**1. 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 products shows a wide range of bioactivities, including cytotoxic [10,11], anti-inflammatory [12], antioxidant [13], antiparasitic [14], and insecticidal [15] properties. Concerning their antimicrobial activity, pristimerin, tingenone, celastrol, and netzahualcoyone (Figure 1) exhibit inhibitory activity against Gram-positive bacteria [8,16,17], and the mode of action of netzahualcoyone against *Bacillus subtilis* and *Escherichia coli* has been investigated [18,19]. Regarding the phenolic nor-triterpenes, studies on their antimicrobial activity, mechanism of action against Gram-positive bacteria, and preliminary structure–activity relationship have been reported [20–25].

**Figure 1.** Most frequently reported antimicrobial celastroloids from Celastraceae species.

As part of an intensive investigation into antimicrobial metabolites from Celastraceae species, we report herein on the minimal inhibitory concentrations (MICs) of five natural phenolic nor-triterpenes (**1**–**5**) and four derivatives (**6**–**9**), three of them reported for the first time, against Gram-positive and Gram-negative bacteria, and the yeas<sup>t</sup> *Candida albicans*. The structure–activity relationship study of compounds **1**–**9** was expanded by the known antimicrobial activity of a series of phenolic nor-triterpenes (**10**–**26**), previously reported by our research group [20–23,25], to deepen our knowledge of the structural requirements for their activity.

#### **2. Materials and Methods**

#### *2.1. 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−<sup>1</sup> deg cm<sup>2</sup> g<sup>−</sup>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. 1H (400 or 500 MHz) and 13C (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 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 <sup>μ</sup>-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].

## *2.2. Phenolic Nor-Triterpenes*

The natural phenolic nor-triterpenes **1** (6-oxopristimerol) [20,26], **3** (7,8-dihydro-6-oxoiguesterol, canarol) [27], **16** (6-oxotingenol) [20], **17** (3-*O*-methyl-6-oxotingenol) [20], and **20** (6-oxoiguesterol) [20] were isolated from the root bark of *Maytenus canariensis*, and compounds **2** (7-hydroxy-6-oxopristimerol) [21,28], **4** (blepharodol) [21], **5** (7α-hydroxyblepharodol) [27], **10** (blepharotriol) [21], **12** (zeylasteral) [27,29], **13** (demethylzeylasteral) [21,29], **14** (zeylaterone) [27,29], **15** (demethylzeylasterone) [21,29], **22** (7- oxoblepharodol) [21], and **25** (6-deoxoblepharodol) [21] from *Maytenus blepharodes*. Derivatives **11** (2,3*O*-dimethoxyblepharodol) [21], **18** (2,3- *O*-dimethyl-6-oxotingenol) [20], **19** (2,3- *O*-diacetoxy-6-oxoting enol) [20], **21** (2,3- *O*-dimethyl-6-oxoiguesterol) [20], **23** (2- *O*-methoxy-7-oxoblepharodol) [21], **24** (pristimerol) [21,30], and **26** (8-epi-6-deoxoblepharodol) [21] were obtained following the methodology previously described [20,21]. The structures of these compounds are depicted in Figure 2. Moreover, the semisynthesis of derivate **6**, not previously described, was achieved by acetylation of pristimerin, a main quinone–methide triterpene isolated from the root bark of *Maytenus* species [27]. In addition, derivatives **7**–**9** were prepared by catalytic reduction and further acetylation of pristimerin. Derivative **7** is a known synthetic compound [31] whose 1H and 13C NMR data have not been previously assigned, whereas derivatives **8** and **9** are reported herein for the first time (Scheme 1, Figures S1–S4.).

