Synthesis and Evaluation on the Fungicidal Activity of S-Alkyl Substituted Thioglycolurils

A series of S-alkyl substituted thioglycolurils was prepared through the alkylation of corresponding thioglycolurils with halogenoalkanes and tested for their fungicidal activity against six phytopathogenic fungi from different taxonomic classes: Venturia inaequalis, Rhizoctonia solani, Fusarium oxysporum, Fusarium moniliforme, Bipolaris sorokiniana, and Sclerotinia sclerotiorum, and two pathogenic yeasts: Candida albicans and Cryptococcus neoformans var. grubii. A number of S-alkyl substituted thioglycolurils exhibited high activity against Venturia inaequalis and Rhizoctonia solani (85–100% mycelium growth inhibition), and moderate activity against other phytopathogens. S-Ethyl substituted thioglycolurils possessed a high activity against Candida albicans. Additionally, the hemolytic and cytotoxic properties of promising derivatives were determined using human red blood cells and human embryonic kidney cells, respectively. Two S-ethyl derivatives possessed both low cytotoxicity against normal human cells and high fungicidal activity against Candida albicans.


Introduction
Parasitic fungi endanger the health of humans or domestic animals and cause damage to crops or ornamental plants. Invasive fungal infections are increasingly recognized as lifethreatening infections in clinics. More than 300 million people suffer from fungal infections, which lead to over 1,350,000 deaths per year [1]. The most common pathogens of mycoses in humans are Aspergillus spp., Candida spp., and Cryptococcus spp. [1,2]. In addition, plant diseases are an important factor in agricultural production, causing significant economic losses [3,4]. Antifungal resistance represents a major challenge and requires a constant updating of the drugs and agrochemicals used [5,6]. In this regard, the development of new effective fungicides is an urgent task [7].
The structures of compounds 4a-f, 5a-f, and 6a-h were confirmed via IR, 1 H NMR, 13 C NMR, and HRMS spectral data. The starting compounds 10a-h have an E configuration around the C=N and C=C bonds [20]. The constants of the vicinal interaction of Recently [20], we have synthesized a series of S-methyl and S-ethyl thioglycoluril derivatives 2, 3 possessing fungicidal activity against the phytopatogenes Rhizoctonia solani, Fusarium oxysporum, Fusarium moniliforme, and Bipolaris sorokiniana (see Figure 1). It was found that S-ethyl derivatives are somewhat more active than S-methyl ones. We assumed that the elongation of the alkyl chain at the S atom from C2 to C3 and C4 would lead to an increase in fungicidal activity. In this paper, we synthesized a series of new S-propyl, S-allyl and S-butyl derivatives of 4-[(E)-((E)-3-phenylallylidene)amino]-5thioxohexahydroimidazo [4,5-d]imidazol-2(1H)-one (4-[(E)-((E)-3-phenylallylidene)amino] thioglycoluril) and evaluated their fungicidal activity. S-allyl thioglycolurils were synthesized to reveal the influence of the unsaturated fragment on the activity.
The structures of compounds 4a-f, 5a-f, and 4a-f were confirmed via IR, 1 H NMR, 13 C NMR, and HRMS spectral data. The starting compounds 10a-h have an E configuration around the C=N and C=C bonds [20]. The constants of the vicinal interaction of protons of the CH=CH-Ar fragment of derivatives 4-6 lie in the range of 15.8-16.1 Hz, which is also characteristic of the trans arrangement of substituents at a double bond.
From the SAR point of view, compounds 4-6 differ in substituents at N(1) and N(3) nitrogen atoms and at the sulfur atom, as well as an aromatic fragment. Substituents at N(1) and N(3) nitrogen atoms do not have a definite effect on the activity of compounds. In some cases, 1,3-dimethyl substituted derivatives 4a, 6a, and 6e were more active than the corresponding 1,3-diethyl derivatives 4b, 6b, and 6f. At the same time, 1,3-diethyl substituted compounds 5f and 6h were more active than the corresponding 1,3-dimethyl derivatives 5e and 6g. In all other pairs of compounds, it is impossible to definitely choose which compound is better.
Among the S-alkyl derivatives, in general, activity increased with an increase in the length of the alkyl chain ( Figure 2, Table 1), which is quite likely due to the increasing lipophilicity with the elongation of the alkyl chain. The activity of S-allyl derivatives 5c-e was at or slightly less than the level of S-propyl derivatives 4c-e (Table 1). From the SAR point of view, compounds 4-6 differ in substituents at N(1) and N(3) nitrogen atoms and at the sulfur atom, as well as an aromatic fragment. Substituents at N(1) and N(3) nitrogen atoms do not have a definite effect on the activity of compounds. In some cases, 1,3-dimethyl substituted derivatives 4a, 6a, and 6e were more active than the corresponding 1,3-diethyl derivatives 4b, 6b, and 6f. At the same time, 1,3-diethyl substituted compounds 5f and 6h were more active than the corresponding 1,3-dimethyl derivatives 5e and 6g. In all other pairs of compounds, it is impossible to definitely choose which compound is better.
