Pyrazole derivatives as photosynthetic electron transport inhibitors: New leads and structure - Activity relationship

Article abstract of DOI:10.1021/jf0500029

Four series of new pyrazoles, namely, 5 4-carboxypyrazolo-3-tert-butylcarboxamide and 6 4-carboxypyrazolo-3-cyclopropylcarboxamide derivatives and 10 pyrazolo[3,4-d][1,3]thiazine-4-one and 9 pyrazolo[3,4-d][1,3]thiazine-4-thione derivatives, were synthesized and screened as potential inhibitors of photosynthetic electron transport. The structures were confirmed by ¹H NMR, elemental, and IR analyses. Their biological activity was evaluated in vitro as the ability to interfere with the light-driven reduction of ferricyanide by isolated spinach chloroplasts. Only a few compounds exhibited excellent inhibitory properties in the micromolar range, comparable to those of commercial herbicides sharing the same target, such as diuron, lenacil, and hexazinone. Nevertheless, most of the remaining molecules exerted a remarkable inhibition in the millimolar range. Combined with previous results on 6 pyrazolo[1,5-a][1,3,5]triazine-2,4-dione and 4 pyrazolo[1,5-c][1,3,5]thiadiazine-2-one derivatives, these data allowed a comprehensive analysis of structure - activity relationship. Molecular modeling studies were undertaken to rationalize the structural determinants of activity in terms of shape, size, and molecular fields. Results suggested that the inhibitory potential of these compounds is associated mainly with their electrostatic properties.

Full text of DOI:10.1021/jf0500029

3848 J. Agric. Food Chem. 2005, 53, 3848−3855
Pyrazole Derivatives as Photosynthetic Electron Transport
Inhibitors: New Leads and Structure Activity Relationship
−
CHIARA B. VICENTINI,† SALVATORE GUCCIONE,# LAURA GIURATO,#
REBECCA CIACCIO,# DONATELLA MARES,‡ AND GIUSEPPE FORLANI*,§
Dipartimento di Scienze Farmaceutiche, Dipartimento delle Risorse Naturali e Culturali, and
Dipartimento di Biologia, Universita` di Ferrara, Italy; and Dipartimento di Scienze Farmaceutiche,
Universita` di Catania, Italy
Four series of new pyrazoles, namely, 5 4-carboxypyrazolo-3-tert-butylcarboxamide and 6 4-car-
boxypyrazolo-3-cyclopropylcarboxamide derivatives and 10 pyrazolo[3,4-d][1,3]thiazine-4-one and
9 pyrazolo[3,4-d][1,3]thiazine-4-thione derivatives, were synthesized and screened as potential
inhibitors of photosynthetic electron transport. The structures were confirmed by 1H NMR, elemental,
and IR analyses. Their biological activity was evaluated in vitro as the ability to interfere with the
light-driven reduction of ferricyanide by isolated spinach chloroplasts. Only a few compounds exhibited
excellent inhibitory properties in the micromolar range, comparable to those of commercial herbicides
sharing the same target, such as diuron, lenacil, and hexazinone. Nevertheless, most of the remaining
molecules exerted a remarkable inhibition in the millimolar range. Combined with previous results on
6 pyrazolo[1,5-a][1,3,5]triazine-2,4-dione and 4 pyrazolo[1,5-c][1,3,5]thiadiazine-2-one derivatives,
these data allowed a comprehensive analysis of structure-activity relationship. Molecular modeling
studies were undertaken to rationalize the structural determinants of activity in terms of shape, size,
and molecular fields. Results suggested that the inhibitory potential of these compounds is associated
mainly with their electrostatic properties.
KEYWORDS: Herbicides; photosynthetic electron transport inhibitors; pyrazole derivatives; structure-
activity relationship
INTRODUCTION
switching from the evaluation of in planta herbicidal efficacy
to target-based in vitro assays (4, 5).
Several reasons exist today for developing new weed control
systems. Pesticides should have a favorable combination of
properties, such as high specific activity, low application rates,
crop tolerance, and low mammalian toxicity. Increasing public
concern for environmental pollution derived from agricultural
practices also requires that herbicides be rapidly degraded by
soilborne microorganisms. Moreover, during the past decade,
intensive and repeated applications of the same active ingredients
caused the selection for and development of herbicide resistance
(1). In most cases, resistance is due to an altered target site (2).
Herbicide-resistant weeds often show a wide pattern of cross-
resistance to both structurally similar herbicides and other
phytochemicals sharing the same target (3). Intensive efforts
have thus been undertaken to discover new compounds with
favorable environmental and safety features to selectively control
weeds. Recently, this aim has been pursued with new strategies,
Many pyrazole derivatives with promising herbicidal proper-
ties have been discovered. Among the active principles already
introduced in the agricultural practice are the inhibitor of
protoporphyrinogen oxidase pyraflufen-ethyl (HRAC group E)
(6), the bleaching herbicides pyrazolynate, pyrazoxyfen, and
benzofenap, which act through the inhibition of 4-hydroxy-
phenylpyruvate dioxygenase (HRAC group F2), the branched-
chain amino acid inhibitors, the sulfonylureas halosulfuron-
methyl, pyrazosulfuron-ethyl, and azimsulfuron (HRAC group
B), the inhibitor of cell division, metazachlor (HRAC group
K3), and the unknown target herbicide difenzoquat (HRAC
group Z). Moreover, pyrimidine-2,4-dione derivatives, including
uracils such as lenacil, terbacil, bromacil, and 1,3,5-triazine-
2,4-dione derivatives, including the triazolinone hexazinone,
have been characterized as photosynthetic electron transport
inhibitors (HRAC group C1) (4, 5).
* Author to whom correspondence should be addressed [fax (39)0532-
249761; e-mail flg@unife.it].
A large number of herbicides act by inhibiting chloroplast
electron transport. In most cases the molecular target was found
to be the plastoquinone-binding site on the D1 protein in the
photosystem II (PSII) reaction center (7). Tight binding to this
domain leads to a block in NADPH production, which is
required for CO2 fixation. This, in turn, induces the formation
† Dipartimento di Scienze Farmaceutiche, via Fossato di Mortara 17-
19, Ferrara.
