Skip to content

Advertisement

  • Research Article
  • Open Access

Design, synthesis and biological activity of hydroxybenzoic acid ester conjugates of phenazine-1-carboxylic acid

Chemistry Central Journal201812:111

https://doi.org/10.1186/s13065-018-0478-2

  • Received: 3 August 2018
  • Accepted: 19 October 2018
  • Published:

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 five 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

Phenazine-1-carboxylic acid (PCA) (1, Fig. 1) is a secondary metabolite isolated from Pseudomonas, Streptomycetes, and a few other bacterial genera from soil or marine habitats [15]. The biological properties of PCA includes antimicrobial [69] antiviral [7], antitumorigenic [812] antitubercular and antileukemic activities [13, 14]. In China, PCA has been registered as a biofungicide against rice sheath blight caused by Rhizoctonia solani, and it is noted for its high efficacy, low toxicity, environmental friendliness and enhancement of crop production [1518]. PCA is also an important precursor for the biosynthesis of ester derivatives [1, 19], some of which show higher fungicidal activity against several phytopathogenic fungi. For instance, compound 6 (Fig. 1) isolated from Pseudomonas, was a more effective derivative against Alternaria alternata and R. solani than PCA [5]. As reported, some synthetic phenazine-1-carboxylate derivatives prepared by chemical modification of the carboxyl group with various alkyl alcohols exhibit strong fungicidal activity against Pyricularia oryzae, and in particular the inhibition of derivative 7 was 100% complete at 8.3 μg/mL [20]. Recently, a series of novel aminophenazine-1-carboxylate derivatives were synthesized and evaluated against five fungi [21], and the results of bioassay showed that compounds 8 and 9 (Fig. 1) could exhibited strong activity against P. piricola with EC50 values of 3.00 μg/mL and 4.44 μg/mL respectively, which were both lower than that of PCA.
Fig. 1
Fig. 1

The structures of PCA and its derivatives

Salicylic acid (SA) (Fig. 2), also known as o-hydroxybenzoic acid which is one of the three isomers of hydroxybenzoic acid, is an important plant growth regulator playing a role in the hypersensitive reaction (HR) and acts as an endogenous signal responsible for inducing systemic acquired resistance in plants [22, 23]. The plants treated with salicylic acid or its derivatives may be able to resist infection by various plant pathogens [2426]. Hydroxybenzoate esters, which are widely used in medicine, foods and cosmetics, have been reported to have various biological activities, such as antimicrobial [2729] antiviral [30, 31], anti-inflammatory and nematicidal activities [32], among others. Accordingly, hydroxybenzoate esters with multiple bioactive chemical structures, have drawn wide attention in the biological and pharmacological fields.
Fig. 2
Fig. 2

The structures of PCA–salicylic acid ester conjugates (5a5e), PCA-3-hydroxybenzoic acid ester conjugates (5f5j) and PCA-p-hydroxybenzoic acid ester conjugates (5k5p)

In this research, considering the potential biological activity of phenazine-1-carboxylic derivatives and that there have been few published studies on the biological activity of phenazine-1-carboxylic phenolic esters, we designed and synthesized 16 novel phenolic ester derivatives of phenazine-1-carboxylic acid (Fig. 2) by a simple esterification reaction of PCA and three types of hydroxybenzoic acids. To enhance the lipophilic properties of the these conjugates, hydroxybenzoic acids were derivatized to its ester with the corresponding CH3(CH2)nOH. The synthetic route of conjugates 5a5p is described in Fig. 3. All these conjugates were evaluated for their fungicidal activity against five phytopathogenic fungi in vitro. Furthermore, the systemic acquired resistance of the most active PCA–SA ester conjugate 5c against rice sheath blight disease was also investigated in rice plants.
Fig. 3
Fig. 3

Synthetic route of target compounds. Reagents and conditions: a oxalyl chloride, CH2Cl2, DMF, reflux, 8 h; b alcohol, reflux, overnight; c hydroxybenzoic acid ester, CH2Cl2, room temperature to reflux, 2 h

Results and discussion

Chemistry

As shown in Fig. 3, three types of hydroxybenzoate esters (4) were first synthesized by a simple esterification reaction with 2-hydroxybenzoic acid, 3-hydroxybenzoic acid or 4-hydroxybenzoic acid as the starting materials. Then treatment of PCA with oxalyl chloride at the reflux temperature in CH2Cl2 solution afforded intermediate 2 after the evaporation of CH2Cl2. The target compound 5a was synthesized by adding intermediate 2 to compound 4a in CH2Cl2 solution, stirred at room temperature for 2 h. PCA–salicylic acid ester conjugates (5a5e), PCA-3-hydroxybenzoic acid ester conjugates (5f5j) and PCA-p-hydroxybenzoic acid ester conjugates (5k5p) were synthesized by this method.

The structures of all conjugates were characterized by 1H NMR and high resolution mass spectroscopy (HRMS) analyses, and the representative conjugate 5d was confirmed by the X-ray crystallographic analysis. The molecular structure of 5d is shown in Fig. 4. The crystal data for 5d: triclinic, space group P21/c, a = 18.130 (3) Å, b = 12.258 (2) Å, c = 8.6490 (14) Å, a = 90°, b = 96.224 (3)°, g = 90°, V = 1910.7 (6) Å3, Z = 4, T = 297 (2) K, μ (Mο) = 0.093 mm−1, Dcalcd. = 1.343 Mg/m3, 14,129 reflections measured (1.130 ≤ 2Ɵ ≤ 26.000°), 3755 unique (R (int) = 0.0316) which were used in all calculations. The final R1 was 0.0408 (I > 2 sigma (I)) and wR2 was 0.1162. Crystallographic data have been deposited with the Cambridge Crystallographic Data Centre, and the deposition number was CCDC 1563918 (Additional file 1).
Fig. 4
Fig. 4

