Development of new fungicides against Magnaporthe grisea: Synthesis and biological activity of pyrazolo[3,4-d][1,3]thiazine, pyrazolo[1,5-c][1,3,5]thiadiazine, and pyrazolo[3,4-d]pyrimidine derivatives

Article abstract of DOI:10.1021/jf0202436

Some pyrazolo[3,4-d]pyrimidin-4(5H)-thione, pyrazolo[3,4-d][1,3]thiazin-4-one/thione, and pyrazolo[1,5-c][1,3,5]thiadiazine-4-one/thione derivatives were synthesized and screened for antifungal activity against the causal agent of rice blast disease, Magnaporthe grisea. In all cases a remarkable inhibition of fungal growth was found in the range from 10 to 200 μg mL^-1. Several compounds were able to control mycelium growth at a rate of 10 μg mL^-1, a concentration at which the reference compound tricyclazole was completely ineffective. At least in the case of the most active substance, at the same dose the growth of seedlings or cultured cells of rice was substantially unaffected. Results allowed definition of structural requirements either to maintain or to enhance mycotoxic activity.

Full text of DOI:10.1021/jf0202436

J. Agric. Food Chem. 2002, 50, 4839−4845 4839
Development of New Fungicides against Magnaporthe grisea:
Synthesis and Biological Activity of Pyrazolo[3,4-d][1,3]thiazine,
Pyrazolo[1,5-c][1,3,5]thiadiazine, and Pyrazolo[3,4-d]pyrimidine
Derivatives
CHIARA B. VICENTINI,*,† GIUSEPPE FORLANI,§ MAURIZIO MANFRINI,†
‡
CARLO ROMAGNOLI,# AND DONATELLA MARES
Dipartimento di Scienze Farmaceutiche, Dipartimento di Biologia, and Dipartimento delle Risorse
Naturali e Culturali, Universita` di Ferrara, I-44100 Ferrara, Italy; and Dipartimento del Museo di
Paleobiologia e dell’Orto Botanico, Universita` di Modena e Reggio Emilia, Italy
Some pyrazolo[3,4-d]pyrimidin-4(5H)-thione, pyrazolo[3,4-d][1,3]thiazin-4-one/thione, and pyrazolo-
[1,5-c][1,3,5]thiadiazine-4-one/thione derivatives were synthesized and screened for antifungal activity
against the causal agent of rice blast disease, Magnaporthe grisea. In all cases a remarkable inhibition
of fungal growth was found in the range from 10 to 200 µg mL-1. Several compounds were able to
control mycelium growth at a rate of 10 µg mL-1, a concentration at which the reference compound
tricyclazole was completely ineffective. At least in the case of the most active substance, at the same
dose the growth of seedlings or cultured cells of rice was substantially unaffected. Results allowed
definition of structural requirements either to maintain or to enhance mycotoxic activity.
KEYWORDS: Rice; blast; Magnaporthe grisea; antifungals; pyrazolo[3,4-d][1,3]thiazine; pyrazolo[1,5-c]-
[1,3,5]thiadiazine; pyrazolo[3,4-d]pyrimidine
INTRODUCTION
approach, the most important is tricyclazole, patended by Eli
Lilly (5). Tricyclazole causes a weakening of fungal walls and
prevents the penetration of plant epidermis by fungal hyphae.
During the past few years a number of other compounds that
inhibit several enzymes in melanin biosynthesis have been
described with a putative ability of preventing blast disease (e.g.
refs 6 and 7). However, because of such an action, these
compounds are ineffective in controlling rice blast if applied
after pathogen penetration (8). Consequently, the development
of new fungicides able to inhibit fungal growth would be of
greatest agronomical value.
Within the framework of a program directed to investigate
the antifungal activity of nitrogen heterocycles, the synthesis
of pyrazolo[3,4-d]pyrimidin-4(5H)-thiones has previously been
reported (9). A screening for antifungal activity showed that
some compounds of this series have a wide spectrum of activity
against phytopathogenic fungi of different taxa and are particu-
larly effective in controlling Pythium ultimum and Corticium
solani (9). With this background, the antifungal activity of
derivatives of pyrazolo[3,4-d]pyrimidin-4(5H)-thiones was in-
vestigated. The first stage was to prepare 6-trifluoromethyl
derivatives (10-12): this was prompted by the reported efficacy
of the trifluoromethyl group in some pesticides (13). In particular
three lead compounds (R ) 3-nitrophenyl, 4-nitrophenyl, and
tert-butyl) showed EC50 and MIC values comparable or only
slightly inferior to those of the reference commercial fungicides
captafol and mancozeb against Sclerotinia minor, C. solani, and
P. ultimum (10, 11, 14). After these results, the introduction of
Plant diseases are estimated to cause yield reduction of almost
20% in the major food and cash crops worldwide (1). Fungicides
remain vital for effective control of fungal plant pathogens; thus,
researchers are continuously developing a diverse range of
products with novel modes of action (2). Rice is the major
human staple crop for almost half of the world’s population,
particularly in East and Southeast Asia, and is of great economic
value in northern Italy. The filamentous fungus Magnaporthe
grisea (T.T. Hebert) Yaegashi & Udagawa, an anamorph of
Pyricularia grisea (Cooke) Sacc., can cause disease in many
species of the grass family. The disease in rice, rice blast, is
considered to be its main fungal disease because of its wide
distribution and destructiveness under favorable conditions (3).
