Actinomycin Analogs From Streptomyces polyantibioticus S4D258: Structural Elucidation and Antiproliferative Activity Against Triple‐Negative Breast Cancer

ABSTRACT

Fifteen new actinomycin analogs were obtained from the marine‐derived Streptomyces polyantibioticus S4D258. Structural elucidation of these compounds was successfully achieved by comprehensive analysis of high‐resolution electrospray ionization mass spectrometry (HR‐ESIMS), nuclear magnetic resonance spectroscopy (NMR), and Marfey's method. Notably, actinomycins H₁–H₅ (1–5) contain L‐serine (L‐Ser), D‐2‐aminobutyric acid (D‐2‐Aba), and L‐2‐methylaminobutyric acid (L‐2‐MeAba) residues, whereas actinomycin H₆ (6) comprises a 5H‐oxazolo[4,5‐b]phenoxazine chromophore. Actinomycins H₇–H₁₅ (7–15) are nine truncated actinomycin analogs only containing one pentapeptidolactone unit. Actinomycins H₁ (1) and H₉ (9) exhibited inhibitory activities against the proliferation of triple‐negative breast cancer MDA‐MB‐231 cells with IC50 values of 28.00 nM and 4.34 µM, respectively. Mechanistic investigation revealed that the above two actinomycin analogs not only induce S‐phase arrest, mitochondrial membrane potential depolarization, and apoptosis, but also trigger ferroptosis, as revealed by an increase in the level of reactive oxygen species, lipid peroxidation, and malondialdehyde. Collectively, our findings enriched the structural diversity of natural actinomycins and inspired the re‐examination of actinomycin analogs as anticancer leads against triple‐negative breast cancer.

Fifteen new actinomycin analogs, named actinomycins H₁–H₁₅, are identified from S. polyantibioticus S4D258. Actinomycins H₇–H₁₅ are truncated actinomycin analogs. Actinomycins H₁–H₆ and H₉ exhibit significant antiproliferative activities against both MDA‐MB‐231 and 4T1 TNBC cells. Mechanistic investigations reveal that actinomycins H₁ and H₉ induce S‐phase arrest, apoptosis, and mitochondrial membrane potential disruption.

1. Introduction

Triple‐negative breast cancer (TNBC) accounts for 15% to 20% of breast cancers [1]. Both aggressive pathology and metastatic tendency indicate the worse prognosis of TNBC, compared with other subtypes of breast cancer [2]. Due to the absence of reliable biomarkers, the clinical treatment of TNBC still relies on cytotoxic agents, including anthracyclines, taxanes, and platinum compounds [3, 4]. However, the clinical benefits of cytotoxic drugs are often curtailed by the emergence of drug resistance, underscoring the urgent need for the development of new drugs with high efficacy, low toxicity, and new mechanism of action [5]. During the recent years, 4‐thiazolidinone, azo‐ether and azo‐sulfone compounds, and thiazolidin‐4‐one derivatives have been synthesized for the development of new anticancer drug leads [6, 7, 8].

Actinomycins, predominantly produced by Streptomyces, are a family of chromopeptide antibiotics, of which the molecule consists of three parts, viz., a conserved tricyclic phenoxazinone core and two pentapeptidolactone loops (α‐ and β‐ring), connected by two amide bonds [9, 10]. These chromopeptide antibiotics exhibited potent antitumor and antibacterial activities. In double‐stranded DNA, the chromophore core of actinomycin inserts between G–C and C–G base pairs, whereas the two pentapeptidolactone loops occupy the minor groove. Collectively, the above modes of action form a stable DNA–ligand complex to block transcription [11]. Actinomycin D, a representative anticancer member of the actinomycin family, remains the cornerstone of combination chemotherapy for malignancies, such as gestational trophoblastic neoplasia, rhabdomyosarcoma, Wilms’ tumor, and Ewing's sarcoma [12, 13, 14, 15]. However, despite its clinical efficacy, dose–limiting toxicity and drug resistance restrict the widespread clinical application of actinomycin D [16, 17]. The structural diversity–oriented natural discovery of actinomycin analogs, combined with rational structural redesign, provides a feasible approach to discover highly effective and low‐toxic anticancer drugs [18].

Since the discovery of the first actinomycin member in 1940, nearly 40 members, all sharing a conserved phenoxazinone chromophore, have been reported. The structural diversity of these members originates from difference in amino acid residue composition of the two pentapeptidolactone rings [19, 20]. Notably, hemiactinomycin, an unusual truncated actinomycin analog containing only a single pentapeptidolactone unit, is an exception, indicating the nonconservation of the canonical framework comprising two pentapeptidolactone units [21]. However, the architectural plasticity of truncated actinomycins remains underexplored.

To find new anti‐TNBC compounds from marine actinomycetes, 15 new actinomycin analogs, named actinomycins H₁–H₁₅ (1–15, Figure 1), were obtained from a marine‐derived Streptomyces polyantibioticus S4D258. These newly obtained actinomycin analogs feature pentapeptidolactone loops incorporating atypical amino acid residues (e.g., 4‐hydroxyproline (Hyp), Ser, 2‐Aba, and 2‐MeAba) or truncated actinomycins containing only a pentapeptidolactone unit. Notably, actinomycins H₁ (1) and H₉ (9) inhibit MDA‐MB‐231 cell proliferation and induce S‐phase arrest. Further mechanistic investigation revealed that the above two actinomycin analogs induce both apoptosis and ferroptosis, highlighting the potential of 1 and 9 as lead structures for novel anti‐TNBC agents. The structure elucidation and anti‐TNBC activities of fifteen actinomycin analogs (1–15) are reported, along with the anticancer mechanistic investigation of actinomycins H₁ (1) and H₉ (9).

FIGURE 1.

Chemical structures of compounds 1–15.

