A sulfur-containing cytidine derivative, its preparation method and application

Abstract

The present invention provides a sulfur-containing cytosine nucleoside derivative, a preparation method thereof, and an application thereof. The sulfur-containing cytosine nucleoside derivative has the following general chemical formula as shown in Formula V; wherein, R is selected from one of methyl, substituted or unsubstituted benzyl; when R is substituted benzyl, the substituent is selected from any one of methoxy, halogen, methyl mercapto, trifluoromethyl, and methyl; n is an integer from 0 to 6. The present invention for the first time discovers that the derivative has good activities against plant pathogenic bacteria, viruses, and promoting plant growth, expands the application scope of sulfur-containing cytosine nucleoside derivatives as pesticides, and solves the problem that the bactericidal ability of commercially available sulfur-containing agricultural fungicides gradually weakens and far fails to reach the expected control effect due to continuous application and poor water solubility, etc.

Description

Technical Field

[0001] The present invention relates to the field of agrochemicals and their applications, and particularly to a sulfur-containing cytidine derivative with bactericidal, antiviral, and plant growth-promoting effects, its preparation method, and its applications. Background Art

[0002] Cytidine is one of the pyrimidine nucleosides that make up nucleic acids. It consists of cytosine (the base part) and ribose (the sugar part), and is formed by the hydrolysis of ribonucleic acid. When treated with nitrous acid, it can form uridine. It is an important raw material for drug synthesis and a biochemical reagent. Existing studies have shown that it can be used as a pharmaceutical intermediate to prepare medical antiviral, anti-tumor drugs such as cytidine diphosphate choline and cytarabine, and is also the core skeleton of the biopesticide ningnanmycin with bactericidal and antiviral effects in the field of agrochemistry.

[0003] Currently, sulfur-containing agricultural fungicides have been widely developed due to their advantages such as high efficiency, low toxicity, broad-spectrum bactericidal activity, low foaming, and low resistance generation. They effectively replace pesticide products such as mercury and copper preparations that have pollution risks. In addition, sulfur is an important component element in plants, and the synthesis of some amino acids such as cysteine and methionine requires the participation of sulfur. Moreover, sulfur can promote the formation of chloroplasts and the absorption of other elements, which is of great significance for the normal growth of crops. Therefore, sulfur-containing fungicides belong to a type of plant protection fungicide with growth-promoting effects.

[0004] Currently, the main sulfur-containing agricultural fungicide products on the market are dithiocarbamate fungicides, thiram fungicides, methylene bisthiocyanate, and isothiazolinone, etc. However, due to continuous application and poor water solubility, the bactericidal ability of these pesticides gradually weakens, far from meeting the expected control effect. Therefore, it is necessary to research and develop a type of sulfur-containing cytidine derivative with stable bactericidal performance and concurrent antiviral and growth-promoting effects. Summary of the Invention

[0005] To solve the problem that the bactericidal ability of sulfur-containing agricultural fungicides on the market gradually weakens and far fails to meet the expected control effect due to continuous application and poor water solubility, the purpose of the present invention is to provide a sulfur-containing cytidine derivative with bactericidal, antiviral, and plant growth-promoting effects, its preparation method, and its applications. The present invention has first discovered that this derivative has good activities against plant pathogens, viruses, and promoting plant growth, expanding the application scope of sulfur-containing cytidine derivatives as pesticides.

[0006] To achieve the above purpose, the technical solution of the present invention is as follows.

[0007] A sulfur-containing cytidine derivative, whose chemical general formula is shown as Formula V below:

[0008]

[0009] Among them, R is selected from one of methyl, substituted or unsubstituted benzyl;

[0010] When R is substituted benzyl, the substituent is selected from any one of methoxy, halogen, methyl mercapto, trifluoromethyl, and methyl;

[0011] n is an integer from 0 to 6.

[0012] Furthermore, R is selected from one of methyl and substituted benzyl;

[0013] When R is substituted benzyl, the substituent is selected from methoxy;

[0014] n is an integer from 0 to 2.

[0015] Furthermore, it is specifically selected from any one of the following compounds;

[0016]

[0017]

[0018] The present invention also provides a preparation method of a sulfur-containing cytosine nucleoside derivative, comprising the following steps:

[0019] S1. Preparation of Compound II

[0020] Compound I, DCC, HOBT are mixed with an organic solvent and stirred at 20 - 35 °C for reaction to obtain Compound II;

[0021] S2. Preparation of Compound IV

[0022] Compound II and Compound III are mixed and stirred at 20 - 35 °C for reaction to obtain Compound IV;

[0023] S3. Preparation of Compound V

[0024] Compound IV, TFA are mixed with an organic solvent and stirred at 20 - 35 °C for reaction. After the reaction is completed, the reaction is quenched with saturated sodium bicarbonate solution, and after post-treatment and purification, Compound V is obtained;

[0025] The specific synthesis route is as follows:

[0026]

[0027] Furthermore, in S1, the mass ratio of Compound I, DCC, and HOBT is 5 - 20:4 - 10:5 - 10.