Plant infection is brought about by the action of specialized
cells called appressoria, generating enormous turgor pressure,
which is translated into an invasive force that allows a narrow
penetration hypha to break plant cuticle (4). With this aim, the
fungus requires and utilizes melanin-derived pressure; thus,
melanin biosynthesis inhibition has been shown to be a
promising biochemical target for the discovery of new selective
fungicides. As to the commercial compounds resulting from this
* Author to whom correspondence should be addressed [fax (39) 0532-
291296; e-mail vcc@unife.it].
† Dipartimento di Scienze Farmaceutiche, Universita` di Ferrara.
§ Dipartimento di Biologia, Universita` di Ferrara.
‡ Dipartimento delle Risorse Naturali e Culturali, Universita` di Ferrara.
# Universita` di Modena e Reggio Emilia.
10.1021/jf0202436 CCC: $22.00 © 2002 American Chemical Society
Published on Web 07/12/2002

4840 J. Agric. Food Chem., Vol. 50, No. 17, 2002
Vicentini et al.
Scheme 1
petroleum ether); IR (KBr, cm-1) νmax 3158, 2992, 1752, 1676, 1571;
1H NMR (CDCl3) δ 1.65 (s, 9H, t-Bu), 2.33 (s, 3H, Me), 6.30 (s, 1H,
CH), 10.31 (br, 1H, NH).
new substituents in the heterocyclic system was planned. The
purpose was to realize the replacement of the 4-thione of
pyrazolopyrimidines with various substituents in order to
investigate the influence of substitution at C4 on antifungal
activity. By combining the obtained results (9-12, 14), we
concluded that key structural requirements for the antifungal
activity of pyrazolo[3,4-d]pyrimidines (1) were (i) a thione
function at position 4, (ii) a trifluoromethyl group at position
6, and (iii) a 3- or 4-nitrophenyl or a tert-butyl group at position
1 (Scheme 1).
In this paper we report the synthesis and evaluation of
biological activity against M. grisea of pyrazolo[3,4-d]pyrimi-
din-4(5H)-thione derivatives (1; Scheme 1) and of the analogues
pyrazolo[3,4-d][1,3]thiazin-4-one/thione (2) and pyrazolo[1,5-
c][1,3,5]thiadiazine-4-one/thione (3). The widely used fungicide
tricyclazole was adopted as a reference compound. The sensitiv-
ity of rice cells and seedlings to the most active substance was
also tested.
4 (R ) phenyl; R′ ) CF3; R′′ ) methyl): yield 74%; mp 180-185
°C (ethyl ether/petroleum ether); IR (KBr, cm-1) νmax 2980, 2830, 1740,
l
1600, 1570, 1510; H NMR (CDCl3) δ 2.27 (s, 3H, Me), 6.47 (s, 1H,
CH), 7.27-7.54 (m, 5H, Ph), 8.47 (br, 1H, NH); 1H NMR (DMSO-d6)
δ 2.25 (s, 3H, Me), 6.37 (s, 1H, CH), 7.46-7.50 (m, 5H, Ph), 11.61
(s, 1H, NH).
4 (R ) 3,4-dichlorophenyl; R′ ) CF3; R′′ ) methyl): yield 71%;
mp 81-84 °C (purified by column chromatography, eluent 1:1 ethyl
acetate/petroleum ether); IR (KBr, cm-1) νmax 2929, 1741, 1578, 1487;
lH NMR (CDCl3) δ 2.32 (s, 3H, Me), 6.52 (s, 1H, CH), 7.22-7.62 (m,
3H, Ph), 8.02 (br, IH, NH).
4 (R ) 3-nitrophenyl; R′ ) CF3; R′′ ) methyl): yield 82%; mp
114-117 °C (ethyl ether/petroleum ether); IR (KBr, cm-1) νmax 3152,
1
1745, 1540; H NMR (CDCl3) δ 2.33 (s, 3H, Me), 6.40 (s, 1H, CH),
7.69-8.31 (m, 5H, Ph + NH).
Method B. A solution of the pertinent acyl chloride (20 mmol) in
methylene chloride (10 mL) was added dropwise to a mixture of the
appropriate 5-amino-1-tert-butylpyrazole (20 mmol) in methylene
chloride (100 mL) and sodium hydrogen carbonate (1.68 g, 20 mmol)
in water (50 mL). After 24 h of stirring at room temperature, the organic
phase was washed with 5% sodium hydrogen carbonate and water and
then anhydrified over anhydrous magnesium sulfate. After removal of
the solvent, the residue was recrystallized from the indicated solvent
to give colorless crystals. By using this procedure the following
compounds were obtained.
