2.4.1. KOL\_18 (TSRR0002\_D-07) *Agelas nakamurai*

Extract KOL\_18 was selected as representative of the group of the four extracts exhibiting an identical bioactivity pattern and similar metabolite composition (Figure 4). Based on the primary activity of the crude extract against *S. aureus*, the corresponding crude extract (1 mg/mL in MeOH) was fractionated in 1 and 2 μL injection volume replicates and rescreened against the same indicator strain. The active fractions were reproduced in both dilutions. The two activity zones, namely fractions 80–81 and 83–84 (Figure S2), could be assigned to partly co-eluting isomeric compounds with an *m*/*z* 422.3283 [M]<sup>+</sup>, corresponding to the molecular formula [C26H40N5] <sup>+</sup>. The compounds showed an UV absorption at 220 and 272 nm. Based on the MS/MS fragmentation pattern, the compounds could be assigned as members of the agelasine A-F family (Figure 5) [9]; since MS/MS fragmentation does not allow for distinction between the different isomeric structures of the diterpene unit. As the name indicates, agelasines are known metabolites of the sponge *Agelas nakamurai*.

The same extract was fractionated against *S. tritici* (injection volume 2 and 5 μL). Both of the replicates showed activity, corresponding to the above-described agelasines. The 5 μL injection volume replicate showed an additional activity zone, namely fraction 69 (Figure S3), which could be assigned to a compound of *m*/*z* 356.2370 [M + H]<sup>+</sup>, corresponding to the molecular formula C18H33N3O2S1. The compound shows UV absorption at 220 nm. Based on the MS/MS fragmentation pattern (Figure S3d), the compound was dereplicated as agelasidine A [10] (Figure 5), also a known metabolite of *A. nakamurai* [11]. The extract was also fractionated against *C. albicans* (2 mg/mL solution, injection volume 2 and 5 μL). Both injection replicates showed activity in the fractions corresponding to the above described agelasines (A-F) and agelasidine A (Figure S4).

Molecular networking analysis revealed the presence of several derivatives (minor compounds) including oxo-agelasines (A-F) of *m*/*z* 436.3073 [M]<sup>+</sup> with a molecular formula of C26H38N5O1, hydroxy-agelasines of *m*/*z* 438.3231 [M]<sup>+</sup> with a molecular formula of C26H40N5O1, and dihydro-hydroxy-agelasines of *m*/*z* 440.3389 [M]<sup>+</sup> with a molecular formula of C26H42N5O1, each present in the extract as a complex mixture of isomers (Figures S5 and S6).

**Figure 5.** Chemical structures of the dereplicated compounds responsible for the activity of the microfractionated samples.