[0028] Furthermore, in S2, the mass ratio of Compound II and Compound III is 5 - 20:4 - 10.

[0029] Further, in S3, the dosage ratio of compound IV to TFA is 0.5 - 5 g: 1 mL.

[0030] Further, in S1 and S3, the organic solvent is any one or a combination of more than one of N,N-dimethylformamide, DCM, acetone, acetonitrile, and dimethyl sulfoxide.

[0031] The present invention also provides an application of a sulfur-containing cytosine nucleoside derivative in the preparation of a drug for inhibiting phytopathogenic fungi and / or plant viruses;

[0032] The phytopathogenic fungi are at least one of Rhizoctonia solani Kühn, Rhizoctonia solani, Rhizoctonia cerealis, Rhizoctonia solani Kühn, Rhizoctonia solani Kühn, Rhizoctonia solani, Alternaria alternata, Colletotrichum capsici, Gibberella zeae, Alternaria solani, Fusarium graminearum, Bipolaris maydis, Fusarium moniliforme, Curvularia lunata, and Fusarium oxysporum;

[0033] The plant virus is Tobacco mosaic virus (TMV).

[0034] The present invention also provides an application of a sulfur-containing cytosine nucleoside derivative in the preparation of a drug for promoting plant growth.

[0035] Further, the test plants for promoting growth are tobacco.

[0036] The beneficial effects of the present invention:

[0037] 1. The sulfur-containing cytosine nucleoside derivative of the present invention has good activities against phytopathogenic bacteria, viruses and promoting plant growth, expanding the application scope of sulfur-containing cytosine nucleoside derivatives as pesticides, and solving the problem that the bactericidal ability of sulfur-containing agricultural fungicides on the market gradually weakens and far fails to reach the expected control effect due to continuous application and poor water solubility.

[0038] 2. The sulfur-containing cytosine nucleoside derivatives of the present invention have good application prospects in inhibiting phytopathogenic fungi and viruses and promoting growth. They have broad-spectrum antibacterial properties, and particularly have significant antibacterial effects against pathogenic fungi of the genus Rhizoctonia such as Rhizoctonia solani, Rhizoctonia solani Kühn, Pectobacterium carotovorum subsp. carotovorum, and Corynespora cassiicola.

[0039] 3. The sulfur-containing cytosine nucleoside derivatives of the present invention can effectively reduce the disease index of tobacco diseases caused by tobacco mosaic virus, and have good promoting effects on the germination and growth of tobacco seeds.

[0040] 4. The sulfur-containing cytosine nucleoside derivatives provided by the present invention can not only sterilize and antiviral but also promote plant growth, thereby saving farmers' expenses and increasing economic income, and also having good ecological and social benefits. Detailed Embodiments

[0041] In order to make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0042] All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts fall within the scope of protection of the present invention.

[0043] Unless otherwise specified, the experimental methods described in the following embodiments are all conventional methods; the reagents and materials, unless otherwise specified, can all be obtained in the market.

[0044] The sulfur-containing cytosine nucleoside derivatives provided by the present invention have the following general chemical formula as shown in Formula V:

[0045]

[0046] Among them, R is selected from one of methyl, substituted or unsubstituted benzyl; when R is substituted benzyl, the substituent is selected from any one of methoxy, halogen, methylthio, trifluoromethyl, and methyl; n is an integer from 0 to 6.

[0047] Specifically selected from any one of the following compounds;

[0048]

[0049] The specific synthesis route is as follows:

[0050]

[0051] Next, we take Compound 1 and Compound 2 as examples to illustrate the preparation and application of the sulfur-containing cytidine derivatives. The preparation methods of Compounds 3-5 are basically the same as those of Compound 1 and Compound 2.

[0052] Example 1

[0053] A preparation method of a sulfur-containing cytidine derivative, comprising the following steps:

[0054] S1. Preparation of Compound II

[0055] Add 8 g of BOC-S-(4-methoxybenzyl)-L-cysteine (Compound I) and 6 g of DCC (dicyclohexylcarbodiimide) to a 50 mL round-bottom flask, then add 7 g of HOBT (1-hydroxybenzotriazole), and add 5 mL of dry N,N-dimethylformamide as a solvent; stir the mixture at 25 °C for 1 h to obtain 1H-benzo[d][1,2,3]triazol-1-yl N-(tert-butoxycarbonyl)-S-(4-methoxybenzyl)-L-cysteine (Compound II).

