Azaphilones from the Red Sea Fungus Aspergillus falconensis

Abstract

The marine-derived fungus Aspergillus falconensis, isolated from sediment collected from the Canyon at Dahab, Red Sea, yielded two new chlorinated azaphilones, falconensins O and P (1 and 2) in addition to four known azaphilone derivatives (3−6) following fermentation of the fungus on solid rice medium containing 3.5% NaCl. Replacing NaCl with 3.5% NaBr induced accumulation of three additional new azaphilones, falconensins Q−S (7−9) including two brominated derivatives (7 and 8) together with three known analogues (10−12). The structures of the new compounds were elucidated by 1D and 2D NMR spectroscopy and HRESIMS data as well as by comparison with the literature. The absolute configuration of the azaphilone derivatives was established based on single-crystal X-ray diffraction analysis of 5, comparison of NMR data and optical rotations as well as on biogenetic considerations. Compounds 1, 3−9, and 11 showed NF-κB inhibitory activity against the triple negative breast cancer cell line MDA-MB-231 with IC₅₀ values ranging from 11.9 to 72.0 µM.

1. Introduction

In the last two decades, marine-derived fungi have gained considerable attention for drug discovery due to their ability to produce a vast diversity of bioactive secondary metabolites [1]. Up until today, hundreds of secondary metabolites have been characterized from marine-derived fungi exhibiting promising biological and pharmacological properties [2,3]. In particular, the diketopiperazine alkaloid halimide, obtained from a marine-derived fungus Aspergillus sp., was the lead structure for the putative anticancer drug plinabulin, which has entered Phase III of clinical trials against non-small cell lung cancer [4,5,6]. Interestingly, genomic sequencing revealed that under conventional culture conditions, many biosynthetic fungal gene clusters remain silent and transcriptionally suppressed [7,8]. Changing the cultivation conditions of fungi may activate silent biosynthetic gene clusters and eventually lead to either upregulation of constitutively present compounds or accumulation of new natural products [9]. The OSMAC (One Strain MAny Compounds) approach, first described by Zeeck et al. [10], represents one of the strategies which triggers diversification of the metabolic profile of fungi by altering the cultivation conditions. Fungi of the genus Aspergillus are rich sources of numerous bioactive secondary metabolites [11,12,13]. Consequently, as a part of our ongoing research on marine-derived fungi [14,15,16], we have investigated the fungus Aspergillus falconensis (formerly known as Emericella falconensis [17]) that was isolated from sea sediment collected at a depth of 25 m from the Canyon at Dahab, Red Sea, Egypt. The previously isolated soil-derived fungus, Emericella falconesis, is known as a producer of anti-inflammatory azaphilone derivatives [18,19,20,21]. Herein, we report the isolation, structure elucidation, and bioactivity of azaphilones obtained from A. falconensis following fermentation of the fungus on solid rice medium that contained either 3.5% NaCl or 3.5% NaBr. Following this approach, we were able to obtain two new chlorinated azaphilones (1 and 2) together with four known azaphilone derivatives (3–6) when the fungus was cultivated in the presence of NaCl and three additional new azaphilone derivatives (7–9) including two brominated analogues (7 and 8) in addition to three known derivatives (10–12) in the presence of NaBr. Compounds 1, 3–9, and 11 were examined with regard to their nuclear factor kappa B (NF-κB) inhibitory activity in the triple negative breast cancer (TNBC) cell line MDA-MB-231. In TNBC, constitutive activation of the proinflammatory NF-κB is associated with tumor aggressiveness [22]. All tested compounds revealed inhibition of NF-κB signaling with IC₅₀ values at two-digit micromolar concentrations.

2. Results and Discussion

After fermentation of A. falconensis on solid rice medium containing 3.5% NaCl (similar to the salinity of sea water), chromatographic separation of the EtOAc extract of the fungus yielded two new chlorinated azaphilone derivatives (1–2). In addition, four known azaphilones (3–6) were identified including falconensins A (3) [18], M (4) [23], N (5) [23], and H (6) [19] by comparison of their spectroscopic data with the literature.

