Synthesis and antimicrobial activity of new ent-kaurene-type diterpenoid derivatives European Journal of Chemistry 14 (4) (2023) 478-485 European Journal of Chemistry ISSN 2153-2249 (Print) / ISSN 2153-2257 (Online) – Copyright © 2023 The Authors – Atlanta Publishing House LLC – Printed in the USA. This work is published and licensed by Atlanta Publishing House LLC – CC BY NC – Some Rights Reserved. https://dx.doi.org/10.5155/eurjchem.14.4.478-485.2478 European Journal of Chemistry View Journal Online View Article Online Synthesis and antimicrobial activity of new ent-kaurene-type diterpenoid derivatives Andrés Eduardo Márquez-Chacón 1,*, Alida Pérez Colmenares 1, Luis Rojas Fermín † 1, Rosa Aparicio 1, Freddy Alejandro Ramos 2, Alfredo Usubillaga 1 and Ysbelia Obregón 1 1 Research Institute, Faculty of Pharmacy and Bioanalysis, University of Los Andes, Mérida, 5101, Venezuela 2 Department of Chemistry, Faculty of Science, National University of Colombia, Bogota, 111321, Colombia * Corresponding author at: Research Institute, Faculty of Pharmacy and Bioanalysis, University of Los Andes, Mérida, 5101, Venezuela. e-mail: andresmqch@gmail.com (A.E. Márquez-Chacón). 10.5155/eurjchem.14.4.478-485.2478 Received: 16 September 2023 Received in revised form: 04 November 2023 Accepted: 12 November 2023 Published online: 31 December 2023 Printed: 31 December 2023 This research consists in the synthesis of ent-kaurene-type diterpenoid derivatives from the new natural product ent-kaur-3-acetoxy-15-ene, to carry out structural modifications on the C3 carbon of the ent-kaurene core by introducing different oxygenated groups, especially esters, in order to probe the structure-activity relationship (SAR) against microorganisms. The structure of the compounds was confirmed by FT-IR, 1H NMR, 13C NMR, and GC-MS. The antimicrobial activity of the synthesized derivatives was evaluated, ent-kaur-3-O-(6’,7’- bibenzyl-oxy-caffeoyl)-15-ene (4) exhibited activity against all tested microorganisms: Staphylococcus aureus (16 mm), Enterococcus faecalis (12 mm), Escherichia coli (13 mm), Klebsiella pneumoniae (10 mm), Pseudomonas aeruginosa (8 mm) and Candida krusei (10 mm). These results reveal a remarkable structure-activity relationship over the C3 carbon of the ent-kaurene core, where the presence of oxygenated groups such as hydroxyl or alkyl esters enhances activity. Diterpenes Drug discovery Antifungal agents Antibacterial activity ent-Kaurene derivatives Structure-activity relationship Cite this: Eur. J. Chem. 2023, 14(4), 478-485 Journal website: www.eurjchem.com 1. Introduction Natural products (NP) and their semi-synthetic analogues have played a vital role in the description and expansion of antimicrobial drugs, especially in the last 20 years [1]. Various terrestrial sources, such as plants, fungi, and lichens, present more than 80% of the described naturally occurring antibiotics. These natural products, together with new synthetic analogues, have confirmed their efficacy as alternatives to antimicrobial agents [2]. In addition, new natural and synthetic compounds have attracted considerable interest in replacing the potency of ineffective antibiotics. Diterpenes are one of the most important classes of NPs, due to their wide range of biological and ecolo- gical activities, such as antimicrobial [3], cytotoxicity with relative selectivity for cancer cells [4], antiparasitic [5], anti-HIV activity [6], anti-inflammatory [7], among others. In particular, there is a notable diterpene skeleton in this class: ent-kaurenes (Figure 1) [8,9]. Ent-kaurene diterpenoids represent an important group of tetracyclic diterpenes that have a long history of research and medical applications in traditional eastern remedies, and have attracted increasing interest since the last century due to their structural diversity and complexity, together with extensive bioactivity profiles such as antitumor, antibacterial, antiviral, and antiinflammatory effects [10]. Ent-kaurene was first isolated from the essential oil of the leaves of the New Zealand kauri (Agathis australis Salisb), a plant locally known as kauri pine [10]. However, plants of the genus Espeletia (subtribe Espeletiinae Cuatrec., Asteraceae), popularly known as "frailejón", which grow at an altitude of 2500 m in the northern Andes of South America and are used by high-moorland inhabitants with well-described medicinal attributes, provide an abundant source of natural products ent-kauranoid [11,12]. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 A B C D Figure 1. Carbon skeleton of an ent-kaurene-type diterpene. Moreover, compounds with slight structural differences can be observed to show very different biological activities in ABSTRACT RESEARCH ARTICLE KEYWORDS https://dx.doi.org/10.5155/eurjchem.14.4.478-485.2478 https://www.eurjchem.com/ https://dx.doi.org/10.5155/eurjchem.14.4.478-485.2478 mailto:andresmqch@gmail.com http://www.eurjchem.com/ https://crossmark.crossref.org/dialog/?doi=10.5155/eurjchem.14.4.478-485.2478&domain=pdf&date_stamp=2023-12-31 Márquez-Chacón et al. / European Journal of Chemistry 14 (4) (2023) 478-485 479 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.4.478-485.2478 several cases [13-16]. It should also be considered that these natural substances can be subjected to chemical modifications by chemical reactions or biotransformations, providing analo- gues that can be even more active [17,18]. Therefore, this is an interesting way to obtain new active compounds, which is important in the search for new antibiotics and treatments to defeat microbes. Natural products research in this field still represents a good prospect for finding new bioactive structures based on which new less expensive drugs can be developed; in recent decades, the use of additive and/or synergistic combina- tions of synthetic drugs and phytochemicals has been increasingly encouraged. Part of the research focuses on the evaluation of biological activities of ent-kaurenes [19,20] considering the semi- synthesis approach [19-22], and some interesting previous results obtained in the research group [23], the decision was made to perform a search for active compounds against bacterial and fungal strains by obtaining semi-synthetic derivatives of the new natural product ent-kaurene: ent-kaur-3- acetoxy-15-ene (1), which was isolated from the extraction of neutral fractions from the leaves of Espeletia semiglobulata Cuatrec. (Asteraceae) [23]. Ent-kaur-3-acetoxy-15-ene (1) [23] was allowed to react with an excess of LiAlH4, as a result of which the hydroxyl derivative was obtained (2), and from its different derivatives were synthesized (3-8). The results of the research on the synthesis, characterization, and antimicrobial activity against some microorganisms of novel ent-kaurane-type diterpenoid derivatives are presented here. 2. Experimental 2.1. Materials and instrumentation All reagents and solvents were obtained from Sigma- Aldrich, Acros Organics, and Fisher Scientific and used as supplied unless otherwise stated. Glassware was oven-dried at 100 °C for 12 hours for moisture-sensitive reactions. Reactions were carried out under Argon using anhydrous solvents. Room temperature refers to ambient temperature. The reactions were monitored by thin layer chromatography (TLC) on