Design, Synthesis and Fungicidal Activity of Novel Sclerotiorin Derivatives

Article abstract of DOI:10.1111/cbdd.12005

Sclerotiorin, a chlorine-containing azaphilone-type natural product, was first isolated from Penicillium sclerotiorum and has been reported to exhibit weak fungicidal activity. Optimization of the substituents at the 3- and 5-positions of the sclerotiorin

Full text of DOI:10.1111/cbdd.12005

ª 2012 Syngenta Ltd
doi: 10.1111/cbdd.12005
Chem Biol Drug Des 2012
Research Article
Design, Synthesis and Fungicidal Activity of
Novel Sclerotiorin Derivatives
Long Lin1, Nick Mulholland2, Shao-Wei
reported to exhibit a wide range of biological activities, such as
inhibition of monoamine oxidase (12), inhibition of the formation of
the P53–MDM2 complex (13), inhibition of the gp120–CD4 binding
reaction (14), inhibition of fatty acid synthase (15), inhibition of lipo-
Huang1, David Beattie2, Dianne Irwin2,
Yu-Cheng Gu2, John Clough2, Qiong-You
Wu1,* and Guang-Fu Yang1
oxygenase (16), as well as having antifungal activity (17,18).
1Key Laboratory of Pesticide & Chemical Biology, College of
Although various potentially beneficial biological activities have
been discovered, the relationship between structure and activity
amongst the azaphilones, as well as their mechanism of action,
remains unclear. Furthermore, most of the azaphilones have been
isolated as secondary metabolites of fungal species, and little
attention has been paid to the modification of structure for the
optimization of antifungal activity. As we know, natural product
leads offer an efficient approach for the discovery and optimization
of new agrochemicals for the control of plant diseases. As part of
our programme for developing fungicides with novel scaffolds (19–
22), we envisioned that sclerotiorin, which had shown weak anti-
fungal activity in Syngenta's screens, might be a useful lead for the
discovery of novel agricultural fungicides.
Chemistry, Central China Normal University, Ministry of Education,
Wuhan 430079, China
2Syngenta, Jealott's Hill International Research Centre, Bracknell,
Berkshire RG42 6EY, UK
*Corresponding author: Qiong-You Wu, qywu@mail.ccnu.edu.cn
Sclerotiorin, a chlorine-containing azaphilone-type
natural product, was first isolated from Penicil-
lium sclerotiorum and has been reported to exhibit
weak fungicidal activity. Optimization of the sub-
stituents at the 3- and 5-positions of the sclerotio-
rin framework was investigated with the aim of
discovering novel fungicides with improved activ-
ity. The design of sclerotiorin analogues involved
replacing the diene side chain with a phenyl group
or an aromatic- or heteroaromatic-containing ali-
phatic side chain. The designed compounds were
synthesized by cycloisomerization and subsequent
oxidation of suitable 2-alkynylbenzaldehydes, in
which a variety of substituents were introduced
using a Sonogashira coupling reaction. The struc-
tures of these newly prepared compounds were
confirmed by 1H and 13C NMR spectroscopy,
HRMS and single-crystal X-ray analysis. The anti-
fungal activity of the synthesized compounds was
evaluated against seven phytopathogenic species.
Compounds 3, 9g and 9h were found to have a
broad spectrum of fungicidal activity, and these
structurally simpler products can be recognized as
lead compounds for further optimization.
Although the nine-carbon aliphatic diene side chain on the bicyclic
core containing a chiral quaternary centre is the common character-
istic of sclerotiorin, some recent research has reported that sclero-
tiorin analogues with substitution other than the long diene chain
displayed interesting biological activities (23–25). For example, mito-
rubrinic acid (23), in which a carboxyvinyl substituent replaces the
diene chain, has been shown to induce formation of chlamydo-
spore-like cells in fungi and also to inhibit trypsin. Furthermore, bul-
garialactone B and related compounds (24) bearing a simple
aliphatic group at the 3-position showed inhibitory activity against
heat shock protein 90 (Hsp90) (Figure 1). These results indicate that
the character of the substitution at 3-position of sclerotiorin and
other azaphilones plays an important role in their observed biologi-
cal activity and provides us with an opportunity to optimize the
structure of sclerotiorin with the aim of discovering novel fungi-
cides. Thus, compound 1, in which the diene side chain has been
removed and replaced with a hydrogen atom, was first designed
and synthesized to determine the effect of the substitution on bio-
logical activity (Figure 1). Compounds 2 and 3, in which the long
aliphatic side chain has been replaced by a phenyl ring, were also
designed and synthesized. In addition, analogues of the type 4, in
which a chain containing oxygen or sulphur atoms links the bicyclic
core with a terminal aromatic ring, were designed and prepared
(the longer side chain in these analogues was expected to mimic
the long aliphatic side chain of sclerotiorin). Finally, examples were
prepared in which the phenyl ring of compounds 2 and 3 was
replaced with the biologically interesting heterocycles pyrimidine or
pyrimidinone. For all of these designed compounds, the bicyclic core
Key words: antifungal activity, azaphilone, natural product, sclero-
tiorin, structural modification
Received 3 March 2012, revised 28 June 2012 and accepted for publi-
cation 3 July 2012
Sclerotiorin was first isolated in 1940 from Penicillium sclerotiorum
as a chlorine-containing fungal pigment (1). It belongs to the class
of natural products known as azaphilones, which have been isolated
from a variety of fungal species (2–5) and feature a highly oxygen-
ated bicyclic core and a chiral quaternary centre that breaks the
aromaticity of the ring system (6–10). To date, over 170 different
natural azaphilones have been identified (11). They have been
1

Lin et al.
Figure 1: The structures of sclerotiorin, mitorubrinic acid, bulgarialactone B and the designed analogues.
of sclerotiorin was retained to mimic the natural parent structure,
and racemic compounds were prepared, ignoring the absolute ste-
reochemistry of the chiral centre of the bicyclic system. Herein, we
report the synthesis and characterization of these sclerotiorin ana-
logues as well as an assessment of their antifungal activities.
aqueous HCl. The mixture was extracted with ethyl acetate, and
the combined organic layers were washed with water and brine,
dried over anhydrous Na2SO4 and filtered and concentrated in
vacuo. The residue was purified by flash chromatography on silica
gel to provide the desired product.
Materials and Methods
General procedure for the conventional
Sonogashira coupling reaction
General techniques
To a mixture of the 2-bromobenzaldehyde 6 (92.4 mg, 0.40 mmol),
an alkyne (0.48 mmol), Pd(PPh3)2Cl2 (14.0 mg, 0.02 mmol) and CuI
(3.8 mg, 0.02 mmol) in 5 mL of anhydrous DMF was added NEt3
(1.2 mmol, 121.2 mg) under an argon atmosphere. The resulting
mixture was heated in an oil bath until TLC analysis indicated that
the starting material had disappeared. The reactant was cooled to
room temperature, diluted with water, neutralized with 1.0 N aque-
ous HCl and extracted with ethyl acetate. The combined organic
layers were washed with water and brine, dried over anhydrous
Na2SO4 and filtered and concentrated in vacuo. The residue was
purified by flash chromatography on silica gel to provide the desired
product.
All chemical reagents were commercially available and treated with
standard methods before use. Solvents were dried and redistilled
1
before use. H NMR spectra were recorded in CDCl3 or DMSO-d6
on a Varian Mercury 400 ⁄ 600 spectrometer, and chemical shifts (d)
are given in ppm relative to tetramethylsilane. 13C NMR spectra
were recorded in CDCl3 on a Varian Mercury 600 (150 MHz) spec-
trometer and (d) are given in ppm relative to the centre line of a
triplet at 77.0 ppm of chloroform-d. The following abbreviations are
used to designate multiplicities: s = singlet, d = doublet, t = triplet,
m = multiplet, br = broad. HRMS were obtained on a Waters
MALDI SYNAPT G2 HDMS equipped with an electrospray source
(Manchester, UK).
