Abstract
In order to find more compounds with antifungal activities, a series of new compounds were designed and synthesized using the natural product of diterpenoid lactones, andrographolide, as the parent nucleus. Target compounds were identified by 1H NMR, 13C NMR and/or HR-MS. The bioassay results demonstrated that compound 4d exhibited inhibition rates of over 60% against six fungal strains at 100 µg/mL, and the EC50 value of 4d against P. piricola was only 9.09 µg/mL. SEM test showed that 4d could damage the mycelia of P. piricola. obviously. Moreover, 4d had no effect on cowpea seed germination, showing high safety.
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Introduction
The harm caused by pathogenic fungi to plants is primarily manifested in the diseases they induce, which constitute approximately 70% to 80% of all plant diseases1. There are many types of pathogenic fungi with such hazards, including powdery mildew, rust, and downy mildew2. These necrotic fungi are notorious for causing significant losses in fields and storage worldwide, as well as diseases that pose a threat to food security3,4.
The most effective antifungal drugs currently available are mainly chemical fungicides5. However, excessive use of fungicides can have an impact on soil respiration, microbial diversity, and enzyme activity6. In addition, the lack of antifungal drugs and the emergence of fungal resistance make it an urgent task to discover and screen new antifungal drugs from chemically synthesized or natural products7.
Compared with chemically synthesized products, natural products have the advantages of wide sources, diverse structures, and relatively low toxicity8,9,10. Whether in their original form or as raw templates for structural optimization to obtain efficient and safe derivatives, natural products are potential sources for antifungal drug libraries11. Traditional Chinese medicine is a resource pool of natural products, and many compounds isolated from Chinese herbal medicine have been proven to have various pharmacological activities, such as antibacterial, anti-tumor, antiviral, and antifungal activities12,13,14.
Andrographolide, extracted from the natural plant Andrographis paniculata in the Acanthaceae family, stands out as a prominent diterpenoid lactone with structural diversity and rich biological activities, which makes them valuable for research and development in the pharmaceutical field. So far, researchers have found that andrographolide and its derivatives have significant advantages (Fig. 1) in antibacterial15, antifungal16, insect resistant17, and other aspects18,19. In in recent years, researchers have found that andrographolide was capable of eliminating mature biofilms and viable cell numbers by up to 99.9% in the C. albicans and C. glabrata20. Moreover, andrographolide loaded nanosuspension can penetrate through the cornea and has significant antibacterial and antifungal activity21. Combination of andrographolide and amphotericin B exhibited antifungal efficacy against A. fumigatus, A. niger, T. mentagrophytes, and C. albicans22.
Andrographolide and its derivatives.
In order to investigate the differential responses of diterpenoid compounds to different fungal species, which is also a continuation of our research, we designed and synthesized a series of 14 substituted andrographolide derivatives based on the preliminary structure-activity relationship of andrographolide and the foundation of previous work23,24, as shown in Fig. 2. Subsequently, the activities all target compounds against eight plant pathogenic fungi were evaluated.
Design idea of target compounds in this study.
Results and discussion
Synthesis
As shown in Fig. 3, a series of 14 aryloxy/amide group substituted andrographolide derivatives 4a-4k were synthesized by structural modification of Andro (1), which was purchased from Nanjing Chemlin Chemical Industry Co., Ltd. Firstly, 3,19 dihydroxy groups were protected to obtain 3,19-isopropylidene andrographolide (intermediate 223) with a yield of 90.2%. Then, a series of compounds 3a-3k with a C-14 position configuration inversion were obtained through Mitsunobu reaction. Finally, in the presence of p-toluenesulfonic acid monohydrate, the protecting groups at positions 3 and 19 were removed to obtain a series of compounds 4a-4k. In this process, a total of 22 andrographolide derivatives were synthesized, including 14 new compounds. All compounds were identified by 1H NMR, 13C NMR, and high-resolution mass spectrometry (HRMS). The specific data, melting point and other information were provided in the supporting information.
Synthetic route of target compounds 3a − 3k and 4a − 4k.
Antifungal activities of compounds against 10 pathogenic fungi in vitro
The preliminary antifungal activity in vitro of compounds 4a–4k was determined by mycelial linear growth rate method at 100 µg/mL. Ten fungi Fusarium graminearum, Alternaria solani, Alternaria brassicae, Alternaria alternata, Curvularia lunata, Colletotrichum gloeosporioides, Fusarium bulbigenum, Valsa mali, Pyricularia oryza and Physalospora piricola were used as the tested fungi, and the commercial fungicide Kresoxim-methyl (KXM) was used as the positive control.
The results listed in Table 1 revealed that all of the 14 aryloxy/amide substituted andrographolides have different degrees of inhibition against 10 tested fungi. Among all the compounds, 4d exhibited relatively high antifungal activity, with higher inhibitory activity against 6 tested fungi than the positive control. In addition, compounds 4b, 4f, 4g and 4k can also significantly inhibit the growth of 10 tested fungi. It is worth noting that the inhibition rate of 4g on C. lunata was 80.9%, much higher than KXM (inhibition rate = 60.9%), and 4k exhibits significant inhibitory activity on A. solani (inhibition rate = 86.5%), which is 1.56 times that of KXM (inhibition rate = 55.4%).
The sensitivity of the 10 tested fungi to the target compound varies, among which A. solani, A. brassicae, C. lunata, C. gloeosporioides, and P. piricola are more sensitive to the target compound. On the contrary, the other five tested fungi had lower sensitivity.
In order to further know the antifungal activity of the compounds and summarize their structure-activity relationship, the median effective concentrations (EC50) of 4 compounds with high activity were determined. The results are listed in Table 2. The results in Table 2 showed that 4g had better antifungal activity against C. lunata (EC50 = 27.1 µg/mL) than the positive control (EC50 = 68.6 µg/mL). It is particularly noteworthy that 4d showed significant activity against P. piricola, with an EC50 value of 9.09 µg/mL, only 35% of KXM.
SEM analysis
We observed the changes of mycelial morphology of P. piricola after 4d treatment by scanning electron microscope (SEM). The results are presented in Fig. 4.
Analysis of hyphal morphology of P. piricola treated with 4 d at 15 µg/mL. (A, B) Control group and (C, D) treated group.
It is obvious that the mycelium arrangement of the control group is regular, with a plump mycelial morphology, and a relatively smooth mycelial surface. On the contrary, the surface of the mycelium becomes dry and wrinkled after 4d treatment, we speculate that which is due to 4d causing dehydration of the mycelium, thereby affecting its growth.
Seed germination experiment
Cowpea seeds were treated with different concentrations of 4d. The effects of compounds on seed germination were evaluated by comparing the germination rate and growth of the treatment group and the control group. The results are shown in Fig. 5.
Effect of compound 4d on seed germination.
It can be clearly seen from Fig. 5 that 4d almost had no effect on the germination rate of seeds at the four concentrations (25, 50, 100, 200 µg/mL). In addition, the growth of the treatment group and the control group were almost the same, and the growth of seeds was not affected at the concentration of 200 µg/mL, indicating that 4d showed high safety.
Discussions----structure-activity relationship
By comparing the data in Table 1 and/or Table 2, some important structure-activity relationships (SAR) of the target compounds could be deduced (Fig. 6). It is obvious that the structural modification has a significant effect on the antifungal activity of the compounds (Andro vs. 4a-4k). First of all, the antifungal activity of the compounds varies with the types of substituents on the benzene ring. Specifically, the introduction of halogen atoms can improve the antifungal activity, especially bromine. On the contrary, the introduction of strong electron withdrawing groups such as cyano and trifluoromethyl did not significantly promote the antifungal activity of the compounds. In addition, the type of ring connected to the amide also affects the antifungal activity of compounds. Compared with other rings, the introduction of pyridine ring can significantly improve the antifungal activity of the compounds (4a vs. 4h-4k).
Structure-activity relationship of target compounds.
Experimental
General
All reagents are commercially available and used directly without further purification. 2,2-dimethoxypropane (99%), pyridinium p-toluenesulfonate (PPTS, AR), triphenylphosphine (AR), p-toluenesulfonic acid monohydrate (98%), diisopropyl azodicarboxylate (DIAD, AR), 2-chlorophenol (98%), 2-cyanophenol (AR), 2-bromophenol (98%), 2-trifluoromethylphenol (97%), 2-iodophenol (98%), 2-fluorophenol (98%), 1,2-cyclopentadienyl imide (98%), 1,2,3,6-tetrahydrophthalimide (98%), 2,3-pyridine diimide (97%), cis-cyclohexane-1,2-diimide (98%), tetrahydrofuran (99.9%), methanol (AR), sodium bicarbonate (AR) Sodium chloride (AR) and anhydrous sodium sulfate (AR) were purchased from Anhui Zesheng Technology Co., Ltd., Phenol (CP) was purchased from Meryer (Shanghai) Chemical Technology Co., Ltd., dichloromethane (AR), ethyl acetate (AR), and petroleum ether (60–90 ℃, AR) were purchased from Sinopharm Chemical Reagent Co., Ltd. Reactions were monitored by thin-layer chromatography (TLC) and a mixture of petroleum ether and ethyl acetate was used as solvent system on GF-254. 1H and13C NMR spectra were recorded at 400 and 101 MHz on a Bruker spectrometer (Bruker, Germany), respectively, using dimethylsulfoxide-d6 (DMSO-d6) or chloroform-d (CDCl3) or benzene-d6 (C6D6) as the solvent. HRMS data were obtained by a High-Resolution mass spectrometry instrument (Thermo Scientific, Q Exactive, America). The melting point was determined using an X-4 binocular microscope melting point apparatus (Gongyi Yuhua Instrument Co., Ltd.).
