Pyrazolo[3,4-c]isothiazole and isothiazolo[4,3-d]isoxazole derivatives as antifungal agents

Abstract

Context: The diseases of plants and humans due to pathogenic fungi are increasing. Among the substances used to combat fungi, the azoles are of primary interest, both in agricultural field both in health. To avoid fungal resistance phenomena, the synthesis and tests of new derivatives are necessary.

Objective: This article discusses the synthesis and the antifungal activity of pyrazolo[3,4-c]isothiazole and isothiazolo[4,3-d]isoxazole derivatives against three fungi that are pathogenic only for plants and two fungi that are opportunistic in humans and plants.

Materials and Methods: The compounds were prepared starting from 2-cyano-3-ethoxy-2-butenethioamide. The antifungal activity of the compounds was determined by measuring the inhibition of growth of the fungi tested at 20, 50, and 100 µg/mL in comparison with the controls.

Results: Results demonstrated that several compounds were able to control the mycelial growth of the tested fungi, even if they showed different sensitivity to the different azole-derivatives. In general Magnaporthe grisea (T.T. Hebert) Yaegashi & Udagawa was the most sensitive fungus, being blocked almost entirely by 4-chloro derivative even at 20 µg/mL, a concentration at which the reference commercial compound tricyclazole was nearly ineffective.

Discussion and conclusion: These findings demonstrate that the pyrazolo[3,4-c]isothiazole derivatives have a wide spectrum of activity on phytopathogenic and opportunistic fungi. In particular the 4-chloro derivative seems to have a great potential as new product to combat M. grisea in the agricultural field.

Keywords::

• Antifungal activity
• isothiazole
• pyrazole
• isoxazole
• pyrazolo[3,4-c]isothiazole
• isothiazolo[4,3-d]isoxazole

Introduction

Fungicides are extensively used to prevent the development of plant diseases. A repeated use of antifungal compounds increases the resistance to the fungicides employed in some cases. This results in the proliferation of plant pathogens. So it is always necessary to identify new classes of antifungal compounds based on structures not previously used.

Among the most important phytopathogenic fungi to be defeated we can find: Magnaporthe grisea (T.T. Hebert) Yaegashi & Udagawa, Pythium ultimum Trow, Sclerotinia minor Fuckel, Fusarium moniliforme Sheldon, and Trichoderma viride (Pers.) Tul.

M. grisea causes diseases in many species of the grass family, including rice, where it yields the rice blast disease (Zeigler et al., 1994). It can attack plants in every development stage, by causing its partial or total decay. S. minor brings vegetables to be seriously damaged, by including rot both in the aerial and in the underground parts (Agrios, 1997). P. ultimum is a polyphagous fungus, able to parasite many agricultural plants; it strikes mostly the young plants that are placed in the hot-humid environment of greenhouses (Hendrix & Campbell, 1973).

In the recent decades some fungi pathogenic for plants represent opportunistic infections also in humans. Immunocompromised patients with invasive fungal infections have increased because of a greater number of AIDS sufferers, transplanted patients and more aggressive anticancer chemotherapeutic regimens. The therapeutic interventions for fungal infections are limited.

The phytopathogen Fusarium, that is a common cause of rot on maize and other grain (Nyvall, 1989), is one of the emerging causes of opportunistic mycoses (Shilabin et al., 2007). Fusarium species, as well as Cryptococcus neoformans (Barchiesi et al., 2003), are among the most commonly encountered etiologic agents of systemic fungal infections in immunocompromised patients. There is a very limited number of active drugs against these fungi. Fusarium infections are very difficult be defeated and their invasive form are often fatal. Moreover, even if few human cases have been identified, Trichoderma causes infections that can develop in patients with scanty immune defences. Trichoderma in fact may cause infections in presence of certain predisposing factors (Groll & Walsh, 2001).

Within the framework of investigation on biologically active heterocycles, we found that pyrazole derivatives are very interesting for their biological properties as agrochemicals and pharmacological agents (Vicentini et al., 1986, 2002, 2004, 2005; Mares et al., 2002, 2004, 2006). It is also known that isothiazole derivatives show antifungal activity (Adibpour et al., 2007).

