Zhu et al. Chemistry Central Journal (2018) 12:111 https://doi.org/10.1186/s13065-018-0478-2 Chemistry Central Journal RESEARCH ARTICLE Open Access Design, synthesis and biological activity of hydroxybenzoic acid ester conjugates of phenazine‑1‑carboxylic acid Xiang Zhu1,2, Linhua Yu2, Min Zhang2, Zhihong Xu2, Zongli Yao2, Qinglai Wu2*, Xiaoying Du2,3* and Junkai Li1,2* Abstract We prepared 16 novel hydroxybenzoic acid ester conjugates of phenazine-1-carboxylic acid (PCA) and investigated their biological activity. Most of the synthesized conjugates displayed some level of fungicidal activities in vitro against fve phytopathogenic fungi. Nine conjugates 5b, 5c, 5d, 5e, 5h, 5i, 5m, 5n and 5o ­(EC50 between 3.2 μg/mL and 14.1 μg/mL) were more active than PCA ­(EC50 18.6 μg/mL) against Rhizoctonia solani. Especially conjugate 5c showed the higher fungicidal activity against Rhizoctonia solani which is 6.5-fold than PCA. And the results of the bioassay indicated that the fungicidal activity of conjugates was associated with their LogP, and the optimal LogP values of the more potent fungicidal activities within these conjugates ranged from 4.42 to 5.08. The systemic acquired resistance induced by PCA–SA ester conjugate 5c against rice sheath blight disease in rice seedlings was evaluated. The results revealed that PCA–SA ester conjugate 5c retained the resistance induction activity of SA against rice sheath blight. Keywords: Phenazine-1-carboxylic acid, Synthesis, Biological activity, Salicylic acid Background [5]. As reported, some synthetic phenazine-1-carboxylate Phenazine-1-carboxylic acid (PCA) (1, Fig. 1) is a sec- derivatives prepared by chemical modifcation of the car- ondary metabolite isolated from Pseudomonas, Strep- boxyl group with various alkyl alcohols exhibit strong tomycetes, and a few other bacterial genera from soil or fungicidal activity against Pyricularia oryzae, and in par- marine habitats [1–5]. Te biological properties of PCA ticular the inhibition of derivative 7 was 100% complete includes antimicrobial [6–9] antiviral [7], antitumo- at 8.3 μg/mL [20]. Recently, a series of novel aminophen- rigenic [8–12] antitubercular and antileukemic activities azine-1-carboxylate derivatives were synthesized and [13, 14]. In China, PCA has been registered as a biofun- evaluated against fve fungi [21], and the results of bioas- gicide against rice sheath blight caused by Rhizoctonia say showed that compounds 8 and 9 (Fig. 1) could exhib- solani, and it is noted for its high efcacy, low toxicity, ited strong activity against P. piricola with EC­ 50 values environmental friendliness and enhancement of crop of 3.00 μg/mL and 4.44 μg/mL respectively, which were production [15–18]. PCA is also an important precur- both lower than that of PCA. sor for the biosynthesis of ester derivatives [1, 19], some Salicylic acid (SA) (Fig. 2), also known as o-hydroxy- of which show higher fungicidal activity against several benzoic acid which is one of the three isomers of phytopathogenic fungi. For instance, compound 6 (Fig. 1) hydroxybenzoic acid, is an important plant growth reg- isolated from Pseudomonas, was a more efective deriva- ulator playing a role in the hypersensitive reaction (HR) tive against Alternaria alternata and R. solani than PCA and acts as an endogenous signal responsible for induc- ing systemic acquired resistance in plants [22, 23]. Te plants treated with salicylic acid or its derivatives may *Correspondence: [email protected]; [email protected]; junkaili@sina. be able to resist infection by various plant pathogens com [24–26]. Hydroxybenzoate esters, which are widely used 2 School of Agriculture, Yangtze University, Jingmi Road 88, Jingzhou 434025, China in medicine, foods and cosmetics, have been reported to Full list of author information is available at the end of the article have various biological activities, such as antimicrobial © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/ publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhu et al. Chemistry Central Journal (2018) 12:111 Page 2 of 10 Fig. 1 The structures of PCA and its derivatives Fig. 2 The structures of PCA–salicylic acid ester conjugates (5a–5e), PCA-3-hydroxybenzoic acid ester conjugates (5f–5j) and PCA-p-hydroxybenzoic acid ester conjugates (5k–5p) [27–29] antiviral [30, 31], anti-infammatory and fungi in vitro. Furthermore, the systemic acquired resist- nematicidal activities [32], among others. Accordingly, ance of the most active PCA–SA ester conjugate 5c hydroxybenzoate esters with multiple bioactive chemical against rice sheath blight disease was also investigated in structures, have drawn wide attention in the biological rice plants. and pharmacological felds. In this research, considering the potential biological activity of phenazine-1-carboxylic derivatives and that Results and discussion there have been few published studies on the biologi- Chemistry cal activity of phenazine-1-carboxylic phenolic esters, As shown in Fig. 3, three types of hydroxybenzoate esters we designed and synthesized 16 novel phenolic ester (4) were frst synthesized by a simple esterifcation reac- derivatives of phenazine-1-carboxylic acid (Fig. 2) by a tion with 2-hydroxybenzoic acid, 3-hydroxybenzoic acid simple esterifcation reaction of PCA and three types of or 4-hydroxybenzoic acid as the starting materials. Ten hydroxybenzoic acids. To enhance the lipophilic prop- treatment of PCA with oxalyl chloride at the refux tem- erties of the these conjugates, hydroxybenzoic acids perature in CH2Cl2 solution aforded intermediate 2 were derivatized to its ester with the corresponding after the evaporation of CH 2Cl2. Te target compound CH3(CH2)nOH. Te synthetic route of conjugates 5a–5p 5a was synthesized by adding intermediate 2 to com- is described in Fig. 3. All these conjugates were evaluated pound 4a in CH 2Cl2 solution, stirred at room tempera- for their fungicidal activity against fve phytopathogenic ture for 2 h. PCA–salicylic acid ester conjugates (5a–5e), PCA-3-hydroxybenzoic acid ester conjugates (5f–5j) and Zhu et al. Chemistry Central Journal (2018) 12:111 Page 3 of 10 B A C Fig. 3 Synthetic route of target compounds. Reagents and conditions: a oxalyl chloride, CH2Cl2, DMF, refux, 8 h; b alcohol, refux, overnight; c hydroxybenzoic acid ester, CH2Cl2, room temperature to refux, 2 h PCA-p-hydroxybenzoic acid ester conjugates (5k–5p) (int) = 0.0316) which were used in all calculations. Te were synthesized by this method. fnal R1 was 0.0408 (I > 2 sigma (I)) and wR2 was 0.1162. Te structures of all conjugates were characterized Crystallographic data have been deposited with the Cam- by 1H NMR and high resolution mass spectroscopy bridge Crystallographic Data Centre, and the deposition (HRMS) analyses, and the representative conjugate 5d number was CCDC 1563918 (Additional fle 1). was confrmed by the X-ray crystallographic analysis. Te molecular structure of 5d is shown in Fig. 4. Te crystal Fungicidal activities data for 5d: triclinic, space group P21/c, a = 18.130 (3) Å, All novel conjugates (5a–5p) were primarily screened b = 12.258 (2) Å, c = 8.6490 (14) Å, a = 90°, b = 96.224 in vitro against fve phytopathogenic fungi, R. solani, A. (3)°, g = 90°, V = 1910.7 (6) Å3, Z = 4, T = 297 (2) K, μ solani, Fusarium oxysporum, Fusaium graminearum and ο −1 3 (M ) = 0.093 mm , Dcalcd. = 1.343 Mg/m , 14,129 refec- P. oryzae, with PCA as a control. Te results of the pre- Ɵ tions measured (1.130 ≤ 2 ≤ 26.000°), 3755 unique (R liminary bioassay are shown in Table 1. We found that Fig. 4 The crystal structure of conjugate 5d Zhu et al. Chemistry Central Journal (2018) 12:111 Page 4 of 10 Table 1 Fungicidal activity of compounds 5a–5p against fve plant fungi in vitro at 50 μg/mL (inhibition rate/%) Compd. R. solani A. solani F. oxysporum F. graminearum P. oryzae 5a 66.2 1.5 11.7 0.5 13.1 0.6 7.3 0.9 34.5 0.9 ± ± ± ± ± 5b 91.6 0.8 30.3 1.6 15.7 1.3 15.9 2.6 32.8 0.0 ± ± ± ± ± 5c 100.0 0.0 13.0 2.3 12.4 0.8 6.5 0.2 37.0 2.7 ± ± ± ± ± 5d 93.5 0.6 12.4 0.9 31.4 2.9 12.3 1.3 27.0 1.2 ± ± ± ± ± 5e 93.1 0.9 15.1 0.6 9.8 0.3 10.9 0.6 27.0 3.9 ± ± ± ± ± 5f 37.3 1.2 45.5 0.3 35.3 3.4 13.0 0.9 72.3 0.0 ± ± ± ± ± 5 g 41.2 0.8 21.3 1.3 16.3 0.9 11.6 2.7 49.6 2.6 ± ± ± ± ± 5 h 93.2 0.3 15.1 0.5 9.8 0.5 10.9 3.4 27.0 0.9 ± ± ± ± ± 5i 100.0 0.0 28.9 1.8 18.3 2.7 10.1 0.5 45.4 1.2 ± ± ± ± ± 5j 45.1 1.0 17.2 2.5 13.1 0.5 11.6 1.9 39.6 1.5 ± ± ± ± ± 5k 33.9 0.9 18.6 0.3 11.1 0.6 8.7 3.5 39.6 3.7 ± ± ± ± ± 5l 42.4 1.2 19.3 0.9 13.7 1.1 13.0 4.4 45.4 0.9 ± ± ± ± ± 5m 100.0 0.0 24.1 1.5 15.7 1.6 10.9 0.8 39.6 0.2 ± ± ± ± ± 5n 98.3 0.2 26.2 0.9 13.7 1.5 8.7 4.6 41.2 0.9 ± ± ± ± ± 5o 93.0 0.2 15.1 0.5 9.8 2.9 10.9 3.3 27.0 0.8 ± ± ± ± ± 5p 44.5 1.2 18.6 0.9 13.7 0.9 0 0.0 34.5 4.9 ± ± ± ± ± PCA 86.2 0.9 85.2 1.2 83.5 1.9 86.1 1.9 92.0 2.7 ± ± ± ± ± Each treatment had three replicates (Mean SD).