Synthesis and Herbicidal Activity against Buffelgrass (Cenchrus ciliaris) of (±)-3-deoxyradicinin

Article abstract of DOI:10.3390/molecules24173193

A novel synthetic strategy for obtainment of (±)-3-deoxyradicinin (2) is reported. This synthetic methodology is more efficient than those previously reported in the literature and also shows higher versatility towards the introduction of different side-chains at both C-7 and C-2. The obtained compound (±)-2 shows phytotoxicity against the grass-weed buffelgrass comparable to that of the natural phytotoxin radicinin (1). Therefore, (±)-2 can constitute a more practical synthetic alternative to 1 as bioherbicide for buffelgrass control.

Full text of DOI:10.3390/molecules24173193

molecules
Article
Synthesis and Herbicidal Activity Against Buffelgrass
(Cenchrus ciliaris) of (±)-3-deoxyradicinin
Giulia Marsico 1, Maria Sabrina Ciccone 1, Marco Masi 2 , Fabrizio Freda 3,
Massimo Cristofaro 3,4, Antonio Evidente 2, Stefano Superchi 1,
*
and Patrizia Scafato 1,
*
1
Department of Sciences, University of Basilicata, Via dell0Ateneo Lucano 10, 85100 Potenza, Italy
Department of Chemical Sciences, University of Naples Federico II, Complesso Universitario Monte
S. Angelo, Via Cintia 4, 80126 Napoli, Italy
2
3
4
BBCA onlus, Via A. Signorelli 105, 00123 Rome, Italy
ENEA C.R. Casaccia, SSPT-BIOAG-PROBIO, Via Anguillarese 301, 00123 Rome, Italy
Correspondence: stefano.superchi@unibas.it (S.S.); patrizia.scafato@unibas.it (P.S.);
Tel.: +39-097-120-6098 (S.S.)
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Vincenzo De Feo
Received: 26 July 2019; Accepted: 28 August 2019; Published: 3 September 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: A novel synthetic strategy for obtainment of (
This synthetic methodology is more efficient than those previously reported in the literature and
also shows higher versatility towards the introduction of different side-chains at both C-7 and C-2.
±
)-3-deoxyradicinin (
2
) is reported.
The obtained compound (
that of the natural phytotoxin radicinin (
alternative to 1 as bioherbicide for buffelgrass control.
±
)-
2
shows phytotoxicity against the grass-weed buffelgrass comparable to
1
). Therefore, ( )- can constitute a more practical synthetic
±
2
Keywords: buffelgrass; phytotoxins; bioherbicides; radicinin; (±)-3-deoxyradicinin
1. Introduction
Fungal phytotoxins constitute an essentially endless source of bioactive metabolites. Their large
structural diversity reflects in a wide variety of bioactivities and potential applications in both medicine
and agriculture. One of the most appealing application of fungal phytotoxins is in the development
of bioherbicides, which show lower or nil toxicity, and then a lower environmental and ecological
impact, than the traditional synthetic pesticides [
promising tool for weed management. The search for new and selective bioherbicides usually starts
with the isolation of phytotoxins from weed pathogens [ ]. This strategy has been followed also for
the search of selective compounds for the control of buffelgrass (Pennisetum ciliare or Cenchrus ciliaris),
a perennial grass-weed native to Africa, the Mediterranean area, and Middle-East [ ], which has
1–3]. For this reason, these are now emerging as a very
3
4
become highly invasive in North America and Australia where it is a menace for the native vegetation,
infesting roadsides and urban landscapes and promoting wildfires. Buffelgrass control is thus an
emerging concern in those countries, where it is now carried out by broad-spectrum herbicides which,
however, can heavily damage non-target native plants [
weed control, the production of phytotoxic metabolites by the buffelgrass foliar pathogens Cochliobolus
australiensis and Pyricularia grisea has been recently investigated [ ]. From the liquid culture of C.
australiensis, the known phytotoxin radicinin ( ) (Figure 1) and a new dihydropyran-pyran-4,5-dione
named cochliotoxin were isolated together with their close metabolites 3-epi-radicinin, radicinol and
3-epi-radicinol [ ]. Radicinin ( ) was first isolated from the fungus Stemphylium radicinum [ ] and its
structure was elucidated some years later by chemical and spectroscopic studies [10] while the absolute
configuration was established by X-ray analysis of its 4-p-bromobenzoate [11]. Compound has been
and cochliotoxin,
5]. Looking for novel bioherbicides for this
6–8
1
6
1
9
1
also found to be produced by many other fungal species [11–17]. Phytotoxins 1
Molecules 2019, 24, 3193; doi:10.3390/molecules24173193
www.mdpi.com/journal/molecules

Molecules 2019, 24, 3193
2 of 10
together with the other compounds isolated from P. grisea [8], were assayed for their phytotoxic activity
on host and non-host indigenous plants. Radicinin (1) demonstrated high target-specific toxicity on
buffelgrass, low toxicity to native plants, and no teratogenic, sub-lethal, or lethal effects on zebrafish
(Brachydanio rerio) embryos [18]. Previous studies had also shown its antifungal, insecticidal and plant
growth regulatory activity, as well as antibiotic activity against Gram-positive bacteria, including
Staphylococcus aureus [19,20]. More recently, the ability of 1 to inhibit Xylella fastidiosa, the causal agent
of many devastating plant diseases, including Pierce0s Disease of grapevine, phony peach disease,
alfalfa dwarf disease, plum leaf scald, citrus variegated chlorosis, and leaf scorch of almond, coffee,
elm, oak, oleander, pear, and sycamore was reported [21]. Although the high and specific bioactivity
of
1
against buffelgrass makes this phytotoxin very promising for the development of bioherbicides
available from fungal sources constitute a real
for the control of this weed, the low quantity of
1
bottleneck for such development, do not allowing open field studies [22]. Furthermore, the large
scale production of fungal natural products requires the use of fermenters where the sticking of
fungal mycelium to walls and blades often results as the most critical problem, strongly affecting the
production yield [23]. For these reasons, we considered worthwhile to develop an efficient synthetic
strategy for the obtainment of
of racemic ( )-3-deoxyradicinin (
the aim to define the effect on phytotoxicity of both the absolute configuration and the presence of
the hydroxy moiety at C-3. In fact, a recent structure-activity relationship study carried out on by
some of us demonstrated that the presence of an -unsaturated carbonyl group at C-4 is essential
1
or some its active analogues. In particular, we faced the synthesis
±
2
) (Figure 1) the immediate biosynthetic precursor of 24] with
1 [
1
α,β
for the phytotoxic activity and that the double bond of the side chain at C-7 also play a role to impart
activity [25]. However, the effect on phytotoxicity of the hydroxy group at C-3 was not ascertained
with certainty. The absolute configuration at C-3 seemed to have some role, because 3-epi-radicinin was
found to be less active, but the effect of the absolute stereochemistry at C-2 was not investigated [25].