In this study, we investigated the activity of analogues of 4-methyl-6-phenyl-6H-pyrazolo[3,4-c]isothiazol-3-amine, which, code-named G8, had previously proved its ability to inhibit the growth of phytopathogenic (Vicentini et al., 1987) and dermatophytic (Romagnoli et al., 1995) fungi. This derivative is potentially therapeutic for infections caused by C. neoformans (Barchiesi et al., 2003). An ultrastructural study suggested a mechanism of action similar to other azoles clinically utilized (Mares et al., 1998).

Now we test the antifungal activity of pyrazolo[3,4-c]isothiazole and isothiazolo[4,3-d]isoxazole derivatives (5a–g and 6, Figure 1) against M. grisea, P. ultimum, S. minor, strictly plant pathogens. We also selected F. moniliforme and T. viride as opportunistic fungi in human and plants.

Figure 1.  Structures of pyrazolo[3,4-c]isothiazole and isothiazolo[4,3-d]isoxazole derivatives (5a–g, 6).

We extend the screening to a wider range of fungi pathogenic for plants, humans and animals. The attractive feature of these compounds is the possibility of a wider spectrum of activity, including both phytopathogenic fungi and fungi of medical interest as well.

Materials and methods

Chemicals

Melting points were determined with a Büchi capillary apparatus and are uncorrected. IR spectra were recorded on a Perkin-Elmer Paragon 500 FT-IR spectrometer using potassium bromide pellets. ¹H NMR spectra were recorded on a Bruker AC200 spectrometer; 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 pyrazolo[3,4-c]isothiazole derivatives 5a–g and the isothiazolo[4,3-d]isoxazole 6 were synthesized in the laboratories of the Department of Pharmaceutical Science, University of Ferrara, as described previously (Vicentini et al., 1987, 1990).

Synthesis of 2-cyano-3-(2-arylhydrazinyl)-2-butenethioamides (2f,g)

2-Cyano-3-ethoxy-2-butenethioamide 1 (3.4 g, 20 mmol) was added to a solution of the pertinent hydrazine (2.16 g, 20 mmol) in chloroform (50 mL). The mixture was refluxed for 20 min. After ice-cooling, the precipitate was collected and crystallized from ethanol.

By using this procedure the following compounds were obtained:

2f (R = 2-Chloro-5-(trifluoromethyl)phenyl): yield 89%; mp 202–203°C; ¹H NMR (DMSO-d₆), δ 2.33 (s, 3H, Me), 7.10–7.65 (m, 3H, Ar), 8.14 (m, 1H, NH₂), 8.82 (br, 1H, ArNH), 8.96 (br, 1H, NH₂), 13.82 (br, 1H, ArNHNH); IR (KBr, cm⁻¹) v_max 3294, 2194, 1604.

2g (R = 3-Chloro-5-(trifluoromethyl)-2-pyridinyl): yield 88%; mp 204–206°C; ¹H NMR (DMSO-d₆), δ 2.27 (s, 3H, Me), 8.05 (br, 1H, NH₂), 8.26-8.52 (m, 2H, Ar), 8.86 (br, 1H, NH₂), 9.95 (br, 1H, ArNH), 13.90 (br, 1H, ArNHNH); IR (KBr, cm⁻¹) v_max 3307, 1608.

Synthesis of 5-amino-3-methyl-1-subsituted-1H-pyrazole-4-carbothioamides (3f,g)

Method A.

A suspension of 2-cyano-3-{2-[2-chloro-5-(trifluoromethyl)phenyl]hydrazinyl}-2-butenethioamide 2f (0.33 g, 1 mmol) in 0.1 N sodium hydroxide (5 mL) was heated to 80–85°. Within a few minutes, the solid was dissolved giving an orange solution from which a white precipitate immediately separated. After ice-cooling, the precipitate was collected and washed with water.

3f (R = 2-Chloro-5-(trifluoromethyl)phenyl): yield 93%; mp 198°C (EtOH); ¹H NMR (CDCl₃), δ 2.52 (s, 3H, Me), 6.56 (br, 2H, NH₂), 6.86 (br, 2H, NH₂), 7.72 (m, 3H, Ar); IR (KBr, cm⁻¹) v_max 3293, 1632, 1589, 1539.

Method B.

