Research Article pubs.acs.org/journal/ascecg
New Dual Functional Salts Based on Cationic Derivative of Plant Resistance InducerBenzo[1.2.3]thiadiazole-7-carbothioic Acid, S‑Methyl Ester Marcin Smiglak,*,†,‡ Rafal Kukawka,‡ Piotr Lewandowski,‡ Marta Budziszewska,§ Aleksandra Obrępalska-Stęplowska,§ Krzysztof Krawczyk,§ Agnieszka Zwolińska,§ and Henryk Pospieszny§ †
Poznan Science and Technology Park, Adam Mickiewicz University Foundation, ul. Rubież 46, 61-612 Poznań, Poland Faculty of Chemistry, Adam Mickiewicz University, ul. Umultowska 89b, 61-614 Poznań, Poland § Institute of Plant Protection, National Research Institute, ul. W. Węgorka 20, 60-318 Poznań, Poland ‡
S Supporting Information *
ABSTRACT: Plant resistance induction is one of the most promising ways to support plants in fights against pathogens, especially viruses, due to the fact there are no plant protection agents acting directly on them. Certain chemicals, including benzo[1.2.3]thiadiazole-7-carbothioic acid, S-methyl ester (BTH) and its derivatives, were discovered as effective inducers of plant immunity. In this article, new BTH derivatives, in the form of organic salts composed of cations based on the plant resistance inducer BTH and anions introduced in order to modify physical (solubility in water, dissolution rate, thermal stability, melting points, pKa and logP values), and biological (antibacterial) properties are presented. The physical properties of resulting salts were altered by, for example, changing water solubility and also through the introduction of a second ion with a biological function (bacteriostatic and bactericidal properties against Gram-positive and Gram-negative bacteria). The major impact this new approach in plant protection may have is that the synthetic plant resistance inducers (used in very low dosages) may in the future become an alternative to the pesticides commonly used in large amounts, thus significantly reducing the use of harmful chemicals in agriculture and their negative impact on the environment and human health. KEYWORDS: Crop protection agents, Sustainable chemistry, Antiviral agents, Systemic acquired resistance, Quaternary ammonium salts, Chemical inducers of plant resistance
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INTRODUCTION
chemical). One of the most frequently used markers of SAR induction is the pathogenesis-related PR1 gene.4,5 The assessment of the expression level of this gene is therefore widely used to study whether plant response associated with SAR was activated.4,6,7 Another characteristic enzymephenylalanine ammonia-lyase (PAL) is a key enzyme of a pathway that leads to SA synthesis.8,9 Viruses are submicroscopic infectious particles composed of a coat protein and a nucleic acid core (genome). The replication cycle of viruses (production of new virus particles) takes places within the hosts (plants) cells by using the cell’s biochemical machinery. Due to that very close relationship between virus and host, mainly on the molecular level, it is very difficult to control these pathogens, and there is no pesticide which can act directly on these pathogens and not be harmful to the host cells at the same time. Thus, inducing a plant’s immune system can be the most effective way in controlling viral diseases.
Benzo[1.2.3]thiadiazole-7-carbothioic acid, S-methyl ester (BTH) and its derivatives have been shown to exhibit slight fungicidal effects in plants.1 Moreover, it was proven in the research by Kunz et al.2 that these compounds can effectively induce the immunity system in plants. Because of their unique properties, BTH and its derivatives became an interesting alternative to fungicides or bactericides that act directly on plant pathogens. Instead, benzo[1.2.3]thiadiazole-7-carbothioic acid, S-methyl ester was found to interact with the plants immune system activating it and setting it at a higher level of alertness, thus preparing the plant for pathogen attack. Such phenomena of the induction of disease resistance in plants is called systemic acquired resistance (SAR) and is one of the most effective biological methods for controlling plant diseases caused by pathogens, especially viruses.3 Systemic induced resistance phenomenon in plants is a natural mechanism that is triggered in plants as a response to pathogen attack, insect attack, or presence of an inducer. In general, SAR in plants is mediated by salicylic acid (SA) and expresses itself through different natural mechanisms that are triggered in a response to pathogen attack or presence of inducers (biological or © XXXX American Chemical Society
