Synthesis and fungicidal activity of phenazine-1-carboxylic triazole derivatives

Article abstract of DOI:10.1080/10286020.2020.1754400

A total of 15 novel-substituted 3-(benzylsulfanyl)-1H-1,2,4-triazol-5-ylamine and 10 novel-substituted 3-benzylmercapto-1,2,4-triazol derivatives were synthesized based on the natural product phenazine-1-carboxylic acid (PCA). Their structures were confirmed by ¹H-NMR, ¹³C-NMR, HRMS, and X-ray. Most substituted 3-benzylmercapto-1,2,4-triazol derivatives displayed very strong fungicidal activity against one or multiple plant pathogens in?vitro and in?vivo. Compounds 8b, 8h, and 8i showed a broad spectrum of fungicidal activity. Further field experiments indicated that compounds 8b, 8c, and 8h displayed better efficacy against rice blast (Pyricularia oryzae) than PCA. These data demonstrate that compounds 8b, 8c, and 8h are promising fungicidal candidates, deserving further studies. (Figure presented.).

Full text of DOI:10.1080/10286020.2020.1754400

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: https://www.tandfonline.com/loi/ganp20
Synthesis and fungicidal activity of phenazine-1-
carboxylic triazole derivatives
Xu-Jun Li, Wei Zhang, Chi-Na Zhao, Qing-Lai Wu, Jun-Kai Li & Zhi-Hong Xu
To cite this article: Xu-Jun Li, Wei Zhang, Chi-Na Zhao, Qing-Lai Wu, Jun-Kai Li & Zhi-Hong Xu
(2020): Synthesis and fungicidal activity of phenazine-1-carboxylic triazole derivatives, Journal of
Asian Natural Products Research, DOI: 10.1080/10286020.2020.1754400
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JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
https://doi.org/10.1080/10286020.2020.1754400
Synthesis and fungicidal activity of phenazine-1-
carboxylic triazole derivatives
ꢀ
ꢀ
Xu-Jun Li , Wei Zhang , Chi-Na Zhao, Qing-Lai Wu, Jun-Kai Li and Zhi-Hong Xu
School of Agriculture, Yangtze University, Jingzhou 434025, China
ABSTRACT
ARTICLE HISTORY
Received 27 November 2019
Accepted 7 April 2020
A total of 15 novel-substituted 3-(benzylsulfanyl)-1H-1,2,4-triazol-
5-ylamine and 10 novel-substituted 3-benzylmercapto-1,2,4-triazol
derivatives were synthesized based on the natural product phena-
zine-1-carboxylic acid (PCA). Their structures were confirmed by
1H-NMR, 13C-NMR, HRMS, and X-ray. Most substituted 3-benzyl-
mercapto-1,2,4-triazol derivatives displayed very strong fungicidal
activity against one or multiple plant pathogens in vitro and
in vivo. Compounds 8b, 8h, and 8i showed a broad spectrum of
fungicidal activity. Further field experiments indicated that com-
pounds 8b, 8c, and 8h displayed better efficacy against rice blast
(Pyricularia oryzae) than PCA. These data demonstrate that com-
pounds 8b, 8c, and 8h are promising fungicidal candidates,
deserving further studies.
KEYWORDS
Derivatives; fungicidal
activity; phenazine-1-
carboxylic acid;
synthesis; triazole
1. Introduction
Phenazine-1-carboxylic acid (PCA; Figure 1) is a microbial-derived antibiotic secreted
by fluorescent pseudomonad [1], which showed antifungal, antitumor, and antileuke-
mia effects [2]. In recent years, it has been widely concerned as high efficiency, low
toxicity, and environmental friendliness. A series of PCA amino acid esters P1
(Figure 1) exhibited strong activity against Rhizoctonia solani. In particular, some
CONTACT Zhi-Hong Xu
Xu-Jun Li and Wei Zhang contributed equally to this work.
x_u_78@sina.com
School of Agriculture, Yangtze University, Jingzhou 434025, China
ꢀ
Supplemental data for this article is available online at https://doi.org/10.1080/10286020.2020.1754400.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
X.-J. LI ET AL.
Figure 1. The structures of PCA and its analogues.
Figure 2. The design of target compounds.
compounds were between 8 and 22 times more effective than PCA [3]. PCA amide
analogue P2 was designed and exhibited strong fungicidal activity against R. solani
than PCA [4]. In previous studies, we found that the fungicidal activity was retained
and phenazine-1-carboxamine substituted showed greater fungicidal activity against
R. solani than PCA [5–8].
Triazoles have received extensive attention in the fields of insecticide, fungicide,
and herbicide [9–12]. Triazoles fungicides are characterized by high efficiency, broad
spectrum, and low residue [13–14]. Many studies showed that the introduced antifun-
gal active fragments to a molecule made it possible to screen compounds with higher
activity and broader antifungal spectrum [15,16].
In this article, we synthesized two series of phenazine-1-carboxylic triazole deriva-
tives 5a–5o and 8a–8j (Figure 2). The synthetic route of target compounds was
showed in Figure 3. Their antifungal activities were evaluated both in vitro and in

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
Figure 3. Synthetic route of target compounds. Reagents and conditions: (a) RCH2Cl, NaOH(4%)
EtOH, reflux, 20 min; (b) oxalyl chloride, CH2Cl2, DMF, reflux, 8 h; (c) pyridine, C4H8O2, 0 ꢁC, 2 h.
vivo and their structure–activity relationship (SAR) was studied. Their antifungal
characters compared with PCA were also discussed.
2. Results and discussion
To determine the stereo structure of the target compounds, a single crystal of the tar-
get compound 8b (Figure 4) was prepared by slow evaporation of a solution of com-
pound 8b in ethyl acetate at room temperature. The fungicidal activities were showed
in Table 1. Results indicated that fungicidal activities in vitro of compounds 5a–5o
were much lower than PCA. Compounds 8a–8i exhibited strong fungicidal activity
against R. solani, Altemaria solani, and Pyricularia oryzae. In particular, compound
8h exhibited more potent fungicidal activities than PCA against R. solani, Fusarium

