1-substituted-5-amino-4-pyrazole acylhydrazone compounds and their use in the prevention and treatment of tobacco mosaic virus disease

Abstract

The application discloses a kind of 1-substituent-5-amino-4-pyrazole acyl hydrazone compounds, it is characterized in that: its structural formula is as follows: wherein R1 is phenyl or tert-butyl group;R2 is phenyl or substituted phenyl group.Series 1-substituent-5-amino-4-pyrazole acyl hydrazone compounds are designed and synthesized in the application, and it is found by in vivo experiment of the compound that the compounds have good treatment, protection and passivation activity on the viral disease caused by tobacco mosaic virus (TMV). And compared with other similar compounds, it shows better treatment activity.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, and in particular to a method for preparing a class of 1-substituted-5-amino-4-pyrazole hydrazone compounds and their application in the treatment of viral diseases after tobacco mosaic virus infection. Background Technology

[0002] Plant viral diseases have remained one of the biggest obstacles to agricultural production in recent decades. Tobacco mosaic virus (TMV) is a typical example of a Tobacco mosaic virus, capable of infecting a range of Solanaceae plants, including tobacco, and affecting over 885 species across 65 families. TMV is one of the most destructive plant viruses, with a wide host range, strong pathogenicity and resistance. While the virus particles are very stable, they retain significant reinfection capacity years after infecting materials (such as leaves or soil). After infecting a plant host, TMV typically migrates from the initial infection area and multiplies throughout the entire plant; the host plant undergoes molecular, cellular, and physiological changes after TMV infection. Researching highly effective, low-toxicity, and low-residue chemical agents is of great significance for controlling diseases caused by Tobacco mosaic virus. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a new type of pyrazolylhydrazone anti-plant virus compound with high therapeutic activity for the prevention and control of plant viral diseases caused by tobacco mosaic virus.

[0004] The technical solution of this invention: a class of 1-substituted-5-amino-4-pyrazole hydrazone compounds, the structural formula of which is as follows: Where R1 is phenyl or tert-butyl; R2 is phenyl or substituted phenyl.

[0005] Preferably, R1 is phenyl or tert-butyl, and R2 is phenyl or substituted phenyl, wherein the substituted phenyl is a single substitution at the ortho, meta, or para position.

[0006] The substituted phenyl group is a single substitution at the ortho, meta, or para position, and the substituent is a halogen, alkyl, trifluoromethyl, or alkoxy group.

[0007] The substituted phenyl group is a single substitution at the ortho, meta, or para position, and the substituent is a halogen, methyl, trifluoromethyl, or alkoxy group.

[0008] A method for preparing a class of 1-substituted-5-amino-4-pyrazole hydrazones, with the following reaction formula:

[0009]

[0010] The application of the aforementioned class of 1-substituted-5-amino-4-pyrazole hydrazone compounds in the prevention and control of plant viral diseases caused by tobacco mosaic virus.

[0011] The beneficial effects of this invention: This invention synthesizes a series of 1-substituted-5-amino-4-pyrazolylhydrazone compounds. In vivo experiments revealed that these compounds exhibit good therapeutic activity against viral diseases caused by tobacco mosaic virus (TMV). Compared with the highly active compounds reported in previous work by our research group, these compounds show significant therapeutic effects against plant viral diseases, providing an important scientific basis for the research and development of new pesticides.

[0012] Instruction manual illustrations

[0013] Figure 1 The therapeutic, protective and therapeutic activities of the compounds against TMV (A-D8, B-D18 and C-D11);

[0014] Figure 2 The effects of different treatments on the growth traits of Nicotiana benthamiana (Mock: no treatment, TMV: inoculated with TMV, TMV+D8: inoculated with TMV followed by spraying with D8 at a concentration of 250 μg / mL, TMV+NNM: inoculated with TMV followed by spraying with ningnanmycin at a concentration of 250 μg / mL). Detailed Implementation

[0015] Synthetic routes for pyrazole hydrazone target compounds:

[0016]

[0017] Starting with tert-butylhydrazine salt or phenylhydrazine, the target compound D was synthesized through ring closure, hydrazinolysis, and substitution.

