Dihydrogibberellin derivative as well as preparation method and application thereof

Abstract

The invention provides a dihydrogibberellin derivative as well as a preparation method and application thereof. The structural formulas of the dihydrogibberellin derivative are shown as a formula (I) and a formula (II), in the formula (I) and the formula (II), R is selected from phenyl, benzyl or naphthyl. According to the preparation method, N-substituted thiosemicarbazide is spliced to a dihydrogibberellin skeleton structure through a Schiff base reaction, and the derivative is synthesized. In addition, the dihydrogibberellin derivative disclosed by the invention has good plant growth inhibition activity. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the technical field of plant growth regulation, and particularly relates to a dihydrogibberellin derivative and a preparation method thereof. Background Art

[0002] Dihydrogibberellin is derived from gibberellic acid (GA3) and is a plant growth inhibitor with application potential. It can play a role in controlling plant growth and preventing lodging in agriculture. Dihydrogibberellin acts as a substrate competitive inhibitor of GA3β-hydroxylase (GA3ox), preventing the conversion of inactive GA9 and GA 20 into bioactive GA4 and GA1, thereby inhibiting the biosynthesis of active gibberellins and causing plant growth to be hindered and plant height to decrease.

[0003] With the development of modern agriculture, more and more plant growth regulation technologies are needed. For example, in Patent Document 1 (CN 116478117 B) of the applicant, a photo-removable protecting group, o-nitrobenzyl, is used as a carboxyl protecting group, and a synthetic route that can be directly used without removing the carboxyl protecting group in the last step of the organic reaction is adopted to achieve the preparation of dihydrogibberellin derivatives and their effective application in light-controlled release. This discovery provides a new idea for the derivation based on the gibberellin skeleton, and the development and application of various derivatives of dihydrogibberellin have also become a hot topic in the research of the field of plant growth regulation technology. Summary of the Invention

[0004] The object of the present invention is to provide a dihydrogibberellin derivative and a preparation method thereof. To achieve this object, the inventors analyzed the crystal structure and action mechanism of the target enzyme that has been resolved, and carried out structural modification at specific sites of the dihydrogibberellin molecule to enhance the binding ability between the inhibitor and the protein, thereby helping to improve the inhibitory effect of the compound on plant growth. Based on this, a series of gibberellin receptor inhibitors derived from gibberellin GA3 as raw materials were designed, and the biological activities of this series of compounds were measured, and some dihydrogibberellin derivatives with excellent activities for regulating plant growth were found.

[0005] In order to study and discover new, highly efficient and safe insecticides, the inventors used rice GA3β-hydroxylase as the target and dihydrogibberellin as the lead compound, protected the carboxyl group with a photo-removable protecting group, and then through a Schiff base reaction, spliced N-substituted thiosemicarbazide onto the dihydrogibberellin skeleton structure to synthesize a series of derivatives with good activities, in order to achieve better plant growth inhibitory activity.

[0006] According to the first aspect of the present invention, there is provided a dihydrogibberellin derivative, the structural formula of which is shown in Formula (I) and Formula (II), and are respectively denoted as CAUZL-A and CAUZL-B;

[0007]

[0008] In formula (I) and formula (II), R is selected from phenyl, benzyl or naphthyl; the phenyl is substituted or unsubstituted phenyl; the substituted phenyl is p-chlorophenyl, p-methylphenyl, p-trifluoromethylphenyl, p-bromophenyl, p-methoxyphenyl, o-fluorophenyl, o-methylphenyl, 3-bromo-5-trifluoromethylphenyl or 4-chloro-2-trifluoromethylphenyl; the naphthyl is 1-methylnaphthyl, 2-methylnaphthyl, methoxynaphthyl, chloronaphthyl, bromonaphthyl, etc.

[0009] In a second aspect of the present invention, a preparation method of the above-mentioned dihydrogibberellin derivative is provided, including the following steps:

[0010] (1) The compound of formula (III) and the compound of formula (IV) undergo a Schiff base reaction under the action of a catalyst and heating conditions to obtain the compound of formula (I);

[0011]

[0012] (2) After adding an oxidant to the compound of formula (I) in a chloroform solvent, an oxidation ring-closing reaction is carried out to obtain the compound of formula (II);

[0013]

[0014] In the above-mentioned formula (IV), R is the same as that in formula (I) and formula (II);

[0015] In the above step 1), when the Schiff base reaction occurs between the compound of formula (III) and the compound of formula (IV), the Schiff base reaction is as follows: in the presence of a catalyst, the carbonyl group is first activated, and then reacted with the compound of formula (IV) under heating conditions to obtain the compound of formula (I);

[0016] The catalyst is p-toluenesulfonic acid, and its dosage is 5%-20% by weight relative to the compound of formula (III);

[0017] The molar ratio of formula (III) to formula (IV) is 1:1 - 1:1.5; preferably 1:1 - 1:1.4;

[0018] The reaction temperature is 80 - 120 °C and the time is 3 - 6 hours.