Figure 1. Structure of radicinin (1) and 3-deoxyradicinin (2).
Our aim was then to develop an efficient synthetic strategy to obtain (
from commercially available products. The synthetic strategy was also designed to be versatile for the
synthesis of analogues of with different side-chains at C-7. Moreover, the transformation of to has
, will also constitute a formal
±
)-2 in high yield, starting
2
2
1
been reported in the literature [26], therefore, a novel approach to ( )-2
±
synthesis of (±)-1.
2. Results and Discussion
2.1. Total Synthesis of (±)-3-deoxyradicinin (2)
To the best of our knowledge, only two approaches to total synthesis of (
in the literature so far. However, the first procedure, reported by Kato et al. in 1969 [26], appears
unpractical, providing ( )- in five steps and <10% overall yield, but from not commercially available
precursors which, in turn, require some additional steps for preparation [27]. On the other hand, the
second one by Suzuki et al. [24], although leading to ( )- in only four steps from triacetic lactone
±
)-2 have been reported
±
2
±
2
methyl ether, still provides only 1% of overall yield. In addition, both approaches appear to be poorly
versatile to other pyrones differently substituted at C-7.
We then decided to apply a different approach, starting from commercially available
2,2,6-trimethyl-4H-1,3-dioxin-4-one (3) (Scheme 1).

Molecules 2019, 24, 3193
3 of 10
Scheme 1. Synthesis of (±)-3-deoxyradicinin (2) and analogue (±)-8.
Accordingly, the dioxinone
THF and trapped with trimethylsilyl chloride (TMSCl), providing the silyl dienolether
after high vacuum distillation (pressure lower than 1.0 mmHg) of the crude product. This yield
may be considered satisfactory, taking into account that the purification of by distillation shall
be carried out on strictly controlled temperature conditions because it decomposes when heated at
3
was enolized with lithium diisopropylamide (LDA) at
−
78 ◦C in
4
in 63% yield
4
temperatures higher than 50 ◦C. The silyl enol ether
conditions in the presence of TiCl4 in dichloromethane at 78 C, affording the alcohol 5a in 61%
yield. Oxidation of 5a with Dess-Martin periodinane gave rise to the corresponding ketone 6a in
4
was reacted◦with crotonaldehyde in Mukaiyama
−
practically quantitative yield. The latter compound spontaneously eliminated acetone and cyclized
upon heating in toluene, providing the hydroxy
with crotonyl chloride, followed by in situ intramolecular Michael addition of the hydroxy moiety,
provided the desired deoxyradicinin ( )- in 50% yield (overall yield 18%). Notably, such synthetic
α-pyrone 7a in 96% yield. Finally, acylation of 7a
±
2
strategy is versatile towards the synthesis of radicinin analogues with different substituents at C-7 and
C-2. In fact, by simply employing a different aldehyde in the Mukaiyama reaction with compound
4
can be introduced a different side-chain at C-7, while the use of a different
chloride in the last step allows to insert a different substituent at C-2. This potentiality was verified
by the synthesis of the methyl-substituted dihydropyran-pyran-4,5-dione ( )- which was obtained
following the same synthetic procedure described for ( )- (Scheme 1). In fact, the Mukaiyama reaction
of with acetaldehyde provided alcohol 5b which, after oxidation to 6b and subsequent cyclization,
α,β-unsaturated acid
±
8
±
2
4
provided the pyrone 7b. The acylation with crotonyl chloride finally provided ( )-8 in similar (17%)
±
overall yield.

