Synthesis and fungicidal activity of lansiumamide A and B and their derivatives

Article abstract of DOI:10.3390/molecules23071499

A efficient 2-step protocol has been applied for the synthesis of Lansiumamide B (N-methyl-N-cis-styryl-cinnamamide, 2) derivatives by various substitution on the amide nitrogen with alkyl, allyl, propargyl, benzyl or ester groups. The structures of nine new compounds were characterized by HRMS,¹H NMR, and¹³C NMR spectra. These compounds were tested in vitro against 10 strains of phytopathogenic fungi and showed a wide antifungal spectrum. The relationship between different substituents on the amide nitrogen and antifungal activity of Lansiumamide B derivatives were compared and analyzed. The result indicates that the length and steric hindrance of N-substitution have a significant impact on biological activities. It is noteworthy that the methyl or ethyl substituent on the amide nitrogen is critical for the antifungal activities.

Full text of DOI:10.3390/molecules23071499

molecules
Article
Synthesis and Fungicidal Activity of Lansiumamide
A and B and Their Derivatives
Huiyou Xu, Ting Chen, Luanbin Huang, Qiuju Shen, Zengwei Lian, Yan Shi, Ming-An Ouyang
and Liyan Song *
Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, Fujian Agriculture and Forestry
University, Fuzhou 350002, China; huiyouxu@126.com (H.X.); fafuchenting@163.com (T.C.);
h17746075243@163.com (L.H.); m18759775477@163.com (Q.S); fafuzengweilian@126.com (Z.L.);
shiyanshelly@163.com (Y.S.); minganouyang@163.com (M.-A.O.)
* Correspondence: songliyan@fafu.edu.cn; Tel.: +86-156-597-743-10
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 29 May 2018; Accepted: 21 June 2018; Published: 21 June 2018
Abstract: A efficient 2-step protocol has been applied for the synthesis of Lansiumamide B
(N-methyl-N-cis-styryl-cinnamamide, 2) derivatives by various substitution on the amide nitrogen
with alkyl, allyl, propargyl, benzyl or ester groups. The structures of nine new compounds were
characterized by HRMS, 1H NMR, and 13C NMR spectra. These compounds were tested in vitro
against 10 strains of phytopathogenic fungi and showed a wide antifungal spectrum. The relationship
between different substituents on the amide nitrogen and antifungal activity of Lansiumamide B
derivatives were compared and analyzed. The result indicates that the length and steric hindrance of
N-substitution have a significant impact on biological activities. It is noteworthy that the methyl or
ethyl substituent on the amide nitrogen is critical for the antifungal activities.
Keywords: antifungal activity; Lansiumamide B; derivatives; SAR; chemical synthesis
1. Introduction
Lansiumamide B (N-methyl-N-cis-styryl-cinnamamide,
from Clausena lansium (Lour.) Skeels by Lin in 1989 [
been reported to exhibit many pharmacological effects including anti-obesity [
anti-necrosis [ ] and anti-inflammatory [ ] bioactivities. Moreover, Lansiumamide B was also evaluated
as agrochemical. Intriguingly, Lansiumamide B performs a wide spectrum of efficient bioactivities,
such as antifeedant activities and cytotoxicity against Spodoptera litura [ ], anti-bacterial activity
against Ralstonia solanacearum [ ], larvicidal activity against mosquito [ ], nematicidal activity against
Bursaphelenehus xylophilus [ 10], insecticidal activity against Plutella xylostella and Mythimna separata,
2
) was firstly isolated from the extracts
]. Since then, Lansiumamide B has
], anti-diabetic [ ],
1
2
3
4
5
6
7
8
9,
acaricidal activity against Tetranychus cinnabarinus, herbicidal activity against Echinochloa crusgalli and
Abutilon theophrasti [11] and fungicidal activity against Colletotrichun gloeosporioides, Fusarium oxysporum
f.sp. cubense [12] and Botrytis cinerea [11]. The biological activities of Lansiumamide B were nicely
reviewed by Wan in 2012 [13].
Structurally, Lansiumamide B (2) was reported as a Z-configuration enamide, which is crucial
to its biological activities [11] and might pose significant synthetic challenges. The combination
of the potent bioactivities and the structural novelty has received considerable synthetic attention.
