Concise Total Synthesis and Antifungal Activities of Fusaric Acid, a Natural Product

Article abstract of DOI:10.3390/molecules25173859

The total synthesis of a natural product alkaloid fusaric acid (FA), which exhibits herbicide, fungicide, insecticide and even diverse notable pharmacological activities, was accomplished in four steps using commercially available materials. The synthesis, based on a unified and flexible strategy using 6-bromonicotinaldehyde as a common intermediate, is concise, convergent, practical and can be carried out on a two-gram scale. This approach could be readily applicable to the synthesis of its analogues. In addition, FA had a wide range of inhibitory activities against 14 plant pathogenic fungi in this study, which demonstrated that as a leading compound, and it has great potential to be further developed as an agricultural fungicide.

Full text of DOI:10.3390/molecules25173859

molecules
Communication
Concise Total Synthesis and Antifungal Activities of
Fusaric Acid, a Natural Product
Bin Bin Huang, Ya Yi Liu, Peng Fei Zhu, Yi Cheng Jiang and Ming-An Ouyang *
Key Laboratory of Biopesticide and Chemical Biology (Ministry of Education), Fujian Agriculture and Forestry
University, Fuzhou 350002, China; bbhuang@fafu.edu.cn (B.B.H.); yayiliu7180@163.com (Y.Y.L.);
z15960505490@163.com (P.F.Z.); jiang1658517698@163.com (Y.C.J.)
* Correspondence: maouyang@hqu.edu.cn; Tel./Fax: +86-0591-87646115
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 4 August 2020; Accepted: 24 August 2020; Published: 25 August 2020
Abstract: The total synthesis of a natural product alkaloid fusaric acid (FA), which exhibits herbicide,
fungicide, insecticide and even diverse notable pharmacological activities, was accomplished in four
steps using commercially available materials. The synthesis, based on a unified and flexible strategy
using 6-bromonicotinaldehyde as a common intermediate, is concise, convergent, practical and can
be carried out on a two-gram scale. This approach could be readily applicable to the synthesis of its
analogues. In addition, FA had a wide range of inhibitory activities against 14 plant pathogenic fungi
in this study, which demonstrated that as a leading compound, and it has great potential to be further
developed as an agricultural fungicide.
Keywords: total synthesis; 5-butylpicolinic acid; natural product; alkaloid; antifungal activity
1. Introduction
Plant diseases are the formidable enemy of agricultural production. According to the statistics of
the Food and Agriculture Organization of the United Nations (FAO), the crop yield loss caused by
plant pathogens is about 10% to 15% of the total output on average annually [1]. The use of fungicides
against plant pathogens is a major means of ensuring crop production, so global agriculture has always
maintained a strong demand for fungicides on the market. In the early 1970s, with the registration
and widespread use of the systemic fungicide benomyl (Benlate), the problem of fungicide resistance
became apparent. Now, significant fungicide resistance is a widespread problem in global agriculture.
In this silent battlefield of fighting against the development of fungicide resistance, the most important
and fundamental weapon that mankind can grasp is to accelerate the research of new structural
compounds that can be used for the agricultural field, and further find new target sites and modes of
action [2].
Fusaric acid (FA), chemically known as 5-butylpicolinic acid, is a natural product alkaloid,
with low to moderate toxicity [
Fusarium heterosporium that parasitizes rice [
diverse notable pharmacological activities, ranging from anti-hypertensive [
activities to anti-cancer effects [ 10] and the treatment of Parkinson’s disease [11
phytotoxin, FA and its derivatives also exhibited marked herbicidal activity [13 14]. It also has certain
insecticidal activity as an important insect phenoloxidase inhibitor [15 16]. In addition, FA showed
strong antifungal activity in both medical and agricultural fields [17,18].
3
], isolated by Yabuta T. in 1934 from the secondary metabolites of
]. A large number of research data showed that FA has
] and anti-viral [
12]. As a kind of
4
5
,6
7,8]
9
,
,
,
,
FA has been discovered for nearly 90 years, during which many synthetic approaches have been
developed. Generally, according to the methods of establishing the target structures, these synthetic
approaches can be classified into two categories: by constructing a pyridine ring via Diels-Alder type
reactions (Scheme 1) [19–22] and by elaborating substituted pyridine intermediates (Scheme 2) [23–28].
Molecules 2020, 25, 3859; doi:10.3390/molecules25173859
www.mdpi.com/journal/molecules

