Synthesis and antifungal activity of 2-hydroxy-4,5-methylenedioxyaryl ketones as analogues of kakuol

Article abstract of DOI:10.1002/cbdv.200900263

In a study aiming to determine the structural elements essential to the antifungal activity of kakuol, we synthesized a series of 2-hydroxy-4,5- methylenedioxyaryl ketones, and we assayed their in vitro antifungal activity. The most sensitive target organisms to the action of these class of compounds were Phytophthora infestans, Phytium ultimum, Cercospora beticola, Cladosporium cucumerinum, and Rhizoctonia solani. Most of the analogs showed a remarkable in vitro activity, and some of them appeared significantly more effective than the natural product. The biological activity was mainly affected by introducing structural modification on the carbonyl moiety of the natural-product molecule. In particular, compound 5a, bearing a C=C bond conjugated to the C=O group, was found active with a MIC value of 10 μg ml^-1 against Cladosporium cucumerinum. The results suggest that 2-hydroxy-4,5-methylenedioxyaryl ketones can be considered promising candidates in the development of new antifungal compounds.

Full text of DOI:10.1002/cbdv.200900263

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
887
Synthesis and Antifungal Activity of 2-Hydroxy-4,5-methylenedioxyaryl
Ketones as Analogues of Kakuol
a
a
a
b
by Loana Musso* ), Sabrina Dallavalle ), Lucio Merlini ), and Gandolfina Farina )
a) Dipartimento di Scienze Molecolari Agroalimentari, Universit a` di Milano, Via Celoria 2,
IT-20133 Milano (phone: þ390250316816; fax: þ390250316801; e-mail: loana.musso@unimi.it)
b) Dipartimento di Protezione dei Sistemi Agroalimentare e Urbano e Valorizzazione delle Biodiversit a` ,
Universit a` di Milano, Via Celoria 2, IT-20133 Milano (phone: þ390250316779; fax: þ390250316748)
In a study aiming to determine the structural elements essential to the antifungal activity of kakuol,
we synthesized a series of 2-hydroxy-4,5-methylenedioxyaryl ketones, and we assayed their in vitro
antifungal activity. The most sensitive target organisms to the action of these class of compounds were
Phytophthora infestans, Phytium ultimum, Cercospora beticola, Cladosporium cucumerinum, and
Rhizoctonia solani. Most of the analogs showed a remarkable in vitro activity, and some of them
appeared significantly more effective than the natural product. The biological activity was mainly
affected by introducing structural modification on the carbonyl moiety of the natural-product molecule.
In particular, compound 5a, bearing a C¼C bond conjugated to the C¼O group, was found active with a
ꢀ
1
MIC value of 10 mg ml against Cladosporium cucumerinum. The results suggest that 2-hydroxy-4,5-
methylenedioxyaryl ketones can be considered promising candidates in the development of new
antifungal compounds.
Introduction. – Innovation in crop protection is essential for sustainability of
agriculture and global food production [1]. In contrast to the growing world population
and the increasing demand for higher quantity and quality of food, the area of farmland
per head of population has decreased dramatically during the last 50 years. In addition,
crop losses due to pests, diseases, and weed damages are still as high as 50% overall.
Although these losses may be attenuated by the use of disease-tolerant cultivars, crop
rotation, or sanification practices, the use of fungicides will remain the predominant
method of crop protection for the foreseeable future.
Despite the availability of effective fungicides [2], new antifungal chemicals are still
needed to obtain improved yields and quality benefits, and to combat pathogens which
show resistance or reduced sensitivity to existing antifungal compounds. In our study
toward the development of new antifungal agents, we focused our interest on naturally
occurring substances with antifungal activity as starting points for the rational design of
new fungicides.
In 2005, Hwang and co-workers [3] isolated kakuol (¼2-hydroxy-4,5-(methyl-
enedioxy)propiophenone¼1-[2-hydroxy-4,5-(methylenedioxy)phenyl]propan-1-one; 1;
Fig. 1) from the rhizomes of Asarum sieboldii (Miq.) Maek, a perennial herb belonging
to the family of Aristolochiaceae, and demonstrated for the first time its antifungal
activity against some important plant pathogens such as Colletotrichum orbiculare,
Rhizoctonia solani, Botrytis cinerea, Phytophthora capsici, and Cladosporium cucu-
merinum.
ꢀ
2010 Verlag Helvetica Chimica Acta AG, Zꢁrich

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Fig. 1. Structure of kakuol (1)
Although Asarum sieboldii had been reported to have antifungal and insecticidal
activity [4][5], the study by Hwang and co-workers was the first to show the fungicidal
activity of the plant component kakuol, which is considered a metabolic oxidation
product of saricin and safrole.
