Structural Requirements for Antimicrobial Activity of Phenolic Nor-Triterpenes from Celastraceae Species

Featured Application: Identifying the structural requirements of phenolic nor-triterpene framework as potential antimicrobial agents. Abstract: The emergence of pathogenic bacteria-resistant strains is a major public health issue. In this regard, natural product sca ﬀ olds o ﬀ er a promising source of new antimicrobial drugs. In the present study, we report the antimicrobial activity against Gram-positive and Gram-negative bacteria and the yeast Candida albicans of ﬁve phenolic nor-triterpenes ( 1 – 5 ) isolated from Maytenus blepharodes and Maytenus canariensis in addition to four derivatives ( 6 – 9 ), three of them reported for the ﬁrst time. Their stereostructures have been elucidated on the basis of spectroscopic analysis, including one-dimensional (1D) and two-dimensional (2D) NMR techniques, spectrometric methods, and comparison with data reported in the literature. To understand the structural basis for the antimicrobial activity of this type of compounds, we have performed an in-depth study of the structure–activity relationship (SAR) of a series of previously reported phenolic nor-triterpenes. The SAR analysis was based on the skeleton framework, oxidation degree, functional groups, and regiosubstitution patterns, revealing that these aspects modulate the antimicrobial activity. group at this position was a carboxylic acid ( 14 ), followed by hydroxyl ( 10 ) and aldehyde ( 12 ) groups, while a methyl group, as in 1 , led to a loss of activity. B. cereus , B. alvei , B. megaterium, and B. pumilus showed similar behavior to B. subtilis ( 14 vs. 12 ); (b) Acetylation and methylation reduce activity, as revealed by comparison of potency of the natural compounds with their corresponding analogues ( 7 vs. 24 , 8 vs. 25 , 9 vs. 26 , 10 vs. 11 , 16 vs. 17 , 18 , 19 , and 20 vs. 21 ), except for compound 6 with an hydroxyl group at C6 ( 6 vs. 7 ). This indicates that the hydroxyl group, which could interact with the receptor as hydrogen-bond-donor (HBD), is the best functional group, suggesting that the hydrophilicity is relevant for the activity; (c) Compounds with an unconjugated double bond at C-7 showed considerable activity (e.g., 6 and 24 ), and reduction at C-7 / C-8 results in a partial loss of antimicrobial activity ( 24 vs. 25 and 26 ); (d) The β -stereochemistry at C-8 increases two-fold the activity with respect to the H-8 α , as shown by comparing the MIC values of 26 and 25 ; (e) A substituent at C-7, in addition to the carbonyl at C-6, is also relevant for the activity, being a ketone preferred to a hydroxyl group ( 22 > 5 vs. 4 ). vs. (b) replacement of the carboxyl group at C-29 its methyl ester to a partial loss of activity ( vs. 12 ); (c) compounds two carboxylic acid groups lacked activity, whereas those with one carboxylic acid showed high activity ( 14 vs. 15 ); (d) the reduction of the double bond at C-7 / C-8 led to less activity ( 24 vs. 25 and 26 against S. epidermidis ); (e) the β -stereochemistry at the C-8 position favored the activity with respect to the H-8 α ( 26 vs. 25 ); (f) a hydroxyl group at C-6 a ﬀ ected the activity against S. epidermidis ( vs. we propose


Introduction
The resistance of common pathogens to standard antibiotic therapies is rapidly becoming a major public health problem all over the world [1], and consequently, there is a need to develop new structural and mechanistic classes of antibiotic agents. In this regard, the development of new antibiotics inspired in natural product scaffolds seems the best short-term solution to address antibiotic resistance [2].
The Celastraceae family is distributed mainly in tropical and subtropical regions of the world including North Africa, South America, and East Asia, and their species have a long history in traditional medicine [3]. The most representative genus in this family is Maytenus, with more than 225 species [4]. In the Amazonian region, species of this genus are well known for their use in the treatment of rheumatism, gastrointestinal diseases, and as an antitumoral for skin cancer [4]. The therapeutic potential of Maytenus species has been mainly attributed to celastroloids, chemotaxonomic markers of the family [5]. The term celastroloid refers to methylenequinone nor-triterpenes with a 24-nor-D:A-friedo-oleanane skeleton. Celastrol [6] and pristimerin [7] are the first and most frequently reported celastroloids, and later on, this term was extended to related phenolic nor-triterpenes [5,8] and their dimer and trimer congeners [9]. This particular class of natural

