*2.2. Inhibition Activity on Human Colon Cancer Cells In Vitro*

The inhibitory effect of the organic extract and fractions of cf. *Neolyngbya* sp. HAINAN-19SEP17-3 were evaluated using HCT116 human colorectal cancer cells (Figure S23A). This allowed for the targeted discovery of new bioactive natural products akin to a published method [43]. Cells were treated for 24 h and analyzed using an XTT cell viability assay to detect fast-acting fractions and compound constituents [44]. It is understood that extended duration exposure (e.g., to 48 or 72 h) will typically increase the observed efficacy or potency of cytotoxicity due to the relatively prolonged accumulation of dead cells. While the crude extract was not cytotoxic at the concentrations tested (200 and 400 μg/mL) in this 24 h experiment, fraction C demonstrated high potency (94–97% mortality) in treated cells versus untreated at both concentrations tested (Figure S23A). After further separation into 6 sub-fractions, a more marked concentration-dependent activity was observed for C3 (Figure S23B). Additional chromatography yielded sub-fractions that were also shown to act concentration-dependently, i.e., C3–5 and C3–7 (Figure S24). While the active fraction C3–7 was observed to be an impure mixture of compounds, fraction C3–5 was found to be a pure molecule (**1**) that was active in this in vitro test model (24 h IC50 = 38 μM), and

noticeably active even after only 8 h of treatment (Figure S25). This sample was thus evaluated further.

To clarify the cell viability decrease following 24 h treatment with C3 and **1** (30 μg/mL), cell cycle distribution analysis was examined. A FACS analysis demonstrated that treatment with C3 and **1** resulted in the accumulation of cells in the sub-G1 phase of the cell cycle at 3.9% and 12.4%, respectively, compared to 2.2% in the untreated (control) cells (Figure 4A). Furthermore, the cells were observed to be accumulating at the G2/M phase following treatment with **1** (34.7% vs. 26.2% in the control), indicating suppression of cell proliferation. Normal, non-cancerous colon cell lines are unavailable. However, the same pattern of cell cycle arrest was not observed when the samples were tested in normal human dermal fibroblasts (NHDF; Figure 4B).

**Figure 4.** In vitro effects of fraction C3 and compound **1** on cell cycle progression after 24 h treatment. Distribution of (**A**) HCT116 human colon cancer cells and (**B**) NHDF normal human dermal fibroblasts at the different cell cycle phases as determined by FACS.

The cell cycle arrest at the G2/M phase accompanied by an accumulation in the sub-G1 phase observed due to treatment with C3 or **1** is suggestive of apoptotic cell death, since this has been reported previously for human colon cancer cells [45]. In order to confirm this hypothesis, HCT116 cells were treated with 30 μg/mL C3 or **1** for 24 h, stained

with FITC labeled Annexin-V and PI, and analyzed by flow cytometry (Figure 5A). The results indicated an increase of approximately 4.4% in apoptotic cells (Q2 + Q4) following treatment with C3, and about 11.3% after exposure to **1**. Annexin/PI double staining analysis of NHDF cells in vitro showed a similar increase in accumulation of apoptotic cells after treatment with fraction C3, of about 4.8%, but a much smaller increase following treatment with compound **1**, of about 1.3%, in comparison to untreated cells (Figure 5B).

**Figure 5.** Annexin-V/PI double staining and flow cytometry evaluation of mechanistic in vitro cytotoxicity of fraction C3 and compound **1** after 24 h treatment of (**A**) HCT116 human colon cancer cells and (**B**) NHDF normal human dermal fibroblasts. For each plot, the lower left quadrant (Q3) represents viable cells, the upper left quadrant (Q1) indicates necrotic cells, the lower right quadrant (Q4) denotes early apoptotic cells, and the upper right quadrant (Q2) represents necrotic or late apoptotic cells.

#### *2.3. Natural Product Structure Elucidation*

Compound **1** was obtained as a white powder and assigned the molecular formula C64H106N8O14 based on a sodium adduct peak in the HRESIMS spectrum at *m*/*z* 1233.7748 [M + Na]+ (calcd. for C64H106N8O14Na+, 1233.7721). This formula indicated that **1** pos-

