Synthesis and bioactivities of Phenazine-1-carboxylic acid derivatives based on the modification of PCA carboxyl group

Article abstract of DOI:10.1016/j.bmcl.2017.03.011

Phenazine-1-carboxylic acid (PCA) as a natural product widely exists in microbial metabolites of Pseudomonads and Streptomycetes and has been registered for the fungicide against rice sheath blight in China. To find higher fungicidal activities compounds and study the effects on fungicidal activities after changing the carboxyl group of PCA, we synthesized a series of PCA derivatives by modifying the carboxyl group of PCA and their structures were confirmed by ¹H NMR and HRMS. Most compounds exhibited significant fungicidal activities in vitro. In particular, compound 6 exhibited inhibition effect against Rhizoctonia solani with EC₅₀ values of 4.35?mg/L and compound 3b exhibited effect against Fusarium graminearum with EC₅₀ values of 8.30?mg/L, compared to the positive control PCA with its EC₅₀ values of 7.88?mg/L (Rhizoctonia solani) and 127.28?mg/L (Fusarium graminearum), respectively. The results indicated that the carboxyl group of PCA could be modified to be amide group, acylhydrazine group, ester group, methyl, hydroxymethyl, chloromethyl and ether group etc. And appropriate modifications on carboxyl group of PCA were useful to extend the fungicidal scope.

Full text of DOI:10.1016/j.bmcl.2017.03.011

Accepted Manuscript
Synthesis and bioactivities of Phenazine-1-carboxylic acid derivatives based on
the modification of PCA carboxyl group
Zhipeng Xiong, Junfan Niu, Hao Liu, Zhihong Xu, Junkai Li, Qinglai Wu
PII:
S0960-894X(17)30248-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.03.011
BMCL 24763
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
15 December 2016
19 February 2017
6 March 2017
Please cite this article as: Xiong, Z., Niu, J., Liu, H., Xu, Z., Li, J., Wu, Q., Synthesis and bioactivities of Phenazine-1-
carboxylic acid derivatives based on the modification of PCA carboxyl group, Bioorganic & Medicinal Chemistry
Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.03.011
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Synthesis and bioactivities of Phenazine-1-carboxylic acid
derivatives based on the modification of PCA
carboxyl group
Zhipeng Xiong, Junfan Niu, Hao Liu, Zhihong Xu, Junkai Li*, Qinglai Wu*
School of Agricultural, Yangtze University, Jingmi Road 88, Jingzhou 434025, China
*Corresponding author. Tel/Fax: +86 716-8066314.
E-mail address: wql106@163.com (Qinglai Wu).
*Corresponding author. Tel/Fax: +86 716-8066314.
E-mail address: junkaili@sina.com (Junkai Li).

Abstract: Phenazine-1-carboxylic acid (PCA) as a natural product widely exists
in microbial metabolites of Pseudomonads and Streptomycetes and has been
registered for the fungicide against rice sheath blight in China. To find higher
fungicidal activities compounds and study the effects on fungicidal activities after
changing the carboxyl group of PCA, we synthesized a series of PCA derivatives by
1
modifying the carboxyl group of PCA and their structures were confirmed by H
NMR and HRMS. Most compounds exhibited significant fungicidal activities in vitro.
In particular, compound 6 exhibited inhibition effect against Rhizoctonia solani with
EC50 values of 4.35 mg/L and compound 3b exhibited effect against Fusarium
graminearum with EC50 values of 8.30 mg/L, compared to the positive control PCA
with its EC50 values of 7.88 mg/L (Rhizoctonia solani) and 127.28 mg/L (Fusarium
graminearum), respectively. The results indicated that the carboxyl group of PCA
could be modified to be amide group, acylhydrazine group, ester group, methyl,
hydroxymethyl, chloromethyl and ether group et.al. And appropriate modifications on
carboxyl group of PCA were useful to extend the fungicidal scope.
Keywords: phenazine-1-carboxylic acid; carboxyl group; fungicidal activity;
derivatives; synthesis
Rice and wheat are the most important crops in the world. Rhizoctonia solani
Kuhn and Fusarium graminearum, the pathogens that cause sheath blight of rice and
Fusariumu head blight of wheat, are the most devastating pathogenic funguses
infecting rice and wheat.1-5 According to the statistics, the funguses can reduce the
grain yield and quality of crops.6-12 Unfortunately, the control of crop diseases caused
by Rhizoctonia solani Kuhn and Fusarium graminearum is still a hard problem12,13
and the intervention of agrochemicals is the most effective method at present.14,15
However, the current chemical-control agents were not fully effective in inhibition of
Rhizoctonia solani Kuhn and Fusarium graminearum.14,15 Hence, it is necessary to
develop more effective novel agents to replace the conventional agrochemicals.15, 16

