Phosphate ester compound containing amide group as well as preparation method and application of phosphate ester compound

Abstract

The invention relates to the technical field of agricultural plant growth regulators, and discloses a phosphate compound containing an amide group as well as a preparation method and application thereof. O-aminopyridine, different substituted benzaldehyde and isocyanide are used as initial raw materials, and ring closing and ring opening are performed to obtain the phosphate compound containing the amide group. And finally, reacting with dialkyl phosphite to obtain the phosphate ester compound containing the amide group. The phosphate ester compound containing the amide group is high in activity, high in stability, simple in synthesis route and low in cost, can serve as an ABA analogue to be combined with abscisic acid receptor (PYL3) protein, can promote plant stomata closing and regulate crop root growth, and can be used as an abscisic acid receptor (PYL3) protein. Further, the drought stress regulation capability of the plants is improved.

Description

Technical Field

[0001] This invention relates to the field of agricultural plant growth regulators, and more specifically, to a phosphate ester compound containing an amide group, its preparation method, and its application. Background Technology

[0002] Abscisic acid (ABA) is a plant hormone and a key factor in plant adaptation to abiotic environmental stresses. Abscisic acid is known to induce stomatal closure, thereby reducing transpiration during periods of high temperature, drought, or salt stress. Given the current climate change leading to intensified global water cycles and increased drought, regulating ABA levels and signaling is a promising approach to improving plant drought tolerance. Although treating crops with exogenous ABA can enhance their drought resistance, its potential for agricultural application is limited. Besides its adverse hormonal effects on seed germination and plant growth, ABA is also photosensitive and rapidly degrades upon contact with plants. Hundreds of ABA analogs have been synthesized in recent years, including several with high activity, but these studies remain largely at the laboratory level, and no ABA analog has yet been applied to agricultural production. Therefore, developing ABA analogs with simple, low-cost, and highly active synthetic routes remains an important measure to address food security.

[0003] Pyrabactin is the first artificially synthesized ABA receptor agonist screened using chemogenetic methods. This provides a new precursor for the research of new ABA receptor activators. Based on the structure of Pyrabactin, its structure was modified using methods such as isosteric electron configuration, molecular docking, and virtual screening, leading to the discovery of active molecules containing sulfonamide structures, such as AM1 (Quinabactin), AMF4, and Cyanabactin. Subsequently, introducing four fluorine atoms into the AM1 structure resulted in AMF4, which, compared to AM1, exhibited significantly enhanced affinity for PYLs and demonstrated superior drought resistance in in vivo experiments. Professor Cutler's team replaced the dihydroquinoline ketone structure in the AM1 structure with cyano-cyclopropylbenzene, obtaining Cyanabactin, which showed high efficiency and sensitivity to PYR1 at nanomolar concentrations and effectively regulated plant water loss. Compound 3CB is a novel amide-based ABA receptor agonist obtained through virtual screening of ABA receptors, containing the active fragment of Cyanabactin in its structure. Based on the structure of 3CB, a cyclopropyl group was introduced at the ortho position of the cyano (-CN) group to increase its hydrophobicity, resulting in Opabactin with even better activity. Its seed germination inhibitory activity is 10 times that of ABA, and its drought resistance activity against wheat and tomato is more persistent than ABA, making it the most active ABA receptor agonist screened to date. Furthermore, by modifying the structure of ABA, analogues including AS6, PAO4, and PANme were obtained, which can block the PYL-PP2C interaction and relieve abscisic acid-inhibited seed germination. Notably, AA1, reported in 2017, can target all Arabidopsis PYLs and reverse the inhibitory effect of ABA on PP2C during the regulatory process by interfering with the PYR / PYLs-PP2C interaction.

[0004] The development of ABA functional analogs has attracted much attention; however, there is a lack of drought-resistant compounds that can balance activity, stability, and have a simple and low-cost synthetic route.

[0005] Therefore, the present invention proposes a phosphate ester compound containing an amide group and its preparation method, which has important practical significance. Summary of the Invention

[0006] In view of this, the present invention proposes a phosphate ester compound containing an amide group to solve the problem of the lack of a drought-resistant compound in the prior art that can take into account activity, stability, simple synthesis route and low cost.

[0007] The chemical structural formula of the phosphate ester compound containing an amide group of the present invention is shown in general formula I: .

[0008] Wherein, R1 is selected from H, 2-Cl, 3-Cl, 2-Br, 3-Br, 2-CH3, 2-OCH3; R2 is selected from -CH2CH3, -CH3, -CH(CH3)2, -CH2CH(CH3)2; R3 is selected from -C6H 11 -C(CH3)3.

[0009] This invention also provides a method for preparing a phosphate ester compound containing an amide group, comprising the following preparation steps: Under the first catalyst, and o-aminopyridine and The mixture was stirred and refluxed in the first solvent to obtain intermediate I; In the presence of a second catalyst, intermediate I was added to a second solvent and the mixture was stirred at room temperature to obtain intermediate II. In the presence of a third catalyst, intermediate II is mixed with dialkyl phosphite and reacted at room temperature to obtain the phosphate compound containing the amide group.

[0010] Furthermore, the chemical structural formula of intermediate I is shown below:

[0011] Furthermore, the chemical structural formula of intermediate II is shown below: .

[0012] Preferably, the first catalyst is lanthanum chloride heptahydrate.

[0013] Preferably, the second catalyst is diethyl iodophenyl ester and p-toluenesulfonic acid.

[0014] Preferably, the third catalyst is DBU.

[0015] Preferably, the first solvent is ethanol.

[0016] Preferably, the second solvent is dichloromethane.

[0017] The present invention also provides the application of amide-containing phosphate ester compounds in the preparation of ABA analogs to enhance the regulation of plants against drought stress.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention synthesizes a phosphate ester compound containing an amide group, which has high activity, high stability, and a simple and low-cost synthetic route.

[0019] 2. The phosphate ester compound containing amide group synthesized in this invention can act as an ABA analog to bind to the abscisic acid receptor (PYL3) protein, thereby promoting stomatal closure, regulating crop root growth, and thus improving the plant's ability to regulate drought stress. Attached Figure Description

[0020] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings: Figure 1 This is an overall synthetic route diagram of phosphate ester compounds containing amide groups provided in the embodiments of the present invention; Figure 2 A synthetic route diagram for intermediate I provided in an embodiment of the present invention; Figure 3 Synthetic route diagram of intermediate II provided in the embodiments of the present invention; Figure 4 A synthetic route for synthesizing phosphate ester compounds containing amide groups using intermediate II, provided in an embodiment of the present invention; Figure 5 The present invention provides a method for determining the IC50 of different concentrations of ABA and C17 on PYL3 using the specific substrate SnRK2.6. 50 Fitted curve; Figure 6 This is a diagram showing the effect of different concentrations of C17 compound on the root length of Arabidopsis thaliana, as provided in the embodiments of the present invention. Figure 7 This is a graph showing the effect of different concentrations of C17 compound on plant leaf temperature, provided in an embodiment of the present invention. Figure 8 This is a graph showing the effect of different concentrations of C17 compound on plant stomata and water loss rate, provided in an embodiment of the present invention. Figure 9 The in vivo drought resistance test diagrams of compounds C14 and C17 provided in the embodiments of the present invention; Figure 10 The diagram shows the effect of the compound of the present invention on biomass and ROS, provided for embodiments of the present invention. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0022] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] This invention provides a phosphate ester compound containing an amide group, the chemical structure of which is shown in general formula I: .

[0027] Wherein, R1 is selected from H, 2-Cl, 3-Cl, 2-Br, 3-Br, 2-CH3, 2-OCH3; R2 is selected from -CH2CH3, -CH3, -CH(CH3)2, -CH2CH(CH3)2; R3 is selected from -C6H 11 (cyclohexyl), -C(CH3)3.