# Dipartimento di Scienze Farmaceutiche, v.le A. Doria 6, Ed. 2, Catania.
‡ Dipartimento delle Risorse Naturali e Culturali, c.so Porta Mare 2,
Ferrara.
§ Dipartimento di Biologia, via L. Borsari 46, Ferrara, Italy.
10.1021/jf0500029 CCC: $30.25
© 2005 American Chemical Society
Published on Web 04/21/2005

PSII-Inhibiting Pyrazole Derivatives
J. Agric. Food Chem., Vol. 53, No. 10, 2005 3849
Scheme 1
of free oxygen radicals, which cause photooxidation of chloro-
phylls and lipids, thus breaking down thylakoid integrity, which
is strictly required for chloroplast functionality (8). A renewed
interest in the development of further compounds sharing this
mode of action is witnessed by numerous papers recently
published on this subject. A group of 3-carboxamido-4-
isoxazolecarboxylic acids (1 and 2) (Scheme 1) was found to
exhibit herbicidal properties, and some of them in the micro-
molar range were able to inhibit the Hill reaction of thylakoids
from wheat (9). Herbicidal amino- and urea-substituted thiazoles
also appear to act via PSII interference. Their relative inhibitory
activity showed that lipophilicity, as modulated by the length
of the hydrocarbon side chain, has a great impact on in vitro
effectiveness. Isopropyl and n-butyl side chains conferred
maximal activity, and this was interpreted as a steric requirement
to reach the QB binding site in the thylakoidal membrane.
However, some discrepancies were found between the results
of in vitro and in vivo tests (10). Also, in the case of herbicidal
2-hydroxy-3-alkyl-1,4-naphthoquinones the length of the 3-n-
alkyl substituent for optimal activity differed between in planta
and in vitro activities (11). Both 2-cyano-3-substituted-py-
ridinemethylaminoacrylates (12) and 2-cyano-3-(2-chlorothiazol-
5-yl)methylaminoacrylates (13), some of which showed excel-
lent herbicidal activities at doses as low as 75 g ha-1, were
found to inhibit PSII electron transport. In a previous paper,
we also reported on some pyrazolo[1,5-a][1,3,5]triazine-2,4-
dione (5) and pyrazolo[1,5-c][1,3,5]thiadiazine-2-one (6) deriva-
tives that exhibited remarkable inhibitory properties at the PSII
level, comparable to those of the reference commercial herbi-
cides lenacil, diuron, and hexazinone (14).
new active principles. In this view, a re-evaluation of those
compounds that had previously shown an ability to interfere
with the photosynthetic electron transport chain may also
provide researchers with useful information.
Here we report the synthesis of some pyrazolo[3,4-d][1,3]-
thiazin-4-one/thione derivatives (3 and 4) and the screening for
the ability to inhibit the Hill reaction in isolated spinach
chloroplasts of compounds 1-4, in comparison with those
compounds already synthesized and tested (5 and 6) (14).
Results, combined with previous data and supported by mo-
lecular modeling, allowed us a thorough analysis of structure-
activity relationships. This led to the definition of molecular
properties, in terms of shape, electronics, and hydrophobic
complementarity to the macromolecular site, which are required
for the design and synthesis of new active principles.
MATERIALS AND METHODS
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-
eter; chemical shifts (δ) are given in parts per million relative to the
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 silica gel (70-230 mesh); for
the flash chromatography technique, silica gel (230-400 mesh) was
employed.
Synthesis. The 3-carboxamido-4-pyrazolecarboxylic acid (1n-r,
2n-s) and 3-carboxamido-4-isoxazolecarboxylic acid (1m, 2m) (16,
17) and the pyrazolo[1,5-a][1,3,5]triazine-2,4-dione (5e-l) and the
pyrazolo[1,5-c][1,3,5]thiadiazine-2-one (6g-j) (14) derivatives were
prepared according to previously reported procedures.
N-Alkyl/aryl-N′-(4-carboethoxy-3-pyrazolyl)thioureas (8a-j). A mix-
ture of 3-amino-4-carboethoxypyrazole 7 (3.10 g, 20 mmol) and the
appropriate isothiocyanate in toluene (20 mL) was heated under reflux
for 6 h and then cooled. N-Substituted-N′-(4-carboethoxy-3-pyrazolyl)-
thioureas 8a-f,i precipitated spontaneously and were collected as pure
products. After removal of solvent, the residue was shown to be a
mixture of products, and N′-(3-pyrazolyl) thioureas 8g,h,j were isolated
after purification by silica gel chromatography as byproducts.
By using this procedure the following compounds were obtained:
8a (R ) 3-trifluoromethylphenyl): yield, 75%; mp, 169.3-169.7 °C;
IR (KBr, cm-1), νmax 3351, 1666, 1589, 1534; 1H NMR (DMSO-d6), δ
Until recently, a structure-activity relationship analysis that
allowed a rational molecular design of new PSII-inhibiting
herbicides by structure modeling was limited because a suf-
ficiently high-resolution X-ray structure of photosystem II was
not available. The reaction center of purple photosynthetic
bacteria, which is homologous to PSII, served as a model, but
increasing evidence showed that there is significant variation
between the two QB sites. However, the structure of PSII of
the cyanobacterium Thermosynechococcus elongatus has been
recently described at 3.5 Å resolution, revealing its molecular
architecture and details of the binding sites for cofactors (15).
This result opens up highly interesting prospects of developing

3850 J. Agric. Food Chem., Vol. 53, No. 10, 2005
Vicentini et al.
1.30 (t, 3H, Me, J ) 7.2 Hz), 4.29 (q, 2H, CH2, J ) 7.2 Hz), 7.61-
8.15 (m, 4H, Ph), 8.45 (s, 1H, CH), 9.61 (s, 1H, NH), 11.55 (s, 1H,
NH), 13.53 (s, 1H, NH).
