Novel 4-Chromanone-Derived Compounds as Plant Immunity Inducers against CMV Disease in Passiflora spp. (Passion Fruit)

This study involved the design and synthesis of a series of novel 4-chromanone-derived compounds. Their in vivo anti-cucumber mosaic virus (CMV) activity in field trials against CMV disease in Passiflora spp. was then assessed. Bioassay results demonstrated that compounds 7c and 7g exhibited remarkable curative effects and protection against CMV, with inhibition rates of 57.69% and 51.73% and 56.13% and 52.39%, respectively, surpassing those of dufulin and comparable to ningnanmycin. Field trials results indicated that compound 7c displayed significant efficacy against CMV disease in Passiflora spp. (passion fruit) after the third spraying at a concentration of 200 mg/L, with a relative control efficiency of 47.49%, surpassing that of dufulin and comparable to ningnanmycin. Meanwhile, nutritional quality test results revealed that compound 7c effectively enhanced the disease resistance of Passiflora spp., as evidenced by significant increases in soluble protein, soluble sugar, total phenol, and chlorophyll contents in Passiflora spp. leaves as well as improved the flavor and taste of Passiflora spp. fruits, as demonstrated by notable increases in soluble protein, soluble sugar, soluble solid, and vitamin C contents in Passiflora spp. fruits. Additionally, a transcriptome analysis revealed that compound 7c primarily targeted the abscisic acid (ABA) signaling pathway, a crucial plant hormone signal transduction pathway, thereby augmenting resistance against CMV disease in Passiflora spp. Therefore, this study demonstrates the potential application of these novel 4-chromanone-derived compounds as effective inducers of plant immunity for controlling CMV disease in Passiflora spp. in the coming decades.

Keywords: 4-chromanone; plant immune inducer; Passiflora spp.; anti-CMV activity; ABA signaling pathway

Passiflora spp. (passion fruit) is highly nutritious and possesses an exquisite sweet taste, making it widely cherished by the majority of consumers [[1]]. However, with the extensive cultivation of passion fruit in China, diseases have proliferated across all provinces due to the introduction, sale, and propagation of infected seedlings [[2]]. Among these diseases, cucumber mosaic virus (CMV) disease stands out as one of the primary viral infections affecting passion fruit plants [[2]]. This disease significantly diminishes both production and quality within the Passiflora spp. industry. To date, a wide variety of commercial antiviral agents targeting Passiflora spp. CMV disease have been reported on the market including chitosan oligosaccharide, dufulin, and ningnanmycin [[4]]. While traditional chemical antiviral agents have proven effective in treating Passiflora spp. CMV disease, their usage also raises numerous environmental concerns [[5]]. Therefore, there is an urgent need to focus on developing new innovative and promising antiviral agents for Passiflora spp. CMV disease in the coming decades.

The utilization of natural product pesticides for plant disease management represents a pioneering approach toward achieving sustainable agricultural development in the 21st century and beyond [[6]]. Rich structures, target species specificity, unique modes of action, and biodegradability make natural products an inspiring source for the discovery of lead compounds in pesticides [[7]]. The exploration of novel active components and the development of new pesticides through structural modifications of natural products are crucial strategies [[8]]. Chromone, a botanical active component, and its derivatives are widely distributed in various plant parts such as roots, flowers, pericarps, and stems, exhibiting a broad spectrum of biological properties including antifungal, antibacterial, antiviral, and anticancer properties [[9], [11], [13], [15]]. 4-Chromanone, which belongs to the chromone compound family, exhibits a wide range of significant biological and pharmaceutical activities, including anticancer, antioxidant, antifungal, and antibacterial activities [[17]]. Numerous studies have demonstrated that structural alterations in 4-chromanone, particularly at the 2 or 3 position, offer significant diversity which facilitates the development of novel active molecules [[18], [20]]. Meanwhile, in our previous work, a series of novel 4-chromanone derivatives (Figure 1) were synthesized and employed for the management of plant bacterial and fungal diseases; our bioassay screening results indicated that the target compounds exhibited moderate-to-potent antibacterial activities against Xanthomonas axonopodis pv. citri and Xanthomonas oryzae pv. oryzicolaby; however, they demonstrated weaker inhibitory effects on Mucor bainieri, Mucor fragilis, and Trichoderma atroviride [[22]].

To further expand upon our previous investigation and develop novel lead compounds with enhanced bioactivity, the objective of this study was to substitute a thioether group with a sulfone group in order to synthesize novel 4-chromanone-derived compounds containing a sulfone moiety (Figure 1). Subsequently, these compounds were subjected to laboratory antiviral activity assays and field trial tests against Passiflora spp. CMV disease.

The synthesis of compounds 7a–7o, as depicted in Figure 2, was accomplished through a five-step reaction route involving substitution, Michael addition, cyclization, condensation, and oxidation reactions, following established protocols [[22]]. The structures of the target compounds 7a–7o were confirmed through analyses using 1H NMR, 13C NMR, and HRMS techniques. The representative data for the target compounds 7a–7o are provided below. As an illustration, the integration of 1H NMR spectra confirms the presence of hydrogen atoms in compound 7c, which is consistent with the proposed structure. A double-doublet (dd) peak at 5.59 ppm and two dd peaks at 3.22 and 3.16 ppm were associated with the presence of OCH and CH2 in the 4-chromanone group, respectively. Similar results were also reported in our previous study [[22]].