The pioneering works of Taylor [14], Maier [15] and Fürstner [16] furnished the enamide unit as a
mixture of E/Z isomers. In 2010, Goo
of Lansiumamide A through the use of a ruthenium-catalysed hydroamination of phenylacetylene and
cinnamamide. In 2014, Marquez [18] developed another synthetic route to afford Lansiumamide A (
βen [17] documented the stereoselective and one-step synthesis
1
)
Molecules 2018, 23, 1499; doi:10.3390/molecules23071499
www.mdpi.com/journal/molecules

Molecules 2018, 23, 1499
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as a single double bond isomer in four steps and 20% overall yield. Then NaH-mediated methylation
on the amide nitrogen simply provided the desired Lansiumamide B.
Phytopathogenic fungi causes severe losses in agricultural production. Thus, the efficient control
of plant fungal disease is of great importance for food production worldwide [19]. The commercially
available fungicides have been extensively used in currant agriculture and lead to the growing
resistance of phytopathogenic fungi [20]. And the discovery and development of new antifungal
agents would be necessary to provide new arsenals for the battle against phyto-fungal disease.
Natural product derived agrochemicals have received considerable attention from researchers owing
to their higher efficiency, lower mammalian toxicity, and environmental compatibility [21].
Lansiumamide B is proven to be a promising lead compound for the development of novel
antifungal agents [11,12]. Some Lansiumamide B-based derivatives were designed and synthesized
by Wan and coworkers [22]. However, most of them showed lower antifungal activities against
Colletotrichum gloeosporioides than the lead compound (Lansiumamide B). Thus, the Z-enamide moiety
was further proven to be essential for the antifungal activities [22]. These results prompted us to
figure out the structure-activity relationship (SAR) of Lansiumamide B derivatives and extend the
modification of Lansiumamide B with the aim of securing more potent antifungal agents. Herein,
we report a new series of Lansiumamide B derivatives containing various amide nitrogen substitutions
(Figure 1) and their antifungal activities. The SAR between the title compounds and their lead
compound (Lansiumamide B) is also compared and discussed.
Figure 1. Lead compounds and design strategy for the title compounds.
2. Results and Discussion
2.1. Chemical Syntheses
The synthetic route of compounds
hydroamination of commercially available cinnamamide and phenylacetylene according to
Goo en s protocol gave Lansiumamide A ( ) in a single step and an almost quantitative yield.
Final BuLi-mediated substitution on the N-H moiety of Lansiumamide A ( ) afforded Lansiumamide
B ( ) and its derivatives in good to excellent yields (73% to 89%). The synthetic route is shown in
Scheme 1. The structures of 3 were firmly established by HRMS, 1H NMR, and 13C NMR spectra.
1, 2 and 3 is outlined in Scheme 1. Ruthenium-catalyzed
β
'
1
1
2
3

Molecules 2018, 23, 1499
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Scheme 1. General synthetic procedure for title compounds.
2.2. Fugicidal Activity
The in vitro antifungal activities of compounds
1, 2, 3a to 3i were screened against ten plant
pathogenic fungi at 100 g/mL. The results listed in Tables 1 and 2 show all the compounds display
µ
the activity in varying degrees against each of the test fungi. Although part or most of the synthetic
compounds are less active than their lead compound (Lansiumamide B), some structure-activity
relationships can still be discovered. Firstly, the amide N-H moiety is detrimental to the bioactivities,
which could be avoided by substitution on the amide nitrogen. Secondly, increasing the substituent
from methyl to ethyl group would decrease the antifungal activity slightly. There are exceptions and
it becomes more efficient to Thanatephorus cucumeris, Fusarium solani, and Alternaria solani. As the
substituent continues to increase from the propyl to butyl group, the corresponding biological activities
descend gradually. Furthermore, we notice that as the substituent in compounds 3d, 3f, 3g, 3i grows
bulkier, the antifungal activity comes down rapidly. Thirdly, similar steric hindrance would result in
similar inhibitory rate. For example, the n-propyl and allyl group come to almost the same antifungal
activities, as does the propargyl group except that it becomes more powerful against Botrytis cinerea
and Fusahum graminearum than the ethyl group. Finally, the ester group would not help to elevate the
antifungal activity. Noteworthy is that all of our synthetic compounds exhibit much higher inhibitory
activity against Alternaria solani than the positive control carbendazim.
Table 1. Preliminary antifungal activities of all title compounds against five fungi at 100 µg/mL.