4
X.-J. LI ET AL.
Figure 4. The X-ray crystal structure of 8b.
Table 1. Inhibitory rates of PCA derivatives against six pathogenic fungi in vitro.
Compounda R. solani (%) F. graminearum (%) A. solani (%) F. oxysporm (%) P. oryzae (%) P. capsici (%)
PCA
5a
5b
5c
5d
5e
5f
5g
5h
5i
74.1 1.0
24.1 3.8
40.0 2.1
19.4 2.9
4.7 8.1
11.0 0.8
8.5 0.9
4.7 0.4
9.4 0.3
10.0 0.8
1.3 1.5
6.6 0.4
4.1 1.3
0.6 1.1
6.0 0.7
7.5 0.5
9.7 0.5
0.9 1.1
11.3 1.8
3.5 0.5
8.2 3.1
35.7 1.9
29.8 0.5
50.8 0.5
53.3 0.2
6.6 2.2
24.1 0.8
50.5 0.6
79.3 1.8
46.7 0.8
39.5 0.5
68.8 0.7
3.0 2.0
1.7 4.0
1.0 1.1
3.7 2.0
4.0 0.9
1.0 3.1
14.6 3.2
4.0 2.2
36.9 0.5
11.3 1.4
36.6 1.4
10.3 4.7
49.2 6.8
2.7 1.3
58.2 0.2
3.8 0.6
11.1 0.4
9.2 0.7
5.7 1.4
3.1 0.7
5.7 0.0
6.9 0.8
8.0 0.7
13.0 0.7
10.0 1.3
10.3 0.6
4.2 0.6
11.5 0.6
7.3 0.9
7.7 0.4
44.8 0.4
35.2 0.2
50.2 3.2
47.9 0.2
22.6 0.7
18.8 0.3
36.4 0.2
52.5 5.6
54.4 0.2
44.1 0.6
64.3 0.2
7.5 1.1
14.3 0.5
13.2 0.5
5.0 0.5
1.1 2.7
9.6 1.8
7.9 0.7
2.9 1.6
21.1 1.7
3.2 1.1
12.5 1.7
7.1 0.4
17.1 0.7
12.9 1.1
16.4 0.5
66.1 1.1
70.7 1.1
83.6 2.5
78.6 1.1
46.4 0.9
33.2 0.6
43.6 2.1
82.5 0.7
68.6 0.6
64.6 0.4
49.6 2.5
0.0 0.0
1.1 4.7
0.0 0.0
0.0 0.0
0.0 2.3
3.7 2.5
0.0 0.0
0.4 2.7
0.4 4.2
0.0 0.0
1.1 3.3
0.0 1.2
0.0 0.0
0.0 0.0
0.0 0.0
50.4 1.4
25.9 1.4
56.9 2.4
44.5 1.4
0.0 0.0
3.3 2.1
43.8 0.2
90.1 0.6
73.7 1.1
14.6 14.4
20.6 2.6
27.6 0.7
31.2 0.6
29.4 5.0
45.3 2.1
39.4 0.3
42.9 1.2
36.5 4.2
47.1 1.6
28.8 1.3
39.4 1.5
71.8 1.9
85.9 2.3
78.8 1.3
65.9 1.6
52.3 1.6
42.9 0.3
62.3 0.4
82.9 0.9
82.4 0.8
64.7 0.6
5j
5k
5l
5m
5n
5o
8a
8b
8c
8d
8e
8f
1.0 1.1
64.8 0.7
61.8 0.5
70.4 0.4
66.5 0.2
54.8 2.5
46.2 6.4
47.2 0.3
69.4 0.4
66.1 1.0
67.8 0.4
8g
8h
8i
8j
aAt the concentration of 200 lM.
Notes: Each treatment had three replicates (mean SD). PCA was used as the positive control. The bold part show
that the EC50 values of compounds had been determined.
graminearum, A. solani, P. oryzae, and Phytophthora capsici with EC50 values of 43.3,
129.5, 46.1, 61.2, and 35.7 lM, respectively (Table 2). The preliminary SAR of the tar-
get compounds showed when the substituents were in m-position, the fungicidal

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
Table 2. Fungicidal activities of several compounds against five pathogenic fungi in vitro.
EC50(lM)
Compounda
R. solani
F. graminearum
A. solani
P. oryzae
P. capsici
PCA
8b
8c
8d
8h
8i
71.4 0.1
46.1 0.4
75.7 0.1
NT
43.3 0.5
26.1 0.7
1191.5 0.6
61.8 0.1
NT
68.1 0.1
NT
74.8 0.1
NT
77.1 0.1
82.3 0.3
61.2 0.2
NT
81.2 0.3
NT
NT
NT
35.7 1.6
NT
NT
NT
129.5 0.0
46.4 0.1
NT
NT
NT
Notes: Each treatment had three replicates (mean SD). PCA was used as the positive control. NT means not tested.
Table 3. Control efficiencies of tested compounds against P. oryzae in green house.
Compound
PCA
Concentration (mg/L)
Control efficiency (%)
25
50
25
50
25
50
25
50
25
50
59.4 0.7c
70.4 3.2b
46.5 2.3d
73.4 2.1ab
75.4 0.4ab
80.6 0.9ab
72.6 3.2ab
78.6 0.8ab
76.5 2.4ab
81.5 2.5a
8a
8b
8c
8h
Notes: Data are mean SD. Different letters in the same column indicate significant difference at P < 0.05 level by
Duncan’s new multiple range test.
activities rank as F > CH3 > Cl (8d > 8a > 8f). For the fluorine substituent benzyl
group compounds, the fungicidal activities displayed o-F > m-F > p-F (8c > 8d > 8e).
In particular, when isopropyl was introduced (8h) to R, the fungicidal activities
against all the tested pathogenic fungi increased remarkably, especially against P. cap-
sici, much higher than other substituted group and PCA. Significantly, compounds
8a–8j exhibited much higher fungicidal activities against all the tested pathogenic
fungi than the other series of compounds 5a–5o.
On the basis of fungicidal results in vitro, compounds 8a, 8b, 8c, and 8h were
selected for further bioassay for controlling rice blast (P. oryzae) on living plants in
greenhouse (Table 3). Results showed that compounds 8b, 8c, and 8h exhibited high
control efficiency with more than 72% at the concentration of 25 mg/L, which was
better than PCA (59.4%).
In summary, we have designed and synthesized two series of PCA triazoles deriva-
tives, and assayed their in vitro and in vivo fungicidal activity against six fungi.
Compounds 8a–8j exhibited great fungicidal activities against six pathogenic fungi
in vitro at the concentration of 200 lM. Particularly, compound 8h exhibited great
fungicidal activity against P. capsici with inhibitory rate at 90%. Compounds 8a, 8b,
8c, and 8h exhibited high control efficiency against rice blast in vivo at concentration
of 25 and 50 mg/L. The SAR of the target compounds revealed that the compounds
have greater bioactivity when PCA connected with 1,2,4-triazole directly as well as
different types of substituent group of benzyl and the substituent position play
important roles in fungicidal activities. The result provide lead compounds for the
further structural optimization.