Synthesis
General procedure for the synthesis of compounds 3a − 3k and 4a − 4k
To the solution of andrographolide (9.95 g, 28.4 mmol) and 2,2-dimethoxypropane (24 mL, 195.3 mmol) in 20 mL of anhydrous dichloromethane, PPTS (0.71 g, 2.8 mmol) was added and the reaction mixture was heated at 40 ℃. The reaction was monitored by TLC (petroleum ether/ethyl acetate = 1:1) and then treated with ethyl acetate and sat. NaHCO3 after the reaction was complete. The organic phase was washed with brine, dried over anhydrous Na2SO4, and then filtered organic solution was evaporated to dryness. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 1:1) to produce compound 2. Compound 2 (3.94 g, 10.1 mmol), PPh3 (3.97 g, 15.1 mmol) and RH (15.1 mmol) were dissolved in 30 mL of anhydrous THF under N2 atmosphere. The solution was cooled to 0 ℃ and then treated with DIAD (3 mL, 15.1 mmol) in 5 mL of anhydrous THF. The reaction was stirred overnight at room temperature after being stirred at 0 ℃ for 1 h. After distilling off the volatile solvents, the residue was dissolved in ethyl acetate and washed with brine about 5 times and dried over anhydrous Na2SO4. The filtered organic solution was evaporated to dryness and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 5:1) to give 3a-3k with 32.4%–51.0% yield. Compounds 3a-3k (5.0 mmol) were added in 15 mL of methanol and then treated with p-toluenesulfonic acid monohydrate (0.10 g, 0.5 mmol) at 20 ℃ for 30 min. Diluted by ethyl acetate and washed with sat. NaHCO3, brine, the organic phase was dried over anhydrous Na2SO4, filtered, evaporated by a rotavap to dryness. Compounds 4a-4k were purified by silica gel column chromatography (petroleum ether/ethyl acetate = 10:7) with 65.5%–86.7% yield. The1H and13C NMR spectra data, HR-MS data and melting points of compounds 2, 3a-3k and 4a-4k are shown below, and all the spectra are collected in the supporting information.
14-(R)-® (phenoxy)−3,19-isopropylidene andrographolide (3a). Yield, 51.0%; white powder; m.p. 153–155 °C; 1H NMR (400 MHz, CDCl3) δ (ppm) 7.39–7.28 (m, 2H, 3’-H and 5’-H), 7.13 (td, J = 7.4, 1.8 Hz, 1H, 2’-H), 7.05 (tt, J = 7.5, 1.0 Hz, 1H, 6’-H), 6.88–6.80 (m, 2 H, 4’-H and 12-H), 5.53 (d, J = 5.7 Hz, 1H, 14-H), 4.87 (d, J = 1.6 Hz, 1H, 17-H), 4.60 (dd, J = 10.7, 5.8 Hz, 1H, 15-H), 4.46 (s, 1H, 17-H), 4.40 (dd, J = 10.7, 2.0 Hz, 1H, 15-H), 3.91 (d, J = 11.6 Hz, 1H, 19-H), 3.42 (dd, J = 8.3, 3.9 Hz, 1H, 19-H), 3.14 (d, J = 11.5 Hz, 1H, 3-H), 2.57–2.46 (m, 1H, 7-H), 2.45–2.26 (m, 2 H, 7-H and 11-H), 1.95 (dd, J = 27.6, 12.1 Hz, 2 H, 6-H and 9-H), 1.88–1.77 (m, 1H, 11-H), 1.75–1.67 (m, 1H, 6-H), 1.64 (dd, J = 11.8, 6.0 Hz, 1H, 2-H), 1.51 (dd, J = 8.0, 5.4 Hz, 1H, 1-H), 1.37 (s, 3 H, acetonylidene-CH3), 1.34 (s, 3 H, acetonylidene-CH3), 1.31–1.19 (m, 3 H, 2-H, 1-H and 5-H), 1.17 (s, 3 H, 20-CH3), 0.87 (s, 3 H, 18-CH3); 13C NMR (101 MHz, C6D6) δ 168.8 (16-C), 157.0 (1’-C), 149.6 (12-C), 148.1 (8-C), 130.1 (3’-C and 5’-C), 126.0 (13-C), 122.2 (4’-C), 115.9 (2 C, 2’-C and 6’-C), 108.0 (17-C), 99.4 (acetonylidene-C), 75.4 (3-C), 71.5 (15-C), 70.5 (14-C), 64.2 (19-C), 55.8 (9-C), 51.3 (5-C), 38.5 (4-C), 38.3 (10-C), 37.8 (7-C), 33.7 (1-C), 26.7 (2-C), 26.1 (acetonylidene-CH3), 25.7 (acetonylidene-CH3), 25.3 (6-C), 24.8 (11-C), 23.3 (20-C), 16.6 (18-C); ESI-HRMS: m/z 489.2658 [M + Na]+, calcd for C29H38NaO5, 489.2617.
14-(R)-® (2’-chloro-phenoxy)−3,19-isopropylidene andrographolide (3b). Yield, 48.1%; white powder; m.p. 155.8–159.2 °C; 1H NMR (400 MHz, C6D6) δ 7.22 (td, J = 7.3, 1.8 Hz, 1H, 3’-H), 7.12 (dd, J = 8.0, 1.7 Hz, 1H, 5’-H), 6.76–6.70 (m, 1H, 12-H), 6.52 (td, J = 7.7, 1.4 Hz, 1H, 6’-H), 6.19–6.12 (m, 1H, 4’-H), 4.90 (dd, J = 4.6, 1.8 Hz, 1H, 14-H), 4.83 (d, J = 1.5 Hz, 1H, 17-H), 4.42 (d, J = 1.5 Hz, 1H, 15-H), 3.84–3.78 (m, 2 H, 17-H and 15-H), 3.67–3.61 (m, 1H, 19-H), 3.42 (dd, J = 7.6, 3.7 Hz, 1H, 19-H), 3.07 (d, J = 11.5 Hz, 1H, 3-H), 2.25–2.09 (m, 3 H, 7-CH2 and 11-H), 1.80–1.66 (m, 2 H, 6-H and 9-H), 1.65–1.57 (m, 1H, 11-H), 1.52–1.44 (m, 1H, 6-H), 1.41 (s, 3 H, acetonylidene-CH3), 1.40–1.31 (m, 2 H, 2-H and 1-H), 1.36 (s, 3 H, acetonylidene-CH3), 1.10 (s, 3 H, 20-CH3), 1.02–0.89 (m, 3 H, 2-H, 1-H and 5-H), 0.84 (s, 3 H, 18-CH3); 13C NMR (101 MHz, C6D6) δ 168.37 (16-C), 152.43 (1’-C), 150.31 (12-C), 148.18 (8-C), 131.06 (3’-C), 127.78 (5’-C), 125.35 (13-C), 124.58 (2’-C), 123.13 (4’-C), 116.05 (6’-C), 107.83 (17-C), 99.30 (acetonylidene-C), 75.25 (3-C), 72.76 (15-C), 70.05 (14-C), 64.13 (19-C), 55.90 (9-C), 51.02 (5-C), 38.38 (4-C), 38.15 (10-C), 37.62 (7-C), 33.53 (1-C), 26.52 (2-C), 25.94 (acetonylidene-CH3), 25.79 (acetonylidene-CH3), 25.19 (6-C), 24.62 (11-C), 23.21 (20-C), 16.52 (18-C); ESI-HRMS: m/z 523.2222(35Cl) 525.2192(37Cl), [M + Na]+, calcd for C29H37ClNaO5, 523.2227(35Cl) 525.2198(37Cl).
14-(R)-® (2’-cyano-phenoxy)−3,19-isopropylidene andrographolide (3c). Yield, 49.7%; white powder; m.p. 154.6–154.7 °C; 1H NMR (400 MHz, DMSO-d6) δ ppm 7.83 (dd, J = 7.7, 1.6 Hz, 1H, 5’-H), 7.77–7.69 (m, 1H, 3’-H), 7.28 (d, J = 8.6 Hz, 1H, 4’-H), 7.19 (t, J = 7.5 Hz, 1H, 6’-H), 7.11 (t, J = 7.1 Hz, 1H, 12-H), 6.01 (d, J = 5.2 Hz, 1H, 14-H), 4.87 (s, 1H, 17-H), 4.74 (dd, J = 11.0, 5.5 Hz, 1H, 15-H), 4.65 (s, 1H, 17-H), 4.40 (d, J = 11.0 Hz, 1H, 15-H), 3.79 (d, J = 11.6 Hz, 1H, 19-H), 3.19–3.14 (m, 1H, 19-H), 3.05 (d, J = 11.6 Hz, 1H, 3-H), 2.47–2.28 (m, 3 H, 7-CH2 and 11-H), 1.99 (t, J = 9.7 Hz, 2 H, 6-H and 9-H), 1.72–1.59 (m, 2 H, 11-H and 6-H), 1.46–1.30 (m, 2 H, 2-H and 1-H), 1.26 (s, 3 H, acetonylidene-CH3), 1.21 (s, 3 H, acetonylidene-CH3), 1.24–1.14 (m, 2 H, 2-H and 1-H), 1.07 (s, 3 H, 20-CH3), 0.98–0.88 (m, 1H, 5-H), 0.77 (s, 3 H, 18-CH3). 13C NMR (126 MHz, DMSO-d6) δ 168.78 (16-C), 158.07 (1’-C), 150.78 (12-C), 147.59 (8-C), 135.27 (5’-C), 134.24 (3’-C), 125.17 (13-C), 122.11 (4’-C), 116.22 (2’-CN), 113.90 (6’-C), 108.19 (17-C), 101.32 (2’-C), 98.18 (acetonylidene-C), 75.56 (3-C), 72.04 (15-C), 70.67 (14-C), 62.71 (19-C), 56.04 (9-C), 51.11 (5-C), 38.21 (4-C), 37.14 (10-C), 36.87 (7-C), 33.49 (1-C), 27.33 (2-C), 25.68 (acetonylidene-CH3), 25.25 (acetonylidene-CH3), 25.06 (6-C), 24.51 (11-C), 22.66 (20-C), 15.71 (18-C). ESI-HRMS: m/z 514.2564, [M + Na]+, calculated for C30H37NO5Na, 514.2569.