[0028] Specifically, the phosphate ester compounds containing amide groups of the present invention are preferably the specific compounds in Table 1: Table 1. Specific structures of phosphate ester compounds containing amide groups

[0029] like Figure 1 As shown, the present invention also provides a method for preparing a phosphate ester compound containing an amide group, comprising the following preparation steps: Under the first catalyst, and o-aminopyridine and The mixture was stirred and refluxed in the first solvent to obtain intermediate I; In the presence of a second catalyst, intermediate I was added to a second solvent and the mixture was stirred at room temperature to obtain intermediate II. In the presence of a third catalyst, intermediate II is mixed with dialkyl phosphite and reacted at room temperature to obtain the phosphate compound containing the amide group.

[0030] The chemical structural formula of intermediate I is shown below: .

[0031] The chemical structural formula of intermediate II is shown below: .

[0032] The first catalyst is preferably lanthanum chloride heptahydrate; The second catalyst is preferably diethyl iodophenyl ester and p-toluenesulfonic acid; The third catalyst is preferably DBU; The first solvent is preferably ethanol; The second solvent is dichloromethane.

[0033] Specifically, (1) the preparation of intermediate I (e.g. Figure 2 As shown): Using lanthanum chloride heptahydrate as a catalyst, substituted benzaldehyde and o-aminopyridine as raw materials, intermediate I is generated by stirring and refluxing with isocyanocyclohexane or tert-butylisocyanate in ethanol.

[0034] (3) Preparation of intermediate II (e.g.) Figure 3 As shown): Using intermediate 1 as raw material, diethyl iodophenyl ester and p-toluenesulfonic acid as catalysts, and dichloromethane as solvent, the reaction was stirred at room temperature to generate intermediate II through ring opening.

[0035] (3) Preparation of phosphate ester compounds containing amide groups (e.g.) Figure 4 As shown): using intermediate II as a raw material and DBU as a catalyst, the target product, an amide phosphate compound, is rapidly generated from dialkyl phosphite at room temperature in a solvent-free environment.

[0036] Specifically, the method for preparing the phosphate ester compound containing an amide group according to the present invention includes the following steps (using benzaldehyde and isocyancyclohexane as examples of raw materials): (1) Weigh 9.16 mmol (1.0 eq) benzaldehyde and 9.16 mmol (1.0 eq) o-aminopyridine and measure 9.16 mmol (1.0 eq) isocyanocyclohexane into a 50 mL round-bottom flask, add 20 mL of ethanol, heat and stir, then add 9.16 μmol (0.1 eq) lanthanum chloride heptahydrate, reflux to close the ring to generate dihydroimidazole[1,2-a]pyridine amine compounds, heat and stir under reflux for 3 h, and use thin-layer chromatography (TLC) (V petroleum ether:V ethyl acetate = 3:1) to track the reaction progress. After the reaction is completed, cool to room temperature, remove the solvent under reduced pressure to obtain crude product, and purify by column chromatography to obtain intermediate I.

[0037] (2) Weigh 2.73 mmol (1.0 eq) of intermediate I and 8.18 mmol (3.0 eq) of p-toluenesulfonic acid into a 50 mL single-necked flask, use 20 mL of dichloromethane as solvent, stir at room temperature and add 2.73 mmol (1.0 eq) of iodobenzene acetate. The reaction is carried out for about 1 hour. The reaction progress is monitored by thin-layer chromatography (TLC) (V petroleum ether:V ethyl acetate = 3:1). After the reaction is completed, column chromatography is used to purify and obtain intermediate II.

[0038] (3) 1.30 mmol (1.0 eq) of intermediate II and 1.30 mmol (1.0 eq) of dialkyl phosphite and 130 μmol (0.1 eq) of DBU were added to a 25 mL single-necked flask and reacted rapidly at room temperature without solvent to generate the target amide phosphate ester compound. The reaction was carried out for 5 min. The reaction progress was tracked by thin-layer chromatography (TLC) (V petroleum ether:V ethyl acetate = 1:1). After the reaction was completed, the target amide phosphate ester compound was obtained by column chromatography purification.

[0039] Understandably, using o-aminopyridine, benzaldehyde with different substitutions, and isocyanate as initial raw materials, the reaction proceeds through ring closure and ring opening, and finally reacts with dialkyl phosphite to obtain amide phosphate compounds. The reaction synthesis route is simple and inexpensive.

[0040] Example 1: Synthesis of 2-(cyclohexylamino)-2-oxo-1-phenylethyl diethyl phosphate (1) (wherein (1) indicates that the target product of this example is the compound numbered 1 in Table 1, and the meaning of subsequent examples is similar):

[0041] S1: Weigh 972.08 mg (9.16 mmol, 1.0 eq) benzaldehyde and 862.10 mg (9.16 mmol, 1.0 eq) o-aminopyridine and measure 1.14 mL (9.16 mmol, 1.0 eq) isocyancyclohexane into a 50 mL round-bottom flask. Add 20 mL of ethanol, heat and stir, then add 42.20 mg (9.16 μmol, 0.1 eq) lanthanum chloride heptahydrate. Reflux to close the ring and generate dihydroimidazole[1,2-a]pyridine amine compounds. Heat and stir under reflux for 3 h, and perform thin-layer chromatography (TLC) (V 石油醚 :V 乙酸乙酯 =3:1) The reaction was followed up. After the reaction was completed, the mixture was cooled to room temperature and the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by column chromatography to obtain 2.36 g of intermediate I, with a yield of 87.81%.

[0042] S2: Weigh 800 mg (2.73 mmol, 1.0 eq) of intermediate I and 1.41 g (8.18 mmol, 3.0 eq) of p-toluenesulfonic acid into a 50 mL single-necked flask. Use 20 mL of dichloromethane as solvent. Add 878.21 mg (2.73 mmol, 1.0 eq) of iodobenzene acetate while stirring at room temperature. React for about 1 hour, and then analyze the reaction using thin-layer chromatography (TLC) (V... 石油醚 :V 乙酸乙酯 The reaction was monitored using a ratio of 3:1. After the reaction was completed, the intermediate II was purified by column chromatography to obtain 580.13 mg, with a yield of approximately 90%.

[0043] S3: 300.0 mg (1.30 mmol, 1.0 eq) of intermediate II and 180.0 mg (1.30 mmol, 1.0 eq) of dialkyl phosphite, along with 32.54 mg (130 μmol, 0.1 eq) of DBU, were added to a 25 mL single-necked flask. The mixture was rapidly reacted at room temperature in a solvent-free environment to generate the target amide phosphate compound. The reaction was carried out for 5 min, and the mixture was analyzed by thin-layer chromatography (TLC) (V... 石油醚 ∶V 乙酸乙酯 The reaction was monitored at a ratio of 1:1. After the reaction was completed, column chromatography was used to purify the target amide phosphate compound, yielding 440.9 mg in 92% yield. The spectral data are as follows: 2-(cyclohexylamino)-2-oxo-1-phenylethyl diethyl phosphate (1): 1 H-NMR (400 MHz, CDCl3): δ 7.45 (dd, 2H Ar-H, J = 7.4, 2.0 Hz,), 7.37 (dd, 3H, Ar-H, J = 4.9,2.3 Hz,), 6.58 (d, 1H, NH,J = 8.0 Hz,), 5.60 (d, 1H, CO-H, J = 8.7 Hz,),4.10 – 4.01 (m, 3H,CH3CH2), 3.96 – 3.90 (m, 1H, CH3CH2), 3.83 – 3.77 (m, 1H,-CH(CH2)5), 1.97 – 1.87 (m, 2H, -CH(CH2)5), 1.77 – 1.70 (m, 2H, -CH(CH2)5), 1.63(dd, 1H, -CH(CH2)5, J = 9.1, 3.7 Hz), 1.42 – 1.33 (m, 2H, CH3CH2), 1.28 – 1.24(m, 4H, CH3CH2), 1.24 – 1.20 (m, 5H, -CH(CH2)5). 13 C-NMR (100 MHz, CDCl3): δ167.3 (d, J = 6.9 Hz), 136.2 (d, J = 3.3 Hz), 129.1, 128.6, 127.2, 78.9(d, J = 6.5 Hz), 64.3 (d, J = 5.6 Hz), 48.2, 32.9 (d, J = 4.7 Hz), 25.4, 24.8, 15.9 (dd, J = 6.8, 3.4 Hz). 31 P-NMR (162 MHz, CDCl3): δ -2.48. HRMS(ESI): Calculated for C 18 H 29 O5NP [M+H] + : 307.17779, found: 307.17694. Example 2: Synthesis of 1-(2-chlorophenyl)-2-(cyclohexylamino)-2-oxoethyl diethyl phosphate (2)