A suspension of 2-cyano-3-{2-[3-chloro-5-(trifluoromethyl)-2-pyridinyl]hydrazinyl}-2-butenethioamide 2g (0.33 g, 1 mmol) in xylene (50 mL) was refluxed for 6 h, affording a mixture of 5-amino-3-methyl-1-[3-chloro-5-(trifluoromethyl)-2-pyridinyl]-1H-pyrazole-4-carbothioamide 3g and 5-amino-3-methyl-1-[3-chloro-5-(trifluoromethyl)-2-pyridinyl]-1H-pyrazole-4-carbonitrile 4g. The solution was then evaporated and the required pyrazole-4-carbothioamide 3g and the byproduct 4 were isolated after purification by silica gel chromatography (eluent ethyl acetate: petroleum ether 3:7).

3g (R = 3-Chloro-5-(trifluoromethyl)-2-pyridinyl): yield 60%; mp 159–160°C; ¹H NMR (CDCl₃), δ 2.53 (s, 3H, Me), 6.83 (br, 2H, NH₂), 7.96 (br, 2H, NH₂), 8.14-8.64 (m, 2H, Ar); IR (KBr, cm⁻¹) v_max 3313, 1625, 1610, 1550.

4g (R = 3-Chloro-5-(trifluoromethyl)-2-pyridinyl): yield 25%; mp 200–201°C; ¹H NMR (CDCl₃), δ 2.33 (s, 3H, Me), 5.78 (br, 2H, NH₂), 8.16–8.62 (m, 2H, Ar); IR (KBr, cm⁻¹) v_max 3365, 2211, 1630, 1558.

Synthesis of 4-methyl-6-substituted-6H-pyrazolo[3,4-c]isothiazol-3-amines (5f,g)

Hydrogen peroxide (40%) (0.08 mL, 0.9 mmol) was added to an ice-cooled solution of the pertinent pyrazole-4-carbothioamide 3 (0.6 mmol) in pyridine (0.66 mL). The solution was stirred at 0° for 2 h and then at room temperature for 24 h. The solvent was then evaporated and the required pyrazolo[3,4-c]isothiazole and the byproduct 4 were isolated by purification by silica gel chromatography.

5f (R = 2-Chloro-5-(trifluoromethyl)phenyl): yield 60%; mp 155–157°C (purified by column chromatography; eluent ethyl acetate/petroleum ether 1:1); ¹H NMR (DMSO-d₆), δ 2.39 (s, 3H, Me), 7.75 (br, 2H, NH₂), 7.85–7.91 (m, 3H, Ar); IR (KBr, cm⁻¹) v_max 3286, 3139, 1592, 1577, 1507.

4f (R = 2-Chloro-5-(trifluoromethyl)phenyl): yield 28%; mp 180–181°C (purified by column chromatography; eluent ethyl acetate/petroleum ether 1:1); ¹H NMR (CDCl₃), δ 2.33 (s, 3H, Me), 4.45 (br, 2H, NH₂), 7.72–7.76 (m, 3H, Ar); IR (KBr, cm⁻¹) v_max 3337, 2225, 1651, 1574, 1541.

5g (R = 3-Chloro-5-(trifluoromethyl)-2-pyridinyl): yield 85%; mp 195–196°C (purified by flash column chromatography; eluent ethyl acetate/petroleum ether 1:1); ¹H NMR (DMSO-d₆), δ 2.39 (s, 3H, Me), 7.76 (br, 2H, NH₂), 8.62-8.86 (m, 2H, Ar); IR (KBr, cm⁻¹) v_max 3160, 1636, 1587, 1519.

4g (R = 3-chloro-5-(trifluoromethyl)-2-pyridinyl): yield 10% (purified by flash column chromatography; eluent ethyl acetate/petroleum ether 1:1).

Biological tests

Test organisms

The fungi used as test organism during this study were the strictly phytopathogens Magnaporthe grisea (T.T. Hebert) Yaegashi & Udagawa, strain n°64413 [American Type Culture Collection (ATCC)]; Pythium ultimum Trow, strain n°58812 (ATCC), Sclerotinia minor Fuckel; and opportunistic fungi Fusarium moniliforme Sheldon, strain n° 36541 (ATCC); Trichoderma viride (Pers.) Tul.