[0018] Preparation of intermediates

[0019] Preparation of ethyl 1-(substituent)-5-amino-pyrazole-4-carboxylate (B)

[0020]

[0021] In a round-bottom flask, a substituted hydrazine salt (70.9 mmol), ethyl ethoxymethylidene (59.1 mmol), and ethanol (25 mL) were added. The mixture was heated to reflux and the reaction was monitored by TLC until complete. After cooling, the ethanol in the flask was removed by vacuum distillation. The crude product was dissolved in ethyl acetate and washed 2–3 times with saturated brine. The organic phase was dried over anhydrous sodium sulfate. Ethyl acetate was filtered off, evaporated to dryness, and purified by column chromatography to obtain intermediates B1 and B2. The physicochemical and spectroscopic data are as follows:

[0022] 1-tert-butyl-5-amino-pyrazole-4-carboxylic acid ethyl ester (B1): pale yellow solid, yield 85%, melting point 53–54℃; 1 H NMR(500MHz,CDCl3)δ:7.53(s,1H,NC H ),5.25(s,2H,N H 2), 4.22(q, J = 7.1 Hz, 2H, OC) H 2CH3),1.59(s,9H,t-butyl),1.29(t,J=7.1Hz,3H,OCH2C H 3). 13 C NMR(126MHz, CDCl3)δ:164.99,149.21,137.51,97.03,59.33,58.82,28.81,14.44.HRMS(ESI):calcd forC 10 H 17 N3O2[M+H] + ,212.1321; found 212.1387.

[0023] 1-Phenylacetyl-5-amino-pyrazole-4-carboxylic acid ethyl ester (B2): pale yellow solid, yield 92%, melting point 91–92℃; 1 HNMR(400MHz,CDCl3)δ:7.71(s,1H,NC H ),7.51–7.42(m,4H,phenyl H),7.38–7.31(m,1H,phenyl H),5.34(s,2H,N H 2), 4.25(q, J = 7.1 Hz, 2H, OC) H 2CH3), 1.32(s, 3H, OCH2C H 3); 13 C NMR(101MHz, CDCl3)δ:164.41,148.92,140.47,137.43,129.57,127.93,123.61,95.95,59.50,14.38.HRMS(ESI):calcd for C 12 H 13 N3O2[M+H] + ,232.1008; found 232.1074.

[0024] Preparation of 1-substituted-5-amino-pyrazole-4-acylhydrazide (C)

[0025]

[0026] In a round-bottom flask, ethyl 1-substituted-5-amino-4-pyrazolium carboxylate (37.1 mmol) and hydrazine hydrate (5 mL), and methanol (50 mL) were added. The reaction was monitored by TLC until complete, then stopped. The mixture was extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to give intermediates C1 and C2. The physicochemical and spectroscopic data are as follows:

[0027] 1-tert-butyl-5-amino-pyrazole-4-acylhydrazide (C1): yellow solid, yield 61%, melting point 169–170 °C; 1 HNMR(500MHz,DMSO-d6)δ:8.98(s,1H,N H ),7.59(s,1H,NC H ),6.09(s,2H,N H 2), 4.18(d, J = 10.0 Hz, 2H, NHN H 2), 1.51(s,9H,t-butyl). 13 C NMR(126MHz,DMSO-d6)δ:164.99,149.21,137.51,97.03,59.33,58.82,28.81,14.44.HRMS(ESI):calcd for C8H 15 N5O[M+H] + ,198.1277; found 198.1344.

