Antifungal agents. Part 4: Synthesis and antifungal activities of novel indole[1,2-c]-1,2,4-benzotriazine derivatives against phytopathogenic fungi in vitro

Article abstract of DOI:10.1016/j.ejmech.2010.10.022

A series of novel indole[1,2-c]-1,2,4-benzotriazine derivatives were obtained by a modified Sandmeyer reaction in the presence of tert-butylnitrite (t-BuONO). As compared with hymexazol, a commercially available agricultural fungicide, at the concentration of 50 μg/mL, two indole[1,2-c]-1,2,4- benzotriazines, 5h and 5k, exhibited the more promising and pronounced antifungal activities in vitro against five phytopathogenic fungi. It clearly demonstrated that introduction of appropriate substituents on the indolyl ring of indole[1,2-c]-1,2,4-benzotriazine (5a) would lead to the more potent derivatives.

Full text of DOI:10.1016/j.ejmech.2010.10.022

European Journal of Medicinal Chemistry 46 (2011) 364e369
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journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Antifungal agents. Part 4: Synthesis and antifungal activities of novel
indole[1,2-c]-1,2,4-benzotriazine derivatives against phytopathogenic
fungi in vitro
,
b
,
a
Hui Xu a , Ling-ling Fan
*
a Laboratory of Pharmaceutical Design and Synthesis, College of Sciences, Northwest A&F University, Xinong Road 22#, Yangling, Shaanxi 712100, China
b State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou 730000, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel indole[1,2-c]-1,2,4-benzotriazine derivatives were obtained by a modified Sandmeyer
reaction in the presence of tert-butylnitrite (t-BuONO). As compared with hymexazol, a commercially
available agricultural fungicide, at the concentration of 50 mg/mL, two indole[1,2-c]-1,2,4-benzotriazines,
5h and 5k, exhibited the more promising and pronounced antifungal activities in vitro against five
phytopathogenic fungi. It clearly demonstrated that introduction of appropriate substituents on the
indolyl ring of indole[1,2-c]-1,2,4-benzotriazine (5a) would lead to the more potent derivatives.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Received 21 July 2010
Received in revised form
19 October 2010
Accepted 20 October 2010
Available online 27 October 2010
Keywords:
Indole[1,2-c]-1,2,4-benzotriazine
Cyclization
Sandmeyer reaction
Antifungal activity
Phytopathogenic fungi
1. Introduction
benzotriazine moiety, and wanted to investigate their antifungal
activities against phytopathogenic fungi.
Phytopathogenic fungi, which are hard to control, easily infect
many crops, therefore development of new compounds that
effectively inhibit those agricultural diseases is still highly desir-
able. The indole moiety (I, Fig. 1) represents an important structural
component associated with a variety of alkaloids [1,2] and wide-
ranging biological activities, such as antiviral activities [3e7],
antitumor activities [8,9], antimicrobial activities [10,11], anti-
tuberculosis activities [12], and antifungal activities [13]. Mean-
while, the benzotriazine derivatives (II, Fig. 1) have also gained
widespread interest due to their broad potential activities [14].
Recently, fragment-based lead discovery has emerged as a more
rational and focused approach for molecular modification and drug
design. As a part of our ongoing program aimed at the discovery
and development of compounds with superior bioactivities
[15e18], consequently, in this article we designed and prepared
some novel indole[1,2-c]-1,2,4-benzotriazine derivatives (5aek,
Fig. 1) by combinatorial optimization of the indole unit with the
2. Results and discussion
2.1. Chemistry
As shown in Scheme 1, 5-bromoindole (1b) was prepared from
indole (1a) in the presence of ethanol and 27% aqueous sodium
bisulfite, followed by reaction with acetic anhydride and bromine
[19]. Then 1b reacted with sodium methoxide in the presence of
cuprous iodide (CuI) and N,N-dimethylformamide (DMF) to give
5-methoxyindole (1c). As outlined in Scheme 2, indole derivatives
(1aeh) firstly reacted with 2-nitrophenyl halides (2a and b) by the
SNAr reaction mediated with cesium carbonate (Cs2CO3) to yield
3aej [20], which were subsequently reduced to 4aej in the pres-
ence of stannous chloride dihydrate (SnCl2$2H2O). Finally, indole
[1,2-c]-1,2,4-benzotriazine derivatives (5aej) were obtained from
4aej by a modified Sandmeyer reaction via the intramolecular
cyclization in the presence of tert-butylnitrite (t-BuONO). Com-
pound 5k was synthesized starting from indole as described in
Scheme 3. At first, 1a reacted with DMF in the presence of phos-
phorus chloride oxide (POCl3) to produce 3-formylindole (1i) [21],
which further reacted with 2-fluoronitrobenzene by the SNAr
* Corresponding author. Laboratory of Pharmaceutical Design and Synthesis,
College of Sciences, Northwest A&F University, Xinong Road 22#, Yangling, Shaanxi
712100, China. Tel./fax: þ86 29 87091952.