[0056] The amounts prepared and the volume of the reaction vessel are scaled up or down in accordance with the corresponding ratio.

[0057] S2. Preparation of Compound IV

[0058] Add 8 g of 1H-benzo[d][1,2,3]triazol-1-yl N-(tert-butoxycarbonyl)-S-(4-methoxybenzyl)-L-cysteine (Compound II) to 8 g of cytidine (Compound III), and stir at 25 °C for 1 h to obtain tert-butyl ((R)-1-((1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-3-((4-methoxybenzyl)thio)-1-oxopropan-2-yl)carbamate (Compound V).

[0059] The amounts prepared and the volume of the reaction vessel are scaled up or down in accordance with the corresponding ratio.

[0060] S3. Preparation of Compound V

[0061] Add 1 g of tert-butyl ((R)-1-((1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-3-((4-methoxybenzyl)thio)-1-oxopropan-2-yl)carbamate (Compound IV) to a 50 mL round-bottom flask, evacuate, and add 5 mL of dichloromethane (DCM) and 1 mL of trifluoroacetic acid (TFA).

[0062] The mixture was stirred at 25 °C for 2 h. The progress of the reaction was monitored by silica gel thin-layer chromatography, using ethyl acetate: petroleum ether = 1:3 as the chromatography solvent.

[0063] The reaction was quenched with saturated sodium bicarbonate solution and extracted with ethyl acetate. The ethyl acetate layer was separated. The ethyl acetate phase was washed successively with water and saturated sodium chloride, and separated and concentrated under vacuum.

[0064] The mixture was purified by phenyl chromatography column with ACN / H 2 O (v / v = 1 / 3), separated and concentrated under vacuum to obtain (R)-2-amino-N-(1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-3-((4-methoxybenzyl)thio)propanamide (Compound V).

[0065] The amount of Compound V prepared and the volume of the reaction vessel were scaled up or down in accordance with the corresponding ratio.

[0066] The physical and chemical parameters and structural parameters of Compound V are shown in Table 1.

[0067] Table 1 Physical and chemical parameters and structural parameters of the sulfur-containing cytidine compounds of the examples of the present invention

[0068]

[0069]

[0070] Example 2

[0071] A preparation method of a sulfur-containing cytosine nucleoside derivative, comprising the following steps:

[0072] S1. Preparation of Compound II

[0073] 20 g of BOC-S-(4-methoxybenzyl)-L-cysteine (Compound I) and 10 g of DCC (dicyclohexylcarbodiimide) were added to a 50 mL round-bottom flask, and then 10 g of HOBT (1-hydroxybenzotriazole) was added. 10 mL of dry N,N-dimethylformamide was added as the solvent; the mixture was stirred at 35 °C for 2 h to obtain 1H-benzo[d][1,2,3]triazol-1-yl N-(tert-butoxycarbonyl)-S-(4-methoxybenzyl)-L-cysteine (Compound II).

[0074] The amount prepared and the volume of the reaction vessel were scaled up or down in accordance with the corresponding ratio.

[0075] S2. Preparation of Compound IV

[0076] To 20 g of 1H-benzo[d][1,2,3]triazol-1-yl N-(tert-butoxycarbonyl)-S-(4-methoxybenzyl)-L-cysteine (Compound II), 10 g of cytidine (Compound III) was added, and the mixture was stirred at 35 °C for 1 h to obtain tert-butyl ((R)-1-((1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-3-((4-methoxybenzyl)thio)-1-oxopropan-2-yl)carbamate (Compound V).

[0077] The amounts prepared and the volumes of the reaction vessels were scaled up or down in corresponding proportions.

[0078] S3. Preparation of Compound V

[0079] 5 g of tert-butyl ((R)-1-((1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-3-((4-methoxybenzyl)thio)-1-oxopropan-2-yl)carbamate (Compound IV) was added to a 50 mL round-bottom flask, evacuated, and 5 mL of dichloromethane (DCM) and 1 mL of trifluoroacetic acid (TFA) were added.

[0080] The mixture was stirred at 35 °C for 3 h. The progress of the reaction was monitored by silica gel thin-layer chromatography using ethyl acetate:petroleum ether = 4:7 as the eluent.

[0081] The reaction was quenched with saturated sodium bicarbonate solution and extracted with ethyl acetate, and the ethyl acetate layer was separated. The ethyl acetate phase was washed successively with water and saturated sodium chloride, and separated and concentrated under vacuum.