Received: February 26, 2016
A
DOI: 10.1021/acssuschemeng.6b00398 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
Research Article
ACS Sustainable Chemistry & Engineering BTH is a bicyclic, aromatic (with 10 π electrons)10 molecule that contains a six-membered aromatic ring fused to a fivemembered ring with two nitrogen atoms and one sulfur atom in molecule.11 Due to the fact that the nitrogen atoms in the molecule have free electron pairs, it is possible to subject them to SN2 alkylation reaction, for example, with alkyl halides in order to form ammonium salt derivatives of benzothiadiazole. Such salts, due to introduction of a counterion, can form derivatives with modified physical and chemical properties. Moreover, as BTH molecules have no antibacterial activity, the introduction of another biologically active ion may result in the formation of salts with dual functionality, i.e., resistance inducer and antibacterial agent. Since 1880, when Menschutkin synthesized the first quaternary ammonium salts,12 the popularity of these compounds has been rising. The biggest interest in the recent decades revolves around organic salts in the form of ionic liquids (ILs), which are chemical compounds composed of anions and cations with melting points below 100 °C.13 The main interest in such compounds comes from the fact that ionic liquids are promising materials for many applications.14−18 Moreover, due to the fact that both ions in an ionic liquid can be independently modified and, for example, biological functions can be introduced, in recent years ionic liquids have begun to be investigated in such fields as pharmaceuticals.19 Although this tunability is, of course, limited and one cannot always predict the properties of the prepared salt, it has been shown that certain anions and cations “transmit” their properties into product molecules. As was described by Rogers et al.,20 ILs can be organized into three main groups: ILs with (i) unique targeted physical properties (Generation 1), (ii) adjusted chemical properties (Generation 2), and (iii) tunable biological properties (Generation 3). This possibility to tune biological properties to form dual functional salts led to the development of many examples of biologically active ionic liquids,21 for example, salts with a combined anesthetic ion with an emollient ion (improving surface contact), analgesic ion with antibacterial ion, anesthetic ion and antiarrhythmic ion,22 or sweetener ion with antibacterial ion.23 The application of the concept of ionic liquids in the field of plant protection against pests is not widely explored, but recent literature has begun to show more examples of such applications. Recent studies on herbicidal ILs carried out by Pernak et al. concentrated on the synthesis of various derivatives of commonly known herbicides.24−26 Another application for the use of dual functional ILs is in the preparation of novel antifungal agents. As reported by Bica et al.,27 novel fungicide forms of thiabendazole and imazalil with increased rain persistence and activity against potato tuber diseases were synthesized and analyzed. In contrast to herbicides and fungicides, our group is working on cationic and anionic derivatives of benzo[1.2.3]thiadiazole, S-methyl ester, which is very effective plant resistance inducer.28,29 In this paper, we report the synthesis and determination of the physical properties (thermal stabilities, melting point, dissolution rate, pKa, and logP values) and antibacterial properties of nine new cationic derivatives of benzo[1.2.3]thiadiazole-7-carbothioic acid, S-methyl ester in the form of 3methyl-benzo[1.2.3]thiadiazolium salts. Our aim was to modify neutral benzo[1.2.3]thiadiazole-7-carbothioic acid, S-methyl ester by derivatizing it to a salt form and then introducing the second function either being a biologically active ion or
solubility-altering ion and to the determine physical and biological properties of the resulting salts. As a result, we identified new salts that meet this dual functionality criteria and, most importantly, maintained their plant resistance inducer properties.
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RESULTS AND DISCUSSION We have successfully synthesized nine new salts based on 3methyl-benzo[1.2.3]thiadiazole-7-carbothioic acid, S-methyl ester ([BTHMe]+) (Figure 1) and 3-methyl-benzo[1.2.3]-
Figure 1. Methods for obtaining methylated derivatives of BTH (1). “∗”: synthesis of the compound was reported previously.28
