WO2002051811A1

WO2002051811A1 - Derivatives of acetic acid for increasing abiotic stress tolerance of cultivated plants

Info

Publication number: WO2002051811A1
Application number: PCT/HU2001/000152
Authority: WO
Inventors: Jeno Szilbereky; Gáborné BARLA SZABÓ; Gábor GALIBA
Original assignee: Sinnex Muszaki Fejleszto Es Tanacsado Kft
Current assignee: Sinnex Muszaki Fejleszto Es Tanacsado Kft
Priority date: 2000-12-22
Filing date: 2001-12-20
Publication date: 2002-07-04
Anticipated expiration: 2003-06-22
Also published as: HUP0005002A2; HU0005002D0

Abstract

The present invention relates to the use of the compounds of the following general formula (I) and their agriculturally acceptable salts, wherein R1 is a phenyl group or a five- or six-membered unsaturated heterocyclic ring containing nitrogen as heteroatom; R2 is a hydrogen atom or a lower alkyl group; X is an amino group optionally substituted with one or two lower alkyl groups; or a halogen atom, preferably chlorine or bromine; Y is a hydroxyl group, a lower alkoxy group, an amino group optionally substituted with a five- or six-membered saturated heterocyclic ring containing two identical or different heteroatoms selected from nitrogen and oxygen atoms or with a lower carboxyalkyl group, or an amino group the substituents of which are replacing both of its hydrogen atoms and are forming with the enclosed nitrogen atom a five- or six-membered heterocyclic group containing two nitrogen atoms, where the other nitrogen atom is optionally substituted by a lower alkyl group; as well as to the use of compositions containing these compounds or their agriculturally acceptable salts and methods for increasing abiotic stress tolerance of cultivated plants, inhibiting the senescence of seeds and enhancing their vigour under stress conditions. The invention also relates to a narrower group of the compounds of the above general formula which are new; in these compounds the meaning of the substituents are as defined above with the proviso that if R2 is a hydrogen atom or a lower alkyl group, X is a halogen atom and Y is a lower alkoxyl group, then R1 can only be a five- or six-membered unsaturated heterocyclic ring containing nitrogen as a heteroatom.

Description

DERIVATINES OF ACETIC ACID AND COMPOSITIONS THEREOF

Technical Field

The present invention relates to new acetic acid derivatives of the following general formula:

(la) and their agriculturally acceptable salts, wherein

R¹ is a phenyl group or a five- or six-membered unsaturated heterocyclic ring containing nitrogen as heteroatom;

R² is a hydrogen atom or a lower alkyl group;

X is an amino group optionally substituted with one or two lower alkyl groups; or a halogen atom, preferably chlorine or bromine;

Y is a hydroxyl group, a lower alkoxy group, an amino group optionally substituted with a five- or six-membered saturated heterocyclic ring containing two identical or different heteroatoms selected from nitrogen and oxygen atoms or with a lower carboxyalkyl group, or an amino group the substituents of which are replacing both of its hydrogen atoms and are forming with the enclosed nitrogen atom a five- or six-membered heterocyclic group containing two nitrogen atoms, where the other nitrogen atom is optionally substituted by a lower alkyl group; with the proviso that if R² is a hydrogen atom or a lower alkyl group, X is a halogen atom and Y is a lower alkoxy group, then R¹ can only be a five- or six-membered unsaturated heterocyclic ring containing nitrogen as a heteroatom.

In addition, the present invention relates to the use of compounds of the following general formula:

(I) wherein R¹ is a phenyl group or a five- or six-membered unsaturated heterocyclic ring containing nitrogen as heteroatom; R is a hydrogen atom or a lower alkyl group;

X is an amino group optionally substituted with one or two lower alkyl groups; or a halogen atom, preferably chlorine or bromine; Y is a hydroxyl group, a lower alkoxy group, an amino group optionally substituted with a five- or six-membered saturated heterocyclic ring containing two identical or different heteroatoms selected from nitrogen and oxygen atoms or with a lower carboxyalkyl group, or an amino group the substituents of which are replacing both of its hydrogen atoms and are forming with the enclosed nitrogen atom a five- or six-membered heterocyclic group containing two nitrogen atoms, where the other nitrogen atom is optionally substituted by a lower alkyl group; and to that of the agriculturally acceptable salts of the above compounds, as well as compositions containing such compounds or their agriculturally acceptable salts, and methods for increasing abiotic stress tolerance of cultivated plants, and for inhibiting the senescence of seeds and enhancing their vigour under stress conditions.

Background Art

The extremities of the weather, namely cold, frost and drought cause serious stress effects on cultivated plants. These abiotic conditions damage cultivated plants in different ways and to different extent which may result in decreased yield, thus, significant agricultural losses might occur. The methods worked out to improve the resistance of plants against the extremities of the weather are greatly varied.

Hungarian Patent Application No. P9700792 discloses in details the effects of the different extremities of the weather on the development of plants. The same patent application describes the use of hydroxylamine derivatives. According to the description, these compounds bring the protective system of the plants into action as a response to extreme weather conditions, and as a result of the induced metabolic processes the cold, frost and drought tolerances of the plants improve. The drawback of this procedure which hampers the protection of plants against the extremities of the weather is that plants respond differently to the extremities of the weather and the circumstances of the occurrence of said extremities (e.g. time, extent) are unpredictable.

According to US 4 828 602, to protect the plants against drought the surface of the plants is coated with a thin polymer film thereby limiting their transpiration. The disadvantages of the procedure are that the coating permanently limits the transpiration of the plants, furthermore, the local coating can be achieved with high amount of manual work, only.

The use of growth regulating hormones or their chemical analogues having the same effect, usually in a combination assuring improved effect is known in the art. Such methods are discussed e.g. in WO 96 08481 Al and EP 327309 Al.

The disadvantage of the hormones is that they affect the hormone system of the plants and modify, for instance, the hormonal equilibrium and the metabolic processes in the plants, therefore, they may result in unpredictable physiological changes. Thus, these compounds can only be applied in limited areas, with special care, after running greater number of special preliminary experiments than usual.

Therefore, the use of more simple, hormonally neutral compounds is often preferred.