aluminum-supported silica gel and visualized by exposure to 240-nm UV light and/or exposure to basic phosphomolybdic acid solution followed by heating. Flash column chromate- graphy was performed with Merck PLC Silica Gel 60 using commercial solvents. All in vacuo evaporations were conducted at reduced pressure using a Büchi rotary evaporator. Infrared spectra were recorded on a PerkinElmer Spectrum Two FTIR, 10.03.06 version, in KBr disks. Maximum absorption (ν) are reported in wavenumbers (cm−1). GC-MS was performed on a Hewlett-Packard MSD 5973 instrument equipped with a fused silica column with 5% phenylmethyl polysiloxane (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 until reaching the final temperature of 300 °C and reported as m/z. NMR spectroscopy (1H, 13C, APT, COSY, HSQC, HMBC) was recorded at 25 °C with a Bruker-Advance Neo 400 (1H at 400 MHz and 13C at 100 MHz) in deuterated solvents, CDCl3. NMR chemical shifts (δ) are quoted in parts per million (ppm) referencing the residual proton or the carbon peak of the solvent (CDCl3: δH = 7.26 and δC = 77.1). The coupling constants are reported in hertz (Hz). Data for 1H spectra are presented as chemical shift (δ, ppm), integration and multiplicity (b, broad; s, singlet; d, doublet; t, triplet; q, quartet; quin, quintet; sept, septet; m, multiplet; or as a combination (e.g., dd, dt, etc.)). All carbon spectra were obtained by the attached proton test (APT). 2.2. Synthesis Ent-kaur-3-acetoxy-15-ene (1) was allowed to react with an excess of LiAlH4, as a result of which the hydroxyl derivative was obtained (2), and different derivatives were synthesized from it: one derivative by oxidation of this hydroxyl group (3) and the rest were acetoxy-alkylated derivatives (4-8) by esterification reactions with different organic acids. The synthetic pathway of these ent-kaurene derivatives is repre- sented in Scheme 1. Ent-kaur-3-acetoxy-15-ene (1) [23] was allowed to react with an excess of LiAlH4, as a result of which the hydroxyl derivative was obtained (2) and its different derivatives were synthesized: one derivative by oxidation of this hydroxyl group (3), and the rest were acetoxy-alkylated derivatives (4-8) by esterification reactions with different organic acids. The results of the research on the synthesis, characterization, and anti- microbial activity against some microorganisms of novel ent- kaurane-type diterpenoid derivatives are presented here. 2.2.1. Synthesis of ent-kaur-3-hydroxy-15-ene (2) derivative 2.44 mmol of ent-kaur-3-acetoxy-15-ene (1) were intro- duced into a round bottom flask under an argon atmosphere and afterward dissolved in 160 mL of diethyl ether. Subse- quently, an excess of LiAlH4 (26.31 mmol), was added and it was left under continuous stirring in an argon atmosphere at room temperature for 8 h. Subsequently, the sample was carefully treated with a 1 M HCl solution until the appearance of a white precipitate. The mixture was filtered and the aqueous phase was extracted several times with diethyl ether (3 × 30 mL). The combined organic extracts were dried over MgSO4, filtered, and concentrated in vacuum. As a result, a white solid was obtained, which was purified by column chromatography using a mixture of hexane:AcOEt (4:1) as an eluent to obtain compound 2. Ent-kaur-3-hydroxy-15-ene (2): Color: White crystals. Yield: 792.00 mg, 99 %. M.p.: 127-129 °C. FT-IR (KBr, ν, cm-1): 3392 (O-H, alcohol), 3040 (=C-H, alkene), 2925 (C-H), 1656 (C=C, alkene), 1045 (C-O, alcohol). 1H NMR (400 MHz, CDCl3, δ, ppm): 5.06 (s, 1H, CH, H15), 3.57 (s, 1H, OH, H21), 3.15 (m, 1H, CH-OH, H3), 1.86 (m, 1H, CH, H13), 1.80 (s, 3H, CH3, H17), 1.56 (m, 2H, CH2, H1), 1.54 (t, 2H, CH2, H2), 1.49 (m, 2H, CH2, H6), 1.48 (m, 2H, CH2, H12), 1.47 (m, 1H, CH, H5), 1.43 (m, 2H, CH2, H11), 1.33 (t, 2H, CH2, H14), 1.29 (m, 2H, CH2, H7), 0.82 (s, 3H, CH3, H18), 0.80 (s, 3H, CH3, H19), 0.78 (m, 1H, CH, H9), 0.73 (s, 3H, CH3, H20). 13C NMR (100 MHz, CDCl3, δ, ppm): 139.78 (C, C16), 124.47 (CH, C15), 79.06 (CH-OH, C3), 55.41 (CH, C5), 52.40 (CH, C9), 47.79 (CH, C13), 41.68 (C, C8), 41.49 (CH2, C14), 39.68 (C, C10), 37.86 (C, C4), 33.01 (CH2, C6), 32.69 (CH2, C7), 31.27 (CH2, C1), 28.22 (CH3, C18), 27.28 (CH2, C2), 26.75 (CH2, C12), 23.37 (CH3, C17), 22.70 (CH3, C19), 21.07 (CH2, C11), 17.66 (CH3, C20). MS (EI, m/z (%)): 288 (M+, 15.0), 273.2 (M+-CH3, 40.2), 271.2 (M+-OH, 38.1), 218.1 (M+- C5H11, 80.7), 189.2 (M+-C6H11O, 33.2), 161.2 (M+-C8H15O, 29.4). 2.2.2. Synthesis of ent-kaur-3-oxo-15-ene (3) derivative An equivalent of compound 2 (0.25 mmol) was dissolved in 10 mL of tetrahydrofuran (THF) and 4 equivalents of stabilized 2-iodoxybenzoic acid (SIBX) (1.01 mmol). The reaction mixture was continuously stirred in an argon atmosphere at room temperature for 48 hours. The mixture obtained was washed with water and extracted several times with diethyl ether (3 × 30 mL). The combined organic extracts were dried over MgSO4, filtered, and concentrated in vacuum. The crude product was purified through silica gel using hexane:AcOEt (99:1) as an eluent to give compound 3. 480 Márquez-Chacón et al. / European Journal of Chemistry 14 (4) (2023) 478-485 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.4.478-485.2478 O O HO O O O O OO O O OHOOC O O O OBr (1) (2) (3) (4) (5) (7) (6) (8) LiAlH4 Et2O 25°C x 8h H3O+ SIBX exc. THF 25°C x 48h COOH BnO BnO DCC, DMAP CH2Cl2 25°C x 48h DCC, DMAP CH2Cl2 25°C x 48h COOH BrCOOHHOOC exc. DCC, DMAP CH2Cl2 25°C x 48h COOH HOOC HOOC (2) exc. DCC, DMAP CH2Cl2 25°C x 48h DCC, DMAP CH2Cl2 25°C x 48h 3 3 2' 3' 1''' 1'' 2' 1' 1' 6' 1'2' 3' 4' 1' 2' 2' 1' 4 2 19 19 4 18 15 17 1 5 79 13 14 16 18 19 20 3' 4' 17 5'6' 7' 8' 9' 2'' 3''4'' 5'' 4'' 3'' 4' 4' 5' 5' 4 17 O O O Scheme 1. Synthetic pathway of ent-kaurene derivatives. Ent-kaur-3-oxo-15-ene (3): Color: White crystalline solid. Yield: 72.00 mg, 98 %. M.p.: 131-132 °C. FT-IR (KBr, ν, cm-1): 3041 (=C-H, alkene), 2927 (C-H), 1709 (C=O, ketone), 1639 (C=C, alkene). 1H NMR (400 MHz, CDCl3, δ, ppm): 0.79 (m, 1H, CH, H9), 0.81 (s, 3H, CH3, H20), 1.06 (s, 3H, CH3, H19), 1.08 (s, 3H, CH3, H18), 1.26 (s, 1H, CH, H5), 1.28 (m, 2H, CH2, H7), 1.34 (t, 2H, CH2, H14), 1.43 (m, 2H, CH2, H11), 1.48 (m, 2H, CH2, H12), 1.49 (m, 2H, CH2, H6), 1.58 (m, 2H, CH2, H1), 1.90 (s, 3H, CH3, H17), 1.97 (s, 1H, CH, H13), 2.71 (t, 2H, CH2, H2), 5.16 (s, 1H, CH, H15). 13C NMR (100 MHz, CDCl3, δ, ppm): 216.86 (C=O, C3), 139.87 (C, C16), 124.34 (CH, C15), 55.40 (CH, C9), 52.54 (CH, C5), 48.05 (CH, C13), 47.58 (C, C4), 41.69 (C, C8), 39.72 (CH2, C14), 37.68 (C, C10), 35.72 (CH2, C7), 34.37 (CH2, C2), 33.58 (CH2, C1), 28.16 (CH3, C18), 26.89 (CH2, C12), 23.33 (CH3, C17), 21.67 (CH2, C6), 20.92 (CH3, C19), 19.79 (CH2, C11), 17.63 (CH3, C20). MS (EI, m/z (%)): 286 (M+, 15.11), 257.2 (M+-C2H6, 40.2), 247.2 (M+-C3H4, 38.1), 218.2 (M+- C3H7, 38.6), 189.2 (M+-C6H10O, 69.4). 