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    • 专利摘要:
    Chitin deacetylase inhibitors and their use as agricultural fungicides, arthropocides and nematicides. Method for identifying a compound with potential pesticidal activity against a chitin-containing organism, said compound being a CDA inhibitor comprising: a) obtain for said compound a value of the discriminant function DF1, in which: DF1 = (-10.295 x GATS4m) + (35.124 GGI8) + 6.917 where: - GATS4m: Geary autocorrelation index - lag4/weighted by atomic masses, - GGI8: topological load index of order 8 and b) verifying if the DF1 value obtained in (a) is between 0 and +14, said compound is identified as a potential inhibitor of the CDA enzyme. c) carry out an experimental analysis of the compound according to step b), which comprises putting said compound in contact with a pathogen and observing if there is growth of this. Use of a chemical compound with pesticidal activity against a chitin-containing organism selected from fungi, arthropods, fungi and nematodes. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2794348A1
    申请号:ES201930440
    申请日:2019-05-17
    公开日:2020-11-17
    发明作者:Garcia Alejandro Perez；Cruz Jesús Martinez；Ricardo Zanni；Hinojosa Diego Francisco Romero；Ortuno Dolores Fernanez；Llompart María Galvez；Domenech Ramón Garcia；Alvarez Jorge Galvez
    申请人:Universidad de Malaga；Universitat de Valencia；
    IPC主号:
    专利说明:

    [0001] Chitin deacetylase inhibitors and their use as agricultural fungicides, arthropocides and nematicides
    [0003] BACKGROUND OF THE INVENTION
    [0005] Chemical control has been essential in preventing losses due to plant diseases. Chitin is the main component of the exoskeleton of arthropods and of the cell walls of fungi; it is also present in the eggs and intestinal linings of nematodes. Chitin is considered a safe target for fungicides, insecticides or nematicides, since chitin is absent in plants and mammals1. Chitin is also a known inducer of immune responses in plants. As a consequence of the enzymatic activity of plant chitinases, small oligomers of chitin are released. These oligomers can be recognized by plant receptors such as CERK1, promoting the activation of the chitin-specific signaling cascade. In response, pathogens have developed strategies to overcome chitin detection. One of these strategies is the conversion of chitin in the cell wall into chitosan by the action of chitin deacetylase (CDA - chitin deacetylase -). This enzyme catalyzes the hydrolysis of the N-acetamido group in the N-acetylglucosamine units of chitin to convert it into chitosan, the deacetylated derivative of chitin, a poor substrate for chitinases and a compound with inductive activity significantly lower than chitin2,3 .
    [0007] In the particular case of fungi, the fungicides marketed for disease control in agricultural crops are under pressure. The best examples of fungal diseases in which chemicals play a key role in disease management are powdery mildews. Among the economically important crops affected by powdery mildew are cereals, vines, and many horticultural crops and ornamental plants. The problem of resistance to fungicides in powdery mildews is well illustrated in the case of the cucurbit pathogen Podosphaera xanthii4. In southern Spain, resistance to the most popular anti-powdery mildew fungicides5-7 has been described, with multi-resistant isolates in the most intense cultivation areas7. Therefore, there is a pressing demand to identify and develop new phytosanitary products.
    [0008] "Morphological and functional analysis of the interaction of Podosphaera xanthii -cucurbits" is the title of the doctoral thesis of one of the inventors of this invention.8 This document describes a study whose result identifies chitin deacetylase (CDA) as a key protein for the pathogenesis, since gene silencing or its inhibition with different carboxylic acids activates the immunity triggered by chitin and blocks fungal development, making it an ideal target for the development of new phytosanitary products. However, it does not describe a method for detect inhibitors as set forth in the present invention.
    [0010] Insecticide resistance is also a very serious problem for agriculture and farmers are also demanding new tools to control the most important pests. CN105462996A refers to a technology based on gene silencing of a gypsy moth chitin deacetylase gene, a gene segment of a gypsy moth chitin deacetylase gene, and a dsRNA dsRNA of that gene segment and the application of that gene. DsRNA to interfere with the development process of the insect and cause an altered metamorphosis.
    [0012] Similarly, CDA appears to be also an important protein for nematodes9. In a paper using the model nematode Caenorhabditis elegans, it was shown that disruption of CDA genes caused a delay in the development time of the nematode. They also demonstrated the presence of homologues in many other Nematoda species, including important plant and vertebrate parasitic nematodes, concluding that CDA may be a valid target for the development of intervention tools against parasitic nematodes.
    [0014] One of the most promising computer-aided drug design methods is molecular topology. Contrary to the rest of the quantitative structure-activity relationship (QSAR) methods, the methodology allows a fast and accurate prediction of many biological and physicochemical properties. Defined as part of mathematical chemistry, molecular topology is basically related to the association between molecules and graphs, in such a way that it allows describing molecular structures through graphotheoretical indices. Furthermore, it deals with the connectivity of atoms in molecules and not with geometric characteristics such as angles, distances, or three-dimensional structure, which is common in standard / conventional approaches. In this way, graph theory and the surrounding disciplines stand as basic tools of molecular topology. Following this approach, obtained excellent results in the design and selection of new drugs in different medical fields.
    [0016] Starting from the structure-activity analysis on the effect of some known carboxylic acids on the fungal CDA protein, a combined strategy of molecular topology and experimental tests was developed to identify new inhibitors of chitin deacetylase (CDA). This strategy consisted of a selection by virtual screening of more than 3,000,000 molecules belonging to various databases and an additional experimental analysis of the most interesting molecules, which allowed the elaboration of a useful method for the identification of molecules to control pests, such as such as fungi, insects or nematodes, based on the inhibition of CDA.
    [0018] DESCRIPTION OF THE INVENTION
    [0020] The term "fungicide" in this application refers to the toxic effect produced by the compound when brought into contact with a fungus. This effect may or may not be mediated by a plant.
    [0022] The remaining terms in this specification have the general meaning commonly ascribed in the field.
    [0024] The present invention refers to a method for the identification of a compound with potential pesticidal activity against chitin-containing organisms, said compound being an inhibitor of the enzyme chitin deacetylase (CDA), comprising:
    [0025] a) Obtain for said compound a value that results from applying the discriminant function DF1, which is:
    [0027] DF1 = (-10.295 x GATS4m) (35.124 x GGI8) 6.917
    [0028] where:
    [0030] - GATS4m: Geary's autocorrelation index - lag4 / weighted by atomic masses,
    [0032] - GGI8: topological load index of order 8,
    [0034] b) check if the DF1 value obtained in (a) for said compound is between 0 and 14. If the value is between 0 and 14, said compound is identified as a potential inhibitor of the CDA enzyme.
    [0035] According to additional particular embodiments of the method, as defined above:
    [0037] - step (a) further comprises obtaining for said compound a predictive value of the inhibitory activity Log (lnh%), where:
    [0039] Log (lnh%) = 0.814 - 0.018 x T (N..N) 9.552 x JGI2
    [0040] where:
    [0042] - T (N..N) is the topological distance expressed as the number of edges between two consecutive nitrogen, and
    [0044] - JGI2 is the topological average load index of order 2;
    [0046] - step (b) further comprises verifying if the value of Log (lnh%) obtained in (a) for said compound is between 1 and 2. If the value is between 1 and 2, said compound is identified as a potential inhibitor of the CDA enzyme.
    [0048] According to additional particular embodiments of the method, as defined above:
    [0050] - step (a) further comprises obtaining for said compound a value for the discriminant function DF2, in which:
    [0052] DF2 = 21.022 - (19.407 x GGI10) - (5.104 x SFige) - (7.911 x GATS3e)
    [0053] where:
    [0055] - GGI10: topological load index of order 10,
    [0057] - Seige: Eigenvalue sum of the eigenvalue of electronegativity.
    [0059] - GATS3e: Geary autocorrelation index -lag3 / weighted by Sanderson atomic electronegativities; and.
    [0061] - step (b) further comprises verifying if the DF2 value obtained in (a) for said compound is between 0 and 6. If the value is between 0 and 6, said compound is identified as a potential inhibitor of the CDA enzyme.