3d (R ) 3-chlorophenyl): yield, 98%; mp, 241-243 °C; IR (KBr,
1
cm-1), νmax 3069, 1673, 1567, 1541; H NMR (DMSO-d6), δ 7.14-
7.62 (m, 4H, Ph), 7.96 (s, 1H, CH), 10.70 (br, 1H, NH), 13.69 (br, 1H,
NH).
8b (R ) 4-bromophenyl): yield, 87%; mp, 211-213.8 °C; IR (KBr,
1
cm-1), νmax 3220, 1663, 1575, 1539; H NMR (DMSO-d6), δ 1.30 (t,
3e (R ) 2-chlorophenyl): yield, 76%; mp, 213-215 °C; IR (KBr,
1
3H, Me, J ) 7.2 Hz), 4.29 (q, 2H, CH2, J ) 7.2 Hz), 7.56-7.62 (m,
4H, Ph), 8.45 (s, 1H, CH), 9.54 (s, 1H, NH), 11.45 (s, 1H, NH), 13.51
(s, 1H, NH).
cm-1), νmax 3071, 1693, 1560, 1531; H NMR (DMSO-d6), δ 7.35-
7.62 (m, 4H, Ph), 7.92 (s, 1H, CH), 10.50 (br, 1H, NH), 13.52 (br, 1H,
NH).
3f (R ) 4-nitrophenyl): yield, 39%; mp, 208-210 °C; IR (KBr,
8c (R ) 4-chlorophenyl): yield, 78%; mp, 194-196 °C; IR (KBr,
1
cm-1), νmax 3401, 3312, 1662, 1537, 1495; H NMR (DMSO-d6), δ
1
cm-1), νmax 3220, 1662, 1575, 1537; H NMR (DMSO-d6), δ 1.30 (t,
8.01-8.27 (m, 4H, Ph), 8.15 (s, 1H, CH), 11.06 (br, 1H, NH), 13.70
3H, Me, J ) 7.0 Hz), 4.30 (q, 2H, CH2, J ) 7.0 Hz), 7.44-7.70 (m,
4H, Ph), 8.45 (s, 1H, CH), 9.54 (s, 1H, NH), 11.45 (s, 1H, NH), 13.51
(s, 1H, NH).
(br, 1H, NH).
3g (R ) ethyl): yield, 70%; mp, 246-249.7 °C; IR (KBr, cm-1),
ν
max 3310, 1672, 1574, 1544; 1H NMR (DMSO-d6), δ 1.16 (t, 3H, Me,
8d (R ) 3-chlorophenyl): yield, 84%; mp, 183.5-184.2 °C; IR (KBr,
1
cm-1), νmax 3272, 1660, 1583, 1537; H NMR (DMSO-d6), δ 1.30 (t,
J ) 7.2 Hz), 3.34-3.46 (m, 2H, CH2), 7.86 (s, 1H, CH), 8.66 (br, 1H,
NH), 13.33 (br, 1H, NH).
3H, Me, J ) 7.2 Hz), 4.30 (q, 2H, CH2, J ) 7.2 Hz), 7.28-7.90 (m,
4H, Ph), 8.45 (s, 1H, CH), 9.56 (s, 1H, NH), 11.50 (s, 1H, NH), 13.53
(s, 1H, NH).
3h (R ) butyl): yield, 87%; mp, 175.7-177.3 °C; IR (KBr, cm-1),
1
νmax 3312, 1660, 1542; H NMR (DMSO-d6), δ 0.89 (t, 3H, Me, J )
7.2 Hz), 1.30-1.58 (m, 4H, 2CH2), 3.32-3.42 (m, 2H, CH2), 7.86 (s,
8e (R ) 2-chlorophenyl): yield, 80%; mp, 173.8-176 °C; IR (KBr,
1
cm-1), νmax 3275, 1656, 1587, 1539; H NMR (DMSO-d6), δ 1.31 (t,
1H, CH), 8.65 (br, 1H, NH), 13.25 (br, 1H, NH).
3i (R ) benzyl): yield, 30%; mp, 154.3-155.9 °C; IR (KBr, cm-1),
3H, Me, J ) 7.2 Hz), 4.30 (q, 2H, CH2, J ) 7.2 Hz), 7.60-8.10 (m,
4H, Ph), 8.46 (s, 1H, CH), 9.63 (s, 1H, NH), 11.49 (s, 1H, NH), 13.56
(s, 1H, NH).
ν
max 3192, 1677, 1551; 1H NMR (DMSO-d6), δ 4.60 (d, 2H, CH2, J )
5.8 Hz), 7.23-7.35 (m, 5H, Ph), 7.86 (s, 1H, CH), 9.19 (br, 1H, NH),
13.34 (br, 1H, NH).
8f (R ) 4-nitrophenyl): yield, 98%; mp, 211-213 °C; IR (KBr,
1
cm-1), νmax 3247, 1656, 1595, 1539; H NMR (DMSO-d6), δ 1.30 (t,
3j (R ) cyclohexyl): yield, 84%; mp, 169.4 °C; IR (KBr, cm-1),
1
3H, Me, J ) 7.2 Hz), 4.28 (q, 2H, CH2, J ) 7.2 Hz), 8.02-8.30 (m,
4H, Ph), 8.45 (s, 1H, CH), 9.71 (s, 1H, NH), 11.73 (s, 1H, NH), 13.56
(s, 1H, NH).
νmax 3254, 2930, 1724, 1645; H NMR (DMSO-d6), δ 1.10-1.92 (m,
10H, cyclohexyl), 3.89 (m, 1H, cyclohexyl), 7.84 (s, 1H, CH), 8.55
(d, 1H, NH, J ) 7.4 Hz), 13.25 (br, 1H, NH).