[0082] The mixture was purified by phenyl chromatography column with ACN / H 2 O (v / v = 2 / 6), separated and concentrated under vacuum to obtain (R)-2-amino-N-(1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-3-((4-methoxybenzyl)thio)propanamide (Compound V).

[0083] The amounts of Compound V prepared and the volumes of the reaction vessels were scaled up or down in corresponding proportions.

[0084] The physical and chemical parameters and structural parameters of Compound V are shown in Table 1.

[0085] Example 3

[0086] A preparation method of a sulfur-containing cytosine nucleoside derivative, comprising the following steps:

[0087] S1. Preparation of Compound II-1

[0088] Add 15 g of BOC-S-(4-methoxybenzyl)-L-cysteine (Compound I) and 6 g of DCC (dicyclohexylcarbodiimide) to a 50 mL round-bottom flask, then add 10 g of HOBT (1-hydroxybenzotriazole) and 5 mL of dry N,N-dimethylformamide as a solvent, and stir the mixture at 30 °C for 1 h to obtain 1H-benzo[d][1,2,3]triazol-1-yl N-(tert-butoxycarbonyl)-S-(4-methoxybenzyl)-L-cysteine (Compound II).

[0089] The preparation amount and the volume of the reaction vessel are enlarged or reduced in accordance with the corresponding ratio.

[0090] S2. Preparation of Compound IV

[0091] Add 10 g of cytosine nucleoside (Compound III) to 15 g of 1H-benzo[d][1,2,3]triazol-1-yl N-(tert-butoxycarbonyl)-S-(4-methoxybenzyl)-L-cysteine (Compound II), and stir at 30 °C for 2 h to obtain tert-butyl ((R)-1-((1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-3-((4-methoxybenzyl)thio)-1-oxopropan-2-yl)carbamate (Compound V).

[0092] The preparation amount and the volume of the reaction vessel are enlarged or reduced in accordance with the corresponding ratio.

[0093] S3. Preparation of Compound V

[0094] Add 5 g of tert-butyl ((R)-1-((1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-3-((4-methoxybenzyl)thio)-1-oxopropan-2-yl)carbamate (Compound IV) to a 50 mL round-bottom flask, evacuate, and add 5 mL of dichloromethane (DCM) and 1 mL of trifluoroacetic acid (TFA).

[0095] Stir the mixture at 30 °C for 3 h. Monitor the progress of the reaction by silica gel thin-layer chromatography, using ethyl acetate:petroleum ether = 2:4 as the chromatography solution.

[0096] The reaction was quenched with saturated sodium bicarbonate solution and extracted with ethyl acetate, and the ethyl acetate layer was separated. The ethyl acetate phase was washed successively with water and saturated sodium chloride, and separated and concentrated under vacuum.

[0097] The mixture was purified by phenyl chromatography column with ACN / H2O (v / v = 2 / 5), separated and concentrated under vacuum to obtain (R)-2-amino-N-(1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-3-((4-methoxybenzyl)thio)propanamide (Compound V).

[0098] The amount of Compound V prepared and the volume of the reaction vessel were scaled up or down in corresponding proportion.

[0099] The physical and chemical parameters and structural parameters of Compound V are shown in Table 2.

[0100] Table 2 Physical and chemical parameters and structural parameters of sulfur-containing cytidine compounds in the examples of the present invention

[0101]

[0102]

[0103] Example 4

[0104] A preparation method of a sulfur-containing cytosine nucleoside derivative, comprising the following steps:

[0105] S1. Preparation of Compound II-1

[0106] 5 g of BOC-S-(4-methoxybenzyl)-L-cysteine (Compound I) and 4 g of DCC (dicyclohexylcarbodiimide) were added to a 50 mL round-bottom flask, and then 5 g of HOBT (1-hydroxybenzotriazole) and 5 mL of dry N,N-dimethylformamide were added as solvents. The mixture was stirred at 30 °C for 1 h to obtain 1H-benzo[d][1,2,3]triazol-1-yl N-(tert-butoxycarbonyl)-S-(4-methoxybenzyl)-L-cysteine (Compound II).

[0107] The amount prepared and the volume of the reaction vessel were scaled up or down in corresponding proportion.

[0108] S2. Preparation of Compound IV

[0109] 4 g of cytidine (Compound III) was added to 1H-benzo[d][1,2,3]triazol-1-yl N-(tert-butoxycarbonyl)-S-(4-methoxybenzyl)-L-cysteine (Compound II) under 5 g, and the mixture was stirred at 30 °C for 2 h to obtain tert-butyl ((R)-1-((1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-3-((4-methoxybenzyl)thio)-1-oxopropan-2-yl)carbamate (Compound V).