Molecules 2019, 24, 3193
4 of 10
With (
±
)-
2
in hand, we considered worthwhile to try its conversion to (
±
)-
1
following the
oxyacetylation procedure described in the literature by Kato et al. [26] (Scheme 2). Accordingly, ( )-2
was treated with lead tetracetate in acetic acid, stirring at 100 ◦C for 2h. Contrary to what reported in
±
the literature, where obtainment of only 3-acetoxy radicinin was claimed, we got 6:4 trans:cis mixture of
(
±
)-radicinin acetate 9a and 3-epi-(
pyranopyrandione 10 (74%) as the main product of the reaction. The latter probably formed from
)- through spontaneous acetic acid elimination. The low yield obtained, the low diastereoselectivity
±
)-radicinin acetate 9b (19% overall) together with the unsaturated
(±
9
observed and, most importantly, the large formation of 10 as a by-product in the present reaction makes
necessary a further optimization of such synthetic transformation, possibly trying different reagents for
the α-oxidation of 3-deoxyradicinin (2). This type of study is out of the scope of the present research
and will be developed successively. However, the availability of the radicinin derivative 10 prompted
us to test also this compound in the phytotoxicity assays on C. ciliaris (vide infra).
Scheme 2. Acetoxylation of ( )-3-deoxyradicinin (2) with Pb(OAc)4 (bold and dashed lines in figure
±
refer to the relative configuration).
2.2. Phytotoxicity Assays on C. ciliaris
Compounds (
±
)-
2
and 10 were tested by buffelgrass (C. ciliaris) leaf puncture assay at 2.5 × 10−3
M
as reported in Materials and Methods section and their activity was evaluated in comparison with that
showed by the natural metabolite radicinin (1) previously reported by Masi et al. [18] (Figure 2).
Figure 2. Results of leaf puncture bioassay on buffelgrass (C. ciliaris) at concentration of 2.5
for the synthetic derivatives (±)-2 and 10 and the natural compound 1.
×
10−3
M
As inferred from Figure 2, the activity of compound (
radicinin (2S,3S)- , while the activity of the pyranopyrandione 10 is slightly lower. The lack of the
hydroxy group on C-3 in seems to suggest, contrary to previous observations [25], that this moiety is
not essential for the compounds phytotoxicity. Moreover, the comparable activity of racemic and
non-chiral compound 10 with optically active (2S,3S)- , highlight that the absolute configuration of the
±
)-2 is comparable to that of the natural
1
2
2
1

Molecules 2019, 24, 3193
5 of 10
methyl group at C-2 also has a minor influence on phytotoxicity. As far as absolute configuration at C-3
is concerned, previous results have shown that in the presence of a hydroxy moiety on this stereocenter
its stereochemistry results very important to impart a strong activity considering the significant
reduction in phytotoxicity showed by (2S,3R)-3-epi-radicinin in comparison with (2S,3S)-1 [25].
The obtained results highlight that derivatives like (
±
)-2 and 10, much simpler to be prepared
than optically active radicinin ( ) can be considered as valid alternative to this phytotoxin as possible
1
bioherbicides against buffelgrass. Moreover, the possibility to employ, with similar results, racemic or
non-chiral compounds in place of enantiopure ones is a great advantage for the preparation of the
large amounts of compounds necessary for the field test of bioherbicides. In fact, it is obviously much
simpler to prepare on large scale a racemic compound than an optically active one, which usually
require to employ expensive chiral reagents or catalysts.
3. Materials and Methods
3.1. General Experimental Procedures
1H (400 MHz) and 13C (100 MHz) NMR spectra were recorded in CDCl3 or DMSO-d6 on a Varian
INOVA 400 spectrometer, using TMS as an internal standard (see Supplementary Materials). GC/MS
spectra were recorded on a HP 6890 gas chromatograph, equipped with a HP-5975 mass spectrometric
detector. HRESI mass spectra were recorded on Agilent Technologies 1100 LC/MS TOF instrument.
Analytical TLC was performed on silica gel 60 Macherey–Nagel sheets. The spots were visualized
by exposure to UV radiation (254 nm) and/or by spraying the plates with a potassium permanganate
solution. Column chromatography was performed using silica gel (Merck, Kieselgel 60, (60–230 mesh).
THF was freshly distilled before its use on sodium benzophenone ketyl under nitrogen atmosphere.
Diisopropylamine (DIPA) and CH2Cl2 were dried by distillation over calcium hydride and stored under
a nitrogen atmosphere. Trimethylsilyl chloride (TMSCl), crotonaldehyde, and crotonoyl chloride were
distilled and stored under argon atmosphere before their use. Commercially available n-butyllithium
(Aldrich) was a 2.5 M solution in hexanes. The other analytical grade solvents and commercially
available reagents were used without further purification.
3.2. ((2,2-Dimethyl-4-methylene-4H-1,3-dioxin-6-yl)oxy)-trimethylsilane (4)
A solution of DIPA (5.4 mL, 1.1 eq) in THF was stirred under nitrogen atmosphere and cooled to
0 ◦C, then n-butyllithium 2.5 M (15.4 mL, 1.1 eq) was slowly added in about 10 min and the mixture
was stirred at the same temperature for another 45 min. Afterwards, the pale yellow solution was
cooled to 3 (4.65 mL, 35 mmol) was added in 5 min.
78 ◦C and 2,2,6-trimethyl-4H-1,3-dioxin-4-one
−
After 1h, also freshly distilled TMSCl (5.33 mL, 1.2 eq) was added in about 10 min; the dark orange
mixture was stirred for 1 h and then slowly heated to room temperature. The mixture was filtered
under reduced pressure, the solid was washed with hexanes and the filtrate concentrated by distillation
under a reduced pressure. The crude product was purified by distillation under a high vacuum, using
a rotary pump (pressure less than 1 mmHg) and heating to 50◦C. The pure silyl dienolether
(4.76 g, 63%) corresponded to the collected fraction having bp = 32–34 ◦C.