2. Results and Discussion

Compound 1, a red amorphous powder, had the molecular formula C₆₁H₈₄N₁₂O₁₆ as deduced by analysis of the positive HR‐ESIMS ion at m/z 1241.6220 (calcd for [M + H]⁺, 1241.6201) (Figure S2). The NMR data of 1 indicated the presence of a phenoxazinone chromophore and two pentapeptidolactone moieties (Tables 1 and 3; Figures S3–S9). The extended sequence of amino acid residues in the α‐pentapeptidolactone ring of 1 consists of valine (Val), proline (Pro), sarcosine (Sar), 2‐MeAba, and threonine (Thr), whereas the β‐pentapeptidolactone ring comprises Val, Pro, Sar, N‑methylvaline (MeVal), and Thr. ¹H–¹H COSY correlations between H‐2/H₂‐3 and H₂‐3/H₃‐4 within the 2‐MeAba residue, along with HMBC correlations between H‐2/N‐Me, H‐2/C‐1, H‐2/C‐3, H₃‐4/C‐2, H₃‐4/C‐3, and N‐Me/C‐2, established the presence of the 2‐MeAba residue unit in the α‐ring (Figure 2). The residues were assigned as D‐Val, L‐MeVal, L‐2‐MeAba, L‐Thr, L‐Pro, and non‐chiral Sar by Marfey's analyses (Figures S10–S13). The characteristic UV_max absorption of 1 closely resembled that of actinomycin D (UV_max 239 and 442 nm in 1 vs. UV_max 241 and 442 nm in actinomycin D), indicating the preservation of the phenoxazinone chromophore scaffold [22]. ¹H–¹H COSY correlations between H‐7/H‐8, along with HMBC correlations between H‐7/C‐5a, H‐7/C‐9, H‐8/C‐6, H‐8/C‐9a, H‐8/C‐13, H₃‐11/C‐5a, H₃‐11/C‐6, H₃‐11/C‐7, H₃‐12/C‐3, H₃‐12/C‐4, and H₃‐12/C‐4a, established a 2‐amino‐4,6‐dimethyl‐3H‐phenoxazin‐3‐one chromophore (Figure 2), consistent with that of actinomycin D [22]. The connection of α‐ and β‐pentapeptidolactone rings to the phenoxazinone chromophore of 1 was confirmed by HMBC cross‐peaks from NHs of two L‐Thr residues to C‐13 and C‐14, respectively. Collectively, the above findings established the structure of 1, named actinomycin H₁, which represents a new actinomycin analog incorporating an L‐2‐MeAba residue in the α‐ring.

TABLE 1.

¹³C NMR data for compounds 1–6 (150 MHz, δ _C in ppm).