As all we know, it is an effective method to develop new green agrochemicals by
introducing known active sub-structures in natural products. Phenazine-1-carboxylic
acid (PCA) (Figure1) is a very important compound widely existed in microbial
metabolites of Pseudomonads and Streptomycetes, which has been proved having
antifungal, antitumor and antileukemia effects.17,18 In recent years, PCA can be
produced by several pseudomonads and be of great significance in agricultural
application because of inhibition effects against several soil-borne fungal
phytopathogens.19-22 It has been registered as the biofungicide “Shenqinbactin” in
China and is noted for its high fungicidal efficiency, low toxicity to human and
animals, friendliness to environment and improvement of crop production.23-27
Phenazine-1-carboxylic hydrazine (Figure1), one analogue of PCA, is also derived
from metabolites of Pseudomonads and has shown potent fungicidal activities against
Botrytis cinerea, Powdery mildew, Rhizoctonia solani, Fusarium oxysporum,
Phytophthora capsici and Sclerotinia sclerotiorum with the EC50 value of 0.56 mg/L,
0.85 mg/L, 0.28 mg/L, 1.88 mg/L, 1.20 mg/L and 1.52 mg/L, respectively.28
Phenazine-1-carboxylic esters have been reported having excellent fungicidal
activities against magnaporthe oryzae.29 In our previous studies, we have synthesized
a series of PCA-amino acid ester conjugates (Figure1) by conjugating PCA with
different amino-acid esters and found that some of them showed better results against
Rhizoctonia solani Kuhn than PCA.30
Figure 1.The structures of PCA and its analogues.
In consideration of the excellent fungicidal activities of the above compounds
and the structural feature of PCA, we synthesized a series of PCA derivatives by
modifying the carboxyl group of PCA and the fungicidal activities of all the
synthesized compounds were tested. In the present study, we found potent fungicidal

activities compounds and discussed the primary structure-activity relationship.
Synthesis of the PCA derivatives was achieved in Scheme 1. Treatment of PCA
with oxalyl chloride at reflux temperature in CH2Cl2 solution afforded intermediate 2
after the evaporation of CH2Cl2;30 Subjecting 2 to the corresponding amines led to
3a-3d31,32 and to the hydrazides led to 4a-4b.28 Compounds 2 was treated with the
corresponding alcohols to afford the esters 5a-5c;29 Reduction of PCA methyl ester
(5a) or ethyl ester (5b) with lithium aluminum hydride afforded
phenazin-1-ylmethanol 7,33 which was reacted with 2-chloropropanoyl-chloride in
CH2Cl2 led to phenazin-1-ylmethyl-2-chloropropanoate 9 or with thionyl chloride
afforded 1-(chloromethyl)-phenazine 10.34 Meanwhile, Compounds 6 was obtained by
the reduction of compounds 5a-5b with aluminum chloride as catalyst.35 Oxidization
of phenazin-1-ylmethanol 7 with potassium dichromate in acetone afforded
phenazine-1-carbaldehyde 8.36
Treating 1-(chloromethyl)-phenazine 10 with sodium alkoxide led to the
corresponding 1-(alkoxymethyl)-phenazines 11a-11b or with amines gave
1-(phenazin-1-yl)-methanamines 12a-12c.34 (Analysis data of all the synthesized
compounds are available in Supplementary Information).
R1
R2
R1
CH2NHR6
CH3
CON
CH2N
CHO
R2
(e)
N
N
N
N
N
N
N
N
6
N
N
8
3a~3d
12a~12c
(f)
(k)
(c)
COOR4
CH2OR5
(h)
CH2Cl
CH2OH
COOH
COCl
N
N
N
N
N
N
N
(e)
(g)
(b)
(j)
(a)
N
N
N
N
N
2
11a~11b
10
1
7
5a~5c
(d)
(i)
CONHNHR3
CH2O
N
N
Cl
O
N
N
4a~4b
9
12a: R6=H
11a: R5=CH3
11b: R5=C2H5
4a: R3=H
3a: R1=H, R2=H
5a: R4=CH3
5b: R4=C2H5
12b: R6=CH3
4b: R3=t-Bu
3b: R1=H, R2=CH3
3c: R1=H, R2=i-Pr
3d: R1=H, R2=C6H5
12c: R6=i-Pr
5c: R4=3-C8H17
Scheme 1 Synthetic route of target compounds. Reagents and conditions: (a) Oxalyl chloride,
CH2Cl2, DMF, reflux, 8h; (b) Alcohol, 20℃ to reflux, 30min; (c) Amine, CH2Cl2, 0℃ to reflux,