General Procedures
Optical rotations were measured on a Perkin Elmer 241 automatic polarimeter in CHCl3 at 25 °C, and the [α]D values are given in 10 −1 deg cm 2 g −1 . UV spectra were obtained in absolute EtOH on a JASCO V-560 instrument. IR (film) spectra were measured in CHCl3 on a Bruker IFS 55 spectrophotometer. 1 H (400 or 500 MHz) and 13 C (100 or 125 MHz) NMR spectra were recorded on Bruker Avance 400 or 500 spectrometers; chemical shifts are given in ppm and coupling constants in hertz. Samples were dissolved (CDCl3: δH 7.26, δC 77.0). EI-MS and EI-HRMS were recorded on a Micromass Autospec spectrometer. Silica gel 60 (particle size 15-40 µ m) for column chromatography and silica gel 60 F254 for analytical (TLC) and preparative thin-layer chromatography (PTLC) were purchased from Macherey-Nagel. Sephadex LH-20 was obtained from Pharmacia Biotech. Shimadzu high-performance liquid chromatography (HPLC) equipment consisted of a pump LKB 2248 solvent delivery module, SPD-6V detector set at 254 nm, using a semipreparative silica gel column (Waters µ -Porosil ® , 15 cm × 1.6 mm, particle size 10 µ m). The mobile phase consisted of a mixture of n-hexane-EtOAc (8:2) in isocratic mode with a flow rate of 9 mL/min. The degree of purity of the compounds was over 95%, as indicated by a single peak in HPLC and NMR. All solvents used were of analytical grade (Panreac), and the reagents, used without purification, were purchased from Sigma-Aldrich. Pristimerin, used as starting material, was isolated from the root bark of M. blepharodes and M. canariensis, as previously described [20,21].

General Procedures
Optical rotations were measured on a Perkin Elmer 241 automatic polarimeter in CHCl 3 at 25 • C, and the [α] D values are given in 10 −1 deg cm 2 g −1 . UV spectra were obtained in absolute EtOH on a JASCO V-560 instrument. IR (film) spectra were measured in CHCl 3 on a Bruker IFS 55 spectrophotometer. 1 H (400 or 500 MHz) and 13 C (100 or 125 MHz) NMR spectra were recorded on Bruker Avance 400 or 500 spectrometers; chemical shifts are given in ppm and coupling constants in hertz. Samples were dissolved (CDCl 3 : δ H 7.26, δ C 77.0). EI-MS and EI-HRMS were recorded on a Micromass Autospec spectrometer. Silica gel 60 (particle size 15-40 µm) for column chromatography and silica gel 60 F254 for analytical (TLC) and preparative thin-layer chromatography (PTLC) were purchased from Macherey-Nagel. Sephadex LH-20 was obtained from Pharmacia Biotech. Shimadzu high-performance liquid chromatography (HPLC) equipment consisted of a pump LKB 2248 solvent delivery module, SPD-6V detector set at 254 nm, using a semipreparative silica gel column (Waters µ-Porosil ® , 15 cm × 1.6 mm, particle size 10 µm). The mobile phase consisted of a mixture of n-hexane-EtOAc (8:2) in isocratic mode with a flow rate of 9 mL/min. The degree of purity of the compounds was over 95%, as indicated by a single peak in HPLC and NMR. All solvents used were of analytical grade (Panreac), and the reagents, used without purification, were purchased from Sigma-Aldrich. Pristimerin, used as starting material, was isolated from the root bark of M. blepharodes and M. canariensis, as previously described [20,21].

Preparation of Compound 6
To a stirred solution of pristimerin (31.0 mg) in pyridine (0.15 mL), anhydride acetic (0.1 mL) and a catalytic amount of 4-dimethylamino-pyridine were added. The resulting orange-red solution was stirred for 14 h at room temperature, until TLC showed complete conversion. This yellow solution was concentrated under reduced pressure, and the residue was purified by preparative TLC using n-hexane-EtOAc (1:1) as eluent to afford compound 6 (14.0 mg, 37.0%).