2.2.3. Synthesis of ent-kaur-3-acetoxyalkylated derivatives (4-8) A compound 2 equivalent was placed in a round bottom flask and dissolved in 10 mL of CH2Cl2, with 1.5 equivalents of 3.4-bibenzyloxy-caffeic acid (0.52 mmol), 3 equivalents of dicyclohexylcarbodimide (DCC, 1.04 mmol) and 3 equivalents of N,N-dimethylaminopyridine (DMAP, 1.04 mmol). The reac- tion mixture was left stirring at room temperature for 48 hours under an atmosphere of argon, while being continuously monitored by TLC. The resulting mixture was washed with water and extracted with CH2Cl2 (3 x 20 mL). The organic phase was dried with MgSO4, filtered, and concentrated. The crude was purified through silica gel using hexane:AcOEt (96:4) as eluent, yielding compound 4: Color: solid white crystalline. Yield: 143.40 mg, 47.80 %. M.p.: 130-131 °C. A similar procedure was performed for the synthesis of compounds 5-8, maintaining the same conditions and the purification proce- dure described above. For compound 5: 1 equiv. compound 2 was dissolved in 5 mL of CH2Cl2, with 3 equiv. of p-bromo- phenylacetic acid (0.939 mmol), 3 equiv. DCC, and 3 equiv. DMAP. Color: white. Yield: 40.00 mg, 45 %. M.p.: 145-146 °C. Compound 6: 1 equiv. compound 2 was dissolved in 10 mL of CH2Cl2, with 6 equiv. of succinic acid (2.08 mmol), 5 equiv. DCC, and 5 equiv. DMAP. Color: white. Yield: 57.00 mg, 57 %. M.p.: 185-186 °C. Compound 7: 2 equiv. compound 2 (0.35 mmol) was dissolved in 10 mL of CH2Cl2, with 1 equiv. of succinic acid (0.17 mmol), 3 equiv. DCC, and 3 equiv. DMAP. Color: white. Yield: 49.00 mg, 50 %. M.p.: 190-191 °C. Compound 8: 1 equiv. compound 2 was dissolved in 10 mL of CH2Cl2, with 4 equiv. of kaurenic acid (1.04 mmol), 6 equiv. DCC (1.04 mmol), and 6 equiv. DMAP (1.04 mmol). Color: white. Yield: 140.00 mg, 40 %. M.p.: 173 -174 °C. The synthetic pathway and structure of the compounds are given below (Scheme 1). Ent-kaur-3-O-(6’,7’-bibenzyl-oxy-caffeoyl)-15-ene (4): FT-IR (KBr, ν, cm-1): 3090 (=C-H, alkene), 2946 (C-H), 1702 (C=O, ester carbonyl), 1633 (C=C, alkene), 1261 (C-O, ester), 843 (=C- H, alkene). 1H NMR (400 MHz, CDCl3, δ, ppm): 0.73 (s, 3H, CH3, H20), 0.79 (m, 1H, CH, H9), 0.80 (s, 3H, CH3, H19), 0.81 (s, 3H, CH3, H18), 1.30 (m, 2H, CH2, H7), 1.34 (t, 2H, CH2, H14), 1.43 (m, 2H, Márquez-Chacón et al. / European Journal of Chemistry 14 (4) (2023) 478-485 481 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.4.478-485.2478 CH2, H11), 1.47 (m, 2H, CH2, H12), 1.48 (m, 2H, CH2, H2), 1.50 (m, 2H, CH2, H6), 1.51 (m, 1H, CH, H5), 1.55 (m, 2H, CH2, H1), 1.83 (s, 3H, CH3, H17), 1.87 (s, 1H, CH, H13), 4.66 (t, 1H, CH-COO, H3), 5.16 (s, 1H, CH, H15), 5.21 (s, 2H, -O-CH2-Ar, H1’’’), 5.22 (s, 2H, -O-CH2- Ar, H1’’), 6.26 (d, 1H, -CH=CH-COO-, H2’), 6.93 (s, 1H, -CH=CH-Ar, H9’), 6.95 (s, 1H, -CH=CH-Ar, H8’), 7.16 (s, 1H, -CH=CH-Ar, H5’), 7.35 (m, 2H, 2CH-Ar, H5’’), 7.40 (m, 2H, 2CH-Ar, H4’’), 7.41 (m, 2H, 2CH-Ar, H6’’), 7.44 (m, 2H, 2CH-Ar, H7’’),7.45 (m, 2H, 2CH-Ar, H3’’), 7.56 (d, 1H, -CH=CH-COO-, H3’). 13C NMR (100 MHz, CDCl3, δ, ppm): 167.15 (-COO, C1’), 151.03 (-C=, C7’), 149.10 (-C=, C6’), 144.12 (=CH, C3’), 139.78 (=C-CH3, C16), 136.91 (2C, C2’’), 128.71 (2CH, C3’’), 128.70 (2C, C7’’), 128.19 (-C=, C4’), 128.09 (2C, C6’’), 127.48 (2C, C5’’), 127.32 (2CH, C4’’), 124.35 (CH=CH, C15), 122.98 (=CH, C5’), 117.01 (=CH-, C2’), 114.40 (=CH, C9’), 113.81 (-C=, C8’), 80.84 (CH-COO, C3), 71.49 (CH2-O, C1’’), 71,11 (CH2-O, C1’’’).55.44 (CH, C9), 52.50 (CH, C5), 48.02 (CH, C13), 40.18 (C, C8), 39.85 (C, C10), 39.67 (CH2, C14), 38.11 (C, C4), 36.60 (CH2, C7), 33.00 (CH2, C1), 28.16 (CH3, C18), 26.66 (CH2, C12), 23.57 (CH2, C2), 23.41 (=C- CH3, C17), 22.70 (CH3, C19), 21.10 (CH2, C6), 18.47 (CH2, C11), 17.66 (CH3, C20). MS (EI, m/z (%)): 341.2 (M+-C20H18O2, 15.11), 289.1 (M+-C26H41O2, 10.21), 218.5 (M+-C7H10O2, 68.5), 189.2 (M+-C9H14O2, 38.6). Ent-kaur-3-O-(carboxymethyl-2’-p-bromophenyl)-15-ene (5): FT-IR (KBr, ν, cm-1): 3036 (=C-H, alkene), 2924 (C-H), 1733 (C=O, ester carbonyl), 1659 (C=C, alkene), 1255 (C-O, ester), 1136 (Ar-Br), 804 (p-Ar-H). 1H NMR (400 MHz, CDCl3, δ, ppm): 0.76 (s, 3H, CH3, H20), 0.78 (m, 1H, CH, H9), 0.80 (s, 3H, CH3, H19), 0.81 (s, 3H, CH3, H18), 1.32 (m, 2H, CH2, H7), 1.35 (t, 2H, CH2, H14), 1.44 (m, 2H, CH2, H11), 1.47 (m, 2H, CH2, H2), 1.48 (m, 2H, CH2, H12), 1.51 (m, 1H, CH, H5), 1.52 (m, 2H, CH2, H6), 1.53 (m, 2H, CH2, H1), 1.91 (s, 3H, CH3, H17), 1.98 (m, 1H, CH, H13), 3.59 (s, 2H, CH, H5’).4.51 (t, 1H, CH-COO, H3), 5.13 (s, 1H, CH, H15), 7.20 (d, 2H, -CH2-COO, H2’), 7.45 (d, 2H, =CH-, H4’). 13C NMR (100 MHz, CDCl3, δ, ppm): 170.89 (COO, C1’), 139.78 (=C-, C16), 133.53 (C-CH2- COO, C3’), 131.73 (2CH-, C5’), 131.17 (2CH-, C4’), 124.42 (=CH-, C15), 121.13 (Br-CH=, C6’), 81.56 (CH-COO, C3), 55.36 (CH, C9), 52.51 (CH, C5), 47.75 (CH, C13), 41.52 (Ar-CH2-COO, C2’), 40.18 (C, C8), 39.67 (CH2, C14), 38.52 (C, C10), 37.94 (C, C4), 35.65 (CH2, C7), 32.97 (CH2, C1), 28.16 (CH3, C18), 26.65 (CH2, C12), 23.67 (CH2, C2), 23.38 (CH3-C=, C17), 21.42 (CH3, C19), 21.01 (CH2, C6), 18.40 (CH2, C11), 17.65 (CH3, C20). MS (EI, m/z (%)): 405.2 (M+- Br, 18.2), 367.2 (M+-C3H4Br, 25.3), 297.2 (M+-CH5OBr, 47.2), 218.2 (M+-C5H11Br, 80.5), 189.2 (M+-C14H18O2Br, 48.7). Ent-kaur-3-O-succinyl-15-ene (6): FT-IR (KBr, ν, cm-1): 3368 (-O-H, carboxylic acid), 3036 (=C-H, alkene), 2943 (C-H), 1710 (C=O, carboxylic acid carbonile), 1719 (-O-C=O, ester carbonyl), 1637 (C=C, alkene), 1378 (C-O), 1082 (C-O), 843 (=C-H). 1H NMR (400 MHz, CDCl3, δ, ppm): 0.73 (s, 3H, CH3, H20), 0.79 (m, 1H, CH, H9), 0.80 (s, 3H, CH3, H19), 0.81 (s, 3H, CH3, H18), 1.33 (m, 2H, CH2, H7), 1.34 (t, 2H, CH2, H14), 1.45 (m, 2H, CH2, H11), 1.46 (m, 2H, CH2, H2), 1.49 (m, 2H, CH2, H12), 1.51 (m, 2H, CH2, H6), 1.53 (m, 1H, CH, H5), 1.54 (m, 2H, CH2, H1), 1.83 (s, 3H, CH3-C=, H17), 1.86 (s, 1H, CH, H13), 2.57 (t, 2H, CH2-COO, H2’), 2.61 (t, 2H, CH2-COOH, H3’), 4.47 (t, 1H, CH-COO, H3), 5.05 (s, 1H, -CH=, H15), 11.4 (s, 1H, H-OOC, H4’). 13C NMR (100 MHz, CDCl3, δ, ppm): 177.94 (COOH, C4’),171.96 (COO, C1’), 139.77 (=C-, C16), 124.42 (=CH-, C15), 81.72 (CH-COO, C3), 55.40 (CH, C9), 52.50 (CH, C5), 47.75 (CH, C13), 40.15 (C, C8), 9.67 (CH2, C14), 38.55 (C, C10), 37.90 (C, C4), 35.68 (CH2, C7), 32.97 (CH2, C1), 29.46 (CH2-COO, C2’), 29.15 (CH2-COOH, C3’), 28.15 (CH3, C18), 26.64 (CH2, C12), 23.65 (CH2, C2), 23.35 (CH3-C=, C17), 21.55 (CH3, C19), 21.06 (CH2, C6), 18.36 (CH2, C11), 17.66 (CH3, C20). MS (EI, m/z (%)): 388 (M+, 17.7), 342.2 (M+-HCOOH, 19.3), 218.2 (M+-C8H13O3, 89.6), 189.2 (M+-C9H14O2, 40.6), 161.0 (M+-C8H16O2, 18.4), 147.2 (M+- C9H18O2, 30.4). Di-ent-kaur-3,3’-O-succinyl-15,15’-diene (7): FT-IR (KBr, ν, cm-1): 2993 (=C-H, alkene), 2944 (C-H), 1731 (-O-C=O, éster carbonyl), 1631 (C=C, alkene), 1133 (C-O), 836 (=C-H). 1H NMR (400 MHz, CDCl3, δ, ppm): 0.73 (s, 6H, 2CH3, H20), 0.79 (m, 2H, 2CH, H9), 0.80 (s, 6H, 2CH3, H19), 0.81 (s, 6H, 2CH3, H18),1.33 (m, 4H, 2CH2, H7), 1.35 (m, 4H, 2CH2, H14), 1.44 (m, 4H, 2CH2, H11), 1.47 (m, 4H, 2CH2, H12), 1.48 (m, 4H, 2CH2, H2), 1.51 (m, 4H, 2CH2, H6), 1.52 (m, 2H, 2CH, H5), 1.54 (m, 4H, 2CH2, H1), 1.82 (s, 6H, 2CH3, H17), 1.88 (s, 2H, 2CH, H13), 2.57 (s, 4H, 2CH2, H2’’), 4.47 (t, 2H, 2CH, H3), 5.05 (s, 2H, 2CH=, H15). 13C NMR (100 MHz, CDCl3, δ, ppm): 172.16 (2COO, C1’), 139.78 (2C=, C16), 124.46 (2CH=, C15), 81.37 (2CH, C3), 55.40 (2CH, C9), 52.51 (2CH, C5), 47.99 (2CH, C13), 40.17 (2C, C8), 39.67 (2CH2, C14), 38.58 (2C, C10), 37.94 (2C, C4), 35.68 (2CH2, C7), 33.00 (2CH2, C1), 29.80 (2CH2, C2’),28.16 (2CH3, C18), 26.63 (2CH2, C12), 23.57 (2CH2, C2), 23.38 (2CH3, C17), 21.55 (2CH3, C19), 21.06 (2CH2, C6), 18.38 (2CH2, C11), 17.66 (2CH3, C20). MS (EI, m/z (%)): 468 (M+-C14H21, 34.6), 426.2 (M+-C17H27, 35.5), 342.2 (M+-C21H30O2, 40.7), 327.2 (M+-C22H33O2, 39.3). Ent-kaurenic anhydride (8): FT-IR (KBr, ν, cm-1): 3069 (=C- H, alkene), 2934 (C-H), 1789 (-O-C=O, asymmetric vibration of ester carbonyl), 1729 (-O-C=O, symmetric vibration of ester carbonyl), 1657 (C=C, alkene), 1378 (C-O, anhydride), 887 (=C- H). 