[0044] The only difference from Example 1 is that benzaldehyde in step S1 is replaced with 2-chlorobenzaldehyde, and in step S3, intermediate II reacts with diethyl phosphite to obtain compound (2), with a yield of 92%. The spectral data are as follows: 1-(2-Chlorophenyl)-2-(cyclohexylamino)-2-oxoethyl diethyl phosphate (2): 1 H-NMR (400 MHz, CDCl3): δ 7.46 (dd, 1H, Ar-H, J = 5.7, 3.7 Hz), 7.40 (dd, 1H, Ar-H, J = 5.9,3.4 Hz), 7.31 – 7.28 (m, 2H, Ar-H), 6.67 (d, 1H, NH, J = 7.9 Hz), 6.01 (d, 1H, CO-H, J = 8.8 Hz), 4.10 – 4.03 (m, 3H, CH3CH2), 3.97 – 3.91 (m, 1H,CH3CH2), 3.86 – 3.80 (m, 1H, -CH(CH2)5), 1.93 (d, 2H, 1H,-CH(CH2)5, J = 5.4Hz), 1.73 (dd, 2H, -CH(CH2)5, J = 9.1, 3.9 Hz), 1.62 (dd, 1H, -CH(CH2)5, J =9.0, 3.8 Hz), 1.40 – 1.33 (m, 2H, CH3CH2), 1.26 (t, J = 7.1 Hz, 4H, CH3CH2),1.22 (dd, J = 7.5, 6.8 Hz, 5H, -CH(CH2)5). 13 C-NMR (100 MHz, CDCl3): δ 167.3(d, J = 6.9 Hz), 136.2 (d, J = 3.3 Hz), 129.1, 128.6, 127.2, 78.9 (d, J =6.5 Hz), 64.3 (d, J = 5.6 Hz), 48.2, 32.9 (d, J = 4.7 Hz), 25.4 , 24.8 ,15.9 (dd, J = 6.8, 3.4 Hz). 31P-NMR (162 MHz, CDCl3): δ -2.53. HRMS (ESI): Calculated for C 18 H 28 O5NClP [M+H] + : 404.13881, found: 404.13727. Example 3: Synthesis of 1-(3-chlorophenyl)-2-(cyclohexylamino)-2-oxoethyl diethyl phosphate (3)

[0045] The only difference from Example 1 is that benzaldehyde in step S1 was replaced with 3-chlorobenzaldehyde, and in step S3, intermediate II reacted with diethyl phosphite to obtain compound (3), with a yield of 91.23%. The spectral data are as follows: 1-(3-chlorophenyl)-2-(cyclohexylamino)-2-oxoethyl diethyl phosphate (3): 1 H-NMR(400 MHz, CDCl3) δ 7.46 (s, 1H, Ar-H), 7.36 7.30 (m, 3H, Ar-H), 6.58 (d, 1H, NH, J =8.0 Hz), 5.56 (d, 1H, CO-H, J = 8.8 Hz), 4.13 – 3.99 (m, 4H, CH3CH2), 3.78(m, 1H, -CH(CH2)5), 1.96 – 1.86 (m, 2H, CH(CH2)5), 1.77 – 1.69 (m, 2H, - CH(CH2)5), 1.67 –1.60 (m, 1H, - CH(CH2)5), 1.41 1.33 (m, 2H, CH3CH2), 1.31 – 1.27(m, 4H, CH3CH2), 1.26 – 1.19 (m, 5H, - CH(CH2)5). 13 C-NMR (101 MHz, CDCl3) δ166.7 (d, J = 6.6 Hz), 138.1 (d, J = 3.5 Hz), 134.5 , 129.9 , 129.2 , 127.2 ,125.4 , 78.0 (d, J = 6.4 Hz), 64.5 (d, J = 5.7 Hz), 48.3, 32.9 (d, J = 2.5Hz), 25.4, 24.8, 16.0 (dd, J = 6.8, 1.6 Hz). 31P-NMR (162 MHz, CDCl3) δ 2.45.HRMS (ESI): Calculated for C 18 H 28 O5NClP [M+H] + 404.13881, found: 404.13791. Example 4: Synthesis of 1-(4-chlorophenyl)-2-(cyclohexylamino)-2-oxoethyl diethyl phosphate (4)

[0046] The only difference from Example 1 is that benzaldehyde in step S1 is replaced with 4-chlorobenzaldehyde, and in step S3, intermediate II reacts with diethyl phosphite to obtain compound (4), with a yield of 92%. The spectral data are as follows: 1-(4-Chlorophenyl)-2-(cyclohexylamino)-2-oxoethyl diethyl phosphate (4): 1 H-NMR (400 MHz, CDCl3) δ 7.39 – 7.35 (m, 2H, Ar-H), 7.34 – 7.30 (m, 2H, Ar-H), 6.58 (d, 1H,NH, J = 8.2 Hz), 5.54 (d, 1H, CO-H, J = 8.8 Hz), 4.10 – 3.92 (m, 4H, CH3CH2),3.78 – 3.71 (m, 1H, - CH(CH2)5), 1.86 (dd, 2H, CH(CH2)5, J = 23.5, 12.0 Hz),1.73 – 1.66 (m, 2H, - CH(CH2)5), 1.59 (dd, 1H, CH(CH2)5, J = 9.3, 3.6 Hz), 1.37 – 1.29 (m, 2H, - CH(CH2)5), 1.24 (dd, 3H, - CH(CH2)5, J = 5.3, 4.1 Hz), 1.18 (ddd, 6H, CH3CH2, J = 14.6, 9.5, 1.9 Hz). 13C-NMR (101 MHz, CDCl3) δ 166.9(d, J = 6.6 Hz), 134.9, 134.8 (d, J = 3.5 Hz), 128.8, 128.5, 78.1 (d, J = 6.4Hz), 64.4 (dd, J = 5.6, 2.1 Hz), 48.3, 32.9 (d, J = 5.3 Hz), 25.4, 24.7, 16.0 (d, J = 6.7 Hz). 31 P -NMR (162 MHz, CDCl3) δ -2.47. HRMS (ESI): Calculated forC 18 H 28 O5NClP [M+H] + : 404.13881, found: 404.13760. Example 5: Synthesis of 1-(2-bromophenyl)-2-(cyclohexylamino)-2-oxoethyl diethyl phosphate (5)