Data for 6-fluoro-N-(5-(methylsulfonyl)-1,3,4-thiadiazol-2-yl)-4-oxochromane-2-carboxamide (7a). Yellow solid, mp 226–228 °C, yield 52%; 1H NMR (400 MHz, DMSO-d6, ppm) δ: 13.81 (s, 1H, CONH), 7.54–7.45 (m, 2H, Ar-H), 7.26 (dd, J = 8.0, 4.0 Hz, 1H, Ar-H), 5.59 (dd, J = 8.0, 4.0 Hz, 1H, OCH), 3.56 (s, 3H, CH3), 3.22 (dd, J = 16.0, 4.0 Hz, 1H, CH2), 3.16 (dd, J = 16.0, 8.0 Hz, 1H, CH2); 13C NMR (100 MHz, DMSO-d6, ppm) δ: 189.4 (C=O), 168.6 (C=O), 163.1 (Ar-C–F, d, J = 102.0 Hz), 158.5 (Ar-C), 156.2 (Ar-C, d, J = 10.0 Hz), 124.3 (Ar-C, d, J = 25.0 Hz), 121.8 (Ar-C), 120.8 (Ar-C, d, J = 7.0 Hz), 111.6 (Ar-C), 111.4 (Ar-C), 76.0 (CH2), 43.8 (OCH), 38.5 (CH3); HRMS (ESI) [M + Na]+ calcd for C13H10FN3O5S2: 393.99381, found 393.99308.

Data for N-(5-(ethylsulfonyl)-1,3,4-thiadiazol-2-yl)-6-fluoro-4-oxochromane-2-carboxamide (7b). Yellow solid, mp 215–217 °C, yield 50%; 1H NMR (400 MHz, DMSO-d6, ppm) δ: 13.81 (s, 1H, CONH), 7.55–7.45 (m, 2H, Ar-H), 7.26 (dd, J = 8.0, 4.0 Hz, 1H, Ar-H), 5.59 (dd, J = 8.0, 4.0 Hz, 1H, OCH), 3.66 (q, J = 8.0, 4.0 Hz, 2H, CH2CH3), 3.22 (dd, J = 16.0, 4.0 Hz, 1H, CH2), 3.16 (dd, J = 16.0, 8.0 Hz, 1H, CH2), 1.25 (t, J = 4.0 Hz, 3H, CH2CH3); 13C NMR (100 MHz, DMSO-d6, ppm) δ: 189.4 (C=O), 168.6 (C=O), 162.4 (Ar-C-F, d, J = 88.0 Hz), 158.5 (Ar-C), 156.3 (Ar-C), 124.3 (Ar-C, d, J = 25.0 Hz), 121.7 (Ar-C, d, J = 6.0 Hz), 120.7 (Ar-C, d, J = 8.0 Hz), 111.6 (Ar-C), 111.4 (Ar-C), 76.0 (CH2), 50.2 (OCH), 38.5 (CH2), 7.4 (CH3); HRMS (ESI) [M + Na]+ calcd for C14H12FN3O5S2: 408.00946, found 408.00934.

Data for 6-fluoro-4-oxo-N-(5-(propylsulfonyl)-1,3,4-thiadiazol-2-yl)chromane-2-carboxamide (7c). Yellow solid, mp 220–221 °C, yield 47%; 1H NMR (400 MHz, DMSO-d6, ppm) δ: 13.81 (s, 1H, CONH), 7.55–7.45 (m, 2H, Ar-H), 7.26 (dd, J = 8.0, 4.0 Hz, 1H, Ar-H), 5.59 (dd, J = 8.0, 4.0 Hz, 1H, OCH), 3.64 (t, J = 8.0 Hz, 2H, CH2CH2CH3), 3.22 (dd, J = 16.0, 4.0 Hz, 1H, CH2), 3.16 (dd, J = 16.0, 8.0 Hz, 1H, CH2), 1.76–1.67 (m, 2H, CH2CH2CH3), 0.96 (t, J = 4.0 Hz, 3H, CH2CH2CH3); 13C NMR (100 MHz, DMSO-d6, ppm) δ: 189.4 (C=O), 168.6 (C=O), 162.8 (Ar-C), 162.4 (Ar-C), 158.5 (Ar-C), 156.3 (Ar-C), 156.2 (Ar-C, d, J = 13.0 Hz), 124.3 (Ar-C, d, J = 24.0 Hz), 121.7 (Ar-C), 120.7 (Ar-C), 111.6 (Ar-C), 111.4 (Ar-C), 76.0 (CH2), 56.8 (OCH), 38.5 (CH2), 16.5 (CH2), 12.9 (CH3); HRMS (ESI) [M + Na]+ calcd for C15H14FN3O5S2: 422.02511, found 422.02494.

Data for N-(5-(benzylsulfonyl)-1,3,4-thiadiazol-2-yl)-6-fluoro-4-oxochromane-2-carboxamide (7d). White solid, mp 245–246 °C, yield 60%; 1H NMR (400 MHz, DMSO-d6, ppm) δ: 13.78 (s, 1H, CONH), 7.53–7.45 (m, 2H, Ar-H), 7.35–7.34 (m, 3H, Ar-H), 7.29–7.23 (m, 3H, Ar-H), 5.56 (dd, J = 8.0, 4.0 Hz, 1H, OCH), 5.05 (s, 2H, SO2CH2), 3.19 (dd, J = 16.0, 4.0 Hz, 1H, CH2), 3.14 (dd, J = 16.0, 8.0 Hz, 1H, CH2); 13C NMR (100 MHz, DMSO-d6, ppm) δ: 189.4 (C=O), 170.4 (C=O), 168.6 (Ar-C), 162.4 (Ar-C-F, d, J = 123.0 Hz), 158.6 (Ar-C), 156.6 (Ar-C), 131.8 (Ar-C), 129.2 (Ar-C, d, J = 33.0 Hz), 127.5 (Ar-C), 124.3 (Ar-C, d, J = 25.0 Hz), 121.8 (Ar-C), 121.7 (Ar-C), 120.9 (Ar-C), 120.8 (Ar-C), 120.7 (Ar-C), 111.6 (Ar-C), 111.4 (Ar-C), 76.1 (CH2), 61.3 (OCH), 38.5 (CH2); HRMS (ESI) [M + Na]+ calcd for C19H14FN3O5S2: 470.02511, found 470.02449.