14-(R)-® (2’-bromo-phenoxy)−3,19-isopropylidene andrographolide (3d). Yield, 44.3%; white powder; m.p. 161.4–161.9 °C; 1H NMR (400 MHz, DMSO-d6) δ ppm 7.64 (dd, J = 7.9, 1.2 Hz, 1H, 3’-H), 7.44–7.35 (m, 1H, 5’-H), 7.16 (d, J = 8.1 Hz, 1H, 4’-H), 7.04 (t, J = 7.3 Hz, 1H, 6’-H), 6.98 (t, J = 7.6 Hz, 1H, 12-H), 5.91 (d, J = 5.0 Hz, 1H, 14-H), 4.86 (s, 1H, 17-H), 4.70 (dd, J = 10.9, 5.3 Hz, 1H, 15-H), 4.62 (s, 1H, 17-H), 4.35 (d, J = 10.9 Hz, 1H, 15-H), 3.78 (d, J = 11.6 Hz, 1H, 19-H), 3.17 (dd, J = 8.8, 3.4 Hz, 1H, 19-H), 3.04 (d, J = 11.6 Hz, 1H, 3-H), 2.44–2.25 (m, 3 H, 7-CH2 and 11-H), 1.99 (t, J = 10.4 Hz, 2 H, 6-H and 9-H), 1.72–1.57 (m, 2 H, 11-H and 6-H), 1.43–1.30 (m, 2 H, 2-H and 1-H), 1.26 (s, 3 H, acetonylidene-CH3), 1.21 (s, 3 H, acetonylidene-CH3), 1.25–1.14 (m, 2 H, 2-H and 1-H), 1.07 (s, 3 H, 20-CH3), 0.93 (dt, J = 16.1, 8.2 Hz, 1H, 5-H), 0.76 (s, 3 H, 18-CH3); 13C NMR (101 MHz, DMSO-d6) δ 168.98 (16-C), 152.85 (1’-C), 150.16 (12-C), 147.67 (8-C), 133.53 (3’-C), 129.15 (5’-C), 125.66 (13-C), 123.12 (4’-C), 114.97 (6’-C), 111.66 (2’-C), 108.19 (17-C), 98.18 (acetonylidene-C), 75.49 (3-C), 71.89 (15-C), 70.97 (14-C), 62.74 (19-C), 55.84 (9-C), 50.96 (5-C), 38.15 (4-C), 37.15 (10-C), 36.96 (7-C), 33.43 (1-C), 27.33 (2-C), 25.65 (acetonylidene-CH3), 25.24 (acetonylidene-CH3), 25.06 (6-C), 24.49 (11-C), 22.64 (20-C), 15.78 (18-C); ESI-HRMS: m/z 567.1765(79Br) 569.1757(81Br), [M + Na]+, calculated for C29H37BrO5Na, 567.1722(79Br) 569.1702(81Br).
14-(R)-® (2’-trifluoromethyl-phenoxy)−3,19-isopropylidene andrographolide (3e). Yield, 33.8%; white powder; m.p. 62.2–62.6 °C; 1H NMR (400 MHz, DMSO-d6) δ 7.69 (t, J = 7.7 Hz, 2 H, 3’-H and 5’-H), 7.37 (d, J = 8.4 Hz, 1H, 4’-H), 7.19 (t, J = 7.6 Hz, 1H, 6’-H), 6.96 (t, J = 6.7 Hz, 1H, 12-H), 6.09 (d, J = 5.0 Hz, 1H, 14-H), 4.85 (s, 1H, 17-H), 4.71 (dd, J = 11.0, 5.4 Hz, 1H, 15-H), 4.54 (s, 1H, 17-H), 4.31 (d, J = 11.0 Hz, 1H, 15-H), 3.78 (d, J = 11.6 Hz, 1H, 19-H), 3.22 (dd, J = 8.6, 3.4 Hz, 1H, 19-H), 3.04 (d, J = 11.6 Hz, 1H, 3-H), 2.43–2.35 (m, 2 H, 7-CH2), 2.32 (d, J = 12.5 Hz, 1H, 11-H), 1.92 (dd, J = 14.3, 9.9 Hz, 2 H, 6-H and 9-H), 1.74–1.58 (m, 2 H, 11-H and 6-H), 1.39 (s, 1H, 2-H), 1.45–1.32 (m, 2 H, 1-H and 2-H), 1.26 (s, 3 H, acetonylidene-CH3), 1.21 (s, 3 H, acetonylidene-CH3), 1.18–1.14 (m, 2 H, 1-H and 5-H), 1.05 (s, 3 H, 20-CH3), 0.76 (s, 3 H, 18-CH3); 13C NMR (101 MHz, DMSO-d6) δ 168.84 (16-C), 154.22 (d, J = 1.1 Hz, 1’-C), 150.39 (12-C), 147.59 (8-C), 134.27 (5’-C), 127.25 (dd, J = 9.9, 4.8 Hz, 3’-C), 125.18 (13-C), 123.57 (d, J = 272.4 Hz, 2’-C), 121.27 (4’-C), 117.92 (q, J = 30.2 Hz, 2’-CF3), 114.58 (6’-C), 108.32 (17-C), 98.27 (acetonylidene-C), 75.35 (3-C), 71.49 (15-C), 70.76 (14-C), 62.84 (19-C), 55.41 (9-C), 51.08 (5-C), 37.97 (4-C), 37.22 (10-C), 36.98 (7-C), 33.30 (1-C), 27.02 (2-C), 25.65 (acetonylidene-CH3), 25.29 (acetonylidene-CH3), 24.97 (6-C), 24.38 (11-C), 22.63 (20-C), 15.85 (18-C); ESI-HRMS: m/z 557.2489, [M + Na]+, calculated for C30H37F3O5Na, 557.2491.
14-(R)-® (2’-iodo-phenoxy)−3,19-isopropylidene andrographolide (3f). Yield, 47.8%; white powder; m.p. 179.7–179.8 °C; 1H NMR (400 MHz, DMSO-d6) δ ppm 7.83 (dd, J = 7.7, 1.4 Hz, 1H, 3’-H), 7.45–7.34 (m, 1H, 5’-H), 7.05 (t, J = 7.9 Hz, 2 H, 4’-H and 6’-H), 6.82 (t, J = 7.2 Hz, 1H, 12-H), 5.90 (d, J = 4.9 Hz, 1H, 14-H), 4.86 (s, 1H, 17-H), 4.70 (dd, J = 10.9, 5.3 Hz, 1H, 15-H), 4.64 (s, 1H, 17-H), 4.32 (d, J = 10.9 Hz, 1H, 15-H), 3.78 (d, J = 11.6 Hz, 1H, 19-H), 3.18 (dd, J = 8.6, 3.1 Hz, 1H, 19-H), 3.04 (d, J = 11.6 Hz, 1H, 3-H), 2.46–2.26 (m, 3 H, 7-CH2 and 11-H), 2.10–1.97 (m, 2 H, 6-H and 9-H), 1.71–1.55 (m, 2 H, 11-H and 6-H), 1.42–1.30 (m, 3 H, 2-CH2 and 1-H), 1.26 (s, 3 H, acetonylidene-CH3), 1.21 (s, 3 H, acetonylidene-CH3), 1.19–1.14 (m, 1H, 1-H), 1.08 (s, 3 H, 20-CH3), 0.98–0.89 (m, 1H, 5-H), 0.76 (s, 3 H, 18-CH3); 13C NMR (101 MHz, DMSO-d6) δ 169.00 (16-C), 155.20 (1’-C), 149.96 (12-C), 147.69 (8-C), 139.58 (3’-C), 129.85 (5’-C), 125.76 (13-C), 123.54 (4’-C), 113.57 (6’-C), 108.16 (17-C), 98.19 (acetonylidene-C), 87.32 (2’-C), 75.42 (3-C), 71.69 (15-C), 70.96 (14-C), 62.78 (19-C), 55.73 (9-C), 50.76 (5-C), 38.18 (4-C), 37.19 (10-C), 36.94 (7-C), 33.42 (1-C), 27.29 (2-C), 25.59 (acetonylidene-CH3), 25.37 (acetonylidene-CH3), 25.04 (6-C), 24.51 (11-C), 22.67 (20-C), 15.87 (18-C); ESI-HRMS: m/z 615.1582, [M + Na]+, calculated for C29H37IO5Na, 615.1583.
14-(R)-® (2’-fluoro-phenoxy)−3,19-isopropylidene andrographolide (3g). Yield, 49.6%; white powder; m.p. 157.8–158.3 °C; 1H NMR (500 MHz, DMSO-d6) δ 7.29 (ddd, J = 11.6, 8.1, 1.5 Hz, 1H, 4’-H), 7.23 (td, J = 8.3, 1.6 Hz, 1H, 3’-H), 7.18 (td, J = 8.1, 1.1 Hz, 1H, 5’-H), 7.09–7.04 (m, 1H, 6’-H), 6.96 (td, J = 7.1, 1.1 Hz, 1H, 12-H), 5.83 (d, J = 5.2 Hz, 1H, 14-H), 4.84 (s, 1H, 17-H), 4.66 (dd, J = 10.9, 5.3 Hz, 1H, 15-H), 4.54 (s, 1H, 17-H), 4.40 (dd, J = 10.9, 1.1 Hz, 1H, 15-H), 3.80 (d, J = 11.6 Hz, 1H, 19-H), 3.24 (dd, J = 8.9, 4.0 Hz, 1H, 19-H), 3.06 (d, J = 11.6 Hz, 1H, 3-H), 2.37–2.25 (m, 3 H, 7-CH2 and 11-H), 1.99–1.88 (m, 2 H, 6-H and 9-H), 1.77–1.69 (m, 1H, 11-H), 1.67–1.60 (m, 1H, 6-H), 1.45 (ddd, J = 17.4, 8.1, 5.2 Hz, 1H, 2-H), 1.41–1.34 (m, 1H, 1-H), 1.28 (s, 3 H, acetonylidene-CH3), 1.22 (s, 3 H, acetonylidene-CH3), 1.21–1.12 (m, 2 H, 2-H and 1-H), 1.08 (s, 3 H, 20-CH3), 1.06–0.99 (m, 1H, 5-H), 0.77 (s, 3 H, 18-CH3); 13C NMR (126 MHz, DMSO-d6) δ 168.97 (16-C), 152.52 (d, J = 244.1 Hz, 2’-C), 150.06 (12-C), 147.58 (8-C), 144.21 (d, J = 10.6 Hz, 1’-C), 125.55 (13-C), 125.02 (d, J = 3.8 Hz, 5’-C), 122.83 (d, J = 7.0 Hz, 4’-C), 117.53 (6’-C), 116.55 (d, J = 18.0 Hz, 3’-C), 108.23 (17-C), 98.19 (acetonylidene-C), 75.53 (3-C), 72.70 (15-C), 71.01 (14-C), 62.75 (19-C), 55.57 (9-C), 51.25 (5-C), 38.07 (4-C), 37.14 (10-C), 36.99 (7-C), 33.49 (1-C), 27.29 (2-C), 25.70 (acetonylidene-CH3), 25.11 (acetonylidene-CH3), 25.09 (6-C), 24.48 (11-C), 22.64 (20-C), 15.70 (18-C); ESI-HRMS: m/z 507.2520, [M + Na]+, calculated for C29H37FO5Na, 507.2523.