Several analogous propiophenones, such as 2-methoxy-4,5-(methylenedioxy)pro-
piophenone (methylkakuol) [6–8], 2-methoxy-3,4-(methylenedioxy)propiophenone
[
9], and 2,4,5-trimethoxypropiophenone [10], were recently isolated from Asarum,
Anethum, and Piper species, respectively, but their antifungal activity was not
investigated. Although some data related to antifungal activity of kakuol and
methylkakuol have been reported in a recent patent [11], nothing has been published
so far about the synthesis and the determination of a structure–activity relationship
(
SAR) for a series of kakuol analogues. This lack of information prompted us to
investigate this promising class of small molecules.
The results reported in the present study give an insight into some structural
features affecting antifungal activity of kakuol.
Results and Discussion. – 1. Synthesis. In an effort to define the structural elements
essential to the antifungal activity, we have altered kakuol at single sites assumed to be
critical for function: the phenol group, and the methylenedioxy and the acyl moieties.
Two different strategies were applied to obtain kakuol and the series of 2-
hydroxyaryl ketones; the first one involved Fries-type rearrangement [12] of
appropriate phenol esters (Method A, Scheme 1); the second one exploited the
reaction of highly coordinating metal phenolates with appropriate acyl chlorides to
afford in a single step ortho-acyl phenols [13] (Method B, Scheme 2). For each
compound, the strategy was chosen depending on the availability of starting materials
and with the aim to minimize the number of synthetic steps.
Scheme 1. Synthesis of Kakuol (1) and 4,5-Dimethoxypropiophenone (4)
a) BF ·Et O, propanoic anhydride, 45 min, 808; yield 73% for 1, 42% for 4.
3
2

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
889
Scheme 2. Synthesis of 2-Hydroxy-4,5-methylenedioxyaryl Ketones 5a–5i
a) MeMgBr, Et O, 45 min, r.t. b) RCOCl, toluene, r.t., 16 h; see Exper. Part for yields. c) Propanoyl
2
chloride, toluene, r.t., yield 22% for 8, 46% for 9.
Endo et al. [11] reported a two-step synthesis of kakuol in a 26% overall yield. In
our synthetic approach, kakuol (1) was obtained in a single step by reacting sesamol (¼
3
,4-(methylenedioxy)phenol; 2) with BF ·Et O in propanoic anhydride with a 73%
3 2
yield (Scheme 1). Following the same procedure, compound 4 was obtained from 3,4-
dimethoxyphenol (3) in 42% yield.
The compounds bearing different chains linked to the CO moiety were obtained by
reacting magnesium 3,4-(methylenedioxy)phenolates with appropriate acyl chlorides
affording in a single step the ortho-acyl phenols 5a–5i (Scheme 2). Commercially
unavailable acyl chlorides were prepared according to reported methods [14–16].
In some cases, besides the desired ortho-acyl phenols, we observed the formation of
the corresponding esters 6. The presence of these compounds in the reaction mixture
made the purification of desired ortho-acyl phenols very difficult, and, in some cases, a
further purification by crystallization after flash chromatography was needed. The best
results were obtained for 5a, obtained in good yield (66%) and without the formation of
the corresponding ester, and for 5i, obtained in 45% yield, together with a small amount
of the ester (16%).
For the synthesis of compound 8, phenol 7 was prepared in 86% yield from 2,3-
dihydro-1,4-benzodioxine-6-carbaldehyde by Bayer–Villiger oxidation [17] and imme-
diate hydrolysis of the formate. The acylation of the magnesium phenolate of 7 afforded
compound 8 in only 22% yield together with 46% of ester 9.
Compound 10 was obtained from 1 by methylation with MeI and K CO in acetone
2
3
[
18] (Scheme 3). The synthesis of compound 11 was performed by reacting kakuol (1)
with NH OH·HCl [15] and AcONa in MeOH, and thione 12 was prepared in 13%
2
yield by refluxing a solution of 1 in toluene with Lawessonꢂs reagent [19] for 7 h.

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Scheme 3. Synthesis of Compounds 10, 11, and 12
a) MeI, K CO , acetone, 48%. b) NH OH·HCl, AcONa, MeOH, reflux, 14 h; 80%. c) Lawessonꢂs
2
3
2
reagent, toluene, reflux, 12 h; 13%.
2
. Biological Activity. To integrate the scarce literature data reporting kakuol
biological activity, synthetic 1 was tested against the following pathogens: Phytoph-
thora infestans, Pythium ultimum, Botrytis cinerea, Cercospora beticola, Rhizoctonia
solani, and Cladosporium cucumerinum. As reported in the Table, compound 1
exhibited a broad spectrum of activity and led to a good reduction of mycelial growth
ꢀ
1
on P. ultimum, R. solani, B. cinerea (ca. 70% at 62.5 mg ml ). It was less efficient on C.
ꢀ
1
ꢀ1
beticola (49% at 62.5 mg ml ) and C. cucumerinum (30% at 62.5 mg ml ).
To define the most relevant structural features affecting in vitro antifungal activity,
we first examined the role of the free phenol group. The methoxy derivative 10
presented values of mycelial growth inhibition comparable to those of kakuol (1) with
ꢀ
1
the exception for P. ultimum (29% at 62.5 mg ml ; Table 1). This result suggests that
the presence of the OH group is not crucial for the activity.