MATERIALS AND METHODS
Chemicals. Melting points were determined with a Bu¨chi 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-
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. Column
chromatography was performed using Merck silica gel (70-230 mesh);
for the flash chromatography technique, silica gel (230-400) mesh
was employed.
Syntheses. Compounds 1a-d (11), 5 (R ) t-Bu; R′ ) Ph, CH2Ph;
R′′ ) Me) (15), 6 (R′) 4-ClPh, 4-BrPh; R′′ ) Me) (16), and 2i, 2k,
2m, and 3a (15) were prepared according to the methods previously
reported by us.
N-(5-Pyrazolyl)carboxamides (4). Method A. A solution of the
appropriate 5-aminopyrazole (10 mmol) in trifluoroacetic anhydride
(5 mL) was heated at 100 °C for 1 h. After cooling, the solution was
evaporated to give a crude solid, which was purified by column
chromatography or by recrystallization from the indicated solvent. By
using this procedure the following compounds were obtained.
4 (R ) tert-butyl; R′ ) CF3; R′′ ) methyl): yield 71%; mp 92-95
°C (purified by column chromatography, eluent 1:1 ethyl acetate/
4 (R′ ) phenyl, R′′ ) H; R ) t-Bu): yield 55%; mp 200-201 °C
1
(ethyl ether); IR (KBr, cm-1) νmax 3299, 2991, 1660, 1557, 1511; H
NMR (CDCl3) δ 1.68 (s, 9H, t-Bu), 6.39 (s, 1H, CH), 7.47-7.57 (m,
3H, Ph), 7.60 (s, 1H, CH), 7.71 (br, 1H, NH).
4 (R′, R′′ ) phenyl; R ) t-Bu): yield 69%; mp 187-190 °C (ethyl
1
ether); IR (KBr, cm-1) νmax 3227, 2988, 1652, 1552, 1523; H NMR
(CDCl3) δ 1.70 (s, 9H, t-Bu), 6.69 (s, 1H, CH), 7.25-7.92 (m, 10H,
2Ph), 10.86 (br, 1H, NH).
N-(5-Pyrazolyl)thiocarboxamides (5). Lawesson’s reagent (2.02 g,
5 mmol) was added to a solution of the appropriate carboxamide 4 (5
mmol) in hexamethylphosporamide (12.5 mL) at 80 °C. The mixture
was kept under stirring at 80 °C until no more of the starting material
could be detected by TLC (5-6 h). The solution was then diluted with
water and extracted with ethyl acetate. The organic layer was extracted
with 1 N sodium hydroxide (3 × 50 mL), and the aqueous layer was
acidified to pH 5 with 10% hydrochloric acid and then extracted with
ethyl acetate (3 × 50 mL). After drying over anhydrous magnesium

New Fungicides against Magnaporthe grisea
J. Agric. Food Chem., Vol. 50, No. 17, 2002 4841
sulfate, the solvent was removed and the solid residue was purified by
column chromatography to give a pale yellow crystalline product.
5 (R ) tert-butyl; R′ ) CF3; R′′ ) methyl): yield 57%; oil (purified
by column chromatography, eluent 17:2:1 methylene chloride/methanol/
removal of the solvent, the solid residue was purified by column
chromatography to give a white crystalline product.
2e (R ) 3-nitrophenyl; R′ ) CF3; R′′ ) methyl): yield 20%; mp
l08-110 °C (purified by column chromatography, eluent 1:1 ethyl
1
toluene); IR (near, cm-1) νmax 3258, 2984, 1557; H NMR (CDCl3) δ
1
acetate/petroleum ether); IR (KBr, cm-1) νmax 2927, 1690, 1537; H
1.63 (s, 9H, t- Bu), 2.03 (s, 3H, Me), 6.30 (s, 1H, CH), 10.50 (br, 1H,
NH).
NMR (CDCl3) δ 2.71 (s, 3H, Me), 7.70-8.94 (m, 4H, Ph).
Cleavage of the tert-Butyl Group from 5 (R ) t-Bu) and from
2f)h. A solution of 5 (R ) t-Bu), 2i, or 2j (2 mmol) in formic acid
(12 mL) was heated under reflux until dealkylation was completed (1
h). The solution was evaporated to give a solid, which was taken up
with water (10 mL) and extracted with ethyl acetate (3 × 15 mL).
After drying over anhydrous magnesium sulfate, the solvent was
removed and the resulting solid was recrystallized from the indicated
solvent to give a white crystalline product.
5 (R ) phenyl; R′ ) CF3; R′′ ) methyl): yield 39%; mp 220-223
°C (purified by column chromatography, eluent 17:2:1 methylene
chloride/methanol/toluene); IR (KBr, cm-1) νmax 2791, 1567, 1533,
1
1502; H NMR (CDCl3) δ 2.36 (s, 3H, Me), 7.03 (s, 1H, CH), 7.43-
7.51 (m, 5H, Ph), 9.5 (br, 1H, NH).
