C ring may be dispensable for β-carboline: Design, synthesis, and bioactivities evaluation of tryptophan analog derivatives based on the biosynthesis of β-carboline alkaloids

Article abstract of DOI:10.1016/j.bmc.2015.08.016

According to our previous work and the latest research on the biosynthesis of β-carboline, and using the reverse thinking strategy, tryptophan, the biosynthesis precursor of β-carboline alkaloids, and their derivatives were synthesized, and their biological activities and structure-activity relationships were studied. This bioassay showed that these compounds exhibited good inhibitory activities against tobacco mosaic virus (TMV); especially (S)-2-amino-3-(1H-indol-3-yl)-N-octylpropanamide (4) (63.3 ± 2.1%, 67.1 ± 1.9%, 68.7 ± 1.3%, and 64.5 ± 3.1%, 500 μg/mL) exhibited the best antiviral activity both in vitro and in vivo. Compound 4 was chosen for the field trials and the acute oral toxicity test, the results showed that the compound exhibited good anti-TMV activity in the field and low acute oral toxicity. We also found that these compounds showed antifungal activities and insecticidal activities.

Full text of DOI:10.1016/j.bmc.2015.08.016

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
C ring may be dispensable for b-carboline: Design, synthesis,
and bioactivities evaluation of tryptophan analog derivatives
based on the biosynthesis of b-carboline alkaloids
⇑
⇑
Yuanqiong Huang, Yongxian Liu, Yuxiu Liu, Hongjian Song , Qingmin Wang
State Key Laboratory of Elemento-Organic Chemistry, Research Institute of Elemento-Organic Chemistry, Collaborative Innovation Center of Chemical Science and
Engineering (Tianjin), Nankai University, Tianjin 300071, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
According to our previous work and the latest research on the biosynthesis of b-carboline, and using the
reverse thinking strategy, tryptophan, the biosynthesis precursor of b-carboline alkaloids, and their
derivatives were synthesized, and their biological activities and structure–activity relationships were
studied. This bioassay showed that these compounds exhibited good inhibitory activities against tobacco
mosaic virus (TMV); especially (S)-2-amino-3-(1H-indol-3-yl)-N-octylpropanamide (4) (63.3 2.1%,
Received 25 June 2015
Revised 3 August 2015
Accepted 13 August 2015
Available online xxxx
67.1 1.9%, 68.7 1.3%, and 64.5 3.1%, 500 lg/mL) exhibited the best antiviral activity both in vitro
Keywords:
b-Carboline
Tryptophan
Biosynthesis
Anti-TMV
and in vivo. Compound 4 was chosen for the field trials and the acute oral toxicity test, the results showed
that the compound exhibited good anti-TMV activity in the field and low acute oral toxicity. We also
found that these compounds showed antifungal activities and insecticidal activities.
Ó 2015 Published by Elsevier Ltd.
Fungicide
1. Introduction
tryptophan derivatives also have great value in the fields of biology
and medicine.12–15
In our previous work, series of tetrahydro-b-carboline deriva-
tives were found to exhibit excellent bioactivities.1–3 As we all know,
b-carbolines are synthesized in organisms using tryptophan as pre-
cursor via the catalysis of enzymes. Recently, Ju and co-workers4
successfully parsed the biosynthetic machinery of b-carboline alka-
loids arrived from the microorganisms in deep-sea. They found
these marinacarbolines are biosynthesized using tryptophan and
acetate as precursors via enzyme-catalyzed Pictet–Spengler cycliza-
tion (Fig. 1); tryptophan can also be used as precursor of biosynthe-
sis for other indole alkaloids.5,6
Although there are a lot of research on the biology activities of
the important biosynthetic precursor, there is little study focus on
its application in the field of pesticides. As we have found tetrahy-
dro-b-carboline derivatives exhibited excellent pesticidal activity,
we wonder whether their biosynthetic precursor (tryptophan)
exhibited similar activities. Based on the above points, firstly,
tryptophan and their ester, amides derivatives were synthesized
(1–22); then, the ureido moiety was introduced into the trypto-
phan derivatives (23–27R) as we have found that it was beneficial
to increase the anti-TMV activity of b-carboline derivatives when
increasing hydrogen bond donor;16 lastly, in order to investigate
the influence of the amino acid part on the activities, the ureido
and ester moieties were transformed to cyclothioureas structure
via the cyclization reaction (28–32). The bioactivities and struc-
ture–activity relationships (SAR) of these derivatives were studied
for the first time (Fig. 2).
In addition to being important synthetic precursor in biosynthe-
sis, L-tryptophan is one of the eight kinds of essential amino acids
in the human body, which participates in the synthesis of various
proteins and in the regulation of metabolic network. For the past
decades,
but also eutherapeutic drug for tristimania, insomnia, hyperten-
sion, dermatitis and schizophrenia, etc.7,8
-Tryptophan, as one of
L-tryptophan is used as not only intravenous nutrition,
D
non-protein amino acids, also has a certain value, especially in
the pharmaceutical industry; it is an important synthetic precursor
of anticancer agent and immune inhibitor.9–11 Furthermore, some
2. Results and discussion
2.1. Synthesis
Using tryptophan as starting material, compound 1 and 2 could
be synthesized via a classical esterification by reacting with thionyl
chloride and ethanol (Scheme 1a). Compound 3 could be obtained
⇑
Corresponding authors. Tel./fax: +86 (0)222 350 3952 (Q.W.).
E-mail address: wangqm@nankai.edu.cn (Q. Wang).
http://dx.doi.org/10.1016/j.bmc.2015.08.016
0968-0896/Ó 2015 Published by Elsevier Ltd.
Please cite this article in press as: Huang, Y.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.08.016

2
Y. Huang et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
R
Table 1. To our delight, most of them exhibited good antiviral
activity. For the in vitro activity, as we can see, tryptophan itself
showed moderate antiviral activity and the chirality ( ) affected
-tryptophan (33.3 2.3%, 500 g/mL)
-tryptophan (37.6 3.0%, 500 g/mL).
showed similar antiviral activities
(35.9 2.3% and 38.4 2.0%, respectively, at 500 g/mL) as - or
-tryptophan. However, when we changed the ester group to
O
OH
O
N
H
N
O
N
H
N
O
N
H
L/D
a R = H
b R = OH
c R = OMe
O
slightly: the in vitro activity of
was slightly lower than that of
L
l
l
McbA
OH
McbB
McbC
OH
McbB
D
NH2
N
O
N
H
N
H
O
Compounds
1
and
2
NH
N
H
l
L
N
N
H
N
D
N
H
O
amide group, most of these compounds exhibited desirable anti-
TMV activity in vitro. Compounds 4, 9, 11, 14, 15, 19, 20, 23, 31S,
and 31R showed obviously higher inhibition than that of ribavirin
O
Figure 1. Biosynthesis of marinacarbolines using tryptophan as a precursor.
at 500 and 100
lg/mL; especially, the activity of compound 4
reached 31.5 2.4% at 100
l
g/mL, it means increasing liposolubil-
O
ity is beneficial to the anti-TMV activity of these compounds. All
these results above proved that the introduction of the amide
group to the tryptophan skeleton was advantageous to the antivi-
ral activities.
Additionally, the substituents of the amide moiety had a signifi-
cant impact on anti-TMV activity. Firstly, when R1 was hydrogen
and R2 was an aliphatic substituent, the longer and less branched
the aliphatic chain was, the higher the anti-TMV activity was,
R2
X
NH2 R1
N
H
X = O, N
O
O
O
O
O
biosynthesis
OH
simplification
derivatization
XR1
HN
HN
NH2
N
N
N
H
H
N
H
starting material
R4
R
L/D-tryptophan
b-carboline
O
N
S
R5
HN
N
compound 4 (63.3 2.1%, 500
lg/mL) displayed obviously higher
H
anti-TMV activity than compound 3 (46.6 1.2%, 500
lg/mL). It
Figure 2. Design of target compounds.
was noteworthy that the existence of NAH bond of the amide
moiety is of crucial importance: the activity of compound 7
from (S)-methyl 2-amino-3-(1H-indol-3-yl)propanoate by reaction
with n-butylamine which also acted as solvent (Scheme 1b). How-
ever, other amines does not apply to this reaction conditions.
Instead, compounds 5–22 were synthesized (Scheme 1c) via car-
bodiimide (such as EDCI) mediated condensation and the subse-
quent deprotection. Compounds 23–27R were obtained via the
reaction of methyl 2-amino-3-(1H-indol-3-yl)propanoate with
isocyanates. When methyl 2-amino-3-(1H-indol-3-yl)propanoate
reacted with isothiocyanates, the cyclization compounds 28–32
could be obtained.
(26.1 1.7%, 500
(41.4 3.4%, 500
l
g/mL) was reduced sharply. Compound
8
lg/mL) containing a propargyl exhibited the
same anti-TMV activity as ribavirin; compound 9 (47.7 2.7%,
500 g/mL) containing a saturated carbocycle exhibited obviously
l
higher antiviral activities than ribavirin; However, when the sub-
stituent was changed to a saturated heterocycle, the activity of
compound 10 (38.8 1.5%, 500
lg/mL) was reduced. Furthermore,
it was noteworthy that compound 21 (23.7 2.1%, 500
l
g/mL)
showed much lower inhibitory effect compared with those of com-
pounds 3–9. These results above meant that the introduction of
heteroatom was adverse to the anti-TMV activity. On the other
hand, when R1 was hydrogen and R2 was a benzyl, compound 11
2.2. Antiviral activities
(26.3 2.0% and 48.6 2.5% at 100
tively) exhibited good inhibitory effect; In contrast, compound12
lg/mL and 500 lg/mL, respec-
The results of antiviral activities, in vitro and in vivo (inactiva-
tion, curative, and protection), of these compounds are listed in
(0 and 38.7 1.7% at 100
lg/mL and 500 lg/mL, respectively)
O
O
O
O
O
N
a
c
b
OR
OR
OH
ROH
OMe
NH2
NH2
NH2
NH2
H
SOCl2
N
H
NH2
N
H
N
H
NH2
N
H
RT
N
H
3
R = methyl,
R = ethyl (
L/D-tryptophan
2
R = ethyl (
)
1
)
4
n-octyl ( );
O
R
1 = H; R2
=
14
(S)-1-phenylethyl ( );
O
O
O
isopropyl (5);
phenethyl (15);
R2
EDCI, DMAP,
NEt3, R1NHR2
R1
OH
HCl
6
16 ;
4-methoxybenzyl ( )
tert-butyl ( );
(Boc)2O, NaOH
THF, H2O, RT
N
OH
NHBoc
N
R
8
NH2 R1
prop-2-ynyl ( );
NH2
17
2,4-dimethoxybenzyl ( );
dioxane
N
H
2
N
H
NHBoc
cyclohexyl (9);
18 ;
4-chlorobenzyl (
)
N
H
DCM
N
H
10
(tetrahydrofuran-2-yl)methyl ( );
4-20
9
3-chlorobenzyl (1 );
L-tryptophan
11
benzyl ( );
20);
2-chlorobenzyl (
R1 = ethyl; R2 = ethyl ( );
12
phenyl ( );
7
3)
thiazol-2-yl (1
;
O
O
O
O
EDCI, DMAP,
R3
R3
OH
NEt3,RNH2
Pd/C, H2
MeOH
CbzCl, NaOH
H2O, RT
N
H
OH
NHCbz
N
R3 = 2-hydroxyethyl ( );
21
NH2
H
NH2
N
H
NHCbz
DCM
N
N
H
pyridin-3-ylmethyl (22);
N
H
H
L-tryptophan
21-22
d
R4NCO, DMAP
NEt3
R4 = n-Bu ( );
23
NH
26
cyclopentyl ( );
O
24
i-Pr ( );
27S
Bn (
);
R4
O
cyclohexyl (25); Bn (27R)
N
N
H
O
DCM
H
O
OMe
O
NH2
R5NCS, DMAP
NEt3
R5
N
H
N
R5
=
28
i-Pr ( );
31S
Bn ( );
HN
29
31R
cyclohexyl ( ); Bn (
cyclopentyl (30); Ph (32)
);
S
DCM
N
H
Scheme 1. Synthesis of compounds 1–32.