Compound 1 was isolated as a yellow oil. Its HRESIMS analysis showed an isotope pattern characteristic for two chlorine atoms in the molecule at m/z 511/513/515 (9:6:1), corresponding to the molecular formula C₂₄H₂₄Cl₂O₈ with 12 degrees of unsaturation. The UV pattern and NMR data of 1 (

Table 1. ¹³C NMR data of compounds 1, 2, 7, 8, and 9 in CDCl₃.

No.	1 a,c	2 a,c	7 b	8 a,c	9 b
1	67.8, CH2	68.2, CH2	68.5, CH2	68.4, CH2	68.0, CH2
3	160.2, C	168.2, C	160.5, C	160.2, C	160.2, C
4	102.4, CH	100.9, CH	102.8, CH	102.6 CH	102.8, CH
4a	149.4, C	149.2, C	150.3, C	149.9, C	148.9, C
5	116.6, CH	115.7, CH	116.6, CH	116.6, CH	117.1, CH
6	192.3, C	192.7, C	193.8, C	193.4, C	193.3, C
7	83.4, C	83.4, C	85.6, C	85.4, C	82.5, C
8	70.1, CH	70.4, CH	69.8, CH	69.7, CH	70.7, CH
8a	38.1, CH	38.0, CH	37.8, CH	37.5, CH	38.2, CH
9	17.9, CH3	18.2, CH3	16.8, CH3	16.6, CH3	18.2, CH3
10	124.9, CH	36.4, CH2	125.4, CH	125.3, CH	125.2, CH
11	134.1, CH	20.0, CH2	133.9, CH	133.6, CH	133.9, CH
12	18.2, CH3	13.6, CH3	18.4, CH3	18.2, CH3	18.4, CH3
1′	164.3, C	164.4, C	165.3, C	164.9, C	166.2, C
2′	122.2, C	122.3, C	117.4, C	117.2, C	115.5, C
3′	152.8, C	152.9, C	156.2, C	157.3, C	159.0, C
4′	112.9, C	112.8, C	97.1, CH	93.8, CH	96.9, CH
5′	149.2, C	149.8, C	154.0, C	155.8, C	157.6, C
6′	117.0, C	117.1, C	105.3, C	106.6, C	109.2, CH
7′	134.3, C	134.5, C	137.4, C	138.1, C	140.0, C
8′	16.9, CH3	17.1, CH3	20.2, CH3	20.1, CH3	19.6, CH3
8-OAc	170.0, C	170.1, C			169.7, C
	20.5, CH3	20.8, CH3			20.8, CH3
3′-OMe	62.4, CH3	62.6, CH3	56.5, CH3	56.3, d CH3	56.0, CH3
5′-OMe				56.4, d CH3

^a Measured at 150 MHz; ^b Measured at 175 MHz; ^c Data extracted from the HSQC (Heteronuclear Single Quantum Coherence) and HMBC spectra; ^d interchangeable.

and

Table 2. ¹H NMR data of compounds 1, 2, 7, 8, and 9 in CDCl₃.

