4.1 General

Optical rotation was measured on a JASCO P-1010 polarimeter. IR spectra were obtained in a Nicolet iZ10 Thermo Scientific spectrophotometer (each compound was dissolved in a minimum amount of solvent, and a drop of solution was added to the AgCl IR plates). NMR experiments were performed on a Bruker AVANCE II 400 MHz instrument. Multiplicity determinations (HSQC-DEPT) and 2D spectra (COSY, HSQC, HMBC, and NOESY) were obtained using standard Bruker software. Chemical shifts are expressed in ppm (δ) units using tetramethylsilane as the standard. Exact mass spectra were obtained on a Bruker microTOF-Q II mass spectrometer, equipped with an ESI source operating in positive mode. Antimicrobial activity measurements were determined in a microplate absorbance reader (Synergy HT- Biotek. 2012). Chromatographic separations were performed by column chromatography and vacuum on silica gel 60 (0.063–0.200 mm) and preparative TLC on silica gel 60 F254 (0.2 mm thick) plates. The presence of compounds was revealed by anisaldehyde reagents.

4.2 Plant material

Plants were collected in different locations of Córdoba Province, Argentina, from November 2021 to March 2022. Plants were selected according to their availability, accessibility, and previous reports on antimicrobial activity or chemical content. The plant material was identified by Gloria Barboza (IMBIV-CONICET, Córdoba, Argentina), and a voucher specimen was deposited on the Museo Botánico de Córdoba, Universidad Nacional de Córdoba (under the herbarium codes shown in the Table S1 in Supplementary Information).

4.3 Crude extracts and fractionation

The different vegetal materials collected were air-dried, powdered, and extracted at room temperature by three extraction cycles by EtOH 96% (10 g of vegetal powder in 200 mL × 3) and the solvent was evaporated under reduced pressure. Afterwards, 200 mg of extracts were resuspended in 100 mL water (8:2) and partitioned with n-hexane (3 × 80 mL). Then the aqueous phase was partitioned and extracted with EtOAc (3 × 80 mL). Finally, the hexane and EtOAc were independently dried (Na2SO4), filtered, and evaporated to dryness.

The selected EtOAc plant extracts with promising antimicrobial activity were fractionated by flash chromatography. Elution with n-hexane/ EtOAc mixtures of increasing polarity (100:0–0:100) and EtOAc/MeOH (100:0–0:100) were grouped in four fractions (F1-F4). The structures of the compounds were determined by a combination of 1- and 2-dimensional NMR spectroscopic methods, together with the exact masses and isotopic distribution using HRMS spectrometry. All compounds were determined to be > 95% pure by 1H NMR spectroscopy. See supplementary information for detailed NMR data and structural elucidation.

4.4 Isolation of metabolites from Xanthium species4.4.1 Isolation of xanthanolides from X. spinosum

774 g of X. spinosum branches conditioned and extracted as described above yielded 94 g of dry extract. Partitioning this residue with n-hexane and EtOAc yielded 15.7 g and 32.9 g, respectively. Subsequently, in order to explore the chemical content (in terms of yield), 25 mg of EtOAc extract was subjected to preparative TLC purification with a mixture of EtOAc and Hex (3:1) resulting in 5.6 mg of xanthatin, 2.9 mg of isoxanthanol, 2.0 mg of 8-epi-isoxanthanol, 2.1 mg of ivalbin and 1.6 mg of 8-epi-ivalbin.

Xanthatin: 1H NMR (CDCl3, 400.13 MHz): 7.07 d (1H, J = 16.0 Hz, H-2), 6.28 dd (1H, J = 9.2; 3.4 Hz, H-5), 6.20 d (1H, J = 16.0 Hz, H-3), 6.20 d (1H, J = 3.0 Hz, H2-13a), 5.48 d (1H, J = 3.0 Hz, H2-13b), 4.29 ddd (1H, J = 12.3; 10.1; 2.6 Hz, H-8),3.08 m (1H, H-10), 2.79 ddd (1H, J = 16.7; 9.0; 2.6 Hz, H2-6a), 2.55 m (1H, H-7), 2.38 ddd (1H, J = 12.7; 4.0; 2.7 Hz, H2-9a), 2.30 s (3H, H3-15), 2.22 m (1H, H2-6b), 1.85 td (1H, J = 12.7; 3.6 Hz, H2-9b),1.16 d (3H, J = 7.5 Hz, H3-14).13C NMR (CDCl3, 100.03 MHz): 198.6 (C, C-4),169.9 (C, C-12),148.4 (CH, C-2),144.9 (C, C-1),138.2 (C, C-11),137.8 (CH, C-5),124.8 (CH, C-3),118.8 (CH2, C-13), 81.4 (CH, C-8), 47.5 (CH, C-7), 36.7 (CH2, C-9), 29.2 (CH, C-10), 27.9 (CH3, C-15), 27.2 (CH2, C-6), 18.9 (CH3, C-14). 1H NMR identical with literature reported by Yuan et al. [27].

Isoxanthanol: 1H NMR (CDCl3, 400.13 MHz): 6.16 d (1H, J = 3.2 Hz, H2-13a), 5.75 dd (1H, J = 5.6; 3.6 Hz, H-5), 5.43 d (1H, J = 3.2 Hz, H2-13b), 4.94 m (1H, H-4), 4.29 ddd (1H, J = 12.5; 10.4; 3.0 Hz, H-8), 4.12 dd (1H, J = 6.9; 6.7 Hz, H-2), 2.80 m (1H, H-10), 2.51 m (1H, H2-6a), 2.46 m (1H, H-7), 2.32 m (1H, H2-9a), 2.10 ddd (1H, J = 15.1; 11.2; 3.2 Hz, H2-6b), 2.03 s (3H, H3-17), 1.94 m (1H, H2-3a), 1.66 m (1H, H2-3b), 1.66 m (1H, H2-9b), 1.26 d (3H, J = 6.2 Hz, H3-15), 1.18 d (3H, J = 7.3 Hz, H3-14).13C NMR (CDCl3, 100.03 MHz): 170.7 (C, C-16),170.0 (C, C-12), 149.3 (C, C-1), 139.5 (C, C-11),124.4 (CH, C-5),118.3 (CH2, C-13), 82.2 (CH, C-8), 76.8 (CH, C-2), 69.2 (CH, C-4), 47.3 (CH, C-7), 41.9 (CH2, C-3), 36.9 (CH2, C-9), 29.0 (CH, C-10),25.4 (CH2, C-6), 21.4 (CH3, C-17), 20.5 (CH3, C-15), 19.9 (CH3, C-14). 1H NMR identical with literature reported by Marco et al. [29].