Among the S-alkyl derivatives, in general, activity increased with an increase in the length of the alkyl chain ( Figure 2, Table 1), which is quite likely due to the increasing lipophilicity with the elongation of the alkyl chain. The activity of S-allyl derivatives 5c-e was at or slightly less than the level of S-propyl derivatives 4c-e (Table 1). The aromatic fragment also affected the activity of the tested compounds. Compounds containing a phenyl ring with an electron-donating methoxy group possessed moderate to remarkable activity (4e, 5e, 5f, 6e, 6f). Introducing an electron-withdrawing nitro group in the ortho-position of the aromatic fragment led to a significant reduction in activity (4c,d, 5c,d, 6c,d). The most active compounds are among the derivatives containing an unsubstituted phenyl or furyl ring (4a, 6a, 6g, 6h) ( Figure 3, Table 1). Perhaps, besides the electron effects of the aryl ring substituents, the steric ones can affect the activity. The aromatic fragment also affected the activity of the tested compounds. Compounds containing a phenyl ring with an electron-donating methoxy group possessed moderate to remarkable activity (4e, 5e, 5f, 6e, 6f). Introducing an electron-withdrawing nitro group in the ortho-position of the aromatic fragment led to a significant reduction in activity (4c,d, 5c,d, 6c,d). The most active compounds are among the derivatives containing an unsubstituted phenyl or furyl ring (4a, 6a, 6g, 6h) ( Figure 3, Table 1). Perhaps, besides the electron effects of the aryl ring substituents, the steric ones can affect the activity.
Thus, we found some structure-activity correlations: (i) an increase in the length of the alkyl chain at the sulfur atom leads to an increase in activity; and (ii) depending on the arylmethylidene fragments, compounds could be arranged according to decreasing activity as follows: Some S-alkyl derivatives, both new and earlier synthesized [20], were assessed for microbiological activity by COADD (the Community for Antimicrobial Drug Discovery) [27][28][29][30][31]. S-Methyl (2a,b,e), S-ethyl (3a,b,d,e,g), S-propyl (4a,d) and S-butyl thioglycolurils (6b) were tested for inhibitory activity against two yeast fungi (Candida albicans ATCC 90028 and Cryptococcus neoformans var. grubii ATCC 208821). All the tested compounds did not possess significant activity against Cryptococcus neoformans in a concentration of 32 µg mL −1 (<50% mycelium growth inhibition, MIC values >32 µg mL −1 for 3a,b,e,g). The results of the analysis of activity against Candida albicans are shown in Table 2. Thus, we found some structure-activity correlations: (i) an increase in the length of the alkyl chain at the sulfur atom leads to an increase in activity; and (ii) depending on the arylmethylidene fragments, compounds could be arranged according to decreasing activity as follows: Some S-alkyl derivatives, both new and earlier synthesized [20], were assessed for microbiological activity by COADD (the Community for Antimicrobial Drug Discovery) [27][28][29][30][31]. S-Methyl (2a,b,e), S-ethyl (3a,b,d,e,g), S-propyl (4a,d) and S-butyl thioglycolurils (6b) were tested for inhibitory activity against two yeast fungi (Candida albicans ATCC 90028 and Cryptococcus neoformans var. grubii ATCC 208821). All the tested compounds did not possess significant activity against Cryptococcus neoformans in a concentration of 32 μg mL −1 (< 50% mycelium growth inhibition, MIC values >32 μg mL −1 for 3a,b,e,g). The results of the analysis of activity against Candida albicans are shown in Table 2.
Among the tested S-alkyl derivatives, only S-ethyl thioglycolurils 3a,b,e,g exhibited high fungicidal activity against Candida albicans. S-Methyl 2a,b,e, S-propyl 4a,d, S-butyl thioglycolurils 6b, and S-ethyl thioglycoluril 3d with a nitrophenyl ring as an aromatic fragment were inactive. For the more potent compounds, 3a,b,e,g, minimum inhibitory concentrations and cytotoxicity on human embryonic kidney cells (HEK-293, ATCC CRL-1573, CC50) and human red blood cells (RBC, HC10) were additionally determined. Compounds 3b and 3e were found to exhibit higher cytotoxicity than fungicidal activity, while two compounds, 3a and 3g, displayed potent activity along with low cytotoxicity towards HEK-293 and RBC cells.  of compounds 2a,b,e, 3a,b,d,e,g, 4a,d, and 6b against C. albicans.   2a,b,e, 3a,b,d,e,g, 4a,d, and 6b against C. albicans. Thus, we found some structure-activity correlations: (i) an increase in the length of the alkyl chain at the sulfur atom leads to an increase in activity; and (ii) depending on the arylmethylidene fragments, compounds could be arranged according to decreasing activity as follows: Ph-substituted ≥ Fu-substituted > 2-MeOC6H4-substituted >> 2-NO2C6H4-substituted. Some S-alkyl derivatives, both new and earlier synthesized [20], were assessed for microbiological activity by COADD (the Community for Antimicrobial Drug Discovery) [27][28][29][30][31]. S-Methyl (2a,b,e), S-ethyl (3a,b,d,e,g), S-propyl (4a,d) and S-butyl thioglycolurils (6b) were tested for inhibitory activity against two yeast fungi (Candida albicans ATCC 90028 and Cryptococcus neoformans var. grubii ATCC 208821). All the tested compounds did not possess significant activity against Cryptococcus neoformans in a concentration of 32 μg mL −1 (< 50% mycelium growth inhibition, MIC values >32 μg mL −1 for 3a,b,e,g). The results of the analysis of activity against Candida albicans are shown in Table 2.
Among the tested S-alkyl derivatives, only S-ethyl thioglycolurils 3a,b,e,g exhibited high fungicidal activity against Candida albicans. S-Methyl 2a,b,e, S-propyl 4a,d, S-butyl thioglycolurils 6b, and S-ethyl thioglycoluril 3d with a nitrophenyl ring as an aromatic fragment were inactive. For the more potent compounds, 3a,b,e,g, minimum inhibitory concentrations and cytotoxicity on human embryonic kidney cells (HEK-293, ATCC CRL-1573, CC50) and human red blood cells (RBC, HC10) were additionally determined. Compounds 3b and 3e were found to exhibit higher cytotoxicity than fungicidal activity, while two compounds, 3a and 3g, displayed potent activity along with low cytotoxicity towards HEK-293 and RBC cells.  of compounds 2a,b,e, 3a,b,d,e,g, 4a,d, and 6b against C. albicans. The minimum inhibitory concentration (MIC) and cytotoxicity on human embryonic kidney cells (HEK-293, CC50) and human red blood cells (RBC, HC10) are expressed in μg mL −1 .