Please cite this article in press as: Huang, Y.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.08.016

Y. Huang et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
Table 1
In vitro and in vivo antiviral activity of compounds L/D-tryptophans, 1–32 against TMV
Compd
Concn (
lg/mL)
In vitro inhibition rate (%)
In vivo
Inactivation effect (%)
Curative effect (%)
Protection effect (%)
Ribavirin
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
40.0 1.3
12.9 1.1
33.3 2.3
0
37.6 3.0
0
35.9 2.3
8.6 2.0
34.8 1.2
0
46.6 1.2
0
63.3 2.1
31.5 2.4
45.8 1.4
8.4 1.2
40.3 1.5
18.2 1.1
26.1 1.7
0
37.4 2.1
13.7 2.3
34.1 1.2
10.0 1.1
39.1 2.1
13.1 1.3
42.5 3.2
17.3 1.5
43.0 1.3
15.1 1.3
56.7 1.5
23.6 1.5
67.1 1.9
26.3 1.3
49.5 2.1
11.4 1.1
42.0 2.2
16.8 1.4
29.7 2.3
0
36.2 1.9
16.9 1.8
32.5 2.5
0
38.5 2.5
16.2 1.4
33.0 1.6
8.2 1.4
37.3 2.7
11.7 2.3
36.3 1.2
15.1 2.3
37.3 2.1
18.2 3.1
48.2 2.7
17.0 2.0
64.5 3.1
24.0 1.2
47.1 1.8
6.9 2.3
43.8 1.8
12.1 1.5
32.3 1.9
0
38.7 2.5
7.6 1.4
48.2 1.7
19.0 1.1
38.1 2.1
11.6 1.2
46.1 2.7
13.2 1.9
48.9 2.3
6.4 1.2
40.4 1.2
16.7 1.5
46.1 3.3
23.9 1.3
51.3 2.8
17.6 1.9
28.9 2.1
0
L
-Tryptophan
-Tryptophan
36.2 1.5
10.3 1.5
37.0 1.7
12.0 1.2
39.2 3.0
15.9 2.1
50.3 3.1
17.5 1.2
68.7 1.3
29.8 3.0
53.4 2.3
18.5 2.1
45.4 2.9
19.7 1.0
28.6 2.7
0
45.1 1.9
15.9 1.0
42.6 1.5
12.6 1.7
35.9 1.2
14.4 2.0
54.6 3.7
12.8 1.4
44.2 1.4
13.0 1.1
45.9 2.4
11.7 1.2
44.6 1.7
12.4 2.4
51.8 4.0
22.9 2.1
34.0 1.3
0
D
1
2
3
4
5
6
7
8
41.4 3.4
0
44.2 2.1
0
9
47.7 2.7
17.8 1.0
38.8 1.5
7.9 1.1
48.6 2.5
26.3 2.0
38.7 1.7
0
45.4 2.0
10.8 1.3
40.0 3.0
8.7 1.4
52.3 3.2
17.4 1.2
46.5 1.2
16.6 2.0
46.7 1.9
9.8 1.0
50.0 2.1
18.9 2.0
48.4 2.0
18.2 1.0
33.7 1.5
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27S
27R
28
29
30
31S
31R
32
38.2 1.1
0
51.1 3.2
19.6 1.4
50.2 2.3
23.5 1.4
30.5 1.3
0
40.0 1.7
0
29.8 2.3
0
37.7 1.2
0
39.4 1.9
7.6 1.0
43.5 2.5
9.9 1.1
50.0 2.8
23.2 2.0
48.3 1.3
13.2 1.0
27.7 2.4
0
44.0 1.5
13.7 1.0
53.3 3.8
26.9 1.3
48.6 1.1
17.3 1.2
23.7 2.1
0
31.4 2.1
0
58.1 2.5
14.9 1.4
32.5 1.5
0
47.2 2.6
8.8 2.1
43.3 1.9
9.8 2.0
49.2 1.9
14.7 1.0
48.6 2.3
0
49.5 1.4
15.3 1.3
48.6 2.1
20.1 1.6
46.2 1.9
21.3 1.3
31.5 1.9
0
48.0 2.1
20.4 2.0
51.1 1.6
18.3 1.3
45.3 3.0
18.8 1.8
29.0 1.5
0
32.9 1.3
0
34.6 1.9
0
38.3 2.3
0
51.3 1.9
17.2 1.7
40.0 1.6
15.4 1.2
46.1 2.1
19.2 1.4
50.5 2.0
21.4 1.5
51.3 2.3
10.8 1.4
39.8 2.5
10.1 1.1
43.1 1.9
14.5 1.2
40.1 2.1
16.6 1.4
47.5 1.5
12.2 1.2
50.0 2.3
19.6 2.1
57.6 3.8
28.1 1.7
43.7 2.3
11.3 1.2
53.4 3.1
23.3 2.0
36.2 2.3
7.3 1.0
43.5 2.4
14.8 1.4
46.2 1.4
16.3 1.1
43.8 2.7
15.4 1.2
41.6 1.7
15.0 1.5
40.6 1.3
8.3 1.5
46.2 2.6
10.4 1.2
42.3 3.0
18.0 1.0
53.8 2.1
21.7 1.4
49.8 1.9
25.4 2.3
41.7 2.1
20.0 1.7
47.1 2.1
21.6 1.1
34.7 1.3
10.0 1.7
52.8 1.6
23.9 1.3
55.8 4.0
27.1 1.9
46.2 1.3
7.6 1.9
46.9 1.0
11.7 1.7
48.0 2.1
17.2 1.0
42.8 1.7
11.5 1.2
50.9 2.1
21.4 2.1
45.5 1.5
12.2 1.0
51.4 1.5
20.0 1.1
48.0 1.8
22.3 1.3
35.7 2.3
0
45.0 1.4
6.9 1.2
38.7 1.3
21.5 1.5
56.8 3.1
25.4 1.4
53.3 2.3
15.2 1.2
38.9 1.8
0
The bold values were used to highlight that these compounds showed good activities.
Please cite this article in press as: Huang, Y.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.08.016

4
Y. Huang et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Table 2
Fungicidal activities of L/D-tryptophans, compounds 1, 3, 4, and 6–32 against fourteen kinds of phytopathogens
Compd Fungicidal activity (%)/50 mg/kg
F.C.a
C.H.
P.P.
A.S.
F.G.
F.M.
S.S.
P.C.
R.C.
40.5
B.M.
28.0
W.A.
24.0
P.I.
8.7
R.S.
1.4
B.C.
4.5
4.5
18.2
18.2
42.4
45.5
9.5
9.5
46.2
35.3
23.5
7.7
6.1
41.7
66.7
L-Tryptophan
69.2
15.4
15.2
43.2
28.0
20.0
17.4
0.0
D-Tryptophan
1
3
4
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27S
27R
28
29
30
31S
31R
32
18.2
38.5
13.6
13.6
13.6
15.4
4.5
26.9
30.8
30.8
23.1
4.5
4.5
4.5
9.1
27.3
0.0
9.1
30.8
9.1
10.5
10.5
7.9
18.4
26.3
57.9
13.2
71.1
31.6
15.8
10.5
13.2
<50
100
18.2
58.8
36.4
9.1
30.3
83.3
54.5
45.5
81.8
55.6
57.6
69.4
55.6
50.0
69.4
54.5
51.5
48.5
36.4
36.4
21.2
48.5
97.2
48.5
33.3
-2.0
0.0
14.3
45.0
52.4
19.0
33.3
15.0
19.0
0.0
46.2
62.1
53.8
38.5
53.8
44.8
53.8
65.5
27.6
20.7
27.6
38.5
69.2
46.2
53.8
46.2
53.8
46.2
34.5
46.2
17.9
26.8
17.9
53.6
26.8
35.7
21.4
75.0
35.7
14.3
26.8
25.0
100
29.4
21.4
47.1
29.4
35.3
21.4
29.4
21.4
21.4
14.3
14.3
35.3
29.4
41.2
35.3
41.2
35.3
35.3
21.4
29.4
7.1
7.1
7.1
7.1
7.1
10.7
3.6
35.7
28.6
14.3
10.7
10.7
<50
100
17.3
82.4
75.0
11.5
48.1
58.8
11.5
76.5
35.3
82.4
64.7
28.8
30.8
9.6
9.1
62.2
88.2
54.1
40.5
56.8
60.8
54.1
47.1
52.9
62.7
52.9
56.8
51.4
43.2
54.1
62.2
43.2
59.5
52.9
29.7
34.3
27.1
25.7
31.4
27.1
37.1
34.3
84.3
74.3
24.3
18.6
34.3
28.0
32.0
48.0
32.0
32.0
16.0
24.0
16.0
20.0
24.0
20.0
32.0
36.0
36.0
32.0
52.0
24.0
36.0
24.0
32.0
8.1
16.0
33.3
48.0
36.0
32.0
22.2
28.0
18.5
22.2
22.2
18.5
32.0
32.0
36.0
32.0
52.0
28.0
32.0
25.9
24.0
12.9
16.1
16.1
9.7
21.7
36.0
34.8
26.1
26.1
12.0
21.7
28.0
24.0
28.0
16.0
17.4
17.4
21.7
17.4
43.5
13.0
30.4
32.0
8.7
23.5
23.5
8.8
35.3
8.8
35.3
17.6
67.6
29.4
5.9
27.8
32.9
36.1
1.4
29.2
13.2
9.7
26.3
13.2
22.4
13.2
1.4
0.0
11.1
1.4
33.3
1.4
8.3
13.2
8.3
10.5
7.1
7.1
7.1
7.1
10.7
3.6
35.7
28.6
14.3
10.7
10.7
52.8
72.7
61.1
50.0
69.4
4.5
47.2
4.5
9.1
78.9
78.8
15.2
12.1
18.4
36.4
7.9
23.7
36.8
18.4
36.4
48.5
15.2
21.2
81.8
21.2
81.8
23.7
3.0
19.6
19.6
25.5
29.4
23.5
29.4
43.1
68.6
52.9
23.5
27.5
49.0
<50
72.7
29.4
27.3
23.5
35.3
35.3
35.3
27.3
36.4
27.3
36.4
54.5
0.0
27.3
41.2
9.1
10.3
24.1
13.8
6.9
10.3
17.2
20.7
41.4
58.6
10.3
10.3
17.2
<50
0.0
30.0
5.0
13.6
0.0
33.3
33.3
23.8
33.3
57.1
23.8
38.1
20.0
14.3
18.5
25.9
22.2
33.3
29.6
22.2
48.1
63.0
44.4
37.0
14.8
18.5
<50
66.7
69.4
61.1
61.1
75.0
52.8
69.4
9.1
55.6
10.3
35.2
35.2
42.3
28.2
39.4
32.4
88.7
39.4
35.2
31.0
35.2
<50
1.9
46.2
26.9
44.2
94.1
3.8
14.8
11.1
11.1
77.8
12.3
16.0
24.7
85.2
17.3
12.3
12.3
21.0
100
2.7
16.2
16.2
16.2
10.8
16.2
35.1
45.9
13.5
13.5
21.6
0.0
0.0
25.8
16.1
25.8
38.7
38.7
12.9
16.1
19.4
39.2
29.4
92.2
78.4
39.2
33.3
39.2
<50
8.8
23.5
100
86.4
Carbendazim
Chlorothalonil
100
100
100
91.3
100
91.3
100
100
73.3
100
73.3
<50
<50
100
100
The bold values were used to highlight that these compounds showed good activities.
a
F.C.: Fusarium oxysporiumf. sp. cucumeris; C.H.: Cercospora arachidicola Hori; P.P.: Physalospora piricola; A.S.: Alternaria solani; F.G.: Fusarium graminearum; F.M.: Fusarium
moniliforme; S.S.: Sclerotinia sclerotiorum; P.C.: Phytophthora capsici; R.C.: Rhizoctonia cerealis; B.M.: Bipolaris maydis; W.A.: Watermelon-anthracnose; P.I.: Phytophthora infestans;
R.S.: Rhizoctonia solani; B.C.: Botrytis cinerea.
