Biology, Medicine, & Natural Product Chemistry ISSN 2089-6514 (paper) Volume 12, Number 1, April 2023 | Pages: 151-157 | DOI: 10.14421/biomedich.2023.121.151-157 ISSN 2540-9328 (online) A New ent-kaurene Diterpenoid Isolated from Leaves of Espeletia semiglobulata Cuatrec. and its Potential Antimicrobial Activity Andrés Márquez1,*, Alida Pérez1, †Luis Rojas1, Rosa Aparicio1, Freddy Ramos2, Ysbelia Obregón1, Alfredo Usubillaga1 1Research Institute, Faculty of Pharmacy and Bioanalysis, University of Los Andes, Mérida 5101, Venezuela. 2Department of Chemistry, Faculty of Science, National University of Colombia, Bogotá D.C. 111321, Colombia. Corresponding author* andresmqch@gmail.com Manuscript received: 03 December, 2022. Revision accepted: 16 January, 2023. Published: 24 January, 2023. Abstract The fraction of the neutral extraction of the leaves of Espeletia semiglobulata Cuatrec. was subjected to chromatographic separation, it yielded four ent-kaurene type diterpenoids: three known [ent-kaur-16-en-19-al (I), ent-kaur-18-nor-16-en-4-ol (III), ent-kaur-16-en-19-ol (IV)] and a new one elucidated as ent-kaur-3-acetoxy-15-ene (II), based on the physicochemical and spectroscopic data of FTIR, GC-MS, and 1D and 2D NMR. These compounds were subjected to antimicrobial bioassay studies. This new ent-kaurene showed a significant inhibition potential against the growth of gram negative bacterial strains [Escherichia coli (ATCC 25922): 8 mm, Klebsiella pneumoniae (ATCC 23357): 10 mm, Pseudomonas aeruginosa (ATCC 27853): 8 mm], it also showed inhibition against the growth of fungal strain (Candida krusei: 8 mm), at a 2 mg/mL concentration. The compounds (I), (III) and (IV) failed to show any significant results in the antimicrobial screening against five bacterial strains [Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 29212), Klebsiella pneumoniae (ATCC 23357), Escherichia coli (ATCC 25922) y Pseudomonas aeruginosa (ATCC 27853)] and one fungal strain [Candida krusei (ATCC 6558)]. These results reveal a remarkable natural structure-activity relationship of the ent-kaurene core regarding the C-3 position (A ring of perhydrophenanthrene unit), whose oxygenation or addition of a hydrogen bond acceptor or donor group, improves the antimicrobial activity. Keywords: Diterpene; ent-kaurene; Asteraceae; Natural Products; Antimicrobial Activity. Abbreviations: NMR – Nuclear Magnetic Resonance; 1H NMR – Proton Nuclear Magnetic Resonance; 13C NMR – Carbon Nuclear Magnetic Resonance; 13C-APT – attached proton test; COSY- Correlation spectroscopy; HSQC – Heteronuclear single quantum correlation spectroscopy; HMBC – Heteronuclear multiple bond correlation spectroscopy; IR – Infrared spectroscopy; GC-MS – Gas chromatography–Mass Spectrometry; mp – melting point; MH – Müeller-Hinton; DMSO – Dimethyl Sulfoxide; EtOAc – Ethyl Acetate. INTRODUCTION Bioactive natural products have played an important role in treating and preventing human diseases. These are important sources for new drugs and also good lead compounds suitable for further modification during drug development (Atanasov et al., 2021). The terpenoids are among the most important natural compounds known for their medicinal value (Kamran et al., 2022; Proshkina et al., 2020); within them, ent-kauranes represent an important group of tetracyclic diterpenes that possesses a wide spectrum of biological and pharmaceutical activities such as antimicrobial, antiparasitic, insect antifeedant, cytotoxic, antitumour, anti-HIV, steroidogenic, antifertility, hypotensive, antiinflamatory, among others (Ghisalberti, 1997). This type of diterpene is extensively found in different species of Espeletiinae (Asteraceae), a family of resinous plants, popularly known as “Frailejon”, that grow at an altitude of 2500 m in the Andes of Northern South America. These plants are used by the inhabitants of the high moors to treat asthma and rheumatic conditions (Usubillaga et al., 2003; Aparicio et al., 2013). Ent-kaurenes have been identified in some studies about the Espeletia semiglobulata Cuatrec. Species. It grows above an altitude of 3900 m at the “Piedras Blancas” Moor, which is part of Sierra La Culata, located near the