14-(R)-® (cis-1’, 2’, 3’, 6’-tetrahydrophthalimide)−3,19-isopropylidene andrographolide (3h). Yield, 34.3%; white powder; m.p. 211.7–213.4 °C; 1H NMR (400 MHz, DMSO-d6) δ 6.43 (td, J = 5.9, 4.6, 2.3 Hz, 1H, 12-H), 5.92–5.79 (m, 2 H, 4’-H and 5’-H), 5.46 (d, J = 8.4 Hz, 1H, 14-H), 4.79 (s, 1H, 17-H), 4.57 (t, J = 9.4 Hz, 1H, 15-H), 4.34 (s, 1H, 17-H), 4.19 (dd, J = 9.9, 3.1 Hz, 1H, 15-H), 3.85 (d, J = 11.6 Hz, 1H, 19-H), 3.40 (dd, J = 8.8, 4.0 Hz, 1H, 8’-H), 3.23 (ddd, J = 9.7, 7.6, 2.4 Hz, 1H, 9’-H), 3.13 (td, J = 7.9, 2.4 Hz, 1H, 19-H), 3.09 (d, J = 11.8 Hz, 1H, 3-H), 2.42 (dq, J = 15.8, 2.7 Hz, 1H, 7-H), 2.33 (ddd, J = 12.9, 6.2, 3.0 Hz, 2 H, 7-H and 11-H), 2.20 (dt, J = 15.5, 8.5 Hz, 2 H, 3’-H and 6’-H), 2.11–2.00 (m, 2 H, 3’-H and 6’-H), 1.93 (tt, J = 13.4, 5.7 Hz, 3 H, 6-H, 9-H and 11-H), 1.69 (dp, J = 16.1, 5.3 Hz, 2 H, 6-H and 2-H), 1.47 (dt, J = 12.9, 6.0 Hz, 1H, 1-H), 1.33 (s, 3 H, acetonylidene-CH3), 1.30–1.16 (m, 3 H, 2-H, 1-H and 5-H), 1.25 (s, 3 H, acetonylidene-CH3), 1.11 (s, 3 H, 20-CH3), 0.80 (s, 3 H, 18-CH3); 13C NMR (101 MHz, DMSO-d6) δ 179.15 (2’-C), 178.83 (7’-C), 168.87 (16-C), 147.64 (12-C), 145.09 (8-C), 127.88 (4’-C), 127.50 (5’-C), 124.45 (13-C), 108.35 (17-C), 98.29 (acetonylidene-C), 75.51 (3-C), 68.29 (15-C), 62.88 (19-C), 54.80 (9-C), 51.15 (5-C), 45.45 (8’-C), 38.85 (9’-C), 38.18 (4-C), 37.79 (14-C), 37.25 (10-C), 37.04 (7-C), 33.74 (1-C), 27.28 (2-C), 25.71 (2 C, acetonylidene-CH3), 25.17 (6-C), 24.59 (11-C), 22.97 (2 C, 4’-C and 5’-C), 22.70 (20-C), 15.86 (18-C); ESI-HRMS: m/z 546.2815, [M + Na]+, calculated for C31H41NO6Na, 546.2832.
14-(R)-® (ciscyclohexa-1’, 2’-dimethylformamide)−3,19-isopropylidene andrographolide (3i). Yield, 32.4%; white powder; m.p. 143.6–145.8 °C; 1H NMR (400 MHz, DMSO-d6) δ 6.49 (td, J = 6.5, 2.3 Hz, 1H, 12-H), 5.50 (d, J = 7.9 Hz, 1H, 14-H), 4.82 (s, 1H, 17-H), 4.58 (t, J = 9.4 Hz, 1H, 15-H), 4.38 (s, 1H, 17-H), 4.24 (dd, J = 10.0, 3.0 Hz, 1H, 15-H), 3.84 (d, J = 11.6 Hz, 1H, 19-H), 3.39 (dd, J = 8.7, 4.0 Hz, 1H, 19-H), 3.08 (d, J = 11.6 Hz, 1H, 3-H), 3.01 (q, J = 6.8 Hz, 1H, 8’-H), 2.93 (q, J = 7.1 Hz, 1H, 9’-H), 2.33 (dd, J = 13.0, 3.5 Hz, 1H, 7-H), 2.18 (t, J = 7.1 Hz, 2 H, 7-H and 11-H), 1.96 (p, J = 7.8, 6.5 Hz, 2 H, 6-H and 9-H), 1.92–1.77 (m, 2 H, 3’-H and 6’-H), 1.76–1.62 (m, 4 H, 4’-H, 5’-H, 11-H and 6-H), 1.51–1.36 (m, 4 H, 3’-H, 6’-H, 2-H and 1-H), 1.35–1.30 (m, 1H, 2-H), 1.32 (s, 3 H, acetonylidene-CH3), 1.29–1.17 (m, 4 H, 4’-H, 5’-H, 1-H and 5-H), 1.24 (s, 3 H, acetonylidene-CH3), 1.11 (s, 3 H, 20-CH3), 0.81 (s, 3 H, 18-CH3); 13C NMR (126 MHz, DMSO-d6) δ 178.55 (2’-C), 178.30 (7’-C), 168.93 (16-C), 147.59 (12-C), 144.94 (8-C), 124.75 (13-C), 108.39 (17-C), 98.31 (acetonylidene-C), 75.43 (3-C), 68.47 (15-C), 62.89 (19-C), 54.70 (9-C), 51.08 (5-C), 45.16 (8’-C), 39.05 (9’-C), 38.78 (4-C), 37.80 (14-C), 37.25 (10-C), 37.00 (7-C), 33.77 (1-C), 27.22 (2-C), 25.69 (acetonylidene-CH3), 25.14 (acetonylidene-CH3), 24.96 (6-C), 24.56 (11-C), 23.84 (3’-C), 22.76 (6’-C), 22.71 (20-C), 21.36 (2 C, 4’-C and 5’-C), 15.89 (18-C); ESI-HRMS: m/z 548.2972, [M + Na]+, calculated for C31H43NO6Na, 548.2988.
14-(R)-® (1’, 2’-cyclopentadiimide)−3,19-isopropylidene andrographolide (3j). Yield, 37.4%; white powder; m.p. 229.1–231.2 °C; 1H NMR (400 MHz, DMSO-d6) δ ppm 6.52–6.42 (m, 1H, 12-H), 5.47 (d, J = 7.8 Hz, 1H, 14-H), 4.81 (s, 1H, 17-H), 4.56 (t, J = 9.4 Hz, 1H, 15-H), 4.37 (s, 1H, 17-H), 4.23 (dd, J = 10.0, 3.0 Hz, 1H, 15-H), 3.83 (d, J = 11.6 Hz, 1H, 19-H), 3.39 (dd, J = 8.4, 3.9 Hz, 1H, 19-H), 3.25 (dd, J = 11.8, 7.8 Hz, 1H, 7’-H), 3.20–3.13 (m, 1H, 6’-H), 3.08 (d, J = 11.6 Hz, 1H, 3-H), 2.32 (d, J = 12.9 Hz, 1H, 7-H), 2.11 (t, J = 8.7 Hz, 2 H, 7-H and 11-H), 1.95 (dd, J = 17.4, 12.1 Hz, 2 H, 6-H and 9-H), 1.90–1.76 (m, 5 H, 11-H, 6-H, 3’-H, 5’-H and 4’-H), 1.74–1.61 (m, 3 H, 4’-H, 3’-H and 5’-H), 1.44–1.35 (m, 1H, 2-H), 1.32 (s, 3 H, acetonylidene-CH3), 1.28–1.24 (m, 2 H, 1-H and 2-H), 1.24 (s, 3 H, acetonylidene-CH3), 1.23–1.13 (m, 2 H, 1-H and 5-H), 1.10 (s, 3 H, 20-CH3), 0.81 (s, 3 H, 18-CH3); 13C NMR (101 MHz, CDCl3) δ 178.78 (2 C, 2’-C and 6’-C), 169.04 (16-C), 147.04 (12-C), 146.50 (8-C), 123.86 (13-C), 108.72 (17-C), 99.46 (acetonylidene-C), 75.74 (3-C), 68.71 (15-C), 64.15 (19-C), 56.01 (9-C), 51.79 (5-C), 46.13 (7’-C), 45.24 (6’-C), 45.10 (4-C), 38.29 (14-C), 38.14 (10-C), 37.63 (7-C), 34.30 (1-C), 30.72 (3’-C), 30.50 (5’-C), 26.75 (2-C), 26.12 (4’-C), 25.46 (acetonylidene-CH3), 25.26 (acetonylidene-CH3), 25.05 (6-C), 24.75 (11-C), 23.31 (20-C), 16.60 (18-C); ESI-HRMS: m/z 534.2826, [M + Na]+, calculated for C30H41NO6Na, 534.2832.