E-mail address: orgxuhui@nwsuaf.edu.cn (H. Xu).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.10.022

H. Xu, L. Fan / European Journal of Medicinal Chemistry 46 (2011) 364e369
365
2.2. Antifungal activity
1
A
R
B
N
N
N
The antifungal activities of 11 novel indole[1,2-c]-1,2,4-benzo-
triazine derivatives (5aek) against five phytopathogenic fungi
(i.e., Fusarium graminearum, Alternaria alternata, Pyricularia oryzae,
Fusarium oxysporum f. sp. vasinfectum, and Alternaria brassicae)
Combinatorial optimization
C
1
R
N
H
D
2
R
were investigated at the concentration of 50
mg/mL in vitro
I
N
N
by poisoned food technique [22]. Hymexazol, a commercially
available agricultural fungicide, was used as a positive control at
2
R
5a-k
N
50
mg/mL.
As outlined in Table 1, among all the derivatives, compounds 5h
II
and 5k generally exhibited the more promising and pronounced
antifungal activities than hymexazol. Through a comparative study
on the relationship between the chemical structures and antifungal
activities of 5aek (SAR), some interesting results were described as
follows: (1) In general, introduction of the phenylaminocarbonyl
substituent on the D-ring of 5a or 5d gave 5i or 5j, the cor-
responding antifungal activities of which were decreased as
compared with 5a and 5d, respectively. For example, the inhibition
Fig. 1. Design strategy of the target compounds 5aek.
reaction to give 3k. Then 3k was reduced to 4k by SnCl2$2H2O, and
4k was further reduced to 4l with sodium borohydride (NaBH4). At
last, 4l reacted with t-BuONO to give 5k via the intramolecular
cyclization. However, the diazonium salt of 4k did not cyclize to
produce 5l at all. The structures of the target compounds were well
characterized by 1H NMR, 13C NMR, m.p., HRMS or MS.
a
b
c
SO Na
H O
2
SO Na
3
3
N
.
.
N
N
Ac
1/2H O
2
H
H
1a
Br
MeO
d
N
H
N
H
1b
1c
Scheme 1. Synthesis of compounds 1b and 1c. Reagents and conditions: (a) 27% aq. NaHSO3, EtOH, rt, 20 h, 98%; (b) Ac2O, 70 ꢀC, 2 h, then 90 ꢀC, 0.5 h, 89%; (c) Br2, H2O, 0e5 ꢀC, 1 h,
then rt, 1 h, 74%; (d) MeONa, DMF, CuI, reflux, 6 h, 98%.
4
3
X
1
2
R
NO
R1
R1
2
A
7
B
1
N
N
a
c
N
R
b
+
1 N
NO
N
2
C
N
H
NH2
2
R
2a-b
D
1a-h
5a-j
4a-j
3a-j
1a: R1 = H
2
R
R2
R2
1b: R1 = 5-Br
2a: X = F, R2 = H
2b: X = Cl, R2 = CONHPh
1c: R1 = 5-OMe
1d: R1 = 3-Me
1e: R1 = 3-Br
1f: R1 = 4-Me
1g: R1 = 6-Me
1h: R1 = 7-Me
5a: R1 = H, R2 = H; 5b: R1 = 5-Br, R2 = H
5c: R1 = 5-OMe, R2 = H; 5d: R1 = 3-Me, R2 = H
5e: R1 = 3-Br, R2 = H; 5f: R1 = 4-Me, R2 = H
5g: R1 = 6-Me, R2 = H; 5h: R1 = 7-Me, R2 = H
5i: R1 = H, R2 = CONHPh; 5j: R1 = 3-Me, R2 = CONHPh
Scheme 2. The synthetic route of compounds 5aej. Reagents and conditions: (a) Cs2CO3, DMSO, 40 ꢀC, 2e8 h, 21e99%; (b) SnCl2$2H2O, EtOAc, reflux, 1e4 h, 44e93%; (c) t-BuONO,
MeCN, rt, 1/6e22 h, 24e99%.
CHO
CHO
CHO
c
d
b
a
N
N
NO
2
NH
N
H
N
2
H
3k
1a
1i
4k
e
CH OH
2
CH OH
2
CHO
e
N
N
N
N
N
N
N
NH
2
4l
5k
5l
Scheme 3. The synthetic route of compound 5k. Reagents and conditions: (a) POCl3, DMF, 0 ꢀC, 20 min, then 40 ꢀC, 1 h, then 20% aq. NaOH, 0 ꢀC, then reflux, 6 h, 96%; (b) Cs2CO3,
2-fluoronitrobenzene, DMSO, 40 ꢀC, 5 h; (c) SnCl2$2H2O, EtOAc, reflux, 22 h, 29% (from 1i to 4k); (d) NaBH4, MeOH, rt, 1 h, 78%; (e) t-BuONO, MeCN, rt, 0.5 h, 66%.

366
H. Xu, L. Fan / European Journal of Medicinal Chemistry 46 (2011) 364e369
Table 1
4. Experimental
Antifungal activities of compounds 5aek at 50 mg/mL.