NO.	Pentapeptidolactone (α‐ring, δ C)						NO.	Pentapeptidolactone (β‐ring, δ C)
	1 a	2 a	3 a	4 a	5 a	6 b		1 a	2 a	3 a	4 a	5 a	6 b
Thr/ 1	168.4 qC	168.6 qC	168.7 qC	168.6 qC	169.2 qC	169.0 qC	Thr/ 1	169.1 qC	168.9 qC	169.2 qC	169.0 qC	168.7 qC	169.1 qC
Ser 2	55.4 CH	55.4 CH	53.3 CH	55.4 CH	55.5 CH	53.8 CH	Ser 2	55.0 CH	55.0 CH	55.0 CH	55.0 CH	52.9 CH	55.0 CH
3	75.6 CH	75.2 CH	65.2 CH2	75.3 CH	75.3 CH	71.8 CH	3	75.2 CH	75.6 CH	75.1 CH	75.1 CH	65.5 CH2	72.1 CH
4	18.0 CH3	17.6 CH3		18.0 CH3	17.8 CH3	16.8 CH3	4	17.3 CH3	17.7 CH3	17.9 CH3	17.6 CH3		16.3 CH3
Val/ 1	173.5 qC	173.4 qC	173.2 qC	173.3 qC	172.9 qC	170.5 qC	Val 1	173.4 qC	173.4 qC	173.3 qC	173.5 qC	172.9 qC	170.6 qC
2‐Aba 2	59.0 CH	59.0 CH	58.9 CH	54.2 CH	58.9 CH	57.3 CH	2	58.9 CH	58.8 CH	58.9 CH	58.8 CH	58.0 CH	57.3 CH
3	31.6 CH	31.7 CH	31.7 CH	26.2 CH	31.7 CH	30.0 CH	3	32.0 CH	31.9 CH	31.6 CH	32.0 CH	31.3 CH	30.1 CH
4	19.4 CH3	19.4 CH3	19.4 CH3	10.1 CH3	19.4 CH3	19.0 CH3	4	19.5 CH3	19.4 CH3	19.5 CH3	19.4 CH3	19.4 CH3	19.0 CH3
5	19.1 CH3	19.2 CH3	19.1 CH3		19.0 CH3	18.5 CH3	5	19.2 CH3	19.2 CH3	19.1 CH3	19.2 CH3	19.0 CH3	18.5 CH3
Pro 1	173.9 qC	173.6 qC	173.5 qC	173.6 qC	173.2 qC	172.9 qC	Pro/ 1	173.5 qC	173.9 qC	173.8 qC	173.9 qC	173.8 qC	172.9 qC
2	56.4 CH	56.3 CH	56.3 CH	56.3 CH	56.6 CH	54.6 CH	Hyp 2	56.4 CH	56.6 CH	56.3 CH	56.5 CH	56.3 CH	54.7 CH
3	31.4 CH2	31.1 CH2	31.2 CH2	31.4 CH2	31.1 CH2	30.9 CH2	3	31.1 CH2	31.4 CH2	31.3 CH2	31.3 CH2	41.0 CH2	31.1 CH2
4	23.0 CH2	23.2 CH2	23.1 CH2	23.1 CH2	23.1 CH2	22.6 CH2	4	23.2 CH2	23.0 CH2	23.4 CH2	23.3 CH2	69.6 CH	22.7 CH2
5	47.8 CH2	47.5 CH2	47.8 CH2	47.8 CH2	47.7 CH2	46.3 CH2	5	47.5 CH2	47.7 CH2	47.5 CH2	47.5 CH2	55.2 CH2	46.3 CH2
Sar 1	166.6 qC	166.5 qC	166.5 qC	167.7 qC	166.5 qC	166.9 qC	Sar 1	166.7 qC	166.6 qC	166.4 qC	167.8 qC	166.1 qC	167.0 qC
2	51.6 CH2	51.6 CH2	51.6 CH2	51.5 CH2	51.4 CH2	51.1 CH2	2	51.6 CH2	51.5 CH2	51.5 CH2	51.5 CH2	51.5 CH2	51.1 CH2
N‐Me	34.9 CH3	35.0 CH3	35.1 CH3	35.2 CH3	35.1 CH3	34.2 CH3	N‐Me	35.1 CH3	35.0 CH3	34.9 CH3	35.4 CH3	35.3 CH3	34.3 CH3
MeVal/ 1	168.7 qC	167.7 qC	168.4 qC	166.6 qC	167.6 qC	168.0 qC	MeVal/ 1	167.8 qC	168.8 qC	167.7 qC	166.7 qC	169.1 qC	168.0 qC
2‐MeAba 2	67.1 CH	71.6 CH	71.5 CH	71.5 CH	70.8 CH	69.5 CH	2‐MeAba 2	71.4 CH	66.9 CH	71.3 CH	71.5 CH	71.5 CH	69.5 CH
3	21.4 CH	27.0 CH	27.1 CH	27.1 CH	28.0 CH	26.3 CH	3	27.0 CH	21.5 CH	27.4 CH	27.0 CH	27.1 CH	26.4 CH
4	11.1 CH3	21.9 CH3	22.0 CH3	21.8 CH3	22.0 CH3	20.9 CH3	4	21.8 CH3	11.1 CH3	21.8 CH3	21.8 CH3	21.8 CH3	21.0 CH3
5		19.3 CH3	19.2 CH3	19.2 CH3	19.3 CH3	18.6 CH3	5	19.3 CH3		19.3 CH3	19.2 CH3	19.2 CH3	18.7 CH3
N‐Me	38.9 CH3	39.5 CH3	39.5 CH3	39.4 CH3	39.5 CH3	38.5 CH3	N‐Me	39.4 CH3	38.7 CH3	39.5 CH3	39.4 CH3	39.5 CH3	38.6 CH3
	Chromophore (δ C)
1	101.9 qC	102.0 qC	102.2 qC	101.9 qC	99.5 qC	99.9 qC	9a	129.3 qC	129.3 qC	130.0 qC	129.3 qC	129.2 qC	129.8 qC
2	147.8 qC	147.8 qC	147.8 qC	147.8 qC	149.3 qC	132.4 qC	10a	146.1 qC	146.0 qC	146.0 qC	146.0 qC	146.5 qC	133.9 qC
3	179.3 qC	179.3 qC	179.1 qC	179.3 qC	179.1 qC	144.4 qC	11	15.2 CH3	15.2 CH3	15.4 CH3	15.2 CH3	15.2 CH3	14.5 CH3
4	113.6 qC	113.6 qC	113.7 qC	113.6 qC	113.8 qC	111.7 qC	12	7.9 CH3	8.0 CH3	8.0 CH3	8.0 CH3	8.0 CH3	8.6 CH3
4a	145.3 qC	145.2 qC	145.2 qC	145.3 qC	145.7 qC	142.9 qC	13	166.3 qC	166.2 qC	164.5 qC	166.1 qC	166.8 qC	166.4 qC
5a	140.6 qC	140.6 qC	140.7 qC	140.6 qC	140.6 qC	140.2 qC	14	166.3 qC	166.5 qC	166.3 qC	166.5 qC	166.7 qC	164.9 qC
6	127.7 qC	127.7 qC	128.6 qC	127.7 qC	128.5 qC	127.7 qC	15						155.7 qC
7	130.4 CH	130.4 CH	130.1 CH	130.4 CH	130.4 CH	122.3 CH	16						186.1 qC
8	126.1 CH	126.1 CH	126.9 CH	126.1 CH	126.4 CH	122.1 CH	17						33.5 CH2
9	132.8 qC	132.8 qC	130.1 qC	132.7 qC	131.3 qC	113.6 qC	18						27.1 CH2
							19						173.4 qC

^a Measured in CDCl₃.

^b Measured in DMSO‐d _6.

TABLE 3.

¹H NMR data for compounds 1–6 (600 MHz, δ _H in ppm, J in Hz).