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The reported syntheses using Diels–Alder reactions either must be carried out under harsh conditions,
or substances such as selenium dioxide (SeO2) [21 23 29], gaseous hydrogen chloride (HCl) [19],
,
,
potassium permanganate (KMnO4) [24] and magnesium amalgam [24], which are both toxic to human
body and harmful to the environment, were used, and these methods are not suitable for large-scale
synthesis. Substituted pyridine intermediates designed and elaborated can readily facilitate the
synthesis of FA and its analogs. Song et al. [27] prepared the key intermediate benzyl 5-bromopicolinate
from raw material 2,5-dibromopyridine, and then synthesized FA through Negishi coupling, catalytic
hydrogenation reaction and recrystallization purification. Tung et al. [28] prepared the key intermediates
methyl (or ethyl) 5-bromopicolinate, then synthesized FA and its analogies via two-step reaction of
Suzuki coupling and hydrolysis. We have practiced these two routes and found that the yield of
Negishi and Suzuki coupling reactions was not satisfactory (<15%) due to the ester moieties. Hence,
a more practical and scalable approach with an easily accessible reagent is still highly desirable to
synthesize FA and its analogues to facilitate further antifungal activity evaluations.
Scheme 1. Previous work for total synthesis of fusaric acid (FA) by constructing pyridine ring via
Diels-Alder type reaction.

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Scheme 2. Previous work for total synthesis of FA by elaborating substituted pyridine intermediate.
We envisioned that the sequential reactions, such as Wittig and carbonylation reactions, at C(2)
and C(5) positions of the pyridine ring would enable us to establish a concise and flexible route to a
range of FA and its derivatives from a template 6-bromonicotinaldehyde, the commercially available
compound (Scheme 3).
The objective of this study was to develop a concise, practical and scalable synthesis of FA and to
facilitate further determinations of the FA inhibitory activities against several plant pathogenic fungi.
In the meantime, we wish to reveal the great potential which FA has for further developing as an
agricultural fungicide.

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Saponification
Carbonylation
O
OH
N
N
N
Br
O
O
2
3
fusaric acid (1)
Hydrogenation
O
Wittig
H
N
Br
N
Br
5
4
Br-
P+
Scheme 3. Synthetic plan for FA.
2. Results and Discussion
2.1. Total Synthesis of FA
Our synthesis (Scheme 4) began with the preparation of 2-bromo-5-(but-1-en-1-yl)pyridine
) from commercially available 6-bromonicotinaldehyde ( ) and n-propyltriphenyl-phosphonium
bromide by using the Wittig reaction [30], followed by a hydrogenation reaction [31] to afford
2-bromo-5-butylpyridine ( ). Once 2-bromo-5-butylpyridine ( ) was in hand, a carbonylation
reaction [32] was carried out immediately and methyl 5-butylpicolinate ( ) was obtained. To our
delight, all the reactions had run very well and afforded the expected end-product FA ( ) in high
yield after a saponification reaction [33]. It is noteworthy that the synthetic route is reliable, practical
and gram scale for the desired FA ( ), of which the total yield was 58% over only four steps and all
(4
5
3
3
2
1
1
spectroscopic data for our synthetic samples were in good agreement with those reported for the
corresponding natural products.
Scheme 4. Total synthesis of FA.