5 (R ) 3,4-dichlorophenyl; R′ ) CF3; R′′ ) methyl): yield 14%;
mp 203-204 °C (purified by column chromatography, eluent 1:1 ethyl
1
acetate/petroleum ether); IR (KBr, cm-1) νmax 2924, 1577, 1483; H
6 (R′ ) CF3; R′′ ) methyl): yield 64%; mp 186-188 °C (ethyl
NMR (CDC13) δ 2.35 (s, 3H, Me), 6.80 (s, 1H, CH), 7.22-7.59 (m,
3H, Ph), 9.29 (br, 1H, NH).
1
ether); IR (KBr, cm-1) νmax 3280, 2775, 1597, 1560, 1491; H NMR
(CDCl3) δ 2.33 (s, 3H, Me), 5.6 (br, 1H, NH), 5.19 (s, 1H, CH), 11.00
(br, 1H, NH).
5 (R ) 3-nitrophenyl; R′ ) CF3; R′′ ) methyl): yield 40%; mp
178-180 °C (purified by column chromatography, eluent 8:2 ethyl
1
acetate/petroleum ether); IR (KBr, cm-1) νmax 2924, 1535; H NMR
6 (R′ ) phenyl, R′′ ) H): yield 94%; mp 191-3 °C (ethyl acetate);
IR (KBr, cm-1) νmax 3402, 2922, 1583, 1483; 1H NMR (CDCl3) δ 7.36-
7.88 (m, 5H, Ph), 7.56 (s, 1H, CH), 8.05 (s, 1H, CH), 10.86 (br, 1H,
NH).
(CDCl3) δ 2.35 (s, 3H, Me), 6.64 (s, 1H, CH), 7.66-8.32 (m, 4H, Ph),
9.30 (br, 1H, NH).
5 (R′ ) phenyl, R′′ ) H; R ) t-Bu): yield 74%; oil (purified by
column chromatography, eluent 1:1 ethyl acetate/petroleum ether); IR
6 (R′, R′′ ) phenyl): yield 71%; mp 204-206 °C (ethyl ether); IR
(KBr, cm-1) νmax 3417, 2918, 1597, 1490; 1H NMR (DMSO-d6) δ
7.37-7.64 (m, 11H, CH + 2Ph), 12.18 (s, 1H, NH), 13.29 (s, 1H,
NH).
1
(near, cm-1) νmax 3377, 2988, 1549, 1470; H NMR (CDCl3) δ 1.67
(s, 9H, t-Bu), 6.37 (s, 1H, CH), 7.46-7.56 (m, 3H, Ph), 7.54 (s, 1H,
CH), 8.55 (br, 1H, NH).
5 (R′, R′′ ) phenyl; R ) t-Bu): yield 73%; mp 101-104 °C (purified
by column chromatography, eluent 17:2:1 methylene chloride/methanol/
toluene); IR (KBr, cm-1) νmax 3147, 2982, 1553, 1519; 1H NMR
(CDCl3) δ 1.71 (s, 9H, t-Bu), 6.70 (s, 1H, CH), 7.25-7.90 (m, 10H,
2Ph), 8.58 (br, 1H, NH).
6-Substituted Pyrazolo[3,4-d][1,3]thiazin-4-thiones (2a)d,f,h).
Thiophosgene (0.41 mL, 5.5 mmol) was added to a suspension of
thiocarboxamide 5 (5 mmol) in anhydrous toluene (100 mL), placed
in a round-bottom flask equipped with a Vigreaux reflux condenser.
The mixture was heated under reflux until hydrogen chloride evolution
ceased (∼5 h). After removal of the solvent, the solid residue was
purified by column chromatography to give a yellow crystalline product.
2a (R ) tert-butyl; R′ ) CF3; R′′ ) methyl): yield 19%; mp 133-
135 °C (purified by column chromatography, eluent 3:7 ethyl acetate/
petroleum ether); IR (KBr, cm-1) νmax 2977, 1563, 1515, 1472; 1H NMR
(CDCl3) δ 1.76 (s, 9H, t-Bu), 2.70 (s, 3H, Me).
2j (R′ ) benzyl; R′′ ) methyl): yield 92%; mp 199-200 °C (toluene);
IR (KBr, cm-l) νmax 3200-2700, 1570, 1490, 1460; 1H NMR (DMSO-
d6) δ 2.59 (br, 3H, Me), 4.14 (br, 2H, CH2), 7.32 (br, 5H, Ph), 14.21
(br, 1H, NH).
2l (R′ ) phenyl; R′′ ) methyl): yield 93%; mp 249-250 °C (ethyl
acetate); IR (KBr, cm-1) νmax 3160-2600, 1630, 1560; 1H NMR
(DMSO-d6) δ 2.63 (br, 3H, Me), 7.58 (m, 3H, Ph), 7.98 (d, J ) 7.2
Hz, 2H, Ph).