Preparation of 6-ethynyl-2,4-dihydroxy-3-
Preparation of the designed compounds
methylbenzaldehyde 5a
To a mixture of the 2-bromobenzaldehyde 6 (924 mg, 4 mmol),
PdCl2(PPh3)2 (140 mg, 0.2 mmol) and CuI (38 mg, 0.2 mmol) in 40 mL
anhydrous NEt3 was added ethynyltrimethylsilane (0.49 g, 5 mmol)
(Scheme 1). The resulting mixture was stirred at room temperature
for 2 h. The reaction mixture was diluted with water, neutralized with
1.0 N aqueous HCl and extracted with ethyl acetate. The combined
organic layers were washed with water and brine, dried over anhy-
drous Na2SO4 and filtered and concentrated in vacuo. The residue
was purified by flash chromatography on silica gel (petroleum ether ⁄ -
EtOAc 12:1) to provide the trimethylsilylethynylbenzaldehyde 14. The
General procedure for the microwave-assisted
Sonogashira coupling reaction
To a mixed solution of 1,4-dioxane (4 mL) and H2O (1 mL) were suc-
cessively added the 2-bromobenzaldehyde 6 (92.4 mg, 0.40 mmol),
Pd(PPh3)2Cl2 (14.0 mg, 0.02 mmol), CuI (3.8 mg, 0.02 mmol), NEt3
(1.6 mmol, 162 mg) and the appropriate alkyne (0.48 mmol). The
resulting mixture was sealed in a microwave tube and irradiated at
100 ꢀC for the indicated period of time. The reactant was cooled to
room temperature, diluted with water and neutralized with 1.0 N
2
Chem Biol Drug Des 2012

Novel Sclerotiorin Derivatives
TMS
HO
HO
HO
Br
b
a
TMS
CHO
CHO
CHO
OH
14
OH
5a
OH
6
Scheme 1: Reagents and conditions: (A) 5 mol % PdCl2(PPh3)2, 5 mol% CuI, Et3N, room temperature, 2h, 92% (B) 1.5 equiv. K2CO3,
MeOH, rt, 45 min, 81%.
1
obtained compound 14 (4 mmol) was dissolved in 20 mL of dry meth-
anol, and dry K2CO3 (828 mg, 6 mmol) was added. The resulting mix-
ture was stirred at room temperature until TLC analysis indicated that
the starting material had disappeared. The reaction mixture was
diluted with water, neutralized with 1.0 N aqueous HCl and extracted
with ethyl acetate. The combined organic layers were washed with
water and brine, dried over anhydrous Na2SO4 and filtered and con-
centrated in vacuo. The residue was purified by flash chromatography
on silica gel (petroleum ether ⁄ EtOAc 10:1) to provide the product 5a.
Data for 8c, m.p. 95–97 ꢀC; H NMR (600 MHz, CDCl3) d 1.55 (s,
3H), 3.87 (br, 1H), 4.22 (s, 2H), 4.64 (s, 2H), 5.59 (s, 1H), 6.40 (s,
1H), 7.34–7.40 (m, 5H), 7.86 (s, 1H); 13C NMR (150 MHz, CDCl3) d
28.3, 67.1, 73.4, 83.5, 106.4, 108.9, 116.0, 127.8, 128.2, 128.6,
136.5, 142.9, 152.4, 158.1, 195.4, 196.2; HRMS (MALDI) calcd. for
C18H16O5 [M + Na]+ 335.0895, found 335.0877.
1
Data for 8d, m.p. 177–178 ꢀC; H NMR (600 MHz, CDCl3) d 1.54
(s, 3H), 2.30 (s, 3H), 2.62 (s, 3H), 4.91 (dd, J = 16.2 Hz, 12.6 Hz,
2H), 5.59 (s, 1H), 6.24 (s, 1H), 6.27 (s, 1H), 7.83 (s, 1H); 13C NMR
(150 MHz, CDCl3) d 22.9, 23.5, 28.2, 83.7, 107.5, 110.1, 110.5,
116.1, 141.9, 152.1, 154.2, 158.0, 161.5, 163.2, 195.0, 196.1; HRMS
(MALDI) calcd. for C17H16N2O5 [M + Na]+ 351.0957, found 351.0946.
1
Data for 14, H NMR (600 MHz, DMSO-d6) d 0.26 (s, 9H), 1.97 (s,
3H), 6.60 (s, 1H), 10.06 (s, 1H), 11.11 (br, 1H), 12.26 (s, 1H); 13C
NMR (150 MHz, DMSO-d6) d 193.9, 162.8, 162.2, 124.8, 112.7,
112.5, 112.3, 100.8, 99.9, 7.4, )0.39; HRMS (MALDI) calcd. for
C13H16O3Si [M + H]+ 249.0947, found 249.0944.
1
Data for 8e, m.p. 111–113 ꢀC; H NMR (600 MHz, CDCl3) d 1.26 (s,
3H), 2.74 (s, 3H), 3.91 (br, 1H), 4.97 (s, 2H), 5.62 (s, 1H), 6.37 (s,
1H), 6.79 (s, 1H), 7.83 (s, 1H); 13C NMR (150 MHz, CDCl3) d 23.3,
44.5, 83.8, 107.8, 110.8, 111.2, 116.0, 119.1, 120.9, 141.7, 151.2,
151.4, 152.2, 153.0, 161.0, 161.4, 195.1, 196.3; HRMS (MALDI)
calcd. for C17H13F3N2O5 [M + Na]+ 405.0674, found 405.0591.
1
Data for 5a, m.p.109–111 ꢀC; H NMR (600 MHz, CDCl3) d 2.13 (s,
3H), 3.36 (s. 1H), 5.68 (br, 1H), 6.61 (s, 1H), 10.24 (s, 1H), 12.34 (s, 1H).
General procedure for the preparation of
azaphilones 8aꢀ8g
1
Data for 8f, m.p. 176–178 ꢀC, H NMR (600 MHz, CDCl3) d 1.54 (s,
To a mixture of the alkynylbenzaldehyde 5 (0.5 mmol) and AgNO3
(4.25 mg, 0.025 mmol) or Au(OAc)3 (9.35 mg, 0.025 mmol) were
added 2.0 mL 1,2-dichloroethane and 200 lL trifluoroacetic acid,
and the mixture was stirred at room temperature until TLC monitor-
ing indicated the disappearance of the material 5 (about 5 min). To
the resulting mixture was added 2-iodoxybenzoic acid (IBX, 155 mg,
0.55 mmol) and tetrabutylammonium iodide (9.25 mg, 0.025 mmol),
and the reaction mixture was stirred at room temperature for a fur-
ther 1 h and then quenched with saturated Na2S2O3 and extracted
three times with ethyl acetate. The organic extracts were combined,
washed with brine, dried over Na2SO4 and concentrated. Purifica-
tion by flash chromatography on silica gel afforded compounds
8aꢀ8g.
3H), 2.46 (s, 6H), 4.12 (dd, J = 15.6 Hz, 16.8Hz, 2H), 4.59 (br, 1H), 5.54
(s, 1H), 6.51 (s, 1H), 6.82 (s, 1H), 7.89 (s, 1H); 13C NMR (150 MHz,
CDCl3) d 23.7, 28.4, 31.6, 83.5, 105.9, 110.1, 115.6, 116.7, 143.6,
153.0, 158.3, 167.6, 168.0, 195.5, 196.3; HRMS (MALDI) calcd. for
C17H16N2O4S [M + Na]+ 367.0728, found 367.0660.
