Extraction as well as isolation and characterization of sissortrin (1) from Trifolium baccarinii were performed by a method previously described by Tsamo et al. (2021) [8] .

Column chromatography was carried out on silica gel 230 - 400 mesh, Merck

(Merck, Darmstadt, Germany), 70 - 230 mesh (Merck) (VWR, France). Thin-layer chromatography (TLC) was carried out on Merck pre-coated silica gel (60 F254) aluminium foil (Merck) and the spots were visualized by an UV lamp multiband UV-254/365 nm (Model UVGL-58, Upland, CA 91786, USA) followed by spraying with 50% H₂SO₄ followed by heating at 100˚C.

The ¹H and ¹³C-NMR spectra were recorded on a Bruker Avance III 600 spectrometer equipped with a cryoplatform (¹H at 500 or 600 MHz and ¹³C at 125 or 150 MHz). 2D NMR experiments were performed using standard Bruker microprograms (Xwin-NMR version 2.1 software). All chemical shifts (δ) were reported in parts per million (ppm) using the residual solvent signals as secondary reference relatively to TMS (δ = 0) as an internal standard, while the coupling constants (J) were given in hertz (Hz). Deuterated solvents, methanol (CD₃OD) and dimethyl sulfoxide (DMSO-d₆) were used as solvents for the NMR experiments.

High resolution mass spectra were obtained with QTOF (Quadrupole Time of Flight) Compact Spectrometer (Bruker, Germany) equipped with a HRESI source. The spectrometer was operated in positive and negative modes (mass range: 50 - 1500, with a scan rate of 1.00 Hz) with automatic gain control to provide high-accuracy mass measurements within 0.4 ppm deviation using Na formate as calibrant. The following parameters were used for experiments: spray voltage of 4.5 kV, capillary temperature of 200˚C. Nitrogen was used as sheath gas (4 L/min).

The nitration reaction of 200 mg of compound 1 led to two products, 1a and 1b, with masses of 7.10 mg and 8.20 mg respectively, while the 100 mg acetylation reaction of the same compound led to a single product, 1c with a mass of 110.6 mg, giving a yield of 80.34%. A study of the literature reveals that the presence of a methoxyl group in 4' promotes nitration on the ring A, in particular in positions 6 and 8 of the 5,7-dihydroxylated flavonoids [11] . However, position 8 being the most favored [12] which was not the case for our reaction. The low yield obtained in the first reaction is justified by the fact that it proceeds in several steps, in particular in three steps (with loss of the osidic part) and it resulted in more than one product as presented in Scheme 1. In contrast, in the second reaction there is no loss of the osidic moiety.

Compound 1b obtained from the nitration reaction was isolated as a yellow powder soluble in methanol. Its HR-ESI-MS shows the pseudomolecular ion peak [M + H]⁺ at m/z 330.0609 a.m.u corresponding to the molecular formula C₁₆H₁₂NO₇ (calculated mass for C₁₆H₁₂NO₇: 330.0608 a.m.u) contain twelve degrees of unsaturation. The mass difference observed between 1b and the genin of 1 (i.e. 284) is 45 a.m.u, which corresponds to the mass of a nitro group (46 a.m.u) minus one unit (corresponding to the substituted proton). This observation allows us to affirm that 1b comes from the mono-nitration of 1 after hydrolysis. The ¹H NMR data of this derivative is close to the substrate 1 but however with some dissimilarities (Table 1). The main difference is the disappearance of the signals attributable to the osidic protons in 1b. We also observe the disappearance of the aromatic H-6 proton signal, resulting in the appearance of the H-8 proton signal as a singlet at δ_H 6.36 (1H, s).

Similarly, a comparison of its ¹³C NMR data with that of 1 (Table 2) further confirms the disappearance of the osidic part and the nitration of position 6. Indeed, a concomitant interpretation of the ¹³C NMR and HSQC spectra of 1b allows us to see that the single aromatic proton of ring A is carried by the carbon at δ_C 96.0 ppm characteristic of the carbon in position 8 of 5,7-dihydroxylated flavonoids [13] [14] . Indeed, it has been established that in these systems (5,7-dihydroxylated), the proton H-8 is more deshielded than H-6 while its carbon resonates at slightly stronger fields (around 98.0 ppm) than that of H-6 (generally around 100.0 ppm). In addition, the presence of the meta nitro group results in a sight shielding of the C-8 carbon which appears at slightly stronger fields (96.0 ppm). From the aforementioned data, the structure of compound 1b was established as 6-nitrobiochanin A.

Compound 1a obtained from the nitration reaction was isolated as a yellow powder soluble in methanol. Its HR-ESI-MS shows the pseudomolecular ion peak [M + H]⁺ at m/z 330.0566 a.m.u corresponding to the molecular formula C₁₆H₁₂NO₇ (calculated mass for C₁₆H₁₂NO₇: 330.0608 a.m.u) contain twelve degrees of unsaturation. Its ¹H and ¹³C NMR data are identical to those of 8-nitrobiochanin A which was previously reported by Al-Maharik and Jaradat in 2020 [12] .