6
X.-J. LI ET AL.
3. Experimental
3.1. General experimental procedures
The melting points were determined on a WRR melting point apparatus (Shanghai
1
Jingke Industrial Co. Ltd., Shanghai, China) and modified. H-NMR spectrum was
recorded in CDCl3 solution on a Bruker 400 MHz spectrometer (Bruker Co.,
Switzerland), using tetramethyl silane as an internal standard and chemical shift val-
ues (d) were given in parts per million (ppm). MS data were obtained using an
APEX IV Fourier Transform Mass Spectrometry (Bruker Co., Basel, Switzerland).
Thin-layer chromatography was performed on silica gel 60 F254 (Qingdao Marine
Chemical Ltd., Qingdao, China). Column chromatography (CC) was performed over
silica gel (200–300 mesh, Qingdao Marine Chemical Ltd.). All fungi were obtained
from the College of Agricultural, Yangtze University (Jingzhou, China). Chemicals
and solvents were purchased from commercial suppliers and were used without fur-
ther purification. Solvents and reagents were abbreviated as follow: absolute ethyl
alcohol (EtOH), dichloromethane (DCM), dimethylformamide (DMF), ethyl acetate
(EtOAc), petroleum ether (PE), pyridine (Py), hydrochloric acid (HCl), sodium bicar-
bonate (NaHCO3), potassium carbonate (K2CO3), and sodium hydroxide (NaOH).
3.2. General procedure for synthesis of benzyl-substituted 3-amino-1H-1,2,4-
triazole-5-thiols
About 20 mmol 3-amino-1H-1,2,4-triazole-5-thiol (2) and 20 ml 4% NaOH aqueous
solution were added in 50 ml EtOH. The reaction was stirred at reflux temperature
until the color become light and 24 mmol of benzyl chloride was added. Then, the
reaction was stirred at reflux temperature for another 20 min. The mixture was
quenched with DCM, and filtered to get a white solid (3) which was used in next
step without purification.
3.3. General procedure for synthesis of phenazine-1-carbonyl chloride
About 10 mmol PCA (1) and 0.1 mmol DMF were added in 30 ml dry DCM and
stirred at 0 ꢁC, and then a solution of 15 mmol oxalyl chloride in 20 ml dry DCM was
dropped. The reaction was stirred at reflux temperature for 8 h. The intermediate (4)
was afforded after evaporation of DCM and excessive oxalyl chloride, then dissolved
in 10 ml dry DCM and used in next step without purification [6, 7].
3.4. General procedure for synthesis of phenazine-1-carboxylic triazole
derivatives (5a–5o and 8a–8j)
About 10 mmol benzyl-substituted 3-amino-1H-1,2,4-triazole-5-thiols (3) and appro-
priate amount of pyridine were added in 100 ml dry EtOAc and stirred at 0 ꢁC, then
the solution of phenazine-1-carbonyl chloride (4) (20 mmol in 20 ml dry DCM) was
added slowly. The reaction was stirred at room temperature for 2 h. The mixture was
evaporated under vacuum and dissolved in DCM, then quenched with 4% K2CO3

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
aqueous solution and 4% hydrochloric acid. The organic layer was dried over
Na2SO4, filtered and concentrated in vacuo. The afforded crude was purified by silica
gel CC (petroleum ether:ethyl acetate ¼ 2:1) to give pure compounds 5a–5o [17].
Compounds 8a–8j were synthesized by the similar method.
3.4.1. N-(5-(benzylthio)-1H-1,2,4-triazol-3-yl)phenazine-1-carboxamide (5a)
1
Yellow solid; yield: 75.3%; m. p. 235.1–236.2 ꢁC. H-NMR (400 MHz, CDCl3) d 8.47
(dd, J ¼ 8.8, 1.3 Hz, 1H, Phenazine-H), 8.25–8.35 (m, 1H, Phenazine-H), 8.14–8.23
(m, 1H, Phenazine-H), 7.94–8.04 (m, 2H, Phenazine-H), 7.90 (ddd, J ¼ 11.9, 8.4,
1.7 Hz, 2H, Phenazine-H), 7.04 (d, J ¼ 7.1 Hz, 1H, Ph-H), 6.94–7.00 (m, 4H, Ph-H),
6.64 (s, 2H, 1,2,4-triazole-H, CO-NH), 3.89 (s, 2H, Ph-CH2). 13C-NMR (101 MHz,
DMSO-d6) d 167.3, 161.4, 158.1, 143.7, 143.0, 142.3, 140.2, 138.4, 134.8, 132.3, 132.2,
131.9, 130.6, 130.0, 129.9, 129.7, 128.9 (ꢂ2), 128.2 (ꢂ2), 127.1, 34.1. HRESIMS: m/z
413.1177 [M þ H]þ (calcd for C22H16N6OS, 413.1179).
3.4.2. N-(5-((2-methylbenzyl)thio)-1H-1,2,4-triazol-3-yl)phenazine-1-carboxamide (5b)
1
Yellow solid; yield: 78.2%; m. p. 198.3–199.9 ꢁC. H-NMR (400 MHz, CDCl3) d 8.46
(dd, J ¼ 8.8, 1.3 Hz, 1H, Phenazine-H), 8.26–8.34 (m, 1H, Phenazine-H), 8.18 (s, 1H,
Phenazine-H), 8.02 (d, J ¼ 1.3 Hz, 1H, Phenazine-H), 7.94 (d, J ¼ 8.8 Hz, 1H,
Phenazine-H), 7.89 (d, J ¼ 8.8 Hz, 2H, Phenazine-H), 6.81–6.92 (m, 3H, Ph-H), 6.70
(d, J ¼ 7.1 Hz, 1H, Ph-H), 6.62 (s, 2H, 1,2,4-triazole-H, CO-NH), 3.89 (s, 2H, Ph-
CH2), 2.16 (s, 3H, C-CH3). 13C-NMR (101 MHz, CDCl3) d 167.8, 161.9, 157.7, 143.8,
143.5, 142.4, 140.7, 136.6, 134.5, 133.6, 132.7, 131.3, 131.0, 130.2, 130.1, 129.9, 129.6,
129.6, 129.1, 127.4, 125.6, 33.0, 19.0. HRESIMS: m/z 427.1335 [M þ H]þ (calcd for
C23H18N6OS, 427.1336).
3.4.3. N-(5-((3-methylbenzyl)thio)-1H-1,2,4-triazol-3-yl)phenazine-1-carboxamide (5c)
1
Yellow solid; yield: 67.7%; m. p. 213.5–214.6 ꢁC. H-NMR (400 MHz, CDCl3) d 8.46
(dd, J ¼ 8.8, 1.3 Hz, 1H, Phenazine-H), 8.25–8.34 (m, 1H, Phenazine-H), 8.19 (s, 1H,
Phenazine-H), 8.01 (d, J ¼ 1.3 Hz, 1H, Phenazine-H), 7.95 (d, J ¼ 8.8 Hz, 1H,
Phenazine-H), 7.89 (d, J ¼ 8.8 Hz, 2H, Phenazine-H), 6.82–6.90 (m, 3H, Ph-H), 6.71
(d, J ¼ 7.1 Hz, 1H, Ph-H), 6.64 (s, 2H, 1,2,4-triazole-H, CO-NH), 3.88 (s, 2H, Ph-
CH2), 2.18 (s, 3H, C-CH3). 13C-NMR (101 MHz, CDCl3) d 167.8, 161.9, 157.7, 143.8,
143.5, 142.4, 140.7, 137.9, 136.9, 133.5, 132.7, 131.3, 131.0, 130.2, 130.0, 129.6, 129.4,
129.0, 128.0, 127.9, 125.8, 35.0, 21.2. HRESIMS: m/z 427.1335 [M þ H]þ (calcd for
C23H18N6OS, 427.1336).
3.4.4. N-(5-((4-methylbenzyl)thio)-1H-1,2,4-triazol-3-yl)phenazine-1-carboxamide (5d)
1
Yellow solid; yield: 82.1%; m. p. 201.1–202.7 ꢁC. H-NMR (400 MHz, CDCl3) d 8.47
(dd, J ¼ 8.7, 1.4 Hz, 1H, Phenazine-H), 8.26–8.31 (m, 1H, Phenazine-H), 8.20 (d,
J ¼ 8.4 Hz, 1H, Phenazine-H), 8.03 (dd, J ¼ 6.6, 1.3 Hz, 1H, Phenazine-H), 7.94–7.99
(m, 1H, Phenazine-H), 7.83–7.93 (m, 2H, Phenazine-H), 6.96 (dt, J ¼ 14.2, 7.1 Hz,
2H, Ph-H), 6.86 (d, J ¼ 7.1 Hz, 1H, Ph-H), 6.78 (t, J ¼ 7.5 Hz, 1H, Ph-H), 6.69 (s, 2H,
1,2,4-triazole-H, CO-NH), 3.89 (s, 2H, Ph-CH2), 2.05 (s, 3H, C-CH3). 13C-NMR
(101 MHz, DMSO-d6) d 167.4, 161.4, 158.1, 143.7, 143.1, 142.3, 140.3, 136.2, 135.2,