1
Data for 8g, m.p. 164–166 ꢀC; H NMR (600 MHz, CDCl3) d 1.60
(s, 3H), 3.98 (br, 1H), 5.70 (s, 1H), 6.78 (s, 1H), 7.54–7.50 (m, 3H),
7.75 (d, J = 6.6 Hz 2H), 8.04 (s, 1H); 13C NMR (150 MHz, CDCl3) d
196.1, 195.5, 157.3, 152.6, 143.8, 131.6, 130.0, 129.1, 125.6, 115.8,
106.8, 106.2, 83.5, 28.5; HRMS (MALDI): Calcd for C16H12O4
[M + Na]+ 291.0633, Found 291.0638.
1
Data for 8a, m.p. 147–149 ꢀC; H NMR (600 MHz, CDCl3) d 1.57 (s,
General procedure for the bromination of
azaphilones 8aꢀ8g
3H), 3.65 (br, 1H), 5.60 (s, 1H), 6.35 (d, 1H, J = 5.4 Hz), 7.12 (d, 1H,
J = 5.4 Hz), 7.89 (s, 1H); 13C NMR (150 MHz, CDCl3) d 28.3, 83.7,
106.4, 112.6, 116.5, 142.2, 148.4, 152.6, 195.5, 196.3; HRMS
(MALDI) calcd. for C10H8O4 [M + Na]+ 215.0320, found 215.0340.
To a solution of one of the azaphilones 8aꢀ8g (0.4 mmol) in 5 mL
of CH2Cl2 was added NBS (106.8 mg, 0.6 mmol). The resulting mix-
ture was stirred at room temperature until TLC monitoring indicated
the disappearance of starting material. The reaction mixture was
washed with water, dried over anhydrous Na2SO4 and filtered and
concentrated in vacuo. Purification by flash chromatography on sil-
ica gel afforded the brominated azaphilones.
Data for 8b, m.p. 129–131 ꢀC; 1H NMR (600 MHz, CDCl3) d 1.57
(s, 3H), 3.89 (br, 1H), 4.75 (s, 2H), 5.62 (s, 1H), 6.50 (s, 1H), 6.95–
6.96 (m, 2H), 7.04–7.07 (m, 1H), 7.33–7.34 (m, 2H), 7.90 (s, 1H); 13C
NMR (150 MHz, CDCl3) d 28.2, 65.2, 83.6, 106.9, 109.3, 114.6,
116.1, 122.2, 129.7, 142.5, 152.3, 156.6, 157.3, 195.3, 196.2; HRMS
(MALDI) calcd. for C17H14O5 [M + Na]+ 321.0739, found 321.0763.
1
Data for 1b, m.p. 128–130 ꢀC; H NMR (600 MHz, CDCl3) d 1.60
(s, 3H), 3.86 (br, 1H), 6.86 (d, J = 5.4 Hz, 1H), 7.35 (d, J = 5.4 Hz,
Chem Biol Drug Des 2012
3

Lin et al.
1H), 7.91 (s, 1H); 13C NMR (150 MHz, CDCl3): d 28.4, 84.2, 101.5,
112.3, 116.9, 140.4, 150.3, 151.8, 190.4, 194.0; HRMS (MALDI)
calcd. for C10H7BrO4 [M + Na]+ 292.9425, found 292.9473.
127.9, 128.3, 128.6, 136.4, 138.6, 151.6, 160.0, 190.0, 193.8; HRMS
(MALDI) calcd. for C18H15ClO5 [M + Na]+ 369.0506, found 369.0508.
1
Data for 9e, m.p.: 76–78 ꢀC; H NMR (600 MHz, CDCl3) d 1.56 (s,
1
Data for 9b, m.p.: 95–97 ꢀC; H NMR (600 MHz, CDCl3) d 1.25 (s,
3H), 2.47 (s, 6H), 4.16 (dd, J = 15.0 Hz, 18.0 Hz, 2H), 6.81 (s, 1H),
7.05 (s, 1H), 7.92 (s, 1H); 13C NMR (150 MHz, CDCl3) d 23.7, 28.4,
31.9, 84.0, 107.7, 109.3, 115.3, 116.7, 139.3, 152.3, 160.1, 167.6,
168.0, 190.0, 193.8; HRMS (ESI) calcd. for C17H15ClN2O4S [M + H]+
379.0519, found 379.0554.
3H), 4.83 (s, 2H), 6.97 (s, 1H), 6.98–6.99 (m, 2H), 7.05–7.07 (m, 1H),
7.33–7.36 (m, 2H), 7.91 (s, 1H); 13C NMR (150 MHz, CDCl3) d 28.3,
65.5, 84.2, 102.3 109.3, 114.7, 115.5, 122.4, 129.8, 140.7, 151.6,
157.3, 158.8, 190.3, 193.8; HRMS (MALDI) calcd. for C17H13BrO5
[M + Na]+ 398.9844, found 398.9841.
1
Data for 9g, H NMR (600 MHz, CDCl3) d 1.63 (s, 3H), 3.96 (br, 1H),
1
Data for 9d, m.p. 104–106 ꢀC; H NMR (600 MHz, CDCl3) d 1.57
7.22 (s, 1H), 7.58–7.53 (m, 3H), 7.83–7.82 (m, 2H), 8.08 (s, 1H); HRMS
(s, 3H), 3.94 (br, 1H), 4.29 (s, 2H), 4.67 (s, 2H), 6.88 (s, 1H), 7.34–
7.38 (m, 5H), 7.86 (s, 1H); 13C NMR (150 MHz, CDCl3) d 28.3, 67.2,
73.6, 84.1, 101.7, 108.8, 116.4, 127.9, 128.3, 128.6, 136.4, 141.0,
151.6, 160.3, 190.3, 193.9; HRMS (MALDI) calcd. for C18H15BrO5
[M + Na]+ 413.0001, found 413.0030.
(MALDI): Calcd for C16H11ClO4 [M + Na]+ 325.0244, Found 325.0240.
General procedure for the preparation of
acetylated azaphilones 4
To a stirred solution of an azaphilone 9 (0.4 mmol) and acetic anhy-
dride (1.6 mL) in 5 mL CH2Cl2 were added Et3N (0.8 mmol) and 4-
(dimethylamino)pyridine (0.4 mmol) at 0 ꢀC. The resulting mixture
was stirred at 0 ꢀC until TLC analysis indicated the disappearance
of the starting material. The solvent was removed under reduced
pressure. The residue was purified by flash chromatography on sil-
ica gel to provide the acetylated product 4.
1
Data for 9f, m.p. 139–141 ꢀC; H NMR (600 MHz, CDCl3) d 1.56 (s,
3H), 2.47 (s, 6H), 4.17 (dd, J = 15.0 Hz, 25.8 Hz, 2H), 6.82 (s, 1H),
7.07 (s, 1H), 7.90 (s, 1H); 13C NMR (150 MHz, CDCl3) d 23.8, 28.5,
31.9, 84.1, 101.0, 110.4, 116.1, 116.8, 141.6, 152.4, 160.4, 167.7,
168.0, 190.3, 193.9; HRMS (MALDI) calcd. for C17H15BrN2O4S
[M + Na]+ 444.9834, found 444.9834.
1
1
Data for 9h, m.p.: 142–144 ꢀC; H NMR (600 MHz, CDCl3) d 1.63
Data for 4a, m.p. 157–159 ꢀC; H NMR (600 MHz, CDCl3) d 1.57 (s,
(s, 3H), 3.96 (br, 1H), 7.26 (s, 1H), 7.59–7.53 (m, 3H), 7.83 (d,
J = 7.2 Hz, 2H), 8.05 (s, 1H); HRMS (MALDI): Calcd for C16H11BrO4
[M + Na]+ 368.9738, found 368.9748.