4.1. Chemistry
Compd Antifungal activities (inhibition %)
F. graminearum P. oryzae
F. oxysporum A. alternata A. brassicae
f. sp.
4.1.1. General remarks
vasinfectum
All solvents and reagents (except 1b, 1c, and 1i) were used as
obtained from commercial sources without further purification.
Thin-layer chromatography (TLC) and silica gel column chroma-
tography were used with silica gel 60 GF254 and 200e300 mesh,
respectively (Qingdao Haiyang Chemical Co., Ltd., China). Melting
points were determined on a digital melting-point apparatus
and were uncorrected. Proton nuclear magnetic resonance spectra
(1H NMR) and carbon-13 nuclear magnetic resonance spectra
5a
5b
5c
5d
5e
5f
5g
5h
20.5 (ꢃ0.4)
11.2 (ꢃ0.6)
15.8 (ꢃ1.3)
21.1 (ꢃ0.4)
14.0 (ꢃ0.4)
10.2 (ꢃ1.5)
21.4 (ꢃ0.4)
60.6 (ꢃ0.4)
11.1 (ꢃ0.4)
2.5 (ꢃ0.7)
80.1 (ꢃ0.6)
56.5 (ꢃ0.4)
0
4.4 (ꢃ1.2) 36.7 (ꢃ1.2)
2.7 (ꢃ0.7) 11.2 (ꢃ1.2)
13.4 (ꢃ1.2) 24.9 (ꢃ0.5)
10.9 (ꢃ1.0) 10.1 (ꢃ0.5)
18.1 (ꢃ1.7) 8.5 (ꢃ0)
15.5 (ꢃ1.2) 20.1 (ꢃ1.9)
16.8 (ꢃ0.6) 14.2 (ꢃ0.9)
9.2 (ꢃ0.6) 5.5 (ꢃ1.0)
18.5 (ꢃ1.7) 23.4 (ꢃ0.6)
75.3 (ꢃ0.6) 75.6 (ꢃ0.5)
1.1 (ꢃ0.6) 1.8 (ꢃ1.2)
1.1 (ꢃ1.3) 4.1 (ꢃ1.6)
54.3 (ꢃ0.6) 57.2 (ꢃ0.5)
64.8 (ꢃ0.6) 53.5 (ꢃ0.5)
13.8 (ꢃ2.2) 21.9 (ꢃ0.7)
7.3 (ꢃ0.7) 16.1 (ꢃ1.2)
0.2 (ꢃ0.7) 16.1 (ꢃ1.2)
3.0 (ꢃ0.7) 9.9 (ꢃ1.4)
16.3 (ꢃ1.3) 17.2 (ꢃ0.7)
72.6 (ꢃ1.4) 54.3 (ꢃ1.0)
3.8 (ꢃ1.3) 16.5 (ꢃ1.2)
5i
5j
(
13C NMR) were recorded on Bruker Avance DMX 400 or 500 MHz
0
10.1 (ꢃ0.7)
and 125 MHz instruments, respectively, using TMS as the internal
standard and CDCl3 or DMSO-d6 as the solvent. High-resolution
mass spectra (HRMS) were carried out with APEX II Bruker 4.7T AS
instrument. Electrospray ion trap mass spectrometry (ESI-MS) was
carried out with Thermo Scientific LCQ Fleet mass spectrometer.
5k
Hym
Acetone
43.1 (ꢃ1.2) 74.2 (ꢃ1.2)
56.4 (ꢃ1.2) 63.0 (ꢃ0.6)
0
0
0
0
rates of 5a and 5i at 50 mg/mL against F. graminearum, P. oryzae,
F. oxysporum f. sp. vasinfectum, A. alternata, and A. brassicae
4.1.2. Synthesis of 5-bromoindole (1b)
Compound 1b was synthesized according to the literature [23].
were 20.5%/11.1%, 4.4%/3.8%, 36.7%/16.5%, 13.4%/1.1%, and 24.9%/
1.8%, respectively; the inhibition rates of 5d and 5j at 50 mg/mL
Yield: 74%, white solid, m.p. 90e92 ꢀC (lit. 88e90 ꢀC [23]); 1H
NMR (500 MHz, CDCl3) d: 6.49 (1H, s), 7.19 (1H, s), 7.23e7.28 (2H,
against F. graminearum, P. oryzae, F. oxysporum f. sp. vasinfectum,
A. alternata, and A. brassicae were 21.1%/2.5%, 7.3%/0%, 16.1%/10.1%,
15.5%/1.1%, and 20.1%/4.1%, respectively. (2) When the methyl group
was introduced at the 7th position on the A-ring of 5a to give 5h,
the corresponding antifungal activities of 5h increased sharply as
m), 7.77 (1H, s), 8.14 (1H, s, NH); ESI-MS m/z: 194.16 ([M ꢁ 1]ꢁ,
100%,), 196.03 ([M ꢁ 1]ꢁ, 88%).