    [0063] According to further particular embodiments, the method as defined above comprises after step (b):
    [0065] - c) carrying out an experimental analysis of the compound identified as a potential inhibitor of the CDA enzyme according to step (b), which comprises putting said compound in contact with a pathogen, preferably a fungal pathogen, and more preferably a fungal pathogen in the presence of a plant and observe if there is growth of this.
    [0067] According to a more particular embodiment, the pathogen is a fungus located on a plant, even more particularly Podosphaera xanthii. According to another more particular embodiment, the pest is an insect even more particularly Galleria mellonella.
    [0069] According to additional particular embodiments, the method as defined above further comprises performing a docking experiment with a fragment of the CDA enzyme comprising the active site thereof and the compound identified as an inhibitor of the enzyme chitin deacetylase. . Said compound will show an affinity value lower than -7 kcal / mol, by virtue of its ability to interact with the active site of the CDA enzyme. In particular, the CDA enzyme used for the docking experiment is a fungal CDA enzyme, preferably the CDA from Colletotrichum lindemuthianum.
    [0071] According to additional particular embodiments, the method defined above comprises a docking experiment with the entire CDA enzyme, of the compound identified as an inhibitor of the enzyme chitin deacetylase, in which said compound shows two or three hydrogen bonds with two or three different amino acids located within the active site of the CDA enzyme. According to a more particular embodiment, the CDA enzyme used for the docking experiment is a fungal CDA enzyme, more preferably the CDA from Colletotrichum lindemuthianum. According to another more particular embodiment, the CDA enzyme used for the docking experiment is an insect CDA enzyme, more preferably the Bombyx mori CDA .
    [0072] The method defined above is useful to identify compounds to combat pests in which the pests are those organisms that contain chitin, such as fungi, arthropods or nematodes.
    [0074] The present invention also relates to the use of chemical compounds with pesticidal activity against a chitin-containing organism selected from fungi, arthropods and nematodes, identified by the method defined above.
    [0076] According to a particular embodiment, the invention refers to the use of a chemical compound with pesticidal activity against an organism that contains chitin selected from fungi, arthropods and nematodes, characterized in that:
    [0077] - said compound has a DF1 value between 0 and 14,
    [0079] - preferably said compound also has a value of Log (lnh%) between 1 and 2;
    [0080] - more preferably, said compound also has a DF2 value between 0 and 6.
    [0081] According to a more particular embodiment, the invention relates to the use of a chemical compound with pesticidal activity against a chitin-containing organism selected from fungi, arthropods and nematodes, as defined above, characterized in that said compound shows an affinity value lower than - 7 kcal / mol by virtue of its ability to interact with the active site of the CDA enzyme.
    [0083] According to specific embodiments, the chemical compound detected with pesticidal activity against a chitin-containing organism is selected from:
    [0085] - (1-methyl-2,4-dioxo-1,4,6,7-tetrahydro [1,3] thiazol [2,3f] purin-3 (2H) yl) acetic acid
    [0086] - (3 - [(1,3-dimethyl-2,4,6-trioxotetrahydro-5 (2H) -pyrimidinylene) methyl] phenoxy) acetic acid,
    [0087] - 2- (2-methoxy-4 - ([(5Z) -2,4,6-trioxo-1- (prop-2-en-1-yl) -1-3-diazinan-5-ylidene] methyl acid ) phenoxy) acetic,
    [0089] - 5- [4- (2-hydroxyethoxy) -3-methoxybenzylidene] -1,3-dimethyl-2,4,6 (1H, 3H, 5H) -pyrimidinetrione,
    [0090] - 7- [2-hydroxy-3- (4-morpholinyl) propyl] -1,3-dimethyl-3,7-dihydro-1 H-purine-2,6-dione,
    [0092] - 2-amino-7-methyl-5-oxo-4- [4- (trifluoromethoxy) phenyl] -4H, 5H-pyrano [4,3-b] pyran-3-carbonitrile,
    [0094] - N- {4- [3- (2,6-dimethyl-4-morpholinyl) -2,5-dioxo-1-pyrrolidinyl] phenyl} acetamide,
    [0096] - 2- (1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl) -N- (2-methoxy-1-methylethyl) acetamide,
    [0098] - N-cyclopropyl-2- (1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-9H-purin-9-yl) acetamide
    [0099] - 5-imino-1- (2-methyl-5-nitrophenyl) -3-phenylidantoin,
    [0101] - 5-imino-1- (4-methyl-5-nitrophenyl) -3-phenylidantoin,
    [0103] - 3- {2,5-dioxo-3 - [(5Z) -4-oxo-5- (phenylmethylidene) -2-sulfanylidene-1,3-thiazolidin-3-yl] pyrrolidin-1-yl} propanoic acid
    [0105] - 2- (10,12-dioxo-9 - {[(1,3-thiazol-2-yl) carbamoyl] methyl} -7-thia-9,11-diazatricyclo [6.4.0.02,6] dodeca-1 acid (8), 2 (6) -dien-11-yl) acetic,
    [0107] - 2- {4 - [(1,3-dimethyl-4,6-dioxo-2-sulfanylidene-1,3-diazinan-5-ylidene) methyl] phenoxy} acetic acid,
    [0108] - 2 - ({[(5E) -4,6-dioxo-1- [4- (propan-2-yl) phenyl] -2-sulfaniM, 4,5,6-tetrahydropyrimidin-5-ylidene] methyl} acid amino) -3- (1 H-imidazol-4-yl) propanoic,
    [0110] - 2 - [(5E) -4-oxo-5 - {[2- (prop-2-en-1-yloxy) phenyl] methylidene} -2-sulfanylidene-1,3-thiazolidin-3-yl] pentanedioic acid ,
    [0112] - 2- {1 - [(3,4-difluorophenyl) methyl] -3-oxopiperazin-2-yl} acetic acid,
    [0114] - 2- {3 - [(2-chloro-6-fluorophenyl) methyl] -2,4,5-trioxoimidazolidin-1-yl} acetic acid,
    [0116] - {2 - [(1,3-dimethyl-4,6-dioxo-2-thioxotetrahydro-5 (2H) -pyrimidinylidene) methyl] phenoxy} acetic acid and
    [0118] - 3-benzyl-1,7-dimethyl-7,9-dihydro-1 H-purin-2,6,8 (3H) -trione.
    [0120] Table 1 lists 20 compounds with pesticidal activity predicted by molecular topology, as well as their respective DF1, Log (inh%) and DF2 values.
    [0121]
    [0122]
    [0126] Other fungal pathogens include the Ascomycota and Basidiomycota classes, as well as pathogens of the Oomycetes class, on any cereal, vine, fruit tree, horticultural, fiber and / or ornamental crops. These pathogens include: Ascomycetes, pathogens included in the order Erysiphales such as Blumeria graminis, Erysiphe necator, Erysiphe polygoni, Leveillula taurica, Podosphaera aphanis and Podosphaera xanthii, and other pathogens including Alternaría solani, Botrytis cineicoum, Cercospoletico gramineraum, Cercospoletico graminera columbarium, Cercospolaminera gramineico. , Fusarium oxysporum, Gaeumannomyces graminis, Magnaporthe grisea, Monilia fructicola fijensis Mycosphaerella, Phomopsis viticola, Pyrenophora teres, Rhizopus stolonifer, Rynchosporium secalis, Sclerotinia sclerotiorum, Septoria tritici, Venturia inaequalis and Verticillium dahliae; Basidiomycetes, pathogens of the order Uredinales such as Puccinia spp. ( Puccinia striiformis, Puccinia hordei, Puccinia graminis) and Uromyces spp. ( Uromyces viciae-fabae, Uromyces betae), pathogens in the order Ustilaginales such as Ustilago maydis and Ustilago tritici, and other pathogens including Armillaria mellea, Rhizoctonia solani, and Sclerotium rolfsii; Oomycetes, including Phytophthora pathogens such as Phytophthora infestans and Phytophthora cinnamomi, Pythium pathogens such as Pythium aphanidermatum, and Peronosporaceae family pathogens such as Plasmopara viticola, Pseudoperonospora cubensis, and Bremia lactucae; and other genera and species closely related to these pathogens.