8
X.-J. LI ET AL.
134.8, 132.2, 132.1, 131.9, 130.6, 129.9, 129.7 (ꢂ2), 128.9 (ꢂ2), 128.6 (ꢂ2), 33.8, 20.8.
HRESIMS: m/z 427.1334 [M þ H]þ (calcd for C23H18N6OS, 427.1335).
3.4.5. N-(5-((4-methoxybenzyl)thio)-1H-1,2,4-triazol-3-yl)phenazine-1-carboxamide (5e)
1
Yellow solid; yield: 56.6%; m. p. 184.6–185.3 ꢁC. H-NMR (400 MHz, CDCl3) d 8.46
(dd, J ¼ 8.8, 1.3 Hz, 1H, Phenazine-H), 8.27–8.32 (m, 1H, Phenazine-H), 8.20 (dd,
J ¼ 8.4, 1.1 Hz, 1H, Phenazine-H), 8.03 (dd, J ¼ 6.6, 1.3 Hz, 1H, Phenazine-H),
7.99–7.93 (m, 1H, Phenazine-H), 7.90 (ddd, J ¼ 10.2, 8.4, 4.0 Hz, 2H, Phenazine-H),
6.91 (t, J ¼ 7.9 Hz, 1H, Ph-H), 6.59–6.73 (m, 4H, Ph-H, 1,2,4-triazole-H, CO-NH),
6.53 (d, J ¼ 7.5 Hz, 1H, Ph-H), 3.91 (s, 2H, Ph-CH2), 3.68 (s, 3H, C-OCH3). 13C-
NMR (101 MHz, DMSO-d6) d 167.3, 161.4, 159.3, 158.1, 143.6, 143.0, 142.3, 140.2,
139.7, 134.6, 132.2, 132.1, 132.0, 130.5, 130.2, 129.9, 129.6, 129.3, 121.1, 114.7, 112.6,
55.3, 34.2. HRESIMS: m/z 443.1284 [M þ H]þ (calcd for C23H18N6O2S, 443.1285).
3.4.6. N-(5-((2-fluorobenzyl)thio)-1H-1,2,4-triazol-3-yl)phenazine-1-carboxamide (5f)
1
Yellow solid; yield: 68.3%; m. p. 182.9–183.9 ꢁC. H-NMR (400 MHz, CDCl3) d 8.47
(dd, J ¼ 8.4, 1.7 Hz, 1H, Phenazine-H), 8.30 (dd, J ¼ 8.8, 0.8 Hz, 1H, Phenazine-H),
8.18 (dd, J ¼ 8,4, 1.2 Hz, 1H, Phenazine-H), 8.02 (dd, J ¼ 6.6, 1.3 Hz, 1H, Phenazine-
H), 7.93 (ddd, J ¼ 8.4, 7.5, 3.1 Hz, 2H, Phenazine-H), 7.84–7.90 (m, 1H, Phenazine-
H), 6.98 (ddd, J ¼ 7.1, 5.7, 2.6 Hz, 1H, Ph-H), 6.85–6.68 (m, 4H, Ph-H, 1,2,4-triazole-
H, CO-NH), 6.55 (td, J ¼ 7.5, 0.8 Hz, 1H, Ph-H), 3.91 (s, 2H, Ph-CH2). 13C-NMR
(101 MHz, DMSO-d6) d 167.4, 161.0, 158.1, 146.2, 143.7, 143.0, 142.7, 142.3, 140.2,
134.7, 132.2, 131.9, 131.1, 130.6, 130.0, 129.8, 127.5, 125.1, 125.0, 123.9, 115.4, 27.7.
HRESIMS: m/z 431.1085 [M þ H]þ (calcd for C22H15FN6OS, 431.1085).
3.4.7. N-(5-((3-fluorobenzyl)thio)-1H-1,2,4-triazol-3-yl)phenazine-1-carboxamide (5g)
1
Yellow solid; yield: 76.8%; m. p. 209.4–210.3 ꢁC. H-NMR (400 MHz, CDCl3) d 8.47
(dd, J ¼ 8.4, 1.7 Hz, 1H, Phenazine-H), 8.26–8.32 (m, 1H, Phenazine-H), 8.15–8.21
(m, 1H, Phenazine-H), 7.98 (ddd, J ¼ 15.5, 7.5, 4.4 Hz, 2H, Phenazine-H), 7.89 (dtd,
J ¼ 8.0, 6.6, 3.5 Hz, 2H, Phenazine-H), 6.88–6.94 (m, 1H, Ph-H), 6.62–6.78 (m, 5H,
Ph-H, 1,2,4-triazole-H, CO-NH), 3.86 (s, 2H, Ph-CH2). 13C-NMR (101 MHz, DMSO-
d6) d 167.3, 163.2, 161.1, 158.1, 143.6, 143.0, 142.3, 141.4, 140.2, 134.6, 132.2, 131.9,
130.5, 130.1, 129.9, 129.8, 124.9, 115.7, 115.5, 114.1, 113.9, 33.6. HRESIMS: m/z
431.1085 [M þ H]þ (calcd for C22H15FN6OS, 431.1085).
3.4.8. N-(5-((4-fluorobenzyl)thio)-1H-1,2,4-triazol-3-yl)phenazine-1-carboxamide (5h)
1
Yellow solid; yield: 75.7%; m. p. 202.1–203.2 ꢁC. H-NMR (400 MHz, CDCl3) d 8.47
(d, J ¼ 8.8 Hz, 1H, Phenazine-H), 8.30 (d, J ¼ 9.3 Hz, 1H, Phenazine-H), 8.17 (d,
J ¼ 8.4 Hz, 1H, Phenazine-H), 8.01 (d, J ¼ 5.7 Hz, 1H, Phenazine-H), 7.84–7.96 (m,
3H, Phenazine-H), 6.85 (dd, J ¼ 8.4, 5.3 Hz, 2H, Ph-H), 6.55–6.68 (m, 4H, Ph-H,
1,2,4-triazole-H, CO-NH), 3.83 (s, 2H, Ph-CH2). 13C-NMR (101 MHz, DMSO-d6) d
167.3, 161.3, 158.1, 143.7, 143.0, 142.3, 140.2, 134.8, 134.7, 133.5, 132.3, 132.2, 131.9,
130.9, 130.8, 130.6, 130.0, 129.9, 129.8, 115.0, 114.7, 33.2. HRESIMS: m/z 431.1084
[M þ H]þ (calcd for C22H15FN6OS, 431.1085).