Position	1 a	2 a	3 a	4 a	5 a	6 b
Pentapeptidolactone (α‐ring, δ H)
Thr/ 2	4.48 dd (6.3, 2.5)	4.51 d (4.6)	4.25 br s	4.48 d (4.3)	4.95 dd (7.2, 2.3)	4.99 d (9.6)
Ser 3	5.13 m	5.22 m	4.76 d (11.0) 4.32 d (11.2)	5.19 m	5.28 m	5.28 m
4	1.26 d (5.7)	1.26 d (4.3)		1.26 d (5.8)	1.30 d (6.1)	1.29 d (6.2)
NH	7.17 d (6.5)	7.18 d (6.4)	8.28 d (3.3)	7.27 d (6.7)	7.24 d (7.4)	7.27 d (8.7)
Val/ 2	3.58 m	3.53 m	3.52 m	3.81 m	3.61 m	3.52 m
2‐Aba 3	2.22 m	2.20 m	2.17 m	1.84 m	2.12 m	1.89 m
4	0.91 d (6.5)	0.88 d (6.2)	0.90 d (5.3)	0.98 overlapped	0.95 d (7.0)	0.71 d (6.2)
5	1.13 d (6.7)	1.12 d (5.4)	1.14 d (6.5)		1.17 d (6.7)	0.95 d (6.7)
NH	8.10 d (5.5)	8.13 d (5.7)	8.18 d (5.1)	8.04 d (5.7)	7.75 d (6.3)	8.46 d (5.0)
Pro 2	5.98 d (9.1)	6.03 d (9.0)	6.01 d (9.1)	5.92 d (9.0)	6.08 d (9.1)	6.26 d (7.5)
3	2.96, 1.86 m	2.94, 1.80 m	2.72, 2.16 m	2.99, 2.67 m	2.57, 1.82 m	2.09, 1.74 m
4	2.28, 2.08 m	2.29, 2.04 m	2.24, 2.16 m	2.30, 2.11 m	2.22, 2.07 m	1.91, 1.66 m
5	3.82, 3.73 m	3.95, 3.73 m	3.77, 3.72 m	3.85, 3.77 m	3.78, 3.76 m	3.48, 3.27 m
Sar 2	4.68 d (17.2) 3.66 d (17.1)	4.71 d (16.9) 3.63 d (16.8)	4.67 d (17.5) 3.61 d (17.7)	4.76 d (17.1) 3.64 d (17.2)	4.68 d (17.4) 3.64 d (17.0)	4.81 d (17.9) 4.11 d (17.7)
N‐Me	2.88 s	2.86 s	2.87 s	2.90 s	2.91 s	2.77 s
MeVal/ 2	2.98 m	2.66 overlapped	2.69 overlapped	2.69 overlapped	2.85 d (8.8)	3.23 d (9.4)
2‐MeAba 3	1.97 m	2.67 m	2.68 m	2.67 m	2.64 m	2.55 m
4	0.83 t (7.3)	0.97 d (5.0)	0.96 d (5.8)	0.97 overlapped	1.02 d (6.5)	0.98 d (6.4)
5		0.74 d (5.3)	0.74 d (6.3)	0.76 d (5.6)	0.78 d (6.8)	0.80 d (6.7)
N‐Me	2.95 s	2.93 s	2.91 s	2.95 s	2.95 s	3.22 s
Pentapeptidolactone (β‐ring, δ H)
Thr 2	4.62 dd (6.2, 2.2)	4.60 d (3.8)	4.62 dd (6.5, 2.2)	4.61 d (4.0)	4.18 br s	4.90 d (8.8)
Ser/ 3	5.17 m	5.11 m	5.20 m	5.19 m	4.64 dd (11.8, 2.2) 4.46 d (11.1)	5.17 m
4	1.26 d (5.7)	1.25 d (4.7)	1.25 d (6.4)	1.27 d (6.0)		1.20 d (6.2)
NH	7.79 d (6.0)	7.75 d (5.9)	7.51 d (6.4)	7.81 d (6.2)	8.45 d (3.0)	9.38 d (8.0)
Val 2	3.55 m	3.61 m	3.58 m	3.58 m	3.57 m	3.52 m
3	2.15 m	2.16 m	2.17 m	2.21 m	2.12 m	1.89 m
4	0.89 d (6.9)	0.92 d (6.5)	0.90 d (5.3)	0.94 overlapped	0.89 d (6.4)	0.71 d (6.2)
5	1.11 d (6.4)	1.13 d (4.4)	1.11 d (6.6)	1.14 d (6.2)	1.10 d (6.7)	0.94 d (6.6)
NH	7.95 d (6.0)	7.95 d (5.7)	7.96 d (4.5)	8.07 d (6.0)	8.12 d (4.7)	8.33 d (5.0)
Pro/ 2	6.04 d (9.1)	5.97 d (9.0)	6.05 d (9.1)	6.01 d (9.0)	5.87 t (7.5)	6.26 d (7.5)
Hyp 3	2.65, 1.80 m	2.68, 1.85 m	2.65, 1.83 m	2.99, 2.67 m	2.47, 2.22 m	2.09, 1.74 m
4	2.28, 2.08 m	2.08, 2.27 m	2.30, 2.08 m	2.30, 2.11 m	4.62 m	1.91, 1.66 m
5	3.99, 3.73 m	3.85, 3.73 m	3.96, 3.75 m	3.96, 3.77 m	4.34, 3.86 m	3.48, 3.27 m
Sar 2	4.77 d (17.5) 3.61 d (17.1)	4.74 d (16.9) 3.63 d (16.8)	4.74 d (16.9) 3.65 d (17.4)	4.79 d (17.1) 3.68 d (17.2)	4.73 d (17.2) 3.65 d (17.0)	4.76 d (17.9) 4.08 d (17.7)
N‐Me	2.86 s	2.88 s	2.89 s	2.90 s	2.88 s	2.76 s
MeVal/ 2	2.67 overlapped	2.99 m	2.75 d (9.7)	2.69 overlapped	2.68 overlapped	3.17 d (9.4)
2‐MeAba 3	2.65 m	1.95 m	2.68 m	2.67 m	2.64 m	2.55 m
4	0.95 d (5.9)	0.83 t (7.3)	1.00 d (6.1)	0.98 overlapped	0.97 d (5.8)	0.98 d (6.4)
5	0.73 d (6.0)		0.75 d (6.5)	0.77 d (5.6)	0.75 d (6.3)	0.81 d (6.7)
N‐Me	2.89 s	2.92 s	2.92 s	2.91 s	2.95 s	3.14 s
Chromophore (δ H)
7	7.36 d (7.5)	7.37 d (7.7)	7.39 d (7.6)	7.39 d (7.7)	7.35 d (7.6)	6.63 d (8.0)
8	7.64 d (7.7)	7.65 d (7.6)	7.94 d (7.7)	7.68 d (7.7)	7.64 d (7.8)	7.08 d (7.9)
11‐Me	2.55 s	2.55 s	2.57 s	2.58 s	2.54 s	2.08 s
12‐Me	2.25 s	2.25 s	2.26 s	2.28 s	2.24 s	2.25 s
17						3.40 m
18						2.71 m
NH2‐2					6.77 s, 9.31 s
NH‐10						12.04 s
OH‐19						12.39 s

^a Measured in CDCl₃.

