Synthesis and antifungal evaluation of PCA amide analogues

Article abstract of DOI:10.1080/10286020.2018.1461843

To improve the physical and chemical properties of phenazine-1-carboxylic acid (PCA) and find higher antifungal compounds, a series of PCA amide analogues were designed and synthesized and their structures were confirmed by ¹H NMR, HRMS, and X-

Full text of DOI:10.1080/10286020.2018.1461843

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://tandfonline.com/loi/ganp20
Synthesis and antifungal evaluation of PCA amide
analogues
Chuan Qin, Di-Ya Yu, Xu-Dong Zhou, Min Zhang, Qing-Lai Wu & Jun-Kai Li
To cite this article: Chuan Qin, Di-Ya Yu, Xu-Dong Zhou, Min Zhang, Qing-Lai Wu & Jun-Kai Li
(2018): Synthesis and antifungal evaluation of PCA amide analogues, Journal of Asian Natural
Products Research, DOI: 10.1080/10286020.2018.1461843
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Journal of asian natural Products research, 2018
https://doi.org/10.1080/10286020.2018.1461843
Synthesis and antifungal evaluation of PCA amide analogues
Chuan Qina, Di-Ya Yua, Xu-Dong Zhoub, Min Zhanga, Qing-Lai Wua and Jun-Kai Lia
aschool of agricultural, Yangtze university, Jingzhou, 434023, china; bschool of Pharmaceutical sciences,
Wenzhou Medical university, Wenzhou, 325023, china
ABSTRACT
ARTICLE HISTORY
received 18 december 2017
accepted 3 april 2018
To improve the physical and chemical properties of phenazine-
1-carboxylic acid (PCA) and find higher antifungal compounds, a
series of PCA amide analogues were designed and synthesized and
KEYWORDS
1
their structures were confirmed by H NMR, HRMS, and X-ray. Most
Phenazine-1-carboxylic acid
(Pca); amide; analogue;
antifungal activity; synthesis
compounds showed some antifungal activities in vitro. Particularly,
compound 3d exhibited inhibition effect against Pyriculariaoryzac
Cavgra with EC50 value of 28.7 μM and compound 3q exhibited effect
against Rhizoctonia solani with EC50 value of 24.5 μM, more potently
active than that of the positive control PCA with its EC50 values of
37.3 μM (Pyriculariaoryzac Cavgra) and 33.2 μM (Rhizoctonia solani),
respectively.
COOH
Pseudomonads
N
R1
N
O
R2
N
n
PCA
N
N
Metabolites
Fungicide
CONH2
N
N
Target compounds
Phenazine-1-carboxamide
or Streptomycetes
Higher fungicidal activities
1. Introduction
Phenazine-1-carboxylic acid (PCA) (Figure 1) is a very promising natural product occur-
ring in microbial metabolites of Streptomycetes and Pseudomonads, which has been proved
having antifungal, antitumor, and antileukemia effects [1,2]. Particularly, in recent years,
PCA has received significant attention extensively because of its remarkable inhibition
effects against many soil-borne fungal phytopathogens in agricultural application [3–6]. It
has been registered as the biofungicide “Shenqinbactin” in China and is noted for its high
antifungal efficiency, low toxicity to human and animals, friendliness to environment, and
improvement of crop production [7–11]. But PCA is difficult to dissolve in any solvents
CONTACT Qing-lai Wu
wql106@163.com; Jun-Kai li
junkaili@sina.com
© 2018 informa uK limited, trading as taylor & francis Group

2
C. QIN ET AL.
COOH
CONH2
N
N
N
N
Phenazine-1-carboxylicacid(PCA)
Phenazine-1-carboxamide
Figure 1. the structures of Pca and phenazine-1-carboxamide.
whcich cause it to become hard to be absorbed by the object and produce suitable dosage
form [12]. As a very important class of PCA’s derivatives, phenazine-1-carboxamines (Figure
1) also have excellent antifungal activities [13–17], such as phenazine-1-carboxamide, which
showed more potent activity against Rhizoctonia solani than Carbendazim and equal to
Rifamycin against Xanthomonas oryzae pv. oryzae at the concentration of 5 mg/L [13].