Mycelium Growth
Inhibition, % (C = 32 µg mL −1 ) MIC C.a. HC 10  Among the tested S-alkyl derivatives, only S-ethyl thioglycolurils 3a,b,e,g exhibited high fungicidal activity against Candida albicans. S-Methyl 2a,b,e, S-propyl 4a,d, S-butyl thioglycolurils 6b, and S-ethyl thioglycoluril 3d with a nitrophenyl ring as an aromatic fragment were inactive. For the more potent compounds, 3a,b,e,g, minimum inhibitory concentrations and cytotoxicity on human embryonic kidney cells (HEK-293, ATCC CRL-1573, CC 50 ) and human red blood cells (RBC, HC 10 ) were additionally determined. Compounds 3b and 3e were found to exhibit higher cytotoxicity than fungicidal activity, while two compounds, 3a and 3g, displayed potent activity along with low cytotoxicity towards HEK-293 and RBC cells.

Cytotoxicity Assay
The cytotoxic activity of some new compounds, 4b and 5d-f, was studied at the concentration 10 −5 M against a panel of approximately 60 cancer cell lines derived from nine neoplastic diseases (leukemia, melanoma, lung, colon, CNS, ovarian, renal, prostate, and breast cancers) using the sulforhodamine B method of the National Cancer Institute Developmental Therapeutic Program (DTP). Some results are presented in Table 3. It was found that compounds 4b and 5d,e did not exhibit cytotoxic activity against all tested cancer cell lines. The mean growth of cell lines was 94.44-95.93%. Compound 5f in a 10 µM concentration was slightly more active inhibiting the cell growth of HL-60(TB), K562, SR (leukemia), MDA-MB-435 (melanoma), and MCF-7, MDA-MB-468 (breast cancer). The growth percentage values for these cell lines were 2.79-45.58 %. On the whole, it can be concluded that synthesized compounds 4b and 5d-f practically do not possess cytotoxicity.