[0110] The amounts prepared and the volumes of the reaction vessels were scaled up or down in corresponding proportions.

[0111] S3. Preparation of Compound V

[0112] 0.5 g of tert-butyl ((R)-1-((1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-3-((4-methoxybenzyl)thio)-1-oxopropan-2-yl)carbamate (Compound IV) was added to a 50 mL round-bottom flask, evacuated, and 5 mL of dichloromethane (DCM) and 1 mL of trifluoroacetic acid (TFA) were added.

[0113] The mixture was stirred at 30 °C for 3 h. The progress of the reaction was monitored by silica gel thin-layer chromatography using ethyl acetate:petroleum ether = 1:2 as the eluent.

[0114] The reaction was quenched with saturated sodium bicarbonate solution and extracted with ethyl acetate, and the ethyl acetate layer was separated. The ethyl acetate phase was washed successively with water and saturated sodium chloride and separated and concentrated under vacuum.

[0115] The mixture was purified by a phenyl chromatography column with ACN / H2O (v / v = 1 / 5), separated and concentrated under vacuum to obtain (R)-2-amino-N-(1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-3-((4-methoxybenzyl)thio)propanamide (Compound V).

[0116] The amounts prepared of Compound V and the volumes of the reaction vessels were scaled up or down in corresponding proportions.

[0117] The physical and chemical parameters and structural parameters of Compound V are shown in Table 2.

[0118] Example 5

[0119] A preparation method of a sulfur-containing cytidine derivative, comprising the following steps:

[0120] S1. Preparation of Compound II-1

[0121] 10 g of BOC-S-(4-methoxybenzyl)-L-cysteine (Compound I) and 7 g of DCC (dicyclohexylcarbodiimide) were added to a 50 mL round-bottom flask, followed by 8 g of HOBT (1-hydroxybenzotriazole) and 8 mL of dry N,N-dimethylformamide as the solvent. The mixture was stirred at 35 °C for 3 h to obtain 1H-benzo[d][1,2,3]triazol-1-yl N-(tert-butoxycarbonyl)-S-(4-methoxybenzyl)-L-cysteine (Compound II).

[0122] The amounts prepared and the volume of the reaction vessel were scaled up or down in corresponding proportions.

[0123] S2. Preparation of Compound IV

[0124] 10 g of 1H-benzo[d][1,2,3]triazol-1-yl N-(tert-butoxycarbonyl)-S-(4-methoxybenzyl)-L-cysteine (Compound II) was added with 10 g of cytidine (Compound III), and the mixture was stirred at 35 °C for 3 h to obtain tert-butyl ((R)-1-((1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-3-((4-methoxybenzyl)thio)-1-oxopropan-2-yl)carbamate (Compound V).

[0125] The amounts prepared and the volume of the reaction vessel were scaled up or down in corresponding proportions.

[0126] S3. Preparation of Compound V

[0127] 1 g of tert-butyl ((R)-1-((1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-3-((4-methoxybenzyl)thio)-1-oxopropan-2-yl)carbamate (Compound IV) was added to a 50 mL round-bottom flask, evacuated, and then 5 mL of dichloromethane (DCM) and 1 mL of trifluoroacetic acid (TFA) were added.

[0128] The mixture was stirred at 30 °C for 3 h. The progress of the reaction was monitored by silica gel thin-layer chromatography, using ethyl acetate:petroleum ether = 1:4 as the eluent.

[0129] The reaction was quenched with saturated sodium bicarbonate solution and extracted with ethyl acetate. The ethyl acetate layer was separated. The ethyl acetate phase was washed successively with water and saturated sodium chloride, and separated and concentrated under vacuum.

[0130] The mixture was purified by phenyl chromatography column with ACN / H2O (v / v = 1 / 5), separated and concentrated under vacuum to obtain (R)-2-amino-N-(1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-3-((4-methoxybenzyl)thio)propanamide (Compound V).

[0131] The amount of Compound V prepared and the volume of the reaction vessel were scaled up or down in accordance with the corresponding ratio.

[0132] The physicochemical parameters and structural parameters of Compound V are shown in Table 2.

[0133] Application Example 1

[0134] Determination of the bactericidal activity of cytosine nucleoside derivatives.

[0135] The bactericidal or bacteriostatic activity of the compounds against various plant pathogens was determined by the mycelial growth rate method. The specific steps were as follows:

[0136] The cytosine nucleoside derivatives (Compound 1 and Compound 2) were quantitatively dissolved in sterile water to prepare a stock solution;

[0137] A quantitative amount of molten PDA medium sterilized at about 50 °C was taken, and the above stock solution was added thereto in proportion so that the final concentrations of the mixed solution were 50 μg / mL and 25 μg / mL respectively to prepare drug-containing PDA plates.

