicity is needed,
despite the biological activity of many of the ILs.5,6 Subsequently,
applications have arisen taking advantage of known biological
activity of several ions that could be made into ILs leading to new
applications. For example, the antimicrobial properties inherent
to some ILs have been recognized as valuable tools for biocide
applications, including the development of bioactive coatings
We sought to study the utility of an IL approach to a major
problem affecting the agricultural economy in Northern Ireland,
diseases of potato tubers. We present herein novel active ILs
composed of the fungicides thiabendazole 1 and imazalil 2
and discuss their activity against potato tuber pathogenic fungi
(Scheme 1).
Thiabendazole and imazalil have an inherent basicity due to
free nitrogen atoms that can easily be protonated. If paired with
an appropriate acid, the acid/base equilibrium lies far enough
to the right to obtain a low-melting, protic IL. For our initial
studies, we chose docusate (bis(2-ethylhexyl) sulfosuccinate) as
a suitable counterion for its ability to form stable, hydrophobic
aInstitute of Applied Synthetic Chemistry, Vienna University of
Technology, 1060, Vienna, Austria
bThe Queen’s University of Belfast, QUILL, School of Chemistry and
Chemical Engineering, Belfast, Northern Ireland, BT9 5AG
cApplied Plant Science and Biometrics Division, Agri-Food and
Biosciences Institute, Newforge Lane, Belfast, UK BT9 5PX
dThe University of Alabama, Department of Chemistry and Center for
Green Manufacturing, Tuscaloosa, AL, 35487, USA.
E-mail: rdrogers@as.ua.edu
† Electronic supplementary information (ESI) available: Synthetical
procedures and analytical data of compound 3 and 4, origin of isolates,
and detailed information onin vivoassays. See DOI: 10.1039/c1gc15170c
2344 | Green Chem., 2011, 13, 2344–2346
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such as hexane or benzene, and also in ethyl acetate or diethyl
ether. On the other hand, good solubility was observed in short-
chain alcohols (MeOH, EtOH), in halogenated solvents, and in
polar-aprotic solvents such as acetone, DMF or DMSO.
Both thiabendazolium and imazalilium docusate 3 and 4 were
obtained as viscous liquids with glass transition temperatures at
◦
◦
-16.3 C and -31.0 C, respectively. However, after some time
both IL formulations solidified into crystalline solids melting at
46.9 or 64.0 ◦C, thus still fitting the current definition of ILs.
When investigating the thermal stability using thermogravimet-
rical analysis (TGA), we observed single-step decompositions
and excellent stabilities of >200 ◦C for both IL fungicides,
indicating that the formation of a low-melting or liquid salt
formulation is not related to limited stability.
Scheme 1 Synthesis of IL fungicide formulations of thiabendazole 1
and imazalil 2 (shown as its sulfate salt).
ILs, as well as its known emollient and skin penetration
features.17–19
We have carried out in vitro tests of the IL thiabendazolium
docusate 3 in comparison to the neutral fungicide using selected
isolates of Fusarium spp., the cause of potato dry rot. F.
coeruleum and F. culmorum are sensitive to thiabendazole
whereas isolates of F. sambucinum are frequently resistant.
Isolates of Phoma spp., the cause of gangrene, and Phytophthora
erythroseptica, the cause of pink rot, were also tested.
Thiabendazolium docusate 3 was prepared as a solution
in EtOH and studied at concentrations of 250 and 25 mM
(equivalent to 50 and 5 mg thiabendazole 1 per liter) in agar
(malt agar for Phoma spp. and potato dextrose agar for the
other organisms) to determine in vitro activity against mycelial
growth of the test organisms. The final concentration of EtOH
in agar was 1% v/v, which gave 0–10% inhibition of mycelial
growth; results were calculated using a 1% ethanol control.
We were pleasantly surprised to see that the activity of
thiabendazole 1 against the Fusarium and Phoma spp. was
retained for the hydrophobic IL formulation (Fig. 1, Table 1).