containing a phenyl was far less effective, the activity was also
reduced when the benzyl was changed to an aryl heterocycle
SAR as the antiviral activities in vitro. Most of the compounds
exhibited excellent antiviral activity, especially the activities of
compounds 3 (56.7 1.5%, 50.3 3.1%, and 48.2 2.7% at 500
(13). Compounds 14(51.1 3.2%, 500
lg/mL) and 15(50.2 2.3%,
500 g/mL) showed the same inhibition as compound 13. In order
l
lg/mL), 4 (67.1 1.9%, 68.7 1.3%, and 64.5 3.1% at 500
lg/mL),
to investigate SAR further, a representative set of compounds 16–
20 containing substituted benzyl structure were synthesized. The
result demonstrated that the electronic effect of substituents on
the benzene ring did have effect on anti-TMV activity. For instance,
it was detrimental to anti-TMV activity when the electron-donating
11 (52.3 3.2%, 54.6 3.7%, and 46.1 2.7% at 500 g/mL), 14
l
(50.0 2.1%, 44.6 1.7%, and 46.1 3.3% at 500
(48.4 2.0%, 51.8 4.0%, and 51.3 2.8% at 500
(48.6 2.1%, 51.1 1.6%, and 50.0 2.8% at 500
l
l
l
g/mL), 15
g/mL), 19
g/mL), 23
(51.3 1.9%, 53.4 3.1%, and 47.1 2.1% at 500
(50.0 2.3%, 53.8 2.1%, and 45.5 1.5% at 500 g/mL), and 31R
(57.6 3.8%, 49.8 1.9%, and 51.4 1.5% at 500 g/mL) were
higher than ribavirin (37.4 2.1%, 36.2 1.9%, and 38.5 2.5% at
500 g/mL). Compound4, containing a long-chain, exhibited the
lg/mL), 31S
group (methoxyl-, 16 (30.5 1.3%, 500
500 g/mL)) was introduced to the benzene ring comparing with
the activity of compounds 18–20(44 1.5%, 53.3 3.8%, and
48.6 1.1% at 500 g/mL, respectively) containing an electron-
l
g/mL), 17 (40 1.7%,
l
l
l
l
l
withdrawing group (chloro-). However, when the benzyl was
changed to pyridin-3-ylmethyl (22), the activity (31.4 2.1%,
500 lg/mL) was greatly reduced. The introduction of the ureido
best antiviral activity both in vitro and in vivo. Considering its good
solubility, stability and simple chemical structure, it is worthy to
conduct further research and exploitation.
moiety was beneficial to the anti-TMV activity; most of the com-
pounds (23–27R) exhibited higher activities than compounds 1
and 2. Especially, the compounds 23 exhibited 58.1 2.5% at
2.3. Fungicidal activities
500 lg/mL. For these compounds which containing cyclothioureas
structure, the substituents of the imide moiety had a significant
impact on anti-TMV activity. Compounds 31S (56.8 3.1% at
500 lg/mL) and 31R (53.3 2.3% at 500 lg/mL) which have a
benzyl on the imide moiety exhibited the best activities.
Further bioassay was conducted to investigate their inactiva-
tion, curative, and protection effect in vivo, which showed similar
Overall, most of these derivatives showed fungicidal activities
against 14 kinds of phytopathogens (Table 2). Especially compound
3 and 29 exhibited more than 70% inhibition to several fungi. Some
of these compounds exhibited fungicidal activities to certain fungi,
for instance, the activities of compound 12 (against Sclerotinia
sclerotiorum) and compound 20 (against Physalospora piricola and
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Y. Huang et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
Table 3
Insecticidal activity of compounds 1, 3, 4, 6–20, and 22–32 against four kinds of insects
Compd
Larvicidal activity (%) at concn (mg/kg)
M. separata
H. armigera
O. nubilalis
C. pipiens pallens
5 mg/kg 2 mg/kg
600/200 mg/kg
600 mg/kg
600 mg/kg
10 mg/kg
1 mg/kg
65
50
45
45
55
35
100
100
70
10
—
—
—
L
-Tryptophan
-Tryptophan
D
1
3
4
6
7
8
9
55
30
20
70
30
65
5
45
15
30
30
5
20
20
25
20
10
65
5
20
100/20
65
50
60
50
70
60
60
50
50
60
50
20
25
50
20
65
30
35
10
25
30
30
30
25
20
25
25
50
5
70
30
65
70
45
45
50
20
65
50
55
65
55
35
15
50
30
25
0
40
20
25
35
5
15
30
20
10
20
50
5
50
20
65
50
55
35
55
35
45
45
50
45
100
75
35
100
65
100
10
100
35
50
55
15
45
35
55
40
20
100
15
100
65
100
65
30
—
—
80
—
70
—
30
—
—
—
—
—
—
—
—
—
50
—
0
—
10
—
40
100
—
20
—
100
—
—
—
—
10
11
12
13
14
15
16
17
18
19
20
22
23
24
25
26
27S
27R
28
29
30
31S
31R
32
—
—
100
100
60
100
45
100
15
100
—
100
60
50
—
100
—
75
The bold values were used to highlight that these compounds showed good activities.
‘—’ not test.
Table 4
2.5. Field trials
Results of anti-TMV activities of field trials in 2014 after 10 days of application of
4amino oligosaccharins, and moroxydine hydrochloride-cupric acetate
As compound 4 exhibited excellent anti-TMV activity both
in vitro and in vivo in laboratory, 1% EC of compound 4 was
employed to evaluate its anti-TMV activity in field trials in Teng-
chong County (Yunnan Province, China) using amino oligosaccha-
rins, moroxydine hydrochloride-cupric acetate, and water as
controls and blank control respectively. The result exhibited that
compound 4 showed the same efficacy as the controls. Table 4
showed part of the results of the field trials.
b
c
Compda
Concn
(gai/ha)
I0eq
Ieq
E
d (%)
4 (1% ME)
100
50
10
—
100
3.82 5.56
5.80 10.55 58.51
2.67 8.02
2.81 12.32
3.33 5.01
66.81
31.49
—
65.70
Blank control
Amino oligosaccharins (5% aqueous
solution)
Moroxydine hydrochloride-cupric
acetate (20% WP)
600
1.23 1.80
66.57
a
b
c
ME: microemulsion, WP: wettable powder.
I0eq: The average disease index of the spraying area before spraying.
Ieq: The average disease index of the spraying area 10 days after the third time
spraying.
d
E: The average control effect 10 days after the third time spraying.
Sclerotinia sclerotiorum) were >80% at 50 mg/kg, which was much
higher than that against other fungi.
2.4. Insecticidal activities
These selected derivatives also showed insecticidal activities
(Table 3). Of which, compounds 27R and 31S exhibited more than
70% activities against culex pipiens pallens at 1 mg/kg. It was inter-
esting that their enantiomers (27S and 31R) exhibited low or no
activity at 5 mg/kg.
Figure 3. Average weight of rats. 4-L means the dosage of compound 4 is 50 mg/kg,
4-H means dosage of compound 4 is 500 mg/kg.
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6
Y. Huang et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
(490 mL) was added di-tert-butyl dicarbonate (23.50 g,
107.7 mmol) at room temperature, and the resulting solution
was stirred at room temperature for 36 h. THF was removed under
reduced pressure, and the aqueous layer was acidified with citric
acid to pH = 4. The mixture was extracted with ethylacetate, and
concentrated to give the product as a white solid (14.90 g) in quan-
titative yield: 1H NMR (400 MHz, DMSO-d6):d 11.15 (s, 1H, COOH),
3
8.17 and 8.14 (s, 1H, NH), 7.59 (d, JHH = 8.0 Hz, 1H, Ar-H), 7.36–
3
7.29 (m, 1H, Ar-H), 7.19 (t, JHH = 7.2 Hz, 1H, Ar-H), 7.11 (t,
Figure 4. Autopsy results of the rats.
3JHH = 7.2 Hz, 1H, Ar-H), 6.95 and 6.81 (s, 1H, Ar-H), 6.26 and
5.10 (d, JHH = 7.6 Hz, 1H, O@CANH), 4.71–4.61 and 4.45–4.40 (m,
3
1H, CH), 3.39–3.22 and 3.12–3.01 (m,2H, CH2), 1.42 and 1.27 (s,
9H, CH3).
2.6. Rat acute oral toxicity of compound 4
To balance the toxicity versus efficacy of compound 4, we next
tested its tolerated dose (50 mg/kg and 500 mg/kg) using a group
of rats (female rats and male rats). The test was conducted using
our previously reported methods,17 and monitoring morbidity
(body weight loss) and mortality in the meantime. There was no
obvious weight difference between the two doses (50 mg/kg and
500 mg/kg) (Fig. 3). After dissected, all rats showed no abnormal
state under the macroscopic observation (Fig. 4). The results above
proved that compound 4 display low toxicity to rats.
4.1.2. Synthesis of (S)-2-(benzyloxycarbonylamino)-3-(1H-
indol-3-yl)propanoic acid19
To a stirred mixture of L-tryptophan (20.07 g, 98.04 mmol) and
NaOH (9.41 g, 235.25 mmol) in water (250 mL) was added drop-
wise benzyl chloroformate (20.18 g, 117.65 mmol) in an ice bath.
The mixture was allowed to stir overnight at room temperature,
then acidified to pH = 1 with hydrochloric acid, and filtered. The
cake was washed with cold water and dried to give the product
as a white solid (21.02 g) in 64% yield: 1H NMR (300 MHz,
3
DMSO-d6): d 10.85 (s, 1H,COOH), 7.54 (d, JHH = 7.8 Hz, 1H, Ar-H),
3. Conclusions
3
7.40–7.20 (m, 6H, Ar-H), 7.14 (s, 1H, Ar-H), 7.06 (t, JHH = 7.5 Hz,
3
1H, Ar-H), 6.96 (t, JHH = 7.5 Hz, 1H, Ar-H), 4.97 (s, 2H, CH2),
In conclusion, according to our previous study on the activities
of b-carboline and their derivatives and the latest research work on
their biosynthesis, and using the reverse thinking strategy, trypto-
phan and its derivatives containing amide or ester moiety were
designed and synthesized via simplifying the b-carboline alkaloid.
Most of the target compounds showed good anti-TMV activity both
in vitro and in vivo (inactivation, curative, and protection) in the
laboratory, activities of compounds 3, 4, 11, 14, 15, and 19 were
much higher than that of ribavirin and the relevant SAR was sum-
marized. Meanwhile, the field trials of compound 4 further demon-
strated its antiviral efficacy against TMV, and the rat acute oral
toxicity test showed low toxicity. Because of the outstanding fea-
tures, compound 4 is a promising candidate to develop into an
inhibitor of plant virus. In addition, these compounds also showed
fungicidal activity and insecticidal activity. For example, com-
pound 3 showed more than 70% inhibition against five kinds of
phytopathogens at 50 mg/kg, and 27R and 31S exhibited more
than 70% activities against culex pipiens pallens at 1 mg/kg. The sub-
stituents on nitrogen of these compounds containing cycloth-
ioureas structure played important role in the anti-TMV activity.
These compounds containing benzyl group (31S and 31R)
exhibited the best activities.
3
2
4.25–4.10 (m, 1H, CH), 3.21 (dd, JHH = 4.2 Hz, JHH = 14.4 Hz, 1H,
CH2), 2.99 (dd, JHH = 9.0 Hz, JHH = 14.4 Hz, 1H, CH2). 13C NMR
(100 MHz, DMSO-d6): d 174.0, 173.9, 155.8, 137.1, 136.0, 128.3,
127.6, 127.5, 123.6, 120.7, 118.2, 111.3, 110.5, 65.1, 55.4, 27.1.
3
2
4.1.3. Synthesis of (S)-methyl 2-amino-3-(1H-indol-3-yl)-
propanoate
To a stirred solution of
L-tryptophan(10 g, 49.02 mmol) in
methanol (150 mL) was added dropwise sulfur dichloride(10 mL)
in an ice bath, the reaction solution was heated at reflux for 4 h.
After cooling and evaporation of methanol, the residue was parti-
tioned between ethyl acetate (80 mL) and a saturated solution of
NaHCO3 (50 mL). The organic layer was washed with saturated
brine, dried over Na2SO4 and concentrated to give a brown solid
(9.16 g) in 86% yield: mp = 90–91 °C; 1H NMR (300 MHz, CDCl3):
3
d 8.47 (s, 1H, Ar-NH), 7.60 (d, JHH = 7.8 Hz, 1H, Ar-H), 7.32 (d,
3
3JHH = 8.1 Hz, 1H, Ar-H), 7.18 (t, JHH = 7.2 Hz, 1H, Ar-H), 7.11 (t,
3
3JHH = 7.2 Hz, 1H, Ar-H), 7.00 (s, 1H, Ar-H), 3.82 (dd, JHH = 4.8 Hz,
3
7.5 Hz, 1H, CH), 3.71 (s, 3H, CH3), 3.28 (dd, JHH = 4.5 Hz,
3
2
2JHH = 14.4 Hz, 1H, CH2), 3.04 (dd, JHH = 7.5 Hz, JHH = 14.4 Hz, 1H,
CH2), 1.67 (s, 2H, NH2). 13C NMR (100 MHz, CDCl3): d 175.7,
136.3, 127.3, 123.2, 121.9, 119.3, 118.6, 111.3, 110.5, 54.9, 52.0,
30.7.