2-Substituted Pyrazolo[1,5-c][1,3,5]thiadiazine-4-ones (3b)d,f,g).
Trichloromethyl chloroformate (0.6 mL, 5 mmol) was added to a
solution of the pertinent 6 (5 mmol) in anhydrous tetrahydrofuran (50
mL). After 10 h of stirring at room temperature, the solvent was
removed and the solid residue was purified by column chromatography.
3b (R′ ) p-bromophenyl; R′′ ) methyl): yield 22%; mp 243-245
°C (purified by column chromatography, eluent 3:7, ethyl acetate/
petroleum ether); IR (KBr, cm-l) νmax 3433, 1736, 1591, 1575; 1H NMR
(CDCl3) δ 2.48 (s, 3H, Me), 6.62 (s, 1H, CH), 7.65 (d, J ) 8.6 Hz,
2H, Ph), 7.85 (d, J ) 8.6 Hz, 2H, Ph).
3c (R′ ) p-chlorophenyl; R′′ ) methyl): yield 25%; mp 229-231
°C (purified by column chromatography, eluent 3:7, ethyl acetate/
petroleum ether); IR (KBr, cm-l) νmax 3447, 1727, 1596, 1583; 1H NMR
(CDCl3) δ 2.48 (s, 3H, Me), 6.61 (s, 1H, CH), 7.50 (d, J ) 8.6 Hz,
2H, Ph), 7.93 (d, J ) 8.6, 2H, Ph).
2b (R ) phenyl; R′ ) CF3; R′′ ) methyl): yield 28%; mp 120-124
°C (purified by column chromatography, eluent 3:7 ethyl acetate/
petroleum ether); IR (KBr, cm-1) νmax 3448, 1559, 1520, 1471; 1H NMR
(CDCl3) δ 2.73 (s, 3H, Me), 7.40-7.81 (m, 5H, Ph).
2c (R ) 3,4-dichlorophenyl; R′ ) CF3; R′′ ) methyl): yield 48%;
mp 138-140 °C (purified by column chromatography, eluent 1:1 ethyl
acetate/petroleum ether); IR (KBr, cm-1) νmax 3435, 1554, 1522, 1468;
1H NMR (CDCl3) δ 2.79 (s, 3H, Me), 7.52-8.13 (m, 3H, Ph).
2d (R ) 3-nitrophenyl; R′ ) CF3; R′′ ) methyl): yield 78%; mp
105-108 °C (purified by flash column chromathography, eluent 3:7
ethyl acetate/petroleum ether); IR (KBr, cm-1) νmax 3111, 2926, 1537,
3d (R′) CF3; R′′ ) methyl): yield 53%; mp 120-123 °C (purified
by flash chromatography, eluent 1:9 ethyl acetate/petroleum ether); IR
1
(KBr, cm-l) νmax 3097, 1708, 1585; H NMR (CDCl3) δ 2.51 (s, 3H,
1
1470; H NMR (CDCl3) δ 2.81 (s, 3H, Me), 7.70-8.92 (m, 4H, Ph).
Me), 6.84 (s, 1H, CH).
2f (R ) tert-butyl; R′ ) benzyl; R′′ ) methyl): yield 71%; mp 81-
82 °C (purified by flash column chromatography, eluent 0.4:9.6 ethyl
3f (R′ ) phenyl, R′′ ) H): yield 25%; mp 168-171 °C (purified by
column chromatography, eluent 3:7, ethyl acetate/petroleum ether); IR
1
acetate/petroleum ether); IR (KBr, cm-1) νmax 1560, 1470, 1220; H
1
(KBr, cm-1) νmax 3350, 1715, 1575; H NMR (CDCl3) δ 6.60 (s, 1H,
NMR (DMSO-d6) δ 1.60 (s, 9H, t-Bu), 2.53 (s, 3H, Me), 4.19 (s, 2H,
CH2), 7.2-7.4 (m, 5H, Ph).
2h (R ) tert-butyl; R′ ) phenyl; R′′ ) methyl): yield 75%; mp 169-
170 °C (purified by flash column chromatography, eluent 0.2:9.8 ethyl
acetate/petroleum ether); IR (KBr, cm-1) νmax 1540, 1510, 1470, 1220;
1H NMR (CDCl3) δ 1.63 (s, 9H, t-Bu), 2.71 (s, 3H, Me), 7.50-7.54
(m, 3H, Ph), 8.00-8.20 (m, 2H, Ph).
6-Trifluoromethylpyrazolo[3,4-d][1,3]thiazin-4-one (2e). Trichlo-
romethyl chloroformate (0.6 mL, 5 mmol) was added to a suspension
of thiocarboxamide 5 (5 mmol) in anhydrous toluene (100 mL), placed
in a round-bottom flask equipped with a Vigreaux reflux condenser.
The mixture was stirred at room temperature for 30 min and then heated
under reflux until hydrogen chloride evolution ceased (∼5 h). After
CH), 7.53-8.08 (m, 5H, Ph), 7.57 (s, 1H, CH).
