Synthetic pyrazole derivatives as growth inhibitors of some phytopathogenic fungi

Article abstract of DOI:10.1021/jf072077d

The present study was carried out to investigate the antifungal activity of pyrazole/isoxazole-3-carboxamido-4-carboxylic acids, 4-oxo-5-substituted pyrazolo[3,4-d]pyrimidine-6-thiones, and N-alkyl/aryl-N′-(4-carbethoxy-3- pyrazolyl)thioureas against Pyth

Full text of DOI:10.1021/jf072077d

J. Agric. Food Chem. 2007, 55, 10331–10338 10331
Synthetic Pyrazole Derivatives as Growth Inhibitors
of Some Phytopathogenic Fungi
CHIARA B. VICENTINI,*,† CARLO ROMAGNOLI,‡ ELISA ANDREOTTI,‡ AND
§
DONATELLA MARES
Dipartimento di Scienze Farmaceutiche, Università di Ferrara, via Fossato di Mortara 17, Ferrara,
Italy, Dipartimento di Biologia ed Evoluzione, Università di Ferrara, via Ercole d’Este 32, Ferrara,
Italy, and Dipartimento del Museo di Paleobiologia e dell’Orto Botanico, Università di Modena e
Reggio Emilia, via le Caduti in Guerra 127, Reggio Emilia, Italy
The present study was carried out to investigate the antifungal activity of pyrazole/isoxazole-3-
carboxamido-4-carboxylic acids, 4-oxo-5-substituted pyrazolo[3,4-d]pyrimidine-6-thiones, and N-alkyl/
aryl-N′-(4-carbethoxy-3-pyrazolyl)thioureas against Pythium ultimum, Botrytis cinerea, and Mag-
naporthe grisea. The results on growth inhibition showed differences in the sensitivity of the three
fungi to the tested substances, and in general P. ultimum was shown to be the most sensitive. On all
phytopathogens the best results within the pyrazole/isoxazolecarboxamide series are given by
the compounds with the carboxamide and carboxylic groups in positions 3 and 4; the presence of
these groups seems to be critical for biological activity in this series of compounds. Among the
pyrazolopyrimidines the derivative supplied with the benzylic group was the most active on the three
fungi and in particular against P. ultimum. Several compounds belonging to the thiourea series are
able to inhibit selectively M. grisea at 50 and 10 µg mL-1, doses at which the reference commercial
compound tricyclazole had low or no effect.
KEYWORDS: Antifungals; pyrazole-3-carboxamido-4-carboxylic acids; isoxazole-3-carboxamido-4-car-
boxylic acids; 4-oxo-5-substituted pyrazolo[3,4-d]pyrimidine-6-thiones; N-alkyl/aryl-N′-(4-carbethoxy-3-
pyrazolyl)thioureas; Pythium ultimum; Botrytis cinerea; Magnaporthe grisea
INTRODUCTION
Pyrazole and isoxazole fungicides have attracted attention
from many industrial companies. This led to the commercial
introduction of hymexazole, rabenzazole, and pyraclostrobin.
Hymexazole is used to control soil-borne diseases caused by
Pythium, Fusarium, Aphanomyces, Corticium, and Typhula
spp. (2, 3). Rabenzazole inhibits the synthesis of ꢀ-tubulin during
mitosis of fungi (2, 6). Pyraclostrobin, a strobilurin-mitochon-
drial electron transport inhibitor, shows broad spectrum activity
on anthracose-inducing fungi and on Alternaria, downyn
mildew, Cercospora leaf spot, rust, powdery mildew, Septoria,
Phytophthora, Rhizoctonia, and Pythium (3–5) (Figure 1).
Molecules with both pyrazole and carboxamide moieties led
to the synthesis of several antifungals (7–10). Furametpyr (3, 4),
5-chloro-N-(1,3-dihydro-1,1,3-trimethylisobenzofuran-4-yl)-1,3-
dimethylpyrazole-4-carboxamide, is a fungicide used to control
rice sheat blight (11–13). Penthiopyrad, N-[2-(1,3-dimethylbu-
tyl)-3-thienyl]-1-methyl-3-(trifluoromethyl)-1H-pyrazole-4-car-
boxamide, shows activity on strobilurin and DMI resistant
diseases (gray mold, powdery mildew, apple scab, rusts,
Rhizoctonia, Botrytis) (3–5) (Figure 1).
