Phytoactive Aryl Carbamates and Ureas as Cytokinin-like Analogs of EDU

Abstract

Ureas, carbamates and oxamates are rather common structural motifs. They are present in both natural and synthetic compounds that exhibit a wide spectrum of biological activity. These derivatives of carbonic and oxalic acids are regularly employed as the basic structural elements in hybrid molecule synthesis, as well as in organic synthesis in general. A series of unsymmetrical hybrid compounds (E1–E4) has been synthesized, with core imidazolidin-2-one and aryl moieties as urea and carbamate derivatives. Plant growth regulatory activity of these compounds was studied with respect to their influence on germination, growth and development of wheat (Triticum aestivum L.) seeds in laboratory and field tests. Their effect on drought resistance concentrations as low as 4 × 10⁻⁷ M was established. Compounds E1 and E4 have shown higher growth-regulating activity than standard thidiazuron and CCC.

1. Introduction

Over the last decades of climate change, the content of tropospheric ozone (O₃) in the middle latitudes of the northern hemisphere has been increasing at a yearly rate of 0.5–2 per cent [1]. Ground-level ozone buildup reduces crop yields due to increased oxidative stress [2]. For forty years, N-[2-(2-oxo-l-imidazolidinyl)ethyl]-N′-phenyiurea (EDU) (Figure 1) has been successfully used to protect plants against O₃.

EDU mitigates the harmful effects of oxidative stress, improves photosynthesis, chlorophyll fluorescence, growth, and increases biomass in treated plants. The mechanisms of action that can explain its protective properties remain unclear [3], in addition its action varies between and within species. The use of EDU in agriculture increases the yield of corn [1], rice [2,4,5], peanut [6], as well as biomass and total dissolved sugar, protein and proline in peas and other legumes [7,8,9]. The impact of O₃ on wheat is manifested by stress and associated yield reduction. EDU improves growth, nutritional quality and yield of wheat [10,11,12,13]. In addition, EDU protects seedlings of Japanese larch [14,15] and willow [3], which are grown as bioenergy crops throughout the world.

In most experiments [16], EDU is used as a foliar spray or as a soil drench [3]. However, when introduced to soil, EDU can accumulate and exhibit toxicity to soil microorganisms. Surface treatment is a good alternative, but its effectiveness depends on atmospheric precipitations affecting the absorption of chemicals. A screening trial confirmed that a foliar spray of 500 ppm (5 × 10⁻⁴ M) EDU provided optimal protections for flowers, herbaceous plants and some types of tree seedlings. This EDU content provides optimal protection for flowers, herbaceous plants and some types of tree seedlings. The EDU concentration in soil applications can be in the range of 200–400 ppm. Such products have the greatest positive effect on crops grown in the field: they reduce damage to the plant foliage and slow down the aging process. The slowdown in aging can be explained the fact that EDU exhibits cytokinine-like activity at a concentration of 2 × 10⁻³ M [17]. This is demonstrated by comparing EDU with kinetine (Kin) at a concentration of 2.3 × 10⁻⁵. In terms of its biological properties, EDU is close to natural adenine-type cytokinins such as Kin [18].

Considering the cytokinin-like compounds, the derivatives of aryl- and heteroarylureas belong to the most promising group. Previously, we have synthesized EDU analogs and studied their antiproliferative and cytoprotective activity [19]. Chemical modification of cytokinin analogs can lead to enhanced proliferative effects coupled with an inversion of their activity.

The aim of our research was to investigate the phytoactivity of new substituted imidazolidinones (E1–E3) in comparison with EDU (E4) in laboratory and field tests, namely, to study their effect on the growth and development of wheat (Triticum aestivum L.) seeds.

2. Materials and Methods

2.1. Chemicals

Arylureas and arylcarbamates N-[2-(2-oxoimidazolidin-1-yl)ethyl]-N′- (3-chlorophenyl)urea (E1), 2-(2-oxoimidazolidin-1-yl)ethyl-N-(3-chlorophenyl) carbamate (E2), 2-(2-oxoimidazolidin-1-yl) ethyl-N-phenyl carbamate (E3), N-[2-(2-oxo-l-imidazolidinyl)ethyl]-N′-phenyiurea (E4) were synthesized by the reaction of an imidazolidinone substituted amine or alcohol with the corresponding aryl isocyanates in the presence of triethylamine in anhydrous toluene (for aryl ureas) or acetonitrile (for aryl carbamates), as described in [20]. The structures of the hybrid compounds (E1–E4) were confirmed by ¹H and ¹³C NMR spectroscopy, mass spectrometry and elemental analysis. The purity of the products was between 95 and 99% as established by HPLC-MS.