Preparation of Compounds 7-9
A mixture of pristimerin (622.0 mg) and Pd/C 5% (100 mg) in acetic acid (15 mL) was stirred under 1 atmosphere of hydrogen for 3 h. The reaction mixture was filtered through a pad of celite, the solution quenched by addition of saturated aqueous sodium bicarbonate solution, and the aqueous residue extracted with dichloromethane (3 × 30 mL). Then, to the crude, pyridine (1.5 mL) dissolved in acetic anhydride (1.5 mL) was added, and the reaction mixture was stirred for 12 h at room temperature. Upon completion of the reaction, the solution was concentrated on a cold finger with liquid nitrogen. The residue was purified by HPLC using n-hexane-ethyl acetate (8:2) as eluent to give compound 7 (21.9 mg, 2.9%, t R = 11.4 min), reported elsewhere [31], and derivatives 8 (76.4 mg, 10.3%, t R = 12.3 min) and 9 (32.1 mg, 4.3%, t R = 12.6 min), not previously reported.  were developed in nutrient broth (NB) or brain heart infusion broth (for E. faecalis and M. smegmatis containing 0.06% Tween 80), and the yeast was cultured in Sabouraud liquid medium at 37 • C. All culture media were purchased from Oxoid. The minimum inhibitory concentrations (MICs) were determined for each compound in triplicate by broth microdilution method (range 0.08-40 µg/mL) in 96 well microtitre plates, according to the M07-A9 of the CLSI (Clinical and Laboratory Institute) [32]. Wells with the same proportions of dimethyl sulfoxide (DMSO) were used as controls and never exceeded 1% (v/v). The starting microorganism concentration was approximately 1-5 × 10 5 CFU/mL, and growth was monitored by measuring the increase in optical density (OD) at 550 nm (OD 550 ) with a microplate reader (Multiskan Plus II, Tritertek, Huntsville, AL, USA) and viable count in agar plates. The MIC was defined as the lowest concentration of compound that completely inhibits growth of the organisms compared with that of the control after incubation time.

Chemistry
Compounds 1-5, 10, 12-17, 20, 22, and 25 ( Figure 2) were isolated, purified, and characterized in our laboratory from two Maytenus species, M. blepharodes and M. canariensis, as previously described [20,21,27]. Derivatives 11, 18, 19, 21, 23, 24, and 26, achieved by acetylation or methylation from natural compounds, are described by González et al. [20] and Rodríguez et al. [21]. Moreover, derivatives 6-9 were prepared from pristimerin (Scheme 1). The semisynthesis of 6 was achieved by acetylation of pristimerin, and derivatives 7-9 were obtained by catalytic reduction and further acetylation of pristimerin, as described in the experimental section. Their structures were greatly aided by comparison of their spectroscopic data with those previously reported for related compounds 24-26 [21]. Even so, a complete set of two-dimensional (2D) NMR spectra (COSY, ROESY, HSQC and HMBC) was acquired for the new derivatives to gain the complete assignment of the 1 H and 13 C NMR resonances (see Experimental Section, Figures S1-S4). Derivative 7 was identified as the previously described 2,3-diacetylpristimerol [31], however, its 1 H and 13 C NMR assignments have not been previously reported. Moreover, derivatives 6, 8, and 9 are described herein for the first time, and their structures were elucidated as described below.
Compounds 8 and 9, both with the molecular formula C 34 H 48 O 6 (EI-HRMS), differ from that of 7 by the presence of two additional hydrogen atoms. The most remarkable difference in their NMR spectra, when compared to that of compound 7, was the absence of the double bond C-7-C-8. Thus, in the 13 C NMR spectra of 8 and 9, the signals assigned to C-7 and C-8 resonated in the region of aliphatic carbons (δ C 18.2 and 43.5 in 8, and δ C 30.5 and 56.0 in 9). Moreover, differences between 8 and 9 were also observed for the chemical shifts of Me-25 (δ H 1.60, δ C 27.3 in 8, and δ H 1.46, δ C 36.8 in 9) and Me-26 (δ H 0.88, δ C 15.9 in 8, and δ H 1.21, δ C 25.8 in 9). Analysis of 2D COSY, HSQC, and HMBC spectra allowed us to define their core structures. The ROE effects of H-8/Me-25 and Me-26 observed in a ROESY experiment for compound 8, and those of H-8/Me-27 observed for compound 9, further supported the stereochemical assignment for epimers 8 and 9. Their structures were also confirmed by chemical correlations; hydrolysis of 8 and 9 yielded compounds whose spectroscopic data were identical to those previously reported for 6-deoxoblepharodol (25) and 8-epi-6-deoxoblepharodol (26) [21], respectively. Consequently, the structure of compounds 8 and 9 were deduced to be 2,3-diacetyl-6-deoxoblepharodol and 2,3-diacetyl-8-epi-6-deoxoblepharodol, respectively.