[0028] 1-Phenylacetyl-5-amino-pyrazole-4-acylhydrazide (C2): yellow solid, yield 75%, melting point 182–183 °C; 1 H NMR(400MHz, CDCl3)δ:9.16(s,1H,N H ),7.89(s,1H,NC H ),7.59–7.48(m,4H,phenyl H),7.38(t,J=7.1Hz,1H,phenyl H),6.33(s,2H,N H 2), 4.27(s,2H,NHN) H 2); 13 C NMR (101MHz, CDCl3) δ: 164.88, 149.36, 138.54 (d, J = 22.5Hz), 129.85, 127.53, 123.52, 96.55; HRMS (ESI): calcdfor C 10 H 11 N5O[M+H] + ,218.0964; found 218.1031.

[0029] Synthesis of target compound D

[0030] (1) Taking the synthesis of target compounds D1 and D10 as an example

[0031]

[0032] Formula 1: Structural formula of target compound D1

[0033] In a round-bottom flask, C (5.1 mmol) and anhydrous ethanol (15 mL) were added and stirred for 2 hours. Benzaldehyde (5.1 mmol) was then added and the mixture was stirred at room temperature for 3 hours. The reaction was monitored by TLC. The precipitated solid was filtered, the filter cake was washed with ice-cold ethanol, and the solid was dried to give D1, a white solid with a yield of 89% and a melting point of 204-205 °C. 1 H NMR(500MHz,DMSO-d6)δ:11.18(d,J=3.4Hz,1H,N H ), 8.46–7.75 (m, 2H, NC) H ),7.67(d,J=7.4Hz,2H,phenyl H),7.52–7.31(m,3H,phenylH),6.67–6.12(m,2H,N H 2), 1.55(s,9H,t-butyl); 13 C NMR(126MHz,DMSO-d6)δ:134.69,129.55,128.91,126.69,58.10,28.36.HRMS(ESI):calcd for C 15 H 19 N5O[M+H] + ,286.1623; found 286.1857.

[0034]

[0035] Formula 2: Structural formula of target compound D10

[0036] In a round-bottom flask, C (5.1 mmol) and anhydrous ethanol (15 mL) were added and stirred for 2 hours. Benzaldehyde (5.1 mmol) was then added and the mixture was stirred at room temperature for 3 hours. The reaction was monitored by TLC. The precipitated solid was filtered, the filter cake was washed with ice-cold ethanol, and the solid was dried to give D10, a white solid with a yield of 77% and a melting point of 212-213 °C. 1 H NMR(500MHz,DMSO-d6)δ:11.33(d,J=13.3Hz,1H,N H ), 8.48–7.98 (m, 2H, NC) H),7.71(d,J=7.4Hz,2H,phenyl H),7.61–7.53(m,4H,phenylH),7.44(dt,J=25.6,7.3Hz,4H,phenyl H),6.66(dd,J=97.2,1.3Hz,2H,N H 2); 13 C NMR(101MHz,DMSO-d6)δ:138.51,135.08,130.19,130.00,129.46,127.86,127.28,123.92.HRMS(ESI):calcd for C 17 H 15 N5O[M+H] + ,306.1277; found 306.1337.

[0037] The synthesis methods for other target compounds are similar to those for target compounds D1 and D10.

[0038] (2) Structure and physicochemical data of the target compound

[0039]

[0040] Formula 3: Structural formula of target compound D2

[0041] D2, white solid, yield 66%, melting point 228-229℃; 1 H NMR(500MHz,DMSO-d6)δ:11.10(d,J=22.3Hz,1H,N H ), 8.33–7.68(m,2H,NC) H ),7.56(d,J=7.6Hz,2H,phenyl H),7.25(d,J=7.9Hz,2H,phenyl H),6.71–6.12(m,2H,N H 2), 2.33(s, 3H, C H 3), 1.55(s,9H,t-butyl); 13 CNMR(126MHz,DMSO-d6)δ:139.27,131.97,129.62,126.67,58.09,28.36,21.05.HRMS(ESI):calcd for C 16 H 21 N5O[M+H] + ,300.1811; found 300.1779.

