Andrographolide Derivatives, Their Synthesis and Applications

Abstract

This invention relates to the field of antibacterial agent technology, specifically to an andrographolide derivative, its synthesis method, and its application. By structurally modifying andrographolide, a series of 14-aryloxy / amide-substituted derivatives were synthesized. First, the 3,19-dihydroxy group was protected to obtain 3,19-isopropylidene andrographolide. Subsequently, a series of compounds with C-14 configuration inversion were obtained through the Mitsunobu reaction. Finally, the protecting group at the 3,19-position was removed in the presence of p-toluenesulfonic acid monohydrate to obtain the target compound. The andrographolide derivative provided in the above technical solution showed an inhibition rate of over 60% against six tested fungi at a concentration of 100 μg / mL, and its activity was superior to the positive control azoxystrobin. Simultaneously, it significantly disrupted the mycelial morphology of *Pyrrosia lingua* and had no effect on cowpea seed germination, demonstrating high safety.

Description

Technical Field

[0001] This invention relates to the field of antibacterial agent technology, specifically to an andrographolide derivative, its synthesis method, and its application. Background Technology

[0002] Plant pathogenic fungi primarily harm crops by causing diseases, accounting for approximately 70%–80% of all plant diseases. These pathogenic fungi are diverse, including powdery mildew, rust, and downy mildew. These necrotic fungi not only cause significant losses in the field and during storage but also threaten food security. Currently, the most effective antifungal agents are mainly chemical fungicides; however, overuse of fungicides can affect soil respiration, microbial diversity, and enzyme activity. Furthermore, the scarcity of antifungal agents and the emergence of fungal resistance make the development of novel antifungal drugs from chemical synthesis or natural products an urgent priority.

[0003] Compared with chemically synthesized products, natural products have advantages such as wide availability, structural diversity, and relatively low toxicity. Whether using natural products directly or optimizing their structure to obtain highly effective and safe derivatives, natural products are an important potential source of antifungal drugs. Traditional Chinese medicine is a treasure trove of natural products, and many compounds isolated from it have been shown to possess various pharmacological activities, including antibacterial, antitumor, antiviral, and antifungal properties. Andrographolide, extracted from the plant *Andrographis paniculata* (Acanthaceae family), is an important diterpenoid lactone compound. Its diverse structures and broad biological activities make it of significant research value in the pharmaceutical field. Studies have shown that andrographolide and its derivatives have significant advantages in antibacterial, antifungal, and insecticidal activity (e.g.,...). Figure 1 (As shown). There is an urgent need to develop andrographolide derivatives with better antibacterial, antifungal, and insecticidal effects. Summary of the Invention

[0004] The purpose of this invention is to provide a andrographolide derivative, its synthesis method, and its application, which can effectively solve the problems involved in the background art.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0006] An andrographolide derivative, with the molecular formula:

[0007]

[0008] In Equation I and Equation II, R is selected from...

[0009] Preferably, R in Formula I and Formula II is selected from...

[0010] Preferably, the molecular formula of the andrographolide derivative is:

[0011]

[0012] And R in the formula is

[0013] Furthermore, this invention also relates to a method for synthesizing andrographolide derivatives, the synthetic route of which is as follows:

[0014]

[0015] Where R is selected from

[0016] In addition, the present invention also relates to the application of andrographolide derivatives in the preparation of plant antifungal drugs; and the application of andrographolide derivatives prepared by the above-mentioned synthesis method in the preparation of plant antifungal drugs.

[0017] Preferably, the andrographolide derivatives have inhibitory effects on the following plant pathogens: Fusarium graminearum, Alternaria solani, Alternaria brassicae, Alternata alternata, C. lunata, C. gloeosporioides, Fusarium bulbigenum, V. mali (the causal agent of apple rot), P. oryza (the causal agent of rice blast), and P. piricola.

[0018] The andrographolide derivatives, their synthesis methods, and applications provided in the above technical solutions have synthesized new compounds with antifungal activity. The synthesized compounds showed an inhibition rate of over 60% against six test fungi at a concentration of 100 μg / mL, and their activity was superior to that of the positive control azoxystrobin. At the same time, they could significantly disrupt the mycelial morphology of P. piricola and had no effect on cowpea seed germination, demonstrating high safety. Attached Figure Description

[0019] Figure 1 Andrographolide and its derivatives are existing compounds.

[0020] Figure 2 Scanning electron micrographs of the hyphal morphology of *Pyroderma pyrifolia* before and after 4 days of treatment with andrographolide derivatives.

[0021] Figure 3 Photos of cowpea seeds treated with different concentrations of andrographolide derivatives for 4 days.

[0022] Figure 4 For compound 3c 1 1H NMR spectrum (solvent: DMSO-d6);

[0023] Figure 5 For compound 3d 1 1H NMR spectrum (solvent: DMSO-d6);

[0024] Figure 6 For compound 3e 13 C10 NMR spectrum (solvent: DMSO-d6);

[0025] Figure 7 For compound 3f 1 1H NMR spectrum (solvent: DMSO-d6);

[0026] Figure 8 3g of compound 13 C10 NMR spectrum (solvent: DMSO-d6);

[0027] Figure 9 For compound 3j 1 1H NMR spectrum (solvent: DMSO-d6);

[0028] Figure 10 For compound 3k 1 1H NMR spectrum (solvent: DMSO-d6);

[0029] Figure 11 For compound 4c 1 1H NMR spectrum (solvent: DMSO-d6);

[0030] Figure 12 For compound 4c 13 C10 NMR spectrum (solvent: DMSO-d6);

[0031] Figure 13 For compound 4d 1 1H NMR spectrum (solvent: DMSO-d6);

[0032] Figure 14 For compound 4d 13 C10 NMR spectrum (solvent: DMSO-d6);

[0033] Figure 15 For compound 4e 1 1H NMR spectrum (solvent: DMSO-d6);

[0034] Figure 16 For compound 4e 13C10 NMR spectrum (solvent: DMSO-d6);

[0035] Figure 17 For compound 4f 1 1H NMR spectrum (solvent: DMSO-d6);

[0036] Figure 18 For compound 4f 13 C10 NMR spectrum (solvent: DMSO-d6);

[0037] Figure 19 4g of compound 1 1H NMR spectrum (solvent: DMSO-d6);

[0038] Figure 20 4g of compound 13 C10 NMR spectrum (solvent: DMSO-d6);

[0039] Figure 21 For compound 4j 1 1H NMR spectrum (solvent: DMSO-d6);

[0040] Figure 22 For compound 4j 13 C10 NMR spectrum (solvent: DMSO-d6);

[0041] Figure 23 For compound 4k 1 1H NMR spectrum (solvent: DMSO-d6);

[0042] Figure 24 For compound 4k 13 C10 NMR spectrum (solvent: DMSO-d6). Detailed Implementation

[0043] To make the objectives and advantages of this invention clearer, the invention will be specifically described below with reference to embodiments. It should be understood that the following text is merely used to describe one or more specific embodiments of the invention and does not strictly limit the scope of protection specifically claimed by the invention.