4 [28]
1H-NMR (400 MHz, CDCl3):
δ 0.27 (s, 9H, Si(CH3)3), 1.55 (s, 6H, C(CH3)2), 3.88 (s, 1H, C=CHaH),
4.07 (s, 1H, C=HHb), 4.65 (s, 1H, C=CH). 13C-NMR (100 MHz, CDCl3):
δ 0.24, 24.5, 76.6, 84.9, 102.5,
151.8, 153.3.
3.3. (E)-6-(2-hydroxypent-3-en-1-yl)-2,2-dimethyl-4H-1,3-dioxyn-4-one (5a)
A solution of crotonaldehyde (2.02 mL, 24.4 mmol, 1.1 eq) in 90 mL of CH2Cl2 was cooled
to −78 ◦C under a nitrogen atmosphere, TiCl4 (2.43 mL, 22.2 mmol, 1.0 eq) and silyl dienolether
4
(4.76 g, 22.2 mmol) was slowly added in sequence. After 1.5 h of stirring at the same temperature,
the mixture was quenched with 40 mL of a saturated solution of NaHCO3 and the organic phase
separated. The aqueous phase was again extracted with CH2Cl2, the combined organic phases were

Molecules 2019, 24, 3193
6 of 10
dried over anhydrous sodium sulfate and the solvent eliminated under reduced pressure. The crude
product was purified by column chromatography on silica gel (first n-hexane:ethyl acetate 7:3 and then
n-hexane:EtOAc 1:1), obtaining pure alcohol 5a [29] (2.87 g) in 61% yield.
1H-NMR (400 MHz, CDCl3):
δ 1.69 (s, 3H, CCH3CH3), 1.70 (s, 3H, CCH3CH3), 1.71 (d, J = 7.2 Hz, 3H,
CH=CHCH3), 2.40-2.48 (m, 2H, CH2CHOH), 4.36 (bq, J = 6.4 Hz, 1H, CHOH), 5.32 (s, 1H, C=CH),
5.51 (dd, J = 15.2 Hz, 7.2 Hz, 1H, CH=CHCH3), 5.71–5.78 (m, 1H, CH=CHCH3). 13C-NMR (100 MHz,
CDCl3): δ 17.6, 24.9, 25.3, 41.5, 69.8, 95.2, 106.6, 128.5, 132.3, 161.1, 168.5.
3.4. 6-(2-Hydroxypropyl)-2,2-dimethyl-4H-1,3-dioxyn-4-one (5b)
Following the same procedure as above, but starting from freshly distilled acetaldehyde, alcohol
5b [30] was obtained in 62% yield.
1H-NMR (400 MHz, CDCl3):
δ 1.27 (d, 3H, J = 6.0 Hz, CH-CH3), 1.70 (s, 6H, C(CH3)2), 1.86 (br. s, 1H,
OH), 2.38 (d, 2H, J = 6.4 Hz, CH-CH2), 4.09–4.13 (m, 1H, CH-CH2), 5.33 (s, 1H, C=CH). 13C-NMR
(100 MHz, CDCl3): δ 23.5, 24.8, 25.2, 43.2, 65.2, 95.0, 106.6, 161.3, 169.0.
3.5. (E)-2,2-dimethyl-6-(2-oxopent-3-en-1-yl)-4H-1,3-dioxyn-4-one (6a)
Dess-Martin periodinane (2.50 g, 1.3 eq) was added in small portions to a solution of alcohol 5a
(940 mg, 4.43 mmol) in 20 mL of CH2Cl2 and the solution was stirred at room temperature, monitoring
the conversion by TLC analysis. After 1.5 h, the mixture was diluted with 120 mL of diethyl ether and
24 mL of a saturated solution of sodium bicarbonate containing 5% sodium thiosulfate and stirred
for further 20 min. The organic layer was separated, dried over anhydrous sodium sulfate and then
concentrated under reduced pressure. The crude product was purified by column chromatography on
silica gel (first n-hexane:EtOAc 7:3 and then n-hexane:EtOAc 1:1), to afford pure ketone 6a [29] (912 mg,
98% yield).
1H-NMR (400 MHz, CDCl3):
δ 1.71 (s, 6H, C(CH3)2), 1.95 (d, J = 6.8 Hz, 3H, CH=CHCH3), 3.46 (s, 2H,
COCH2), 5.36 (s, 1H, C=CH), 6.17 (d, J = 15.6 Hz, 1H, CH=CHCH3), 6.90–6.97 (m, 1H, CH=CHCH3).
13C-NMR (100 MHz, CDCl3): δ 18.4, 25.0, 44.6, 96.6, 107.1, 130.8, 145.4, 160.7, 165.0, 192.3.
3.6. 2,2-Dimethyl-6-(2-oxopropyl)-4H-1,3-dioxyn-4-one (6b)
Following the same procedure as above ketone 6b, [30] was obtained in 98% yield.
1H-NMR (400 MHz, CDCl3):
δ 1.72 (s, 6H, C(CH3)2), 2.25 (s, 3H, CO-CH3), 3.35 (s, 2H, CO-CH2),
5.36 (s, 1H, C=CH). 13C-NMR (100 MHz, CDCl3): δ 25.0, 30.1, 47.9, 96.7, 107.2, 160.6, 164.3, 184.3.