^b Measured in DMSO‐d _6.

FIGURE 2.

¹H–¹H COSY and key HMBC correlations of compounds 1, 6, 7, 11, 14, and 15.

Compound 2 provided the molecular formula C₆₁H₈₄N₁₂O₁₆ as determined by the HR‐ESIMS data (Figure S15). The NMR data of 2 indicated the presence of a phenoxazinone chromophore and two pentapeptidolactone moieties (Tables 1 and 3; Figures S16–S22). The extended sequence of amino acid residues in the α‐pentapeptidolactone ring of 2 consists of Val, Pro, Sar, MeVal, and Thr, whereas the β‐pentapeptidolactone ring comprises Val, Pro, Sar, 2‐MeAba, and Thr. ¹H–¹H COSY correlations between H‐2/H₂‐3 and H₂‐3/H₃‐4 within the 2‐MeAba residue, along with HMBC correlations between H‐2/N‐Me, H‐2/C‐1, H‐2/C‐3, H₃‐4/C‐2, H₃‐4/C‐3, and N‐Me/C‐2, established the 2‐MeAba residue in the β‐ring. The residues were assigned as D‐Val, L‐MeVal, L‐2‐MeAba, L‐Thr, L‐Pro, and non‐chiral Sar by Marfey's analyses (Figure S23). The characteristic UV_max absorption and detailed NMR analyses established the chromophore of 2 as identical to that of 1. Collectively, the above findings established the structure of 2, named actinomycin H₂, as a new actinomycin analog incorporating an L‐2‐MeAba residue in the β‐ring. The positioning of the L‐2‐MeAba residue within the α‐ versus β‐pentapeptidolactone ring might be derived from the substrate promiscuity‌ of nonribosomal peptide synthetase (NRPS).

Compound 3 provided the molecular formula C₆₁H₈₄N₁₂O₁₆ as determined by the HR‐ESIMS data (Figure S25). The NMR data of 3 indicated the presence of a phenoxazinone chromophore and two pentapeptidolactone moieties (Tables 1 and 3; Figures S26–S32). The extended sequence of amino acid residues in the α‐pentapeptidolactone ring of 3 consists of Val, Pro, Sar, MeVal, and Ser, whereas the β‐pentapeptidolactone ring comprises Val, Pro, Sar, MeVal, and Thr. ¹H–¹H COSY correlations between NH‐2/H‐2 within the Ser residue, together with HMBC correlations between H‐2/C‐1 and H₂‐3/C‐2, indicated the presence of the Ser residue unit in the α‐ring. The residues were assigned as D‐Val, L‐MeVal, L‐Ser, L‐Thr, L‐Pro, and non‐chiral Sar by Marfey's analyses (Figures S33–S34). Collectively, the above findings established the structure of 3, named actinomycin H₃, as a new actinomycin analog incorporating an L‐Ser residue in the α‐ring.

Compound 4 afforded the molecular formula C₆₁H₈₄N₁₂O₁₆ as determined by the analysis of the HR‐ESIMS data (Figure S36). The NMR data of 4 indicated the presence of a phenoxazinone chromophore and two pentapeptidolactone moieties (Tables 1 and 3; Figures S37–S43). The extended sequence of amino acid residues in the α‐pentapeptidolactone ring of 4 consists of 2‐Aba, Pro, Sar, MeVal, and Thr, whereas the β‐pentapeptidolactone ring comprises Val, Pro, Sar, MeVal, and Thr. ¹H–¹H COSY correlations between NH‐2/H‐2, H‐2/H₂‐3, and H₂‐3/H₃‐4 within the 2‐Aba residue, along with HMBC correlations between NH‐2/C‐2, NH‐2/C‐3, H‐2/C‐1, H‐2/C‐3, H‐2/C‐4, H₂‐3/C‐1, H₂‐3/C‐2, H₂‑3/C‐4, H₃‐4/C‑2, and H₃‑4/C‐3, indicated the presence of the 2‐Aba residue unit in the α‐ring. The residues were assigned as D‐2‐Aba, D‐Val, L‐MeVal, L‐Thr, L‐Pro, and non‐chiral Sar by Marfey's analyses (Figures S44–S45). Collectively, the above findings established the structure of 4, named actinomycin H₄, as a new actinomycin analog incorporating a D‐2‐Aba residue in the α‐ring.

Compound 5 afforded the molecular formula C₆₁H₈₄N₁₂O₁₇ as determined by the HR‐ESIMS data (Figure S47). The NMR data of 5 indicated the presence of a phenoxazinone chromophore and two pentapeptidolactone moieties (Tables 1 and 3; Figures S48–S54). The extended sequence of amino acid residues in the α‐pentapeptidolactone ring of 5 consists of Val, Pro, Sar, MeVal, and Thr, whereas the β‐pentapeptidolactone ring comprises Val, Hyp, Sar, MeVal, and Ser. ¹H–¹H COSY correlations between H‐2/H₂‐3, H₂‐3/H‐4, and H‐4/H₂‐5 within the Hyp residue, along with HMBC correlations between H‐2/C‐3, H‐2/C‐4, H₂‐3/C‐2, H₂‐3/C‐4, H₂‐5/C‐3, and H₂‐5/C‐4, indicated the Hyp residue unit in the β‐ring. Likewise, ¹H–¹H COSY correlations between NH‐2/H‐2 within the Ser residue, along with HMBC correlations between H‐2/C‐1 and H₂‐3/C‐2, corroborated the Ser residue unit in the β‐ring. The residues were assigned as D‐Val, L‐MeVal, L‐Ser, L‐Thr, L‐Hyp, L‐Pro, and non‐chiral Sar by Marfey's analyses (Figures S55–S56). Collectively, the above findings established the structure of 5, named actinomycin H₅, as a new actinomycin analog incorporating L‐Ser and L‐Hyp residues in the β‐ring.