8g (R ) ethyl): yield, 8%; mp, 133 °C (purified by column
Synthesis of 6-Substituted Pyrazolo[3,4-d][1,3]thiazin-4-thiones (4a-
j). The pertinent 6-substituted pyrazolo[3,4-d][1,3]thiazin-4-one 3a-j
(2 mmol) and phosphorus pentasulfide (0.49 g, 2.2 mmol) were
vigorously refluxed under stirring in anhydrous xylene (24 mL) for 7
h. After cooling, the precipitate was collected and purified by column
chromatography.
chromatography; eluent, ethyl acetate/petroleum ether, 3:7, 8:2; IR (KBr,
1
cm-1), νmax 3244, 1659, 1578, 1539; H NMR (DMSO-d6), δ 1.16-
1.33 (m, 6H, 2Me), 3.58-3.64 (m, 2H, CH2,), 4.27 (q, 2H, CH2, J )
7.2 Hz), 8.38 (s, 1H, CH), 9.25 (s, 1H, NH), 9.66 (br t, 1H, NH), 13.33
(s, 1H, NH).
8h (R ) butyl): yield, 7%; mp, 100-102 °C (purified by column
chromatography; eluent, ethyl acetate/petroleum ether, 2:8, 3:7, 8:2);
IR (KBr, cm-1), νmax 3247, 1663, 1588, 1540; 1H NMR (DMSO-d6), δ
0.91 (t, 3H, Me, J ) 7.2 Hz), 1.24-1.62 (m, 7H, Me + 2CH2), 3.55-
3.64 (m, 2H, CH2), 4.27 (q, 2H, CH2, J ) 7.2 Hz), 8.38 (s, 1H, CH),
9.26 (s, 1H, NH), 9.71 (t, 1H, NH, J ) 5.1 Hz), 13.34 (s, 1H, NH).
By using this procedure the following compounds were obtained:
4a (R ) 3-trifluoromethylphenyl): yield, 45%; mp, 285-287 °C
(purified by column chromatography; eluent ethyl acetate/petroleum
ether, 1:1); IR (KBr, cm-1), νmax 3083, 1552, 1489; 1H NMR (DMSO-
d6), δ 7.48-8.02 (m, 4H, Ph), 8.16 (s, 1H, CH), 11.02 (br, 1H, NH),
13.83 (br, 1H, NH).
8i (R ) benzyl): yield, 40%; mp, 184.3-191.2 °C; IR (KBr, cm-1),
4b (R ) 4-bromophenyl): yield, 55%; mp, 298-300 °C (purified
by column chromatography, eluent ethyl acetate:/petroleum ether, 3:7);
IR (KBr, cm-1), νmax 3051, 1562, 1487; 1H NMR (DMSO-d6), δ 7.56-
7.73 (m, 4H, Ph), 8.10 (s, 1H, CH), 10.87 (br, 1H, NH), 13.76 (br, 1H,
NH).
1
νmax 3289, 1656, 1625, 1583, 1542; H NMR (DMSO-d6), δ 1.29 (t,
3H, Me, J ) 7.0 Hz), 4.27 (q, 2H, CH2, J ) 7.0 Hz), 4.87 (d, 2H,
CH2, J ) 5.6 Hz), 7.30-7.37 (m, 5H, Ph), 8.37 (s, 1H, CH), 9.38 (s,
1H, NH), 10.04 (t, 1H, NH, J ) 5.4 Hz), 13.32 (s, 1H, NH).
8j (R ) cyclohexyl): yield, 8%; mp, 141-141.6 °C (purified by
column chromatography, eluent ethyl acetate/petroleum ether, 3:7, 8:2);
IR (KBr, cm-1), νmax 3244, 2931, 1669, 1585, 1539; 1H NMR (DMSO-
d6), δ 1.25-1.98 (m, 13H, Me + cyclohexyl), 4.13 (m, 1H, CH,
cyclohexyl), 4.27 (q, 2H, CH2, J ) 7.2 Hz), 8.38 (s, 1H, CH), 9.22 (s,
1H, NH), 9.74 (d, 1H, NH, J ) 7.8 Hz), 13.36 (s, 1H, NH).
Synthesis of 6-Substituted Pyrazolo[3,4-d][1,3]thiazin-4-ones (3a-
j). A solution of 8 (0.6 g) in 98% sulfuric acid (4 mL) was stirred at
room temperature for 4 days and then poured into ice water. The
precipitate was collected and purified by column chromatography, eluent
ethyl acetate/petroleum ether 1:1.
4c (R ) 4-chlorophenyl): yield, 55%; mp, 158-162 °C (purified
by column chromatography, eluent ethyl acetate:/petroleum ether 3:7);
IR (KBr, cm-1), νmax 2917, 1560, 1491; 1H NMR (DMSO-d6), δ 7.44-
7.78 (m, 4H, Ph), 8.10 (s, 1H, CH), 10.88 (br, 1H, NH), 13.77 (br, 1H,
NH).
4d (R ) 3-chlorophenyl): yield, 35%; mp, 215-219 °C (purified
by column chromatography, eluent ethyl acetate/petroleum ether: 2:8);
IR (KBr, cm-1), νmax 2918, 1598, 1560, 1492; 1H NMR (DMSO-d6), δ
7.20-7.97 (m, 4H, Ph), 7.98 (s, 1H, CH), 10.89 (br, 1H, NH), 13.83
(br, 1H, NH).
4e (R ) 2-chlorophenyl): yield, 35%; mp, 162-167 °C (purified
by column chromatography; eluent ethyl acetate/petroleum ether 3:7,
8:2); IR (KBr, cm-1), νmax 3319, 1562, 1539, 1496; 1H NMR (DMSO-
d6), δ 7.36-7.62 (m, 4H, Ph), 8.07 (s, 1H, CH), 10.72 (br, 1H, NH),
13.60 (br, 1H, NH).
By using this procedure the following compounds were obtained:
3a (R ) 3-trifluoromethylphenyl): yield, 80%; mp, 239-241 °C;
IR (KBr, cm-1), νmax 3095, 1669, 1557; 1H NMR (DMSO-d6), δ 7.46-
8.07 (m, 4H, Ph), 8.17 (s, 1H, CH), 10.87 (br, 1H, NH), 13.75 (br, 1H,
NH).