Data for 6-fluoro-N-(5-((4-fluorobenzyl)sulfonyl)-1,3,4-thiadiazol-2-yl)-4-oxochromane-2-carboxamide (7e). White solid, mp 254–256 °C, yield 40%; 1H NMR (400 MHz, DMSO-d6, ppm) δ: 13.79 (s, 1H, CONH), 7.53–7.45 (m, 2H, Ar-H), 7.35–7.32 (m, 2H, Ar-H), 7.27–7.17 (m, 3H, Ar-H), 5.57 (dd, J = 8.0, 4.0 Hz, 1H, OCH), 5.08 (s, 2H, SO2CH2), 3.22–3.12 (m, 2H, CH2); 13C NMR (100 MHz, DMSO-d6, ppm) δ: 189.4 (C=O), 168.6 (C=O), 162.4 (Ar-C-F, d, J = 139.0 Hz), 161.6 (Ar-C), 156.3 (Ar-C), 134.0 (Ar-C, d, J = 9.0 Hz), 124.4 (Ar-C, d, J = 25.0 Hz), 123.9 (Ar-C), 123.9 (Ar-C), 121.8 (Ar-C), 121.7 (Ar-C, d, J = 7.0 Hz), 120.7 (Ar-C, d, J = 7.0 Hz), 116.2 (Ar-C), 116.0 (Ar-C), 111.6 (Ar-C), 111.4 (Ar-C), 76.1 (CH2), 60.3 (OCH), 38.5 (CH2); HRMS (ESI) [M + Na]+ calcd for C19H13F2N3O5S2: 488.01569, found 488.01468.

Data for 6-chloro-N-(5-(methylsulfonyl)-1,3,4-thiadiazol-2-yl)-4-oxochromane-2-carboxamide (7f). Yellow solid, mp 266–268 °C, yield 62%; 1H NMR (400 MHz, DMSO-d6, ppm) δ: 13.82 (s, 1H, CONH), 7.70–7.66 (m, 2H, Ar-H), 7.26–7.23 (m, 1H, Ar-H), 5.62 (dd, J = 8.0, 4.0 Hz, 1H, OCH), 3.55 (s, 3H, CH3), 3.24 (dd, J = 16.0, 4.0 Hz, 1H, CH2), 3.17 (dd, J = 16.0, 8.0 Hz, 1H, CH2); 13C NMR (100 MHz, DMSO-d6, ppm) δ: 189.0 (C=O), 168.5 (C=O), 163.6 (Ar-C), 162.6 (Ar-C), 158.6 (Ar-C), 136.5 (Ar-C), 126.7 (Ar-C), 125.6 (Ar-C), 122.2 (Ar-C), 120.9 (Ar-C), 76.0 (CH2), 43.8 (OCH), 38.5 (CH3); HRMS (ESI) [M + Na]+ calcd for C13H10ClN3O5S2: 409.96426, found 409.96430.

Data for 6-chloro-N-(5-(ethylsulfonyl)-1,3,4-thiadiazol-2-yl)-4-oxochromane-2-carboxamide (7g). Yellow solid, mp 249–251 °C, yield 54%; 1H NMR (400 MHz, DMSO-d6, ppm) δ: 13.82 (s, 1H, CONH), 7.70–7.66 (m, 2H, Ar-H), 7.26–7.23 (m, 1H, Ar-H), 5.62 (dd, J = 8.0, 4.0 Hz, 1H, OCH), 3.66 (q, J = 16.0, 8.0 Hz, 2H, CH2CH3), 3.24 (dd, J = 16.0, 4.0 Hz, 1H, CH2), 3.17 (dd, J = 16.0, 8.0 Hz, 1H, CH2), 1.25 (t, J = 4.0 Hz, 3H, CH2CH3); 13C NMR (100 MHz, DMSO-d6, ppm) δ: 189.0 (C=O), 168.6 (C=O), 162.9 (Ar-C), 162.0 (Ar-C), 158.6 (Ar-C), 136.5 (Ar-C), 126.7 (Ar-C), 125.6 (Ar-C), 122.2 (Ar-C), 120.9 (Ar-C), 76.1 (CH2), 50.2 (OCH), 38.5 (CH2), 7.4 (CH3); HRMS (ESI) [M + Na]+ calcd for C14H12ClN3O5S2: 423.97991, found 423.97918.

Data for 6-chloro-4-oxo-N-(5-(propylsulfonyl)-1,3,4-thiadiazol-2-yl)chromane-2-carboxamide (7h). Yellow solid, mp 183–184 °C, yield 50%; 1H NMR (400 MHz, DMSO-d6, ppm) δ: 13.81 (s, 1H, CONH), 7.70–7.66 (m, 2H, Ar-H), 7.26–7.23 (m, 1H, Ar-H), 5.62 (dd, J = 8.0, 4.0 Hz, 1H, OCH), 3.63 (t, J = 8.0 Hz, 2H, CH2CH2CH3), 3.24 (dd, J = 16.0, 4.0 Hz, 1H, CH2), 3.17 (dd, J = 16.0, 8.0 Hz, 1H, CH2), 1.76–1.66 (m, 2H, CH2CH2CH3), 0.96 (t, J = 4.0 Hz, 3H, CH2CH2CH3); 13C NMR (100 MHz, DMSO-d6, ppm) δ: 189.0 (C=O), 168.5 (C=O), 162.8 (Ar-C), 162.5 (Ar-C), 158.6 (Ar-C), 136.5 (Ar-C), 126.7 (Ar-C), 125.6 (Ar-C), 122.2 (Ar-C), 120.9 (Ar-C), 76.0 (CH2), 56.8 (OCH), 38.5 (CH2), 16.5 (CH2), 12.9 (CH3); HRMS (ESI) [M + Na]+ calcd for C15H14ClN3O5S2: 437.99556, found 437.19301.