3H), 2.18 (s, 3H), 4.82 (s, 2H), 6.97–6.99 (m, 2H), 7.02 (s, 1H), 7.06–
7.08 (m, 1H), 7.34–7.37 (m, 2H), 7.92 (s, 1H); 13C NMR (150 MHz,
CDCl3) d 20.0, 22.2, 65.4, 84.5, 104.4, 109.9, 114.7, 115.8, 122.3,
129.8, 139.6, 152.7, 157.3, 158.3, 170.0, 186.4, 191.5; HRMS (ESI):
calcd. for C19H15BrO6 [M + H]+ 419.0130, found 419.0160.
General procedure for the chlorination of
azaphilones 8aꢀ8g
1
Data for 4b, m.p. 155–157 ꢀC; H NMR (400 MHz, CDCl3) d 1.56
To a solution of one of the azaphilones 8aꢀ8g (0.4 mmol) in 5 mL
CH3CN was added NCS (80.1 mg, 0.6 mmol). The resulting mixture
was stirred at 30 ꢀC until TLC analysis indicated the disappearance
of the starting material. The reaction mixture was diluted with
water and extracted with ethyl acetate, and the combined organic
layers were washed with water and brine, dried over anhydrous
Na2SO4 and filtered and concentrated in vacuo. Purification by flash
chromatography on silica gel afforded the chlorinated azaphilones.
(s, 3H), 2.18 (s, 3H), 4.28 (s, 2H), 4.67 (s, 2H), 6.87 (s, 1H), 7.37–
7.40 (m, 5H), 7.90 (s, 1H); 13C NMR (100 MHz, CDCl3) d 20.0, 22.2,
67.2, 73.4, 84.6, 106.7, 111.7, 115.0, 127.9, 128.3, 128.6, 136.5,
137.5, 152.7, 159.4, 170.0, 186.3, 191.4; HRMS (MALDI) calcd. for
C20H17ClO6 [M + Na]+ 411.0611, found 411.0623.
1
Data for 4c, m.p. 173–175 ꢀC; H NMR (400 MHz, CDCl3) d 1.56 (s,
3H), 2.18 (s, 3H), 4.28 (s, 2H), 4.67 (s, 2H), 6.92 (s, 1H), 7.34–7.41
(m, 5H), 7.88 (s, 1H); 13C NMR (100 MHz, CDCl3) d 20.0, 22.3, 67.2,
73.5, 84.5, 104.1, 109.5, 115.8, 128.0, 128.4, 128.7, 136.5, 139.9,
152.8, 159.7, 170.1, 186.4, 191.7; HRMS (ESI) calcd. for C20H17BrO6
[M + H]+ 433.0287, found 433.0323.
1
Data for 1a, m.p. 127–129 ꢀC; H NMR (600 MHz, CDCl3) d 1.61 (s,
3H), 3.88 (br, 1H), 6.82 (d, J = 6.0 Hz, 1H), 7.33 (d, J = 6.0 Hz, 1H),
7.93 (s, 1H); 13C NMR (150 MHz, CDCl3): d 28.4, 84.2, 109.7, 109.9,
116.1, 138.0, 149.9, 151.8, 190.1, 193.8; HRMS (ESI) calcd. for
C10H7ClO4 [M + H]+ 227.0111, found 227.0128.
1
Data for 4d, m.p. 159–161 ꢀC; H NMR (400 MHz, CDCl3) d 1.53
(s, 3H), 2.16 (s, 3H), 2.48 (s, 6H), 4.16 (dd, J = 6.6 Hz, 15.0 Hz, 2H),
6.80 (s, 1H), 7.09 (s, 1H), 7.90 (s, 1H); 13C NMR (100 MHz, CDCl3) d
20.0, 22.3, 23.8, 31.9, 84.5, 103.4, 110.8, 115.4, 116.7, 140.5, 153.4,
159.8, 167.6, 168.1, 170.1, 186.4, 191.6; HRMS (ESI) calcd. for
C19H17BrN2O5S [M + H]+ 465.0120, found 465.0130.
1
Data for 9a, m.p. 61–63 ꢀC; H NMR (600 MHz, CDCl3) d 1.61 (s,
3H), 4.83 (s, 2H), 6.97 (s, 1H), 6.98–6.99 (m, 2H), 7.06–7.09 (m, 1H),
7.35–7.37 (m, 2H), 7.95 (s, 1H); 13C NMR (150 MHz, CDCl3) d 28.4,
65.4, 84.2, 106.7 110.6, 114.7, 115.8, 122.4, 129.8, 138.4, 151.6,
157.2, 158.5, 190.1, 193.7; HRMS (ESI) calcd. for C17H13ClO5
[M + H]+ 333.0530, found 333.0550.
Preparation of 3-bromo-4,6-dihydroxy-5-methyl-
2-(2-phenylethynyl)benzaldehyde 12
1
Data for 9c, m.p. 57–59 ꢀC; H NMR (600 MHz, CDCl3) d 1.58 (s, 3H),
4.28 (s, 2H), 4.67 (s, 2H), 6.85 (s, 1H), 7.36–7.40 (m, 5H), 7.89 (s, 1H);
To a solution of compound 5g (0.252 g, 1 mmol) in 4 mL of aceto-
nitrile was added NBS (0.25 g, 1.4 mmol). The reaction mixture was
13C NMR (150 MHz, CDCl3) d 28.2, 67.2, 73.5, 84.1, 106.2, 110.0, 115.6,
4
Chem Biol Drug Des 2012

Novel Sclerotiorin Derivatives
stirred for 2 h at room temperature (progress of the reaction was
monitored by TLC), and 30 mL of ethyl acetate was added. The
resulting mixture was washed with water (2 · 20 mL) and brine
and dried over anhydrous Na2SO4. The solvent was removed under
reduced pressure, and the residue was purified by flash chromatog-
raphy on silica gel to give 12 as a yellow solid (0.298 g, 90%).
surface of the agar. Samples (10 lL) at the appropriate concentra-
tion (200 ppm and 60 ppm for Phytophthora infestans, 100 ppm for
Septoria tritici and Uromyces viciae-fabae) were dispensed onto the
surface of each leaf piece. Each rate was carried out in duplicate
or triplicate.
Spore suspensions of the pathogen species were made up to the
necessary rate (approximately 150 000 sporangia ⁄ mL for Phytoph-
thora infestans, 1 000 000 conidia ⁄ mL for Septoria tritici and
0.3 mg spores ⁄ mL for Uromyces viciae-fabae) and applied to the
treated leaf pieces using a handheld spray gun. Lids were placed
on the plates, and they were stored under appropriate controlled
environment conditions for between 5 and 14 days, depending on
the test species.
1H NMR (600 MHz, DMSO-d6) d 13.31 (s, 1H), 10.73 (br, 1H), 10.22
(s, 1H), 7.65–7.63 (m, 2H), 7.46–7.44 (m, 3H), 2.09 (s, 3H); HRMS
(MALDI): Calcd for C16H11BrO3 [M + H]+ 330.9970, Found 330.9941.
Preparation of 2,4-dihydroxy-3,5-dimethyl-6-(2-
phenylethynyl)benzaldehyde 13
Compound 12 (66 mg, 0.20 mmol), Pd(PPh3)4 (35 mg, 0.03 mmol)
and Sn(CH3)4 (108 mg, 0.60 mmol) were suspended in 3 mL of DMF
under argon. The vessel was sealed and heated at 140 ꢀC by irradi-
ation with microwaves for 20 min. The resulting mixture was
poured into water and extracted three times with ethyl acetate.
The combined organic extracts were washed with brine and dried
over anhydrous Na2SO4. The solvent was removed under reduced
pressure, and the residue was purified by flash chromatography on
silica gel to give 13 as a yellow solid (35.7 mg, 67%).