4g (R ) ethyl): yield, 70%; mp, 109-112 °C (purified by column
3b (R ) 4-bromophenyl): yield, 80%; mp, 253-255 °C; IR (KBr,
chromatography, eluent ethyl acetate/petroleum ether 1:1); IR (KBr,
1
cm-1), νmax 3406, 3312, 1658, 1608, 1560, 1531; H NMR (DMSO-
1
cm-1), νmax 3207, 3279, 1560; H NMR (DMSO-d6), δ 1.16 (t, 3H,
d6), δ 7.52-7.75 (m, 4H, Ph), 7.97 (s, 1H, CH), 10.66 (br, 1H, NH),
13.66 (br, 1H, NH).
Me, J ) 7.2 Hz), 3.37-3.44 (m, 2H, CH2), 8.01 (s, 1H, CH), 8.94 (br,
1H, NH), 12.40 (v br, 1H, NH).
3c (R ) 4-chlorophenyl): yield, 79%; mp, 223-225 °C; IR (KBr,
cm-1), νmax 3056, 1669, 1538; 1H NMR (DMSO-d6), δ 7.41-7.82 (m,
4H, Ph), 8.04 (s, 1H, CH), 10.64 (br, 1H, NH), 13.63 (br, 1H, NH).
4h (R ) butyl): yield, 77%; mp, 193.2 °C (purified by flash column
chromatography; eluent ethyl acetate/petroleum ether 2:8, 1:1); IR (KBr,
1
cm-1), νmax 3173, 2905, 1558, 1530; H NMR (DMSO-d6), δ 0.90 (t,

PSII-Inhibiting Pyrazole Derivatives
J. Agric. Food Chem., Vol. 53, No. 10, 2005 3851
3H, Me, J ) 7.2 Hz), 1.23-1.62 (m, 4H, 2CH2), 3.33-3.39 (m, 2H,
CH2), 8.01 (s, 1H, CH), 8.92 (br, 1H, NH), 13.44 (br, 1H, NH).
4i (R ) benzyl): yield, 83%; mp, 195-198 °C (purified by column
chromatography; eluent ethyl acetate/petroleum ether 3:7); IR (KBr,
yphenyl)-1-phenylpyrazole (database entry: BIRFOX) were exported
to .mol2 files, read, and modified by the fragment library in the software
MACROMODEL (version. 8.1) as implemented in the MAESTRO suite
(21, 22) and used as the building blocks for compounds 1-4, whereas
the atrazine structure as extracted from the pdb entry 1DXR was used
as the building block for compounds 5 and 6. After each export, the
atom potential types and bond orders were carefully checked to evaluate
their correctness with respect to the intended structure prior to the
Macromodel (21, 22), SPARTAN (23), and HINT (24) sessions (e.g.,
compound 3f, where the NO2 needed to be set in SPARTAN as
dianionic). The .mol2 files were saved as .prj files for MACROMODEL,
and a minimization (2000 steps, convergence 0.001) was carried out
using the default static Merck Molecular Force Field as implemented
in MACROMODEL 8.1 (21, 22). Default options were used with the
Polak-Ribiere Conjugate Gradient scheme, until a gradient of 0.001
kcal Å-1 was reached. To search the conformational space, 2000MC
steps were performed on each starting conformation. Least-squares
superposition of all non-hydrogen atoms was used to eliminate duplicate
conformations. An energy cutoff of 12.5 kcal mol-1 (50.0 kJ mol-1),
high enough to map the conformational space of these rigid molecules
including the bioactive conformation, was applied to the search results.
Aqueous and in vacuo conformational analyses were performed using
the Monte Carlo Multiple Minimum Search protocol as implemented
in the MACROMODEL (21, 22) software. Because in the Monte Carlo
approach the dynamic of a molecule is simulated by randomly changing
dihedral angle rotations or atom positions, the trial conformation is
accepted if its energy has decreased from the previous one. If the energy
level is higher, there are various criteria to select the calculated
conformer or not. In our simulations, all of the dihedral angles of single
linear bonds were allowed to move freely, and the conformation was
accepted if the energy was lower than that of the previous conformation
or within a fixed energy window as selector. The lowest energy
conformers were further optimized using the multiple minimization
protocol as implemented in MACROMODEL 8.1 (21, 22). The minima
were exported to .mol2 and saved as .spinput files for the SPARTAN
(log Ps and electrostatic potential calculations) using the semiempirical
model RHF/PM3/MMFF94s as in SPARTAN (23). Slightly different
values but a similar trend were obtained using the semiempirical model
RHF/AM1/MMFF94s as in SPARTAN (23). Compound (2p) was
calculated in its negatively charged (carboxylate) form, assuming it
was similar to that in the biological medium. Electrostatic potential
ramp (kcal/mol) was observed to change from red (more negative
values) to blue (higher values). The .mol2 files were used as such for
the HINT (log P calculations and hydropathicity maps) (24) calculations,
for which all of the HINT settings were left as default.
1
cm-1), νmax 3182, 1576, 1554; H NMR (DMSO-d6), δ 4.63(d, 2H,
CH2, J ) 5.4 Hz), 7.21-7.45 (m, 5H, Ph), 8.03 (s, 1H, CH), 9.42 (br,
1H, NH), 13.50 (br, 1H, NH).
4j (R ) cyclohexyl): yield, 34%; mp, 193-196 °C (purified by flash
column chromatography; eluent ethyl acetate/petroleum ether 3:7); IR
1
(KBr, cm-1), νmax 3223, 2927, 1554, 1487; H NMR (DMSO-d6), δ
1.15-2.15 (m, 10H, cyclohexyl), 4.17 (m, 1H, cyclohexyl), 7.98 (s,
1H, CH), 8.93 (br d, 1H, NH), 13.43 (br, 1H, NH).
Biological Tests. EValuation of Pyrazole DeriVatiVes as Inhibitors
of the Photosynthetic Electron Transport by the Hill Reaction.