city of Mérida, Venezuela. ent-kaur-16-en-19-oic acid has been identified on the fraction of acid extraction of the leaves of this plant as the most abundant component and known for its diverse biological properties (Sosa-Sequera et al., 1996; Cavalcanti et al., 2005; Kim et al., 2016; Zhang et al., 2017; Cotoras et al., 2004; Mongelli et al., 2002), and ent-kauran-19-oic acid, ent-kaur-9(11)-16-dien-19- oic acid, ent-kaur-15α-isovaleroxy-16-en-19-oic acid, ent-kaur-15α-hidroxy-16-en-19-oic acid, ent-kauran- 16α-hidroxy-19-oic acid, and ent-kaur-15-en-19-oic acid have been identified as the less abundant components. On the other hand, ent-kaur-16-en-19-al and ent-kaur- https://doi.org/10.14421/biomedich.2023.121.151-157 152 Biology, Medicine, & Natural Product Chemistry 12 (1), 2023: 151-157 16-en-19-ol have been identified as majority components in the fraction of neutral extraction, and ent- kaur-16α-hidroxy-16-en-19-al, ent-kaur-18-nor-16-en-4- ol, ent-kaur-16-en-19-ol acetate have been identified as minority components without any biological activity reported on any of them (Usubillaga et al., 1988; Aparicio et al., 2013). In this study, we are reporting the isolation, structural identification, and antimicrobial activity of a new ent-kaurene-type terpenoid natural product (II) from neutral fraction of Espeletia semiglobulata Cuatrec. leaves, also, the evaluation of antimicrobial activity of some known ent-kaurenes coming from this neutral extraction is being reported for the first time. MATERIALS AND METHODS General methods Melting points were determined on a Fisher-Johns melting point apparatus. IR spectra were measured on a Perkin Elmer Spectrum two, 10.03.06 version, as KBr disks. NMR spectra was recorded with a Bruker- UltraShieldTM 400 MHz instrument for solutions in CDCl3. GC-MS were performed on a Hewlett-Packard MSD 5973 instrument fitted with a 5 % phenylmethyl polysiloxane fused-silica column (HP-5MS, 30 m, 0.25 mm, film thickness 0.25 μm). The initial analysis temperature was 250 °C, which was increased at 5 °C/min. to a final temperature of 300 °C. Analytical thin-layer chromatography was performed on E. Merck aluminum-backed silica gel foils (F254). Flash chromatography was performed on silica gel E. Merck grade 60, 63-200 mesh, by gradient elution with hexane and hexane-EtOAc mixtures. Plant Collection Leaves of Espeletia semiglobulata Cuatrec. were collected at 3900 m of altitude in “Piedras Blancas” Moor and along the road to Piñango, about 13 Km from the mountain’s peak “Pico del Aguila”. Voucher specimens (AU30 and AU21) were deposited at the Faculty of Pharmacy and Bioanalysis Herbarium, University of Los Andes (MERF Herbarium). Extraction and neutral fraction obtainment Espeletia semiglobulata Cuatrec. leaves were dried at 40 °C during 48 hours and they were grinded. The grinded material (200 g) was extracted at room temperature with a mixture of hexane/diethyl ether (3:1, v/v) for a period of two days. The solvents were removed under reduced pressure and the solid residues were dissolved in hexane/EtOAc, and shaken with 5 % aqueous NaOH. The aqueous phase was taken to pH 3 by careful addition of concentrated HCl and shaken with hexane to obtain the acid fraction. The original hexane-diethyl ether solution, left over after the alkaline treatment surplus, was mixed with active charcoal and boiled for 10 minutes. A neutral fraction of 25 g was obtained after an open column chromatography treatment. Isolation of kaurene diterpenes Most of the leaves neutral fraction was submitted to flash chromatography over silica gel. The column (A) was eluted with hexane and hexane/AcOEt mixtures and 100 mL fractions were collected, which were constantly motorized by TLC. Fractions 1-35 eluted with hexane yielded 840 mg of a mixture that contained plant waxes and (I). Fractions 36-39 eluted with hexane rendered 500 mg of pure (I) as a white solid, mp 113-116 ºC, identical to an authentic sample of ent-kaur-16-en-19-al (mp,1H-NMR, 13C-NMR) obtained from Espeletia semiglobulata (Usubillaga et al., 1988; Aparicio et al., 2013). The elution continued with hexane (40-45 fractions), it yielded 150 mg of a mixture of (I) and (II). The elution