4. Experimental
4.1. Chemistry
Compounds 1 and 2 were synthesized using similar procedure
as (S)-methyl 2-amino-3-(1H-indol-3-yl)propanoate.
1H NMR spectra were obtained at 400 MHz using a Bruker
AV400 spectrometer in CDCl3 or DMSO-d6 solution with tetram-
ethylsilane as the internal standard. HRMS data were obtained
on an FTICR-MS instrument (Ionspec 7.0 T). The melting points
were determined on an X-4 binocular microscope melting point
apparatus and are uncorrected. All anhydrous solvents were dried
and purified by standard techniques. The synthetic routes were
given in Scheme 1.
4.1.4. Data for (S)-ethyl 2-amino-3-(1H-indol-3-yl)propanoate
(1)
This compound was obtained as a yellow solid in 87% yield:
mp = 58–60 °C; 1H NMR (300 MHz, CDCl3) d 8.10 (s, 1H, Ar-NH),
3
3
7.64 (d, JHH = 7.6 Hz, 1H, Ar-H), 7.37 (d, JHH = 8.0 Hz, 1H, Ar-H),
3
3
7.20 (t, JHH = 7.2 Hz, 1H, Ar-H), 7.13 (t, JHH = 7.6 Hz, 1H, Ar-H),
3
7.08 (s, 1H, Ar-H), 4.16 (q, JHH = 7.2 Hz, 2H, OCH2CH3), 3.82 (dd,
3JHH = 5.2 Hz, JHH = 7.6 Hz, 1H, CH), 3.29 (dd, JHH = 4.5 Hz,
2
3
2JHH = 14.4 Hz, 1H, CH2), 3.05 (dd, JHH = 8.0 Hz, JHH = 14.4 Hz, 1H,
3
2
CH2), 1.24 (t, JHH = 7.2 Hz, 3H, OCH2CH3).13
C NMR (CDCl3,
3
4.1.1. Synthesis of (S)-2-(tert-butoxycarbonylamino)-3-(1H-
indol-3-yl)propanoic acid18
100 MHz) d 175.2, 136.3, 127.4, 123.1, 122.0, 119.3, 118.7, 111.2,
110.8, 61.0, 54.9, 30.6, 14.1; HRMS (ESI) calcd for C13H17N2O2 [M
+H]+ 233.1285, found 233.1289.
To a mixture of
L-tryptophan (10.00 g, 49.02 mmol) and NaOH
(4.30 g, 107.5 mmol) in a mixed solvent of THF (450 mL) and H2O
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Y. Huang et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7
4.1.5. Data for (R)-ethyl 2-amino-3-(1H-indol-3-yl)propanoate
(2)
118.9, 111.4, 111.0, 55.4, 39.3, 31.8, 30.3, 29.4, 29.3, 29.2, 26.9,
22.7, 14.1. HRMS (ESI) calcd for C19H30N3O [M+H]+ 316.2383, found
316.2384.
Compounds 5–20 were synthesized using similar procedure as
compound 4.
This compound was obtained as a yellow solid in 84% yield:
mp = 58–60 °C; 1H NMR (300 MHz, CDCl3) d 8.10 (s, 1H, Ar-NH),
3
3
7.64 (d, JHH = 7.6 Hz, 1H, Ar-H), 7.37 (d, JHH = 8.0 Hz, 1H, Ar-H),
3
3
7.20 (t, JHH = 7.2 Hz, 1H, Ar-H), 7.13 (t, JHH = 7.6 Hz, 1H, Ar-H),
3
7.08 (s, 1H, Ar-H), 4.16 (q, JHH = 7.2 Hz, 2H, OCH2CH3), 3.82 (dd,
4.1.8. Data for (S)-2-amino-3-(1H-indol-3-yl)-N-isopropylpro-
panamide (5)
3JHH = 5.2 Hz, JHH = 7.6 Hz, 1H, CH), 3.29 (dd, JHH = 4.5 Hz,
2
3
2JHH = 14.4 Hz, 1H, CH2), 3.05 (dd, JHH = 8.0 Hz, JHH = 14.4 Hz, 1H,
This compound was obtained as a yellow solid in 76% yield:
3
2
CH2), 1.24 (t, JHH = 7.2 Hz, 3H, OCH2CH3);13
C NMR (CDCl3,
mp = 113–115 °C; 1H NMR (400 MHz, CDCl3): d 8.71 (s, 1H, Ar-
3
3
3
100 MHz) d 175.2, 136.3, 127.4, 123.1, 122.0, 119.3, 118.7, 111.2,
110.8, 61.0, 54.9, 30.6, 14.1; HRMS (ESI) calcd for C13H17N2O2 [M
+H]+ 233.1285, found 233.1289.
NH), 7.65 (d, JHH = 7.6 Hz, 1H, Ar-H), 7.37 (d, JHH = 8.0 Hz, 1H,
3
Ar-H), 7.18 (t, JHH = 7.6 Hz, 1H, Ar-H), 7.14–7.05 (m, 2H, Ar-H
and O@CANH), 7.04 (s, 1H, Ar-H), 4.17–4.04 (m, 1H, CH), 3.67
3
3
(dd, JHH = 8.8 Hz, 4.4 Hz, 1H, CH), 3.37 (dd, JHH = 4.0 Hz,
3
2
4.1.6. Synthesis of ((S)-2-amino-N-butyl-3-(1H-indol-3-yl)-
propanamide) (3)
2JHH = 14.4 Hz, 1H, CH2), 2.90 (dd, JHH = 8.8 Hz, JHH = 14.4 Hz, 1H,
CH2), 1.56 (s, 2H, NH2), 1.10 (t, JHH = 6.4 Hz, 6H, CH3). 13C NMR
3
(S)-Methyl
2-amino-3-(1H-indol-3-yl)propanoate
(0.40 g,
(100 MHz, CDCl3): d 173.9, 136.5, 127.5, 123.2, 122.1, 119.4,
118.9, 111.5, 111.3, 55.6, 40.8, 30.8, 22.7, 22.7. HRMS (ESI) calcd
for C14H20N3O [M+H]+ 246.1601, found 246.1605.
1.83 mmol) was added to butan-1-amine (10 mL) and this reaction
solution was stirred for 2 h at room temperature. The solvent was
removed, the residue was partitioned between dichloromethane
(20 mL) and H2O (15 mL). The combined organic layer was dried
over Na2SO4 and concentrated to give yellow oil (0.33 g) in 70%
yield: 1H NMR (300 MHz, CDCl3): d 8.14 (s, 1H, Ar-NH), 7.69 (d,
4.1.9. Data for (S)-2-amino-N-tert-butyl-3-(1H-indol-3-yl)-
propanamide (6)
This compound was obtained as a yellow solid in 71% yield:
3JHH = 7.8 Hz, 1H, Ar-H), 7.38 (d, JHH = 7.8 Hz, 1H, Ar-H), 7.21 (t,
mp = 53–55 °C; 1H NMR (400 MHz, CDCl3): d 8.81 (s, 1H, Ar-NH),
3
3JHH = 7.5 Hz, 1H, Ar-H), 7.13 (t, JHH = 7.5 Hz, 1H, Ar-H), 7.08 (s,
7.64 (d, JHH = 8.0 Hz, 1H, Ar-H), 7.36 (d, JHH = 8.0 Hz, 1H, Ar-H),
3
3
3
3
3
1H, Ar-H), 3.71 (dd, JHH = 9.0 Hz, 3.9 Hz, 1H, CH), 3.40 (dd,
7.17 (t, JHH = 7.6 Hz, 1H, Ar-H), 7.11 (s, 1H, O@CANH), 7.08 (t,
3JHH = 3.9 Hz, JHH = 14.4 Hz, 1H, CH2), 3.25 (q, JHH = 6.6 Hz, 1H,
3JHH = 7.6 Hz, 1H, Ar-H), 7.04 (s, 1H, Ar-H), 3.63 (dd, JHH = 8.4 Hz,
2
3
3
3
2
3
2
CH2), 2.91 (dd, JHH = 9.0 Hz, JHH = 14.4 Hz, 1H, CH2), 1.57–1.20
4.4 Hz, 1H, CH), 3.34 (dd, JHH = 4.4 Hz, JHH = 14.4 Hz, 1H, CH2),
2.20 (br, 2H, NH2), 1.31 (s, 9H, CH3). 13C NMR (100 MHz, CDCl3):
d 173.8, 136.5, 127.5, 123.4, 122.1, 119.5, 118.9, 111.4, 55.9, 50.6,
30.6, 28.7. HRMS (ESI) calcd for C15H22N3O [M+H]+ 250.1757, found
250.1756.
(m, 4H, CH2CH2), 0.91 (t, JHH = 7.5 Hz, 3H, CH3). 13C NMR
3
(100 MHz, CDCl3): d 174.7, 136.4, 127.5, 123.1, 122.2, 119.5,
119.0, 111.8, 111.3, 55.7, 38.8, 31.6, 30.9, 20.1, 13.8. HRMS (ESI)
calcd for C15H22N3O [M+H]+ 260.1757, found 260.1760.
4.1.7. Synthesis of (S)-2-amino-3-(1H-indol-3-yl)-N-octylpro-
panamide (4)20
4.1.10. Data for (S)-2-amino-N,N-diethyl-3-(1H-indol-3-yl)-
propanamide (7)
To a stirred solution of (S)-2-(tert-butoxycarbonylamino)-3-
(1H-indol-3-yl)propanoic acid(0.50 g, 1.64 mmol), 4-dimethy-
laminopyridine (DMAP, 0.40 g, 3.28 mmol) and triethylamine
(0.33 g, 3.28 mmol) in dichloromethane (40 mL) was added
1-ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride
(EDCI, 0.63 g, 3.28 mmol) in one portion. After the mixture was
stirred at 0 °C for 0.5 h, n-octylamine (0.32 g, 2.46 mmol) was
added to the above mixture and the mixture was then kept at room
temperature for another 8 h until the reaction was complete
detected by TLC. A saturated solution of ammonium chloride
(30 mL) was added to the reaction, and the layers were separated.
The organic phase was washed with saturated brine, dried over
Na2SO4 and concentrated. The crude product was chro-
matographed on silica gel (petroleum ether/ethyl acetate 2:1) to
give a yellow solid. Then a mixture of HCl (4 M, 4 mL)/dioxane
(4 mL) was added, and the mixture was stirred for 3 h at room tem-
perature. After the solvent was removed under reduced pressure,
the residue was resolved in H2O (20 mL) and the pH was adjusted
to around 10 by progressively adding solid Na2CO3. The aqueous
solution was extracted with ethyl acetate (25 mL ꢀ 4), and the
combined organic layer was washed with saturated brine, dried
over Na2SO4, and concentrated to give the expected compound 4
(0.39 g) as yellow oil in 75% yield: 1H NMR (300 MHz, CDCl3): d
This compound was obtained as yellow oil in 62% yield: 1H NMR
3
(400 MHz, CDCl3): d 8.66 (s, 1H,NH), 7.57 (d, JHH = 7.6 Hz, 1H, Ar-
H), 7.36 (d, 3JHH = 8.0 Hz, 1H, Ar-H), 7.18 (t, 3JHH = 7.2 Hz, 1H, Ar-H),
3
7.11 (t, JHH = 7.2 Hz, 1H, Ar-H), 7.05 (s, 1H, Ar-H), 3.96 (t,
3
2
3JHH = 6.8 Hz, CH), 3.45 (dd, JHH = 6.8 Hz, JHH = 13.6 Hz, 1H, CH2),
3
2
3.31–3.01 (m, 4H, CH2CH3), 2.94 (dd, JHH = 7.6 Hz, JHH = 14.0 Hz,
1H, CH2), 2.46 (br, 1H, NH2), 1.13–0.94 (m, 6H, CH2CH3). 13C
NMR (100 MHz, CDCl3): d 174.1, 136.4, 127.5, 123.3, 122.0, 119.4,
118.4, 111.4, 51.7, 41.6, 40.5, 32.4, 14.6, 13.0. HRMS (ESI) calcd
for C15H22N3O [M+H]+ 260.1758, found 260.1756.