[0047] The only difference from Example 1 is that benzaldehyde in step S1 is replaced with 2-bromobenzaldehyde, and in step S3, intermediate II reacts with diethyl phosphite to obtain compound (5), with a yield of 91%. The spectral data are as follows: 1-(2-bromophenyl)-2-(cyclohexylamino)-2-oxoethyl diethyl phosphate (5): 1H-NMR (400 MHz, CDCl3) δ 7.57 (d, 1H, Ar-H, J = 8.0 Hz), 7.44 – 7.41 (m, 1H, Ar-H), 7.32 (t,1H, Ar-H, J = 7.5 Hz), 7.20 (dd, 1H, Ar-H, J = 11.6, 5.0 Hz), 6.61 (d, 1H, NH, J = 7.8 Hz,), 6.00 (d, 1H, CO-H, J = 8.9 Hz), 4.09 – 4.03 (m, 3H, CH3CH2), 3.95–3.89 (m, 1H, CH3CH2), 3.81 –3.76 (m, 1H, -CH(CH2)5), 1.941.88 (m, 2H, -CH(CH2)5), 1.71 (dd, 2H, - CH(CH2)5, J= 9.2, 3.8 Hz), 1.60 (dd, 1H, CH(CH2)5, J =9.0, 3.7 Hz), 1.37 – 1.30 (m, 2H, CH3CH2), 1.25 (t, J= 7.1 Hz, 4H, CH3CH2),1.20(t, 5H, - CH(CH2)5, J = 7.1 Hz). 13 C-NMR (101 MHz, CDCl3) δ 166.5 (d, J =6.9 Hz), 136.1 (d, J = 3.4 Hz), 133.2, 130.5, 129.5, 127.8, 123.7, 77.7(d, J = 6.4 Hz), 64.4 (t, J = 5.7 Hz), 48.4, 32.8 (d, J = 3.1 Hz), 25.5, 24.7, 15.9 (dd, J = 9.4, 6.9 Hz). 31 P -NMR (162 MHz, CDCl3) δ -2.53. HRMS(ESI): Calculated for C 18 H 28 O5NBrP [M+H] + :448.08830, found: 448.08694. Example 6: Synthesis of 1-(3-bromophenyl)-2-(cyclohexylamino)-2-oxoethyl diethyl phosphate (6)

[0048] The only difference from Example 1 is that benzaldehyde in step S1 is replaced with 3-bromobenzaldehyde, and in step S3, intermediate II reacts with diethyl phosphite to obtain compound (6) in 91% yield. The spectral data are as follows: 1-(3-bromophenyl)-2-(cyclohexylamino)-2-oxoethyl diethyl phosphate (6): 1 H-NMR (400 MHz, CDCl3) δ 7.50 – 7.47 (m, 2H, Ar-H), 7.31 (d, 2H, Ar-H, J = 8.4 Hz), 6.54 (d,1H, NH, J = 8.2 Hz), 5.52 (d, 1H, CO-H, J = 8.8 Hz), 4.01 (dddd, 4H,(CH3CH2)2, J = 21.6, 17.8, 7.6, 2.6 Hz), 3.78 – 3.71 (m, 1H, CH(CH2)5), 1.94 –1.86 (m, 2H, - CH(CH2)5), 1.70 (td, 2H, - CH(CH2)5, J = 8.6, 4.2 Hz), 1.63 – 1.58 (m, 1H, - CH(CH2)5), 1.37 – 1.30 (m, 2H, CH3CH2), 1.28 – 1.24 (m, 4H, CH3CH2), 1.23 – 1.17 (m, 5H, – CH(CH2)5). 13 C-NMR (101 MHz, CDCl3) δ 166.9 (d,J = 6.6 Hz), 135.3 (d, J = 3.5 Hz), 131.9 , 128.7 , 123.2 , 78.2 (d, J = 6.4Hz), 64.4 (d, J = 5.6 Hz), 48.3, 32.9 (d, J = 4.3 Hz), 25.4, 24.8, 16.0 (d, J = 6.8 Hz). 31 P -NMR (162 MHz, CDCl3) δ -2.43. HRMS (ESI): Calculated forC 18 H 28 O5NBrP [M+H] + :448.08830, found: 448.08707. Example 7: Synthesis of 1-(4-bromophenyl)-2-(cyclohexylamino)-2-oxoethyl diethyl phosphate (7)

[0049] The only difference from Example 1 is that benzaldehyde in step S1 is replaced with 4-bromobenzaldehyde, and in step S3, intermediate II reacts with diethyl phosphite to obtain compound (7) in 91% yield. The spectral data are as follows: 1-(4-bromophenyl)-2-(cyclohexylamino)-2-oxoethyl diethyl phosphate (7): 1 H-NMR (400 MHz, CDCl3) δ 7.50 – 7.47 (m, 2H, Ar-H), 7.31 (d, 2H, Ar-H, J = 8.4 Hz), 6.54 (d,1H, NH, J = 8.2 Hz), 5.52 (d, 1H, CO-H, J = 8.8 Hz), 4.01 (dddd, 4H,(CH3CH2)2, J = 21.6, 17.8, 7.6, 2.6 Hz), 3.78 – 3.71 (m, 1H, CH(CH2)5), 1.94 –1.86 (m, 2H, - CH(CH2)5), 1.70 (td, 2H, - CH(CH2)5, J = 8.6, 4.2 Hz), 1.63 – 1.58 (m, 1H, - CH(CH2)5), 1.37 – 1.30 (m, 2H, CH3CH2), 1.28 – 1.24 (m, 4H, CH3CH2), 1.23 – 1.17 (m, 5H, – CH(CH2)5). 13 C-NMR (101 MHz, CDCl3) δ 166.9 (d,J = 6.6 Hz), 135.3 (d, J = 3.5 Hz), 131.9 , 128.7 , 123.2 , 78.2 (d, J = 6.4Hz), 64.4 (d, J = 5.6 Hz), 48.3, 32.9 (d, J = 4.3 Hz), 25.4, 24.8, 16.0 (d, J = 6.8 Hz). 31 P-NMR (162 MHz, CDCl3) δ -2.43. HRMS (ESI): Calculated forC 18 H 28 O5NBrP [M+H] + :448.08830, found: 448.08707. Example 8: Synthesis of diethyl 2-(cyclohexylamino)-1-(4-fluorophenyl)-2-oxoethyl phosphate (8)

[0050] The only difference from Example 1 is that benzaldehyde in step S1 is replaced with 4-fluorobenzaldehyde, and in step S3, intermediate II reacts with diethyl phosphite to obtain compound (8), with a yield of 89%. The spectral data are as follows: 2-(cyclohexylamino)-1-(4-fluorophenyl)-2-oxoethyl phosphate diethyl ester (C8): 1 H-NMR (400 MHz, CDCl3): δ 7.38 – 7.33 (m, 2H, Ar-H), 7.01 – 6.96 (m, 2H, Ar-H), 6.51 (d, 1H,NH, J = 8.2 Hz), 5.50 (d, 1H, CO-H, J = 8.7 Hz), 4.03 – 3.87 (m, 4H, (CH3CH2)2), 3.76 – 3.68 (m, 1H, - CH(CH2)5), 1.89 – 1.81 (m, 2H, – CH(CH2)5), 1.70 – 1.62 (m, 2H, – CH(CH2)5)., 1.58 – 1.52 (m, 1H, - CH(CH2)5), 1.34 1.25(m, 2H, CH3CH2), 1.21 – 1.17 (m, 4H, CH3CH2), 1.15 (dt, 5H, - CH(CH2)5, J =4.8, 3.0 Hz). 13 C-NMR (100 MHz, CDCl3): δ 167.1 (d, J = 6.9 Hz), 163.1 (d, J =248.0 Hz), 132.2 (t, J = 3.3 Hz), 129.1 (d, J = 8.4 Hz), 115.6 (d, J = 21.8Hz), 78.2 (d, J = 6.4 Hz), 64.3 (d, J = 5.6 Hz), 48.2, 32.9 (d, J = 7.0 Hz), 25.4, 24.8, 16.0 (d, J = 6.8 Hz). 31 P NMR (162 MHz, CDCl3): δ -2.41. HRMS(ESI): Calculated for C 18 H 28O5NFP [M+H] + : 388.16836, found: 388.16693. Example 9: Synthesis of diethyl 2-(cyclohexylamino)-2-oxo-1-(o-tolyl)ethyl phosphate (9)

