## 3 Results and Discussion
### Structure-Activity Relationship Analysis
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 \(\beta\)-stereochemistry at C-8 increases two-fold the activity with respect to the H-8\(\alpha\), 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**). 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 \(\beta\)-stereochemistry at the C-8 position favored the activity with respect to the H-8\(\alpha\) (**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-6\(\alpha\)-hydroxy-pristimerol, that would have a zeyslasterone 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). Figure 4: Most relevant structural features for phenolic nor-triterpenes against the most sensitive microorganism, _Bacillus subtilis_. 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, \(\text{clog}\ P\) values [34] of this series of compounds were calculated, including that of the hypothetical compound, 4-carboxy-6\(\alpha\)-hydroxy-pristimerol (\(\text{clog}\ P\) 4.88), and are shown in Table 1. Slight increases in lipophilicity of **24** (\(\text{clog}\ P\) 7.09) and **25** and **26** (\(\text{clog}\ P\) 6.41) by acetylation (7, **8** and **9**, respectively) (\(\text{clog}\ P\) 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 \(\text{clog}\ P\) 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.