According to WO 92/08350 Al, tefrahydrofurfuryl alcohol, tetrahydrofurfuryl a ine or a combination thereof is used to increase the cold resistance of plants. In Hungarian Patent No. 181241 secunder or tertier beta-hydroxyethylamines, or the corresponding quaternary ammonium salts are recommended to enhance the tolerance of plants against cold and frost. However, the compounds used in these methods need to be applied several times, also when the cold is over, preferably regularly, all-around the surface of the plant, therefore, these solutions are disadvantageous from application technique aspects. The main disadvantage of these applications is that they are particularly labour- intensive.

In US 4 451 281 and DE 28 08 317 compounds similar to the compounds of the present invention are disclosed which stimulate the growth of cultivated plants and can be applied in numerous special areas of plant protection as selective antidotes of different herbicides.

To improve the viability (vigour) of plants is especially important in the early phase of development (germination) and during flowering because plants are the most vulnerable under abiotic stress conditions in these periods.

Stimulating the growth of cultivated plants is possible as early as the sowing time and several methods are known in the art.

According to US 5 244 487, adenine derivatives are applied that increase the activity of plant cytokines thereby stimulating the germinating ability of the plants and the development of new shoots, moreover increase the yield of crop.

According to US 5 512 069, also the improved production of plant cytokines (e.g. zeatin) is used to achieve the above-mentioned effect and the impregnation of seeds with a natural substance, PPFM (pink pigmented facultative methylotroph) is disclosed.

According to US 4 919 703, oxadiazol derivatives are used to stimulate plant growth.

In US 4 409 910 an agrotechnical method is disclosed, according to which a thin stripe of soil is removed from the site to be sowed and conditioned with sufficient nutrient and vitamin additives. Seeds are sown into this stripe containing the treated and returned soil.

According to US 5 415 672, seeds are coated with clavibacter microorganisms that effect the germination of the seeds advantageously.

According to US 5 518 989, in the course of corn seed cultivation the vigour of new seeds is increased by defoliating the whole plant in a certain phase before pollination.

The disadvantages of the above cited solutions are that some of them are cumbersome or labour-intensive to implement and also, a number of them requires application of expensive and/or sensitive materials which makes these solutions economically unprofitable. The common feature of the above cited solutions which is also their common drawback that they achieve the desired effect under normal physiological conditions of plant cultivation and not under stress conditions. Disclosure of the Invention

In the course of our investigations active ingredients have been developed that increase the physiological vigour of plants and their tolerance against environmental stress effects. Plants treated with these methods suffer less injuries as a consequence of environmental stress effects arising under real cultivating conditions. This is particularly important in the case of culturing hybrid seeds because in the production of hybrid seeds so- called inbred lines are involved which are especially sensitive. Conditioning physiologically such plants may result not only in increased yield of crop but it can also significantly enhance the biological value of the seed crop (germinating capability, seed vigour), and also decrease its deterioration during long-term storage.

At the same time an effort was made to find a solution where the active ingredients are hormonally neutral and non-toxic, the material utilisation and thus the chemical load of the environment is minimal, the application technology is simple and, therefore, especially economic.

We found that compounds of the following general formula:

(I) wherein

R¹ is a phenyl group or a five- or six-membered unsaturated heterocyclic ring

^■ containing nitrogen as heteroatom;

R² is a hydrogen atom or a lower alkyl group;

Y is a hydroxyl group, a lower alkoxy group, an amino group optionally substituted with a five- or six-membered saturated heterocyclic ring containing two identical or different heteroatoms selected from nitrogen and oxygen atoms or with a lower carboxyalkyl group, or an amino group the substituents of which are replacing both of its hydrogen atoms and are forming with the enclosed nitrogen atom a five- or six-membered heterocyclic group containing two nitrogen atoms, where the other nitrogen atom is optionally substituted by a lower alkyl group; are more beneficial to enhance cold, frost and drought tolerance of plants than the compounds used earlier.

Of the compounds according to the invention those in which

R¹ is a phenyl or pyridil group; R² is a hydrogen atom or a lower alkyl group; X is an amino group or a halogen atom; Y is a hydroxyl, lower alkoxy or substituted amino group are preferred.

Especially beneficial compounds are those in which

R¹ is a pyridil group; R² is a hydrogen atom; X is an amino group or a chlorine atom; Y is a hydroxyl or substituted amino group.

The most beneficial compound according to the invention is [(pyridin-3- carboximidoyl)aminooxy] acetic acid.

Agriculturally acceptable salts of the compounds according to the invention formed with organic or inorganic acids are, for example, hydrochlorides, sulphates, nitrates, phosphates, acetates.

Of the compounds of the formula (I), those compounds of the following general formula

(la) wherein R¹ is a phenyl group or a five- or six-membered unsaturated heterocyclic ring containing nitrogen as heteroatom;

R^l is a hydrogen atom or a lower alkyl group; X is an amino group optionally substituted with one or two lower alkyl groups; or a halogen atom, preferably chlorine or bromine;

Y is a hydroxyl group, a lower alkoxy group, an amino group optionally substituted with a five- or six-membered saturated heterocyclic ring containing two identical or different heteroatoms selected from nitrogen and oxygen atoms or with a lower carboxyalkyl group, or an amino group the substituents of which are replacing both of its hydrogen atoms and are forming with the enclosed nitrogen atom a five- or six-membered heterocyclic group containing two nitrogen atoms, where the other nitrogen atom is optionally substituted by a lower alkyl group; with the proviso that if R² is a hydrogen atom or a lower alkyl group, X is a halogen atom and Y is a lower alkoxy group, then R¹ can only be a five- or six-membered unsaturated heterocyclic ring containing nitrogen as a heteroatom; are new.

Brief Description of the Drawings

Fig. 1 is the representation of the root and shoot length of cold tolerant Dunghan Shaly (DuSh) and cold sensitive Javae and CAN8603 rice types at 18 °C after 21 days of germination. Treatments: blank column - control; bright column - 1 mg SIN- 5/100 seeds; dark column - 15 mg SIN-5/100 seeds. Results marked by *, ** and *** are significantly differ from control sample if *, p<0.05, **, p<0.01 and *** , pO.OOl, respectively.

Fig. 2 is the representation of the number of baby fruits after drought test. Before the water deprivation period plants were sprayed with different concentrations of aqueous solutions of SIN-5: control (DO) sprayed with water; Dl: 5 mg/1; D2: 10 mg/1; D3: 25 mg/1. Result marked by * significantly differs from the control if p < 0.05.

