含1,3,4-噻 (噁) 二唑吩嗪-1-甲酰胺類衍生物的合成與殺菌活性

何 敏, 古景文, 李司丞, 向緒穩, 姜 珊, 崔紫寧*,,2

(1. 亞熱帶農業生物資源保護與利用國家重點實驗室，群體微生物研究中心，廣東省微生物信號與作物病害防控重點實驗室，植物保護學院，華南農業大學，廣州 510642；2. 嶺南現代農業科學與技術廣東省實驗室，廣州 510642)

0 Introduction

Phenazine-1-carboxylic acid (PCA), the bioactive product in Pseudomonas fluorescens isolated from plant roots, has been registered as a biocide "Shenqinmycin" in China to control rice sheath blight. PCA plays a key role in inhibiting fungal infection and contributing to ecological balance[1]. The first activity research of PCA was carried out as a well known microbial pesticide, and then it be applied in medicine field, including antiviral[2], antitumorigenic[3], antitubercular[4]and antibacteria[5]. Due to its broad-spectrum biological activity, low toxicity and ecological friendliness, PCA also has been widely used in the field of agriculture[6],for the control of fungal diseases caused by Fusarium oxysporum[7], Rhizoctonia solani[8], Botrytis cinerea[9]and Fusarium graminearum[10].

Nitrogen-containing heterocycles, display extremely extensive and excellent biological activities,have long been concerned in medicinal chemistry.Recently, our group has been committed to the bioactivity development of nitrogen-based heterocyclic derivatives, such as 1,3-thiazolidine-2-thione[11],tetrahydroquinoline[12], 3,5-dimethylpyrazole[13],thiazolidin-2-cyanamide[14]and 2,4-disubstituted oxazole[15]. Furthermore, 1,3,4-thiadiazole and 1,3,4-oxadiazole are known important class of fivemembered heterocyclics, with worth expecting biological activities, such as insecticidal[16], fungicidal[17-18],herbicidal[19-20]and anticancer activities[21-22]. In our previous work, 1,3,4-thiadiazole derivatives have very good fungicidal activities[23]. The most promising candidate showed EC50value of 5.7 μg/mL against Phytophthora infestans, about two-ninth and one-fifth of the positive controls of bupidoline and carbendazim,respectively. In addition, another study about 1,3,4-oxadiazole derivatives showed in vivo fungicidal activity against B. cinerea and R. solanii at 500 μg/mL obviously[24].

It is well known that amide bonds have been widely recognized as the common type of bond in drug molecules[25]. In this work, according to the reasonable structure optimization and amide bond link mode, two series of PCA derivatives containing 1,3,4-thia(oxa)diazol were designed and synthesized to obtain more promissing fungicidal agents (Scheme 1).All title compounds were final product and characterized by mass spectrometry, elemental analysis, and nuclear magnetic resonance spectroscopy, and their fungicidal activities were evaluated. The preliminary structureactivity relationships of these compounds will be elucidated. The synthetic routes of compounds I1-I28were shown in Scheme 2.

1 Materials and methods

1.1 Instrumental analysis

The melting points were determined with a Cole-Parmer melting point apparatus while the thermometer was uncorrected.1H NMR spectra were recorded on Bruker Avance DRX spectrometer at 600 MHz.Analytical thin-layer chromatography (TLC) was carried out on precoated plates (silica gel 60 F254),and spots were visualized with ultraviolet (UV) light.Elemental analysis was carried out with a Flash EA 1 112 elemental analyzer and was performed at the laboratories of the Institute of Chemistry, Chinese Academy of Sciences. All strains were provided by Institute of Plant Protection, Chinese Academy of Agricultural Sciences.

1.2 Synthetic procedures

1.2.1 General procedure for the synthesis of 2-amino-5-aryl-1,3,4-thiadiazole A mixture of thiosemicarbazide (10 mmol) and aryl substituted carboxylic acid (10 mmol), and phosphorus oxychloride(5 mL) was refluxed for 0.5 hours. Upon completion of the reaction, turned off the heat and cooled to room temperature. Ice water (100 mL) was added to system very slowly till complete decomposition of POCl3then the mixture was basified to pH 8 by 50% NaOH solution. The formed precipitate was filtered, washed with water, and dried to afford the corresponding 1,3,4-thiadiazol-2-amines.