[0051] The only difference from Example 1 is that benzaldehyde in step S1 was replaced with 2-methylbenzaldehyde, and step S3 involved the reaction of intermediate II with diethyl phosphite to obtain compound (9), with a yield of 91.06%. The spectral data are as follows: 2-(cyclohexylamino)-2-oxo-1-(o-tolyl)ethyl phosphate diethyl ester (9): 1 H-NMR (400 MHz, CDCl3) δ 7.23 (t, 3H, Ar-H, J = 6.2 Hz), 7.15 (d, 1H, Ar-H, J = 6.7 Hz), 6.56 (d, 1H, NH, J = 8.2 Hz), 5.53 (d, 1H, CO-H, J = 8.7 Hz), 4.09 – 3.99 (m, 3H, (CH3CH2)2), 3.95 – 3.88 (m, 1H, (CH3CH2)2), 3.80 (ddd, 1H, (CH3CH2)2J =14.9, 10.7, 4.4 Hz), 2.35 (s, 3H, (CH3CH2)2), 1.97 – 1.88 (m, 2H, - CH(CH2)5),1.76 – 1.69 (m, 2H, - CH(CH2)5), 1.65 – 1.59 (m, 1H, – CH(CH2)5), 1.40 – 1.32(m, 2H, CH3CH2), 1.25 (t, J = 7.1 Hz, 4H, CH3CH2), 1.20 (dd, 5H, CH(CH2)5, J =12.9, 5.8 Hz). 13 C-NMR (100 MHz, CDCl3) δ 166.9, 161.0, 149.7, 143.4 (d, J =3.9 Hz), 137.7 (d, J = 3.6 Hz), 137.1, 132.4, 132.3, 129.3 (d, J = 34.0 Hz),128.8, 126.8, 122.2 (d, J = 273.4 Hz). 45.0, 40.6, 19.9, 16.5.31 P-NMR (162MHz, CDCl3) δ 2.46. HRMS (ESI): Calculated for C 19 H 31 O5NP [M+H] + : 384.19344,found:384.19202. Example 10: Synthesis of diethyl 2-(cyclohexylamino)-2-oxo-1-(m-tolyl)ethyl phosphate (10)

[0052] The only difference from Example 1 is that benzaldehyde in step S1 is replaced with m-methylbenzaldehyde, and in step S3, intermediate II reacts with diethyl phosphite to obtain compound (10), with a yield of 91%. The spectral data are as follows: 2-(cyclohexylamino)-2-oxo-1-(m-tolyl)ethyl phosphate diethyl ester (10): 1 H-NMR (400 MHz, CDCl3): δ 7.23 (t, 3H, Ar-H, J = 6.2 Hz), 7.15 (d, 1H, Ar-H, J = 6.7 Hz), 6.56 (d, 1H, NH, J = 8.2 Hz,), 5.53 (d, 1H, CO-H, J = 8.7 Hz,), 4.09 – 3.99(m, 3H, (CH3CH2)2), 3.95 – 3.88 (m, 1H, (CH3CH2)2), 3.80 (ddd, 1H, -CH(CH2)5, J = 14.9, 10.7, 4.4 Hz,), 2.35 (s, 3H, CH3), 1.97 – 1.88 (m, 2H, -CH(CH2)5), 1.76 – 1.69 (m, 2H, -CH(CH2)5), 1.65 – 1.59 (m, 1H, -CH(CH2)5), 1.40 – 1.32(m, 2H, CH3CH2), 1.25 (t, 4H, CH3CH2 J = 7.1 Hz), 1.20 (dd, 5H, -CH(CH2)5, J =12.9, 5.8 Hz). 13C-NMR (100 MHz, CDCl3): δ 166.9, 161.0, 149.7, 143.4 (d, J =3.9 Hz), 137.7 (d, J = 3.6 Hz), 137.1, 132.4, 132.3, 129.3 (d, J = 34.0 Hz),128.8, 126.8, 122.2 (d, J = 273.4 Hz). 45.0, 40.6, 19.9, 16.5. 31 P-NMR (162MHz, CDCl3): δ -2.46. HRMS (ESI): Calculated for C 19 H 31 O5NP [M+H] + :384.19344,found:384.19202 Example 11: Synthesis of diethyl 2-(cyclohexylamino)-2-oxo-1-(p-tolyl)ethyl phosphate (11)

[0053] The only difference from Example 1 is that benzaldehyde in step S1 is replaced with p-methylbenzaldehyde, and in step S3, intermediate II reacts with diethyl phosphite to obtain compound (11), with a yield of 91%. The spectral data are as follows: 2-(cyclohexylamino)-2-oxo-1-(p-tolyl)ethyl phosphate diethyl ester (C 11 ): 1 H-NMR (400 MHz, CDCl3): δ 7.32 (d, 2H, Ar-H, J = 8.1 Hz), 7.17 (d, 2H, Ar-H, J = 7.9 Hz,), 6.53 (d, 1H, NH, J = 8.3 Hz), 5.54 (d, 1H, CO-H, J = 8.7 Hz), 4.10 – 3.98(m, 3H, CH3CH2)2), 3.94 – 3.87 (m, 1H, (CH3CH2)2), 3.84 – 3.75 (m, 1H, -CH(CH2)5), 2.34 (s, 3H, CH3), 1.91 (dd, 2H, -CH(CH2)5, J= 19.3, 15.0 Hz), 1.77 –1.68 (m, 2H, -CH(CH2)5), 1.62 – 1.59 (m, 1H, -CH(CH2)5), 1.40 – 1.32 (m, 2H,CH3CH2), 1.28 – 1.23 (m, 4H, CH3CH2), 1.23 – 1.17 (m, 5H, -CH(CH2)5). 13 C-NMR (100 MHz, CDCl3): δ 167.5, 138.9, 133.2, 129.3, 127.1, 78.9 (d, J = 6.6Hz), 64.2 (d, J = 5.5 Hz), 48.1, 32.9 (d, J = 4.8 Hz), 25.4, 24.8, 21.2, 15.9 (dd, J = 6.9, 4.0 Hz). 31 P-NMR (162 MHz, CDCl3): δ -2.44. HRMS (ESI): Calculated for C 19 H 31 O5NP [M+H] + :384.19344, found:384.19238. Example 12: Synthesis of 2-(cyclohexylamino)-1-(2-nitrophenyl)-2-oxoethyl ester phosphate (12)