Fig. 3 is the representation of the number of pods on soybean plants following the drought period. Before the water deprivation period plants were sprayed with different concentration aqueous solutions of compound SIN-5: control (DO): sprayed with water; Dl: 10 mg/1; D2: 25 mg/1; D3: 50 mg/1. Result marked by * significantly differs from the control ifp < 0.05. Fig. 4 is the representation of the effect of coating corn caryopses with SIN-5 on fresh weight of corn plantlets after cultivation in gradient chamber for 37 days.

Modes for Carrying out the Invention

For the preparation of the compounds of formulae (I) and (la), for example, the following methods can be used: a) A compound of the general formula

- wherein R¹, X and Y are as defined for general formula (I) - or an acid-additional salt thereof is treated with a diazoting agent known er se (see Werner, Chemische Berichte 26, p. 1567, and Werner, Chemische Berichte 28, p. 1374).

As diazoting agents alkali metal nitrites (potassium or sodium nitrite) or alkyl nitrites (e.g. isoamyl nitrite) in the presence of a hydrogen halide (hydrochloric acid, hydrogen bromide, etc.) can be used. The reaction is carried out at a temperature between -5 °C and 10 °C, then the mixture is stirred until the decomposition of the transiently formed diazonium salt, preferably for 60 to 180 minutes. b) A compound of the general formula

- wherein R¹ and X are as defined for general formula (I) - in the presence of a solvent (preferably a lower alcohol) is reacted with a compound of the general formula

R-Z (IN) wherein R is -CH₂-C(=O)-Y, wherein Y is the same as in formula (I) and Z is a leaving group, preferably CI or Br. The reaction is carried out at the boiling point of the solvent in the presence of potassium tert-butylate or potassium hydroxide (Organic Reactions, 1953, Vol. 7, pages 343 and 373, Journal fur Praktische Chemie 66, p 353., Liebigs Ann. 250, p. 165). c) If a compound wherein Y is an optionally substituted amino group is to be produced, the corresponding amine of the formula Y-H is reacted with a compound of the general formula

- wherein R , X and Z are as defined above. The reaction is carried out in a halogenated solvent, in the presence of an acid binding agent. Such reactions are discussed for example in Zabicky's Chemistry of Amides, p. 73-185 (Interscience, New York, 1970).

The compounds according to the invention of the general formula (I) are equally suitable for the treatment of the seed, the seedling and the fully developed plant. The compounds are applied to the plants or to the seeds by the general methods of plant protection.

According to the invention, in the process to improve the tolerance of cultivated plants against the extremities of the weather, the plant or its seed is treated with an acetic acid derivative of the general formula (I), wherein the substituents are as defined above, or with an agriculturally acceptable salt thereof. The compound is applied in aqueous solution or as a preparation that contains usual auxiliary materials, in addition to the active ingredient, e.g. those aiding the coating, spraying, distribution and absorption of the active agent.

The dose and application concentration of the active ingredients of general formula (I) depend on the species or type of the plant to be protected and on the method of use.

If the cold or frost tolerance of plants is to be improved preferably the seeds of the plants are treated with the compounds of the general formula (I). The seed is coated with a preparation suitable for coating, containing the active ingredient, it is preferably pearled, optionally pelleted or simply treated with the aqueous solution of the active ingredient, for example soaked in the aqueous solution of a compound of the general formula (I) or its salt.

The coating of the seeds is preferably achieved by pelleting with pelleting agents that contain a compound of the general formula (I) or its salt in 0.01-1 g/litre concentration and the usual ingredients and auxiliary materials. The pelleting agents may contain additional active ingredients beside the compounds of the general formula (I), for example fungicides, additives promoting germination and microelements. The pelleting agent is applied in small volume, for instance, when treating rice, capsicum, soya-bean or corn seed, 1 ml pearling agent is used per 100 seeds which is dried onto the seeds uniformly during constant stirring.

For soaking 1-200 mg/litre concentration aqueous solution of the compounds of the general formula (I) is used.

According to a further procedure to increase cold tolerance of plants, the plant is sprayed with a dispersion medium containing a compound of the general formula (I) or a salt thereof before or at the entry of the cold season.

1-500 mg/litre concentration aqueous solution of the active ingredient is used for spraying. The spraying solution may contain additional active ingredients to the compounds of the general formula (I), such as fungicides and materials aiding spraying.

To increase the drought tolerance of plants, the plants are sprayed with a spraying preparation containing a compound of the general formula (I) before or at the entry of the dry season.

The preparations according to the invention that can be used to increase the resistance of cultivated plants against weather extremities contain a compound of the general formula (I) in an amount of 0.001-95 % by mass, wherein the substituents are as defined above, and also agriculturally acceptable solid or liquid vehicles and/or auxiliary materials.

The preparation usually contains water as vehicle. The aqueous solution of the active ingredient is preferably a concentrate which should be diluted with water before use to the concentration given above. Preferably the aqueous solutions contain surfactants, the solutions for treating seeds contain materials aiding pelleting or pearling, for example film forming materials. In addition to surfactants the dispersion media may contain agents improving adhesion or spreading, light protecting agents, if desired, stabilising agents and other additives as auxiliary agents. For spraying purposes water dilutable ULN concentrates, emulsifyable concentrates, wettable powders, soluble granulates or microgranulates can be prepared. These preparations preferably contain anionic or non-ionic detergents to assist dilution with water. Solid preparations preferably contain kaolin, diatomaceous earth or dolomite, but may contain any other solid vehicle generally used in such products. For preparing microgranulates preferably perlit is used as a vehicle. The composition according to the invention can be applied together with other pesticides or can be even combined with them, provided that their active ingredient is compatible with the active ingredient of the composition according to the invention. In the case of such applications the treatment with a composition according to the invention does not require additional processes, it can be carried out together with other protective treatments of cultivated plants.

Two especially important advantages of the use of compounds according to the invention or their agriculturally acceptable salts are pointed out below. a) Improving yield: SC (single cross) hybrid corn seeds are produced by using inbred lines. These plants are extremely sensitive to environmental effects as a result of inbreeding which can seriously effect the amount of the seed crop. Thus, it is of great importance to achieve a better resistance of plants against environmental stresses and a higher yield of seed. b) Producing improved quality seed: seed is significantly distinguished from ordinary plant product. The market value of a hybrid corn seed is 15-20 times greater than that of the same amount of corn feed. This difference is valid as long as the quality (germinating ability, vigour) is adequate. Thus, the main objective of seed production is to achieve the best quality and to avoid deterioration during storage.