3g (R′, R′′ ) phenyl): yield 96%; mp 223-225 °C (purified by
column chromatography, eluent 3:7, ethyl acetate/petroleum ether); IR
1
(KBr, cm-1) νmax 3350, 1710, 1680, 1580, 1550; H NMR (CDCl3) d
7.11 (s, 1H, CH), 7.46-8.04 (m, 10H, 2Ph).
2-Trifluoromethylpyrazolo[1,5-c][1,3,5]thiadiazine-4-thione (3e).
Thiophosgene (0.60 mL, 6 mmol) was added dropwise to a heterogen-
eous mixture of dealkylated thioamide 6 (R′ ) CF3) (6 mmol) in water
(45 mL) with vigorous stirring. After 3 h of stirring at room temperature,
the precipitate was collected, washed with water, and dissolved in ethyl
acetate. After drying over magnesium sulfate, the solvent was removed
to give a solid, which was purified by column chromatography.

4842 J. Agric. Food Chem., Vol. 50, No. 17, 2002
Vicentini et al.
3e (R′ ) CF3; R′′ ) methyl): yield 45%; mp 75-78 °C (purified by
separated by the least-significant difference test. When differences are
reported, they are at the 99% confidence level (P ) 0.01).
column chromatography, eluent 1:1, ethyl acetate/petroleum ether); IR
1
(KBr, cm-l) νmax 3089, 1593; H NMR (CDCl3) δ 2.54 (s, 3H, Me),
6.89 (s, 1H, CH).
RESULTS AND DISCUSSION
Fungal Growth Conditions and Evaluation of Antifungal Activ-
ity. Magnaporthe grisea (T.T. Hebert) Yaegashi & Udagawa, ATCC
64413 strain, was purchased from American Type Culture Collection
(Rockville, MD) and maintained at 4 °C as agar slants on potato
dextrose agar (PDA; Difco, Detroit, MI).
Synthesis. The preparative route to the target products is
outlined in Scheme 2. N-(5-Pyrazolyl)carboxamides (4) were
obtained by acylation of the corresponding 5-aminopyrazoles.
Thiation of 4 with Lawesson’s reagent provided the thiocar-
boxamides (5). Heating under reflux of equimolar amounts of
5 and thiophosgene or trichloromethyl chloroformate gave
satisfactory yields of the target 6-trifluoromethylpyrazolo[3,4-
d][1,3]thiazin-4-thiones (2a-d,f,h) and -ones (2e,g,i), respec-
tively. The 1-tert-butyl derivatives (2f-i) were converted by
heating in formic acid into the dealkylated homologues (2j-
m). Following the same procedure the N-(1-tert-butyl-5-
pyrazolyl)thiocarboxamides (5, R ) t-Bu) were converted into
the homologues (6). As expected, the reaction of 6 with
trichloromethyl chloroformate or thiophosgene proceeded
smoothly to afford pyrazolo[1,5-c][1,3,5]thiadiazine-4-ones
(3a-d,f,g) and thione (3e).
Biological Activity. The ability of these compounds to inhibit
the growth of M. grisea was evaluated at rates of 10-200 µg
mL-1, a range in which the reference compound tricyclazole
was found to exert an increasing effect. Results are summarized
in Table 1. The first set of compounds, pyrazolo[3,4-d]-
pyrimidine derivatives with R ) tert-Bu and R ) Ph, were
marginally effective at all tested doses. On the contrary, the
presence of a chlorophenyl or a nitrophenyl group (compounds
1c,d) significantly improved their antifungal activity, which in
both cases was higher than that of tricyclazole. Remarkably, at
concentrations >50 µg mL-1 compound 1c achieved total
control of fungal growth.
Among pyrazolo[3,4-d][1,3]thiazine derivatives, compounds
2a-d have exactly the same substituents as the previous group.
In this case also, the presence of a tert-butyl or a phenyl at
position 1 resulted in a lower effectiveness than those of
compounds bearing 3,4-chlorophenyl or 3-nitrophenyl groups,
so their presence seems to be critical for biological activity.
Interestingly, compound 2d was able to exert a 71% inhibition
of fungal growth at concentrations as low as 10 µg mL-1, a
dose at which tricyclazole was completely ineffective. The
replacement of X ) S in compound 2d with X ) O, giving
rise to compound 2e, significantly reduced the inhibitory effect.
A similar result was obtained when R′ ) CF3 was changed to
a phenyl or a CH2Ph group in compounds 2h-m. However,
the lack of a dose-effect relationship for some compounds of
this series may suggest that in the aqueous environment required
for fungal growth their solubility (and thus the availability) may
be low. The recorded effect may thus be ascribed to the small
concentration able to dissolve into the culture medium.