4.1.3. Synthesis of 5-methoxyindole (1c)
Firstly sodium (1.18 g, 51.3 mmol) reacted with absolute meth-
anol (12 mL) to produce sodium methoxide, which was mixed
directly with 1b in the presence of cuprous iodide (CuI, 1.97 g,
103 mmol) and N,N-dimethylformamide (DMF, 21.2 mL), and the
mixture was refluxed for 6 h. Then the mixture was filtered and the
filtrate was concentrated in vacuo, followed by addition of 10% aq.
NaOH (40 mL). The solution was extracted with EtOAc (30 mL ꢂ 3).
Finally, the organic phases were combined, dried over Na2SO4, and
purified by silica gel column chromatography to give 5-methoxy-
indole (1c).
compared with 5a. The inhibition rates of 5a and 5h at 50 mg/mL
against F. graminearum, P. oryzae, F. oxysporum f. sp. vasinfectum,
A. alternata, and A. brassicae were 20.5%/60.6%, 4.4%/72.6%,
36.7%/54.3%, 13.4%/75.3%, and 24.9%/75.6%, respectively. On the
contrary, introduction of the methyl group, bromo atom or
methoxy group at other position on the A- or B-ring of 5a produced
5d, 5f, 5g, 5b, 5e, or 5c, the corresponding antifungal activities
of which were not increased to some extent as compared with 5a.
(3) The hydroxymethyl group on the B-ring of 5k was very
crucial for the antifungal activities. For example, when the
hydroxymethyl group was introduced on the B-ring of 5a to give
5k, the corresponding antifungal activities of 5k increased
obviously as compared with 5a. The inhibition rates of 5a and
Yield: 98%, white solid, m.p. 59e61 ꢀC (56e57 ꢀC [23]); 1H
NMR (500 MHz, CDCl3) d: 3.86 (3H, s, OCH3), 6.47 (1H, s), 6.85
(1H, dd, J ¼ 8.5, 2.0 Hz), 7.11 (1H, s), 7.15 (1H, t, J ¼ 2.5 Hz), 7.25
(1H, d, J ¼ 9.0 Hz), 8.03 (1H, s, NH); ESI-MS m/z: 146.20
([M ꢁ 1]ꢁ, 73%).
5k at 50 mg/mL against F. graminearum, P. oryzae, F. oxysporum f.
sp. vasinfectum, A. alternata, and A. brassicae were 20.5%/80.1%,
4.4%/43.1%, 36.7%/74.2%, 13.4%/54.3%, and 24.9%/57.2%, respec-
tively. While other functional group (e.g., methyl group or bromo
atom) was introduced on the B-ring of 5a, the corresponding
compounds did not display the potent antifungal activities (5d
and 5e).
4.1.4. Synthesis of 3-formylindole (1i)
To DMF (7 mL), phosphorus oxychloride (POCl3, 1 mL) was
added dropwise at 0 ꢀC. The mixture was stirred for 20 min, then
a solution of indole (10 mmol) in DMF (3 mL) was added dropwise.
After the mixture was stirred at 35 ꢀC for 1 h, ice was added, fol-
lowed by 20% aq. sodium hydroxide (NaOH), and the mixture was
refluxed for 6 h. On cooling, the mixture was poured into ice water,
and the precipitated product was collected, washed by water, and
dried.
3. Conclusion
In summary, 11 novel indole[1,2-c]-1,2,4-benzotriazine deriva-
tives were synthesized by a modified Sandmeyer reaction in the
presence of tert-butylnitrite, and evaluated in vitro for their
antifungal activities against five phytopathogenic fungi at the
Yield: 96%, pink solid, m.p. 190e192 ꢀC (190e192 ꢀC [24]);
1H NMR (400 MHz, DMSO-d6):
d 9.93 (s, 1H, CHO), 8.29 (s, 1H), 8.08
(d, J ¼ 8.0 Hz,1H), 7.50 (d, J ¼ 8.0 Hz,1H), 7.20e7.28 (m, 2H); ESI-MS,
concentration of 50 mg/mL. Among all the derivatives, compounds
m/z (%) 145 (Mþ, 96).
5h and 5k generally exhibited the more promising and pronounced
antifungal activities than hymexazol, a commercially available
agricultural fungicide. It clearly demonstrated that introduction
of appropriate substituents on the indolyl ring of indole[1,2-c]-
1,2,4-benzotriazine (5a) would lead to the more potent derivatives.
It implied that 5h and 5k might be considered as new promising
lead candidates for further design and synthesis of agricultural
fungicides.
4.1.5. General procedures for the synthesis of N-(2-aminophenyl)
indole derivatives (4aej)
Compounds 3aej were prepared as described in our previous
paper [20]. A mixture of 3aej (1 equiv.) and stannous chloride
dihydrate (SnCl2$2H2O, 5 equiv.) in ethyl acetate (EtOAc,10 mL) was
refluxed for 1e4 h under N2. The reaction mixture was alkalinized
to pH ¼ 8 or 9 with the saturated NaHCO3, and the solution was

H. Xu, L. Fan / European Journal of Medicinal Chemistry 46 (2011) 364e369
367
filtered to remove the precipitate. Then the organic phase was
separated from the water phase, and the latter was extracted with
EtOAc (20 mL ꢂ 2). Finally, the organic phases were combined,
dried over Na2SO4, and purified by silica gel column chromatog-
raphy to give 4aej.