4.1.11. Data for (S)-2-amino-3-(1H-indol-3-yl)-N-(prop-2-ynyl)-
propanamide (8)
This compound was obtained as yellow oil in 71% yield: 1H NMR
(300 MHz, DMSO-d6): d 10.85 (s, 1H, NH), 8.29 (s, 1H, O@CANH),
3
3
7.56 (d, JHH = 7.5 Hz, 1H, Ar-H), 7.33 (d, JHH = 8.1 Hz, 1H, Ar-H),
3
7.15 (s, 1H, Ar-H), 7.06 (t, JHH = 7.5 Hz, 1H, Ar-H), 6.97 (t,
3JHH = 7.2 Hz, 1H, Ar-H), 3.86 (s, 2H, N-CH2), 3.46 (dd, 3JHH = 7.8 Hz,
4.8 Hz, 1H, CH), 3.17–2.97 (m, 2H, CCH and CH2), 2.74 (dd,
3JHH = 8.1 Hz, JHH = 14.1 Hz, 1H, CH2). 13C NMR (100 MHz, DMSO-
2
d6): d 174.5, 136.2, 127.4, 123.8, 120.9, 118.5, 118.2, 111.3, 110.5,
81.3, 72.8, 55.3, 31.0, 27.9. HRMS (ESI) calcd for C14H16N3O [M
+H]+ 242.1288, found 242.1291.
3
8.49 (s, 1H, Ar-NH), 7.64 (d, JHH = 7.8 Hz, 1H, Ar-H), 7.35 (d,
3JHH = 8.1 Hz, 1H, Ar-H), 7.32–7.24 (m, 1H, O@CANH), 7.17 (t,
3JHH = 7.5 Hz, 1H, Ar-H), 7.13–7.05 (m, 2H, Ar-H), 3.79 (dd,
3JHH = 7.5 Hz, 4.5 Hz, 1H, CH), 3.36 (dd, 3JHH = 3.9 Hz, 2JHH = 14.7 Hz,
4.1.12. Data for (S)-2-amino-N-cyclohexyl-3-(1H-indol-3-yl)-
propanamide (9)
This compound was obtained as a yellow solid in 78% yield:
3
1H, CH2), 3.24–3.08 (m, 2H, N-CH2), 2.97 (dd, JHH = 8.4 Hz,
mp = 58–60 °C; 1H NMR (400 MHz, CDCl3): d 8.81 (s, 1H, Ar-NH),
2JHH = 14.4 Hz, 1H, CH2), 2.76 (br, 2H, NH2), 1.45–1.34 (m, 2H,
7.63 (d, JHH = 7.6 Hz, 1H, Ar-H), 7.35 (d, JHH = 8.0 Hz, 1H, Ar-H),
3
3
3
3
CH2), 1.34–1.13 (m, 10H, CH2), 0.87 (t, JHH = 6.6 Hz, 3H, CH3). 13C
7.22–7.11 (m, 2H, O@CANH and Ar-H), 7.07 (t, JHH = 7.6 Hz, 1H,
NMR (100 MHz, CDCl3): d 173.8, 136.4, 127.5, 123.5, 122.2, 119.5,
Ar-H), 7.03 (s, 1H, Ar-H), 3.83–3.63 (m, 2H, CH), 3.35 (dd,
Please cite this article in press as: Huang, Y.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.08.016

8
Y. Huang et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
2
3
3JHH = 3.6 Hz, JHH = 14.4 Hz, 1H, CH2), 2.91 (dd, JHH = 8.8 Hz,
2JHH = 14.4 Hz, 1H, CH2), 2.20 (s, 2H, NH2), 1.89–1.73 (m, 2H,
CH2), 1.73–1.49 (m, 3H, CH2), 1.40–1.22 (m, 2H, CH2), 1.21–1.00
(m, 3H, CH2). 13C NMR (100 MHz, CDCl3): d 173.6, 136.5, 127.5,
123.4, 122.1, 119.4, 118.9, 111.4, 111.3, 55.4, 47.8, 33.0, 32.9,
4.1.17. Data for (2S)-2-amino-3-(1H-indol-3-yl)-N-((S)-1-
phenylethyl)propanamide (14)
This compound was obtained as a yellow solid in 75% yield:
mp = 91–92 °C; 1H NMR (400 MHz, CDCl3): d 8.18 (s, 1H,NH),
3
3
7.69 (d, JHH = 8.0 Hz, 1H, Ar-H), 7.53 (d, JHH = 8.0 Hz, 1H,
3
30.7, 25.5, 24.8. HRMS (ESI) calcd for
286.1914, found 286.1914.
C
17H24N3O [M+H]+
O@CANH), 7.37 (d, JHH = 8.0 Hz, 1H, Ar-H), 7.34–7.24 (m, 5H,
Ph-H), 7.21 (t, JHH = 8.0 Hz, 1H, Ar-H), 7.13 (t, JHH = 7.6 Hz, 1H,
3
3
4
Ar-H), 7.04 (d, JHH = 1.6 Hz, 1H, Ar-H), 5.17–5.07 (m, 1H, CH),
3
3
4.1.13. Data for (2S)-2-amino-3-(1H-indol-3-yl)-N-((tetrahydro-
furan-2-yl)methyl)propanamide (10)
3.71 (dd, JHH = 8.8 Hz,4.0 Hz, 1H, CH), 3.40 (dd, JHH = 4.0 Hz,
2JHH = 14.4 Hz, 1H, CH2), 2.96 (dd, JHH = 8.8 Hz, JHH = 14.4 Hz, 1H,
3
2
This compound was obtained as yellow oil in 80% yield: 1H NMR
CH2), 1.60 (s, 1H, NH2), 1.42 (d, JHH = 6.8 Hz, 3H, CH3). 13C NMR
3
3
(400 MHz, CDCl3): d 8.45 (s, 1H, Ar-NH), 7.67 (d, JHH = 7.6 Hz, 1H,
(100 MHz, CDCl3): d 173.8, 143.5, 136.4, 128.6, 127.5, 127.2,
126.2, 123.1, 122.3, 119.7, 119.0, 111.8, 110.3, 55.5, 48.3, 30.8,
22.0. HRMS (ESI) calcd for C19H22N3O [M+H]+ 308.1758, found
308.1764.
3
Ar-H), 7.63–7.50 (m, 1H, O@CANH), 7.37 (d, JHH = 8.0 Hz, 1H,
3
Ar-H), 7.19 (t, JHH = 7.6 Hz, 1H, Ar-H), 7.15–7.08 (m, 1H, Ar-H),
7.05 (t, 4JHH = 2.8 Hz, 1H, Ar-H), 3.98–3.88 (m, 1H, O-CH), 3.86–3.78
(m, 1H, O-CH2), 3.76–3.69 (m, 2H, O-CH2 and CH), 3.59–3.49 (m,
3
2
1H, N-CH2), 3.39 (dd, JHH = 4.0 Hz, JHH = 14.4 Hz, 1H, CH2), 3.23–
3.12 (m, 1H, N-CH2), 2.90 and 2.89 (dd, 3JHH = 9.2 Hz, 2JHH = 14.4 Hz,
1H, CH2), 1.99–1.90 (m, 1H, CH2), 1.90–1.80 (m, 2H, CH2), 1.78 (br,
2H, NH2), 1.55–1.44 (m, 1H, CH2). 13C NMR (100 MHz, CDCl3): d
175.1 and 175.0, 136.5, 127.5 and 127.5, 123.2, 122.2, 119.5, 119.0,
111.7, 111.3, 77.8 and 77.8, 68.1 and 68.1, 55.7 and 55.6, 43.0 and
43.0, 30.9 and 30.8, 28.7 and 28.7, 25.9. HRMS (ESI) calcd for
4.1.18. Data for (S)-2-amino-3-(1H-indol-3-yl)-N-phenethylpro-
panamide (15)
This compound was obtained as yellow oil in 75% yield: 1H NMR
3
(400 MHz, CDCl3): d 8.44 (s, 1H, NH), 7.64 (d, JHH = 6.8 Hz, 1H,
O@CANH), 7.35 (d, 3JHH = 7.2 Hz, 1H, Ar-H), 7.33–7.05 (m, 8H, Ar-H
and Ph-H), 7.02 (s, 1H, Ar-H), 3.74–3.63 (m, 1H, CH), 3.56–3.40 (m,
2H, CH2), 3.39–3.26 (m,1H, CH2), 2.98–2.85 (m, 1H, CH2), 2.06 (br,
1H, NH2), 1.79–1.65 (m, 2H, CH2). 13C NMR (100 MHz, CDCl3): d
174.6, 139.0, 136.4, 128.8, 128.5, 127.5, 126.4, 123.2, 122.2, 119.6,
119.0, 111.5, 111.3, 55.6, 40.3, 35.7, 30.7. HRMS (ESI) calcd for
C
16H22N3O2 [M+H]+ 288.1707, found 288.1708.
4.1.14. Data for (S)-2-amino-N-benzyl-3-(1H-indol-3-yl)-
propanamide (11)
C
19H22N3O [M+H]+ 308.1758, found 308.1764.
This compound was obtained as a yellow solid in 73% yield:
mp = 41–43 °C; 1H NMR (400 MHz, DMSO-d6): d 10.90 (s, 1H,NH),
4.1.19. Data for (S)-2-amino-3-(1H-indol-3-yl)-N-(4-methoxy-
benzyl)propanamide (16)
3
8.45 (s, 1H, O@CANH), 7.59 (d, JHH = 6.8 Hz, 1H, Ar-H), 7.35 (d,
3JHH = 7.2 Hz, 1H, Ar-H), 7.30–7.10 (m, 6H, Ph-H and Ar-H), 7.07
This compound was obtained as yellow oil in 80% yield: 1H NMR
3
3
3
(t, JHH = 7.6 Hz, 1H, Ar-H), 6.98 (t, JHH = 7.2 Hz, 1H, Ar-H), 4.35–
4.19 (m, 1H, CH2), 3.89 (br, 1H, NH2), 3.65–3.56 (m, 1H, CH),
(400 MHz, CDCl3): d 8.73 (s, 1H,NH), 7.63 (d, JHH = 7.2 Hz, 1H,
3
Ar-H), 7.55 (s, 1H, O@CANH), 7.34 (d, JHH = 7.6 Hz, 1H, Ar-H),
3
2
3
3.20–3.07 (m, 2H, CH2), 2.85 (dd, JHH = 7.2 Hz, JHH = 14.0 Hz, 1H,
CH2). 13C NMR (100 MHz, DMSO-d6): d 173.6, 139.3, 136.3, 128.2,
127.4, 127.2, 126.7, 124.0, 120.9, 118.6, 118.3, 111.4, 110.0, 55.1,
42.0, 30.6. HRMS (ESI) calcd for C18H20N3O [M+H]+ 294.1601, found
294.1606.
7.16 (t, JHH = 6.8 Hz, 1H, Ar-H), 7.13–7.03 (m, 3H, Ph-H and
3
Ar-H), 6.97 (s, 1H, Ar-H), 6.80 (d, JHH = 7.2 Hz, 2H, Ph-H), 4.34 (d,
4JHH = 2.8 Hz, 1H, CH2), 3.85–3.63 (m, 4H, CH3 and CH), 3.36 (d,
3JHH = 13.2 Hz, 1H, CH2), 3.01–2.86 (m, 1H, CH2), 1.77 (br, 1H,
NH2). 13C NMR (100 MHz, CDCl3): d 174.8, 158.9, 136.5, 130.5,
129.1, 123.4, 122.1, 119.5, 118.9, 114.0, 111.4, 111.4, 55.6, 55.3,
42.7, 30.9. HRMS (ESI) calcd for C19H22N3O2 [M+H]+ 324.1707,
found 324.1697.