2h; (d) Amines, CH2Cl2, 0℃ to room temperature, 4h; (e) LiAlH4, THF, 0℃ to reflux, 4h; (f)
AlCl3, LiAlH4, THF, 0℃ to reflux, 8h; (g) SOCl2, CH2Cl2, reflux, 6h; (h) K2Cr2O7, 20%H2SO4,
actone, room temperature to reflux, 1h; (i) Chloroacetyl chloride, CH2Cl2, 0℃ to reflux, 2h; (j)
Sodium alkoxide, alcohol, 60~80℃, 3h; (k) Amine, CH2Cl2, room temperature to reflux, 6h.
In a standard primary screen, all the synthesized compounds 3a-12c were
evaluated for their fungicidal activity in vitro against Rhizoctonia solani, Fusarium
graminearum, Walnut root rot, Rhizoctonia solani Kühn, and Sclerotinia sclerotiorum
(Lib.) de Bary by using a plate method (PDA medium).30,37 The results of preliminary
bioassay showed that all compounds exhibited certain fungicidal activities against the
pathogenic fungi at the concentration of 50 mg/L. In particular, compound 3a, 3b, 4a,
5a, 5b, 6, 7, 10 and 11b exhibited bioactivity more potent than PCA against
Rhizoctonia solani and Fusarium graminearum, and the result indicated that the
carboxyl group of PCA could be modified to amide group, acylhydrazine group, ester
group, methyl, hydroxymethyl, chloromethyl and ether group. By comparing the
structures of all compounds, it was found that the derivatization of the PCA with CH3
and CH2OH instead of the groups with large steric hindrance enhanced the activities.
When comparing the inhibitory activity of the amides derivatives, the bioassay results
showed the activities rank as 3a>3b>PCA>3c>3d and the hydrophobicity of the
amines played an important role in fungicidal activities. The activities enhanced as the
hydrophobicity of the amines increased appropriately among a certain level.
Compounds 3a, 11b exhibited the most potent fungicidal activities against
Rhizoctonia solani with the inhibitory rates of 96.6%, 98.6% at the concentration of
50 mg/L, and better than PCA. In addition, the fungicidal activities increased
significantly when carboxyl group of PCA was modified to be amide group or ether
group, which provided helpful research ideas for the design of new members of this
type derivatives as well as biological studies of these molecules and their analogues.
Interestingly, PCA had poor fungicidal activity against Fusarium graminearum, but
most of the compounds exhibited better fungicidal activities against Fusarium
graminearum than PCA. In particular, compound 3a, 3b exhibited the most potent
bioactivity against Fusarium graminearum with the inhibitory rate of 100% at the
concentration of 50 mg/L, which indicated that the appropriate modifications on