3.7. (E)-4-hydroxy-6-(propen-1-yl)-2H-piran-2-one (7a)
Ketone 6a (912 mg, 4.34 mmol) was dissolved in 40 mL of toluene and the solution was heated to
100 ◦C for 0.5–1 h. During this time, a very thin white precipitate was formed. After cooling to room
temperature, toluene was removed at reduced pressure and the solid was washed with diethyl ether
1
and a small amount of chloroform. H-NMR analysis in DMSO-d6, being it insoluble in chloroform,
confirmed it as pure pyranodienone 7a [29] (632 mg, 96% yield).
1H-NMR (400 MHz, DMSO-d6)
C=CH), 6.15 (d, J = 16.8 Hz, 1H, CH=CHCH3), 6.41–6.49 (m, 1H, CH=CHCH3), 11.63 (s, 1H, OH).
δ 1.82 (d, J = 6.4 Hz, 3H, CH=CHCH3), 5.22 (s, 1H, C=CH), 5.98 (s, 1H,
13C NMR (100 MHz, DMSO-d6)
δ 23.2, 94.6, 104.9, 128.5, 138.9, 164.1, 168.1, 175.5. MS(EI): m/z 152 (51,
M+), 137 (50), 124 (34), 69 (100), 55 (11).
3.8. 4-Hydroxy-6-(methyl)-2H-piran-2-one (7b)
Following the same procedure as above, 4-hydroxypyranone 7b [31] was obtained in 92% yield.

Molecules 2019, 24, 3193
7 of 10
1H-NMR (400 MHz, DMSO-d6):
δ
2.12 (s, 3H, C=C-CH3), 5.17 (s, 1H, C=CH), 5.92 (s, 1H, CO-CH),
11.58 (s, 1H, OH). 13C-NMR (100 MHz, DMSO-d6): δ 35.9, 93.3, 105.4, 168.5, 169.1, 175.7.
3.9. (E)-2-methyl-7-(prop-1-en-1-yl)-2,3-dihydropyrane[4,3-b]pyran-4,5-dione ((±)-3-deoxyradicinin; (±)-2)
Under a nitrogen atmosphere, TiCl4 (0.234 mL, 213 mmol, 1.8 eq) and crotonoyl chloride (0.170 mL,
1.77 mmol, 1.5 eq) were dropwise added in sequence to a solution of pyranodienone 7a (180 mg,
1.18 mmol) in 2 mL of CHCl2CHCl2. The mixture was first stirred for 15 min at room temperature
and then heated to 100 ◦C for 3 h. After cooling to room temperature, the mixture was poured into
water/ice (10 mL) and extracted with ethyl acetate. The organic layer was washed with brine and
concentrated under reduced pressure. The crude product was purified by column chromatography on
silica gel (n-hexane:acetone 1:1), to afford (±)-2 [24] (130 mg) in 50% yield.
1H-NMR (400 MHz, CDCl3):
2.59–2.70 (m, 2H, COCH2), 4.73–4.80 (m, 1H, OCHCH3), 5.83 (s, 1H, C=CH), 6.02 (d, J = 15.6 Hz, 1H,
δ 1.54 (d, J = 6.4 Hz, 3H, CHCH3), 1.95 (d, J = 6.8 Hz, 3H, CH=CHCH3),
CH=CHCH3), 6.89–6.97 (m, 1H, CH=CHCH3). 13C-NMR (100 MHz, CDCl3):
δ 18.7, 20.3, 43.7, 53.4,
98.1, 100.2, 122.7, 140.0, 157.2, 163.4, 175.9, 186.4. MS(EI): m/z 220 (44, M+), 205 (100), 179 (24), 111 (17),
69 (77). HRESIMS (+) m/z 221.0810 [M + H]+ (calcd for C12H13O4 221.0814).
3.10. 2,7-Dimethyl-2,3-dihydropyrane[4,3-b]pyran-4,5-dione ((±)-8)
Following the same procedure as above, the compound analogue (
45% yield.
±
)-8 [32] was obtained in
1H-NMR (400 MHz, CDCl3):
δ 1.54 (d, J = 6.8 Hz, 3H, CH-CH3), 2.27 (s, 3H, CH=C-CH3), 2.59–2.68
(m, 2H, CH-CH2), 4.74–4.79 (m, 1H, CH-CH2), 5.90 (s, 1H, C=CH). 13C-NMR (100 MHz, CDCl3):
δ 20.2,
20.7, 29.2, 43.7, 95.1, 99.6, 157.8, 168.7, 175.9, 186.4. MS(EI): m/z 194 (28, M+), 179 (100), 153 (16), 69 (61),
41 (16). HRESIMS (+) m/z 195.0651 [M + H]+ (calcd for C10H11O4 195.0657).
3.11. Oxidation of (±)-3-deoxyradicinin (2) with Pb(OAc)4
A mixture of compound 2 (40 mg, 0.18 mmol), Pb(OAc)4 (120 mg, 1.5 eq) and acetic acid (3 mL)
was stirred and heated to 100 ◦C for 2h. The mixture was then cooled to room temperature, poured
into water/ice and extracted with dichloromethane. The organic layer was washed with a 5% NaHCO3
solution and brine and concentrated under reduced pressure. By column chromatography on silica gel
(n-hexane: acetone 1:1) of the residue, two fractions were eluted, corresponding to a 6:4 mixture of
trans:cis (±)-radicinin acetate 9 [21] (9.5 mg, 19%) and the pyranopyrandione 10 [26] (29 mg, 74%).