The invention is demonstrated with the following examples, without limiting the scope of protection.

Example 1

Preparation of [(Pyridine-3-carboximidoyl)aminooxy] -acetic acid (SIN-5)

10.0 g (72.9 mmol) of nicotinamide oxime is dissolved in 100 ml of 2-propanol, then 16.4 g (146.0 mmol) of potassium-tert-butylate is added. After 30 minutes of stirring 10.1 g (72.9 mmol) of bromoacetic acid dissolved in 20 ml of 2-propanol is added and the reaction mixture is boiled for another 4 hours under stirring. The precipitate is filtered and dried to constant weight (19.2 g). The dry material is dissolved in 100 ml of water, then 10.5 g (54.7 mmol) of citric acid monohydrate is added. The precipitate is filtered and washed with a small amount of water and dried. It is recrystallized from ethanol and then from water using charcoal and dried in desiccator over phosphorus pentoxide to yield an amount of 7.2 g (50.6 %) of product, m.p.: 161-163 °C. 1H-NMR (CDCl₃+DMSO-d₆): 8.9 (1H, m, pyridine 2-H), 8.62 (1H. m, pyridine 6-H), 8.0 (1H, m, pyridine 4-H), 7.35 (1H, m, pyridine 5-H), 5.9 (2H, bs, NH₂), 4.6 (2H, s, OCH₂). IR (KBr): 3360, 3320, 2910, 2400, 1720, 1650, 1430, 1390, 1250, 1230, 1120, 1040, 1015, 940, 895, 840, 825, 700, 640, 518 c f¹.

Example 2

Preparation of [(Chloropyridine-3-yl-methylene)aminooxy]-acetic acid (SIN 3)

2.0 g (10.26 mmol) of [(pyridin-3-carboximidoyl)aminooxy]-acetic acid is dissolved in 100 ml of 1:1 mixture of concentrated hydrochloric acid and water under stirring at 0-5 °C and 2.83 g (41.04 mmol) of sodium nitrite dissolved in 15 ml of water is added dropwise at the same temperature. After further stirring for 2 hours the pH of the reaction mixture is adjusted to 6 by adding 1 N sodium hydroxide solution, the precipitate is filtered and dried. It is recrystallized from water with charcoal and dried in desiccator over phosphorous pentoxide. This gives a final yield of 1.4 g (63.6%), m.p.:151-153 °C.

IR (KBr): 3410, 3060, 2920, 2740, 2480, 1742, 1590, 1570, 1550, 1460, 1430, 1400, 1360, 1240, 1120, 1070, 1040, 1000, 920, 825, 715, 690, 625, 590, 480 cm^"1.

Example 3

Preparation of [(Chloropyridine-3-yl-methylene)aminooxy] -acetic acid methyl ester (SIN-

10)

0.8 g (3.7 mmol) of [(chloropyridine-3-yl-methylene)aminooxy]-acetic acid is dissolved in a 2:1 mixture of dichloromethane and methanol, and diazomethane in dichloromethane solution is added in excess under stirring. After 30 minutes the diazomethane excess is decomposed with acetic acid and the solution is evaporated under reduced pressure. The residue is dissolved in 50 ml of chloroform, washed with 10 % sodium hydrogen carbonate solution, then with water, and finally with saturated saline solution, the organic phase is separated, dried on magnesium sulphate, filtered and evaporated. The evaporation residue is purified by column chromatography with silica gel, to obtain 0.51 g (60.0%) of pale yellow oil.

IR (KBr): 2950, 2851, 2361, 2343, 1761, 1591, 1475, 1438, 1413, 1383, 1338, 1264, 1223, 1094, 1035, 1024, 984, 960, 902, 821, 723, 708, 698, 420 cm^-1. ^' Example 4

Preparation of N-Morpholine-4-yl-2-[(chloropyridine-3-yl-methylene)aminooxy]-acetamide

(SIN-7) a) Preparation of acid chloride solution:

0.8 g (3.72 mmol) of [(chloropyridine-3-yl-methylene)aminooxy] -acetic acid with 5.42 ml (74.3 mmol) of thionyl chloride is boiled in 30 ml of chloroform for 2 hours under stirring. After evaporating the solution yet 50 ml of dioxane solvent is evaporated off the residue, twice. The residue is dissolved in 15 ml of chloroform and the acid chloride solvent obtained is used for the subsequent reactions. b) Reaction with 4-aminomorpholine:

0.36 ml (3.72 mmol) of 4-aminomorpholine dissolved in 5 ml chloroform is added dropwise to the acid chloride solution prepared above in the presence of an equivalent amount of triethylamine at 20 °C under stirring. After stirring for 2 hours the reaction mixture is washed with 3% acetic acid solution, then with 10 % sodium carbonate solution, then with water, and finally with saturated saline solution. The residue obtained after evaporation is crystallized from ethanol with charcoal to obtain 0.54 g (48.6 %) of product, m.p.: 179-182 °C.

1H-NMR (CDC1₃): 9.05 (1H, m, pyridine 2-H), 8.77 and 8.62 (1H, m, pyridine 6-H), 8.18 and 8.15 (1H, m, pyridine 4-H), 7.42 and 7.35 (1H, m, pyridine 5-H), 7.0 and 6.58 (1H, bs, amide NH), 5.15 and 4.85 (1H, s, OCH₂), 3.92 (4H, m, morpholine OCH₂), 2.95 (4H, m, morpholine NCH₂).

IR (KBr): 3415, 3200, 3060, 2960, 2860, 2820, 1670, 1580, 1560, 1480, 1450, 1420, 1335, 1260, 1240, 1105, 1070, 1040, 1020, 1000, 970, 955, 900, 860, 800, 760, 700, 665, 645, 610, 590 cm-¹.

Example 5

Preparation of [2-[(chloropyridine-3-yl-methylene)aminooxy]acetylamino]-acetic acid (SIN

8)

This compound is prepared with the procedure described in Example 4. a) The acid chloride is prepared with the procedure described in Example 4a. b) Reaction with 0.28 (3.72 mmol) of aminoacetic acid (glycine): In the preparation procedure the product is isolated with adjusting the pH value to 6 to obtain ayield of 0.35 g (41.7 %) of product, m.p.: 183-186 °C.

