Reactions of Nitroxides, Part 17. Synthesis, Fungistatic and Bacteriostatic Activity of Novel Five- and Six-Membered Nitroxyl Selenoureas and Selenocarbamates

The reactions of 3-isoselenocyanato-2,2,5,5-tetramethylpyrrolidine-1-oxyl, 3-isoselenocyanatomethyl-2,2,5,5-tetramethyl-3-pyrrolidine-1-oxyl, and 4-isoselenocyanato-2,2,6,6-tetramethylpiperidine-1-oxyl with selected amines and alcohols give the corresponding novel nitroxyl selenoureas and selenocarbamates, all bearing a nitroxyl moiety. Synthesized selenoureas and selenocarbamates show significant activity against pathogenic fungi and bacteria. In contrast to piperidine nitroxides, pyrrolidine, five-membered nitroxyl selenoureas and selenocarbamates show excellent antifungal and antibacterial activity against pathogenic fungi and bacteria, respectively.


Introduction
In our previous paper [1], we synthesized nitroxyl radicals containing a tellurium atom and evaluated their antifungal activity. As a part of our continuing interest in the synthesis and evaluation of the biological activity of the compounds containing chalcogen atoms, the activity of organoselenium compounds bearing nitroxyl moieties is discussed in the present paper.
The predominant amount of synthesized nitroxyl selenoureas and selenocarbamates proved to be unstable at elevated temperatures. Compounds generally underwent decomposition upon heating during the melting temperature measurement. As a result of the decomposition, wide ranges of melting temperatures were observed. The purity of the synthesized compounds was evaluated by means of HPLC.
The predominant amount of synthesized nitroxyl selenoureas and selenocarbamates proved to be unstable at elevated temperatures. Compounds generally underwent decomposition upon heating during the melting temperature measurement. As a result of the decomposition, wide ranges of melting temperatures were observed. The purity of the synthesized compounds was evaluated by means of HPLC.
In order to confirm the molecular mass, ESI MS was performed for the almost all compounds under investigations (except of a selenourea 4b and selenocarbamates 9a and 10a-10c). The molecular masses were confirmed by the observation m/z M + 23 (100%) signals.
Exact molecular masses were confirmed by means of HR ESI MS (HR EI MS in the case of selenourea 4b and selenocarbamates 9a and 10a-10c).

Fungistatic and Bacteriostatic Activity of Selenoureas 4-8 and Selenocarbamates 9,10
All synthesized selenoureas 4-8 and selenocarbamates 9, 10 were tested for the herbicidal, insecticidal, acaricidal, antifungal, and antibacterial activities. No herbicidal, insecticidal, and acaricidal activities were observed. Significant fungistatic and bacteriostatic activities were found.  The investigated selenium containing nitroxides 4-8 and 9, 10 were tested in vitro against the basic set of phytopathogenic fungi: Botrytis cinerea, Fusarium culmorum, Phytophthora cactorum, and Rhizoctonia solani at the concentration of 200 mg/L, and for the selected, active compounds, at the concentration of 20 mg/L. The selenoureas 4-8 and selenocarbamates 9, 10 were also tested in vivo against Blumeria graminis as a phytopathogenic fungi, however, no tested compounds showed satisfactory activity against this species.
In order to enlarge the set of phytopathogenic fungi, the selected, active selenouraeas 4-8 and selenocarbamates 9, 10 were also tested at the concentration of 20 mg/L against the phytopathogenic fungi Alternaria alternata, Fusarium oxysporum, Phytophtora infestans, and against Ascosphaera apis (causing chalkbrood disease in honey bees).
The bacteriostatic activity of selenoureas 4-8 and selenocarbamates 9, 10 was tested for phytopathogenic bacteria Erwinia carotovora sub. atraseptica, Pseudomonas phaseolicola, Pseudomonas lachrymans, Pseudomonas syringae at concentration of 100 mg/L. The results of the fungistatic activity were presented in Tables 1 and 2 The results of the bacteriostatic activity were presented in Tables 3 and 4.   Almost all nitroxyl selenoureas 4-8 (except 4e-cyclododecyl derivative-see below) showed 100% activity against at least one fungus of the basic set of the tested fungi at the basic concentration of 200 mg/L.
Molecules 2019, 24, 2457 8 of 20 As noted above almost all "100%" compounds at the basic concentration of 200 mg/L were tested against the basic set of fungi (B. cinerea, F. culmorum, P. cactorum, and R. solani) at the concentration of 20 mg/L and against the additional set of fungi (A. alternata, F. oxysporum, P. infestans, and A. apis) also at the concentration of 20 mg/L. No tested nitroxyl compounds attained 100% in tests with the additional set of fungi. However, against the basic set of fungi significant amount of the tested nitroxyl selenoureas were active also at the concentration of 20 mg/L at the same 100% level. Nitroxyl selenourea 4c was active at the concentration of 20 mg/L at 100% level (MIC ≤ 20) against two species: B. cinerea and P. cactorum. Nitroxyl selenoureas 4d, 4h, 5a-5d, 5g, 5h, 7 were active at the concentration of 20 mg/L at 100% level (MIC ≤ 20) against P. cactorum.
The different size of alkyl and cycloalkyl fragments present in the nitroxyl selenoureas 4-8, prompted us to estimate the potential correlation between the observed fungicidal activity and the calculated octanol-water partition coefficient (clog P, HyperChem 7 software, Hypercube Inc., Gainesville, Fl, USA). Linear dependence between the average fungicidal activity (at 200 mg/L) vs. clog P was observed for the series of five-membered nitroxides 4a-4g (R 2 = 0.95). Interestingly, the analogous dependence for the similar series 5a-5g was not observed.
5g showed activity at concentration of <100 mg/L against three of four bacteria species. 4a, 4d, 4g, 5f showed activity at concentration of <100 mg/L against two of four bacteria species. 4f showed activity at concentration of <100 mg/L against one of four bacteria species. Nitroxyl selenocarbamate 9b showed activity at concentration of <100 mg/L against three of four tested bacteria species. Nitroxyl selenocarbamate 10d showed activity at concentration of <100 mg/L against one of four tested bacteria species.