CDCl3) d: 2.39 (3H, s, CH3), 3.83 (2H, br. s, NH2), 6.99 (1H, s),
7.11e7.26 (6H, m), 7.37e7.42 (3H, m), 7.64e7.66 (3H, m), 7.90 (1H, s,
NH); ESI-MS m/z: 342.11 ([M þ 1]þ, 100%).
4.1.6. Synthesis of N-(2-aminophenyl)-3-hydroxymethylindole (4l)
N-(2-Nitrophenyl)-3-formylindole (3k) was prepared as de-
scribed in our previous paper [20].
4.1.5.1. N-(2-Aminophenyl)indole (4a). Yield: 81%, colorless oil (lit.
colorless oil [25]); 1H NMR (500 MHz, CDCl3)
d: 3.43 (2H, br. s, NH2),
Yield: 98%, yellow solid; m.p. 126e128 ꢀC; 1H NMR (500 MHz,
6.68 (1H, d, J ¼ 3.0 Hz), 6.82e6.87 (2H, m), 7.14e7.26 (6H, m), 7.68
CDCl3)
d
: 7.05 (1H, d, J ¼ 8.0 Hz), 7.28e7.34 (1H, m), 7.36e7.37 (1H,
(1H, d, J ¼ 7.5 Hz); ESI-MS m/z: 209.15 ([M þ 1]þ, 100%).
m), 7.60 (1H, dd, J ¼ 8.0, 1.0 Hz), 7.70e7.74 (1H, m), 7.81e7.85 (2H,
m), 8.14 (1H, dd, J ¼ 8.0, 1.0 Hz), 8.36 (1H, d, J ¼ 7.5 Hz), 10.11 (1H, s,
CHO); ESI-MS m/z: 267.06 ([M þ 1]þ, 100%).
4.1.5.2. N-(2-Aminophenyl)-5-bromoindole (4b). Yield: 93%, color-
less oil; 1H NMR (500 MHz, CDCl3)
d: 3.53 (2H, br. s, NH2), 6.61
Compound 4k was prepared as the same procedure as 4aej. As
4k was not very stable, it was used directly for the next step.
Sodium borohydride (NaBH4, 2.25 mmol) was added dropwise to
a stirred solution of 4k (1.5 mmol) in methanol (5 mL) at 0 ꢀC. After
adding, the mixture was stirred at room temperature for 1 h. Then
the mixture was concentrated in vacuo, followed by addition of
water (20 mL). The solution was extracted with EtOAc (30 mL ꢂ 3).
Finally, the organic phases were combined, dried over Na2SO4, and
purified by silica gel column chromatography to give 4l.
(1H, d, J ¼ 3.0 Hz), 6.82e6.87 (2H, m), 7.00 (1H, d, J ¼ 8.5 Hz), 7.15
(1H, dd, J ¼ 7.5, 1.5 Hz), 7.19 (1H, d, J ¼ 3.0 Hz), 7.23e7.27 (2H, m),
7.80 (1H, d, J ¼ 8.0 Hz); ESI-MS m/z: 286.96 ([M þ 1]þ, 100%), 289.03
([M þ 1]þ, 88%).
4.1.5.3. N-(2-Aminophenyl)-5-methoxyindole (4c). Yield: 64%, color-
less oil [26]; 1H NMR (500 MHz, CDCl3)
d: 3.36 (2H, br. s, NH2), 3.85
(3H, s, OCH3), 6.59 (1H, d, J ¼ 3.0 Hz), 6.82e6.85 (3H, m), 7.02 (1H,
d, J ¼ 9.0 Hz), 7.14e7.23 (4H, m); ESI-MS m/z: 239.18 ([M þ 1]þ,
100%).
Yield: 78%, colorless oil; 1H NMR (500 MHz, CDCl3)
d: 3.83 (2H,
br. s, NH2), 4.93 (2H, s, CH2), 6.83e6.87 (2H, m), 7.14e7.24 (6H, m),
7.77e7.79 (1H, m); ESI-MS m/z: 221.14 ([M þ 1 ꢁ H2O]þ, 85%).
4.1.5.4. N-(2-Aminophenyl)-3-methylindole (4d). Yield: 85%, color-
less oil [27]; 1H NMR (500 MHz, CDCl3)
d: 2.38 (3H, s, CH3), 3.58
4.1.7. General procedures for the synthesis of indole[1,2-c]-
1,2,4-benzotriazine derivatives (5aek)
(2H, s, NH2), 6.80e6.86 (2H, m), 6.98 (1H, d, J ¼ 0.5 Hz), 7.10e7.23
(5H, m), 7.62 (1H, dd, J ¼ 6.5, 1.5 Hz); ESI-MS m/z: 223.14 ([M þ 1]þ,
100%).