8-epi-Isoxanthanol: [α] 25L: −16.5 (c 0.003, DCM). IR (dry film) 3464, 2964, 2932, 1761, 1734, 1373, 1242 cm−1. 1H NMR (CDCl3, 400.13 MHz): 6.28 d (1H, J = 3.2 Hz, H2-13a), 5.74 dd (1H, J = 8.9; 5.8 Hz, H-5), 5.53 d (1H, J = 3.2 Hz, H2-13b), 4.96 m (1H, H-4), 4.61 ddd (1H, J = 11.4; 8.6; 2.5 Hz, H-8), 4.10 dd (1H, J = 7.9; 6.2 Hz, H-2), 3.34 m (1H, H-7), 2.60 m (1H, H-10), 2.45 m (1H, H2-6a), 2.30 m (1H, H2-6b), 2.08 m (1H, H2-9a), 2.04 s (3H, H3-17), 1.97 m (1H, H2-3a), 1.83 m (1H, H2-9b), 1.71 m (1H, H2-3b), 1.29 d (3H, J = 6.3 Hz, H3-15), 1.20 d (3H, J = 6.9 Hz, H3-14). 13C NMR (CDCl3, 100.03 MHz): 170.6 (C, C-16), 170.2 (C, C-12), 147.7 (C, C-1), 138.7 (C, C-11), 122.4 (CH, C-5), 121.8 (CH2, C-13), 78.8 (CH, C-8), 73.6 (CH, C-2), 69.2 (CH, C-4), 42.2 (CH2, C-3), 41.4 (CH, C-7), 36.9 (CH2, C-9), 32.9 (CH, C-10), 26.1 (CH2, C-6), 21.9 (CH3, C-14), 21.3 (CH3, C-17), 20.4 (CH3, C-15). HRESIMS m/z [M + Na]+ 331.1528 (calcd for C17H24NaO+, 331.1516).

Ivalbin: 1H NMR (CDCl3, 400.13 MHz): 6.15 d (1H, J = 3.3 Hz, H2-13a), 5.85 dd (1H, J = 9.0; 3.5 Hz, H-5), 5.44 d (1H, J = 3.3 Hz, H2-13b), 4.30 dd (1H, J = 6.9; 6.7 Hz, H-2), 4.30 m (1H, H-8), 4.07 m (1H, H-4), 2.85 m (1H, H-10), 2.52 ddd (1H, J = 15.7; 9.0; 2.5 Hz, H2-6a), 2.43 m (1H, H-7), 2.32 m (1H, H2-9a), 2.11 ddd (1H, J = 15.7; 11.8; 4.1 Hz, H2-6b), 1.68 m (1H, H2-3a), 1.68 m (1H, H2-9b), 1.55 m (1H, H2-3b), 1.23 d (3H, J = 6.2 Hz, H3-15), 1.18 d (3H, J = 7.3 Hz, H3-14).13C NMR (CDCl3, 100.03 MHz): 170.1 (C, C-12), 150.1 (C, C-1), 139.5 (C, C-11), 123.4 (CH, C-5), 118.3 (CH2, C-13), 82.4 (CH, C-8), 79.7 (CH, C-2), 68.8 (CH, C-4), 48.6 (CH, C-7), 43.7 (CH2, C-3), 37.0 (CH2, C-9), 29.4 (CH, C-10), 25.1 (CH2, C-6), 24.4 (CH3, C-15),19.6 (CH3, C-14). 1H NMR identical with literature reported by Marco et al. [29].

8-epi-ivalbin: [α] 25D: + 10.3 (cc 0.004, DCM). IR (dry film) 3404, 2958, 2919, 2851, 1739 cm−1. 1H NMR (CDCl3, 400.13 MHz): 6.28 d (1H, J = 3.2 Hz, H2-13a), 5.82 dd (1H, J = 8.8; 5.8 Hz, H-5), 5.53 d (1H, J = 3.2 Hz, H2-13b), 4.62 ddd (1H, J = 11.4; 8.5; 2.5 Hz, H-8), 4.27 dd (1H, J = 10.2; 2.2 Hz, H-2), 4.08 m (1H, H-4), 3.35 m (1H, H-7), 2.64 m (1H, H-10), 2.47 m (1H, H2-6a), 2.32 m (1H, H2-6b), 2.08 m (1H, H2-9a), 1.84 m (1H, H2-9b), 1.71 m (1H, H2-3a), 1.59 dt (1H, J = 14.5; 2.5 Hz, H2-3b), 1.25 d (3H, J = 6.2 Hz, H3-15), 1.19 d (3H, J = 6.8 Hz, H3-14). 13C NMR (CDCl3, 100.03 MHz): 170.2 (C, C-12), 148.5 (C, C-1), 138.8 (C, C-11), 122.0 (CH, C-5), 121.9 (CH2, C-13), 79.2 (CH, C-8), 76.7 (CH, C-2), 69.1 (CH, C-4), 44.6 (CH2, C-3), 41.6 (CH, C-7), 37.0 (CH2, C-9), 33.4 (CH, C-10), 26.1 (CH2, C-6), 24.4 (CH3, C-15), 21.9 (CH3, C-14). HRESIMS m/z [M + Na]+ 289.1416 (calcd for C15H22NaO4+, 289.1410).