sessed 16 degrees of unsaturation. The 1H and 13C NMR data of **1** (Table 1) were suggestive of a lipopeptide scaffold with seven sets of signals characteristic of amino acid α protons, as well as two aromatic rings, two oxygenated methylenes, three oxygenated methines, one methoxy and three *N*-methyl groups, along with many alkyl moieties and eight amide carbonyls. The region measured from *δ*<sup>H</sup> 3.8 to 4.9 ppm had sufficient peak resolution to nucleate seven amino acid and derivative substructures that were able to be constructed using 1D and 2D NMR data. For example, an "α proton" signal at *δ*<sup>H</sup> 3.91 (H-2) was connected to a carbon at *δ*<sup>C</sup> 52.4 (C-2) with the evidence of a peak in the 1H-13C HSQC spectrum. After examination of the 1H-1H COSY spectrum and HSQC data, this methine was determined to be adjacent to an oxygenated methylene group, CH2-1 (*δ*<sup>C</sup> 62.7, *δ*<sup>H</sup> 3.35), and a benzylic methylene group, CH2-3 (*δ*<sup>C</sup> 35.6, *δ*<sup>H</sup> 2.77 and 2.53). The assignment of the aromatic ring connected to C-3 was completed by further inclusion of long-range coupling data obtained from the 1H-13C HMBC spectrum. As shown in Figure 6, this *para*-methoxysubstituted phenyl group was characterized by correlations observed between H2-3 and C-5/9, H-5/9 and C-7, H3-7-*O*-Me and C-7, as well as H-6/8 and C-4. The planar structure of this subunit was thus established as 2-amino-3-(4-methoxyphenyl)propan-1-ol; "Amp".

**Figure 6.** Selected correlations used to determine the planar structure of wenchangamide A (**1**). Red single-sided arrows represent cross peaks from the 1H-13C HMBC spectrum. Black bolded bonds show protons correlated in the 1H-1H COSY and TOCSY spectra.

> Much of the remaining NMR data for **1** could be further assigned to a series of standard or *N*-methyl amino acid residues that were determined by similar methods as for the Amp group, including two Ile residues, an *N*-Me-Gln, *N*-Me-Phe, *N*-Me-Ile, and Ser. Several of the aliphatic groups had partially overlapping signals in the 1H NMR spectrum, e.g., H2-12 (*δ*<sup>H</sup> 1.97 and 1.61) and H-42 (*δ*<sup>H</sup> 1.61), as well as H2-13 (*δ*<sup>H</sup> 1.90 and 1.83) and H-35 (*δ*<sup>H</sup> 1.9), which complicated their assignment using NMR data from the COSY or even 1H-1H TOCSY spectra. However, these groups were differentiated and assigned conclusively by the resolution of their corresponding signals in the HSQC and HSQC-TOCSY spectra, e.g., C-12 (*δ*<sup>C</sup> 23.8) and C-42 (*δ*<sup>C</sup> 24.3), as well as C-13 (*δ*<sup>C</sup> 31.3) and C-35 (*δ*<sup>C</sup> 32.6). Since the signals from TOCSY and HSQC-TOCSY result from extended or even complete 1H-1H spin system couplings, the signals observed from the well-resolved region (*δ*<sup>H</sup> 3.8 to 4.9 ppm) in the f2 dimension were sufficient to support the assignment of the structural subunits described above. Each of the three *N*-methyl groups was able to be assigned to a defined amino acid residue based on correlations observed in the HMBC spectrum, i.e., from H3-11-*N*-Me (*δ*<sup>H</sup> 2.42) to C-11 (*δ*<sup>C</sup> 56.0), H3-16-*N*-Me (*δ*<sup>H</sup> 2.89) to C-16 (*δ*<sup>C</sup> 54.0), and H3-34 *N*-Me (*δ*<sup>H</sup> 2.94) to C-34 (*δ*<sup>C</sup> 59.9). Amide NH protons were similarly able to be assigned by correlations observed in the COSY and TOCSY spectra, i.e., from 2-NH (*δ*<sup>H</sup> 7.27) to H-2, 25-NH (*δ*<sup>H</sup> 8.04) to H-25 (*δ*<sup>H</sup> 4.49), 31-NH (*δ*<sup>H</sup> 7.53) to H-31 (*δ*<sup>H</sup> 4.23), and 40 NH (*δ*<sup>H</sup> 8.15) to H-40 (*δ*<sup>H</sup> 4.72). As further shown in Figure 6, the sequence of amide or "peptide" bonds was able to be deduced from the HMBC correlations observed between *N*-Me, NH, and "α proton" signals to the carbonyl of the adjacent residue. The sequence order of these structural subunits was further supported by characteristic amide bond "y" fragmentation masses that were detected in the MS/MS spectrum of **1** (Figure 7).

