SK289396B6 - Crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea, preparations containing it and its use - Google Patents

Abstract

The invention relates to the crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea (PTU), its use in agriculture, horticulture, forestry and plant biotechnology in the cultivation of plants and preparations containing this substance. The crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea exhibits improved biological activity compared to the amorphous form.

Description

Field of technology

The invention relates to a crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea (PTU), its use in agriculture, horticulture, forestry and plant biotechnology in the cultivation of plants and preparations containing this substance.

State of the art

Cytokinins are plant signaling substances (phytohormones) that play a central role in the regulation of cell division in plant cells and in numerous plant developmental processes. Cytokinins were discovered by F. Skoog, C. Miller and co-workers in the 1950s as factors that promote cell division (cytokinesis). The first cytokinin discovered was an adenine derivative called kinetin (6-furfurylaminopurine), which was isolated as a product of DNA degradation. The first common natural cytokinin identified was isolated from immature corn kernels and named zeatin (chemical name: 6-(4-hydroxy-3-methylbut-2-enylamino)purine). Several other cytokinins with similar heterocyclic structures are now known. Cytokinins have a wide range of biological effects.

Cytokinins were discovered as factors promoting cell division and, due to their morphogenetic activities, their use has been widely extended in in vitro cultures of higher plants (plant biotechnology), where they allow de novo formation of organs and/or whole plants, as well as organogenesis and/or somatic embryogenesis. In tissue culture, some urea derivatives, such as N-phenyl-N'-(2-chloro-4-pyridyl)urea (CPPU) and N-phenyl-N'-1,2,3-thiadiazol-5-ylurea (thidiazuron, TDZ), are increasingly used in comparison to naturally occurring cytokinins, showing improved cytokinin-like activity, although their structure is unrelated to adenine derivatives. These synthetic compounds and some of their derivatives have been shown to have an effect at the cytokinin receptor level, regardless of their different molecular structure, are not degraded by cytokinin oxidase CKX, and positively affect endogenous cytokinin concentrations in plants (Nisler et al. 2016, 2021). In addition, it has recently been reported that some urea derivatives slow down leaf senescence, which is a cytokinin-mediated effect, without affecting cytokinin levels (Nisler et al. 2018). One of the urea derivatives is l-phenyl-3-(4H-l,2,4-triazol-4-yl)urea (Suleymanov wt al. bioconf/20225200012 FIES 2022,12, 2022; US patent 3,836,350). So far, it has been prepared using processes that lead to the formation of non-crystalline forms that are difficult to clean.

The present invention aims to develop a crystalline water-soluble form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea with increased activity, particularly suitable for biotechnological and agricultural use.

The essence of the invention

The essence of the present invention is a crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea, showing increased biological activity in plant bioassays and on plants.

The subject of the present invention is a crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea (PTU) of formula (I),

which has characteristic reflections in the X-ray powder diffraction spectrum measured with CuKα radiation: 8.2; 12.2; 18.8; 21.0; 26.4; 30.8 ±0.2° 2-theta. More preferably, it has further characteristic reflections: 15.4; 16.5; 20.6; 22.0; 24.5; 28.1 ±0.2° 2-theta.

1-Phenyl-3-(4H-1,2,4-triazol-4-yl)urea (PTU) in this crystalline form crystallizes in the orthorhombic system with the space group specification Pca21.

In the present invention, based on the investigation of the single crystal structure, it was confirmed that the crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea has the molecular formula C9H9N5O1. Molecular formula calculated: C9H9N5O1, measured: C9H9N5O1; molecular weight (m.w.) calculated: 203.21, measured: 203.20.

The crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea according to the invention has a melting point of 231-232°C.

The crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea according to the invention is prepared by recrystallization of crude 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea from a C1-C4 alcohol, preferably methanol, ethanol or isopropanol, or from water. Crude (amorphous) 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea is preferably prepared by the reaction of phenyl isocyanate and 4-amino-1,2,4-triazole in tetrahydrofuran (THF).

The crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea (PTU) according to the present invention exhibits increased biological activity compared to the amorphous form in a wide range of biological activities including antioxidant, anti-senescence, promoting phloem loading, influencing the formation of the above-ground part of the plant and the root, pro-differentiation activity, which are particularly useful in agricultural, horticultural, forestry and biotechnological applications (e.g. for the treatment of plant tissues, cells, organs and whole plants). The PTU compound (and preparations containing it) have minimal or no toxicity. This allows their use in a wide range of applications. The exact reason for the increased biological activity has not yet been identified, it may be related to crystallization purification from specific impurities or to physical changes during crystallization.

The subject of the invention is the use of the crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea according to the invention for protecting plant cells in vivo and in vitro against oxidative and electrophilic stress.

The invention also provides the use of the crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea according to the invention as an antioxidant for inhibiting lipid, protein and DNA peroxidation in plants in vivo or in vitro.

This invention further relates to the use of the crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea according to the invention for increasing yield, improving rooting, increasing the number of tillers, increasing multiplication and delaying senescence in the production of useful plants and agricultural crops, in particular cereals (wheat, barley, rice, corn, rye, oats, sorghum and related species), beets (sugar and fodder beets), pome fruits, stone fruits and small (soft) fruits (apples, pears, plums, peaches, almonds, cherries, strawberries, blueberries), legumes (beans, lentils, peas, soybeans), oilseeds (rapeseed, mustard, poppy, olives, sunflowers, coconut, castor oil, cocoa fruits, peanuts), cucurbits (cucumbers, pumpkins, melons), fiber plants (cotton, flax, hemp, jute), citrus (oranges, lemons, grapefruits, mandarins), vegetables (spinach, cinnamon, camphor) or plants such as tobacco, nuts, eggplant, sugarcane, tea, grapevine, hops, bananas and natural rubber and medicinal plants as well as ornamental plants. Crops include those plants that have been shown to be tolerant to various groups of growth regulators/factors using conventional breeding methods or genetic engineering methods.

The invention also provides the use of the crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea according to the invention as a growth-regulating and pro-differentiation factor. These properties can be used to optimize conditions in plant tissue culture in vitro. Specifically for in vitro include somatic embryogenesis, micropropagation, androgenesis, gynogenesis, suspension and protoplast cultures.

The crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea (PTU) of formula (I) can, according to the present invention, be further used in plant tissue cultures to suppress undesirable physiological disorders, such as hyperhydricity, growth tip necrosis, chimera formation, inhibition of root formation during acclimation and early senescence.

The invention also provides the use of the crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea according to the invention for inhibiting or delaying senescence of plant cells, tissues, organs and whole plants.

The invention also provides the use of the crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea according to the invention in agriculture, horticulture, forestry and biotechnology for inhibiting, delaying or reducing adverse effects associated with senescence, both in vivo and in vitro, these uses also include improving the overall appearance and condition of plants, in particular plant epidermal and mesophyll cells, and also for inhibiting, delaying or reducing senescence, yellowing, loss of chloroplasts and/or chlorophyll.