[0019] In the above step (2), the oxidant is manganese dioxide, and its dosage is 5 - 20 equivalents of the compound of formula (I), and the reaction time is 2 - 5 hours.

[0020] In the third aspect of the present invention, there is provided an application of a dihydrogibberellin derivative as described in the first aspect of the present invention as a gibberellin GA3β-hydroxylase inhibitor.

[0021] In the fourth aspect of the present invention, there is provided a plant growth regulator, and its active ingredient is the dihydrogibberellin derivative as described in the first aspect of the present invention.

[0022] In a specific case, the dosage form of the plant growth regulator is a pharmaceutically acceptable dosage form; the dosage forms include emulsifiable concentrates, wettable powders, suspensions, powders, soluble powders, aqueous solutions, water-dispersible powders, smoke agents, granule agents or seed coating agents.

[0023] The plant growth regulator herein can be used in Arabidopsis thaliana and cereal crops such as corn, wheat, and rice.

[0024] Advantages of the present invention

[0025] Taking rice gibberellin GA3β-hydroxylase as a biological target, the present invention synthesizes novel-structured dihydrogibberellin derivatives. The dihydrogibberellin derivatives of the present invention have good plant growth inhibitory activity, and the root length of Arabidopsis thaliana and the elongation of the second leaf sheath hypocotyl of rice of some compounds exceed those of the control agent dihydrogibberellin. The compounds described in the present invention have the application potential of plant growth retardants.

[0026] In addition, when the dihydrogibberellin derivatives of the present invention are used as plant growth regulators, they at least have the effects of delaying plant growth, inhibiting stem elongation, shortening plant internodes, improving plant stress resistance, promoting leaf growth, promoting flower stem differentiation or increasing plant yield, and are more effective especially when the plants are Arabidopsis thaliana and cereal crops such as corn, wheat, and rice. Description of the drawings

[0027] Figure 1 It is the nuclear magnetic resonance hydrogen spectrum of compound CAUZL-A01.

[0028] Figure 2 It is the nuclear magnetic resonance carbon spectrum of compound CAUZL-A01.

[0029] Figure 3 It is the nuclear magnetic resonance hydrogen spectrum of compound CAUZL-B01.

[0030] Figure 4 It is the nuclear magnetic resonance carbon spectrum of compound CAUZL-B01. Detailed implementation manners

[0031] The present invention will be described below through specific examples. It should be noted that the descriptions and examples given here are only for describing the specific implementation manners of the present invention to make it easier for those skilled in the art to understand the present invention, and they are not intended to limit the scope of the present invention.

[0032] In the following examples, the experimental methods are conventional methods unless otherwise specified; the reagents and materials can be obtained from commercial sources unless otherwise specified.

[0033] Preparation of Compound CAUZL-A01, R = 4-Cl-Ph in Example 1

[0034]

[0035] Synthesis of Compound of Formula (IV)

[0036] (1) Thionyl chloride (4 mL, 22 mmol) and 20 mL of dichloromethane were added to a 50 mL round-bottom flask. p-Chloroaniline (2.56 g, 20 mmol) and triethylamine solution (3.2 mL, 22 mmol) were dissolved in 5 mL of dichloromethane and added dropwise into the flask under nitrogen protection and in an ice bath. After stirring for 1 h, the solvent was removed by distillation under reduced pressure to obtain 3.70 g of p-chlorophenyl isothiocyanate with a yield of 99%. The obtained product was directly used as a reactant (2.2 g, 13 mmol) and dissolved in 20 mL of ethanol. A mixture of 5 mL of hydrazine hydrate (130 mmol) and water was slowly added dropwise to the system with vigorous stirring. A solid precipitated rapidly in the system. After stirring for 30 min, the mixture was filtered to obtain N-(4-chlorophenyl)thiocarbazide, and 2.6 g of the compound shown in Formula (IV) was obtained with a yield of 98%.