Data for N-(5-(benzylsulfonyl)-1,3,4-thiadiazol-2-yl)-6-chloro-4-oxochromane-2-carboxamide (7i). Yellow solid, mp 244–245 °C, yield 45%; 1H NMR (400 MHz, DMSO-d6, ppm) δ: 13.79 (s, 1H, CONH), 7.69–7.66 (m, 2H, Ar-H), 7.36–7.22 (m, 6H, Ar-H), 5.59 (dd, J = 8.0, 4.0 Hz, 1H, OCH), 5.05 (s, 2H, SO2CH2), 3.21 (dd, J = 16.0, 4.0 Hz, 1H, CH2), 3.15 (dd, J = 16.0, 8.0 Hz, 1H, CH2); 13C NMR (100 MHz, DMSO-d6, ppm) δ: 189.0 (C=O), 168.5 (C=O), 163.0 (Ar-C), 161.8 (Ar-C), 158.6 (Ar-C), 136.5 (Ar-C), 131.8 (Ar-C), 129.4 (Ar-C), 129.1 (Ar-C), 127.5 (Ar-C), 126.7 (Ar-C), 125.6 (Ar-C), 122.2 (Ar-C), 120.9 (Ar-C), 76.0 (CH2), 61.3 (OCH), 38.5 (CH2); HRMS (ESI) [M + Na]+ calcd for C19H14ClN3O5S2: 461.99906, found 461.99923.

Data for 6-chloro-N-(5-((4-fluorobenzyl)sulfonyl)-1,3,4-thiadiazol-2-yl)-4-oxochromane-2-carboxamide (7j). White solid, mp 256–257 °C, yield 40%; 1H NMR (400 MHz, DMSO-d6, ppm) δ: 13.80 (s, 1H, CONH), 7.69–7.66 (m, 2H, Ar-H), 7.35–7.32 (m, 2H, Ar-H), 7.26–7.17 (m, 3H, Ar-H), 5.60 (dd, J = 8.0, 4.0 Hz, 1H, CH), 5.08 (s, 2H, SO2CH2), 3.22 (dd, J = 16.0, 4.0 Hz, 1H, CH2), 3.16 (dd, J = 16.0, 8.0 Hz, 1H, CH2); 13C NMR (100 MHz, DMSO-d6, ppm) δ: 189.0 (C=O), 168.5 (C=O), 163.6 (Ar-C-F, d, J = 103 Hz), 161.6 (Ar-C), 158.6 (Ar-C), 136.5 (Ar-C), 134.0 (Ar-C, d, J = 9.0 Hz), 126.7 (Ar-C), 125.6 (Ar-C), 123.9 (Ar-C), 122.2 (Ar-C), 120.9 (Ar-C), 116.2 (Ar-C), 116.0 (Ar-C), 76.1 (CH2), 60.3 (OCH), 38.5 (CH2); HRMS (ESI) [M + Na]+ calcd for C19H13ClFN3O5S2: 503.98614, found 503.98554.

Data for 6-methyl-N-(5-(methylsulfonyl)-1,3,4-thiadiazol-2-yl)-4-oxochromane-2-carboxamide. (7k). White solid, mp 240–242 °C, yield 48%; 1H NMR (400 MHz, DMSO-d6, ppm) δ: 13.77 (s, 1H, CONH), 7.55 (d, J = 4.0 Hz, 1H, Ar-H), 7.44 (dd, J = 8.0 Hz, 4.0 Hz, 1H, Ar-H), 7.08 (d, J = 8.0 Hz, Ar-H), 5.54 (dd, J = 8.0 Hz, 4.0 Hz, 1H, CH), 3.55 (s, 3H, CH3), 3.16 (dd, J = 16.0, 4.0 Hz, 1H, CH2), 3.12 (dd, J = 16.0, 4.0 Hz, 1H, CH2), 2.28 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6, ppm) δ: 190.0 (C=O), 168.9 (C=O), 163.5 (Ar-C), 162.6 (Ar-C), 157.9 (Ar-C), 137.8 (Ar-C), 131.7 (Ar-C), 126.2 (Ar-C), 120.8 (Ar-C), 118.3 (Ar-C), 75.9 (CH2), 43.8 (OCH), 38.8 (CH3), 20.4 (CH3); HRMS (ESI) [M + Na]+ calcd for C14H13N3O5S2: 390.018883, found 390.01893.

Data for N-(5-(ethylsulfonyl)-1,3,4-thiadiazol-2-yl)-6-methyl-4-oxochromane-2-carboxamide (7l). White solid, mp 206–207 °C, yield 51%; 1H NMR (400 MHz, DMSO-d6, ppm) δ: 13.77 (s, 1H, CONH), 7.55 (s, 1H, Ar-H), 7.44 (dd, J = 8.0, 4.0 Hz, 1H, Ar-H), 7.08 (d, J = 8.0 Hz, 1H, Ar-H), 5.53 (dd, J = 8.0, 4.0 Hz, 1H, OCH), 3.65 (q, J = 16.0 Hz, 8.0 Hz, 2H, CH2CH3), 3.16 (dd, J = 16.0, 4.0 Hz, 1H, CH2), 3.11 (dd, J = 16.0, 8.0 Hz, 1H, CH2), 2.28 (s, 3H, CH3), 1.24 (t, J = 4.0 Hz, 3H, CH2CH3); 13C NMR (100 MHz, DMSO-d6, ppm) δ: 190.0 (C=O), 168.9 (C=O), 162.9 (Ar-C), 161.9 (Ar-C), 158.0 (Ar-C), 137.8 (Ar-C), 131.7 (Ar-C), 126.2 (Ar-C), 120.8 (Ar-C), 118.3 (Ar-C), 75.9 (CH2), 50.2 (OCH), 38.8 (CH2), 20.4 (CH3), 7.4 (CH3); HRMS (ESI) [M + Na]+ calcd for C15H15N3O5S2: 404.03453, found 404.03410.

