**2. Results and Discussion**

#### *2.1. Botanical Identity of Machaerium sp. (Rimachi 12161)*

The regional floras and other relevant publications were consulted for possible botanical identification of the species [7,10]. The voucher specimen (Rimachi 12161) was confirmed to belong to the genus *Machaerium*, based on the morphological features and available field information such as habitat, leaf, inflorescence, and fruit characters. The leaves showed partial similarities with those of *M. leiophyllum* var. *leiophyllum* and *M. glabrum*. However, complete identification of the specimen was not possible due to a lack of information on necessary diagnostic features, such as presence or absence of spines, features of stipules, and floral characters. It is possible that the specimen could represent an un-described taxon. The description (vide infra) is based on a single herbarium specimen and the associated collection information available from the original collection.

The *Machaerium* sp. (12161) plant was found to grow on sandy soils in open forests in Maynas, near Loreto, Peru, at an altitude of about 140–160 m. It is a woody liana with cylindrical stems, imparipinnate leaves with 17–21 leaflets, green flowers in axillary panicles, and 1-seeded samaroid fruits with a terminal wing showing reticulate venations.

#### *2.2. Phytochemical Constituents*

The dried EtOH extract of the stem bark was fractionated with *n*-hexane, followed by dichloromethane (DCM), and resulted in the isolation of compounds **1**–**5** (Figure 1 and Figure S1) (see the Experimental Section).

**Figure 1.** Structures of the isolated compounds.

Chromatographic separation of the DCM fraction 20–24 that led to the isolation of compound **1**, showed a single peak upon LC–MS analysis, which showed a protonated molecular ion peak at m/z 379.1906 [M + H]<sup>+</sup> in its ESI–HRMS, suggesting the molecular formula C28H29O3. A careful analysis of the 1H and 13C NMR spectra (Table 1), and 2D NMR COSY, HMQC, HMBC, and NOESY spectra (Figures S2–S11) suggested that the compound was a mixture of C-5 epimers **1a** and **1b** (Figure 2).


**Table 1.** 1H and 13C NMR (in CDCl3) data for epimeric compounds **1a** and **1b**.

a 1H Spectra recorded at 400 MHz, and b 13C spectra recorded at 100 MHz.

**Figure 2.** Structure of the epimeric compound (**1a**) (major) and (**1b**) (minor) in mixture.

Moreover, the NMR data were found to be partially comparable with the machaeridiol C (**11**) [6], suggesting the presence of a benzofuran side chain (δ<sup>H</sup> 7.18–7.52) (4H) attached to the substituted resorcinol moiety. The HMBC spectrum (**1a**; Figure 2) showed <sup>2</sup>*J*- and <sup>3</sup>*J* correlations between the H-8at δ<sup>H</sup> 6.89 (brs) and the two sp2-hybridized carbons at C-4 and C-7- (δ<sup>C</sup> 130.6 and 154.9, respectively), supporting the attachment of C-4 of the resorcinol unit to the C-7 of benzofuran ring. The 1H NMR spectrum also showed signals at δ<sup>H</sup> 6.83 (2H) for two identical protons (H-3 and H-5- ), suggesting the presence of C-1- ,2- ,4- ,6- -tetra substituted resorcinol ring with two oxygenated carbons at C-2 and C-6- (δ<sup>C</sup> 155.3 and 155.4). In addition, the 1H NMR spectrum (**1a**) showed signals at δ<sup>H</sup> 4.0 (1H, m, H-5) and 5.32 (1H, dd, J = 1.2, 7.6 Hz, H-3) for the tetrahydrofuran ring, and at δ<sup>H</sup> 1.02 (3H, d, J = 7.2 Hz, H-11) for a Me-group. The HMBC (Figure 3) spectrum showed correlations between the H-3 (δ<sup>H</sup> 5.32) and the three sp<sup>2</sup> hybridized carbons at C-1- , C-2- , and C-6- (δ<sup>C</sup> 113.3, 155.3, and 155.4, respectively), supporting the attachment of the tetrahydrofuran ring at C-3 (δ<sup>C</sup> 77.7) to C-1 position. The HMBC spectrum also showed cross peaks between the methyl protons H-11 (δ<sup>H</sup> 1.02) and C-1, C-2, and C-5 (δ<sup>C</sup> 35.2, 41.8, and 83.1), supporting the attachment of the methyl (C-11, δ<sup>C</sup> 15.5) to the tetrahydrofuran at C-1 (δ<sup>C</sup> 35.2). The 1H, 13C, and 2D NMR spectra supported the presence of the 2-methylbut-2-ene unit. This was confirmed by the HMBC spectrum, which showed correlations between the H-6 at (δ<sup>H</sup> 2.4) and the three carbons at C-5, C-7, and C-8 (δ<sup>C</sup> 83.1, 120.0, and 134.5, respectively), confirming the attachment of the methylbut-2-ene unit to the tetrahydrofuran moiety at C-5. The relative configurations at C-1, C-3, and C-5 of the tetrahydrofuran were assigned via careful analysis of NOESY correlations for **1a**. In the NOESY spectrum (assigned for **1a**), H-5 (δ<sup>H</sup> 4.0) showed correlation with H-1 (δ<sup>H</sup> 2.4) and H-3 (δ<sup>H</sup> 5.32), indicating the cofacial (β)-orientation of the three groups. Additionally, NOESY showed cross peaks between H-1 (δ<sup>H</sup> 2.4.) and H-3 (δ<sup>H</sup> 5.32), which supported their presence in the same plane of the molecule like H-5. On the other hand, such nOe signals were not evident in the NOESY of the minor compound **1b**. Moreover, in its 1H NMR spectrum, H-5 and H-3 were deshielded at δ<sup>H</sup> 4.21 (1H, m) and 5.59 (1H, dd, J = 12.0, 8.0 Hz), respectively, for the tetrahydrofuran ring, and at δ<sup>H</sup> 1.02 (3H, d, J = 7.2 Hz, H-11) for an Me-group. Moreover, a CD analysis for this compound revealed a weak spectrum in the range of 250–500 nm, which is reflective of the epimeric nature of the compound. Based on the foregoing discussion and comparing the NMR data with compound **11** [6], the structure **1a** and **1b** were determined for machaerifurogerol and 5-*epi*-machaerifurogerol, respectively.