Regarding the modification of the methylenedioxy moiety, we prepared the
ethylenedioxy derivative 8 and the 4,5-dimethoxy derivative 4. Both of them were less
active than kakuol on all the tested pathogens at the same concentration range.
Transforming the CO group of kakuol (1) into an oxime, 11, led to a decrease of
antifungal activity against B. cinerea, C. cucumerinum, and R. solani; on the contrary, in
the case of P. infestans, P. ultimum, and C. beticola, the inhibition of mycelial growth
was quite similar to that of 1. Moreover, the substitution of the CO with the CS group
(cf. 12) led to a decrease of activity against the tested organisms.
More useful information was obtained from compounds with a modified chain
(
compounds 5a–5i). In particular, the introduction of a C¼C bond in the a-position to
the CO gave compound 5a with increased activity. In fact, it almost completely
inhibited the mycelial growth of the tested pathogens at 62.5 mg ml . Only B. cinerea
ꢀ
1
ꢀ
1
was less sensitive to this compound (64% growth inhibition at 125 mg ml ).
The results listed in the Table showed that the introduction of a Me group at the a-
position of the CO group (i.e., 5b) generally did not affect the activity compared to
compound 5a; on the other hand, the introduction of two Me groups (i.e., 5c) led to a
substantial decrease of activity. As regards the relevance of the position of the C¼C

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
891
Table. Growth Inhibition [%] Induced by Compounds 1–12
Conc. [mg mlꢀ1] P. infestans P. ultimum B. cinerea C. beticola C. cucumerinum R. solani
1
0
8
4
5
5
5
5
5
5
5
5
5
1
2
125
41
31
54
25
27
16
30
24
100
92
100
100
19
14
0
82
70
57
29
44
45
26
20
100
100
100
100
19
15
0
83
70
80
51
22
15
18
13
64
32
64
5
49
49
59
33
–
40
49
44
97
91
79
66
32
30
0
73
28
–
–
–
–
–
–
100
100
100
100
8
5
0
0
16
8
48
42
35
33
10
0
0
0
42
29
47
–
81
71
90
75
23
11
44
30
100
100
100
93
29
22
0
62.5
1
125
62.5
125
62.5
125
62.5
a
b
c
d
e
f
125
62.5
125
62.5
125
20
14
0
62.5
62.5
31.25
0
0
0
0
0
125
31
31
65
45
47
40
15
9
48
27
85
60
80
77
37
17
24
14
87
79
56
100
–
18
26
62
50
25
24
0
41
41
58
51
37
33
33
13
20
20
51
41
33
–
66
60
79
72
38
32
14
3
28
22
69
63
68
–
62.5
125
62.5
g
h
i
125
62.5
125
62.5
0
125
4
3
16
15
53
42
35
–
62.5
1
1
125
59
41
42
100
–
62.5
100
100
25
a
Euparen )
Sideral )
Tetraconazole ) 10
a
100
–
–
100
–
100
–
100
a
–
–
a) Euparen (toluilfluanide; 50%), sideral (procimidone; 50%), and tetraconazole; the reported standard
concentrations referred to the pure compounds.
bond in the side chain, compound 5e was much less active than 5a, and more active than
5
c. Thus, we can hypothesize that the presence of a C¼C bond conjugated to the CO
group; i.e., 5a vs. 1 and 5e, plays an important role, most likely due to the increased
reactivity of the molecule to nucleophilic addition.
Moreover, the drop of antifungal activity in compound 5c, which bears two Me
groups at the C¼C bond in b-position, could be due either to an unfavorable steric
hindrance or to an increased lipophilicity.
To have a further insight into the relevance of lipophilicity, we synthesized
compounds 5d, 5g, 5f, and 5i. The lipophilicity values of the compounds 1 and 5a–5i,
expressed as logarithm of partition coefficient in octan-1-ol/H O (log P) were
2
calculated according to Ghose and Crippenꢂs fragmentation method [20] using

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
ChemDraw Ultra Vers. 5.0 [21]. As previously reported (Table), compounds 1 (log P
.39), 5a (log P 1.42), and 5b (log P 1.77) showed higher antifungal activities than the
more liphophilic 5c (log P 1.96) and 5e (log P 1.95). The cyclopropyl derivative 5f
log P 1.46) exhibited activity quite similar to kakuol (1) on a wide range of organisms.
1
(
The introduction of a lipophilic Ph residue (i.e., 5i; log P 2.63) led to a decrease of
antifungal activity. Conversely, the introduction of a MeO in the chain (i.e., 5h; log P
0.40), led to a much less active compound. This substituent lowered the lipophilicity of
the compound causing a detrimental effect on antifungal activity.
Due to its very poor solubility, the lauroyl derivative 5d (log P 4.73) was tested at
ꢀ
1
lower concentrations (62.5 and 31.25 mg ml ) than the other compounds, and was
found completely inactive against all the tested pathogens.