thiadiazole-7-carbothioic acid ([BTHCOOH(Me)]+) (Figure 2) cations with anions which modify physical and biological properties in regard to the starting neutral derivatives benzo[1.2.3]thiadiazole-7-carbothioic acid, S-methyl ester (BTH (1)) and benzo[1.2.3]thiadiazole-7-carboxylic acid
Figure 2. Methods of obtaining methylated derivatives of BTHCOOH (11). B
DOI: 10.1021/acssuschemeng.6b00398 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
Research Article
ACS Sustainable Chemistry & Engineering
Table 1. Results of Thermal Stabilities, Melting Points, Solubility Experiments, logP, and pKa Determination of Obtained Compounds compound BTH (1) [BTHMe][MeSO4] (2)28 [BTHMe][I] (3) [BTHMe][OTf] (4) [BTHMe][Cl] (5) [BTHMe][NTf2] (6)28 [BTHMe][Doc] (7)28 [BTHMe][MES] (8) [BTHMe][LS] (9) BTHCOOH (11) [BTHCOO(Me)]± (12) [BTHCOOH(Me)] [MeSO4] (13) [BTHCOOH(Me)] [I] (14) [BTHCOOH(Me)] [NTf2] (15) [BTHCOOH(Me)] [Doc] (16)
melting point, Tpeak [°C] 134.7 118.7 112.9 159.5 124.9 87.8 not observed 131.4 103.6 263.3 201.8 204.7 177.6 205.9 not observed
thermal stability, T5% onset [°C] 147.2 143.9 133.4 195.0 135.9 199.1 134.3 177.6 174.1 198.2 209.1 205.7 139.6 211.3 207.4
relative dissolution rateg 1.0 30.0a 7.3a 50.0a 30.0a 6.1a 3.5a 37.5a 9.4a 1.0 37.5b 33.3b 12.0b 4.9b 1.6b
solubilty in water [g/L] −3
7 × 10 >10 >10 >10 >10 0.18c >10 >10 >10 5.4 × 10−3 >10 >10 >10 6.3c >10
logP
pKa
2.93 0.32 0.59 0.32 0.31 2.04 3.48 3.50 2.95 10g/L in comparison to 7 × 10−3g/L for BTH and 5.4 × 10−3g/L for BTHCOOH. Only in the case of salts (6) and (15) with the hydrophobic anion [NTf2]− is the solubility substantially lower than in the case of other salts (0.18 g/L for [BTHMe][NTf2] (6) and 6.3 g/L for [BTHCOOH(Me)][NTf2] (15)). Figure 4 shows the dissolution rates of the starting material BTH and its alkylated derivatives with simple anions. From the results, it is shown that [BTHMe][OTf] (4) dissolved 50 times faster, [BTHMe][MeSO4] (2) and [BTHMe][Cl] (5) both ∼30 times faster, and [BTHMe][I] (3) 7.3 times faster than
neutral BTH (1). Salts derivatized with organic functional anions show similar characteristics (Table 1). A lower dissolution rate was observed for [BTHMe][LS] (9), but it still dissolved 9.4 times faster than the starting material. The slowest to dissolve of all the cationic derivatives were [BTHMe][NTf2] (6) and [BTHMe][Doc] (7), which have shown dissolution rates, respectively, 6.1 and 3.5 times faster than BTH. A similar dependence as for BTH derivatives was observed for the BTHCOOH derivatives (Supporting Information, Figure S2). It is worth noting that the compounds with the hydrophobic anions ([BTHCOOH(Me)][NTf2] (15)) dissolved faster than the derivatives with the nonhydrophobic anions. Octanol/Water Partition Coefficient. The SAR-inducing properties of the prepared substances can also depend on their solubility in lipids (allowing penetration of the active substance through the wax on the leaf surface to the interior of the plant) and solubility in water (transport in the interior of the plant). The octanol/water partition coefficient (logP) is a convenient criterion for predicting the likeliness of new substances to be transported into the plants. The logP values were determined for all of the reported compounds by using the standard OECD procedure utilizing the reverse phase HPLC method.33 Except for salts (6)−(9), logP values of all new salts were relatively low (∼0.3), and thus, their penetration into the plant can be limited. These results correspond to limited induction of resistance in plants treated with those substances. The relatively high logP values were recorded only for BTH derivatives with bulky organic anions. Among them are [BTHMe][NTf2] (6) and [BTHMe][Doc] (7), which induced a plants’ resistance at highest recorded level. It is worth noting that in the case of BTHCOOH derivatives, its salts, even with organic anions, had a low value of logP. Also the logP value for the starting compound BTHCOOH (11) was lower than that for BTH (1), which also explains a lower biological activity BTHCOOH compared with BTH as presented in an earlier publication.29 pKa Values. It is very important for cultivating plants to maintain an appropriate range of pH in the soil. A too high or too low pH of the soil can reduce the quality and quantity of crop yields or in extreme cases can cause phytotoxic effects. Therefore, determination of pKa values, using a conductometric method, for compounds (13)−(16) with dissociable protons D
DOI: 10.1021/acssuschemeng.6b00398 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
Research Article
ACS Sustainable Chemistry & Engineering