[0044] Andrographolide derivatives were synthesized using the following synthetic method:

[0045]

[0046] In the formula, R is They correspond to the letters ak respectively.

[0047] Example 1: A series of 14-aryloxy / amide-substituted derivatives, 4a-4k, were synthesized by structural modification of andrographolide. First, the 3,19-dihydroxy group was protected to give 3,19-isopropylidene andrographolide in 90.2% yield. Subsequently, a series of compounds 3a-3k with C-14 configuration inversion were obtained by the Mitsunobu reaction; finally, the protecting group at the 3,19-position was removed in the presence of p-toluenesulfonic acid monohydrate to obtain the target compounds 4a-4k.

[0048] The amounts of each raw material, calculated by molar ratio, are as follows: andrographolide: 2,2-dimethoxypropane ≈ 1:7; PPTS is the catalytic amount (andrographolide: PPTS = 1:0.1); compound 2: triphenylphosphine: DIAD: RH = 1:1.5:1.5:1.5; compound 3a-3k: p-toluenesulfonic acid monohydrate = 1:0.1 (normal catalytic amount is sufficient).

[0049] Specifically, andrographolide (9.95 g, 28.4 mmol) and 2,2-dimethoxypropane (24 mL, 195.3 mmol) were dissolved in 20 mL of anhydrous dichloromethane (DCM), and pyridinium p-toluenesulfonate (PPTS, 0.71 g, 2.8 mmol) was added. The reaction was carried out at 40 °C. After the reaction was completed by TLC monitoring, the mixture was treated with ethyl acetate and saturated sodium bicarbonate solution. The organic phase was washed with brine, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1:1) to give compound 2. Under nitrogen protection, compound 2 (3.94 g, 10.1 mmol), triphenylphosphine (PPh3, 3.97 g, 15.1 mmol), and RH (15.1 mmol) were dissolved in 30 mL of anhydrous tetrahydrofuran. The solution was cooled to 0°C, and 5 mL of anhydrous tetrahydrofuran solution of diisopropyl azodicarbonate (DIAD, 3 mL, 15.1 mmol) was slowly added dropwise. The mixture was stirred at 0°C for 1 hour, then stirred overnight at room temperature. After evaporating the solvent, the residue was dissolved in ethyl acetate, washed five times with brine, and dried over anhydrous sodium sulfate. After filtration, the residue was evaporated to dryness, and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 5:1) to give 3a-3k in yields of 32.4%–51.0%. Compound 3a-3k (5.0 mmol) was dissolved in 15 mL of methanol, and p-toluenesulfonic acid monohydrate (0.10 g, 0.5 mmol) was added. The mixture was stirred at 20°C for 30 minutes. The solution was diluted with ethyl acetate, washed with saturated sodium bicarbonate solution and brine, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10:7) to give 4a-4k in yields of 65.5%–86.7%.

[0050] All compounds were tested. 1 H NMR,13 The structure was confirmed by C10 NMR and high-resolution mass spectrometry (HR-MS). 1 H NMR and 13 CNMR spectra were measured using a Bruker NMR spectrometer (400 MHz and 101 MHz) with deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), or deuterated benzene (C6D6) as solvents. High-resolution mass spectrometry (HR-MS) data were obtained using a Thermo Scientific Q Exactive mass spectrometer. Melting points were determined using an X-4 micromelting point apparatus.

[0051] 14-(S)-3,19-Isopropylidene andrographolide (Compound 2):

[0052] White powder, melting point 145.2-146.1℃, yield 90.2%; 1 H NMR(400MHz,DMSO-d6)δppm 6.63(t,J=6.4Hz,1H),5.74(d,J=6.0Hz,1H),4.93(t,J=5.8Hz,1H),4.86(s,1H),4.69( s,1H),4.41(q,J=10.0,6.0Hz,1Hz),4.04(dd,J=9.8,1.8Hz,1H),3.89(d,J=11.6Hz,1H) ,3.42(dd,J=9.2,3.6Hz),3.12(d,J=11.6Hz),2.54-2.50(m,1H),2.40-1.88(m,3H),1. 78-1.63(m.3H),1.34(s,3H),1.26(s,3H),1.34-1.15(m,3H),1.14(s,3H),0.88(s,3H).

[0053] 14-(R)-phenoxy-3,19-isopropylidene andrographolide (compound 3a):

[0054] White powder, melting point 153-155℃, yield 51.0%; 1H NMR(400MHz, CDCl3)δ(ppm)7.39–7.28(m,2H),7.13(td,J=7.4,1.8Hz,1H),7.05(tt,J=7.5,1.0Hz,1H),6.88–6.80(m,2H),5.53(d,J=5.7H z,1H),4.87(d,J=1.6Hz,1H),4.60(dd,J=10.7,5.8Hz,1H),4.46(s,1H),4.40(dd,J=10.7,2.0Hz,1H),3.91(d,J=11.6Hz,1H),3.42(dd,J= 8.3,3.9Hz,1H),3.14(d,J=11.5Hz,1H),2.57–2.46(m,1H),2.45–2.26(m,2H),1.95(dd,J=27.6,12.1Hz,2H),1.88–1.77(m,1H),1.75–1.6 7(m,1H),1.64(dd,J=11.8,6.0Hz,1H),1.51(dd,J=8.0,5.4Hz,1H),1.37(s,3H),1.34(s,3H),1.31–1.19(m,3H),1.17(s,3H),0.87(s,3H). 13 C NMR(101MHz,C6D6)δ168.8,157.0,149.6,148.1,130.1,126.0,122.2,115.9,108.0,99.4,75.4,71.5, 70.5,64.2,55.8,51.3,38.5,38.3,37.8,33.7,26.7,26.1,25.7,25.3,24.8,23.3,16.6.ESI-HRMS:m / z 489.2658[M+Na] + calcd for C 29 H 38 NaO5,489.2617.

[0055] 14-(R)-(2'-chlorophenoxy)-3,19-isopropylidene andrographolide (compound 3b):

[0056] White powder, melting point 155.8-159.2℃, yield 48.1%; 1H NMR (400MHz, C6D6) δ7.22(td,J=7.3,1.8Hz,1H),7.12(dd,J=8.0,1.7Hz,1H),6.76–6.70(m,1H),6.52(td,J=7.7, 1.4Hz,1H),6.19–6.12(m,1H),4.91(s,1H),4.83(d,J=1.5Hz,1H),4.42(d,J=1.5Hz,1H),3.84–3.78(m,2H),3.67 –3.61(m,1H),3.42(dd,J=7.6,3.7Hz,1H),3.07(d,J=11.5Hz,1H),2.25–2.09(m,3H),1.74(dq,J=13.4,6.5,6.0H z,2H),1.61(d,J=10.2Hz,1H),1.53–1.44(m,1H),1.43–1.31(m,8H),1.10(s,3H),1.02–0.89(m,3H),0.84(s,3H). 13 C NMR(101MHz,C6D6)δ168.37,152.43,150.31,148.18,131.06,127.78,125.35,124.58,123.13,116.05,107.83,99.30,75.25,7 2.76,70.05,64.13,55.90,51.02,38.38,38.15,37.62,33.53,26.52,25.94,25.79,25.19,24.62,23.21,16.52.ESI-HRMS:m / z 523.2227,[M+Na] + ,calcdfor C 29 H 37 ClNaO5,523.2233.