1.2.2 General procedure for the synthesis of 2-amino-5-aryl-1,3,4-oxadiazole Substituted benzoyl chloride obtained by reacting corresponding substituted benzoic acid (10 mmol) with dichlorosulfoxide(10 mL) for 2 h was slowly added to semicarbazide hydrochloride (10 mmol), triethylamine (20 mmol)and acetonitrile (30 mL) at low temperature, and then the reaction was transferred to room temperature stirring for 6 h. Reaction liquid filtration. The solid was heated with phosphorus oxychloride and toluene for reflux for 3 h. When the reaction liquid was cooled to room temperature, 100 mL ice water was added to quench excess phosphorus oxychloride, and 50% NaOH solution was used to adjust the pH to 8.0.The formed precipitate was filtered, washed with water, and dried to afford the corresponding 1,3,4-oxadiazole-2-amines.

1.2.3 General synthetic procedure for the title compounds I1-I28The title compounds were obtained by reaction of the key intermediates with phenazine-1-formyl chloride, 1 or 2, potassium carbonate and acetonitrile. The reaction was transferred to room temperature and stirred for 1 h.After the reaction, dichloromethane and water were added to extract, and the organic layer was concentrated to obtain the crude product. The product was purified by column chromatography (40 mm × 250 mm) on silica gel using dichloromethane and methanol V/V 95 : 5) as the eluent to yield the title compounds I1-I28.

1.3 Fungicidal activity

1.3.1 In vitro fungicidal activity In vitro fungicidal activities of the compounds I1-I28were tested against M. oryzae, B. cinerea, P. capsici, R. solani and F.graminearum, using a plate method (PDA medium)[28-29].Their relative inhibition rate (%) was determined using the mycelium growth rate method[30-31]. The PCA was assessed under the same conditions as the positive control. After the mycelia grew completely,the diameters of the mycelia were measured and the inhibition rate was calculated according to the formula (1).

In the formula: I is the inhibition rate, %; D1is the average diameter of mycelia in the blank test, and D2is the average diameter of mycelia in the presence of those compounds[32].

1.3.2 In vivo fungicidal activity Because the in vitro fungicidal activities of the compounds with electron-donating groups were generally better than the compounds with electron-withdrawing groups, we chose the compounds with electron-donating groups for the further experiments. Using the pot culture test[33], twelve compounds with electron-donating groups and two compounds without substituent were selected for in vivo fungicidal activitytests against five phytopathogenic fungi including M. oryzae,B. cinerea, P. capsici, R. solani and F. graminearum.Compounds were prepared into a solution with a concentration of 500 μg/mL. PCA was assessed under the same conditions as the positive control. The culture plates were cultivated at (24 ± 1) °C. The rice,cucumber, pepper and wheat seeds were soaked in water for 2 h at 50 °C and then kept moist for 24 h at 28 °C in an incubator. When the radicles were 0.5 cm,the seeds grow in plastic pots containing a 1 : 1(V/V)mixture of vermiculite and peat. Cucumber, pepper and wheat were at the stage of two seed leaves and rice was at the stage of three seed leaves. Tested compounds were confected to 2.5% EC (emulsifiable concentration) formulations, in which pesticide emulsifier 600 (2.125%) and pesticide emulsifier 500(0.375%) were the additives, DMSO (0.1%) was the solvent, and xylene was the co-solvent. The formulation was diluted to 500 μg/mL by water. The pathogenic fungi were inoculated to the surface of seed leaves and then the solution of title compounds was sprayed using a hand sprayer. Three replicates for each treatment were conducted. After inoculation, the plants were maintained at (24 ± 1) °C and above 80%relative humidity. When the untreated plant (blank control) fully developed symptoms, the fungicidal activity was assessed. To determine the average disease index, the area of inoculated leaves covered by disease symptoms was evaluated and compared to that of untreated ones. The relative control efficacy of compounds compared to the blank assay was calculated by using the following formula (2).

In the formula, Eris relative control efficacy, ICKis the average disease index during the blank assay and IPTis the average disease index after treatment during testing.

2 Results and discussion

2.1 Synthesis of the title compounds

Physical and chemical data of the title compounds I1-I28were listed in Table 1.1H NMR were listed in Table 2.