It’s well known that the appropriate oil–water amphiphilicity of a compound is a very
important factor to influence its efficacy [18,19]. To improve the physical and chemical
properties of phenazine-1-carboxylic acid (PCA) and find higher antifungal compounds,
we have designed and synthesized a series of PCA amide analogues, including fifeen novel
compounds and three known compounds (3a [16], 3p [14] and 3q [20]). Different amines,
such as aromatic amines, heterocyclic amines, aryl aliphatic amines and diamines, were
taken into consideration respect to structure and chemical diversity. e antifungal activities
of all the synthesized compounds were tested and the primary structure–activity relation-
ship was discussed.
2. Results and discussion
Synthesis of the phenazine-1-carboxamines was achieved in Scheme 1. PCA was treated
with oxalyl chloride at reflux temperature in CH2Cl2 solution for 8 hours, and intermediate
2 was afforded afer the evaporation of CH2Cl2 [21,22]. 2 was allowed to react with the cor-
responding amines to give 3a–3r [14,16]. (Analysis data of all the synthesized compounds
are available in Supplementary Information).
To determine the stereo structure of the target compounds, a single crystal of the target
compound 3p (n = 2, R1 = H, R2 = Ph) was prepared by slow evaporation of a solution
of compound 3p in ethyl acetate at room temperature. As shown in Figure 2, the crystal
data for 3p: triclinic, space group P-1, a = 11.283(2) Å, b = 11.499(2) Å, c = 14.726(3)
Å, a = 68.211(3)°, b = 74.173(3)°, g = 76.611(3)°, V = 1688.6(6) Å3, Z = 2, T = 296(2) K,
μ(Mο)=0.085 mm−1, Dcalcd. = 1.323 Mg/m3,12124 reflections measured (1.522 ≤ 2Ɵ ≤ 25.499°),
6207 unique (R(int) = 0.0269) which were used in all calculations. e final R1 was 0.0520
(I > 2 sigma (I)) and wR2 was 0.1541. Crystallographic data have been deposited with the
Cambridge Crystallographic Data Centre, and the deposition number was CCDC 1563916.
e antifungal activities of all target compounds in vitro were screened against Rhizoctonia
solani, Fusarium oxysporum, Alternaria solani, Fusarium graminearum, and Pyricularia
oryzac Cavgra [23,24], and the Log Kow value of all target compounds were calculated by
the ALOGPS 2.1 program, as shown in Table 1. e preliminary bioassay results showed
that all compounds exhibited moderate antifungal activities against the pathogenic fungi at
the concentration of 200 μM (this concentration of PCA equal to 44.8 mg/L). In particular,
compounds 3d and 3 g showed great antifungal activities against Pyricularia oryzac Cavgra

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
Figure 2. the X-ray crystal structure of 3p.
with inhibition rate of more than 85%, and compounds 3 m and 3q exhibited excellent
antifungal activities against Rhizoctonia solani with inhibition rate of more than 90%. When
n = 0, compared to 3a (R1 = H, R2 = Ph), R2 is substituted phenyl or heterocyclic group,
such as pyridinyl, isoxazolyl, and benzothiazolyl, or the H atom of NH substituted by CH3
or CH2COOC2H5, the majority of the tested antifungal activities increased to some degree,
especially compounds 3d showed the best activity (94.5%) against Pyricularia oryzac Cavgra
which was higher than PCA .e results showed that the value of “n” also affected the
activities of the target compounds. N = 0 (3a, 3b, 3c) and n = 1 (3j, 3 k, 3 l) have equiva-
lent antifungal activities, however when n increased to 2, the compounds (3p) exhibited
the best activities, and the antifungal activities declined as n = 3 (3r). Significantly, the F
atom of aryl group can help to increase the antifungal activities, such as 3d, 3 m and 3n.