With the successful introduction of carboxamides as fungi-
cides with systemic activity by the U.S. Rubber Co. in 1966, a
new chapter in the history of plant protection began. For the
first time it was possible to control important crop diseases in
barley and wheat (1).
Within this chemical class of fungicides, variation of mech-
anism of action can be observed. Carboxamides benodanil,
boscalid, carboxin, fenfuram, flutolanil, mepronil, oxycarboxin,
thifluzamide, furametpyr, and penthiopyrad are fungitoxic
because they inhibit the succinate dehydrogenase complex, in
the respiratory electron chain, leading to inhibition of aspartate
and glutamate synthesis (2–5). The biochemical mode of action
of diclocymet and carpropamid appears to be based on inhibition
of dehydratase in melanin biosynthesis (3, 4). The inhibition of
ATP production is proposed as the mode of action of the
thiophene carboxamide silthiofam; the target of the thiazole
derivative ethaboxam is unknown (3, 4).
* Author to whom correspondence should be addressed: fax, (39)
0532 455953; e-mail, vcc@unife.it.
Within the framework of investigation on biologically active
heterocycles, we found that pyrazole and isoxazole derivatives
are interesting for their biological properties as antifungal
agents (14–22).
† Dipartimento di Scienze Farmaceutiche, Università di Ferrara.
‡ Dipartimento del Museo di Paleobiologia e dell’Orto Botanico,
Università di Modena e Reggio Emilia.
§ Dipartimento di Biologia ed Evoluzione, Università di Ferrara.
10.1021/jf072077d CCC: $37.00
2007 American Chemical Society
Published on Web 11/15/2007

10332 J. Agric. Food Chem., Vol. 55, No. 25, 2007
Vicentini et al.
Figure 1
Figure 4
Figure 5
Figure 2
Figure 3
Among pyrazoles, pyrazolo[3,4-d]pyrimidine-4(5H)-thiones
were effective in controlling Pythium ultimum and Rhizoc-
tonia solani (23). In particular, the activity of trifluoromethyl
derivatives was comparable to that of the reference com-
mercial fungicides captafol and mancozeb (24, 25). Pyra-
zolo[3,4-d]pyrimidine-4(5H)-thione, pyrazolo[3,4-d][1,3]thiazin-
4-one/thione, and pyrazolo[1,5-c][1,3,5]thiadiazin-4-one/
thione derivatives were screened for antifungal activity
against the causal agent of rice blast disease, Magnaporthe
grisea; some of these compounds caused a remarkable
inhibition of fungal growth (17). Among these derivatives
7-methyl-2-phenylpyrazolo[1,5-c][1,3,5]thiadiazin-4-one in-
hibited the fungus even at the lowest dose tested (10 µg
mL-1), at which the reference commercial fungicide tricy-
clazole was completely ineffective.
Against the same fungus, more recently even pyrazolo[1,5-
a][1,3,5]triazine-2,4-dione, pyrazolo[1,5-c][1,3,5]thiadiazin-2-
one, and pyrazolo[3,4-d][1,3]thiazin-4-one/thione derivatives
were tested; several compounds showed greater inhibition than
tricyclazole, providing potential new chemicals for control of
M. grisea infections (20) (Figure 2).
Figure 6
accomplished by us (26); the compounds are referred to here
as in a previous paper (Figure 4) where they showed a low
herbicidal activity, being efficient only at high doses, in the
millimolar range (28).
Since pyrazole-4-carboxylic acid esters were already reported
to be fungicides (27), the corresponding 4-ester of the pyrazole-
3-carboxamide (3n) and of pyrazole-3,4-dicarboxylic acid (4n)
here are tested because it is expected that they also exhibit
similar activity (Figure 5).
