The phosphonate 3a in turn reacts with hydrazine under reflux with methanol to form the hydrazonophosphonates 4a-c (Fig. 2, Table 1) Elleuch et al [23 (b)].
I-2 Antifungal activity of the acetate family:
MBH acetate adducts are synthetized in a single step by the reaction between MBH alcohols and the acetic anhydride in presence of trimethylamine and DMAP catalysts, the reaction time was just 30 min. MBH acetates synthesis is cheaper and rapid.
The majority of synthetized MBH acetates acquire an antifungal activity which depends on the radical group.
a-Antifungal activity against Trichoderma reesei strain:
The antifungal activity of newly synthetized acetates having different radical groups was screened against Trichoderma reesei and showed that the growth inhibition zone diameters (d) of theses adducts exhibited varying degrees of antifungal activities against several plant pathogenic fungi tested. This activity depends on the radical group. In fact, the MBH adduct 2a, a simple acetate with six-membered carbons (R = H), inhibited only the Trichoderma reesei strain growth with an inhibition zone diameter d = 2.5 ± 0. This activity is lower than that of compound 2g which is a simple five-membered carbons acetate, with a significant inhibitory effect (at p ≤ 0.05) (d = 4.85 ± 0.07) on the growth of Trichoderma reesei (Fig. 3). In addition, the five-membered carbons acetate (R = Me) 2i (d = 3.9 ± 0.14) showed a significant antifungal effect compared to the effect of the six-membered carbons acetate (R = Me) 2b with d = 3.55 ± 0.07 (Table 2).
As well, comparing the different inhibition zone diameters (d) of the MBH six membered carbons acetates with different radical groups, it is noted that the antifungal effect depends on the radical group. When R = Me, d(2b) = 3.55 ± 0.07 ≥ d(2a) = 2.5 ± 0 when R = H (Table 2).
Conversely, for MBH five-membered carbons acetates with different radical group, when R = H (2g), d = 4.85 ± 0.07 and the activity significantly reduced when R = Me (d = 3.9 ± 0.14), same in the case where R = Alkyl, Aryl (Table 2). Thus, the simple five membered carbons acetate (R = H) 2g has the highest inhibition power against Trichoderma reesei (Fig. 3).
b-Antifungal activity against Trichoderma parceramosum strain :
Comparing the results obtained of the antifungal activity against Trichoderma parceramosum, it should be noted that there are no significant antifungal effect differences between the five memberedcarbons acetates and theirs homologous (six-membered carbons) as it is shown in Table 2, d(2d(R = Ph)) = 3.35 ± 0.07 ≈ d(2h(R = Ph)) = 3.7 ± 0.14, even for 2i(R = Me), d = 3.55 ± 0.07, and its homologuous 2b(R = Me), d = 3.95 ± 0.07 (Fig. 4; Table 2)
It is also shown that the simple five-membered acetate 2g (R = H) has the highest antifungal potency (d = 4.35 ± 0.21), but this activity is reduced in the case where R = Me or R = Ph.
c- Antifungal activity against Stachybotrys microspora strain:
Likewise, it should be noted that five-membered carbon acetate has the highest Stachybotrys microspora antifungal activity. Radical-group replacement significantly reduces antifungal activity against S. microspora. As shown in the table, when R = H, d = 6.1 ± 0.14, we have the highest antifungal activity. However, the greater the length of the stem group, the less effective the antifungal activity: d (2g(R = H)) = 6.1 ± 0.14 ≥ d (2i(R = CH3)) = 4.1 ± 0.14 ≥ d (2j(R = CH2CH3)) = 3.95 ± 0.07 ≥ d (2k(R = R = CH2CH2CH3)) = 3.55 ± 0.21 (Fig. 5; Table 2); Even in the case of acetate with five membered carbon ring compounds, where R = Ph (phenyl group), causing steric hindrance, 2h shows moderate to good antifungal activity (d (2h) = 3.35 ± 0.21), however for acetate six membered-carbons the phenyl radical group reduces the antifungal activity (d (2d) = 3.6 ± 0.28) to d(2b) = 2.5 ± 0.
d-Antifungal effect of MBH against Fusarium oxysporum:
Against Fusarium oxysporum, the MBH adduct 2g again showed the highest antifungal activity (d = 4.75 ÷ 0.21); Similarly, radical substitution alters the potential for antifungal activity: d (2g(R = H)) = 4.75 ± 0.21 ≥ d (2i(R = CH3)) = 2.6 ± 0 ≥ d (2j(R = CH2CH3)) = 2.3 ± 0 ≥ d (2k(R = CH2CH2CH3)) = 1.4 ± 0 (Fig. 6; Table 2).