The crystal structure of conjugate 5d

Fungicidal activities

All novel conjugates (5a5p) were primarily screened in vitro against five phytopathogenic fungi, R. solani, A. solani, Fusarium oxysporum, Fusaium graminearum and P. oryzae, with PCA as a control. The results of the preliminary bioassay are shown in Table 1. We found that most of conjugates (5a5p) showed low activities against A. solani, F. oxysporum, F. graminearum and P. oryzae Cavara at a concentration of 50 μg/mL, while most conjugates (5a5p) exhibited high activity against R. solani at that rate. The inhibitory activity of 5c, 5e, 5i and 5m was 100%, higher than PCA at 86.2%. To more closely examine preliminary structure–activity relationships (SARs), the conjugates (5a5p) were selected for assessment of EC50 values against Rhizoctonia solani.
Table 1

Fungicidal activity of compounds 5a5p against five 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). The phenazine-1carboxylic acid (PCA) was used as the positive control

The EC50 values against Rhizoctonia solani for all conjugates are presented in Table 2. The results showed that nine conjugates (5b, 5c, 5d, 5e, 5h, 5i, 5m, 5n and 5o) with EC50 values between 3.2 and 14.1 μg/mL exhibited more potent fungicidal activity against Rhizoctonia solani than PCA (EC50 = 18.6 μg/mL). In particular, conjugate 5c with highest fungicidal activity was 6.5-fold more active than PCA.
Table 2

EC50 values against Rhizoctonia solani and octanol–water partition coefficient of conjugates 5a5p

Compd.

EC50 (μg/mL)

Toxicity index

LogP1

5a

48.3

0.43

3.84

5b

14.1

1.48

4.42

5c

3.2

6.50

4.72

5d

9.5

2.19

4.75

5e

12.8

1.63

5.08

5f

96.3

0.22

3.91

5g

68.6

0.30

4.34

5h

9.5

2.19

4.81

5i

4.9

4.24

4.77

5j

56.9

0.37

5.02

5k

138.4

0.15

3.92

5l

70.5

0.30

4.37

5 m

4.5

4.62

4.86

5n

5.6

3.71

4.75

5o

11.8

1.76

5.05

5p

70

0.30

6.46

PCA

18.6

1.00

1.59

1Partition coefficient ‘‘LogP’’ values were calculated using the ALOGPS 2.1 program

The recent study on fungicidal mechanism of PCA indicate that, PCA will promote cell produces poisonous hydroxyl radical and disrupt the normal homeostasis of redox in cells after entering cells through cell walls and cell membranes [19, 33]. It means that a PCA analog with suitable polarity and hydrophobicity can pass through the cell membranes of pathogenic bacteria and fungi more easily and exhibit higher biological activity. As can be seen from Table 2, the fungicidal activities of conjugates were associated with their LogP values. Accordingly, we constructed a mathematical model that described the LogP of conjugates that might be expected to produce high or low levels of fungicidal activity. From Fig. 5, with increasing LogP values, the fungicidal activities of conjugates were also observed to increase. For instance, the LogP values of PCA–salicylic acid ester conjugates were ranked as follows: 5a < 5b < 5c, and the fungicidal activity of conjugates also showed the same ranking. However, the conjugates that exceeded a certain level of LogP values (> 4.72) had decreased fungicidal activity. For instance, the LogP values of PCA–salicylic acid ester conjugates were ranked 5c < 5d < 5e, but the fungicidal activity of conjugates were ranked 5c > 5d > 5e. The same trends also applied to the PCA-3-hydroxybenzoic acid ester conjugates (5f5j) and the PCA-p-hydroxybenzoic acid ester conjugates (5k5p). Through the above analysis, we found that the LogP values of the more potent fungicidal activity within these three types of conjugates ranged from 4.42 to 5.08. Furthermore, conjugates where phenolic ester groups were substituted at different positions did not greatly affect their fungicidal activity.
Fig. 5
Fig. 5

The toxicity index of conjugates 5a5p

Systemic acquired resistance

To evaluate the level of systemic acquired resistance induced by PCA–SA ester conjugates, the disease reduction of the most active PCA–SA ester conjugate 5c was investigated against rice sheath blight disease on rice seedlings following Makandar and others [34, 35]. The results of the study indicated that inoculation with conidia of Rhizoctonia solani onto rice plants treated with SA and conjugate 5c resulted in fewer lesions per leaf sheath as well as reduced blighted leaf area as compared to control plants only receiving distilled water treatment (Fig. 6). Spray treatment with SA and PCA–SA ester conjugate 5c induced resistance to sheath blight disease in rice plants, significantly reducing rice sheath blight disease in rice plants. Compared with the treatments of PCA and water control, combined SA and conjugate 5c treatments had higher induction effects, at 31.0% and 57.0% respectively (Table 3).
Fig. 6
Fig. 6

Protection of rice plants against Rhizoctonia solani by a foliar spray with 200 μmol/L PCA (PCA), 200 μmol/L SA (SA), 200 μmol/L of conjugate 5c (5c) and distilled water (water) 14 days after inoculation with Rhizoctonia solani conidial suspension (105 spore/mL)

Table 3

Induced resistance of rice to rice sheath blight by different inducers treatment

Inducers treatment

Disease (%)

Induced effect (%)

A (PCA)

47.9

12.59

B (SA)

37.3

31.97

C (conjugate 5c)

23.6

56.98

D (water)

54.8

At present, there is extensive research on possible structure–activity relationship of SA and its derivatives for induction of systemic acquired resistance. Safari assessed the potential of some chemical inducers of systemic acquired resistance (SAR) to reduce Alternaria leaf spot disease on tomato in glasshouse trials [26]. The results indicated that, among the salicylate derivatives, the biochemical activators containing electron donating groups are more suitable for inducing disease resistance in tomato crop. Also the structure relationship of 47 mono-substituted and multi-substituted salicylate derivatives with respect to their effects on disease resistance to tobacco mosaic virus and pathogenesis-related protein (PR1) accumulation were evaluated [25]. In this study, using this characteristic of SA, we demonstrated that PCA–SA ester conjugate 5c retained the resistance induction activity of SA against rice sheath blight and had higher induced resistance than SA. However, the relationship between the structures of PCA–SA ester conjugates described here and their induced activities needs further investigation, as well as the mode of action.