With respect to pyrazolo[1,5-c][1,3,5]thiadiazine derivatives,
compound 3a (X ) O, R′ ) Ph) was found to be the most
effective. The activity against M. grisea was reduced by the
presence at position R′ of either larger (bromophenyl, 3b;
chlorophenyl, 3c) or smaller (CF3, 3d) groups. If at position
R′′ was H (3f) or a phenyl group (3g), there was a strong
decrease of activity, in particular in 3g. Contrary to compounds
2, in this case the thione function seems to cause a decrease in
activity, as suggested by the comparison of the effects exerted
by compounds 3d and 3e.
To evaluate biological activity, cultures were obtained by transplant-
ing 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 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, 100, or 200 µg mL-1. The DMSO concentration
in the final solution was adjusted to 0.1%. Controls were set up with
equivalent quantities (0.1%) of DMSO. The growth rate was determined
by measuring daily 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. Percentage inhibition was
expressed as the mean of values obtained in three independent
experiments. The same concentrations of a commercial fungicide,
tricyclazole (Beam, Dow AgroSciences), were also tested. For com-
pound 3a, the most active as fungicide, concentrations <10 µg mL-1
were also used, that is, 5, 2.5, 1, and 0.5 µg mL-1
.
Rice Seedlings and Plant Cell Cultures. Rice (Oryza satiVa L. cv
Maratelli) seeds (obtained from Ente Nazionale Risi, Mortara, Italy)
were surface-sterilized by treatment for 30 min at room temperature
under vacuum in a 1% (v/v) solution of plant preservative mixture
(PPM, Plant Cell Technology, Inc., Washington, DC) containing 0.5
mM MgCl2, followed by 16 h in the same solution under shaking (80
rpm). Then they were allowed to germinate in a growth chamber at 25
( 1 °C under 16-h days (250 µmol m-2 s-1) and 8-h nights in Magenta
vessels on 50 mL of agarized (0.8 wt %/v) half-strength MS salts (17),
pH 6.5, containing 0.1% (v/v) PPM. Callus cultures were initiated from
excised cotyledons placed in Petri dishes onto agarized (0.8% wt/v)
R2 medium (18) supplemented with 20 g L-1 sucrose and 2 mg L-1
2,4-dichlorophenoxyacetic acid (2,4D). For the first 3 months calli were
cultured in complete darkness, with subcultures at 3-week intervals.
Then friable callus pieces were transferred into 0.5-L Erlenmeyer flasks
containing 100 mL of liquid MS medium supplemented with 30 g L-1
sucrose and 2 mg L-1 2,4D, and suspension cultures were incubated
under dim light on a rotary shaker (100 rpm). Subcultures were made
every 14 days by transferring 25-mL aliquots to 100 mL of fresh
medium.
To evaluate the effect of compound 3a on seedling growth, seeds
were germinated aseptically as described on agarized medium to which
the compound had been added as a 10 mg mL-1 solution in DMSO.
The experimental design was a randomized complete block with five
replicates. Each block comprised 12 vessels (each containing eight
seeds) of the following treatments: six rates (0, 1, 5, 10, 20, and 50
µg mL-1) and the corresponding volumes of DMSO. Destructive harvest
was carried out 10 days after germination. Four plants from each
treatment were combined; seed teguments were discarded, and the dry
weight was determined for each sample following drying in an oven
at 90 °C for 48 h. The effect of the same concentrations of compound
3a on exponentially growing cells was measured as described (19).
Briefly, cell samples withdrawn from stock cultures in the late
exponential phase of growth were used to inoculate 100-mL Erlenmeyer
flasks to a density ranging from 1.0 to 1.2 mg mL-1 (dry weight) in a
final volume of 25 mL. The compound was added just after cell density
reached 1.5 mg mL-1. After a further 10 days of incubation, when
untreated controls reached the early stationary phase of growth (∼4.0
mg mL-1), cells were harvested by vacuum filtration, and the dry weight
increase was determined as above. The data, expressed as percentage
of controls treated with DMSO alone, were analyzed by using standard
statistical procedures for analysis of variance and t test. Means were
On the whole, remarkable is the inhibition brought about by
all of the compounds, but mainly by compounds 2d, 2j, and
3a, at the lowest rate tested (10 µg mL-1), one at which
tricyclazole is ineffective. To prevent blast, this fungicide is

New Fungicides against Magnaporthe grisea
J. Agric. Food Chem., Vol. 50, No. 17, 2002 4843
Scheme 2
usually applied to rice plants at a concentration of 200 µg mL-1
.
Table 1. Fungicidal Activity of Compounds 1−3a
The possibility of using 20-fold lower rates might thus represent
a significant reduction in the environmental fallout of agricul-
tural practice. However, such a prospect relies upon the lack of
side effects that may reduce crop productivity. To rule out this
possibility, the phytotoxicity of the most powerful compound,
3a, was evaluated at both the undifferentiated cell and seedlings
levels. The rice cultivar employed, cv. Maratelli, was chosen
because it is highly sensitive to many strains of M. grisea.
Results, depicted in Figure 1, show that at concentrations >10
µg mL-1 the presence of compound 3a in the culture medium
exerted indeed a certain inhibitory effect upon rice growth. This
notwithstanding, in the range of 1-10 µg mL-1 the compound
was able to progressively control fungal proliferation without
any significant effect upon plant and cell culture growth.
