*2.1. Chemical Profiling, Isolation, and Structure Elucidation*

A HPTLC (Figure S3, Supplementary Materials) co-migration with standards and the UPLC-PDA profile (Figure S4, Supplementary Materials) of the acetone extract of *R. conduplicans* suggested the presence of a dozen of visible compounds, among which salazinic acid, usnic acid, sekikaic acid, homosekikaic acid, and divaricatic acid were identified against standards and appeared to represent the most abundant compounds. Initial LC-QToF-MSE analyses of the acetone extract of *R. conduplicans* indicated the presence of depsides, depsidones, and monophenolic acids based on High-Resolution Mass Spectroscopy (HRMS). Molecular formulae for C10–35H10–50O2–15 were generated from mass ranges *m*/*z* 150–750 coupled with the fragment ions and their MS spectral data (accurate mass and fragmentation pattern) and compared to online databases (DNP, Reaxys, SciFinder).

Mass spectrometry (MS) and, particularly, quadruple time-of-flight coupled to Liquid Chromatography (UPLC-Q-ToF-MS) has been widely utilized for profiling metabolites due to its superiority in high-resolution mass, precision, and sensitivity [18], and was helpful to clearly discriminate between the depsides, depsidones, simple phenol acids, dibenzofurans, and hydroxyl fatty acids based on the fragmentation of lichen molecules [16]. Therefore, the acquired TIC of the *R. conduplicans* extracts, obtained within 16 min, were analyzed from spectra obtained in negative mode and, thus, are effective for characterizing trace components (Figure 1). Metabolite assignments were made based on their polarity related to their retention time (Rt) and molecular formulae from accurate molecular weight measurement, along with adducts [M − H]−/ fragment ions and Ring Double Bond Equivalence (RDBE). In the present study, a total of 18 compounds were clearly characterized from

the crude extract of *R. conduplicans* by molecular formulae generated by ToF-MS/MS and MS/MS including their fragmentation profiles, as reported in the literature and presented in Table S1 (Supplementary Materials).

**Figure 1.** TIC of (**A**) *R. conduplicans* acetone extract and (**B**) enriched fraction-4.

Based on the fragmentation of isolates, we have identified compounds (**1**–**5**) and (**7**–**9**) along with atranorin belonging to depsides. The literature clearly indicates that sekikaic acid is an abundant molecule in *Ramalina* species [19]. Sekikaic acid (**1**) is a *m*-depside corresponding to the esterification of two divaricatinic acid units and is found with Rt at 11.98 min and *m*/*z* 417.1547 (C22H25O8) with fragments *m*/*z* 209 and *m*/*z* 225 corresponding to the A ring and B ring, respectively [20]. Compounds **1**, **3**, **5**, **7**, **8,** and **9**, having a common fragment *m*/*z* 209 (Figures S47 and S48, Supplementary Materials), clearly indicate the difference in locating the other Bring. These depsides can be considered as ester derivatives of divaricatinic acid (**11**) while compound **2** is a divaric acid derivative (recognized at Rt 7.50 min, *m*/*z* 195.0657). The other identified monoaromatic compounds correspond to 2,4-di-*O*-methyldivaric acid (**6**), 4-*O*-methyldivaricatic acid (**10**), divaricatinic acid (**11**), olivetolic acid (**12**), divarinolmonomethylether (**13**), and atranol (**14**). In this run, three additional compounds were ionized and fragmented (Rt = 8.53 min, Rt = 11.88 min, and Rt = 13.17 min) and not determined. The fragmentation feature of Compound **5** (*m*/*z* 401.1954 [M − H]<sup>−</sup> (calcd. for [C23H28O6] − 401,1964)) suggested the coupling of a divaricatinic acid moiety to an olivetol monomethylether moiety (Figure S17, Supplementary Materials). Based on these fragmentation studies, we assigned compounds as shown in the Supporting Information section and in Table S1, including the monoaromatic divaric acid, along with the common and already-described atranorin (depside), usnic acid (related to

dibenzofurans), and salazinic acid (a depsidone). The structures were concluded through MS/MS fragmentation patterns and compared with in-house standards.