Scheme 2 below shows the plausible mechanism by which derivatives 1a and 1b would have formed [15] .

Step 1. Hydrolysis

Step 2. Formation of nitronium ion

Step 3. Electrophilic attack of nitronium ion and regeneration of nitric acid

Compound 1c obtained from acetylation reaction was isolated as yellowish powder soluble in dimethyl sulfoxide. The comparison of its ¹H and ¹³C NMR data with those of substrate 1 (Table 1 and Table 2) shows that the main difference was observed in the sugar moiety. Indeed, in addition to the signal of sugar, we pick up the signals attributed to the proton and carbone of acetyl groups. Compound 1c has been identified at 2",3",4",6"-tetraacetylsissotrin previously synthesized by Ying-Jie and Yi-Cheng in 2009 through reaction between 5,7-dihydroxy-4'-methoxyisoflavone and acetobromoglucose [α-D-glucopyranosyl bromide tetraacetate] [16] .

Scheme 3 below shows the mechanism of the acetylation reaction of 1.

Step 1. Formation of acetylpyridinium acetate

Step 2. Nucleophilic attack

The semisynthetic derivatives 1a, 1b and 1c as well as substrate 1 were evaluated for their antibacterial activities (Table 3). This evaluation was carried out on six bacterial strains, three of which were Gram-positive (Staphylococcus aureus ATCC 43300, Methicillin-resistant Staphylococcus aureus 33591 and Staphylococcus aureus CPC) and three Gram-negative (Escherichia coli ATCC 25922, Shighella flexineri CPC and Shighella flexineri NR 518).

Antibacterial cutoff points of pure compounds have been defined as follows: highly active: MIC below 1 µg∙mL⁻¹ (or 2.5 μM), significantly active: 1 ≤ MIC ≤ 10 µg∙mL⁻¹ (or 2.5 ≤ MIC < 25 μM), moderately active: 10 < MIC ≤ 100 µg∙mL⁻¹ (or 25 < MIC ≤ 250 μM), weakly active: 100 < MIC ≤ 1000 µg∙mL⁻¹ (or 250 < MIC ≤ 2500 μM and not active: MIC > 1000 µg∙mL⁻¹ (or >2500 μM) [17] .

Globally, the MIC values ranging from 62.5 µg∙mL⁻¹ to more than 500 µg∙mL⁻¹ depending on compounds and microorganisms. Concerning the substrate 1, from all the strains tested, it shows weak antibacterial activity towards Shighella flexineri CPC (MIC: 250 µg∙mL⁻¹) and Shighella flexineri NR 918 (MIC: 500 µg∙mL⁻¹). It can be seen that derivative 1a was active on all bacteria tested and the best activity (moderate) was observed on Staphylococcus aureus CPC (MIC: 62.5 µg∙mL⁻¹). When tested against on Staphylococcus aureus ATCC 43300 (MIC: 500 µg∙mL⁻¹); Methicillin-resistant Staphylococcus aureus 33591 (MIC: 500 µg∙mL⁻¹); Escherichia coli ATCC 25922 (MIC: 500 µg∙mL⁻¹); Shighella flexineri CPC (MIC: 125 µg∙mL⁻¹) and Shighella flexineri NR 518 (MIC: 250 µg∙mL⁻¹) compound 1a exhibited a weak antibacterial activity and still remain the most active on these strains. All these results are in agreement with the literature which shows that some structural features such as the presence of the sugar moiety or the numbers of hydroxyl, methoxyl groups as well as nitro group had a great influence on their biophysical and biological properties [18] [19] . Unfortunately derivatives 1b and 1c were not active (MIC > 500 µg∙mL⁻¹) against all the strains tested. These observations allow us to state that the hydrolysis followed by the nitration of biochanin A tend to increase the antibacterial

/: not determined; S.a: Staphylococcus aureus ATCC 43300; MRSA3391: Methicillin-resistant Staphylococcus aureus 33591; E.c ATCC 25922: Escherichia coli ATCC 25922; S.f CPC: Shighella flexineri CPC; S.f NR 518: Shighella flexineri NR 518; S.a CPC: Staphylococcus aureus CPC; MIC: Minimum Inhibitory Concentration; Ciprofloxacin: drug reference.

position 8 of the isoflavone. They also allow us to suggest that the hydroxyl activity. This increase is also depending on the position of the nitro group (-NO₂) because as it seems more pronounced when said nitration takes place at groups found on the osidic part would play a role in the antibacterial activity because their removal (by acetylation) led to a total loss of antibacterial activity (MIC ˃ 500 µg∙mL⁻¹) compared to the substrate (MIC: =250 and 500 µg∙mL⁻¹).