Especially compound 3 m showed remarkable activity against Pyricularia oryzac Cavgra
with inhibition rate of 90.6%. In the halogenated compounds, the ortho-position substituted
by halogen (3j, 3 m) was beneficial to increase their antifungal activities.
Meanwhile, it was another very important factor in the influence factors of antifungal
activities against Rhizoctonia solani, hydrophilicity. In the compounds with “n = 1” (3j~3o),
the Log Kow value of compounds plays very important role in antifungal activities, the Log
Kow value showed 3 m < 3n < 3j < 3 l < 3 k < 3o, the antifungal activities against Rhizoctonia
solani exhibited 3 m > 3n > 3j > 3 l > 3 k > 3o, as shown in Figure 3. Compound 3q with
“n = 2” has the higher hydrophilicity than compound 3p, and showed better antifungal
activities against Rhizoctonia solani than compound 3p in which the value of “n” is 2 also.
ese compounds, such as 3d, 3 g, 3 m, and 3q, with inhibitory activities over 85% at
the concentration of 200 μM, were chosen to determine EC50 values [23,24]. e results
were shown in Table 2. Compounds 3d and 3 g showed the antifungal activities against
Pyricularia oryzac Cavgra with the EC50 values of 28.7 and 89.3 μM. Compounds 3 m and
3q exhibited the antifungal activities against Rhizoctonia solani with the EC50 values of
39.6 μM and 24.5 μM. Especially, compound 3d against Pyricularia oryzac Cavgra and 3q
against Rhizoctonia solani were more potently active than that of the positive control PCA
with its EC50 values of 33.2 μM (Rhizoctonia solani) and 37.3 μM (Fusarium graminearum),
respectively.
In this study, a series of phenazine-1-carboxamines were designed, synthesized, and
their structures were confirmed by 1HNMR spectrum, elemental analysis measured with

4
C. QIN ET AL.
Figure 3. relationship between log Kow of 3j~3o and inhibitory rates against Rhizoctonia solani.
R1
N
O
R2
COOH
COCl
n
N
N
N
N
(b)
(a)
N
1
N
2
3a~3r
3j: n=1, R1=H, R2=2-ClPh
1
2
3a:
n=0, R =H, R =Ph
n=1, R1=H, R2=3-ClPh
n=1, R1=H, R2=4-ClPh
3b: n=0, R1=H, R2=PhOPh
3k:
3l:
1
2
3c:
3d:
n=0, R =H, R =2-Cl-4-CF3Ph
3m: n=1, R1=H, R2=2-FPh
1
2
n=0, R =CH3, R =2-FPh
n=1, R1=H, R2=3-FPh
3e: n=0, R1=CH2COOC2H5, R2=Ph
3n:
3o:
n=1, R1=H, R2=PhCHCH3
n=0, R1=H, R2=4-(2-ClPy)
n=0, R1=H, R2=4-(3-ClPy)
3f:
3p: n=2, R1=H, R2=Ph
3g:
n=2, R1=H, R2=NH2
n=3, R1=H, R2=Ph
3h: n=0, R1=H, R2=3-(5-CH3isoxazolyl)
3q:
3r:
COOCH3
n=0, R1=H, R2=
3i:
S
Scheme 1. synthetic route of target compounds. reagents and conditions: (a) oxalyl chloride, ch2cl2,
dMf, reflux, 8 h; (b) amine, ch2cl2, 0 °c to reflux, 2 h.
HRMS and stereo structure determined by X-ray. All the target compounds showed the
antifungal activities in vitro. It is observed that “n” increased to 2 or R2 is substituted phenyl
and heterocyclic group or the H atom of NH substituted by alkyl et al, the majority of the

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
Table 1. inhibitory rates of Pca derivatives against five pathogenic fungi in vitro.