#### 2.2.1. 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 a fford compound **6** (14.0 mg, 37.0%).

6α-Hydroxy-2,3-diacetylpristimerol (**6**). Pale yellow amorphous solid; [ α]<sup>20</sup> D − 39.7 (*c* 0.15, MeOH); UV (EtOH) λmax (log ε) 340 (7.3), 204 (7.4) nm; IR νmax (film) 3517, 2948, 2872, 1770, 1730, 1459, 1373, 1215, 1106, 758 cm<sup>−</sup>1; 1H NMR (500 MHz, CDCl3) δ 0.59 (3H, s, Me-27), 1. 09 (3H, s, Me-28), 1.16 (3H, s, Me-30), 1.24 (3H, s, Me-26), 1.35 (3H, s, Me-25), 2.29 (3H, s, OAc-3), 2.32 (3H, s, Me-23), 2.33 (3H, s, OAc-2), 3.54 (3H, s, OMe-29), 5.06 (1H, dd, *J* = 3.0, 10.8 Hz, H-6), 5.76 (1H, d, *J* = 3.0 Hz, H-7), 7.03 (1H, s, H-1). 13C NMR (125 MHz, CDCl3) δ 12.3 (q, C-23), 18.6 (q, C-27), 20.7 (q, C-26), 21.0 (q, OAc-2), 21.6 (q, OAc-3), 29.1 (t, C-15), 30.2 (t, C-21), 30.4 (t, C-12), 30.9 (t, C-19), 31.2 (s, C-17), 31.9 (q, C-28), 33.1 (q, C-30), 34.5 (t, C-22), 35.1 (t, C-11), 36.9 (t, C-16), 37.4 (q, C-25), 38.3 (s, C-9), 38.6 (s, C-13), 40.8 (s, C-20), 44.6 (d, C-18), 44.8 (s, C-14), 51.9 (q, OMe-29), 66.4 (d, C-6), 116.9 (d, C-1), 117.5 (s, C-7), 128.7 (s, C-4), 130.8 (s, C-5), 139.1 (s, C-3), 142.9 (s, C-2), 151.8 (s, C-10), 159.4 (s, C-8), 168.4 (s, OAc-2), 168.7 (s, OAc-3), 179.2 (s, C-29); EI-MS *m*/*z* (%) 566 [M+] (39), 551 (14), 534 (52), 506 (32), 492 (100), 450 (42), 201 (95); EI-HRMS *m*/*z* 556.3032 (calcd. for C34 H46 O7, 556.3048).

#### 2.2.2. 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%, tR = 11.4 min), reported elsewhere [31], and derivatives **8** (76.4 mg, 10.3%, tR = 12.3 min) and **9** (32.1 mg, 4.3%, tR = 12.6 min), not previously reported.

2,3-Diacetylpristimerol (**7**). Pale yellow amorphous solid; [ α]<sup>20</sup> D − 15.5 (*c* 0.15, MeOH); UV (EtOH) λmax (log ε) 278 (7.3), 203 (7.4), 201 (7.3) nm; IR νmax (film) 2927, 2870, 1775, 1729, 1649, 1371, 1214, 1188, 756 cm<sup>−</sup>1; 1H NMR (500 MHz, CDCl3) δ 0.58 (3H, s, Me-27), 1.08 (3H, s, Me-28), 1.17 (3H, s, Me-30), 1.22 (3H, s, Me-26), 1.35 (3H, s, Me-25), 2.06 (3H, s, Me-23), 2.28 (3H, s, OAc-2), 2.31 (3H, s, OAc-3), 3.06 (1H, d br, *J* = 15.7 Hz, H-6β), 3.32 (1H, dd, *J* = 5.1, 15.7 Hz, H-6 α), 3.55 (3H, s, OMe-29), 5.73 (1H, d, *J* = 5.1 Hz, H-7), 7.00 (1H, s, H-1). 13C NMR (100 MHz, CDCl3) δ 12.5 (q, C-23), 18.2 (q, C-27), 20.4 (q, OAc-2), 20.7 (q, OAc-3), 22.7 (q, C-26), 28.1 (t, C-6), 28.9 (t, C-15), 30.2 (t, C-12), 30.2 (t, C-19), 30.5 (t, C-22), 31.6 (q, C-28), 32.8 (q, C-30), 34.3 (q, C-25), 34.4 (t, C-11), 34.8 (s, C-17), 34.8 (t, C-21), 36.8 (t, C-16), 37.2 (s, C-13), 37.5 (s, C-9), 40.4 (s, C-20), 43.7 (s, C-14), 44.4 (d, C-18), 51.5 (q, OMe-29), 116.7 (d, C-1), 116.9 (d, C-7), 127.8 (s, C-4), 131.5 (s, C-5), 138.0 (s, C-3), 140.6 (s, C-2), 147.5 (s, C-10), 149.0 (s, C-8), 168.4 (s, OAc-2), 168.7 (s, OAc-3), 178.9 (s, C-29); EI-MS *m*/*z* (%): 550 [M+] (1), 535 (8), 475 (16), 322 (7), 229 (17), 149 (9), 57 (100); EI-HRMS *m*/*z* 550.3167 (calcd. for C34 H46 O6, 550.3192).