    [0128] The compounds of the invention are useful for the control of pests such as arthropods, particularly insects, mites and lice, of the house, garden, livestock and agriculture.
    [0130] Additional particular embodiments refer to the use of the compounds identified following the method described above, to control insects in larval stages, preferably of the infraclass Neoptera, which include, but are not limited to, the orders Lepidoptera, Homoptera, Isoptera, Diptera, Orthoptera, Hemiptera and Coleoptera. Most preferably, Drosophila melanogaster and Galleria mellonella, among others.
    [0131] In particular, other representative pests that are controlled by the compounds of the invention include members of the phylum Arthropoda, including mites of the suborders Mesostigmata, Sarcoptiformes, Trombidiformes, and Onchychopalpida; and lice of the orders Anoplura and Mallophaga.
    [0133] Particular embodiments relate to the use of the compounds identified following the method described above to inhibit the growth of the model nematode Caenorhabditis elegans.
    [0135] In particular, the compounds of the invention are useful for the control of parasitic nematodes, including, but not limited to, parasitic nematodes of plants of the order Tylenchida, especially species such as Meloidogyne spp., Heterodera spp., Rotylenchus spp. and Pratylenchus spp. In particular, the parasitic nematodes of mammals, including humans, livestock and pets are also controlled by the compounds of the invention, including the orders Ascaridia, Spirurida and Strongylida, and in particular species such as Trichinella spp., Trichuris spp. and Strongyloides spp.
    [0137] The inventors have developed a method for the identification of compounds to combat agricultural pests and other pests and parasites of non-agricultural origin. Furthermore, the effectiveness of these compounds against fungi, insects and nematodes has been confirmed.
    [0139] BRIEF DESCRIPTION OF THE FIGURES.
    [0141] FIGURE 1 Flow diagram of the experimental tests used for the identification of CDA inhibitors.
    [0143] FIGURE 2 Fungicidal effects of some of the selected compounds against cucurbit powdery mildew ( P. xanthii) in the whole plant test.
    [0145] FIGURE 3 Insecticidal effects of some compounds identified by the molecular topology method in G. mellonella.
    [0147] FIGURE 4 Micrographs of the C. elegans toxicity test performed, showing the effects of some of the compounds identified by the molecular topology method on the development of nematodes.
    [0149] FIGURE 5 Physiological effects on the responses of plants to treatment with the compounds with the best fungicidal activity on melon cotyledons infected with P. xanthii .
    [0150] EXAMPLES
    [0152] Example 1: Development of the molecular topology method and identification of compounds.
    [0154] Several carboxylic acids have been identified as inhibitors of CDA. In particular, EDTA, (GlcNAc) 2, lactic acid and propionic acid. Furthermore, the mechanism of action of EDTA is not linked to its role as a chelating agent, but to its effect as an enzyme inhibitor. Furthermore, all of them were active in concentrations higher than 10 Mm3. To identify new compounds with higher inhibitory activity, an approach based on molecular topology was designed, Figure 1.
    [0155] An approach based on molecular topology was developed starting with the chemical structures of the chemical compounds derived from carboxylic acids, with and without anti-CDA activity, previously determined by experimental means (Table 1) for the identification of new molecules with higher inhibitory activity. From CDA, the chemical structures listed in Table 2 were converted to graphs and then converted to 2D topological descriptors using graph theory. Next, linear discriminant analysis (LDA) is applied to calculate a discriminant function (DF1) 10.
    [0157] DF1 = (-10.295 x GATS4m) (35.124 x GGI8) 6.917
    [0158] where GATS4m is the Geary autocorrelation index - lag4 / weighted by atomic masses; GGI8 is the 8th order topological load index. These parameters were calculated using the DRAGON11 software.
    [0160] Active compounds (A) are those that have a DF 1 value in the range between 0 and 14. Therefore, this model is appropriate for classifying chemical compounds according to their ability to inhibit the CDA enzyme.
    [0170] a Experimental classification of the CDA inhibitory activity of the compounds used as starting material to obtain the first LDA (A, active; I, inactive);
    [0171] b Value of the discriminant function, DF 1 , obtained for each compound (DF 1 = (-10,295 x GATS4m) (35,124 x GGI8) 6,917); where GATS4m is the Geary autocorrelation index - lag4 / weighted by atomic masses; GGI8, topological load index of order 8.
    [0173] c Classification obtained by applying DF 1 (a compound will be chosen as a CDA inhibitor, A, if it has a value of the discriminant function DF1 within the range of 0 to 14; as a non-CDA inhibitor, I, if the value of DF1 is between 0 and -18; and as unclassified, NC, for any other value of DF1).
    [0175] * N-Acetylglucosamine
    [0177] Next, several databases such as SPECS, ChEMBL and eMolecules were analyzed, applying the DF1 function. These databases comprise more than 3,000,000 compounds. If the resulting discriminant value is between 0 and 14, that compound will be considered a CDA inhibitor, while if the discriminant value is outside that range, it will not be considered a CDA inhibitor. Compounds selected from this screen are listed in Table 3. Subsequently, the CDA inhibitory capacity of these compounds was experimentally tested in the laboratory.
    [0184] GATS4m, GGI8 DF1 Prob (Act) Inh (%) exp
    [0186] a Value of the discriminant function, DF1, obtained for each compound (DF1 = (-10,295 x GATS4m) (35,124 x GGI8) 6,917); where GATS4m is the Geary autocorrelation index - lag4 / weighted by atomic masses; GGI8, topological load index of order 8.
    [0188] b Probability with which the model classifies it as active.
    [0189] c Experimental inhibition of CDA (%).
    [0191] Most of the compounds having a DF1 between 0 and 14 were experimentally active against P. xanthii. Table 2, Inh (%) (exp). The experimental results were then used to develop a 10 * 15 multilinear regression analysis to predict the inhibitory activity of the new candidates. Thus, a second predictive equation was obtained with a higher discriminating power than DF1.
    [0193] The correlation equation is:
    [0195] log (Inh%) = 0.814 - 0.018 x T (N..N) 9.552 x JGI2
    [0198] N = 8; R2 = 0.803; Q2 = 0.657; SEE = 0.112; F (2,5) = 10.2; p = 0.02,
    [0200] N, Number of active compounds when tested
    [0202] R2, coefficient of determination;
    [0204] Q2, prediction coefficient (determined by cross validation);
    [0206] SEE, standard error
    [0208] F, Fisher-Snedecor parameter
    [0210] p, statistical significance
    [0212] T (N..N) is the topological distance expressed as the number of edges between two consecutive nitrogens.
    [0214] JGI2 indicates the topological average load index of order 2.
    [0216] These parameters were calculated using the DRAGON11 software.
    [0218] Compounds identified in the first screening, along with their experimental values, are illustrated in Table 4. The molecules that showed some experimental activity were used to develop a multilinear regression analysis. The descriptors used in Function 2 are indicated in Table 3. In the last column, Log (Inh%) Calcd, the predicted value of the CDA inhibitory activity obtained through multilinear regression analysis (Function 2) can be observed. The molecules that have a predicted value of inhibitory activity (Log (Inh%) between 1 and 2, were those that gave positive experimental results.
    [0222]
    [0226]
    [0228] c experimental inhibitory value (log)
    [0229] d calculated inhibitory value (log)
    [0231] A second screen is performed with the second function as described above. This time, a compound will be selected as a possible CDA inhibitor if it has a DF1 discriminant value between 0 and 14 and a predictive value of inhibitory activity (Log (Inh%) between 1 and 2.
    [0233] Therefore, the results of the laboratory tests helped us to obtain new equations that refined the mathematical models. Table 5 shows the list of compounds chosen after the second iteration of the molecular topology. Also included are their calculated DF1 and Log (Inh%) values, as well as the values of the topological indices that appear in both equations.