4.4.2 Isolation of 8-epi-xanthatin from X. cavanillesii

352 g of X. cavanillesii branches conditioned and extracted as described above yielded 44.9 g of dry extract. Partitioning this residue with n-hexane and EtOAc yielded 7.64 g and 15.1 g, respectively. Subsequently, the EtOAc extract was subjected to silica gel column chromatography purification with n-hexane/EtOAc (100:0–0:100) to provide 206 mg of 8-epi-xanthatin.

8-epi-xanthatin: 1H NMR (CDCl3, 400.13 MHz): 6.98 d (1H, J = 16.3 Hz, H-2), 6.32 d (1H, J = 3.3 Hz, H2-13a), 6.20 dd (1H, J = 8.9; 6.3 Hz, H-5), 6.14 d (1H, J = 16.3 Hz, H-3), 5.57 d (1H, J = 3.3 Hz, H2-13b), 4.65 dd (1H, J = 12.4; 10.1; 2.6 Hz, H-8), 3.42 m (1H, H-7), 2.83 m (1H, H-10), 2.59 ddd (1H, J = 18.5; 12.3; 6.3 Hz, H2-6a), 2.50 m (1H, H2-6b), 2.29 s (3H, H3-15), 2.18 ddd (1H, J = 12.4; 8.8; 2.2 Hz, H2-9a), 1.91 ddd (1H, J = 16.2; 12.7; 3.6 Hz, H2-9b), 1.18 d (3H, J = 6.9 Hz, H3-14).13C NMR (CDCl3, 100.03 MHz): 198.6 (C, C-4), 169.8 (C, C-12), 146.4 (CH, C-2), 142.8 (C, C-1), 138.2 (C, C-11),135.6 (CH, C-5), 125.9 (CH, C-3), 122.4 (CH2, C-13), 78.2 (CH, C-8), 41.2 (CH, C-7), 36.3 (CH2, C-9), 31.8 (CH, C-10), 27.7 (CH3, C-15), 27.0 (CH2, C-6), 21.5 (CH3, C-14). 1H NMR identical with literature reported by Kummer et al. [28].

4.5 Derivatization of X. cavanillesii EtOAc extracts4.5.1 Derivatization with ammonium hydroxide

94 mg of the EtOAc extract of X. cavanillesii were dissolved in 3 mL of ammonium hydroxide and stirred for 4 h at room temperature. Afterwards, the reaction crude was extracted with DCM (3 × 10 mL). The combined organic extracts were dried over anhydrous MgSO4 and evaporated, and the resulting residue was purified by preparative TLC (5% of MeOH and 1% of triethylamine in DCM) to yield 2.7 mg of compound 1 (7%).

(3S,3aR,7S,8aR)-3-(Aminomethyl)-7-methyl-6-((E)-3-oxobut-1-en-1-yl)-3,3a,4,7,8,8a-hexahydro-2H-cyclohepta[b]furan-2-one (1). Yellow oil; [α] 25D: + 21.6 (c 0.002, DCM). IR (dry film) 2958, 2926, 1762, 1666, 1363, 1263 cm−1. 1H NMR (CDCl3, 400.13 MHz): 6.97 d (1H, J = 16.0 Hz, H-2), 6.14 m (1H, H-5), 6.12 d (1H, J = 16.0 Hz, H-3), 4.48 ddd (1H, J = 12.2; 8.5; 2.0 Hz, H-8), 2.98 dd (1H, J = 12.4; 4.7 Hz, H2-13a), 2.90 dd (1H, J = 12.4; 5.2 Hz, H2-13b), 2.82 m (1H, H-10), 2.79 m (1H, H-7), 2.45 m (1H, H-11), 2.44 m (2H, H2-6), 2.28 s (3H, H3-15), 2.13 m (1H, H2-9a), 1.96 m (1H, H2-9b), 1.19 d (3H, J = 7.0 Hz, H3-14). 13C NMR (CDCl3, 100.03 MHz): 198.5 (C, C-4),177.6 (C, C-12),146.4 (CH, C-2),142.5 (C, C-1), 135.9 (CH, C-5), 125.7 (CH, C-3), 78.6 (CH, C-8), 48.0 (CH2, C-13), 44.9 (CH, C-11), 39.8 (CH, C-7), 35.2 (CH2, C-9), 31.5 (CH, C-10), 27.5 (CH3, C-15), 27.4 (CH2, C-6), 21.0 (CH3, C-14). HRESIMS m/z [M + H]+ 264.1637 (calcd for C15H22NO3+, 264.1594).

4.5.2 Derivatization with trimethylsilyl azide

To a round bottom flask were added 196 μL of trimethylsilyl azide, 3 mL of DCM and 84 μL of AcOH. Afterwards 73 mg of the EtOAc extract of X. cavanillesii and 9 μL of TEA were added and stirred for 48 h at room temperature. The reaction was quenched by pouring the mixture into ice water; subsequently, the aqueous solution was extracted with AcOEt (3 × 30 mL). Organic layers were combined and washed with saturated NaHCO3 solution and brine, and dried with Na2SO4, filtered, and evaporated. Preparative TLC purification of the crude with a mixture of EtOAc and n-hex (1:1) resulting in 9.7 mg of compound 2 (24%).