of the 46-61 fractions with hexane yielded 1000 mg of pure (II), which was crystallized from hexane/Et2O as white crystalline solid. This compound has a very similar polarity to (I), however, they are not the same (mp, 1H-NMR, 13C-NMR). The elution of the 82-94 fractions with hexane/EtOAc 5 % yielded 300 mg of pure (III), mp 146-148 °C, identical to an authentic sample of ent-kaur-18-nor-16-en-4-ol (mp,1H-NMR, 13C-NMR) obtained from Espeletia nana (Peña et al., 2012). The elution of the 101-106 fractions with hexane/EtOAc 10 % yielded 200 mg of pure (IV), mp 142-144 °C, identical to an authentic sample of ent-kaur- 16-en-19-ol (mp,1H-NMR, 13C-NMR) obtained from Espeletia semiglobulata Cuatrec. (Usubillaga et al., 1988; Aparicio et al., 2013). Finally, the elution using hexane/EtOAc 20 % yielded a complex mixture (21.4 g). Antimicrobial Assay  Antibacterial activity The antibacterial activity was determined by the agar diffusion method with discs. The following microorganisms were tested: Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 29212), Klebsiella pneumoniae (ATCC 23357), Escherichia coli (ATCC 25922) y Pseudomonas aeruginosa (ATCC 27853). An 18 hours culture of each microorganism was used in 2.5 mL Müeller-Hinton (MH) broth at 37 °C. The bacterial inoculum was adjusted with saline physiological solution to the Mc Farland Turbidity Standard N° 0.5 (106-8 CFU/mL). Each inoculum was spread with a swab on the surface of a plate containing Müeller-Hinton agar and then a disc of filter paper (6 mm diameter) previously impregnated with 10 μL of the compound dissolved in DMSO was placed on the surface at a 2 mg/mL concentration for each one [(I), (II), (III), (IV)]. A disc impregnated with DMSO was included as a negative control. In addition, the standard disc of the reference antibiotic was placed as a positive control for each of the microorganism (Oxacillin 1 μg Márquez, et al. – A New ent-kaurene Diterpenoid Isolated from Leaves … 153 for Staphylococcus aureus; Vancomycin 30 μg for Enterococcus faecalis; Tobramycin 10 μg for Escherichia coli; Aztreonam 30 μg for Klebsiella pneumoniae; Cefepime 30 μg for Pseudomonas aeruginosa). After having placed the discs on the Petri plates, they were refrigerated at 4 °C for 24 h (Velasco et al., 2007). The reading of the inhibition halos was performed at 24 h and it was measured (mm) around the disc. All the antibacterial activity trials were done twice. The measurement of the inhibition halos of the antibacterial activity was done thus: C= A-B (C= size of the inhibition halo; A= size of the halo plus the disk of filter paper; B= size of the filter paper disk, 6 mm).  Antifungal activity The antifungal activity was determined by the agar well diffusion method. The microorganism tested was Candida krusei (ATCC 6558), using Fluconazole 100 μg as positive control, and DMSO as negative control. The strain repacked 12 hours before, coming from a savory dextrose agar, it was used to prepare the inoculum which was compared using the Mc Farland Turbidity Standard N°0.5 (106-8 CFU/mL). Each inoculum was spreaded with a swab on the surface of a plate containing Müeller-Hinton agar and then 6 mm diameter holes were opened in it, where 20 μL of the compounds dissolved in DMSO were placed at a 2 mg/mL concentration for each one [(I), (II), (III), (IV)]. One well was included with the DMSO as a negative control and another well with the reference antibiotic as positive control. Then the Petri plates were refrigerated at 4 °C for 24 h (Velasco et al., 2007). The reading of the inhibition halos was performed at 24 h and was measured (mm) around the well. All the antifungal activity trials were done twice. RESULTS AND DISCUSSION Phytochemical analysis and structure elucidation The structures of the ent-kaurene diterpenoids isolated from neutral fraction of E. semiglobulata Cuatrec., leaves are presented on figure 1. Figure 1. Molecular structure of ent-kaurenes from neutral fraction of aerial parts of Espeletia semiglobulata Cuatrec. Compound (I) was isolated as a white powder, mp 113-116 ºC. GC-MS gave [M]+ molecular ion at m/z: 286.1 (calcd. for C20H30O; m/z: 286). FT-IR, vmax (cm - 1), functional group: 2729, v(C-H, aldehyde); 1711, v(C=O); 1655, v(C=C). 