For the artificial media assays, stock cultures of the target species
were grown in appropriate conditions on artificial media in 90-mm
petri dishes. The test pathogens, except for the Pythium dissimile
spore suspensions, were prepared in water from these stock plates,
and they were made into a 3% nutrient agar (final spore concentra-
tions of 10 000 sp ⁄ mL for Gibberella zeae, 15 000 sp ⁄ mL for Botry-
otinia fuckeliana and 20 000 sp ⁄ mL for Alternaria solani). For
Pythium dissimile, a water suspension of fragments of surface myc-
elia was prepared from the stock cultures and made into a 3% agar
as for the other species, adjusted to a final optical density reading
of 0.5 at 425 nm.
1H NMR (600 MHz, CDCl3) d 12.38 (s, 1H), 10.39 (s, 1H), 7.56–7.55
(m, 2H), 7.40–7.38 (m, 3H), 5.53 (s, 1H), 2.41 (s, 3H), 2.17 (s, 3H);
13C NMR (150 MHz, DMSO-d6) d 194.8, 161.0, 160.8, 131.1, 128.5,
128.2, 124.1, 122.3, 120.6, 112.9, 111.6, 98.8, 83.7, 13.6, 7.7; HRMS
(MALDI): Calcd for C17H14O3 [M + H]+ 267.1021, Found 267.0984.
The formulated samples were transferred to the assay plates using
a liquid handling robot. The spore ⁄ agar suspensions (90 lL) were
dispersed into the individual wells of each plate using an auto-
mated liquid handling system to provide the final rates in the media
of 20 ppm (high rate) or 2 ppm (low rate). The plates were then
stored in controlled environments set to appropriate conditions,
until assessment between 5 and 14 days later, depending on the
species. Two replicates were carried out for each rate. The average
scores were used to determine the overall activity level of the com-
pound.
Preparation of 7-hydroxy-5,7-dimethyl-3-
phenylisochroman-6,8-dione 3
A mixture of 13 (16 mg, 0.06 mmol), Au(OAc)3 (3 mg, 0.008 mmol)
and trifluoroacetic acid (1.5 mL) in 1,2-dichloroethane (1.5 mL) was
stirred for 10 min at room temperature. Then IBX (18.6 mg) and
TBAI (2 mg) were added. The progress of the reaction was moni-
tored by TLC, and when it was complete, the mixture was
quenched by the addition of aqueous Na2S2O3, and it was extracted
three times with ethyl acetate. The combined organic extracts were
washed successively with water and brine and then dried with
anhydrous Na2SO4. The solvent was evaporated under reduced
pressure, and the residue was purified by flash chromatography on
silica gel to give 3 as a yellow solid (10 mg, 59%).
Result and Discussion
Chemistry
Although various studies on the synthesis of azaphilones have been
reported, some with stereoselective chemistry (26–28) and others
focused on racemic analogues (13,29–33), this deceptively simple-
looking bicyclic structure still presents significant challenges, espe-
cially when the requirement is to prepare analogues with diverse
substituents at the 3-position. Recently, Porco's (34) group reported
an efficient gold-catalysed cycloisomerization of 2-alkynylbenzalde-
hydes to give 2-benzopyrylium salts that can subsequently be oxi-
dized with IBX to form the azaphilone ring system. In contrast to
the earlier approaches, Porco's method uses readily available alky-
nes to construct the key intermediate 2-alkynylbenzaldehydes, which
can be then transformed into the corresponding azaphilones with
diverse side chains at the 3-position. We realized that this method
would enable us to investigate the effect of modifications at this
position on the activity of sclerotiorin analogues. Thus, we envi-
sioned preparing the core structures 1ꢀ4 by oxidation of the
1H NMR (600 MHz, CDCl3) d 1.58 (s, 3H), 2.18 (s, 3H), 4.05 (br, 1H),
6.83 (s, 1H), 7.53–7.49 (m, 3H), 7.76 (d, J = 7.2 Hz, 2H), 7.93 (s,
1H); 13C NMR (150 MHz CDCl3): d 196.4, 195.6, 156.8, 150.8, 138.8,
131.4, 130.7, 129.1, 125.6, 116.0, 112.5, 104.3, 83.2, 28.7, 10.1;
HRMS (MALDI): Calcd for C17H14O4 [M + Na]+ 305.0790, Found
305.0769.
Biological testing
All testing were undertaken in 96-well microtitre plates. For the
leaf piece assays, 200–300 lL water agar was dispensed into each
well of the assay plates. Leaf pieces (6 mm diameter for tomato
and bean, 6 mm length for wheat) were transferred onto the
Chem Biol Drug Des 2012
5

Lin et al.
appropriate benzopyrylium salts 11, which may be derived from the
alkynylbenzaldehyde 5. Furthermore, the key intermediate alkynyl-
benzaldehydes 5 should be readily accessible using Sonogashira
coupling (Figure 2).
reaction under conventional heating, but none of these efforts
provided satisfactory results (Table 1, entries 1–3). Microwave-
assisted organic synthesis is now widely recognized as a valuable
tool for increasing the rates and improving the yields of organic
reactions, so we next examined the effect of microwave irradia-
tion on this Sonogashira coupling reaction. The solvent effect on
the reaction was first evaluated. We found that THF and 1,4-diox-
ane were poor solvents because the temperature could not be
raised to the desired 100 ꢀC. However, the yield improved to 45%
when the coupling reaction was conducted in DMF. As the pure
solvents did not give satisfactory yields, mixtures of solvents were
considered. When a mixture of THF ⁄ DMF (V ⁄ V = 4:1) was used, a
complex reaction mixture was observed (Table 1, entry 7), but the
yield of 5b increased to 60% when a mixture of 1,4-dioxane and
water in a 4:1 ratio was chosen as the solvent. Furthermore,
increasing the amount of alkyne 7b from 1.2 to 3 equiv. resulted
in a useful increase in the yield to 85%. However, when the cat-
alyst was changed from Pd(PPh3)2Cl2 to Pd(CH3CN)2Cl2, only traces
of the product were detected (Table 1, entry 11). Thus, we chose
the following conditions as optimum for all the subsequent cou-
pling reactions: 5 mol % PdCl2(PPh3)2, 5 mol% CuI, 3 equiv. Et3N
and 3 equiv. alkyne under microwave irradiation at 100 ꢀC for
15 min.
The intermediate 2-bromobenzaldehyde 6 was synthesized accord-
ing to Porco's (34) method, with minor modifications on the larger
scale. With this in hand, the Sonogashira coupling reaction with
ethynyltrimethylsilane was investigated, and it proceeded smoothly
under 5 mol% Pd ⁄ Cu co-catalysis at room temperature to give the
product 14 in 92% isolated yield (Scheme 1). Removal of the
trimethylsilyl group was then carried out using K2CO3 in methanol
at room temperature and provided the desired product 5a. How-
ever, when 1-(prop-2-ynyloxy)benzene (7b) was selected as the
terminal alkyne for the coupling reaction under similar conditions,
the yield of the desired product 5b decreased to 18%, even
when the reaction time was extended to 15 h. Similarly, when 2-
methyl-1-(prop-3-ynyl)-4-trifluoromethyl-pyrimidin-6-one 7e was
used in the coupling reaction, only traces of the expected product
5e could be isolated. Thus, alkyne 7b was chosen as a model
substrate and was used in the coupling reaction with the 2-
bromobenzaldehyde 6 to optimize the reaction conditions. Initially,
we investigated the effect of the catalyst and solvent on the
Figure 2: Retrosynthetic analysis of the designed sclerotiorin analogues.
Table 1: Optimization of the coupling reaction conditionsa
OPh
HO
Br
HO
OPh
CHO
CHO
OH
6
OH
5b
7b
Entry
7b (equiv.)