Moreover, a series of 4-oxo-5-substituted pyrazolo[3,4-
d]pyrimidine-6-thiones (5a–g,i) was synthesized and tested as
potential antifungal agents (Figure 6).
An additional purpose was to extend the screening program
to N-alkyl/aryl-N′-(4-carbethoxy-3-pyrazolyl)thioureas (6a–j),
prepared according to the previously reported procedures (28),
to explore the potential of this further class of compounds as
growth fungal inhibitors. In fact, as results from literature, the
dCdS moiety is as pharmacophore provided with antimycotic
activity (29) (Figure 6).
As tests P. ultimum, B. cinerea, and M. grisea were selected
because they are important phytopathogenic fungi.
Among isoxazoles, 5-aminoisoxazole-4-thiocyanates were
effective in controlling a large number of phytopathogens
including Botrytis cinerea (14) and dermatophytic fungi (15, 16)
(Figure 3).
MATERIALS AND METHODS
The present study was carried out to investigate the antifungal
activity of pyrazole-3-carboxamido-4-carboxylic acids 1n, 1p,
1q, 1r, 2n, and 2s in comparison with isoxazole-3-carboxamido-
4-carboxylic acids 1m and 2m whose synthesis has already been
Chemicals. Melting points were determined with a Buchi capillary
apparatus and are uncorrected. IR spectra were recorded with a Perkin-
Elmer Paragon 500 FT-IR spectrometer using potassium bromide
pellets. 1H NMR spectra were recorded on a Bruker AC200 spectrom-

Pyrazole Derivatives as Inhibitors of Phytopathogenic Fungi
J. Agric. Food Chem., Vol. 55, No. 25, 2007 10333
eter; chemical shifts (δ) are given in parts per million relative to
tetramethylsilane as internal standard. Yields were based on the weight
of the products dried in Vacuo over phosphorus pentoxide. Elemental
analyses (C, H, N, S) were within (0.4 of theoretical values. For the
flash chromatography technique, silica gel (230–400 mesh) was
employed.
Scheme 1
Synthesis. Pyrazole/isoxazole-3-carboxamido-4-carboxylic acids
(1–4) and N-alkyl/aryl-N′-(4-carbethoxy-3-pyrazolyl)thioureas (6a–j)
were synthesized in the laboratories of the Dipartimento di Scienze
Farmaceutiche, University of Ferrara, as described previously
(26, 28).
Synthesis of 4-Oxo-5-substituted Pyrazolo[3,4-d]pyrimidine-6-thiones
(5a–g,i). A suspension of the pertinent N-alkyl/aryl-N′-(4-carbethoxy-
3-pyrazolyl)thiourea (6) (0.6 g) in 4% aqueous sodium hydroxide (4
mL) was refluxed until no more of the starting material could be
detected by TLC (1–2 h). After cooling, the solution was neutralized
with 4% hydrochloric acid and then extracted with ethyl acetate (3 ×
20 mL). After drying over anhydrous magnesium sulfate, the solvent
was removed and the white solid residue was purified by flash column
chromatography.
By using this procedure the following compounds were obtained:
5a (R ) 3-trifluoromethylphenyl): yield 75%; mp 255 °C (purified
by flash column chromatography; eluent ethyl acetate/petroleum ether,
8:2); 1H NMR (DMSO-d6) δ 7.55–7.77 (m, 4H, Ph), 8.62 (s, 1H, CH),
13.50 (s, 1H, NH), 13.77 (s, 1H, NH); IR (KBr, cm-1) νmax 3160, 2945,
1712, 1619, 1542. Anal. Calcd for C12H7F3N4OS: C, 46.15; H, 2.26;
N, 17.94; S, 10.27. Found: C, 46.01; H, 2.32; N, 17.72; S, 10.45.