2,3-Diacetyl-6-deoxoblepharodol (**8**). Pale yellow amorphous solid; [ α]<sup>20</sup> D – 29.5 (*c* 0.20, MeOH); UV (EtOH) λ*max* (log ε) 278 (7.3), 203 (7.3), 201 (7.2) nm; IR ν*max* (film) 2937, 1773, 1730, 1462, 1371, 1213, 1040, 756 cm<sup>−</sup>1; 1H NMR (500 MHz, CDCl3) δ 0.78 (3H, s, Me-27), 0.88 (3H, s, Me-26), 1.10 (3H, s, Me-28), 1.60 (6H, s, Me-25, Me-30), 1.98 (3H, s, Me-23), 2.26 (3H, s, OAc-3), 2.30 (3H, s, OAc-2), 2.59 (1H, dd, *J* = 11.8, 15.7 Hz, H-6 α), 2.60 (1H, dd, *J* = 6.1, 15.7 Hz, H-6 α), 3.59 (3H, s, OMe), 6.89 (1H, s, H-1). 13C NMR (100 MHz, CDCl3) δ 12.5 (q, C-23), 15.9 (q, C-26), 17.3 (q, C-27), 18.2 (t, C-7), 20.4 (q, OAc-3), 20.6 (q, OAc-2), 27.3 (q, C-25), 28.6 (t, C-6), 28.9 (t, C-21), 30.0 (t, C-12), 30.1 (s, C-17), 30.2 (t, C-19), 30.5 (t, C-15), 31.8 (q, C-28), 31.9 (q, C-30), 33.8 (t, C-11), 36.2 (t, C-22), 36.5 (t, C-16), 37.2 (s, C-9), 38.9 (s, C-13), 39.4 (s, C-14), 40.5 (s, C-20), 43.5 (d, C-8), 44.5 (d, C-18), 51.5 (q, OMe-29), 116.2 (d, C-1), 129.4 (s, C-4), 132.7 (s, C-5), 138.0 (s, C-3), 139.9 (s, C-2), 149.7 (s, C-10), 168.4 (s, OAc-3), 168.8 (s, OAc-2), 179.1 (s, C-29); EI-MS *m*/*z* (%) 552 [M+] (10), 510 (49), 495 (5), 468 (100), 453 (15), 249 (26), 190 (9); EI-HRMS *m*/*z* 552.3457 (calcd. for C34 H48 O6, 552.3451).

2,3-Diacetyl-8-*epi*-6-deoxoblepharodol (**9**). Pale yellow amorphous solid; [ α]<sup>20</sup> D – 29.5 (*c* 0.20, MeOH); UV (EtOH) λmax (log ε) 277 (9.4), 203 (9.4), 200 (9.4) nm; IR νmax (film) 2956, 1771, 1728, 1643, 1370, 1214, 1184, 1039, 757 cm<sup>−</sup>1; 1H NMR (500 MHz, CDCl3) δ 0.81 (3H, s, Me-27), 0.88 (2H, m, H-22), 1.08 (3H, s, Me-28), 1.16 (3H, s, Me-30), 1.21 (3H, s, Me-26), 1.42 (1H, m, H-8) 1.46 (3H, s, Me-25), 1.49 (1H, m, H-18), 1.58 (1H, m, H-19β), 1.86 (2H, m, H-16), 2.02 (3H, s, Me-23), 2.21 (2H, m, H-22), 2.26 (3H, s, OAc-3), 2.29 (3H, s, OAc-2), 2.35 (1H, d br, H-19 α), 2.72 (2H, m, *J* = 16 Hz, H-6), 3.56 (3H, s, OMe), 6.98 (1H, s, H-1). 13C NMR (100 MHz, CDCl3) δ 12.6 (q, C-23), 18.9 (q, C-27), 20.4 (q, OAc-2), 20.7 (q, OAc-3), 26.5 (t, C-12), 25.8 (q, C-26), 28.6 (t, C-6), 30.0 (t, C-12), 30.1 (t, C-6), 30.2 (t, C-15), 30.5 (t, C-7), 30.6 (t, C-19), 30.7 (s, C-17), 30.8 (t, C-21), 31.3 (q, C-28), 32.8 (q, C-30), 33.3 (t, C-11), 36.8 (q, C-25), 35.7 (t, C-22), 37.1 (t, C-16), 38.3 (s, C-14), 38.7 (s, C-9), 40.4 (s, C-13), 40.6 (s, C-20), 46.7 (d, C-18), 51.5 (q, OMe-29), 56.0 (d, C-8), 1193 (d, C-1), 128.0 (s, C-4), 135.4 (s, C-5), 137.5 (s, C-3), 140.2 (s, C-2), 148.5 (s, C-10), 168.4 (s, OAc-2), 168.8 (s, OAc-3), 180.7 (s, C-29); EI-MS *m*/*z* (%) 552 [M+] (9), 510 (43), 495 (10), 468 (100), 453 (22), 435 (6), 287 (13), 249 (25), 190 (22); EI-HRMS *m*/*z* 552.3457 (calcd. for C34 H48 O6, 552.3451).