1H-NMR (CDCl₃+DMSO-d₆): 9.05 (1H, m, pyridine 2-H), 8.66 (1H. m, pyridine 6-H), 8.25 (1H, m, pyridine 4-H), 7.38 (1H, m, pyridine 5-H), 7.25 (1H, bs, NH), 4.8 (2H, s, OCH₂), 4.0 (2H, d, NCH₂).

IR (KBr): 3280, 3080, 2450, 1920, 1740, 1680, 1560, 1480, 1410, 1360, 1260, 1220, 1180, 1110, 1070, 1040, 1020, 960, 900, 875, 815, 695, 660, 640, 590, 570 cm^-1. Example 6

Preparation of 1 -(4-methylpiperazine-l -yl)-2-[(chloro-3-phenylmethylene)amino- oxymethylenej-ketone hydrochloride (SIN-9)

This compound is prepared with the procedure described in Example 4. a) The acid chloride is in this case prepared from [chloro-3- phenylmethy lene)aminooxy] -acetic acid. b) Reaction with 0.41 ml (3.72 mmol) of N-methyl-piperazine:

After evaporation the residue is dissolved in ethanol and by the addition of equivalent amount of ethyl alcoholic solution of hydrogen chloride and ether the hydrochloride salt is separated to obtain 0.45 (36.5 %) of product, m.p.: 225-228 °C.

1H-NMR (CDCI₃): 7.81 (2H, m, aromatic protons), 7.5 (2H, m, aromatic protons), 5.05 (2H, bs, OCH₂), 5.05 (2H, bs, OCH₂), 4.4 and 4.1 (4H, m, piperazine), 3.45 and 3.1 (4H, m, piperazine), 2.8 (1H, s, NCH₃).

IR (KBr): 3440, 2920, 2650, 2530, 2430, 1670, 1580, 1560, 1470, 1440, 1400, 1350, 1300, 1270, 1260, 1240, 1220, 1090, 1050, 1020, 1005, 960, 900, 890, 800, 750, 700, 680, 650, 580 cm^-1.

Example 7

Coating seeds with active ingredient

The desired amount of compound SIN-5 is dissolved completely in 1 M sodium hydrogen carbonate. 4 % aqueous solution of polyvinyl-alcohol (PNA) is prepared. In case of pelleting (pearling) corn seed 1 ml of 4 % PNA solution is mixed with 1 ml solution containing SIΝ-5 in 5-15 mg/ml concentration. 200 corn seeds are dampened in the resulting 2 ml solution in Petri-dishes by shaking. Then the coated seeds are allowed to dry overnight at room temperature. In case of crops with smaller seed size (e.g. rice) 1 ml of pearling agent containing 1-15 mg of SIN-5 agent is applied for coating 200 seeds. Example 8

Improving cold tolerance by seed treatment

In this experiment the cold tolerance of corn seeds treated with the active ingredient was studied. The seeds were subjected to low temperature and absence of oxygen. The experiment was carried out with the CSNT (Complex Stressing Vigour Test) method of Barla-Szabό (G. Barla-Szabό and B. Dolinka, Seed Sci. & Technol., (1988) 16: 63-73).

200 seeds were soaked in 150 ml of 10 mg/1 aqueous solution of the active ingredient. The seeds were soaked for 48 hours at 25 °C, then for another 48 hours at 5 °C. After the 96-hour soaking the seeds rolled in wet paper were germinated for 96 hours at a temperature of 25 °C. Each paper roll contained 25 seeds, they were positioned in vertical containers and covered with plastic bags to prevent evaporation. Seeds were kept in dark during the entire experiment.

At the end of the experiment the number of normally germinated and ungerminated seeds was recorded. The shoot length of the normal seedlings was measured and the average length of the 5 longest seedlings was calculated. Those seedlings that were longer than 0.33 times this average were considered high vigour seedlings, those under this value were considered low vigour seedlings.

In the experiments carried out on corn seeds it was found that under the CSNT test conditions the active ingredients studied increased significantly the ratio of high vigour seedlings and the length of shoots of inbred corn lines Mo 17 (Table 1) and HMv 5405 (Table 2), and of hybrid Siloma seeds (Table 3).

Table 1 ive ingredient Rate of high vigour plants (%) Shoot length (mm)

Control 16.0 24.3

SIΝ-3 24.9* 39.9*

SIN-5 25.5* 44.3**

SIN-8 24.0* 38.0*

Table 2

Active ingredient Rate of high vigour plants (%) Shoot length (mm)

Control 72.5 47.5

SIN-3 81.0* 63J*

SIN-5 82.0* 75.0***

SIN-8 80.5* 72.5* Table 3

Active ingredient Rate of high vigour plants (%) Shoot length (mm) Control 58.0 60.6

SIN-3 65.5* 85.6*

SIN-5 75.5* 111.2***

SIN-8 67.0* 106.8***

Results signed by *, ** and *** are significantly differ from control sample, if *, p<0.05, **, p<0.01 and ***, pO.OOl, respectively.

For better understanding of the experimental results it is necessary to mention that the CSNT is developed to predict the minimal expectable germination ratio under extreme conditions. In a given set of seeds the ratio of those seeds that will reliably and well gemrinate in cold spring weather is 90 % if they were proved high vigour by the CSNT, while this value is only 60 % if they showed low vigour under experimental stress conditions. This leads to the conclusion that if an active agent improves the vigour of the seedlings it in fact improves the germinating ratio of the seed on the arable soil at soil temperatures lower than the optimum.

The above experiments unambiguously proved that the compounds of the general formula (I) are able to improve the vigour of the seedlings and the length of the shoot, thereby improving the chance of emergence of the plants, if the weather unpredictedly turns unfavourable after sowing.

Example 9

Improvement of cold tolerance by seed treatment

Rice

Experiments carried out on rice, for which 3 cold sensitive genotypes (Javae 04, Cna 8603 "19", PR 3491 "20") were used, also showed that the active agents have higher effect on the germination and growth of plants on cold sensitive genotypes than on cold tolerant (Dungan Shaly) genotypes. Cold tolerance was measured at 18 °C on rice and the length of the root and the shoot was measured after 21 days of germinating. The caryopses were germinated on the surface of plastic plates covered with filter paper placed into a whatnot. The plates were mounted at an angle of 80 degrees, the caryopses were fastened with wet tissue paper. 20 caryopses were placed on each plate and the number of replicates per treatment was 6. Germination was carried out under continuous illumination.