[0037] Synthesis of Compound of Formula (III)

[0038] (2) Gibberellin GA3 (17.3 g, 50 mmol), anhydrous pyridine (150 mL), acetic anhydride (100 mL) and a catalytic amount of DMAP (0.3 g, 2.5 mmol) were successively added to a 500 mL round-bottom flask and stirred at room temperature for 16 h. After the reaction solution was quenched with water, it was washed with 2M HCl solution to remove residual pyridine and by-product salts. It was extracted with dichloromethane (3×150 mL). The combined organic phases were dried over anhydrous sodium sulfate and concentrated, and purified by recrystallization from a mixed solvent of ethyl acetate / petroleum ether to obtain 20.6 g of white crystalline product 3,13-diacetylated gibberellin with a yield of 99%.

[0039] (3) Dissolve 3,13-diacetylated gibberellin (8.6 g, 20 mmol), EDCI (8.6 g, 30 mmol) and DMAP (0.1 g, 1 mmol) in a 300 mL reaction flask with anhydrous dichloromethane (100 mL). Under nitrogen protection and ice bath conditions, slowly add dropwise a dichloromethane solution (20 mL) of 2-nitrobenzyl alcohol (3.1 g, 20 mmol). After the addition, restore to room temperature and continue the reaction for 3 hours. After the reaction is completed, quench with water and treat successively with 1M HCl solution, dichloromethane (3×100 mL) and saturated brine (2×100 mL). The organic phase is dried and concentrated, and then recrystallized from methanol to obtain 10.7 g of 2-nitrobenzyl esterified product with a yield of 95%.

[0040] (4) Take the 2-nitrobenzyl esterified product (8.7 g, 15 mmol) and add it to a 250 ml round-bottom flask, dissolve it with 100 ml of ethyl acetate, add 0.8 g of Rh-Al2O3, and react overnight under normal pressure hydrogen. After the reaction is completed, filter and recover the catalyst with diatomaceous earth, and distill off the solvent under reduced pressure without further purification to obtain 8.4 g of 1,2,16,17 double bond reduction product with a yield of 98%.

[0041] (5) Dissolve the reduction product in step (4) (8.7 g, 15 mmol) in methanol (100 mL), gradually add dropwise saturated K2CO3 aqueous solution to adjust the pH to 9-10, stir and react for 30 minutes, and then neutralize with 1M HCl. The mixture is extracted with dichloromethane (3×60 mL), washed with saturated brine (2×100 mL), dried and concentrated, and then purified by silica gel column chromatography to obtain 7.5 g of C-3 acetyl deprotected product with a yield of 95%.

[0042] (6) Take the compound in step (5) (7.9 g, 15 mmol) and add it to a 100 mL round-bottom flask, dissolve it in 50 mL of dichloromethane, then add pyridinium chlorochromate (3.2 g, 30 mmol), stir for 5 hours, distill off the solvent under reduced pressure, and separate by column chromatography (petroleum ether / ethyl acetate from 10:1 to 3:1) to obtain the compound of formula (III) (yield 96%).

[0043] Synthesis of the compound of formula (I)

[0044] Dissolve the compound of formula (III) (2.6 g, 5 mmol) and the compound of formula (IV) (1.0 g, 5 mmol) in 20 mL of toluene, add 0.4 g of p-toluenesulfonic acid as a catalyst, and stir at 80 °C for 5 h. Distill off the solvent under reduced pressure, and separate by column chromatography (petroleum ether / ethyl acetate ratio from 20:1 to 5:1) to obtain 3.3 g of the compound of formula (I) with a yield of 93%, denoted as CAUZL-A01.

[0045] Other compounds of the general formula CAUZL-A were all prepared by referring to the above method. Their compound numbers, corresponding substituents, and physicochemical data are shown in Table 1.

[0046] Table 1 Compound numbers, substituents, and physicochemical data of some compounds in the CAUZL-A series

[0047]

[0048] Figure 1 This is the 1H NMR spectrum of compound CAUZL-A01. It can be seen from the figure that the peaks at δ 9.13 ppm and 8.84 ppm are the peaks of the active hydrogens on the thiosemicarbazide structure. There are a total of 8 hydrogens at δ 7.33 - 8.09 ppm, which belong to the hydrogens of the two benzene rings in the structure. These characteristics indicate that p-chlorophenylthiosemicarbazide has been successfully connected to the skeleton. In addition, the 13C NMR spectrum of compound CAUZL-A01 Figure 2 also confirmed this. The newly formed peak near 130 ppm is the signal peak of the benzene ring.