Experimental

Chemicals and instruments

All chemicals and solvents were obtained from commercial suppliers and were used without further purification. The melting points were determined on a WRR melting point apparatus (Shanghai Jingke Industrial Co. Ltd., PR China) and were uncorrected. Thin-layer chromatography (TLC) was performed on silica gel 60 F254 (Qingdao Marine Chemical Ltd., P. R. China). Column chromatography (CC) purification was performed over silica gel (200–300 mesh, Qingdao Marine Chemical Ltd.). 1H NMR spectrum were recorded in CDCl3 solution on a Bruker 600 MHz spectrometer (Bruker Co., Switzerland), using tetramethylsilane (TMS) as an internal standard, and chemical shift values (δ) were given in parts per million (ppm). The following abbreviations were used to designate chemical shift multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiple. MS data were obtained using a APEX IV Fourier-transform mass spectrometry (Bruker).

Synthesis of hydroxybenzoic acid esters

The compound 2-hydroxybenzoic acid (15 mmol) and its corresponding alcohol (30 mL) were added into a 50 mL round-bottom flask, and cooled at 0 °C. An aliquot of 2 mL of 98% H2SO4 was slowly added. The reaction was stirred at reflux temperature for 12 h and monitored by thin-layer chromatography (TLC) until the 2-hydroxybenzoic acid was completely consumed. The mixture was evaporated under vacuum, neutralized with water and 5% NaHCO3 aqueous solution, extracted by ether 3 times, dried over Na2SO4, concentrated in vacuum, and used in next step without purification. The compounds 3-hydroxybenzoic acid esters and p-hydroxybenzoic acid esters were also synthesized by this method.

Synthesis of phenazine-1-carbonyl chloride

Phenazine-1-carboxylic acid (10 mmol) and N,N-dimethylformamide (0.1 mmol) were added in 30 mL of dry CH2Cl2, and cooled at 0 °C. A solution of 15 mmol of oxalyl chloride in 20 mL of dry CH2Cl2 was then slowly added. The reaction was stirred at reflux temperature for 12 h, then cooled to room temperature and evaporated under vacuum. The residue was dissolved in 10 mL of dry CH2Cl2 and used in next step without purification.

General procedure for hydroxybenzoic acid ester conjugates of phenazine-1-carboxylic acid 5a5p

Phenazine-1-carbonyl chloride (10 mmol) dissolved in 10 mL of dry CH2Cl2 was added dropwise to a solution of compound 2-hydroxybenzoic acid methyl ester (10 mmol), and triethylamine (12 mmol) as the attaching acid agent in CH2Cl2, The mixture was stirred at room temperature for 4 h until the reaction was complete (indicated by TLC), then quenched with water and 5% Na2CO3 aqueous solution, dried over Na2SO4, filtered and concentrated in vacuum. The obtained crude extract was purified by recrystallizing from the solution of EtOAc-DCM (1:1) to give pure conjugate 5a. Conjugates 5b5p were also synthesized by this method.

2-(Methoxycarbonyl)phenyl phenazine-1-carboxylate (5a)

Yellow solid; yield: 89.5%; m.p. 141–142 °C; 1H-NMR (600 MHz, CDCl3) δ: 8.69 (d, J = 7.2 Hz, 1H), 8.49 (d, J = 8.8 Hz, 1H), 8.36 (dd, J = 6.0, 3.6 Hz, 1H), 8.28 (dd, J = 6.6, 3.6 Hz, 1H), 8.14 (dd, J = 7.8, 1.2 Hz, 1H), 7.98 (dd, J = 8.4, 7.2 Hz, 1H), 7.94–7.87 (m, 2H), 7.74–7.68 (m, 1H), 7.51 (d, J = 7.8 Hz, 1H), 7.43 (t, J = 7.8 Hz, 1H), 3.86 (s, 3H). HRMS calcd for C21H14N2O4 [M+H]+: 359.1026, found 359.1027.

2-(Ethoxycarbonyl)phenyl phenazine-1-carboxylate (5b)

Yellow solid; yield: 92.3%; m.p. 143–144 °C; 1H-NMR (600 MHz, CDCl3) δ: 8.74–8.69 (m, 1H), 8.48 (dd, J = 8.4, 1.2 Hz, 1H), 8.36 (dd, J = 6.6, 3.6 Hz, 1H), 8.28 (dd, J = 6.6, 3.6 Hz, 1H), 8.14 (dd, J = 7.8, 1.2 Hz, 1H), 7.98 (dd, J = 8.4, 7.2 Hz, 1H), 7.93–7.86 (m, 2H), 7.74–7.66 (m, 1H), 7.50 (d, J = 7.8 Hz, 1H), 7.42 (t, J = 7.8 Hz, 1H), 4.33 (q, J = 7.2 Hz, 2H), 1.27 (t, J = 7.2 Hz, 3H). HRMS calcd for C22H16N2O4 [M+H]+: 373.1183, found 373.1182.