The ability of several compounds to suppress completely the
development of M. grisea hyphae is noteworthy. Tricyclazole
is commonly used to prevent blast in rice fields. It acts by
inhibiting melanin synthesis in appressorial cells, thus weakening
fungal walls and avoiding plant colonization by the pathogen
(20). However, this fungicide is substantially ineffective when
applied on infected plants. On the contrary, the most active
compounds herein described even at lower concentrations might
hinder further pathogen spread in infected plants.
Up to now, no conclusive data have been available about
their mode of action. Due to structural similarities with
previously characterized azoles, one may suppose that they also
may inhibit the synthesis of ergosterol, a main component of
fungal membrane (21, 22), orslike tricyclazolesinhibit the
production of 1,8-dihydroxynaphthalene-derived melanins (7).
The latter hypothesis is strengthened by the results obtained in
vitro with compound 2j, which caused a strong depigmentation
of fungal mycelium (Figure 2). However, this is only indirect
evidence. Moreover, a primary effect upon melanin biosynthesis
µgmL-1
compd
1a
1b
1c
1d
2a
2b
2c
2d
2e
2h
2i
2j
2k
2l
2m
3a
3b
3c
3d
3e
3f
R
R′
R′′
X
10
50
100
200
t-Bu
Ph
3,4-ClPh CF3
CF3
CF3
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
S
S
S
S
S
S
S
S
O
S
O
S
O
S
O
O
O
O
O
S
O
O
25.0
23.0
52.0
32.0
nt
31.0
37.0
35.0
55.0
44.0
58.0
100
95.9
58.3
43.8
91.0 100
3-NO Ph CF3
61.0
nt
nt
78.0
42.7
29.6
2
t-Bu
CF3
CF3
Ph
nt
3,4-ClPh CF3
57.7
71.2
21.9
14.0
14.0
73.0
30.0
14.0
16.0
83.1
89.1
39.6
16.0
14.0
75.0
57.0
15.0
24.0
90.5 100
91.7 100
3-NO Ph CF3
2
3-NO Ph CF3
54.4
16.0
16.0
76.0
77.0
17.0
28.0
81.1
2
t-Bu
t-Bu
H
Ph
Ph
17.0
19.0
77.0
80.0
21.0
30.0
100
74.0
66.0
CH Ph Me
2
H
H
H
CH Ph Me
2
Ph
Ph
Ph
Me
Me
Me
92.4 100
100
4-BrPh Me
4-ClPh Me
CF3
CF3
Ph
47.0
17.0
64.5
48.4
67.0
15.0
0
47.0
58.0
66.0
99.5 100
79.7
73.1
15.0
61.5
62.0
97.9
79.6
70.2
15.0
23.5
Me
Me
H
93.2
75.1
15.0
94.0
3g
tricyclazole
Ph
Ph
a Percentage inhibition of the growth of M. grisea evaluated 5 days after
treatment; values are means of three trials made in triplicate, with SE never
exceeding 5%; nt ) not tested.

4844 J. Agric. Food Chem., Vol. 50, No. 17, 2002
Vicentini et al.
Scheme 3
and cultured cells at high concentrations suggests the possible
occurrence of either other targets or side effects involving other
pathways in cell metabolism. Experiments are currently in
progress to elucidate these aspects.
In any case, several of these compounds proved to possess a
remarkable effectiveness against fungal growth. For pyrazolo-
[3,4-d]pyrimidine and pyrazolo[3,4-d]thiazine derivatives (1, 2)
the structure-activity relationship is consistent with previous
data (11, 14) accounting for a critical role of a trifluoromethyl
group and thione function at positions 6 and 4, respectively
(Scheme 3). Moreover, the current results suggest a 3,4-
dichlorophenyl or 3-nitrophenyl at position 1 as a key structural
requirement for antifungal activity and can indicate that the
substitution of pyrimidine with a thiazine ring acts as amplifier
for the mycotoxic activity. In comparison with the series 1 and
2, the presence of a thiadiazine ring in 3 causes a strong increase
of activity. In particular, with respect to pyrazolo[1,5-c][1,3,5]-
thiadiazine derivatives, compound 3a (X ) O, R′ ) phenyl,
R′′ ) methyl) was found to give very good results. Contrary to
series 1 and 2, in this case the phenyl group and the one function
seem to cause an increase in activity (Scheme 3). The above
conclusions may provide researchers with a lead structure to
be further explored with the aim to develop new fungicides.
Figure 1. Comparison of the effects of compound 3a on the growth of
M. grisea (9) and O. sativa suspension cultured cells (2) or seedlings
(b). Mean values ± SD over at least four replications are reported.
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Received for review February 21, 2002. Revised manuscript received
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grants from the Ministero dell’Universita` e della Ricerca Scientifica e
Tecnologica (MURST) of Italy.
JF0202436