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Another simple method of preparing carboxylic acid by halogenated hydrocarbon is to introduce
the cyano group through the reaction of halogenated hydrocarbon [34] or diazonium salt [35] with
inorganic cyanide before hydrolysis [36]. However, the use of hazardous chemicals such as diazonium
salts or inorganic cyanide is unavoidable. In this study, the methyl ester of FA was prepared by
carbonylation reaction of halogenated hydrocarbons. Although the reaction requires palladium catalyst
and pressure, it is a feasible process route for practical and scalable preparation at one time, especially
if industrial production was required.
Additionally, since n-butyl lithium was used in the Wittig reaction, the reaction should be carried
out at
78 ◦C, which may have adverse effects on the scale-up production. This step of the reaction
−
was also explored preliminarily. When anhydrous dimethyl formamide (DMF) was used as solvent
and n-butyl lithium was replaced by sodium hydride (NaH), the dehydrogenation reaction could be
carried out at −10 ◦C, and the yield of 2-bromo-5-(but-1-en-1-yl)pyridine (4) was about 65%.
2.2. In Vitro Inhibitory Activities of FA against 14 Plant Pathogenic Fungi
The results in Table 1 show that FA had a wide range of inhibitory activities against 14 plant
pathogenic fungi, although the inhibition rates of FA against Valsa mal and Alternaria longipes at
200
µ
g/mL were only 12.93
±
1.18% and 28.57
±
2.04%, respectively. Moreover, FA had the best
inhibitory activities against Thanatephorus cucumeris, Colletotrichum higginsianum and Colletotrichum
gloeosporioides, whose 50% effective concentration (EC50) were 22.42, 31.67 and 90.80 µg/mL, respectively.
Table 1. Toxicity of FA against 14 plant pathogenic fungi.
Toxic Regression
Determination
Coefficient
Plant Pathogenic Fungus
EC50 (95% CL) (µg/mL)
Equation
Colletotrichum higginsianum
Helminthosporium maydis
Pyricularia grisea
Thanatephorus cucumeris
Botrytis cinerea
Gliocladium catenulatum
Colletotrichum gloeosporioides
Pythium aphanidermatum
Valsa mal
31.67 (26.72–37.30)
130.25 (115.68–149.24)
120.39 (107.26–137.04)
22.42 (10.20–34.24)
338.10 (258.80–484.10)
241.31 (193.89–367.29)
90.80 (60.23–171.70)
173.46 (150.11–210.84)
y = 1.954x + 2.068
y = 1.666x + 1.476
y = 1.684x + 1.497
y = 1.334x + 3.199
y = 1.166x + 2.051
y = 2.726x − 1.494
y = 0.969x + 3.103
y = 1.616x + 1.382
–
0.9462
0.9703
0.9999
0.9417
0.9806
0.9990
0.9745
0.9897
Mycosphaerella arachidis
Sclerotinia sclerotiorum
Phytophthora capsici
Rhizoctonia solani
187.60 (174.98–203.80)
190.68 (158.70–262.93)
170.89 (147.58–208.44)
221.17 (174.97–358.79)
y = 3.941x − 3.959
y = 2.703x − 1.163
y = 1.570x + 1.494
y = 2.695x − 1.318
–
0.9530
0.9969
0.9812
0.9893
Alternaria longipes
3. Materials and Methods
3.1. General
Reactions were carried out in flame- or oven-dried glassware under an argon atmosphere,
unless otherwise noted. Tetrahydrofuran (THF) was freshly distilled before use from sodium using
benzophenone as an indicator. All other anhydrous solvents were dried over 3 or 4 Å molecular sieves.
The 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 pre-coated silica gel plates produced by Yantai Jiangyou
silica gel Development Co., Ltd. (Yantai, Shandong Province, China). Flash chromatography was
performed with silica gel 60 (particle size 0.040–0.062 mm) supplied by Qingdao Haiyang Chemical
Co., Ltd. (Qingdao, Shandong Province, China). High resolution mass spectra were measured at
Keecloud Mass Spectrometry Service Company, (Shanghai, China), on an Thermo Scientific LTQ
Orbitrap XL system, Waltham, MA, USA. 1H and 13C-NMR spectra were recorded on a Bruker AVIII-400