The results of the cold tolerance tests are shown in tables 4-7. Table 4 Cold tolerance of rice (Dungan Shaly)

Active agent Root length (mm) Shoot length (mm)

Control 12.24 8.13

SIN-3 13.97* 10.7*

SIN-5 14.24* 13.55*

SIN-8 14.11* 11.32*

Table 5

Cold tolerance of rice (Javae 04)

Active agent Root length (mm) Shoot length (mm)

Control 9.20 4.24

SIN-3 12.12 6.77

SIN-5 13.07* 6.26*

SIN-8 12.91 6.61

Table 6 Cold tolerance of rice (Cna 8603 "19")

Active agent Root length (mm) Shoot length (mm)

Control 5.97 3.11

SIN-3 8.99* 4.97*

SIN-5 10.14* 6.16*

SIN-8 8.28* 4.18*

Table 7

Cold tolerance of rice (PR 3491 "20")

Active agent Root length (mm) Shoot length (mm)

Control 6.0 3.45

SIN-3 8.78 5.34*

SIN-5 9.33* 6.0*

SIN-8 7.86 5.46*

Results signed by * are significant compared to control group if p<0.05.

In an other series of experiment with the same experimental set-up a significant increase in the length of roots and shoots was experienced upon increasing the dose (Figure

1).

Example 10

Improvement of drought tolerance of paprika and soya-bean Based on our previous experiences the plants were subjected to hardening before the use of the active agent: for a few days until the first signs of blighting appeared the plants were not watered. Then the plants were watered again and the active agent was supplied with the irrigation water or was applied to the surface of the plants with spraying. Following this plants were subjected to drought and were watered again and after one week of regeneration time the number of new fruits on the paprika and the number of pods on the soya were recorded.

The description of paprika and soya experiment: paprika and soya seeds were treated with aqueous SIN-5 solutions of different concentration before sowing. The following doses were applied on paprika: DO: control; Dl: 1 mg of SIN-5/100 seeds; D2: 2 mg of SIN-5/100 seeds; D3: 3 mg of SIN-5/100 seeds. Doses applied on soya-beans: DO: control; Dl: 2.5 mg of SIN-5/100 seeds; D2: 5 mg of SIN-5/100 seeds; D3: 10 mg of SIN- 5/100 seeds. Paprika plants were grown in pots, in vegetable mould rich in humus, in growth chamber. The growing conditions were the following: 16/8 h day /night period was applied together with a 25/20 °C thermal cycle. The intensity of the illumination was 220 μmol m^"2 s^"1. Plants were watered daily. In the case of the soya-beans 25/18 °C thermal cycle was applied and the intensity of the illumination was 330 μmol m^" s^" . Watering was suspended for a few days after the appearance of flowers until the first signs of blighting were shown on the leaves. Then the leaves were sprayed with SIN-5, applied in 3 different doses. On the paprika: DO: control (sprayed with water); Dl : 5 mg/1; D2: 10 mg/1; D3: 25 mg/1; while on the soya-bean: DO: control (sprayed with water); Dl: 10 mg/1; D2: 25 mg/1; D3: 50 mg/1 doses were applied. On the following day the plants were watered and then a long dry period followed until the complete blighting of the leaves. Then the plants were watered again and after a recovery period the number of paprika fruits (Figure 2), and the number of soya-bean pods (Figure 3) were recorded. In the case of the paprika the seed treatment of 2 mg SIN-5/100 seeds combined with 25 mg/1 leaf spraying increased the number of fruits significantly. In the case of soya-bean two doses proved to be effective. Significant improvement was recorded with the lowest applied dose combination (2.5 mg SIN-5/100 seeds combined with 10 mg/1 leaf spraying).

Example 11

Improvement of cold tolerance of corn in gradient chamber The experiment was carried out on Mo 17 corn genotype. Caryopses were coated with 5 mg compound to be tested dissolved in 2 ml of 2 % polyvinyl-alcohol before germination, said amount was applied per 100 seeds. Caryopses were germinated for 3 days between wet filter papers, then they were sowed and put into a gradient chamber for 37 days. In the gradient chamber a temperature of 12 to 19 °C was maintained, with 1 °C steps. The cold treatment was followed by a regeneration phase at a temperature of 23/20 °C.

In the end of the experiment the fresh weight (FW) of the plants was measured. The experimental results are summarized in Figure 4.

The intensity of the 16-hour daily illumination was 220 μmol m^'2 s^"1. Plants were sprayed with a 10 mg/1 concentration aqueous SIN-5 solution once at the age of 3 weeks. Before germination caryopses were pelleted: DO control; Dl: 5 mg SIN-5/100 seeds.

Open ground experiments

In the following example results of open ground experiments carried out on early sowing are disclosed which were done to enable us to decide how the investigated compounds affect the growth and yield of plants under cultivating conditions.

Example 12

Rice open ground experiments (cold tolerance)

The experiment was carried out on Janka and Dama genotypes. The compounds investigated were applied with coating onto the plant seeds, by treating 100 seeds with 1 ml coating agent containing 1 mg agent dissolved in 2 % aqueous PNA solution.

The date of sowing was 15 May. During the development of the plants standard cultivation and plant protection procedures were applied. Sowing was carried out with 20 cm row distance and 90 seeds/m. The size of the parcels was 1.5 m². One week after sowing parcels were treated with Stomp 5 1/ha herbicide, and during tillering with 0.80 g/ha Londax. Parcels were flooded when the plants developed 2-3 leaves, until reaching wax ripeness. In the experimental set-up randomised block design with four repeats was arranged. Results were evaluated with one factor variance analysis (AΝONA). The least significant differences (LSD) were calculated at P = 5 %.

The experiment was analysed on the basis of measuring the mass of the crop. Results are presented in Table 8. Table 8 The effect of seed treatment on the yield of Janka and Dama rice types

Active agent Mass of the crop of Janka type Mass of the crop of Dama type

Control 670 820

SIN-3 723* 901*

SIN-5 738* 905*

SIN-8 718* 898*

Example 13

Improvement of average yield of corn at open ground (cold tolerance)

The experiment was carried out on Mo 17 genotype. Before sowing seeds were pelleted with fungicide and insecticide, simultaneously the tested compounds were also applied in the form of 2 % aqueous PNA solution, 2 ml solution per 100 seeds.