P. Oryzac Cavgra
F. graminearum
Compound*
Log Kow
R. solani (%) F. oxysporum (%) A. solani (%)
(%)
(%)
Pca
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
1.59
3.76
5.09
5.10
3.58
3.63
3.73
3.43
3.17
4.51
4.02
4.09
4.08
3.50
3.54
4.92
3.89
1.41
4.30
93.3
53.6
79.8
73.0
74.3
56.8
56.8
59.4
78.2
49.7
79.0
45.7
50.4
90.6
82.8
39.4
61.3
95.8
58.1
85.2
10.0
15.0
30.7
23.9
18.3
17.1
10.0
18.3
22.5
10.0
15.9
18.3
14.1
13.6
14.7
18.3
16.5
17.7
81.3
31.8
34.6
46.1
53.0
32.0
32.9
32.9
35.2
45.9
46.1
41.8
34.6
39.8
39.8
38.6
47.8
43.8
38.6
93.6
23.0
35.8
64.4
94.5
47.9
44.4
85.2
26.4
53.8
33.3
33.3
23.0
74.3
33.3
29.8
58.0
61.5
29.8
49.6
15.3
19.7
10.0
37.5
25.8
10.0
18.8
17.0
21.6
16.4
19.4
18.8
20.5
10.0
24.6
34.6
24.6
17.6
*at the concentration of 200 μM; “log Kow”was calculated by the aloGPs 2.1 program.
Table 2. antifungal activities of 3a, 3b, 6 and 7 against Rhizoctonia solani and Pyricularia oryzac cavgra
in vitro.
Rhizoctonia solani
Pyricularia oryzac Cavgra
Compound
EC50 (μM)
Pca
3d
33.2
nt
nt
39.6
24.5
37.3
28.7
89.3
nt
3g
3m
3q
nt
tested antifungal activities increased. Especially when R2 is F atom substituted aryl group,
they can increase the antifungal activities remarkably. In the halogenated compounds, the
ortho-position substituted by halogen was beneficial to increase their antifungal activities,
and the oil–water amphiphilicity of the target compounds was another very important
factor of antifungal activities against Rhizoctonia solani.
3. Experimental
3.1. General experimental procedures
e melting points are determined on a WRR melting point apparatus (Shanghai Jingke
Industrial Co. Ltd.,Shanghai, China) and are uncorrected. 1H NMR spectra were recorded
in CDCl3 solution on Bruker 600 MHz spectrometer (Bruker Co., Switzerland) and Bruker
400 MHz spectrometer (Bruker Co., Switzerland). MS data were obtained using a APEX
IV Fourier Transform Mass Spectrometry (Bruker Co., Switzerland). Chemicals and sol-
vents were obtained from Sinopharm Chemical Reagent Co.,Ltd (Beijing, China) and were
used without further purification. All fungi were obtained from the School of Agricultural,

6
C. QIN ET AL.
Yangtze University (Jingzhou, China). in-layer chromatography (TLC) was performed
on silica gel 60 F254 (Qingdao Marine Chemical Ltd., Qingdao, China).
3.2. General procedure for synthesis of phenazine-1-carbonyl chloride
To a suspension of 10 mmol of phenazine-1-carboxylic acid and 0.1 mmol of DMF in
30 ml of dry DCM, cooled at 0 °C, a solution of 15 mmol of oxalyl chloride in 20 ml of dry
DCM was added. e reaction was stirred at reflux temperature for 12 h. e mixture was
evaporated under vacuum; the residue was dissolved in 10 ml of dry DCM and used in
next step without purification.
3.3. General procedure for synthesis of PCA amide analogues 3a–3r
To a suspension of 20 mmol of the proper amides and 15 mmol of Et3 N in 30 ml of dry
DCM, cooled at 0 °C, the solution of phenazine-1-carbonyl chloride (10 mmol) in 10 ml
of dry DCM prepared in step 1) was added. e reaction was stirred at reflux temperature
for 4~6 h. e mixture was then quenched with water and 5% Na2CO3 aqueous solution,
dried over Na2SO4, filtered and concentrated in vacuo. e afforded crude was purified by
recrystallizing from the solution of EtOAc–DCM (1:1) to give pure compounds 3a–3r.