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
9
3.4.9. N-(5-((2-chlorobenzyl)thio)-1H-1,2,4-triazol-3-yl)phenazine-1-carboxamide (5i)
1
Yellow solid; yield: 67.4%; m. p. 185.3–186.9 ꢁC. H-NMR (400 MHz, CDCl3) d 8.50
(d, J ¼ 8.8 Hz, 1H, Phenazine-H), 8.32 (d, J ¼ 8.4 Hz, 1H, Phenazine-H), 8.17 (d,
J ¼ 8.8 Hz, 1H, Phenazine-H), 8.04 (d, J ¼ 6.6 Hz, 1H, Phenazine-H), 7.84–8.01 (m,
3H, Phenazine-H), 7.11 (d, J ¼ 8.0 Hz, 1H, Ph-H), 7.04 (s, 2H, 1,2,4-triazole-H, CO-
NH), 6.93 (t, J ¼ 7.9 Hz, 1H, Ph-H), 6.74 (d, J ¼ 7.5 Hz, 1H, Ph-H), 6.61(t, J ¼ 7.5 Hz,
1H, Ph-H), 3.97 (s, 2H, Ph-CH2). 13C-NMR (101 MHz, DMSO-d6) d 167.4, 161.0,
158.2, 143.6, 143.0, 142.3, 140.2, 135.2, 134.7, 133.4, 132.2, 132.0, 131.7, 130.9, 130.5,
130.0, 123.0, 129.8, 129.8, 129.2, 126.7, 32.2. HRESIMS: m/z 447.0790 [M þ H]þ
(calcd for C22H15ClN6OS, 447.0789).
3.4.10. N-(5-((3-chlorobenzyl)thio)-1H-1,2,4-triazol-3-yl)phenazine-1-carboxamide (5j)
1
Yellow solid; yield: 67.3%; m. p. 187.4–189.1 ꢁC. H-NMR (400 MHz, CDCl3) d 8.44
(dd, J ¼ 8.8, 1.3 Hz, 1H, Phenazine-H), 8.27 (dd, J ¼ 8.4, 1.1 Hz, 1H, Phenazine-H),
8.16 (dd, J ¼ 8.4, 1.3 Hz, 1H, Phenazine-H), 8.00 (dd, J ¼ 6.6, 1.7 Hz, 1H, Phenazine-
H), 7.95 (dd, J ¼ 8.8, 6.6 Hz, 1H, Phenazine-H), 7.81–7.91 (m, 2H, Phenazine-H), 7.03
(d, J ¼ 1.7 Hz, 1H, Ph-H), 6.99 (d, J ¼ 7.9 Hz, 1H, Ph-H), 6.83 (t, J ¼ 7.9 Hz, 1H, Ph-
H), 6.74 (d, J ¼ 7.5 Hz, 3H, 1,2,4-triazole-H, CO-NH, Ph-H), 3.82 (s, 2H, Ph-CH2).
13C-NMR (101 MHz, DMSO-d6) d 167.2, 161.2, 158.1, 143.7, 143.0, 142.3, 141.1,
140.2, 134.6, 132.9, 132.3, 132.2, 132.0, 130.6, 130.1, 130.0, 129.9, 129.8, 128.7, 127.5,
127.1, 33.5. HRESIMS: m/z 447.0791 [M þ H]þ (calcd for C22H15ClN6OS, 447.0789).
3.4.11. N-(5-((4-chlorobenzyl)thio)-1H-1,2,4-triazol-3-yl)phenazine-1-carboxamide (5k)
1
Yellow solid; yield: 63.2%; m. p. 203.7–204.9 ꢁC. H-NMR (400 MHz, CDCl3) d 8.48
(dd, J ¼ 8.4, 1.7 Hz, 1H, Phenazine-H), 8.28–8.34 (m, 1H, Phenazine-H), 8.17 (dd,
J ¼ 8.4, 1.1 Hz, 1H, Phenazine-H), 7.95–8.01 (m, 2H, Phenazine-H), 7.86–7.94 (m,
2H, Phenazine-H), 6.83–6.91 (m, 2H, Ph-H), 6.76–6.82 (m, 2H, Ph-H), 6.64 (s, 2H,
1,2,4-triazole-H, CO-NH), 3.81 (s, 2H, Ph-CH2). 13C-NMR (101 MHz, DMSO-d6) d
168.4, 161.1, 158.1, 143.7, 143.0, 142.3, 141.1, 140.2, 134.6, 132.9, 132.3, 132.2, 132.0,
130.6, 130.1, 130.0, 129.9, 129.8, 128.7, 127.5, 127.1, 33.5. HRESIMS: m/z 447.0790
[M þ H]þ (calcd for C22H15ClN6OS, 447.0789).
3.4.12. N-(5-((2,4-dichlorobenzyl)thio)-1H-1,2,4-triazol-3-yl)phenazine-1-carboxamide (5l)
1
Yellow solid; yield: 52.7%; m. p. 202.1–204.2 ꢁC. H-NMR (400 MHz, CDCl3) d 8.46
(d, J ¼ 8.4 Hz, 1H, Phenazine-H), 8.29 (d, J ¼ 8.8 Hz, 1H, Phenazine-H), 8.13 (d,
J ¼ 8.4 Hz, 1H, Phenazine-H), 7.79–8.07 (m, 4H, Phenazine-H), 7.09 (d, J ¼ 1.7 Hz,
1H, Ph-H), 6.89 (s, 2H, 1,2,4-triazole-H, CO-NH), 6.55 (d, J ¼ 8.4 Hz, 1H, Ph-H),
6.42 (d, J ¼ 1.7 Hz, 1H, Ph-H), 3.87 (s, 2H, Ph-CH2). 13C-NMR (101 MHz, DMSO-d6)
d 167.3, 160.8, 158.2, 143.7, 143.0, 142.4, 140.2, 134.7, 134.5, 134.3, 132.8, 132.3,
132.2, 132.1, 132.0, 130.6, 130.0, 129.9, 129.8, 128.6, 126.7, 31.6. HRESIMS: m/z
481.0402 [M þ H]þ (calcd for C22H14Cl2N6OS, 481.0400).
3.4.13. N-(5-((2-nitrobenzyl)thio)-1H-1,2,4-triazol-3-yl)phenazine-1-carboxamide (5m)
1
Yellow solid; yield: 59.5%; m. p. 190.3–191.2 ꢁC. H-NMR (400 MHz, CDCl3) d 8.50
(dd, J ¼ 8.4, 1.7 Hz, 1H, Phenazine-H), 8.32 (dd, J ¼ 8.8, 0.8 Hz, 1H, Phenazine-H),