Thidiazuron or 1-phenyl-3-(1,2,3-thiadiazol-5-yl)urea (TDZ) (CAS 51707-55-2) and Chlorocholinechloride or Chlormequat chloride (CCC) (CAS 999-81-5) were purchased from Sigma-Aldrich. Wheat seeds (Triticum aestivum L.) of the “AGATA” ^® (No. 8853479 [21]) variety, crop 2020, provided by “Zhito” LLC (Oktyabrsky district, Ryazan region, Russia 54.609836° N, 39.80188° E) were used. Wheat seeds (Triticum aestivum L.) were sterilized with 0.2% sodium hydrochloride solution for 10 min before starting tests, washed three times with pure distilled water, dried at a temperature of 30±1 °C for 48 h. The dried seeds were stored at a temperature of 5 ± 1 °C

2.2. Instruments

¹H and ¹³C NMR spectra were obtained using a Bruker DRX-400 NMR spectrometer at 400.13 MHz. DMSO-d6 was used as a solvent and TMS as an internal standard. Chemical shift values were measured with an accuracy of 0.01 ppm; coupling constants are given in Hertz. HPLC-MS was recorded on an xSeries II ICP-MS inductively coupled plasma mass spectrometer (Thermo Scientific Inc., Waltham, MA, USA). Melting points were determined using a Stuart SMP20 instrument (Cole-Palmer, Stone, Staffordshire, UK). Reaction mixture composition qualitative analysis was performed by thin layer chromatography on silica gel (0.015–0.040 mm) aluminum backed TLC plates with an F254 fluorescent indicator (20 × 20 cm) (Merck Millipore, Darmstadt, Germany). “Kieselgel 60” (0.015–0.040 mm, Merck Millipore, Darmstadt, Germany) silica gel was used for chromatographic separation on preparative scale.

Four independent experiment series with identical cameras equipped with phyto-LED UFO lighting-79–01-00 were carried out. Lighting wavelengths of Red 615/Blu 457 nm with an intensity of at least 250 lux were used. The illumination schedule for all the samples was 12/12 h. Experimental conditions were as follows: air relative humidity 50% ± 2%; air temperature 20 °C ± 2 °C; experiment duration 7 days. Dry sterilized seeds (50 pcs) were placed over filter paper in rectangular 75 × 85 mm Petri dishes and sprayed with the aqueous solutions of the investigated compounds. The solution volumes for wheat grain treatment were 0.335 ± 0.003 mL. Vertical spraying was carried out in an isolated box with disposable screen. After spraying, the screen was removed. The surface of the box was disinfected and created with paper napkins. After spraying, the seeds were covered with filter paper and 10 mL of distilled water was poured. Then the Petri dishes with a lid were moved to the growth chamber. The first 24 h of the experiment were conducted in the dark. The seeds were aired every day. Petri dishes were opened for 25 min, and 5–10 mL of distilled water was added so that the seeds did not dry out. On the third day of the experiment, the lids of the Petri dishes were removed so that the shoots grew.

2.3. Laboratory Experiment

Based on the results of a preliminary series of experiments, it is impossible to make an unambiguous conclusion about the advisability of using EDU derivatives (E1–E4) at a concentration of 4 × 10⁻³ M as plant growth regulators. Therefore, for further work it was necessary to clarify the concentration of solutions E1–E4.

Aqueous solutions with concentrations of 4 × 10⁻³ M, 4 × 10⁻⁵ M, 4 × 10⁻⁷ M were prepared for all test compounds E1–E4 and TDZ according to a known technique [22,23].

The test protocol of compounds under study was carried out according to the method [24]. This protocol allows you to analyze experimental data: germination potential, germination, primary root length, number of lateral roots, shoot height and relative water content 120 h after the last watering.