Data for 6-methyl-4-oxo-N-(5-(propylsulfonyl)-1,3,4-thiadiazol-2-yl)chromane-2-carboxamide (7m). White solid, mp 189–190 °C, yield 51%; 1H NMR (400 MHz, DMSO-d6, ppm) δ: 13.76 (s, 1H, CONH), 7.44 (d, J = 8.0 Hz, 1H, Ar-H), 7.08 (d, J = 8.0 Hz, 1H, Ar-H), 5.53 (dd, J = 8.0, 4.0 Hz, 1H, OCH), 3.63 (t, J = 8.0 Hz, 2H, CH2CH2CH3), 3.16 (dd, J = 16.0, 4.0 Hz, 1H, CH2), 3.11 (dd, J = 16.0, 8.0 Hz, 1H, CH2), 2.28 (s, 3H, CH3), 1.76–1.66 (m, 2H, CH2CH2CH3), 0.96 (t, J = 8.0 Hz, 3H, CH2CH2CH3); 13C NMR (100 MHz, DMSO-d6, ppm) δ: 190.0 (C=O), 168.9 (C=O), 162.9 (Ar-C), 162.4 (Ar-C), 158.0 (Ar-C), 137.8 (Ar-C), 131.7 (Ar-C), 126.2 (Ar-C), 120.8 (Ar-C), 118.3 (Ar-C), 75.9 (CH2), 56.8 (OCH), 38.8 (CH2), 20.4 (CH3), 16.5 (CH2), 12.9 (CH3); HRMS (ESI) [M + Na]+ calcd for C16H17N3O5S2: 418.05018, found 418.04983.

Data for N-(5-(benzylsulfonyl)-1,3,4-thiadiazol-2-yl)-6-methyl-4-oxochromane-2-carboxamide (7n). White solid, mp 233–235 °C, yield 41%; 1H NMR (400 MHz, DMSO-d6, ppm) δ: 13.75 (s, 1H, CONH), 7.55 (d, J = 4.0 Hz, 1H, Ar-H), 7.45 (dd, J = 8.0, 4.0 Hz, 1H, Ar-H), 7.35–7.26 (m, 5H, Ar-H), 7.08 (d, J = 8.0 Hz, 1H, Ar-H). 5.05 (s, 2H, SO2CH2), 3.16–3.06 (m, 2H, CH2), 2.28 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6, ppm) δ: 190.0 (C=O), 168.8 (C=O), 163.1 (Ar-C), 161.7 (Ar-C), 157.9 (Ar-C), 137.8 (Ar-C), 131.8 (Ar-C), 129.4 (Ar-C), 129.1 (Ar-C), 127.5 (Ar-C), 126.2 (Ar-C), 120.8 (Ar-C), 118.3 (Ar-C), 75.9 (CH2), 61.3 (OCH), 38.8 (CH2), 20.4 (CH3); HRMS (ESI) [M + Na]+ calcd for C20H17N3O5S2: 466.05018, found 466.04975.

Data for N-(5-((4-fluorobenzyl)sulfonyl)-1,3,4-thiadiazol-2-yl)-6-methyl-4-oxochromane-2-carboxamide (7o). White solid, mp 235–237 °C, yield 41%; 1H NMR (400 MHz, DMSO-d6, ppm) δ: 13.77 (s, 1H, CONH), 7.55 (d, J = 4.0 Hz, 1H, Ar-H), 7.45 (dd, J = 8.0, 4.0 Hz, 1H, Ar-H), 7.35–7.31 (m, 2H, Ar-H), 7.23–7.17 (m, 2H, Ar-H), 7.08 (d, J = 8.0 Hz, 1H, Ar-H). 5.51 (dd, J = 8.0, 4.0 Hz, 1H, OCH), 5.07 (s, 2H, SO2CH2), 3.17–3.06 (m, 2H, CH2), 2.28 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6, ppm) δ: 190.0 (C=O), 168.8 (C=O), 162.3 (Ar-C-F, d, J = 153.0 Hz), 157.9 (Ar-C), 137.8 (Ar-C), 134.0 (Ar-C, d, J = 9.0 Hz), 131.7 (Ar-C), 126.2 (Ar-C), 123.9 (Ar-C), 120.8 (Ar-C), 118.3 (Ar-C), 116.2 (Ar-C), 116.0 (Ar-C), 75.9 (CH2), 60.3 (OCH), 38.8 (CH2), 20.4 (CH3); HRMS (ESI) [M + Na]+ calcd for C20H16FN3O5S2: 484.04076, found 484.04051.

In this study, the anti-CMV activity of target compounds 7a–7o was evaluated at a concentration of 500 mg/L, and the results are recorded in Table 1. The data presented in Table 1 demonstrate that these compounds exhibited moderate to good protection and curative effects against CMV. Notably, compounds 7c and 7g exhibited remarkable protection activity against CMV, with inhibition rates of 57.69% and 56.13%, respectively, surpassing those of dufulin (42.08%) and ningnanmycin (50.13%). Moreover, compounds 7c and 7g demonstrated excellent curative activity against CMV, with inhibition rates of 51.73% and 52.39%, respectively, surpassing those of dufulin (40.78%) and comparable to ningnanmycin (51.35%). A structure–activity relationship (SAR) analysis showed that when R1 was a –F group, it was observed that the compound with a –CH2CH2CH3 group at the R2 substituent group exhibited favorable in vivo protection and curative activities in the following specific orders: 7c > 7a and 7c > 7b; when R1 was a –Cl group, the compound with a –CH2CH3 group at the R2 substituent group showed favorable in vivo protection and curative activities in the following specific orders: 7g > 7f and 7g > 7h.

In order to assess the control efficiency of compounds 7c and 7g against Passiflora spp. CMV disease, a preliminary field efficacy study was conducted at a concentration of 200 mg/L. According to the results presented in Table 2, after the first and second sprayings, both compounds 7c and 7g demonstrated effectiveness in reducing Passiflora spp. CMV disease at a concentration of 200 mg/L, exhibiting comparable relative control efficiencies to those of dufulin and ningnanmycin. Furthermore, after the third spraying, compound 7c exhibited significant efficacy with a relative control efficiency of 47.49% at a concentration of 200 mg/L, surpassing that of dufulin (28.03%) and comparable to ningnanmycin (44.17%). However, following the third spraying, compound 7g demonstrated significant efficacy with a relative control efficiency of 34.82% at a concentration of 200 mg/L, surpassing that of dufulin (28.03%) and falling below that of ningnanmycin (44.17%).