3.3.1. N-Phenylphenazine-1-carboxamide (3a)
Yellow solid in 86% yield [16]; 1H NMR (600 MHz, CDCl3): δ 13.31 (s, 1H), 9.05 (dd, J = 7.2,
1.1 Hz, 1H), 8.39 (dd, J = 8.6, 1.1 Hz, 1H), 8.25 (dd, J = 8.6, 7.2 Hz, 2H), 7.74–8.12 (m, 5H),
7.46 (t, J = 7.8 Hz, 2H), 7.03–7.32 (m, 1H). HRESIMS: m/z 300.1136 [M + H]+ (calcd for
C19H14N3O, 300.1131).
3.3.2. N-(4-Phenoxyphenyl)phenazine-1-carboxamide (3b)
1
Orange yellow solid in 94% yield; m.p.: 193.4–196.0 °C; H NMR (600 MHz, CDCl3): δ
13.29 (s, 1H), 9.05 (d, J = 7.2 Hz, 1H), 8.39 (d, J = 8.4 Hz, 1H), 8.28 (d, J = 8.4 Hz, 1H),
8.23 (d, J = 7.8 Hz, 1H), 7.81–8.02 (m, 5H), 7.32–7.41 (m, 2H), 7.09–7.18 (m, 3H), 7.07
(t, J = 8.4 Hz, 2H). HRESIMS: m/z 392.1393 [M + H]+ (calcd for C25H18N3O2, 392.1394).
3.3.3. N-(2-Chloro-4-(trifluoromethyl)phenyl)phenazine-1-carboxamide (3c)
Yellow solid in 94% yield；m.p. 201.5–203.1 °C; 1H NMR (600 MHz, CDCl3) δ 13.50 (s, 1H),
9.15 (s, 1H), 9.05 (t, J = 7.2 Hz, 1H), 8.44 (t, J = 8.4 Hz, 1H), 8.34–8.41 (m, 1H), 8.25–8.32
(m, 1H), 7.99 (t, J = 8.4 Hz, 1H), 7.88–7.96 (m, 2H), 7.61 (d, J = 8.4 Hz, 1H), 7.38 (dd, J = 8.4,
1.2 Hz, 1H). HRESIMS: m/z 402.0617 [M + H]+ (calcd for C20H12N3OF3Cl, 402.0616).
3.3.4. N-(2-Fluorophenyl)-N-methylphenazine-1-carboxamide (3d)
1
Yellow solid in 93% yield；m.p. 120.1–122.6 °C; H NMR (400 MHz, CDCl3) δ 8.35 (t,
J = 7.6 Hz, 1H), 8.17 (t, J = 7.6 Hz, 1H), 8.12 (d, J = 8.8 Hz, 1H), 7.80–7.98 (m, 3H), 7.70 (t,
J = 8.8 Hz, 1H), 6.94 (dd, J = 8.4, 1.2 Hz, 3H), 6.52 (dd, J = 8.4, 1.2 Hz, 1H), 3.66 (s, 3H).
HRESIMS: m/z 332.1193 [M + H]+ (calcd for C20H15N3OF, 332.1194).

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
3.3.5. Ethyl 2-(N-phenylphenazine-1-carboxamido)acetate (3e)
Yellow solid in 91% yield; m.p. 129.9-132.3 °C; 1H NMR (400 MHz, CDCl3) δ 8.32–8.38 (m,
1H), 8.15–8.21 (m, 1H), 8.11 (dd, J = 8.8, 1.2 Hz, 1H), 7.77–7.92 (m, 3H), 7.62–7.71 (m, 1H),
7.19 – 7.29 (m, 2H), 6.78–7.02 (m, 3H), 4.44–5.66 (m, 2H), 4.34 (q, J = 7.2 Hz, 2H), 1.38
(t, J = 7.2 Hz, 3H). HRESIMS: m/z 386.1500 [M + H]+ (calcd for C23H20N3O3, 386.1499).