[0057] 14-(R)-(2'-cyanophenoxy)-3,19-isopropylidene andrographolide (compound 3c), see reference. Figure 4 :

[0058] White powder, melting point 154.6-154.7℃, yield 49.7%; 1H NMR(400MHz,DMSO-d6)δppm 7.83(dd,J=7.7,1.6Hz,1H),7.77–7.69(m,1H),7.28(d,J=8.6Hz,1H),7.19(t,J=7.5Hz,1H),7.11(t,J=7.1Hz,1H ),6.01(d,J=5.2Hz,1H),4.87(s,1H),4.74(dd,J=11.0,5.5Hz,1H),4.65(s,1H),4.40(d,J=11.0Hz,1H),3.79(d,J =11.6Hz,1H),3.19–3.14(m,1H),3.05(d,J=11.6Hz,1H),2.47–2.28(m,3H),1.99(t,J=9.7Hz,2H),1.72–1.59(m, 2H),1.46–1.30(m,2H),1.26(s,3H),1.21(s,3H),1.24–1.14(m,2H),1.07(s,3H),0.98–0.88(m,1H),0.77(s,3H). 13 C NMR (126MHz, DMSO-d6) δ168.78,158.07,150.78,147.59,135.27,134.24,125.17,122.11,116.22,113.90,108.19,101.32,98.18,75 .56,72.04,70.67,62.71,56.04,51.11,38.21,37.14,36.87,33.49,27.33,25.68,25.25,25.06,24.51,22.66,15.71.ESI-HRMS:m / z 514.2564,[M+Na] + ,calculated for C 30 H 37 NO5Na, 514.2569.

[0059] 14-(R)-(2'-bromophenoxy)-3,19-isopropylidene andrographolide (compound 3d), see reference. Figure 5 :

[0060] White powder, melting point 161.4-161.9℃, yield 44.3%; 1H NMR(400MHz,DMSO-d6)δppm 7.64(dd,J=7.9,1.2Hz,1H),7.44–7.35(m,1H),7.16(d,J=8.1Hz,1H),7.04(t,J=7.3Hz,1H),6.98(t,J=7.6Hz,1H),5.9 1(d,J=5.0Hz,1H),4.86(s,1H),4.70(dd,J=10.9,5.3Hz,1H),4.62(s,1H),4.35(d,J=10.9Hz,1H),3.78(d,J=11.6Hz,1 H),3.17(dd,J=8.8,3.4Hz,1H),3.04(d,J=11.6Hz,1H),2.44–2.25(m,3H),1.99(t,J=10.4Hz,2H),1.72–1.57(m,2H),1 .43–1.30(m,2H),1.26(s,3H),1.21(s,3H),1.25–1.14(m,2H),1.07(s,3H),0.93(dt,J=16.1,8.2Hz,1H),0.76(s,3H); 13 C NMR(101MHz,DMSO-d6)δ168.98,152.85,150.16,147.67,133.53,129.15,125.66,123.12,114.97,111.66,108.19,98.18,75.49 ,71.89,70.97,62.74,55.84,50.96,38.15,37.15,36.96,33.43,27.33,25.65,25.24,25.06,24.49,22.64,15.78; ESI-HRMS:m / z 567.1726,[M+Na] + ,calculated for C 29 H 37 BrO5Na, 567.1722.

[0061] 14-(R)-(2'-trifluoromethylphenoxy)-3,19-isopropylidene andrographolide (compound 3e), see reference. Figure 6 :

[0062] White powder, melting point 62.2-62.6℃, yield 33.8%; 1H NMR (400MHz, DMSO-d6) δ7.69(t,J=7.7Hz,2H),7.37(d,J=8.4Hz,1H),7.19(t,J=7.6Hz,1H),6.96(t,J=6.7Hz,1H),6.09(d ,J=5.0Hz,1H),4.85(s,1H),4.71(dd,J=11.0,5.4Hz,1H),4.54(s,1H),4.31(d,J=11.0Hz,1H),3.78(d,J=11.6Hz,1H),3.2 2(dd,J=8.6,3.4Hz,1H),3.04(d,J=11.6Hz,1H),2.43–2.35(m,2H),2.32(d,J=12.5Hz,1H),1.92(dd,J=14.3,9.9Hz,2H), 1.74–1.58(m,2H),1.39(s,1H),1.45–1.32(m,2H),1.26(s,3H),1.21(s,3H),1.18–1.14(m,2H),1.05(s,3H),0.76(s,3H); 13 C NMR(101MHz,DMSO-d6)δ168.84,154.22(d,J=1.1Hz),150.39,147.59,134.27,127 .25(dd,J=9.9,4.8Hz),125.18,123.57(d,J=272.4Hz),121.27,117.92(q,J=30.2 Hz),114.58,108.32,98.27,75.35,71.49,70.76,62.84,55.41,51.08,37.97,37. 22,36.98,33.30,27.02,25.65,25.29,24.97,24.38,22.63,15.85;ESI-HRMS:m / z 557.2489,[M+Na] + ,calculated for C 30 H 37 F3O5Na, 557.2491.

[0063] 14-(R)-(2'-iodophenoxy)-3,19-isopropylidene andrographolide (compound 3f), see reference. Figure 7 :

[0064] White powder, melting point 179.7-179.8℃, yield 47.8%; 1H NMR(400MHz,DMSO-d6)δppm 7.83(dd,J=7.7,1.4Hz,1H),7.45–7.34(m,1H),7.05(t,J=7.9Hz,2H),6.82(t,J=7.2Hz,1H),5.90(d,J=4.9Hz ,1H),4.86(s,1H),4.70(dd,J=10.9,5.3Hz,1H),4.64(s,1H),4.32(d,J=10.9Hz,1H),3.78(d,J=11.6Hz,1H), 3.18(dd,J=8.6,3.1Hz,1H),3.04(d,J=11.6Hz,1H),2.46–2.26(m,3H),2.10–1.97(m,2H),1.71–1.55(m,2H), 1.42–1.30(m,3H),1.26(s,3H),1.21(s,3H),1.19–1.14(m,1H),1.08(s,3H),0.98–0.89(m,1H),0.76(s,3H); 13 C NMR(101MHz,DMSO-d6)δ169.00,155.20,149.96,147.69,139.58,129.85,125.76,123.54,113.57,108.16,98.19,87.32,75.42, 71.69,70.96,62.78,55.73,50.76,38.18,37.19,36.94,33.42,27.29,25.59,25.37,25.04,24.51,22.67,15.87; ESI-HRMS:m / z 615.1582,[M+Na] + ,calculated for C 29 H 37 IO5Na, 615.1583.