[0054] The only difference from Example 1 is that benzaldehyde in step S1 was replaced with p-2-nitrobenzaldehyde, and in step S3, intermediate II reacted with diethyl phosphite to obtain compound (12), with a yield of 90.16%. The spectral data are as follows: 2-(cyclohexylamino)-1-(2-nitrophenyl)-2-oxoethyl phosphate (12): 1 H-NMR (400 MHz, CDCl3) δ 8.02 (d, 1H, Ar-H, J = 8.1 Hz), 7.74 – 7.71 (m, 1H, Ar-H), 7.69 –7.64 (m, 1H, Ar-H), 7.55 – 7.51 (m, 1H, Ar-H), 6.78 (d, 1H, NH, J= 7.8 Hz), 6.40 (d, 1H, CO-H, J = 9.5 Hz), 4.17 – 4.06 (m, 4H, CH3CH2)2), 3.79 – 3.73 (m,1H, -CH(CH2)5), 1.91 (dd, 2H, -CH(CH2)5, J = 12.1, 2.8 Hz), 1.75 – 1.69 (m,2H, -CH(CH2)5), 1.63 – 1.58 (m, 1H, -CH(CH2)5), 1.39 – 1.33 (m, 2H, CH3CH2),1.30 (d, 4H, CH3CH2, J = 6.5 Hz), 1.27 (d, J = 7.0 Hz, 5H, -CH(CH2)5). 13 C-NMR (101 MHz, CDCl3) δ 165.8 (d, J = 5.7 Hz), 148.07 , 133.4 , 131.1 (d, J = 4.4Hz), 129.4 (d, J = 30.9 Hz), 124.9, 74.5 (d, J = 5.7 Hz), 64.7 (dd, J = 5.9,2.4 Hz), 48.6, 32.7, 25.4, 24.6, 16.0 (d, J = 6.6 Hz). 31 P-NMR (162 MHz, CDCl3) δ -1.68. HRMS (ESI): Calculated for C 18 H 28 O7N2P [M+H] + : 415.16286, found:415.16165. Example 13: Synthesis of 2-(cyclohexylamino)-1-(3-nitrophenyl)-2-oxoethyl phosphate (13)

[0055] The only difference from Example 1 is that benzaldehyde in step S1 was replaced with p-2-nitrobenzaldehyde, and in step S3, intermediate II reacted with diethyl phosphite to obtain compound (13), with a yield of 90.76%. The spectral data are as follows: 2-(cyclohexylamino)-1-(3-nitrophenyl)-2-oxoethyl phosphate: 1 H-NMR (400 MHz, CDCl3): δ 8.02 (d, 1H, Ar-H, J = 8.1 Hz,), 7.74 – 7.71 (m, 1H, Ar-H), 7.69 – 7.64 (m,1H, Ar-H), 7.55 – 7.51 (m, 1H, Ar-H), 6.78 (d, 1H, NH, J = 7.8 Hz), 6.40 (d, 1H, CO-H, J = 9.5 Hz), 4.17 – 4.06 (m, 4H, (CH3CH2)2), 3.79 – 3.73 (m, 1H, -CH(CH2)5), 1.91 (dd, 2H, -CH(CH2)5, J = 12.1, 2.8 Hz), 1.75 – 1.69 (m, 2H, -CH(CH2)5), 1.63 – 1.58 (m, 1H, -CH(CH2)5), 1.39 – 1.33 (m, 2H, CH3CH2), 1.30 (d, J = 6.5 Hz, 4H, CH3CH2), 1.27 (d, 5H, -CH(CH2)5, J = 7.0 Hz). 13 C-NMR (101 MHz, CDCl3) δ 165.8 (d, J = 5.7 Hz), 148.1 , 133.4 , 131.1 (d, J = 4.4 Hz), 129.4(d, J = 30.9 Hz), 124.9, 74.5 (d, J = 5.7 Hz), 64.7 (dd, J = 5.9, 2.4 Hz),48.6, 32.7, 25.4, 24.7, 16.0 (d, J = 6.6 Hz). 31 P-NMR (162 MHz, CDCl3) δ -2.24. HRMS (ESI): Calculated for C 18 H 28 O7N2P [M+H] +: 415.16286, found:415.16174. Example 14: Synthesis of 2-(cyclohexylamino)-1-(4-nitrophenyl)-2-oxoethyl phosphate (14)

[0056] The only difference from Example 1 is that benzaldehyde in step S1 was replaced with p-4-nitrobenzaldehyde, and in step S3, intermediate II reacted with diethyl phosphite to obtain compound (14), with a yield of 90.76%. The spectral data are as follows: 2-(cyclohexylamino)-1-(4-nitrophenyl)-2-oxoethyl phosphate: 1 H-NMR (400 MHz, CDCl3): δ 8.17 (d, 2H, Ar-H, J = 8.8 Hz), 7.60 (d, 2H, Ar-H, J = 8.7 Hz), 6.56 (d, 1H, NH, J = 8.1 Hz), 5.62 (d, 1H, CO-H, J = 9.0 Hz), 4.09 – 3.96 (m, 4H, (CH3CH2)2), 3.72 – 3.64 (m, 1H, -CH(CH2)5), 1.90 – 1.83 (m, 1H, -CH(CH2)5), 1.76 (dd, J = 12.2, 2.1 Hz, 1H, -CH(CH2)5), 1.70 – 1.60 (m, 2H, -CH(CH2)5), 1.58 – 1.52 (m, 1H, -CH(CH2)5), 1.34 – 1.26 (m, 2H, CH3CH2), 1.25 – 1.21 (m,4H, CH3CH2), 1.21 – 1.13 (m, 5H, -CH(CH2)5). 13 C-NMR (100 MHz, CDCl3): δ 166.1(d, J = 6.1 Hz), 148.1, 143.0 (d, J = 3.8 Hz), 127.8 , 123.7 , 77.5 (d, J =6.2 Hz), 64.7 (dd, J = 5.6, 3.9 Hz), 48.5, 32.8, 25.4, 24.7 (d,J = 2.6Hz), 16.1 (d, J = 6.6 Hz). 31 P-NMR (162 MHz, CDCl3): δ -2.26. HRMS (ESI): Calculated for C 18 H 28 O7N2P [M+H] + : 415.16286, found: 415.16168. Example 15: Synthesis of 2-(cyclohexylamino)-1-(3-methoxyphenyl)-2-oxoethyl ester phosphate (15)

[0057] The only difference from Example 1 is that benzaldehyde in step S1 is replaced with p-3-methoxybenzaldehyde, and in step S3, intermediate II reacts with diethyl phosphite to obtain compound (15) in 90% yield. The spectral data are as follows: 2-(cyclohexylamino)-1-(3-methoxyphenyl)-2-oxoethyl phosphate: 1 H-NMR (400 MHz, CDCl3): δ 7.29 (t, 1H, Ar-H, J = 7.9 Hz), 7.04 (d, 1H, Ar-H, J = 7.7 Hz),7.00 – 6.98 (m, 1H, Ar-H,), 6.91 – 6.90 (m, 1H, Ar-H,), 6.89 – 6.88 (m, 1H,NH), 6.54 (d, 1H, CO-H, J = 8.3 Hz), 5.55 (d, 1H, (CH3CH2)2, J= 8.8 Hz),4.11 – 4.01 (m, 3H, (CH3CH2)2), 3.98 – 3.91 (m, 1H, -CH(CH2)5), 3.81 (s, 3H,-OCH3), 3.81 – 3.75 (m, 1H, -CH(CH2)5), 1.97 – 1.87 (m, 2H, -CH(CH2)5), 1.77 –1.70 (m, 2H, -CH(CH2)5), 1.66 – 1.60 (m, 1H, -CH(CH2)5), 1.42 – 1.33 (m, 2H,CH3CH2), 1.30 – 1.25 (m, 4H, CH3CH2), 1.25 – 1.19 (m, 5H, -CH(CH2)5). 13 C-NMR (100 MHz, CDCl3): δ 167.2 (d, J = 6.8 Hz), 159.7, 137.6 (d, J = 3.4 Hz),129.7, 119.4, 114.8, 112.5, 78.9 (d, J = 6.5 Hz), 64.3 (d, J = 5.6 Hz),55.3, 48.2, 32.9 (d, J = 4.7 Hz), 25.4, 24.8, 16.0 (dd, J = 6.9, 3.5 Hz). 31 P-NMR (162 MHz, CDCl3): δ -2.51. HRMS (ESI): Calculated for C 19 H 31 O6NP [M+H] + : 400.18835, found: 400.18732. Example 16: Synthesis of 2-(tert-butylamino)-2-oxo-1-phenylethyl phosphate (16)