10
X.-J. LI ET AL.
8.15 (dd, J ¼ 8.4, 0.8 Hz, 1H, Phenazine-H), 7.97–8.03 (m, 2H, Phenazine-H),
7.85–7.96 (m, 2H, Phenazine-H), 7.79 (d, J ¼ 7.9 Hz, 1H, Ph-H), 7.08 (ddd, J ¼ 8.4,
5.7, 3.1 Hz, 1H, Ph-H), 6.75–6.82 (m, 2H, Ph-H), 6.70 (s, 2H, 1,2,4-triazole-H, CO-
NH), 4.22 (s, 2H, Ph-CH2). 13C-NMR (101 MHz, Chloroform-d) d 167.8, 161.6,
157.6, 147.9, 143.8, 143.5, 142.4, 140.6, 133.8, 133.5, 132.6, 132.5, 131.9, 131.4, 131.1,
130.2, 129.6, 129.3, 129.1, 128.0, 124.8, 32.3. HRESIMS: m/z 458.1028 [M þ H]þ
(calcd for C22H15N7O3S, 458.1030).
3.4.14. N-(5-((4-isopropylbenzyl)thio)-1H-1,2,4-triazol-3-yl)phenazine-1-carboxamide (5n)
1
Yellow solid; yield: 83.7%; m. p. 220.6–222.1 ꢁC. H-NMR (400 MHz, CDCl3) d 8.45
(d, J ¼ 8.4 Hz, 1H, Phenazine-H), 8.28 (d, J ¼ 8.2 Hz, 1H, Phenazine-H), 8.20 (d,
J ¼ 8.0 Hz, 1H, Phenazine-H), 8.01 (d, J ¼ 6.2 Hz, 1H, Phenazine-H), 7.80–7.97 (m,
3H, Phenazine-H), 6.89–6.65 (m, 6H, Ph-H, 1,2,4-triazole-H, CO-NH), 3.84 (s, 2H,
Ph-CH2), 2.71 (dt, J ¼ 13.7, 6.6 Hz, 1H, Isopropyl-H), 1.11 (d, J ¼ 7.1 Hz, 6H, C-
C2H6). 13C-NMR (101 MHz, DMSO-d6) d 167.3, 161.5, 158.1, 147.3, 143.7, 143.1,
142.4, 140.3, 135.8, 134.9, 132.3, 132.2, 131.8, 130.7, 129.9, 129.0, 125.9, 40.6, 40.4,
40.2, 40.0, 39.3, 33.7, 33.4, 24.1. HRESIMS: m/z 455.1646 [M þ H]þ (calcd for
C25H22N6OS, 455.1649).
3.4.15. N-(5-((4-(tert-butyl)benzyl)thio)-1H-1,2,4-triazol-3-yl)phenazine-1-carboxamide (5o)
1
Yellow solid; yield: 74.5%; m. p. 230.2–231.4 ꢁC. H-NMR (400 MHz, CDCl3) d 8.47
(dd, J ¼ 8.8 1.3 Hz, 1H, Phenazine-H), 8.27–8.33 (m, 1H, Phenazine-H), 8.20–8.26 (m,
1H, Phenazine-H), 8.03 (dd, J ¼ 6.6, 1.7 Hz, 1H, Phenazine-H), 7.94–7.99 (m, 1H,
Phenazine-H), 7.91 (td, J ¼ 8.0, 1.7 Hz, 2H, Phenazine-H), 6.97 (d, J ¼ 8.4 Hz, 2H, Ph-
H), 6.85 (d, J ¼ 8.4 Hz, 2H, Ph-H), 6.69 (s, 2H, 1,2,4-triazole-H, CO-NH), 3.88 (s, 2H,
Ph-CH2), 1.21(s, 9H, C-C3H9). 13C-NMR (101 MHz, DMSO-d6) d 167.3, 161.5, 158.1,
149.6, 143.7, 143.1, 142.4, 140.3, 135.5, 134.9, 132.4, 132.3, 131.8, 130.7, 130.0, 128.7,
124.8, 40.6, 40.4, 40.2, 40.0, 39.7, 39.5, 34.4, 33.5, 31.4. HRESIMS: m/z 469.1804
[M þ H]þ (calcd for C26H24N6OS, 469.1805).
3.4.16. (3-((3-Methylbenzyl)thio)-1H-1,2,4-triazol-1-yl)(phenazin-1-yl)methanone (8a)
1
Yellow solid; yield: 81.6%; m. p. 149.7–150.6 ꢁC. H-NMR (400 MHz, CDCl3) d 9.02
(s, 1H, 1,2,4-triazole-H), 8.52 (dd, J ¼ 8.8, 1.3 Hz, 1H, Phenazine-H), 8.27–8.32 (m,
1H, Phenazine-H), 8.08–8.14 (m, 2H, Phenazine-H), 7.98 (dd, J ¼ 8.8, 6.6 Hz, 1H,
Phenazine-H), 7.84–7.95 (m, 2H, Phenazine-H), 6.95 (d, J ¼ 8.0 Hz, 2H, Ph-H), 6.85
(d, J ¼ 8.0 Hz, 2H, Ph-H), 4.07 (s, 2H, Ph-CH2), 2.24 (s, 3H, C-CH3). 13C-NMR
(101 MHz, DMSO-d6) d 166.7, 164.0, 163.8, 148.3, 143.7, 143.0, 142.4, 140.6, 136.6,
134.6, 133.3, 132.5, 132.4, 132.3, 132.1, 130.5, 129.9, 129.7, 129.4, 129.1, 129.0, 34.8,
21.0. HRESIMS: m/z 412.1223 [M þ H]þ (calcd for C23H17N5OS, 412.1227).
3.4.17. (3-((4-Methylbenzyl)thio)-1H-1,2,4-triazol-1-yl)(phenazin-1-yl)methanone (8b)
1
Yellow solid; yield: 70.5%; m. p. 159.6–160.7 ꢁC. H-NMR (400 MHz, CDCl3) d 9.02
(s, 1H, 1,2,4-triazole-H), 8.52 (dd, J ¼ 8.8, 1.3 Hz, 1H, Phenazine-H), 8.30 (dd, J ¼ 8.4,
0.8 Hz, 1H, Phenazine-H), 8.08–8.12 (m, 2H, Phenazine-H), 7.98 (dd, J ¼ 8.8, 6.6 Hz,
1H, Phenazine-H), 7.89 (ddd, J ¼ 16.0, 8.8, 1.5 Hz, 2H, Phenazine-H), 6.95 (d,