The uses of the crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea according to the invention generally involve the application of an effective amount of a compound of formula (I) to cells (e.g. in tissue culture), plant organelles, plant parts or whole plants. The compounds of formula (I) can be applied alone or - usually - in the form of a preparation with carriers and/or excipients.

The invention further includes agricultural and biotechnological preparations containing the crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea (PTU) and at least one excipient. For this purpose, the preparations are preferably formulated as solid active ingredient concentrates, suspensions, dispersions, soluble powders, granules, oil suspensions and also coated formulations, e.g. with polymeric substances. The preparations can be sterilized and/or can contain further excipients of a neutral nature, such as preservatives, stabilizers, wetting agents or emulsifiers, solubilizing agents, as well as fertilizers, trace element donors or other means to achieve special effects.

In this text, the term in vivo means the use of a whole plant or part of a plant capable of independent life. The term in vitro means the cultivation of cell (or tissue) cultures outside the organism from which they originate. In a figurative sense, this then means in a test tube, Erlenmeyer flask, Petri dish, and other laboratory containers where something can be grown or cultivated.

Preparations

Preparations containing the compound of formula (I) (active ingredient) in crystalline form, and if necessary, one or more excipients, are prepared by known methods, for example by mixing or grinding the active ingredient with excipients, such as e.g. solvents or solid carriers. Surface-active substances (surfactants) may be added to the preparations. Suitable surface-active substances are non-ionic, cationic and/or anionic surfactants and mixtures thereof having good emulsifying, dispersing and wetting properties. Examples of suitable anionic, non-ionic and cationic wetting agents are summarized, for example, in WO 97/34485.

Also suitable for the preparation of compositions containing the compound 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea (PTU) of formula (I) in crystalline form according to the invention are surfactants commonly used in the formulation of compositions, which are described, for example, in "McCutcheon's Detergents and Emulsifiers Annual MC Publishing Corp., Ridgewood New Jersey, 1981, Stach, H., "Tensid-Taschenbuch, Carl Hanser Verlag, MunichNienna, 1981. Ash, "Encyclopedia of Surfactants, Vol-lll, Chemical Publishing Co., New York, 1980-81.

The application form of the preparation contains by weight from 0.1 to 99% (w/w), in particular from 0.1 to 95% (w/w) of the active ingredient corresponding to the substance of formula (I), while also containing from 5 to 99.9% of a mixture of additives or carriers, depending on the methods of application, and optionally also containing by weight from 0.1 to 25% of a wetting agent.

Commercial products are usually prepared in the form of powders, wettable powders, granules, coated granules, suspension concentrates. The composition may therefore also contain other additives such as fillers, binders, stabilizers, e.g. vegetable oils or epoxidized vegetable oils (epoxidized palm oil 0;1, rapeseed or olive oil), defoamers, e.g. silicone oil, preservatives, stabilizers, wetting agents or emulsifiers, viscosity agents, binders, adhesives, as well as fertilizers and other active ingredients. Preparations with the following composition are preferably used: (% = weight percentage).

Emulsion concentrates active ingredient: 1 to 90%, preferably 5 to 20% wetting agent: 1 to 30%, preferably 10 to 20% liquid carrier: 5 to 94%, preferably 60 to 85%

Powder active ingredient: 0.1 to 10%, preferably 0.1 to 5% solid carrier: 99.9 to 90%, preferably 99.9 to 95%

Suspension concentrates

active ingredient: 5 to 75%, preferably 10 to 50% water: 94 to 24%, preferably 88 to 30% wetting agent: 1 to 40%, preferably 2 to 30% Wetting powders active ingredient: 0.5 to 90%, preferably 1 to 80% wetting agent: 0.5 to 20%, preferably 1 to 15% solid carrier: 5 to 95%, preferably 15 to 90% Granules active ingredient: 0.1 to 30%, preferably 0.1 to 15% solid carrier: 99.9 to 70%, preferably 99.9 to 85%

Various methods and technologies are used to use the compound of formula (I) in crystalline form, or compositions containing it, such as the following.

(i) Seed coating

a) Coating the seed with a wettable powder formulation of the compound of formula (I) in crystalline form, by stirring in a container until a uniform distribution is achieved on the surface of the seeds (dry coating). In such a procedure, approximately 1 to 500 g of active ingredient (4 g to 2 kg of wettable powder) is used per 100 kg of seed.

b) Coating the seed with an emulsified or suspension concentrate of the compound of formula (I) in crystalline form, in accordance with method a).

c) Seed coating by soaking the seeds for 1 to 72 h in a liquid containing from 100 to 1000 ppm of the compound of formula (I) formed by dissolving the crystalline form of the substance of formula (I) and preferably subsequently drying the seeds (immersion coating).

In general, the substance according to the invention is used in an amount of from 1 to 1000 g, preferably from 5 to 250 g, per 100 kg of seeds, but depending on the methodology, which also allows the addition of other active substances or micronutrients; the determined concentration limits can move up or down (repeated coating).

ii) Application into the seed furrow

The compound of formula (I) in crystalline form is applied to the open furrow sown with the seed in the form of a wettable powder or granules. The seed furrow is then closed.

Overview of images in drawings

Fig. 1 shows the crystal structure of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea (PTU) determined by single crystal X-ray structural analysis. It is clear from the figure that this is a urea derivative, where one nitrogen atom is substituted with 1,2,4-triazole and a phenyl group is attached to the second nitrogen atom. PTU crystallizes in the orthorhombic system with the space group specification Pca21.

Fig. 2 shows the XRPD pattern of the crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea (PTU).

Fig. 3 shows the XRPD pattern of the amorphous form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea (PTU) prepared according to the prior art.

Fig. 4A shows a comparison of the XRPD pattern of the crystalline and amorphous forms of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea (PTU) and Fig. 4B is an enlargement of this comparative XRPD pattern.

Fig. 5 shows the XRPD pattern of the crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea (PTU) with the corresponding calculated 2 theta diffraction angle values.

Fig. 6 shows the XRPD record of the crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea (PTU), recrystallized from 4 different solvents (ethanol, methanol, isopropanol and water) in comparison with the crude product isolated after synthesis (PTU) - a section of the record with characteristic diffractions. The record demonstrates that the substance PTU crystallizes from all the solvents mentioned in a single identical stable crystalline modification.

Fig. 7 shows the antisenescence activity of a solution prepared from the crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea (PTU) compared to a solution from the amorphous form of PTU in a senescence bioassay on excised wheat leaf segments cultured in the dark for 4 days. The activity is further compared to the DMSO control and to the most widely used classical cytokinin 6-benzylaminopurine (BAP).

Fig. 8 shows the results of testing the crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea (PTU) compared to the amorphous form of PTU in an amaranth bioassay. The activity (betacyanin dye concentration) is compared against DMSO control, cytokinin standard 6-benzylaminopurine (BAP) and adenine (cytokinin precursor).