5b (R ) 4-bromophenyl): yield 73%; mp 300–305 °C (purified by
flash column chromatography; eluent ethyl acetate/petroleum ether, 8:2);
1H NMR (DMSO-d6) δ 7.18–7.64 (m, 4H, Ph), 8.60 (s, 1H, CH), 13.44
(s, 1H, NH), 13.75 (s, 1H, NH); IR (KBr, cm-1) νmax 3140, 2949, 1685,
1609, 1542. Anal. Calcd for C11H7BrN4OS: C, 40.88; H, 2.18; N, 17.34;
S, 9.92. Found: C, 40.75; H, 2.35; N, 17.48; S, 9.78.
5c (R ) 4-chlorophenyl): yield 70%; mp 290–293 °C (purified by
flash column chromatography; eluent ethyl acetate/petroleum ether, 8:2);
1H NMR (DMSO-d6) δ 7.25–7.51 (m, 4H, Ph), 8.60 (s, 1H, CH), 13.44
(s, 1H, NH), 13.75 (s, 1H, NH); IR (KBr, cm-1) νmax 3142, 2938, 1708,
1612, 1544. Anal. Calcd for C11H7ClN4OS: C, 47.40; H, 2.53; N, 20.10;
S, 11.50. Found: C, 47.62; H, 2.43; N, 19.98; S, 11.42.
5d (R ) 3-chlorophenyl): yield 65%; mp 310–315 °C (purified by
flash column chromatography; eluent ethyl acetate/petroleum ether, 8:2);
1H NMR (DMSO-d6) δ 7.21–7.49 (m, 4H, Ph), 8.60 (s, 1H, CH), 13.50
(s, 1H, NH), 13.75 (s, 1H, NH); IR (KBr, cm-1) νmax 3137, 2914, 1730,
1613, 1585, 1551. Anal. Calcd for C11H7ClN4OS: C, 47.40; H, 2.53;
N, 20.10; S, 11.50. Found: C, 47.25; H, 2.62; N, 20.02; S, 11.33.
5e (R ) 2-chlorophenyl): yield 82%; mp 264–269 °C (purified by
flash column chromatography; eluent ethyl acetate/petroleum ether, 8:2);
1H NMR (DMSO-d6) δ 7.41–7.59 (m, 4H, Ph), 8.63 (s, 1H, CH), 13.53
(s, 1H, NH), 13.82 (s, 1H, NH); IR (KBr, cm-1) νmax 3138, 2936, 1700,
1612, 1542. Anal. Calcd for C11H7ClN4OS: C, 47.40; H, 2.53; N, 20.10;
S, 11.50. Found: C, 47.54; H, 2.61; N, 20.02; S, 11.36.
Fungal Growth Conditions and Evaluation of Antifungal Activ-
ity. As tests, the following phytopathogenic fungi were employed: P.
ultimum Trow, ATCC 58812 strain, B. cinerea (Pers.) Micheli, ATCC
48339 strain, and M. grisea (T. T. Hebert) Yaegashi & Udagawa, ATCC
64413 strain; all strains were purchased from the American Type Culture
Collection (ATCC), Rockville, MD. The fungi were maintained at 4 °C
as agar slants on potato dextrose agar (PDA; Difco, Detroit, MI).
To evaluate the ability of the compounds to inhibit fungal growth,
cultures of each fungus were obtained by transplanting mycelium disks,
10 mm in diameter, from a single culture in stationary phase. These
were incubated at 26 ( 1 °C on PDA (pH 5.6 ( 0.2) on thin sterile
sheets of cellophane (BeP Italia, Gorizia, Italy) until the logarithmic
phase of growth was reached and then transferred to Petri dishes
containing the medium supplemented with the compound to be tested.
Each compound was dissolved into dimethyl sulfoxide (DMSO), and
a proper dilution was aseptically added to the medium at 45 °C to obtain
a final concentration of 10, 50, and 100 µg mL-1. The DMSO
concentration in the final solution was adjusted to 0.1%. Control media
contained equivalent quantities (0.1%) of DMSO. The growth rate was
determined by measuring daily the colony diameter for 5 days after
the transport of the fungus onto dishes containing the substance to be
tested. Three replicates were used for each concentration. Results were
expressed as percentage of growth in untreated controls and are the
means of at least three independent experiments. As positive controls
for comparison the same concentrations (10, 50, and 100 µg mL-1) of
standard commercial fungicide specific for each fungus were also tested:
pyraclostrobin (Fluka) for P. ultimum, benodanil (Fluka) for B. cinerea,
and tricyclazole (Beam, Dow AgroSciences) for M. grisea.