1H NMR (400 MHz, CDCl3, δ, ppm): 0.73 (s, 6H, 2CH3, H20),0.78 (m, 2H, 2CH, H9), 0.80 (s, 6H, 2CH3, H18), 0.83 aβ(axi) x 2 - 1.95 bα(equ) x 2 (m, 4H, 2CH2, H1), 1.11 (d, 2H, 2CH, H5), 1.30 (m, 4H, 2CH2, H7), 1.40 (m, 4H, 2CH2, H11), 1.41 (m, 4H, 2CH2, H2), 1.50 (m, 4H, 2CH2, H6), 1.59 (m, 4H, 2CH2, H12), 2.05 (m, 4H, 2CH2, H15), 2.08 (m, 4H, 2CH2, H14), 2.19 (m, 4H, 2CH2, H3), 2.64 (s, 2H, 2CH, H13), 4.74 Ha x2- 4.80 Hb x2 (s, 4H, 2=CH, H17). 13C NMR (100 MHz, CDCl3, δ, ppm): 173.55 (2COO-, C19), 155.77 (2C=CH2, C16), 103.10 (2CH2=C, C17), 57.27 (2CH, C5), 54.98 (2CH, C9), 48.92 (2CH2, C15), 45.39 (2C-COO-, C4), 43.96 (2CH, C13), 43.78 (2C, C8), 41.31 (2CH2, C1), 41.30 (2CH2, C14), 40.60 (2CH2, C7), 39.60 (2C, C10), 33.10 (2CH2, C12), 33.00 (2CH2, C3), 28.06 (2CH3, C18), 21.92 (2CH2, C6), 18.97 (2CH2, C2), 18.44 (2CH, C11), 16.40 (2CH3, C20). MS (EI, m/z (%)): 256.2 (M+-C21H30O3, 37.6), 241.2 (M+- C22H33O3, 39.9), 157.0 (M+- C28H45O3, 10.6). 2.3. Antibacterial activity Antibacterial activity was determined using the paper-disk- diffusion agar method [24,25]. The following microorganisms were tested: Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 29212), Klebsiella pneumoniae (ATCC 23357), Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853). An 18-hour culture of each microorganism in 2.5 mL of Müeller-Hinton (MH) broth at 37 °C was used. The bacterial inoculum was adjusted with physio- logical saline solution to the Mc Farland turbidity standard N ° 0.5 (1×108 CFU/mL). Each inoculum was spread with a swab on the surface of a plate containing Müeller-Hinton agar and then a filter paper disk (6 mm diameter) previously impregnated with 10 μL of the compound dissolved in DMSO at a concent- ration of 2 mg/mL for each (1, 2, 3, 4, 5, 6, 7, 8) was placed on the surface. A disc impregnated with DMSO was included as a negative control. Furthermore, the standard disc of the reference antibiotic was placed as a positive control for each microorganism (Oxacillin® 1 μg 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 placing the discs on the Petri plates, they were refrigerated at 4 °C for 24 h [24]. Inhibition halos were read at 24 h and measured (mm) around the disc. All tests were performed in duplicate. The measurement of inhibition halos for antibacterial activity was done as follows: 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). 2.4. Antifungal activity Antifungal activity was determined by the paper-disk- diffusion agar method [24]. The microorganism tested was Candida krusei (ATCC 6558), using Fluconazole 100 μg as a 482 Márquez-Chacón et al. / European Journal of Chemistry 14 (4) (2023) 478-485 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.4.478-485.2478 positive control, and dimethylsulfoxide (DMSO) as a negative control. The strain had been repacked 12 hours before it came to a salted dextrose agar and it was used to prepare the inoculum which was compared to the Mc Farland Turbidity Standard N° 1.5 (108 CFU/mL). Each inoculum was spread 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 (1, 2, 3, 4, 5, 6, 7, 8). One well with DMSO was included as a negative control and one well with the reference antibiotic as a positive control. The Petri plates were then refrigerated at 4 °C for 24 h [26]. Inhibition halos were read at 24 h and measured (mm) around the well. All tests were performed in duplicate. 3. Results and discussion 3.1. Chemistry In recent decades, the introduction of ester functional groups into organic compounds has become increasingly common in drug design and development, as ester substitution can greatly improve the solubility, bioavailability, and absorption of drugs [27]. In this context, previous studies on the biological activity of ent-kaurene-type diterpenoids and triterpenes reported that the presence of oxygenated groups such as hydroxyls or esters on the C-3 carbon of the ent-kaurene core, as well as in triterpenes (whose core is largely constituted by a decahydronaphthalene ring, such as the ent-kaurene core with rings A and B), increases biological activity [28-30]. Therefore, in this study, structural modifications were perfor- med on the carbon C-3 of the ent-kaurene core of compound 1 (Scheme 1) by introducing different oxygenated groups, espe- cially esters, to investigate the structure–activity relationship (SAR) of the derivatives ent-kaur-3-acetoxyalkylated and further enhance their antimicrobial power. The structure of the synthetized derivatives was characterized by IR, 1H NMR, 13C NMR, and GC-MS. The natural product ent-kaur-3-acetoxy-15-ene (1) was first subjected to a reduction reaction of the acetate group using LiAlH4, forming the C3-hydroxylated derivative ent-kaur-3- hydroxy-15-ene (2). FT-IR analysis showed evidence of this transformation by the appearance of a characteristic absorp- tion band of alcohol (3392 cm-1, O-H) and the disappearance of the characteristic absorption bands of the ester group [1735 cm-1 (O−C=O) and 1243 cm-1 (O−C)] [23]. In the 1H NMR spectrum, the disappearance of the singlet of the methyl protons of the ester group at δ 1.98 ppm and the appearance of a small singlet at δ 3.57 ppm of the proton of a secondary hydroxyl group, as well as the high-field shift of the signal coming from the H3 methine proton at δ 3.15 ppm is mainly observed compared to compound 1. The 13C NMR spectrum confirms the presence of 20 carbons, mainly the disappearance of the carbonyl signal of the ester group at δ 171.17 ppm is observed, together with the methyl signal of the same terminal ester at δ 21.47 ppm, both signals belonging to the starting compound (1) [23], and the shift to a high field of the methine carbon at δ 79.06 ppm. On the other hand, its mass spectrum showed the molecular ion peak at m/z 288 [M+] consistent with their molecular formula with five unsaturations. The derivative 3, ent-kaur-3-oxo-15-ene, was obtained through an oxidation reaction of the hydroxyl group of compound 2 using SIBX as an oxidizing agent. FT-IR showed the characteristic band of the carbonyl group at 1709 cm-1, which is an absent band compared to the IR spectrum of compound 2, as well as the disappearance of the hydroxyl group signal. In the 1H NMR spectrum, the multiplet corresponding to the methyne proton H3 at δ 3.15 ppm disappears compared to the 1H NMR of 2, as well as the small downfield singlet of the hydroxyl group at δ 3.57 ppm. The low-field shift of the H2 methylene proton signal at δ 2.71 ppm is also notable, which is due to the effective oxidation of the hydroxyl group to carbonyl. The 13C NMR spectrum confirms the presence of 20 carbons, mainly showing the shift to the low field of carbon C3 at δ 216.86 ppm with respect to compound 2, as well as an increase in the δ of methylene C2 at δ 34.37 ppm and the carbon quaternary C4 at δ 47.58 ppm. The GC-MS study showed the molecular ion peak at m/z 286 [M+] consistent with their molecular formula with six unsaturations. Acetoxy-alkylated derivatives (4-8) were obtained by Steglich Esterification between compound 2 and variation of different carboxylic acids (Scheme 1), using DCC as reaction activator and DMAP as catalyst. In view of the FT-IR spectra, the absence of the peak corresponding to -OH and the appearance of two characteristic absorption bands in the range of 1730- 1740 cm-1 (-O-C=O) and 1300-1000 cm-1 (C-O) indicated that compound 2 (alcohol) had been converted into products (esters). Specifically, derivative 4 shows a smaller absorption shift compared to the carbonyl band (1702 cm-1, CO-C=C-) due to being conjugated, and a more intense band corresponding to the alkene group stands out (1633 cm-1, C=C). For derivative 5, in addition to the presence of bands of the ester group at 1733 cm-1 (-O-C=O) and 1255 cm-1 (C-O), a band at 1136 cm-1 charac- teristic of the halogen-aromatic ring bond (Br-Ar) is also shown, and at 804 cm-1 the band indicates the para disposition of this halogen on the aromatic ring. In derivative 6, the presence of a carboxyl group is confirmed by observing a characteristic broad absorption band of the O-H bond at 3368 cm-1 and a charac- teristic carbonyl band at 1710 cm-1, as well as the presence of ester group bands at 1719 cm-1 (-O-C=O), 1378 cm-1 (C-O, tension vibration) and 1082 cm-1 (C-O, bending vibration). Compound 7 showed bands of the ester group at 1731 cm-1 (-O- C=O) and 1133 cm-1 (C-O); and in derivative 8, the presence of characteristic bands of the anhydride group at 1789 cm-1 (>C=O, asymmetric vibration), 1729 cm-1 (>C=O, symmetric vibration) and 1378 cm-1 (C-O of anhydride) stands out, confirming the formation of this functional group in the structure. Concerning the 1H NMR spectrum, the downfield shift of the H3 proton in compounds 4-8 in contrast to compound 2 confirms the transformation of the OH group. Derivative 4 showed the H3 signal at δ 4.66 ppm, two singlets at δ 5.22 ppm and 5.21 ppm attributed to the methylene protons H1’’ and H1’’’, respectively, and two doublets at δ 6.26 ppm and δ 7.56 ppm corresponding to the methynic protons H2’ and H3’, respectively, which lie on the conjugated C=C double bond; a set of signals corresponding to the aromatic rings of the 3,4-bibenzyloxy- caffeoyl system are also observed. Compound 5 showed the methyne H3 signal at δ 4.51 ppm, a singlet signal at δ 3.59 ppm attributed to the methylene protons H2’, and two doublets are observed at δ 7.20 ppm and δ 7.45 ppm at a low field, attributed to the phenyl protons H4' and H5', respectively. Compound 6 exhibited the methyne H3 signal at δ 4.47 ppm, and two strong triplets at δ 2.57 ppm and δ 2.61 ppm attributed to the methylene protons H2' and H3', respectively, corresponding to the alkyl part of succinic acid. The derivative 7 displayed the methyne H3 signal at δ 4.47 ppm, and an intense singlet at δ 2.57 ppm attributed to the methylene protons H2'' and H2''', which exhibit a single signal due to molecular symmetry. The proton spectrum of compound 8 differs mainly from compound 2 in the following: the H13 methine proton is at δ 2.64 ppm; H3 proton is a doublet and is at δ 2.19 ppm; the vinyl proton H15 singlet disappears at δ 5.06 ppm and is now found at δ 2.05 ppm as a multiplet; the signal of the H19 methyl protons disappears; H17 protons are diasterotopic and show two singlets at δa17 4.74 ppm and δb17 4.80 ppm. These results reveal that the esterification reaction between compound 2 and ent-kaurenic acid did not take place; in contrast, the 1H NMR spectrum of this product is consistent with the 1H NMR spectrum of ent-kaurenic acid [31]. Márquez-Chacón et al. / European Journal of Chemistry 14 (4) (2023) 478-485 483 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.4.478-485.2478 Table 1. Antibacterial activity of compound 1 and its synthetic derivatives (2-8) expressed as diameters of the inhibition halo (mm) #. Bacterial strains Compounds Positive control 1 2 3 4 5 6 7 8 OX VA TO AZ CF Staphylococcus aureus IA 10 IA 16 IA IA 11 IA *23 ̶ ̶ ̶ ̶ Enterococcus faecalis IA 10 12 12 8 10 10 IA ̶ *19 ̶ ̶ ̶ Escherichia coli 8 IA 10 13 8 IA 10 IA ̶ ̶ *26 ̶ ̶ Klebsiella pneumoniae 10 8 10 10 8 8 10 8 ̶ ̶ ̶ *34 ̶ Pseudomonas aeruginosa 8 10 8 8 10 10 10 10 ̶ ̶ ̶ ̶ *32 # IA: inactive; OX: Oxacillin®; VA: Vancomycin®; TO: Tobramycin®; AZ: Aztreonam®; CF: Cefepime®. * Halo of inhibition in mm for the positive control groups [24,25]. Table 2. Antifungal activity of compound 1 and its synthetic derivatives (2-8) expressed as inhibition halo diameters (mm) #. Fungal strains Compounds Positive control 1 2 3 4 5 6 7 8 FLU (100 μg) Candida krusei 8 10 IA 12 IA 10 8 IA *15 # IA: inactive; FLU: Fluconazole®. * Halo of inhibition in mm for the control groups [26]. The assignment of 13C NMR spectra of carbon atoms presented in these acetoxy-alkylated derivatives showed the following. Compound 4 contains 43 carbons, highlighting the presence of two signals at δ 71.49 ppm and δ 71.11 ppm corresponding to the carbons of the methylenes C1'' and C1''', respectively; two signals at δ 144.12 ppm and δ 117.12 ppm corresponding to the carbons that make up the conjugated double bond C3' and C2', respectively; ester formation is confirmed when the signal corresponding to the quaternary carbon C1' (δ 167.15 ppm, >C=O) is observed in the low field; and a set of signals that are consistent with the carbons that make up the aromatic rings of the 3,4-bibenzyloxy-caffeoyl system. The mass spectrum confirms the structure by showing two abundant ions at m/z 341.2 (M+-C20H18O2) and m/z 289.1 (M+-C26H41O2), which correspond to the breaking of the molecule in half. In derivative 5, the presence of 28 carbons is confirmed, emphasizing the presence of two signals at δ 121.13 ppm and δ 133.53 ppm corresponding to the quaternary carbons C3' and C6', respectively, and two signals at δ 131, 17 ppm and δ 131.73 ppm corresponding to the C4' and C5' carbons, respectively; which make up the aromatic ring from p-bromo- phenylacetic acid; in addition, ester formation is confirmed by observing the signal corresponding to the C1' carbonyl carbon (δ 170.89 ppm, >C=O) in the low field. The mass spectrum confirms the structure by showing an abundant ion at m/z 405.2 (M+-Br). The presence of 24 carbons is confirmed in compound 6; the presence in a high field of two signals at δ 29.46 ppm and δ 29.15 ppm stands out with respect to C2' and C3' methylenes, respectively; and the two signals at δ 171.96 ppm and δ 177.94 ppm attributed to the C1' carbonyl carbon of the ester group and the C4' carboxylic carbon, respectively, confirming the presence of both functional groups in the structure of this derivative. The mass spectrum confirms the structure showing the molecular ion peak at m/z 388.2 [M+] consistent with their molecular formula with seven unsaturations. Compound 7 highlights 22 carbon signs, showing the presence of a signal at δ 29.80 ppm at a high field corresponding to methylenes C2', and at a low field, a signal at δ 172.16 ppm corresponding to the carbonyl carbons C1' of the ester group. The mass spectrum demonst- rates the formation of a dimeric structure by showing three abundant ions at m/z 468 (M+-C14H21, 34.6), 426.2 (M+-C17H27, 35.5), and 342.2 (M+-C21H30O2, 40.7), consistent with their molecular formula with twelve unsaturation. Compound 8 presents quantifiable signals for 19 carbons, highlighting: C3 signal at δ 33.00 ppm, C15 carbon at δ 48.92 ppm, C16 at δ 155.77 ppm, C17 at δ 103.10 ppm, and C19 at δ 173.55 ppm, which is found at δ 184.72 ppm in 13C NMR spectrum of ent-kaurenic acid [30]. The similarity of these data with those reported in the bibliography for 13C NMR of ent-kaurenic acid [32], and also the displacement obtained from the C19 signal indicate that this carboxylic acid reacted with itself, forming a symmetrical anhydride (dimer). This is confirmed with the mass spectrum, where three abundant ions are shown at m/z 256.2 (M+- C21H30O3, 37.6), m/z 241.2 (M+- C22H33O3, 39.9), and m/z 157.0 (M+- C28H45O3, 10.6), consistent with their molecular formula with twelve unsaturation. The formation of this collateral product could be explained by the steric hindrance exerted by the bulky structure of ent-kaurenic acid on the alkoxide (also bulky) formed from compound 2, which is a weaker nucleophile than the carboxylate formed from the same acid. Therefore, the formation of the anhydride occurs. According to Steglich et al., when poor nucleophiles are used in Steglich esterification, side product formation prevails [33]. 