Fig. 9 shows the results of testing the crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea (PTU) compared to the amorphous form of PTU in a callus bioassay using cytokinin-dependent tobacco callus. The cell proliferation activity of PTU is compared against DMSO control, cytokinin standard 6-benzylaminopurine (BAP) and adenine (a precursor of cytokinins).

Examples of embodiments of the invention

The following examples serve to illustrate the invention without limiting its scope. Unless otherwise stated, all percentages are based on the weight of the compounds.

Example 1

Preparation of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea (PTU) - process according to US 3,836,350 (prior art)

Phenyl isocyanate (1.19 g; 0.01 mol) was added to a 50 °C stirred warm solution of 4-amino-1,2,4-triazole (0.59 g; 0.007 mol) in THF (25 ml) and the resulting reaction mixture was refluxed for 1 hour. It was then allowed to cool spontaneously to room temperature with stirring. The precipitated product was filtered off, washed with THF (3 x 5 ml) and dried at 60 °C to constant weight.

Yield: 1.60-1.80 g (78-88%) amorphous white powder; HPLC-UV/VIS: 98%+; MH = 203.20; ESI-MS: [M+H]+ = 204.22 (100%), 205.30 (28%)

The crude PTU product thus obtained was recrystallized from various organic solvents. Lower alcohols (C1-C4) proved to be very advantageous. In the present case, the following solvents were chosen: methanol, ethanol and isopropanol, preferably ethanol.

The actual crystallization process is as follows.

The crude PTU product (500 mg) was dissolved in methanol (20 ml) or ethanol (20 ml) or isopropanol (110 ml) at boiling (reflux temperature of the selected solvent). After the formation of a homogeneous solution without visible solid particles, the solution was allowed to cool spontaneously with stirring to 20 °C for 12 hours. The precipitated crystalline product was filtered off, washed with the corresponding cooled (+5 °C) solvent (3 x 5 ml) and dried in a hot air oven at 60 °C to constant weight.

Thus, 191 mg (from methanol), 341 mg (from ethanol) and 440 mg (from isopropanol) of recrystallized PTU, suitable for biological applications, were obtained.

In all selected cases of crystallization, an identical crystalline modification was obtained, characterized in Example 3 by X-ray powder diffraction (XRPD). The XRPD record of the crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea (PTU) recrystallized from 4 different solvents is shown in Fig. 6. The crystalline form of PTU prepared in this way is precisely crystallographically defined, its preparation is easily reproducible both in laboratory and pilot scale and is validable. The PTU obtained by this procedure has an advantageous solubility in water, which is necessary for all biological application spheres, preferably for agricultural applications, and a surprising increase in biological activity compared to the amorphous form.

Example 2

Determination of the melting point of the prepared crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea (PTU)

The melting point of crystalline PTU was determined on a Buchi Melting point B-540 apparatus and compared with the melting point of the amorphous form of PTU prepared according to the prior art (Example 1). The melting point range was determined for the crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea to be 231-232°C and for the amorphous form to be 230-231°C.

Example 3

X-ray powder diffraction (XRPD) of the crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea (PTU)

Powder X-ray diffraction studies were performed on a Bruker D8 Advance ECO diffractometer with Cu K-alpha radiation and SSD160 detector. Approximately 5 mg of sample was gently pressed into an XRPD sample holder. The sample was then loaded into the instrument in transmission mode and analyzed using the tabulated experimental conditions, see below. The XRPD pattern of the crystalline form of PTU (Example 2) is shown in Fig. 2, the XRPD pattern of the amorphous form of PTU (Example 1) is shown in Fig. 3, and a comparison of both XRPD patterns of the crystalline and amorphous forms of PTU is shown in Fig. 4A and 4B (magnified detail of the XRPD pattern).

XRPD measurement conditions Scan axis gonio initial position [°2th.] 4.0000 end position [°2th.] 40.00 scan size [°2th.] 0.0100 scan time [s] 0.3 (48) sec scan type continuous offset [°2th.] 0.0000 type of divergence slit fixed size of the divergence slit [°] 0.3000 sample length [mm] 10.00 measurement temperature [°C] 25.00 anode material Cu K-alpha1 [a] 1.54060 K-alpha2 [a] 1.54443 K-beta [a] 1.39225 K-a2/k-a1 ratio 0.50000 generator setting 40mA, 25kV goniometric radius [mm] 250.00 dist. focus - divergent slits [mm] 110.00 Ni Kbeta filter yes incident beam monochromator no spinning no

The tables show the relative radiation intensity, d-spacing (inter-hole distance) and corresponding 2Θ angles for the crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea (PTU).

Table 1 Relative intensities of diffracted radiation, angles, d-spacing obtained by powder diffraction for the crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea (PTU), angle [°], d-spacing [Å], intensity, rel. intensity [%]

Angle [°] d-spacing [Ä] Intensity Relative intensity [%] 8,251 10.70725 70,500 96.70 12,206 7.24512 37,100 50.90 15.41 5.7453 4,810 6.60 16,484 5.37346 5,320 7.30 18,025 4.9173 1,310 1.80 18,804 4.71526 15,400 21.10 19,831 4.47339 829 1.10 20,394 4.35124 2,870 3.90 20,573 4.31368 4,910 6.70 21,037 4.21964 72,800 100.00 21,968 4.04286 4,720 6.50 22,475 3.95282 1,910 2.60 24,496 3.63109 4,660 6.40 24,842 3.5812 765 1.10 25,098 3.54525 2,410 3.30 26.41 3.37209 38,700 53.10 27,444 3.24728 1,110 1.50 28,086 3.17449 6,580 9.00 28,365 3.14394 1,110 1.50 29,964 2.97974 389 0.50 30,811 2.89968 7,330 10.10 31,291 2.85626 2,760 3.80 32,268 2.77202 1,290 1.80 32,615 2.74329 937 1.30 33,291 2.68914 853 1.20 34,276 2.61408 966 1.30 34,818 2.5746 153 0.20 35,839 2.5036 160 0.20 36.33 2.47088 138 0.20 37,074 2.42295 412 0.60 38,088 2.36077 1,210 1.70 38,295 2.34849 886 1.20 38,608 2.33016 304 0.40 39.22 2.29516 306 0.40

Example 4

1H and ¹³ C nuclear magnetic resonance (NMR) of the crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea (PTU)

1H NMR was measured on a JEOL 500 SS at 300 K and 500.13 MHz. Samples were prepared by dissolving the compounds in DMSO-d6. Tetramethylsilane (TMS) was used as an internal standard. Chemical shift calibration is based on the residual solvent peak in 1H DMSO-D6, which is 2.50 ppm. Each sample was prepared at a concentration of approximately 8 mg.

1H-NMR (500 MHz, DMSO-D6) δ 7.02-6.99 (m, 1H), 7.30-7.27 (m, 2H), 7.46 (d, J = 8.9 Hz, 2H), 8.66 (s, 2H), 9.55 (s, 1H), 9.83 (s, 1H);

¹³ C-NMR (126 MHz, DMSO-D6) δ 118.71, 122.57, 128.69, 138.77, 144.49, 153.76.