1H-NMR (400 MHz, CDCl3) δH: 9.73 (H1-19, s, CHO), 4.74 (H1-17a, s, =CH), 4.79 (H1- 17b, s, =CH), 2.64 (H1-13, s, C-H), 2.05 (H2-15, m, CH2), 0.98 (H3-18, s, CH3). 13C-NMR (100 MHz, CDCl3) δC: 205.94 (C-19, CHO), 103.24 (C-17, =CH2), 43.75 (C-13, CH), 49.05 (C-15, CH2), 24.29 (C-18, CH3). This data completely matches with the spectral data reported in the bibliographic references (Usubillaga et al., 1988; Aparicio et al., 2013), this leads us to the conclusion, the compound (I) is an ent-kaurene-type diterpene called ent-kaur-16-en-19-al, commonly known as kaurenal. This compound was first isolated and characterized as a natural product of Espeletia grandiflora Humb. et Bompl. (Piozzi et al., 1971). It is particularly abundant in species of Espeletia genus (Morales et al., 1973; Bohlmann et al., 1980; Usubillaga et al., 1988), and in fact, it has already been isolated and reported from E. semiglobulata Cuatrec. leaves (Usubillaga et al., 1988; Aparicio et al., 2013). Compound (II) was obtained as a crystalline white powder. The analysis of its 13C NMR and GC-MS data (m/z 330 [M]+) deduced its molecular formula C22H34O2, which suggested 6 degrees of unsaturation. The IR spectrum showed the absorption bands of ester group [1735 cm-1 (O−C=O) and 1243 cm-1 (O−C)] and typical bands of alkene group [3040 cm-1 (=C−H) and 1661 cm- 1 (C=C)]. The 1H NMR (400 MHz, CDCl3) and 13C NMR (CDCl3, 100 MHz) data (Table 1) demonstrated the signals of thirty-four protons and twenty-two carbons sorted by attached proton test (13C-APT) and heteronuclear single quantum coherence (HSQC) spectra, which can be classified as: A terminal ester group [δH/δC: 1.98 (H3-22, s, CH3) / 21.47 (C-22, CH3); 171.17 (C-21, >C=O)], a trisubstituted alkene group [δH/δC: 5.06 (H1-15, s, =CH) / 124.47 (C-15, =CH); 139.78 (C-16, >C<)], four methyl groups [δH/δC: 0.81 (H3-18, s, CH3) / 28.22 (C-18, CH3); 0.80 (H3-19, s, CH3) / 22.70 (C-19, CH3); 0.73 (H3-20, s, CH3) / 22.70 (C-20, CH3)] including an olefinic methyl [δH/δC: 1.80 (H3-17, s, CH3) / 23.37 (C-17, CH3)], a methinic proton bonded to a carbon bearing heteroatom (deshielded proton) [δH/δC: 4.50 (H1-3, t, O−CH) / 80.84 (C-3, O−CH)] seven sp3 methylene [δH/δC: 1.55 (H2-1, m, CH2) / 33.01 (C-1, CH2); 1.57 (H2-2, t, CH2) / 23.52 (C- 2, CH2); 1.49 (H2-6, m, CH2) / 21.07 (C-6, CH2); 1.30 (H2-7, m, CH2) / 36.69 (C-7, CH2); 1.48 (H2-11, m, CH2) / 18.39 (C-11, CH2); 1.54 (H2-12, m, CH2) / 26.75 (C-12, CH2); 1.35 (H2-14, t, CH2) / 41.49 (C-14, CH2)], three sp3 methines [δH/δC: 1.51 (H1-5, m, CH) / 52.51 (C-5, CH); 0.79 (H1-9, m, CH) / 55.41 (C-9, CH); 1.86 (H1-13, m, CH) / 47.79 (C-13, CH)], and three sp3 quaternary carbon [δC: 37.86 (C-4); 40.17 (C-8); 39.68 (C-10)]. Detailed analysis of these NMR data suggested the presence of a typical ent-kaurene-type diterpenic 154 Biology, Medicine, & Natural Product Chemistry 12 (1), 2023: 151-157 skeleton [perhydrophenanthrene unit (A, B and C rings) fused to a cyclopentane unit (D ring) formed by a two- carbon bridge between C-8 and C-13 (Figure 1)] (Batista et al., 2005). This partial structure was further confirmed by correlations spectroscopy (COSY) and heteronuclear multiple bond correlation (HMBC), which has been shown in Figure 2. By analysis of the COSY correlations, it was possible to identify six structural areas (a - f) of this compound. The fact that the terminal ester group [C-21 (δC 171.17, O-C=O)/C-22 (δC 21.47, CH3); H3-22(δH 1.98, s, CH3)]; where the terminal arrangement was confirmed by the HMBC correlation between the methylic protons H3-22 and carbonyl group C-21 (δC 171.17, C=O)] was on C-3 (δC 142.7) was confirmed by the HMBC correlation from methinic proton H1-3 (δH 4.50, t, CH) to ester carbonyl group C- 21 (δC 171.17, >C=O), as well as, the correlation of H1-3 with methylene C-2 (δC 23.52, CH2) and quaternary carbon C-4 (δC 37.86, >C<). The presence of two methyls on C-4 was confirmed by its HMBC correlation with H3-18 (δH 0.81, s, CH3) and H3-19 (δH 0.80, s, CH3), in addition to the HMBC correlation of these methylic prontons with methine C-3 (δC 80.84, O-CH), as well as, by the correlations: H3-19 (δH 0.80, s, CH3) / C-5 (δC 52.51, CH) and H1-5 (δH 1.51, m, CH) / C-4 (δC 37.86, >C<). The mutual correlations of both methyls, both by HMBC [H3-18 (δH 0.81, s, CH3) / C-19 (δC 22.70, CH3); H3-19 (δH 0.80, s, CH3) / C-18 (δC 28.22, CH3)] and by COSY [H3-18 (δH 0.81, s, CH3) / H3-19 (δH 0.80, s, CH3)] also confirmed the vicinal arrangement of them on C-4. The existence of one methyl on C-10 was verified by the HMBC correlation from methylic protons H3-20 (δH 0.73, s, CH3) to quaternary carbon C-10 (δC 39.68, >C<) and methylene C-1 (δC 33.01, CH2). On the other hand, the presence of an endocyclic C-C double bond between C-15 and C-16, and the presence of an olefinic methyl on C-16 (D ring) were confirmed by HMBC correlations between methylic prontons H3-17 (δH 1.80, s, CH3) with olefinic methine C-15 (δC 124.47, =CH) and methine C-13 (δC 47.79, CH), and at the same time, the methinic proton H1-13 (δH 1.86, m, CH) correlates with quaternary carbon C-16 (δC 139.78, =C<) and methylene C-12 (δC 26.75, CH2). This partial structure has also been confirmed by COSY correlation from olefinic methyl protons H3-17 (δH 1.80, s, CH3) to olefinic methine proton H1-15 (δH 5.06, s, =CH), and vice versa, as well as, the mutual correlation between olefinic methyl protons H3-17 with methinic proton H1-13 (δH 1.86, m, CH), and this last, mutual correlated with methylenic ptotons H2-14 (δH 1.35, t, CH2) and H2-12 (δH 1.54, m, CH2). Finally, the central ring of ent-kaurene core (B ring) was confirmed by the HMBC correlations between methinic proton H1-9 (δH 0.79, m, CH) with quaternary carbon C-10 (δC 39.68, >C<), C-8 (δC 40.17, >C<) and methylene C-11(δC 18.39, CH2), as well as, this ring was confirmed by COSY correlations between methinic proton H1-9 with H1-5 (δH 1.51, m, CH), H2-7 (δH 1.30, m, CH2) and H2-11 (δH 1.48, m, CH2). Based on the spectroscopic analysis performed, the isolation of an ent-isokaurene-type diterpene is confirmed, especially ent-kaur-3-acetoxy-15-ene, which have also be called 3- acetoxy-isokaurene, this being a natural product reported for the first time. Compound (III) was isolated as a white powder, mp 146-148 °C. GC-MS analysis shown [M]+ molecular ion at m/z: 274.3 (calcd. for C19H30O; m/z: 274). FT-IR, vmax (cm-1), functional group: 3500, v(O-H, alcohol); 1600, v(C=C). 1H-NMR (400 MHz, CDCl3) δH: 4.76 (H1-17a, s, =CH), 4.82 (H1-17b, s, =CH), 3.65 (H1-18, s, OH), 2.66 (H1-13, t, C-H), 2.16 (H2-15, m, CH2), 1.20 (H3-18, s, CH3), 1.11 (H3-20, s, CH3). 13C-NMR (100 MHz, CDCl3) δC: 79.31 (C-4, >COH), 44.20 (C-13, CH), 49.40 (C-15, CH2), 102.93 (C-17, =CH2), 21.52 (C-18, CH3), 17.74 (C-20, CH3). This data completely matches with the spectral data reported in the bibliographic references (Bohlmann et al., 1980; Peña et al., 2012), this leads us to the conclusion that the compound III) is an ent-kaurene-type diterpene called ent-kaur-18-nor-16-en-4-ol, commonly known as ruilopeziol. This compound has only been isolated and reported as a natural product of Ruilopezia linden, Coespeletia lutescens, and Espeletia nana Cuatrec. (Bohlmann et al., 1980; Peña et al., 2012), therefore, its isolation and characterization from E. semiglobulata Cuatrec. leaves is a new discovery. Márquez, et al. – A New ent-kaurene Diterpenoid Isolated from Leaves … 155 Table 1. 1H-NMR (CDCl3, 400 MHz), 13C-NMR (CDCl3, 100 MHz), COSY ( 1H↔1H) (CDCl3, 600 MHz) and HMBC [ 1H (600 MHz) → 13C (100 MHz), CDCl3] of compound (II) (ent-kaur-3-acetoxy-15-ene). Position δ 1H (ppm), mult. δ 13C (ppm), Type COSY ( 1H↔1H) HMBC ( 1H→13C) 1 1.55, m 33.01, (>CH2) H2-1 ↔ H2-2 -- 2 1.57, t 23.52,(>CH2) H2-2 ↔ H2-1 H2-2 → C-3 3 4.50, t 80.84, (O-CH-) H1-3 ↔ H2-2 H1-3 → C-2, C-4, C-21 4 -- 37.86, (>C<) -- -- 5 1.51, m 52.51, (>CH-) H1-5 ↔ H3-19 H1-5 → C-4, C-19, C-6 6 1.49, m 21.07, (>CH2) -- H2-6 → C-5 7 1.30, m 36.69, (>CH2) -- H2-7 → C-8 8 -- 40.17, (>C<) -- -- 9 0.79, m 55.41, (>CH-) H1-9 → H2-7, H1-5, H2-11 H1-9 → C-10, C-11, C-8 10 -- 39.68, (>C<) -- -- 11 1.48, m 18.39, (>CH2) -- H2-11 → C-12 12 1.54, m 26.75, (>CH2) H2-12 ↔ H1-13 H2-12 → C-13, C-11 13 1.86, m 47.79, (>CH-) H1-13 ↔ H2-12, H3-17 H1-13 → C-17, C-16, C-12 14 1.35, t 41.49, (>CH2) H2-14 ↔ H1-13 -- 15 5.06, s 124.47, (=CH) H1-15 ↔ H3-17 