3.2. Antimicrobial activity In this study, the antibacterial activity of the synthetic derivatives of compound 1 (2-8) was evaluated using the disk diffusion method (Kirby-Bauer). The results obtained are summarized in Table 1 and are expressed as the diameters of the inhibition halo reported in millimeters. The synthesized compounds evaluated showed specific antibacterial activity against some bacteria used. Compounds 4 and 7 showed antibacterial activity by generating inhibition halos against all tested, compound 4 being the one generating the highest inhibition halos (Staphylococcus aureus: 16 mm; Enterococcus faecalis: 12 mm; Escherichia coli: 13 mm; Klebsiella pneumoniae: 10 mm and Pseudomonas aeruginosa: 8 mm). Compound 2 was found to be inactive only against Escherichia coli, while compounds 3, 5, 6, and 8 were found to be inactive only against Staphylococcus aureus. Finally, compound 6 was found to be active against Enterococcus faecalis (10 mm), Klebsiella pneumoniae (8 mm), and Pseudomonas aeruginosa (10 mm). However, the antifungal activity of the synthetic derivatives was also evaluated through the well diffusion method on Müller-Hinton agar (Kirby-Bauer). The results obtained are summarized in Table 2 and are expressed as the diameters of the inhibition halo reported in millimeters. Some of the synthetic compounds evaluated showed specific antifungal activity against Candida krusei. Compounds 2: 10 mm, 4: 12 mm, 6: 10 mm, and 7: 8 mm were active, compound 4 being the most active of all (12 mm). On the contrary, compounds 3, 5, and 8 did not present inhibition halos. These results of antimicrobial activity reveal a notable relationship between the structural modification of the C3 carbon of the ent-kaurene core and the activity, which, in general terms, is an improvement. Previous studies on the structure-activity relationship of ent-kaurenes have identified that the pharmacological action of these compounds is based on the perhydrophenanthrene unit (rings A, B, and C) fused with a cyclopentane unit (ring D) formed by a bridge of two carbons between C8 and C13 (Figure 1), this skeleton confers lipophilic properties that are essential to cross membranes and occupy 484 Márquez-Chacón et al. / European Journal of Chemistry 14 (4) (2023) 478-485 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.4.478-485.2478 hydrophobic pockets in the target cell [34]. In addition, various authors describe some specific structural requirements for the biological activity of ent-kaurenes and terpenes based on a decalin ring: 1) a substituted decalin system, capable of inser- ting itself into a lipophilic region of the cell membrane, and 2) a hydrophilic or relatively hydrophilic fragment that contains a hydrogen bond donor or acceptor group capable of interacting with acceptor or donor groups in the membrane (mostly phosphorylated groups), as an "anchoring" group; this way, these compounds will have the ability to cross the phospholipid membrane and cause cell damage to the microorganism [35,36]. In this regard, the synthetic ent-kaurenes tested in this work present structural characteristics very similar to those described above: the substituted perhydrophenanthrene unit is fused with a cyclopentane unit, which makes them possess an essential lipophilic character to cross the cell membrane. However, each compound possesses distinctive characteristics: compounds 4, 5, 6, and 7 have ester groups in C3 whose hydrophilicity is low; however, they present a well-defined negative dipole moment in the carbonyl oxygen atom that allows them to accept hydrogen bonds (anchor group) with the hydrophilic part of the membrane phospholipids. In addition, each of these compounds has acidic protons or another reactive system that also allow them to interact with the hydrophilic portion of membrane phospholipids. Compound 4, aside from having five acidic protons (H3, H2', H3', H1'', and H1'''), also shows an α,β-unsaturated system whose reactivity is very high and causes electron donor or acceptor dipole moments that are highly defined, making the compound more susceptible to form hydrogen bonds with the phosphorylated groups of the cell membrane. This could be the reason why this compound turned out to be the most active against all of the microorganisms tested. Unlike the others, compound 2 has a hydroxyl group in C3, which is a hydrophilic group that donates hydrogen bonds responsible for "anchoring" with the phosphorylated hydrogen bond acceptor groups of the cell membrane. On the contrary, compound 3 has a ketone in C3, which is a non-polar functional group that confers lipophilicity to the molecule; however, it has a negative dipole moment defined by the oxygen atom, which allows it to be an acceptor of hydrogen bonds with the hydrophilic part of the phospholipids, and it anchors itself to the cell membrane and exerts antibacterial activity. Finally, compound 8 turned out to be slightly active against bacteria and inactive against the fungus, possibly due to the high lipophilicity of the molecule conferred by the two ent-kaurene core. According to Urzua et al., the high addition of alkyl moieties to the decalinic system causes a decrease in biological activity due to excess lipophilicity that hinders molecular anchorage with the hydrophilic part of phospholipids [33]. 