No.	1 a	2 a	7 b	8 a	9 b
1	4.34, dd (11.0, 5.0); 3.96, dd (13.0, 11.0)	4.29, dd (11.0, 5.1); 3.93, dd (13.2, 11.0)	4.77, dd (10.5, 5.1); 3.84, dd (13.0, 10.5)	4.77, dd (10.6, 5.0); 3.84, dd (13.0, 10.6)	4.32, dd (10.8, 5.0); 3.93, dd (13.0, 10.8)
4	5.59, s	5.53, s	5.56, s	5.57, s	5.57, s
5	5.86, d (1.7)	5.79, d (1.9)	5.80, d (1.6)	5.80, d (1.8)	5.85, d (1.9)
8	6.11, d (10.7)	6.09, d (10.7)	4.74, d (10.7)	4.75, d (10.7)	6.11, d (10.7)
8a	2.98, dddd (13.0, 10.7, 5.0, 1.7)	2.94, dddd (13.2, 10.7, 5.1, 1.9)	2.88, dddd (13.0, 10.7, 5.1, 1.6)	2.88, dddd (13.0, 10.7, 5.0, 1.8)	2.96, dddd (13.0, 10.7, 5.0, 1.9)
9	1.56, s	1.56, s	1.47, s	1.47, s	1.54, s
10	5.91, dq (15.4, 1.3)	2.20, m	5.90, dq (15.4, 1.4)	5.90, dd (15.4, 1.5)	5.90, dd (15.3, 1.6)
11	6.45, dq (15.4, 7.0)	1.58, m	6.47, dq (15.4, 7.0)	6.46, dd (15.4, 7.0)	6.42, dd (15.3, 7.0)
12	1.88, dd (7.0, 1.3)	0.94, t (7.4)	1.87, dd (7.0, 1.4)	1.88, dd (7.0, 1.5)	1.87, dd (7.0, 1.6)
4′			6.52, s	6.38, s	6.23, s
6′					6.22, s
8′	2.50, s	2.50, s	2.48, s	2.51, s	2.39, s
8-OAc	2.17, s	2.16, s			2.15, s
3′-OMe	3.83, s	3.83, s	3.83, s	3.92, c s	3.74, s
5′-OH	6.04, s	6.04, s
5′-OMe				3.89, c s

^a Measured at 600 MHz; ^b Measured at 700 MHz; ^c interchangeable.

) were similar to those of the co-isolated known falconensin M (4), which was previously reported from Emericella falconensis [23], suggesting 1 to be an azaphilone derivative [24]. However, the ¹H NMR spectrum of 1 displayed the signal of an additional methyl group at δ_H 2.07 (s) when compared to 4. The Heteronuclear Multiple Bond Correlation (HMBC) correlations from the protons of this additional methyl and H-8 to a carbonyl carbon at δ_C 170.0 together with the obvious deshielded chemical shift of H-8 (δ_H 6.11) indicated the replacement of the hydroxy group by an acetoxy group at C-8 in 1. Detailed analysis of the 2D NMR spectra of 1 indicated that the remaining substructure of 1 was identical to that of 4. The large values of ³J_H-8,H-8a (10.7 Hz) and ³J_H-1b,H-8a (13.0 Hz) and the small value of ³J_H-1a,H-8a (5.0 Hz) obtained from the ¹H NMR spectrum of 1, indicated trans-diaxial orientation between H-8a (δ_H 2.98) and H-8 and between H-8a and H-1b (δ_H 3.96) and cis-orientation between H-8a and H-1a (δ_H 4.34). The NOE correlations from H-8a to H-1a and Me-9 (δ_H 1.56) as well as between H-8 and H-1b confirmed that H-1a, H-8a and Me-9 were on the same side of the ring (α-oriented) whereas H-1b and H-8 were on the opposite side (β-oriented). Thus, compound 1 was elucidated as 8-O-acetyl analogue of 4, representing a new falconensin derivative, for which the trivial name falconensin O is proposed. Incubation of putative precursors of 1 such as 4 in EtOAc over three days failed to generate the corresponding acetates, thus ruling out the possibility that the acetylated azaphilones isolated in this study are artefacts generated during chromatographic workup.

The molecular formula of compound 2 was deduced as C₂₄H₂₆Cl₂O₈, containing two additional protons when compared to 1. Investigation of the ¹H and ¹³C NMR data of 2 (Table 1 and Table 2) demonstrated the close structural similarity between 1 and 2 except for signals of the side chain at C-3. In particular, the resonances for the olefinic methine protons at δ_H 5.91 (H-10) and 6.45 (H-11) of 1 were replaced by two methylene groups at δ_H 2.20 (H₂-10) and 1.58 (H₂-11) in 2. The COSY correlations between H₂-10/H₂-11/Me-12 together with the HMBC correlations from H₂-11 to C-3 (δ_C 168.2) and from H₂-10 to C-3 and C-4 (δ_C 100.9) confirmed the attachment of a n-propyl moiety at C-3 in 2. The remaining substructure of 2 was determined to be identical to that of 1 by comparison of the 2D NMR data of 1 and 2. The similar coupling constants and NOE relationships between 1 and 2 indicated that both share the same relative configuration.