Compound 6 had the molecular formula C₆₇H₉₀N₁₂O₁₉ as deduced by analysis of the HR‐ESIMS data (Figure S58). The NMR data of 6 indicated the presence of a tetracyclic chromophore and two pentapeptidolactone moieties (Tables 1 and 3; Figures S59–S65). The extended sequence of amino acid residues in both α‐ and β‐pentapeptidolactone rings of 6 consists of Val, Pro, Sar, MeVal, and Thr. The residues were assigned as D‐Val, L‐MeVal, L‐Thr, L‐Pro, and non‐chiral Sar by Marfey's analyses (Figure S66). ¹H–¹H COSY correlations between H‐7/H‐8, along with HMBC correlations between H‐7/C‐5a, H‐7/C‐9, H‐8/C‐6, H‐8/C‐9a, H‐8/C‐13, H₃‐11/C‐5a, H₃‐11/C‐6, and H₃‐11/C‐7, indicated a monomethyl‐substituted benzene ring. HMBC cross‐peaks from an exchangeable proton (δ _H 12.04 s) to C‐1, C‐4a, C‐5a, and C‐9 revealed the 10‐NH group. Those from the methyl protons (δ _H 2.25 s) to C‐3, C‐4, and C‐4a corroborated the 12‐Me assignment (Figure 2). The downfield‐shifted C‐15 (δ _C 155.7 ppm) revealed the presence of a C‐15 imine group. Thus, the tetracyclic chromophore of 6 was concluded to be 5H‐oxazolo[4,5‐b]phenoxazine. This scaffold is analogous to that of the previously reported actinomycin D₁ [23]. Notably, the UV_max absorption of 6 differed from that of actinomycin D₁ (UV_max 255 and 450 nm in 6 vs. 252 and 409 nm in actinomycin D₁) and a molecular weight increase of 100 Da in 6 compared to that of actinomycin D₁ suggested an additional side chain on the oxazolo‐phenoxazine scaffold. ¹H–¹H COSY correlations between H₂‐17/H₂‐18, along with HMBC cross‐peaks between H₂‐17/C‐16, H₂‐17/C‐19, H₂‐18/C‐16, and H₂‐18/C‐19, supported a 4‐oxobutanoic acid unit (Figure 2). Collectively, the above findings established the structure of 6, named actinomycin H₆, as a new actinomycin analog featuring a 5H‐oxazolo[4,5‐b]phenoxazine backbone and bearing a 4‐oxobutanoic acid side chain.

Compound 7 had the molecular formula C₃₇H₄₉N₇O₁₁ as established by the HR‐ESIMS data (Figure S68). The NMR data of 7 indicated the presence of a bicyclic chromophore and a pentapeptidolactone moiety (Tables 2 and 4; Figures S69–S75). ¹H–¹H COSY correlations between H‐2/H‐3, along with HMBC cross‐peaks between H‐2/C‐4, H‐2/C‐6, H‐2/C‐13, H‐3/C‐1, H‐3/C‐5, H₃‐11/C‐3, H₃‐11/C‐4, H₃‐11/C‐5, indicated a monomethyl‐substituted benzene ring. HMBC cross‐peaks from an exchangeable proton (δ _H 11.91 s) to C‐1, C‐5, C‐6, C‐8, and C‐9 revealed the 10‐NH group. The downfield‐shifted C‐9 (δ _C 156.2 ppm) revealed a C‐9 carbonyl group. Collectively, the above findings corroborated the presence of a morpholin‐3‐one motif. HMBC correlations between H₃‐12/C‐8, H₃‐12/C‐14, H₃‐12/C‐15, NH₂‐16/C‐15, and NH₂‐16/C‐16 corroborated the presence of a 3‐methyl‐2‐oxobut‐3‐enamide side chain attached at C‐8 of the morpholin‐3‐one motif (Figure 2). Thus, the chromophore of 7 was concluded to be an 8‐methyl‐2H‐benzo[b][1,4]oxazin‐3(4H)‐one scaffold bearing a 3‐methyl‐2‐oxobut‐3‐enamide side chain. The extended sequence of amino acid residues in the pentapeptidolactone ring of 7 consists of Val, Pro, Sar, MeVal, and Thr. The residues were assigned as D‐Val, L‐MeVal, L‐Thr, L‐Pro, and non‐chiral Sar by Marfey's analyses (Figure S76). Collectively, the above findings established the structure of 7, named actinomycin H₇, as a new actinomycin analog featuring only one pentapeptidolactone moiety and a benzoxazinone chromophore scaffold.

TABLE 2.

¹³C NMR data for compounds 7–15 (150 MHz, δ _C in ppm).