[0058] The only difference from Example 1 is that isocyanocyclohexane in step S1 is replaced with tert-butyl isocyanate, and in step S3, intermediate II reacts with diethyl phosphite to obtain compound (16) in 90% yield. The spectral data are as follows: 2-(tert-butylamino)-2-oxo-1-phenylethyl phosphate: 1H NMR (400 MHz, CDCl3): δ 7.45(dd, 2H, Ar-H, J = 7.3, 1.9 Hz), 7.38 (dd, 3H, Ar-H, J = 8.1, 2.5 Hz), 6.52(s, 1H, NH), 5.51 (d, 1H, CO-H, J = 8.6 Hz), 4.01 (dddd, 4H, (CH3CH2)2, J =24.9, 17.6, 10.0, 2.9 Hz), 1.39 (s, 9H, -C(CH3)3), 1.27 (d, 3H, CH3CH2, J =7.1 Hz), 1.22 (d, 3H, CH3CH2, J = 7.1 Hz). 13 C NMR (100 MHz, CDCl3): δ 167.45(d, J = 6.7 Hz), 136.32 (d, J = 3.4 Hz), 129.00, 128.63, 127.19, 79.14 (d, J = 6.6 Hz), 64.25 (d, J = 5.5 Hz), 51.52 , 28.67 , 15.98 (d, J = 7.0 Hz). 31 P-NMR (162 MHz, CDCl3): δ -2.51. HRMS (ESI): Calculated for C 16 H 27 O5NP [M+H] + : 344.16214, found: 344.16190. Example 17: Synthesis of 2-(tert-butylamino)-2-oxo-1-(m-tolyl)ethyl ester phosphate (17)

[0059] The only difference from Example 16 is that benzaldehyde in step S1 was replaced with 3-methylbenzaldehyde, and in step S3, intermediate II reacted with diethyl phosphite to obtain compound (17), with a yield of 90%. The spectral data are as follows: 2-(tert-butylamino)-2-oxo-1-(m-tolyl) ethyl phosphate:1 H-NMR (400 MHz, CDCl3): δ 7.20 – 7.15 (m, 3H, Ar-H), 7.08 (d, 1H, Ar-H, J = 7.0 Hz), 6.41 (s, 1H, NH), 5.38 (d, 1H, CO-H, J = 8.6 Hz), 4.02 – 3.84 (m, 4H, (CH3CH2)2), 2.28 (s,3H, CH3), 1.31 (s, 9H, -C(CH3)3), 1.18 (t, 3H, CH3CH2, J = 6.1 Hz), 1.14 (dd,3H, CH3CH2, J = 9.6, 3.0 Hz). 13 C-NMR (100 MHz, CDCl3): δ 167.6 (d, J = 6.7Hz), 138.3, 136.2 (d, J = 3.4 Hz), 129.8, 128.5, 127.9, 124.1, 79.2 (d, J = 6.6 Hz), 64.2 (dd, J = 5.6, 2.3 Hz), 51.5, 28.7, 21.4, 15.9 (dd, J =6.9, 1.7 Hz). 31 P-NMR (162 MHz, CDCl3): δ -2.48. HRMS (ESI): Calculated forC 16 H 27 O5NP [M+H] + : 358.17779, found: 358.17712. Example 18: Synthesis of 2-(tert-butylamino)-2-oxo-1-(p-tolyl)ethyl phosphate (18)

[0060] The only difference from Example 16 is that benzaldehyde in step S1 was replaced with 4-methylbenzaldehyde, and in step S3, intermediate II reacted with diethyl phosphite to obtain compound (18), with a yield of 92%. The spectral data are as follows: 2-(tert-butylamino)-2-oxo-1-(p-tolyl)ethyl phosphate:1 H-NMR (400 MHz, CDCl3): δ 7.33 (d, 2H, Ar-H, J = 8.1 Hz), 7.19 (d, 2H, Ar-H, J = 7.9 Hz), 6.50 (s,1H, NH), 5.47 (d, 1H, CO-H, J = 8.6 Hz), 4.13 – 3.95 (m, 4H, (CH3CH2)2), 2.36(s, 3H, -CH3), 1.39 (s, 9H, -C(CH3)3), 1.28 – 1.25 (m, 3H, CH3CH2), 1.25 – 1.22(m, 3H, CH3CH2). 13 C-NMR (100 MHz, CDCl3): δ 167.6 (d, J = 6.7 Hz), 138.9, 133.4 (d, J = 3.4 Hz), 129.3, 127.1, 79.1 (d, J = 6.6 Hz), 64.2 (dd, J =5.6, 2.0 Hz), 51.5, 28.7, 21.3, 16.0 (d, J = 6.8 Hz). 31 P-NMR (162 MHz, CDCl3): δ -2.51. HRMS (ESI): Calculated for C 17 H 29 O5NP [M+H] + : 358.17779, found: 358.17703. The synthesis methods of the remaining compounds 19 to 46 in Table 1 differ from those in Example 1 only in that the raw materials in steps S1 and S3 are replaced with the groups described in Table 1 to obtain compounds 19 to 46.

[0061] Test Example 1: 2-(tert-butylamino)-2-oxo-1-(m-tolyl)ethyl ester phosphate prepared in Example 17 against receptor protein IC 50 Measurement (1) Protein expression and extraction: Add 1 µL of the digestion product to pre-prepared E. coli BL21 competent cells and place on ice for 30 min; heat shock accurately in a 42°C water bath for 90 s, then quickly transfer to ice and incubate on ice for another 3 min; add 0.9 mL of LB liquid medium under aseptic conditions and incubate at 37°C at low speed for 1 h; spread 200 µL of the suspension onto LB solid medium (with added ampicillin) and incubate upside down at 37°C for 12–16 h; screen for positive clones. PYLs and HAB1 were induced to express in E. coli. Single colonies were selected and placed in 50 mL of LB broth (containing ampicillin) and incubated in a constant temperature shaking incubator (220 rpm, 37 °C) for 14 h. 50 mL of the bacterial culture was then pipetted into 1000 mL of LB broth and incubated under the same conditions. When the OD600 value was approximately 0.8, 1 mL of 1 mmol / L IPTG was added and the culture was continued with shaking (160 rpm, 16 °C) for approximately 14 h. The culture was then centrifuged (4000 rpm, 15 min, 4 °C). The cells were then lysed using lysis buffer (usually 30 mL) and thoroughly disrupted using an autoclave (usually 3 times). The cells were then centrifuged (16,000 rpm, 4 °C, 25 min) and the supernatant was collected. Protein purification: ① The protein was purified using a nickel affinity chromatography column; ② The target protein obtained from the previous nickel affinity chromatography step was concentrated to the loading volume at 4℃, and then purified using a molecular sieve. Finally, the purified protein was detected by SDS-PAGE polyacrylamide gel electrophoresis.

[0062] (2) Determination of enzyme activity: HAB1 belongs to the PP2C dephosphorylase protein family. Therefore, this experiment selected two phosphate substrates to determine enzyme activity. One is a phosphorylated peptide substrate (HSQPKpSTVGTP), which is derived from HAB1's specific substrate SnRK2.6. When ABA or its functional analogue is not added, PYL can form a complex with HAB1, releasing phosphate groups from the peptide. The released phosphate groups can be detected using a Phosphate Assay Kit. The specific steps for hydrolyzing phosphorylated peptide substrates are as follows: A. Preparation of the reaction system: The reaction system contains 50 nM HAB1, 250 nM PYLs, different concentrations of compounds, and reaction buffer (1 mM EGTA, 250 mM Imidazole, 25 mM MgCl2, 0.5 mM BSA, 0.04 ml 2-mercaptoethanol, pH 7.2). Incubate at room temperature for 30 min (Note: The experiment must adhere to the principle of single variable; also, because PP2C is unstable and easily inactivated, it must be added last).