When 3.5% NaCl as a constituent of the rice medium was replaced with 3.5% NaBr, a profound change in the metabolic profile of the fungus was observed. In total, two new brominated azaphilone derivatives (7 and 8), one new non-halogenated azaphilone (9), in addition to the known falconensins K (10) [23] and I (11) [23] were obtained.

The ¹H and ¹³C NMR spectra of 7 (Table 1 and Table 2) were comparable to those of the co-isolated known compound, falconensin K (10) [23]. The HRESIMS data of 7 established the molecular formula C₂₂H₂₃BrO₇, differing from that of 10 by the replacement of the chlorine with a bromine atom. The HMBC correlations from Me-8′ (δ_H 2.48) to C-2′ (δ_C 117.4), C-6′ (δ_C 105.3), and C-7′ (δ_C 137.4), from H-4′ to C-2′, C-6′, C-3′ (δ_C 156.2), and C-4′ (δ_C 154.0), from 3′-OMe (δ_H 3.83) to C-3′ together with the NOE correlation between 3′-OMe and Me-9 (δ_H 1.47) indicated the position of the bromine atom at C-6′ in 7. The remaining azaphilone core structure including the relative configuration of 7 was confirmed to be identical to that of 10 after further inspection of the 2D NMR spectra of 7. Thus, the structure of 7 was elucidated as shown (Figure 1), and the trivial name falconensin Q is proposed for this compound.

Falconensin R (8) was isolated as a yellow oil. Its molecular formula was determined as C₂₃H₂₅BrO₇ by HRESIMS, containing an additional methyl group when compared to 7. The NMR data of 8 (Table 1 and Table 2) were similar to those of 7 except for the appearance of two methoxy groups at δ_C 56.4 and 56.3, and at δ_H 3.89 (5′-OMe) and 3.92 (3′-OMe) instead of one methoxy group in 7. The location of the additional methoxy group at C-5′ in 8 was confirmed by the HMBC correlations from 3′-OMe to C-3′ (δ_C 157.3), from 5′-OMe to C-5′ (δ_C 155.8), and from H-4′ to C-2′ (δ_C 117.2), C-6′ (δ_C 106.6), C-3′ and C-5′, as well as based on the NOE correlations from H-4′ to 3′-OMe and 5′-OMe.

The molecular formula of 9 was determined as C₂₄H₂₆O₈ by HRESIMS, indicating 42 amu more than that of the co-isolated known falconensin I (11) (C₂₂H₂₄O₇) [23]. Comparison of the ¹H and ¹³C NMR spectra revealed that the structure of 9 was closely related to that of 11, except for the appearance of an additional acetoxy group at δ_H 2.15 and δ_C 20.8 and 169.7 in 9. The location of this additional acetoxy group at C-8 was confirmed by the COSY correlations between H-1ab/H-8a/H-8 and the HMBC correlations from H-8 to the carbon of the additional acetoxy group. The similar NOE correlations of 9 and 11 suggested that they shared the same configuration. Thus, compound 9 was elucidated as 8-O-acetyl analogue of falconensin I (11).

The specific optical rotation values of 1, 2, 7, 8, and 9 are positive (+99 − +233), which is in agreement with the positive values reported for other known falconensin derivatives with 7R, 8S, and 8aS configuration, suggesting that the new compounds share the same absolute configuration as the known derivatives [18,20,25]. Although the absolute configuration of other falconensin derivatives had been determined previously by Mosher’s reaction and observation of Cotton effects of CD curves [18,20,25], no crystal structure had so far been reported for these compounds. To independently assign the absolute configuration of the isolated compounds, a single crystal X-ray diffraction analysis through anomalous dispersion of the known falconensin N (5), for which crystals of sufficient quality were obtained, was conducted. Herein, we present for the first time the crystal structure of falconensin N (5) (Figure 2). From the single-crystal structure refinement, the absolute structure assignment was based on the Flack parameter of 0.016(5) (Table S1) [26,27,28,29]. The crystal structure of falconensin N (5) is in agreement with the previously reported absolute configuration [18,20,25]. Based on these data, all falconensin derivatives isolated in this study are suggested to share the same 7R, 8S, and 8aS absolute configuration.