(3S,3aR,7S,8aR)-3-(Azidomethyl)-7-methyl-6-((E)-3-oxobut-1-en-1-yl)-3,3a,4,7,8,8a-hexahydro-2H-cyclohepta[b]furan-2-one (2). Yellow oil. [α] 25D: + 31.2 (c 0.006, DCM). IR (dry film) 2963, 2930, 2107, 1770, 1716, 1452, 1365, 1277, 1184 cm−1. 1H NMR (CDCl3, 400.13 MHz): 6.97 d (1H, J = 16.1 Hz, H-2), 6.14 m (1H, H-5), 6.13 d (1H, J = 16.1 Hz, H-3), 4.53 ddd (1H, J = 12.4; 8.7; 1.9 Hz, H-8), 3.68 brd (2H, J = 4.9 Hz, H2-13), 2.85 m (1H, H-10), 2.84 m (1H, H-7), 2.51 m (1H, H-11), 2.50 m (2H, H2-6), 2.30 s (3H, H3-15), 2.16 m (1H, H2-9a), 1.95 m (1H, H2-9b), 1.20 d (3H, J = 7.0 Hz, H3-14).13C NMR (CDCl3, 100.03 MHz): 198.6 (C, C-4), 175.3 (C, C-12), 146.1 (CH, C-2), 142.9 (C, C-1),135.3 (CH, C-5), 125.9 (CH, C-3), 78.5 (CH, C-8), 49.6 (CH2, C-13), 44.6 (CH, C-11), 40.0 (CH, C-7), 35.1 (CH2, C-9), 31.6 (CH, C-10), 27.6 (CH3, C-15), 27.3 (CH2, C-6), 21.2 (CH3, C-14). HRESIMS m/z [M + HN2]+ 262.1446 (calcd for C15H20NO3+, 262.1438).

4.5.3 Derivatization with hydroxylammonium chloride

To a solution of EtOAc extract (130 mg) in MeOH (3 mL) were added 38 mg of [NH₄OH]Cl and the reaction mixture stirred for 24 h at room temperature. After, 5 mL of H2O was added, and the pH of the reaction was adjusted to 7 with a saturated NaHCO3 solution. MeOH was evaporated and the aqueous phase was extracted with DCM (3 × 30 mL). Organic layers were combined and dried with Na2SO4, filtered, and evaporated. Preparative TLC purification was carried out with a mixture of EtOAc and n-hex (1:1), resulting in 17.0 mg of product 3 (26%) and 20.6 mg of product 3’ (32%).

(3aR,7S,8aR)-6-((1E,3E)-3-(Hydroxyimino)but-1-en-1-yl)-7-methyl-3-methylene-3,3a,4,7,8,8a-hexahydro-2H-cyclohepta[b]furan-2-one (3). Grayish oil; [α] 25D: + 28.1 (c 0.004, DCM). IR (dry film) 3335, 2960, 2932, 2873, 1765, 1272 cm−1. 1H NMR (CDCl3, 400.13 MHz): 6.36 d (1H, J = 16.5 Hz, H-2), 6.30 d (1H, J = 3.4 Hz, H2-13a), 6.22 d (1H, J = 16.5 Hz, H-3), 6.92 dd (1H, J = 9.0; 6.3 Hz, H-5), 5.55 d (1H, J = 3.0 Hz, H2-13b), 4.65 ddd (1H, J = 12.2; 8.8; 2.2 Hz, H-8), 3.39 m (1H, H-7), 2.86 m (1H, H-10), 2.55 ddd (1H, J = 19.8; 12.8; 6.2 Hz, H2-6a), 2.42 m (1H, H2-6b), 2.14 m (1H, H2-9a), 2.02 s (3H, H3-15), 1.88 m (1H, H2-9b), 1.18 d (3H, J = 6.9 Hz, H3-14).13C NMR (CDCl3, 100.03 MHz): 169.9 (C, C-12), 156.8 (C, C-4), 143.6 (C, C-1), 138.5 (C, C-11), 136.3 (CH, C-2), 128.8 (CH, C-5), 124.5 (CH, C-3), 121.6 (CH2, C-13), 78.4 (CH, C-8), 41.6 (CH, C-7), 36.6 (CH2, C-9), 31.8 (CH, C-10), 26.9 (CH2, C-6), 21.6 (CH3, C-14), 9.55 (CH3, C-15). HRESIMS m/z [M + Na]+ 284.1248 (calcd for C15H19NNaO3+, 284.1257).

(3aR,7S,8aR)-6-((1E,3Z)-3-(Hydroxyimino)but-1-en-1-yl)-7-methyl-3-methylene-3,3a,4,7,8,8a-hexahydro-2H-cyclohepta[b]furan-2-one (3’). Greenish oil; [α] 25D: + 50.0 (c 0.005, DCM). IR (dry film) 3285, 2960, 2933, 1762, 1616, 1373, 1274 cm−1. 1H NMR (CDCl3, 400.13 MHz): 6.96 d (1H, J = 16.6 Hz, H-3), 6.41 d (1H, J = 16.6 Hz, H-2), 6.30 d (1H, J = 3.2 Hz, H2-13a), 6.00 dd (1H, J = 8.9; 6.3 Hz, H-5), 5.55 d (1H, J = 3.2 Hz, H2-13b), 4.65 dd (1H, J = 14.4; 9.0; 5.0 Hz, H-8), 3.39 m (1H, H-7), 2.93 m (1H, H-10), 2.57 ddd (1H, J = 19.8; 12.8; 6.2 Hz, H2-6a), 2.44 m (1H, H2-6b), 2.16 m (1H, H2-9a), 2.02 s (3H, H3-15), 1.89 m (1H, H2-9b),1.21 d (3H, J = 6.9 Hz, H3-14).13C NMR (CDCl3, 100.03 MHz): 169.9 (C, C-12),153.4 (C, C-4),143.8 (C, C-1),139.3 (CH, C-2),138.5 (C, C-11),130.9 (CH, C-5),122.1 (CH2, C-13), 115.4 (CH, C-3),78.4 (CH, C-8), 41.4 (CH, C-7), 36.6 (CH2, C-9), 31.8 (CH, C-10), 26.9 (CH2, C-6), 21.6 (CH3, C-14),16.8 (CH3, C-15). HRESIMS m/z [M + H]+ 262.1439 (calcd for C15H20NO3+, 262.1438).

4.5.4 Derivatization with propargylamine

To 213 mg of EtOAc extract in 4 mL of MeOH was added 86 μL propargylamine, and the reaction mixture stirred for 28 h at room temperature. The solvent was evaporated and the residue purified by column chromatography with n-hexane/EtOAc mixtures of increasing polarity (100:0–0:100) to yield 68.2 mg (56%) of compound 4.