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
11
J ¼ 8.0 Hz, 2H, Ph-H), 6.85 (d, J ¼ 8.0 Hz, 2H, Ph-H), 4.07 (s, 2H, Ph-CH2), 2.23 (s,
3H, C-CH3). 13C-NMR (101 MHz, CDCl3) d 164.6, 164.3, 145.6, 143.9, 143.5, 142.4,
140.8, 137.0, 133.6 (ꢂ2), 132.2, 131.5, 131.3, 130.5, 130.1, 129.7, 129.0, 128.9 (ꢂ2),
128.7 (ꢂ2), 35.5, 21.0. HRESIMS: m/z 412.1230 [M þ H]þ (calcd for
C23H17N5OS, 412.1227).
3.4.18. (3-((2-Fluorobenzyl)thio)-1H-1,2,4-triazol-1-yl)(phenazin-1-yl)methanone (8c)
1
Yellow solid; yield: 62.8%; m. p. 135.5–136.9 ꢁC. H-NMR (400 MHz, CDCl3) d 8.98
(dd, J ¼ 7.1, 1.3 Hz, 1H, Phenazine-H), 8.54 (dd, J ¼ 8.8, 1.3 Hz, 1H, Phenazine-H),
8.35 (d, J ¼ 8.4 Hz, 1H, Phenazine-H), 8.27–8.31 (m, 1H, Phenazine-H), 8.17 (s, 1H,
1,2,4-triazole-H), 7.96–8.06 (m, 3H, Phenazine-H), 7.35 (td, J ¼ 7.9, 1.7 Hz, 1H, Ph-
H), 7.18–7.25 (m, 1H, Ph-H), 7.00–7.07 (m, 2H, Ph-H), 4.40 (s, 2H, Ph-CH2). 13C-
NMR (101 MHz, CDCl3) d 166.2, 162.1, 159.6, 145.6, 144.1, 143.4, 140.1, 139.8, 137.5,
135.2, 133.3, 131.8, 131.0, 130.3, 130.1, 129.5, 128.0, 124.8, 124.2, 115.6, 115.4, 30.4.
HRESIMS: m/z 416.0976 [M þ H]þ (calcd for C22H14FN5OS, 416.0976).
3.4.19. (3-((3-Fluorobenzyl)thio)-1H-1,2,4-triazol-1-yl)(phenazin-1-yl)methanone (8d)
1
Yellow solid; yield: 57.2%; m. p. 144.2–145.7 ꢁC. H-NMR (400 MHz, CDCl3) d 9.01
(s, 1H, 1,2,4-triazole-H), 8.50 (dd, J ¼ 8.8, 1.3 Hz, 1H, Phenazine-H), 8.27
(d, J ¼ 8.4 Hz, 1H, Phenazine-H), 8.02–8.10 (m, 2H, Phenazine-H), 7.93–7.99 (m, 1H,
Phenazine-H), 7.87 (d, J ¼ 23.1 Hz, 2H, Phenazine-H), 6.93–7.01 (m, 1H, Ph-H),
6.69–6.87 (m, 3H, Ph-H), 4.05 (s, 2H, Ph-CH2). 13C-NMR (101 MHz, DMSO-d6) d
163.9, 163.5, 161.0, 148.6, 143.7, 142.9, 142.4, 140.9, 140.6, 133.3, 132.5, 132.3, 130.4,
130.3, 129.9, 129.7, 125.2, 116.0, 115.8, 114.5, 114.3, 34.5. HRESIMS: m/z 416.0974
[M þ H]þ (calcd for C22H14FN5OS, 416.0976).
3.4.20. (3-((4-Fluorobenzyl)thio)-1H-1,2,4-triazol-1-yl)(phenazin-1-yl)methanone (8e)
1
Yellow solid; yield: 59.4%; m. p. 146.3–147.1 ꢁC. H-NMR (400 MHz, CDCl3) d 9.04 (s,
1H, 1,2,4-triazole-H), 8.54 (dd, J¼ 8.8, 1.3 Hz, 1H, Phenazine-H), 8.29 (d, J¼ 8.4 Hz, 1H,
Phenazine-H), 8.09 (dd, J¼ 6.6, 1.1 Hz, 1H, Phenazine-H), 7.98–8.02 (m, 1H, Phenazine-
H), 7.90–7.98 (m, 2H, Phenazine-H), 7.83–7.87 (m, 1H, Phenazine-H), 7.74
(d, J¼ 8.8 Hz, 2H, Ph-H), 7.09 (d, J¼ 8.8 Hz, 2H, Ph-H), 4.09 (s, 2H, Ph-CH2).
13C-NMR (101 MHz, CDCl3) d 164.5, 163.4, 146.8, 145.6, 144.8, 143.9, 143.3, 142.2,
140.6, 133.7, 132.2, 131.7, 131.6, 131.0, 129.8, 129.7, 129.3 (ꢂ2), 129.0, 123.2 (ꢂ2), 34.7.
HRESIMS: m/z 416.0974 [M þ H]þ (calcd for C22H14FN5OS, 416.0976).
3.4.21. (3-((3-Chlorobenzyl)thio)-1H-1,2,4-triazol-1-yl)(phenazin-1-yl)methanone (8f)
1
Yellow solid; yield: 68.2%; m. p. 165.8–166.9 ꢁC. H-NMR (400 MHz, CDCl3) d 9.04
(s, 1H, 1,2,4-triazole-H), 8.52 (dd, J ¼ 8.8, 1.3 Hz, 1H, Phenazine-H), 8.27–8.32
(m, 1H, Phenazine-H), 8.09 (ddd, J ¼ 9.7, 7.9, 1.3 Hz, 2H, Phenazine-H), 7.99
(dd, J ¼ 8.8, 6.6 Hz, 1H, Phenazine-H), 7.84–7.95 (m, 2H, Phenazine-H), 7.15
(d, J ¼ 2.2 Hz, 1H, Ph-H), 7.06 (dd, J ¼ 5.7, 3.5 Hz, 1H, Ph-H), 6.89–6.97 (m, 2H,
Ph-H), 4.05 (s, 2H, Ph-CH2). 13C-NMR (101 MHz, CDCl3) d 164.2, 163.9, 145.6,
143.9, 143.5, 142.3, 140.8, 139.0, 134.0, 133.7, 132.0, 131.5, 131.4, 131.3, 130.0, 129.7,