[0042]

[0043] Formula 4: Structural formula of target compound D3

[0044] D3, white solid, yield 76%, melting point 224-225℃; 1 H NMR(500MHz,DMSO-d6)δ:11.42(s,1H,N H ), 8.79–7.34 (m, 6H, NC) H ,phenyl H),6.47(d,J=83.9Hz,2H,N H 2), 1.55(s,9H,t-butyl); 13 C NMR(126MHz,DMSO-d6)δ:132.78,131.91,130.94,129.95,127.69,126.55,58.15,28.34.HRMS(ESI):calcd for C 15 H 18 N5OCl[M+H] + ,319.1200; found 319.1266.

[0045]

[0046] Formula 5: Structural formula of target compound D4

[0047] D4, white solid, yield 71%, melting point 223-224℃; 1 H NMR(500MHz,DMSO-d6)δ:11.32(d,J=3.7Hz,1H,N H ), 8.35–7.33 (m, 6H, NC) H ,phenyl H), 6.66–6.21(m,2H,N H 2), 1.54(s,9H,t-butyl); 13 C NMR(126MHz,DMSO-d6)δ:136.97,133.68,130.79,129.16,125.97,125.35,58.14,28.34.HRMS(ESI):calcd for C 15 H 18 N5OCl[M+H] + ,319.1200; found 319.1266.

[0048]

[0049] Formula 6: Structural formula of target compound D5

[0050] D5, white solid, yield 80%, melting point 248-249℃; 1 H NMR(500MHz,DMSO-d6)δ:11.23(d,J=2.5Hz,1H,N H ), 8.33–7.42 (m, 6H, NC) H ,phenyl H), 6.60–6.21(m,2H,N H 2), 1.54(s,9H,t-butyl); 13 C NMR(126MHz,DMSO-d6)δ:133.90,133.64,130.07,129.13,128.98,128.32,58.12,28.35.HRMS(ESI):calcd for C 15 H 18 N5OCl[M+H] + ,319.1200; found 319.1266.

[0051]

[0052] Formula 7: Structural formula of target compound D6

[0053] D6, white solid, yield 63%, melting point 247-248℃; 1 H NMR(500MHz,DMSO-d6)δ:11.36(s,1H,N H ),8.25(dd,J=86.3,71.8Hz,2H,NC H ),7.88(d,J=8.1Hz,2H,phenyl H),7.79(d,J=8.3Hz,2H,phenyl H),6.77–6.20(m,2H,N H 2), 1.55(s,9H,t-butyl); 13 C NMR (126MHz, DMSO-d6) δ: 139.18, 129.65 (d, J = 31.5Hz), 127.73, 126.28, 125.77, 123.60, 58.63, 28.82; 19 FNMR(471MHz,DMSO-d6)δ:–60.99(s).HRMS(ESI):calcd for C 16 H 18 N5OF3[M+H] + ,354.1463; found 354.1528.

[0054]

[0055] Formula 8: Structural formula of target compound D7

[0056] D7, white solid, yield 65%, melting point 212-213℃; 1 H NMR(500MHz,DMSO-d6)δ:11.19(d,J=0.8Hz,1H,N H ), 8.42–7.65(m,2H,NC H ),7.52–6.91(m,4H,phenyl H),6.45(dd,J=100.0Hz,1.8Hz,2H,N H 2), 3.79(s, 3H, OC) H 3), 1.55(s,9H,t-butyl); 13 C NMR(126MHz,DMSO-d6)δ:159.58,136.13,130.03,58.12,55.14,28.36.HRMS(ESI):calcd for C 16 H 21 N5O2[M+H] + ,316.1695; found 316.1759.