(3S,3aR,7S,8aR)-7-Methyl-6-((E)-3-oxobut-1-en-1-yl)-3-((prop-2-yn-1-ylamino)methyl)-3,3a,4,7,8,8a-hexahydro-2H-cyclohepta[b]furan-2-one (4). Yellow oil; [α] 25D: + 7.4 (c 0.003, DCM). IR (dry film) 32,980, 2957, 2928, 1762, 1665, 1595, 1361, 1258, 1179, 1019, 984 cm−1. 1H NMR (CDCl3, 400.13 MHz): 6.97 d (1H, J = 16.2 Hz, H-2), 6.13 dd (1H, J = 8.9; 6.6 Hz, H-5), 6.12 d (1H, J = 16.2 Hz, H-3), 4.49 ddd (1H, J = 12.4; 8.6; 1.9 Hz, H-8), 3.44 t (2H, J = 2.5 Hz, H2-1´), 2.94 ddd (2H, J = 22.2; 12.1; 6.4 Hz, H2-13), 2.82 m (1H, H-10), 2.80 m (1H, H-7), 2.50 m (1H, H-11), 2.47 m (2H, H2-6), 2.28 s (3H, H3-15), 2.22 m (1H, H-2´), 2.14 m (1H, H2-9a), 1.96 m (1H, H2-9b), 1.19 d (3H, J = 6.9 Hz, H3-14).13C NMR (CDCl3, 100.03 MHz): 198.6 (C, C-4), 177.5 (C, C-12), 146.4 (CH, C-2), 142.6 (C, C-1), 135.8 (CH, C-5),125.6 (CH, C-3), 81.5 (C, C-1´), 78.5 (CH, C-8), 72.0 (CH, C-2´), 47.2 (CH2, C-13), 44.7 (CH, C-11), 40.2 (CH, C-7), 38.4 (CH2, C-16), 35.1 (CH2, C-9), 31.6 (CH, C-10), 27.6 (CH3, C-15), 27.4 (CH2, C-6), 21.1 (CH3, C-14). HRESIMS m/z [M + H]+ 302.1753 (calcd for C18H24NO3+, 302.1751).

4.5.5 Extract derivatization with potassium hydroxide

To a round bottom flask was added 2 ml of DCM and 68.5 mg of KOH and stirred for 30 min at room temperature. Afterwards, 150 mg of the EtOAc extract was dissolved in 2 mL of DCM was added and stirred for another 2 h. The solvent was evaporated, 5 ml of water was added, and pH neutralized with a 4 M HCl solution. Then, the aqueous phase was extracted with EtOAc (3 × 30 mL). The combined organic extracts were dried over anhydrous MgSO4 and evaporated, and the resulting residue was purified by preparative TLC (EtOAc/n-hexane, 3:2) to yield 24.9 mg of compound 6 (31%).

(3S,3aR,7S,8aR)-3-(Methoxymethyl)-7-methyl-6-((E)-3-oxobut-1-en-1-yl)-3,3a,4,7,8,8a-hexahydro-2H-cyclohepta[b]furan-2-one (6). Yellow oil; [α] 25D: + 27.6 (c 0.013, DCM). IR (dry film) 2928, 2877, 1770, 1671, 1363, 1189, 1118 cm−1. 1H NMR (CDCl3, 400.13 MHz): 6.97 d (1H, J = 16.3 Hz, H-2), 6.14 m (1H, H-5), 6.12 d (1H, J = 16.3 Hz, H-3), 4.49 ddd (1H, J = 12.4; 8.8; 2.2 Hz, H-8), 3.65 d (2H, J = 4.6 Hz, H2-13), 3.36 s (3H, H3-1´), 2.91 m (1H, H-7), 2.82 m (1H, H-10), 2.49 m (1H, H-11), 2.48 m (2H, H2-6), 2.28 s (3H, H3-15), 2.13 ddd (1H, J = 13.8; 7.5; 2.2 Hz, H2-9a), 1.96 m (1H, H2-9b), 1.18 d (3H, J = 7.0 Hz, H3-14).13C NMR (CDCl3, 100.03 MHz): 198.6 (C, C-4), 176.2 (C, C-12), 146.6 (CH, C-2),142.5 (C, C-1),136.2 (CH, C-5),125.6 (CH, C-3), 78.9 (CH, C-8), 70.2 (CH2, C-13), 59.1 (CH3, C-1´), 45.5 (CH, C-11), 39.8 (CH, C-7), 35.1 (CH2, C-9), 31.5 (CH, C-10), 27.6 (CH3, C-15), 27.5 (CH2, C-6), 21.3 (CH3, C-14). HRESIMS m/z [M + Na]+ 301.1419 (calcd for C16H22NaO4+, 301.1410).

4.5.6 Derivatization with m-chloroperoxybenzoic acid

To a solution of EtOAc extract (146 mg) in DCM (3 mL) at 0 °C was added 153 mg of MCPBA, and the reaction mixture was stirred for 24 h at room temperature. Subsequently, the solvent was evaporated, and the residue redissolved in EtOAc, washed with an aqueous Na2S2O3 solution, then with NaHCO3 and finally with H2O. The organic layer was dried with Na2SO4, filtered, and evaporated. Preparative TLC purification (EtOAc/n-hex, 1:1), resulting in 26.1 mg of 7 (31%) and 14.6 mg of compound 8 (13%).