12
X.-J. LI ET AL.
129.4, 129.0, 128.8, 127.4, 126.8, 35.0. HRESIMS: m/z 432.0683 [M þ H]þ (calcd for
C22H14ClN5OS, 432.0680).
3.4.22. (3-((2,4-Dichlorobenzyl)thio)-1H-1,2,4-triazol-1-yl)(phenazin-1-yl)methanone (8g)
1
Yellow solid; yield: 43.9%; m. p. 179.2–181.1 ꢁC. H-NMR (400 MHz, CDCl3) d 9.04
(s, 1H, 1,2,4-triazole-H), 8.53 (dd, J ¼ 8.8, 1.3 Hz, 1H, Phenazine-H), 8.30 (dd, J ¼ 8.8,
0.8 Hz, 1H, Phenazine-H), 8.11 (dd, J ¼ 6.6, 1.3 Hz, 1H, Phenazine-H), 7.96–8.05 (m,
2H, Phenazine-H), 7.92 (dd, J ¼ 8.4, 1.7 Hz, 1H, Phenazine-H), 7.87 (dd, J ¼ 8.8,
1.7 Hz, 1H, Phenazine-H), 7.18 (d, J ¼ 2.2 Hz, 1H, Ph-H), 6.85 (d, J ¼ 8.4 Hz, 1H, Ph-
H), 6.65 (dd, J ¼ 8.4, 2.2 Hz, 1H, Ph-H), 4.12 (s, 2H, Ph-CH2). 13C-NMR (101 MHz,
CDCl3) d 164.4, 164.0, 145.6, 143.9, 143.4, 142.3, 140.7, 134.8, 133.9, 133.5, 133.3,
132.3, 131.6, 131.5, 131.3, 130.1, 123.0, 129.7, 129.2, 129.0, 126.6, 32.8. HRESIMS: m/z
466.0289 [M þ H]þ (calcd for C22H13Cl2N5OS, 466.0291).
3.4.23. (3-((4-Isopropylbenzyl)thio)-1H-1,2,4-triazol-1-yl)(phenazin-1-yl)methanone (8h)
1
Yellow solid; yield: 57.8%; m. p. 132.5–133.9 ꢁC. H-NMR (400 MHz, CDCl3) d 9.02
(s, 1H, 1,2,4-triazole-H), 8.53 (dd, J ¼ 8.8, 1.3 Hz, 1H, Phenazine-H), 8.30
(d, J ¼ 8.4 Hz, 1H, Phenazine-H), 8.08–8.15 (m, 2H, Phenazine-H), 7.96–8.03 (m, 1H,
Phenazine-H), 7.85–7.95 (m, 2H, Phenazine-H), 6.98 (d, J ¼ 8.0 Hz, 2H, Ph-H), 6.91
(d, J ¼ 8.0 Hz, 2H Ph-H), 4.09 (s, 2H, Ph-CH2), 2.78 (d, J ¼ 6.6 Hz, 1H, Isopropyl-H),
1.17 (d, J ¼ 6.6 Hz, 6H, C-C2H6). 13C-NMR (101 MHz, CDCl3) d 166.1, 148.4, 144.1,
143.4, 140.1, 139.9, 137.5, 135.2, 134.1, 133.3, 131.8, 130.3 (ꢂ2), 130.1 (ꢂ2), 128.8
(ꢂ2), 128.0, 126.8, 124.9 (ꢂ2), 37.0, 33.8, 23.9 (ꢂ2). HRESIMS: m/z 440.1543
[M þ H]þ (calcd for C25H21N5OS, 440.1540).
3.4.24. (3-((4-(Tert-butyl)benzyl)thio)-1H-1,2,4-triazol-1-yl)(phenazin-1-yl)methanone (8i)
1
Yellow solid; yield: 57.5%; m. p. 182.4–183.4 ꢁC. H-NMR (400 MHz, CDCl3) d 9.02
(s, 1H, 1,2,4-triazole-H), 8.53 (dd, J ¼ 8.8, 1.3 Hz, 1H, Phenazine-H), 8.30
(d, J ¼ 8.4 Hz, 1H, Phenazine-H), 8.08–8.15 (m, 2H, Phenazine-H), 7.95–8.01 (m, 1H,
Phenazine-H), 7.85–7.95 (m, 2H, Phenazine-H), 6.98 (d, J ¼ 8.0 Hz, 2H, Ph-H), 6.91
(d, J ¼ 8.0 Hz, 2H, Ph-H), 4.27 (s, 1H, Ph-CH2), 4.08 (s, 1H, Ph-CH2), 1.26
(d, J ¼ 6.6 Hz, 4H, C-C4H9), 1.10 (d, J ¼ 6.6 Hz, 5H, C-C4H9). 13C-NMR (101 MHz,
DMSO-d6) d 166.7, 164.0, 163.9, 145.0, 148.3, 143.7, 143.0, 142.4, 140.9, 140.6, 140.0,
134.8, 134.1, 133.3, 132.6, 130.9, 130.5, 129.9, 129.0, 128.9, 128.8, 128.4, 125.6, 125.2,
34.6, 31.5. HRESIMS: m/z 476.1518 [M þ H]þ (calcd for C26H23N5OS, 476.1516).
3.4.25. (3-((Naphthalen-2-ylmethyl)thio)-1H-1,2,4-triazol-1-yl)(phenazin-1-yl)metha-
none (8j)
1
Yellow solid; yield: 76.5%; m. p. 129.8–131.2 ꢁC. H-NMR (400 MHz, CDCl3) d 9.05
(s, 1H, 1,2,4-triazole-H), 8.51 (dd, J ¼ 8.8, 1.3 Hz, 1H Phenazine-H), 8.29
(d, J ¼ 8.5 Hz, 1H, Phenazine-H), 8.13 (dd, J ¼ 12.2, 5.3 Hz, 2H, Naphthyl-H), 7.95–7.99
(m, 1H, Naphthyl-H), 7.93 (dd, J ¼ 5.7, 4.0 Hz, 2H, Phenazine-H), 7.86–7.90 (m, 1H,
Phenazine-H), 7.83–7.79 (m, 1H, Phenazine-H), 7.69 (d, J ¼ 8.4 Hz, 1H, Phenazine-H),
7.44–7.49 (m, 2H, Naphthyl-H), 7.21 (d, J ¼ 8.0 Hz, 1H, Naphthyl-H), 7.14
(d, J ¼ 8.0 Hz, 1H, Naphthyl-H), 4.61 (s, 2H, Naphthyl-CH2). 13C-NMR (101 MHz,

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
13
CDCl3) d 166.7, 164.0, 163.9, 150.0, 148.3, 143.7, 143.0, 142.4, 140.9, 140.6, 140.0,
134.8, 134.1, 133.3, 132.6, 130.9, 130.5, 129.9, 129.0, 128.9, 128.8, 128.4, 125.6, 125.2,
34.6, 31.5. HRESIMS: m/z 448.1231 [Mþ H]þ (calcd for C26H17N5OS, 448.1227).
3.5. Antifungal activity
All synthesized compounds were screened for their antifungal activities in vitro
against six fungi, including R. solani, A. solani, Fusarium oxysporum, F. graminearum,
P. oryzae, and P. capsici by using the mycelium growth rate method [16]. The com-
pound was dissolved in 1 ml acetone, diluted to 5 ml with 0.1% Tween 80 aqueous
solution, and then added to a sterile potato medium (PDA, 45 ml). The compounds
used for the preliminary screening were tested at a concentration of 200 lM (PCA
concentration ¼ 44.8 mg/L). The screening was repeated three times for each sample.
The test fungi were inoculated in the center of the Petri dish and incubated at
28 1 ꢁC. When growing to two-thirds of the culture dishes, the diameter of each col-
ony of the three replicates was measured four times by the cross-surrounding
method, and the data was statistically analyzed using DPS7.05 software, and the cor-
rected inhibition rate was calculated by the following formula.
Relative inhibition rate (%) ¼ [(C ꢃ T)/(C ꢃ 7 mm)] ꢂ 100% (where C represents
the average diameter of mycelia in the blank test, T represents the average diameter
of mycelia on treated PDA media).
On the basis of the fungicidal results in vitro, compounds 8a, 8b, 8c, and 8h
against P. oryzae in vivo were performed on living plants in greenhouse. A total of 12
seeds of rice were germinated and put in a plastic pots of 18 cm diameter, and then
the plastic pots were kept in a greenhouse and cultivated at 26～28 ꢁC. The tested
compounds were sprayed over the plant at the end of tillering period at the given
dose and PCA as the contrast. The relative control efficiencies of compounds com-
pared to the blank assay was calculated via the following equation: relative control
efficiency (%) ¼ (CK ꢃ PT)/CK ꢂ 100% (where CK is the average disease index dur-
ing the blank assay and PT is the average disease index after treatment with tested
compounds). All experiments were replicated three times.
Acknowledgments
We thank the Key Laboratory of Natural Pesticide and Chemical Biology, Ministry of
1
Education, South China Agricultural University for HRMS and H NMR spectra.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
The authors acknowledge financial support by State Key Laboratory of the Discovery and
Development of Novel Pesticide (2017NYRD02), the National Key R&D Program of China
(2018YFD0200500) and the National Natural Science Foundation of China (NO. 31672069).

14
X.-J. LI ET AL.
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