[0138] Then, a pathogen agar disc with a diameter of 5 mm was inoculated into the center of the petri dish and cultured in an incubator at 28 °C. After the colonies in the corresponding blank control had grown sufficiently, the colony diameters of each treatment were measured by the cross method, and the average value was taken.

[0139] The mixed medium of PDA and sterile water was used as the blank control, and jinggangmycin aqueous solution at 200 μg / mL and 100 μg / mL was used as the control. The test was repeated 3 times.

[0140] The inhibition rate of the test compound against the growth of the pathogen was calculated by the following formula:

[0141] Net growth amount = average colony diameter - agar disc diameter;

[0142] Inhibition rate (%) = [(net growth amount of the control colony - net growth amount of the treated colony) / net growth amount of the control colony] × 100%.

[0143] The test plant pathogens include fungi of the genus Rhizoctonia, such as Rhizoctonia solani Kühn causing sheath blight of rice, Rhizoctonia solani causing sheath blight of maize, Rhizoctonia cerealis causing sheath blight of wheat, Rhizoctonia solani Kühn causing black scurf of potato, Rhizoctonia solani Kühn causing brown rot of Chinese cabbage, Rhizoctonia solani causing target spot of tobacco, Alternaria alternata causing brown spot of tobacco, Colletotrichum capsici causing anthracnose of pepper, Gibberella zeae causing scab of wheat, Alternaria solani causing early blight of potato, Fusarium graminearum causing root rot of wheat, Bipolaris maydis causing southern leaf blight of maize, Fusarium moniliforme causing bakanae disease of rice, Curvularia lunata causing curvularia leaf spot of maize, and Fusarium oxysporum causing fusarium wilt of cucumber.

[0144] Using compound 1 (L01) prepared in Example 1 and compound 2 (L02) prepared in Example 3 as the test compounds, and validamycin aqueous solution as the control agent; the inhibitory effects of the test compounds and agents on plant pathogenic fungi are shown in Table 3.

[0145] Table 3 Inhibitory effects of the test compounds and agents on plant pathogenic fungi

[0146]

[0147] The results in Table 3 show that the cytosine nucleoside derivatives provided in the examples of the present invention have certain inhibitory activities against the test strains, and there is a concentration dependence.

[0148] Among them, the inhibitory effects on six Rhizoctonia pathogens, namely Rhizoctonia solani Kühn causing sheath blight of rice, Rhizoctonia solani causing sheath blight of maize, Rhizoctonia cerealis causing sheath blight of wheat, Rhizoctonia solani Kühn causing black scurf of potato, Rhizoctonia solani Kühn causing brown rot of Chinese cabbage, and Rhizoctonia solani causing target spot of tobacco, are particularly significant. The inhibition rate of compound L02 at 50 μg / mL against Rhizoctonia solani Kühn causing sheath blight of rice is as high as 92.59%; the inhibition rates of compounds L01 and L02 at 25 μg / mL against the six Rhizoctonia pathogens are both greater than 55%, and are superior to the control agent.

[0149] Application Example 2

[0150] Determination of the activity of cytosine nucleoside derivatives against Tobacco mosaic virus (TMV) in Nicotiana glutinosa.

[0151] The antiviral activity of cytosine nucleoside derivatives against Tobacco mosaic virus (TMV) in Nicotiana glutinosa was determined by the local lesion method, and the specific steps were as follows:

[0152] The virus particles were purified according to the method of Gooding and Hebert (1967) and stored at 4 °C for later use.

[0153] Compound L01 (Compound 1 prepared in Example 1) and Compound L02 (Compound 2 prepared in Example 3) were mixed with the virus venom to make the final concentration of the agent reach 50 μg / mL and 25 μg / mL, and then left standing for 30 min.

[0154] Then, Nicotiana glutinosa plants at about the six-leaf stage with consistent growth were selected, and the mixture of the agent and the venom was inoculated onto the leaves by the method of rubbing inoculation. Four days later, the number of local lesions was recorded and the inhibition rate was calculated.

[0155] The aqueous solution of ningnanmycin at 100 μg / mL and 50 μg / mL was used as the control, and the treatment with an equal amount of clear water was used as the blank control. The above experiments were repeated 3 times.

[0156] The inhibition rate of the test compounds and the control agents against Tobacco mosaic virus disease was calculated by the following formula:

[0157] Inhibition rate (%) = [(number of local lesions in the control - number of local lesions in the treatment) / number of local lesions in the control] × 100%;

[0158] Compound 1 (L01) prepared in Example 1 and Compound 2 (L02) prepared in Example 3 were used as the test compounds, and the aqueous solution of ningnanmycin was used as the control agent; the control effects of the test compounds and the agents against Tobacco mosaic virus disease are shown in Table 4.