(1aS,2S,3aR,6aR,7aS)-2-Methyl-6-methylene-1a-((E)-3-oxobut-1-en-1-yl)octahydro-5H-oxireno[2',3':4,5]cyclohepta[1,2-b]furan-5-one (7). Colorless oil; [α] 25D: + 6.1 (c 0.018, DCM). IR (dry film) 2967, 2932, 1763, 1673, 1627, 1273, 968 cm−1. 1H NMR (CDCl3, 400.13 MHz): 6.74 d (1H, J = 15.8 Hz, H-2), 6.29 m (1H, H2-13a), 6.28 d (1H, J = 15.8 Hz, H-3), 5.66 d (1H, J = 1.9 Hz, H2-13b), 4.61 dd (1H, J = 11.7; 7.4; 4.2 Hz, H-8), 3.30 m (1H, H-7), 3.07 dd (1H, J = 7.7; 4.9 Hz, H-5), 2.29 m (1H, H-10), 2.25 s (3H, H3-15), 2.15 ddd (1H, J = 15.1; 7.8; 3.4 Hz, H2-6a), 2.05 m (1H, H2-6b),1.86 ddd (1H, J = 14.1; 3.9; 2.6 Hz, H2-9a), 1.71 m (1H, H2-9b),1.13 d (3H, J = 6.9 Hz, H3-14).13C NMR (CDCl3, 100.03 MHz): 197.7 (C, C-4),169.0 (C, C-12),146.7 (CH, C-2),139.0 (C, C-11),129.6 (CH, C-3),123.2 (CH2, C-13),79.4 (CH, C-8), 65.8 (CH, C-5), 62.7 (C, C-1), 39.3 (CH, C-7), 32.2 (CH2, C-9), 31.6 (CH, C-10), 31.2 (CH2, C-6), 28.3 (CH3, C-15), 18.8 (CH3, C-14). HRESIMS m/z [M + Na]+ 285.1090 (calcd for C15H18NaO4+, 285.1097).

(2Z,4Z)-4-((3aR,5R,7S,8aR)-5-Hydroxy-7-methyl-3-methylene-2-oxooctahydro-6H-cyclohepta[b]furan-6-ylidene)but-2-en-2-yl 3-chlorobenzoate (8). Colorless oil; [α] 25D: + 44.7 (c 0.009, DCM). IR (dry film) 3491, 2961, 2928, 1768, 1663, 1576, 1426, 1376, 1280, 1263, 1223, 1125, 1004 cm−1. 1H NMR (CDCl3, 400.13 MHz): 8.01 t (1H, J = 1.6 Hz, H-3´), 7.93 dt (1H, J = 7.8; 1.6 Hz, H-7´), 7.67 d (1H, J = 9.0 Hz, H-3),7.59 ddd (1H, J = 8.0; 2.0; 1.0 Hz, H-5´), 7.42 t (1H, J = 7.8 Hz, H-6´), 6.32 d (1H, J = 3.2 Hz, H2-13a), 5.63 d (1H, J = 2.7 Hz, H2-13b), 5.53 d (1H, J = 9.0 Hz, H-2), 5.20 t (1H, J = 7.9 Hz, H-5), 4.58 ddd (1H, J = 12.5; 8.3; 3.2 Hz, H-8), 3.14 m (1H, H-7),2.63 dd (1H, J = 16.1; 8.0 H-10),2.37 ddd (1H, J = 14.0; 6.8; 3.6 Hz, H2-6a), 2.22 m (1H, H2-9a), 2.18 s (3H, H3-15), 2.11 m (1H, H2-6b), 1.81 m (1H, H2-9b),1.33 d (3H, J = 7.3 Hz, H3-14).13C NMR (CDCl3, 100.03 MHz): 169.7 (C, C-12), 168.9 (C, C-4), 164.2 (C, C-1´),152.3 (C, C-1), 138.3 (C, C-11), 135.0 (C, C-2´), 134.9 (C, C-4´), 134.1 (CH, C-5´), 130.0 (CH, C-3´), 129.9 (CH, C-6´), 128.0 (CH, C-7´), 123.7 (CH, C-2), 122.8 (CH2, C-13), 88.7 (CH, C-3), 78.1 (CH, C-8), 68.4 (CH, C-5), 39.1 (CH, C-10), 37.5 (CH, C-7), 35.9 (CH2, C-9), 34.7 (CH2, C-6), 23.3 (CH3, C-14), 20.8 (CH3, C-15). HRESIMS m/z [M + Na + OMe]+ 457.1070 (calcd for C23H27ClO6Na+, 457.1388).

4.5.7 Click reaction of product 4 with benzyl azide.

To a solution of 4 (21 mg, 0.087 mmol) in DMF (3 mL) were added 22 μL of benzyl azide (0.174 equiv), 7 mg of NaAsc (0.035 equiv) and 5 mg of CuSO4 (0.017 equiv). The reaction mixture was stirred for 24 h at room temperature. The work up involved addition of distilled water and extraction with DCM (3 × 30 mL). Preparative TLC purification of the crude with EtOAc resulting in 7.0 mg of product 5 (21%).

(3S,3aR,7S,8aR)-3-((((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)amino)methyl)-7-methyl-6-((E)-3-oxobut-1-en-1-yl)-3,3a,4,7,8,8a-hexahydro-2H-cyclohepta[b]furan-2-on (5). Yellow oil; [α] 25D: + 11.0 (c 0.003, DCM). IR (dry film) 2956, 2932, 1766, 1665, 1622, 1595, 1456, 1360, 1258, 1176, 726 cm−1. 1H NMR (CDCl3, 400.13 MHz): 7.47 s (1H, H-5´), 7.37 m (1H, H-8´), 7.35 m (2H, H-7´), 7.27 m (2H, H-6´), 6.95 d (1H, J = 16.2 Hz, H-2), 6.11 d (1H, J = 16.2 Hz, H-3), 6.08 m (1H, J = 8.9; 6.3 Hz, H-5), 5.50 s (2H, H2-4´), 4.46 m (1H, H-8), 3.95 s (2H, H2-1´), 2.98 dd (1H, J = 12.3; 5.0 Hz, H2-13a), 2.91 dd (1H, J = 12.3; 6.5 Hz, H2-13b), 2.81 m (1H, H-10), 2.73 m (1H, H-7), 2.56 m (1H, H-11), 2.49 m (1H, H2-6a), 2.39 m (1H, J = 14.0; 9.0; 5.1 Hz, H2-6b), 2.28 s (3H, H3-15), 2.12 m (1H, H2-9a),1.95 m (1H, H2-9b),1.17 d (3H, J = 7.0 Hz, H3-14).13C NMR (CDCl3, 100.03 MHz): 198.7 (C, C-4),177.8 (C, C-12),146.4 (CH, C-2),145.7 (C, C-2´),142.5 (C, C-1),136.0 (CH, C-5),129.1 (CH, C-8´),128.9 (CH, C-7´),128.1 (CH, C-6´),125.7 (CH, C-3),121.8 (CH, C-3´), 78.9 (CH, C-8), 54.6 (CH2, C-4´), 47.6 (CH2, C-13), 44.8 (CH2, C-1´), 44.3 (CH, C-11), 40.3 (CH, C-7), 35.0 (CH2, C-9),31.6 (CH, C-10), 27.7 (CH3, C-15), 27.3 (CH2, C-6), 21.1 (CH3, C-14). HRESIMS m/z [M + H]+ 435.2405 (calcd for C25H31N4O3+, 435.2391).