NO.	Pentapeptidolactone (δC)
	7 a	8 a	9 a	10 a	11 a	12 a	13 a	14 a	15 a
Thr 1	171.8 qC	171.3 qC	171.4 qC	170.9 qC	167.9 qC	168.0 qC	168.0 qC	169.1 qC	169.5 qC
2	56.6 CH	56.4 CH	56.3 CH	56.1 CH	54.1 CH	54.1 CH	54.0 CH	53.4 CH	54.5 CH
3	70.7 CH	71.0 CH	70.9 CH	70.9 CH	72.1 CH	72.1 CH	72.1 CH	72.4 CH	71.9 CH
4	16.2 CH3	16.4 CH3	16.2 CH3	16.3 CH3	16.3 CH3	16.4 CH3	16.4 CH3	16.6 CH3	16.3 CH3
Val 1	170.2 qC	170.4 qC	170.3 qC	170.3 qC	170.4 qC	170.4 qC	170.4 qC	170.7 qC	170.4 qC
2	57.9 CH	57.9 CH	57.8 CH	57.7 CH	57.2 CH	57.2 CH	57.2 CH	57.3 CH	57.4 CH
3	30.0 CH	30.1 CH	30.0 CH	30.0 CH	30.0 CH	30.0 CH	30.0 CH	30.0 CH	30.0 CH
4	18.9 CH3	19.1 CH3	18.9 CH3	18.9 CH3	18.9 CH3	18.9 CH3	18.9 CH3	19.0 CH3	19.0 CH3
5	18.5 CH3	18.6 CH3	18.5 CH3	18.5 CH3	18.4 CH3	18.4 CH3	18.4 CH3	18.5 CH3	18.5 CH3
Pro 1	173.1 qC	173.4 qC	173.2 qC	173.3 qC	172.8 qC	172.9 qC	172.9 qC	172.9 qC	172.9 qC
2	55.4 CH	55.4 CH	55.4 CH	55.2 CH	54.8 CH	54.8 CH	54.8 CH	54.5 CH	54.7 CH
3	31.1 CH2	31.5 CH2	31.2 CH2	31.4 CH2	31.2 CH2	31.2 CH2	31.2 CH2	31.3 CH2	31.1 CH2
4	22.5 CH2	22.8 CH2	22.6 CH2	22.7 CH2	22.7 CH2	22.8 CH2	22.8 CH2	22.7 CH2	22.7 CH2
5	46.5 CH2	46.6 CH2	46.5 CH2	46.5 CH2	46.4 CH2	46.4 CH2	46.4 CH2	46.4 CH2	46.3 CH2
Sar 1	167.2 qC	167.1 qC	167.0 qC	167.0 qC	167.0 qC	167.1 qC	167.1 qC	167.1 qC	166.8 qC
2	50.9 CH2	51.1 CH2	50.6 CH2	51.0 CH2	51.0 CH2	51.1 CH2	51.1 CH2	51.2 CH2	51.0 CH2
N‐Me	34.2 CH3	34.4 CH3	34.2 CH3	34.3 CH3	34.3 CH3	34.3 CH3	34.3 CH3	34.3 CH3	34.3 CH3
MeVal 1	168.1 qC	168.1 qC	168.0 qC	168.0 qC	167.8 qC	167.9 qC	168.0 qC	168.1 qC	168.0 qC
2	69.3 CH	69.4 CH	69.3 CH	69.3 CH	69.2 CH	69.2 CH	69.1 CH	69.4 CH	69.4 CH
3	26.4 CH	26.6 CH	26.6 CH	26.4 CH	26.5 CH	26.5 CH	26.5 CH	26.1 CH	26.4 CH
4	21.2 CH3	21.2 CH3	21.1 CH3	21.1 CH3	20.9 CH3	20.9 CH3	20.8 CH3	20.8 CH3	21.0 CH3
5	18.6 CH3	18.7 CH3	18.6 CH3	18.6 CH3	18.7 CH3	18.7 CH3	18.7 CH3	18.6 CH3	18.7 CH3
N‐Me	38.2 CH3	38.1 CH3	37.9 CH3	38.1 CH3	38.4 CH3	38.4 CH3	38.3 CH3	38.4 CH3	38.4 CH3
Chromophore (δ C)
1	122.3 qC	121.0 qC	121.3 qC	120.4 qC	156.9 qC	157.9 qC	159.7 qC	97.5 qC	114.3 qC
2	121.5 CH	121.4 CH	121.5 CH	121.1 CH	160.8 qC	161.5 qC	161.2 qC	183.8 qC	121.2 CH
3	124.6 CH	124.3 CH	124.3 CH	123.9 CH	53.1 CH3			137.4 CH	122.6 CH
4	126.7 qC	127.0 qC	126.8 qC	126.8 qC				141.0 qC	122.7 qC
5	138.6 qC	139.1 qC	138.7 qC	139.5 qC				182.4 qC	142.1 qC
6	122.6 qC	123.7 qC	123.3 qC	124.1 qC				153.2 qC	130.0 qC
7								14.5 CH3
8	139.2 qC	137.8 qC	138.8 qC	136.8 qC				167.5 qC	154.1 qC
9	156.2 qC	155.3 qC	155.0 qC	155.7 qC
10									14.3 CH3
11	15.0 CH3	15.1 CH3	15.0 CH3	15.1 CH3					164.8 qC
12	13.8 CH3	13.9 CH3	13.5 CH3	14.8 CH3
13	166.4 qC	166.7 qC	166.5 qC	166.7 qC
14	121.3 qC	119.4 qC	117.4 qC	123.1 qC
15	191.5 qC	169.6 qC	168.8 qC	169.9 qC
16	163.0 qC		52.1 qC

^a Measured in DMSO‐d _{6 .}

TABLE 4.

¹H NMR data for compounds 7–15 (600 MHz, δ _H in ppm, J in Hz).