To a stirred solution of 4aek (0.5 mmol) in MeCN (2 mL) at room
temperature, a solution of tert-butylnitrite (t-BuONO, 0.75 mmol)
in MeCN (3 mL) was added dropwise. When the reaction was
complete according to TLC analysis, the solvent was evaporated
under reduced pressure to give the residue, to which water (20 mL)
was added. Then the above mixture was extracted by EtOAc
(40 mL ꢂ 3). Subsequently, the combined organic phase was
washed by brine (40 mL), dried over anhydrous Na2SO4, filtered,
concentrated in vacuo and purified by silica gel column chroma-
tography to give 5aek, which were well characterized by 1H NMR,
13C NMR, HRMS, and m.p.
4.1.5.5. N-(2-Aminophenyl)-3-bromoindole (4e). Yield: 87%, color-
less oil; 1H NMR (500 MHz, CDCl3)
d: 3.45 (2H, br. s, NH2), 6.83 (1H,
t, J ¼ 8.0 Hz), 6.87 (1H, d, J ¼ 8.0 Hz), 7.12e7.14 (1H, m), 7.15 (1H, d,
J ¼ 7.5 Hz), 7.23e7.27 (4H, m), 7.62e7.64 (1H, m); ESI-MS m/z:
286.82 ([M þ 1]þ, 100%), 288.86 ([M þ 1]þ, 95%).
4.1.5.6. N-(2-Aminophenyl)-4-methylindole (4f). Yield: 85%, color-
less oil; 1H NMR (500 MHz, CDCl3)
d: 2.60 (3H, s, CH3), 3.40 (2H,
br. s, NH2), 6.70 (1H, d, J ¼ 3.0 Hz), 6.82e6.87 (2H, m), 6.95e6.99
(2H, m), 7.08 (1H, t, J ¼ 7.5 Hz), 7.17e7.25 (3H, m); ESI-MS m/z:
223.14 ([M þ 1]þ, 100%).
4.1.7.1. Indole[1,2-c]-1,2,4-benzotriazine (5a). Yield: 71%, red solid,
m.p. 220e222 ꢀC; 1H NMR (400 MHz, CDCl3)
d: 7.50e7.57 (2H, m),
7.61e7.65 (1H, m), 7.70 (1H, s), 7.80e7.84 (1H, m), 8.06 (1H, d,
4.1.5.7. N-(2-Aminophenyl)-6-methylindole (4g). Yield: 44%, white
J ¼ 8.0 Hz), 8.32 (2H, dd, J ¼ 8.0, 3.2 Hz), 8.49 (1H, d, J ¼ 7.6 Hz); 13C
solid; m.p. 71e72 ꢀC; 1H NMR (500 MHz, CDCl3)
d: 2.41 (3H, s, CH3),
NMR (125 MHz, CDCl3) d: 102.4, 114.2, 115.1, 123.6, 124.7, 126.0,
3.47 (2H, br. s, NH2), 6.62 (1H, d, J ¼ 2.0 Hz), 6.82e6.87 (2H, m), 6.93
(1H, s), 6.98 (1H, d, J ¼ 8.0 Hz), 7.12 (1H, d, J ¼ 3.0 Hz), 7.17e7.19 (1H,
m), 7.22e7.25 (1H, m), 7.55 (1H, d, J ¼ 8.0 Hz); ESI-MS m/z: 223.13
([M þ 1]þ, 100%).
127.6, 128.8, 129.3, 131.8, 133.6, 136.8, 142.3; HRMS-ESI: Calcd. for
C14H9N3 ([M þ H]þ): 220.0869; Found: 220.0873.
4.1.7.2. 5-Bromoindole[1,2-c]-1,2,4-benzotriazine (5b). Yield: 56%,
red solid, m.p. 222e224 ꢀC; 1H NMR (400 MHz, CDCl3)
d: 7.59e7.64
4.1.5.8. N-(2-Aminophenyl)-7-methylindole (4h). Yield: 45%, white
(2H, m), 7.69 (1H, dd, J ¼ 8.8, 1.2 Hz), 7.85 (1H, t, J ¼ 7.6 Hz),
solid; m.p. 123e125 ꢀC; 1H NMR (500 MHz, CDCl3)
d: 2.06 (3H, s,
8.22e8.31 (3H, m), 8.53 (1H, d, J ¼ 7.6 Hz); 13C NMR (125 MHz,
CH3), 3.33 (2H, br. s, NH2), 6.64 (1H, d, J ¼ 3.5 Hz), 6.77e6.80 (2H,
m), 6.90 (1H, d, J ¼ 7.0 Hz), 7.03e7.06 (2H, m), 7.18 (1H, dd, J ¼ 8.0,
3.0 Hz), 7.22e7.25 (1H, m), 7.52 (1H, d, J ¼ 8.0 Hz); ESI-MS m/z:
223.18 ([M þ 1]þ, 100%).