Structure-Activity Relationship Analysis
The influence of substitution patterns of the phenolic nor-triterpenes on their antimicrobial activity was analyzed, taking into account three regions of the triterpene skeleton, the phenolic moiety, the extended B-ring conjugation, and the triterpene scaffold (pristimerin, tingenone, or iguesterin). This analysis revealed the following trends in the structure-activity relationship (SAR) (Figure 4). Regarding the results obtained against Bacillus spp., the SAR studies suggest that: (a) A substituent at C-4 seems essential for the activity. Thus, comparison of the activities (MICs against B. subtilis) of 14, 10, 12, and 1, whose only structural difference is the substituent at C4, showed that the most effective group at this position was a carboxylic acid (14), followed by hydroxyl (10) and aldehyde (12) groups, while a methyl group, as in 1, led to a loss of activity. B. cereus, B. alvei, B. megaterium, and B. pumilus showed similar behavior to B. subtilis (14 vs. 12); (b) Acetylation and methylation reduce activity, as revealed by comparison of potency of the natural compounds with their corresponding analogues (7 vs .  24, 8 vs. 25, 9 vs. 26, 10 vs. 11, 16 vs. 17, 18, 19, and 20 vs. 21), except for compound 6 with an hydroxyl group at C6 (6 vs. 7). This indicates that the hydroxyl group, which could interact with the receptor as hydrogen-bond-donor (HBD), is the best functional group, suggesting that the hydrophilicity is relevant for the activity; (c) Compounds with an unconjugated double bond at C-7 showed considerable activity (e.g., 6 and 24), and reduction at C-7/C-8 results in a partial loss of antimicrobial activity (24 vs. 25 and 26); (d) The β-stereochemistry at C-8 increases two-fold the activity with respect to the H-8α, as shown by comparing the MIC values of 26 and 25; (e) A substituent at C-7, in addition to the carbonyl at C-6, is also relevant for the activity, being a ketone preferred to a hydroxyl group (22 > 5 vs. 4).
activity was analyzed, taking into account three regions of the triterpene skeleton, the phenolic moiety, the extended B-ring conjugation, and the triterpene scaffold (pristimerin, tingenone, or iguesterin). This analysis revealed the following trends in the structure-activity relationship (SAR) (Figure 4). Regarding the results obtained against Bacillus spp., the SAR studies suggest that: (a) A substituent at C-4 seems essential for the activity. Thus, comparison of the activities (MICs against B. subtilis) of 14, 10, 12, and 1, whose only structural difference is the substituent at C4, showed that the most effective group at this position was a carboxylic acid (14), followed by hydroxyl (10) and aldehyde (12) groups, while a methyl group, as in 1, led to a loss of activity. B. cereus, B. alvei, B. megaterium, and B. pumilus showed similar behavior to B. subtilis (14 vs. 12); (b) Acetylation and methylation reduce activity, as revealed by comparison of potency of the natural compounds with their corresponding analogues (7 vs . 24, 8 vs. 25, 9 vs. 26, 10 vs. 11, 16 vs. 17, 18, 19, and 20 vs. 21), except for compound 6 with an hydroxyl group at C6 (6 vs. 7). This indicates that the hydroxyl group, which could interact with the receptor as hydrogen-bond-donor (HBD), is the best functional group, suggesting that the hydrophilicity is relevant for the activity; (c) Compounds with an unconjugated double bond at C-7 showed considerable activity (e.g., 6 and 24), and reduction at C-7/C-8 results in a partial loss of antimicrobial activity (24 vs . 25 and 26); (d) The β-stereochemistry at C-8 increases two-fold the activity with respect to the H-8α, as shown by comparing the MIC values of 26 and 25; (e) A substituent at C-7, in addition to the carbonyl at C-6, is also relevant for the activity, being a ketone preferred to a hydroxyl group (  Furthermore, the results obtained against Staphylococcus spp. suggest that: (a) the presence of a carboxylic acid moiety at C-4 could strongly affect the antimicrobial activity against S. aureus since its substitution by an aldehyde or a methyl group reduced or fully eliminated the activity (14 vs. 12 or 1); (b) moreover, replacement of the carboxyl group at C-29 by its methyl ester led to a partial loss of activity (13 vs. 12); (c) compounds containing two carboxylic acid groups lacked activity, whereas those with one carboxylic acid showed high activity (14 vs. 15); (d) the reduction of the double bond at C-7/C-8 led to less activity (24 vs. 25 and 26 against S. epidermidis); (e) the β-stereochemistry at the C-8 position favored the activity with respect to the H-8α (26 vs. 25); (f) a hydroxyl group at C-6 affected the activity against S. epidermidis (6 vs. 7). Taking into account these structural features, we propose a hypothetical compound, 4-carboxy-6α-hydroxy-pristimerol, that would have a zeylasterone A-ring (as in 14), an unconjugated B-ring (as in 6 and 24), and a pristimerin E-ring as being the most active against Bacillus and Staphylococcus spp. (Figure 5). at C-7/C-8 led to less activity (24 vs. 25 and 26 against S. epidermidis); (e) the β-stereochemistry at the C-8 position favored the activity with respect to the H-8α (26 vs. 25); (f) a hydroxyl group at C-6 affected the activity against S. epidermidis (6 vs. 7). Taking into account these structural features, we propose a hypothetical compound, 4-carboxy-6α-hydroxy-pristimerol, that would have a zeylasterone A-ring (as in 14), an unconjugated B-ring (as in 6 and 24), and a pristimerin E-ring as being the most active against Bacillus and Staphylococcus spp. (Figure 5). Some phenolic compounds primarily target the cytoplasmic membrane due to their hydrophobic nature, and preferentially partition into the lipid bilayer [33], as was observed in our previous works [22][23][24][25]. Therefore, for the purpose of correlating the antimicrobial activity with lipophilicity, clog P values [34] of this series of compounds were calculated, including that of the hypothetical compound, 4-carboxy-6α-hydroxy-pristimerol (clog P 4.88), and are shown in Table 1. Slight increases in lipophilicity of 24 (clog P 7.09) and 25 and 26 (clog P 6.41) by acetylation (7, 8 and 9, respectively) (clog P 7.18 and 6.90) led to a suppression of the antibacterial activity. Moreover, other factors must be taken into account for the expression of the activity, such as the presence of an hydrogen-bond donor (HBD) group, strategically positioned at C-4 (e.g., 14), C-6 (e.g., 6), or at C-2 and C-3 (e.g., 12 and 14) with clog P of 5.04, 5.95, and 5.67, respectively, which seems relevant for the activity. These observations indicated that both lipophilicity and HBD factors are involved in the antimicrobial activity of this type of compound.