14-(R)-® (2’, 3’-pyridine dicarboximide)−3,19-isopropylidene andrographolide (3k). Yield, 37.1%; white powder; m.p. 258.8–259.2 °C; 1H NMR (400 MHz, DMSO-d6) δ 9.03 (d, J = 4.5 Hz, 1H, 4’-H), 8.37 (d, J = 7.1 Hz, 1H, 6’-H), 7.84 (dd, J = 7.6, 5.0 Hz, 1H, 5’-H), 6.64 (dd, J = 6.4, 5.5 Hz, 1H, 12-H), 5.75 (d, J = 8.0 Hz, 1H, 14-H), 4.85 (s, 1H, 17-H), 4.65 (t, J = 9.3 Hz, 1H, 15-H), 4.49 (dd, J = 11.5, 3.8 Hz, 2 H, 17-H and 15-H), 3.74 (d, J = 11.5 Hz, 1H, 19-H), 3.13 (dd, J = 8.6, 3.6 Hz, 1H, 19-H), 3.01 (d, J = 11.5 Hz, 1H, 3-H), 2.35–2.18 (m, 3 H, 7-CH2 and 11-H), 1.85 (td, J = 12.8, 4.9 Hz, 1H, 6-H), 1.68 (d, J = 6.0 Hz, 1H, 9-H), 1.62–1.47 (m, 2 H, 11-H and 6-H), 1.25 (s, 3 H, acetonylidene-CH3), 1.20 (s, 3 H, acetonylidene-CH3), 1.31–1.08 (m, 3 H, 2-CH2 and 1-H), 1.05–0.99 (m, 1H, 1-H), 0.99 (s, 3 H, 20-CH3), 0.75–0.64 (m, 1H, 5-H), 0.70 (s, 3 H, 18-CH3); 13C NMR (101 MHz, DMSO-d6) δ 168.98 (16-C), 165.22 (7’-C), 165.15 (2’-C), 155.20 (8’-C), 151.02 (12-C), 147.37 (8-C), 145.14 (4’-C), 131.58 (6’-C), 128.18 (9’-C), 126.96 (5’-C), 124.31 (13-C), 108.12 (17-C), 98.14 (acetonylidene-C), 75.43 (3-C), 68.73 (15-C), 62.69 (19-C), 54.67 (9-C), 51.20 (5-C), 44.86 (4-C), 37.87 (14-C), 37.02 (10-C), 36.95 (7-C), 33.89 (1-C), 27.31 (2-C), 25.61 (acetonylidene-CH3), 25.10 (acetonylidene-CH3), 24.69 (6-C), 24.35 (11-C), 22.59 (20-C), 15.43 (18-C); ESI-HRMS: m/z 534.2470, [M + Na]+, calculated for C30H36N2O6Na, 543.2471.
14-(R)-® (phenoxy)-andrographolide (4a). Yield, 86.0%; white powder; m.p. 164–166°C; 1H NMR (400 MHz, CDCl3) δ (ppm) 7.40–7.29 (m, 2H, 3’-H and 5’-H), 7.14–7.01 (m, 2 H, 2’-H and 6’-H), 6.89–6.79 (m, 2 H, 4’-H and 12-H), 5.55–5.48 (m, 1H, 14-H), 4.85 (dd, J = 1.9, 1.0 Hz, 1H, 17-H), 4.60 (dd, J = 10.7, 5.9 Hz, 1H, 15-H), 4.44–4.36 (m, 2 H, 17-H and 15-H), 4.17–4.08 (m, 1H, 19-H), 3.40 (dd, J = 11.5, 4.3 Hz, 1H, 19-H), 3.29 (d, J = 10.9 Hz, 1H, 3-H), 2.54–2.44 (m, 1H, 7-H), 2.44–2.36 (m, 1H, 7-H), 2.33–2.23 (m, 1H, 11-H), 2.16 (d, J = 26.3 Hz, 2 H, 3-OH and 19-OH), 1.97 (td, J = 12.7, 10.7, 6.2 Hz, 1H, 6-H), 1.92–1.85 (m, 1H, 9-H), 1.85–1.70 (m, 2 H, 11-H and 6-H), 1.70–1.55 (m, 2 H, 2-H and 1-H), 1.33–1.16 (m, 2 H, 2-H and 1-H), 1.23 (s, 3 H, 20-CH3), 1.11 (td, J = 13.5, 3.7 Hz, 1H, 5-H), 0.58 (s, 3 H, 18-CH3); 13C NMR (101 MHz, C6D6) δ 169.5 (16-C), 156.4 (1’-C), 150.8 (12-C), 147.1 (8-C), 130.0 (2 C, 3’-C and 5’-H), 125.0 (13-C), 122.3 (2 C, 2’-C and 6’-H), 115.7 (4’-C), 108.2 (17-C), 80.4 (3-C), 71.3 (15-C), 71.0 (14-C), 64.1 (19-C), 55.9 (9-C), 55.1 (5-C), 42.8 (4-C), 39.0 (10-C), 37.7 (7-C), 36.8 (1-C), 28.1 (2-C), 25.6 (6-C), 23.7 (11-C), 22.7 (20-C), 15.1 (18-C); ESI-HRMS: m/z 449.2342, [M + Na]+, calcd for C26H34NaO5, 449.2304.
14-(R)-® (2’-chloro-phenoxy)-andrographolide (4b). Yield, 78.3%; white powder; m.p. 178–180 °C; 1H NMR (400 MHz, CD3OD) δ 7.46 (dd, J = 7.9, 1.6 Hz, 1H, 3’-H), 7.33 (ddd, J = 8.3, 7.6, 1.6 Hz, 1H, 5’-H), 7.09 (dddd, J = 23.7, 15.4, 7.7, 1.4 Hz, 3 H, 12-H, 6’-H and 4’-H), 5.82 (d, J = 5.4 Hz, 1H, 14-H), 4.88 (s, 1H, 17-H), 4.67 (dd, J = 10.9, 5.5 Hz, 1H, 15-H), 4.56 (s, 1H, 17-H), 4.39 (dd, J = 10.8, 1.3 Hz, 1H, 15-H), 4.03 (d, J = 11.0 Hz, 1H, 19-H), 3.32–3.29 (m, 3 H, 19-H, 3-H and 7-H), 3.19 (dd, J = 11.9, 3.8 Hz, 1H, 7-H), 2.48–2.25 (m, 3 H, 11-H, 6-H and 9-H), 2.02 (dd, J = 13.5, 11.2 Hz, 2 H, 3-OH and 19-OH), 1.86–1.77 (m, 1H, 11-H), 1.64 (ddd, J = 15.5, 13.3, 3.9 Hz, 1H, 6-H), 1.54–1.44 (m, 2 H, 2-H and 1-H), 1.38–1.21 (m, 2 H, 2-H and 1-H), 1.17 (s, 3 H, 20-CH3), 0.99 (dt, J = 14.8, 4.3 Hz, 1H, 5-H), 0.62 (s, 3 H, 18-CH3). 13C NMR (101 MHz, CD3OD) δ 171.61 (16-C), 153.78 (1’-C), 152.01 (12-C), 149.09 (8-C), 131.98 (3’-C), 129.45 (5’-C), 127.02 (13-C), 124.79 (2’-C), 124.15 (4’-C), 116.70 (6’-C), 108.38 (17-C), 80.76 (3-C), 73.70 (15-C), 72.78 (14-C), 64.92 (19-C), 57.81 (9-C), 55.99 (5-C), 43.59 (4-C), 40.18 (10-C), 38.91 (7-C), 37.74 (1-C), 28.90 (2-C), 26.70 (6-C), 25.17 (11-C), 23.32 (20-C), 15.48 (18-C). ESI-HRMS: m/z 483.1909(35Cl) 485.1879(37Cl), [M + Na]+, calcd for C26H33ClNaO5, 483.1914(35Cl) 485.1885(37Cl).
14-(R)-® (2’-cyano-phenoxy)-andrographolide (4c). Yield, 86.7%; white powder; m.p. 169.2–170.1 °C; 1H NMR(400 MHz, DMSO-d6) δ ppm 7.81 (d, J = 7.5 Hz, 1H, 5’-H), 7.73 (t, J = 7.7 Hz, 1H, 3’-H), 7.28 (d, J = 8.5 Hz, 1H, 4’-H), 7.19 (t, J = 7.5 Hz, 1H, 6’-H), 7.08 (t, J = 7.0 Hz, 1H, 12-H), 6.01 (d, J = 4.5 Hz, 1H, 14-H), 4.99 (d, J = 4.4 Hz, 1H, 15-H), 4.83 (s, 1H, 17-H), 4.75 (dd, J = 11.0, 5.4 Hz, 1H, 15-H), 4.58 (s, 1H, 17-H), 4.39 (d, J = 11.0 Hz, 1H, 19-OH), 4.11 (d, J = 5.5 Hz, 1H, 3-OH), 3.77 (d, J = 10.4 Hz, 1H, 19-H), 3.20 (dd, J = 10.1, 7.9 Hz, 1H, 19-H), 3.05–2.94 (m, 1H, 3-H), 2.48–2.40 (m, 1H, 7-H), 2.39–2.25 (m, 2 H, 7-H and 11-H), 2.03–1.89 (m, 2 H, 6-H and 9-H), 1.71 (d, J = 12.0 Hz, 1H, 11-H), 1.51 (dd, J = 26.0, 12.8 Hz, 2 H, 6-H and 2-H), 1.39–1.23 (m, 2 H, 1-H and 2-H), 1.18 (d, J = 11.5 Hz, 1H, 1-H), 1.05 (s, 3 H, 20-CH3), 0.88 (t, J = 12.3 Hz, 1H, 5-H), 0.56 (s, 3 H, 18-CH3); 13C NMR (101 MHz, DMSO-d6) δ 168.78 (16-C), 158.06 (1’-C), 150.95 (12-C), 147.67 (8-C), 135.21 (5’-C), 134.21 (3’-C), 125.02 (13-C), 122.08 (4’-C), 116.25 (2’-CN), 113.93 (6’-C), 107.73 (17-C), 101.36 (2’-C), 78.33 (3-C), 72.06 (15-C), 70.65 (14-C), 62.54 (19-C), 56.14 (9-C), 54.08 (5-C), 42.15 (4-C), 38.79 (10-C), 37.30 (7-C), 36.13 (1-C), 27.80 (2-C), 25.11 (6-C), 23.91 (11-C), 23.02 (20-C), 14.62 (18-C); ESI-HRMS: m/z 474.2245, [M + Na]+, calculated for C27H33NO5Na, 474.2256.