concentrations for those salts were determined to be at minimum 100 and 101.2 mg/mL, respectively (Supporting Information, Table S1). Phytotoxicity. Substances [BTHMe][MeSO 4 ] (2), [BTHMe][NTf2] (6), [BTHMe][Doc] (7), [BTHMe][MES] (8), [BTHMe][LS] (9), [BTHCOO(Me)] ± (12), [BTHCOOH(Me)][MeSO4] (13), [BTHCOOH(Me)][NTf2] (15), and [BTHCOOH(Me)][Doc] (16) were tested for their phytotoxicity toward plants. Results of the phytotoxicity experiments are shown in Table 2. It is worth noting that the
on the carboxylic acid group was performed. The pKa values for compounds with a simple anion or anions of a strong acid ([BTHCOOH(Me)][MeSO4] (13) and [BTHCOOH(Me)][I] (14)) are lower (pKa ∼ 1.32−1.91) than those for compounds with organic anions ([BTHCOOH(Me)][NTf2] (15) and [BTHCOOH(Me)][Doc] (16)) (pKa ∼ 3.72−3.93). The reason for such a change in the pKa values may come from the fact that anions of weaker acids interact much stronger with the cation core than anions of strong acids (I−, MeSO4−) and this stronger interaction can lead to a decrease in the acidity of the carboxylic acid group on the cation. The values of pKa for the obtained alkylated heterocyclic salts (13)−(16) possessing carboxylic acid groups in the structure of the cation correspond to values reported by Dyson et al.34 for the family of 1-methyl-3-alkylcarboxylic acid imidazolium salts. The pKa values for the salts with the [BTHMe]+ cation ((2)−(9)) could not be determined, as in their structure they do not possess any dissociable protons and thus could not form a conjugated base in water. Antibacterial Activity. Thirteen salts were tested for their direct impact on the bacteria growth in vitro. The minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were determined and are given in Table S1 of the Supporting Information. As a result of the performed tests, it has been determined that 12 substances exhibited some antibacterial activity. Those substances include compounds [BTHMe][MeSO 4 ] (2), [BTHMe][I] (3), [BTHMe][OTf] (4), [BTHMe][Cl] (5), [BTHMe][NTf2] (6), [BTHMe][Doc] (7), [BTHMe][LS] (9), [BTHCOO(Me)]± (12), [BTHCOOH(Me)][MeSO4] (13), [BTHCOOH(Me)][I] (14), [BTHCOOH(Me)][NTf2] (15), and [BTHCOOH(Me)][Doc] (16), (Supporting Information, Table S1). The results suggest that the antibacterial activity of the presented salts is selective, with the most effective salts being [BTHMe][MeSO4] (2), [BTHMe][NTf2] (6), and [BTHMe][Doc] (7). Salts [BTHMe][MeSO4] (2) and [BTHMe][Doc] (7) were effective against all tested human and plant bacterial pathogens, whereas substance [BTHMe][NTf2] (6) was active only against the plant pathogen P. syringae and showed an MIC against S. aureus and P. carotovorum; however, no effect was observed against E. coli. On the other hand, salts [BTHMe][OTf] (4), [BTHMe][Cl] (5), [BTHCOO(Me)]± (12), and [BTHCOOH(Me)][Doc] (16) showed selective activity only against cocci (S. aureus) and rods (P. syringae). Whereas substances [BTHMe][I] (3), [BTHCOOH(Me)][MeSO4] (13), [BTHCOOH(Me)][I] (14), and [BTHCOOH(Me)][NTf2] (15) were selective only for P. syringae, and substance [BTHMe][LS] (9) was only selective for S. aureus. From all the tested salts, only [BTHMe][MES] (8) showed no antibacterial activity at all (Supporting Information, Table S1). As evident from the tests, the best antibacterial properties were expressed by salts with [MeSO4]− and [Doc]− anions: [BTHMe][MeSO4] (2) and [BTHMe][Doc] (7). Such results are not unexpected. The [MeSO4]− anion in an aqueous environment may decompose to MeOH and H2SO4 (both being toxic to organisms), and the docusate anion was already proven in the past as a possible antibacterial anion.35,36 Both MIC and MBC activities against all tested human and plant bacterial pathogens were determined to be at very low levels of concentration of the investigated compounds: 12.5 mg/mL for substance [BTHMe][MeSO4] (2) and 25.3 mg/mL for substance [BTHMe][Doc] (7). Moreover, the phytotoxic
Table 2. Phytotoxicity Effect and Induction Properties after Watering N. tabacum var. Xanthi Plants using Water Solutions of BTH and BTHCOOH Salts
compounda
phytotoxicity effect after watering and spraying
reduction of the number of necrotic spots on leaves treated with salts solution in comparison to control (%)
[BTHMe][MeSO4] (2) [BTHMe][NTf2] (6) [BTHMe][Doc] (7) [BTHMe][MES] (8) [BTHMe][LS] (9) [BTHCOO(Me)]± (12) [BTHCOOH(Me)][MeSO4] (13) [BTHCOOH(Me)][NTf2] (15) [BTHCOOH(Me)][Doc] (16)
strongly phytotoxic slightly phytotoxic slightly phytotoxic slightly phytotoxic slightly phytotoxic phytotoxic phytotoxic phytotoxic phytotoxic