CDCl3) d: 101.5, 114.2, 116.5, 117.3, 125.2, 126.0, 127.2, 127.9, 128.9,
130.4, 132.2, 134.0, 137.1, 142.7; HRMS-ESI: Calcd. for C14H8BrN3
([M þ H]þ): 297.9974; Found: 297.9980.
4.1.7.3. 5-Methoxyindole[1,2-c]-1,2,4-benzotriazine (5c). Yield: 44%,
4.1.5.9. N-(2-Amino-4-phenylaminocarbonylphenyl)indole (4i). Yield:
red solid, m.p. 184e186 ꢀC; 1H NMR (400 MHz, CDCl3)
d: 3.95 (3H, s,
63%, white solid; m.p. 158e160 ꢀC; 1H NMR (500 MHz, CDCl3)
d: 3.32
CH3), 7.28 (1H, s), 7.38 (1H, s), 7.53 (1H, t, J ¼ 7.6 Hz), 7.62 (1H, s),
(2H, br. s, NH2), 6.73 (1H, d, J ¼ 3.0 Hz), 7.14e7.30 (7H, m), 7.37 (1H, t,
J ¼ 8.0 Hz), 7.44 (1H, s), 7.64 (2H, d, J ¼ 8.0 Hz), 7.70 (1H, d,
J ¼ 7.5 Hz), 7.86 (1H, d, J ¼ 15 Hz, NH); ESI-MS m/z: 328.13 ([M þ 1]þ,
100%).
7.79 (1H, t, J ¼ 8.0 Hz), 8.23e8.29 (2H, m), 8.48 (1H, d, J ¼ 8.0 Hz);
13C NMR (125 MHz, CDCl3)
d: 55.8, 101.7, 102.6, 114.0, 116.1, 118.1,
124.5, 124.7, 127.4, 130.2, 131.8, 133.5, 136.9, 143.0, 156.5; HRMS-ESI:
Calcd. for C15H11N3O ([M þ H]þ): 250.0975; Found: 250.0970.
4.1.5.10. N-(2-Amino-4-phenylaminocarbonylphenyl)-3-methylindole
4.1.7.4. 3-Methylindole[1,2-c]-1,2,4-benzotriazine (5d). Yield: 99%,
(4j). Yield: 53%, white solid; m.p. 144e146 ꢀC; 1H NMR (500 MHz,
red solid, m.p. 176e178 ꢀC; 1H NMR (400 MHz, CDCl3)
d: 2.95 (3H, s,

368
H. Xu, L. Fan / European Journal of Medicinal Chemistry 46 (2011) 364e369
CH3), 7.48e7.54 (2H, m), 7.62e7.66 (1H, m), 7.74e7.78 (1H, m), 8.04
(1H, d, J ¼ 8.0 Hz), 8.24 (1H, d, J ¼ 8.4 Hz), 8.30 (1H, d, J ¼ 8.4 Hz),
m), 8.19 (1H, d, J ¼ 7.6 Hz), 8.30 (2H, t, J ¼ 8.4 Hz), 8.46 (1H, dd,
J ¼ 8.0, 1.6 Hz); 13C NMR (125 MHz, DMSO-d6)
d: 53.2, 114.8, 115.5,
8.42 (1H, dd, J ¼ 8.0, 1.2 Hz); 13C NMR (125 MHz, CDCl3)
d: 8.3, 111.8,
116.0, 122.9, 123.9, 125.5, 126.9, 127.1, 128.2, 128.7, 131.5, 135.0,
136.8,138.9; HRMS-ESI: Calcd. for C15H11N3O ([M þ H]þ): 250.0975;
Found: 250.0970.
113.9,114.8,121.7,122.9,124.3,126.2,127.9,128.6,129.0,131.6,133.2,
136.7, 139.7; HRMS-ESI: Calcd. for C15H11N3 ([M þ H]þ): 234.1026;
Found: 234.1022.
4.2. Biological assay
4.1.7.5. 3-Bromoindole[1,2-c]-1,2,4-benzotriazine (5e). Yield: 64%,
red solid, m.p. 254e256 ꢀC; 1H NMR (400 MHz, DMSO-d6)
d:
Eleven 3-acylindole derivatives (5aek) were screened in vitro
for their antifungal activities against five phytopathogenic fungi
(i.e., F. graminearum, A. alternata, P. oryzae, F. oxysporum f. sp.
vasinfectum, and A. brassicae). Potato dextrose agar (PDA) medium
was prepared in the flasks and sterilized. Compounds 5aek were
dissolved in acetone before mixing with PDA, and the concentra-
7.71e7.79 (3H, m), 8.02e8.07 (2H, m), 8.54 (1H, d, J ¼ 7.6 Hz), 8.73
(1H, d, J ¼ 8.4 Hz), 8.80 (1H, d, J ¼ 8.4 Hz); 13C NMR (125 MHz,
DMSO-d6) d: 91.1, 115.4, 116.4, 121.1, 125.0, 126.0, 126.6, 127.4, 127.5,
128.3, 131.6, 135.4, 137.2, 138.2; HRMS-ESI: Calcd. for C14H8BrN3
([M þ H]þ): 297.9974; Found: 297.9976.