2-(Propoxycarbonyl)phenyl phenazine-1-carboxylate (5c)

Yellow solid; yield: 97.5%; m.p. 102–103 °C; 1H-NMR (600 MHz, CDCl3) δ 8.72 (dd, J = 6.6, 1.2 Hz, 1H), 8.48 (dd, J = 8.4, 1.2 Hz, 1H), 8.40–8.31 (m, 1H), 8.32–8.21 (m, 1H), 8.14 (dd, J = 7.8, 1.8 Hz, 1H), 7.98 (dd, J = 8.4, 7.2 Hz, 1H), 7.94–7.79 (m, 2H), 7.73–7.59 (m, 1H), 7.51 (d, J = 7.2 Hz, 1H), 7.43 (dd, J = 11.4, 4.2 Hz, 1H), 4.23 (t, J = 6.6 Hz, 2H), 1.74–1.41 (m, 2H), 0.92 (t, J = 7.2 Hz, 3H). HRMS calcd for C23H18N2O4 [M+H]+: 387.1339, found 387.1338.

2-(Isopropoxycarbonyl)phenyl phenazine-1-carboxylate (5d)

Yellow solid; yield: 90.5%; m.p. 125–126 °C; 1H-NMR (600 MHz, CDCl3) δ 8.73 (d, J = 6.6 Hz, 1H), 8.48 (d, J = 8.4 Hz, 1H), 8.36 (dd, J = 6.6, 3.6 Hz, 1H), 8.27 (dd, J = 6.6, 3.6 Hz, 1H), 8.12 (dd, J = 7.8, 1.2 Hz, 1H), 7.98 (dd, J = 8.4, 7.2 Hz, 1H), 7.93–7.86 (m, 2H), 7.71–7.66 (m, 1H), 7.50 (d, J = 7.8 Hz, 1H), 7.41 (t, J = 7.8 Hz, 1H), 5.30–5.30 (m, 1H), 1.27 (d, J = 6.6 Hz, 6H). HRMS calcd for C23H18N2O4 [M+H]+: 387.1339, found 387.1340.

2-(Butoxycarbonyl)phenyl phenazine-1-carboxylate (5e)

Yellow solid; yield: 94.1%; m.p. 89–90 °C; 1H-NMR (600 MHz, CDCl3) δ 8.72 (dd, J = 6.9, 1.4 Hz, 1H), 8.48 (dd, J = 8.7, 1.4 Hz, 1H), 8.39–8.34 (m, 1H), 8.30–8.25 (m, 1H), 8.13 (dd, J = 7.9, 1.7 Hz, 1H), 7.98 (dd, J = 8.4, 6.6 Hz, 1H), 7.93–7.86 (m, 2H), 7.72–7.67 (m, 1H), 7.51 (dd, J = 7.8, 1.2 Hz, 1H), 7.47–7.37 (m, 1H), 4.27 (t, J = 6.7 Hz, 2H), 1.72–7.57 (m, 2H), 1.42–1.31 (m, 2H), 0.84 (t, J = 7.2 Hz, 3H). HRMS calcd for C24H20N2O4 [M+H]+: 401.1496, found 401.1497.

3-(Methoxycarbonyl)phenyl phenazine-1-carboxylate (5f)

Yellow solid; yield: 95.0%; m.p. 120–121 °C; 1H-NMR (600 MHz, CDCl3) δ 8.48 (t, J = 7.2 Hz, 2H), 8.39–8.34 (m, 1H), 8.30–8.25 (m, 1H), 8.14 (s, 1H), 8.03 (d, J = 7.8 Hz, 1H), 7.98–7.89 (m, 3H), 7.70–7.66 (m, 1H), 7.59 (t, J = 7.8 Hz, 1H), 3.98 (s, 3H). HRMS calcd for C21H14N2O4 [M+H]+: 359.1026, found 359.1027.

3-(Ethoxycarbonyl)phenyl phenazine-1-carboxylate (5g)

Yellow solid; yield: 96.5%; m.p. 109–110 °C; 1H-NMR (600 MHz, CDCl3) δ 8.55–8.41 (m, 2H), 8.39–8.30 (m, 1H), 8.31–8.24 (m, 1H), 8.14 (s, 1H), 8.04 (d, J = 7.8 Hz, 1H), 7.98–7.85 (m, 3H), 7.67 (d, J = 7.8 Hz, 1H), 7.59 (t, J = 7.8 Hz, 1H), 4.44 (q, J = 7.2 Hz, 2H), 1.44 (t, J = 7.2 Hz, 3H). HRMS calcd for C22H16N2O4 [M+H]+: 373.1183, found 373.1182.

3-(Propoxycarbonyl)phenyl phenazine-1-carboxylate (5h)

Yellow solid; yield: 95.2%; m.p. 87–88 °C; 1H-NMR (600 MHz, CDCl3) δ 8.51–8.43 (m, 2H), 8.38–8.32 (m, 1H), 8.27 (dd, J = 6.0, 4.2 Hz, 1H), 8.13 (s, 1H), 8.04 (d, J = 7.8 Hz, 1H), 7.97–7.86 (m, 3H), 7.67 (dd, J = 7.8, 1.2 Hz, 1H), 7.59 (t, J = 7.8 Hz, 1H), 4.34 (t, J = 6.6 Hz, 2H), 1.88–1.82 (m, 1H), 1.06 (t, J = 7.8 Hz, 3H). HRMS calcd for C23H18N2O4 [M+H]+: 387.1339, found 387.1340.