The concentrations causing 50% inhibition (IC50) of in Vitro activity
for selected compounds were obtained by analysis of inhibition curves
of the activity values (%) versus the logarithm of inhibitory concentra-
tion. All calculations were performed by using the nonlinear regression
curve fitting Graph Pad Prism computer program (San Diego, CA). At
least five doses in the inhibitory range were considered.
RESULTS AND DISCUSSION
Synthesis. The preparative route to the target products 6a–j
and 5a–g,i is outlined in Scheme 1. N-Alkyl/aryl-N′-(4-
carbethoxy-3-pyrazolyl)thioureas 6 were obtained by reaction
of 3-amino-4-carbethoxypyrazole (7) with the appropriate
isothiocyanate, according to previously reported procedures (28).
High yields of 6 have been obtained in the reaction of 7 with
aryl isothiocyanates, but in the reaction of 7 with ethyl, butyl,
and cyclohexyl isothiocyanate, the N′-(3-pyrazolyl)thioureas
6g,h,j were isolated after purification by silica gel chromatog-
raphy as byproducts. Cyclization of 6 with 4% aqueous sodium
hydroxide provided the required 4-oxo-5-substituted pyra-
zolo[3,4-d]pyrimidine-6-thiones (5a–g,i) (Scheme 1).
Biological Activity. The ability of these 28 compounds to
inhibit the growth of P. ultimum, B. cinerea, and M. grisea was
evaluated at concentrations of 10, 50, and 100 µg mL-1 in
comparison with their untreated controls and with the reference
compounds for each fungus: pyraclostrobin, benodanil, and
tricyclazole, respectively.
5f (R ) 4-nitrophenyl): yield 78%; mp 328–333 °C (purified by
flash column chromatography; eluent methylene chloride/methanol/
1
toluene, 17:2:1); H NMR (DMSO-d6) δ 7.55–8.33 (m, 4H, Ph), 8.63
(s, 1H, CH), 13.56 (s, 1H, NH), 13.80 (s, 1H, NH); IR (KBr, cm-1
)
νmax 3278, 1718, 1615, 1589, 1542. Anal. Calcd for C11H7N5O3S: C,
45.67; H, 2.44; N, 24.21; S, 11.09. Found: C, 45.49; H, 2.59; N, 24.09;
S, 10.94.
5g (R ) ethyl): yield 70%; mp 254–258 °C (purified by flash column
1
chromatography; eluent ethyl acetate/petroleum ether, 8:2); H NMR
(DMSO-d6) δ 1.17 (t, 3H, J ) 7.0 Hz, Me), 4.41 (q, 2H, J ) 7.0 Hz,
CH2), 8.53 (s, 1H, CH), 13.23 (s, 1H, NH), 13.70 (s, 1H, NH); IR
(KBr, cm-1) νmax 3151, 1703, 1623, 1546. Anal. Calcd for C7H8N4OS:
C, 42.84; H, 4.11; N, 28.55; S, 16.34. Found: C, 42.66; H, 4.24; N,
28.37; S, 16.23.
5i (R ) benzyl): yield 72%; mp 253 °C (purified by flash column
1
chromatography; eluent ethyl acetate/petroleum ether, 8:2); H NMR
(DMSO-d6) δ 5.65 (s, 2H, CH2), 7.20–7.30 (m, 5H, Ph), 8.56 (s, 1H,
CH), 13.40 (s, 1H, NH), 13.77 (s, 1H, NH); IR (KBr, cm-1) νmax 3390,
1702, 1627, 1543. Anal. Calcd for C12H10N4OS: C, 55.80; H, 3.90; N,
21.69; S, 12.41. Found: C, 55.96; H, 4.02; N, 21.48; S, 12.23.