Compounds
Average Inhibition Rate ± SD (%) (n = 3)
Thanatephorus
cucumeris
Sclerotinia
sclerotiorum
64.57 ± 0.50
87.52 ± 2.49
66.55 ± 0.85
58.23 ± 1.36
43.61 ± 1.35
62.45 ± 1.03
64.90 ± 1.17
35.96 ± 0.95
26.98 ± 1.28
18.02 ± 0.37
25.00 ± 1.72
100
Botrytis
Phytophthora
sojae
Fusahum
graminearum
18.09 ± 0.58
76.63 ± 0.50
49.90 ± 0.55
30.52 ± 1.21
20.83 ± 1.12
41.67 ± 1.82
62.04 ± 0.89
17.08 ± 1.39
6.74 ± 0.44
26.95 ± 0.66
27.09 ± 0.76
100
No.
R
cinerea
1
2
H
Me
53.89 ± 0.19
91.20 ± 0.87
93.33 ± 0.66
63.94 ± 1.14
42.41 ± 0.95
70.80 ± 0.94
64.28 ± 0.63
27.09 ± 1.08
80.60 ± 0.46
68.08 ± 1.14
37.16 ± 0.96
19.03 ± 0.76
40.45 ± 0.85
77.70 ± 1.43
29.50 ± 0.87
32.97 ± 1.98
27.23 ± 0.27
49.18 ± 0.93
100
33.08 ± 0.61
79.20 ± 1.76
62.16 ± 0.57
49.41 ± 1.60
36.57 ± 0.78
60.19 ± 1.48
47.34 ± 0.57
53.94 ± 0.78
19.47 ± 1.43
21.75 ± 0.62
59.01 ± 0.71
100
3a
3b
3c
3d
3e
3f
3g
3h
3i
Et
n-Pr
n-Bu
allyl
propargyl
2-methylallyl 47.13 ± 0.49
prenyl
CH2CO2Et
benzyl
40.02 ± 1.58
27.34 ± 1.25
5.07 ± 0.81
100
Carbendazim

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Table 2. Preliminary antifungal activities of all title compounds against another five fungi at 100
µg/mL.
Compounds
Average Inhibition Rate ± SD (%) (n = 3)
RhizoctoniasolaniGloeosporium
Fusarium
solani
Alternaria
solani
Pyricularia
grisea
No.
R
kuehn
piperatum
18.43 ± 0.94
66.21 ± 0.76
60.40 ± 1.47
46.92 ± 0.56
43.27 ± 1.24
53.03 ± 0.60
65.22 ± 1.46
47.56 ± 0.89
13.44 ± 0.27
25.38 ± 2.01
48.30 ± 1.21
100
1
2
H
Me
27.25 ± 0.37
71.23 ± 1.20
49.81 ± 0.34
47.97 ± 1.71
33.99 ± 1.26
48.98 ± 0.87
47.79 ± 0.71
15.94 ± 0.72
63.81 ± 0.23
67.40 ± 0.23
38.86 ± 1.08
33.14 ± 1.71
52.29 ± 1.71
51.51 ± 0.55
30.73 ± 1.05
27.30 ± 0.65
26.44 ± 0.55
19.37 ± 0.34
100
33.72 ± 0.46
52.52 ± 0.24
60.83 ± 0.35
41.86 ± 0.99
29.99 ± 0.35
48.89 ± 0.68
55.69 ± 0.69
35.06 ± 0.44
23.89 ± 1.26
45.50 ± 1.53
27.31 ± 1.15
1.25 ± 0.37
17.76 ± 0.83
51.82 ± 1.30
45.32 ± 0.39
43.03 ± 1.05
11.50 ± 0.96
30.67 ± 2.04
47.40 ± 0.29
9.09 ± 1.44
15.50 ± 0.91
10.59 ± 0.97
32.94 ± 0.70
100
3a
3b
3c
3d
3e
3f
3g
3h
3i
Et
n-Pr
n-Bu
allyl
propargyl
2-methylallyl 35.17 ± 1.72
prenyl
CH2CO2Et
benzyl
31.87 ± 1.44
31.31 ± 2.02
20.82 ± 1.07
100
Carbendazim
3. Experimental Section
3.1. General Information
Reactions were carried out in oven or flame-dried glassware under a nitrogen atmosphere,
unless otherwise noted. Tetrahydrofuran (THF) was freshly distilled before use from sodium using
benzophenone as indicator. Dichloromethane was freshly distilled before use from calcium hydride
(CaH2). All other anhydrous solvents were dried over 3 Å or 4 Å molecular sieves. Solvents used in
workup, extraction and column chromatography were used as received from commercial suppliers
without prior purification. Reactions were magnetically stirred and monitored by thin layer
chromatography (TLC, 0.25 mm) on Liangchen pre-coated silica gel plates. Flash chromatography
1
was performed with silica gel 60 (particle size 0.040–0.062 mm) supplied by Liangchen. H and 13
C
NMR spectra were recorded on a Bruker AVIII-400 spectrometer (400 MHz for 1H, 100 MHz for 13C)
(Bruker BioSpin, Rheinstetten, Germany). Chemical shifts are reported in parts per million (ppm) as
values relative to the internal chloroform (7.26 ppm for 1H and 77.16 ppm for 13C). Abbreviations for
signal coupling are as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet. High resolution
mass spectra were measured at Keecloud Mass Spectrometry Service Company (Shanghai, China) on
either a Thermo Scientific LTQ Orbitrap XL system or a Bruker solariX System.