[0057]

[0058] Formula 9: Structural formula of target compound D8

[0059] D8, white solid, yield 68%, melting point 221-222℃; 1 H NMR(500MHz,DMSO-d6)δ:11.16(s,1H,N H ), 8.35–7.66 (m, 4H, NC) H ,phenyl H),7.28(t,J=8.8Hz,2H,phenyl H),6.43(d,J=90.9Hz,2H,N H 2), 1.55(s,9H,t-butyl); 13 C NMR (126MHz, DMSO-d6) δ: 164.26, 162.29, 131.79, 129.28 (d, J = 8.1Hz), 116.43 (d, J = 21.9Hz), 58.60, 28.85; 19 F NMR(471MHz,DMSO-d6)δ–111.29(s).HRMS(ESI):calcd for C 15 H18 N5O F [M+H] + ,304.1559; found 304.1495.

[0060]

[0061] Formula 10: Structural formula of target compound D9

[0062] D9, white solid, yield 55%, melting point 242-243℃; 1 H NMR(500MHz,DMSO-d6)δ:11.04(d,J=27.1Hz,1H,N H ),8.29–7.90(ddd,J=102.1,60.3,55.6Hz,2H,NC H ),7.61(d,J=8.3Hz,2H,phenyl H),7.01(d,J=8.7Hz,2H,phenyl H),6.43(d,J=111.0Hz,2H,N H 2), 3.79(s, 3H, OC) H 3), 1.55(s,9H,t-butyl); 13 C NMR(126MHz,DMSO-d6)δ:160.42,130.03,128.24,127.26,114.42,58.08,55.34,28.37.HRMS(ESI):calcd for C 16 H 21 N5O2[M+H] + ,316.1695; found316.1761.

[0063]

[0064] Formula 11: Structural formula of target compound D11

[0065] D11, white solid, yield 69%, melting point 186-187℃; 1 H NMR(500MHz,DMSO-d6)δ:11.27(d,J=5.0Hz,1H,N H ), 8.37–8.01(m,2H,NC H ),7.62–7.52(m,6H,phenyl H),7.41(t,J=7.3Hz,1H,phenyl H),7.27(d,J=7.5Hz,2H,phenyl H),6.65(d,J=102.3Hz,2H,N H2), 2.34(s, 3H, C H 3); 13 C NMR(101MHz,DMSO-d6)δ:139.96,138.54,132.37,130.01,127.85,127.28,123.91,21.57.HRMS(ESI):calcd for C 18 H 17 N5O[M+H] + ,320.1433; found 320.1494.

[0066]

[0067] Formula 12: Structural formula of target compound D12

[0068] D12, white solid, yield 85%, melting point 222-223℃; 1 H NMR(500MHz,DMSO-d6)δ:11.59(s,1H,N H ), 8.80–8.04(m,2H,NC H ),8.04–7.94(m,1H,phenyl H),7.55(m,5H,phenyl H),7.42(m,3H,phenyl H),6.68(d,J=82.5Hz,2H,N H 2); 13 C NMR(126MHz,DMSO-d6)δ:138.46,133.38,132.32,131.58,130.49,130.01,128.26,127.90,127.13,123.93.HRMS(ESI):calcd for C 17 H 14 N5OCl[M+H] + ,340.0887; found 340.0949.

[0069]

[0070] Formula 13: Structural formula of target compound D13

[0071] D13, white solid, yield 90%, melting point 203-204℃; 1 H NMR(400MHz,DMSO-d6)δ:11.47(s,1H,N H ), 8.18(d, J = 74.4.0 Hz, 2H, NC H),7.83–7.27(m,9H,phenyl H),6.82–6.42(m,2H,N H 2); 13 C NMR(101MHz,DMSO-d6)δ:137.96,136.86,133.71,130.82,129.49,129.29,127.38,126.11,125.42,123.42.HRMS(ESI):calcd for C 17 H 14 N5OCl[M+H] + ,340.0887; found 340.0947.