The plots of percent growth inhibition induced by compounds 1, 5a–5c, and 5e–5i
ꢀ
1
(
at 125 mg ml ) vs. log P for C. cucumerinum and C. beticola (Fig. 2) showed that the
most active compounds have a log P value in the range of 1.4–1.8 (compounds 5a, 5b, 1,
and 5f), the only exception being compound 5g that maintained a moderate antifungal
activity, despite its quite high log P value (2.66).
Fig. 2. Percent growth inhibition of a) C. cucumerinum and b) C. beticola induced by compounds 1, 5a–
ꢀ
1
5
c, and 5e–5i (at 125 mg ml ) vs. log P

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
893
Among the tested compounds, 5a appeared to be the most promising one. For this
reason, it was tested at lower concentrations in order to evaluate the minimum
inhibitory concentration (MIC) for the most sensitive organisms (P. infestans, P.
ultimum, C. beticola, C. cucumerinum, and R. solani). The lowest MIC value for
ꢀ
1
compound 5a (10 mg ml ) was observed against C. cucumerinum. A slightly higher
ꢀ
1
MIC value (ca. 25 mg ml ) was found against the oomycetes P. infestans and P.
ultimum, whereas R. solani and C. beticola were found less sensitive (MIC values of
ꢀ
1
ꢀ1
5
0 mg ml and >100 mg ml , resp.).
Observations at the optical microscope of C. cucumerinum conidia, inoculated on
ꢀ
1
PDA (potato dextrose agar) amended with 5a at 25 and 12.5 mg ml , showed absence
of germination. Moreover, when the same conidia were transferred on new, not
amended PDA, they were again unable to germinate. This means that 5a is featured
with a high and persistent fungicidal activity interfering with spore germination,
differently from the standard compound tetraconazole. This latter does not interfere
with the spore germination but stops the mycelial growth, as expected for a compound
belonging to the sterol biosynthesis inhibitors (SBI).
A long-lasting effect of 5a was also observed in P. infestans and P. ultimum mycelial
growth, while, in cultures treated with the standard Euparen (tolylfluanide) at the same
ꢀ
1
concentration (100 mg ml ), mycelial growth was visible after 9-d treatment.
ꢀ
1
Preliminary in vivo studies showed that 5a inhibited, at 1000 mg ml , 60% of wheat
rust and 80% of tomato downy mildew.
Conclusions. – The results obtained suggest that 2-hydroxy-4,5-methylenedioxyaryl
ketones can be considered promising candidates in the development of new antifungal
compounds. In particular, compound 5a, bearing a C¼C bond conjugated to the CO
group, showed a remarkable in vitro antifungal activity. Further biological tests and
structure–activity relationship studies on this new series of compounds are currently
underway.
The authors gratefully acknowledge Dr. Luigi Mirenna, Department of Biology and Agronomy of
ISAGRO Research, for the in vivo tests, Mr. Elio Burrone for the in vitro test, and the University of
Milano (FIRST 2004-5 funds) for financial support.
Experimental Part
General. All reagents and solvents were reagent-grade or were purified by standard methods before
use. Solvents were routinely distilled prior to use; anh. THF and Et O were obtained by distillation from
2
Na-benzophenone ketyl; dry CH Cl and toluene were obtained by distillation from CaCl . All reactions
2
2
2
requiring anh. conditions were performed under a positive N2 flow, and glassware was oven-dried.
Isolation and purification of the compounds were performed by flash column chromatography (FCC) on
silica gel 60 (SiO ; 230–400 mesh). Anal. TLC: Fluka TLC plates (silica gel 60 F254, aluminium foil).
2
1
M.p.: Stuart Scientific SMP3 instrument; uncorrected. H-NMR Spectra: in CDCl (if not otherwise
3
stated) at r.t. on a Bruker AMX-300 spectrometer operating at 300 MHz; chemical shifts (d) and coupling
constants (J) were reported in ppm and in Hz, resp. HR-MS: Finnigan MAT-TSQ70 spectrometer; in
m/z (rel. %).
General Synthetic Procedures. Method A. Propanoic anhydride (10.1 mmol) and BF
3 2
·Et O
(
7.1 mmol) were added to a soln. of the appropriate phenol (3.6 mmol), and the mixture was heated
at 808. The mixture was then cooled to r.t., and H O (3 ml) was added. After extraction with CH Cl and
2
2
2
evaporation of the org. solvent, the crude material was purified by FCC.

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Method B. To a suspension of Mg beads (5.5 mmol) in dry Et O (5 ml), MeI (5.5 mmol) was added
2
under stirring at 30–358. When the Mg was completely dissolved, a soln. of phenol (3.62 mmol) in dry
Et O (5 ml) was added, and the resulting mixture was stirred for 1 h. The solvent was removed in vacuo,
2
and dry toluene (10 ml) was added. The appropriate acyl chloride (3.62 mmol) was added dropwise to the
resulting slurry at 08, and the yellow suspension was stirred at r.t. The mixture was poured into sat. aq.
NH Cl and then extracted three times with AcOEt. The soln. was dried (Na SO ), the solvent was
4
2
4
evaporated under reduced pressure, and the crude material was purified by FCC on SiO2.