3.2. Synthetic Procedures
3.2.1. General Synthetic Procedure for Lansiumamide A (1)
Lansiumamide A (1) was synthesized according to Gooβen’s protocol [17]. An oven-dried
flask was charged with the trans-cinnamamide (147 mg, 1.00 mmol), bis(2-methallyl)-cycloocta-1,5-
diene-ruthenium(II) (16.0 mg, 0.05 mmol), 1,4-bis(dicyclohexylphosphinobutane (27.0 mg, 0.06 mmol),
and ytterbium triflate (24.8 mg, 0.04 mmol) and flushed with nitrogen. Subsequently, dry DMF
(3 mL), phenylacetylene (204 mg, 2.00 mmol),◦and water (108
µL, 6.00 mmol) were added via syringe.
The resulting solution was stirred for 6 h at 60 C. The reaction mixtures were poured into aq NaHCO3
(30 mL). The resulting mixture was extracted with EtOAc (3
were washed with water (20 mL) and brine (20 mL), dried over Na2SO4, and concentrated under
reduced pressure. The residue was purified by flash chromatography on silica gel using eluents
×
20 mL), the combined organic layers
(petroleum ether/ethyl acetate = 5/1) to give the Lansiumamide A (1) (241 mg, 0.97 mmol) as a light
yellow solid. Note: reaction on a larger scale (>1 mmol) resulted in lower yield. Therefore, the reaction
1
on a 1.0 mmol scale was repeated five times to obtain 1.20 grams of Lansiumamide A (
(400 MHz, CDCl3)
1
). H NMR
δ
: 7.79 (d, J = 9.2 Hz, 1H), 7.73 (d, J = 15.6 Hz, 1H), 7.38–7.30 (m, 10H), 7.13 (dd,

Molecules 2018, 23, 1499
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J = 10.4, 9.2 Hz, 1H), 6.37 (d, J = 15.6 Hz, 1H), 5.83 (d, J = 10.4 Hz, 1H). 13C NMR (100 MHz, CDCl3)
: 163.22, 143.13, 135.77, 134.49, 130.19, 129.18 (2 C), 128.91 (2 C), 128.07 (2 C), 128.02 (2 C),
127.06, 122.32, 119.49, 110.72.
δ
×
×
×
×
3.2.2. General Synthetic Procedure for Lansiumamide B (2) the Title Compounds 3
A flame-dried round bottle under nitrogen atmosphere was charged with Lansiumamide A
(100 mg, 0.4 mmol) and anhydrous tetrahydrofuran (THF, 2 mL). The solution was cooled to
78 ◦C
−
and n-butyllithiu◦m (0.25 mL, 1.6 M in hexane, 0.4 mmol) was added dropwise. The reaction mixture
◦
was warmed to 0 C, stirred for additional 30 min and then cooled to
−
78 C. The corresponding iodide
or bromide (0.6 mmol) was added dropwise to the cold solution. The reaction mixture was then allowed
to gradually warm to room temperature and stirred overnight. Aqueous saturated NH4Cl solution
(5 mL) was added under vigorous stirring to quench the reaction. The volatiles (mainly THF) were
removed under reduced pressure and the aqueous phase was extracted with diethyl ether (3 × 10 mL).