Example 5

Solubility of the crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea (PTU) in water

The solubility of the substance in water was determined according to the following protocol.

A supersaturated solution was prepared by dissolving the crystalline form of PTU in water. It was filtered to remove visible undissolved particles and a saturated solution of PTU in water was obtained. The saturated solution was diluted 1:10,000 and measured on an Alliance Waters 2690 HPLC instrument with a chromatographic column of 2.1 mm diameter and 150 mm length with a C18 Symmetre sorbent with a porosity of 5 pm. The sample was dissolved in a mobile phase (MeOH: HCOONH4 - 1:9). The sample was eluted in a methanol gradient (10 to 90%, over 35 min) at pH 4 and a flow rate of 0.3 ml/min. The absorption of the compounds was detected in the UV region of 210 to 400 nm. The peak area was determined 2729231 and compared with the peak areas at previously measured specific concentrations of the crystalline form of PTU in water. The obtained peak area was compared with the peak areas at certain specific concentrations of the substance in water, and thus the resulting concentration of the saturated PTU solution was determined.

This means that the PTU concentration was approximately determined to be 5.3 mM (1.077 g/l), which is an order of magnitude better solubility in water than that described for other free cytokinin bases, see table below.

Free agency database Concentration of saturated solution [mM] o-topolin 0.03 m-topolín 0.06 benzylaminopurine 0.37 6-(3-Methoxybenzylamino)purine 0.21 kinetin 0.14 PTU 5.3

Example 6

Stability studies of the crystalline form of PTU using powder X-ray diffraction

The stability of the sample was tested by storing 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea in crystalline form under air at 25 °C. No change in structure or color was observed after 30 days of storage. After these 30 days, the powder diffractogram was measured and no change in structure (change in the diffractogram) was recorded. The crystalline form is therefore stable.

Example 7

Disproportionation studies of the crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea A mg sample of crystalline 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea was dissolved in 1 ml of distilled water, left in solution for 48 hours, and then crystallized and the XRPD diffractogram was measured to detect any disproportionation changes. No disproportionation was observed in the diffractogram, the substance did not change its structure.

Example 8

Single crystal X-ray structural analysis of the crystalline form of PTU and description of the molecular and crystal structure of the crystalline form of PTU

Preparation of single crystals: Crystals for single crystal X-ray analysis were grown from solutions of PTU (ca. 20 mg) dissolved in methanol (2 cm ³ ). The solution was left to evaporate the solvent freely for 7 days, after which time translucent crystals were formed, from which suitable representatives for X-ray single crystal analysis were selected.

Single crystal X-ray diffraction: Elongated translucent crystals were selected for analysis. Diffraction data were measured using a Supernova four-ring microfocus diffractometer with Cu/Kα radiation collimated by thin-film mirrors, using a closed microfocus X-ray tube, equipped with an Atlas S2 CCD detector, operating at 95 K. The conditions and results are detailed in the following protocol. single crystal:

Bond accuracy: C-C = 0.0016 A; wavelength = 1.54184

Cell: a = 9.942(2) b = 10.373(2) c = 9.472(2)

Angles: alpha = 90 beta = 90 gamma = 90

Temperature: 95 K

Volume

Space group

Hall group

Compound formula

Molecular formula Molecular weight

Dx,g cm ^-3

From

Mu (mm-1)

F000

F000' h,k,lmax

Nref

Tmin, Tmax

Darkness

Calculated Measured 976.8(3) 976.9(3) Pc and 21 Pc and 21 P 2c -2ac P -2xac;-2y C9 H9 N5 O C9 H9 N5 O C9 H9 N5 O C9 H9 N5 O1 203.21 203.20 1.382 1.382 4 4 0.812 0.812 424.0 424.0 425.33 12,12,11 12,12,11 1964[1047] 1921 0.919,0.966 0.742,1.000 0.853

Correction method = # Measured T limits: Tmin = 0.742 Tmax = 1.000

Absolute correction = GAUSSIAN

Data completeness = 1.83/0.98 theta(max) = 73.370

R(reflection) = 0.0255(1894) wR2(reflection) = 0.0707(1921)

S = 1.497 Npar = 143

The crystal structure of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea (PTU) was determined by single crystal X-ray diffraction analysis using a Supernova four-ring microfocus diffractometer. Fig. 1 shows that it is a urea derivative in which one nitrogen atom is substituted with 1,2,4-triazole and the other nitrogen atom is bound to a phenyl group. PTU crystallizes in the orthorhombic system with the space group specification Pca21.

Example 9

Ability to scavenge free radicals using the ORAC (oxygen radical absorbance capacity) method

The ORAC assay utilizes the ability of AAPH [2,2'-azobis(2-amidinopropane)dihydrochloride] to form peroxyl radicals when heated in the presence of sufficient oxygen. These radicals quench and thereby reduce the fluorescence of the probe. The ability of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea (PTU) and reference cytokinins to scavenge free radicals in vitro was determined using the ORAC (Oxygen Radical Absorbance Capacity) method. 100 µl of fluorescein (500 mM) and 25 µl of an aqueous solution of PTU or reference substance were pipetted into each well of a 96-well microtiter plate preheated to 37 °C. The reaction itself was initiated by the addition of 25 µl of 250 mM AAPH [2,2-azobis(2-amidinopropane)dihydrochloride]. After 5 s of shaking, fluorescence (Ex. 485 nm, Em. 510 nm) was read every 3 min for 90 min using an Infinite 200 instrument (TECAN, Switzerland). The area under the curve was used to express the antioxidant capacity relative to trolox, which is used as a standard. Substances with an ORAC activity greater than 1 are more effective than trolox, which is a hydrophilic equivalent of vitamin E. Trolox, a water-soluble analogue of vitamin E, is known for its antioxidant activity, which has also been shown to be responsible for its protective activity in human cells. The ORAC assay, which is generally used to determine antioxidant activity, showed that several cytokinins, such as kinetin and 6-benzylaminopurine (BAP), were associated with some antioxidant activity expressed in trolox equivalents (TE). It has been shown that PTU in the form of a solution obtained by dissolving the crystalline form of PTU is a much more effective substance.