-(6-Hydroxy-1,3-benzodioxol-5-yl)propan-1-one (1). Compound 1 was obtained from 3,4-(meth-
1
ylenedioxy)phenol according to Method A. FCC (hexane/AcOEt 95 :5): 516 mg (73%) of 1. M.p. 109–
1
1
2
108 (yellow crystals). H-NMR: 13.09 (s, OH); 7.08 (s, 1 arom. H); 6.44 (s, 1 arom. H); 5.97 (s, OCH O);
2
þ
þ
.88 (q, J¼7.4, MeCH ); 1.21 (t, J¼7.4, MeCH ). HR-EI-MS: 194.0581 (M , C H O ; calc. 194.0584).
2
2
10 10
4
1
-(2-Hydroxy-4,5-dimethoxyphenyl)propan-1-one (4). Compound 4 was obtained by Method A. The
1
crude material was purified by FCC (hexane/Et O 1:1): 4 (245 mg, 42%). M.p. 118–1198. H-NMR
2
(
(
(D )DMSO): 12.53 (s, OH); 7.29 (s, 1 arom. H); 6.54 (s, 1 arom. H); 3.82 (s, MeO); 3.76 (s, MeO); 3.05
6
þ
þ
q, J¼7.1, MeCH ); 1.09 (t, J¼7.1, MeCH ). HR-EI-MS: 210.0893 (M , C H O
4
; calc. 210.0897).
2
2
11 14
1-(6-Hydroxy-1,3-benzodioxol-5-yl)prop-2-en-1-one (5a). Compound 5a was obtained by Method B,
starting from sesamol (2; 1.50 g, 10.86 mmol) and acryloyl chloride. The crude material was purified by
1
FCC (hexane/AcOEt 95 :5): 5a (1.37 g, 66%). M.p. 117–1188. H-NMR: 13.49 (s, OH); 7.13 (s, 1 arom.
H); 7.13 (dd, J¼10.42, 16.7, 1 H of CH¼CH ); 6.52 (dd, J¼1.49, 16.7, 1 H of CH¼CH ); 6.48 (s, 1 arom.
2
2
þ
þ
4
H); 5.99 (s, OCH O); 5.90 (dd, J¼1.5, 10.4, 1 H of CH¼CH ). HR-EI-MS: 192.0424 (M , C H O ;
2
2
10
8
calc. 192.0428).
-(6-Hydroxy-1,3-benzodioxol-5-yl)-2-methylprop-2-en-1-one (5b). Compound 5b was obtained by
1
Method B starting from 2 (500 mg, 3.62 mmol) and 2-methylacryloyl chloride. The crude material was
1
purified by FCC (hexane/AcOEt 95 :5): 415 mg of a mixture of 5b (23% yield determined by H-NMR)
1
and 6b (1,3-benzodioxol-5-yl 2-methylprop-2-enoate; 32% yield determined by H-NMR of the mixture);
crystallization of this mixture with Et O/hexane gave 5b (100 mg, 13%).
2
1
Data of 5b. M.p. 78–798. H-NMR: 12.87 (s, OH); 7.12 (s, 1 arom. H); 6.97 (s, 1 arom. H); 6.30 (s, 1 H
þ
of C¼CH ); 5.99 (s, OCH O); 5.75 (s, 1 H of C¼CH ); 2.02 (s, Me). HR-EI-MS: 206.0580 (M ,
2
2
2
þ
C H O
4
; calc. 206.0584).
1
1
10
1
Data of 6b. H-NMR: 6.77 (d, J¼8.6, 1 arom. H); 6.10 (d, J¼2.6, 1 arom. H); 6.05 (dd, J¼8.6, 2.6, 1
arom. H); 5.99 (s, OCH O); 5.60 (s, 1 H of C¼CH ); 5.30 (s, 1 H of C¼CH ); 2.02 (s, Me).
2
2
2
1
-(6-Hydroxy-1,3-benzodioxol-5-yl)-3-methylbut-2-en-1-one (5c). Compound 5c was obtained by
Method B starting from 2 (500 mg, 3.62 mmol) and 3,3-dimethylacryloyl chloride. The crude material was
purified by FCC (hexane/AcOEt 95 :5) to afford 386 mg of a mixture of 5c (29% yield determined by
1
H-NMR of the mixture) and 6c (1,3-benzodioxol-5-yl 3-methylbut-2-enoate; 19% yield determined by
1
H-NMR); crystallization of this mixture with Et O/hexane gave 5c (192 mg, 24%). M.p. 104–1068.
2
1
H-NMR: 13.64 (s, OH); 7.11 (s, 1 arom. H); 6.51–6.53 (m, CH¼CMe ); 6.44 (s, 1 arom. H); 5.96 (s,
2
þ
þ
4
OCH O); 2.16 (d, J¼1.1, Me); 2.00 (d, J¼1.1, Me). HR-EI-MS: 220.0738 (M , C H O ; calc.
2
12 12
2
20.0741).