Conclusions
In summary, the antimicrobial activity of five phenolic nor-triterpenes isolated from two Maytenus species and those of four derivatives revealed that compound 6 showed significant activity against Gram-positive bacteria, higher than cephotaxime, used as positive control. In order to understand and further optimize the structural requirements for effective inhibition of bacterial growth in vitro, an extensive SAR analysis of a series of nor-triterpene phenols was performed. This study suggests that the phenolic moiety and carboxyl group at C-4 on the A-ring, a nonconjugated double bond system on the B-ring, and the ring E, characteristic of pristimerin series, all contribute to the antimicrobial effectiveness. Based on these findings, we propose a hypothetical lead compound, 4-carboxy-6α-hydroxy-pristimerol. This comprehensive SAR study supports the future rational design of antimicrobial agents based on the phenolic nor-triterpene scaffold. Some phenolic compounds primarily target the cytoplasmic membrane due to their hydrophobic nature, and preferentially partition into the lipid bilayer [33], as was observed in our previous works [22][23][24][25]. Therefore, for the purpose of correlating the antimicrobial activity with lipophilicity, clog P values [34] of this series of compounds were calculated, including that of the hypothetical compound, 4-carboxy-6α-hydroxy-pristimerol (clog P 4.88), and are shown in Table 1. Slight increases in lipophilicity of 24 (clog P 7.09) and 25 and 26 (clog P 6.41) by acetylation (7, 8 and 9, respectively) (clog P 7.18 and 6.90) led to a suppression of the antibacterial activity. Moreover, other factors must be taken into account for the expression of the activity, such as the presence of an hydrogen-bond donor (HBD) group, strategically positioned at C-4 (e.g., 14), C-6 (e.g., 6), or at C-2 and C-3 (e.g., 12 and 14) with clog P of 5.04, 5.95, and 5.67, respectively, which seems relevant for the activity. These observations indicated that both lipophilicity and HBD factors are involved in the antimicrobial activity of this type of compound.

Conclusions
In summary, the antimicrobial activity of five phenolic nor-triterpenes isolated from two Maytenus species and those of four derivatives revealed that compound 6 showed significant activity against Gram-positive bacteria, higher than cephotaxime, used as positive control. In order to understand and further optimize the structural requirements for effective inhibition of bacterial growth in vitro, an extensive SAR analysis of a series of nor-triterpene phenols was performed. This study suggests that the phenolic moiety and carboxyl group at C-4 on the A-ring, a nonconjugated double bond system on the B-ring, and the ring E, characteristic of pristimerin series, all contribute to the antimicrobial effectiveness. Based on these findings, we propose a hypothetical lead compound, 4-carboxy-6α-hydroxy-pristimerol. This comprehensive SAR study supports the future rational design of antimicrobial agents based on the phenolic nor-triterpene scaffold. Funding: This study was supported by SAF2015-65113-C2-1-R and RTI2018-094356-B-C21 Spanish MINECO co-funded by the European Regional Development Fund (FEDER) projects.