**Figure 3.** Key 2D NMR COSY, HMBC, and NOESY correlations of compound **1a.**

During the course of isolation, four isoflavonoid derivatives (**2**–**5**) and previously reported machaeriols (**6–8**) and machaeridiols (**10**–**12**) were isolated from the DCM partition of the stem bark and leaves extracts. However, the minor compound **9** could not be isolated due to a paucity of material. Compounds **2** and **3** were identified as known isoflavons (+)-vestitol and 7-*O*-methylvestitol, while **4** and **5** were identified as known peterocarpans. (+)-medicarpin and 3,8-dihydroxy-9-methoxy-pterocarpan, respectively, previously reported from *Machaerium vestitum* and Cuban propolis [10,11]. Compounds **4** and **5** were also reported from Cuban propolis [11]. The 1H and 13C NMR spectroscopic data (see Table S1) of compounds **2**–**5** were in agreement with those reported [11,12]. In addition, examination of the leaves of *Machaerium* sp. also yielded compounds **6**–**8** and **10**–**12**, as well as their presence in the root extract. The identities of compounds **6**–**12** were established by NMR spectra and by direct comparison with authentic samples (TLC, HPLC/ LC–MS).

#### *2.3. Antimicrobial Activity against Gram-Positive Species and Fungi*

The availability of machaeriols A-C (**6**–**8**) and machaeridiols (**10**–**12**) [5,6] offered the opportunity to carry out a comprehensive investigation of antimicrobial activity. Among the tested fractions, DCM-25-32 (enriched with compounds **8** and **10**–**12**) and DCM-10-15 (enriched with compound **12**) were the most active against bacteria *S. aureus*, and methicillin-resistant *S. aureus* (MRSA), and the fungi *Candida glabrata*, *C. krusei*, and *Cryptococcus neoformans*, with IC50 values of <0.8, <0.8, <0.8, 5.35, <0.8 μg/mL, and <0.8, 1.95, 3.0, 6.07, 12.58 μg/mL, respectively (Table 2). Antibacterial activities of **6**–**8, 10**–**12**, and a mixture **10** + **11** (1:1) were evaluated against methicillin-resistant *S. aureus* (ATCC 1708, 1696, and 1717), the ex vivo MRSA XEN31 strain, and vancomycin-resistant *Enterococci* (VRE; *Enterococcus faecium* ATCC 700221), low-level VRE (*E. faecalis* ATCC 51299), and the vancomycin-sensitive strain (VSA; *E. faecalis* ATCC 29212) (Tables 3 and 4). Compound **11** and mixture **10** + **11** (1:1) showed the most potent activity against MRSA BAA 1696, BAA 1708, BAA 1717, and BBA 33591 with IC50/MIC/MBC values of 0.43/1.25/5 μg/mL, 0.38/1.25/1.25 μg/mL, 0.38/1.25/2.5 μg/mL, 0.71/1.25/1.25 μg/mL; and 0.41/1.25/10 μg/mL, 0.34/1.25/1.25 μg/mL, 0.39/1.25/1.25 μg/mL and 0.61/1.25/10 μg/mL, respectively. On the other hand, compound **8** and mixture **10** + **11** were found to be the most potent against *E. faecium* ATCC 700221 and *E. faecalis* ATCC 51299, (VRE) and *E. faecalis* ATCC 29212 (VSE) with IC50/MIC/MBC of 0.48/1.25/2.5 μg/mL, 1.02/1.25/5 μg/mL and 1.16/2.5/2.5 μg/mL; and 0.49/1.25/2.5 μg/mL, 0.70/1.25/5 μg/mL, and 0.72/1.25/5 μg/mL, respectively (Tables 3 and 4). The activities of compounds **6**–**8** and **10**–**12** were found to be more potent than ciprofloxacin and vancomycin against the MRSA BBA 1708 strain, while **6**, **8**, and **10** + **11** were more active against VRE 700221 than the positive controls.