Cytotoxicity Assay
The cytotoxic activity of some new compounds, 4b and 5d-f, was studied at the concentration 10 −5 M against a panel of approximately 60 cancer cell lines derived from nine neoplastic diseases (leukemia, melanoma, lung, colon, CNS, ovarian, renal, prostate, and breast cancers) using the sulforhodamine B method of the National Cancer Institute Developmental Therapeutic Program (DTP). Some results are presented in Table 3. It was found that compounds 4b and 5d,e did not exhibit cytotoxic activity against all tested cancer cell lines. The mean growth of cell lines was 94.44-95.93%. Compound 5f in a 10 μM concentration was slightly more active inhibiting the cell growth of HL-60(TB), K562, SR (leukemia), MDA-MB-435 (melanoma), and MCF-7, MDA-MB-468 (breast cancer). The growth percentage values for these cell lines were 2.79-45.58 %. On the whole, it can be concluded that synthesized compounds 4b and 5d-f practically do not possess cytotoxicity.

General Information
Melting points were determined in open glass capillaries on a Gallenkamp (Sanyo) melting point apparatus. IR spectra were recorded on a Bruker ALPHA instrument on KBr pellets. High resolution mass spectra (HRMS) were measured on a Bruker micrOTOF II instrument using electrospray ionization (ESI). The measurements were completed in a positive ion mode (interface capillary voltage-4500 V) or in a negative ion mode (3200 V), with a mass range from m/z 50 to m/z 3000 Da, and external or internal calibration was conducted with electrospray calibrant solution (Fluka). A syringe injection was used for solutions in acetonitrile or methanol (flow rate 3 µL/min). Nitrogen was applied as a dry gas; the interface temperature was set at 180 • C. 1 H and 13 C NMR spectra were recorded on Bruker AV-300 (300.13 MHz (1H)) and Bruker AM-300 (300.13 and 75.47 MHz, respectively) spectrometers using DMSO-d 6 as a solvent and referenced to the residual solvent peak. The chemical shifts are reported in ppm (δ); multiplicities are indicated by s (singlet), d (doublet), t (triplet), m (multiplet). Coupling constants, J, are reported in hertz. The analysis of compound 4f was carried out on a 1100 LS/MSD (Agilent Technologies) chromate-mass spectrometer equipped with an ELSD PL-ELS-1000 mass detector, with detection at 254 nm. The column used was Onyx monolithic C18, 50 × 4.6 mm. The flow rate was 3.75 mL min -1 and the eluent gradient was "A" (2.0 min)-"B" (0.6 min)-"A" (0.2 min) (A-0.1% F 3 CCOOH, 2.5% MeCN in H 2 O, B-0.1% F 3 CCOOH in MeCN).

Cytotoxicity assay against 60 Cancer Cell Lines at the National Cancer Institute
The initial assessment of cytotoxic activity was performed using an NCI 60 set of sixty human tumor cell lines derived from nine tumor diseases according to the National Cancer Institute's Division of Drug Evaluation protocol at a single dose (10 -5 mol L -1 ) [32]. The percentage growth number is an increase in the number of cells compared to the control, wherein the cells were not treated with the test substance. This allows the detection of both growth inhibition (values from 0 to 100) and lethality (values less than 0). A value of 100 means no growth inhibition.

Conclusions
The fungicidal activity of a series of S-alkyl substituted N-(3-arylallylidene)aminothioglycolurils was studied, and structure-activity relationships were established. Compounds exceeding or comparable to the well-known fungicidal agent triadimefon were identified. The S-alkyl-N-(3-arylallylidene)aminothioglycolurils with an unsubstituted phenyl or furyl ring were the most effective at inhibiting the growth of phytopathogenic fungi. Inhibitory activity increased with an increase in the length of the alkyl chain at the S atom. Compounds containing an ethyl substituent at the S atom demonstrated elevated activity against yeast Candida albicans. The hemo-and cytotoxicity tests showed that 5-ethylthio-1,3-dimethyl