Subsequently, the acetone extract was subjected to column chromatography to give eight fractions (I to VIII). An LC–MSE analysis of all fractions revealed the presence of depsides in III–VI fractions (Figures S4–S6, Supplementary Materials). Thus, the targeted isolation and purification of III–VI fractions yielded the isolation of one new depside (**5**), along with other known depsides (**1**–**4** and**7**–**9**) and monoaromatic compounds (**6** and **10**–**14**). The spectra and fragmentation patterns of these molecules were shown in the Supporting Information section (Figures S7–S46, Supplementary Materials).

The structures of the isolated compounds (Figure 2) were determined by a combination of spectroscopic data (HRESIMS, 1H and 13C NMR) and in comparison with the reported literature data. They were identified as sekikaic acid (**1**) [21], 4 -*O*-methylnorhomosekikaic acid (**2**) [22], homosekikaic acid (**3**) [22], hyperhomosekikaic acid (**4**) [23], 2,4-dimethyldivaric acid (**6**) [24], divaricatic acid (**7**) [25], decarboxydivaricatic acid (**8**) [26], decarboxystenosporic acid (**9**) [26], methyldivaricatinate (**10**) [24], divaricatinic acid (**11**) [21], olivetolic acid (**12**) [27], divarinolmonomethylether (**13**) [21], and atranol (**14**) [28].

**Figure 2.** Isolated compounds (**1**–**14**) from *Ramalina conduplicans* Vain.

Compound **5** was isolated as white amorphous powder and identified as a new compound. Its molecular formula was established as C23H29O6 based on a HRESIMS ion at *m*/*z* 401.1954 [M − H]<sup>−</sup> (calcd. for [C23H28O6] <sup>−</sup>, 401.1964). The 1H and 13C NMR data of **5** (Table 1) showed the presence of four aromatic protons, (δ<sup>H</sup> 6.53 (d, *J* = 1.8 Hz, 1H), 6.51 (d, *J* = 1.8 Hz, 1H), 6.46 (d, *J* = 2.6 Hz), and 6.40 (d, *J* = 2.6 Hz); δ<sup>C</sup> 120.0, 110.7, 105.4, and 100.8) and one ester carbonyl (δ<sup>C</sup> 170.3). In addition, two methoxyl groups (δ<sup>H</sup> 3.86 (3H, s), 3.81 (3H, s)) and two n-alkane side chains of two methylene groups that were adjacent to a benzene ring (δ<sup>H</sup> 3.02–2.93 (m, 2H), 2.62–2.51 (m, 2H)) were also distinguished from the NMR spectra, respectively (Table S2, Supplementary Materials). These spectral features, together with the characteristic ester carbonyl group at C-7 (δ<sup>C</sup> 170.3) in the 13C NMR spectrum, strongly imply that **5** is a depside-type derivative [16,29].


**Table 1.** NMR data of compound **5** (400 & 100 MHz, acetone-*d6*) \*.

\* = values are assigned with the comparison of sekikaic acid data and COSY/NOESY correlations.

A comparison of 1H NMR and 13C NMR data from**5** with those of 4 -*O*-methylnorhomosekikaic acid, which were isolated from the same species, indicated an overall similarity, except for the absence of a COOH group and the presence of two additional methylenes. This reasoning was further supported by its 13C NMR spectrum, which showed the absence of a carbonyl COOH group, and its 1H NMR spectrum indicated the presence of an additional aromatic proton at 6.53 (d, *J* = 1.8 Hz, 1H). A comprehensive analysis of 2D NMR (COSY, and HSQC) data, especially the 1H–1H COSY spectrum, revealed two discrete spin systems, including -CH-CH2-CH3- (from H-1, H-2 and H-3) and -CH-CH2-CH2- CH2-CH3 (from H-1 to 5), as drawn with bold lines in Figure 3. The position of the

*n*-pentyl group at C-6 and *n*-propyl chain at C-6 was confirmed on the basis of the NOESY correlations (H-1/H-5 , H-1/H-1 and H-1/H-5) (Figure 3) and in comparison with the sekikaic acid data. In addition, the MS/MS spectrum of **5** showed (Figure 4) product ions *m*/*z* 209, thereby indicating the breakage of the C–O bond between two aromatic rings supported by the fragments at *m*/*z* 165 and 137. Based on these spectral characteristics, the structure of **5** was established and trivially named as decarboxyhomosekikaic acid.

**Figure 3.** Key COSY and NOESY correlations of compound **5**.

**Figure 4.** MS/MS spectrum and proposed fragmentation of compound **5**.