Molecules 2020, 25, 3859
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1
spectrometer (400 MHz for H, 100 MHz for 13C), (Rheinstetten, German). Chemical shifts were
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. The visualization of NMR spectra could be found in the Supplementary Materials.
3.2. Total Synthesis of FA
3.2.1. Synthesis of 2-Bromo-5-(but-1-en-1-yl)pyridine (4)
To a solution of n-propyltriphenyl-phosphonium bromide (9.25 g, 24.0 mmol) in anhydrous THF
(25 mL) was added n-butyllithium (13.75 mL, 22.0 mmol, 1.6 M in hexane) dropwise at
78 ◦C and under
−
an argon atmosphere. The reaction mixture was stirred for 15 min and then heated to 0 ◦C for at least
1 h. After the reaction mixture was cooled to −78 ◦C, the solution dissolved 6-bromonicotinaldehyde
(5) (3.72 g, 20.0 mmol) in anhydrous THF (25 mL) was added. It was stirred for 15 min and then heated
to room temperature (RT); the reaction mixture was stirred at RT until the complete consumption
of starting material was observed by TLC (3 h). Aqueous saturated ammonium chloride (NH4Cl)
solution (10 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 dichloromethane
(3 × 20 mL). The combined organic fractions were washed with water and brine, dried over anhydrous
sodium sulfate (Na2SO4) and evaporated under reduced pressure. The residue was purified by
flash column chromatography on silica gel using eluents (petroleum ether/ethyl acetate = 20/1) to
afford 2-bromo-5-(but-1-en-1-yl)pyridine (4) as a colorless oil (3.28 g, 77% yield). Major diastereomer.
1H-NMR (400 MHz, CDCl3)
δ 8.26 (s, 1H), 7.45–7.43 (m, 2H), 6.26 (dt, J = 11.6, 1.5 Hz, 1H), 5.83 (dt,
J = 11.6, 7.4 Hz, 1H), 2.31–2.25 (m, 2H), 1.06 (t, J = 7.5 Hz, 3H). 13C-NMR (100 MHz, CDCl3)
δ
150.1,
1
139.7, 138.4, 138.1, 132.7, 127.6, 123.5, 22.1, 14.3. Minor diastereomer. H-NMR (400 MHz, CDCl3)
δ
8.29 (d, J = 2.5 Hz, 1H), 7.53 (dd, J = 8.1, 2.5 Hz, 1H), 7.40 (d, J = 8.1, 1H), 6.39–6.29 (m, 2H), 2.25–2.21
(m, 2H), 1.11–1.10 (m, 3H). 13C-NMR (100 MHz, CDCl3)
26.3, 13.4.
δ 148.1, 139.7, 136.3, 135.1, 133.0, 127.9, 124.1,
3.2.2. Synthesis of 2-Bromo-5-butylpyridine (3)
A round bottom flask was charged with 2-bromo-5-(but-1-en-1-yl)pyridine (4) (3.28 g, 15.5 mmol),
then evacuated and backfilled with argon. After ethyl acetate (30 mL) and 10% Pd/C (1.65 g, 1.55 mmol)
were added under argon, the argon in the round bottom flask was replaced by hydrogen for at least
10 min. It was stirred at RT under a hydrogen atmosphere until the complete consumption of starting
material was observed by TLC (15 min). The resulting suspension was filtered through a compacted
diatomaceous earth layer eluting with dichloromethane (3
and evaporated under reduced pressure to afford a colorless oil. It was used directly as crude (3) in the
×
20 mL). The combined filtrate was collected
next step immediately.
3.2.3. Synthesis of Methyl 5-butylpicolinate (2)
The crude (3
), palladium (II) acetate (0.067 g, 0.30 mmol), 1,10-Bis(diphenylphosphino)ferrocene
(DPPF; 0.333 g, 0.60 mmol), trimethylamine (TEA; 2.98 g, 29.5 mmol) and methanol (12 mL, 294.8 mmol)
were dissolved in DMF (30 mL) and the solution was stirred under carbon monoxide (CO; 1.5 MPa) at
100 ◦C overnight. The reaction mixture was cooled to RT before most of the volatile components were
removed under reduced pressure. Saturated sodium chloride (NaCl) solution (100 mL) was added and
then the mixture was shaken vigorously. The solution was extracted with ethyl acetate (3 × 30 mL)
,
and the combined organic layers were washed with water and brine, dried (Na2SO4), filtered and
concentrated by evaporating under reduced pressure. The resulting residue was purified by flash
chromatography on silica gel using eluents (petroleum ether/ethyl acetate = 10/1) to afford the desired
methyl 5-butylpicolinate (
CDCl3) 8.55 (d, J = 1.8 Hz, 1H), 8.05 (d, J = 8.0 Hz, 1H), 7.64 (dd, J = 8.0, 1.8 Hz, 1H), 3.99 (s, 3H), 2.68
2
) (2.56 g, 85% yield over 2 steps) as a colorless oil. 1H-NMR (400 MHz,
δ