The ATCC fungi were purchased from ATCC Rockville, Maryland; T. viride and S. minor were purchased from SIAPA-ISAGRO, Milano, Italy. The strains were maintained at 4°C as agar slants on potato dextrose agar (PDA) (Oxoid Ltd., UK).

Evaluation of antifungal activity

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 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 dimethylsulfoxide (DMSO), and a proper dilution was aseptically added to warm (45°C) medium to obtain a final concentration of 20, 50 and 100 µg/mL. 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 colony diameter for 5 days after the transport of the fungus onto dishes containing the substance to be tested (kept as 0 time). The radial growth was compared to that of the fungal strains cultured in medium not amended with the antifungal compounds. As positive controls, for comparison, the same concentrations (20, 50, and 100 µg/mL) of 5a (G8) were used. Three replicates were made for each concentration. Results were expressed as percentage of growth in untreated controls and are means of at least three independent experiments.

Results and discussion

Synthesis

The compounds 5f, 5g were prepared by applying the same procedure that we had previously investigated for the synthesis of pyrazolo[3,4-c]isothiazoles 5a–e (Vicentini et al., 1987). The preparative route to the target products is outlined in Scheme 1.

Scheme 1.  Synthesis of new pyrazolo[3,4-c]isothiazole derivatives.

2-Cyano-3-(2-arylhydrazinyl)-2-butenethioamides 2f, g were readily available by condensing 2-cyano-3-ethoxy-2-butenethioamide 1 (McCall, 1962) with monosubstituted hydrazines. Compounds 2 were cyclized to 5-amino-3-methyl-1-substituted-1H-pyrazole-4-carbothioamides 3f, g by heating few minutes in sodium hydroxide (Method A) or by refluxing in xylene for 6 h (Method B). The 5-amino-3-methyl-1H-pyrazole-4-carbonitrile 4 was obtained as byproduct.

Good yields of pyrazolo[3,4-c]isothiazoles 5f, g were obtained by treating 3 with hydrogen peroxide in pyridine at 0°. The synthesis involved the concomitant oxidation of both the amino and thiocarbamoyl functions of the aromatic ring, and leads to ring closure with formation of the fused isothiazole. A possible drawback is the competitive dehydrosulfurization effected by the oxidant on the thiocarbamoyl function, which is thereby converted into a cyano group. In effect, we found that the oxidation of 3 afforded a mixture of pyrazolo[3,4-c]isothiazoles and 3-methyl-4-cyano-5-aminopyrazoles 4 under the experimented conditions which we employed. Analytical and spectral data of all compounds agree with the proposed structures.

Biological activity

We study new pyrazolo[3,4-c]isothiazole and isothiazolo[4,3-d]isoxazole derivatives with different functional groups at various locations, to see their potential antifungal activity, as the location of the substituent on the molecule backbone seems to play crucial role in determining the antifungal activity of the compounds. Almost all of the synthesized compounds exhibited some degree of toxicity toward the filamentous fungi tested (Figure 2). Results were expressed in comparison with their untreated controls and with the reference compound 5a (4-methyl-6-phenyl-6H-pyrazolo[3,4-c]isothiazol-3-amine). The last one, already code-named G8, was the founder of the chemical family of the novel compounds we have tested here and had been previously studied on fungi pathogenic for plants (Vicentini et al., 1987) and humans (Romagnoli et al., 1995; Mares et al., 1998).

Figure 2.  Antifungal activity of the compounds 5a-g and 6 expressed as percentage inhibition of growth on test organisms. The fungi were cultured on PDA in presence of 20, 50, and 100 µg/mL (empty, dotted, and full bars, respectively). Values are means of three trials made in triplicate, Y-error bars indicate standard deviation.

Remarkable differences indicating dissimilar sensitivities to the compounds were observed among the tested fungi. The less susceptible fungi are the oomycete Pythium and the ascomycetes Trichoderma and Fusarium. They seem not to suffer from the treatment with some compounds in fact: P. ultimum and T. viride showed an inhibition = 0 after treatment with the 5c compound; Trichoderma, Fusarium and Sclerotinia are quite insensitive to 3-methyl-4-aminoisothiazolo[4,3-d]isoxazole 6.