14-(R)-® (2’-bromo-phenoxy)-andrographolide (4d). Yield, 84.2%; white powder; m.p. 149.0–149.9 °C; 1H NMR (400 MHz, DMSO-d6) δ ppm 7.64 (d, J = 7.7 Hz, 1H, 3’-H), 7.40 (t, J = 7.6 Hz, 1H, 5’-H), 7.16 (d, J = 8.1 Hz, 1H, 4’-H), 7.00 (dt, J = 14.8, 7.3 Hz, 2 H, 6’-H and 12-H), 5.90 (d, J = 4.3 Hz, 1H, 14-H), 4.98 (d, J = 2.9 Hz, 1H, 15-H), 4.82 (s, 1H, 17-H), 4.70 (dd, J = 10.8, 5.2 Hz, 1H, 15-H), 4.55 (s, 1H, 17-H), 4.34 (d, J = 10.9 Hz, 1H, 3-OH), 4.11 (d, J = 4.8 Hz, 1H, 19-OH), 3.76 (d, J = 10.8 Hz, 1H, 19-H), 3.19 (dd, J = 10.0, 7.1 Hz, 1H, 19-H), 3.00 (d, J = 10.9 Hz, 1H, 3-H), 2.42–2.22 (m, 3 H, 7-CH2 and 11-H), 1.95 (d, J = 11.0 Hz, 2 H, 6-H and 9-H), 1.70 (d, J = 12.2 Hz, 1H, 11-H), 1.56–1.38 (m, 2 H, 6-H and 2-H), 1.37–1.22 (m, 2 H, 1-H and 2-H), 1.17 (d, J = 12.5 Hz, 1H, 1-H), 1.04 (s, 3 H, 20-CH3), 0.93–0.81 (m, 1H, 5-H), 0.54 (s, 3 H, 18-CH3); 13C NMR (101 MHz, DMSO-d6) δ 169.08 (16-C), 152.91 (1’-C), 150.39 (12-C), 147.77 (8-C), 133.59 (3’-C), 129.19 (5’-C), 125.56 (13-C), 123.21 (4’-C), 115.13 (6’-C), 111.80 (2’-C), 107.79 (17-C), 78.35 (3-C), 72.03 (15-C), 71.01 (14-C), 62.65 (19-C), 55.94 (9-C), 54.04 (5-C), 42.19 (4-C), 38.78 (10-C), 37.45 (7-C), 36.10 (1-C), 27.86 (2-C), 25.17 (6-C), 23.93 (11-C), 23.07 (20-C), 14.73 (18-C); ESI-HRMS: m/z 505.1552(79Br) 507.1551(81Br), [M + H]+, calculated for C26H34BrO5, 505.1590(79Br) 507.1569(81Br).
14-(R)-® (2’-trifluoromethyl-phenoxy)-andrographolide (4e). Yield, 82.2%; white powder; m.p. 83.3–83.4 °C; 1H NMR (400 MHz, DMSO-d6) δ ppm 7.68 (t, J = 7.5 Hz, 2 H, 3’-H and 5’-H), 7.36 (d, J = 8.5 Hz, 1H, 4’-H), 7.18 (t, J = 7.6 Hz, 1H, 6’-H), 6.94 (t, J = 6.6 Hz, 1H, 12-H), 6.08 (d, J = 4.9 Hz, 1H, 14-H), 5.01 (d, J = 4.9 Hz, 1H, 15-H), 4.81 (s, 1H, 17-H), 4.70 (dd, J = 11.0, 5.4 Hz, 1H, 15-H), 4.48 (s, 1H, 17-H), 4.30 (d, J = 11.0 Hz, 1H, 3-OH), 4.10 (dd, J = 7.5, 2.7 Hz, 1H, 19-OH), 3.77 (dd, J = 10.9, 2.7 Hz, 1H, 19-H), 3.20 (dd, J = 10.8, 7.7 Hz, 1H, 19-H), 3.02 (dt, J = 8.8, 4.2 Hz, 1H, 3-H), 2.46–2.26 (m, 3 H, 7-CH2 and 11-H), 1.88 (dd, J = 18.6, 6.8 Hz, 2 H, 6-H and 9-H), 1.76–1.66 (m, 1H, 11-H), 1.59–1.46 (m, 1H, 6-H), 1.41 (d, J = 10.0 Hz, 2 H, 2-H and 1-H), 1.36–1.22 (m, 1H, 2-H), 1.03 (s, 3 H, 20-CH3), 1.13–0.91 (m, 2 H, 1-H and 5-H), 0.54 (s, 3 H, 18-CH3); 13C NMR (101 MHz, DMSO-d6) δ 168.89 (16-C), 154.22 (d, J = 1.3 Hz, 1’-C), 150.61 (12-C), 147.69 (8-C), 134.30 (5’-C), 127.28 (d, J = 5.1 Hz, 3’-C), 125.04 (13-C), 123.59 (d, J = 272.6 Hz, 2’-C), 121.33 (4’-C), 117.89 (q, J = 30.3 Hz, 2’-CF3), 114.72 (6’-C), 107.89 (17-C), 78.36 (3-C), 71.54 (15-C), 70.74 (14-C), 62.55 (19-C), 55.48 (9-C), 54.23 (5-C), 42.16 (4-C), 38.58 (10-C), 37.37 (7-C), 36.06 (1-C), 27.77 (2-C), 25.11 (6-C), 23.88 (11-C), 23.07 (20-C), 14.66 (18-C); ESI-HRMS: m/z 517.2153 [M + Na]+, calculated for C27H33F3O5Na, 517.2178.
14-(R)-® (2’-iodo-phenoxy)-andrographolide (4f). Yield, 81.7%; white powder; m.p. 177.6–178.3 °C; 1H NMR (400 MHz, DMSO-d6) δ ppm 7.83 (dd, J = 7.7, 1.5 Hz, 1H, 3’-H), 7.44–7.36 (m, 1H, 5’-H), 7.06 (d, J = 7.6 Hz, 1H, 4’-H), 7.02 (t, J = 7.4 Hz, 1H, 6’-H), 6.83 (td, J = 7.6, 1.0 Hz, 1H, 12-H), 5.88 (d, J = 5.1 Hz, 1H, 14-H), 4.97 (d, J = 4.9 Hz, 1H, 15-H), 4.83 (s, 1H, 17-H), 4.70 (dd, J = 10.9, 5.4 Hz, 1H, 15-H), 4.57 (s, 1H, 17-H), 4.31 (d, J = 10.9 Hz, 1H, 3-OH), 4.10 (dd, J = 7.5, 2.8 Hz, 1H, 19-OH), 3.76 (dd, J = 10.9, 2.8 Hz, 1H, 19-H), 3.19 (dd, J = 10.8, 7.6 Hz, 1H, 19-H), 3.01 (dt, J = 9.4, 4.4 Hz, 1H, 3-H), 2.44–2.35 (m, 1H, 7-H), 2.28 (ddd, J = 18.7, 11.0, 5.8 Hz, 2 H, 7-H and 11-H), 1.98 (t, J = 11.6 Hz, 2 H, 6-H and 9-H), 1.77–1.67 (m, 1H, 11-H), 1.56–1.47 (m, 1H, 6-H), 1.46–1.39 (m, 1H, 2-H), 1.31 (dd, J = 13.0, 3.6 Hz, 2 H, 1-H and 2-H), 1.25 (d, J = 12.7 Hz, 1H, 1-H), 1.05 (s, 3 H, 20-CH3), 0.88 (dd, J = 13.4, 10.2 Hz, 1H, 5-H), 0.54 (s, 3 H, 18-CH3); 13C NMR (101 MHz, DMSO-d6) δ 169.01 (16-C), 155.22 (1’-C), 150.10 (12-C), 147.75 (8-C), 139.58 (3’-C), 129.82 (5’-C), 125.61 (13-C), 123.57 (4’-C), 113.72 (6’-C), 107.70 (17-C), 87.36 (2’-C), 78.23 (3-C), 71.80 (15-C), 70.93 (14-C), 62.58 (19-C), 55.74 (9-C), 53.82 (5-C), 42.17 (4-C), 38.76 (10-C), 37.37 (7-C), 36.08 (1-C), 27.82 (2-C), 25.19 (6-C), 23.90 (11-C), 22.97 (20-C), 14.71 (18-C); ESI-HRMS: m/z 575.1266 [M + Na]+, calculated for C26H34IO5Na, 575.1270.
14-(R)-® (2’-fluoro-phenoxy)-andrographolide (4g). Yield, 81.9%; white powder; m.p. 160.2–160.3 °C; 1H NMR (500 MHz, DMSO-d6) δ 7.28 (ddd, J = 11.6, 8.1, 1.5 Hz, 1H, 4’-H), 7.23 (td, J = 8.3, 1.6 Hz, 1H, 3’-H), 7.18 (td, J = 8.0, 1.0 Hz, 1H, 5’-H), 7.09–7.03 (m, 1H, 6’-H), 6.94 (td, J = 7.1, 1.2 Hz, 1H, 12-H), 5.82 (d, J = 5.2 Hz, 1H, 14-H), 4.98 (d, J = 4.9 Hz, 1H, 15-H), 4.80 (s, 1H, 17-H), 4.65 (dd, J = 10.9, 5.4 Hz, 1H, 15-H), 4.48 (s, 1H, 17-H), 4.39 (dd, J = 10.9, 1.1 Hz, 1H, 3-OH), 4.09 (dd, J = 7.5, 2.9 Hz, 1H, 19-OH), 3.77 (dd, J = 11.0, 2.9 Hz, 1H, 19-H), 3.21 (dd, J = 10.9, 7.6 Hz, 1H, 19-H), 3.04 (dt, J = 11.5, 4.3 Hz, 1H, 3-H), 2.34–2.23 (m, 3 H, 7-CH2 and 11-H), 1.94–1.85 (m, 2 H, 6-H and 9-H), 1.74–1.67 (m, 1H, 11-H), 1.52 (dt, J = 12.4, 7.4 Hz, 1H, 6-H), 1.45–1.38 (m, 2 H, 2-H and 1-H), 1.30 (qd, J = 13.1, 4.1 Hz, 1H, 2-H), 1.08 (dd, J = 12.7, 2.5 Hz, 1H, 1-H), 1.04 (s, 3 H, 20-CH3), 0.94 (dd, J = 13.5, 9.7 Hz, 1H, 5-H), 0.55 (s, 3 H, 18-CH3); 13C NMR (126 MHz, DMSO-d6) δ 168.98 (16-C), 152.56 (d, J = 244.2 Hz, 2’-C), 150.21 (12-C), 147.68 (8-C), 144.21 (d, J = 10.7 Hz, 1’-C), 125.42 (13-C), 124.99 (d, J = 3.7 Hz, 5’-C), 122.82 (d, J = 6.9 Hz, 4’-C), 117.59 (6’-C), 116.54 (d, J = 18.1 Hz, 3’-C), 107.76 (17-C), 78.30 (3-C), 72.73 (15-C), 70.97 (14-C), 62.52 (19-C), 55.68 (9-C), 54.27 (5-C), 42.15 (4-C), 38.65 (10-C), 37.39 (7-C), 36.15 (1-C), 27.76 (2-C), 24.98 (6-C), 23.89 (11-C), 23.02 (20-C), 14.58 (18-C); ESI-HRMS: m/z 467.2203 [M + Na]+, calculated for C26H33FO5Na, 467.2210.