Nitroxyl Selenocarbamates 9a-9d; Reaction of the Nitroxyl Isoselenocyanates 1a, 1b with either Sodium Methoxide or Sodium Ethoxide; a General Procedure
A sodium methoxide solution was prepared by dissolving metallic sodium (0.050 g, 0.00217 mol) in either methanol or ethanol (5 mL). The sodium methoxide (ethoxide) solution (3.5 mL, 0.0015 mol), respectively, was added dropwise to the solution of the isoselenocyanate 1a or 1b (0.001 mol) in methanol or ethanol (5 mL). The reagents were stirred for 1 h at room temperature. The solvent was evaporated under reduced pressure. The residue was purified by column chromatography using benzene:methanol 9:1 or benzene:ethanol 9:1, respectively, as a mobile phase. A chromatographically purified product was triturated with hexane. A crystalline precipitate was filtered off to give the appropriate nitroxyl selenocarbamate 9a-9d (Scheme 3).

Nitroxyl Selenocarbamates 9a-9d; Reaction of the Nitroxyl Isoselenocyanates 1a, 1b with either Sodium Methoxide or Sodium Ethoxide; a General Procedure
A sodium methoxide solution was prepared by dissolving metallic sodium (0.050 g, 0.00217 mol) in either methanol or ethanol (5 mL). The sodium methoxide (ethoxide) solution (3.5 mL, 0.0015 mol), respectively, was added dropwise to the solution of the isoselenocyanate 1a or 1b (0.001 mol) in methanol or ethanol (5 mL). The reagents were stirred for 1 h at room temperature. The solvent was evaporated under reduced pressure. The residue was purified by column chromatography using benzene:methanol 9:1 or benzene:ethanol 9:1, respectively, as a mobile phase. A chromatographically purified product was triturated with hexane. A crystalline precipitate was filtered off to give the appropriate nitroxyl selenocarbamate 9a-9d (Scheme 3).

Nitroxyl Selenocarbamates 9a-9d; Reaction of the Nitroxyl Isoselenocyanates 1a, 1b with either Sodium Methoxide or Sodium Ethoxide; a General Procedure
A sodium methoxide solution was prepared by dissolving metallic sodium (0.050 g, 0.00217 mol) in either methanol or ethanol (5 mL). The sodium methoxide (ethoxide) solution (3.5 mL, 0.0015 mol), respectively, was added dropwise to the solution of the isoselenocyanate 1a or 1b (0.001 mol) in methanol or ethanol (5 mL). The reagents were stirred for 1 h at room temperature. The solvent was evaporated under reduced pressure. The residue was purified by column chromatography using benzene:methanol 9:1 or benzene:ethanol 9:1, respectively, as a mobile phase. A chromatographically purified product was triturated with hexane. A crystalline precipitate was filtered off to give the appropriate nitroxyl selenocarbamate 9a-9d (Scheme 3).

Antifungal Activity Assays
Fungitoxicity of the tested compounds against phytopathogenic fungi was assessed in vitro using agar growth medium poison technique. PDA media in 100 mm Petri plates containing the acetone solutions of the tested compounds in the defined concentrations were infected with agar disks with thin mycelium of fungi cultures and allowed the solvent to evaporate. Linear growth of each colony was determined after 3-5 days. The effect of each compound on mycelial growth was

Antifungal Activity Assays
Fungitoxicity of the tested compounds against phytopathogenic fungi was assessed in vitro using agar growth medium poison technique. PDA media in 100 mm Petri plates containing the acetone solutions of the tested compounds in the defined concentrations were infected with agar disks with thin mycelium of fungi cultures and allowed the solvent to evaporate. Linear growth of each colony was determined after 3-5 days. The effect of each compound on mycelial growth was

Antifungal Activity Assays
Fungitoxicity of the tested compounds against phytopathogenic fungi was assessed in vitro using agar growth medium poison technique. PDA media in 100 mm Petri plates containing the acetone solutions of the tested compounds in the defined concentrations were infected with agar disks with thin mycelium of fungi cultures and allowed the solvent to evaporate. Linear growth of each colony was determined after 3-5 days. The effect of each compound on mycelial growth was

Antifungal Activity Assays
Fungitoxicity of the tested compounds against phytopathogenic fungi was assessed in vitro using agar growth medium poison technique. PDA media in 100 mm Petri plates containing the acetone solutions of the tested compounds in the defined concentrations were infected with agar disks with thin mycelium of fungi cultures and allowed the solvent to evaporate. Linear growth of each colony was determined after 3-5 days. The effect of each compound on mycelial growth was assessed by calculating the percentage of growth reduction, where: percentage of linear growth reduction = [(colony diameter of the control plate -colony diameter of the tested plate)/(colony diameter of the control plate)] × 100.

Antibacterial Activity Assays
Antibacterial tests were performed by dilution method on a solid support. The test results were read after 48 h incubation of the plates at 25 • C with bacterial strains. The antibacterial activity of the compounds was expressed in terms of the minimum growth inhibitory concentration of the test strain (MIC) in mg/L. Plant pathogenic strains: Erwinia carotovora, Pseudomonas phaseolicola Pseudomonas lachrymans, Pseudomonas syringae, were used.