The size of the parcels was 9.2 m x 3.75 m, the density of the sowing was 5 seeds/m. The experimental arrangement was randomised block design with four repeats. Plants were sowed 4-8 cm deep, depending on the moisture of the soil, in 30 cm distance between plants, with 45 cm row distance. The date of sowing was 8 May. The number of emerged plants was recorded.

The crop was harvested when the moisture content of the seeds decreased under 28 %. At harvesting the mass of the plant and that of the crop were measured. The measured data was evaluated with one factor variance analysis (AΝONA). The least significant differences (LSD) were calculated at P = 5 %. Results are presented in Table 9 and 10.

As a result of the seed treatment the number of emerged plants per parcel increased.

Table 9 Number of emerged plants per parcel

Active agent Number of emerged control 81.5

SIN-3 95.8*

SIN-5 100.5*

SIN-8 92.0* Table 10

Amount of yield per parcel

Active agent yield/parcel control 10.29

SIN-3 11.30

SIN-5 12.81*

SIN-8 12.10*

Example 14

Production of hybrid corn seed

Treatments with SIN-5 were carried out on commercially produced lands of two SC hybrid seeds. Female parent seeds and the plants twice during their development were treated. Compound SIN-5 was applied for coating seeds in the following concentrations:

1. Half dose: 5 mg of SIN-5 dissolved in 2 ml of 2 % aqueous PVA solution per 100 seeds.

2. Full dose: 10 mg of SIN-5 dissolved in 2 ml of 2 % aqueous PVA solution per 100 seeds.

3. Double dose: 20 mg of SIN-5 dissolved in 2 ml of 2 % aqueous PVA solution per 100 seeds.

Beside seed treatment plants were sprayed with the SIN-5-solution (50 mg of SIN-5 was dissolved in 2 1 water and this amount was used per parcel) before tasseling. One parcel consisted of four 6 m long row. The distance of the rows was 91 cm. In the case of hybrids the distance between plants was 20 cm which corresponds to a density of 48-50000 plants/hectare. In the case of inbred lines the distance between plants was 16 cm which corresponds to a density of 60-62000 plants/hectare.

Plants treated with the full dose gave higher seed yield. The quality of the seed also improved significantly which was especially observable after one year of storage. Results are summarized in Table 11.

Table ll

Effect of treatment of hybrid corn seed with SIN-5 on the yield and the quality of seed

Hybrid A kg/parcel Test on new seeds Test on seeds after one year of storage Treatment Mean Emerging Cold test CSVT Emerging Cold test CSVT

Control 12.7 99 97 62 96 69 42

Half dose 13.4 99 98 68 98 71 53*

Full dose 14.5* 99 97 65 97 67 53*

Double 12.6 100 98 64 94 66 48 dose

LDS 1.73 2 3 7 3 7 8

Hybrid B kg/parcel Test on new seeds Test on seeds after one year of storage

Treatmen Mean Emerging Cold test CSVT Emerging Cold test CSVT

Control 16.2 97 96 83 94 85 53

Half dose 18.5* 98 96 87 92 79 49

Full dose 17.6 98 96 91* 95 81 60*

Double 16.8 97 97 89* 98* 91* 72* dose

LDS 1.94 3 3 6 3 5 8

Example 15

Chemical treatment of new and aged seeds

The biological value (germinating capability, vigour) of newly produced seeds is generally good. During storage the quality of the seed deteriorates first slowly and later with an increasing rate while its vitality also decreases. A stand originating from fresh seeds can produce even 10-15% more than that consisting of genetically identical and of the same number of plants but originating from aged seeds. Deterioration of germinating ability and vigour can be slowed with different procedures one of which could be a chemical treatment.

New, 2 and 4 year old seeds of a corn SC hybrid and those of a new inbred line were treated with compound SIN-5, with the aim of eliminating the effect of physiological deterioration of older seeds. The applied concentration: 10 mg of SIN-5 in 2 ml of 2 % aqueous PVA solution per 100 seeds. The pollen viability highly depends on the stress effects of the environment. The yield could be significantly limited by the decreased pollen viability. Table 12

Effect of treatment of new and stored corn seeds with SIN-5 on the amount of crop and on the viability of the pollen. The mass of the crop is given in kg/parcel, the viability of the pollen is given in %.

Age of Control Treated with SIN-5

Test l seed Crop kg/parcel Pollen viabilit

SC 0 7.1 60 8.0* 85

2 6.8 65 6.9* 80 hybrid 4 6.6 60 7.9* 60

LSD 0.05% 0,84

Inbred 0 4.7 60 4.5 85

2 3.2 85 4.5* 95 line 4 3.7 80 4.3* 80

LSD 0.05% 0,59

Example 16 Spraying solution

Spraying solution of the following composition is prepared: compound SIN-5 20 parts of weight sodium-laurilsulphate 3 parts of weight sodium-ligninsulphonate 6 parts of weight water 63 parts of weight kaolin 8 parts of weight

Example 17 Spraying solution

Spraying solution of the following composition is prepared: compound SIN-5 20 parts of weight alkyl-arylsulphonate 5 parts of weight water 75 parts of weight

Example 18 Coating agent

Coating agent of the following composition is prepared: compound SIN-5 0.25 parts of weight 2 % aqueous solution of polyvinyl-alcohol 99.75 parts of weight

The coating agent is applied in 0.01-0.02 % by mass of the seed.

Example 19 Granulate

A granulate of the following composition is prepared: compound SIN-5 10 parts of weight limestone grist 64 parts of weight ethyleneglycol 3 parts of weight high dispersity silicic acid 4 parts of weight sodium-ligninsulphonate 4 parts of weight water 15 parts of weight

The mixture of the components is grinded in a hammering mill to a grain size of less then 5 μm.