4.6 Isoxanthanol esterification’s with carboxylic acids4.6.1 Derivatization with cinnamic acid

To a round-bottom flask, 24 mg (0.078 mmol) of isoxanthanol was added and dissolved in DCM (3 mL). Separately, 15 mg (0.102 mmol) of cinnamic acid was dissolved in DCM (1 mL) and added stepwise to the previous solution at 0°C with continuous stirring. Subsequently, 21 mg (0.102 mmol) of DCC and 2 mg (0.011 mmol) of DMAP, both dissolved in DCM (1 mL), were added to the reaction mixture at 0°C. The mixture was stirred at room temperature overnight. Afterwards, EtOAc and a concentrated solution of NH₄Cl was added, followed by filtration through Celite. Then, the organic layer was washed with a concentrated solution of NH₄Cl and brine. Organic layer was dried with Na₂SO₄, filtered and evaporated. Preparative TLC purification of the crude with (EtOAc/n-hex, 1:1) resulting in 17.2 mg of product 9 (50%).

(1S,3S)-3-acetoxy-1-((3aS,7S,8aS)-7-methyl-3-methylene-2-oxo-3,3a,4,7,8,8a-hexahydro-2H-cyclohepta[b]furan-6-yl)butyl cinnamate (9). Yellow oil; [α] 25D: + 12.9 (c 0.007, DCM). IR (dry film) 2974, 2934, 2863, 1771, 1732, 1715, 1637, 1248 cm−1. 1H NMR (CDCl3, 400.13 MHz): 7.69 d (1H, J = 16.0 Hz, H-20), 7.53 m (2H, H-22,22´), 7.39 m (3H, H-23,23´,24), 6.42 d (1H, J = 16.0 Hz, H-19), 6.16 d (1H, J = 3.2 Hz, H2-13a), 5.93 dd (1H, J = 9.1; 3.4 Hz, H-5), 5.44 d (1H, J = 3.0 Hz, H-13b), 5.34 t (1H, J = 7.3 Hz, H-2), 4.90 m (1H, H-4), 4.29 td (1H, J = 11.3;2.8 Hz, H-8), 2.86 m (1H, H-10), 2.51 m (H, H-6a), 2.48 m (H, H-7), 2.34 ddd (1H, J = 12.7; 4.4; 3.0 Hz, H-9a), 2.18 m (H, H-3a), 2.15 m (H, H-6b), 2.07 s (3H, H3-17), 1.80 ddd (1H, J = 14.1; 7.4; 5.0 Hz, H-3b), 1.72 td (H, J = 12.3;3.5 Hz, H-9b), 1.26 d (3H, J = 6.2 Hz, H3-15), 1.11 d (3H, J = 7.3 Hz, H3-14). 13C NMR (CDCl3, 100.03 MHz): 170.5 (C, C-16), 170.0 (C, C-12), 166.1 (C, C-18), 145.4 (CH, C-20), 144.8 (C, C-1), 139.3 (C, C-11), 134.3 (C, C-21), 130.5 (CH, C-24´), 128.9 (2CH, C-23, 23´), 128.2 (2CH, C-22, 22´), 127.5 (CH, C-5), 118.5 (CH2, C-13), 117.9 (CH, C-19), 82.0 (CH, C-8), 78.0 (CH, C-2), 67.9 (CH, C-4), 48.0 (CH, C-7), 39.2 (CH2, C-3), 37.0 (CH2, C-9), 29.3 (CH, C-10), 25.5 (CH2, C-6), 21.3 (3C, CH3-17), 20.2 (3H, CH3-15), 19.4 (3H, CH3-14). HRESIMS m/z [M + Na]+ 461.1944 (calcd for C26H30NaO6+, 461.1935).

4.6.2 Derivatization X 4-oxo-4-(p-tolyl)butanoic acid

To a round-bottom flask, 28 mg (0.091 mmol) of isoxanthanol was added and dissolved in 3 mL of DCM. Separately, 23 mg (0.118 mmol) of 4-oxo-4-(p-tolyl)butanoic acid was dissolved in 1 mL of DCM and added stepwise to the previous solution at 0°C with continuous stirring. Subsequently, 24 mg (0.118 mmol) of DCC and 2 mg (0.014 mmol) of DMAP, both dissolved in 1 mL of DCM, were added to the reaction mixture at 0°C. The mixture was stirred at room temperature overnight. The reaction mixture was then quenched by adding EtOAc and a concentrated solution of NH₄Cl, followed by filtration through Celite. The organic layer was washed with a concentrated NH₄Cl solution and brine. After drying the organic layer over Na₂SO₄, it was filtered and evaporated. Preparative TLC purification of the crude with (EtOAc/n-hex, 1:1) resulting in 19.7 mg of product 10 (49%).