The combined organic fractions were washed with water and brine, dried over anhydrous MgSO4
and evaporated under reduced pressure. The residue was purified by flash column chromatography
on silica gel using eluents (petroleum ether/ethyl acetate = 10/1) to afford the desired compound.
The copies of 1H NMR and 13C NMR could be found in the Supplementary Materials.
2
. 85 mg, 81% yield; light yellow solid. 1H NMR (400 MHz, CDCl3)
7.39–7.31 (m, 10H), 6.96 (d, J = 15.6 Hz, 1H), 6.53 (d, J = 8.8 Hz, 1H), 6.27 (d, J = 8.8 Hz, 1H), 3.12 (s, 3H).
13C NMR (100 MHz, CDCl3)
: 166.45, 142.70, 135.17, 134.42, 129.72, 128.85, 128.74 (2 C), 128.68
δ: 7.67 (d, J = 15.6 Hz, 1H),
δ
×
(4 × C), 128.10, 127.95 (2 × C), 125.08, 118.30, 34.67.
3a. 92 mg, 83% yield; light yellow oil. H NMR (400 MHz, CDCl3)
(m, 10H), 6.89 (d, J = 15.6 Hz, 1H), 6.34 (s, 2H), 3.65 (q, J = 7.2 Hz, 2H), 1.20 (t, J = 7.2 Hz, 3H). 13C NMR
1
δ: 7.58 (d, J = 15.6 Hz, 1H), 7.32–7.24
(100 MHz, CDCl3)
128.22, 127.86 (2
278.1545, found 278.1550.
δ
: 165.73, 142.44, 135.30, 134.32, 129.54, 128.67 (2
×
C), 128.64 (2
× C), 128.59 (3 × C),
×
C), 127.14, 118.84, 41.92, 13.15. HRMS (ESI) m/z calculated for C19H20NO [M + H]+
1
3b. 91 mg, 78% yield; light yellow oil. H NMR (400 MHz, CDCl3)
(m, 10H), 6.91 (d, J = 15.6 Hz, 1H), 6.36 (d, J = 8.8 Hz, 1H), 6.31 (d, J = 8.8 Hz, 1H), 3.55 (t, J = 7.6 Hz,
2H), 1.66 (dq, J = 14.8, 7.6 Hz, 2H), 0.90 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3)
: 165.85,
C), 128.22, 127.87 (2 C),
δ: 7.59 (d, J = 15.6 Hz, 1H), 7.36–7.28
δ
142.47, 135.31, 134.29, 129.55, 128.68 (2
127.68, 126.89, 118.73, 48.85, 21.31, 11.59. HRMS (ESI) m/z calculated for C20H22NO [M + H]+ 292.1701,
×
C), 128.65 (2
×
C), 128.61 (2
×
×
found 292.1697.
1
3c. 92 mg, 75% yield; light yellow oil. H NMR (400 MHz, CDCl3)
δ
: 7.59 (d, J = 15.6 Hz, 1H), 7.34–7.26
(m, 10H), 6.90 (d, J = 15.6 Hz, 1H), 6.36 (d, J = 8.8 Hz, 1H), 6.31 (d, J = 8.8 Hz, 1H), 3.58 (t, J = 7.6 Hz,
1H), 1.61 (dt, J = 14.8, 7.6 Hz, 2H), 1.32 (dq, J = 14.8, 7.2 Hz, 2H), 0.90 (t, J = 7.2 Hz, 3H). 13C NMR
(400 MHz, CDCl3)
δ
: 165.82, 142.45, 135.31 (2
×
C), 129.54, 128.68 (2
×
C), 128.65 (2
× C), 128.60 (2 × C),
128.21, 127.87 (2 × C), 127.68, 126.84, 118.79, 46.96, 30.12, 20.36, 13.84. HRMS (ESI) m/z calculated for
C21H24NO [M + H]+ 306.1858, found 306.1855.
3d. 103 mg, 89% yield; light yellow oil. 1H NMR (400 MHz, CDCl3)
δ: 7.61 (d, J = 15.6 Hz, 1H),
7.34–7.26 (m, 10H), 6.91 (d, J = 15.6 Hz, 1H), 6.37 (d, J = 10.0 Hz, 1H), 6.32 (d, J = 10.0 Hz, 1H), 5.87 (ddt,
J = 10.0, 8.0, 5.6, 1H), 5.14 (dd, J = 10.0, 8.0 Hz, 2H), 4.21 (d, J = 5.6 Hz, 2H). 13C NMR (100 MHz, CDCl3)
δ
: 165.81, 142.93, 135.20, 134.29, 132.78, 129.67, 128.72 (2
127.92 (2 C), 127.17, 127.05, 118.45, 117.84, 49.45. HRMS (ESI) m/z calculated for C20H19NNaO [M +
Na]+ 312.1364, found 312.1365.