P. ultimum: no radial growth was observed when 5c was present in the medium, only 5a and 5f (at the higher dose) inhibited growth more than 80%. Interestingly 5f led an inhibition near to the 60% at 20 µg/mL, thus resulting on this fungus better than the reference compound 5a at the lowest dose.

Trichoderma showed no inhibition after treatment with 5c and 6 compounds and none of the new pyrazole derivatives reached the inhibition of the 5a. T. viride is an interesting fungus that can be plant pathogen, by causing green mold rot of onion, but it can also be human pathogen, by giving infections in the immunocompromised patients (Groll & Walsh, 2001). It is also a bio-fungicide: it is used on seeds and soil treatment for suppression of various diseases caused by other fungal pathogens (Brown & Bruce, 1999a,b). On the whole, Trichoderma seemed to be the most resistant fungus to the new substances here tested.

The other opportunistic fungus, F. moniliforme, is quite insensitive to the isothiazolo[4,3-d]isoxazole derivative 6. It was mildly affected by the treatment with the most of the new pyrazole derivatives: the higher inhibition (≈ 70%) of radial growth was done by the 5a and 5d compounds. Our results show that the two opportunistic fungi Trichoderma and Fusarium are especially drug-resisting, confirming the difficulty to find new active drugs against these microorganisms.

Very good on S. minor are the growth inhibitions exerted by 5a–d treatment in particular, whereas the 6 compound was entirely inefficient at all concentrations. The mycelium of this fungus looks typically concentric for the presence of black sclerotia. The treatment with some of the new compounds (with 5a in particular) yields cultures lighter and devoid of sclerotia (Figure 3). The visual observation of cultures in Petri plates can suggest us that the treatment with these pyrazole derivatives not only blocks the fungal growth but also does not allow the formation of resistance spores. This fact can be of interest for a possible future use of the compounds in the struggle against this important pathogen. S. minor can infect many species of Dicotyledonous plants such as chicory, soybean, cucumber, cabbage, tomato and many others flower and vegetable crops mainly (Agrios, 1997).

Figure 3.  Cultures of Sclerotinia minor control and treated with compound 5a (G8) at 100 µg/mL: in the control are evident black sclerotia, which are instead absent after treatment with 5a (plate on the right).

M. grisea appeared to be the most sensitive fungus, resulting in almost total inhibition by 5a and 5b used at all three doses and 5d at the highest concentration, while 5d, 5f, and 5g inhibited radial growth by more than 50% even at 20 and 50 µg/mL. Figure 4 shows the comparison between the fungal growth inhibition exerted by 5a, 5b and tricyclazole (the reference commercial compound) used at the same doses. This last fungicide is commonly used in agriculture against Magnaporthe to prevent blast in rice fields. It acts by inhibiting melanin synthesis in appressorial cells, thus weakening fungal walls and avoiding plant colonization (Sisler, 1986). The mode of action of tricyclazole is pointed out by a strong depigmentation of the mycelium. On the contrary, in our experiments, after treatment with the pyrazolo[3,4-c]isothiazole derivatives, the mycelium exhibited a darker pigmentation than the controls (Figure 5) indicating a mode of action different from tricyclazole. Even if no conclusive data are available, we think that the new compounds here tested could act as the previously characterized azoles, that inhibit the growth by blocking the synthesis of ergosterol, a main component of fungal membrane (Van den Bossche, 1990). The high capacity of curbing radial growth on Magnaporthe by five new compounds used at the lowest dose (20 µg/mL), by 5a and 5b in particular, might represent a significant reduction in the environmental fall-out of agricultural practice, since tricyclazole is usually applied at a dose of 200 µg/mL.

Figure 4.  Comparison between the antifungal activity on M. grisea of the compounds 5a–5b and the reference compound, tricyclazole, treated at the same doses.

Figure 5.  Cultures of M. grisea control and treated with compound 5b (G8-1) at 20, 50, and 100 µg/mL: the mycelia of the treated samples exhibited a very darker pigmentation than the control.

Based on the results obtained by the various compound variously substituted, it appears that the most powerful compound was 5a, attesting that the pyrazolo[3,4-c]isothiazole with R = phenyl is a compound very interesting for possible future developments.