Table 3 Oxygen Radical Absorbance Capacity (ORAC) data expressed in trolox equivalents (TE) on an equimolar basis

Average (TE) SD (n = 3) kinetin (K) 1,201 0.54 6-Benzylaminopurine (BAP) 0.402 0.12 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea (PTU) 2,143 1.07

Example 10

Antisenescence activity of a new crystalline form of PTU in a senescence assay on wheat leaf segments cultivated in the dark (senescence delay - delay of leaf aging)

Seeds of winter wheat Triticum aestivum cv. Hereward were washed under running water for 24 h, and then sown in vermiculite saturated with Knop's nutrient solution. The seed containers were placed in an air-conditioned growth chamber with a 16-h photoperiod (light intensity 50 mmol.m ^-2 .s ^-1 ) and a temperature of 15 °C. After 7 days, the seedlings had developed the first flag leaf and the second leaf was starting to grow. Apical sections of approximately 35 mm in length were taken from the first leaves of 5 plants each, which were shortened so that their weight was exactly 100 mg. The basal ends of these 5 leaf segments were placed in the wells of polystyrene microtiter plates containing 150 ml of a solution of the test substance (PTU or reference cytokinin). The plates were placed in a plastic box lined with filter paper, which was saturated with water to maximize air humidity. After 96 hours of incubation in the dark at 25 °C, leaf sections were removed and chlorophyll extracted in 5 ml of 80% ethanol by heating at 80 °C for 10 min. The sample volume was then made up to 5 ml by adding 80% ethanol. The absorbance of the extracts was measured at 665 nm. As controls, chlorophyll extracts from leaves and leaf tips incubated in deionized water were also measured. The calculated values are the average of 5 replicates and the entire experiment was repeated at least 2 times. In each experiment, the activity of PTU (the solution formed by dissolving the crystalline form vs. the amorphous form) was tested and compared with the activity of the cytokinin 6-benzylaminopurine (BAP), which is known to have very high cytokinin activity. The tested cytokinins were dissolved in dimethyl sulfoxide (DMSO) and the stock solution was made up to 10 ^-3 M with water. This stock solution was further diluted with the test medium in the concentration range of 10 -8 to 10 -4 M. The final concentration of DMSO in the medium did not exceed 0.2% and at this concentration did not affect the biological activity of the test. IC50 values were determined for the standard, 6-benzylaminopurine (BAP, the cytokinin most commonly used in plant biotechnology and agriculture) and for the crystalline and amorphous forms of PTU (Table 4).

The tested compound PTU of formula (I) has a positive effect on delaying senescence in wheat leaf segments in the dark. The solution formed by dissolving the crystalline form of PTU significantly surpassed the anti-senescence activity of the solution formed by dissolving the amorphous form of PTU, both forms of PTU also surpassed BAP (a cytokinin standardly used in plant biotechnology and agriculture). The quantitative effect on slowing down senescence after 4 days is also remarkable, because after application of the solution from the crystalline form of PTU, only 10.4% of chlorophyll was degraded at 10 ^-4 M concentration, while in the case of the solution from the amorphous form of PTU, 18.8% degradation and in the case of BAP even a 44.5% reduction in chlorophyll content in the leaves (see Fig. 7).

Table 4 Effect on delaying senescence in decapitated leaf segments of Triticum aestivum cv. Hereward in the dark

Substance Senescence bioassay (IC50, pM)/s. d. 6-Benzylaminopurine (BAP) 10.5 ± 2.2 crystalline 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea 0.8 ± 0.8 amorphous 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea 5.41 ± 0.13

Example 11

Antisenescence activity of crystalline form of PTU tested in senescence assay on wheat leaf segments under light exposure

The ability to slow down chlorophyll degradation in wheat leaves was also tested under light conditions. The experiment was performed as described in Example 10, with the following modifications - incubation of leaves with compounds was performed under a light period of 8/16 (dark/light - pmol.m ^-2 .s ^-1 ) at 22 °C for 7 days. As shown in Table 5, the crystalline PTU solution had stronger positive effects on delaying senescence in wheat leaf segments under light conditions compared to the amorphous PTU solution and compared to benzylaminopurine (BAP).

Table 5 Effect on delaying senescence in decapitated leaf segments of Triticum aestivum cv. Hereward in light

No. Substance Senescence Bioassay (IC50, pM)/s.d. 1 6-Benzylaminopurine (BAP) 12.5 (±3.3) 2 crystalline 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea (PTU) 0.42 (±0.22) 3 amorphous 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea (PTU) 2.16 (±0.41)

Example 12

Protection against membrane lipid peroxidation in wheat

A typical manifestation accompanying senescence is an increase in the level of reactive oxygen species and subsequent peroxidation of membrane lipids. Therefore, the levels of malonyldialdehyde (MDA), an oxidative marker of membrane lipid damage, were determined in detached wheat leaves exposed to PTU or BAP for four days in the dark, as described in Example 10. MDA levels were determined using thiobarbituric acid (TBA). 100 mg of fresh plant material was homogenized using an oscillating mill (MM301, Retsch, Germany), with 1 ml of 80% methanol. The crude extract was centrifuged at 10,000 xg for 5 min, a 100 μl aliquot of the supernatant was shaken with 100 μl of 0.5% (w/v) TBA containing 0.1% (w/v) trichloroacetic acid, and the resulting solution was incubated for 30 min at 95°C. The samples were quickly cooled on ice and centrifuged for 5 min at 1,000 xg. The absorbance of the supernatant was measured at 532 nm with correction at 600 nm, and the amount of MDA-TBA complex formed was calculated from the extinction coefficient of 155 mM- ¹ cm- ¹ . A solution prepared from the crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea significantly reduces membrane lipid peroxidation compared to the untreated control and BAP-treated samples (Table 6 - values express the content of MDA, which is a product of membrane lipid decomposition).

Table 6 Effect on membrane lipid peroxidation during senescence of cut leaves of Triticum aestivum cv. Hereward in the dark

No. Test substance MDA (nmol/g FW) 0 untreated control 21.7 (±2.2) 1 6-Benzylaminopurine (BAP) 19.2 (±1.4) 2 crystalline 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea 13.8 (±1.1)

Example 13

Testing a new crystalline form of PTU in an amaranth bioassay

The amaranth bioassay was also used to study cytokinin activity in the following modification. Seeds of Amaranthus caudatus var. Atropurpurea were surface sterilized with 10% N-chlorobenzenesulfonamide (w/v) for 10 min, and then washed 5 times with deionized water. The seeds were placed in 15 cm Petri dishes with filter paper saturated with distilled water. After 72 h of cultivation at 25 °C in the dark, the roots were removed from the seedlings. These explants containing 2 cotyledons and a hypocotyl were placed in 5 cm Petri dishes on 2 layers of filter paper saturated with 1 ml of incubation medium containing 10 pmol Na2HPO4-KH2PO4, pH 6.8, 5 pmol tyrosine and the test substance. 20 explants were placed per dish. All manipulations were performed under green light in a dark chamber. After 48 hours of incubation at 25 °C in the dark, betacyanin was extracted by freezing the explants in 3.33 pM acetic acid. The betacyanin concentration was determined by comparing the absorbances at 537 and 620 nm and calculated using the formula □A = A537nm - A620nm. The values were plotted in □A concentration dependence graphs. Five replicates were prepared for each tested concentration and the test was repeated at least 2 times. 6-benzylaminopurine (BAP), which is known to have very high cytokinin activity, was used as a control in the experiment. The test substances (forms) were dissolved in dimethyl sulfoxide (DMSO) and the stock solution was made up to 10 ^-3 M with water. This stock solution was further diluted with the test medium in the concentration range of 10 ^-8 to 10 ^-4 M. The final concentration of DMSO in the medium did not exceed 0.2% and at this concentration did not affect the biological activity of the test. The 10 ^-4 M concentration of the control substance 6-benzylaminopurine (BAP) was postulated as 100% of the biological activity. The solution prepared from crystalline PTU showed higher activity in the amaranth bioassay than the control cytokinin BAP, namely 107.03% of the BAP activity, while the solution prepared from amorphous PTU showed 87.97% (Fig. 8).