The MBH six-membered carbons acetate exhibit less antifungal activity.
e-Antifungal activity against Fusarium solani, Penicillium occitanis and Aspergillus niger strains.
Fusarium solani and Penicillium occitanis were inhibited by the five-membered MBH acetate adducts to different degrees depending on the radical. 2g (R = H) exhibits the highest antifungal activity, in contrast, when R = Me, Alkyl or Ph, the respective compounds (2i, 2j and 2h) share rather moderate antifungal potency (Table 3).
It should be noted that the five-membered MBH acetate adduct (R = H) (2g) was the only compound able to inhibit Aspergillus niger growth (Table 3).
Compared to the antifungal activity of nystatin which is a potent commercial antifungal product, most of the compounds tested exhibited moderate to strong inhibitory effects. Simple acetate 2g also appears to have the highest antifungal activity against Fusarium solani and Aspergillus niger.
All the MHB products are tested at (100µL/well) while Nystatin was tesed at (30µL/well) from the same concentration (10− 1M).
Compred to the control nystatin, compounds 2i, 2j, 2h, 2k and 2a, displayed varying antifungal activity, while 2g exhibited a broad spectrum of biological activity on all tested fungi.
f-Antifungal activity against Aspergillus flavus, Alternaria alternata and Fusarium aethiopicum strains.
As shown in Table 4, Fusarium aethiopicum, Alternaria alternata and Aspergillus flavus strains, newly isolated in our laboratory, were inhibited by five membered MBH acetate adducts (R = H) (2g) and simple six membered carbons acetate (R = H), the antifungal activity becomes weaker when the radical is longer (R=(CH2)2CH3) or even zero against Alternaria alternata. Of the three fungi, Alternaria alternata and Aspergillus flavus strains are more sensitive to 2g. Comparing the antifungal activity of 2g and standard nystatin, 2g maight have a more promising future as a fungicide.
All the MHB products are tested at (100µL/well) while Nystatin was tesed at (30µL/well) from the same concentration 10− 1M). Despite of the difference of the tested volume, the simple acetate with six membered carbons had good antifungal activity against Fusarium aethiopicum, Alternaria alternata and Aspergillus flavus strains. 2k exerted weak antifungal activity. However the antifungal activity of the acetate with five membered carbons was better than those of 2k and 2a excepted against Fusarium aethiopicum that seems more sensible to 2a. Results suggest that the introduction alkyl reduces the activity.
g- Assessment of fungicidal activity of 2g compound:
In order to deepen our investigations, we determined the fungicidal activity of the synthesized compound 2g, which manifested itself effectively at 1mM against Aspergillus niger and Penicillium occitanis. As shown in Fig. 7, the simple acetate 2g is able to inhibit 100% fungal growth.
Screening of antifungal activity demonstrated the ability of MBH adducts to inhibit fungal growth. Indeed, among these adducts the alcohol 1d(R = Ph) and MBH acetates were the only antifungal products. Thus, the antifungal activity varied depending on the fonctionnel group, with the five-membered carbons acetate with the H radical group being the most active against all tested fungi.
The introduction of radical substituents in the ortho position of the n-membered carbon acetate promoted increased or decreased antifungal activity depending on the core of the compound. Notably, compound 2g is lipophilic (absence of OH group), which suggests that this property could facilitate its penetration into the fungal cell and thus promote its activity [33, 34]. The acetate functional group would be responsible for the antifungal effect and the radical H would ensure the effectiveness of its antifungal power.