H1-15 → C-17 16 -- 139.78, (=C<) -- -- 17 1.80, s 23.37, (-CH3) H3-17 ↔ H1-15, H1-13 H3-17 → C-13, C-15, C-16 18 0.81, s 28.22, (-CH3) H3-18 ↔ H3-19 H3-18 → C-3, C-4, C-19 19 0.80, s 22.70, (-CH3) H3-19 ↔ H3-18, H1-5 H3-19 → C-3, C-4, C-5, C-18 20 0.73, s 17.66, (-CH3) -- H3-20 → C-1, C-10 21 -- 171.17, (>C=O) -- -- 22 1.98, s 21.47, (-CH3) -- H3-22 → C-21 Figure 2. Selective 1H↔1H COSY (▬) and 1H→13C HMBC (▬) correlations of (II). Compound (IV) was isolated as a white powder, mp 142-144 °C. GC-MS analysis shown [M]+ molecular ion at m/z: 288.3 (calcd. for C20H32O; m/z: 288). FT-IR, vmax (cm-1), functional group: 3406, v(O-H, alcohol); 1026, v(C-O); 1600, v(C=C). 1H-NMR (400 MHz, CDCl3) δH: 4.73 (H1-17a, s, =CH), 4.79 (H1-17b, s, =CH), 3.44 (H1-19a, dd, CH2), 3.74 (H1-19b, dd, CH2), 2.63 (H1-13, t, C-H), 2.06 (H2-15, m, CH2), 1.01 (H3-18, s, CH3), 0.96 (H3-20, s, CH3). 13C-NMR (100 MHz, CDCl3) δC: 65.60 (C-19, CH2OH), 155.90 (C-16, >C=), 103.00 (C-17, =CH2), 27.10 (C-18, CH3), 18.21 (C-20, CH3), 44.20 (C-13, CH), 49.10 (C-15, CH2). By comparing this experimental data with the spectroscopic data reported in the bibliography (Bohlmann et al., 1980; Piozzi et al., 1971), it is evident that this compound is an ent-kaurene-type diterpene called ent- kaur-16-en-19-ol, commonly known as kaurenol. Currently, this diterpene is considered a widely distributed product in the Asteraceae family, since it has been isolated in different species, including the species of the Espeletia genus (Seaman et al., 1990), like Espeletia semiglobulata Cuatrec. (Usubillaga et al., 1988; Aparicio et al., 2013). Antimicrobial Activity The isolated compounds, ent-kaur-16-en-19-al (I), ent- kaur-3-acetoxy-15-ene (II), ent-kaur-18-nor-16-en-4-ol (III) and ent-kaur-16-en-19-ol (IV) were evaluated for in vitro growth inhibitory potential against different bacterial [Staphylococcus aureus (ATCC 25923), 156 Biology, Medicine, & Natural Product Chemistry 12 (1), 2023: 151-157 Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 23357), Pseudomonas aeruginosa (ATCC 27853)] and fungal [Candida krusei (ATCC 6558)] strains, according to the previously described procedure to agar diffusion method with discs (strain of bacteria), and agar well diffusion method, (strain of fungi). The results in Table 2, shows that all the compounds are inactive againt the bacterial and fungal strains tested, except compound (II), which showed growth inhibition against gram-negative bacterial strains (Escherichia coli: 8 mm, Klebsiella pneumoniae: 10 mm, Pseudomonas aeruginosa: 8 mm), as well as it showed growth inhibition against the fungal strain (Candida krusei: 8 mm), at a concentration of 2 mg/mL. These results reveal a remarkable natural structure- activity relationship of the ent-kaurene core with regarding the C-3 position (A ring of perhydrophenanthrene unit), whose oxygenation or structural modification apparently, improves the antimicrobial activity. Similar results have been found synthetically, by preparing derivatives of hydroxylated ent-kaurenes at C-3, improving their biological activity (Ohkoshi et al., 2004). Moreover, the evaluation of the relationship between structure and antimicrobial activity of some diterpenes suggested that the presence of a substituted decalin skeleton (essential hydrophobic portion for crossing membranes), and a hydrophilic region bearing a donor or acceptor group able to interact with hydrogen-bond acceptor/donor groups on the membrane were necessary for their insertion in a phospholipid bilayer model. This feature enables the diterpenes to cross the bacterial cell membrane causing cell damage (Urzúa et al., 2008). In this context, the ent-kaurenes tested have the structural characteristics described above: a substituted decalinic system that confers a lipophilic character, and a hydrophilic region bearing a hydrogen-bond donor or acceptor group. However, the compounds (I), (III) and (IV), which possess these polar groups in the C-4 position, they are sterically limited to form hydrogen- bond with acceptor/donor groups on the microorganism membrane, and therefore cannot cause damage to it. On the contrary, compound (II) has this moderately hydrophilic