[0065] 14-(R)-(2'-fluorophenoxy)-3,19-isopropylidene andrographolide (compound 3g), reference Figure 8 :

[0066] White powder, melting point 157.8-158.3℃, yield 49.6%; 1H NMR (500MHz, DMSO-d6) δ7.29(ddd,J=11.6,8.1,1.5Hz,1H),7.23(td,J=8.3,1.6Hz,1H),7.18(td,J=8.1,1.1Hz,1H),7.09–7.04(m,1H),6.96( td,J=7.1,1.1Hz,1H),5.83(d,J=5.2Hz,1H),4.84(s,1H),4.66(dd,J=10.9,5.3Hz,1H),4.54(s,1H),4.40(dd,J=10.9,1.1Hz,1H),3.80(d,J=1 1.6Hz,1H),3.24(dd,J=8.9,4.0Hz,1H),3.06(d,J=11.6Hz,1H),2.37–2 .25(m,3H),1.99–1.88(m,2H),1.77–1.69(m,1H),1.67–1.60(m,1H),1. 45(ddd,J=17.4,8.1,5.2Hz,1H),1.41–1.34(m,1H),1.28(s,3H),1.22( s,3H),1.21–1.12(m,2H),1.08(s,3H),1.06–0.99(m,1H),0.77(s,3H); 13 C NMR (126MHz, DMSO-d6) δ168.97,152.52(d,J=244.1Hz),150.06,147.58,144.21(d,J= 10.6Hz),125.55,125.02(d,J=3.8Hz),122.83(d,J=7.0Hz),117.53,116.55(d,J=18.0 Hz),108.23,98.19,75.53,72.70,71.01,62.75,55.57,51.25,38.07,37.14,36.99,33 .49,27.29,25.70,25.11,25.09,24.48,22.64,15.70; ESI-HRMS:m / z507.2520,[M+Na] + ,calculated for C 29 H 37 FO5Na,507.2523.

[0067] 14-(R)-(cis-1',2',3',6'-tetrahydrophthalimide)-3,19-isopropylidene andrographolide (compound 3h):

[0068] White powder, melting point 211.7-213.4℃, yield 34.3%;1 H NMR (400MHz, DMSO-d6) δ6.43(td,J=5.9,4.6,2.3Hz,1H),5.92–5.79(m,2H),5.46(d,J=8.4Hz,1H),4.79(s,1H),4.57(t,J=9.4Hz,1H),4.34(s,1H), 4.19(dd,J=9.9,3.1Hz,1H),3.85(d,J=11.6Hz,1H),3.40(dd,J=8.8,4.0H z,1H),3.23(ddd,J=9.7,7.6,2.4Hz,1H),3.13(td,J=7.9,2.4Hz,1H),3.0 9(d,J=11.8Hz,1H),2.42(dq,J=15.8,2.7Hz,1H),2.33(ddd,J=12.9,6.2, 3.0Hz,2H),2.20(dt,J=15.5,8.5Hz,2H),2.11–2.00(m,2H),1.93(tt,J=1 3.4,5.7Hz,3H),1.69(dp,J=16.1,5.3Hz,2H),1.47(dt,J=12.9,6.0Hz,1H ),1.33(s,3H),1.30–1.16(m,3H),1.25(s,3H),1.11(s,3H),0.80(s,3H). 13 CNMR(101MHz,DMSO-d6)δ179.15,178.83,168.87,147.64,145.09,127.88,127.50,124.45,108.35,98.29,75.51,68.29,62.88,54.8 0,51.15,45.45,38.85,38.18,37.79,37.25,37.04,33.74,27.28,25.71,25.17(2C),24.59,22.97(2C),22.70,15.86.ESI-HRMS:m / z 546.2815,[M+Na] + ,calculated for C 31 H 41 NO6Na, 546.2832.

[0069] 14-(R)-(ciscyclohexane-1',2'-dimethylformamide)-3,19-isopropylidene andrographolide (compound 3i):

[0070] White powder, melting point 143.6-145.8℃, yield 32.4%; 1H NMR (400MHz, DMSO-d6) δ6.49(td,J=6.5,2.3Hz,1H),5.50(d,J=7.9Hz,1H),4.82(s,1H),4.58(t,J=9.4Hz,1H),4.38(s,1 H),4.24(dd,J=10.0,3.0Hz,1H),3.84(d,J=11.6Hz,1H),3.39(dd,J=8.7,4.0Hz,1H),3.08(d,J=11.6Hz,1H),3.01(q,J=6 .8Hz,1H),2.93(q,J=7.1Hz,1H),2.33(dd,J=13.0,3.5Hz,1H),2.18(t,J=7.1Hz,2H),1.96(p,J=7.8,6.5Hz,2H),1.92–1 .77(m,2H),1.76–1.62(m,4H),1.51–1.36(m,4H),1.35–1.17(m,5H),1.32(s,3H),1.24(s,3H),1.11(s,3H),0.81(s,3H); 13 CNMR(126MHz,DMSO-d6)δ178.55,178.30,168.93,147.59,144.94,124.75,108.39,98.31,75.43,68.47,62.89,54.70,51.08,45.16, 39.05,38.78,37.80,37.25,37.00,33.77,27.22,25.69,25.14,24.96,24.56,23.84,22.76,22.71,21.36(2C),15.89; ESI-HRMS:m / z 548.2972,[M+Na] + ,calculated for C 31 H 43 NO6Na, 548.2988.

[0071] 14-(R)-(1',2'-cycloglutamylimide)-3,19-isopropylidene andrographolide (compound 3j), see reference. Figure 9 :

[0072] White powder, melting point 229.1-231.2℃, yield 37.4%; 1H NMR(400MHz,DMSO-d6)δppm 6.52–6.42(m,1H),5.47(d,J=7.8Hz,1H),4.81(s,1H),4.56(t,J=9.4Hz,1H),4.37(s,1H),4.23(dd,J=10.0,3.0 Hz,1H),3.83(d,J=11.6Hz,1H),3.39(dd,J=8.4,3.9Hz,1H),3.25(dd,J=11.8,7.8Hz,1H),3.20–3.13(m,1H),3.0 8(d,J=11.6Hz,1H),2.32(d,J=12.9Hz,1H),2.11(t,J=8.7Hz,2H),1.95(dd,J=17.4,12.1Hz,2H),1.90–1.76(m,5 H),1.74–1.61(m,3H),1.44–1.35(m,1H),1.32(s,3H),1.24(s,4H),1.28–1.13(m,7H),1.10(s,3H),0.81(s,3H); 13 C NMR(101MHz, CDCl3)δ178.78(2C),169.04,147.04,146.50,123.86,108.72,99.46,75.74,68.71,64.15,56.01,51.79,46.13,45.24,45 .10,38.29,38.14,37.63,34.30,30.72,30.50,26.75,26.12,25.46,25.26,25.05,24.75,23.31,16.60;ESI-HRMS:m / z534.2826,[M+Na] + ,calculated for C 30 H 41 NO6Na, 534.2832.