(E)-2-methyl-4,5-dioxo-7-(prop-1-en-1-yl)-2,3,4,5-tetrahydropyrano[4,3-b]pyran-3-yl acetate (9a
+ 9b
1
mixture). H-NMR (400 MHz, CDCl3):
δ
1.45 (d, J = 6.4 Hz, 3H, CHCH3, 9b), 1.55 (d, J = 6.4 Hz, 3H,
9b), 2.17 (s, 3H, COCH3, 9b), 2.21 (s, 3H, COCH3,
9a), 4.69–4.75 (m, 1H, CHCH3, 9a), 4.85–4.92 (m, 1H, CHCH3, 9b), 5.25 (d, J = 10.8 Hz, 1H,CHOAc, 9a),
CHCH3, 9a), 1.97 (d, J = 7.2 Hz, 3H, CH=CHCH3, 9a
+
5.49 (d, J = 3.6 Hz, 1H, CHOAc, 9b), 5.85 (s, 1H, C=CH, 9a
+
9b), 6.04 (dd, J = 15.2 Hz, 1.6 Hz, 1H,
CH=CHCH3, 9a + 9b), 6.93–7.00 (m, 1H, CH=CHCH3, 9a + 9b).
1
(E)-2-methyl-7-(prop-1-en-1-yl)pyrano[4,3-b]pyran-4,5-dione (10). H-NMR (400 MHz, CDCl3):
δ 1.97
(d, J = 7.2 Hz, 3H, CH=CHCH3), 2.30 (s, 3H, CH=CCH3), 6.06 (s, 1H, CH=CCH3), 6.07 (d, J = 15.6 Hz,
1H, CH=CHCH3), 6.17 (s, 1H, C=CH), 6.90–6.99 (m, 1H, C=CH-CH3). 13C-NMR (100 MHz, CDCl3):
δ 18.7, 19.6, 96.7, 105.7, 115.7, 122.3, 139.8, 156.9, 162.2, 163.6, 169.3, 174.3. HRESIMS (+) m/z 219.0653
[M + H]+ (calcd for C12H11O4 219.0657).
3.12. Leaf Puncture Bioassays
The synthetic derivatives (
buffelgrass (C. ciliaris) following a procedure previously reported [22
with that of natural 18]. Accordingly, compounds were first dissolved in MeOH (final concentration
±
)-
2
and 10 were assayed at 2.5
×
10−3 M for phytotoxicity on leaves of
25] and their activity compared
,
1
[

Molecules 2019, 24, 3193
8 of 10
4%) and stock solutions using sterile distilled water were then prepared. An incision of ca. 3 mm was
made on the adaxial surface of each leaf section of 3 cm with an insulin needle. The leaf sections were
placed in groups of six on the surface of a water-saturated filter paper in each of four petri dishes.
Five leaf sections in each petri dish were tested with the solution containing the compound, while
one leaf section was used as a negative control (4% MeOH only). A droplet (10 µL) of the appropriate
solution was applied over each needle incision using a micropipette. The dishes were sealed with
parafilm and incubated at 24 ◦C for 3 days in a temperature-regulated chamber under a photoperiod
of 14–10 h (light/dark). After three days of treatment, necrotic lesion development was evaluated by
removing the petri dish cover, placing a glass disc on the leaf sections to flatten them into a single
plane, and photographing each dish with its leaf sections. Each acquired image was then analyzed
with the software ImageJ to measure the necrotic area caused by the solution.
3.13. Statistical Analyses
Statistical analyses were carried out using the GraphPad Prism 8 software. Data were represented
as the mean
±
standard deviation and analyzed for statistical significance using ordinary one-way or
two-ways analysis of variance (ANOVA) and multiple comparisons. For all test, P < 0.5 was considered
to indicate a statistically significant difference.
4. Conclusions
A novel efficient synthetic strategy for obtainment of ( )-3-deoxyradicinin (2) has been developed.
±
This synthetic methodology not only is more efficient than those previously reported in the literature,
but it also shows higher versatility towards the introduction of different side-chains at both C-7 and C-2
as shown with the synthesis of the analogue (
against the grass-weed buffelgrass comparable to that of the natural phytotoxin radicinin (
)- can constitute a more practical synthetic alternative to as bioherbicide for buffelgrass control.
In fact, while is available only in very small amount by fungal culture, ( )- can be obtained in much
±
)-
8
. The obtained compound (
±
)-2
shows phytotoxicity
1
). Therefore,
(±
2
1
1
±
2
larger quantities by organic synthesis thus allowing to carry out extensive studies on its phytotoxic
activity and even open field tests. These results are then of interest for both synthetic and natural
products chemistry as well as for the nowadays forefront research on bioherbicides for weed control
in agriculture.
Supplementary Materials: The following are available online. 1H-NMR and 13C-NMR spectra of compounds
(±)-2, 4, 5a,b, 6a,b, 7a,b, (±)-8, (±)-9a,b, 10.
Author Contributions: All the authors contributed in the design of the study, the analysis of the data and writing
of the manuscript. P.S. and S.S. performed the conceptualization, acquired the funding and wrote a draft of the
manuscript; P.S., G.M. and M.S.C. carried out the syntheses; F.F. performed the bioassays and M.M. analyzed the
data; M.M., M.C. and A.E. finalized the draft and revised the manuscript.