Results, summarized in Tables 1–3, were expressed as percent
of growth in untreated controls, taken at the fifth day from the

10334 J. Agric. Food Chem., Vol. 55, No. 25, 2007
Vicentini et al.
Table 1. Percentage Inhibition Rate after 5 Days of Treatment with New Compounds 1–4 on P. ultimum, B. cinerea, and M. grisea and Relative Standard
Specific Fungicidesa
a Growth was evaluated as described in Materials and Methods either in the absence or in the presence of pyrazole derivatives at concentrations ranging from 10 to
100 µg mL-1. Each sample was carried out in triplicate, and values were expressed as percentage of untreated controls.
transplant of the fungi; an example graph showing the growth
lines of a fungus untreated and treated with one of the most
active compound and the specific reference compound during
the 5 days of the experiment is shown in Figure 7.
For the most active substances were carried out also the IC50,
the concentrations causing 50% inhibition of in Vitro activity
(Table 4).
P. ultimum. Considering P. ultimum the reference compound
is pyraclostrobin. For the cyclopropylcarboxamides (Table 1)
the replacement of the carboxyisoxazole moiety with a car-
boxypyrazole resulted in a loss of activity (2m > 2s > 2n).
The methyl ester 3n showed lower inhibition whereas 4n was
completely ineffective when compared to the corresponding
carboxamide 2n.
When in position 3 there is a tert-butylcarboxamide group,
the efficacy of the pyrazole-3-tert-butylcarboxamide 1n (R )
CH3) and isoxazole-3-tert-butylcarboxamide 1m is comparable.
Interesting is the insertion of a substituent in position 1 of
pyrazole. The presence of a phenyl, 4-chlorophenyl, or 2,6-
dichloro-4-trifluoromethylphenyl group at position 1 (com-
pounds 1p, 1q, and 1r) significantly improved the antimycotic
activity of the compounds. The phenyl derivative 1p was able
to exert an inhibition of fungal growth higher than 80% at
concentrations of 50 and 100 µg mL-1; remarkable is also the
inhibition near 50% exerted by the 4-chlorophenyl derivative
1q at the lowest dose tested (10 µg mL-1).
The pyrazolo[3,4-d]pyrimidine derivative 5a [5-(3-trifluo-
romethyl)phenyl] was able to exert a dose-dependent inhibition
of fungal growth (Table 2).
The replacement of the (3-trifluoromethyl)phenyl group with
a 4-bromophenyl, 4-chlorophenyl, 4-nitrophenyl, or 3-chlo-
rophenyl group resulted in a gradual decrease of activity (5b >
5c > 5f > 5d) or in a total inefficacy with 2-chlorophenyl (5e)
and ethyl (5g) groups. Instead, the presence of a benzyl group
in position 5 (compound 5i) significantly improved the activity.
The efficacy of the thioureas 6e, 6c, 6d, 6f, and 6j (R )
2-chlorophenyl, 4-chlorophenyl, 3-chlorophenyl, 4-nitrophe-
nyl, and cyclohexyl) is comparable at the highest dose varying
from 60.8% to 70%. Remarkable is the inhibition brought

Pyrazole Derivatives as Inhibitors of Phytopathogenic Fungi
J. Agric. Food Chem., Vol. 55, No. 25, 2007 10335
Table 2. Percentage Inhibition Rate after 5 Days of Treatment with New Compounds 5 on P. ultimum, B. cinerea, and M. grisea and Relative Standard
Specific Fungicidesa
a Growth was evaluated as described in Materials and Methods either in the absence or in the presence of pyrazole derivatives at concentrations ranging from 10 to
100 µg mL-1. Each sample was carried out in triplicate, and values were expressed as percentage of untreated controls.
by the 2-chloropheyl derivative 6e and cyclohexyl derivative
6j at the lowest concentration tested (Table 3).
Nevertheless, all compounds gave an inhibition rate inferior
to that of the reference compound pyraclostrobin at the same
doses (Tables 1–3). The compounds selected for IC50 were 1p,
1q, and 5i (Table 4); they showed an antifungal activity in the
micromolar range, but it is not comparable with that of the
reference compound pyraclostrobin.