3-(Butoxycarbonyl)phenyl phenazine-1-carboxylate (5i)

Yellow solid; yield: 95.5%; m.p. 97–98 °C; 1H-NMR (600 MHz, CDCl3) δ 8.47 (d, J = 7.8 Hz, 2H), 8.39–8.31 (m, 1H), 8.29–8.23 (m, 1H), 8.15–8.09 (m, 1H), 8.03 (d, J = 7.8 Hz, 1H), 7.97–7.85 (m, 3H), 7.66 (dd, J = 7.8, 2.0 Hz, 1H), 7.58 (t, J = 7.8 Hz, 1H), 5.37–5.25 (m, 1H), 1.41 (d, J = 6.6 Hz, 6H). HRMS calcd for C23H18N2O4 [M+H]+: 387.1339, found 387.1340.

3-(Butoxycarbonyl)phenyl phenazine-1-carboxylate (5j)

Yellow solid; yield: 95.2%; m.p. 87–88 °C; 1H-NMR (600 MHz, CDCl3) δ 8.48 (dd, J = 7.8, 3.6 Hz, 2H), 8.39–8.33 (m, 1H), 8.31–8.25 (m, 1H), 8.12 (s, 1H), 8.04 (d, J = 7.8 Hz, 1H), 7.98–7.90 (m, 3H), 7.67 (dd, J = 7.8, 2.4 Hz, 1H), 7.59 (t, J = 7.8 Hz, 1H), 4.39 (t, J = 6.6 Hz, 2H), 1.83–1.77 (m, 2H), 1.56–1.48 (m, 2H), 1.01 (t, J = 7.2 Hz, 3H). HRMS calcd for C24H20N2O4 [M+H]+: 401.1496, found 401.1495.

4-(Methoxycarbonyl)phenyl phenazine-1-carboxylate (5k)

Yellow solid; yield: 95.0%; m.p. 164–165 °C; 1H-NMR (600 MHz, CDCl3) δ 8.54–8.43 (m, 2H), 8.37–8.33 (m, 1H), 8.31–8.26 (m, 1H), 8.24–8.18 (m, 2H), 7.97–7.90 (m, 3H), 7.57–7.51 (m, 2H), 3.97 (s, 3H). HRMS calcd for C21H14N2O4 [M+H]+: 359.1026, found 359.1025.

4-(Ethoxycarbonyl)phenyl phenazine-1-carboxylate (5l)

Yellow solid; yield: 98.1%; m.p. 123–125 °C; 1H-NMR (600 MHz, CDCl3) δ 8.51–8.46 (m, 2H), 8.38–8.32 (m, 1H), 8.31–8.26 (m, 1H), 8.21 (t, J = 5.4 Hz, 2H), 7.98–7.89 (m, 3H), 7.53 (t, J = 5.4 Hz, 2H), 4.43 (q, J = 7.2 Hz, 2H), 1.44 (t, J = 7.2 Hz, 3H). HRMS calcd for C22H16N2O4 [M+H]+: 373.1183, found 373.1182.

4-(Propoxycarbonyl)phenyl phenazine-1-carboxylate (5m)

Yellow solid; yield: 98.1%; m.p. 95 °C; 1H-NMR (600 MHz, CDCl3) δ 8.61–8.40 (m, 2H), 8.41–8.30 (m, 1H), 8.31–8.27 (m, 1H), 8.27–8.15 (m, 2H), 8.02–7.82 (m, 3H), 7.64–7.46 (m, 2H), 4.33 (t, J = 6.6 Hz, 2H), 1.89–1.79 (m, 2H), 1.07 (t, J = 7.2 Hz, 3H). HRMS calcd for C23H18N2O4 [M+H]+: 387.1339, found 387.1340.

4-(Butoxycarbonyl)phenyl phenazine-1-carboxylate (5n)

Yellow solid; yield: 97.5%; m.p. 119–120 °C; 1H-NMR (600 MHz, CDCl3) δ 8.52–8.44 (m, 2H), 8.36–8.31 (m, 1H), 8.30–8.25 (m, 1H), 8.23–8.18 (m, 2H), 7.96–7.89 (m, 3H), 7.55–7.51 (m, 2H), 5.33–5.28 (m, 1H), 1.41 (d, J = 6.6 Hz, 6H). HRMS calcd for C23H18N2O4 [M+H]+: 387.1339, found 387.1340.

4-(Butoxycarbonyl)phenyl phenazine-1-carboxylate (5o)

Yellow solid; yield: 99.0%; m.p. 89–90 °C; 1H-NMR (600 MHz, CDCl3) δ 8.53–8.39 (m, 2H), 8.36–8.31 (m, 1H), 8.29–8.25 (m, 1H), 8.24–8.19 (m, 2H), 7.96–7.87 (m, 3H), 7.56–7.51 (m, 2H), 4.38 (t, J = 6.6 Hz, 2H), 1.88–1.76 (m, 2H), 1.57–1.48 (m, 2H), 1.02 (t, J = 7.2 Hz, 3H). HRMS calcd for C24H20N2O4 [M+H]+: 401.1496, found 401.1497.

4-(Octyloxycarbonyl)phenyl phenazine-1-carboxylate (5p)

Yellow solid; yield: 97.1%; m.p. 57–59 °C; 1H-NMR (600 MHz, CDCl3) δ 8.60–8.40 (m, 2H), 8.43–8.31 (m, 1H), 8.31–8.24 (m, 1H), 8.25–8.18 (m, 2H), 8.02–7.85 (m, 3H), 7.63–7.46 (m, 2H), 4.36 (t, J = 6.6 Hz, 2H), 1.88–1.76 (m, 2H), 1.53–1.43 (m, 2H), 1.42–1.26 (m, 8H), 0.91 (t, J = 6.6 Hz, 3H). HRMS calcd for C28H28N2O4 [M+H]+: 457.2122, found 457.2123.

