Synthesis and bioactivities of phenazine-1-carboxylic piperazine derivatives

Abstract Phenazine-1-carboxylic acid (PCA) as a natural product which has significant inhibition effects against many soil-borne fungal phytopathogens in agricultural application and has been registered in China as the fungicide against rice sheath blight. In order to find new higher fungicidal activities lead compounds and develop new eco-friendly agrochemicals, we introduced substructure piperazines which also have high biological activity into PCA, designed and synthesized a series of phenazine-1-carboxylic piperazine derivatives, and their structures were confirmed by 1H NMR and HRMS. Most compounds exhibited certain in vitro fungicidal activities. In particular, Compounds 5r exhibited the activity against all the tested pathogenic fungi, such as Rhizoctonia solani, Alternaria solani, Fusarium oxysporum, Fusarium graminearum, Pyricularia oryzac Cavgra, with the EC50 value of 24.6μM, 42.9μM, 73.7μM, 73.8μM, 34.2μM, respectively, more potent activities than PCA (33.2μM, 81.5μM, 186.5μM, 176.4μM, 37.3μM). This result provided a highly active lead compound for the further structure optimization design. Graphical Abstract


Introduction
Phenazine-1-carboxylic acid (PCA) (Figure 1) is a very important natural product, which has been proved having antifungal, antitumor and antileukemia effects (Laursen and Nielsen 2004), widely existed in microbial metabolites of Streptomycetes and Pseudomonads. Particularly, in recent years, PCA gained extensively attention because of remarkable inhibition effects against many soil-borne fungal phytopathogens in agricultural application (Padaria et al. 2016;Pan et al. 2017). PCA is also found friendliness to environment, low toxicity to human and animals and improvement of crop production, for that it has been registered in China as the biofungicide "Shenqin bactin" (He L et al. 2008;Arseneault et al. 2014). In our previous studies, we have found that phenazine-1-carboxamine (EC50 ¼ 6.66 mg/L) and N-methylphenazine-1-carboxamine (EC50 ¼ 7.09 mg/L) showed better fungicidal activity against Rhizoctonia solani than PCA (EC50 ¼ 7.88 mg/L), and exhibited excellent fungicidal activity against Fusarium graminearum (EC 50 = 12.67 mg/L, EC 50 = 8.30 mg/L) which PCA had poor fungicidal activity (EC 50 = 127.28 mg/L). In our another studies, a series of PCA-amino acid ester conjugates ( Figure 1) were synthesized, and found that some conjugates exhibited the fungicidal activities against Rhizoctonia solani 2 5 times as much as that of PCA (Niu et al. 2016).
Piperazine is a symmetrical diamine which has perfect bioactivities and used in bioactive molecular design. Piperazine analogues have extensive pesticide biological activities. Early in 1948 (Quesadaocampo et al. 2016), 1-(4-isopropylbenzyl) piperazine ( Figure 2) had been used to control fungi and bacterium on apple tree and had significant effect. A series of N-substituted Piperazines I (Figure 2), displayed excellent antifungal activities of 100% against Leptosphaeria nodorum and Mycosphaerella graminicola at the concentration of 25 mg/L (Chai L et al. 2008). Wang Baolei (Wang BL et al. 2010) has reported that trifluoromethyl-substituted 1,2,4-Triazole piperazine derivatives II ( Figure 2) exhibited good antifungal activities, several compounds showed more fungicidal activities against Pseudoperonospora cubensis than commercial chemicals Dimethomorph at the concentration of 500 mg/L. As above mentioned, PCA is an important and active natural product. In order to find a new lead compound with high biological activity and develop new eco-friendly agrochemicals, we introduced substructure piperazines which also have high biological activity into PCA (Figure 3), designed and synthesized a series of phenazine-1-carboxylic piperazine derivatives. Structures of the target compounds were characterized by 1 H-NMR and HR-MS. Most compounds exhibited certain in vitro fungicidal activities. In particular, Compounds 5r exhibited the activity against all the tested pathogenic fungi, with the EC 50 value of 24.6lM, 42.9lM, 73.7lM, 73.8lM, 34.2lM, respectively, more potent activities than PCA (33.2lM, 81.5lM, 186.5lM, 176.4lM, 37.3lM).