[0063] Add the peptide substrate to the reaction system (B), mix well, and incubate at 30 °C for 30 min.

[0064] Add Phosphate Assay Kit colorimetric solution to the reaction system, mix well, and then add to a 96-well plate. (Note: Ensure there are no air bubbles).

[0065] D used an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance of reaction systems containing different concentrations of compounds at 630 nm and calculated the IC50 value of the compounds.

[0066] (3) Test results are as follows Figure 5 As shown: More than 47 derivatives were synthesized, and their in vitro activity was detected using a specific substrate (SnRK2.6 phosphopeptide). Among them, 27 showed good activity against PYLs3 in the activity test.

[0067] Test Example 2: Determination of Plant Physiological Activities 1. Plant physiological experiments (live experiments) mainly consist of root length measurement, infrared phenotype, and stomatal regulation experiments such as drought stress.

[0068] The root length determination method was as follows: First, Arabidopsis seeds were disinfected and cleaned, then sown in 1 / 2 MS medium and cultured for 5 days. After culturing in the medium, they were transferred to 1 / 2 MS medium containing 50 μM ABA analogue, with 7 soybean seedlings in each treatment. Then, the petri dishes were placed vertically in an incubator, and the length of the wild-type taproot was measured after 4 days to determine the effect of ABA analogue on plant roots.

[0069] In the infrared phenotypic experiment, wild-type Arabidopsis seeds, after being disinfected and cleaned, were first sown on 1 / 2 MS medium and cultured for 7 days. They were then transplanted into nutrient pots containing nutrient soil, with one plant per pot. The seedlings were allowed to recover for 40 days at a temperature of 20℃ and a photoperiod of 8h light / 16h darkness. They were then treated with a drug, while the control group received the same DMSO treatment. The compound was delivered to the Arabidopsis leaves through spraying and other means. Changes in the temperature of the soybean leaves were detected using an infrared camera.

[0070] The drought stress experiment began with disinfecting and cleaning wild-type Arabidopsis seeds, which were cultured on 1 / 2 MS medium for 7 days. Six seedlings were then transplanted into pots containing a measured amount of nutrient soil. The seedlings were allowed to acclimate for 25 days at 20°C with a photoperiod of 8 hours of light and 16 hours of darkness. The soil was then watered until saturated, and the plants were treated with a pesticide. The control group received the same DMSO treatment, with treatments repeated every 3 days. After the control group plants wilted, they were rehydrated and cultured for another 2 days. The mortality rate of the plants was then recorded.

[0071] 2. Test results are as follows Figure 6-10 As shown: Depend on Figure 6 As shown, different concentrations (12.5 μM, 25 μM, 50 μM, 100 μM) of the compound prepared in Embodiment 17 of this invention (hereinafter referred to as C17), ABA as the treatment group, and DMSO as the control group were used to conduct experiments on Arabidopsis seedlings to verify the effect of the compound on the early growth stage. As shown in the figure, C17 at medium and low concentrations (12.5 μM, 25 μM, 50 μM) promoted the growth of Arabidopsis, especially in promoting lateral root growth. When Arabidopsis seedlings were treated with 25 μM and 50 μM C17, compared with the DMSO group, it was found that not only was root length growth promoted, but also lateral root growth was promoted. This conclusion proves that seedlings treated with the above concentrations can better promote root growth, thereby improving the crop's ability to absorb water and thus improving its drought resistance.

[0072] like Figure 7 As shown, using chili peppers and green beans as model crops, there was no temperature difference in the initial stage, but after treatment with C17 or ABA for 24 / 48 hours, a significant temperature increase was observed compared to the control group. Crucially, C17 maintained its efficacy for 3-4 days at low concentrations (30 μM), indicating superior chemical stability.

[0073] This invention further evaluates water conservation by measuring pore opening size and water loss, as shown in the following figures. Figure 8 As shown, we verified the drug's ability to promote stomatal closure by measuring the stomatal conductance and leaf water loss rate of green beans. Figure 8 As shown in the figure, the stomata of the treatment groups (ABA, C17) were clearly closed. Although the stomatal conductance was slightly higher than that of ABA, it was significantly lower than that of DMSO. This indicates that C17 can induce stomatal closure in plants. Furthermore, the water loss rate of plant leaves after application was measured. Figure 8 This also demonstrates the effect of drugs on inducing stomatal closure in plants.

[0074] Based on the above conclusions, to further verify the effectiveness of C17 in drought resistance, we conducted an in vivo drought resistance experiment using Arabidopsis thaliana as a model plant. The results are as follows: Figure 9 As shown, plants treated with C14 (the compound prepared in Example 14 of this invention, hereinafter referred to as C14) and C17 exhibited significantly improved drought resistance. After 25 days of rehydration, the survival rate of the treatment groups (treated with C14, C17, and ABA) was significantly higher than that of the control group (DMSO), and there was no significant difference between the treatment groups treated with C14 and C17 and the treatment group treated with ABA. The above experiments demonstrate that C14 and C17 are potential drought-resistant compounds similar to ABA.

[0075] However, because the leaves of plants treated with C14 showed obvious whitening, in order to verify the toxicity of C17 to plants and its mechanism of action, this invention also determined the effects of the compound on growth status and ROS. The results are as follows: Figure 10 As shown, growth analysis of *Nicotiana benthamiana* 20 days after treatment revealed that, after C17 application, there was no significant reduction in stem fresh weight and taproot length, but root fresh weight increased by approximately 50%. ROS staining, malondialdehyde (MDA), and peroxidase (POD) assays of *Nicotiana benthamiana* leaves showed that drought-induced ROS accumulation was reduced and MDA and POD activities were increased in plants treated with C17.

[0076] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A phosphate ester compound containing an amide group, characterized in that, Its chemical structural formula is shown in general formula I: ； Wherein, R1 is selected from H, 2-Cl, 3-Cl, 2-Br, 3-Br, 2-CH3, 2-OCH3; R2 is selected from -CH2CH3, -CH3, -CH(CH3)2, -CH2CH(CH3)2; R3 is selected from -C6H 11 -C(CH3)3.

2. A method for preparing the phosphate ester compound containing an amide group as described in claim 1, characterized in that, The preparation steps include the following: Under the first catalyst, and o-aminopyridine and The mixture was stirred and refluxed in the first solvent to obtain intermediate I; In the presence of a second catalyst, intermediate I was added to a second solvent and the mixture was stirred at room temperature to obtain intermediate II. In the presence of a third catalyst, intermediate II is mixed with dialkyl phosphite and reacted at room temperature to obtain the phosphate compound containing the amide group.

3. The method for preparing the phosphate ester compound containing an amide group according to claim 2, characterized in that, The chemical structural formula of intermediate I is shown below: 。 4. The method for preparing the phosphate ester compound containing an amide group according to claim 3, characterized in that, The chemical structural formula of intermediate II is shown below: 。 5. The method for preparing the phosphate ester compound containing an amide group according to claim 4, characterized in that, The first catalyst is lanthanum chloride heptahydrate.

6. The method for preparing the phosphate ester compound containing an amide group according to claim 5, characterized in that, The second catalyst is diethyl iodophenyl ester and p-toluenesulfonic acid.

7. The method for preparing the phosphate ester compound containing an amide group according to claim 6, characterized in that, The third catalyst is DBU.

8. The method for preparing the phosphate ester compound containing an amide group according to claim 7, characterized in that, The first solvent is ethanol.

9. The method for preparing the phosphate ester compound containing an amide group according to claim 8, characterized in that, The second solvent is dichloromethane.

10. The use of the amide-containing phosphate compound as described in claim 1 in the preparation of ABA analogs to enhance the regulation of plant drought stress.

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