4.1.15. Data for (S)-2-amino-3-(1H-indol-3-yl)-N-phenylpro-
panamide (12)
This compound was obtained as a yellow solid in 77% yield:
mp = 48–49 °C; 1H NMR (400 MHz, DMSO-d6): d 10.87 (s, 1H,NH),
7.66–7.56 (m, 3H, Ar-H and Ph-H), 7.36–7.26 (m, 3H, Ar-H and
4.1.20. Data for (S)-2-amino-N-(2,4-dimethoxybenzyl)-3-(1H-
indol-3-yl)propanamide (17)
4
Ph-H), 7.17 (d, JHH = 2.0 Hz, 1H, Ar-H), 7.09–7.01 (m, 2H, Ar-H
This compound was obtained as yellow oil in 69% yield: 1H NMR
(400 MHz, CDCl3): d 8.24 (s, 1H,NH), 7.66 (d, 3JHH = 8.0 Hz, 1H, Ar-H),
7.52 (t, 3JHH = 5.2 Hz, 1H, O@CANH), 7.35 (d, 3JHH = 8.0 Hz, 1H, Ar-H),
7.19 (t, 3JHH = 7.6 Hz, 1H, Ar-H), 7.13 (d, 3JHH = 8.0 Hz, 1H, Ph-H), 7.11
3
3
and Ph-H), 6.96 (t, JHH = 7.6 Hz, 1H, Ar-H), 3.66 (dd, JHH = 7.2 Hz,
3
2
5.6 Hz, 1H, CH), 3.16 (dd, JHH = 5.2 Hz, JHH = 14.0 Hz, 1H, CH2),
3
2
2.89 (dd, JHH = 7.6 Hz, JHH = 14.0 Hz, 1H, CH2), 1.56 (br, 2H,
NH2), 1.10 (t, JHH = 6.4 Hz, 3H, CH3). 13C NMR (100 MHz, DMSO-
(t, JHH = 7.6 Hz, 1H, Ar-H), 6.98 (d, JHH = 1.2 Hz, 1H, Ar-H),
3
3
4
4
3
d6): d 173.5, 138.8, 136.2, 128.7, 127.4, 123.8, 123.2, 120.9, 119.3,
6.47–6.38 (m, 2H, Ph-H), 4.37 (dd, JHH = 4.0 Hz, JHH = 5.6 Hz, 2H,
CH2), 3.80 (s, 3H, CH3), 3.76 (s, 3H, CH3), 3.70 (dd, JHH = 8.8 Hz,
4.4 Hz, 1H, CH2), 3.38 (dd, JHH = 4.4 Hz, JHH = 14.4 Hz, 1H, CH2),
2.90 (dd, JHH = 8.8 Hz, JHH = 14.4 Hz, 1H, CH2), 1.58 (br, 1H,
NH2).13C NMR (100 MHz, CDCl3): d 174.4, 160.4, 158.6, 136.4,
130.3, 127.5, 123.1, 122.2, 119.6, 119.1, 119.0, 111.9, 111.2, 103.9,
98.6, 55.8, 55.4, 55.3, 38.4, 30.9. HRMS (ESI) calcd for C20H24N3O3
[M+H]+ 354.1812, found 354.1806.
3
118.5, 118.2, 111.3, 110.3, 56.1, 30.7. HRMS (ESI) calcd for
3
2
C
17H18N3O [M+H]+ 280.1444, found 280.1446.
3
2
4.1.16. Data for (S)-2-amino-3-(1H-indol-3-yl)-N-(thiazol-2-yl)-
propanamide (13)
This compound was obtained as a yellow solid in 76% yield:
mp = 74–76 °C; 1H NMR (400 MHz, DMSO-d6): d 10.86 (s, 1H,NH),
3
7.58 (d, JHH = 7.6 Hz, 1H, Ar-H), 7.46 (s, 1H, thiazole-H), 7.32 (d,
3JHH = 7.6 Hz, 1H, Ar-H), 7.20 (s, 1H, thiazole-H), 7.13 (s, 1H,
4.1.21. Data for (S)-2-amino-N-(4-chlorobenzyl)-3-(1H-indol-3-
yl)propanamide (18)
3
3
Ar-H), 7.05 (t, JHH = 6.8 Hz, 1H, Ar-H), 6.94 (t, JHH = 6.8 Hz, 1H,
Ar-H), 5.33 (br, 1H, NH2), 3.85–3.74 (m, 1H, CH), 3.14 (dd,
This compound was obtained as yellow oil in 83% yield: 1H NMR
(400 MHz, CDCl3): d 8.43 (s, 1H,NH), 7.70–7.59 (m, 2H, Ar-H and
2
3
3JHH = 5.2 Hz, JHH = 13.6 Hz, 1H, CH2), 2.91 (dd, JHH = 7.2 Hz,
2JHH = 13.6 Hz, 1H, CH2). 13C NMR (100 MHz, CDCl3): d 173.9,
158.7, 138.3, 137.1, 128.1, 124.0, 123.1, 120.5, 119.4, 114.2,
112.1, 111.5, 56.0, 31.1. HRMS (ESI) calcd for C14H15N4OS [M+H]+
287.0961, found 287.0964.
3
O@CANH), 7.36 (d, JHH = 8.0 Hz, 1H, Ar-H), 7.24–7.16 (m, 3H,
Ar-H and Ph-H), 7.14–7.03 (m, 3H, Ph-H and Ar-H), 7.00 (s, 1H,
Ar-H), 4.37 (t, 4JHH = 5.2 Hz, 3JHH = 5.2 Hz, 1H, CH2), 3.75 (dd, 3JHH
=
3
2
8.4 Hz, 4.4 Hz, 1H, CH2), 3.37 (dd, JHH = 4.4 Hz, JHH = 14.4 Hz, 1H,
Please cite this article in press as: Huang, Y.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.08.016

Y. Huang et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
9
3
2
3
CH2), 2.99 (dd, JHH = 8.4 Hz, JHH = 14.4 Hz, 1H, CH2), 1.60 (br, 1H,
NH2). 13C NMR (100 MHz, CDCl3): d 174.9, 137.0, 136.5, 133.1,
129.0, 128.7, 127.5, 123.2, 122.3, 119.7, 119.0, 111.5, 111.3, 55.5,
42.4, 30.8. HRMS (ESI) calcd for C18H19N3OCl [M+H]+ 328.1211,
found 328.1210.
3H, Py-H and O@CANH), 7.56 (d, JHH = 8.0 Hz, 1H, Ar-H), 7.48–
3
7.43 (m, 1H, Py-H), 7.35 (d, JHH = 8.0 Hz, 1H, Ar-H), 7.26 (dd,
3JHH = 4.8 Hz, 7.6 Hz, 1H, Py-H), 7.14 (d, JHH = 2.0 Hz), 7.06 (t,
4
3JHH = 7.6 Hz, 1H, Ar-H), 6.97 (t, JHH = 7.6 Hz, 1H, Ar-H), 4.28 (t,
3
4JHH = 2.4 Hz, 2H, Py-CH2), 3.52 (dd, JHH = 5.2 Hz, 7.6 Hz, 1H, CH),
3
3
2
3.09 (dd, JHH = 5.2 Hz, JHH = 14.0 Hz, 1H, CH2), 2.80 (dd,
2
4.1.22. Data for (S)-2-amino-N-(3-chlorobenzyl)-3-(1H-indol-3-
yl)propanamide (19)
3JHH = 7.6 Hz, JHH = 14.0 Hz, 1H, CH2). 13C NMR (100 MHz, DMSO-
d6): d 174.9, 148.8, 148.0, 136.4, 135.1, 135.1, 127.5, 124.0, 123.5,
121.0, 118.6, 118.4, 111.5, 110.5, 55.6, 40.4, 31.1. HRMS (ESI) calcd
for C17H19N4O [M+H]+ 295.1553, found 295.1558.
This compound was obtained as yellow oil in 79% yield: 1H NMR
(400 MHz, CDCl3): d 8.39 (s, 1H,NH), 7.72–7.60 (m, 2H, Ar-H and
O@CANH), 7.37 (d, 3JHH = 8.0 Hz, 1H, Ar-H), 7.23–7.14 (m, 4H, Ar-H
3
3
and Ph-H), 7.11 (t, JHH = 7.6 Hz, 1H, Ar-H), 7.05 (d, JHH = 5.6 Hz,
4.1.26. Synthesis of (S)-methyl 2-(3-butylureido)-3-(1H-indol-3-
yl)propanoate (23)
1H, Ph-H), 7.01 (d, 3JHH = 0.8 Hz, 1H, Ar-H), 4.42–4.36 (m, 1H, CH2),
3
3
3.76 (dd, JHH = 8.0 Hz, 4.0 Hz, 1H, CH2), 3.38 (dd, JHH = 4.4 Hz,
A mixture of (S)-methyl 2-amino-3-(1H-indol-3-yl)propanoate
(0.60 g, 2.75 mmol), NEt3 (0.3 mL), n-butyl isocyanate (0.33 g,
3.3 mmol) in dichloromethane (40 mL) were stirred at rt overnight.
The mixture was concentrated in vacuum when the reaction was
complete detected by TLC, and the crude product was chro-
matographed on silica gel (petroleum oil/ethyl acetate 30:1) to
give compound 23 (0.65 g) as oil in 85% yield: 1H NMR
2JHH = 14.4 Hz, 1H, CH2), 3.01 (dd, JHH = 8.4 Hz, JHH = 14.4 Hz, 1H,
CH2), 1.52 (br, 1H, NH2). 13C NMR (100 MHz, CDCl3): d 175.0,
140.6, 136.4, 134.4, 129.9, 127.7, 127.5, 127.5, 125.9, 123.3, 122.3,
119.7, 118.9, 111.5, 111.4, 55.6, 42.6, 30.8. HRMS (ESI) calcd for
3
2
C
18H19N3OCl [M+H]+ 328.1211, found 328.1210.
3
4.1.23. Data for (S)-2-amino-N-(2-chlorobenzyl)-3-(1H-indol-3-
yl)propanamide (20)
(400 MHz, CDCl3) d 8.68 (s, 1H, Ar-NH), 7.50 (d, JHH = 8.0 Hz, 1H,
Ar-H), 7.30 (d, JHH = 8.0 Hz, 1H, Ar-H), 7.14 (t, JHH = 7.6 Hz, 1H,
3
3
This compound was obtained as yellow oil in 77% yield: 1H NMR
(400 MHz, CDCl3): d 8.35 (s, 1H,NH), 7.90–7.56 (m, 2H, Ar-H and
O@CANH), 7.52–6.86 (m, 8H, Ar-H and Ph-H), 4.53 (s, 1H, CH2),
3.86–3.62 (m, 1H, CH), 3.54–3.25 (m, 1H, CH2), 3.11–2.82 (m, 1H,
CH2), 1.56 (br, 1H, NH2). 13C NMR (100 MHz, CDCl3): d 174.9,
136.4, 135.8, 133.7, 129.9, 129.5, 128.8, 127.5, 127.0, 123.2,
122.3, 119.6, 118.9, 111.6, 111.3, 55.7, 41.1, 30.8. HRMS (ESI) calcd
for C18H19N3OCl [M+H]+ 328.1211, found 328.1208.
Ar-H), 7.07 (t, JHH = 7.6 Hz, 1H, Ar-H), 6.90 (d, JHH = 2.0 Hz, 1H,
Ar-H), 5.27 (br, 1H, C@ONH), 4.94 (br, 1H, C@ONH), 4.79 (dd,
3
3
3JHH = 13.2 Hz, JHH = 5.6 Hz, 1H, CHC@O), 3.62 (s, 3H, OCH3), 3.22
3
3
(d, JHH = 5.2 Hz, 2H, CH2CH), 2.97 (br, 1H, CH2NH), 1.37–1.15 (m,
4H, CH2), 0.93–0.77 (m, 3H, CH3); 13C NMR (100 MHz, CDCl3) d
174.0, 157.9, 136.2, 127.6, 123.2, 122.0, 119.4, 118.5, 111.4,
109.8, 53.7, 52.2, 40.2, 32.0, 28.1, 19.9, 13.7; HRMS (ESI) calcd for
C
17H23N3O3 [M+H]+ 318.1812, found 318.1816.
Compounds 24–27R were synthesized using the similar proce-
4.1.24. Synthesis of (S)-2-amino-N-(2-hydroxyethyl)-3-(1H-
indol-3-yl)propanamide (21)21
dure as compound 23.
To a stirred solution of (S)-2-(benzyloxycarbonylamino)-3-(1H-
indol-3-yl)propanoic acid(0.50 g, 1.48 mmol), DMAP (0.36 g,
1.48 mmol) and triethylamine (0.30 g, 2.96 mmol) in dichloro-
methane (40 mL) was added EDCI (0.57 g, 2.96 mmol) in one por-
tion. The mixture was stirred at 0 °C for 0.5 h, ethanolamine
(0.13 g, 2.13 mmol) was added to the above mixture and then kept
at room temperature for another 8 h, the reaction was complete
detected by TLC. A saturated solution of ammonium chloride
(30 mL) was added to the reaction, and the layers were separated.