The presence of Cl in position 4 of the phenyl (5b) in general reduced the activity in comparison with the unsubstituted compound (5a) when tested at the two higher doses (50 and 100 µg/mL); on the contrary it improved the antifungal effects at the lower concentration. This happened in all the phytopathogens, except for T. viride. The different position of Cl on the phenyl (R = 2-chlorophenyl) of the molecule 5c reduced the inhibitory effect in comparison with 5b (R = 4-chlorophenyl); complete inactivity of 5c was observed on P. ultimum and T. viride.

The insertion in the molecule of a second chlorine atom (5d) resulted in an increase of the inhibitory effects when compared to the corresponding compound having the Cl only in position 2 (5c), but not in comparison to 5b, having Cl in position 4. Thus, it can suggest that the position of the chlorine atom in the molecule, not the presence of one or two Cl, is important for the activity. This hypothesis seems to be confirmed by the data obtained from 5e, that is substituted with Cl in position 2 and 5; this compound was found to be only marginally effective. Therefore, by comparing the compounds 5b–5e, it is fundamental the insertion of the chlorine substituent in position 4, in order to obtain very good results. The presence of a Cl in position 2 both in monosubstituted (5c) and in bisubstituted (5d, e) compounds, leads to a lowered activity, with different results in conformity with the fungal strain and of the compound’s concentrations used.

The efficacy of the trifluoromethyl group in some pesticides (Pavlath, 1986; Poli et al., 1989; Vicentini et al., 1989), was reported also for the effectiveness of pyrazolo derivatives as antifungal substances in the previous works (Vicentini et al., 1989, 2002; Pavlath, 1986; Poli et al., 1989; Mares et al., 2000). For this reason, the 5f and 5g molecules were synthesized here. Nevertheless, only a small improvement of the antifungal activity was achieved by the insertion of a trifluoromethyl group in position 5 (5f), as suggested by the comparison between 5f and 5e.

Finally, the 3-methyl-4-aminoisothiazolo[4,3-d]isoxazole (6) resulted to be the scantest compound among all of the tested substances; it proved to be ineffective on four out of five tested fungi.

The experimental data of this work showed that some of this novel group of compounds sharing the pyrazolo[3,4-c]isothiazole scaffold have a good activity in vitro against fungi of different taxa, including both phytopathogenic and opportunistic strains. The wide action spectrum of pyrazole derivatives attests to their interesting activities, that already have been shown in phytopharmaceutic (Vicentini et al., 1987) and antidermatophytic field (Romagnoli et al., 1995; Mares et al., 1998) and against systemic mycoses, those caused by Cryptococcus neoformans (Barchiesi et al., 2003) in particular.

Among the tested fungi, the most susceptible were those that are strictly pathogen of plants. Interesting are also the results on opportunistic fungi, in particular on F. moniliforme, which causes common systemic infections to farm-workers. Nevertheless, none of the compounds inhibited entirely radial growth of Fusarium and Trichoderma, confirming that they are especially drug-resisting fungi and the difficulty of finding new active drugs against these microorganisms.

In Conclusion, very important is the almost total block of the growth exerted at low doses by some new compounds on M. grisea, that is a real calamity in rice plantations and cause of huge economic damages for the agriculture (Zeigler et al., 1994). In particular, the compound 5b (R= 4-chlorophenyl) caused an almost complete growth inhibition on Magnaporthe, not only at higher doses but also at the lowest of 20 μg mL⁻¹. Given the importance of this pathogen of rice and as this new molecule is ten times more active than the commercial product widely used in agriculture (tricyclazole), 5b can be a potential lead to be considered for practical use. The use of 5b at 20 µg/mL might represent a significant reduction in the environmental fall-out of agricultural practice, since tricyclazole is usually applied at a dose of 200 µg/mL. Future analyses are in progress to assess the phytotoxic effects of the 5b compound.

On the whole, the results of this work emphasize the potential of some of these new pyrazole derivatives for fungicide development.

Acknowledgements

We thank Dr. Vittoria Fabbri and Dr. Matteo Carli for their skilfull technical assistance. This research has been supported by grants of University of Ferrara (FAR2009) and MIUR Italy (PRIN: 2008 – n. 20082L3NFT_003).

Declaration of interest

The authors report no declaration of interest. The authors alone are responsible for the content and writing of the paper.