Catalyst (5 mol %)
Solvent
Time (min)
Yield (%)b
1c
2c
3d
4
5
6
7
8
9
10
11
1.2
1.2
1.2
1.2
1.2
1.2
1.2
1.2
1.2
3.0
3.0
PdCl2(PPh3)2
PdCl2(PPh3)2
Pd (PPh3)4
DMF
THF
DMF
THF
1,4-dioxane
DMF
THF ⁄ DMF (4:1)
1,4-dioxane ⁄ H2O
1,4-dioxane ⁄ H2O
1,4-dioxane ⁄ H2O
1,4-dioxane ⁄ H2O
900
600
900
18
10
20
–
–
e
PdCl2(PPh3)2
PdCl2(PPh3)2
PdCl2(PPh3)2
PdCl2(PPh3)2
PdCl2(PPh3)2
PdCl2(PPh3)2
PdCl2(PPh3)2
PdCl2(CH3CN)2
–
–
e
10
10
10
15
15
15
45
Complex
60
65
85
Trace
aThe reaction was conducted using 5 mol % PdCl2(PPh3)2, 5 mol% CuI and three equiv. Et3N under microwave irradiation at 100 ꢀC unless otherwise noted.
bIsolated yield.
cThe reaction was conducted using 5 mol % PdCl2(PPh3)2, 5 mol% CuI and three equiv. Et3N under conventional heating at 100 ꢀC.
dThe reaction was conducted using 5 mol % PdCl2(PPh3)2, 5 mol% CuI and three equiv. Et3N under conventional heating at 100 ꢀC.
eThe reaction was stopped by the reactor system because the temperature could not reach the setting value.
6
Chem Biol Drug Des 2012

Novel Sclerotiorin Derivatives
With the optimum reaction conditions in hand, we next conducted
the Sonogashira coupling reaction between 2-bromobenzaldehyde 6
and several functionalized terminal acetylenes (Table 2) and found
that aryloxy-, benzyloxy- and heterocycle-substituted acetylenes all
tolerated the reaction conditions and afforded the desired coupling
products in good yields.
tion of the compounds 1a, 1b and 9a. We therefore designed an
alternative route to circumvent this problem, as shown in
Scheme 2. In this route, the halogen atom was introduced earlier in
the sequence, prior to the cycloisomerization and oxidation steps,
and led to the desired products in higher yields. Thus, the isolated
yields of 1a, 1b and 9a were improved from 15%, 32% and 30%
to 49%, 50% and 53%, respectively.
The subsequent cycloisomerization with catalytic Lewis acid (AgNO3
or Au(OAc)3) in a mixed solvent of 1,2-dichloroethane and trifluoro-
acetic acid (10:1) afforded the pyrylium salts, which were oxidized
without isolation using o-iodoxybenzoic acid (IBX) to give the aza-
philones 8 in moderate yields (Table 3). Generally, the intermediate
2-alkynylbenzaldehydes 5 were converted smoothly into the corre-
sponding azaphilones, and the nature of the substituents on the
carbon–carbon triple bond did not dramatically affect the yield of
azaphilones. However, it is worth noting that a very low yield was
observed when the simple 2-ethynylbenzaldehyde 5a was used as
the starting material under similar reaction conditions. In this case,
we changed the catalyst from AgNO3 to Au(OAc)3 in the cycloiso-
merization step, and a satisfactory yield was obtained.
Synthesis of the analogues 3 with a methyl group at the 5-position
and an additional phenyl substituent was accomplished to further
probe the structure–activity relationships. We initially planned to
construct the skeleton of compound 3 by direct methylation of 9h.
However, treatment of 9h with tetramethyl stannane in the pres-
ence of a palladium catalyst failed to produce compound 3. Thus,
an alternative synthetic route was designed and is illustrated in
Scheme 3. The synthesis began with the bromination of the 2-alky-
nylbenzaldehyde 5g by treating it with four equivalents of aqueous
HBr in the presence of IBX to give the congested alkynylbenzalde-
hyde 12. Palladium-catalysed cross-coupling with tetramethyl stann-
ane then successfully led to compound 13, which underwent the
cycloisomerization and oxidation procedure to produce the required
product 3.
Compound 8 was then converted into the corresponding 5-haloge-
nated product 9 by treating it with NCS or NBS in dichloromethane
at room temperature, and some of the halogenated compounds
were transformed into the acetylated derivatives by treatment with
acetic anhydride in the presence of triethylamine and DMAP
(Table 4). The structure of compounds 8 and their equivalent halo-
genated compounds 9 and the acetylated compounds 4 were con-
firmed by their NMR spectra and HRMS analysis. The structures of
4a and 8e were further confirmed by carrying out an X-ray struc-
ture analysis (Figure 3).
Fungicidal activity
The fungicidal activity of compounds 1–4 and their precursors 8b-
g and 9a-h were evaluated against seven phytopathogenic spe-
cies, in a combination of assays conducted in artificial media or on
leaf pieces (Table 5). In general, any activity observed was
restricted to the assays in artificial media and rarely extended to
pathogens tested on leaf pieces. Furthermore, although the most
active compounds were effective against a range of species, the
activity tended to lack potency and was rarely observed at the
lower rates tested.
During this research, we noticed that some halogenation reactions
suffered from the problem of low yield, especially for the prepara-
Table 2: Microwave-assisted synthesis of 2-alkynylbenzaldehydes 5a
R1
HO
Br
HO
R1
CHO
CHO
OH
6
OH
5
7
O
O
O
N
HO
HO
HO
N
CHO
OH
5b, 85%
CHO
CHO
OH
5d, 85%
OH
5c, 89%
O
N
N
S
N
HO
HO
HO
N
CF3
CHO
CHO
OH
5g, 85%
CHO
OH
5f, 62%
OH
5e, 80%
aThe reaction was run using 5 mol % PdCl2(PPh3)2, 5 mol% CuI and three equiv. Et3N under microwave irradiation at 100 ꢀC.
Chem Biol Drug Des 2012
7

Lin et al.
Table 3: Synthesis of 8a–f via cycloisomerization and oxidation
R
O
R
HO
AgNO3 or Au(OAc)3
IBX, TBAI
O
DCE/TFA (10:1)
CHO
HO
O
OH
8a-8g
5a-5f
O
N
O
O
O
O
O
O
O
N
HO
O
HO
HO
O
HO
O
O
O
O
O
8a, 33%
8b, 41%
8c, 45%
8d, 46%
N
O
O
O
O
S
N
N
HO
CF3
O
HO
O
HO
O
N
O
O
O
8g, 63%
8e, 23%
8f, 36%
Table 4: Synthesis of compounds 3 and 9 via halogenation and acetylationa
X
X
O
AcO
R
O
R
O
R
(AcO)2O, NEt3
DMAP
NXS
O
HO
H3C
O
HO
H3C
O
H3C
O
4 or 2
O
8
O
1 or 9
Yield (%)
Entry
R
X
1 or 9
4 or 2
b
1
2
3
4
5
6
7
8
9
H
H
Cl
Br
Cl
Br
Cl
Br
Cl
Br
Cl
Br
1a, 15
1b, 32
9a, 30
9b, 71
9c, 76
9d, 68
9e, 59
9f, 43
9g, 60
9h, 70
–
–
–
C6H5-OCH2-
C6H5-OCH2-
C6H5-CH2OCH2-
C6H5-CH2OCH2-
(4,6-di-Me-pyrimidin-2-yl)-SCH2-
(4,6-di-Me-pyrimidin-2-yl)-SCH2-
C6H5-
C6H5-
4a, 45
4b, 62
4c, 60
–
4d, 65
2a, 50
2b, 56
10
aThe halogenation was conducted by employing 1.5 equiv. NBS in dichloromethane at room temperature or 1.5 equiv. NCS in acetonitrile at 30 ꢀC.
b–indicates not synthesized.