The presence of soluble proteins in plants is essential for enhancing cellular water retention, providing cellular protection, and bolstering plant disease resistance [[24]]. The role of soluble sugars in the life cycle of plants is pivotal as they not only serve as a primary source of energy and metabolic intermediates for plant growth and development but also play a crucial role as signaling molecules [[25]]. Phenol compounds are classified as polyhydroxyl secondary metabolites that exhibit notable antioxidant activity and have been found to be closely associated with enhancing plant resistance against diseases [[26]]. The content of chlorophyll, the photosynthetic pigment in green plants, is intricately associated with photosynthesis and proliferation, as well as factors that hinder chlorophyll synthesis, resulting in leaf chlorosis to enhance photosynthesis and strengthen plant defense against microbial infections [[27]]. The results depicted in Figure 3 demonstrate that compound 7c could effectively enhance resistance against Passiflora spp. CMV disease by significantly increasing the soluble protein, soluble sugar, total phenol, and chlorophyll contents.

In recent years, with the growing emphasis on personal health, consumers have increasingly demanded fruits with higher nutritional quality. It is widely acknowledged that key indicators of nutritional quality, such as soluble protein, soluble sugar, soluble solid, and vitamin C contents, play a pivotal role in determining the flavor and taste of fruits while also influencing consumer purchasing decisions [[28]]. Soluble protein is of paramount importance to fruit growers as it plays a crucial role in enhancing the flavor and taste of fruits, thereby stimulating consumer purchases [[29]]. Soluble sugar, as one of the primary determinants influencing fruit quality, exerts a direct impact on the sweetness of fresh fruits while also serving as a precursor for synthesizing other compounds related to quality, including organic acids, anthocyanins, and aroma compounds [[30]]. Soluble solids, a crucial parameter in the fruit ripening process and economic benefits, are closely related to fruit flavor and optimal harvest time [[31]]. The nutritional component vitamin C is a paramount vitamin for human nutrition that is sourced from fruits [[32]]. The results depicted in Figure 4 demonstrate that treatment with compound 7c significantly enhances the contents of soluble protein, soluble sugar, soluble solid, and vitamin C in Passiflora spp. fruits, which not only improves the nutritional quality but also enhances the flavor and taste of Passiflora spp. fruits.

The cDNA libraries of Passiflora spp. leaves treated with compound 7c and the CK group were analyzed using the Illumina HiSeq platform to investigate gene expression information at the transcriptome level [[33]]. The transcriptome sequencing data were evaluated for a valid ratio, Q20 and Q30 base proportions, and GC content, with summarized results presented in Table 3. Based on the findings from Table 3, it can be concluded that the sequencing of both samples exhibited exceptional quality, with valid ratios > 98%, proportions of Q20 bases > 98%, proportions of Q30 bases > 93%, and GC contents > 43%, indicating suitability for a subsequent bioinformatics analysis [[34]].

The results, as depicted in Figure 5 and Table S1, demonstrate that a total of 652 DEGs were identified, with 246 upregulated and 406 downregulated genes observed in the compound 7c treatment group compared to the CK group.

Meanwhile, a functional annotation of these DEGs was performed using a GO analysis to gain insights into their biological significance. As depicted in Figure 6 and Table S2, the GO analysis revealed the enrichment of 150 GO functions by the DEGs, encompassing 77 BP (cellular process (GO:0009987), metabolic process (GO:0008152), biological regulation (GO:0065007), the regulation of biological process (GO:0050789), response to stimulus (GO:0050896), etc.), 43 CC (cell (GO:0005623), cell part (GO:0044464), organelle (GO:0043226), membrane (GO:0016020), organelle part (GO:0044422). etc.), and 30 MF (binding (GO:0005488), catalytic activity (GO:0003824), transporter activity (GO:0005215), molecular function regulator (GO:0000988), signal transducer activity (GO:0004871), etc.) categories.

In addition, Figure 7 and Table S3 show that the enriched KEGG pathways of the DEGs were photosynthesis (ko00195), photosynthesis antenna proteins (ko00196), porphyrin and chlorophyll metabolism (ko00860), carbon fixation in photosynthetic organisms (ko00710), glyoxylate and dicarboxylate metabolism (ko00630), carbon metabolism (ko01200), gap junction (ko04540), apoptosis (ko04210), glycine, serine, and threonine metabolism (ko00260), the metabolism of xenobiotics by cytochrome P450 (ko00980), drug metabolism—cytochrome P450 (ko00982), phagosome (ko04145), arginine and proline metabolism (ko00330), glutathione metabolism (ko00480), methane metabolism (ko00680), plant hormone signal transduction (ko04075), ascorbate and aldarate metabolism (ko00053), carotenoid biosynthesis (ko00906), etc.

In recent years, it has become evident that the interplay among various plant hormones regulates both plant growth and disease resistance. Additionally, plant hormone signal transduction may also play a role in the mechanisms underlying diverse pesticide applications and disease control techniques employed for crop protection [[35]]. The abscisic acid (ABA) signaling pathway is a pivotal plant hormone that regulates the response to abiotic stress and significantly impacts plants' ability to defend against various pathogens [[36]]. The PYL and PP2C proteins, which are essential components of the ABA receptor-coupled core signaling pathway, were found to be significantly enriched in the plant hormone signal transduction pathway and were specifically annotated within the ABA signaling pathway [[37]]. In this study, a transcriptome analysis revealed that compound 7c elicited an upregulation of PYL gene expression and a downregulation of PP2C gene expression. Therefore, it is speculated that 4-chromanone-derived compounds could mediate the ABA signaling pathway, thereby enhancing resistance to Passiflora spp. CMV disease (Figure 8). Numerous studies have demonstrated that when external stress triggers the production or release of ABA in plants, ABA binds to the PYR/PYL receptor, forming a complex that inhibits PP2C to facilitate the dissociation of SnRK2 from the PP2C complex, allowing it to phosphorylate downstream transcription factors and activate the ABA signaling pathway [[38], [40]].