2.2 Fungicidal activity

2.2.1 In vitro fungicidal activity All the title compounds were primarily screened in vitro against five phytopathogenic fungi, F. graminearum, P.capsici, R. solani, M. oryzae and B. cinerea, with PCA as control. The results of the preliminary bioassay were shown in Table 3. The results showed that all title compunds with EC50values between 33.25 and 99.45 μg/mL, which exhibited better fungicidal activities against F. graminearum than PCA(EC50= 128.54 μg/mL).

Table1 Physical and chemical data of title compounds I1-I28

In particular, compounds I8, I11and I22, which had more potential fungicidal activities against F.graminearum, had about 3-4 times the activity of PCA.Although the compounds had no significant inhibitory activities against P. capsici, I22(EC507.24 g/mL)still achieved the same level as PCA (EC507.26 g/mL).In addition, compound I8(EC50= 8.64 μg/mL)showed similar fungicidal activity to PCA (EC50=7.56 μg/mL) against R. solani. Unfortunately, in the other two phytopathogenic fungi M. oryzae and B.cinerea, the title compounds failed to exceed the activity of PCA, but compound I22still showed promising activity (EC50= 16.56 μg/mL against M.oryzae, EC50= 45.08 μg/mL against B. cinerea) to PCA (EC50= 12.63 μg/mL, EC50= 19.76 μg/mL).Thus, compound I22may be a candidate for a broad spectrum of fungicidal agents, while compound I8was a specific candidate against F. graminearum and R. solani.

2.2.2 In vivo fungicidal activity To further confirms the bioactivity of the title compounds, in vivo fungicidal activities against five fungi were assessed and the results were presented in Table 4.Tendency of the results was nearly in consistent with that of the in vitro bioactivity. For F. graminearum,the bioactivities of all compounds were between 40.46% and 58.69%, which were better than PCA.Especially, for compounds I8(58.69%) and I22

(55.37%), the control effect was about twice of that of PCA (25.14%) at 500 μg/mL. Moreover, the fungicidal activity of compound I8(91.34%) against R. solani was similar to that of PCA (90.25%). The in vivo fungicidal activity of compound I22(94.23%,76.38%, 85.94%) against P. capsici, R. solani and M.oryzae were better than or similar to that of PCA(93.48%, 90.25%, 92.18%). These results further confirmed the broad spectrum and specific fungicidal activity of compounds I8and I22.

Table2 1H NMR of title compounds I1-I28

Table3 EC50 values (μg/mL) against M. oryzae, B. cinerea, P. capsici, R. solani and F. graminearum

Table4 Fungicidal activity of compounds against five plant fungi in vivo at 500 μg/mL (inhibition rate/%)

2.3 Structure-activity relationship

In addition, we discussed preliminary structureactivity studies on these PCA derivatives according to the bioactivity data against F. graminearum. In the substituted compounds, -CH3and -CH3O (I8, I9, I10,I11, I12, I13, I22, I23, I24, I25, I26, I27) substituent showed better activities than Cl and F substituent (I2,I3, I4, I5, I6, I7, I16, I17, I18, I19, I20, I21). In a word,introducing electron-withdrawing group into benzene ring was not conducive to the activity of the compounds, and obviously reduces their biological activity. While introducing electron-donating group was conducive to improving their bioactivity. At the same time, the substitution of the same substituent at different positions on the benzene ring also affects the activity of the compounds. The substitution position of the same substituent on the benzene ring was in the order of o＞p＞m according to the bioactivity. The preliminary structure-activity relationship study will provide the theoretical basis for the study of these compounds.

3 Conclusions

In summary, a series of novel structures of phenazine-1-carboxylic acid derivatives containing 1,3,4-thiadiazole and 1,3,4-oxadiazole were designed.The title compounds were characterized by mass spectrometry, elemental analysis, and nuclear magnetic resonance spectroscopy. All the title compounds exhibited significant in vitro fungicidal activities and in vivo fungicidal activities against phytopathogenic fungi. Especially compounds I8(X=S, R=2-OCH3) and I22(X=O, R=2-OCH3) had better fungicide activities than that of PCA for F.graminearum. Compound I22exhibited broad spectra fungicidal activities, while compound I8was a candidate against F. graminearum and R. solani specifically. The stucture-activity relationship results showed that the introduction of electron-withdrawing group into benzene ring was not favor to the activity of the compounds, while the introduction of electrondonating group was conducive to improving the activity of the compounds. At the same time, the substitution position on the benzene ring were in the order of o＞p＞m according to the bioactivity. These results can be used to guide the further structural modification of these compounds.