[0072]

[0073] Formula 14: Structural formula of target compound D14

[0074] D14, white solid, yield 86%, melting point 208-209℃; 1 H NMR(500MHz,DMSO-d6)δ:11.41(d,J=24.8Hz,1H,N H ), 8.18(d, J = 118.2 Hz, 2H, NC H ),7.73(d,J=8.4Hz,2H,phenyl H),7.56(m,6H,phenyl H),7.42(t,J=7.2Hz,1H,phenyl H),6.65(d,J=93.6Hz,2H,N H 2); 13 C NMR(101MHz,DMSO-d6)δ:138.40,134.48,133.95,129.94,129.47,128.86,127.82,123.85.HRMS(ESI):calcd for C 17 H 14 N5OCl[M+H] + ,340.0887; found 340.0950.

[0075]

[0076] Formula 15: Structural formula of target compound D15

[0077] D15, white solid, yield 81%, melting point 221-222℃; 1 H NMR(500MHz,DMSO-d6)δ:11.53(s,1H,N H),8.24(dd,J=113.3,29.7Hz,2H,NC H ),7.86(dd,J=51.6,7.8Hz,4H,phenyl H),7.66–7.50(m,4H,phenyl H),7.42(dd,J=11.2,4.3Hz,1H,phenyl H),6.69(d,J=79.0Hz,2H,N H 2); 13 C NMR (126MHz, DMSO-d6) δ: 139.10, 138.45, 129.98 (d, J = 12.2Hz), 127.91 (d, J = 10.8Hz), 126.35, 125.78, 123.97, 123.62; 19 F NMR(471MHz,DMSO-d6)δ:–61.02.HRMS(ESI):calcd for C 18 H 14 N5OF3[M+H] + ,374.1150; found 374.1208.

[0078]

[0079] Formula 16: Structural formula of target compound D16

[0080] D16, white solid, yield 68%, melting point 106-107℃; 1 H NMR(500MHz,DMSO-d6)δ:11.36(s,1H,N H ),8.19(dd,J=122.1,7.3Hz,2H,NC H ),7.70–7.50(m,4H,phenyl H),7.47–7.32(m,4H,phenyl H),7.32–7.21(m,2H,phenyl H),7.00(dd,J=8.0,2.2Hz,1H,phenyl H),6.66(dd,J=94.9,1.1Hz,2H,N H 2), 3.81(s, 3H, C H 3); 13 C NMR(101MHz,DMSO-d6)δ:160.03,138.42,136.46,130.50,129.93,127.81,123.87.HRMS(ESI):calcd for C 18 H 17 N5O2[M+H] +,336.1382; found 336.1442.

[0081]

[0082] Formula 17: Structural formula of target compound D17

[0083] D17, white solid, yield 65%, melting point 226-227℃; 1 H NMR(500MHz,DMSO-d6)δ:11.37(d,J=28.5Hz,1H,N H ),8.17(dd,J=131.8,23.0Hz,2H,NC H ),7.74–7.48(m,6H,phenyl H),7.41(t,J=7.3Hz,1H,phenyl H),7.03(d,J=8.1Hz,2H,phenyl H),6.64(d,J=105.7Hz,2H,N H 2); 13 C NMR (101MHz, DMSO-d6) δ: 164.60, 162.14, 138.51, 131.69, 130.00, 129.41, 127.86, 123.91, 116.61; 19 F NMR(471MHz,DMSO-d6)δ:–111.08.HRMS(ESI):calcd forC 17 H 14 N5OF[M+H] + ,324.1182; found324.1243.

[0084]

[0085] Formula 18: Structural formula of target compound D18

[0086] D18, white solid, yield 47%, melting point 115-116℃; 1 H NMR(500MHz,DMSO-d6)δ:11.20(d,J=27.9Hz,1H,N H ),8.17(dd,J=131.8,23.0Hz,2H,NC H ),7.68–7.52(m,6H,phenyl H),7.41(t,J=7.3Hz,1H,phenyl H),7.03(d,J=8.1Hz,2H,phenyl H),6.64(d,J=105.7Hz,2H,N H 2), 3.80(s, 3H, C)H 3); 13 C NMR(101MHz,DMSO-d6)δ:160.96,138.47,129.92,128.77,127.76,127.58,123.81,114.87,55.74.HRMS(ESI):calcd for C 18 H 17 N5O2[M+H] + ,336.1382; found 336.1441.