tion of test compounds in the medium was fixed at 50 mg/mL. The
4.1.7.6. 4-Methylindole[1,2-c]-1,2,4-benzotriazine (5f). Yield: 67%,
medium was then poured into sterilized Petri dishes. All types of
fungi were incubated in PDA at 28 ꢃ 1 ꢀC for 5 d to get new
mycelium for the antifungal assays, and a mycelia disk of approx-
imately 5 mm diameter cut from culture medium was picked up
with a sterilized inoculation needle and inoculated in the center of
the PDA Petri dishes. The inoculated Petri dishes were incubated at
28 ꢃ 1 ꢀC for 4 d. Acetone without any compounds mixed with
PDA was served as the control, while hymexazol, a commercially
available agricultural fungicide, was used as a positive control at
red solid, m.p. 212e214 ꢀC; 1H NMR (400 MHz, CDCl3)
d: 2.75 (3H, s,
CH3), 7.28 (1H, d, J ¼ 6.8 Hz), 7.49e7.56 (2H, m), 7.69 (1H, s), 7.78
(1H, t, J ¼ 8.0 Hz), 8.12 (1H, d, J ¼ 8.8 Hz), 8.30 (1H, d, J ¼ 7.2 Hz),
8.47 (1H, d, J ¼ 6.4 Hz); 13C NMR (125 MHz, CDCl3)
d: 19.3, 100.9,
112.4, 114.2, 123.4, 124.6, 126.2, 127.6, 129.0, 129.1, 131.6, 133.2,
133.4, 136.9, 142.1; HRMS-ESI: Calcd. for C15H11N3 ([M þ H]þ):
234.1026; Found: 234.1022.
4.1.7.7. 6-Methylindole[1,2-c]-1,2,4-benzotriazine (5g). Yield: 54%,
50 mg/mL. For each treatment, three replicates were conducted. The
red solid, m.p. 144e146 ꢀC; 1H NMR (400 MHz, CDCl3)
d: 2.66 (3H, s,
radial growths of the fungal colonies were measured and the data
were statistically analyzed. The inhibitory effects of the test
compounds on these fungi in vitro were calculated by the formula:
CH3), 7.33 (1H, d, J ¼ 8.0 Hz), 7.51 (1H, t, J ¼ 7.6 Hz), 7.64 (1H, s), 7.77
(1H, t, J ¼ 8.4 Hz), 7.92 (1H, d, J ¼ 8.4 Hz), 8.09 (1H, s), 8.29 (1H, d,
J ¼ 8.4 Hz), 8.45 (1H, d, J ¼ 8.0 Hz); 13C NMR (125 MHz, CDCl3)
d:
22.9, 102.4, 114.2, 114.5, 123.2, 124.5, 125.8, 126.8, 127.6, 129.7, 131.5,
133.3, 136.5, 136.8, 142.3; HRMS-ESI: Calcd. for C15H11N3
([M þ H]þ): 234.1026; Found: 234.1033.
Inhibition rate ð%Þ ¼ ðC ꢁ TÞ ꢂ 100=C
where C represents the diameter of fungi growth on untreated PDA,
and T represents the diameter of fungi on treated PDA.
4.1.7.8. 7-Methylindole[1,2-c]-1,2,4-benzotriazine (5h). Yield: 24%,
red solid, m.p. 126e128 ꢀC; 1H NMR (400 MHz, CDCl3)
d: 2.82 (3H, s,
Acknowledgments
CH3), 7.39 (1H, d, J ¼ 6.8 Hz), 7.45 (1H, t, J ¼ 7.6 Hz), 7.51 (1H, d,
J ¼ 7.6 Hz), 7.62 (1H, s), 7.68 (1H, t, J ¼ 8.0 Hz), 7.83e7.89 (2H, m),
This work was financially in part supported by the National
Natural Science Foundation of China (no. 31071737), the Program
for New Century Excellent University Talents, State Education
Ministry of China (NCET-06-0868), the Fok Ying Tong Education
Foundation for Young Talents (no. 121032), and the State Key
Laboratory of Applied Organic Chemistry, Lanzhou University. We
also thank Prof. H.L. Zhang for the NMR experiments.
8.44 (1H, d, J ¼ 8.0 Hz); 13C NMR (125 MHz, CDCl3)
d: 23.2, 103.6,
118.2, 120.6, 124.4, 124.5, 124.8, 128.4, 129.0, 129.8, 130.5, 130.6,
132.2, 137.9, 143.2; HRMS-ESI: Calcd. for C15H11N3 ([M þ H]þ):
234.1026; Found: 234.1031.
4.1.7.9. Indole[1,2-c]-7-phenylaminocarbonyl-1,2,4-benzotriazine (5i).
Yield: 70%, red solid, m.p. 310e312 ꢀC; 1H NMR (400 MHz, DMSO-
d6) d: 7.14e7.18 (1H, m), 7.39e7.43 (2H, m), 7.62e7.65 (1H, m), 7.74
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d
: 5.59
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