Chemistry
The synthetic route of phenazine-1-carboxylic piperazine derivatives is shown in Figure 4. Treatment of piperazine 2 with RCH 2 Cl and K 2 CO 3 as base in acetonitrile solution at reflux temperature afforded monosubstituted piperazines 3. Reacted phenazine-1-carbonyl chloride 4 which prepared by reacting PCA with oxalyl chloride with monosubstituted piperazine 3 by using triethylamine as base at room temperature giving the target compounds (Zhu et al. 2018) (analysis data of target compounds are presented in Supplementary data).

Fungicidal activities
The in vitro fungicidal activities of all the target compounds have been determined against five pathogenic fungi, such as Rhizoctonia solani, Alternaria solani, Fusarium oxysporum, Fusarium graminearum and Pyricularia oryzac Cavgra, at a concentration of 200 lM, using a plate method (PDA medium) (Niu et al. 2016;Shentu et al. 2014). The commercial fungicide Shenqinbactin (PCA) was assessed under the same conditions as positive controls. The results of preliminary bioassay showed that all compounds exhibited certain fungicidal activities against the pathogenic fungi at the concentration of 200 lM (This concentration of PCA equal to 44.8 mg/L) ( Table 1). In particular, compound 5r exhibited bioactivity more potent than PCA against all the tested pathogenic fungi with the rate more than 90%. The structure-activity relationship of the target compounds showed some regularity, although most of them were less effective than PCA. In the compounds which contain methyl substituent benzyl group, the fungicidal activities displayed p-CH 3 > o-CH 3 > m-CH 3 (5c > 5a > 5b). When the compounds all are p-methyl substituent, the bioassay results showed the activities rank as CH 3 > C 2 H 5 > CH(CH 3 ) 2 (5c > 5d > 5e). However, the fungicidal activities indicated m-Cl > o-Cl > p-Cl (5i > 5 h > 5j) in the compounds which contain chlorine substituent benzyl group. The substituent is also ortho-position, fluorine substituent compounds showed higher activities than chlorine substituent compounds. In the para-position substituted compounds, CH 3 O substituent (5f) showed the best fungicidal activities, and in the meta-position substituted compounds, Cl atom substituent (5i) exhibited better activities than CH 3 (5b), NO 2 (5k) and CF 3 (5l) substituent. Replaced phenyl with naphthyl (5 m, 5n), the change of the fungicidal activities was not obvious, and increased the length of carbon chain (5o, 5p) or introduced into the chain of -CH 2 O-(5s) showed the same results. Significantly, when introduced morpholinyl (5r) to R, the fungicidal activities against all the tested pathogenic fungi increased   (Zhu et al. 2018) remarkably (100% for R. solani, 98.3% for A. solani, 90.3% for F. oxysporum, 90.7% for F. graminearum and 100% for P. oryzac Cavgra, respectively), much higher than other substituted group and also higher than PCA, the result provided a highly active lead compound for the further structural optimum design.
Compounds 5r was chosen to determine EC 50 values (Niu et al. 2016;Shentu et al. 2014) against all the tested pathogenic fungi, because 5r showed fungicidal activities with the inhibitory rate of over 90% against the five pathogenic fungi at the concentration of 200 lM. The results were shown in Table 2. Compounds 5r exhibited the activity against all the tested pathogenic fungi with the EC 50 value of 24.6lM, 42.9lM, 73.7lM, 73.8lM, 34.2lM, respectively, more potent activities than PCA (33.2lM,81.5lM,186.5lM,176.4lM,37.3lM).

Conclusion
In summary, a series of phenazine-1-carboxylic piperazine derivatives were designed, synthesized and their structures were confirmed by 1 H-NMR spectrum and elemental analysis measured with HRMS. All the target compounds showed certain in vitro fungicidal activities against the pathogenic fungi at the concentration of 200 lM.  24.6 ± 0.8 42.9 ± 2.1 73.7 ± 1.6 73.8 ± 2.2 34.2 ± 3.3 a at the concentration of 200 lM. Notes: Each treatment had three replicates (Mean ± SD). The phenazine-1-carboxylic acid (PCA) was used as the positive control.
The structure-activity relationship of the target compounds revealed that the type of substituent group of benzyl, the substituent position and replace phenyl with heterocyclic group et al were the very important factors of fungicidal activities. Particularly, compound (5r) which R was morpholinyl showed the best fungicidal activities against all the tested pathogenic fungi, higher than PCA, the result provided a highly active lead compound for the further structural optimum design.