14-(R)-® (cis-1’, 2’, 3’, 6’-tetrahydrophthalimide)-andrographolide (4h). Yield, 65.5%; white powder; m.p. 188.6–189.6 °C; 1H NMR (500 MHz, DMSO-d6) δ 6.43 (ddd, J = 9.2, 4.3, 2.3 Hz, 1H, 12-H), 5.84 (t, J = 3.4 Hz, 2 H, 4’-H and 5’-H), 5.45 (dq, J = 8.1, 2.5 Hz, 1H, 14-H), 5.04 (d, J = 4.8 Hz, 1H, 15-H), 4.76 (s, 1H, 17-H), 4.61–4.52 (m, 1H, 15-H), 4.30 (s, 1H, 17-H), 4.17 (dd, J = 9.9, 3.1 Hz, 1H, 3-OH), 4.11 (dd, J = 7.6, 2.9 Hz, 1H, 19-OH), 3.82 (dd, J = 10.9, 2.9 Hz, 1H, 19-H), 3.28–3.18 (m, 3 H, 8’-H, 9’-H and 19-H), 3.13 (td, J = 9.2, 8.6, 2.6 Hz, 1H, 3-H), 2.45–2.38 (m, 1H, 7-H), 2.30 (ddt, J = 14.3, 12.1, 2.7 Hz, 2 H, 7-H and 11-H), 2.25–2.15 (m, 2 H, 3’-H and 6’-H), 2.10–2.03 (m, 1H, 3’-H), 1.99 (q, J = 8.9, 7.4 Hz, 1H, 6’-H), 1.91 (td, J = 13.6, 13.1, 4.4 Hz, 2 H, 6-H and 9-H), 1.76–1.69 (m, 1H, 11-H), 1.62 (tt, J = 12.9, 5.8 Hz, 2 H, 6-H and 2-H), 1.48 (dt, J = 13.2, 3.5 Hz, 1H, 1-H), 1.32 (qd, J = 13.0, 4.1 Hz, 1H, 2-H), 1.21–1.11 (m, 2 H, 1-H and 5-H), 1.07 (s, 3 H, 20-CH3), 0.57 (s, 3 H, 18-CH3); 13C NMR (101 MHz, DMSO-d6) δ 179.17 (2’-C), 178.87 (7’-C), 168.91 (16-C), 147.69 (12-C), 145.11 (8-C), 127.85 (4’-C), 127.49 (5’-C), 124.34 (13-C), 107.87 (17-C), 78.36 (3-C), 68.34 (15-C), 62.61 (19-C), 54.95 (9-C), 54.24 (5-C), 45.46 (8’-C), 42.24 (9’-C), 38.82 (4-C), 38.39 (14-C), 38.16 (10-C), 37.44 (7-C), 36.40 (1-C), 27.78 (2-C), 25.00 (6-C), 23.96 (11-C), 23.06 (4’-C), 22.97 (5’-C), 22.94 (20-C), 14.67 (18-C); ESI-HRMS: m/z 506.2503, [M + Na]+, calculated for C28H37NO6Na, 506.2519.
14-(R)-® (ciscyclohexa-1’, 2’-dimethylformamide)-andrographolide (4i). Yield, 72.8%; white powder; m.p. 179.1–182.3 °C; 1H NMR (400 MHz, DMSO-d6) δ 6.50 (ddd, J = 7.6, 4.8, 2.2 Hz, 1H, 12-H), 5.58–5.44 (m, 1H, 14-H), 5.05 (d, J = 4.8 Hz, 1H, 15-H), 4.80 (s, 1H, 17-H), 4.58 (dd, J = 10.0, 8.7 Hz, 1H, 15-H), 4.35 (s, 1H, 17-H), 4.24 (dd, J = 10.0, 3.0 Hz, 1H, 3-OH), 4.13 (dd, J = 7.5, 2.9 Hz, 1H, 19-OH), 3.87–3.77 (m, 1H, 19-H), 3.23 (ddd, J = 19.8, 10.8, 6.3 Hz, 2 H, 19-H and 3-H), 2.98 (dq, J = 33.7, 7.2 Hz, 2 H, 8’-H and 9’-H), 2.31 (dt, J = 12.6, 3.4 Hz, 1H, 7-H), 2.24–2.06 (m, 2 H, 7-H and 11-H), 1.97–1.87 (m, 2 H, 6-H and 9-H), 1.85–1.79 (m, 1H, 3’-H), 1.77–1.66 (m, 3 H, 6’-H, 4’-H and 5’-H), 1.65–1.55 (m, 2 H, 11-H and 6-H), 1.52–1.40 (m, 3 H, 3’-H, 6’-H and 2-H), 1.40–1.28 (m, 3 H, 1-H, 2-H and 4’-H), 1.28–1.22 (m, 2 H, 5’-H and 1-H), 1.17–1.12 (m, 1H, 5-H), 1.08 (s, 3 H, 20-CH3), 0.59 (s, 3 H, 18-CH3); 13C NMR (101 MHz, DMSO-d6) δ 178.53 (2’-C), 178.31(7’-C), 168.95 (16-C), 147.62 (12-C), 144.95 (8-C), 124.63 (13-C), 107.89 (17-C), 78.31 (3-C), 68.50 (15-C), 62.58 (19-C), 54.86 (9-C), 54.21 (5-C), 45.14 (8’-C), 42.22 (9’-C), 39.04 (4-C), 38.80 (14-C), 38.42 (10-C), 37.40 (7-C), 36.48 (1-C), 27.80 (2-C), 24.77 (6-C), 23.95 (11-C), 23.88 (3’-C), 23.03 (6’-C), 22.74 (20-C), 21.40 (4’-C), 21.36 (5’-C), 14.68 (18-C); ESI-HRMS: m/z 508.2660, [M + Na]+, calculated for C28H39NO6Na, 508.2675.
14-(R)-® (1’, 2’-cyclopentadiimide)-andrographolide (4j). Yield, 77.1%; white powder; m.p. 231.2–232.3 °C; 1H NMR (400 MHz, DMSO-d6) δ ppm 6.48 (t, J = 5.6 Hz, 1H, 12-H), 5.47 (d, J = 8.1 Hz, 1H, 14-H), 5.06 (s, 1H, 3-OH), 4.79 (s, 1H, 17-H), 4.57 (t, J = 9.3 Hz, 1H, 15-H), 4.34 (s, 1H, 17-H), 4.24 (dd, J = 10.0, 2.9 Hz, 1H, 15-H), 4.14 (s, 1H, 19-OH), 3.82 (d, J = 10.9 Hz, 1H, 19-H), 3.29–3.22 (m, 2 H, 19-H and 7’-H), 3.21–3.13 (m, 2 H, 6’-H and 3-H), 2.31 (d, J = 12.4 Hz, 1H, 7-H), 2.16–2.01 (m, 2 H, 7-H and 11-H), 1.97–1.78 (m, 6 H, 6-H, 9-H, 11-H, 6-H, 3’-H and 5’-H), 1.78–1.66 (m, 2 H, 4’-CH2), 1.65–1.53 (m, 2 H, 3’-H and 5’-H), 1.42 (d, J = 13.0 Hz, 1H, 2-H), 1.37–1.27 (m, 1H, 1-H), 1.25–1.11 (m, 3 H, 2-H, 1-H and 5-H), 1.08 (s, 3 H, 20-CH3), 0.59 (s, 3 H, 18-CH3); 13C NMR (101 MHz, DMSO-d6) δ 179.12 (2’-C), 179.01 (6’-C), 168.92 (16-C), 147.61 (12-C), 144.85 (8-C), 124.61 (13-C), 107.87 (17-C), 78.34 (3-C), 68.38 (15-C), 62.61 (19-C), 54.83 (9-C), 54.23 (5-C), 45.46 (7’-C), 44.95 (6’-C), 44.65 (4-C), 42.23 (14-C), 39.52 (10-C), 38.42 (7-C), 37.42 (1-C), 36.45 (3’-C), 29.69 (5’-C), 29.53 (2-C), 27.83 (4’-C), 24.78 (6-C), 23.98 (11-C), 23.04 (20-C), 14.69 (18-C); ESI-HRMS: m/z 494.2508 [M + Na]+, calculated for C27H37NO6Na, 494.2519.
14-(R)-® (2’, 3’-pyridine dicarboximide)-andrographolide (4k). Yield, 78.5%; white powder; m.p. 162.2–162.8 °C; 1H NMR (400 MHz, DMSO-d6) δ ppm 9.01 (dd, J = 5.0, 1.4 Hz, 1H, 4’-H), 8.33 (dd, J = 7.7, 1.4 Hz, 1H, 6’-H), 7.89 (d, J = 1.7 Hz, 1H, 5’-H), 7.82 (dd, J = 7.7, 5.0 Hz, 1H, 12-H), 5.07–5.01 (m, 1H, 3-OH), 4.98 (d, J = 4.8 Hz, 1H, 14-H), 4.96–4.83 (m, 3 H, 17-H, 15-H and 17-H), 4.74 (s, 1H, 15-H), 4.07 (dd, J = 7.6, 2.7 Hz, 1H, 19-OH), 3.79 (dd, J = 10.9, 2.8 Hz, 1H, 19-H), 3.20 (dd, J = 10.9, 7.8 Hz, 1H, 19-H), 3.12–3.03 (m, 1H, 3-H), 2.53–2.47 (m, 1H, 7-H), 2.31 (d, J = 12.3 Hz, 1H, 7-H), 2.14–2.03 (m, 1H, 11-H), 1.82–1.72 (m, 1H, 6-H), 1.71–1.62 (m, 2 H, 9-H and 11-H), 1.61–1.51 (m, 3 H, 6-H, 2-H and 1-H), 1.29 (qd, J = 12.9, 3.8 Hz, 1H, 2-H), 1.11 (dd, J = 12.6, 1.9 Hz, 1H, 1-H), 0.99 (s, 3 H, 20-CH3), 0.93 (dd, J = 13.4, 9.6 Hz, 1H, 5-H), 0.64 (s, 3 H, 18-CH3); 13C NMR (101 MHz, DMSO-d6) δ 172.35 (16-C), 166.00 (7’-C), 165.94 (2’-C), 155.17 (8’-C), 150.91 (12-C), 150.22 (4’-C), 147.38 (8-C), 131.67 (6’-C), 129.87 (9’-C), 128.17 (5’-C), 126.89 (13-C), 106.56 (17-C), 78.21 (3-C), 70.63 (15-C), 62.66 (19-C), 53.81 (9-C), 50.99 (5-C), 45.05 (4-C), 42.24 (14-C), 38.61 (10-C), 37.72 (7-C), 35.99 (1-C), 27.86 (2-C), 24.14 (6-C), 24.12 (11-C), 22.87 (20-C), 15.00 (18-C); ESI-HRMS: m/z 503.2160 [M + Na]+, calculated for C27H32N2O6Na, 503.2158.