B. cinerea. For this fungus the reference compound is
benodanil.
Among the first set of compounds, the cyclopropyl derivatives
2m, 2s, and 2n were marginally effective at all tested doses
(Table 1).
On the contrary the presence of the tert-butyl group improved
the antifungal activity of the pyrazole derivatives (1n–r) but
not that of the isoxazole derivative (1m). Middling inhibitions
(around 50%) resulted from the 4-chlorophenyl derivative 1q
at all doses; remarkable (45.6% at 10 µg mL-1) and higher to
that one of benodanil (equal to 6.34% at 10 µg mL-1) was the
inhibition at the lowest concentration.
Among the pyrazolo[3,4-d]pyrimidines, only 5e (2-chlorophe-
nyl) was able to exert a good inhibition of growth at all of the
concentrations; in particular, noteworthy is the inhibition brought
at the lowest rate tested (Table 2). The benzyl, 4-chlorophenyl,
and 3-chlorophenyl derivatives 5i, 5c, and 5d showed only low
activities at 100 µg mL-1, about 30% inhibition, whereas they
were as ineffective at the lower doses as were the other
compounds belonging to the same series.
The efficacy of the thioureas 6d (R ) 3-chlorophenyl) and
6i (R ) benzyl) at the highest dose is comparable, but only 6d
showed remarkable inhibition at the lowest dose tested
(Table 3).
The 4-chlorophenyl, 4-nitrophenyl, and 2-chlorophenyl de-
rivatives 6c, 6f, and 6e caused an inhibition of 46.2%, 37.8%,
and 36.0%, respectively.
The compounds selected to evaluate the IC50 were 5e and
6d; the last showed a value even lower than that of benodanil,
suggesting a good activity against B. cinerea (Table 4 and
Figure 7).
M. grisea. For M. grisea the specific reference compound
tricyclazole here is used at concentrations of 10, 50, and 100
µg mL-1, a range in which all new compounds were tested. At
these doses tricyclazole exerts an increasing inhibition effect,
even if it does not achieve the fungal 100% inhibition, that is
reached in the normal agricultural practice where, as a rule, it
is used at 200 µg mL-1 (2). The mechanism of action of this

10336 J. Agric. Food Chem., Vol. 55, No. 25, 2007
Vicentini et al.
Table 3. Percentage Inhibition Rate after 5 Days of Treatment with New Compounds 6 on P. ultimum, B. cinerea, and M. grisea and Relative Standard
Specific Fungicidesa
a Growth was evaluated as described in Materials and Methods either in the absence or in the presence of pyrazole derivatives at concentrations ranging from 10 to
100 µg mL-1. Each sample was carried out in triplicate, and values were expressed as percentage of untreated controls.
compound has been studied on M. grisea, where it prevents
penetration of plant epidermis by weakening the fungal walls
by inhibiting pigmentation of the hyphae (30). In our previous
work some pyrazole derivatives showed two different mecha-
nisms of action: one similar to that of other azoles, by inhibiting
hyphal growth, and the other similar to that of tricyclazole, by
inhibiting pigmentation of the mycelium (17). For this reason
on this fungus we evaluated both inhibition of growth and
depigmentation.
Under the experimental conditions employed, the com-
pounds belonging to pyrazole/isoxazolecarboxamides (1 and
2) and their esters (3n and 4n) in general showed a low
efficiency (Table 1).
On the contrary, the thiourea 6e (R ) 2-chlorophenyl)
showed a good activity higher than the one of the reference
compound at all of the doses (Table 3). Noteworthy for all
of the compounds of this series, except 6h (R ) butyl), is
the ability of inhibit the growth of M. grisea even at the
lowest dose. The compounds selected for IC50 were 6a and
6e, both showing values lower than that of tricyclazole, in
particular, 6e (Table 4).
A parameter that we considered in this work was the capacity
of the new azoles to inhibit the color of the mycelium.
Subsequent visual observation showed that tricyclazole inhibits
the pigmentation of the hyphae, whereas all of the new
compounds 1–6 were ineffective.