(1S,3S)-3-acetoxy-1-((3aS,7S,8aS)-7-methyl-3-methylene-2-oxo-3,3a,4,7,8,8a-hexahydro-2H-cyclohepta[b]furan-6-yl)butyl 4-oxo-4-(p-tolyl)butanoate (10). Yellow oil; [α] 25D:−33.6 (c 0.006, DCM). IR (dry film) 2975, 2932, 2860, 2360, 1770, 1736, 1686, 1249 cm−1. 1H NMR (CDCl3, 400.13 MHz): 7.86 d (2H, J = 8.2 Hz, H-24,24´), 7.25 d (2H, J = 8.2 Hz, H-23,23´), 6.15 d (1H, J = 3.2 Hz, H-13a), 5.85 dd (1H, J = 9.1; 3.4 Hz, H-5), 5.43 d (1H, J = 3.0 Hz, H-13b), 5.23 t (1H, J = 7.2 Hz, H-2), 4.85 m (1H, H-4), 4.29 td (1H, J = 11.2;2.8 Hz, H-8), 3.27 t (2H, J = 6.2 Hz, H2-20), 2.78 m (1H, H-10), 2.74 m (2H, H2-19), 2.50 m (1H, H-6a), 2.47 m (1H, H-7), 2.41 s (3H, H3-25), 2.31 ddd (1H, J = 12.7, 4.4, 3.1, H-9a), 2.24 m (1H, H-3a), 2.09 m (1H, H-6b), 2.04 m (1H, H-3b), 2.03 s (3H, H3-17), 1.68 m td (1H, J = 12.8;3.5 Hz, H-9b), 1.24 d (3H, J = 6.3 Hz, H3-15), 1.13 d (3H, J = 7.3 Hz, H3-14). 13C NMR (CDCl3, 100.03 MHz): 197.4 (C, C-21), 172.0 (C, C-18), 170.4 (C, C-16), 169.8 (C, C-12), 144.5 (C, C-1), 144.1 (C, C-25), 139.0 (C, C-11), 128.1 (C, C-24), 129.3 (CH, C-23), 127.5 (CH, C-5), 118.3 (CH2, C-13), 82.1 (CH, C-8), 78.1 (CH, C-2), 67.8 (CH, C-4), 47.9 (CH, C-7), 39.1 (CH2, C-3), 37.0 (CH2, C-9), 33.1 (CH2, C-20), 29.5 (CH, C-10), 28.3 (CH2, C-19), 25.3 (CH2, C-6), 21.6 (CH3, C-26), 21.2 (CH3, C-17), 20.1 (CH3, C-15), 19.3 (CH3, C-14). HRESIMS m/z [M + Na]+ 505.2205 (calcd for C28H34NaO7+, 505.2197).

4.7 Biological assays4.7.1 Bacterial strains

Staphylococcus aureus (ATCC 25923), methicillin resistant S. aureus (ATCC 43300), Escherichia coli (ATCC 25922), Candida albicans ATCC 10231, C. tropicalis ATCC 66029, and Cryptococcus. neoformans ATCC 66031 were purchased from Bioartis SRL (Buenos Aires, Argentina). Bacterial strains were stored at − 80 °C in tryptic soy (TS) broth (Oxoid Ltd., Basingstoke, Hampshire, U.K.) with added 20% glycerol. Strains were revived by plating TS agar and incubated at 36 °C 24–48 h. In the case of yeast species, the same procedure was followed using Sabouraud glucose (SG) broth.

4.7.2 Determination of minimum inhibitory concentration (MIC)

The MIC of the extracts, fractions and isolated compounds were determined in multiwell plates by the standard broth microdilution method described by the Clinical and Laboratory Standards Institute (CLSI) [42]. Briefly, 1:2 serial dilutions of each sample to be evaluated were prepared in 100 µL of Mueller Hinton (MHB) broth (Oxoid Ltd., Basingstoke, Hampshire, U.K.), ranging from 1 to 0.008 mg/mL. 100 μL of a standardized bacterial inoculum (105 CFU/mL) were added to each well. The MIC values were recorded as the lowest concentration of the compound at which no signs of growth were observed, based on the OD625 value of less than 0.05 after 24 h of incubation. Meanwhile, antibiotics ampicillin was included as control using the following concentration window: 0.1 to 0.5 × 10–4 mg/mL. For the MIC evaluation of yeast-like species, SG broth was used. The multiwell plates were incubated for 48–72 h, and growth determination was based on OD530 nm values, with ketoconazole included as a positive control (1.5 × 10−2 to 1.2 × 10–4 mg/mL). In all cases, negative and microbial growth controls were performed with 1% DMSO.

4.7.3 Minimum bactericide (MBC) and fungicidal (MFC) concentration

Briefly, from the MIC determination, a known aliquot was removed from the wells corresponding to MIC, 2xMIC and 4xMIC and seeded in TS agar plates. The lowest concentration of compounds that provided no bacterial growth after 24 h of incubation at 36 °C was the MBC. For the determination MFC, subculture was carried out on SG agar plates.

4.7.4 Modulation of the antimicrobial activity assay

For the evaluation of pure compounds as antibiotic and antifungal modulators, the MICs of ampicillin, linezolid, ketoconazole, and amphotericin B were determined in the presence or absence of 8-epi-xanthine at a subinhibitory concentration (1/2 MIC). The concentrations of the antibiotic or antifungal agent tested were consistent with the MIC determination assay. In the wells containing a combination of antimicrobial drugs and 8-epi-xanthine, a fixed amount of the latter was added to ensure that the final concentration was consistent with its MIC/2. The microplates were incubated at 37°C for 24–48 h, and growth was determined by reading the OD625 or OD350 nm for S. aureus and C. albicans, respectively. The analyzed metabolite was considered a “potentiator” of antimicrobial activity when the combined MIC was lower than the MIC of the antimicrobial drug and “non-potentiator” when no changes were observed (combined MIC ≥ antimicrobial MIC). Minor variations in MIC were not considered significant [43].