Biological assays

Compounds were screened for their in vitro fungicidal activity against Rhizoctonia solani, Fusaium graminearum, Altemaria solani, Fusarium oxysporum, Sclerotinia sclerotiorum and Pyricularia oryzae with the mycelium growth rate test.

The method for testing the primary biological activity was performed aseptically with pure cultures. Synthesized compounds were dissolved in 100% acetone, and the solutions were diluted with aqueous 1% Tween 80 and were then added to sterile potato dextrose agar (PDA). The target final concentration of each compound was 50 μg/mL. The control blank assay was performed with 1 mL of sterile water. Mycelial plugs 6 mm in diameter were obtained with a cork borer and placed on the amended PDA. The culture plates were incubated at 28 °C. The diameter of the mycelia was measured after 72 h. Acetone in sterile aqueous 1% Tween 80 served as the negative control, whereas phenazine-1-carboxylic acid served as positive controls. Each sample was screened with three replicates, and each colony diameter of the three replicates was measured four times. All statistical analysis was performed using EXCEL 2010 software. The log dose–response curves allowed determination of the EC50 for the bioassay using probit analysis. The 95% confidence limits for the range of EC50 values were determined by the least-square regression analysis of the relative growth rate (% control) against the logarithm of the compound concentration. The relative inhibition rate of the circle mycelium compared to blank assay was calculated via the following equation:
$${\text{Relative}}\;{\text{inhibition}}\;{\text{rate}}\;\left( \% \right) = \left[ {{{\left( {{\text{CK}} - {\text{PT}}} \right)} \mathord{\left/ {\vphantom {{\left( {{\text{CK}} - {\text{PT}}} \right)} {\left( {{\text{CK}} - 6\;{\text{mm}}} \right)}}} \right. \kern-0pt} {\left( {{\text{CK}} - 6\;{\text{mm}}} \right)}}} \right] \times 100\%$$
where CK is the extended diameter of the circle mycelium during the blank assay; and PT is the extended diameter of the circle mycelium during testing.

Plant materials and fungal growth condition

Seeds of rice (Feng liang you xiang No. 1), with high rates of germination, were grown in plastic pots of 20 cm diameter and kept in a greenhouse under a temperature of 26–28 °C, with 10 plant per pot. After 4 weeks the four-leaf stage plants were used in the experiments. Rhizoctonia solani was cultured for 4 days at 28 °C on potato dextrose agar (PDA), under aseptic conditions. Spore concentration was adjusted with sterile distilled water to 105 spores/mL.

Chemical treatment of plants

Chemical treatments of plants were carried out as described by Makandar and others [3435]. Briefly, a stock solution of 10 mmol/L for testing conjugate 5c (highest fungicidal activity against Rhizoctonia solani) was prepared in water and diluted to a final concentration of 200 μmol/L. Rice plants at the four-leaf of the similar size were sprayed with a concentration of 200 μmol/L of test conjugate 5c, PCA and of salicylic acid (SA). A blank water control was also applied under the same conditions. There were four treatments as follows: (1) PCA, (2) SA, (3) conjugate 5c, and water-treated control. Each treatment consisted of three pots each containing 10 rice seedlings, and were arranged in a completely randomized design and replicated four times. In all treatments, spraying was done 24 h prior to inoculation.

Fungal inoculation and disease rating

Plants were treated with chemicals and 24 h later, point inoculations of rice leaf sheaths were done with needle injection of 10 μL of the 105 spores/mL suspension at the four-leaf stage of seedlings of rice. For each replication of each treatment, 30 leaf sheaths were inoculated. The inoculated plants were covered with black plastic bags and kept in a growth room maintained at 90% relative humidity near 90% at 26–28 °C for 24 h. Plants were evaluated for rice sheath blight disease as percent leaf sheath infected with Rhizoctonia solani at 14 days after inoculation. All statistical analyses were performed using EXCEL 2010 software. The disease reduction was calculated as follows:
$${\text{Disease}}\;{\text{reduction}}\;\left( \% \right) = \left[ {{{\left( {{\text{CK}} - {\text{PT}}} \right)} \mathord{\left/ {\vphantom {{\left( {{\text{CK}} - {\text{PT}}} \right)} {\text{CK}}}} \right. \kern-0pt} {\text{CK}}}} \right] \times 100\%$$
where CK is the percent disease in inoculated plants treated with water while PT is the disease rating for inducer treatments.

Conclusions

In summary, we prepared 16 novel hydroxybenzoic acid ester conjugates of phenazine-1-carboxylic acid and investigated their biological activity. Most of the synthetic conjugates displayed some level of fungicidal activity in vitro against five phytopathogenic fungi. In particular, nine conjugates 5b, 5c, 5d, 5e, 5h, 5i, 5m, 5n and 5o (EC50 values were between 3.2 μg/mL and 14.1 μg/mL) were more active than PCA (EC50 value was 18.6 μg/mL) against Rhizoctonia solani, and conjugate 5c had the highest fungicidal activity, 6.5-fold greater than PCA. The results of the bioassay indicated that the fungicidal activity of conjugates is associated with their LogP, and the optimal LogP values of the more potent fungicidal activity within these conjugates ranged from 4.42 to 5.08. The test of systemic acquired resistance against rice sheath blight disease in rice seedlings revealed that PCA–SA ester conjugate 5c retains the resistance induction activity of SA to rice sheath blight, and has higher activity than SA. Meanwhile, the mechanism of systemic acquired resistance against rice sheath blight in rice seedlings by PCA–SA ester conjugate 5c will be the focus of our next study.