[0073] 14-(R)-(2',3'-pyridinediimide)-3,19-isopropylidene andrographolide (compound 3k), see reference. Figure 10 :

[0074] White powder, melting point 258.8-259.2℃, yield 37.1%; 1H NMR (400MHz, DMSO-d6) δ9.03(d,J=4.5Hz,1H),8.37(d,J=7.1Hz,1H),7.84(dd,J=7.6,5.0Hz,1H),6.64(dd,J=6.4,5 .5Hz,1H),5.75(d,J=8.0Hz,1H),4.85(s,1H),4.65(t,J=9.3Hz,1H),4.49(dd,J=11.5,3.8Hz,2H),3.74(d,J=11.5Hz ,1H),3.13(dd,J=8.6,3.6Hz,1H),3.01(d,J=11.5Hz,1H),2.35–2.18(m,3H),1.85(td,J=12.8,4.9Hz,1H),1.68(d,J =6.0Hz,1H),1.62–1.47(m,2H),1.25(s,3H),1.20(s,3H),1.31–1.08(m,3H),1.01(s,1H),0.99(s,3H),0.70(s,4H); 13 CNMR(101MHz,DMSO-d6)δ168.98,165.22,165.15,155.20,151.61,151.02,147.37,145.14,131.58,128.18,126.96,124.31,108.12,98.14,75 .43,68.73,62.69,54.67,51.20,44.86,37.87,37.02,36.95,33.89,27 .31,25.61,25.10,24.69,24.35,15.43;ESI-HRMS:m / z534.2470,[M+Na] + ,calculated for C 30 H 36 N₂O₆Na, 543.2471.

[0075] 14-(R)-phenoxyandrographolide (compound 4a):

[0076] White powder, melting point 164-166℃, yield 86.0%; 1H NMR(400MHz, CDCl3)δ(ppm)7.40–7.29(m,2H),7.14–7.01(m,2H),6.89–6.79(m,2H),5.55–5.48(m,1H),4.85(dd,J=1.9,1 .0Hz,1H),4.60(dd,J=10.7,5.9Hz,1H),4.44–4.36(m,2H),4.17–4.08(m,1H),3.40(dd,J=11.5,4.3Hz,1H),3.29(d,J=10. 9Hz,1H),2.54–2.44(m,1H),2.44–2.36(m,1H),2.33–2.23(m,1H),2.16(d,J=26.3Hz,3H),1.97(td,J=12.7,10.7,6.2Hz,1 H),1.92–1.85(m,1H),1.85–1.70(m,2H),1.70–1.55(m,2H),1.33–1.16(m,5H),1.11(td,J=13.5,3.7Hz,1H),0.58(s,3H). 13 C NMR(101MHz,C6D6)δ169.5,156.4,150.8,147.1,130.0,125.0,122.3,115.7,108.2,80.4,71.3,71.0, 64.1,55.9,55.1,42.8,39.0,37.7,36.8,28.1,25.6,23.7,22.7,15.1.ESI-HRMS:m / z449.2342,[M+Na] + calcd for C 26 H 34 NaO5,449.2304.

[0077] 14-(R)-(2'-chlorophenoxy)andrographolide (compound 4b):

[0078] White powder, melting point 178-180℃, yield 78.3%; 1H NMR (400MHz, CD3OD) δ7.46(dd,J=7.9,1.6Hz,1H),7.33(dddd,J=8.3,7.6,1.6Hz,1H),7.09(dddd,J=23.7,15.4,7.7,1.4Hz,3H ),5.82(d,J=5.4Hz,1H),4.88(s,1H),4.67(dd,J=10.9,5.5Hz,1H),4.56(s,1H),4.39(dd,J=10.8,1.3Hz,1H),4.03(d,J=11.0 Hz,1H),3.29(s,1H),3.19(dd,J=11.9,3.8Hz,1H),2.48–2.25(m,3H),2.02(dd,J=13.5,11.2Hz,2H),1.86–1.77(m,1H),1.64 (ddd,J=15.5,13.3,3.9Hz,1H),1.54–1.44(m,2H),1.38–1.21(m,2H),1.17(s,3H),0.99(dt,J=14.8,4.3Hz,1H),0.62(s,3H). 13 C NMR (101MHz, CD3OD) δ171.61,153.78,152.01,149.09,131.98,129.45,127.02,124.79,124.15,116.70,108.38,80.76,73.70 ,72.78,64.92,57.81,55.99,43.59,40.18,38.91,37.74,28.90,26.70,25.17,23.32,15.48.ESI-HRMS:m / z483.1914,[M+Na] + calcd for C 26 H 33 ClNaO5, 483.1915.

[0079] 14-(R)-(2'-cyanophenoxy)andrographolide (compound 4c), see reference Figure 11 and Figure 12 :

[0080] White powder, melting point 169.2-170.1℃, yield 86.7%; 1H NMR(400MHz,DMSO-d6)δppm 7.81(d,J=7.5Hz,1H),7.73(t,J=7.7Hz,1H),7.28(d,J=8.5Hz,1H),7.19(t,J=7.5Hz,1H),7.08(t,J=7.0Hz,1H),6.01(d,J=4.5Hz,1H) ,4.99(d,J=4.4Hz,1H),4.83(s,1H),4.75(dd,J=11.0,5.4Hz,1H),4.58(s,1H),4.39(d,J=11.0Hz,1H),4.11(d,J=5.5Hz,1H),3.77(d, J=10.4Hz,1H),3.20(dd,J=10.1,7.9Hz,1H),3.05–2.94(m,1H),2.48–2.40(m,1H),2.39–2.25(m,2H),2.03–1.89(m,2H),1.71(d,J=12 .0Hz,1H),1.51(dd,J=26.0,12.8Hz,2H),1.39–1.23(m,2H),1.18(d,J=11.5Hz,1H),1.05(s,3H),0.88(t,J=12.3Hz,1H),0.56(s,3H); 13 C NMR(101MHz,DMSO-d6)δ168.78,158.06,150.95,147.67,135.21,134.21,125.02,122.08,116.25,113.93,107.73,101.36,78.33, 72.06,70.65,62.54,56.14,54.08,42.15,38.79,37.30,36.13,27.80,25.11,23.91,23.02,14.62; ESI-HRMS:m / z474.2245,[M+Na] + ,calculated for C 27 H 33 NO5Na, 474.2256.