It is difficult to draw a detailed structure–activity relationship for
the synthesized azaphilone derivatives on the basis of the present
biological data; however, some interesting results have been
observed. Most importantly, the spectrum of antifungal activity was
improved by the incorporation of a halogen (chlorine or bromine) or
methyl group at the 5-position and a phenyl group at the 3-position
of the sclerotiorin framework. The resulting compounds, 3, 9g and
9h, all gave strong antifungal activity in artificial media against Py-
thium dissimile, Alternaria solani and Gibberella zeae, with 3 and
9g in particular displaying activity at the low rate of 2 ppm against
G. zeae and P. dissimile, respectively. Compound 3 was also active
on the leaf piece assay against Uromyces viciae-fabae. In contrast,
2a and 2b, the O-acetylated products of 9g and 9h, were much
less active than their corresponding unprotected analogues, which
demonstrates that the free hydroxyl group plays an important role
in the observed activity.
Compound 8g, the non-halogenated precursor of 9g and 9h, also
showed a much narrower spectrum of antifungal activity, which
may be attributed to the absence of a substituent at the 5-position.
8
Chem Biol Drug Des 2012

Novel Sclerotiorin Derivatives
A
B
Figure 3: X-ray structures of 4a and 8e.
X
R
R
X
HO
HO
R
O
O
AgNO3 or Au(OAc)3
IBX, TBAI
NXS
HO
DCE/TFA (10:1)
CHO
CHO
O
OH
OH
5a or 5b
10a, X = Cl, R = H, 80%
9a, X = Cl, R = H, 49%
10b, X = Br, R = H, 85%
9b, X = Br, R = H,50%
10c, X = Cl, R = C6H5-OCH2-, 63%
9c, X = Cl, R = C6H5-OCH2-, 53%
Scheme 2: Synthesis of azaphilones 9a, 9b and 9c.
Furthermore, compound 1b, the analogue of 9h without the phenyl
group at the 3-position, did not exhibit any antifungal activity in
these assays, demonstrating the importance of the substituent at
the 3-position for activity.
that these more complex side chains do not represent useful start-
ing points for further structural modification.
Conclusion
Compounds 9a–9f, which have substituents at the 3-position such
as -CH2OPh, -CH2OCH2Ph or chains terminating in heterocycles such
as pyrimidine or pyrimidinone, were found to be far less active than
the simple phenyl-bearing compounds 9g and 9h, and it follows
In summary, we have synthesized a series of novel azaphilone ana-
logues by replacement of the diene side chain of sclerotiorin with
hydrogen, phenyl and aliphatic substituents. A practical approach to
Chem Biol Drug Des 2012
9

Lin et al.
CH3
Ph
Br
Ph
Ph
HO
HO
HO
a
b
H3C
CHO
H3C
CHO
H3C
OH
5g
CHO
OH
13
OH
12
c
Scheme 3: Reagents and con-
ditions: (A) four equiv. HBr (aq.),
1.5 equiv. IBX, r.t., 80% (B) SnMe4,
Pd(PPh3)4, DMF, MW, 140 ꢀC,
20min, 67% c) (i) Au(OAc)3,
DCE ⁄ TFA (10:1) (ii) IBX, TBAB, 40%
over two steps.
CH3
Br
O
Ph
O
Ph
b
O
O
HO
HO
O
3
O
9h
Table 5: Fungicidal activity of the synthesized compoundsa
Pib
St
Uvf
Pd
As
Bf
Gz
Pi
St
Uvf
Pd
As
Bf
Gz
No
200 ⁄ 60c
100
100
20 ⁄ 2
20 ⁄ 2
20 ⁄ 2
20 ⁄ 2
No
200 ⁄ 60c
100
100
20 ⁄ 2
20 ⁄ 2
20 ⁄ 2
20 ⁄ 2
2a
2b
4a
4b
4c
4d
8b
8c
8d
8e
8f
0 ⁄ –a
0 ⁄ –
0
99
49
0
0
0
0
0
0
0
0
0
27
0
27
0
0
0
0
27
0
99 ⁄ 77
99 ⁄ 99
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
8g
1b
9a
9b
9d
9e
9f
0 ⁄ 0
0
99
49
49
0
0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0
0
18
0
0
0
0
0
0
0
0
27
0
0
0
0
0
0
0
99 ⁄ 49
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
99 ⁄ 99
99 ⁄ 77
99 ⁄ 0
99 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
99 ⁄ 27
77 ⁄ 0
27 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
27 ⁄ 0
0 ⁄ 0
99 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
99 ⁄ 0
99 ⁄ 0
99 ⁄ 99
0 ⁄ 0
0
0
0
0
0
0
0
0
0 ⁄ 0
99 ⁄ 49
99 ⁄ 27
0 ⁄ 0
0 ⁄ 0
0 ⁄ 0
9g
9h
3
Scld
0
0
99
NCH
0
aMean scores across replicates, where 0 means 0–49% control of pathogen; 55 means 50–80% control of pathogen; 99 means 81–100 control of pathogen;
and '–' indicates that the compound was untested at that rate. NCH indicates that no assessment was possible because of herbicidal effects on the leaf piece.
bKey: Pi, Phytophthora infestans (tested on tomato leaf pieces); St, Septoria tritici (tested on wheat leaf pieces); Uvf, Uromyces viciae-fabae (tested on bean
leaf pieces); Pd, Pythium dissimile; As, Alternaria solani; Bf, Botryotinia fuckeliana; Gz, Gibberella zeae (all tested on artificial media).
cRates in ppm.
dSclerotiorin.
these compounds has been developed by employing metal-catalysed
cycloisomerization and subsequent oxidation of suitable 2-alkynyl-
benzaldehydes, in which a variety of desired substituents were
introduced using a Sonogashira coupling reaction.
for further optimization of fungicidal activity. Further structural mod-
ifications to the sclerotiorin framework are underway, with a view
to developing a full understanding of the structural requirements
for optimal fungicidal activity.
The antifungal activity of the synthesized compounds was assessed.
Where activity was observed, it tended to be in the assays conducted
on artificial media. While several of the test compounds (notably 2a,
2b, 8b, 8c, 8g) showed good activity against Pythium dissimile, it
was the compounds 3, 9g and 9h that gave the broadest spectrum
of activity. The following conclusions can be drawn: (i) Activity is
improved by the presence of a halogen or methyl substituent at the 5-
position and a phenyl group at the 3-position. (ii) The free hydroxyl
group at the 7-position is important for maintaining antifungal activity.
(iii) Aliphatic- or heterocycle-containing aliphatic side chains at the 3-
position appear to be detrimental to antifungal activity.
Acknowledgments
This research was supported in part by Syngenta and the NSFC
(No. 21002038). We thank Mrs Dianne Irwin and the biology team
at Syngenta for their kind help in screening the compounds for bio-
logical activity.
References
1. Curtin T.P., Reilly J. (1940) Sclerotiorine, a chlorinated metabolic
product of Penicillium sclerotiorum van Beyma. Nature;7:
335–335.
Compounds 3, 9g and 9h, which have much better antifungal
activity and simpler structures than sclerotiorin, are inspiring leads
10
Chem Biol Drug Des 2012

Novel Sclerotiorin Derivatives
2. Huang H., Feng X., Xiao Z., Liu L., Li H., Ma L., Lu Y., Ju J., She
Z., Lin Y. (2011) Azaphilones and p-terphenyls from the man-
grove endophytic fungus Penicillium chermesinum (ZH4-E2) Iso-
lated from the South China Sea. J Nat Prod;74:997–1002.
3. Gao S.S., Li X.M., Zhang Y., Li C.S., Cui C.M., Wang B.G. (2011)
Comazaphilones A–F, azaphilone derivatives from the marine
sediment-derived fungus Penicillium commune QSD-17. J Nat
Prod;74:256–261.
4. Arunpanichlert J., Rukachaisirikul V., Sukpondma Y., Phongpaichit
S., Tewtrakul S., Rungjindamai N., Sakayaroj J. (2010) Azaphilone
and isocoumarin derivatives from the endophytic fungus Penicil-
lium sclerotiorum PSU-A13. Chem Pharm Bull;58:1033–1036.