Funding: This research was funded by Project PON RI 2014-2020 BIOFEEDSTOCK (ARS01_00985), Programme
STAR 2017, financially supported by UniNA and Compagnia di San Paolo, grant number E62F16001250003 and by
BBCA onlus Foundation, Rome, Italy.
Acknowledgments: Antonio Evidente is associated with “Istituto di Chimica Biomolecolare del CNR”, Pozzuoli,
Italy. The authors thank Jacopo Guardascione for synthetic help in the preparation of compound (±)-8.
Conflicts of Interest: The authors declare no conflicts of interest.
References
1.
2.
3.
Dayan, F.E.; Duke, S.O. Natural compounds as next generation herbicides. Plant Physiol. 2014, 166, 1090–1105.
[CrossRef] [PubMed]
Gerwick, B.C.; Sparks, T.C. Natural products for pest control: An analysis of their role, value and future.
Pest Man. Sci. 2014, 70, 1169–1185. [CrossRef] [PubMed]
Cimmino, A.; Masi, M.; Evidente, M.; Superchi, S.; Evidente, A. Fungal phytotoxins with potential herbicidal
activity: Chemical and biological characterization. Nat. Prod. Rep. 2015, 32, 1629–1653. [CrossRef] [PubMed]

Molecules 2019, 24, 3193
9 of 10
4.
5.
6.
Abella, S.R.; Chiquoine, L.P.; Backer, D.M. Ecological characteristics of sites invaded by buffelgrass (Pennisetum
ciliare). Invasive Plant. Sci. Manag. 2012, 5, 443–453. [CrossRef]
Bean, T.M. Tools for Improved Management of Buffelgrass in the Sonoran Desert. Ph.D. Thesis, The University
of Arizona, Phoenix, AZ, USA, 2014.
Masi, M.; Meyer, S.; Clement, S.; Cimmino, A.; Cristofaro, M.; Evidente, A. Cochliotoxin, a
dihydropyranopyran-4,5-dione, and its analogues produced by Cochliobolus australiensis display phytotoxic
activity against buffelgrass (Cenchrus ciliaris). J. Nat. Prod. 2017, 80, 1241–1247. [CrossRef] [PubMed]
Masi, M.; Meyer, S.; Clement, S.; Pescitelli, G.; Cimmino, A.; Cristofaro, M.; Evidente, A. Chloromonilinic acids
C and D, two phytotoxic tetrasubstituted 3-chromanonacrylic acids isolated from Cochliobolus australiensis
with potential herbicidal activity against buffelgrass (Cenchrus ciliaris). J. Nat. Prod. 2017, 80, 2771–2777.
[CrossRef]
7.
8.
9.
Masi, M.; Meyer, S.; G
órecki, M.; Mandoli, A.; Di Bari, L.; Pescitelli, G.; Cimmino, A.; Cristofaro, A.;
Clement, S.; Evidente, A. Pyriculins A and B, two monosubstituted hex-4-ene-2,3-diols and other phytotoxic
metabolites produced by Pyricularia grisea isolated from buffelgrass (Cenchrus ciliaris). Chirality 2017
29, 726–736. [CrossRef]
,
Clarke, D.D.; Nord, F.F. Radicinin: A new pigment from Stemphylium radicinum. Arch. Biochem. Biophys. 1953
,
45, 469–470. [CrossRef]
10. Grove, J.F. Metabolic products of Stemphylium radicinum. Part I. Radicinin. J. Chem. Soc. 1964, 3234–3239.
[CrossRef]
11. Robeson, D.J.; Gray, G.R.; Strobel, G.A. Production of the phytotoxins radicinin and radicinol by Alternaria
chrysanthemi. Phytochemistry 1982, 21, 2359–2362. [CrossRef]
12. Nukina, M.; Marumo, S. Radicinol, a new metabolite of Cochliobolus lunata, and absolute stereochemistry of
radicinin. Tetrahedron Lett. 1977, 37, 3271–3272. [CrossRef]
13. Tal, B.; Robeson, D.J.; Burke, B.A.; Aasen, A.J. Phytotoxins from Alternaria helianti: Radicinin and the
structures of deoxyradicinol and radianthin. Phytochemistry 1985, 24, 729–731. [CrossRef]
14. Noordeloos, M.E.; De Gruyter, J.; Van Eijl, G.W.; Roeijmans, H.J. Production of dendritic crystals in pure
cultures of Phoma and Ascochyta and its value as a taxonomic character relative to morphology, pathology
and cultural characteristics. Micol. Res. 1993, 97, 1343–1350. [CrossRef]
15. Kadam, S.; Poddig, J.; Humphrey, P.; Karwowski, J.; Jackson, M.; Tennent, S.; Fung, T.; Hochlowski, J.;
Rasmussen, R.; McAlpine, J. Citrine hydrate and radicinin: Uman rinovirus 3-C protease inhibitors discovered
in target-directed microbial screen. J. Antib. 1994, 47, 836–839. [CrossRef] [PubMed]
16. Nakajima, H.; Ishida, T.; Otsuka, Y.; Hamasaki, T.; Ichinoe, M. Phytotoxins and related metabolites produced
by Bipolaris coicis, the pathogen of job0s tears. Phytochemistry 1997, 45, 41–45. [CrossRef]