[0081] 14-(R)-(2'-bromophenoxy)andrographolide (compound 4d), reference Figure 13 and Figure 14 :

[0082] White powder, melting point 149.0-149.9℃, yield 84.2%; 1H NMR(400MHz,DMSO-d6)δppm 7.64(d,J=7.7Hz,1H),7.40(t,J=7.6Hz,1H),7.16(d,J=8.1Hz,1H),7.00(dt,J=14.8,7.3Hz,2H),5.90(d,J=4.3Hz,1H),4.9 8(d,J=2.9Hz,1H),4.82(s,1H),4.70(dd,J=10.8,5.2Hz,1H),4.55(s,1H),4.34(d,J=10.9Hz,1H),4.11(d,J=4.8Hz,1H),3. 76(d,J=10.8Hz,1H),3.19(dd,J=10.0,7.1Hz,1H),3.00(d,J=10.9Hz,1H),2.42–2.22(m,3H),1.95(d,J=11.0Hz,2H),1.70( d,J=12.2Hz,1H),1.56–1.38(m,2H),1.37–1.22(m,2H),1.17(d,J=12.5Hz,1H),1.04(s,3H),0.93–0.81(m,1H),0.54(s,3H); 13 C NMR(101MHz,DMSO-d6)δ169.08,152.91,150.39,147.77,133.59,129.19,125.56,123.21,115.13,111.80,107.79,78 .35,72.03,71.01,62.65,55.94,54.04,42.19,38.78,37.45,36.10,27.86,25.17,23.93,23.07,14.73; ESI-HRMS:m / z 505.1552( 79 Br)507.1551( 81 Br), [M+H] + ,calculated for C 26 H 34 BrO5,505.1590( 79 Br)507.1569( 81 Br).

[0083] 14-(R)-(2'-trifluoromethylphenoxy)andrographolide (compound 4e), reference Figure 15 and Figure 16 :

[0084] White powder, melting point 83.3-83.4℃, yield 82.2%; 1H NMR(400MHz,DMSO-d6)δppm 7.68(t,J=7.5Hz,2H),7.36(d,J=8.5Hz,1H),7.18(t,J=7.6Hz,1H),6.94(t,J=6.6Hz,1H),6.08(d,J=4.9Hz,1H),5.01(d,J=4. 9Hz,1H),4.81(s,1H),4.70(dd,J=11.0,5.4Hz,1H),4.48(s,1H),4.30(d,J=11.0Hz,1H),4.10(dd,J=7.5,2.7Hz,1H),3.77(dd, J=10.9,2.7Hz,1H),3.20(dd,J=10.8,7.7Hz,1H),3.02(dt,J=8.8,4.2Hz,1H),2.46–2.26(m,3H),1.88(dd,J=18.6,6.8Hz,2H) ,1.76–1.66(m,1H),1.59–1.46(m,1H),1.41(d,J=10.0Hz,2H),1.36–1.22(m,1H),1.03(s,3H),1.13–0.91(m,2H),0.54(s,3H); 13 C NMR (101MHz, DMSO-d6) δ168.89, 154.22 (d, J = 1.3Hz), 150.61, 147.69, 134. 30,127.28(d,J=5.1Hz),125.04,123.59(d,J=272.6Hz),121.33,117.89(q ,J=30.3Hz),114.72,107.89,78.36,71.54,70.74,62.55,55.48,54.23,42 .16,38.58,37.37,36.06,27.77,25.11,23.88,23.07,14.66;ESI-HRMS:m / z 517.2153[M+Na] + ,calculated for C 27 H 33 F3O5Na, 517.2178.

[0085] 14-(R)-(2'-iodophenoxy)andrographolide (compound 4f), reference Figure 17 and Figure 18 :

[0086] White powder, melting point 177.6-178.3℃, yield 81.7%; 1¹H NMR (400MHz, DMSO-d⁶) δppm 7.83(dd,J=7.7,1.5Hz,1H),7.44–7.36(m,1H),7.06(d,J=7.6Hz,1H),7.02( t,J=7.4Hz,1H),6.83(td,J=7.6,1.0Hz,1H),5.88(d,J=5.1Hz,1H),4.97(d, J=4.9Hz,1H),4.83(s,1H),4.70(dd,J=10.9,5.4Hz,1H),4.57(s,1H),4.31( d,J=10.9Hz,1H),4.10(dd,J=7.5,2.8Hz,1H),3.76(dd,J=10.9,2.8Hz,1H), 3.19(dd,J=10.8,7.6Hz,1H),3.01(dt,J=9.4,4.4Hz,1H),2.44–2.35(m,1H) ,2.28(ddd,J=18.7,11.0,5.8Hz,2H),1.98(t,J=11.6Hz,2H),1.77–1.67(m, 1H),1.56–1.47(m,1H),1.46–1.39(m,1H),1.31(dd,J=13.0,3.6Hz,2H),1.2 5(d,J=12.7Hz,1H),1.05(s,3H),0.88(dd,J=13.4,10.2Hz,1H),0.54(s,3H); 13 CNMR(101MHz,DMSO-d6)δ169.01,155.22,150.10,147.75,139.58,129.82,125.61,123.57,113.72,107.70,87.36,78.23,71. 80,70.93,62.58,55.74,53.82,42.17,38.76,37.37,36.08,27.82,25.19,23.90,22.97,14.71;ESI-HRMS:m / z575.1266[M+Na] + ,calculated for C 26 H 34 IO5Na, 575.1270.

[0087] 14-(R)-(2'-fluorophenoxy)andrographolide (compound 4g), reference Figure 19 and Figure 20 :

[0088] White powder, melting point 160.2-160.3℃, yield 81.9%; 1H NMR (500MHz, DMSO-d6) δ7.28 (ddd, J=11.6, 8.1, 1.5Hz, 1H), 7.23 (td, J=8.3, 1. 6Hz,1H),7.18(td,J=8.0,1.0Hz,1H),7.09–7.03(m,1H),6.94(td,J=7.1,1.2Hz ,1H),5.82(d,J=5.2Hz,1H),4.98(d,J=4.9Hz,1H),4.80(s,1H),4.65(dd,J=10 .9,5.4Hz,1H),4.48(s,1H),4.39(dd,J=10.9,1.1Hz,1H),4.09(dd,J=7.5,2.9H z,1H),3.77(dd,J=11.0,2.9Hz,1H),3.21(dd,J=10.9,7.6Hz,1H),3.04(dt,J= 11.5,4.3Hz,1H),2.34–2.23(m,3H),1.94–1.85(m,2H),1.74–1.67(m,1H),1.52 (dt,J=12.4,7.4Hz,1H),1.45–1.38(m,2H),1.30(qd,J=13.1,4.1Hz,1H),1.08( dd,J=12.7,2.5Hz,1H),1.04(s,3H),0.94(dd,J=13.5,9.7Hz,1H),0.55(s,3H); 13 C NMR(126MHz,DMSO-d6)δ168.98,152.56(d,J=244.2Hz),150.21,147.68,144 .21(d,J=10.7Hz),125.42,124.99(d,J=3.7Hz),122.82(d,J=6.9Hz),117.59 ,116.54(d,J=18.1Hz),107.76,78.30,72.73,70.97,62.52,55.68,54.27,4 2.15,38.65,37.39,36.15,27.76,24.98,23.89,23.02,14.58;ESI-HRMS:m / z 467.2203[M+Na] + ,calculated for C 26 H 33 FO5Na,467.2210.