Example 14

Testing the effect on cell division of plant cells

The stimulatory effect of PTU was tested in a callus bioassay using cytokinin-dependent tobacco callus. This cytokinin-dependent tobacco callus of Nicotiana tabacum L. cv. Wisconsins 38 was maintained at 25 °C on modified Murashige-Skoog (MS) medium containing per liter: 4 pmol nicotinic acid, 2.4 pmol pyridoxine hydrochloride, 1.2 pmol thiamine, 26.6 pmol glycine, 1.37 pmol glutamine, 1.8 pmol myo-inositol, 30 g sucrose, 8 g agar, 5.37 pmol α-naphthylacetic acid and 0.5 pmol 6-benzylaminopurine. Subcultures were performed every 3 weeks. Fourteen days before the start of the bioassay, the callus was transferred to medium without 6-benzylaminopurine (BAP). The growth-stimulating activity was determined based on the increase in fresh callus mass after 4 weeks of cultivation. Five replicates were prepared for each tested concentration and the test was repeated at least 2 times. In each experiment, 6-benzylaminopurine, which is known for its high cytokinin activity, was used as a control substance. The tested forms of PTU were dissolved in dimethyl sulfoxide (DMSO) and the stock solution was supplemented with water to 10 ^-3 M. This stock solution was further diluted with the test medium in the concentration range of 10 ^-8 to 10 ^-4 M. The final concentration of DMSO in the medium did not exceed 0.2% and at this concentration did not affect the biological activity of the test. From the obtained data, the maximum effective concentration of the tested form and its relative efficiency at this concentration were again calculated (Table 7). The 10 ^-5 M concentration of the control substance 6-benzylaminopurine was postulated as 100% biological activity.

The crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea (PTU) of formula (I) showed significant cytokinin activity in the callus bioassay compared to classical cytokinin. PTU exhibits advantageous cell-stimulating properties that can be significantly utilized in tissue cultures and plant biotechnology, since PTU does not exhibit the characteristic inhibitory activity of BAP in the callus bioassay at higher concentrations (10 ^-4 M) and thus has practical application in a much wider range of concentrations.

Table 7 Effect of new PTUs on the growth of cytokinin-dependent tobacco callus Nicotiana tabacum L. Cv. Wisconsins 38

No. Test compound Maximum effective concentration (mol.l ^-1 ) Activity (%) [10' ⁵ mol.r ¹ BAP = 100%] 1 6-Benzylaminopurine (BAP) 10' ⁵ 100 (±11.4) 2 adenine 10' ⁵ 10.4(±0.3) 3 crystal 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea 10' ⁴ 98.3 (±10.2) 4 amorphous 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea 10 4 93.6 (±8.4)

Example 15

Effect on root growth of Arabidopsis thaliana

Arabidopsis thaliana (ecotype Columbia) seeds were sterilized with 70% ethanol containing 0.1% Triton X-100, and then washed with 70% ethanol containing 0.01% Triton X-100. The seeds were then transferred to square vertical Petri dishes (11 seeds per dish) on solid medium containing MS (2.15 g/l), MES (0.5 g/l), sucrose (1 g/l), agar (11 g/l), buffered to pH 5.8, and various concentrations of the test substances. The substances were dissolved in DMSO, and 0.05% DMSO was used as a control solution, respectively. After three days of incubation in the dark at 4 °C, the seeds germinated and the plants were further grown in growth chambers (fluorescent light with an intensity of 150 pE m ^-2 s ^-1 , humidity 55%, 16 h day/8 h night, 22 °C). After 7 and 14 days, the length of the primary root was evaluated using Scion Image software (Scion Corporation, Frederick, MD, USA). The number of lateral roots was counted under a binocular magnifying glass. Application of PTU to MS medium had no negative effect on root elongation in the range of 1 pM to 100 nM. In contrast, classical cytokinins such as transzeatin, BAP (control), kinetin, meta- and ortho-topolin and isopentenyladenine, as known from the prior art, significantly inhibited root elongation and branching even at nanomolar concentrations. In addition, PTU slightly increased the number of established lateral roots. Arabidopsis seedlings grown on MS medium containing PTU did not show noticeable changes (reduction) in the number and size of established leaves, in contrast to cytokinins (Table 8).

Tab. 8 Effect of PTU compared to classical cytokinins on Arabidopsis root growth - number of lateral roots and length of main root

Test compound Concentration with the highest activity (mol.l ^-1 ) Length of the main root Number of lateral roots Activity (%) [10'7 mol.1' ¹ BAP = 100%] trans-zeatin 10'7 88.5 (±16) 92.1 (±12) meta-topolin 10'7 94.8 (±14) 89.8 (±10) ortho-topolin 10’7 42.7 (±17) 59.4 (±12) kinetin 10’7 68.6 (±12) 73.9 (±10) isopentenyladenine 10’7 45.7 (±16) 62.8 (±12) 6-(3-Methoxybenzylamino)purine 10’7 69.3 (±19) 76.5 (±14) PTU 10’7 218.4 (±28) 237.8 (±21)

Example 16

In vitro cytotoxic activity (calcein AM metabolism)

Since toxic compounds negatively affect the metabolic processes of cells, many standard cytotoxicity assays are based on measuring the rate of metabolism of various artificial substrates. The resulting viability is then quantified, for example, by spectrometry. The assays can be easily modified for use in 96-well plates. A microtiter assay based on the quantification of Calcein AM metabolism was used to evaluate the cytotoxicity of PTU according to the invention. The assay is widely used in drug testing programs and in viability testing. In living cells, Calcein AM is enzymatically hydrolyzed and the accumulation of the resulting calcein is indicated by a green color.

The following cell lines - RPMI 8226 (multiple myeloma), CEM (T-lymphoblastic leukemia), K562 (chronic myeloid leukemia), HL-60 (promyelocytic leukemia), MCF-7 (breast adenocarcinoma), (malignant melanoma), HOS (human osteosarcoma), and BJ (human foreskin fibroblasts) - were obtained from the American Type Culture Collection (Manassas, VA, USA). These cells were maintained in standard DMEM or RPMI medium (Sigma, MO, USA) supplemented with 2 mM heat-inactivated fetal bovine serum (10%).

L-glutamine and streptomycin (1%) under standard cell culture conditions (37°C, 5% CO2 in a humidified environment) and subcultured two or three times a week using a standard trypsinization procedure.