    [0234]
    [0240] 1 Before starting the in vitro tests with the compounds obtained in the second iteration by molecular topology, a docking experiment was carried out . Details are explained in Example 1.3. After that, the compounds listed in Table 4 were experimentally tested.
    [0242] Although the last 8 molecules in Table 5 had no activity in the experimental results, they were taken into account to obtain a new discriminant function (DF2), in which those 8 compounds are introduced as inactive.
    [0244] Finally, a final discriminant equation (DF2) is obtained:
    [0246] DF2 = 21.022 - (19.407 x GGI10) - (5.104 x SFige) - (7.911 x GATS3e)
    [0247] N = 35 A = 0.450 F (3.31) = 12.3 p <0.00001
    [0248] where N is the number of compounds;
    [0249] F is the Fisher-Snedecor parameter;
    [0250] A is Wilks lambda; and
    [0251] p is the statistical significance.
    [0253] where:
    [0255] - GGI10: topological load index of order 10,
    [0257] - Seige: Eigenvalue sum of the eigenvalue of electronegativity.
    [0259] - GATS3e: Geary autocorrelation index -lag3 / weighted by Sanderson atomic electronegativities,
    [0261] these parameters were obtained using the DRAGON11 software,
    [0263] Table 6 shows the list of the molecules detected in molecular topology approaches 1 and 2 (Table 2 and Table 4, respectively) classified according to DF2 values.
    [0265] A compound will be chosen as a CDA inhibitor if, in addition to the conditions imposed by DF1 and Log (Inh%), they show a DF2 value between 0 and 6 and a docking affinity value lower than -7 kcal / mol.
    [0268]
    [0269]
    [0270]
    [0273] c Value of the discriminant function, DF2 = 21.022 - (19.407 * GGI10) - (5.104 * Seige) - (7.911 * GATS3e), and
    [0274] d Activity probability based on DF2 results.
    [0276] Example 1.2: Experimental validation of the compounds identified by a molecular topology method as antioid fungicides.
    [0278] Compounds identified by molecular topology screening were first tested for fungicidal activity in zucchini cotyledon discs against two isolates of P. xanthii , isolates 2086 and SF60. A previously described leaf disc test was used for the fungicide susceptibility test5 with minor modifications. Before applying the treatments, the cotyledon discs were inoculated in the center with conidia of P. xanthii and incubated for 24 h at 25 ° C and a light / dark cycle of 16 h light / 8 h dark. . After this incubation, the discs were immersed in the solution of the corresponding compound (1 or 100 µM) or an aqueous solution of 1% acetone that was also included as a negative control, and were incubated under the same conditions for 7 days. After incubation, the surface of the leaf covered by the fungus was evaluated by digital analysis. For this analysis, the images were captured with a digital camera with a fixed distance from the discs of 20 cm. The images were analyzed using the free Java Image image processing software to calculate the area covered by powdery mildew symptoms on each disk.
    [0282]
    [0283]
    [0288] Table 7 shows the fungicidal effect of the compounds identified by the molecular topology method on the development of cucurbit powdery mildew ( P. xanthii) in the leaf disk test. Several compounds showed greater than 50% efficacy in reducing disease symptoms compared to the negative control (water). However, the response was different depending on the fungal strain and the concentration used.
    [0290] Example 1.3: Molecular docking of a fungal ADC to the identified compounds
    [0292] In order to confirm the specific binding of the compounds identified in the screening against the CDA enzyme, a molecular docking experiment was performed against a fragment of a fungal CDA protein that comprised the active site. This filter was only applied to compounds that had passed the previous three filters. Those compounds with a value lower than -7 kcal / mol in the docking study or were considered active.
    [0294] A docking experiment was performed with a CDA fragment from Colletotrichum lindemuthianum, which contains the active center and where the binding to the compounds occurs. This protein was chosen due to its abundant description in the literature12. The protein structure (2IW0) was obtained from the Protein Databank (PDB). To perform the docking analysis, the AutoDock Vina program was used.
    [0295] The applied coordinates are detailed below:
    [0296] - receiver = cda.pdbqt
    [0297] - ligand = xxxx.pdbqt
    [0298] - out = all.pdbqt
    [0299] - center_x = 13.3
    [0300] - center_y = 12.6
    [0301] - center_z = 11.3
    [0302] - size_x = 36
    [0303] - size_y = 36
    [0304] - size_z = 36
    [0307] - exhaustiveness = 8
    [0309] These are the values that represent the 3D space in which the ligand appears to be the best match to fit the protein. The area comprises the active site of CDA, in which the Zn atom and other essential amino acids for CDA activity are present. The docking results returned by AutoDock Vina indicated that the acid compound (3 - [(1,3-dimethyl-2,4,6-trioxotetrahydro-5 (2H) -pyrimidinylene) methyl] phenoxy) acetic acid had the best affinity results as a CDA inhibitor. This molecule forms several hydrogen bonds with amino acids in the active site of CDA. The compounds tested in this test and the results obtained are shown in Table 8.
    [0313]
    [0314] Example 2: Fungicide sensitivity tests on plants and fruits
    [0315] Seedling test against Cucurbit powdery mildew
    [0317] The leaf disk test is a good test for a first screening of the efficacy of fungicidal compounds against powdery mildew. However, the predicted fungicidal activity of these compounds is presumably not associated with their toxicity but rather with their ability to activate chitin-triggered immunity. Since the distribution of the inoculum may be crucial for the efficacy of this response, we decided to test the compounds in a plant assay using a dispersed inoculum. Therefore, a seedling test was then performed using P. xanthii strain 2086 and only one concentration (100 µM).
    [0319] For the seedling test, 2-week-old melon seedlings were used. Plants were inoculated by spraying a suspension of P. xanthii conidia (1 * 104 conidia / ml) to the point of runoff. Twenty-four hours after inoculation, the leaves were sprayed with the compound solution (100 µM). A 1% acetone aqueous solution was also included as a negative control. The plants were kept in a growth chamber on a 16 h light / 8 h dark cycle at 25 ° C for 12 days. After this incubation, disease symptoms were assessed by digital analysis as indicated above.
    [0322]
    [0323]
    [0326] Table 9 shows the fungicidal effect of the compounds identified by the molecular topology method on the powdery mildew of cucurbits P. xanthii. The efficacy of the compound was determined by the Abbott formula and statistical differences were calculated using Fisher's least significant difference (LSD) method. Generally speaking, the efficacy of the compounds was increased compared to the leaf disk test. The compounds with the most significant fungicidal activity on P. xanthii according to the statistical analysis carried out were:
    [0328] - (1-methyl-2,4-dioxo-1,4,6,7-tetrahydro [1,3] thiazol [2,3f] purin-3 (2H) yl) acetic acid, - (3 - [( 1,3-dimethyl-2,4,6-trioxotetrahydro-5 (2H) -pyrimidinylene) methyl] phenoxy) acetic,
    [0329] - 2- (2-methoxy-4 - ([(5Z) -2,4,6-trioxo-1- (prop-2-en-1-yl) -1-3-diazinan-5-ylidene] methyl acid ) phenoxy) acetic,
    [0330] - 3- {2,5-dioxo-3 - [(5Z) -4-oxo-5- (phenylmethylidene) -2-sulfanylidene-1,3-thiazolidin-3-yl] pyrrolidin-1-yl} propanoic acid,
    [0331] - 7- [2-hydroxy-3- (4-morpholinyl) propyl] -1,3-dimethyl-3,7-dihydro-1 H-purine-2,6-dione, and - 5- [4- (2 -hydroxyethoxy) -3-methoxybenzylidene] -1,3-dimethyl-2,4,6 (1H, 3H, 5H) -pyrimidinetrione.
    [0333] Compounds with the best potential as fungicides according to the seedling test were further tested in plant and fruit trials to determine their fungicidal potential against three main fungal diseases, Cucurbit powdery mildew ( P. xanthii ), the gray rot of tomato ( Botrytis cinerea) and the green rot of citrus ( Penicillium digitatum).
    [0335] Plant test against cucurbit powdery mildew
    [0337] Melon plants 6 weeks old were used for the plant test. For this test, the inoculation of the pathogen, the application of the compounds (100 µM) and the evaluation of the symptoms, were carried out as described above for the seedling test.