4. Conclusion A series of seven new ent-kaurene-type diterpenes was synthesized from the new natural product ent-kaur-3-acetoxy- 15-ene (1). Structural elucidation was performed using FT-IR, 1H NMR, 13C NMR, and GC-MS spectra. The synthesized compounds were evaluated for their antibacterial and antifungal activities and showed specific activity against some bacterial and fungal strains. Ent-kaur-3-O-(6’,7’-bibenzyl-oxy- caffeoyl)-15-ene (4) exhibited the higher antibacterial activity against all microorganisms tested: Staphylococcus aureus (16 mm), Enterococcus faecalis (12 mm), Escherichia coli (13 mm), Klebsiella pneumoniae (10 mm), Pseudomonas aeruginosa (8 mm) and Candida krusei (10 mm). These results reveal a remarkable structure-activity relationship on the C3 carbon of the ent-kaurene core, where the presence of oxygenated groups such as hydroxyl or alkyl esters enhances activity. However, this activity will depend (aside from the fundamental decalin system) on the type and number of hydrophilic or relatively hydrophilic fragments on C3 containing a hydrogen bond donor or acceptor group capable of interacting with acceptor or donor groups in the phospholipid cell membrane, as an "anchor" group, traversing and causing cell damage to the micro- organism. Acknowledgements All authors thank the Research Institute 'Dr. Alfredo Nicolás Usubillaga del Hierro', Faculty of Pharmacy and Bioanalysis, University of Los Andes, Mérida 5101, Venezuela for its support. The Research Institute authors are grateful to Prof. Freddy Ramos from the Department of Chemistry, Faculty of Science, National University of Colombia, Bogotá, 111321, Colombia, for NMR and MS equipment. Also, all authors are grateful to the MSc. Ana Deixy Flores from the Clinical Institute 'Santa Filomena', Microbiology Laboratory, Mérida 5101, Venezuela, for antimicrobial assays. Disclosure statement Conflict of interests: The authors declare that they have no conflict of interest. Ethical approval: All ethical guidelines have been adhered. Sample availability: Samples of the compounds are available from the author. CRediT authorship contribution statement Conceptualization: Andrés Eduardo Márquez, Alida Pérez Colmenares, Luis Rojas Fermín, Rosa Aparicio, Alfredo Usubillaga; Methodology: Andrés Eduardo Márquez, Alida Pérez, Luis Rojas, Rosa Aparicio; Software: Andrés Eduardo Márquez, Alida Pérez, Freddy Ramos, Rosa Aparicio; Validation: Andrés Eduardo Márquez, Alida Pérez, Rosa Aparicio, Freddy Ramos; Formal Analysis: Andrés Eduardo Márquez, Alida Pérez, Rosa Aparicio, Freddy Ramos; Investigation: Andrés Eduardo Márquez, Alida Pérez, Rosa Aparicio; Resources: Andrés Eduardo Márquez, Alida Pérez, Freddy Ramos, Rosa Aparicio; Data Curation: Andrés Eduardo Márquez, Alida Pérez; Writing - Original Draft: Andrés Eduardo Márquez, Alida Pérez, Rosa Aparicio; Writing - Review and Editing: Andrés Eduardo Márquez, Alida Pérez, Rosa Aparicio, Ysbelia Obregón; Visualization: Andrés Eduardo Márquez, Alida Pérez, Rosa Aparicio, Ysbelia Obregón; Supervision: Alida Pérez, Luis Rojas, Rosa Aparicio. Funding Program for the Support of Research Groups (ADG) of the Council of Scientific, Humanistic, Technological, and Artistic Development. University of Los Andes, Mérida, Venezuela. Research Group: Natural Products and Medicinal Chemistry. Research Institute - Natural Products Section. ORCID and Email Andrés Eduardo Márquez-Chacón andresmqch@gmail.com https://orcid.org/0000-0002-8529-505X Alida Pérez Colmenares alidaperezc@gmail.com https://orcid.org/0000-0001-8910-4663 Luis Rojas Fermín rojasfermin33@gmail.com https://orcid.org/0000-0003-4508-1927 Rosa Aparicio apariciorosa12@gmail.com https://orcid.org/0000-0002-5020-0954 Freddy Alejandro Ramos faramosr@unal.edu.co https://orcid.org/0000-0002-9271-7193 Alfredo Usubillaga usubi80@gmail.com https://orcid.org/0000-0002-2913-5684 Ysbelia Obregón ysbeliaobregon@gmail.com https://orcid.org/0000-0001-6152-6696 References [1]. Atanasov, A. G.; the International Natural Product Sciences Taskforce; Zotchev, S. B.; Dirsch, V. M.; Supuran, C. T. Natural products in drug mailto:andresmqch@gmail.com https://orcid.org/0000-0002-8529-505X mailto:alidaperezc@gmail.com https://orcid.org/0000-0001-8910-4663 mailto:rojasfermin33@gmail.com https://orcid.org/0000-0003-4508-1927 mailto:apariciorosa12@gmail.com https://orcid.org/0000-0002-5020-0954 mailto:faramosr@unal.edu.co https://orcid.org/0000-0002-9271-7193 mailto:usubi80@gmail.com https://orcid.org/0000-0002-2913-5684 mailto:ysbeliaobregon@gmail.com https://orcid.org/0000-0001-6152-6696 Márquez-Chacón et al. / European Journal of Chemistry 14 (4) (2023) 478-485 485 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.4.478-485.2478 discovery: advances and opportunities. Nat. Rev. Drug Discov. 2021, 20, 200–216. [2]. Abdel-Razek, A. S.; El-Naggar, M. E.; Allam, A.; Morsy, O. M.; Othman, S. I. Microbial natural products in drug discovery. Processes (Basel) 2020, 8, 470. [3]. Porto, T. S.; Simão, M. R.; Carlos, L. Z.; Martins, C. H. G.; Furtado, N. A. J. C.; Said, S.; Arakawa, N. S.; dos Santos, R. A.; Veneziani, R. C. S.; Ambrósio, S. R. Pimarane-type diterpenes obtained by bio- transformation: Antimicrobial properties against clinically isolated gram-positive multidrug-resistant bacteria. Phytother. Res. 2013, 27, 1502–1507. [4]. Miron-Lopez, G.; Bazzocchi, I. L.; Jimenez-Diaz, I. A.; Moujir, L. M.; Quijano-Quiñones, R.; Quijano, L.; Mena-Rejon, G. J. Cytotoxic diterpenes from roots of Crossopetalum gaumeri, a Celastraceae species from Yucatan Peninsula. Bioorg. Med. Chem. Lett. 2014, 24, 2105–2109. [5]. Bou, D. D.; Tempone, A. G.; Pinto, É. G.; Lago, J. H. G.; Sartorelli, P. Antiparasitic activity and effect of casearins isolated from Casearia sylvestris on Leishmania and Trypanosoma cruzi plasma membrane. Phytomedicine 2014, 21, 676–681. [6]. Pardo-Vargas, A.; Ramos, F. A.; Cirne-Santos, C. C.; Stephens, P. R.; Paixão, I. C. P.; Teixeira, V. L.; Castellanos, L. Semi-synthesis of oxygenated dolabellane diterpenes with highly in vitro anti-HIV-1 activity. Bioorg. Med. Chem. Lett. 2014, 24, 4381–4383. [7]. Jiang, K.; Chen, L.-L.; Wang, S.-F.; Wang, Y.; Li, Y.; Gao, K. Anti- inflammatory Terpenoids from the Leaves and Twigs of Dysoxylum gotadhora. J. Nat. Prod. 2015, 78, 1037–1044. [8]. de S. Vargas, F.; D. O. de Almeida, P.; Aranha, E.; de A. Boleti, A.; Newton, P.; de Vasconcellos, M.; Junior, V.; Lima, E. Biological Activities and Cytotoxicity of Diterpenes from Copaifera spp. Oleoresins. Molecules 2015, 20, 6194–6210. [9]. Miranda, M. M.; Panis, C.; da Silva, S. S.; Macri, J. A.; Kawakami, N. Y.; Hayashida, T. H.; Madeira, T. B.; Acquaro, V. R.; Nixdorf, S. L.; Pizzatti, L.; Ambrósio, S. R.; Cecchini, R.; Arakawa, N. S.; Verri, W. A.; Conchon Costa, I.; Pavanelli, W. R. Kaurenoic acid possesses leishmanicidal activity by triggering a NLRP12/IL-1β/cNOS/NO pathway. Mediators Inflamm. 2015, 2015, 1–10. [10]. Zhu, L.; Huang, S.-H.; Yu, J.; Hong, R. Constructive innovation of approaching bicyclo[3.2.1]octane in ent-kauranoids. Tetrahedron Lett. 2015, 56, 23–31. [11]. Usubillaga, A.; Romero, M.; Aparicio, R. Kaurenic acid in Espeletiinae. Acta Hortic. 2003, 129–130. [12]. Aparicio Z., R. L.; Villasmil, T.; Peña, A.; Rojas S., J. C.; Usubillaga, A. Estudio fitoquímico de las hojas de Espeletia semiglobulata Cuatrec. Revista de la Facultad de Farmacia 2014, 55(2), 2–5, http://www.saber.ula.ve/handle/123456789/38474 (accessed Oct 10, 2023). [13]. de Andrade, B. B.; Moreira, M. R.; Ambrosio, S. R.; Furtado, N. A. J. C.; Cunha, W. R.; Heleno, V. C. G.; Silva, A. N.; Simão, M. R.; da Rocha, E. M. P.; Martins, C. H. G.; Veneziani, R. C. S. Evaluation of ent-kaurenoic acid derivatives for their anticariogenic activity. Nat. Prod. Commun. 