Molecules 2020, 25, 3859
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(t, J = 7.6 Hz, 2H), 1.67–1.56 (m, 2H), 1.42–1.30 (m, 2H), 0.93 (t, J = 7.3 Hz, 3H). 13C-NMR (100 MHz,
CDCl3) δ 165.9, 150.1, 145.6, 142.4, 136.8, 125.1, 52.9, 33.1, 32.9, 22.3, 13.9.
3.2.4. Synthesis of FA (1)
Methyl 5-butylpicolinate (2) (2.56 g, 13.2 mmol) was mixed with lithium hydrate (LiOH; 1.58 g,
66.0 mmol) in a 30 mL methanol/water (v/v = 1:1) mixed solvent, and then it was stirred at RT. After
TLC analysis indicated complete consumption of the starting material (1 h), the volatiles (mainly
methanol) were removed under reduced pressure. The pH of the resulting suspension was adjusted to
about 2 with 1.0 M HCl. The aqueous phase was extracted with dichloromethane until no product
was observed in the aqueous phase by TLC. The combined organic layers were dried over anhydrous
Na2SO4, filtered and evaporated under reduced pressure. The desired FA (1) was obtained as a white
solid (2.12 g, 89% yield). Mp = 99–101 ◦C. (lit. 99–100.5 ◦C) [22] 1H-NMR and 13C-NMR of the synthetic
1
material were identical to those reported for the natural product [22]: H-NMR (400 MHz, CDCl3)
δ
10.79 (s, 1H), 8.59 (s, 1H), 8.15 (d, J = 7.8 Hz, 1H), 7.73 (d, J = 7.5 Hz, 1H), 2.70 (t, J = 7.5 Hz, 2H),
1.73–1.51 (m, 2H), 1.45–1.25 (m, 2H), 0.92 (t, J = 7.3 Hz, 3H). 13C-NMR (100 MHz, CDCl3)
δ 165.8, 148.1,
145.2, 143.1, 138.4, 124.4, 33.1, 32.9, 22.4, 13.9. HRMS (ESI) m/z calculated for C10H13NO2 [M + H]+
180.10191, found 180.10147.
3.3. In Vitro Inhibitory Activities of FA against 14 Plant Pathogenic Fungi
The antifungal activities were evaluated against 14 pathogenic fungi, namely, C. higginsianum,
H. maydis, P. grisea, T. cucumeris, B. cinerea, G. catenulatum, C. gloeosporioides, P. aphanidermatum, V. mal,
M. arachidis, S. sclerotiorum, P. capsici, R. solani, and A. longipes, by the poison plate technique [37–39].
FA was dissolved in chromatographic grade methanol (2 mL) to prepare a 10 mg/mL stock solution.
A proper amount of stock solution was precisely transferred with a pipette, and mixed with sterilized
and melted potato dextrose agar (PDA; 49 mL). The corresponding amount of chromatographic grade
methanol and sterile distilled water were added to make the total volume of 50 mL with 2% methanol
content. FA was tested at five double-declining concentratio◦ns (e.g., 200, 100, 50, 25, and 12.5 µg/mL).
Meantime, all fungi were cultivated in PDA at 28.0
mycelium for the identification of antifungal activity. Then, mycelia dishes of approximately 5 mm
diameter were cut from the culture medium. A mycelium was obtained using a germ-free inoculation
±
0.5 C for 5 days in darkness to make new
needle and inoculated reversely in the center of the PDA plate aseptically. The inoculated plates
◦
were incubated at 28.0
±
0.5 C for 2–7 days in darkness. Chromatographic-grade methanol in
sterile distilled water served as the control. At least three biological replicates and controls were
used for each concentration. Radial growth of the fungal colonies was measured, and the data
were statistically analyzed. Inhibitory activities of FA in vitro on these fungi were calculated by
the formula I (%) = [(C − T)/(C − 5.00)] × 100, where C represents the diameter of fungal growth on
untreated PDA (blank PDA with 2% methanol), T represents the diameter of fungal growth on treated
PDA, and I represents the inhibition rate. Finally, the corresponding toxic regression equation, EC50,
95% confidence interval and determination coefficient were obtained by using the software package
SPSS 22.0.
4. Conclusions
In summary, we have developed a total synthesis of naturally occurring alkaloid FA in four
steps with a 58% yield from 6-bromonicotinaldehyde (5), by taking advantage of Wittig reaction,
hydrogenation, carbonylation and saponification. The synthesis is concise, convergent, practical and
can be carried out on a two-gram scale. Since this synthesis is based on a unified and flexible strategy
using 6-bromonicotinaldehyde (5) as a common intermediate, it is readily applicable to analogue
synthesis. Furthermore, the results from in vitro antifungal activities of FA reveal that FA, as a leading
compound, has great potential to be further researched and developed as an agricultural fungicide.
Further exploration of the strategy for the generation of novel analogues for biological evaluation is

Molecules 2020, 25, 3859
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ongoing in our laboratory. As a kind of phytotoxin, FA can cause some plants to wilt or inhibit root
elongation, such as bananas [40 41], tomatoes [13], tobacco [42 43], maize [44], and watermelons [45 46],
,
,
,
etc. In order to ensure the plants of agricultural interest being safe, novel FA analogues for further
biological evaluation should be evaluated as to whether the analogues might cause phytotoxicity at an
effective dose. Relevant study work is also ongoing in our laboratory.
Supplementary Materials: The following are available online. Copies of 1H and 13C of new compounds.
Author Contributions: Conceived and designed the experiments: M.-A.O. and B.B.H. Wrote the paper: B.B.H.
Performed the experiments: B.B.H., Y.Y.L., P.F.Z. and Y.C.J. Contributed reagents/materials/analysis tools/scientific
funds: M.-A.O. and B.B.H. All authors have read and agreed to the published version of the manuscript.
Funding: This study was supported by the Educational and Scientific Research Project for Young and Middle-aged
Teachers of Education Department of Fujian Province (No. JAT170167). The funder had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript.
Acknowledgments: The authors are grateful for the financial support from the funding.
Conflicts of Interest: The authors report no conflict of interest.
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Sample Availability: Samples of the compounds 1, 2, 4 and 5 are available from the authors.
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