1
-(6-Hydroxy-1,3-benzodioxol-5-yl)dodecan-1-one (5d). Compound 5d was obtained by Method B
starting from 2 (400 mg, 2.90 mmol) and dodecanoyl chloride. The crude material was purified by FCC
hexane/AcOEt 95 :5) to give 550 mg of a mixture of 5d and 6d (1,3-benzodioxol-5-yl dodecanoate);
crystallization of this mixture with Et O/hexane gave of 5d (136 mg, 15%), together with 6d (393 mg,
(
2
3
6%).
Data of 5d. M.p. 83–84.08. H-NMR: 13.18 (s, OH); 7.08 (s, 1 arom. H); 6.44 (s, 1 arom. H); 5.97 (s,
1
OCH O); 2.82 (t, J¼7.4, COCH ); 1.79–1.60 (m, CH ); 1.45–1.15 (m, 8 CH ); 0.87 (t, J¼6.7, Me).
2
2
2
2
1
Data of 6d. H-NMR: 6.75 (d, J¼7.8, 1 arom. H); 6.57 (d, J¼1.8, 1 arom. H); 6.50 (dd, J¼7.8, 1.8, 1
arom. H); 5.97 (s, OCH O); 2.50 (t, J¼7.4, COCH ); 1.77–1.55 (m, CH ); 1.45–1.15 (m, 8 CH ); 0.75–
2
2
2
2
0
.95 (m, Me).
1-(6-Hydroxy-1,3-benzodioxol-5-yl)pent-4-en-1-one (5e). Compound 5e was obtained by Method B
starting from 2 (500 mg, 3.62 mmol) and pent-4-enoyl chloride. The crude material was purified by FCC
1
(
hexane/AcOEt 95 :5) to provide 287 mg of a mixture of 5e (26% yield determined by H-NMR of the

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
895
1
mixture) and 6e (1,3-benzodioxol-5-yl pent-4-enoate, 16% yield determined by H-NMR); crystallization
of this mixture with Et O/hexane gave 5e (150 mg, 19%).
2
1
Data of 5e. M.p. 74.6–76.78. H-NMR: 13.09 (s, OH); 7.07 (s, 1 arom. H); 6.44 (s, 1 arom. H); 5.97 (s,
OCH O); 5.95–5.77 (m, CH¼CH ); 5.09 (ddd, J¼1.5, 3.3, 17.1, 1 H, CH¼CH ); 5.02 (ddd, J¼1.5, 3.3,
2
2
2
1
2
1.9, 1 H, CH¼CH ); 2.95 (t, J¼7.1, CH CH CH¼CH ); 2.57–2.42 (m, CH CH CH¼CH ). HR-EI-MS:
2
2
2
2
2
2
2
þ þ
20.0738 (M , C H O ; calc. 220.0740).
12
12
4
1
Data of 6e. H-NMR: 6.74 (d, J¼8.5, 1 arom. H); 6.53 (d, J¼2.2, 1 arom. H); 6.50 (dd, J¼8.5, 2.2, 1
arom. H); 5.97 (s, OCH O); 5.87–5.72 (m, CH¼CH ); 5.17–4.92 (m, CH¼CH ); 2.70–2.55 (m,
2
2
2
CH
2
CH
2
CH¼CH
2
); 2.52–2.35 (m, CH
2
CH
2
CH¼CH
2
).
Cyclopropyl(6-hydroxy-1,3-benzodioxol-5-yl)methanone (5f). Compound 5f was obtained by
Method B starting from 2 (500 mg, 3.62 mmol) and cyclopropylcarbonyl chloride. The crude material
was purified by FCC (hexane/AcOEt 95 :5) to obtain 5f (111 mg, 15%). In this case, the crude material
1
was a very complex mixture, and it was not possible to verify, by H-NMR spectra, the presence of ester
1
derived from O-acylation reaction. M.p. 838 (dec.). H-NMR: 13.24 (s, OH); 7.25 (s, 1 arom. H); 6.45 (s, 1
arom. H); 5.99 (s, OCH O); 2.55–2.40 (m, CH); 1.28–1.40 (m, CH ); 0.95–1.25 (m, CH ). HR-EI-MS
2
2
2
þ
þ
(
pos.): 206.0581 (M , C H O ; calc. 206.0584).
11 10
4
1-(6-Hydroxy-1,3-benzodioxol-5-yl)-2,2-dimethylpropan-1-one (5g). Compound 5g was obtained by
Method B starting from 2 (500 mg, 3.62 mmol) and pivaloyl chloride. The crude material was purified by
1
FCC (hexane/AcOEt 95 :5) to yield 5g (74 mg, 9%). M.p. 938. H-NMR: 13.06 (s, OH); 7.37 (s, 1 arom.
þ
þ
H); 6.47 (s, 1 arom. H); 5.96 (s, OCH O); 1.41 (s, Me C). HR-EI-MS: 222.0894 (M , C H O ; calc.
2
3
12 14
4
2
22.0897).