[0087] Bioactivity assay method for target compounds

[0088] Antiviral activity test

[0089] Using the tobacco mosaic virus model plant *Nicotiana sicerata*, the in vivo activity of the target compounds was determined by the half-leaf necrotic spot method, and the antiviral activity of all compounds at a concentration of 500 μg / mL was tested.

[0090] Test materials and buffer solutions

[0091] Test virus: Tobacco mosaic virus (TMV), purchased from the Wuhan Institute of Virology, Chinese Academy of Sciences, and stored on common tobacco K326.

[0092] The tested tobacco species was Nicotiana glutinosa, a host for tobacco mosaic virus necrotic spot.

[0093] Test reagents: the synthesized compound (provided in this experiment); the control reagent was 2% Ningnanmycin aqueous solution, purchased from Heilongjiang Qianger Biochemical Technology Development Co., Ltd.

[0094] Phosphate buffer: 0.2 mol / L phosphate buffer (PBS) at pH 7.0; 0.01 mol / L phosphate buffer (PBS) at pH 7.0.

[0095] Anti-TMV in vivo activity screening

[0096] Therapeutic activity of compounds against TMV

[0097] Select tobacco plants with 5-6 leaves of uniform growth, remove the terminal leaflets, wash off the soil, and dry them. Sprinkle the leaves with carborundum and rub the virus solution onto the leaves using an oil painting brush. After 30 minutes of inoculation, rinse the leaves with clean water and dry them. Apply the agent to the left half of the leaf and the corresponding amount of solvent to the right half as a control. Then, culture them in a light incubator at a controlled temperature of 27±1℃ and a light intensity of 10000 Lux. Observe and record the number of necrotic spots after 2-3 days. Two plants per treatment, each with 3-4 leaves, were repeated three times.

[0098] Compound's protective activity against TMV

[0099] Select 5-6 leafy tobacco plants with uniform growth. Apply the agent to the left side of the leaf with a brush, and apply the corresponding amount of solvent to the right side of the leaf as a control. Incubate in a light incubator at 27±1℃ and 10000 Lux for 24 hours. Then, rub the virus-infused solution onto the leaves sprinkled with carborundum using a brush. After inoculation, wait 30 minutes and then rinse the leaves with clean water. Incubate again in a light incubator at 27±1℃ and 10000 Lux. Observe and record the number of necrotic spots after 2-3 days. Use 2 plants per treatment, each with 3-4 leaves, and repeat 3 times.

[0100] Compound passivation activity against TMV

[0101] Select 5-6 leafy tobacco plants with uniform growth. Mix the appropriate dose of the agent with the virus extract and stir thoroughly for 30 minutes. Then, rub the mixture onto the left side of the leaf sprinkled with carborundum. Use an oil painting brush to rub the virus solution onto the right side of the leaf. Rinse with clean water 30 minutes after inoculation. Incubate in a light incubator at a temperature of 27±1℃ and a light intensity of 10000 Lux. Observe and record the number of necrotic spots after 2-3 days. Two plants are planted per agent treatment, with each plant having 3-4 leaves.

[0102] Results Analysis

[0103] When obvious necrotic spots appear on the control half-leaf, the spots can be counted about 2-3 days after the experiment. The number of necrotic spots on the left and right halves of each leaf should be recorded separately, and the ratio of the test compound to tobacco mosaic virus should be calculated using the following formula.

[0104] S = (YZ) / Y × 100%

[0105] Where: S is the ratio of the compound to TMV;

[0106] Y represents the number of necrotic spots in the control group (left half of the leaf), in units of: spots;

[0107] Z represents the number of necrotic spots in the compound-treated group (right half of the leaf), in units of individual spots.

[0108] The mean and deviation of the inhibition rate of each drug against tobacco mosaic virus were calculated. Each treatment used its counterpart as a control, and a treatment with commercial ningnanmycin was set up as a comparison.