17. Pryor, B.M.; Gilbertson, R.L. Relationships and taxonomic status of Alternaria radicina, A. carotincultae, and
A. petroselini based upon morphological, biochemical, and molecular characteristics. Mycologia 2002, 94, 49–61.
[CrossRef]
18. Masi, M.; Freda, F.; Sangermano, F.; Calabrò, V.; Cimmino, A.; Cristofaro, M.; Meyer, S.; Evidente, A.
Radicinin, a fungal phytotoxin as a target-specific bioherbicide for invasive buffelgrass (Cenchrus ciliaris)
control. Molecules 2019, 24, 1086. [CrossRef] [PubMed]
19. Li, S.; Shao, M.W.; Lu, Y.H.; Kong, L.C.; Jiang, D.H.; Zhang, Y.L. Phytotoxic and antibacterial metabolites
from Fusarium proliferatum ZS07 isolated from the gut of long-horned grasshoppers. J. Agric. Food Chem.
2014, 62, 8997–9001. [CrossRef]
20. Hussain, H.; Kliche-Spory, C.; Al-Harrasi, A.; Al-Rawahi, A.; Abbas, G.; Green, I.R.; Schulz, B.; Krohn, K.;
Shah, A. Antimicrobial constituents from three endophytic fungi. Asian Pac. J. Trop. Med. 2014, 7, S224–S227.
[CrossRef]
21. Aldrich, T.J.; Rolshausen, P.E.; Roper, M.C.; Reader, J.M.; Steinhaus, M.J.; Rapicavoli, J.; Vosburg, D.A.;
Maloney, K.N. Radicinin from Cochliobolus sp. inhibits Xylella fastidiosa, the causal agent of Pierce0s Disease
of grapevine. Phytochemistry 2015, 116, 130–137. [CrossRef]
22. Masi, M.; Meyer, S.; Clement, S.; Cimmino, A.; Evidente, A. Effect of cultural conditions on the production of
radicinin, a specific fungal phytotoxin for buffelgrass (Cenchrus ciliaris) biocontrol, by different Cochlioboulus
australiensis strains. Nat. Prod. Res. 2019. [CrossRef] [PubMed]
23. Abdul Manan, M.; Webb, C. Estimating fungal growth in submerged fermentation in the presence of solid
particles based on colour development. Biotech. Biotechnol. Equipm. 2018, 32, 618–627. [CrossRef]

Molecules 2019, 24, 3193
10 of 10
24. Suzuki, M.; Sakuno, E.; Ishihara, A.; Tamura, J.I.; Nakajima, H. Conversions of deoxyradicinin to radicinin and
of radicinin to 3-epi-radicinin in the phytopathogenic fungus Bipolaris coicis. Phytochemistry 2012, 75, 14–20.
[CrossRef] [PubMed]
25. Masi, M.; Freda, F.; Clement, S.; Cimmino, A.; Cristofaro, M.; Meyer, S.; Evidente, A. Phytotoxic activity
and structure–activity relationships of radicinin derivatives against the invasive weed buffelgrass (Cenchrus
ciliaris). Molecules 2019, 24, 2793. [CrossRef] [PubMed]
26. Kato, K.; Hirata, Y.; Yamamura, S. Syntheses of (
±
)-radicinin and ( )-dihydroradicinin. J. Chem. Soc. 1969,
±
15, 1997–2002. [CrossRef]
27. Yamamura, S.; Kato, K.; Hirata, Y. The reaction of 6-carboxymethyl-4-methoxy-2-pyrone with acetic anhydride.
Chem. Comm. 1968, 1580. [CrossRef]
28. Grunwell, J.R.; Karipides, A.; Wigal, J.C.; Heinzman, S.V.; Parlow, J.; Surso, J.A.; Claytone, L.; Fleitz, F.J.;
Daffner, M.; Stevens, J.E. Theformaloxidativeadditionofelectron-richtransoiddienestobromonaphthoquinones.
J. Org. Chem. 1991, 56, 91–95. [CrossRef]
29. Bach, T.; Kirsch, S. Synthesis of 6-substituted 4-hydroxy-2-pyrones from aldehydes by addition of an
acetoacetate equivalent, Dess–Martin oxidation and subsequent cyclization. Synlett 2001, 1974–1976.
[CrossRef]
30. Fang, Z.; Clarkson, G.J.; Wills, M. Asymmetric reduction of 2,2-dimethyl-6-(2-oxoalkyl/oxoaryl)-1,3-
dioxin-4-ones and application to the synthesis of (+)-yashabushitriol. Tetrahedron Lett. 2013, 54, 6834–6837.
[CrossRef]
31. Demuner, A.J.; Moreira Valente, V.M.; Almeida Barbosa, L.C.; Rathi, A.H.; Donohoe, T.J.; Thompson, A.L.
Synthesis and phytotoxic activity of new pyridones derived from 4-hydroxy-6-methylpyridin-2(1H)-one.
Molecules 2009, 14, 4973–4986. [CrossRef]
32. Zehnder, L.R.; Dahly, J.W.; Hsung, R.P. Lewis acid mediated condensation reactions of
α
,
β
-unsaturated acids
with 4-hydroxy-2-pyrones. A concise structural assignment of Fleischmann0s
α,α-bispyrone and Praill’s
α,γ-bispyrone. Tetrahedron Lett. 2000, 41, 1901–1905. [CrossRef]
Sample Availability: Samples of the compounds (±)-2, (±)-8, and 10 are available from the authors.
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
©
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).