Only the 4-chlorophenyl derivative 1q was able to exert a
20.9% inhibition of fungal growth at a concentration of 10 µg
mL-1, a dose at which tricyclazole was completely ineffective,
and at 50 µg mL-1 caused an inhibition equal to 31.5%, that is
slightly higher than that of the reference compound.
Even the results of the pyrazolopyrimidine derivatives on
Magnaporthe are scanty: in the series only the pyrazolo[3,4-
d]pyrimidine 5i (R ) benzyl) was active, and its action is
comparable to that of tricyclazole at 50 µg mL-1 (Table 2).
In conclusion, the results on fungal growth inhibition (Tables
1–3) and IC50 evaluation (Table 4) showed differences in the
sensitivity of the three fungi to the substances tested, P. ultimum
being in general the most inhibited.
Within the compounds of the pyrazole/isoxazolecarboxamide
series on all fungi compounds 1p and 1q were found to give
the best results. The presence of the carboxamide and carboxylic
groups in positions 3 and 4 seems to be critical for biological
activity. In our previous work carboxamides 1 and 2 were tested

Pyrazole Derivatives as Inhibitors of Phytopathogenic Fungi
J. Agric. Food Chem., Vol. 55, No. 25, 2007 10337
Figure 7. B. cinerea treated with 6d and benodanil.
Table 4. IC50 of Selected Compounds
Against B. cinerea the pyrazolopyrimidine derivative 5e (R
) 2-chlorophenyl) and thioureidic derivative 6d (R ) 3-chlo-
rophenyl) are to be underlined because they showed a good
P. ultimum
B. cinerea
M. grisea
activity at 50 and 100 µg mL-1, but, especially at 10 µg mL-1
,
a
a
a
compound
1p
1q
compound
compound
IC50 (µM)
IC50 (µM)
IC50 (µM)
they showed inhibitions much higher than those of the reference
compound benodanil.
79
38
5e
6d
83
31
6a
6e
224
48
5i
pyraclostrobin
66
,10
These results can be interesting for the future possibility of
using in agricultural practices chemical synthetic substances
against two widespread phytopathogenic fungi (M. grisea and
B. cinerea) at doses much lower than those currently used and,
thus, more ecocompatible. Screening in depth on the compound
phytotoxicity will be made for the most active compound in
each group.
benodanil
66
tricyclazole
474
a The concentrations causing 50% inhibition (IC50) of in vitro activity were
obtained by analysis of inhibition curves of the activity values (%) versus the
logarithm of inhibitory concentration. All calculations were performed by using the
nonlinear regression curve fitting Graph Pad Prism computer program (San Diego,
CA).
In spite of the different responses given by the fungi, the
present results are encouraging for further studies to clarify the
mechanism of action of the most active compounds.
as possible herbicides and showed in general a low ability to
act as photosynthetic electron inhibitors and only at millimolar
concentrations (28); thus, their use as agrochemicals seems to
be not satisfactory. Among the same compounds tested as
antifungals the most interesting is 1q, which is the only one
with both herbicidal (IC50 ) 0.95 mM on isolated chloroplasts)
and antifungal activity (IC50 ) 38 µM on P. ultimum). But,
whereas the herbicidal activity is evinced at millimolar con-
centration, the antifungal activity is evinced in the micromolar
range, at doses lower (about 25 times) than those of the
herbicidal. From these preliminary data we think that its use as
an antifungal could be not precluded, because the effect against
the fungus is substantial at concentrations at which it is
ineffective against plants.
ACKNOWLEDGMENT
We gratefully acknowledge Prof. Alessandro Dalpiaz for
helpful discussions and suggestions. We thank Dr. Nicola
Battistella and Dr. Enkeleida Borekai for skillful technical
assistance.
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Received for review July 11, 2007. Revised manuscript received
September 24, 2007. Accepted September 25, 2007. This work was
supported by research grants from MURST (Ministero dell’Università
e della Ricerca Scientifica e Tecnologica) of Italy.
JF072077D