**Figure 7.** Selected MS/MS fragmentation ions observed that supported the amino acid and derivative residue sequence in the planar structure of wenchangamide A (**1**).

All of the NMR data that remained unassigned was proposed to result from a polyhydroxylated fatty acid moiety (FA), since this corresponded to three oxygenated methines, six downfield methylenes, and three alkyl methyl groups and one carbonyl. Due to diagnostic HMBC correlations from both H-40 and 40-NH to the remaining unassigned carbonyl (*δ*<sup>C</sup> 171.0; C-45), the attachment point for this structural subunit was able to be assigned to the nitrogen of the Leu-2 residue. Further HMBC correlations to C-45 were observed from a deshielded methylene (*δ*<sup>H</sup> 2.22 and 2.13, *δ*<sup>C</sup> 43.8; CH2-46) and a more deshielded, oxygenated, methine (*δ*<sup>H</sup> 3.87, *δ*<sup>C</sup> 65.7; CH-47). This allowed for the generation of a growing alkyl carbon chain that was able to be extended by COSY correlations, i.e., from CH-47 to CH2-48 (*δ*<sup>H</sup> 1.23 and 0.92) and then CH-49 (*δ*<sup>H</sup> 1.76), as well as signals in the HMBC spectrum, including from H2-46 to C-48 (*δ*<sup>C</sup> 45.2) and H-47 to C-49 (*δ*<sup>C</sup> 25.6). CH-49 was connected to and had a COSY correlation with a methyl group (*δ*<sup>H</sup> 0.82, *δ*<sup>C</sup> 20.3; CH3-50). This PKS-like subunit, –CH2–(CH–OH)–CH2–(CH–CH3)–, was found to repeat two more times in the linear alkyl chain of the FA moiety, and terminated the molecule with an alkyl methyl group (*δ*<sup>H</sup> 0.85, *δ*<sup>C</sup> 14.2; CH3-60) adjacent to a penultimate methylene unit (*δ*<sup>H</sup> 1.35 and 1.26, *δ*<sup>C</sup> 18.6; CH2-59). In sum, this yielded the planar structure of **1** as shown in Figure 6. Compound **1** is a new natural product, here assigned the trivial name wenchangamide A due to the location of the geographical collection site that yielded this discovery.

The structure of **1** has many features that resemble minnamide A, a cyanobacterial natural product recently reported from a sample of *Okeania hirsuta* that was collected in Minna island, Okinawa Prefecture, Japan [46]. However, noteworthy differences in the structures (Figure 8) include a different length polypeptide core scaffold, where minnamide A has an *N*-Me-Val–Ser–*N*-Me-Val moiety instead of the *N*-Me-Phe group present in **1**, as well as a longer fatty acid tail that contains an additional PKS-like repeating unit described above (repeats 3x in **1** and 4x in minnamide A). Accordingly, the molecular weight of minnamide A is 238 Da higher than that of **1**, and these molecules have significantly different MS/MS spectra due to the multiple structural differences. However, the hydrolysis of an aliquot of **1**, and subsequent analysis by chiral HPLC along with standard compounds, supported the assignment of the same configuration for all shared amino acid residues and derivatives from minnamide A, specifically (*S*)-Amp, *N*-Me-L-Gln, D-Leu-1, D-Ser, *N*-Me-D-*allo*-Ile, and L-Leu-2. Comparison of the NMR data obtained for **1** in pyridine-*d*5 (see Supplementary Table S1) with published values for minnamide further supported these assignments [46]. The *N*-Me-Phe residue (present in **1** and absent in minnamide A) was determined to be that of the L form by the same protocol. It is hypothesized that the configuration of the repeating PKS-like subunits of the fatty acid chain in **1** match with those reported for minnamide A; however, this has not been established empirically in the present study. In total, this information was used to assign the absolute configuration of the peptide core scaffold of **1** as presented in Figures 1 and 8.


**Table 1.** 1H and 13C NMR Spectroscopic Data for **1** in DMSO-*d*<sup>6</sup> *a,b*.

*<sup>a</sup>* Data recorded at 298 K, 600 MHz (1H) and 150 MHz (13C). *<sup>b</sup>* Assignments supported by 2D NMR. *<sup>c</sup>* Signal partially overlapped.

**Figure 8.** Structural comparison of wenchangamide A (**1**) and minnamide. Shared structural motifs are drawn in black. Differences in **1** are highlighted in red. Differences in minnamide are highlighted in blue. Configurations from the shared part of the FA residue in **1** are hypothesized to match those of minnamide.