Antifungal activity in vitro
The antifungal activities of the target compounds in vitro were determined according to the method reported in the literature1. Dissolve the compound (20 mg) in 10 mL of 5% DMSO, and then mix the solution with the sterilized potato dextrose agar (PDA) medium (190 mL) to obtain the medicated medium. Fungus disks (d = 5 mm) were placed on the PDA medium and cultured for 72 h. The inhibition rate was calculated by measuring the diameter of each colony. The commercial antifungal agent KXM was used as the positive control, and each test was parallel for 3 times. The same method was used for the determination of EC50 values. The test compounds were prepared into 100, 80, 60, 40, 20, 10, 5 µg/mL in the medium, and the corresponding inhibition rates were determined. The data were analyzed by PRISM software ver. 7.00 (Graphpad Software Inc., San Diego, CA) to calculate EC50 values.
SEM analysis
SEM was carried out according to the methods reported in the literature25. The mycelia were fixed overnight in 4% glutaraldehyde at 4 ℃. Then the fixed sample was rinsed with 0.1 M phosphate buffer solution (PBS, pH 6.8) for 4 times, about 10 min each time. The rinsed mycelia were dehydrated with 10%, 30%, 50%, 70%, 80% and 90% (V/V) ethanol orderly, and then dehydrated with 100% ethanol for three times. The dehydrated sample can be observed by scanning electron microscope (Hitachi s-4800) after CO2 vacuum drying and gold spraying.
Seed germination experiment
According to the method reported in literature26, the test compound was dissolved in 5% DMSO and prepared into 200, 100, 50 and 25 µg/mL solutions. The mature seeds of Vigna unguiculata (NO. 8 Shenzhou, Jiangxi Agricultural Seed Co., Ltd.) were soaked in the above four concentrations of drug solution for 12 h. The seeds that absorbed the drug solution were transferred to the Petri dish with distilled water and placed in the incubator at 25 ℃ and 80% humidity in the dark.
Conclusions
Eleven final products were obtained through a series of reactions using Andro as the starting material in this study. The antifungal activity indicates that most of the 14 aryloxy/amide substituted andrographolides synthesized in this study exhibit significant antifungal activities. Among them, 4d exhibited high antifungal activity in vitro, with inhibition rates exceeding 60% against 6 tested fungi at 100 µg/mL. And the EC50 value of 4d against P. piricola was only 9.09 µg/mL, 35% of KXM. Moreover, 4d can cause serious damage to fungal mycelium and showed high safety through seed germination experiment. Therefore, 4d was identified as a promising lead scaffold. SAR studies have shown that aryloxy containing adjacent halogen atoms, especially the introduction of bromine atoms or 2,3-pyridinediimide groups are beneficial to improve antifungal activity. It can also provide reference for the subsequent structural modification of Andro. The antifungal mechanisms of these compounds can be further studied in the future, which also provides assistance for the development of natural product antibiotics.
Data availability
Data is provided within the manuscript and supporting information.
References
Wang, Y. et al. First report of fusarium tricinctum causing root rot on Chinese Dwarf Cherry (Cerasus humilis) in China. Plant. Dis. 108 (1), 213 (2024).
Mishra, M., Srivastava, A., Singh, A., Pandey, G. C. & Srivastava, G. An overview of symbiotic and pathogenic interactions at the fungi-plant interface under environmental constraints. Front. Fungal Biol. 5, 1363460 (2024).
Luciano-Rosario, D. et al. Mold in, mold out: harvest bins harbor viable inoculum that can be reduced using novel sanitation methods to manage blue mold decay of apples. Postharvest Biol. Tec. 221, 113323 (2025).
Dwivedi, M., Singh, P. & Pandey, A. K. Botrytis fruit rot management: what have we achieved so far? Food Microbiol. 122, 104564 (2024).
Toda, M., Beer, K. D., Kuivila, K. M., Chiller, T. M. & Jackson, B. R. Trends in agricultural Triazole fungicide use in the united states, 1992–2016 and possible implications for antifungal-resistant fungi in human disease. Environ. Health Persp. 129 (5), 055001 (2021).
Wang, Z. K. et al. Effect of fungicides on soil respiration, microbial community, and enzyme activity: A global meta-analysis (1975–2024). Ecotoxicol. Environ. Saf. 289, 117433 (2025).
Sun, P. Z., Sun, S. X., Kong, W. L. & Li, S. K. In pursuit of lead innovation: pharmaceutically important and distinct amide-free succinate dehydrogenase inhibitors. J. Med. Chem. 68 (2), 1051–1067 (2025).
Abed-Ashtiani, F. et al. Plant tonic, a plant-derived bioactive natural product, exhibits antifungal activity against rice blast disease. Ind. Crop Prod. 112, 105–112 (2017).
Bai, Y. B. et al. Synthesis and antifungal activity of derivatives of the natural product Griseofulvin against phytopathogenic fungi. J. Agric. Food Chem. 71 (16), 6236–6248 (2023).
Dai, P. et al. Design, synthesis, antifungal activity, and 3D-QASR of novel oxime ether-containing coumarin derivatives as potential fungicides. J. Agric. Food Chem. 72 (11), 5983–5992 (2024).
Armengol, E. S., Harmanci, M. & Laffleur, F. Current strategies to determine antifungal and antimicrobial activity of natural compounds. Microbiol. Res. 252, 126867 (2021).
Orellana-Paucar, A. M. Turmeric essential oil constituents as potential drug candidates: a comprehensive overview of their individual bioactivities. Molecules 29 (17), 4210 (2024).
Liang, J. J. et al. Biological activities and secondary metabolites from sophora tonkinensis and its endophytic fungi. Molecules 27 (17), 5562 (2022).
Xiao, H. X. et al. Lycorine and organ protection: review of its potential effects and molecular mechanisms. Phytomedicine 104, 154266 (2022).
Li, F. et al. Discovery and preliminary SAR of 14-aryloxy-andrographolide derivatives as antibacterial agents with immunosuppressant activity. RSC Adv. 8 (17), 9440–9456 (2018).
Dafur, G. S., Harun, A., Kub, T. N. T., Bakar, R. A. & Harun, A. A systematic review on the antimicrobial activity of Andrographolide. J. Microbiol. Biotechnol. 35, e2408028 (2025).
Xu, M. et al. Evaluation of andrographolide-based analogs derived from Andrographis paniculata against Mythimna separata walker and Tetranychus cinnabarinus boisduval. Bioorg. Chem. 86, 28–33 (2019).
Gao, J. et al. Inhibition of AIM2 inflammasome-mediated pyroptosis by Andrographolide contributes to amelioration of radiation-induced lung inflammation and fibrosis. Cell. Death Dis. 10 (12), 957 (2019).
Yen, C. C. et al. W. Andrographolide attenuates oxidized LDL-induced activation of the NLRP3 inflammasome in bone marrow-derived macrophages and mitigates HFCCD-induced atherosclerosis in mice. AM. J. Chin. Med. 51 (1), 129–147 (2023).
Žiemytė Miglė, Rodríguez-Díaz, J. C., Ventero-Martín María, P., Alex, M. & Ferrer, M. D. Real-time monitoring of biofilm growth identifies Andrographolide as a potent antifungal compound eradicating Candida biofilms. Biofilm 5, 100134 (2023).
Mansuk, A. G. & Pachpute, T. S. Comprehensive assessment of transcorneal permeation, antimicrobial, and antifungal activities of andrographolide-loaded nanosuspension: in vitro and in vivo studies. J. Drug Deliv Ther. 13 (6), 35–42 (2023).
Dafur, G. S. et al. Antifungal effects of Andrographolide and its combination with amphotericin B against selected fungal pathogens. PeerJ 13, e19544 (2025).
Liu, Z. Y. et al. Synthesis and discovery of Andrographolide derivatives as non-steroidal farnesoid X receptor (FXR) antagonists. RSC Adv. 4 (26), 13533–13545 (2014).
Guo, B. J. et al. Andrographolide derivative ameliorates dextran sulfate sodium-induced experimental colitis in mice. Biochem. Pharmacol. 163, 416–424 (2019).
Yang, S. S. et al. Design, synthesis, and antifungal activity of hydrazone schiff bases containing cinnamaldehyde moieties. Phytochem Lett. 65, 53–57 (2025).
Zhang, Y. H. et al. Discovery of N-phenylpropiolamide as a novel succinate dehydrogenase inhibitor scaffold with broad-spectrum antifungal activity on phytopathogenic fungi. J. Agric. Food Chem. 71 (8), 3681–3693 (2023).
Funding
The research is financially supported by Research Project of Basic Science (Natural Science) in Colleges and Universities of Jiangsu Province (NO. 23KJD350004), 2024 Taizhou Vocational Education Federation (Taizhou Vocational Education Group) - Taizhou Polytechnic College Joint Research Project (NO. 2024BZD02), 2023 Jiangsu Province University “Blue Project” Excellent Teaching Team and 2024 College Students’ Innovation and Entrepreneurship Cultivation Plan Project (NO. TZY2024024).
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Zhuyun Liu and Zhuqing Liu conceived the research. Zhuyun Liu and Shanshan Yang wrote manuscript. Zhuyun Liu, Shanshan Yang, Jierui Ma, Yunying Sha, Yuyan Zhou, Zhuqing Liu and Lizhong Wang performed all experiments. All authors designed experiments, analyzed data, edited and approved the manuscript.
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Liu, Z., Yang, S., Ma, J. et al. Design, synthesis, and antifungal activity of 14-aryloxy/amide substituted andrographolide derivatives. Sci Rep 15, 41906 (2025). https://doi.org/10.1038/s41598-025-25907-3
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DOI: https://doi.org/10.1038/s41598-025-25907-3