[0159] Table 4 Test on the control effect of different agents against Tobacco mosaic virus disease

[0160]

[0161] The results in Table 4 show that the inhibitory effects of Compounds L01 and L02 on the local lesions caused by Tobacco mosaic virus in Nicotiana glutinosa were comparable, and were significantly better than the control agent ningnanmycin; the inhibition rate of local lesions still reached about 70% under the treatment of 25 μg / mL L01 and L02.

[0162] Application Example 3

[0163] Field test of cytosine nucleoside derivatives and determination of the control effect against Tobacco mosaic virus disease.

[0164] As a major disease of tobacco, the occurrence, prevalence and damage of tobacco mosaic virus disease often affect the yield and quality of tobacco leaves, resulting in significant losses. Therefore, it is particularly important to research and develop new antiviral agents.

[0165] The field antiviral assay was conducted at the tobacco experimental base in Fengcheng City, Dandong City, Liaoning Province in mid-June 2022. The tested variety was Liaoyan 17.

[0166] Compound L01 (Compound 1 prepared in Example 1) and Compound L02 (Compound 2 prepared in Example 3) were respectively formulated into 50 μg / mL solutions, which were sprayed on tobacco seedlings and in the field, respectively, with two consecutive applications at an interval of 7 days. Spraying 100 μg / mL ningnanmycin aqueous solution was used as a control, and spraying water was used as a blank control.

[0167] Each chemical treatment was applied to 150 plants with 3 replicates. Two weeks after the second spraying, the test plots were investigated by the 5-point sampling method, and 30 plants were surveyed per point.

[0168] According to the national industry standard GB / T23222-2008 Classification and investigation methods for tobacco diseases and pests, the disease index was statistically analyzed and the control effect was calculated.

[0169] The disease grading criteria are as follows:

[0170] Grade 0 - The whole plant is disease-free;

[0171] Grade 1 - The veins of the heart leaf are clear or slightly mosaic, and the plant shows no obvious dwarfing;

[0172] Grade 3 - 1 / 3 of the leaves are mosaic but not deformed, or the plant is dwarfed to more than 3 / 4 of the normal plant height;

[0173] Grade 5 - 1 / 3 to 1 / 2 of the leaves are mosaic, or a few leaves are deformed, or the main vein turns black, or the plant is dwarfed to 2 / 3 to 3 / 4 of the normal plant height;

[0174] Grade 7 - 1 / 2 to 2 / 3 of the leaves are mosaic, or deformed, or necrosis occurs on the side of the main vein, or the plant is dwarfed to 1 / 2 to 2 / 3 of the normal plant height;

[0175] Grade 9 - All the leaves of the whole plant are mosaic, severely deformed or necrotic, or the diseased plant is dwarfed to more than 1 / 2 of the normal plant height.

[0176] The field disease index and control effect of the tested compounds and the control agent against tobacco mosaic virus disease were calculated by the following formula:

[0177] Disease index = [Σ (number of diseased plants at each level × value of that disease level) / total number of plants surveyed × highest disease level value] × 100;

[0178] Control effect = (disease index of the control plot - disease index of the treated plot) / disease index of the control plot.

[0179] Table 5 Field control effect of different pesticides on tobacco mosaic virus disease

[0180] Agent treatment Average disease index Control effect % L01 3.65±0.38 64.18±3.34 L02 3.52±0.52 65.46±4.67 Ningnanmycin aqueous solution 4.26±±0.29 58.19±3.54 Water control 10.19±±1.03 ——

[0181] The results in Table 5 show that the investigation two weeks after the second application of the pesticide showed (Table 4): the best field control effect was the cytosine nucleoside derivative L02, with a control effect of 65.46%, followed by L01, with a control effect of 64.18%. The difference in control effect between the two was small, but it was significantly better than the control agent Ningnanmycin.

[0182] Application Example 4

[0183] Determination of plant growth promoting activity of cytosine nucleoside derivatives.

[0184] (1) Germination rate determination:

[0185] After the seeds of Liaoyan 17 were disinfected with alcohol, they were soaked in 25 μg / mL of the compounds L01 and L02 described in the invention for 20 hours, and then placed on a damp gauze in a culture dish. After 7 days of moisturizing culture at 28°C, the germination rate of the seeds was tested. Twenty seeds were treated in each dish, and the process was repeated 3 times. 0.1 μg / mL of 0.01% brassinolide was used as the control, and water treatment was used as the blank control.