-(6-Hydroxy-1,3-benzodioxol-5-yl)-2-methoxyethanone (5h). Compound 5h was obtained by
1
Method B starting from 2 (250 mg, 1.81 mmol) and 2-methoxyacetyl chloride. The crude material was
purified by FCC (hexane/Et O 3 :1) to afford 116 mg of a mixture of 5h (22% yield determined by
2
1
H-NMR of the mixture) and 6h (1,3-benzodioxol-5-yl methoxyacetate, 9% yield determined by
1
H-NMR); crystallization of this mixture from Et
2
O/hexane gave 5h (69 mg,18%).
1
Data of 5h. M.p. 1258. H-NMR: 12.69 (s, OH); 7.25 (s, 1 arom. H); 7.04 (s, 1 arom. H); 5.98 (s,
þ
þ
OCH O); 4.54 (s, MeOCH ); 3.49 (s, MeOCH ). HR-EI-MS: 210.0530 (M , C H O
5
; calc. 210.0533).
2
2
2
10 10
1
Data of 6h. H-NMR: 6.67 (d, J¼8.5, 1 arom. H); 6.63 (d, J¼2.2, 1 arom. H); 6.63 (dd, J¼8.5, 2.2, 1
arom. H); 5.98 (s, OCH O); 4.24 (s, MeOCH ); 3.51 (s, MeOCH ).
2
2
2
(
6-Hydroxy-1,3-benzodioxol-5-yl)(phenyl)methanone (5i). Compound 5i was obtained by Method B
starting from 2 (500 mg, 3.62 mmol) and benzoyl chloride. The crude material was purified by FCC
(
hexane/Et O 3 :1) to give 5i (438 mg, 45%), and 200 mg of a mixture of phenol and ester 6i (1,3-
2
1
benzodioxol-5-yl benzoate; 72 :28 determined by H-NMR).
1
Data of 5i. M.p. 105–1068. H-NMR: 13.00 (s, OH); 7.70–7.61 (m, 2 arom. H); 7.60–7.42 (m, 3 arom.
þ
þ
H); 6.92 (s, 1 arom. H); 6.05 (s, 1 arom. H); 6.00 (s, OCH O). HR-EI-MS: 242.0581 (M , C H O ; calc.
2
14 10
4
2
42.0584).
1
Data of 6i. H-NMR: 8.45–8.05 (m, 2 arom. H (Ph)); 7.67–7.35 (m, 3 arom. H (Ph)); 6.80 (d, J¼7.8, 1
arom. H); 6.72 (d, J¼2.6, 1 arom. H); 6.22 (dd, J¼7.8, 2.6, 1 arom. H); 6.00 (s, OCH O).
2
1-(2,3-Dihydro-7-hydroxy-1,4-benzodioxin-6-yl)propan-1-one (8). Compound 8 was obtained by
Method B starting from 3,4-(ethylenedioxy)phenol (196 mg, 1.29 mmol) and propanoyl chloride. The
crude material was purified by FCC (hexane/Et O 8 :2) to give 8 (59 mg, 22% yield) together with
2
compound 9 (2,3-dihydro-1,4-benzodioxin-6-yl propanoate; 46%).
1
Data of 8. M.p. 111–1128. H-NMR ((D )DMSO): 12.02 (s, OH); 7.40 (s, 1 arom. H); 6.40 (s, 1 arom.
6
H); 4.35–4.28 (m, 2 H of OCH CH O); 4.25–4.07 (m, 2 H of OCH CH O); 3.00 (q, J¼7.1, MeCH );
2
2
2
2
2
þ
þ
4
1
.06 (t, J¼7.1, MeCH ). HR-EI-MS: 208.0738 (M , C H O ; calc. 208.0741).
2
11 12
1
Data of 9. H-NMR: 6.80 (d, J¼8.9, 1 arom. H); 6.61 (d, J¼2.6, 1 arom. H); 6.54 (dd, J¼8.9, 2.6, 1
arom. H); 4.37–4.17 (m, OCH CH O); 2.53 (q, J¼7.4, MeCH ); 1.23 (t, J¼7.4, MeCH ).
2
2
2
2
1
-(6-Methoxy-1,3-benzodioxol-5-yl)propan-1-one (10). To a soln. of 1 (90 mg, 0.46 mmol) in acetone
(
2 ml), K CO (65 mg, 0.47 mmol) and MeI (162 ml, 2.6 mmol) were added, and the mixture was refluxed
2
3
for 16 h. The mixture was washed with H O and brine, the soln. was dried (Na SO ), and solvent was
2
2
4
removed in vacuo. The crude material was purified by FCC (hexane/Et O 95 :5) to give 10 (46 mg, 48%).
2

8
96
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
1
M.p. 88–898. H-NMR: 7.31 (s, 1 arom. H); 6.52 (s, 1 arom. H); 5.97 (s, OCH O); 3.85 (s, MeO); 2.94 (q,
2
þ
þ
4
J¼7.4, MeCH ); 1.13 (t, J¼7.4, MeCH ). HR-EI-MS: 208.0738 (M , C H O ; calc. 208.0741).