Approximately 10,000 cells in 80 µl of medium were seeded into a 96-well microtiter plate. After 12 hours of incubation, test compounds were added in 20 µl aliquots. Control cultures were treated with DMSO alone. The final concentration of DMSO in the medium did not exceed 0.5%. Serial, three-fold dilutions (six in total, highest concentration in incubations 166 pM) of each compound were tested. After 72 hours of incubation, Calcein AM solution (Molecular Probes) was added to a final concentration of pg 1/ml and the cells were incubated for an additional hour. Free calcein fluorescence was then quantified using a Fluoroscan Ascent fluorometer (Microsystems) and the percentage of surviving cells in each well was calculated by dividing the OD obtained from each cell exposed to the cells by the mean OD obtained from the control wells x 100%. Finally, IC50 values (concentration causing 50% decrease in cellular esterase) were calculated for each compound, generated from dose-response curves (Kryštof et al., 2005, Bioorg. Med. Chem. Lett. 12, 32833286). The IC50 values reported here are the averages obtained from at least three independent measurements, where individual replicate values fell within 20% of the mean. Growth inhibition was calculated using the following equation:

IC50 = (mean FD cytokinin-exposed well — mean FD blank)/(mean FD control well - mean FD blank) x 100%

The IC50 value, the drug concentration causing a 50% reduction in Calcein AM conversion, was calculated from the obtained dose-response curves.

The cytotoxicity of the compounds was tested on a panel of cell lines of different histogenetic and species origin.

Zero cytotoxicity is a basic requirement for the use of these substances in biotechnological and agricultural applications. To evaluate the cytotoxic activity, classical cytokinins and PTU were tested on panels containing cell lines of different histogenetic and species origin (Table 9). It was shown that for all tested tumor lines the effect of PTU was zero and also for non-malignant cell lines, i.e. NIH3T3 fibroblasts and normal human lymphocytes; all were resistant to this effect of PTU. In contrast, some “classical cytokinins” represented by 6-substituted purines (known from the prior art) are cytotoxic. The crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea (PTU) of formula (1) shows zero toxicity to normal and tumor cells at concentrations higher than 166.7 pM and is therefore much more suitable for agricultural and biotechnological applications than "classical cytokinins" (6-substituted purine derivatives).

Table 9 Cytotoxic activity of PTU compared to classical cytokinins expressed as IC50 values in the 3-day Calcein-AM assay; values presented are the averages of at least 3 independent measurements, where individual replicates fall within 20% of the mean.

Cell line used/Gl50 (pmol/l) Compound HOC K-562 MCF7 B16-F0 NIH-3T3 G-361 CEM HL60 kinetin > 166.7 164.1 > 166.7 > 166.7 155.1 isopentenyladenine > 166.7 146.9 > 166,.7 > 166.7 92.2 > 166.7 benzyladenine > 166.7 138.9 166.1 > 166.7 > 166.7 > 166.7 trans-zeatin > 166.7 > 166.7 > 166.7 > 166.7 meta-topolin > 166.7 128.4 > 166.7 90.6 > 166.7 > 166.7 90.1 79.2 ortho-topolin > 166.7 >166.7 > 166.7 150 > 166.7 103.4 69.2 78 adenine >166.7 > 166.7 > 166.7 > 166.7 > 166.7 PTU > 166.7 > 166.7 > 166.7 > 166.7

Example 17

Ames test

The crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea of formula (I) was tested for mutagenicity by bacterial reverse mutation assay. The assay was based on the EU B.13/14 Mutagenicity Reverse mutation test using bacteria, which is an analogue of the OECD Test Guideline No. 471 methodology. Four indicator strains of Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and one strain of Escherichia coli WP2 uvrA were used. The crystalline form of PTU was dissolved in dimethyl sulfoxide (DMSO) and was tested at doses of 10 to 1000 pg per plate, which was applied to the plates in a volume of 0.1 ml. The experiments were performed with metabolic activation with rat liver supernatant and a mixture of cofactors as well as without metabolic activation. The working procedure was carried out according to the documents Method B.13/14, Mutagenicity - Reverse mutation test using bacteria, Council Regulation (EC) No.440/2008. Published in O. J. L 142, 2008 and according to the methodology OECD Test Guideline 471, Bacterial Reverse Mutation Test. Adopted July 21, 1997. When tested in the above arrangement, the test substance was found to be non-mutagenic for all test strains used with metabolic activation as well as without metabolic activation.

Example 18

Foliar application of PTU extends the lifespan of Arabidopsis thaliana under drought stress conditions and reduces yield losses.

Young Arabidopsis thaliana plants, grown in small pots in clay, were sprayed with 0.01 pM aqueous solution of PTU at the 4 true leaf stage (within 14-20 days after sowing). Control plants were sprayed with aqueous solutions of DMSO (c = 0.01% (v/v)) and silwet (c = 0.001% (v/v)). Control plants were further divided into watered and plants with reduced watering (drought stressed). Plants treated with PTU also had reduced watering (drought stressed). All plants were grown until their natural death. Control watered plants lived on average for 85 days before they spontaneously withered. Their seed yield was on average 141 (±22) mg. Control plants with reduced watering lived on average only 61 days and the average yield was 95 (±14) mg. Plants treated with PTU and with reduced watering lived on average 75 days and their average yield was 117 (±16) mg. These data clearly show (Table 10) that PTU significantly increases the yield and viability of Arabidopsis plants that have been exposed to environmental stress.

Table 10 Foliar application of PTU reduces seed yield losses in Arabidopsis thaliana

Yield (mg) Yield(%) Benefit watered control 141±22 100% reduced fill control 95 ±14 67% plants with reduced watering + 0.01 pM PTU 117 ±16 83% + 16%

Example 19

Application of PTU increases barley seed yield in field conditions

The field experiment investigated the effect of PTU on barley seed yield and phenotype. All field experiments were conducted according to the criteria valid for good agricultural practice, i.e. six plots of 10 ^m2 were used for one variant. The crop was not further treated with any other growth regulator (only PTU) or fungicide. The crystalline form of PTU was applied to the seeds by dry coating at a concentration of 10 pM before sowing. The barley seeds treated in this way were grown in the field in 2016 and the yield of these plants was compared with the untreated control (Table 10). The treated plants had a seed yield of 105.3% compared to the control (100%). Phenotypic analysis further showed that the higher yield could be due to the higher number of tillers, which the treated plants had by 14% more than the control plants.

The effect of foliar application of PTU on barley yield was also tested in field conditions. PTU (solution prepared from crystalline form) was applied to barley leaves at a concentration of 5 pM (in a volume of 200 l per hectare) in two different periods - BBCH 21 - 23 and BBCH 51. Both applications led to an increase in seed yield, to 106.3% and 106.8%, respectively, compared to the control (100%), (Table 11).

Table 11 Application of PTU increases barley seed yield in field conditions.

Growth phase Yield(%) Benefit Control 100% 10 pM PTU (seed application) 105.3% + 5.3% 5 pM PTU (foliar application) BBCH 21 - 23 106.3% + 6.3% 5 pM PTU (foliar application) BBCH 51 106.8% + 6.8%

Example 20

Application of PTU increases the yield of winter rapeseed under field conditions.