    [0339] Fruit test against gray rot of tomato and green rot of citrus
    [0342] Commercial tomatoes and oranges were used in these trials1314. Eight hours after inoculating the fruits with 30 jl of a spore solution (1x103 spores / ml) of the corresponding pathogen, the treatments were applied by immersing the fruits in the solutions of the compounds (100 | jM) for 1 min in the in the case of tomatoes or 2 min in the case of oranges. A 1% acetone aqueous solution was also included as a negative control. The fruits were incubated in boxes and in the dark and at the appropriate temperature (22 ° C for tomatoes or 25 ° C for oranges) for 5-6 days until the development of symptoms in the negative controls. Symptoms of the disease (fruit surface covered by fungal growth) were evaluated by digital analysis as indicated above.
    [0344] Figure 2 shows the fungicidal effect of some of the selected compounds against cucurbit powdery mildew ( P. xanthii) in the plant test. Melon leaves show a strong reduction in the number of powdery mildew colonies on the leaves treated with the selected compounds.
    [0351] Table 10 shows the fungicidal effect of the selected compounds in plant and fruit tests against three important fungal plant diseases, cucurbit powdery mildew ( P. xanthii), tomato gray rot ( B. cinerea ) and green rot of tomato . citrus ( P. digitatum).
    [0358] The three compounds showed outstanding and significant disease suppression effects on P. xanthii and B. cinerea according to the statistical analysis performed (Table 11). Against P. digitatum only 2- (2-methoxy-4 - ([(5Z) -2,4,6-trioxo-1- (prop-2-en-1-yl) -1-3-diazinan- 5-ylidene] methyl) phenoxy) acetic showed a significant inhibitory effect.
    [0360] Toxicity tests on B. cinerea and P. digitatum
    [0362] To separate the fungicidal activity mediated by the plant by the fungicidal activity in vitro (toxicity) of the compounds used in the fruit tests,
    [0364] - (1-methyl-2,4-dioxo-1,4,6,7-tetrahydro [1,3] thiazol [2,3f] purin-3 (2H) yl) acetic acid
    [0365] - (3 - [(1,3-dimethyl-2,4,6-trioxotetrahydro-5 (2H) -pyrimidinylene) methyl] phenoxy) acetic acid,
    [0366] - 2- (2-methoxy-4 - ([(5Z) -2,4,6-trioxo-1- (prop-2-en-1-yl) -1-3-diazinan-5-ylidene] methyl acid ) phenoxy) acetic,
    [0368] the toxicity of such compounds was tested in vitro against B. cinerea and P. digitatum. In these tests, the fungi were grown in 1 ml of PDB (Potato Dextrose Broth) in 24-well plates, which were supplemented with the different compounds to reach final concentrations ranging from 0 to 200 µM. The wells were inoculated with 30 µl of spore suspensions (104 conidia / ml) and the plates were incubated for 72 h at 22 ° C for B. cinerea or at 25 ° C in the case of P. digitatum.
    [0369] No inhibitory effect was observed compared to negative controls with the three compounds tested in the range of concentrations tested.
    [0371] Example 3: Toxicity tests on insects
    [0373] Toxicity test for Gallería mellonella
    [0375] To analyze the effect of the different compounds on the larvae of the wax moth G. mellonella 15, six larvae were injected with 20 µl of 150 µM solutions of the different compounds. A 1.5% acetone aqueous solution was included as a negative control. After incubation, the number of pupae and moths formed was counted. The morphology of the resulting moths was also examined.
    [0377] Figure 3 shows that the most dramatic effect is related to the formation of the moth that either did not form or resulted in the formation of abnormal adults with deformed wings, a phenotype previously associated with the silencing of a CDA gene in the moth beetle. potato Leptinotarsa decemlineata 16.
    [0380] CN 5 m <i -
    [0381]
    [0382] Table 12 shows the insecticidal effect of the compounds identified by the molecular topology method on the larvae of the wax moth G. mellonella. Generally speaking, the compounds did not affect pupal formation, but some of them had a significant effect on metamorphosis (moth formation).
    [0389] The compounds with significant insecticidal activity on G. mellonella according to the statistical analysis performed (Table 13) were:
    [0391] - (3 - [(1,3-dimethyl-2,4,6-trioxotetrahydro-5 (2H) -pyrimidinylene) methyl] phenoxy) acetic acid,
    [0392] - 5-imino-1- (2-methyl-5-nitrophenyl) -3-phenylidantoin and
    [0393] - 5-imino-1- (4-methyl-5-nitrophenyl) -3-phenylidantoin.
    [0395] Drosophila melanogaster toxicity assay
    [0397] Toxicity against D. melanogaster was tested using the liquid food feeding test17. To perform the toxicity test, 25 fly larvae were placed in 20 ml plastic containers containing 0.5 ml of liquid food and the corresponding compound at a concentration of 150 µM. A 1.5% aqueous acetone solution was also included as a negative control. During incubation, the number of pupae formed was recorded, as well as the number of adults (flies) that resulted.
    [0398]
    [0399]
    [0401] 4 Table 14 shows the insecticidal effect of the compounds identified by a molecular topology method on the larvae of the fruit fly D. melanogaster. Overall, most of the compounds significantly reduced fly formation.
    [0406] Although many of the compounds have a strong effect on the formation of adult flies (9 of 20), the compounds with significant insecticidal activity in the formation of pupae and adult flies according to the statistical analysis performed (Table 15) were:
    [0408] - (3 - [(1,3-dimethyl-2,4,6-trioxotetrahydro-5 (2H) -pyrimidinylene) methyl] phenoxy) acetic acid,
    [0409] - 7- [2-hydroxy-3- (4-morpholinyl) propyl] -1,3-dimethyl-3,7-dihydro-1 H-purine-2,6-dione, - 5-imino-1- (2 -methyl-5-nitrophenyl) -3-phenylidantoin and
    [0410] - 5-imino-1- (4-methyl-5-nitrophenyl) -3-phenylidantoin.
    [0412] Example 4 Toxicity test for Caenorhabditis elegans
    [0414] To investigate toxicity on nematodes, the C. elegans 18 plate toxicity assay was used. To perform the assay, a 75 µl drop of a 1 mM solution of the corresponding compound was added to each well on the agar surface. , to reach a final concentration in the culture medium of 150 jM. As a negative control, a 10% acetone aqueous solution (final concentration of 1.5% acetone) was also included. The drops were allowed to dry at room temperature for 30 min. Subsequently, each well was inoculated with 40 µl of the washed and filtered nematode population. After five days of incubation at 20 ° C, each well was washed with 100 µl of buffer M9. Next, ten 3 µl drops were examined under an optical microscope to count the number of nematodes in the different larval stages (L1, L2-L3 and L4).
    [0416] Figure 4 shows some micrographs of the C. elegans toxicity test carried out with the effects of some compounds identified by the molecular topology method on the development of nematodes. Compared to the negative control (water), the compounds caused a clear reduction in nematode formation.
    [0417]
    [0420] Table 16 shows the nematicidal effect of the compounds identified by the molecular topology method on C. elegans. Most of the compounds showed a strong effect on C. elegans development, which is consistent with the developmental delay phenotype associated with disruption of CDA9-encoding genes.