Intermediates 2–6 were prepared using 4-substituted phenol as the starting material, following the established methods described in Figure 2 [[22]].

As depicted in Figure 2, the target compounds 7a–7o were prepared according to the reported method [[23]]. Intermediate 6 (0.01 mol), acetic acid (10 mL), and ammonium molybdate (0.5 mmol), dissolved in 30% H2O2 (0.03 mol), were mixed in a 25 mL round-bottomed flask, followed by a reaction at room temperature for 1–4 h. Subsequently, the resulting mixture was poured into 50 mL of distilled water. Following filtration, the crude products were subjected to recrystallization with ethanol in order to obtain the pure target compounds 7a−7o.

The curative activities and protection of the target compounds 7a–7o against CMV in Chenopodium amaranticolor (C. amaranticolor) plants were assessed using the half-leaf method at a concentration of 500 mg/L, and the inhibition rate I (%) of the anti-CMV activity was calculated using the established method [[41]].

(1) $$\mathrm{Inhibition}\mathrm{rate}\mathrm{I}(\%)=\frac{\mathrm{Average}\mathrm{local}\mathrm{lesion}\mathrm{number}\mathrm{of}\mathrm{control}\mathrm{group}-\mathrm{Average}\mathrm{local}\mathrm{lesion}\mathrm{number}\mathrm{smeared}\mathrm{with}\mathrm{compounds}}{\mathrm{Average}\mathrm{local}\mathrm{lesion}\mathrm{number}\mathrm{of}\mathrm{control}}\times 100\%$$

Leaves were selected from plants of the same age belonging to C. amaranticolor species. Subsequently, whole leaves were inoculated with crude CMV virus at a concentration of 6 × 10−3 mg/mL on both sides after previously treatment with silicon carbide. After 0.5 h, the leaves were washed and dried. Then, the target compound solutions were applied to the left side of the leaves while solvents were applied to the right side as a control measure. The number of local lesions was recorded 3 to 4 days post inoculation. Three replications were conducted for each compound in order to ensure result reliability.

Leaves of C. amaranticolor plants of the same age were selected. The compound solutions were applied to the left side of the leaves, while solvents were applied to the right side as a control. After 12 h, crude CMV (concentration of 6 × 10−3 mg/mL) was inoculated on both sides of the leaves at equal concentrations after prior scattering with silicon carbide. Following a 0.5 h interval, the leaves were rinsed with water and subsequently dried. The number of localized lesions was recorded between 3 and 4 days post inoculation. Each experiment involving each compound was conducted in triplicate to ensure result reliability.

In 2023, field trials were conducted in Zhenning City (Guizhou Province, China) to evaluate the efficacy of compounds 7c and 7g against Passiflora spp. CMV disease at a concentration of 200 mg/L. Sterile distilled water was used as the CK group, while dufulin and ningnanmycin were employed as positive controls at the same concentration. Each group was set up with 4 plots, and the 5 groups were set up with a total of 20 plots, each of which was 15 m2, and the total land area was 300 m2. A total of three sprayings were conducted, with an interval of seven days between each spraying. The incidence in the community was assessed prior to spraying and then on the seventh day after each spraying. Three plants were randomly selected for investigation, and approximately 15 leaves were observed per plant. The disease index of the leaves corresponding to each disease grade was recorded based on the grading standard for diseased leaves. The following formula was used to calculate the control efficiencies of compounds 7c and 7g as well as dufulin and ningnanmycin against Passiflora spp. CMV disease on the 7th day after each of the three sprayings [[42]]. In the equation, C is the disease index of the CK group and T is the disease index of the treatment group. Each experiment was performed in triplicate to ensure result reliability.

(2) $$\mathrm{Control}\mathrm{efficiency}\mathrm{I}(\%)=\frac{\mathrm{C}-\mathrm{T}}{\mathrm{C}}\times 100\%$$

To investigate the impact of compound 7c on the nutritional composition of Passiflora spp. leaves, we determined the levels of soluble protein, soluble sugar, total phenol, and chlorophyll in Passiflora spp. leaves collected on the 7th day after the third spraying, using established methods [[43], [45]]. Additionally, we conducted an analysis of the levels of soluble protein, soluble sugar, soluble solid, and vitamin C in Passiflora spp. fruits collected on the 7th day after the third spraying, following established protocols [[43], [47]]. Each experiment was conducted in triplicate to ensure result reliability.

To accurately measure a specific quantity (0.1 g) of Passiflora spp. leaves or fruits in triplicate, pulverize them thoroughly with liquid nitrogen and transfer the ground material into a test tube. Add a precise volume of distilled water to the test tube and centrifuge it at 12,000 rpm and 4 °C for 10 min to collect the supernatant as the protein extract. Take an appropriate amount of the extract and mix it with 5 mL of Coomassie Brilliant Blue solution on a vortex oscillator for a complete reaction over a period of 2 min. Measure the absorbance at 595 nm to calculate the soluble protein content using a standard curve generated using bovine serum protein.

The Passiflora spp. leaves or fruits (0.1 g) were accurately weighed in triplicate and fully ground with a small amount of liquid nitrogen in a mortar. Subsequently, 2.5 mL of distilled water was added, followed by boiling and filtration to obtain the soluble sugar extract. A certain volume of the extract was transferred into a test tube, along with distilled water, an anthranone–ethyl acetate mixed solution, and concentrated sulfuric acid. The mixture was then boiled in water for 1 min and cooled to room temperature, and the absorbance at 630 nm was measured. Finally, the content of soluble sugar was determined using a standard curve constructed from a sucrose standard solution.