[0049] Example 2 Preparation of compound CAUZL-B01, R = 4-Cl-Ph

[0050]

[0051] The compound of formula (I) (3.5 g, 5 mmol) was added to a 50 mL round-bottom flask, dissolved in 20 mL of chloroform, and manganese dioxide (4.35 g, 50 mmol), the oxidant, was added. After stirring for 3 hours, the oxidant was filtered off with diatomaceous earth, and the solvent was removed by distillation under reduced pressure. The compound of formula (II) (3.36 g, yield 95%) was obtained by gradient separation by column chromatography (the ratio of petroleum ether / ethyl acetate was from 20:1 to 5:1), denoted as CAUZL-B01.

[0052] Other compounds of the general formula CAUZL-B were all prepared by referring to the above method. Their compound numbers, corresponding substituents, and physicochemical data are shown in Table 2.

[0053] Table 2 Compound numbers, substituents, and physicochemical data of some compounds in the CAUZL-B series

[0054]

[0055] Figure 3 This is the 1H NMR spectrum of compound CAUZL-B01. It can be seen from the figure that the signal peaks of the two active hydrogens of thiosemicarbazide disappear, indicating the formation of the thiadiazole structure. At the same time, from Figure 4 the 13C NMR spectrum of CAUZL-B01 also further confirmed the successful occurrence of the reaction. Through the oxidative cyclization reaction, the compound of formula (II) was obtained.

[0056] Example 3 Determination of the Arabidopsis growth regulatory activity of compounds of general formula CAUZL-A and CAUZL-B

[0057] Determination method (Arabidopsis root length method): The elongation length of Arabidopsis roots was used as an indicator. The Arabidopsis variety was Columbia wild type (Arabidopsis thaliana). Different concentrations of the dihydrogibberellin derivative liquid medicine of the present invention were mixed with the melted half-strength MS medium to prepare a medicated medium with a final concentration of 100 μM. Five-day-old Arabidopsis seedlings were transferred to the medicated medium and after 7 days of cultivation, the elongation of Arabidopsis root length was measured. The activity data of the compounds of general formula CAUZL-A and CAUZL-B series in inhibiting root length are shown in Table 3.

[0058] Table 3 Inhibition rate (%) of the root length of Arabidopsis by CAUZL-A and B series compounds at a concentration of 100 μmol / L

[0059] Compound Inhibition rate Compound Inhibition rate CAUZL-A01 24.3％ CAUZL-A12 41.9％ CAUZL-A02 32.1％ CAUZL-B01 12.6％ CAUZL-A03 23.9％ CAUZL-B02 23.0％ CAUZL-A04 34.1％ CAUZL-B03 19.5％ CAUZL-A05 59.4％ CAUZL-B04 13.4％ CAUZL-A06 21.0％ CAUZL-B05 23.4％ CAUZL-A07 28.6％ CAUZL-B06 21.0％ CAUZL-A08 23.4％ CAUZL-B07 23.2％ CAUZL-A09 34.7％ CAUZL-B08 20.4％ CAUZL-A10 10.2％ CAUZL-B09 31.5％ CAUZL-A11 14.1％

[0060] As can be seen from Table 3, both the compounds of CAUZL-A and CAUZL-B series have obvious inhibitory effects on the elongation of Arabidopsis root length. Among them, the compounds of CAUZL-A series show better inhibitory effects, and the inhibition rates are all above 10%. Among the compounds in the same series, the compounds with electron-donating group substitutions on the benzene ring (A02, A07, A09) have better activity than those with electron-withdrawing substitutions. The growth inhibition rates of compounds CAUZL-A05 and CAUZL-A12 on Arabidopsis root length reached 59.4% and 41.9% respectively. Therefore, it can be used as a reference when selecting inhibitors for different crops.

[0061] Example 4 Determination of the rice growth regulatory activity of compounds of general formula CAUZL-A and CAUZL-B

[0062] Determination method (rice dropping method): The length of the hypocotyl under the second leaf sheath of rice was used as an indicator. The rice variety was wild rice Nipponbare (Oryza sativa L.). When the tip of the second leaf of rice just emerged, the dihydrogibberellin derivative liquid medicine with a final concentration of 100 μM was dropped between the coleoptile and the leaf sheath of rice, and 1 μL was dropped on each plant. After 7 days of cultivation, the length of the hypocotyl under the second leaf sheath was measured. The activity data of the compounds of general formula CAUZL-A and CAUZL-B series in inhibiting root length are shown in Table 4.