    [0430] All the compounds tested showed some level of toxicity at least on the larval stage. The compounds with the strongest nematicidal activity according to the statistical analysis performed (Table 17) were:
    [0431] - (1-methyl-2,4-dioxo-1,4,6,7-tetrahydro [1,3] thiazol [2,3f] purin-3 (2H) yl) acetic acid, - 5-imino-1- (2-methyl-5-nitrophenyl) -3-phenylidantoin,
    [0432] - 2- (10,12-dioxo-9 - {[(1,3-thiazol-2-yl) carbamoyl] methyl} -7-thia-9,11-diazatricyclo [6.4.0.02,6] dodeca-1 acid (8), 2 (6) -dien-11-yl) acetic,
    [0433] - 2- {4 - [(1,3-dimethyl-4,6-dioxo-2-sulfanylidene-1,3-diazinan-5-ylidene) methyl] phenoxy} acetic acid,
    [0434] - 2 - ({[(5E) -4,6-dioxo-1- [4- (propan-2-yl) phenyl] -2-sulfanyl-1,4,5,6-tetrahydropyrimidin-5-ylidene] acid methyl} amino) -3- (1H-imidazol-4-yl) propanoic and - 2- {1 - [(3,4-difluorophenyl) methyl] -3-oxopiperazin-2-yl} acetic acid
    [0436] Example 5: Determining the mode of action as inhibitors of CDA
    [0438] Compounds with the best fungicidal potential were tested for their mode of action as CDA inhibitors. Two different experiments were performed to provide direct (enzymatic assay) and indirect (plant assay) evidence of inhibition of a fungal CDA.
    [0440] Assay in plant on tissues with the gene CERK1 s Ilenced
    [0442] If inhibition of fungal CDA in planta causes activation of chitin-activated immunity and suppression of fungal growth, application of CDA inhibitors to leaf tissues in which the chitin receptor gene CERK1 is silenced, It shouldn't have any effect on fungal growth. Melon CERK1 gene silencing was performed using 2-week-old melon cotyledons and the ATM-HIGS ( Agrobacterium- mediated host-induced gene silencing) assay 19. Twenty-four hours after the agroinfiltration, the plants were inoculated with a suspension of P. xanthii conidia (1 * 104conidia / ml) and 24 h later, the leaves were sprayed with the compound solution (100 µM). The plants were then kept in a growth chamber on a 16 h light / 8 h dark cycle at 25 ° C and subsequently examined for activation of defense responses and development of disease symptoms. Twelve days after inoculation, disease symptoms were assessed by
    [0445] digital analysis as above. On the other hand, 72 h after inoculation, the in situ accumulation of hydrogen peroxide (H 2 O 2 ) was studied by histochemical analysis, following the DAB20 consumption method.
    [0447] Figure 5 shows the physiological effects of melon cotyledon treatments with the compounds with the best fungicidal activity. As previously shown with EDTA, the compounds induced a strong inhibition of fungal growth (Fig. 5A) as a consequence of the strong activation of the production of reactive oxygen species (ROS reactive oxygen species) (Fig. 5B). This plant response can be suppressed by silencing the CERK1 chitin receptor gene . In these plants, fungal growth is restored in the presence of the compounds (Fig. 5A) and ROS production is clearly reduced (Fig. 5B). The results show the relationship between the fungicidal activity of the compounds and the activation of immunity activated by chitin in the host plant, results that can be explained by the inhibition of CDA activity by the compounds and the subsequent perception of non-deacetylated chitin oligomers. by plant receptors.
    [0449] Chitin deacetylase activity assay
    [0451] For this assay, two CDA proteins identified in P. xanthii (PxCDA1 and PxCDA2) were expressed in vitro in E. coli. Recombinant proteins with a polyhistidine tag at the N-terminus were produced following standard procedures. The enzymatic activity was determined using the fluorescamine method, using colloidal chitin as substrate12. To test the CDA inhibitory activity of the selected compounds, the enzymatic reaction was carried out in the absence or presence of the compounds at concentrations of 10 and 100 µM. The reaction mixtures were incubated for 45 min at 37 ° C. After incubation, reactions were stopped with 0.4M borate buffer (pH 9.0). After recording the data, the percentages of inhibition of enzyme activity were calculated.
    [0453]
    [0456] Table 18 shows the results of the CDA enzyme activity inhibition experiments carried out with two CDA proteins from P. xanthii , PxCDA1 and PxCDA2, expressed in E. coli and exposed to the compounds with the best fungicidal activity. At 100 µM, the compounds induced a strong inhibition of the enzymatic activity of CDA proteins in a range of 75 to 93%.
    [0458] The sequences for PxCDA1 and PxCDA2 have been deposited with GenBank under accession numbers KX495502 and KX495503.
    [0460] Docking of fungal and insect CDA proteins to selected compounds
    [0461] To map the interactions between the best fungicidal and insecticidal compounds and CDA proteins and to identify potential binding sites for these compounds, docking experiments were performed . For these experiments, complete CDA proteins of the fungal agent responsible for the anthracnose bean Colletotnchum lindemunthianum and the silkworm Bombyx morí were used, unlike the docking experiment described in Example 1.3 in which only the active site of C. lindemunthianum. For this, the option "blind docking" was chosen, in which the complete CDA protein was used. Protein structures were used
    [0464] corresponding data, 2IW0 and 5Z34, obtained from the Protein Databank (PDB), and the SwissDock web server. Docking was carried out using the "Accurate" parameter and the default parameters were used for the rest of the parameters, without a defined region of interest (blind coupling). Five compounds were used in these analyzes, the three selected with the best fungicidal potential
    [0466] - (1-methyl-2,4-dioxo-1,4,6,7-tetrahydro [1,3] thiazol [2,3f] purin-3 (2H) yl) acetic acid, - (3 - [( 1,3-dimethyl-2,4,6-trioxotetrahydro-5 (2H) -pyrimidinylene) methyl] phenoxy) acetic and
    [0467] - 2- (2-methoxy-4 - ([(5Z) -2,4,6-trioxo-1- (prop-2-en-1-yl) -1-3-diazinan-5-ylidene] methyl acid ) phenoxy) acetic.
    [0469] and two of the compounds identified with notable insecticidal activity
    [0470] - 5-imino-1- (2-methyl-5-nitrophenyl) -3-phenylidantoin and
    [0471] - 5-imino-1- (4-methyl-5-nitrophenyl) -3-phenylidantoin.
    [0478] Table 19 shows the docking analysis to a fungal CDA. Only compounds with fungicidal activity were able to form two hydrogen bonds with two different amino acid residues, Y145 and L146, resulting in a favorable binding site. Since Y145 is one of the catalytic residues of the active site of the fungal enzyme21, the docking results appear consistent with the previous data, confirming that these compounds are true inhibitors of the fungal CDA enzyme.
    [0485] Similarly, Table 20 shows the docking analysis to the insect CDA. In this case, only compounds with insecticidal activity were capable of forming two or three hydrogen bonds with residues K508 and G512. These residues are part of the active site pocket of the B. mori22 CDA enzyme , suggesting that these compounds may interfere with enzyme activity.
    [0487] Taken together, the docking results provide computational evidence supporting the specific activity of these molecules as fungicidal or insecticidal compounds, through their specific binding to a fungal or insect CDA enzyme, reinforcing their activities as specific CDA inhibitors.
    [0488] BIBLIOGRAPHY
    [0490] 1. Gow et al. 2017. Microbiology Spectrum 5: FUNK-0035-2016.
    [0491] 2. Blair et al. 2006. Biochemistry 45: 9416-9426.
    [0492] 3. Sánchez-Vallet et al. 2015. FEMS Microbiology Reviews 39: 171-183.
    [0493] 4. Pérez-García et al. 2009. Molecular Plant Pathology 10: 153-160.
    [0494] 5. Fernández-Ortuño et al. 2006. European Journal of Plant Pathology 115: 215.
    [0495] 6. López-Ruiz et al. 2010. Pest Management Science 66: 801-808.
    [0496] 7. Bellón-Gómez et al. 2015. Pest Management Science 71: 1407-1413.
    [0497] 8. Martínez Cruz, 2016. PhD Thesis, Morphological and functional analysis of the interaction of Podosphaera Xanthii - cucurbits.
    [0498] 9. Heustis et al. 2012. PLoS ONE 7 (7): e40426.
    [0499] 10. Introduction to Linear Regression Analysis, John Wiley & Sons, 2015. J.
    [0500] Machuca, A. Briales, G. Labrador, P.
    [0501] 11. R. Todeschini, V. Consonni, A. Mauri, M. Pavan, DRAGON-software for the Calculation of Molecular Descriptors, Version 5.4, Talete srl, Milan, Italy, 2004.
    [0502] 12. Blair et al. 2006. Biochemistry 45: 9416.