×
C), 128.69 (2
×
C), 128.61 (2
×
C), 128.29,
×
1
3e. 95 mg, 83% yield; light yellow oil. H NMR (400 MHz, CDCl3)
(m, 10H), 6.86 (d, J = 15.6 Hz, 1H), 6.42 (s, 2H), 4.39 (s, 2H), 2.23 (s, 1H). 13C NMR (100 MHz, CDCl3)
: 165.75, 143.53, 135.03, 134.04, 129.83, 128.80 (2 C), 128.71 (2 C), 128.54 (2 C), 128.46, 128.03,
δ: 7.61 (d, J = 15.6 Hz, 1H), 7.33–7.24
δ
×
×
×

Molecules 2018, 23, 1499
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127.98 (2 × C), 126.48, 117.91, 78.73, 71.98, 35.82. HRMS (ESI) m/z calculated for C20H18NO [M + H]+
288.1388, found 288.1383.
1
3f. 99 mg, 82% yield; light yellow oil. H NMR (400 MHz, CDCl3)
δ: 7.64 (d, J = 15.6 Hz, 1H), 7.35–7.26
(m, 10H), 6.96 (d, J = 15.6 Hz, 1H), 6.37 (d, J = 8.8 Hz, 1H), 6.32 (d, J = 8.8 Hz, 1H), 4.87 (s, 1H), 4.74
(s, 1H), 4.18 (s, 2H), 1.71 (s, 3H). 13C NMR (100 MHz, CDCl3)
: 165.97, 143.12, 140.38, 135.19, 134.29,
129.70, 128.72 (2 C), 128.70 (2 C), 128.64 (2 C), 128.27, 127.94 (2 C), 127.20, 126.93, 118.30,
112.68, 52.05, 20.40. HRMS (ESI) m/z calculated for C21H22NO [M + H]+ 304.1701, found 304.1704.
δ
×
×
×
×
3g. 100 mg, 79% yield; light yellow oil. 1H NMR (400 MHz, CDCl3)
: 7.58 (d, J = 15.2 Hz, 1H),
7.34–7.25 (m, 10H), 6.88 (d, J = 15.2 Hz, 1H), 6.32 (s, 2H), 5.28 (t, J = 7.2 Hz, 1H), 4.21 (d, J = 7.2 Hz, 2H),
1.69 (s, 3H), 1.62 (s, 3H). 13C NMR (100 MHz, CDCl3)
: 165.70, 142.47, 136.54, 135.32, 134.43, 129.53,
δ
δ
128.65 (4
×
C), 128.58 (2
×
C), 128.18, 127.86 (2
×
C), 127.56, 127.03, 119.31, 118.75, 44.56, 25.73, 17.95.
HRMS (ESI) m/z calculated for C22H24NO [M + H]+ 318.1858, found 318.1859.
1
3h. 98 mg, 73% yield; light yellow oil. H NMR (400 MHz, CDCl3)
δ
: 7.71 (d, J = 15.6 Hz, 1H), 7.41–7.32
(m, 10H), 6.96 (d, J = 15.6 Hz, 1H), 6.61 (d, J = 8.8 Hz, 1H), 6.29 (d, J = 8.8 Hz, 1H), 4.23 (s, 2H),
4.18 (q, J = 7.2 Hz, 2H), 1.25 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3)
: 169.02, 166.94, 144.01,
C), 127.76, 125.69, 117.52, 61.30, 48.97, 14.16.
δ
134.94, 134.02, 129.98, 128.79 (6
×
C), 128.42, 128.10 (2
×
HRMS (ESI) m/z calculated for C21H22NO3 [M + H]+ 336.1600, found 336.1606.