[0109] In vivo activity data of the target compound against TMV

[0110] Based on the experimental steps described above, the bioactivity of all target compound pyrazolylhydrazone derivatives against tobacco mosaic virus (TMV) was tested using the half-leaf necrotic spot method. The results are shown in Tables 1 and 2.

[0111] Table 1. Results of anti-TMV activity tests of pyrazolylhydrazone derivatives D1-D18 at a concentration of 500 μg / mL. a

[0112] serial number Protective activity (%) Therapeutic activity (%) Passivation activity (%) D1 39.5±6.9 46.5±2.9 43.5±7.1 D2 39.9±8.0 45.1±3.3 47.0±8.3 D3 17.8±2.3 0 47.6±7.8 D4 8.5±6.9 28.0±3.2 0 D5 39.4±8.9 39.4±2.9 0 D6 0 60.2±3.1 61.0±5.3 D7 0 25.4±5.0 16.6±5.4 D8 54.1±5.8 67.4±3.5 33.0±5.8 D9 49.0±7.3 58.5±3.3 63.6±4.2 D10 50.6±8.8 44.5±6.9 9.6±4.1 D11 46.1±9.1 40.1±3.1 75.7±7.1 D12 0 10.7±5.3 10.4±5.0 D13 16.4±7.1 0 21.8±4.3 D14 37.5±9.2 37.4±7.5 23.3±8.6 D15 0 38.0±4.0 42.6±5.3 D16 0 22.6±4.1 35.8±6.4 D17 50.9±9.1 58.6±8.2 35.8±4.8 D18 55.1±9.2 60.0±4.8 37.7±6.6 Ningnanmycin 70.6±6.5 66.1±2.7 80.9±4.7

[0113] “a” indicates that each experiment is repeated three times.

[0114] Table 2 EC50 of the therapeutic activity of pyrazolylhydrazone derivatives against TMV 50 a

[0115] serial number <![CDATA[EC 50 (μg / mL)]]> Poisoning equation <![CDATA[R 2 ]]> D6 211.3±3.4 y = 0.8925x + 2.9250 0.980 D8 139.0±6.3 y = 0.9385x + 2.9887 0.961 D9 265.6±6.7 y = 0.9887x + 2.6031 0.993 D17 192.6±8.1 y = 1.0827x + 2.3436 0.989 D18 284.1±7.7 y = 0.9619x + 2.7344 0.992 Ningnanmycin 152.3±5.9 y = 0.9420x + 2.9439 0.971

[0116] “a” indicates that each experiment is repeated three times.

[0117] The in vivo activity tests of the target compounds against tobacco mosaic virus at a concentration of 500 μg / mL, as shown in Table 1, indicate that some target compounds exhibited certain inactivation and protective activities, while several showed good therapeutic activity. Specifically, the therapeutic activities of D6 (60.2%), D9 (58.5%), D17 (58.6%), and D18 (60.0%) were slightly lower than those of ningnanmycin (66.1%), while the therapeutic activity of D8 (67.4%) was similar to that of ningnanmycin. Table 2 shows the EC50 values ​​of the target compounds' therapeutic activities against tobacco mosaic virus. 50 Based on the values, compound D8 (139.0 μg / mL) showed better therapeutic activity than ningnanmycin (152.3 μg / mL), thus demonstrating promising research and development potential.

Claims

1. A class of 1-substituted-5-amino-4-pyrazole hydrazone compounds, characterized in that: The structural formula is as follows: wherein R1 is tert-butyl and R2 is 4-fluorophenyl.

2. The method for preparing a class of 1-substituted-5-amino-4-pyrazole hydrazone compounds as described in claim 1, characterized in that: The reaction formula is as follows: 。 3. The application of the 1-substituted-5-amino-4-pyrazole hydrazone compounds as described in claim 1 in the prevention and control of tobacco viral diseases caused by tobacco mosaic virus.

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