The organic phase was washed with saturated brine, dried over
Na2SO4 and concentrated. The crude product was chro-
matographed on silica gel (dichloromethane/methanol 30:1) to
give a white solid. Then methanol (30 mL) and Pd/C (0.02 g, 10%)
was added, and the solution was purged with H2 overnight at room
temperature. The mixture was filtered through a Celite pad, and
the filtrate was concentrated to give compound 21 (0.15 g) as
brown oil in 41% yield: 1H NMR (400 MHz, DMSO-d6): d 10.97 (s,
1H,NH), 8.33 (t, 3JHH = 5.2 Hz, 1H, O@CANH), 7.63 (d, 3JHH = 7.6 Hz,
4.1.27. Data for (S)-methyl 3-(1H-indol-3-yl)-2-(3-
isopropylureido)propanoate (24)
This compound was obtained as a yellow solid in 80% yield:
mp = 58–61 °C; 1H NMR (400 MHz, DMSO-d6) d 10.91 (s, 1H, Ar-
3
3
NH), 7.44 (d, JHH = 8.0 Hz, 1H, Ar-H), 7.34 (d, JHH = 8.0 Hz, 1H,
3
Ar-H), 7.15–7.02 (m, 2H, Ar-H), 6.98 (t, JHH = 7.6 Hz, 1H, Ar-H),
6.01 (d, 3JHH = 7.6 Hz, 1H, CONH), 5.96 (d, 3JHH = 8.0 Hz, 1H, CONH),
4.45 (dd, JHH = 13.6 Hz,3JHH = 6.4 Hz, 1H, C@OCH), 3.68–3.59 (m,
3
1H, CH), 3.57 (s, 3H, OCH3), 3.14–2.99 (m, 2H, CH2), 1.00 (d,
3JHH = 6.8 Hz, 6H, CH3); 13C NMR (100 MHz, DMSO-d6) d 173.9,
157.2, 136.5, 127.7, 124.2, 121.4, 118.8, 118.6, 111.8, 109.6, 53.8,
52.1, 41.3, 28.3, 23.7, 23.6; HRMS (ESI) calcd for C16H21N3O3 [M
+H]+ 304.1656, found 304.1662.
4.1.28. Data for (S)-methyl 2-(3-cyclohexylureido)-3-(1H-indol-
3-yl)propanoate (25)
This compound was obtained as a yellow solid in 81% yield:
mp = 84–87 °C; 1H NMR (400 MHz, DMSO-d6) d 10.90 (s, 1H,
3
4
3
3
1H, Ar-H), 7.35 (d, JHH = 7.6 Hz, 1H, Ar-H), 7.19 (d, JHH = 2.0 Hz,
Ar-NH), 7.44 (d, JHH = 8.0 Hz, 1H, Ar-H), 7.33 (d, JHH = 8.0 Hz, 1H,
3
3
3
1H, Ar-H), 7.08 (t, JHH = 7.2 Hz, 1H, Ar-H), 6.99 (t, JHH = 7.2 Hz,
Ar-H), 7.12–7.02 (m, 2H, Ar-H), 6.97 (t, JHH = 7.6 Hz, 1H, Ar-H),
6.08 (d, JHH = 8.0 Hz, 1H, NH), 5.98 (d, JHH = 8.0 Hz, 1H, NH), 4.45
3
3
3
1H, Ar-H), 5.91 (br, 2H,NH2), 4.77 (s, 1H, OH), 3.72 (dd, JHH = 8.0 -
3
3
Hz, 5.6 Hz, 1H, CH), 3.45–3.35 (m, 2H, CH2), 3.25–3.05 (m, 3H, CH2),
2.94 (dd, 3JHH = 8.0 Hz, 2JHH = 14.4 Hz, 1H, CH2). 13C NMR (100 MHz,
DMSO-d6): d 171.2, 136.2, 127.2, 124.3, 121.0, 118.4, 118.3, 111.3,
108.6, 59.6, 53.7, 41.5, 28.9. HRMS (ESI) calcd for C13H18N3O2 [M
+H]+ 248.1394, found 248.1397.
(dd, JHH = 13.6 Hz, JHH = 6.4 Hz, 1H, C@OCH), 3.57 (s, 3H, OCH3),
3.33–3.27 (br, 1H, CH), 3.14–2.98 (m, 2H, CH2), 1.80–0.94 (m,
10H, cyclohexyl–CH2); 13C NMR (100 MHz, DMSO-d6) d 173.4,
156.6, 136.1, 127.3, 123.6, 120.9, 118.3, 118.1, 111.3, 109.1, 53.3,
51.6, 47.6, 33.2, 33.1, 27.8, 25.2, 24.3; HRMS (ESI) calcd for
Compounds 22 were synthesized using the similar procedure as
compound 21.
C
19H26N3O3 [M+H]+ 344.1969, found 344.1975.
4.1.29. Data for (S)-methyl 2-(3-cyclopentylureido)-3-(1H-indol-
3-yl)propanoate (26)
4.1.25. Data for (S)-2-amino-3-(1H-indol-3-yl)-N-(pyridin-3-
ylmethyl)propanamide (22)
This compound was obtained as a yellow solid in 82% yield:
This compound was obtained as a yellow solid in 49% yield: 1
H
mp = 60–62 °C; 1H NMR (400 MHz, DMSO-d6) d 10.91 (s, 1H,
3
3
NMR (400 MHz, DMSO-d6): d 10.89 (s, 1H, Ar-NH), 8.50–8.40 (m,
Ar-NH), 7.44 (d, JHH = 8.0 Hz, 1H, Ar-H), 7.34 (d, JHH = 8.0 Hz,
Please cite this article in press as: Huang, Y.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.08.016

10
Y. Huang et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
2
3
1H, Ar-H), 7.13–7.03 (m, 2H, Ar-H), 6.97 (t, 3JHH = 7.6 Hz, 1H, Ar-H),
(m, 1H, CH(CH2)2), 3.18 (dd, JHH = 15.2 Hz, JHH = 4.4 Hz, 1H,
3
3
2
3
6.17 (d, JHH = 7.2 Hz, 1H, NH), 5.93 (d, JHH = 8.0 Hz, 1H, NH), 4.46
CH2), 3.07 (dd, JHH = 14.8 Hz, JHH = 4.4 Hz, 1H, CH2), 1.53 (m,
4H), 1.08–0.55 (m, 4H); 13C NMR (100 MHz, DMSO-d6) d 183.3,
175.1, 136.2, 127.7, 124.6, 121.2, 119.1, 118.7, 111.5, 107.1, 59.0,
53.6, 28.5, 27.6, 26.4, 25.8, 25.7, 25.2; HRMS (ESI) calcd for
3
3
(dd, JHH = 14.0 Hz, JHH = 6.4 Hz, 1H, C@OCH), 3.89–3.75 (m, 1H,
CH), 3.57 (s, 3H, OCH3), 3.15–2.99 (m, 2H, CH2), 1.81–1.40 (m,
8H, cyclopentyl-CH2); 13C NMR (100 MHz, DMSO-d6) d 173.4,
156.9, 136.1, 127.3, 123.7, 120.9, 118.3, 118.1, 111.3, 109.1, 53.3,
51.6, 50.8, 50.8, 33.0, 32.8, 27.8, 23.1; HRMS (ESI) calcd for
C
18H21N3OS [M+H]+ 328.1478, found 328.1694.
C
18H23N3O3 [M+H]+ 330.1812, found 330.1815.
4.1.34. Data for (S)-5-((1H-indol-3-yl)methyl)-3-cyclopentyl-2-
thioxoimidazolidin-4-one (30)
4.1.30. Data for (S)-methyl 2-(3-benzylureido)-3-(1H-indol-3-
yl)-2-(3-isopropylureido)propanoate (27S)
This compound was obtained as a yellow solid in 83% yield:
mp = 155–158 °C; 1H NMR (400 MHz, DMSO-d6) d 10.89 (s, 1H,
3
This compound was obtained as a white solid in 74% yield:
Ar-NH), 10.33 (s, 1H, NH), 7.52 (d, JHH = 7.6 Hz, 1H, Ar-H), 7.29
mp = 151–153 °C; 1H NMR (400 MHz, DMSO-d6) d 10.92 (s, 1H,
(d, JHH = 8.0 Hz, 1H, Ar-H), 7.07–7.00 (m, 2H, Ar-H), 6.95 (t,
3
3
Ar-NH), 7.46 (d, JHH = 7.6 Hz, 1H, Ar-H), 7.38–7.17 (m, 6H, Ar-H),
3JHH = 7.6 Hz, 1H, Ar-H), 4.66–4.52 (m, 1H, CH(CH2)2), 4.44 (t,
3
2
3
7.11 (d, JHH = 2.4 Hz, 1H, Ar-H), 7.09–7.04 (m, 1H, ArH), 7.01–
3JHH = 4.0 Hz, 1H, CHNH), 3.19 (dd, JHH = 14.8 Hz, JHH = 4.4 1H,
3
2
6.95 (m, 1H, Ar-H), 6.62 (t, JHH = 6.0 Hz, 1H, NHCH2), 6.26 (d,
CH2), 3.09 (dd, JHH = 15.2 Hz,3JHH = 4.4, 1H,CH2),1.92–1.27 (m,
3JHH = 8.0 Hz, 1H, C@ONH), 4.54–4.46 (m, 1H, CHNH), 4.19 (d,
3JHH = 6.0 Hz, 2H, CH2CH), 3.58 (s, 3H, OCH3), 3.17–3.00 (m, 2H,
CH2); 13C NMR (100 MHz, DMSO-d6) d 173.8, 157.9, 141.1, 136.5,
128.7, 127.7, 127.4, 127.7, 124.2, 121.4, 118.9, 118.6, 111.8,
109.6, 54.0, 52.2, 43.2, 28.3; HRMS (ESI) calcd for C20H21N3O3 [M
+H]+ 352.1656, found 352.1657.
8H), 1.21–1.11 (m, 1H); 13C NMR (100 MHz, DMSO-d6) d 183.1,
174.3, 135.7, 127.2, 124.1, 120.8, 118.5, 118.2, 111.0, 106.8, 58.6,
53.2, 27.7, 27.2, 25.9, 24.7, 24.6; HRMS (ESI) calcd for C17H19N3OS
[M+H]+ 314.1322, found 314.1613.
4.1.35. Data for (S)-5-((1H-indol-3-yl)methyl)-3-benyl-2-
thioxoimidazolidin-4-one (31S)
4.1.31. Data for (R)-methyl 2-(3-benzylureido)-3-(1H-indol-3-
yl)-2-(3-isopropylureido)propanoate (27R)
This compound was obtained as a yellow solid in 86% yield:
mp = 185–188 °C; 1H NMR (400 MHz, DMSO-d6) d 10.94 (s, 1H,
3
This compound was obtained as a yellow solid in 75% yield:
Ar-NH), 10.55 (s, 1H, NH), 7.58 (d, JHH = 8.0 Hz, 1H, Ar-H), 7.37
mp = 190–193 °C; 1H NMR (400 MHz, DMSO-d6) d 10.95 (s, 1H,
(d, 3JHH = 8.0 Hz, 1H, Ar-H), 7.06 (m, 5H, Ar-H), 6.96 (t, 3JHH = 7.2 Hz,
3
3
3
Ar–NH), 10.56 (s, 1H, NH), 7.58 (d, JHH = 7.6 Hz, 1H, Ar-H), 7.37
1H, Ar-H), 6.56 (d, JHH = 7.6 Hz, 2H, Ar-H), 4.68 (d, JHH = 3.6 Hz,
3
(d, JHH = 8.0 Hz, 1H, Ar-H), 7.15–6.99 (m, 5H, Ar-H), 6.96 (t,
1H, CHNH), 4.66 (s, 2H, CH2), 3.27 (dd, 2JHH = 14.8 Hz, 3JHH = 4.0 Hz,
3
3JHH = 6.4 Hz, 1H, Ar-H), 6.56(d, JHH = 6.8 Hz, 1H, Ar-H), 6.26 (d,
1H, CH2CH), 3.17 (dd, 2JHH = 14.8 Hz, 3JHH = 4.0 Hz, 1H, CH2CH); 13
C
3JHH = 8.0 Hz, 1H, C@ONH),4.67 (m, 3H, CHNH+CH2Ph), 3.58 (s,
NMR (100 MHz, DMSO-d6) d 182.9, 174.7, 136.3, 136.3, 128.4,
127.8, 127.0, 126.6, 124.8, 121.4, 119.0, 118.9, 111.7, 107.4, 60.2,
43.4, 26.0; HRMS (ESI) calcd for C19H17N3OS [M+H]+ 336.1165,
found 336.1167.