Declarations

Authors’ contributions

The current study is an outcome of constructive discussion with JL and XZ. XZ synthesized the compounds and carried out most of the bioassay experiments. LY, MZ and ZY did part of the bioassay experiments. XZ took part in the compound structural elucidation and bioassay experiments. ZX and QW carried out some structure elucidation experiments. JL was the principle investigator of the project and provided the research funding. XD is the co-corresponding author for this work. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Availability of data and materials

We have presented all our main data in the form of tables and figures. Meanwhile, all the copies of 1H NMR and HRMS for the title compounds were presented in the Additional file.

Funding and acknowledgements

The authors gratefully acknowledge Grants from the National Natural Science Foundation of China (No. 31672069) and Natural Science Foundation of Hubei Province (No. 2014CFA105).

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/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://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Hubei Collaborative Innovation Centre for Grain Industry, Yangtze University, Jingmi Road 88, Jingzhou, 434025, China
(2)
School of Agriculture, Yangtze University, Jingmi Road 88, Jingzhou, 434025, China
(3)
Engineering Research Center of Ecology and Agricultural Use of Wetland, Ministry of Education, Yangtze University, Jingmi Road 88, Jingzhou, 434025, China

References

  1. Laursen JB, Nielsen J (2004) Phenazine natural products: biosynthesis, synthetic analogues, and biological activity. Chem Rev 104:1663View ArticleGoogle Scholar
  2. Guttenberger N, Blankenfeldt W, Breinbauer R (2017) Recent developments in the isolation, biological function, biosynthesis, and synthesis of phenazine natural products. Bioorg Med Chem 25:6149–6166View ArticleGoogle Scholar
  3. Messenger AJM, Turner JM (1983) Phenazine-1, 6-dicarboxylate and its dimethyl ester as precursors of other phenazines in bacteria. FEMS Microbiol Lett 18:65–68View ArticleGoogle Scholar
  4. Pusecker K, Laatsch H, Helmke E, Weyland H (1997) Dihydrophencomycin methyl ester, a new phenazine derivative from a marine Streptomycetet. J Antibiot 50:479–483View ArticleGoogle Scholar
  5. Puopolo G, Masi M, Raio A, Andolfi A, Zoina A, Cimmino A, Evidente A (2013) Insights on the susceptibility of plant pathogenic fungi to phenazine-1-carboxylic acid and its chemical derivatives. Nat Prod Res 27:956–966View ArticleGoogle Scholar
  6. Bigge CF, Elslager EF, French JC, Graham BD, Hokanson GC, Mamber SW, Smitka TA, Tunac JB, Wilton JH (1987) Antimicrobial and antitumor phenazine carboxaldehydes and derivatives. US 4657909Google Scholar
  7. Palchykovska LG, Vasylchenko OV, Platonov MO, Kostina VG, Babkina MM, Tarasov OA, Starosyla DB, Samijlenko SP, Rybalko SL, Deriabin OM, Hovorun DM (2012) Evaluation of antibacterial and antiviral activity of N-arylamides of 9-methyl and 9-methoxyphenazine-1-carboxylic acids - inhibitors of the phage T7 model transctiption. Biopolym Cell 28:477–485View ArticleGoogle Scholar
  8. Udumula V, Endres JL, Harper CN, Jaramillo L, Zhong HA, Bayles KW, Conda-Sheridan M (2016) Simple synthesis of endophenazine G and other phenazines and their evaluation as anti-methicillin-resistant Staphylococcus aureus agents. Eur J Med Chem 125:710–721View ArticleGoogle Scholar
  9. Rewcastle GW, Denny WA, Baguley BC (1987) Potential antitumor agents. 51. Synthesis and antitumor activity of substituted phenazine-1-carboxamides. J Med Chem 30:843–851View ArticleGoogle Scholar
  10. Mekapati SB, Denny WA, Kurupa A, Hanscha C (2001) QSAR of anticancer compounds. bis(11-oxo-11H-indeno[1,2-b]quinoline-6-carboxamides), bis(phenazine-1-carboxamides) and bis(naphthalimides). Bioorg Med Chem 9:2757–2762View ArticleGoogle Scholar
  11. Spicer JA, Gamage SA, Rewcastle GW, Finlay GJ, Bridewell DJA, Baguley BC, Denny WA (2000) Bis(phenazine-1-carboxamides): structure-activity relationships for a new class of dual topoisomerase I/II-directed anticancer drugs. J Med Chem 43:1350–1358View ArticleGoogle Scholar
  12. Gamage SA, Rewcastle GW, Baguley BC, Charltonb PA, Denny WA (2006) Phenazine-1-carboxamides: structure-cytotoxicity relationships for 9-substituents and changes in the H-bonding pattern of the cationic side chain. Bioorg Med Chem 14:1160–1168View ArticleGoogle Scholar
  13. Gupta A, Jaiswal A, Prachnad S (2014) Quantitative structure activity relationship analysis of N-substituted phenazine-1-carboxamides analogs as anti-mycobacterial agents. Int J Pharm Life Sci 5:3230–3240Google Scholar
  14. Logua AD, Palchykovska LH, Kostina VH, Sanna A, Meleddu R, Chisu L, Alexeeva IV, Shved AD (2009) Novel N-aryl- and N-heteryl-phenazine-1-carboxamides as potential agents for the treatment of infections sustained by drug-resistant and MDR Mycobacterium tuberculosis. Int J Antimicrob Agents 33:223–229View ArticleGoogle Scholar