Photosynthetically active thylakoid membranes were isolated from
market spinach (Spinacea oleracea L.) leaves. Deveined plant material
was resuspended in 5 mL g-1 of ice-cold 20 mM N-tris(hydroxymethyl)-
methylglycine (Tricine)-NaOH buffer (pH 8.0) containing 10 mM
NaCl, 5 mM MgCl2, and 0.4 M sucrose and homogenized for 30 s in
a blender at maximal speed. The homogenate was filtered through
surgical gauze, and the filtrate was centrifuged at 4 °C for 1 min at
500g; the supernatant was further centrifuged for 10 min at 1500g.
Pelleted chloroplasts were osmotically swollen by resuspension in
sucrose-lacking buffer. The suspension was immediately diluted 1:1
with sucrose-containing buffer, kept on ice in the dark, and used within
a few hours from the preparation. Following proper dilution with 80%
(v/v) acetone, the absorbance of each sample was determined at 645
and 663 nm, and the chlorophyll content was calculated on the basis
of Arnon’s formula. The rate of photosynthetic electron transport was
measured by the following light-driven ferricyanide reduction. Aliquots
of membrane preparations corresponding to 20 µg of chlorophyll were
incubated at 24 °C in 1-mL cuvettes containing 20 mM Tricine-NaOH
buffer (pH 8.0), 10 mM NaCl, 5 mM MgCl2, 0.2 M sucrose, and 1
mM K3Fe(CN)6. The assay was initiated by exposure to saturating light
(800 µmol m-2 s-1), and the rate of ferricyanide reduction was measured
at 30-s intervals for 10 min against an exact blank at 420 nm. Activity
was calculated over the linear portion of the curve from a molar
extinction coefficient of 1000 M-1 cm-1. Under the adopted conditions,
the value for untreated controls was 65.8 ( 2.0 nmol of ferricyanide
reduced s-1 (mg of chlorophyll)-1. Pyrazole derivatives were dissolved
in 0.1 M NaOH, and then the pH was brought to a value ranging from
8.0 to 8.5 with 1 M HCl; when appropriate, the solutions were further
diluted with water. Their effect upon oxygen evolution was evaluated
in parallel assays in which the compounds were added to the reaction
mixture at concentrations ranging from 100 nM to 1 mM. Each dose
was carried out at least in triplicate, and results were expressed as
percentage of untreated controls. Reference herbicides were initially
dissolved in water (hexazinone and lenacil) or acetone (diuron) and
then water-diluted; in the case of the latter compound, controls received
the same amounts of solvent. The concentrations causing 50% inhibition
(IC50) of in vitro activity were estimated utilizing the linear regression
equation of activity values plotted against the logarithm of inhibitor
concentration. At least four doses in the inhibitory range were
considered. Confidence limits were computed according to the method
of Snedecor and Cochran (18).
RESULTS AND DISCUSSION
Synthesis. The preparative route to the target products 3a-j
and 4a-j is outlined in Scheme 2. N-Alkyl/aryl-N′-(4-carboet-
hoxy-3-pyrazolyl)thioureas 8 were obtained by reaction of
3-amino-4-carboethoxypyrazole 7 with the appropriate isothio-
cyanate. High yields of 8 have been obtained in the reaction of
7 with arylisothiocyanates, but, in the reaction of 7 with ethyl,
butyl, and cyclohexyl isothiocyanate, the N′-(3-pyrazolyl)-
thioureas 8g,h,j were isolated after purification by silica gel
chromatography as byproducts. Cyclization of 8 with 98%
sulfuric acid provided the required 6-substituted pyrazolo-[3,4-
d][1,3]thiazin-4-ones 3. Thiation of 3 with P2S5 gave the
corresponding 6-substituted pyrazolo-[3,4-d][1,3]thiazin-4-
thiones 4.
Biological Activity. 4-Carboxypyrazolo-3-tert-butylcarboxa-
mide and 4-carboxypyrazolo-3-cyclopropylcarboxamide deriva-
tives (1 and 2) were synthesized on the basis of results
previously described, concerning the biological activity of
isoxazoledicarboxylic acid derivatives (9). Among the com-
pounds tested in that study, some 4-carboxyisoxazole-3-car-
boxamides had been reported as potent inhibitors of the
photosynthetic electron transport at the PSII level, with IC50
Molecular Modeling and Analysis of Structure-Activity Rela-
tionship. The Cambridge Structural Database (CSD) search (version
5.25, November 2003; update July 3, 2004) and structure retrieval was
run on a PC working under Windows XP by the software CONQUEST
(version 1.6) (19, 20). All of the conformational and modeling studies
were carried out on SGI-OCTANE and -R-5000 O2 workstations
operating under IRIX 6.5.+ using the software Macromodel (version
8.1) as implemented in the version 5.1 of the MAESTRO suite (21,
22), SPARTAN (version 2002) (23), and HINT2.35S (24) as imple-
mented in SYBYL 6.9 (25). During the review process a new version
of SYBYL (7.0 update 1), comprehensive of the new HINT 3.09S,
was released. Provided that nothing is conceptually changed, slight
numerical differences can be registered with respect to the HINT log
P values reported in this paper.
The CSD files of dimethyl-1-(3-chloro-4-methylphenyl)pyrazole-3,4-
dicarboxylate (database entry: BANQUD) and 4-cyano-3-(4-methox-

3852 J. Agric. Food Chem., Vol. 53, No. 10, 2005
Vicentini et al.
Scheme 2
Table 1. In Vitro Effects of 4-Carboxypyrazolo-3-tert-butylcarboxamide
and 4-Carboxypyrazolo-3-cyclopropylcarboxamide Derivatives on
Ferricyanide Reduction by Functionally Intact Chloroplasts Isolated
from S. oleracea Leavesa
values ranging from 0.3 to 30 µM. In fact, the reference
compounds 4-carboxyisoxazole-3-isopropylcarboxamide (2m)
and 4-carboxyisoxazole-3-tert-butylcarboxamide (1m) were
found to interfere with the light-driven ferricyanide reduction
by isolated spinach thylakoids. Under the experimental condi-
tions employed, they showed a lower efficiency, with IC50 values
of 100 and 290 µM, respectively (Table 1). Nevertheless, the
replacement of the carboxyisoxazole moiety with a carboxy-
pyrazole (as in compound 2s) resulted in a loss of activity.