    [0503] 13. Fernández-Ortuño et al., 2013 Plant Dis. 97 (6): 848.
    [0504] 14. Holmes & Eckert et al, 1994. 89 (9): 716-21.
    [0505] 15. Dalton, J.P., et al. 2017. Front Cell Infect Microbiol. 7:99.
    [0506] 16. Wu JJ, et al. 2019 Insect Mol Biol. 28 (1): 52-64.
    [0507] 17. Gasque, G., et al. 2013. Sci Rep. 3: 2120.
    [0508] 18. Xiong, H., et al. 2017. Sci Rep. 7: 9839.
    [0509] 19. Martínez-Cruz, J., et al 2014. Fungal Genet. Biol. 71: 21-31.
    [0510] 20. Thordal-Christensen, H., et al. 1997. Plant J. 11: 1187-1194.
    [0511] 21. Aragunde H. et al. 2018. Int J Mol Sci. 30; 19 (2).
    [0512] 22. Liu L, et al. J Biol Chem. 2019. 12; 294 (15): 5774-5783
    1
    权利要求:
    Claims (25)
    [1]
    1. Method for the identification of a compound with potential pesticidal activity against chitin-containing organisms, said compound being an inhibitor of the enzyme chitin deacetylase (CDA), comprising:
    a) obtain for said compound a value for the discriminant function DF1, in which:
    DF1 = (-10.295 x GATS4m) (35.124 x GGI8) 6.917
    where:
    - GATS4m is the Geary autocorrelation index - lag4 / weighted by atomic masses,
    - GGI8 is the topological load index of order 8; and
    b) Check if the DF1 value obtained in (a) for said compound is between 0 and 14, where if said value is between 0 and 14 said compound is identified as a potential inhibitor of the CDA enzyme.
    [2]
    2. Method according to claim 1 in which:
    -step (a) further comprises obtaining for said compound a predictive value of Log inhibitory activity (lnh%), where:
    Log (lnh%) = 0.814 - 0.0184 x T (N..N) 9.552 x JGI2
    where:
    - T (N..N) is the topological distance, expressed as the number of edges between two consecutive nitrogen atoms,
    - JGI2 is the topological average load index of order 2; and
    -step b) additionally consists in checking if the value of Log (lnh%) obtained in (a) for said compound is between 1 and 2, in which case said compound is identified as a potential inhibitor of the CDA enzyme.
    [3]
    3. Method according to claim 2 in which:
    -step (a) further comprises obtaining for said compound a value of the discriminant function DF2, in which:
    DF2 = 21.022 - (19.407 x GGI10) - (5.104xSFige) - (7.911 x GATS3e)
    2
    in which:
    - GGI10 is the topological load index of order 10,
    - SFige is the sum eigenvalue of the eigenvalue of electronegativity,
    - GATS3e is the Geary autocorrelation index -lag3 / weighted by Sanderson atomic electronegativities; and
    step b) comprises checking if the value obtained for said compound in step (a) is between 0 and 6, in which case said compound is identified as a potential inhibitor of the CDA enzyme.
    [4]
    4. Method according to any one of the preceding claims comprising after step (b):
    - c) carrying out an experimental analysis of the compound identified as a potential inhibitor of the CDA enzyme according to step b), which comprises putting said compound in contact with a pathogen and observing if there is growth of this.
    [5]
    5. Method according to claim 4 wherein the pathogen can be selected from fungi and insects, preferably Podosphaera xanthii or Galleria mellonella.
    [6]
    6. Method according to any one of the preceding claims, which comprises carrying out a docking experiment on a CDA enzyme with a compound identified in step (b) as a potential inhibitor of the CDA enzyme due to its ability to interact with the site. active enzyme CDA.
    [7]
    Method according to claim 4, in which a fragment containing the active site of the CDA enzyme of C. lindemuthianum is used for the docking experiment.
    [8]
    8. Method according to any one of the preceding claims in which the chitin-containing organism is selected from fungi, arthropods and nematodes.
    [9]
    9. Use of chemical compounds with pesticidal activity against an organism that contains chitin selected from fungi, arthropods and nematodes detected by the method defined in any one of claims 1 to 8.
    [10]
    10. Use of a chemical compound according to claim 9 against a chitin-containing organism selected from fungi, arthropods and nematodes, characterized in that said compound:
    - i) has a DF1 value between 0 and 14,
    DF1 = (-10.295 x GATS4m) (35.124 x GGI8) 6.917
    where:
    - GATS4m is the Geary autocorrelation index - lag4 / weighted by atomic masses, and
    - GGI8 is the topological load index of order 8.
    [11]
    11. Use of a chemical compound according to claim 10 against a chitin-containing organism selected from fungi, arthropods and nematodes, characterized in that said compound:
    - ii) has a value of Log (lnh%) between 1 and 2,
    Log (lnh%) = 0.814 - 0.018 x T (N..N) 9.552 x JGI2
    where:
    - T (N..N) is the topological distance expressed as the number of edges between two consecutive nitrogen, and
    - JGI2 is the topological average load index of order 2.
    [12]
    12. Use of a chemical compound according to claim 11 against an organism that contains chitin selected from fungi, arthropods and nematodes, characterized in that said compound:
    - iii) has a DF2 value between 0 and 6,
    DF2 = 21.022 - (19.407 x GGI10) - (5.104 x Seige) - (7.911 x GATS3e) where:
    4
    - GGI10 is the topological load index of order 10,
    - Seige is the sum eigenvalue of the eigenvalue of electronegativity, and
    - GATS3e is the Geary autocorrelation index -lag3 / weighted by Sanderson atomic electronegativities.
    [13]
    13. Use of a chemical compound according to claim 12 against an organism that contains chitin selected from fungi, arthropods and nematodes, characterized in that said compound interacts with the active site of the CDA enzyme of C. lindemuthianum in a docking experiment .
    [14]
    Use according to one of claims 10 to 13, in which the chemical compound is selected from:
    - (1-methyl-2,4-dioxo-1,4,6,7-tetrahydro [1,3] thiazol [2,3f] purin-3 (2H) yl) acetic acid
    - (3 - [(1,3-dimethyl-2,4,6-trioxotetrahydro-5 (2H) -pyrimidinylene) methyl] phenoxy) acetic acid,
    - 2- (2-methoxy-4 - ([(5Z) -2,4,6-trioxo-1- (prop-2-en-1-yl) -1-3-diazinan-5-ylidene] methyl acid ) phenoxy) acetic,
    - 5- [4- (2-hydroxyethoxy) -3-methoxybenzylidene] -1,3-dimethyl-2,4,6 (1H, 3H, 5H) -pyrimidinetrione,
    - 7- [2-hydroxy-3- (4-morpholinyl) propyl] -1,3-dimethyl-3,7-dihydro-1 H-purine-2,6-dione,
    - 2-amino-7-methyl-5-oxo-4- [4- (trifluoromethoxy) phenyl] -4H, 5H-pyrano [4,3-b] pyran-3-carbonitrile,
    - N- {4- [3- (2,6-dimethyl-4-morpholinyl) -2,5-dioxo-1-pyrrolidinyl] phenyl} acetamide,
    - 2- (1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl) -N- (2-methoxy-1-methylethyl) acetamide,
    - N-cyclopropyl-2- (1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-9H-purin-9-yl) acetamide
    - 5-imino-1- (2-methyl-5-nitrophenyl) -3-phenylidantoin,
    - 5-imino-1- (4-methyl-5-nitrophenyl) -3-phenylidantoin,
    - 3- {2,5-dioxo-3 - [(5Z) -4-oxo-5- (phenylmethylidene) -2-sulfanylidene-1,3-thiazolidin-3-yl] pyrrolidin-1-yl} propanoic acid
    - 2- (10,12-dioxo-9 - {[(1,3-thiazol-2-yl) carbamoyl] methyl} -7-thia-9,11-diazatricyclo [6.4.0.02,6] dodeca-1 acid (8), 2 (6) -dien-11-yl) acetic,
    - 2- {4 - [(1,3-dimethyl-4,6-dioxo-2-sulfanylidene-1,3-diazinan-5-ylidene) methyl] phenoxy} acetic acid,
    - 2 - ({[(5E) -4,6-dioxo-1- [4- (propan-2-yl) phenyl] -2-sulfanyl-1,4,5,6-tetrahydropyrimidin-5-ylidene] acid methyl} amino) -3- (1 H-imidazol-4-yl) propanoic,
    - 2 - [(5E) -4-oxo-5 - {[2- (prop-2-en-1-yloxy) phenyl] methylidene} -2-sulfanylidene-1,3-thiazolidin-3-yl] pentanedioic acid ,
    - 2- {1 - [(3,4-difluorophenyl) methyl] -3-oxopiperazin-2-yl} acetic acid,
    - 2- {3 - [(2-chloro-6-fluorophenyl) methyl] -2,4,5-trioxoimidazolidin-1-yl} acetic acid,
    - {2 - [(1,3-dimethyl-4,6-dioxo-2-thioxotetrahydro-5 (2H) -pyrimidinylidene) methyl] phenoxy} acetic acid and
    - 3-benzyl-1,7-dimethyl-7,9-dihydro-1 H-purin-2,6,8 (3H) -trione.