[0063] Table 4 Inhibition rate (%) of the hypocotyl elongation of rice by CAUZL-A and B series compounds at a concentration of 100 μmol / L

[0064] Compound Inhibition rate Compound Inhibition rate CAUZL-A01 26.1％ CAUZL-A12 39.5％ CAUZL-A02 34.6％ CAUZL-B01 13.5％ CAUZL-A03 21.4％ CAUZL-B02 32.0％ CAUZL-A04 28.2％ CAUZL-B03 19.5％ CAUZL-A05 46.2％ CAUZL-B04 16.4％ CAUZL-A06 27.5％ CAUZL-B05 23.5％ CAUZL-A07 45.2％ CAUZL-B06 29.0％ CAUZL-A08 30.4％ CAUZL-B07 32.0％ CAUZL-A09 35.6％ CAUZL-B08 14.5％ CAUZL-A10 19.2％ CAUZL-B09 33.0％ CAUZL-A11 22.5％

[0065] As can be seen from Table 4, both the compounds of CAUZL-A and CAUZL-B series have obvious inhibitory effects on the elongation of the hypocotyl under the second leaf sheath of rice, and their inhibition rates are all above 13%. Among them, the overall inhibition rate of the compounds of CAUZL-A series is better. The inhibition rates of the two best compounds, CAUZL-A05 and CAUZL-A12, on the growth of rice reach 46.2% and 39.5% respectively. The above data can be used as a reference when selecting inhibitors for different crops.

[0066] In summary, the dihydrogibberellin derivatives of the present invention have good plant growth inhibitory activity. Some compounds exceed the control agent dihydrogibberellin in terms of the root length of Arabidopsis thaliana and the elongation of the hypocotyl under the second leaf sheath of rice. Therefore, the compounds of the present invention have the application potential of plant growth retardants.

[0067] The description of the above embodiments is only used to help understand the method of the present invention and its core idea. It should be noted that for those of ordinary skill in the art of this technology, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A dihydrogibberellin derivative, whose structural formula is shown as formula (I) and formula (II); Among them, In formula (I) and formula (II), R is selected from phenyl, benzyl or naphthyl.

2. The dihydrogibberellin derivative according to claim 1, wherein, The phenyl is a substituted or unsubstituted phenyl; the substituted phenyl is p-chlorophenyl, p-methylphenyl, p-trifluoromethylphenyl, p-bromophenyl, p-methoxyphenyl, o-fluorophenyl, o-methylphenyl, 3-bromo-5-trifluoromethylphenyl or 4-chloro-2-trifluoromethylphenyl; the naphthyl is 1-methylnaphthyl, 2-methylnaphthyl, methoxynaphthyl, chloronaphthyl, bromonaphthyl.

3. A method for preparing the dihydrogibberellin derivative according to claim 1 or 2, characterized in that, It includes the following steps: (1) Heating the compound of formula (III) and the compound of formula (IV) under the action of a catalyst to carry out a Schiff base reaction to obtain the compound of formula (I); (2) Dissolving the compound of formula (I) in chloroform, adding an oxidant, and then carrying out an oxidative cyclization reaction to obtain the compound of formula (II). In the above formula (IV), R is the same as that in the above formula (I) and formula (II).

4. The preparation method according to claim 3, characterized in that, The catalyst is p-toluenesulfonic acid, and its dosage is 5%-20% by weight relative to the compound of formula (III).

5. The preparation method according to claim 3, characterized in that, The molar ratio of the formula (III) to the formula (IV) is 1:1 - 1:1.

5.

6. The preparation method according to claim 3, wherein, In the step (1), the reaction temperature is 80 - 120 °C, and the reaction time is 3 - 6 hours.

7. The preparation method according to claim 3, characterized in that, In the step (2), the oxidant is manganese dioxide, and its dosage is 5 - 20 equivalents relative to the compound of formula (I).

8. An application, using the dihydrogibberellin derivative described in claim 1 as a gibberellin GA3 β-hydroxylase inhibitor.

9. A plant growth regulator, whose active ingredient is the dihydrogibberellin derivative described in claim 1.

10. According to the plant growth regulator described in claim 9, the dosage forms of the regulator include emulsifiable concentrate, wettable powder, suspension, powder, soluble powder, aqueous solution, water dispersible powder, smoke agent, granule or seed coating agent.

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