3.3.6. N-(2-Chloropyridin-4-yl)phenazine-1-carboxamide (3f)
Yellow solid in 96% yield; m.p. 242.2–243.7 °C; 1H NMR (600 MHz, CDCl3) δ 13.78 (s, 1H),
9.04 (dd, J = 7.2, 1.2 Hz, 1H), 8.49 (dd, J = 8.4, 1.2 Hz, 1H), 8.38 (d, J = 5.4 Hz, 1H), 8.34 (d,
J = 8.4 Hz, 1H), 8.27 (d, J = 8.4 Hz, 1H), 7.91–8.17 (m, 4H), 7.73 (dd, J = 5.4, 1.8 Hz, 1H).
HRESIMS: m/z 335.0695 [M + H]+ (calcd for C18H12N4OCl, 335.0694).
3.3.7. N-(3-Chloropyridin-4-yl)phenazine-1-carboxamide (3g)
1
Yellow solid in 94% yield; m.p. 206.2–208.1 °C; H NMR (600 MHz, CDCl3) δ 13.52 (s,
1H), 9.12 (dd, J = 8.1, 1.5 Hz, 1H), 9.05 (dd, J = 7.1, 1.5 Hz, 1H), 8.38–8.53 (m, 2H), 8.29 (t,
J = 9.3 Hz, 1H), 8.21 (dd, J = 7.1, 1.5 Hz, 1H), 7.86–8.07 (m, 3H), 7.37 (dd, J = 8.1, 1.5 Hz,
1H). HRESIMS: m/z 335.0695 [M + H]+ (calcd for C18H12N4OCl, 335.0694).
3.3.8. N-(5-Methylisoxazol-3-yl)phenazine-1-carboxamide (3h)
Yellow solid in 95% yield; m.p.>250 °C; 1H NMR (600 MHz, CDCl3) δ 13.88 (s, 1H), 9.07
(d, J = 7.2 Hz, 1H), 8.50 (d, J = 8.4 Hz, 1H), 8.45 (d, J = 8.4 Hz, 1H), 8.32 (d, J = 8.4 Hz, 1H),
7.89–8.12 (m, 3H), 6.98 (s, 1H), 2.51 (s, 3H). HRESIMS: m/z 305.1032 [M + H]+ (calcd for
C17H13N4O2, 305.1033).
3.3.9. Methyl 2-(phenazine-1-carboxamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-
carboxylate (3i)
Red solid in 78% yield; m.p. 199.1–200.6 °C; 1H NMR (400 MHz, CDCl3) δ 14.91 (s, 1H),
9.01– 9.15 (m, 2H), 8.47 (dd, J = 8.8, 1.6 Hz, 1H), 8.25–8.34 (m, 1H), 7.90–8.04 (m, 3H), 4.03
(s, 3H), 2.88 (t, J = 4.8 Hz, 2H), 2.76 (t, J = 4.8 Hz, 2H), 1.87 (t, J = 3.2 Hz, 4H). HRESIMS:
m/z 418.1216 [M + H]+ (calcd for C23H20N3O3S, 418.1220).
3.3.10. N-(2-Chlorobenzyl)phenazine-1-carboxamide (3j)
Yellow solid in 97% yield; m.p. 168.4–170.0 °C; 1H NMR (400 MHz, CDCl3) δ 11.60 (s, 1H),
9.03 (dd, J = 7.2, 1.6 Hz, 1H), 8.40 (dd, J = 8.8, 1.6 Hz, 1H), 8.25–8.34 (m, 1H), 8.16–8.24
(m, 1H), 7.87–8.02 (m, 3H), 7.67 (dd, J = 7.2, 2.4 Hz, 1H), 7.40–7.51 (m, 1H), 7.26–7.31 (m,
2H), 5.00 (d, J = 6.0 Hz, 2H). HRESIMS: m/z 348.0895 [M + H]+ (calcd for C20H15N3OCl,
348.0898).