Azaphilones constitute a class of fungal metabolites possessing various biological activities, including antiviral, antibacterial, antioxidant, hypolipidemic, cytotoxic, and anti-inflammatory properties [24,30]. For falconensins in particular, anti-inflammatory activity against 12-O-tetradecanoylphorbol-13-acetate-induced inflammatory ear edema in mice had been reported [21]. Compounds 1, 3–9, and 11 were evaluated for their anti-inflammatory activity in the triple negative breast cancer cell line NF-κB-MDA-MB-231. The IC₅₀ values were calculated based on inhibition of the NF-κB-dependent luciferase activity and revealed NF-κB blockade for all compounds (

Table 3. IC₅₀ values (µM) of the compounds tested in MDA-MB-231 cells for NF-κB inhibition and inhibition of cell viability. ^a

Compound	NF-κB inhibition	Cell viability inhibition
Falconensin O (1)	15.7 ± 0.7	> 200
Falconensin A (3)	53.2 ± 21.4	89.7 ± 9.1
Falconensin M (4)	56.5 ± 8.3	> 200
Falconensin N (5)	71.0 ± 7.3	> 200
Falconensin H (6)	72.0 ± 28.1	> 400
Falconensin Q (7)	11.9 ± 2.1	> 200
Falconensin R (8)	14.6 ± 1.7	126.8 ± 5.4
Falconensin S (9)	20.1 ± 5.6	> 200
Falconensin I (11)	19.5 ± 2.5	> 400

^aAverage IC₅₀ of at least three individual experiments ± SD tested in the concentration range of 400 µM to 0.78 µM (3, 6, and 11) and 200 µM to 0.78 µM (1, 4, 5, 7, 8, and 9) in a two-fold serial dilution.

). To exclude that cytotoxicity caused reduction of NF-κB activity, cell viability was determined in parallel. Compounds 1, 4–7, 9, and 11 did not influence cell viability within the selected concentration range, whereas compound 8 reduced cell viability with an IC₅₀ of 126.8 ± 5.4 µM, thus showing about 9-times higher potency in NF-κB-blockade compared to its cytotoxicity. Compound 3 with an IC₅₀ of 89.7 ± 9.1 µM in the cytotoxicity assay only showed around 2-times higher potency in NF-κB-blockade. Plotting the pIC₅₀, which is calculated as the negative decadic logarithm of IC₅₀, of the cell viability against the pIC₅₀ of NF-κB inhibition, illustrates a greater anti-inflammatory potential of the examined compounds compared to their cytotoxic potential (Figure S40). In conclusion, inhibition of NF-κB signaling in the TNBC cell line MDA-MB-231 could be induced by 1, 3 − 9, and 11, with 7 being the most potent compound.