**Table 2.** Antimicrobial activity (IC50 in μg/mL) of *Machaerium* DCM fractions a.

<sup>a</sup> IC50 is the concentration causing 50% growth inhibition; MIC (minimum inhibitory concentration) is the lowest concentration that allows no growth; MFC (minimum fungicidal concentration) or MBC (minimum bactericidal concentration) is the lowest concentration at kills the test organism; <sup>b</sup> Vancomycin sensitive, <sup>c</sup> Low-level vancomycin resistant. <sup>d</sup> Vancomycin resistant strain; -: not active at the highest test concentration of 20 μg/mL.


**Table 3.** Anti-MRSA activities (in μg/mL) of compounds **6**–**8** and **10**–**12**.

NT not tested.

**Table 4.** Anti-VRE activities (in μg/mL) of compounds **6**–**8** and **10**–**12**.


<sup>a</sup> vancomycin sensitive; <sup>b</sup> low-level vancomycin resistant; <sup>c</sup> vancomycin resistant.

## *2.4. Antimicrobial Combination Studies*

In light of the strong antimicrobial activity of the DCM fraction 25–32, which is enriched with compounds **8**, **10**–**12** (Table 3), a combination study using an in vitro Checkerboard method [13,14] was carried out for machaeriol (**7** or **8**) and machaeridiol (**11** or **12**), to evaluate the synergy of combination treatment against the strains of MRSA and *Enterococcus* (VRE) (Table 4). Among these compounds, a combination of machaeridiol B (**12**; at MIC 5 μg/mL) and machaeriol C (**8**; at <sup>1</sup> <sup>2</sup> MIC 1.25 μg/mL) exhibited a potent activity, with the MIC values of 0.156 and 0.625 μg/mL exhibiting a >32- and >8-fold reduction of MICs, compared to those observed for **12**, against MRSA 1708 and MRSA 1717 strains. When these two compounds were tested with an inverse concentration (i.e., MIC of **8**; 2.5 μg/mL + <sup>1</sup> 2 MIC of **12**; 2.5 μg/mL), a strong synergism was also observed, but to a lesser extent. When tested against VRE (*E. faecium* 700221), this combination showed synergism with the MIC values of 1.25 μg/mL, a >4-fold reduction of MIC compared to **12** (Table 5).

Isobologram showing synergistic activity of the combination of compounds **8** and **12** in MRSA 1708 (red) and 1717 (blue) are presented in Figure 4. The green series represents the additivity line of compounds **8** and **12** (green dots represent the MIC of each compound alone; the green line represents all possible additive combinations). The red (MRSA 1708) and blue (1717) dots represent the combination of compounds **8** and **12**, and show that they fell below the additivity line (the combination of the compounds produces a synergistic effect beyond additivity). This synergism between machaeriol (HHDBP) and machaeridiol (seco-HHDBP) could be due to different molecular targets affected by these two molecules. A combination study of compounds **8** and **12** with antibiotics, either methicillin or ciprofloxacin, did not show any additive or synergistic effects.


**Table 5.** Combination study (MIC in μg/mL) <sup>a</sup> of compounds **7**, **8, 11**, and **12** by Checkerboard assay against MRSA and VRE.

<sup>a</sup> In general, when the MIC of each compound decreased ≥4X in the presence of the other, it is considered synergistic; reduction of MIC in parentheses; <sup>b</sup> Vancomycin-sensitive *Enterococcus faecalis*; <sup>c</sup> Low-level vancomycin-resistant *Enterococcus faecium*; <sup>d</sup> Vancomycin-resistant *Enterococcus faecium*; *<sup>e</sup>* Diacetylphloroglucinol; NT: Not tested.

**Figure 4.** Isobologram of combination of compounds **8** and **12** in MRSA 1708 (red) and 1717 (blue). The green series represents the additivity line of compounds **8** and **12** green dots represent the MIC of each compound alone; the green line represents all possible additive combinations). The red (MRSA 1708) and blue (1717) dots represent the combination of compounds **8** and **12**, and show that they fell below the additivity line (the combination of compounds produces a synergistic effect beyond additivity).