  15. Commare RR, Nandakumar R, Kandan A, Suresh S, Bharathi M, Raguchander T, Samiyappan R (2002) Pseudomonas fluorescens based bio-formulation for the management of sheath blight disease and leaffolder insect in rice. Crop Prot 21:671–677View ArticleGoogle Scholar
  16. Zhou Q, Su J, Jiang H, Huang X, Xu Y (2010) Optimization of phenazine-1-carboxylic acid production by a gacA/qscR-inactivated Pseudomonas sp. M18GQ harboring pME6032Phz using response surface methodology. Appl Microbiol Biot 86:1761–1773View ArticleGoogle Scholar
  17. Ye L, Zhang H, Xu H, Zou Q, Cheng C, Dong D, Xu Y, Li R (2010) Phenazine-1-carboxylic acid derivatives: design, synthesis and biological evaluation against Rhizoctonia solani Kuhn. Bioorg Med Chem Lett 20:7369–7371View ArticleGoogle Scholar
  18. Su JJ, Zhou Q, Zhang HY, Li YQ, Huang XQ, Xu YQ (2010) Medium optimization for phenazine-1-carboxylic acid production by a gacA qscR double mutant of Pseudomonas sp. M18 using response surface methodology. Bioresour Technol 101:4089–4095View ArticleGoogle Scholar
  19. Chin-A-Woeng TFC, Bloemberg GV, Lugtenberg BJJ (2003) Phenazines and their role in biocontrol by Pseudomonas bacteria. New Phytol 157:503–523View ArticleGoogle Scholar
  20. Li B, Lu L, Sun Q, Zhu QD, Li ZN, Wang G (2016) CN105418518AGoogle Scholar
  21. Wang MZ, Xu H, Yu SJ, Feng Q, Wang SH, Li ZM (2010) Synthesis and fungicidal activity of novel aminophenazine-1-carboxylate derivatives. J Agric Food Chem 58:3651–3660View ArticleGoogle Scholar
  22. Gaffney T, Friedrich L, Vernooij B, Negrotto D, Nye G, Uknes S, Ward E, Kessmann H, Ryals J (1993) Requirement of salicylic acid for the induction of systemic acquired resistance. Science 261:754–756View ArticleGoogle Scholar
  23. Poóra P, Takács Z, Bela K, Czékus Z, Szalai G, Tari I (2017) Prolonged dark period modulates the oxidative burst and enzymatic antioxidant systems in the leaves of salicylic acid-treated tomato. J Plant Physiol 213:216–226View ArticleGoogle Scholar
  24. Bernsdorff F, Döring AC, Gruner K, Schuck S, Bräutigam A, Zeier J (2016) Pipecolic acid orchestrates plant systemic acquired resistance and defense priming via salicylic acid-dependent and -independent pathways. Plant Cell 28:102–129PubMedGoogle Scholar
  25. Silverman FP, Petracek PD, Heiman DF, Fledderman CM, Warrior P (2005) Salicylate activity. 3. Structure relationship to systemic acquired resistance. J Agric Food Chem 53:9775–9780View ArticleGoogle Scholar
  26. Safari S, Soleimani MJ, Mohajer A, Fazlikhani L (2013) Possible structure-activity profile of salicylate derivatives: their relationship on induction of systemic acquired resistance. J Agric Technol 9:1215–1225Google Scholar
  27. Cueva C, Moreno-Arribas MV, Martín-Álvarez PJ, Bills G, Vicente MF, Basilio A, Rivas CL, Requena T, Rodríguez JM, Bartolome B (2010) Antimicrobial activity of phenolic acids against commensal, probiotic and pathogenic bacteria. Res Microbiol 161:372–382View ArticleGoogle Scholar
  28. Chong KP, Rossall S, Atong M (2009) In vitro antimicrobial activity and fungitoxicity of syringic acid, caffeic acid and 4-hydroxybenzoic acid against Ganoderma Boninense. J Agric Sci 1:15–20Google Scholar
  29. Kosová M, Hrádková I, Mátlová V, Kadlec D, Šmidrkal J, Filip V (2015) Antimicrobial effect of 4-hydroxybenzoic acid ester with glycerol. J Clin Pharm Ther 40:436–440View ArticleGoogle Scholar
  30. Özçelik B, Kartal M, Orhan I (2011) Cytotoxicity, antiviral and antimicrobial activities of alkaloids, flavonoids, and phenolic acids. Pharm Biol 49:396–402View ArticleGoogle Scholar
  31. Flausino OA Jr, Dufau L, Regasini LO, Petrônio MS, Silva DH, Rose T, Bolzani VS, Reboud-Ravaux M (2012) Alkyl hydroxybenzoic acid derivatives that inhibit HIV-1 protease dimerization. Curr Med Chem 19:4534–4540View ArticleGoogle Scholar
  32. Manuja R, Sachdeva S, Jain A, Chaudhary J (2013) A comprehensive review on biological activities of P-hydroxy benzoic acid and its derivatives. Int J Pharm Sci Rev Res 22:109–115Google Scholar
  33. Price-Whelan A, Dietrich LEP, Newman DK (2007) Pyocyanin alters redox homeostasis and carbon flux through central metabolic pathways in Pseudomonas aeruginosa PA14. J Bacteriol 189:6372–6381View ArticleGoogle Scholar
  34. Makandar R, Nalam VJ, Lee H, Trick HN, Dong Y, Shah J (2012) Salicylic acid regulates basal resistance to Fusarium head blight in wheat. Mol Plant Microbe Interact 25:431–439View ArticleGoogle Scholar
  35. Sorahinobar M, Niknam V, Ebrahimzadeh H, Soltanloo H, Behmanesh M, Enferadi ST (2016) Central role of salicylic acid in resistance of wheat against Fusarium graminearum. J Plant Growth Regul 35:477–491View ArticleGoogle Scholar

Copyright

© The Author(s) 2018

Advertisement