3. Materials and Methods

3.1. General Experimental Procedures

Optical rotations were measured using a Jasco P-2000 polarimeter. The compounds were dissolved in optically pure solvents Uvasol^® (spectroscopic grade solvents, Merck). 1D and 2D NMR spectra were recorded on Bruker Avance III 300 or 600 or 700 MHz NMR spectrometers (Bruker BioSpin, Rheinstetten, Germany). Low-resolution mass spectra were recorded with an Ion-Trap-API Finnigan LCQ Deca (Thermo Quest) mass spectrometer, while high-resolution mass data were measured on a FTHRMS-Orbitrap (Thermo-Finnigan) mass spectrometer. HPLC analysis was conducted using a Dionex UltiMate-3400 SD with an LPG-3400SD pump coupled to a photodiode array detector (DAD3000RS) and employing a Knauer Eurospher C₁₈ analytical column (125 × 4 mm i.d., 5 μm). Purification of the compounds was performed using semipreparative HPLC on the VWR Hitachi Chromaster HPLC system, 5160 Pump; 5410 UV detector; Eurosphere-100 C₁₈, 300 mm × 8 mm i.d., 10 µm, Knauer, Germany) with MeOH and H2O as eluents utilizing a flow rate of 5 mL/min. Column chromatography was performed using different stationary phases including Merck MN silica gel 60 M (0.04−0.063 mm), silica gel 60 RP-18 (40–63 µm), and Sephadex LH-20 (Merck). TLC plates precoated with silica gel 60 F₂₅₄ (Merck) were used for monitoring fractions resulting from column chromatography. Detection of spots on the TLC was done by UV absorption at 254 and 365 nm followed by anisaldehyde spray reagent.

3.2. Fungal Material

The fungus was isolated from marine sediment which was collected at a depth of 25 m from the Canyon at Dahab, Red Sea, Egypt in November 2016. The fungus was identified as A. falconensis (GenBank accession No. MN905375) through amplification and sequencing of the internal transcribed spacer region including the 5.8S ribosomal DNA following by a subsequent BLAST search in NCBI as described before [31]. A deep-frozen specimen of the fungal strain has been deposited in one of the author’s laboratory (P.P.).

3.3. Fermentation, Extraction, and Isolation

Initial fermentation of the fungus was conducted in ten 1L Erlenmeyer flasks. To each flask, 100 g of rice (Oryza Milchreis), 100 mL of demineralized water, and 3.5 g of NaCl were added. Thereafter, the flasks were autoclaved at 121 °C for 20 min and after cooling to room temperature, the fungus was inoculated on the rice medium. Fermentation of the fungus was continued under static conditions for 21 days at 20 ℃ until the fungus had totally overgrown the medium. Then, each flask was extracted with 600 mL EtOAc. After overnight soaking in EtOAc, the rice medium was cut into small pieces and shaken for 8 h at 150 rpm followed by evaporation of EtOAc, yielding around 16 g of EtOAc extract. For the OSMAC experiment, the same cultivation and extraction procedures of the initial cultivation were conducted except for the replacement of the added 3.5% NaCl with 3.5% NaBr and the cultivation on three flasks instead of ten. Eventually, the OSMAC experiment yielded approximately 2 g of EtOAc extract.

The initial crude extract of the large scale cultivation of the fungus obtained in presence of NaCl (16 g) was fractionated by vacuum liquid chromatography (VLC) on silica gel as a stationary phase utilizing a step gradient of solvents consisting of mixtures of n-hexane/EtOAc and CH₂Cl₂/MeOH, to yield 12 fractions (V1 to V12). Fraction V4 (874 mg) was further separated by Sephadex LH20 column chromatography using CH₂Cl₂-MeOH (1:1) as mobile phase affording eight subfractions (V4-S1 to V4-S8). Subfraction V4-S4 (162.8 mg) was purified by semi-preparative HPLC using acetonitrile-H₂O containing 0.1% formic acid (from 60:40 to 95:5 in 22 min) to afford 1 (1.8 mg), 2 (1.5 mg), and 3 (11.8 mg). Subfraction V4-S5 (139.6 mg) was further purified by semi-preparative HPLC using gradient elution with acetonitrile-H₂O containing 0.1% formic acid (60:40 to 95:5 in 22 min) affording 4 (6.3 mg) and 5 (2.2 mg). Fraction V6 (686.1 mg) was submitted to RP-VLC column using H₂O-MeOH gradient elution to yield 10 subfractions (V6-R1 to V6-R10). Subfraction V6-R7 (110 mg) was further purified using semi-preparative HPLC with a gradient of MeOH-H₂O containing 0.1% formic acid (67:33 to 80:20 in 20 min) to afford 6 (15.1 mg).