[0186] (2) Hydroponic seedling test:

[0187] Yunyan 87 seeds were soaked with 25μg / mL compounds L01 and L02, respectively, and sown in hydroponic seedling trays containing growth solution after germination. After tobacco seedlings emerged, they were sprayed with 25μg / mL compounds L01 and L02 again. When the tobacco grew to 5-7 true leaves, the maximum leaf length and aboveground fresh weight were measured. Each treatment had 50 plants, repeated 3 times, with brassinolide as the control and water treatment as the blank control.

[0188] Table 6 Effects of different treatments on tobacco seedling growth

[0189] Agent treatment Germination rate % Maximum leaf width cm Maximum leaf length cm Fresh weight of aboveground part g L01 90.32±5.38 8.22±2.05 13.24±3.13 7.02±1.18 L02 89.62±4.31 9.33±1.13 14.54±2.87 7.96±0.99 Brassinolide 93.55±5.09 10.52±2.18 15.34±3.03 8.59±0.96 Water control 80.92±4.33 5.26±0.12 8.28±1.12 6.05±1.51

[0190] The results in Table 6 show that the germination rate of the seeds treated with the compounds L01 and L02 of the invention basically reached about 90%, which was significantly higher than that of the water control, and the maximum leaf width, maximum leaf length and aboveground fresh weight of the plants were also significantly higher than those of the blank control group, and slightly lower than those of the brassinolide treatment group. In summary, the treatment with compounds L01 and L02 can make tobacco seedlings grow better and significantly promote tobacco growth.

[0191] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A sulfur-containing cytosine nucleoside derivative, characterized in that, its chemical general formula is shown as the following formula V: wherein, R is selected from one of substituted or unsubstituted benzyl; when R is substituted benzyl, the substituent is selected from any one of methoxy, halogen, methyl mercapto, trifluoromethyl, methyl; n is an integer from 0 to 6.

2. The sulfur-containing cytosine nucleoside derivative according to claim 1, characterized in that, when R is substituted benzyl, the substituent is selected from methoxy; n is an integer from 0 to 2.

3. The sulfur-containing cytosine nucleoside derivative according to claim 1, characterized in that, it is specifically selected from any one of the following compounds; 4. A preparation method of the sulfur-containing cytosine nucleoside derivative according to claim 1, characterized in that, it includes the following steps: S1. Preparation of compound II Mix compound I, DCC, HOBT with an organic solvent, and stir and react at 20 - 35 °C to obtain compound II; S2. Preparation of compound IV Mix compound II and compound III, and stir and react at 20 - 35 °C to obtain compound IV; S3. Preparation of compound V Mix compound IV, TFA with an organic solvent, and stir and react at 20 - 35 °C. After the reaction is completed, quench the reaction with saturated sodium bicarbonate solution, and obtain compound V through post-treatment and purification; The specific synthetic route is as follows:

5. The preparation method of the sulfur-containing cytosine nucleoside derivative according to claim 4, characterized in that, in S1, the mass ratio of compound I, DCC, HOBT is 5 - 20:4 - 10:5 - 10.

6. The preparation method of the sulfur-containing cytosine nucleoside derivative according to claim 4, characterized in that, in S2, the mass ratio of compound II and compound III is 5 - 20:4 - 10.

7. The preparation method of the sulfur-containing cytosine nucleoside derivative according to claim 4, characterized in that, in S3, the dosage ratio of compound IV and TFA is 0.5 - 5 g:1 mL.

8. The preparation method of the sulfur-containing cytosine nucleoside derivative according to claim 4, characterized in that, in S1, the organic solvent is N,N-dimethylformamide; in S3, the organic solvent is DCM.

9. An application of the sulfur-containing cytosine nucleoside derivative according to claim 1 in the preparation of a drug for inhibiting plant pathogenic fungi and / or plant viruses; the plant pathogenic fungi are at least one of Rhizoctonia solani of rice, Rhizoctonia solani of corn, Rhizoctonia solani of wheat, Rhizoctonia solani of potato, Pectobacterium carotovorum subsp. atrosepticum of Chinese cabbage, Corynespora cassiicola of tobacco, Alternaria alternata of tobacco, Colletotrichum capsici of chili pepper, Fusarium graminearum of wheat, Alternaria solani of potato, Bipolaris sorokiniana of wheat, Bipolaris maydis of corn, Fusarium moniliforme of rice, Curvularia lunata of corn, Fusarium oxysporum f. sp. cucumerinum of cucumber; the plant virus is Tobacco mosaic virus.

10. An application of the sulfur-containing cytosine nucleoside derivative according to claim 1 in the preparation of a drug for promoting plant growth.

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