[0089] 14-(R)-(cis-1',2',3',6'-tetrahydrophthalimide)andrographolide (4h):

[0090] White powder, melting point 188.6-189.6℃, yield 65.5%; 1 H NMR(500MHz,DMSO-d6)δ6.43(ddd,J=9.2,4.3,2.3Hz,1H),5.84(t,J=3.4Hz,2 H),5.45(dq,J=8.1,2.5Hz,1H),5.04(d,J=4.8Hz,1H),4.76(s,1H),4.61–4.52 (m,1H),4.30(s,1H),4.17(dd,J=9.9,3.1Hz,1H),4.11(dd,J=7.6,2.9Hz,1H), 3.82(dd,J=10.9,2.9Hz,1H),3.28–3.18(m,3H),3.13(td,J=9.2,8.6,2.6Hz,1 H),2.45–2.38(m,1H),2.30(ddt,J=14.3,12.1,2.7Hz,2H),2.25–2.15(m,2H), 2.10–2.03(m,1H),1.99(q,J=8.9,7.4Hz,1H),1.91(td,J=13.6,13.1,4.4Hz,2 H),1.76–1.69(m,1H),1.62(tt,J=12.9,5.8Hz,2H),1.48(dt,J=13.2,3.5Hz,1 H),1.32(qd,J=13.0,4.1Hz,1H),1.21–1.11(m,2H),1.07(s,3H),0.57(s,3H); 13 C NMR(101MHz,DMSO-d6)δ179.17,178.87,168.91,147.69,145.11,127.85,127.49,124.34,107.87,78.36,68.34,62.61,54.95,54.24 ,45.46,42.24,38.82,38.39,38.16,37.44,36.40,27.78,25.00,23.96,23.06,22.97,22.94,14.67;ESI-HRMS:m / z506.2503,[M+Na] + ,calculated for C 28 H 37 NO6Na, 506.2519.

[0091] 14-(R)-(cis-1',2'-dimethylcyclohexylformamide)andrographolide (compound 4i):

[0092] White powder, melting point 179.1-182.3℃, yield 72.8%;1 H NMR(400MHz, DMSO-d6)δ7.64(ddd,J=6.8,5.5,1.6Hz,1H),6.50(ddd,J=7.6,4.8,2.2Hz,1H),5.58–5.44(m,1H),5.05 (d,J=4.8Hz,1H),4.80(s,1H),4.58(dd,J=10.0,8.7Hz,1H),4.35(s,1H),4.24(dd,J=10.0,3.0Hz,1H),4.13(dd,J=7 .5,2.9Hz,1H),3.87–3.77(m,1H),3.23(ddd,J=19.8,10.8,6.3Hz,2H),2.98(dq,J=33.7,7.2Hz,2H),2.31(dt,J=12. 6,3.4Hz,1H),2.24–2.06(m,2H),1.97–1.87(m,2H),1.85–1.54(m,6H),1.52–1.22(m,8H),1.08(s,3H),0.59(s,3H); 13 CNMR(101MHz,DMSO-d6)δ178.53,178.31,168.95,147.62,144.95,124.63,107.89,78.31,68.50,62.58,54.86,54.21,45.1 4,42.22,39.04,38.80,38.42,37.40,36.48,27.80,24.77,23.95,23.88,23.03,22.74,21.40,21.36,14.68; ESI-HRMS:m / z 508.2660,[M+Na] + ,calculated for C 28 H 39 NO6Na, 508.2675.

[0093] 14-(R)-(1',2'-cycloglutamylimide)andrographolide (compound 4j), reference Figure 21 and Figure 22 :

[0094] White powder, melting point 231.2-232.3℃, yield 77.1%; 1H NMR(400MHz,DMSO-d6)δppm 6.48(t,J=5.6Hz,1H),5.47(d,J=8.1Hz,1H),5.06(s,1H),4.79(s,1H),4.57(t,J=9.3Hz,1 H),4.34(s,1H),4.24(dd,J=10.0,2.9Hz,1H),4.14(s,1H),3.82(d,J=10.9Hz,1H),3.29–3 .13(m,4H),2.31(d,J=12.4Hz,1H),2.16–2.01(m,2H),1.97–1.78(m,6H),1.78–1.53(m,4H ),1.42(d,J=13.0Hz,1H),1.37–1.27(m,1H),1.25–1.11(m,3H),1.08(s,3H),0.59(s,3H); 13 C NMR(101MHz,DMSO-d6)δ179.12,179.01,168.92,147.61,144.85,124.61,107.87,78.34,68.38,62.61,54.83,54.23,45.46,44 .95,44.65,42.23,39.52,38.42,37.42,36.45,29.69,29.53,27.83,24.78,23.98,23.04,14.69; ESI-HRMS:m / z494.2508[M+Na] + ,calculated for C 27 H 37 NO6Na, 494.2519.

[0095] 14-(R)-(pyridine-2',3'-diimide)andrographolide (compound 4k), reference Figure 23 and Figure 24 :

[0096] White powder, melting point 162.2-162.8℃, yield 78.5%; 1H NMR(400MHz,DMSO-d6)δppm 9.01(dd,J=5.0,1.4Hz,1H),8.33(dd,J=7.7,1.4Hz,1H),7.89(d,J=1.7Hz,1H),7.82(dd,J=7.7,5.0Hz,1H),5.07–5.01(m,1H) ,4.98(d,J=4.8Hz,1H),4.96–4.83(m,3H),4.74(s,1H),4.07(dd,J=7.6,2.7Hz,1H),3.79(dd,J=10.9,2.8Hz,1H),3.20(dd,J= 10.9,7.8Hz,1H),3.12–3.03(m,1H),2.53–2.47(m,1H),2.31(d,J=12.3Hz,1H),2.14–2.03(m,1H),1.82–1.72(m,1H),1.71–1. 51(m,5H),1.29(qd,J=12.9,3.8Hz,1H),1.11(dd,J=12.6,1.9Hz,1H),0.99(s,3H),0.93(dd,J=13.4,9.6Hz,1H),0.64(s,3H); 13 C NMR(101MHz,DMSO-d6)δ172.35,166.00,165.94,155.17,150.91,150.22,147.38,131.67,129.87,128.17,126.89,106.56,78.21, 70.63,62.66,53.81,50.99,45.05,42.24,38.61,37.72,35.99,27.86,24.14,24.12,22.87,15.00; ESI-HRMS:m / z503.2160[M+Na] + ,calculated for C 27 H 32 N₂O₆Na, 503.2158.