The germination potential of wheat seeds was determined 24 h after the start of the experiment according to Equation (1) [25]:

$$\mathrm{Germination}\mathrm{potential}\left(\%\right)=\frac{\mathrm{Number}\mathrm{of}\mathrm{germination}\mathrm{seeds}1\mathrm{d}}{\mathrm{Number}\mathrm{of}\mathrm{total}\mathrm{seed}}\times 100$$

(1)

Seed germination was calculated by Equation (2) [26] after the end of the experiment:

$$\mathrm{Germination}\left(\%\right)=\frac{\mathrm{Number}\mathrm{of}\mathrm{germination}\mathrm{seeds}7\mathrm{d}}{\mathrm{Number}\mathrm{of}\mathrm{total}\mathrm{seed}}\times 100$$

(2)

Fifteen shoots were randomly selected from Petri dishes to determine the root length and the shoot height, as well as the relative water content (RWC). The last watering of the shoots was carried out 96 h after the start of the experiment (on the fourth day of the experiment).

RWC was determined within three days after the end of the experiment according to Equation (3) [27]:

$$\mathrm{RWC}\left(\%\right)=\frac{\left(\mathrm{FW}-\mathrm{DW}\right)}{\left(\mathrm{TW}-\mathrm{DW}\right)}\times 100$$

(3)

where: FW = fresh weight; TW = turgid weight; DW = dry weight.

For laboratory experiments and field tests Thidiazuron or 1-Phenyl-3-(1,2,3-thiadiazol-5-yl)urea (TDZ) (CAS 51707-55-2) and Chlorocholinechloride or Chlormequat chloride(CCC) (CAS 999-81-5) were used as standard substances.

2.4. Field Test

Field tests were carried out in 2022 at the experimental site of “Zhito” LLC in Oktyabrsky district, Ryazan region, Russia, 54.609836° N, 39.80188° E. The seeds of “AGATA” ^® (No. 8853479 [21]) were sown at the rate of 300 seeds/m² (10.8 ± 0.07 g) in a sod-podzolic, heavy loamy soil (humus content—1.7%, nitrate nitrogen—7.0 mg/kg, ammonia nitrogen—1.8 mg/kg, pH of salt extract—5.3, mobile phosphorus—176 mg/kg, mobile potassium—198 mg/kg). Mineral fertilizers were not added before sowing. The area of the experimental plots was 100 m², and the number of replicates repetition was fourfold. The experimental plot was divided into twelve equal strips, alternating and not in contact with each other. The scheme of the field diagram is a rectangle that was divided into squares (Figure S2).

Wheat seeds were planted manually after spraying according to the method [21,26]. For each plot, wheat seeds, as well as shoots in the tillering and bobbing phases, were sprayed with 1.92 ± 0.25 mL solution of test compounds and CCC in concentration 4 × 10⁻³ M according to BASF recommendations [28]. The phenological phases [29] of the development of spring wheat are presented in the Supplementary Materials (III.S).

The weather conditions for the spring of 2022 had a positive effect on the spring wheat germination. The atmospheric precipitations and optimal temperature in May 2022 promoted the production of balanced shoots. The weather conditions for the tillering and tube releasing phases were mostly beneficial as well: the temperatures in the second and third weeks of June was moderately warm and the moisture was sufficient. That led to the growth of additional stems of the wheat plants. The average monthly temperature in June 2022 was 19.1 °C, with a total of 155 mm of precipitation. July 2022 was characterized by moderately warm temperatures with some rain, which is in general accordance with the long-term averages. The grain was ripe by the end of the first week of August. At this time, the humidity was moderate, with precipitation of 20 mm, and the air temperature fluctuated around 25.6 °C.

2.4.1. Crude Grain Gluten Content

The content of gluten in the grain was determined according to a manual hand washing method [30,31]. The results are shown in Table 2.

2.4.2. Protein Mass Percentage

The protein content in the grain was determined by the Kjeldahl method as described in [32,33]. The results are shown in Table 3.

2.5. Statistical Analysis

Statistical analysis was performed with Microsoft Excel and STATISTICA 13.3 TRIAL programs (StatSoft, Russia). ANOVA variance analysis was applied for the data comparison. Differences from the control (water) at p ≤ 0.05 were considered to be significant.