2
2
11 12
6
-[(1E)-N-Hydroxypropanimidoyl]-1,3-benzodioxol-5-ol (11). To a suspension of NH OH·HCl
2
(
31 mg, 0.45 mmol) and AcONa (41 mg, 0.49 mmol) in MeOH (3 ml), 1 (80 mg, 0.41 mmol) was added,
and the mixture was refluxed for 6 h. After the addition of a second portion of NH OH·HCl (31 mg,
2
0
.45 mmol) and AcONa (41 mg, 0.49 mmol), the mixture was refluxed for 8 h. The solvent was
evaporated, and the crude material was dissolved in AcOEt. The org. phase was washed with H O, dried
2
(
Na SO ), and the solvent was removed in vacuo. The crude material was purified by FCC (hexane/
2 4
1
AcOEt 9 :1) to give 11 (69 mg, 80%). M.p. 174–1768. H-NMR ((D )DMSO): 11.87 (s, OH); 11.25 (s,
6
C¼NOH); 7.01 (s, 1 arom. H); 6.48 (s, 1 arom. H); 5.95 (s, OCH O); 2.71 (q, J¼7.4, MeCH ); 1.02 (t, J¼
2
2
þ
þ
4
7
.4, MeCH ). HR-EI-MS: 209.0690 (M , C H NO ; calc. 209.0693).
2
1
0
1
1
1-(6-Hydroxy-1,3-benzodioxol-5-yl)propane-1-thione (12). To a soln. of 1 (100 mg, 0.5 mmol) in dry
toluene (3 ml) Lawessonꢂs reagent (121 mg, 0.3 mmol) was added. The mixture was refluxed for 7 h; in
spite of further addition of Lawessonꢂs reagent, the reaction was not complete. The solvent was
evaporated and the crude material was purified by FCC (hexane/AcOEt 9 :1) to give 12 (13 mg, 13%).
1
H-NMR: 14.41 (s, OH); 7.24 (s, 1 arom. H); 6.53 (s, 1 arom. H); 5.99 (s, OCH O); 3.25 (q, J¼7.4,
2
þ
MeCH ); 1.36 (t, J¼7.4, MeCH ). GC/MS: 210 (100, M ), 181 (56), 147 (89), 119 (22), 91 (33), 69 (89),
2
2
þ
þ
5
5 (22). HR-EI-MS: 210.0353 (M , C H O S ; calc. 210.0356).
10 10 3
Biological Assays. The antifungal activity was tested in vitro on Phytophthora infestans (Mont.) de
Bary, Pythium ultimum Trow, Botrytis cinerea Pers., Cercospora beticola Sacc., Cladosporium
cucumerinum Ellis & Arthur, Rhizoctonia solani J. G. Kˇhn.
Radial Fungal-Growth Assay. The activity of the compounds was assayed as growth inhibition by the
commonly used ꢃpoisoned foodꢂ technique. Different solns. of each compound were prepared by
dissolving the appropriate amount of compound in DMSO plus Tween 20 (2%). Equal volumes of
DMSO containing diluted compounds were added to sterile cool agar media (Potato Dextrose Agar
Difco) to give suitable concentrations for each substance.
A zero-concentration treatment, containing the same percentage of DMSO and Tween 20 to ensure
equivalent concentrations of these components in all the treatments, was prepared for each strain. The
final DMSO concentration did not exceed 0.3% of the final volume in both control and treated cultures.
Compound-amended agar medium was dispersed aseptically onto 9-cm-diameter plastic Petri dishes
(
10 ml/dish). Each dish was inoculated with two mycelial discs cut from the periphery of actively growing
colonies. Two replicates were used for each concentration, together with controls containing toxicant-free
medium. The growth inhibition was calculated from mean differences between treated and control
cultures as a percentage of the latter. The results were compared with those obtained with three standard
fungicides, tolylfluanide, procymidon, and tetraconazole. The growth was determined after 24 h of
incubation at 248 for P. infestans and P. ultimum, 3 d for B. cinerea, 6 d for R. solani, and 9 d for C. beticola
and C. cucumerinum.
In vivo Assays. The tests of direct protecting activity of the compounds were performed on the
following pathogen-host combinations: Puccinia graminis/wheat cv Irnerio (wheat rust) and Phytoph-
thora infestans/tomato cv Marmande (downy mildew). Both surfaces of plant leaves were treated with a
ꢀ
1
2
0% (v/v) aq. soln. of acetone of the selected compounds (1000 mg ml ). Inoculation with a suitable
conidia suspension was performed 7 d after the treatment on tomato and 24 h after the treatment on
wheat. The area of inoculated leaves covered by disease symptoms was assessed on a 100-point scale from
0
to 100, in which 100 corresponded to completely infected leaves and 0 to not visible symptoms. The
results were compared with those obtained with two standard fungicides in commercially available
formulations, benalaxil and tetraconazole, and the activity was expressed as percent inhibition of
infection in comparison with untreated control.
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Received August 27, 2009