3.3.11. N-(3-Chlorobenzyl)phenazine-1-carboxamide (3k)
Yellow solid in 95% yield; m.p. 167.1–168.8 °C; 1H NMR (400 MHz, CDCl3) δ 11.59 (s, 1H),
9.03 (dd, J = 7.2, 1.6 Hz, 1H), 8.39 (dd, J = 8.8, 1.6 Hz, 1H), 8.24–8.33 (m, 1H), 8.13–8.24
(m, 1H), 7.79–8.04 (m, 3H), 7.58–7.79 (m, 1H), 7.38–7.58 (m, 1H), 7.20–7.38 (m, 2H), 4.99
(d, J = 6.0 Hz, 2H). HRESIMS: m/z 348.0896 [M + H]+ (calcd for C20H15N3OCl, 348.0898).

8
C. QIN ET AL.
3.3.12. N-(4-Chlorobenzyl)phenazine-1-carboxamide (3l)
1
Yellow solid in 94% yield; m.p. 159.3–160.8 °C; H NMR (400 MHz, CDCl3) δ 11.31 (d,
J = 9.6 Hz, 1H), 9.01–9.12 (m, 1H), 8.35–8.48 (m, 1H), 8.19–8.34 (m, 1H), 7.96–8.11 (m,
2H), 7.78–7.96 (m, 2H), 7.48 (t, J = 8.0 Hz, 2H), 7.39 (d, J = 8.4 Hz, 2H), 4.89 (t, J = 4.8 Hz,
2H). HRESIMS: m/z 348.0896 [M + H]+ (calcd for C20H15N3OCl, 348.0898).
3.3.13. N-(2-Fluorobenzyl)phenazine-1-carboxamide (3 m)
Yellow solid in 92% yield; m.p. 136.3–138.0 °C; 1H NMR (400 MHz, CDCl3) δ 11.58 (s, 1H),
9.04 (dd, J = 7.2, 1.2 Hz, 1H), 8.42 (dd, J = 8.8, 1.2 Hz, 1H), 8.27–8.34 (m, 1H), 8.14–8.23
(m, 1H), 7.90–8.05 (m, 3H), 7.55–7.66 (m, 1H), 7.29–7.36 (m, 1H), 7.13–7.21 (m, 2H), 4.97
(d, J = 5.6 Hz, 2H). HRESIMS: m/z 332.1191 [M + H]+ (calcd for C20H15N3OF, 332.1194).
3.3.14. N-(3-Fluorobenzyl)phenazine-1-carboxamide (3n)
Yellow solid in 93% yield; m.p. 161.0–163.0 °C; 1H NMR (400 MHz, CDCl3) δ 11.48 (s, 1H),
9.06 (dd, J = 7.2, 1.6 Hz, 1H), 8.44 (dd, J = 8.8, 1.6 Hz, 1H), 8.26–8.35 (m, 1H), 8.06–8.14
(m, 1H), 8.01 (dd, J = 8.8, 7.2 Hz, 1H), 7.86–7.96 (m, 2H), 7.28–7.43 (m, 3H), 6.99–7.10 (m,
1H), 4.93 (d, J = 5.6 Hz, 2H). HRESIMS: m/z 332.1187 [M + H]+ (calcd for C20H15N3OF,
332.1194).
3.3.15. N-(1-Phenylethyl)phenazine-1-carboxamide (3o)
1
Yellow solid in 91% yield; m.p. 161.6–163.7 °C; H NMR (400 MHz, CDCl3) δ 11.56 (d,
J = 6.8 Hz, 1H), 9.02 (dd, J = 7.2, 1.6 Hz, 1H), 8.40 (dd, J = 8.8, 1.6 Hz, 1H), 8.25–8.33 (m,
1H), 8.07– 8.14 (m, 1H), 7.90–8.01 (m, 3H), 7.61 (d, J = 7.6 Hz, 2H), 7.45 (t, J = 7.6 Hz,
2H), 7.34 (t, J = 7.6 Hz, 1H), 5.45–5.64 (m, 1H), 1.80 (d, J = 6.8 Hz, 3H). HRESIMS: m/z
328.1438 [M + H]+ (calcd for C21H18N3O, 328.1444).