3. Results and Discussion

3.1. Laboratory Experiments

The current study was carried out over two years, 2021 and 2022, from March to the end of October. It has been previously demonstrated that EDU in low concentrations promotes the growth and development of plants [34,35]. Our preliminary results showed (Table S1) that the tested EDU compounds (E1–E4) at a concentration of 4 × 10⁻³ M can be used as growth regulators. To research the effect of the tested compounds at low concentrations, as well as for a correct comparison with TDZ, solutions of 4 × 10⁻⁵ M and 4 × 10⁻⁷ M were additionally prepared.

It is known that TDZ is able to activate cytokinin receptors and has stronger antiaging properties than trans-zeatin (tZ), kinetin (Kin), and 6-benzylaminopurine (BAP) [36,37]. It also promotes shoot growth and increases fruit size [37]. Wheat seeds were pre-treated with tested compounds (E1–E4) and TEDS according to the previously describe method [24]. The results of the experiments are shown in

Table 1. The results of laboratory test of compounds E1–E4 and TDZ.

	Concentration, M	Germination Potential %	Germination, %	Primary Root Length, cm	Number of Roots	Lateral Roots Length, cm	Shoot Height, cm	Relative Water Content RWC, %
“Control”	0	56	75	-	4	6.5	12.4	17.62
E1	4 × 10−3	55	86	9.9	5	6.2 *	13.1	31.86
	4 × 10−5	51	89	10.8	5	7.4 **	12.6	24.00
	4 × 10−7	57	84	11.6	5	7.5	13.4	23.07
E2	4 × 10−3	54	91	10.3	5	7.2 *	12.6	20.23
	4 × 10−5	58	86	10.2	5	7.2 **	13.6	31.64
	4 × 10−7	64 **	87	9.1	6	6.4	13.7	19.37
E3	4 × 10−3	56	78	10.6	5	6.7 *	13.9	19.54
	4 × 10−5	53	81	10.0	6	7.0 **	12.6	20,54
	4 × 10−7	55	84	11.3	6	7.2	14.5	25.39
E4	4 × 10−3	51	81	10.1	5	6.5	12.5	18.59
	4 × 10−5	56	88	10.3	6	6.4 **	13.6	19.76
	4 × 10−7	58	86	10.8	5	7.0	13.6	31.50
TDZ	4 × 10−3	45	83	-	4	4.0	12.3	23.85
	4 × 10−5	49	90	-	4	5.1	13.1	33.55
	4 × 10−7	56	89	-	4	5.8	11.7	23.63

“Control”—Seeds put in a Petri dish and treated only with distilled water were taken as control. *—Statistically significant difference from the control (water) at p < 0.05. **—Statistically significant difference from the control (water) at p < 0.01.

.

Seed sprouting is a key stage in the plant life cycle. The first 24 h of the experiment were conducted in the dark to mimic the environment of the natural seed development in the soil. Seed germination potential (Gp) was calculated after 24 h according to Equation (1). Root length (1–3 mm) is a criterion for estimating Gp as the root penetrates the seed membranes [25]. The results for germination potential and germination rate are shown in Figure 2.

Germination (G) of the seeds was calculated on the seventh day using Equation (2), as recommended by the International Seed Testing Association [26].

For compounds E1, E4 and TDZ at a concentration of 4 × 10⁻⁷ M, the Gp values are maximum in their subgroup and are comparable with Gp of the control sample ~(56%). The exceptions are E2 and E3. Seeds treated with E2 at a concentration of 4 × 10⁻⁷ and E3 at a concentration of 4 × 10⁻³ showed Gp values 8% higher than the control for E2 and comparable to the control for E3.

Seeds treated with E1, E4 and TDZ at a concentration of 4 × 10⁻⁵ M show G values ~89 ± 1%, which is 14% higher than that of the control. Thus, urea derivatives show the same upward trend in results.

The G values of seeds treated with carbamate—E2 (91% at a concentration of 4 × 10⁻³ M) and E3 (84% at a concentration of 4 × 10⁻⁷ M), are 16% and 9%, respectively, higher than the control. In this case, there is a different reaction of plants depending on the concentration of substances relative to each other.