Interestingly, however, the insertion of substituents in position
1 restored at least in part the ability to interfere with the Hill
reaction. The methyl (1n and 2n) and p-chlorophenyl (1q and
2q) derivatives showed maximal activity, with IC50 values
ranging from 0.4 to 1.2 mM, whereas the presence of a tert-
butyl substituent (1o and 2o) was ineffective (Table 1). Due to
the higher concentration needed for Hill reaction inhibition, these
compounds seem not to be satisfactory for use as agrochemicals.
More promising results were obtained with the other two
groups of pyrazole derivatives. In previous work, we described
the features of a few pyrazolo[1,5-a][1,3,5]triazine-2,4-dione
(5) and pyrazolo[1,5-c][1,3,5]thiadiazine-2-one (6) derivatives,
some of which showed excellent in vitro inhibitory activity
against the PSII, comparable to those of commercial reference
herbicides (14). The potential of a number of new pyrazolo-
[3,4-d][1,3]thiazine-4-ones and pyrazolo[3,4-d][1,3]thiazine-4-
thiones was then investigated. All compounds tested were found
to inhibit ferricyanide reduction by spinach thylakoid mem-
branes, with IC50 values ranging in most cases from 10-6 to
10-4 M (Tables 2 and 3). As to the analysis of the scaffold
effect (thiazine-4-one versus thiazine-4-thione), the comparison
between compounds that have exactly the same substituents
strongly suggested that the replacement of the oxygen moiety
with sulfur acts to amplify the inhibitory effect. In general, the
IC50 for a given pyrazolo[3,4-d][1,3]thiazine-4-thione (Table
3) was, in fact, 2-100-fold lower than that of its thiazine-4-
one counterpart (Table 2). In the latter group, maximal activity
was shown by the compound bearing the benzyl substituent (3i),
with an IC50 of 11 µM. Among thiazine-4-thiones, more than
half of the compounds (4a,b,h-j) showed IC50 values lower
than 10 µM, and maximal efficacy was related to the presence
of a cyclohexyl substituent (4j). The efficacy of 4j in inhibiting
the Hill reaction (IC50 ) 0.64 µM) is comparable to those
a Activity was measured as described under Materials and Methods either in
the absence or in the presence of pyrazole derivatives at concentrations ranging
from 30
µM to 1 mM. Each sample was carried out at least in triplicate, and
values were expressed as percentage of untreated controls. b The concentrations
causing 50% inhibition (IC50) of in vitro activity were estimated as described under
Materials and Methods utilizing the linear regression equation of the activity values
plotted against the logarithm of inhibitor concentration. Confidence limits were
computed according to the method of Snedecor and Cochran (18).
previously found (14) for commercial herbicides, such as diuron,
lenacil, and hexazinone (IC50 ) 0.27, 0.08 and 0.11 µM,
respectively). The positive effect of the sulfur moiety might be
interpreted as a function of the corresponding increase in
lipophilicity. However, this may not be the case, because
lipophilicity does not appear to be a general descriptor of the
activity (vide infra). Alternatively, a slightly different target
might be supposed for each chemotype.
Because the method employed, which measures the reduction
of an electron acceptor (the ferricyanide) downstream from PSI,
does not distinguish the exact target in the electron transport
chain, further trials were performed with the two most active
compounds (4j and 3i) by the addition of 2 µM 2,5-dibromo-
3-methyl-6-isopropyl-p-benzoquinone, a cytochrome b6f inhibi-
tor. The subsequent inclusion in the reaction mixture of 0.1 mM
phenylenediamine restored electron transfer from water to
ferricyanide that excludes PSI. The ability of 4j and 3i to inhibit
this transfer at similar rates (data not shown) indicated that PSI
is not involved and that, as expected and previously determined
for compounds 5 and 6 (14), PSII is the target of their action.
Structure-Activity Relationships. An analysis of structural
requirements for the inhibitory activity against PSII was then
performed, including data previously obtained in the case of
the pyrazolo[1,5-a][1,3,5]triazine-2,4-dione and pyrazolo[1,5-
c][1,3,5]thiadiazine-2-one derivatives 5 and 6 (14; Table 4).
Compounds 1o, 2o, 2p, 2r, 3f, 3h, 4a, 4h, 5e, 5h, 5j, 6h, and
6j, spanning a range of >6 orders of magnitude (from active to
inactive), were selected. Most of them are not conformationally
flexible, and only a few derivatives in the active series show

PSII-Inhibiting Pyrazole Derivatives
J. Agric. Food Chem., Vol. 53, No. 10, 2005 3853
Figure 1. SPARTAN 02 transparent electrostatic surface maps of active (4j and 6j) versus less active (3h) and inactive (2p) compounds. Effective
molecules in the investigated series (tube representation) have a positive electrostatic surface [4j, from 62.343 (red) to 32.835 (blue); 6j, from 58.445
(red) to 41.954 (blue)] as a common motif, whereas the ineffective compound 2p (tube representation as carboxylate, see text) shows a strong negative
surface [from 185.108 (red) to 21.899 (blue)]. The moderately active compound 3h [from 57.674 (red) to 29.576 (blue)] presents an intermediate
electrostatic character. Electrostatic potentials are in kcal/mol. E-plot values are expressed as e/au3.