carboxyl group of PCA were useful to extend the fungicidal scope and the derivatives
with amide groups showed a good prospect in developing new fungicidal agents.
Table 1
Inhibitory rates of PCA derivatives against five pathogenic fungi in vitro
Rhizoctonia
solani
(%)
Fusarium
graminearum
(%)
Walnut root
rot
Rhizoctonia
solani Kühn
(%)
Sclerotinia
sclerotiorum
(%)
Compound*
(%)
PCA
3a
3b
3c
83.3
96.6
83.5
75.0
70.6
89.8
70.1
88.8
83.9
54.4
93.3
88.8
76.5
30.9
85.4
79.4
98.6
77.2
81.3
72.6
32.9
66.9
68.5
58.2
37.3
33.3
34.7
26.0
33.3
34.0
26.8
53.8
59.7
62.7
30.3
44.3
39.1
41.3
45.0
47.8
42.3
50.9
11.6
100
57.9
46.3
100
57.1
45.5
52.7
36.4
23.2
39.0
22.0
2.5
3d
4a
4b
5a
5b
5c
58.9
28.2
7.5
68.5
29.5
40.5
50.7
31.2
10.8
34.3
29.5
11.6
38.4
26.0
5.8
75.3
37.2
20.7
6
85.0
47.6
28.1
7
45.2
52.8
12.4
8
17.8
39.5
8.3
9
58.9
38.1
26.5
10
11a
11b
12a
12b
12c
63.7
37.3
14.9
49.3
33.8
12.4
33.6
40.7
26.5
35.6
52.2
33.8
29.5
38.9
21.6
*at the concentration of 50 mg/L
Compounds 3a, 3b, 6 and 7 were chosen to determine EC50 values30,37 against
Rhizoctonia solani and Fusarium graminearum, because these compounds showed
fungicidal activities with the inhibitory rate of over 75% against the two pathogenic
fungi at the concentration of 50 mg/L. Compound 11b was also chosen to determine
EC50 value because of its best fungicidal activities against Rhizoctonia solani. The
results were shown in Table 2. Especially, compound 6 exhibited the best activity
among all the target compounds against Rhizoctonia solani with the EC50 value of
4.35 mg/L and compound 3b exhibited the best activity among all the target
compounds against Fusarium graminearum with the EC50 value of 8.30 mg/L. Their

activities were more potent than that of the positive control PCA with its EC50 values
of 7.88 mg/L (Rhizoctonia solani) and 127.28 mg/L (Fusarium graminearum),
respectively.
Table 2
Fungicidal activities of 3a, 3b, 6 and 7 against Rhizoctonia solani and Fusarium graminearum in
vitro
Rhizoctonia solani
Fusarium graminearum
EC50 (mg/L)
Compound
PCA
3a
3b
6
7.88 1.82
6.66 0.72
7.09 0.75
4.35 0.83
9.98 1.64
8.68 1.26
127.28 5.86
12.67 1.63
8.30 1.35
21.44 3.36
41.82 4.34
51.36 3.21
7
11b
In summary, a series of PCA derivatives were designed, synthesized and their
structures were confirmed by 1HNMR spectrum and elemental analysis measured with
HRMS. All the compounds exhibited excellent fungicidal activities in vitro. It is
observed that the carboxyl group of PCA could be modified to be amide group,
acylhydrazine group, ester group, methyl, hydroxymethyl, chloromethyl and ether
group. In addition, the derivatization of the PCA with CH3 and CH2OH instead of the
groups with large steric hindrance enhanced the activities. The structure–activity
analysis of the amides revealed that the hydrophobicity of the amines seemed to be
crucial for the fungicidal activities. And appropriate modifications on carboxyl group
of PCA were useful to extend the fungicidal scope.
Acknowledgments
The authors gratefully acknowledge the financial support from Natural Science
Foundation of China (No. 31672069) and Natural Science Foundation of Hubei
Province (No. 2014CFA105). We also thank the Key Laboratory of Natural Pesticide
and Chemical Biology, Ministry of Education, South China Agricultural University

for HRMS and 1H NMR spectrums.
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Graphical abstract
Synthesis and bioactivities of Phenazine-1-carboxylic acid derivatives
based on the modification of PCA carboxyl group
Zhipeng Xiong, Hao Liu, Junfan Niu, Zhihong Xu, Junkai Li*, Qinglai Wu*
School of Agricultural, Yangtze University, Jingmi Road 88, Jingzhou 434025, China
Pseudomonads
Streptomycetes
CH2OR5
CONHNHR3
CH2Cl
R1
N
N
CON
R2
N
N
COOH
N
N
N
CH3
N
N
N
COOR4
N
N
N
N
CH2OH
CH2O
N
N
N
Cl
O
CH2NHR6
CHO
N
N
N
N
N
The synthesis and bioactivities of Phenazine-1-carboxylic acid derivatives based on the
modification of PCA carboxyl group were studied systematically. To find higher fungicidal
activities compounds and study the effects on fungicidal activities after changing the
carboxyl group of PCA, we synthesized a series of Phenazine-1-carboxylic acid derivatives by
modifying the carboxyl group of PCA as much as possible. The results of structure–activity
analysis indicated that the carboxyl group of PCA could be modified to be amide group,
acylhydrazine group, ester group, methyl, hydroxymethyl, chloromethyl and ether group et.al.
And appropriate modification on carboxyl group of PCA was useful to extending the fungicidal
scope.