3.3.16. N-Phenethylphenazine-1-carboxamide (3p)
Yellow solid in 89% yield [14]; m.p. 110.5–112.4 °C; 1H NMR (400 MHz, CDCl3) δ 11.03 (s,
1H), 9.03 (dd, J = 7.2, 1.6 Hz, 1H), 8.38 (dd, J = 8.8, 1.6 Hz, 1H), 8.25 (dd, J = 8.4, 1.2 Hz,
1H), 7.97 (dd, J = 8.4, 7.2 Hz, 1H), 7.78–7.93 (m, 2H), 7.63–7.69 (m, 1H), 7.26–7.43 (m,
5H), 4.03-4.08 (m, 2H), 3.12 (t, J = 6.8 Hz, 2H). HRESIMS: m/z 328.1441 [M + H]+ (calcd
for C21H18N3O, 328.1444).
3.3.17. N-(2-Aminoethyl)phenazine-1-carboxamide (3q)
Yellow solid in 65% yield [20]; m.p. 171.9–173.7 °C; 1H NMR (600 MHz, CDCl3) δ 11.22
(s, 1H), 8.99 (dd, J = 7.2, 1.2 Hz, 1H), 8.38 (dd, J = 8.4, 1.2 Hz, 1H), 8.22–8.30 (m, 2H), 7.96
(dd, J = 8.4, 7.2 Hz, 1H), 7.88–7.94 (m, 2H), 3.73-3.81 (m, 2H), 3.12 (t, J = 6.0 Hz, 2H).
HRESIMS: m/z 267.1241 [M + H]+ (calcd for C15H15N4O, 267.1243).
3.3.18. N-(3-Phenylpropyl)phenazine-1-carboxamide (3r)
Yellow solid in 87% yield; m.p. 124.0–125.6 °C; 1H NMR (400 MHz, CDCl3) δ 11.08 (s, 1H),
9.04 (dd, J = 7.2, 1.6 Hz, 1H), 8.41 (dd, J = 8.8, 1.6 Hz, 1H), 8.29–8.35 (m, 1H), 8.19–8.25
(m, 1H), 7.92–8.03 (m, 3H), 7.29–7.35 (m, 4H), 7.18–7.25 (m, 1H), 3.73-3.82 (m, 2H),
2.92 (t, J = 8.0 Hz, 2H), 2.12– 2.22 (m, 2H). HRESIMS: m/z 342.1596 [M + H]+ (calcd for
C22H20N3O, 342.1601).

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
9
3.4. Bioassays of fungicidal activities
e in vitro fungicidal activities of all target compounds against Rhizoctonia solani, Fusarium
graminearum, Walnut root rot, Rhizoctonia solani Kühn, and Sclerotinia sclerotiorum were
evaluated and screened by the mycelium growth rate method [23,24]. A quantity of 5 ml of
prepared solutions containing the compounds was added to the sterile potato dextrose agar
(PDA, 45 ml), and the compounds were tested at a concentration of 200 μM. Acetone in
sterile aqueous 1% Tween 60 served as the negative control. Before antifungal assaying, all
fungi species were cultivated for 2–5 days in PDA at 25 1 °C to produce new mycelium.
en, the culture medium was cut into mycelia dishes of about 6 mm diameter which were
picked up with a sterilized inoculating needle to inoculate in the center of the PDA plates.
Every treatment with three replicates was incubated at 25 1 °C in a bioclean environ-
ment for 2–7 days from different fungi. e hypha diameter was measured four times by
the cross-bracketing method, and the data were statistically analyzed. e inhibitory rates
(I) of the target compounds were calculated by the following formula: I (%) = [(C − T)/
(C − 6)] × 100, where C represents the average diameter (mm) of mycelia on untreated
PDA, and T represents the average diameter (mm) of fungi on treated PDA.
Acknowledgments
We thank the Key Laboratory of Natural Pesticide and Chemical Biology, Ministry of Education,
South China Agricultural University for HRMS and 1H NMR spectra.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
is research was financially supported by Natural Science Foundation of China [grant number
31672069].
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