Wheat is the world’s most cultivated crop. Drought and heat waves are the major factors limiting yields in most agricultural regions. A robust root system plays an important role in plant adaptation to drought [38,39]. It is known that the root system consists of two types of roots: the primary root (PR) and lateral roots (LRs).

For the shoots that were treated with E1–E4, the formation of both the main root and the lateral roots is observed, in contrast to the control and shoots treated with TDZ. It is possible because the tested compounds are similar to the cytokinin compounds inhibiting the initiation of lateral root growth and elongation of the primary root [40,41].

Shoots treated with TDZ do not have a main root, due to the inhibitory ability of TDZ at high concentrations of 4 × 10⁻³ M (Figure 2a); a similar result was described by the authors [42]. As the concentration of TDZ decreased from 4 × 10⁻⁵ (Figure 2b) to 4 × 10⁻⁷ M (Figure 2c), the length of the roots increased, but primary root formation was not observed.

Plants in natural and agricultural conditions are constantly exposed to stress. To cope with water scarcity, plants have evolved acclimatization and adaptation mechanisms, including stress avoidance and resistance to dehydration. Relative water content (RWC) in leaves is a simple way to assess the water status of plants at a given point in time. RWC reflects the balance between water entering leaf tissues and transcription, i.e., the rate of water movement through the plant [43]. The higher the RWC value, the less susceptible the plants are to drought. The RWC was calculated using Equation (3) [27]. The results of the research are presented in Table S2.

In this study, we have identified different RWC rates of wheat shoots treated with test compounds (E1–E4) and TDZ under water-deficient conditions (Figure S1). The relative water content RWC was determined 120 h after the last watering, to monitor the change in RWC under conditions of water deficiency. Shoots treated with E1 at a concentration of 4 × 10⁻³ M, E2 and TDZ at a concentration of 4 × 10⁻⁵ M and E4 at a concentration of 4 × 10⁻⁷ M had a minimum degree of wilting and a greatest RWC of more than 30% compared to the control (17.62%).

Thus, the compounds studied can be used to improve the drought tolerance of wheat plants at relatively low concentrations from 10⁻⁵ M down to 10⁻⁷ M.

3.2. Field Test

Field tests were carried out simultaneously with laboratory experiments using seeds of spring wheat “AGATA” ^® (No. 8853479 [21]. Chlorocholine chloride (CCC) was used as a standard in this experiment.

CCC is known to be a growth regulator and is used to reduce the risk of lodging in crops by shortening the internodes, increasing stem strength and evenness [44,45]. In addition, its use increases the root system mass the resistance of the plants to stress. It is known that spraying wheat shoots with growth regulators at the phenological stages of “tillering” and “booting” promotes the development of the root system and increases the resistance of plants to lodging [29]. Therefore, in the field experiment, additionally at these phenological stages, wheat shoots were treated with solutions of the tested compounds (E1–E4), as well as CCC. The results of the field studies are summarized in

Table 2. The results of field tests for the compounds E1–E4.

	Plant Height, cm	Number of Plants, pcs/m2	Number of Productive Stems, pcs/m2	Spike Length, cm	Quantity Per Ear, pcs.		Weight of Grain per Ear, g
					Spikelets	Grains
Control	54.6 ±2.79	238 ±10.5	274 ±6.95	8.6 ±0.31	23.0 ±1.41	23.7 ±1.7	0.85 ±0.028
E1	56.7 ±1.81	256 ** ±8.44	343 ** ±9.17	8.1 ±0.12	22.7 ±0.51	25.5 ±2.08	0.87 ±0.043
E2	52.5 ±4.11	245 * ±10.5	304 ** ±7.59	8.4 ±0.48	22.3 ± 0.18	20.4 ± 1.91	0.86 ± 0.022
E3	51.0 ±3.44	248 ** ±1.02	332 ** ±4.52	8.9 ±0.17	22.2 ±0.25	24.7 ±3.32	0.88 ±0.031
E4	48.1 ±0.80	240 ±8.13	299 ** ±9.43	8.9 * ±0.39	22.5 ±0.57	19.6 ±3.39	0.82 ±0.058
CCC	53.8 ±4.66	254 ±9.29	307 ±7.02	8.55 ±0.42	22.0 ±0.81	16.7 ±1.97	0.83 ±0.069

*—Statistically significant difference from the control (water) at p < 0.05. **—Statistically significant difference from the control (water) at p < 0.01.