The EtOAc extract (2 g) obtained from the OSMAC experiment with addition of 3.5% NaBr, was also fractionated by VLC on silica gel as described before to give 13 fractions (BrV1 to BrV13). Subsequent purification of BrV5 (157 mg), BrV6 (32 mg), and BrV10 (140 mg) using Sephadex LH20 column chromatography with CH₂Cl₂-MeOH (1:1) as mobile phase and semi-preparative HPLC with a gradient of MeOH-H₂O containing 0.1% formic acid to afford 7 (3.1 mg), 8 (2.1 mg), 9 (1.2 mg), 10 (0.9 mg), and 11 (2.0 mg).

Falconensin O (1): Yellow oil; ${\left[\alpha \right]}_D^{20}$ +176 (c 0.2, MeOH); UV (MeOH) λ_max 355 and 206 nm; ¹H and ¹³C NMR data, Table 1 and Table 2; HRESIMS m/z 511.0923 [M + H]⁺ (calcd for C₂₄H₂₅Cl₂O₈, 511.0921).

Falconensin P (2): Yellow amorphous powder; ${\left[\alpha \right]}_D^{20}$ +233 (c 0.2, MeOH); UV (MeOH) λ_max 329 and 216 nm; ¹H and ¹³C NMR data, Table 1 and Table 2; HRESIMS m/z 513.1073 [M + H]⁺ (calcd for C₂₄H₂₇Cl₂O₈, 513.1077).

Falconensin Q (7): Yellow oil; ${\left[\alpha \right]}_D^{20}$ +182 (c 0.2, MeOH); UV (MeOH) λ_max 354 and 205 nm; ¹H and ¹³C NMR data, Table 1 and Table 2; HRESIMS m/z 479.0702 [M + H]⁺ (calcd for C₂₂H₂₄BrO₇, 479.0700).

Falconensin R (8): Yellow oil; ${\left[\alpha \right]}_D^{20}$ +99 (c 0.2, MeOH); UV (MeOH) λ_max 353 and 206 nm; ¹H and ¹³C NMR data, Table 1 and Table 2; HRESIMS m/z 493.0853 [M + H]⁺ (calcd for C₂₃H₂₆BrO₇, 493.0856).

Falconensin S (9): Yellow oil; ${\left[\alpha \right]}_D^{20}$ +105 (c 0.2, MeOH); UV (MeOH) λ_max 355 and 201 nm; ¹H and ¹³C NMR data, Table 1 and Table 2; HRESIMS m/z 443.1700 [M + H]⁺ (calcd for C₂₄H₂₇O₈, 443.1700).

3.4. Crystallographic Analysis of Compound 5

Crystals were obtained by solvent evaporation (MeOH). Data Collection: compound 5 was measured with a Bruker Kappa APEX2 CCD diffractometer with a microfocus tube and Cu-Kα radiation (λ = 1.54178 Å). APEX2 was used for data collection, SAINT for cell refinement and data reduction [32], and SADABS for experimental absorption correction [33]. The structure was solved by intrinsic phasing using SHELXT [34], and refinement was done by full-matrix least-squares on F² using SHELXL-2016/6 [35]. The hydrogen atoms were positioned geometrically (with C-H = 0.95 Å for aromatic CH, 1.00 Å for aliphatic CH, 0.99 Å for CH₂, and 0.98 Å for CH) and refined using riding models (AFIX 43, 13, 23, 137, respectively), with Uiso(H) = 1.2 Ueq(CH, CH₂), and 1.5 Ueq(CH₃). The absolute structure configuration of 5 was solved using anomalous dispersion from Cu-Kα, resulting in a Flack parameter of 0.016(5) using Parsons quotient method. All graphics were drawn using DIAMOND [36]. The structural data have been deposited in the Cambridge Crystallographic Data Center (CCDC No. 1976223).

3.5. Triple Negative Breast Cancer Studies

3.5.1. Cell Culture and Chemicals

Culture medium and supplements were purchased from Gibco (Fisher Scientific, Schwerte, Germany). Cell plates were obtained from Greiner bio-one (Frickenhausen, Germany). Cells were grown and incubated in a humidified 5% CO₂ atmosphere at constant 37 °C. The metastatic breast cancer cell line, MDA-MB-231, was purchased from the European Collection of Authenticated Cell Cultures (ECACC) (Salisbury, UK). Sub-culturing was performed in RPMI 1640 medium (#21875-034) supplemented with 15% (v/v) fetal calf serum (FCS) and 1% (v/v) penicillin-streptomycin (pen-strep) (10,000 U/mL). The MDA-MB-231 cell line stably expressing a plasmid with the NF-κB response element and the gene sequence for the firefly-luciferase protein (NF-κB- MDA-MB-231) was purchased from Signosis (Santa Clara, CA, USA; #SL-0043). For selection, 100 µg/mL hygromycin B (Life Technologies, Darmstadt, Germany; #10687010) was applied in high glucose DMEM (#41966-029) supplemented with 10% (v/v) FCS, 1% (v/v) pen-strep. Starvation medium for the NF-κB inhibition assay contained 1% (v/v) FCS, 1% (v/v) pen-strep, and 100 µg/mL hygromycin B. Cell detachment occurred by trypsinization in 0.25% trypsin-EDTA and cell counting was performed at 1:1 (v/v) dilution in Erythrosin B (BioCat, Heidelberg, Germany; #L13002) using the LUNA II automated cell counter (BioCat). Compounds 1, 4, 5, 7, 8, and 9 were dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 10 mM, whereas the compounds 3, 6, 11 were dissolved at 20 mM in DMSO. Further dilutions in cell culture medium contained maximal 2% DMSO.

3.5.2. NF-κB Inhibitory Assay

3 × 10⁴ NFκB-MDA-MB-231 cells were seeded in total 100 µL medium per well in a 96-well plate (Greiner; #655098). On the next day, medium was exchanged, and triplicates were pre-incubated for 20 min without (negative control) or with compounds in total 100 µL starvation medium. Final concentration of the compounds ranged a twofold serial dilution starting from 400 µM (compound 3, 6, and 11) or 200 µM (compound 1, 4, 5, 7, 8, and 9) to 0.78 µM. To activate NF-κB signaling, untreated cells (positive control) or compound treated cells were subsequently stimulated for 24 h with 20 ng/mL TNFα (Peprotech, Hamburg, Germany; #300-01A). Finally, cell lysis and measurement were done according to the manufacturer’s instruction of the Luciferase Assay System (Promega, Mannheim, Germany; #E1500). Injection of equal volume of luciferase substrate with 10 s integration time and subsequent luminescence measurement was performed using the Spark^® microplate reader (TECAN, Männedorf, Switzerland).

3.5.3. Cell Viability Assay

Using the CyBio^® Well vario pipetting robot (Analytik Jena, Jena, Germany; #OL3381-24-730), 18 µL of the MDA-MB-231 cell suspension (2.8 × 10⁵ cells/mL) were seeded per well in a 384-well plate (Greiner; #781074) and incubated for 24 h. The final concentration of the compounds ranged in twofold serial dilution steps starting from 400 µM (compound 3, 6, and 11) or 200 µM (compound 1, 4, 5, 7, 8, and 9) to 0.78 µM. After 24 h compound stimulation in quadruples, cell lysis and measurement were done as prescribed in the manufacturer’s instruction of the CellTiter-Glo^® Luminescent Cell Viability Assay (Promega; #G7570). In short, it was applied equal volume of the CellTiter-Glo^® reagent and luminescence was measured using the Spark^® microplate reader (TECAN).

3.5.4. Statistical Analysis

Data of the NF-κB inhibitory assay and cell viability assay represent at least three individual experiments and were analyzed using GraphPad Prism (GraphPad Software, San Diego, USA; Version 8.1.2). For the NF-κB inhibitory assay, data below the relative light unit (RLU) of the negative control were excluded for further analysis. Half maximal inhibitory concentration (IC₅₀) values were determined by nonlinear regression analysis based on the dose–response inhibition calculation “log(inhibitor) vs. response–variable slope (four parameters)” without curve fitting.