    [15]
    15. Use according to one of claims 10 to 13 in which the chemical compound is selected from those having a purine group:
    - (1-methyl-2,4-dioxo-1,4,6,7-tetrahydro [1,3] thiazol [2,3f] purin-3 (2H) yl) acetic acid,
    - 7- [2-hydroxy-3- (4-morpholinyl) propyl] -1,3-dimethyl-3,7-dihydro-1 H-purine-2,6-dione,
    - 2- (1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl) -N- (2-methoxy-1-methylethyl) acetamide,
    - N-cyclopropyl-2- (1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-9H-purin-9-yl) acetamide and,
    - 3-benzyl-1,7-dimethyl-7,9-dihydro-1 H-purin-2,6,8 (3H) -trione.
    [16]
    16. Use according to one of claims 10 to 13 in which the chemical compound is selected from those having a pyrimidine group:
    - (3 - [(1,3-dimethyl-2,4,6-trioxotetrahydro-5 (2H) -pyrimidinylene) methyl] phenoxy) acetic acid,
    - 5- [4- (2-hydroxyethoxy) -3-methoxybenzylidene] -1,3-dimethyl-2,4,6 (1H, 3H, 5H) -pyrimidinetrione,
    - 2 - ({[(5E) -4,6-dioxo-1- [4- (propan-2-yl) phenyl] -2-sulfanyl-1,4,5,6-tetrahydropyrimidin-5-ylidene] acid methyl} amino) -3- (1 H-imidazol-4-yl) propanoic and
    - {2 - [(1,3-dimethyl-4,6-dioxo-2-thioxotetrahydro-5 (2H) -pyrimidinylidene) methyl] phenoxy} acetic acid.
    [17]
    17. Use according to one of claims 10 to 13 in which the chemical compound is selected from those having an imidazole group:
    - 5-imino-1- (2-methyl-5-nitrophenyl) -3-phenylidantoin,
    - 5-imino-1- (4-methyl-5-nitrophenyl) -3-phenylidantoin and
    - 2- {3 - [(2-chloro-6-fluorophenyl) methyl] -2,4,5-trioxoimidazolidin-1-yl} acetic acid.
    [18]
    18. Use according to one of claims 10 to 13 in which the chemical compound is selected from those having a pyrrolidine group:
    - N- {4- [3- (2,6-dimethyl-4-morpholinyl) -2,5-dioxo-1-pyrrolidinyl] phenyl} acetamide and
    - 3- {2,5-dioxo-3 - [(5Z) -4-oxo-5- (phenylmethylidene) -2-sulfanylidene-1,3-thiazolidin-3-yl] pyrrolidin-1-yl} propanoic acid.
    [19]
    Use according to one of claims 14 to 18 to control a pest selected from fungi, arthropods, nematodes and any combination of them.
    [20]
    20. Use according to one of claims 10 to 13 to control a pest selected from fungi, arthropods, nematodes and any combination of them, in which the compound is selected from:
    - 2-amino-7-methyl-5-oxo-4- [4- (trifluoromethoxy) phenyl] -4H, 5H-pyrano [4,3-b] pyran-3-carbonitrile,
    - 2- (10,12-dioxo-9 - {[(1,3-thiazol-2-yl) carbamoyl] methyl} -7-thia-9,11-diazatricyclo [6.4.0.02,6] dodeca-1 acid (8), 2 (6) -dien-11-yl) acetic,
    - 2- {4 - [(1,3-dimethyl-4,6-dioxo-2-sulfanylidene-1,3-diazinan-5-ylidene) methyl] phenoxy} acetic acid,
    - 2- (2-methoxy-4 - ([(5Z) -2,4,6-trioxo-1- (prop-2-en-1-yl) -1-3-diazinan-5-ylidene] methyl acid ) phenoxy) acetic,
    - 2 - [(5E) -4-oxo-5 - {[2- (prop-2-en-1-yloxy) phenyl] methylidene} -2-sulfanylidene-1,3-thiazolidin-3-yl] pentanedioic acid and
    - 2- {1 - [(3,4-difluorophenyl) methyl] -3-oxopiperazin-2-yl} acetic acid.
    7
    [21]
    21. Use according to any one of claims 15 to 19 in which the pests are fungi selected from the Ascomycota and Basidiomycota classes, preferably Blumeria graminis, Erysiphe necator, Erysiphe polygoni, Leveillula taurica, Podosphaera aphanis, Podosphaera xanthii, Alternaria solani, Botrytis cinerea, Penicillium digitatum, Cercospora beticola, Colletotrichum graminicola, Fusarium graminearum, Fusarium oxysporum, Gaeumannomyces graminis, Magnaporthe grisea, Monilinia fructicola, Mycosphaerella fijensis, Phomopsis viticola, Pyrenophora, stiltioliferium, Rhizitoria sclerus, Rhynophora, septiniaquinia, septiniaquinia, septiniaquinia Verticillium dahliae; Puccinia striiformis, Puccinia hordei, Puccinia graminis, Uromyces viciae-fabae, Uromyces betae, Ustilago maydis, Ustilago tritici, Armillaria mellea, Rhizoctonia solani, Sclerotium rolfsii, and pathogens Oomycetes, preferably Phytophthora, Pyrophthora infestamatonos, Plasterium aphudo-phytogenium, Plasterium aphudo-phytosis cubensis and Bremia lactucae ..
    [22]
    22. Use according to any one of claims 15 to 20 in which the pests are insects, selected from the orders Lepidoptera, Homoptera, Isoptera, Diptera, Orthoptera, Hemiptera and Coleoptera, preferably G. mellonella or D. melanogaster.
    [23]
    23. Use according to any one of claims 15 to 20 in which the pests are mites selected from the suborders Mesostigmata, Sarcoptiformes, Trombidiformes and Onchychopalpida.
    [24]
    24. Use according to any one of claims 15 to 20 in which the pests are lice selected from the suborders Anoplura and Mallophaga.
    [25]
    25. Use according to any one of claims 15 to 20 in which the pests are parasitic nematodes of plants selected from the order Tylenchida, preferably Meloidogyne spp., Heterodera spp., Rotylenchus spp. and Pratylenchus spp .; and parasitic nematodes of mammals of the orders Ascaridia, Spirurida and Strongylida, preferably Trichinella spp., Trichuris spp. and Strongyloides spp.
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    同族专利:
    公开号 | 公开日
    WO2020234497A1|2020-11-26|
    ES2794348B2|2021-04-14|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    CN113100233B|2021-04-19|2022-02-08|中国农业科学院植物保护研究所|Application of arylhydroxamic acid and derivatives thereof as chitin deacetylase inhibitor and plant antifungal agent|
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    PCT/ES2020/070314| WO2020234497A1|2019-05-17|2020-05-15|Chitin deacetylase inhibitors and use thereof as agricultural fungicides, arthopocides and nematicides|
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