The leaves of Passiflora spp. (0.1 g) were collected in triplicate for each treatment and then sliced into small, uniform pieces using a hole puncher, carefully avoiding the midrib. Subsequently, 50 mg samples were placed in 5 mL of a cold solution consisting of a 2:1 mixture of 85% acetone and 85% ethanol (v/v). These samples were homogenized, incubated at 35 °C for 0.5 h, and finally centrifuged at 6500 rpm for 15 min. Absorbance spectra were recorded at wavelengths of 663 and 645 nm to determine chlorophyll a and chlorophyll b levels, respectively, with reference to a standard solution. Chlorophyll a, chlorophyll b, and total chlorophyll contents were calculated using established methods.

The Passiflora spp. leaves (0.1 g) were accurately weighed in triplicate and added to 2.5 mL of a hydrochloric acid–methanol solution for extraction, which lasted for 20 min. After centrifugation at 8000 rpm for 5 min, the resulting supernatant was collected as the total phenol extract. A certain amount of this extract was transferred into a 15 mL test tube, followed by the addition of 1 mL of a Folin–phenol reagent (diluted) and 2 mL of a sodium carbonate solution. The reaction occurred at room temperature for 30 min. Subsequently, the absorbance value was measured at a wavelength of 760 nm, and the content of total phenols was determined using a standard curve constructed with gallic acid.

Passiflora spp. fruit was accurately weighed (0.1 g) in triplicate and then finely ground in a mortar using an oxalate–EDTA solution. The resulting mixture was carefully transferred into a 100 mL volumetric bottle and filtered to remove any impurities, and a specific amount of passion fruit extract was absorbed. Subsequently, 1mL of metaphosphate–acetic acid solution and a 5% sulfuric acid solution were added to the extract, followed by thorough mixing. Finally, 4 mL of ammonium molybdate solution was added to achieve a constant volume of 50 mL. The absorbance at a 705 nm wavelength was measured, and the vitamin C content was calculated using a standard curve obtained from an ascorbic acid standard solution.

The Passiflora spp. fruits (0.1 g) should be accurately selected for a specific quality, followed by homogenizing the pulp using a homogenizer. The resulting mixture should then be filtered through gauze and analyzed for soluble solids using a handheld refractometer (PAL-BXIACID F5, ATAGO, Tokyo, Japan).

The leaves of Passiflora spp. (15 leaves per plant) were collected and stored in a foam box with liquid nitrogen before they were subjected to transcriptome sequencing. The transcriptome was sequenced by Hangzhou Lianchuan Biological Co., LTD (Hangzhou, China), using the Illumina HiSeq™ 2000 platform (Illumina Inc., San Diego, CA, USA). The raw transcriptome sequencing data have been deposited in the National Center for Biotechnology Information (NCBI) database under the project ID PRJNA1061236. To obtain high-quality reads, Cutadapt software (version: cutadapt-1.9.3) was employed to delete undetermined and low-quality bases [[49]]. Gene expression levels were determined based on mean FPKM values: FPKM < 1 indicated non-expression; 1 ≤ FPKM < 10 indicated low expression; and FPKM ≥ 10 indicated high expression [[50]]. Differentially expressed genes (DEGs) were identified using an R language package at a significance level of adjusted p < 0.05 and −log2FC > 1 [[51]]. Gene ontology (GO), including cellular components (CCs), biological processes (BPs), and molecular function (MF), as well as Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations for DEGs, were obtained from the GO database (http://www.geneontology.org/,23May2023) and KEGG pathway database (http://www.genome.jp/Pathway,23May2023) [[4], [52]].

The nutritional compositions of the Passiflora spp. leaves and fruits were subjected to a statistical analysis using a one-way analysis of variance (ANOVA), followed by the least significance difference test (LSD) in GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA, USA).

In conclusion, our study findings demonstrate that compound 7c effectively controls Passiflora spp. CMV disease and has the potential to be utilized as a plant immune inducer by primarily targeting the ABA signaling pathway to enhance resistance against Passiflora spp. CMV disease. Therefore, our study provides a theoretical basis for the utilization of 4-chromanone-derived compounds as plant immune inducers against Passiflora spp. CMV disease.

Graph: Figure 1 The design idea for the target compounds.

Graph: Figure 2 General protocols for the synthesis of target compounds 7a–7o.

Graph: Figure 3 Effect of compound 7c on the soluble protein (A), soluble sugar (B), total phenol (C), and chlorophyll (D) contents of Passiflora spp. leaves. The nutritional quality of Passiflora spp. leaves was significantly different among various lowercase letters at a significance level of p < 0.05. Error bars refer to mean ± SD (n = 3) values.

Graph: Figure 4 Effect of compound 7c on the soluble protein (A), soluble sugar (B), soluble solid (C), and vitamin C (D) contents of Passiflora spp. fruits. The nutritional quality of Passiflora spp. fruits was significantly different among various lowercase letters at a significance level of p < 0.05. Error bars refer to mean ± SD (n = 3) values.

MAP: Figure 5 Volcanic map of DEGs.

Graph: Figure 6 GO classification of DEGs.

Graph: Figure 7 Top thirty KEGG pathway enrichments of DEGs.

Graph: Figure 8 A model of the ABA signal transduction pathway in Passiflora spp. resistance to CMV disease. Supplementary Materials, compound 7c.

Table 1 In vivo protection and curative activities of the target compounds 7a–7o against CMV at a concentration of 500 mg/L.

Table 2 Field trials of compounds 7c and 7g against Passiflora spp. CMV disease at a concentration of 200 mg/L.

Table 3 A summary of the quality statistics results of the transcriptome sequencing data.

Methodology, T.W. (Tianli Wu) and L.X.; software, L.Y., T.W. (Tao Wang) and B.M.; data curation, T.W. (Tianli Wu) and L.X.; writing—original draft preparation, L.Y.; writing—review and editing, P.L.; project administration, L.Y. and P.L. All authors have read and agreed to the published version of the manuscript.

Not applicable.

Not applicable.

Data are contained within the article and Supplementary Materials.

The authors declare no conflicts of interest.

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/molecules29051045/s1, Table S1: All the identified EDGs; Table S2: All the GO classifications of the DEGs; Table S3. All the KEGG pathway enrichments of the DEGs.