−
−
−
−
−
Table 2. In Vitro Effects of Pyrazolo[3,4-d][1,3]thiazine-4-one
Derivatives on Ferricyanide Reduction by Photosynthetically Active
Thylakoid Membranes Isolated from S. oleraceaa
Table 3. In Vitro Effects of Pyrazolo[3,4-d][1,3]thiazine-4-thione
Derivatives on the Hill Reaction by Functional Chloroplasts Isolated
from S. oleracea Leavesa
a Activity was measured as described under Materials and Methods either in
the absence or in the presence of pyrazole derivatives at concentrations ranging
a Activity was measured as described under Materials and Methods either in
the absence or in the presence of pyrazole derivatives at concentrations ranging
from 1 µM to 1 mM. Each sample was carried out at least in triplicate, and values
from 0.1
µM to 0.1 mM. Each sample was carried out at least in triplicate, and
values were expressed as percentage of untreated controls. b The concentrations
causing 50% inhibition (IC50) of in vitro activity were estimated as described under
Materials and Methods utilizing the linear regression equation of the activity values
plotted against the logarithm of inhibitor concentration. Confidence limits were
computed according to the method of Snedecor and Cochran (18).
were expressed as percentage of untreated controls. b The concentrations causing
50% inhibition (IC50) of in vitro activity were estimated as described under Materials
and Methods utilizing the linear regression equation of the activity values plotted
against the logarithm of inhibitor concentration. Confidence limits were computed
according to the method of Snedecor and Cochran (18).
a quantum mechanical parametrization of a conformationally
dependent hydrophobicity index. The values obtained showed
a similar trend (data not shown), althoughsas expectedsthey
were numerically different due to the different paradigms
underlying the calculation. The Ghose-Crippen model is
parametrized for 110 atom types, including common bondings
of H, C, N, O, S, and the halogens. For a test suite of 494
molecules, it gives a standard deviation of 0.347 and a correlated
coefficient of 0.962. In a test suite of 69 compounds beyond
the original one, it predicts a variance of 0.404. Villar is a model
some degree of conformational freedom, thus making each
analysis straightforward. SPARTAN log Ps were counterchecked
by both of the implemented methods in the SPARTAN 02 suite
[Ghose-Crippen (26) and Villar (27, 28)], which imply that the
hydrophobicity of a molecule can be obtained as the sum of
certain atomic contributions. The former is an atom-based
approach, in which each atom of the molecules is assigned to
a particular class, with additive contributions to the total value
of log P and molar refractivity, whereas the latter is based on

3854 J. Agric. Food Chem., Vol. 53, No. 10, 2005
Vicentini et al.
Table 4. In Vitro Effects of Some Commercial PSII-Inhibiting
Herbicides, Pyrazolo[1,5-a][1,3,5]triazine-2,4-dione, and
pyrazolo[1,5-c][1,3,5]thiadiazine-2-one Derivatives on Light-Driven
Ferricyanide Reduction by Functionally Intact S. oleracea Chloroplastsa
a Data obtained by replotting previously reported results (14).
that works only on semiempirical wave functions with no d
orbitals. The Villar method examines the overlap matrix,
searching for the type and number of lone pairs as well as the
surface area of each atom. It is parametrized for H, C, N, O, F,
S, and Cl (23, 27, 28).
The same trend was observed for the HINT log Ps. HINT is
a novel empirical molecular modeling system that translates the
well-developed Medicinal Chemistry and QSAR formalism of
Log P and hydrophobicity into a free energy interaction model
for all biomolecular systems based on the experimental data
from solvent partitioning. HINT calculates three-dimensional
hydropathic interaction maps that are uniquely instructive for
understanding biomacromolecular structure: substrate/inhibitor/
drug binding to proteins and nucleotides, protein subunit
interactions, and protein folding. The hydrophobic atom con-
stants are calculated using an adaptation of the fragment constant
methods of Leo and Rekker (24, 29, 30). The partition constant,
Log P, is a thermodynamic parameter that, because of its
unprocessed and unbiased experimental nature, contains interac-
tion information specific to the biological environment as well
as solvent effects and entropy. In other terms, it encodes all
significant intermolecular and intramolecular non-covalent
interactions implicated in drug binding or protein folding. Two
types of output data are generated by HINT: tabular lists of
specific atom-atom interactions quantitated by a microinter-
action constant and a variety of graphical maps that reveal (1)
hydropathy, (2) hydropathic interactions, and (3) hydropathy
of an unknown, inferred complementary species (e.g., to design
a substrate to fit a known receptor).
Figure 2. Partition coefficients (log Ps) of selected compounds as related
to their biological activity. Similar trends were obtained with either the
methods in the SPARTAN 02 suite (Ghose−Crippen and Villar) (A) or
HINT-log P (B) values. A HINT transparent hydropatic map of the active
compound 4j (stick representation) is also shown (C): red contours indicate
polar regions, and green contours indicate hydrophobic regions.
compounds show an intermediate electrostatic character, thus
strengthening the conclusion. If the electrostatic potential may
represent to some extent a discriminant factor between active
and inactive series/compounds, the same seems to be untrue
for lipophilicity. Hydropathic interaction indices were found to
be related to the biological activity within the same series, but
did not discriminate between different series (Figure 2A),
because some inactive compounds in series 1 and 2 showed
log P values comparable to those of active compounds in series
4 and 5. Also three-dimensional HINT (hydropatic) maps, which
are based on log P constants, are consistent with this trend
(Figure 2B,C). Further QSAR and modeling studies combining
different computational approaches are currently in progress to
define a pharmacophoric pattern, aiming at the synthesis of more
active chemotypes.
By means of such an approach a good relationship was found
between the electrostatic properties of the molecules and their
activity. All of the active molecules in the investigated series
show a highly positive electrostatic potential as a common motif,
whereas, according to previous studies (10), inactive compounds
share strong negative areas (Figure 1). Moderately active

PSII-Inhibiting Pyrazole Derivatives
J. Agric. Food Chem., Vol. 53, No. 10, 2005 3855
ACKNOWLEDGMENT
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We thank Dr. Eva Segato for skillful technical assistance. We
gratefully acknowledge Prof. Pietro Cozzini (Molecular Model-
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University of Parma) and Wavefunction, Inc., for helpful
discussions and suggestions.
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Received for review January 2, 2005. Revised manuscript received
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Tecnologica (MURST) of Italy.
JF0500029