1
3i. 115 mg, 85% yield; light yellow oil. H NMR (400 MHz, CDCl3)
δ
: 7.64 (d, J = 15.6 Hz, 1H), 7.35–7.23
(m, 15H), 6.92 (d, J = 15.6 Hz, 1H), 6.31 (d, J = 8.8 Hz, 1H), 6.28 (d, J = 8.8 Hz, 1H), 4.78 (s, 2H). 13C NMR
(100 MHz, CDCl3) : 165.99, 143.12, 137.14, 135.20, 134.24, 129.10, 128.75 (4 C), 128.70 (2 C), 128.52
δ
×
×
(4
×
C), 128.34, 127.94 (2
C24H22NO3 [M + H]+ 340.1701, found 340.1728.
×
C), 127.51, 127.41, 127.26, 118.41, 50.03. HRMS (ESI) m/z calculated for
3.3. Bioassays
The antifungal activity of all synthesized compounds 1–3 was tested against ten plant pathogenic
fungi, namely Phytophthora sojae, Sclerotinia sclerotiorum, Thanatephorus cucumeris, Fusahum
graminearum, Pyricularia grisea, Fusarium solani, Rhizoctoniasolani Kuehn, Botrytis cinerea,
Gloeosporium piperatum and Alternaria solani by the poison plate technique.
Compounds were dissolved in DMSO (10 mL) before mixing with Potato Dextrose Agar
(PDA 90 mL) medium. The final concentration of compounds
1–3 in the medium were fixed at
100
µ
g/mL. Ten kinds of fungi were incubated in PDA at 25 ◦C for five days to get a new mycelium
for the antifungal assay, then a mycelia disk of an approximately 0.45 cm diameter cut from the culture
medium was picked up with a sterilized inoculation◦needle and inoculated in the center of the PDA
plate. The inoculated plates were incubated at 25 C for five days. DMSO in sterilized distilled
water served as the control, while carbendazim was used as a positive control for each treatment,
three replicates were carried out. The radial growth of the fungal colonies was measured on the sixth
day and the data were statistically analyzed. The relative control efficacy of compounds compared to
the blank assay was calculated via the following equation: I (%) = [(CK
I is the relative control efficacy, CK is the average disease index during the blank assay, and PT is
the average disease index after treatment during testing. The in vitro inhibiting effects of the test
−
PT/CK)]
×
100%, where
compounds on the fungi were calculated by the formula CV = (A
−
B)/A, where A represents the
diameter of fungi growth on untreated PDA, B represents the diameter of fungi on treated PDA,
and CV represents the rate of inhibition. All of the strains were conserved in the Key Laboratory of
Biopesticide and Chemical Biology, Ministry of Education, Fujian Agriculture and Forestry University
(Fuzhou, China).

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4. Conclusions
In summary, we report the synthesis of a new series of Lansiumamide B derivatives in a simple
and efficient way and the evaluation of their in vitro antifungal activity against ten plant pathogenic
fungi. Most of the synthetic compounds exhibit a potential inhibition activity against all the tested
fungi and some of them are more effective than carbendazim. Some of the compounds, especially 2 and
3a, are identified as the most active and therefore of great potential to be developed as new antifungal
agents. SAR analysis shows that the activity is highly sensitive to the substituent on the amide
nitrogen. The introduction of methyl or ethyl group might enhance the activity, whereas the presence
of N-H or group with larger steric hindrance causes decrease of activity. Therefore, these results are of
great significance for the design, synthesis, and development of novel antifungal reagents containing
Z-enamides. Further structural modification is ongoing in our laboratory, with the aim to secure
compounds with improved antifungal activity.
1
Supplementary Materials: Copies of H, 13C, spectra of new compounds. This material is available free of
charge online.
Author Contributions: H.X. supervised the synthesis steps; T.C., L.H. and Q.S. conducted the synthetic
experiments; Z.L. and Y.S. performed the antifungal experiments; M.-A.O. supervised the antifungal experiments;
H.X. and L.S. wrote the manuscript; L.S. designed the target compound and supervised all part of the work and
approved the final manuscript.
Acknowledgments: Financial support from the National Natural Science Foundation of China (21502018),
Natural Science Foundation of Fujian Province of China (2016J05024), the Fujian Agriculture and Forestry
University Foundation for Outstanding Youth (XJQ201623), Foundation of Pharmaceutical Engineering
Competition Steering Group of Fujian Agriculture and Forestry University (103-111416040) and the Foundation of
Key Laboratory of Biopesticide and Chemical Biology (Keylab2017-01) is gratefully acknowledged.
Conflicts of Interest: The authors declare no conflict of interest.
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