In this experiment, the effect of foliar application of PTU (concentration 1 pM in a volume of 300 l per hectare, solution prepared from crystalline form of PTU) on the yield of winter rapeseed under field conditions was monitored. Field experiments were again carried out according to the criteria valid for good agricultural practice. PTU was applied to plants in two different periods - BBCH 30 and BBCH 33 - 35. In the first case, the rapeseed yield increased to 106.2% compared to the control and in the second case, the seed yield increased to 108.8% compared to the control (100 %), (Table 12). It was further found that the seeds of the treated plants were slightly heavier, the weight of a thousand seeds in the control was 5.38 g. The weight of a thousand seeds in the treated plants was 5.6 g (BBCH 30) and 5.8 g (BBCH 33 - 35). This phenomenon also contributed to the increase in overall seed yield.

Table 12 Application of PTU increases winter rapeseed yield in field conditions.

Growth phase Yield(%) Benefit Control 100% 1 pM PTU (foliar application) BBCH 30 106.2% + 6.2% 1 pM PTU (foliar application) BBCH 33 - 35 108.8% + 8.8%

Example 21

Application of PTU increases wheat seed yield in field conditions.

The effect of PTU application on wheat yield was further monitored under field conditions. The substance was again applied either to wheat seeds before sowing (= seed dressing with PTU at a concentration of 50 pM) or to the leaves (= spraying with PTU at a concentration of 5 pM) at two different times - BBCH 25 and BBCH 51. All field experiments were carried out according to the criteria valid for good agricultural practice. In the case of seed dressing, the yield was increased to 104.0% compared to the control (100%). In the case of foliar application, the yield was increased to 105.9 (BBCH 21) and 105.0% (BBCH 51) compared to the control (100%), (Table 13). Phenotypic analysis revealed that the treated plants had approximately 7% more tillers than the control plants.

Table 13 Application of PTU increases wheat seed yield under field conditions.

Growth phase Yield(%) Benefit Control 100 50 pM PTU (seed application) 104 + 4.0% 5 pM PTU (foliar application) BBCH 25 105.9 + 5.9% 5 pM PTU (foliar application) BBCH 51 105 + 5.0%

Example 22

Preparations

The active ingredient in the preparations is a crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea (PTU) of formula (I).

A1. Wetting powders a) b) c) d) active ingredient 5% 25% 50% 80% sodium lignosulfonate 4% - 3% - sodium lauryl sulfate 2% 3% - 4% sodium diisobutyl naphthalene sulfonate - 6% 5% 6% octylphenol polyglycol ether (7 - 8 mol ethylene oxide) - 1% 2% - highly dispersed silicic acid 1% 3% 5% 10% kaolin 87% 61% 37% -

The active ingredient is thoroughly mixed with the excipients and the mixture is thoroughly ground in a suitable mill. A suspension of any concentration can be obtained by mixing the resulting powder with water.

A2. Coated granules a) b) c) active ingredient 0.1% 5% 15% highly dispersed silicic acid 0.9% 2% 2% inorganic carrier 99.0% 93% 83%

(0.1 - 1 mm) e.g. CaCO3 or SiO2

The active ingredient is dissolved in methylene chloride and sprayed onto the carrier. The solvent is evaporated in vacuo.

A3. Coated granules a) b) c) active ingredient 0.1% 5% 15% polyethylene glycol MW 200 1.0% 2% 3% highly dispersed silicic acid 0.9% 1% 2% inorganic carrier 98.0% 92% 80%

(AE 0.1 - 1 mm) e.g. CaCO3 or SiO2

The finely ground active ingredient is uniformly applied to a carrier moistened with polyethylene glycol in a mixer. This results in dust-free granules.

A4. Extruded granules a) b) c) d) active ingredient 0.1% 3% 5% 15% sodium lignosulfonate 1.5% 2% 3% 4% carboxymethylcellulose 1.4% 2% 2% 2% kaolin 97.0% 93% 90% 79%

The active ingredient is mixed and ground with excipients and the ingredient is moistened with water. The mixture is extruded and dried in a stream of air.

A5. Powders a) b) c) active ingredient 0.1% 1% 5% talc 39.9% 49% 35% kaolin 60.0% 50% 60%

Powder forms for direct use are obtained by grinding the active ingredient with the carrier in a suitable mill.

A6. Suspension concentrate a) b) c) d) active ingredient 3% 10% 25% 50% ethylene glycol 5% 5% 5% 5% 10 nonylphenol polyglycol ether — 1% 2% — (15 mol ethylene oxide) sodium lignosulfonate 3% 3% 4% 5% carboxymethylcellulose 1% 1% 1% 1% 37% aqueous formaldehyde solution 0.2% 0.2% 0.2% 0.2% silicone oil emulsion 0.8% 0.8% 0.8% 0.8% 15 water 86% 78% 64% 38%

The finely ground active ingredient is mixed with excipients. The resulting suspension concentrate allows the preparation of a suspension with the desired concentration by dilution with water.

Claims (9)

PATENT CLAIMS 1. Crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea (PTU) of formula (I) (D with characteristic reflections in the X-ray powder diffraction spectrum measured with CuKα radiation: 8.3; 12.2; 18.8; 21.0; 26.4; 30.8 ±0.2° 2-theta. 2. A crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea according to claim 1 with further characteristic reflections in the X-ray powder diffraction spectrum measured with CuKα radiation: 15.4; 16.5; 20.6; 22.0; 24.5; 28.1 ±0.2° 2-theta. 3. A crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea according to claims 1 or 2 with a melting point of 230 to 231°C. 4. Use of the crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea according to claims 1 to 3 as an antioxidant, anti-senescence, proliferation-stimulating and/or pro-differentiation substance in plant cells, tissues, organs and whole plants. 5. Use of the crystalline form of l-phenyl-3-(4H-l,2,4-triazol-4-yl)urea according to any one of claims 1 to 3 for increasing plant yields, increasing the quality of plant products, increasing plant resistance to stress, accelerating seed setting, increasing the size of seeds and fruits of plants and/or shortening seed germination time, in agriculture, horticulture, forestry and plant biotechnology. 6. Use of the crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea according to claims 1 to 3 for delaying chlorophyll degradation and delaying senescence of plants, plant cells, tissues and/or organs. 7. Use of the crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea according to claims 1 to 3 as an inhibitor of stress and stress-induced senescence in crop production. 8. Antisenescence, antioxidant and/or antistress preparations for whole plants, plant organs and/or plant cells, characterized in that they contain the crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea of formula (I) and at least one excipient. 9. A method for inhibiting senescence and/or stress in plants, in plant organs and/or in plant cells, characterized in that it comprises applying the crystalline form of 1-phenyl-3-(4H-1,2,4-triazol-4-yl)urea according to any one of claims 1 to 3 to the plant or to the plant cells, or to the plant organs.