group (ester) in C-3 position, whose steric hindrance is low and this confers the advantage to interact and establish hydrogen bonds with acceptor/donor groups on the microorganism membrane, being able to cross and cause damage to it. The position and steric hindrance of this hydrogen-bond donor or acceptor group on decalinic system are important characteristics for the biological activity of ent-kaurenes (Urzúa et al., 2008). Table 2. Antimicrobial activity of natural compounds (I)-(IV) expressed as diameters of the inhibition zone (mm). Microorganism Compound Positive control (I) (II) (III) (IV) OX VA TO AZ CF FL Staphylococcus aureus (ATCC 25923) IA IA IA IA *23 - - - - - Enterococcus faecalis (ATCC 29212) IA IA IA IA - *19 - - - - Escherichia coli (ATCC 25922) IA 8 IA IA - - *26 - - - Klebsiella pneumoniae (ATCC 23357) IA 10 IA IA - - - *34 - - Pseudomonas aeruginosa (ATCC 27853) IA 8 IA IA - - - - *32 - Candida krusei (ATCC 6558) IA 8 IA IA - - - - - *15 IA: inactive; OX: Oxacillin®; VA: Vancomycin®; TO: Tobramycin®; AZ: Aztreonam®; CF: Cefepime®; FL: Fluconazole®; *: Inhibition halo in mm, for control groups (CLSI, 2020). CONCLUSIONS Ent-kaurene-type diterpenes, ent-kaur-16-en-19-al (I), ent-kaur-3-acetoxy-15-ene (II), ent-kaur-18-nor-16-en- 4-ol (III), ent-kaur-16-en-19-ol (IV), were isolated from the neutral fraction of Espeletia semiglobulata Cuatrec. leaves, being ent-kaur-3-acetoxy-15-ene (II) a natural product reported for the first time. Compound (II) was the only one that showed antimicrobial activity, but only against gram-negative bacterial strains (Escherichia coli: 8 mm, Klebsiella pneumoniae: 10 mm, Pseudomonas aeruginosa: 8 mm) and fungal strain (Candida krusei: 8 mm), at of 2 mg/mL concentration. These results reveal a remarkable natural structure-activity relationship of the ent-kaurene core with regarding the C-3 position (A ring of perhydrophenanthrene unit), whose oxygenation or addition of a hydrogen-bond acceptor or donor group, improves the antimicrobial activity. Márquez, et al. – A New ent-kaurene Diterpenoid Isolated from Leaves … 157 Acknowledgements: All authors are grateful to the Research Institute “Dr. Alfredo Nicolás Usubillaga del Hierro”, Faculty of Pharmacy and Bioanalysis, University of Los Andes, Mérida 5101, Venezuela for the support. The Research Institute authors are grateful to the Prof. Freddy Ramos from the Department of Chemistry, Faculty of Science, National University of Colombia, Bogotá D.C. 111321, Colombia, for NMR and MS equipment. Besides, all authors are grateful to the MSc. Ana Deixy Flores from the “Instituto Clínico Santa Filomena”, Microbiology Laboratory, Mérida 5101, Venezuela, for the antimicrobial assays. Competing Interests: The authors declare that there are no competing interests. Funding This research was supported by Research Institute “Dr. Alfredo Nicolás Usubillaga del Hierro”, Faculty of Pharmacy and Bioanalysis, University of Los Andes, Mérida 5101, Venezuela. REFERENCES Aparicio, R., Villasmil, T., Peña, A., Rojas, J., & Usubillaga, A. (2013). Estudio fitoquímico de las hojas de Espeletia semiglobulata Cuatrec. Rev Fac Farm., 55(2), 2-5. Atanasov, A.G., Zotchev, S.B., & Dirsch, V.M. (2021). Natural products in drug discovery: advances and opportunities. Nat Rev Drug Discov., 20, 200-216. doi: https://doi.org/10.1038/s41573-020-00114-z Batista, R., García, P., Castro, M., Del Corral, J., Speziali, N., & Oliveira, A. (2007). Methyl ent-16β,17-epoxykauran-19-oate. Act. Crystallogr., 63: 93-94. doi: https://doi.org/10.1107/S1600536807002693 Bohlmann, F., Suding, H., Cuatrecasas, J., King, R. M., & Robinson, H. (1980). Neue diterpene aus der subtribus Espeletiinae. Phytochemistry, 19: 267. Cavalcanti, B.C., Costa-Lotufo, L.V., Moraes, M.O., Burbano, R.R., Silveira, E.R., Cunha, K.M.A., Rao, V.S.N., Moura, D.J, Rosa, R.M., Henriques, J.A.P, & Pessoa, C. (2005). Genotoxicity evaluation of kaurenoic acid, a bioactive diterpenoid present in Copaiba oil. Food Chem. Toxicol., 44(3), 388-392. doi: https://doi.org/10.1016/j.fct.2005.08.011 CLSI: Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; Twenty First Informational supplement. CLSI document M100-S21 (2020). Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania, USA, 19087–1898. Cotoras, M., Folch, C., & Mendoza, L., (2004). Characterization of the antifungal activity on Botrytis cinerea of the natural diterpenoids kaurenoic acid and 3β-Hydroxy-kaurenoic acid. J. Agric. Food Chem., 52(10), 2821-2826. doi: https://doi.org/10.1021/jf030672j Ghisalberti, E., (1997). The biological activity of naturally ocurring kaurene diterpenes. Fitoterapia, 68(4), 303-325. Kamran, S., Sinniah, A., Abdulghani, M.A.M., & Alshawsh, M.A. (2022). Therapeutic Potential of Certain Terpenoids as Anticancer Agents: A Scoping Review. Cancers, 14(5), 1100. doi: https://doi.org/10.3390/cancers14051100 Kim, K.H., Sadikot, R.T., & Joo, M. (2016). Therapeutic effect of ent-kaur-16-en-19-oic acid on neutrophilic lung inflammation and sepsis is mediated by Nrf2, Biochem. Biophys. Res. Commun., 474(3), 534-540. doi: https://doi.org/10.1016/j.bbrc.2016.04.122 Mongelli E., Pomilio A. B., Sánchez J.B., Guerra F.M., & Massanet, G.M. (2002). ent-Kaur-16-en-19-oic acid, a KB cells cytotoxic diterpenoid from Elaeoselinum foetidum, Phytother. Res., 16(4), 387-388. doi: https://doi.org/10.1002/ptr.955 Morales, A., Usubillaga, A., Banerjee, A., & Nakano, T. (1973). Studies on the constituents of Espeletia weddellii. Planta Med., 24: 243-248. doi: https://doi.org/10.1055/s-0028- 1099493 Ohkoshi, E., Kamo, S., Makino, M., & Fujimoto, Y. (2004). ent- kaurenoic acids from Mikania hirsutissima (Compositae). Phytochemistry, 65(7): 885–890. https://doi.org/10.1016/j.phytochem.2004.02.020 Peña, A., Alarcón, L., Baptista, J., Aparicio, R., Villasmil, T., & Usubillaga, A. (2012). A Phytochemical Analysis of Espeletia nana Cuatrec. a midget Espeletiinae from Paramo Ortiz, Venezuela. Avances en Química, 7(3): 187-192. Piozzi, F., Passannanti, S., Paternostro, M., & Sprio, V. (1971). Kaurenoid Diterpenes in Espeletia grandiflora. Phytochemistry, 10: 1164-1166. Proshkina, E., Plyusnin, S., Babak, T., Lashmanova, E., Maganova, F., Koval, L., Platonova, E., Shaposhnikov, M., & Moskalev, A. (2020). Terpenoids as Potential Geroprotectors. Antioxidants, 17(6), 529. doi: https://doi.org/10.3390/antiox9060529 Seaman, F.C., Bohlmann, H., Zdero, C., & Mabry, T.J. (1990). Diterpenes of flowering plants. Compositae (Asteraceae). 551-577. doi: https://doi.org/10.1007/978-1-4612-3274-2 Sosa-Sequera, M., Sosa-Canache, B., Usubillaga, A., Escalona, A., Suarez, O., Reyna, R., & Pascuzzo, C. (1996). Antitumorogenic effect of kaurenic acid on melanoma B16F1, VI. World Conference on Clinical Pharmacology and Therapeutics, Buenos Aires, Argentina. Urzúa, A., Rezende, M.C., Mascayano, C., & Vásquez, L. (2008). A structure-activity study of antibacterial diterpenoids. Molecules, 13(4), 882–891. doi: https://doi.org/10.3390/molecules13040822 Usubillaga, A., & Capra, C. (1988). Chemical constituents of Espeletia semiglobulata. Fitoterapia, 59(5), 383-384. Usubillaga, A., Romero, M., Aparicio, R. (2003). Kaurenic acid in Espeletiinae. Acta Hortic., 597, 129-130. Velasco, J., Rojas, J., Salazar, P., Rodríguez, M., Díaz, T., Morales, A., and Rondón, M. (2007). Antibacterial activity of the essential oil of Lippia oreganoides against multiresistant bacterial strains of nosocomial origin. Nat. Prod. Commun., 2(1):85-88. Doi:https://doi.org/ doi: 10.1177/1934578X0700200117 Zhang, M., Zhao, C., Dai, W., He, J., Jiao, S., & Li, B. (2017). Anti-inflammatory ent-kaurenoic acids and their glycosides from Gochnatia decora. Phytochemistry, 137, 174-181. doi: https://doi.org/10.1016/j.phytochem.2017.02.021 https://doi.org/10.1038/s41573-020-00114-z https://doi.org/10.1107/S1600536807002693 https://doi.org/10.1016/j.fct.2005.08.011 https://doi.org/10.1021/jf030672j https://doi.org/10.3390/cancers14051100 https://doi.org/10.1016/j.bbrc.2016.04.122 https://doi.org/10.1002/ptr.955 https://doi.org/10.1055/s-0028-1099493 https://doi.org/10.1055/s-0028-1099493 https://doi.org/10.1016/j.phytochem.2004.02.020 https://doi.org/10.3390/antiox9060529 https://doi.org/10.1007/978-1-4612-3274-2 https://doi.org/10.3390/molecules13040822 http://dx.doi.org/10.1177/1934578X0700200117 https://doi.org/10.1016/j.phytochem.2017.02.021 THIS PAGE INTENTIONALLY LEFT BLANK