2011, 6(6), 1934578X1100600. [14]. Batista, R.; García, P. A.; Castro, M. A.; Miguel del Corral, J. M.; Speziali, N. L.; de P. Varotti, F.; de Paula, R. C.; García-Fernández, L. F.; Francesch, A.; San Feliciano, A.; de Oliveira, A. B. Synthesis, cytotoxicity and antiplasmodial activity of novel ent -kaurane derivatives. Eur. J. Med. Chem. 2013, 62, 168–176. [15]. Matos, P.; Mahoney, B.; Chan, Y.; Day, D.; Cabral, M.; Martins, C.; Santos, R.; Bastos, J.; Page, P.; Heleno, V. New non-toxic semi-synthetic derivatives from natural diterpenes displaying anti-tuberculosis activity. Molecules 2015, 20, 18264–18278. [16]. Silva, A.; Soares, A. C.; Cabral, M.; de Andrade, A.; da Silva, M.; Martins, C.; Veneziani, R.; Ambrósio, S.; Bastos, J.; Heleno, V. Antitubercular Activity Increase in Labdane Diterpenes from Copaifera Oleoresin through Structural Modification. J. Braz. Chem. Soc. 2016, 28(6), 1106– 1112, http://dx.doi.org/10.21577/0103-5053.20160268 (accessed Oct 10, 2023). [17]. Cordero de Rojas, Y.; Lucena de Ustáriz, M. E.; Araujo, L.; Usubillaga, A.; Rojas, L. B.; Moujir, L. Actividad antibacteriana de diterpenos del kaurano aislados de Coespeletia moritziana (Sch. Bip. ex Wedd.) Cuatrec. Revista de la Facultad de Farmacia 2017, 59(2), 03–07, http://www.saber.ula.ve/handle/123456789/45132 (accessed Oct 10, 2023). [18]. Rios Tesch, N. N.; Villalobos Osorio, D. C.; Rojas, L. B.; Aparicio Z., R. L.; Usubillaga, A.; Mitaine Offer, A. C.; Lacaille Dubois, M. A.; Denis, D.; Peixoto, P.; Laurent, P.; Stéphane, Q. In vivo anti-inflammatory activity of grandiflorenic acid and kaurenic acid isolated from Coespeletia moritziana and Espeletia semiglobulata. Revista de la Facultad de Farmacia 2017, 59 (1), 17–21, http://www.saber.ula.ve/handle/ 123456789/44160 (accessed Oct 10, 2023). [19]. Villasmil, T.; Rojas, J.; Aparicio, R.; Gamboa, N.; Acosta, M. E.; Rodrigues, J.; Usubillaga, A. Antimalarial activity of some kaurenes. Nat. Prod. Commun. 2017, 12, 1934578X1701200. [20]. Peña, A.; Usubillaga, A.; Alarcón Pineda, L. del V.; Velasco Carrillo, J.; Aparicio Z., R. L. Obtención de derivados azufrados del ácido kaurénico y de otros kaurenos substituidos en la posición C-15 y su actividad antibacteriana. Revista de la Facultad de Farmacia 2016, 57 (1), 3–8, http://www.saber.ula.ve/handle/123456789/41936 (accessed Oct 10, 2023). [21]. Ruiz, Y.; Rodrígues, J.; Arvelo, F.; Usubillaga, A.; Monsalve, M.; Diez, N.; Galindo-Castro, I. Cytotoxic and apoptosis-inducing effect of ent-15- oxo-kaur-16-en-19-oic acid, a derivative of grandiflorolic acid from Espeletia schultzii. Phytochemistry 2008, 69, 432–438. [22]. Mora, A. J.; Delgado, G. E.; Vaughan, G. B. M.; Martin, P.; Visbal, T.; Usubillaga, A. A peracetylated glucosyl ester of kaurenic acid. Acta Crystallogr. Sect. E Struct. Rep. Online 2004, 60, o334–o336. [23]. Márquez, A. E.; Pérez, A.; Rojas, L.; Aparicio, R.; Ramos, F.; Obregón, Y.; Usubillaga, A. A New ent-kaurene Diterpenoid Isolated from Leaves of Espeletia semiglobulata Cuatrec. and its Potential Antimicrobial Activity. Biol. Med. Nat. Prod. Chem. 2023, 12, 151–157. [24]. CLSI: Performance standards for antimicrobial susceptibility testing; Twenty-third informational supplement; Clinical & Laboratory Standards Institute, 2013. [25]. Wikler, M. A. Performance standards for antimicrobial disk susceptibility tests; Approved standard; 11th ed.; Clinical & Laboratory Standards Institute, 2012. [26]. Berkow, E. L.; Lockhart, S. R.; Ostrosky-Zeichner, L. Antifungal susceptibility testing: Current approaches. Clin. Microbiol. Rev. 2020, 33, e00069-19. [27]. Abualhasan, M. N.; Al- Masri, M. Y.; Manasara, R.; Yadak, L.; Abu-Hasan, N. S. Anti-inflammatory and anticoagulant activities of synthesized NSAID prodrug esters. Scientifica (Cairo) 2020, 2020, 1–6. [28]. Ohkoshi, E.; Kamo, S.; Makino, M.; Fujimoto, Y. ent-Kaurenoic acids from Mikania hirsutissima (Compositae). Phytochemistry 2004, 65, 885–890. [29]. Rodrı́guez, S.; Garda, H. A.; Heinzen, H.; Moyna, P. Effect of plant monofunctional pentacyclic triterpenes on the dynamic and structural properties of dipalmitoylphosphatidylcholine bilayers. Chem. Phys. Lipids 1997, 89, 119–130. [30]. Herrera, M.; Rodriguez-Rodriguez, R.; Ruiz-Gutierrez, V. Functional properties of pentacyclic triterpenes contained in “orujo” Olive oil. Curr. Nutr. Food Sci. 2006, 2, 45–49. [31]. Anthonsen, T.; Chantharasakul, S.; Raknes, E.; Sørensen, N. A.; Lindberg, A. A.; Ehrenberg, L. Isolation of ent-16-kauren-19-oic acid and ent-16-kauren-19-ol from Abrotanella nivigena Muell. Acta Chem. Scand. 1971, 25, 1925–1927. [32]. Hutchison, M.; Lewer, P.; MacMillan, J. Carbon-13 nuclear magnetic resonance spectra of eighteen derivatives of ent-kaur-16-en-19-oic acid. J. Chem. Soc., Perkin Trans. 1 1984, 2363–2366. [33]. Neises, B.; Steglich, W. Simple method for the esterification of carboxylic acids. Angew. Chem., Int. Ed. Engl. 1978, 17, 522–524. [34]. Batista, R.; García, P. A.; Castro, M. A.; Corral, J. M. M. del; Sanz, F.; Speziali, N. L.; Oliveira, A. B. de Methylent-16β,17-epoxykauran-19- oate. Acta Crystallogr. Sect. E Struct. Rep. Online 2007, 63, o932–o933. [35]. Urzúa, A.; Rezende, M.; Mascayano, C.; Vásquez, L. A structure-activity study of antibacterial diterpenoids. Molecules 2008, 13, 882–891. [36]. Echeverría, J.; Urzúa, A.; Sanhueza, L.; Wilkens, M. Enhanced antibacterial activity of ent-labdane derivatives of salvic acid (7α- hydroxy-8(17)-ent-labden-15-oic acid): Effect of lipophilicity and the hydrogen bonding role in bacterial membrane interaction. Molecules 2017, 22, 1039. Copyright © 2023 by Authors. This work is published and licensed by Atlanta Publishing House LLC, Atlanta, GA, USA. The full terms of this license are available at http://www.eurjchem.com/index.php/eurjchem/pages/view/terms and incorporate the Creative Commons Attribution-Non Commercial (CC BY NC) (International, v4.0) License (http://creativecommons.org/licenses/by-nc/4.0). By accessing the work, you hereby accept the Terms. This is an open access article distributed under the terms and conditions of the CC BY NC License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited without any further permission from Atlanta Publishing House LLC (European Journal of Chemistry). No use, distribution, or reproduction is permitted which does not comply with these terms. Permissions for commercial use of this work beyond the scope of the License (http://www.eurjchem.com/index.php/eurjchem/pages/view/terms) are administered by Atlanta Publishing House LLC (European Journal of Chemistry). http://www.saber.ula.ve/handle/123456789/38474 http://dx.doi.org/10.21577/0103-5053.20160268 http://www.saber.ula.ve/handle/123456789/45132 http://www.saber.ula.ve/handle/%20123456789/44160 http://www.saber.ula.ve/handle/%20123456789/44160 http://www.saber.ula.ve/handle/123456789/41936 http://www.eurjchem.com/index.php/eurjchem/pages/view/terms http://creativecommons.org/licenses/by-nc/4.0 http://www.eurjchem.com/index.php/eurjchem/pages/view/terms 1. Introduction 2. Experimental 2.1. Materials and instrumentation 2.2. Synthesis 2.2.1. Synthesis of ent-kaur-3-hydroxy-15-ene (2) derivative 2.2.2. Synthesis of ent-kaur-3-oxo-15-ene (3) derivative 2.2.3. Synthesis of ent-kaur-3-acetoxyalkylated derivatives (4-8) 2.3. Antibacterial activity 2.4. Antifungal activity 3. Results and discussion 3.1. Chemistry 3.2. Antimicrobial activity 4. Conclusion Acknowledgements Disclosure statement CRediT authorship contribution statement Funding ORCID and Email References PrintField10: PrintField11: PrintField12: PrintField13: PrintField14: PrintField15: PrintField16: PrintField17: PrintField20: PrintField21: PrintField22: PrintField23: PrintField24: PrintField25: PrintField26: PrintField27: