Lynamicins H–P and Indimicin H: Chlorinated Bisindole Pyrroles from a Marine-Derived Streptomyces massiliensis and Their Biological Activities

Abstract

Lynamicins H–P (1–4 and 6–10) and Indimicin H (5), ten previously unreported chlorinated bisindole pyrrole derivatives, along with five known lynamicins A–E (11–15), were isolated from the culture broth of the marine-derived actinomycete Streptomyces massiliensis 213DD-128 obtained from a sediment sample collected in the East Sea, Republic of Korea. The structures of 1–10 were elucidated by a comprehensive analysis of spectroscopic data, including NMR and HRESIMS. The absolute configurations of 5 and 7–9 were determined by comparison of their experimental and calculated electronic circular dichroism (ECD) spectra. Compounds 1–15 were assessed for their antimicrobial and cytotoxic activities. Notably, 10 exhibited approximately 7-fold stronger in vitro antibacterial activity (MIC = 0.5 μg/mL) than vancomycin (MIC = 3.8 μg/mL) against methicillin-resistant Staphylococcus aureus (MRSA) and also showed potent activity against pan-drug susceptible S. aureus (MIC = 0.4 μg/mL), and demonstrated pronounced in vitro cytotoxicity (GI₅₀ = 2.8–10.5 μM) against human cancer cell lines, except for the U266 cell line. Importantly, antibacterial efficacy was further supported in an ex vivo infection model, demonstrating activity beyond conventional in vitro assays. In contrast, 3 showed moderate antibacterial activity against MRSA (MIC = 2.3 μg/mL) compared with other compounds, while exhibiting weak or negligible cytotoxicity against normal human cell lines (MRC-9: GI₅₀ = 21.4 μM; RPMI-1788: GI₅₀ = >30 μM), highlighting its potential as a promising lead scaffold.

Introduction

Natural products (NPs) have been utilized in the treatment of various diseases throughout human history. With continuous advances in science and technology, marine natural products (MNPs) have emerged as an especially rich and attractive source and are increasingly exploited in drug development. Microbes, one of the most prolific sources of MNPs, have been steadily studied for their secondary metabolites up to now. , Following the recent literature, , although the number of reported MNPs derived from microorganisms has slightly decreased because some papers reported structures proposed from mass spectrometric data without full NMR structural analysis, microbes are still recognized as a dominant and indispensable resource.

Indole, an aromatic heterocyclic compound consisting of a benzene ring fused to a pyrrole ring, belongs to alkaloids, which constitute one of the largest classes of NPs. , Their pharmacological effects, including anti-inflammatory, antimicrobial, antiviral, and antitumor activities, along with potential applications as a medication through total synthesis or chemical modification, have already been reviewed in several papers. − In particular, bis and tris-indole alkaloids containing N-heterocyclic linker moieties exhibited potent biological activities. Many FDA-approved antibiotics contain pyrrole motifs, and structural modification of pyrrole-containing scaffolds has proven effective in enhancing antibacterial activity. Several studies have reported bioactive chlorinated bisindole derivatives derived from microorganisms such as Saccharothrix aerocolonigenes (rebeccamycins), Tolypothrix tjipanasensis (tjipanazoles), Marinispora sp. (lynamicins), , and Streptomyces sp. (spiroindimicins, indimicins, and dionemycin). − Lynamicins A–E, bisindole pyrrole derivatives, were also reported to be active against drug-resistant pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE) in 2008. Given that MRSA and VRE continue to pose serious global threats to public health, the discovery and development of antibiotics against these strains remain an urgent priority. Despite extensive studies on lynamicins and related bisindole pyrrole antibiotics, systematic structural diversification through variations in chlorination patterns, oxidation states, and stereochemistry remains limited. Moreover, the biological consequences of such structural variations have not been comprehensively investigated. As part of our ongoing study to discover undescribed bioactive compounds from marine microbes, we isolated Streptomyces massiliensis 213DD-128, which produces streptinone, a new indanone derivative with inhibitory activity against particulate matter (PM)-induced inflammation. The strain was isolated from a sediment sample collected from the East Sea, Republic of Korea, in 2021. In a subsequent large-scale fermentation and chemical investigation, we isolated previously unreported ten bisindole pyrrole derivatives together with lynamicins A–E from the extract of the culture broth. Herein, we report the isolation and structure determination of the undescribed bisindole pyrrole derivatives (1–10), as well as their antimicrobial and cytotoxic activities against six human solid cancer cell lines and seven blood cancer cell lines.

Results and Discussion

Lynamicin H (1) was obtained as an off-white solid. The molecular formula of 1 was determined as C₂₂H₁₆N₃O₂Cl based on the HRESIMS peak at m/z 412.0830 [M + Na]⁺ (calcd for C₂₂H₁₆N₃O₂NaCl, 412.0829), corresponding to 16 degrees of unsaturation. The isotope pattern with a ratio of 3:1 indicated the presence of a chlorine atom (Figure S7). Comprehensive analysis of ¹H and ¹³C NMR spectra (Table ), together with HSQC data, revealed the presence of one carbonyl group, one methoxy group, ten aromatic protons, and two sets of seven degenerate carbon resonances. To distinguish these signals between each carbon and proton, a 2D selective HMBC experiment was conducted (Figure S6). Carbon chemical shift values from C-2′ to C-7a′ were consistent with those of typical indole derivatives. And the ¹H and ¹³C NMR spectra of 1 were very similar to those of lynamicin A (11), which features two indole units symmetrically connected through a pyrrole ring. The connectivity among the methyl ester, the indole, and the chlorinated indole was established based on HMBC correlations from H-5 to C-2, C-3, and C-3″, respectively. These results indicated that 1 contained one less chlorine atom than 11. Thus, the structure of 1 was determined to be a new derivative of lynamicins and named lynamicin H.

1. ¹H (600 MHz) and ¹³C (150 MHz) NMR Data for 1–4 (CD₃OD) and 5 (CDCl₃).

	lynamicin H (1)		lynamicin I (2)		lynamicin J (3)		lynamicin K (4)		indimicin H (5)
position	δC, type	δH, mult. (J in Hz)	δC, type	δH, mult. (J in Hz)	δC, type	δH, mult. (J in Hz)	δC, type	δH, mult. (J in Hz)	δC, type	δH, mult. (J in Hz)
1 (NH)										8.25, s
2	121.0, C		120.9, C		118.1, CH	6.99, s	118.1, CH	6.99, s	120.3, CH	6.81
3	124.5, C		124.5, C		116.5, C		116.8, C		128.4, C
4	121.6, C		121.1, C		116.9, C		116.6, C		114.0, C
5	122.4, CH	7.28, s	122.5, CH	7.27, s	118.0, CH	6.99, s	117.9, CH	6.99, s	116.4, CH	6.80
6	163.7, C		163.6, C
7	51.5, CH3	3.65, s	51.5, CH3	3.66, s
1′ (NH)
2′	125.8, CH	7.07, s	125.9, CH	7.05, s	125.7, CH	6.97, s	125.8, CH	6.94, s	61.3, CH2	3.60, d (8.9); 3.18, d (8.9)
3′	110.7, C		110.6, C		113.0, C		113.0, C		45.0, C
3′-CH3									27.8, CH3	1.53, s
3a′	129.1, C		129.1, C		128.6, C		128.6, C		140.2, C
4′	121.2, CH	7.11, d (8.0)	121.1, CH	7.11, d (7.9)	122.3, CH	7.43, s	122.19, CH	7.46, s	125.1, CH	6.94, s
5′	119.7, CH	6.87, m	119.8, CH	6.83, ddd (7.9, 7.1, 0.9)	125.5, C		125.2, C		120.7, C
6′	122.0, CH	7.01, m	122.1, CH	7.01, ddd (8.1, 7.1, 1.0)	123.4, C		123.5, C		130.6, C
7′	112.1, CH	7.33, d (8.2)	112.2, CH	7.33, d (8.1)	113.6, CH	7.44, s	113.6, CH	7.44, s	110.2, CH	6.49, s
7a′	137.9, C		137.8, C		136.8, C		136.9, C		149.8, C
1″ (NH)										8.01, s
2″	125.5, CH	6.69, s	126.1, CH	6.78, s	125.1, CH	6.92, s	124.3, CH	6.90, s	125.91, CH	6.20, d (2.7)
3″	111.2, C		111.5, C		112.7, C		113.1, C		134.3, C
3a″	129.2, C		128.0, C		129.8, C		127.3, C		111.5, C
4″	119.9, CH	7.36, d (2.0)	121.8. CH	7.36, s	120.4, CH	7.34, d (1.9)	122.21, CH	7.32, d (8.5)	120.9, CH	7.49, s
5″	125.7, C		125.5, C		125.6, C		120.2, CH	6.83, dd (8.5, 1.8)	125.87, C
6″	122.2, CH	6.96, dd (8.5, 2.0)	123.7, C		122.2, CH	6.99 dd (8.5, 1.9)	128.1, C		124.0, C
7″	113.3, CH	7.19, d (8.5)	113.5, CH	7.37, s	113.3, CH	7.26 d (8.5)	111.9, CH	7.30, d (1.8)	112.5, CH	7.37, s
7a″	136.1, C		136.6, C		136.4, C		138.4, C		128.6, C

^aChemical shift value was identified from the HMBC spectrum.

^bOverlapped signals.

Lynamicin I (2) was isolated as an off-white solid. Its molecular formula was deduced to be C₂₂H₁₅N₃O₂Cl₂ by the HRESIMS peak at m/z 446.0435 [M + Na]⁺ (calcd for C₂₂H₁₅N₃O₂NaCl₂, 446.0439), identical to that of lynamicin A (11). The isotope pattern with an intensity ratio of approximately 9:6:1 was diagnostic of two chlorine atoms (Figure S16). The structure of 2 was almost identical to that of 1, differing only by the presence of one additional chlorine atom (Chart ). The distinct singlet proton signals at δ_H 7.36 (s, H-4″) and 7.37 (s, H-7″), in the chlorinated indole moiety, lacking meta coupling, support the assignment of chlorine substituents at C-5″ and C-6″. The position of the dichlorinated indole unit was further confirmed by 2D selective HMBC correlations from H-2″ to C-4 and from H-5 to C-3″ (Figure ).

1.

1.

COSY and HMBC correlations of 1–10.

Lynamicins J and K (3 and 4) were obtained as yellow solids. Their molecular formulas were determined to be C₂₀H₁₂N₃Cl₃ (degree of unsaturation: 15) based on the HRESIMS data. Both 3 and 4 exhibited the same isotope patterns with an intensity ratio of approximately 29:28:9:1 in the HRESIMS (Figures S24 and S31), consistent with the presence of three chlorine atoms. The ¹H and ¹³C NMR spectra of 3 and 4 closely resembled those of 2, except for the absence of a methyl ester group. Notably, subtle but reproducible differences in chemical shift values were observed for the monochlorinated indole moieties (Table ). The positions of chlorine atoms in 3 and 4 were identified by 2D selective HMBC correlations from H-4″ to C-3″/ C-5″ and from H-4″ to C-3″/ C-6″, respectively (Figure ). These correlations established the structures as 5-chloroindole (3) and 6-chloroindole (4). Accordingly, 3 and 4 were identified as positional chlorine isomers.

2.

Experimental (solid line) and calculated (dashed line) ECD spectra of 5 and 7–9.

Indimicin H (5), an off-white solid, exhibited a characteristic isotope pattern corresponding to four chlorine atoms (intensity ratio of 83:109:53:12:1) (Figure S40) in the HRESIMS data. The molecular formula of 5 was deduced as C₂₁H₁₅N₃Cl₄, corresponding to 14 degrees of unsaturation, based on the HRESIMS. The ¹H and ¹³C NMR data (Table ), together with HSQC spectra of 5, revealed the presence of two exchangeable protons, seven aromatic protons, two methylene protons, and one methyl group, as well as 11 aromatic quaternary carbons and one quaternary carbon. The ¹H and ¹³C NMR spectra (Table ) of 5 closely resembled those of 3 and 4, except for the presence of an additional chlorine atom, as well as a methylene and a methyl group originating from an indoline instead of an indole. Two singlet peaks in each benzene ring indicated the presence of the 5,6-dichlorinated substitution pattern. The location of the methyl group was confirmed by HMBC and 2D selective HMBC correlations from H₃-3′-Me to C-2′, C-3′, and C-4′, and from Ha-2′ and Hb-2′ to C-3, C-3′, and C-3′-Me (Figure ). The planar structure of 5 was similar to that of indimicin E, except for the absence of two N-methyl groups and the presence of two additional chlorine atoms. In addition, the specific rotation value ([α]_D ²⁵ −73, c 0.1, CH₃OH) of 5 showed an opposite sign to that of indimicin E ([α]_D ²⁵ +33, c 0.12, CH₃OH), and its CD spectra also exhibited Cotton effects with opposite signs, indicating that 5 possesses the opposite absolute configuration to that of indimicin E.

Lynamicin L (6) was obtained as a pale yellow solid. The molecular formula of 6 was determined as C₂₀H₁₁N₃OCl₄, corresponding to 15 degrees of unsaturation, based on the HRESIMS. Its isotope pattern was also identical to that observed for 5, clearly indicating the presence of four chlorine atoms (Figure S47). The overall spectroscopic features of 6 were similar to those of 1–5. However, the ¹H and ¹³C NMR spectra displayed distinctive signals attributable to a carbonyl group and a methylene unit, together with nearly identical resonances arising from two 5,6-dichlorinated indole moieties (Table ). To resolve ambiguous correlations in ¹H and ¹³C NMR spectra, 2D selective HSQC and HMBC experiments were performed. Of the 15 degrees of unsaturation, 12 were assigned to two bis-chlorinated indole units. The remaining 3 degrees of unsaturation, in combination with a carbonyl group, one methylene, and two quaternary carbons, supported the formation of α,β-unsaturated γ-lactam. The connectivity between the α,β-unsaturated γ-lactam and a chlorinated indole unit was confirmed by key HMBC correlations from H-2″ to C-4 and from H-5 to C-4 (Figure ). These data unambiguously established the structure of 6 as a new lynamicin derivative.

2. ¹H (600 MHz) and ¹³C (150 MHz) NMR Data for 6–10 (CD₃OD).

	lynamicin L (6)		lynamicin M (7)		lynamicin N (8)		lynamicin O (9)		lynamicin P (10)
position	δC, type	δH, mult.	δC, type	δH, mult.	δC, type	δH, mult.	δC, type	δH, mult.	δC, type	δH, mult.
2	177.2, C		175.1, C		175.2, C		174.7, C		174.4, C
3	123.1, C		122.9, C		118.9, C		124.5, C		129.3, C
4	147.2, C		147.8, C		152.2, C		144.7, C		129.3, C
5	50.0, CH2	4.59, s	81.6, CH	6.01, s	91.7, CH	6.24, s	87.2, CH	6.03, s	174.4, C
6							51.6, CH3	3.28, s
2′	129.4, CH	7.52, s	129.9, CH	7.56, s	130.1, CH	7.61, s	130.1, CH	7.61, s	132.3, CH	7.84, s
3′	108.6, C		108.2, C		107.7, C		107.8, C		107.1, C
3a′	127.3, C		127.3, C		127.1, C		127.1, C		126.9, C
4′	122.6, CH	7.07, s	122.8, CH	7.05, s	122.7, CH	7.03, s	122.7, CH	6.98, s	123.3, CH	6.81, s
5′	126.3, C		126.3, C		126.5, C		126.3, C		126.8, C
6′	124.0, C		124.0, C		124.2, C		123.9, C		124.7, C
7′	114.1, CH	7.54, s	114.04, CH	7.52, s	114.2, CH	7.54, s	114.1, CH	7.53, s	114.3, CH	7.54, s
7a′	136.8, C		136.7, C		136.6, C		136.5, C		136.7, C
2″	129.6, CH	7.51, s	130.8, CH	7.68, s	132.5, CH	7.94, s	130.8, CH	7.68, s	132.3, CH	7.84, s
3″	111.5, C		110.6, C		109.6, C		110.3, C		107.1, C
3a″	126.9, C		126.67, C		126.4, C		126.4, C		126.9, C
4″	123.0, CH	7.02, s	123.5, CH	6.93, s	123.6, CH	6.87, s	123.3, CH	6.87, s	123.3, CH	6.81, s
5″	126.2, C		126.70, C		126.9, C		126.7, C		126.8, C
6″	124.9, C		124.5, C		124.8, C		124.5, C		124.7, C
7″	114.3, CH	7.50, s	114.02, CH	7.48, s	114.2, CH	7.50, s	114.1, CH	7.50, s	114.3, CH	7.54, s
7a″	137.1, C		136.9, C		136.9, C		136.8, C		136.7, C

^aChemical shift value was identified from the HMBC spectrum.

^bOverlapped signals.

Lynamicins M and N (7 and 8) were obtained as pale yellow and pale orange solids, respectively. The molecular formulas of 7 and 8 were deduced to be C₂₀H₁₁N₃O₂Cl₄ based on HRESIMS data, and their isotope patterns were characteristic of tetrachlorinated compounds (Figures S55 and S62). A detailed analysis of the NMR data revealed that 7 and 8 share an identical planar structure (Figure and Table ). The structures of 7 and 8 closely resembled lynamicin L (6), except for the presence of a hydroxy group at C-5. These compounds also exhibited structural similarity to tjipanazole J, which was reported to possess an undetermined stereocenter at the hydroxyl-bearing carbon and to exhibit no biological activity. In contrast to tjipanazole, 7 and 8 lack a carbazole moiety formed by aryl–aryl coupling between two pyrrole units and contain two additional chlorine atoms, underscoring their structural novelty. Distinct chemical shift differences were observed for the C-5 hydroxy-bearing carbon in 7 and 8 (δ_H 6.01 and δ_C 81.6 in 7; δ_H 6.24 and δ_C 91.7 in 8), strongly suggesting that these compounds are epimeric at C-5 (Table ). These observations provided the basis for the subsequent determination of their absolute configurations.

Lynamycin O (9) was isolated as a yellow solid. Its molecular formula was determined to be C₂₁H₁₃N₃O₂Cl₄, corresponding to 15 degrees of unsaturation based on HRESIMS data exhibiting a tetrachlorinated isotope pattern (Figure S69). The ¹H and ¹³C NMR data of 9 closely resembled those of 7 and 8 (Table ). Compound 9 was identified as the methylated derivative of 7, bearing a hydroxy group at C-3. Because hydroxy groups in natural products are prone to methylation under isolation conditions, the possibility that 9 is an artifact formed during the isolation process cannot be completely excluded. Nevertheless, considering that methoxylated analogues have been reported from related marine actinomycetes (lynamicins, spiroindimicins, and 6-OMe chromopyrrolic acid derivatives), a biosynthetic origin cannot be ruled out. Accordingly, the structural assignment of 9 is retained, while acknowledging the possibility of artifactual formation.

Lynamicin P (10) was obtained as a red-orange solid, and its HRESIMS spectrum displayed an isotope pattern containing four chlorine atoms, identical to those of 5–9 (Figure S76). The molecular formula of 10 was determined to be C₂₀H₉N₃O₂Cl₄ (16 degrees of unsaturation) based on the HRESIMS data. Compound 10 displayed only half sets of ¹H and ¹³C NMR signals, indicating that 10 has a symmetrical structure. Its overall scaffold was analogous to those of 6–9, except for the substitution at C-5. Unfortunately, no correlations between the carbonyl carbon and adjacent protons were observed in the conventional HMBC experiment optimized for 10 Hz. Therefore, a modified HMBC experiment optimized for ⁿ J _H–C of 5 Hz was carried out to obtain long-range (four- or five-bond) correlations, revealing HMBC correlations from H-2′ to C-2 and from H-4′ to C-3 (Figure ). These observations clearly established the presence of a 1H-pyrrole-2,5-dione moiety. Optically inactive 10 was found to be structurally related to dionemycin, except for the presence of an additional chlorine atom, thereby defining 10 as a new symmetrical tetrachlorinated bisindole pyrrole derivative. The proposed structure was further supported by its characteristic symmetry and consistent spectroscopic features compared with related lynamicin analogues.

The structures of 11–15 were identified as lynamicins A–E (11–15), previously reported, by comparing NMR and MS data with the literature (Figures S77–S89).

The absolute configurations of 5 and 7–9 were determined by comparison of their experimental CD spectra with the calculated ECD spectra of the corresponding conformers (Figure ). The experimental Cotton effects (CEs) of 5 (blue solid line) agreed well with those of the calculated 3′S-conformer (blue dashed line) in the negative CEs (230–290 nm) and positive CEs (300–340 nm) regions. Consequently, the absolute configuration of 5 was assigned to be the S-form. Compounds 7 and 8, which have identical plane structure, showed similar specific rotation values (7: [α]_D ²⁵ −20, c 0.1, CH₃OH; 8: [α]_D ²⁵ −29, c 0.1, CH₃OH), whereas their CD spectra exhibited opposite CEs in the 200–220 and 250–290 nm regions (Figure ), clearly indicating opposite configuration at C-5. The calculated ECD spectra of 5S and 5R conformers were in good agreement with experimental CD spectra of 7 and 8, respectively. Although 7 and 8 exhibited specific rotation values with the same negative sign, their ECD spectra showed clearly opposite CEs in the diagnostically important wavelength regions, supporting opposite absolute configurations at C-5. We also considered the possibility of axial chirality or configurational equilibration at the hydroxyl-bearing stereogenic center. However, no evidence for stable axial chirality was observed in structurally related analogues lacking stereogenic centers. In addition, repeated NMR measurements did not show detectable spectral changes over time, suggesting that rapid equilibration does not occur under the experimental conditions employed. The purity of 7 and 8 was further confirmed by HPLC and NMR analyses prior to optical rotation and ECD measurements. Thus, the absolute configurations at C-5 of 7 and 8 were determined to be 5S and 5R, respectively. The experimental CD spectrum of 9 was similar to that of 7 (Figure ). The wavelength region from 200 to 220 nm, corresponding to the α,β-unsaturated γ-lactam chromophore, , provides the key CEs to determine their absolute configuration. Therefore, the stereocenter at C-5 was assigned the S-configuration. Taken together, the good agreement between experimental and calculated ECD spectra provides strong support for the assigned absolute configurations of 5 and 7–9.

Compounds 1–15 were evaluated for antimicrobial activity against two Gram-positive bacteria (methicillin-resistant S. aureus USA300 and a pan-drug susceptible S. aureus ATCC25923) and two Gram-negative bacterial strains (Pseudomonas aeruginosa PAO1 and Klebsiella pneumoniae KPN ATCC43816). Compounds 1–15 exhibited strong antibacterial activity against Gram-positive bacteria, except for 8, but showed no significant activity against Gram-negative strains (Table ). Among all isolated compounds (1–15), 10 exhibited the strongest antibacterial activity with an MIC value of 0.5 μg/mL against methicillin-resistant S. aureus, approximately 7-fold stronger than the positive control, vancomycin (MIC = 3.8 μg/mL). Compounds 7 and 8, which are S–OH and R–OH epimers, showed a 58- to 80-fold difference in antibacterial potency, clearly demonstrating that stereochemistry at the hydroxyl-bearing carbon critically influences antibacterial activity. In addition, the antimicrobial effect varied depending on the position of methyl substitution. Compound 9, the methylated derivative of 7, also exhibited antibacterial activity comparable to that of 7, whereas previously reported lynamicins F and G, N-methylated in the pyrrole ring, showed no antibacterial activity. These observations indicate that methylation at the pyrrole nitrogen does not affect antibacterial efficacy, whereas methylation at the hydroxyl-bearing carbon does. In addition, methyl substitution at C-3′, accompanied by saturation of C-2′ and C-3′ of the indole moiety, increased antibacterial activity approximately 5- and 2-fold against antibiotic-resistant and pan-drug susceptible strains, compared with the unsaturated analogue (13). Positional chlorine isomers 3 and 4 exhibited comparable antibacterial activities, indicating that chlorine regiochemistry at this position has a limited impact on potency. For compounds exhibiting significant in vitro antibacterial activity, ex vivo efficacy was assessed using the H460 cell line infected with S. aureus USA300 (Table ). Compounds 2, 6, 7, 10, and 12 showed moderate efficacy with EC₉₅ values ranging from 14.2 – 15.0 μg/mL.

3. In Vitro and Ex Vivo Antibacterial Activities of 1–15 .

	MIC50 (μg/mL)				EC50 (μg/mL)	MIC95 (μg/mL)				EC95 (μg/mL)
	Gram-negative bacteria		Gram-positive bacteria			Gram-negative bacteria		Gram-positive bacteria
	P. aeruginosa PAO1	K. pneumoniae KPN ATCC48316	S. aureus ATCC25923	S. aureus USA300	H460 infection assay	P. aeruginosa PAO1	K. pneumoniae KPN ATCC48316	S. aureus ATCC25923	S. aureus USA300	H460 infection assay
1	>128	>128	3.6 (±0.5)	4.0 (±0.1)	11.5 (±0.8)	>128	>128	5.1 (±0.8)	4.9 (±0.2)	25.0 (±2.7)
2	>128	>128	1.4 (±0.05)	2.0 (±0.03)	12.0 (±0.1)	>128	>128	2.5 (±0.06)	2.3 (±0.1)	14.2 (±0.5)
3	>128	>128	1.2 (±0.1)	2.0 (±0.02)	7.5 (±2.1)	>128	>128	2.0 (±0.4)	2.3 (±0.04)	11.8 (±6.6)
4	>128	>128	1.5 (±0.3)	2.0 (±0.01)	6.3 (±6.1)	>128	>128	1.8 (±0.1)	2.4 (±0.02)	42.2 (±28.9)
5	>128	>128	1.9 (±0.04)	1.0 (±0.03)	9.5 (±0.8)	>128	>128	1.9 (±0.2)	3.2 (±0.2)	18.1 (±1.9)
6	>128	>128	1.3 (±0.2)	1.1 (±0.04)	12.6 (±0.2)	>128	>128	1.7 (±0.2)	1.3 (±0.04)	15.0 (±0.3)
7	>128	>128	1.3 (±0.05)	1.9 (±0.01)	7.4 (±0.4)	>128	>128	1.6 (±0.1)	2.2 (±0.02)	14.6 (±0.3)
8	>128	>128	92.2 (±2.9)	102.9 (±2.5)	N.T.	>128	>128	>128	>128	N.T.
9	>128	>128	1.1 (±0.03)	1.7 (±0.04)	6.0 (±4.2)	>128	>128	1.6 (±0.3)	1.8 (±0.1)	22.8 (±2.7)
10	71 (±14.6)	>128	0.3 (±0.03)	0.4 (±0.01)	12.1 (±0.04)	>128	>128	0.4 (±0.02)	0.5 (±0.01)	14.3 (±0.1)
11	>128	>128	1.5 (±0.5)	2.2 (±0.3)	11.1 (±1.1)	>128	>128	3.0 (±1.2)	2.4 (±0.9)	15.4 (±2.4)
12	>128	>128	1.2 (±0.2)	2.0 (±0.1)	10.4 (±2.9)	>128	>128	2.7 (±1.3)	2.3 (±0.1)	14.2 (±0.4)
13	>128	>128	5.4 (±2.5)	4.8 (±1.1)	5.9 (±4.3)	>128	>128	5.7 (±2.8)	15.4 (±1.5)	30.1 (±10.0)
14	>128	>128	3.7 (±0.2)	6.2 (±0.6)	17.7 (±1.1)	>128	>128	12.4 (±1.6)	9.1 (±0.3)	39.7 (±3.3)
15	>128	>128	6.1 (±0.1)	8.6 (±0.4)	19.8 (±0.03)	>128	>128	25.3 (±2.4)	60.9 (±19.2)	48.5 (±5.4)
VCM	N.T.	N.T.	0.7 (±0.01)	0.8 (±0.2)	N.T.	N.T.	N.T.	1.1 (±0.04)	3.8 (±3.4)	N.T.
CST	1.7 (±0.2)	0.8 (±0.1)	N.T.	N.T.	N.T.	1.9 (±0.3)	1.0 (±0.05)	N.T.	N.T.	N.T.
ERY	N.T.	N.T.	N.T.	N.T.	0.4 (±0.2)	N.T.	N.T.	N.T.	N.T.	3.9 (±1.1)

^aVCM, vancomycin; CST, colistin; ERY, erythromycin used as positive controls.

^bN.T. means not tested.

Compounds 2–15 (excluding 4 and 8) were evaluated for their cytotoxicity against six solid cancer cell lines (NUGC-3, stomach; NCI-H23, lung; HCT-15, colon; PC-3, prostate; MDA-MB-231, breast; ACHN, renal), along with a normal cell line (MRC-9, lung fibroblast), and seven blood cancer cell lines (HL-60, acute promyelocytic leukemia; K562, chronic myelogenous leukemia; NALM6, acute lymphoblastic leukemia; Raji, Burkitt’s lymphoma; RPMI-8402, T-cell acute lymphoblastic leukemia; U266, myeloma; WSU-DLCL2, diffuse large B cell lymphoma), along with a normal cell line (RPMI-1788, B lymphocyte) (Tables and ). Among the tested compounds, 7 showed strong overall activity against solid cancer cell lines but weak activity against blood cancer cell lines, suggesting some selectivity. Compound 10 exhibited strong cytotoxicity against both solid and blood cancer cell lines, except for the U266 myeloma cell line. Especially, 10 showed the strongest activity (GI₅₀ = 2.84 μM) against RPMI-8402, T-cell acute lymphoblastic leukemia cells. In contrast, 3 showed moderate antibacterial activity but weak cytotoxicity toward normal cells (MRC-9: GI₅₀ = 21.4 μM; RPMI-1788: GI₅₀ > 30 μM), highlighting its potential as a promising lead scaffold with improved therapeutic selectivity. Taken together, although not all compounds could be fully evaluated because of their limited availability and their precise mechanisms of action remain unclear, the diverse bisindole pyrrole scaffolds described herein provide valuable structural templates and biological insights for the development of new antibacterial and anticancer agents beyond conventional in vitro antibacterial screening.

4. Growth Inhibition (GI₅₀, μM) of 2–15 (Except for 4 and 8) against Solid Cancer Cell Lines.

	cell line
	MRC-9	NUGC-3	NCI-H23	HCT-15	PC-3	MDA-MB-231	ACHN
2	6.2 (±0.2)	4.6 (±0.3)	5.4 (±0.3)	6.0 (±0.0)	6.2 (±0.3)	6.0 (±0.1)	5.6 (±0.4)
3	21.4 (±0.5)	10.2 (±2.5)	14.5 (±2.6)	13.5 (±1.8)	13.1 (±2.8)	16.0 (±1.7)	14.0 (±1.9)
5	7.1 (±0.1)	5.2 (±0.4)	6.3 (±0.3)	5.4 (±1.0)	5.6 (±0.0)	6.8 (±0.0)	5.5 (±0.1)
6	16.2 (±2.4)	9.1 (±1.4)	13.7 (±1.1)	13.8 (±2.4)	15.6 (±1.4)	15.8 (±0.5)	14.6 (±1.2)
7	6.0 (±0.6)	4.1 (±0.0)	4.7 (±0.6)	4.7 (±0.8)	5.1 (±0.4)	5.7 (±0.2)	5.2 (±0.1)
9	7.7 (±0.7)	5.7 (±0.2)	5.2 (±0.1)	5.7 (±0.3)	6.3 (±0.5)	5.2 (±1.0)	5.3 (±0.1)
10	6.5 (±0.4)	4.9 (±0.3)	5.8 (±0.4)	4.9 (±0.1)	5.4 (±1.1)	5.6 (±0.2)	5.9 (±0.1)
11	6.2 (±0.8)	4.0 (±0.1)	5.8 (±1.2)	6.1 (±0.7)	6.2 (±0.6)	6.3 (±0.1)	5.8 (±0.0)
12	17.7 (±1.4)	11.2 (±1.2)	16.3 (±1.5)	15.6 (±3.0)	15.8 (±0.9)	14.6(±2.4)	15.8 (±1.6)
13	10.4 (±0.6)	9.6 (±0.6)	9.7 (±1.9)	9.9 (±1.8)	10.1 (±0.6)	11.2 (±0.7)	11.3 (±1.0)
14	16.6 (±0.1)	11.6 (±1.0)	17.2 (±3.5)	16.4 (±2.4)	19.8 (±0.0)	14.8 (±2.1)	18.5 (±1.3)
15	19.9 (±1.9)	10.7 (±0.3)	17.5 (±0.9)	16.9 (±0.1)	18.7 (±0.6)	16.6 (±0.0)	17.2 (±0.7)
ADR	0.3 (±0.0)	0.2 (±0.0)	0.3 (±0.0)	0.3 (±0.0)	0.3 (±0.0)	0.2 (±0.0)	0.3 (±0.1)

^aADR, adriamycin used as a positive control.

^bNormal cell line.

5. Growth Inhibition (GI₅₀, μM) of 2–15 (Except for 4 and 8) against Blood Cancer Cell Lines.

	cell line
	RPMI-1788	HL-60	K562	NALM6	Raji	RPMI-8402	U266	WSU-DLCL2
2	18.0 (±0.6)	27.3 (±0.0)	15.7 (±0.2)	21.5 (±0.2)	18.2 (±0.8)	13.3 (±0.2)	>30	16.9 (±0.6)
3	>30	>30	28.5 (±2.0)	>30	>30	>30	>30	>30
5	18.2 (±0.6)	17.0 (±0.4)	9.1 (±0.4)	12.4 (±0.2)	17.0 (±0.9)	8.9 (±0.0)	>30	15.8 (±0.5)
6	>30	>30	23.6 (±0.3)	>30	>30	22.9 (±0.7)	>30	25.4 (±1.3)
7	17.9 (±0.3)	27.5 (±2.8)	17.2 (±0.1)	15.2 (±0.1)	21.5 (±1.2)	12.9 (±0.8)	>30	18.5 (±1.2)
9	>30	>30	27.9 (±1.3)	14.5 (±1.0)	26.5 (±1.6)	16.7 (±0.2)	>30	18.2 (±2.1)
10	9.2 (±0.3)	4.0 (±1.1)	3.3 (±0.1)	3.0 (±0.2)	8.3 (±1.2)	2.8 (±0.2)	>30	10.5 (±0.2)
11	18.4 (±0.9)	14.9 (±1.7)	13.8 (±0.0)	18.2 (±0.9)	17.4 (±0.1)	12.4 (±0.8)	>30	13.9 (±0.3)
12	18.2 (±0.7)	>30	17.7 (±0.3)	25.0 (±0.7)	19.7 (±0.7)	17.0 (±0.0)	>30	15.2 (±1.2)
13	14.6 (±0.1)	18.5 (±0.1)	7.8 (±0.0)	10.0 (±0.8)	11.7 (±0.1)	10.2 (±0.1)	>30	12.2 (±0.2)
14	21.1 (±1.0)	>30	17.6 (±0.4)	29.7 (±3.4)	8.0 (±0.2)	19.4 (±0.8)	>30	16.4 (±0.4)
15	>30	>30	>30	>30	>30	>30	>30	22.5 (±0.2)
ADR	0.01 (±0.00)	0.02 (±0.00)	0.1 (±0.0)	0.01 (±0.00)	0.02 (±0.00)	0.1 (±0.0)	0.05 (±0.01)	0.01 (±0.00)

^aADR, adriamycin used as a positive control.

^bNormal cell line.

Experimental Section

General Experimental Procedures

Specific rotations were measured with a Rudolph Analytical Autopol III S2 polarimeter. IR spectra were obtained on a JASCO FT/IR-4100. UV spectra were recorded with a Shimadzu UV-1650PC spectrophotometer, and CD spectra were measured with a JASCO J-1500 polarimeter at the Gyeongnam Bio and Antiaging Core Facility Center for Changwon National University in Changwon, Republic of Korea. NMR spectroscopic data were obtained using a Bruker AVANCE III 600 spectrometer with a 3 mm probe operating at 600 MHz (¹H) and 150 MHz (¹³C). Chemical shifts were referenced to solvent peaks for CD₃OD (δ_H 3.31 and δ_C 49.15 ppm) and CDCl₃ (δ_H 7.24 and δ_C 77.23 ppm). LRESIMS data were collected by a Thermo Scientific UltiMate 3000 system equipped with an ISQ EM single quadrupole mass spectrometer. HRESIMS data were obtained with a Waters SYNPT G2 Q-TOF mass spectrometer at the Korea Basic Science Institute (KBSI) in Cheongju, Republic of Korea. HPLC was performed using a PrimeLine binary pump with a Shodex RI-101 refractive index detector and an S3210 variable UV detector with YMC Triart C₁₈ (250 mm × 10 mm, 5 μm). Reversed-phase silica gel (YMC-Gel ODS-A, 12 nm, S-75 μm) was used for vacuum and flash column chromatography. The solvents used were either HPLC grade or distilled prior to use.

Strain Material and Fermentation

Streptomyces massiliensis 213DD-128 (GenBank No. OR807412) was isolated from a sediment sample collected from the East Sea, Republic of Korea, in April 2021. Detailed information on the isolation procedure for the strain was described in our previous study. The seed and mass cultures of the strain 213DD-128 were carried by the Bennett’s (BN) broth medium, which consists of 10 g of glucose, 2 g of tryptone, 1 g of yeast extract, 1 g of beef extract, 5 g of glycerol, and 32 g of artificial sea salt per 1 L of distilled water.

The primary seed culture was performed in a 100 mL flask with 50 mL of BN broth medium at 28 °C for 5 days on a rotary shaker at 120 rpm. For the secondary seed culture, an aliquot (0.2%, v/v) of the primary seed culture broth was transferred into a 2L-flask containing 700 mL of BN broth under aseptic conditions and cultivated under the same conditions. The secondary seed culture broth was inoculated into a 100 L fermenter containing 70 L of the broth medium with 0.05% antifoam. The fermenter was operated at 28 °C, 50 rpm, and an airflow rate of 20 L per minute (LPM) for 19 days.

Extraction and Isolation of 1–15

The fermentation broth (70 L) was extracted twice with an equal volume of ethyl acetate (EtOAc, 70 L × 2). The EtOAc layer was evaporated to obtain a crude extract (ca. 63 g). A part of the extract (5.94 g) was subjected to ODS-A vacuum column chromatography using step gradient elution with MeOH/H₂O (1:4, 2:3, 3:2, 4:1, and 5:0, v/v). Each fraction was divided into three fractions. The first 80% MeOH fraction (497.2 mg) was purified by reversed-phase semipreparative HPLC (YMC Triart C₁₈, 250 mm × 10 mm, 5 μm; 67% MeOH/H₂O; flow rate: 2.0 mL/min; detector: RI and UV: 224 nm) to give 1 (0.8 mg, t _R 21 min), 7 (0.9 mg, t _R 36 min), 8 (0.7 mg, t _R 57 min), 11 (53.4 mg, t _R 39 min), 12 (14.8 mg, t _R 72 min), 14 (2.0 mg, t _R 46 min), and 15 (2.4 mg, t _R 30 min). The second 80% MeOH fraction (1.64 g) was fractionated on ODS-A flash column chromatography with MeOH/H₂O (6:4, 7:3, 8:2, 9:1, and 10:0, v/v), and each elution was collected into three subfractions. The third 60% MeOH subfraction (60.1 mg) was applied to reversed-phase semipreparative HPLC (YMC Triart C₁₈, 250 mm × 10 mm, 5 μm; 50% MeCN/H₂O; flow rate: 2.0 mL/min; detector: RI and UV: 224 nm), affording 7 (3.1 mg, t _R 21 min) and 11 (35.4 mg, t _R 59 min). The first 70% MeOH subfraction (806.8 mg) was subjected to ODS-A vacuum column chromatography followed by step gradient elution with MeCN/H₂O (1:4, 2:3, 3:2, 4:1, and 5:0, v/v). The second 80% MeCN subfraction (98.2 mg) was purified by reversed-phase semipreparative HPLC (YMC Triart C₁₈, 250 mm × 10 mm, 5 μm; 65% MeCN/H₂O; flow rate: 2.0 mL/min; detector: RI and UV: 224 nm) to obtain 3 (0.6 mg, t _R 27 min), 4 (0.4 mg, t _R 29 min), 6 (0.5 mg, t _R 13 min), and 13 (63.5 mg, t _R 36 min). The second 70% MeOH subfraction (573.8 mg) was applied to reversed-phase semipreparative HPLC (YMC Triart C₁₈, 250 mm × 10 mm, 5 μm; 50% MeCN/H₂O; flow rate: 2.2 mL/min; detector: RI and UV: 224 nm) to yield 6 (2.3 mg, t _R 32 min), minor fraction-1 containing 9 and 10 (5.5 mg), and minor fraction-2 containing 2 (6.3 mg). The minor fraction-1 was further purified by reversed-phase HPLC (YMC Triart C₁₈, 250 mm × 10 mm, 5 μm; 47% MeCN/H₂O; flow rate: 2.0 mL/min; detector: RI and UV: 224 nm) to give 9 (1.4 mg, t _R 52 min) and 10 (1.1 mg, t _R 90 min). The minor fraction-2 was purified by reversed-phase HPLC (YMC Triart C₁₈, 250 mm × 10 mm, 5 μm; 45% MeCN/H₂O; flow rate: 2.2 mL/min; detector: RI and UV: 224 nm) to obtain 2 (1.9 mg, t _R 103 min). The third 70% MeOH subfraction (80.8 mg) was applied to reversed-phase HPLC (YMC Triart C₁₈, 250 mm × 10 mm, 5 μm; 50% MeCN/H₂O; flow rate: 2.2 mL/min; detector: RI and UV: 224 nm) to get 5 (7.1 mg, t _R 127 min) and 10 (0.7 mg, t _R 39 min).

Lynamicin H (1): Off-white solid; UV (CH₃OH) λ_max (log ε) 203 (1.62), 223 (1.66), 291 (1.20) nm; IR (CH₃OH) γ_max 3403 (amine), 2925, 1692 (carbonyl), 1452, 1374, 1261 cm^–1; ¹H and ¹³C NMR data, Table ; HRESIMS m/z 412.0830 [M + Na]⁺ (calcd for C₂₂H₁₆N₃O₂NaCl, 412.0829).

Lynamicin I (2): Off-white solid; UV (CH₃OH) λ_max (log ε) 205 (1.88), 223 (1.87), 244 (1.77), 291 (1.44) nm; IR (CH₃OH) γ_max 3406 (amine), 2940, 1689 (carbonyl), 1448, 1374, 1247 cm^–1; ¹H and ¹³C NMR data, Table ; HRESIMS m/z 424.0622 [M + H]⁺ (calcd for C₂₂H₁₆N₃O₂Cl₂, 424.0620) and 446.0435 [M + Na]⁺ (calcd for C₂₂H₁₅N₃O₂NaCl₂, 446.0439).

Lynamicin J (3): Yellow solid; UV (CH₃OH) λ_max (log ε) 208 (2.05), 234 (2.01), 294 (1.31) nm; IR (CH₃OH) γ_max 3423 (amine), 2936, 1450, 1011, 797 cm^–1; ¹H and ¹³C NMR data, Table ; HRESIMS m/z 400.0176 [M + H]⁺ (calcd for C₂₀H₁₃N₃Cl₃, 400.0175).

Lynamicin K (4): Yellow solid; UV (CH₃OH) λ_max (log ε) 208 (2.21), 234 (2.16), 289 (1.52) nm; IR (CH₃OH) γ_max 3420 (amine), 2933, 1569, 1448 cm^–1; ¹H and ¹³C NMR data, Table ; HRESIMS m/z 400.0172 [M + H]⁺ (calcd for C₂₀H₁₃N₃Cl₃, 400.0175).

Indimicin H (5): Off-White solid; [α]_D ²⁵ −73 (c 0.1, CH₃OH); UV (CH₃OH) λ_max (log ε) 210 (2.49), 237 (1.85), 306 (1.18) nm; ECD (1.11 mM, CH₃OH) λ_max (Δε) 326 (+20.2), 253 (−52.9), 215 (+5.1) nm; IR (CH₃OH) γ_max 3441 (amine), 2965, 1456 (C–H alkane) cm^–1; ¹H and ¹³C NMR data, Table ; HRESIMS m/z 450.0099 [M + H]⁺ (calcd for C₂₁H₁₆N₃Cl₄, 450.0098).

Lynamicin L (6): Pale yellow solid; UV (CH₃OH) λ_max (log ε) 203 (1.65), 216 (1.64), 232 (1.65), 293 (1.24) nm; IR (CH₃OH) γ_max 3250 (amide), 2929, 1657 (carbonyl), 1448, 1021 cm^–1; ¹H and ¹³C NMR data, Table ; HRESIMS m/z 449.9732 [M + H]⁺ (calcd for C₂₀H₁₂N₃OCl₄, 449.9734) and 471.9551 [M + Na]⁺ (calcd for C₂₀H₁₁N₃ONaCl₄, 471.9554).

Lynamicin M (7): Pale yellow solid; [α]_D ²⁵ −20 (c 0.1, CH₃OH); UV (CH₃OH) λ_max (log ε) 207 (1.92), 233 (1.90), 290 (1.46), 346 (1.33) nm; ECD (1.07 mM, CH₃OH) λ_max (Δε) 277 (+1.16), 248 (+1.31), 200 (−2.41) nm; IR (CH₃OH) γ_max 3258 (hydroxy), 1675 (carbonyl), 1448, 1106 (C–O stretch) cm^–1; ¹H and ¹³C NMR data, Table ; HRESIMS m/z 487.9504 [M + Na]⁺ (calcd for C₂₀H₁₁N₃O₂NaCl₄, 487.9503).

Lynamicin N (8): Pale orange solid; [α]_D ²⁵ −29 (c 0.1, CH₃OH); UV (CH₃OH) λ_max (log ε) 200 (1.80), 294 (1.21), 348 (1.00) nm; ECD (1.07 mM, CH₃OH) λ_max (Δε) 332 (+0.67), 264 (−0.52), 213 (+1.71) nm; IR (CH₃OH) γ_max 3416 (amine and hydroxy), 2923, 1738 (carbonyl), 1385, 1254 (C–O stretch) cm^–1; ¹H and ¹³C NMR data, Table ; HRESIMS m/z 465.9685 [M + H]⁺ (calcd for C₂₀H₁₂N₃O₂Cl₄, 465.9684) and 463.9527 [M – H]⁻ (calcd for C₂₀H₁₀N₃O₂Cl₄, 463.9527).

Lynamicin O (9): Yellow solid; [α]_D ²⁵ −20 (c 0.1, CH₃OH); UV (CH₃OH) λ_max (log ε) 210 (1.66), 231 (1.62), 291 (1.20), 349 (1.05) nm; ECD (1.04 mM, CH₃OH) λ_max (Δε) 334 (+0.34), 265 (−0.74), 223 (−0.20), 209 (−1.16) nm; IR (CH₃OH) γ_max 3286 (amide), 2925, 1681 (carbonyl), 1448, 1106 cm^–1; ¹H and ¹³C NMR data, Table ; HRESIMS m/z 501.9661 [M + Na]⁺ (calcd for C₂₁H₁₃N₃O₂NaCl₄, 501.9660).

Lynamicin P (10): Red orange solid; UV (CH₃OH) λ_max (log ε) 209 (1.99), 291 (1.34), 355 (1.01), 439 (1.12) nm; IR (CH₃OH) γ_max 3297 (amide), 2933, 1702 (carbonyl), 1530, 1448, 1346 cm^–1; ¹H and ¹³C NMR data, Table ; HRESIMS m/z 463.9524 [M + H]⁺ (calcd for C₂₀H₁₀N₃O₂Cl₄, 463.9527) and 485.9350 [M + Na]⁺(calcd for C₂₀H₉N₃O₂NaCl₄, 485.9347).

Lynamicin A (11): Off-white solid; ¹H NMR (600 MHz, CDCl₃) δ_H 9.38 (1H, d, J = 2.3 Hz, NH-1), 8.07 (1H, d, J = 1.3 Hz, NH-1′), 7.88 (1H, d, J = 1.3 Hz, NH-1″), 7.44 (1H, d, J = 1.7 Hz, H-4″), 7.22 (1H, d, J = 1.7 Hz, H-4′), 7.07 (1H, d, J = 3.1 Hz, H-5), 7.00 (1H, dd, J = 8.6, 1.7 Hz, H-6″), 6.95 (1H, d, J = 8.6 Hz, H-7″), 6.92 (1H, dd, J = 8.6, 1.7 Hz, H-6′), 6.88 (1H, d, J = 8.6 Hz, H-7′), 6.77 (1H, d, J = 2.4 Hz, H-2′), 6.43 (1H, d, J = 2.4 Hz, H-2″), 3.62 (3H, s, H₃-7); ¹³C NMR (150 MHz, CDCl₃) δ_C 161.5 (C-6), 134.3 (C-7a′), 134.2 (C-7a″), 128.1 (C-3a′), 127.8 (C-3a″), 125.9 (C-2′), 125.4 (C-5″), 125.2 (C-5′), 124.0 (C-2″), 122.1 (C-2 and C-6″), 121.9 (C-6′), 121.1 (C-5), 120.2 (C-4), 120.1 (C-3), 119.7 (C-4′), 119.0 (C-4″), 112.4 (C-7″), 112.3 (C-7′), 109.6 (C-3″), 109.1 (C-3′), 51.5 (C-7); LRESIMS m/z 422.07 [M – H]⁻ and 446.05 [M + Na]⁺.

Lynamicin B (12): Off-white solid; ¹H NMR (600 MHz, CDCl₃) δ_H 9.38 (1H, s, NH-1), 8.16 (1H, s, NH-1′), 7.96 (1H, s, NH-1″), 7.47 (1H, s, H-4″), 7.19 (1H, s, H-7″), 7.18 (1H, d, J = 1.9 Hz, H-4′), 7.15 (1H, d, J = 3.1 Hz, H-5), 7.02 (1H, d, J = 8.6 Hz, H-7′), 6.95 (1H, dd, J = 8.6, 1.9 Hz, H-6′), 6.91 (1H, d, J = 2.4 Hz, H-2′), 6.54 (1H, d, J = 2.4 Hz, H-2″), 3.66 (3H, s, H-7); ¹³C NMR (150 MHz, CDCl₃) δ_C 161.5 (C-6), 134.7 (C-7a″), 134.3 (C-7a′), 128.7 (C-3a′), 126.6 (C-3a″), 125.9 (C-2′), 125.6 (C-5″), 125.4 (C-5′), 124.4 (C-2″), 123.7 (C-6″), 122.2 (C-6′), 122.1 (C-2), 121.0 (C-5), 120.7 (C-4″), 120.4 (C-4), 119.9 (C-3), 119.8 (C-4′), 112.7 (C-7″), 112.3 (C-7′), 110.0 (C-3″), 109.3 (C-3′), 51.6 (C-7); LRESIMS m/z 456.05 [M – H]⁻ and 480.12 [M + Na]⁺.

Lynamicin C (13): Orange solid; ¹H NMR (600 MHz, CDCl₃) δ_H 8.42 (1H, s, NH-1), 7.98 (2H, s, NH-1′ and NH-1″), 7.57 (2H, s), 7.36 (2H, s), 7.02 (2H, d, J = 1.8 Hz), 6.86 (2H, d, J = 2.4 Hz); ¹³C NMR (150 MHz, CDCl₃) δ_C 134.9, 127.1, 125.7, 124.1 (C × 2), 123.8, 121.4 (C × 2), 117.5 (C × 2), 115.5, 112.7 (C × 2), 111.5; LRESIMS m/z 432.08 [M – H]⁻.

Lynamicin D (14): Off-white solid; ¹H NMR (600 MHz, CD₃OD) δ_H 7.19 (2H, d, J = 9.0 Hz, H-7′ and H-7″), 6.99 (2H, d, J = 1.8 Hz, H-4′ and H-4″), 6.95 (2H, s, H-2′ and H-2″), 6.92 (2H, dd, J = 9.0, 1.8 Hz, H-6′ and H-6″), 3.70 (6H, s, H-7 and H-9); LRESIMS m/z 480.10 [M – H]⁻, 482.12 [M + H]⁺, and 504.11 [M + Na]⁺.

Lynamicin E (15): Pale yellow solid; ¹H NMR (600 MHz, CD₃OD) δ_H 7.25 (1H, d, J = 7.8 Hz), 7.18 (1H, d, J = 9.0 Hz), 7.07 (1H, d, J = 7.8 Hz), 7.02 (1H, d, J = 1.8 Hz), 6.98 (1H, ddd, J = 8.4, 7.2, 1.2 Hz), 6.92 (1H, dd, J = 9.0, 1.8 Hz), 6.91 (1H, s), 6.87 (1H, s), 6.80 (1H, t, J = 7.8 Hz), 3.69 (3H, s), 3.66 (3H, s); LRESIMS m/z 446.11 [M – H]⁻.

Calculation of ECD Spectra

The primary geometry optimization and conformational searches were conducted with Conflex 8 (Rev. B, Conflex Corp., Tokyo, Japan). Geometry optimization, frequency, and energy calculations for electronic circular dichroism (ECD) were performed using Gaussian 16 (Rev. B.01, Gaussian Corp., USA). Conformational searches were applied with MMFF94s force field with a search limit of 10 kcal/mol. The conformers of 5 and 7–9 were optimized using the ground state method at Hartree–Fock/6–31+G­(d,p) level in the gas phase. The theoretical calculations of ECD spectra were carried out using TD-SCF at B3LYP or CAM-B3LYP/6–31G+(d,p) or dgdzvp level (Nstates = 15, 30, or 50) in methanol with an IEFPCM model. The calculated ECD spectra of each conformer were summed based on their Boltzmann distribution and heat energy using SpecDis 1.71 software. All calculated curves were simulated by SpecDis 1.71 using σ = 0.2–0.4 eV and shifted ± 12 nm to better match the experimental spectra.

Strains and Culture Conditions for Antibacterial Activity Test

For the antibacterial activity test, S. aureus USA300, a methicillin-resistant strain, was purchased from the Nebraska Transposon Mutant Library. The other strains, such as a pan-drug susceptible S. aureus ATCC25923, Pseudomonas aeruginosa PAO1, and Klebsiella pneumoniae KPN ATCC48316, were obtained from the American Type Culture Collection (ATCC). S. aureus strains, P. aeruginosa, and K. pneumoniae were cultivated in Cation-Adjusted Mueller Hinton Broth (CAMHB) at 37 °C. Stock vials containing 25% glycerol were stored in a deep freezer at −80 °C. For each experiment, prepared stocks were inoculated into 10 mL of CAMHB and incubated at 37 °C in a shaking incubator at 180 rpm overnight. For the preliminary screen and dose–response experiments, 100 μL of activated stock was inoculated into 10 mL of fresh CAMHB in tubes and incubated at 37 °C with shaking at 180 rpm for 3 h.

Dose–Response Curve Assay

Antibacterial susceptibility testing was conducted using a modified Clinical and Laboratory Standards Institute (CLSI)-based broth microdilution assay. To accommodate compound-limited screening, assays were performed in 384-well microplates rather than in the conventional 96-well format. Overnight cultures grown in CAMHB were subcultured (100 μL into 10 mL fresh CAMHB) and incubated for 3 h at 37 °C with shaking at 180 rpm to obtain log-phase growing cells. The assay inoculum was prepared by diluting actively growing cultures from a measured density of OD₆₀₀ = 0.5 to calculated dilution targets (0.005 for S. aureus and 0.0005 for P. aeruginosa and K. pneumoniae). This procedure yielded a final bacterial density of approximately 5 × 10⁵ CFU/mL per well.

Test compounds were initially dissolved in 100% DMSO to prepare stock solutions and subsequently diluted with assay medium to ensure that the final DMSO concentration in each well did not exceed 0.5% (v/v), a level confirmed to be noninhibitory to bacterial growth. Assays were performed in 384-well plates using a final volume of 50 μL per well, with a 13-point 2-fold serial dilution starting at 128 μg/mL. After incubation at 37 °C for 18–24 h, bacterial growth was assessed by measuring absorbance at 600 nm using an EnSight multimode plate reader (Revvity, Waltham, MA, USA).

For quantitative analysis of antibacterial activity, percent inhibition was calculated by normalizing bacterial growth between untreated and positive-control conditions. Bacterial growth in the untreated control, representing normal growth in the absence of compound-induced stress, was defined as 0% inhibition, whereas the positive-control condition, in which bacterial growth was suppressed, was defined as 100% inhibition. Using this same normalization scheme, MIC₅₀ and MIC₉₅ were determined from the dose–response curves as the concentrations producing 50% and 95% growth inhibition, respectively, relative to these two reference conditions. Both values were calculated using GraphPad Prism. Assay performance was evaluated using standard quality-control metrics, and the assay showed a Z′ factor of 0.7, with coefficients of variation (CVs) of 8.9% and 1.2% for the untreated and positive-control groups, respectively, indicating acceptable robustness and reproducibility for quantitative analysis.

Effect of Compounds on a Cell Infection Model of S. aureus

To evaluate the ex vivo antibacterial activity of the compounds, an H460 cell-based infection model was used. The H460 cells were cultured in 96-well plates in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere containing 5% CO₂ until full confluence was reached. S. aureus USA300 carrying pCM29 encoding GFP was cultured in CAMHB containing 10 μg/mL chloramphenicol, harvested, washed with phosphate-buffered saline (PBS), and resuspended in RPMI 1640 medium. Precultured H460 cells were infected with S. aureus (1 × 10⁷ CFU) and incubated for 2.5 h. After infection, cells were washed three times with PBS, treated with gentamicin (50 μg/mL in RPMI 1640) for 1 h to remove extracellular bacteria, washed again with PBS, and then treated with serially diluted compounds in RPMI 1640 for 24 h at 37 °C in 5% CO₂. Intracellular bacterial burden was assessed by measuring GFP signal using an EnSight multimode plate reader and an Opera Phenix Plus high-content imaging system (Revvity, Waltham, MA, USA).

For quantitative analysis of ex vivo activity, percent inhibition was calculated using the same normalization strategy applied in the in vitro antibacterial assay. The infected untreated control was defined as 0% inhibition, and the positive-control condition showing maximal suppression of intracellular bacterial signal was defined as 100% inhibition. Based on this normalization, EC₅₀ and EC₉₅ were determined from the dose–response curves as the concentrations producing 50% and 95% reduction of the intracellular bacterial GFP signal, respectively. Both values were calculated using GraphPad Prism.

Confocal Images

Ex vivo infection model images were generated with an Opera Phenix Plus high-content imaging system. Images were obtained by a confocal microscope with a 20× water objective lens. To detect the bacterial fluorescence signal (GFP), the Alexa 488 channel (488 nm laser/500–550 filter) was used for the bacterial GFP signal image.

Cytotoxicity Tests against Human Solid and Blood Cancer Cell Lines

A detailed experimental procedure for cytotoxicity test by SRB assay and CellTiter-Glo assay was carried out as previously described.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.6c02660.

• NMR, HRMS, and LRMS spectra for isolated compounds; geometry optimized conformers; dose–response curves and confocal images in the H460 infection model; and cytotoxicity assay results (PDF)

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

This research was supported by the Korea Institute of Ocean Science and Technology (PEA0314), the Ministry of Oceans and Fisheries, Korea (Grant no. PG54801), and the National Research Foundation (NRF) funded by the Korean government (MSIT) (No. RS-2024–00398073).

The authors declare that a Korean patent application has been filed in connection with the results reported in this work.

The authors declare no competing financial interest.