5. Hsu Y.W., Hsu L.C., Liang Y.H., Kuo Y.H., Pan T.M. (2010) Mona-
philones A-C, three new antiproliferative azaphilone derivatives
18. Yang S.W., Chan T.M., Terracciano J., Loebenberg D., Patel M.,
Gullo V., Chu M. (2009) Sch 1385568, a new azaphilone from
Aspergillus sp. J Antibiot;62:401–403.
19. Huang W., Yang G.F. (2006) Microwave-assisted, one-pot synthe-
ses and fungicidal activity of polyfluorinated 2-benzylthiobenzo-
thiazoles. Bioorg Med Chem;14:8280–8285.
20. Huang W., Zhao P.L., Liu C.L., Chen Q., Liu Z.M., Yang G.F.
(2007) Design, synthesis, and fungicidal activities of new stro-
bilurin derivatives. J Agric Food Chem;55:3004–3010.
21. Chen Q., Zhu X.L., Jiang L.L., Liu Z.M., Yang G.F. (2008)
Synthesis, antifungal activity and CoMFA analysis of novel
1,2,4-triazolo[1,5-a]pyrimidine
Chem;43:595–603.
derivatives.
Eur
J
Med
22. Zhao P.L., Liu C.L., Huang W., Wang Y.Z., Yang G.F. (2007) Syn-
thesis and fungicidal evaluation of novel chalcone-based stro-
bilurin analogues. J Agric Food Chem;55:5697–5700.
from Monascus purpureus NTU 568.
J
Agric Food
Chem;58:8211–8216.
6. Kono K., Tanaka M., Ono Y., Hosoya T., Ogita T., Kohama T.
(2001) S-15183a and b, two new sphingosine kinase inhibitors
from the fungus Zopfiella inermis. J Antibiot;54:415–420.
7. Dong J., Zhou Y., Li R., Zhou W., Li L., Zhu Y., Huang R., Zhang
K. (2006) New nematicidal azaphilones from the aquatic fungus
Pseudohalonectria adversaria YMF1.01019. FEMS Microbiol
Lett;264:65–69.
23. Marsini M.A., Gowin K.M., Pettus T.R.R. (2006) Total synthesis
of (€)-mitorubrinic acid. Org Lett;8:3481–3483.
24. Musso L., Dallavalle S., Merlini L., Bava A., Nasini G., Penco S.,
Giannini G., Giommarelli C., Cesare A.D., Zuco V., Vesci L., Pisa-
no C., Piaz F.D., Tommasi N.D., Zunino F. (2010) Natural and
semisynthetic azaphilones as a new scaffold for Hsp90 inhibi-
tors. Bioorg Med Chem;18:6031–6043.
8. Suzuki S., Hosoe T., Nozawa K., Yaguchi T., Udagawa S.I., Kawai
K.I. (1999) Mitorubrin derivatives on ascomata of some talaromy-
ces species of ascomycetous fungi. J Nat Prod;62:1328–1329.
9. Zhu J.L., Porco J.A. Jr (2006) Asymmetric synthesis of ())-mi-
torubrin and related azaphilone natural product. Org
Lett;8:5169–5171.
10. Quang D.N., Hashimoto T., Tanaka M., Stadler M., Asakawa Y.
(2004) Cyclic azaphilones daldinins E and F from the ascomycete
fungus Hypoxylon fuscum (Xylariaceae). Phytochemistry;65:469–
473.
11. Osmanova N., Schultze W., Ayoub N. (2010) Azaphilones: a class
of fungal metabolites with diverse biological activities. Phyto-
chem Rev;9:315–342.
12. Michael A.P., Grace E.J., Kotiw M., Barrow R.A. (2003) Isochro-
mophilone IX, a novel GABA-containing metabolite isolated from
a cultured fungus, Penicillium sp. Aust J Chem;56:13–15.
13. Clark R.C., Lee S.Y., Boger D.L. (2008) Total synthesis of chloro-
fusin, its seven chromophore diastereomers, and key partial
structures. J Am Chem Soc;130:12355–12369.
14. Matsuzaki K., Tahara H., Inokoshi J., Tanaka H., Masuma R.,
Omura S. (1998) New brominated and halogen-less derivatives
and structure-activity relationship of azaphilones inhibiting
gp120-CD4 binding. J Antibiot;51:1004–1011.
15. Laakso J.A., Raulli R., McElhaney-Feser G.E., Actor P., Underiner
T.L., Hotovec B.J., Mocek U., Cihlar R.L. (2003) CT2108A and B:
new fatty acid synthase inhibitors as antifungal agents. J Nat
Prod;66:1041–1046.
16. Chidananda C., Sattur A.P. (2007) Sclerotiorin, a novel inhibitor
of lipoxygenase from Penicillium frequentans. J Agric Food
Chem;55:2879–2883.
17. Kanokmedhakul S., Kanokmedhakul K., Nasomjai P., Louangsy-
souphanh S., Soytong K., Isobe M., Kongsaeree P., Prabpai S.,
Suksamrarn A. (2006) Antifungal azaphilones from the fungus
Chaetomium cupreum CC3003. J Nat Prod;69:891–895.
25. Quang D.N., Hashimoto T., Stadler M., Radulovic N., Asakawa Y.
(2005) Antimicrobial azaphilones from the fungus Hypoxylon mul-
tiforme. Planta Med;71:1058–1062.
26. Germain A.R., Bruggemeyer D.M., Zhu J.L., Genet C., Brien P.,
Porco J.A. (2011) Synthesis of the azaphilones (€)-Sclerotiorin
and (+)-8-O-methylsclerotiorinamine utilizing (+)-sparteine surro-
gates in copper-mediated oxidative dearomatization. J Org
Chem;76:2577–2584.
27. Zhu J.L., Grigoriadis N.P., Lee J.P., Porco J.A. (2005) Synthesis of
the azaphilones using copper-mediated enantioselective oxida-
tive dearomatization. J Am Chem Soc;127:9342–9343.
28. Qian W.J., Wei W.G., Zhang Y.X., Yao Z.J. (2007) Total synthe-
sis, assignment of absolute stereochemistry, and structural revi-
sion of chlorofusin. J Am Chem Soc;129:6400–6401.
29. Chong R., King R.R., Whalley W.B. (1971) The chemistry of fungi.
Part LXI. The synthesis of (€)-sclerotiorin, of (€)-4,6-dimethylocta-
trans-2,trans-4-dienoic acid, and of an analogue of rotiorin. J
Chem Soc C;356:6–3571.
30. Chong R., King R.R., Whalley W.B. (1971) The chemistry of fungi.
Part LXII. The synthesis of (€)-mitorubrin, a metabolite of Penicil-
lium rubrum. J Chem Soc C;357:1–3575.
31. Suzuki T., Okada C., Arai K., Awall A., Shimizu T., Tanemura K.,
Horaguchi T. (2001) Synthesis of 7-acetyloxy-3,7-dimethy1-7,8-di-
hydro-6H-isochromene-6,8-dione and its analogues. J Heterocycl
Chem;38:1409–1418.
32. Kamino T., Murata Y., Kawai N., Hosokawa S., Kobayashi S.
(2001) Stereochemical control of tertiary alcohol: aldol condensa-
tion of lactate derivatives. Tetra Lett;42:5249–5252.
33. Wei W.G., Yao Z.J. (2005) Synthesis studies toward chloroaza-
philone and vinylogous c-pyridones: two common natural product
core structures. J Org Chem;70:4585–4590.
34. Zhu J.L., Germain A.R., Porco J.A. (2004) Synthesis of azaphil-
ones and related molecules employing cycloisomerization of al-
kynylbenzaldehydes. Angew Chem Int Ed;43:1239–1243.
Chem Biol Drug Des 2012
11