Furthermore, while thiabendazole 1 is not considered active
against the Oomycete pathogen, P. erythroseptica, there is some
evidence that the IL 3 is more active. It is interesting to note that
a simple co-formulation of the neutral active thiabendazole with
As a general strategy, the neutral fungicide was first converted
to the chloride salt by treatment with aqueous hydrochloric
acid (Scheme 1). Subsequent anion metathesis was achieved
by a straightforward metathesis reaction with one equivalent
sodium docusate in a mixture of acetone and water. The newly
formed ILs were then extracted with dichloromethane and, after
extractive removal of inorganic impurities with H2O, isolated
by evaporation in good yields. These suggested techniques can
typically reduce the metal and halide contamination of the final
IL to £ 0.05%. Although ILs based on alkylated thiazolium
cations have been previously described,20 here protonation was
obtained selectively on the benzimidazole heterocyclic system,
thus giving rise to thiabendazolium docusate 3 in 91% yield.
Based on the commercial availability of imazalil as its sulfate
salt, this protocol was slightly modified for the synthesis of
imazalilium docusate 4. Anion metathesis with sodium docusate
was performed in anhydrous chloroform and gave imazalilium
docusate 4 in a similar manner in 61% yield.
As is typical for hydrophobic ILs, these novel fungicide
formulations were insoluble in water, in polar-aprotic solvents
Table 1 Sensitivity of potato tuber pathogens to derivatives of thiabendazole
Inhibition (%)
Isolate
Concentration (mM)
1
3
1-Stearic acid
F. sambucinum ex SASA
F. sambucinum L’gall 53
F. sambucinum L’gall 11
F. coeruleum ex B7/06 T8
F. coeruleum ex B16/08 P31
F. culmorum P13
250
25
250
25
250
25
250
25
250
25
250
25
250
25
250
25
89
22
93
19
76
8
99
99
100
100
100
100
100
95
100
98
51
4
96
19
68
11
83
19
99
99
100
100
100
100
99
94
99
95
97
33
91
89
24
82
20
82
10
99
99
100
100
100
100
99
95
99
97
54
10
34
Phoma exigua 9.1
Phoma foveata BL2 05 T3
Phytophthora erythroseptica BL2/08 P31
Phytophthora erythroseptica Rooster Eire
250
25
250
25
31
8
11
11
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testing showed that fungitoxicity against a range of potato
tuber pathogens was not only retained but enhanced. These
early results might not only lead to a dramatic reduction of
future fungicide applications, but should kick-start ionic liquid
research in crop protection.
Acknowledgements
Mark Wilson is thanked for technical assistance with the in vitro
sensitivity testing.
Notes and references
Fig. 1 In vitro activity of thiabendazolium docusate 3 against F.
coeruleum and F. sambucinum.
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a Concentration required to reduce mycelial growth in vitro by 50%.
a second hydrophobic molecule such as stearic acid – that did
not lead to protonation and IL formation21 – did not enhance the
activity, as was observed for 3, but gave values similar to those of
thiabendazole alone. This behaviour might be explained by the
tendency of hydrophobic ILs to form ion pairs in solution, which
makes them inherently different from a simple co-formulation
of the active compounds with a second hydrophobic neutral.22
In a similar manner, imazalilium docusate 4 was tested at
127, 25, 12.7, 2.5 and 1.27 mM (equivalent to 0.5, 1, 5, 10 and
50 mg imazalil 2 per liter) against some of the same pathogen
isolates. Again, we observed that activity was retained with
EC50 values similar to those obtained with neutral imazalil
(Table 2). Imazalil, an inhibitor of fungal ergosterol synthesis, is
not active against Oomycete pathogens, but the hydrophobic IL
imazalilium docusate 4 showed some limited activity against P.
erythroseptica.
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thiabendazolium and stearic acid did not show any sign of IL or salt
formation. See: K. Bica, J. Shamshina, W. Hough, D. MacFarlane
and R. D. Rogers, Chem. Commun., 2011, 47, 2267–2269.
22 (a) K. J. Fraser, E. I. Izgorodina, M. Forsyth, J. L. Scott and D. R.
MacFarlane, Chem. Commun., 2007, 3817–3819; (b) D. F. Kennedy
and C. J. Drummond, J. Phys. Chem., 2009, 113, 5690–5693.
An IL strategy can be used for novel fungicide formulations
with increased hydrophobicity against potato tuber pathogens.
The basicity inherent in many fungicides, particularly in imi-
dazoles or benzimidazoles allows simple transformation into a
hydrophobic IL form in straightforward metathesis reactions
with hydrophobic anions such as the common emmolient
docusate. In addition to the potential for increased persistence
of these novel hydrophobic fungicide formulations, biological
2346 | Green Chem., 2011, 13, 2344–2346
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The Royal Society of Chemistry 2011
©