2
2
3H, OCH3),3.28 (d, JHH = 15.2 Hz, 1H, CH2CH), 3.18 (d, JHH = 14.8
Hz, 1H, CH2CH); 13C NMR (100 MHz, DMSO-d6) d 183.6, 175.5,
137.0, 129.1, 128.5, 127.7, 127.3, 125.5, 122.1, 119.8, 119.6,
112.4, 108.1, 61.0, 44.1, 26.7; HRMS (ESI) calcd for C20H21N3O3
[M+H]+ 352.1656, found 352.1657.
4.1.36. Data for (R)-5-((1H-indol-3-yl)methyl)-3-benyl-2-
thioxoimidazolidin-4-one (31R)
4.1.32. Synthesis of (S)-5-((1H-indol-3-yl)methyl)-3-isopropyl-
2-thioxoimidazolidin-2,4-dione (28)
This compound was obtained as a yellow solid in 75% yield:
mp = 190–193 °C; 1H NMR (400 MHz, DMSO-d6) d 10.95 (s, 1H,
3
A mixture of (S)-methyl 2-amino-3-(1H-indol-3-yl)propanoate
(0.60 g, 2.75 mmol), NEt3 (0.3 mL), isopropyl isothiocyanates
(0.34 g, 3.3 mmol) in dichloromethane (40 mL) were stirred at
40 °C overnight. The mixture was concentrated in vacuum when
the reaction was complete detected by TLC, and the crude product
was chromatographed on silica gel (petroleum oil/ethyl acetate
30:1) to give compound 23 (0.63 g) as yellow solid in 85% yield:
mp = 166–169 °C; 1H NMR (400 MHz, DMSO-d6) d 10.91 (s, 1H,
Ar-NH), 10.56 (s, 1H, NH), 7.58 (d, JHH = 7.6 Hz, 1H, Ar-H), 7.37
3
(d, JHH = 8.0 Hz, 1H, Ar-H), 7.15–6.99 (m, 5H, Ar-H), 6.96 (t,
3
3JHH = 6.4 Hz, 1H, Ar-H), 6.56 (d, JHH = 6.8 Hz, 2H, Ar-H), 4.67 (m,
2
3H, CHNH+CH2Ph), 3.28 (d, JHH = 15.2 Hz, 1H, CH2CH), 3.18 (d,
2JHH = 14.8 Hz, 1H, CH2CH); 13C NMR (100 MHz, DMSO-d6)
d
183.6, 175.5, 137.0, 129.1, 128.5, 127.7, 127.3, 125.5, 122.1,
119.8, 119.6, 112.4, 108.1, 61.0, 44.1, 26.7; HRMS (ESI) calcd for
C
19H17N3OS [M+H]+ 336.1165, found 336.1165.
3
Ar–NH), 10.31 (s, 1H, NH), 7.52 (d, JHH = 7.6 Hz, 1H, Ar-H), 7.28
3
(d, JHH = 8.0 Hz, 1H, Ar-H), 7.09–6.99 (m, 2H, Ar-H), 6.95 (t,
4.1.37. Data for (S)-5-((1H-indol-3-yl)methyl)-3-phenyl-2-
thioxoimidazolidin-4-one (32)
3JHH = 7.2 Hz, 1H, Ar-H), 4.42 (m, 2H, CHNH+CH(CH3)2), 3.19 (dd,
3
2
2JHH = 14.8 Hz, JHH = 4.0 Hz, 1H, CH2), 3.07 (dd, JHH = 14.8 Hz,
This compound was obtained as a yellow solid in 83% yield:
3JHH = 4.4 Hz, 1H, CH2), 1.17 (d, JHH = 6.8 Hz, 3H, CH3), 0.68 (d,
mp = 176–179 °C; 1H NMR (400 MHz, DMSO-d6) d 10.98 (s, 1H,
3
3JHH = 6.8 Hz, 3H, CH3); 13C NMR (100 MHz, DMSO-d6) d 183.2,
175.1, 136.2, 127.7, 124.7, 121.2, 119.1, 118.7, 111.5, 107.1, 59.0,
45.7, 26.4, 19.1, 18.3; HRMS (ESI) calcd for C15H17N3OS [M+H]+
288.1165, found 288.1164.
Ar-NH), 10.57 (s, 1H, NH), 7.56 (d, JHH = 8.0 Hz, 1H, Ar-H), 7.36
3
3
(d, JHH = 8.0 Hz, 1H, Ar-H), 7.33–7.27 (m, 3H, Ar-H), 7.15 (d,
3
3JHH = 2.0 Hz, 1H, Ar-H), 7.08 (t, JHH = 7.2 Hz, 1H, Ar-H), 6.97 (t,
3JHH = 7.2 Hz, 1H, Ar-H), 6.68–6.62 (m, 2H, Ar-H), 4.72 (t,
2
3
Compounds 29–32 were synthesized using the similar proce-
dure as compound 28.
3JHH = 4.0 Hz, 1H, CHNH), 3.31 (d, JHH = 14.8 Hz, JHH = 4.0 Hz, 1H,
CH2), 3.22 (dd, JHH = 14.8 Hz, JHH = 4.8 Hz, 1H, CH2); 13C NMR
(100 MHz, DMSO-d6) d 182.8, 174.6, 136.3, 133.6, 128.9, 128.8,
127.6, 124.9, 121.4, 119.1, 118.9, 111.7, 107.3, 60.5, 26.8; HRMS
(ESI) calcd for C18H15N3OS [M+H]+ 322.1009, found 328.1010.
2
3
4.1.33. Data for (S)-5-((1H-indol-3-yl)methyl)-3-cyclohexyl-2-
thioxoimidazolidin-4-one (29)
This compound was obtained as a yellow solid in 85% yield:
mp = 69–72 °C; 1H NMR (400 MHz, DMSO-d6) d 10.88 (s, 1H,
4.2. Biological assay
3
Ar-NH), 10.30 (s, 1H, NH), 7.52 (d, JHH = 8.0 Hz, 1H, Ar-H), 7.28
3
(d, JHH = 8.0 Hz, 1H, Ar-H), 7.06–6.99 (m, 2H, Ar-H), 6.95 (t,
The bioassay was repeated in triplicate at 25 1 °C. The error of
the experiments was 5%. Assessments were made on a dead/alive
3
3JHH = 7.2 Hz, 1H, Ar-H), 4.42 (t, JHH = 4.0 Hz, 1H, CHNH), 4.08
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Y. Huang et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
11
basis, and mortality rates were corrected using Abbott’s formula.
4.2.6. Stomach toxicity against oriental armyworm (Mythimna
Evaluations were based on a percentage scale of 0–100, where 0
equals no activity and 100 equals total kill.
separate), cotton bollworm (Helicoverpa armigera), pyrausta
nubilalis (Ostrinia nubilalis)
The stomach toxicities of the title compounds 1, 3, 4, 6–20, and
22–32 against oriental armyworm, cotton bollworm, pyrausta
nubilalis were evaluated by foliar application using the reported
procedure. For the foliar tests, individual corn leaves were placed
on moistened pieces of filter paper in Petri dishes. The leaves were
then sprayed with the test solution and allowed to dry. The dishes
were infested with 10 fourth-instar larvae. Percentage mortalities
were evaluated 4 days after treatment. Each treatment was per-
formed three times.
4.2.1. Antiviral activity of compounds against TMV in vitro
Fresh leaf of the 5–6 growth stage of tobacco inoculated by the
juice-leaf rubbing method (concentration of TMV is
5.88 ꢀ 10ꢁ2
l
g/mL) was cut into halves along the main vein. The
g/mL of the com-
halves were immersed into the solution of 500
l
pounds and double-distilled water for 20 min, respectively, and
then cultured at 25 °C for 72 h. Each compound was replicated at
least 3 times.
4.2.2. Protective effect of compounds against TMV in vivo
The compound solution was smeared on the left side, and the
solvent served as a control on the right side of growing Nicotiana
tabacum L. leaves of the same ages. The leaves were then inocu-
lated with the virus after 12 h. A brush was dipped in TMV of
6 ꢀ 10ꢁ3 mg/mL to inoculate the leaves, which were previously
scattered with silicon carbide. The leaves were then washed with
water and rubbed softly along the nervature once or twice. The
local lesion numbers appearing 3–4 days after inoculation were
counted. There are three replicates for each compound.
4.2.7. Larvicidal activity against mosquito
The larvicidal activity of the title compound 1, 3, 4, 6–20, and
22–32against mosquito were tested. These compounds were pre-
pared to a terminal concentration of 10 and 5 mg/kg by dissolving
them in acetone and adding distilled water. Ten fourth-instar
mosquitolarvae were put into the 10 mL of the test solution and
raised for 8 days; the results were expressed by death percentage.
4.2.8. The field trials
The severity of the virus disease was divided into 0–4 grade. The
disease index and control effect can be calculated by using the fol-
lowing formulae:
4.2.3. Inactivation effect of compounds against TMV in vivo
The virus was inhibited by mixing with the compound solution
at the same volume for 30 min. The mixture was then inoculated
on the left side of the leaves of N. tabacum L., whereas the right side
of the leaves was inoculated with the mixture of solvent and the
virus for control. The local lesion numbers were recorded 3–4 days
after inoculation. There are three replicates for each compound.
Disease index ðIÞ% ¼ ½ ðNn ꢀ nÞ=ðN ꢀ 4Þꢂ ꢀ 100
R
Control effect ð%Þ ¼ ½1 ꢁ ðI0bc ꢀ IÞ=ðIbc ꢀ I0Þꢂ ꢀ 100
where Nn is the number of diseased plants of each relative level val-
ues, n is the relative level values (n = 0–4), N is the total number of
diseased plants, I0bc is the disease index of the blank control area
before spraying, Ibc is the disease index of the blank control area
10 days after the third time spraying, I0 is the disease index of the
spraying area before spraying; And I is the disease index of the
spraying area 10 days after the third time spraying.
4.2.4. Curative effect of compounds against TMV in vivo
Growing leaves of N. tabacum L. of the same ages were selected.
TMV (concentration of 6.0 ꢀ 10ꢁ3 mg/mL) was dipped and inocu-
lated on the whole leaves. Then, the leaves were washed with water
and dried. The compound solution was smeared on the left side, and
the solvent was smeared on the right side for control. The local lesion
numbers were then counted and recorded 3–4 days after inocula-
tion. There are three replicates for each compound.
Acknowledgments
The in vitro and in vivo inhibition rates of the compound were
then calculated according to the following formula (‘av’ means
average, and controls were not treated with compound): inhibition
rate (%) = [(av local lesion number of control ꢁ av local lesion num-
ber of drug-treated)/av local lesion number of control] ꢀ 100%.
This work was supported by the National Natural Science
Foundation of China (21132003, 21421062, 21372131), and the
Specialized Research Fund for the Doctoral Program of Higher
Education (20130031110017) and the ‘111’ Project of Ministry of
Education of China (B06005).
4.2.5. Fungicide activity
References and notes
A stock solution of each compound was prepared at 500 mg/kg
using N,N-dimethylformamide (DMF) as a solvent. A working solu-
tion (50 mg/kg) was then prepared by diluting the stock solution
(0.1 mL) with sterilized water (0.9 mL) in a 10 cm diameter Petri
dish. Potato dextrose agar (PDA, 9 mL) was then added to prepare
the plate. Before the plate solidification, the PDA was thoroughly
mixed by turning around the Petri dish in the sterilized operation
desk 5 times to scatter the compounds in PDA evenly. Then,4 mm
of diameter of fungi cake was inoculated on the plate and cultured
in the culture tank at 24–26 °C. The diameter of fungi spread was
measured 2 days later. Growth inhibition was then calculated
using the corresponding control. Fungi used in this study included
Fusarium oxysporiumf. sp. cucumeris (F.C.); Cercospora arachidicola
Hori (C.H.); Physalospora piricola (P.P.); Alternaria solani (A.S.);
Fusarium graminearum (F.G.); Fusarium moniliforme (F.M.); Sclero-
tinia sclerotiorum (S.S.); Phytophthora capsici (P.C.); Rhizoctonia cere-
alis (R.C.); Bipolaris maydis (B.M.); Watermelon-anthracnose (W.A.);
Phytophthora infestans (P.I.); Rhizoctonia solani (R.S.); Botrytis
cinerea (B.C.).
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