[0097] Example 2: The preliminary antifungal activity of compound 4a-4k against eight plant pathogenic fungi was determined using the mycelial growth rate method at a concentration of 100 μg / mL. The compound (20 mg) was dissolved in 10 mL of 5% dimethyl sulfoxide (DMSO) and mixed with 190 mL of sterilized potato dextrose agar (PDA) medium to prepare a drug-containing medium. Mycelial cakes (d = 5 mm) were placed on PDA medium and incubated at 25°C for 72 hours. The inhibition rate was calculated by measuring the colony diameter. A commercially available fungicide, azoxystrobin, was used as a positive control, and each experiment was repeated in triplicate.

[0098] The tested strains included *Fusarium graminearum*, *Alternaria solani*, *Alternaria brassicae*, *Alternata alternata*, *C. lunata*, *C. gloeosporioides*, *Fusarium bulbiferum*, *V. mali* (the causal agent of apple rot), *P. oryza* (the causal agent of rice blast), and *P. piricola*. The commercially available fungicide pyraclostrobin (KXM) was used as a positive control. The test results are shown in Table 1.

[0099] Table 1. In vitro antifungal activity of compound 4a-4k at a concentration of 100 μg / mL (n = 3, 72 h)

[0100]

[0101]

[0102] Note: FG, Fusarium graminearum; AS, Alternaria alternata; AB, Alternaria brassicae; AA, Alternaria alternata; CL, Curvularia; CG, Colletotrichum gloeosporioides; FB, Fusarium solani; VM, Apple rot fungus; PO, Rice blast fungus; PP, Pyrethrum; KXM is the commercially available fungicide azoxystrobin, used as a positive control.

[0103] Table 1 shows that all 11 compounds exhibited varying degrees of inhibitory activity against the 10 tested fungi. Compound 4d showed the highest antifungal activity, with inhibition rates against all six tested fungi exceeding those of the positive control. Furthermore, compounds 4b, 4f, 4g, and 4k also significantly inhibited the growth of all 10 tested fungi. Notably, 4g showed an inhibition rate of 80.9% against Curvularia, significantly higher than KXM (60%); 4k showed an inhibition rate of 86.5% against Alternaria, 1.56 times that of KXM.

[0104] The 10 tested fungi showed varying sensitivities to the target compound. Among them, Alternaria, Alternaria brassicae, Curvularia, Colletotrichum, and Pyrethrum were more sensitive to the target compound, while the other 5 tested fungi had lower sensitivities.

[0105] Scanning electron microscopy (SEM) analysis: Hyphae were fixed overnight at 4°C with 4% glutaraldehyde. The fixed samples were washed four times with 0.1M phosphate-buffered saline (PBS, pH 6.8), approximately 10 minutes each time. The washed hyphae were then dehydrated sequentially with 10%, 30%, 50%, 70%, 80%, and 90% (v / v) ethanol, followed by three dehydration cycles with 100% ethanol. After dehydration, the samples were vacuum-dried with carbon dioxide and sputter-coated with gold before observation using a Hitachi S-4800 scanning electron microscope.

[0106] The changes in the hyphal morphology of *Pyrrosia lingua* after 4 days of treatment were observed by scanning electron microscopy (SEM). The results are as follows: Figure 2 As shown. From Figure 2 It can be seen that the hyphae in the control group are arranged regularly, with full hyphae and relatively smooth surfaces; while after 4 days of treatment, the hyphae surface becomes dry and wrinkled. This is because 4 days of treatment causes the hyphae to dehydrate, thus affecting their growth.

[0107] Seed germination experiment: The test compound was dissolved in 5% DMSO to prepare solutions of 200, 100, 50, and 25 μg / mL. Mature cowpea seeds (Shenzhou 8) were soaked in the above four concentrations of solutions for 12 hours. After imbibition, the seeds were transferred to petri dishes lined with filter paper, distilled water was added, and the dishes were placed in a constant temperature incubator at 25℃ and 80% humidity in the dark.

[0108] Cowpea seeds were treated with different concentrations of the compound for 4 days. The germination rate and growth of the treated and control groups were compared to evaluate the effect of the compound on seed germination. Results are as follows: Figure 3 As shown. By Figure 3 It was found that at concentrations of 25, 50, 100, and 200 μg / mL, 4 days had no effect on seed germination rate. Furthermore, at a concentration of 200 μg / mL, the growth of the treatment group and the control group was almost identical, and seed growth was unaffected, indicating that 4 days had high safety.

[0109] Structural modifications significantly affect the antifungal activity of compounds. First, the antifungal activity varies with the type of substituents on the benzene ring. Specifically, the introduction of halogen atoms, especially bromine atoms, enhances antifungal activity. Conversely, the introduction of strong electron-withdrawing groups such as cyano and trifluoromethyl groups does not significantly promote antifungal activity. Furthermore, the type of amide linking ring also influences the antifungal activity. Compared to other rings, the introduction of a pyridine ring significantly improves the antifungal activity (e.g., comparing 4a with 4h-4k).

[0110] Of the 14 aryloxy / amide-substituted andrographolide derivatives synthesized in this study, most exhibited significant antifungal activity. Among them, compound 4d demonstrated excellent in vitro antifungal activity, inhibiting all six test fungi at a concentration of 100 μg / mL with an inhibition rate exceeding 60%, and its activity was superior to the positive control azoxystrobin. Furthermore, 4d severely disrupted fungal hyphae, and its high safety was confirmed by seed germination experiments. Structure-activity relationship studies showed that the introduction of an ortho-halogen atom or a 2,3-pyridine diimide group on the benzene ring was beneficial in enhancing antifungal activity. Therefore, 4d can be considered a compound with high antifungal potential. Further research into the antifungal mechanism of such compounds could support the development of natural product antibiotics.

[0111] The embodiments of the present invention have been described in detail above with reference to the examples. However, the present invention is not limited to the above embodiments. For those skilled in the art, after learning the contents described in the present invention, several equivalent changes and substitutions can be made without departing from the principle of the present invention. These equivalent changes and substitutions should also be considered to fall within the protection scope of the present invention.

Claims

1. The application of andrographolide derivatives in the preparation of plant antifungal drugs, wherein the structural formula of the andrographolide derivatives is: Formula I; or Formula II; where, R in Equations I and II are selected from , , , , , , , , , , .

2. The application according to claim 1, characterized in that: R in Equations I and II are selected from , , , , , , .

3. The application according to claim 1, characterized in that, The structural formula of the andrographolide derivative is: Equation II; and R in the equation is selected from , , , , , , , , , , .

4. The application according to claim 3, characterized in that, The structural formula of the andrographolide derivative is: Formula II; And R in the formula is .

5. The application according to claim 1, characterized in that, The synthetic route for andrographolide derivatives is as follows: ; where R is selected from , , , , , , , , , , .

6. The application according to claim 1, characterized in that, Andrographolide derivatives have inhibitory effects on the following plant pathogens: Fusarium graminearum, Alternaria alternata, Alternaria brassicae, Alternaria alternata, Curvularia, Colletotrichum gloeosporioides, Fusarium solani, Fusarium solani, Pyrus pyrifolium, Pyrus pyrifolium, and Pyrus pyrifolium.

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