Example 20 Powder composition

A powder composition of the following composition is prepared:

SIN-5 compound 50 parts of weight polyvinylpyrrolidon 10 parts of weight silicondioxide 25 parts of weight porcelain clay 15 parts of weight

Example 21 Powder composition

A powder composition of the following composition is prepared:

SIN-5 compound 50 parts of weight calcium-ligninsulphonate 5 parts of weight isopropylnaphtalenesulphonate 1 parts of weight silicondioxide 4 parts of weight filling material (kaolin) 40 parts of weight

Claims

Acetic acid derivatives of the general formula:

(la) and their agriculturally acceptable salts, wherein R¹ is a phenyl group or a five- or six-membered unsaturated heterocyclic ring containing nitrogen as heteroatom; R² is a hydrogen atom or a lower alkyl group;

2. The compounds according to claim 1, wherein R¹ is a phenyl or pyridyl group;

R² is a hydrogen atom or a lower alkyl group;

X is an amino group or a halogen atom;

Y is a hydroxyl, lower alkoxy or substituted amino group.

3. The compounds according to claim 2 , wherein R¹ is a pyridyl group; R² is a hydrogen atom;

X is an amino group or chlorine atom;

Y is a hydroxyl or substituted amino group.

4. Use of compounds of the general formula

(I) or their agriculturally acceptable salts, wherein

R¹ is a phenyl group or a five- or six-membered unsaturated heterocyclic ring containing nitrogen as heteroatom; R is a hydrogen atom or a lower alkyl group;

X is an amino group optionally substituted with one or two lower alkyl groups; or a halogen atom, preferably chlorine or bromine; Y is a hydroxyl group, a lower alkoxy group, an amino group optionally substituted with a five- or six-membered saturated heterocyclic ring containing two identical or different heteroatoms selected from nitrogen and oxygen atoms or with a lower carboxyalkyl group, or an amino group the substituents of which are replacing both of its hydrogen atoms and are forming with the enclosed nitrogen atom a five- or six-membered heterocyclic group containing two nitrogen atoms, where the other nitrogen atom is optionally substituted by a lower alkyl group; for increasing abiotic stress tolerance of cultivated plants and for inhibiting the senescence of seeds and enhancing their vigour under stress conditions.

5. Composition for improving abiotic stress tolerance of cultivated plants, for inhibiting the senescence of seeds and for increasing their vigour under stress conditions comprising an acetic acid derivative of the general formula (I) or an agriculturally acceptable salt thereof, wherein the substituents are as defined in claim 4, optionally an agriculturally acceptable solid or liquid vehicle and optionally one or more auxiliary materials.

6. The composition according to claim 5, wherein the substituents in the general formula (I) are as defined in claim 1.

7. The composition according to claim 5, wherein the substituents in the general formula (I) are as defined in claim 2.

8. Process for increasing abiotic stress tolerance of cultivated plants, for inhibiting the senescence of seeds and for increasing their vigour under stress conditions, characterized in that the plant or its seed is treated with an acetic acid derivative of the general formula (I) according to claim 4, an agriculturally acceptable salt thereof or a composition according to claim 5.

9. Process for increasing cold and frost resistance of cultivated plants, characterized in that the plant or its seed is treated with an acetic acid derivative of the general formula (I) according to claim 4, an agriculturally acceptable salt thereof or a composition according to claim 5.

10. The process according to claim 9, characterized in that the seed of the plant is treated with the aqueous solution of an acetic acid derivative of the general formula (I) according to claim 4, of an agriculturally acceptable salt thereof or of a composition according to claim 5.

11. The process according to claim 10, characterized in that the seed of the plant is soaked in an aqueous solution of an acetic acid derivative of the general formula (I) according to claim 4, of an agriculturally acceptable salt thereof or of a composition according to claim 5.

12. The process according to claim 9, characterized in that the seed of the plant is coated with a preparation containing an acetic acid derivative of the general formula (I) or an agriculturally acceptable salt thereof, wherein the substituents are as defined in claim 4, and optionally also other active ingredients and/or materials aiding germination.

13. Process for increasing cold tolerance of cultivated plants, characterized in that before or at the entry of the cold season the plant is sprayed with a spraying agent containing an acetic acid derivative of the general formula (I) or an agriculturally acceptable salt thereof as active ingredient, wherein the substituents are as defined in claim 4.

14. Process for improving drought tolerance of cultivated plants, characterized in that before or at the entry of the dry season the plant is sprayed with a spraying agent containing an acetic acid derivative of the general formula (I) or an agriculturally acceptable salt thereof as active ingredient, wherein the substituents are as defined in claim 4. 33

AMENDED CLAIMS

[received by the International Bureau on 06 June 2002 (06.06.02) original claims 1, 2, amended; remaining claims unchanged (1 page)]

1. Acetic acid derivatives of the general formula:

and their agriculturally acceptable salts, wherein R^l is a five- or six-membered unsaturated heterocyclic ring containing nitrogen as heteroatom; R² is a hydrogen atom or a lower alkyl group; X is an amino group optionally substituted with one or two lower alkyl groups; or a halogen atom, preferably chlorine or bromine;

Y is a hydroxyl group, a tower alkoxy group, an amino group optionally substituted with a five- or six-membered saturated heterocyclic ring containing two identical or different heteroatoms selected from nitrogen and oxygen atoms or with a lower carboxyalkyl group, or an amino group the substituents of which are replacing both of its hydrogen atoms and are forming with the enclosed nitrogen atom a five- or six- membered heterocyclic group containing two nitrogen atoms, where the other nitrogen atom is optionally substituted by a lower alkyl group; with the proviso that if R^l is a pyrrolyl group and X is an unsubstituted amino group, then Y can only be an amino group optionally substituted with a five- or six-membered saturated heterocyclic ring containing two identical or different heteroatoms selected from nitrogen and oxygen atoms or with a lower carboxyalkyl group, or an amino group the substituents of which are replacing both of its hydrogen atoms and are forming with the enclosed nitrogen atom a five- or six-membered heterocyclic group containing two nitrogen atoms, where the other nitrogen atom is optionally substituted by a lower alkyl group.

2, The compounds according to claim 1 , wherein R¹ is a pyridyl group;

R² is a hydrogen atom or a lower alkyl group; X is an amino group or a halogen atom;

Y is a hydroxyl, lower alkoxy or substituted amino group.

3, The compounds according to claim 2 , wherein R¹ is a pyridyl group; 34

STATEMENT UNDER ARTICLE 19(1)

In the case of the above International Patent Application original claims 1 and 2 have been amended under Article 19(1) in such a way that the scope of protection of the amended claims is delimited from the disclosures of the documents cited in the International Search Report. Accordingly, the amendments do not go beyond the disclosure in the International Patent Application as filed.