.

The determining factors in the formation of the yield in the experiment are: the number of surviving plants for harvesting, the density of productive stems and tillering. Plant height was within 48.2–56.7 cm. In some experiments, 240–256 plants per m² were formed. In the control variant, the number of productive stems was 247 pcs/m². The use of various derivatives of EDU (E1–E4) led to an increase in this indicator by~9.0–25.0%. An analysis of the crop structure elements showed that in the experiments that employed the studied compounds, a slight increase in the plant parameters such as ear length, number of spikelets and grains per ear, and mass of grain per ear were shown (the average values are shown in

Table 3. Productivity of field experiments for testing compounds.

Option	Germination from the Site,%	Productivity, kg/m2	Increase, kg/m2	Weight of 1000 Grains, g	Yield kg/4 m2	Crude Gluten Content in Grain, %	Protein Content in Grain, %
Control	78 ±3.57	0.22 ±0.002	-	28.85 ±0.72	0.88	25.0	12.33
E1	85 ** ±3.16	0.335 ** ±0.002	0.115	31.45 ±1.91	1.34	26.5	12.56
E2	81 * ±3.53	0.272 ** ±0.003	0.052	29.93 ±2.09	1.088	25.7	12.43
E3	82 * ±3.51	0.271 ** ±0.002	0.051	29.36 ±2.02	1.084	26.0	12.47
E4	80 ±2.58	0.222 ±0.003	0.002	29.01 ±1.81	0.88	25.2	12.33
CCC	84 ±10.04	0.223 ±0.004	0.003	31.85 * ±1.48	0.89	25.4	12.35

*—Statistically significant difference from the control (water) at p < 0.05. **—Statistically significant difference from the control (water) at p < 0.01.

). The control yield of spring wheat was 0.22 kg/m². In general, the use of EDU analogs on spring wheat during the growing season of 2022 provided yield increases from 0.222 kg/m² to 0.335 kg/m² (Table 3).

The control mass of 1000 grains was 28.85 g, the use of the studied compounds exceeded this metric by ~1.6–8.4%. The content of raw gluten varied within 25.2–26.5%. The greatest value of the raw gluten content in the grain was observed in the experiment with E1 (26.5%), exceeding the control by 6%. The protein mass percentage is one of the most important quality indicators for the wheat grain. Proteins form the structural framework of gluten, determine the nutritional value of wheat products. The protein content in the grain in all experiments was in the range of ~12.3–12.6%.

Figure 3 shows the most common ears of control and treated wheat plants. Meanwhile, the ears of CCC-treated plants and plants of the control group practically do not differ in shape and size. Fuller and well-formed ears demonstrate plants treated with a chloro-substituted analogue of EDU.

The appearance of plants at the time of harvesting was better on plots treated with compounds E1–E4. The number of productive stems, the weight of grain per ear and the weight of 1000 grains are the determining indicators in the formation of the grain yield. The use of EDU derivatives contributed to their increase in comparison with the control variant. The results of the study showed that the use of the studied compounds contributed to an increase in the yield of spring wheat, the maximum increase of which was noted in E1. The total yield increase was 0.115 kg/m², or 52.27% more than the control. Simultaneously with the increase in yield, the use of EDU analogs also contributed to the improvement of the grain quality of spring wheat. The crude gluten content increased by 0.8% (for E4)–6% (for E1) compared to the control.

4. Conclusions

As a result, a use of unsymmetrical hybrid phytoactive arylureas as analogs of cytokinin-like EDUs containing the imidazolidin-2-one and aryl fragment has been developed. Seed germination potential (Gp), germination (G), growth indicators (primery, lateral roots and shoot height) and relative water content (RWC) values were obtained and analyzed for all tested substances. The compounds demonstrated high growth-regulating activity on wheat seeds in the laboratory experiment and in field trials compared to commonly used standards such as CCC and TDZ. The positive effect of these substances on drought resistance was also established even at very low concentrations. New hybrid compounds can be used to increase the resistance of wheat (Triticum aestivum L.) plants to harmful environmental factors.