Position	7 a	8 a	9 a	10 a	11 a	12 a	13 a	14 a	15 a
Pentapeptidolactone (δ H)
Thr 2	4.74 dd (8.3, 2.1)	4.76 dd (8.4, 2.4)	4.76 dd (8.4, 2.3)	4.76 dd (8.2, 1.6)	4.72 dd (9.4, 2.1)	4.70 d (9.2)	4.67 dd (9.6, 2.0)	4.90 d (9.6)	4.94 d (8.7)
3	5.21 m	5.20 m	5.20 m	5.18 m	5.09 m	5.10 m	5.10 m	5.09 m	5.18 m
4	1.21 d (6.3)	1.21 d (6.3)	1.21 d (6.4)	1.21 d (6.2)	1.10 d (6.3)	1.11 d (6.0)	1.09 d (6.4)	1.10 d (6.1)	1.20 d (5.7)
NH	8.71 d (8.2)	8.59 d (8.3)	8.64 d (8.3)	8.54 d (8.3)	7.98 d (9.3)	7.87 d (9.3)	7.98 d (9.6)	9.94 d (9.4)	7.70 d (8.7)
Val 2	3.54 m	3.56 m	3.56 m	3.56 m	3.51 m	3.52 m	3.51 m	3.45 m	3.52 m
3	1.85 m	1.88 m	1.86 m	1.88 m	1.85 m	1.86 m	1.86 m	1.87 m	1.88 m
4	0.70 d (6.7)	0.71 d (6.7)	0.71 d (6.6)	0.70 d (6.6)	0.69 d (6.7)	0.69 d (6.5)	0.69 d (6.7)	0.68 d (6.6)	0.69 d (6.6)
5	0.92 d (6.7)	0.93 d (6.6)	0.93 d (6.7)	0.93 d (6.5)	0.90 d (6.5)	0.90 d (6.5)	0.90 d (6.7)	0.89 d (6.5)	0.93 d (6.5)
NH	8.62 d (4.8)	8.61 d (5.1)	8.61 d (4.8)	8.60 d (5.0)	8.45 d (5.7)	8.46 d (5.3)	8.44 d (5.8)	8.38 d (5.8)	8.50 d (4.4)
Pro 2	6.25 dd (8.6, 2.6)	6.30 dd (8.6, 3.5)	6.28 dd (8.6, 2.9)	6.30 dd (8.4, 2.8)	6.16 dd (8.6, 3.1)	6.17 dd (8.3, 2.4)	6.16 dd (8.6, 3.5)	6.24 dd (8.5, 2.7)	6.21 d (6.1)
3	1.99, 1.79 m	2.16, 1.71 m	2.11, 1.70 m	2.19, 1.73 m	2.18, 1.78 m	2.20, 1.77 m	2.22, 1.77 m	2.16, 1.77 m	2.12, 1.76 m
4	1.86, 1.60 m	1.88, 1.61 m	1.88, 1.63 m	1.89, 1.64 m	1.93, 1.70 m	1.94, 1.71 m	1.93, 1.70 m	1.94, 1.69 m	1.92, 1.68 m
5	3.44, 3.23 m	3.51, 3.23 m	3.48, 3.23 m	3.48, 3.24 m	3.48, 3.27 m	3.49, 3.28 m	3.50, 3.26 m	3.47, 3.26 m	3.46, 3.27 m
Sar 2	5.23 d (18.1) 3.98 d (18.3)	5.27 d (18.1) 3.94 d (18.1)	5.20 overlapped 4.01 d (18.1)	5.28 d (18.0) 3.98 d (18.1)	4.78 d (18.0) 4.10 d (17.9)	4.82 d (17.8) 4.10 d (17.9)	4.83 d (17.8) 4.09 d (17.9)	4.83 d (17.8) 4.08 d (17.9)	4.79 d (17.8) 4.12 d (17.8)
N‐Me	2.74 s	2.76 s	2.76 s	2.76 s	2.75 s	2.75 s	2.75 s	2.75 s	2.75 s
MeVal 2	2.92 d (9.3)	2.99 d (9.3)	2.97 d (9.2)	2.99 d (9.3)	3.24 d (9.3)	3.27 d (9.2)	3.29 d (9.3)	3.25 d (9.4)	3.18 overlapped
3	2.49 m	2.50 m	2.48 m	2.50 m	2.49 m	2.50 m	2.49 m	2.50 m	2.52 m
4	0.97 d (6.5)	0.96 d (6.4)	0.97 d (6.5)	0.96 d (6.5)	0.94 d (6.5)	0.95 d (6.2)	0.94 d (6.5)	0.95 d (6.4)	0.97 d (6.5)
5	0.79 d (6.8)	0.78 d (6.8)	0.79 d (6.9)	0.78 d (6.8)	0.78 d (6.9)	0.78 d (6.1)	0.77 d (6.8)	0.78 d (6.7)	0.79 d (6.8)
N‐Me	2.98 s	3.06 s	3.04 s	3.08 s	3.16 s	3.16 s	3.16 s	3.15 s	3.20 s
Chromophore (δ H)
2	7.04 d (7.8)	7.08 d (7.8)	7.07 d (7.9)	7.08 d (7.8)					7.50 d (7.9)
3	7.01 d (7.9)	6.99 d (8.0)	6.99 d (7.7)	6.97 d (8.0)	3.80 s			6.61 s	7.05 d (7.9)
7								1.96 s
10									2.36 s
11	2.34 s	2.32 s	2.32 s	2.31 s
12	1.99 s	2.03 s	2.03 s	1.99 s
16			3.72 s
NH‐9									11.70 s
NH‐10	11.91 s	11.55 s	11.68 s	11.32 s
NH2‐2							8.04 s, 8.30 s
NH2‐6								8.56 s, 10.30 s
NH2‐15				7.09 s, 7.36 s
NH2‐16	7.91 s, 7.59 s
OH‐15		12.74 s

^a Measured in DMSO‐d ₆.

Compound 8 provided the molecular formula C₃₆H₄₈N₆O₁₁ as determined by the HR‐ESIMS data (Figure S78). The NMR data of 8 indicated the presence of a benzoxazinone chromophore and a pentapeptidolactone moiety (Tables 2 and 4; Figures S79–S85). HMBC cross‐peaks between H₃‐12/C‐8, H₃‐12/C‐14, and H₃‐12/C‐15 corroborated the acrylic acid substituent attached at C‐8 of the morpholin‐3‐one motif. Thus, the chromophore of 8 was concluded to be a benzoxazinone scaffold bearing an acrylic acid side chain. The extended sequence of amino acid residues in the pentapeptidolactone ring of 8 consists of Val, Pro, Sar, MeVal, and Thr. The residues were assigned as D‐Val, L‐MeVal, L‐Thr, L‐Pro, and non‐chiral Sar by Marfey's analyses (Figure S86). Collectively, the above findings established the structure of 8, named actinomycin H₈, as a new actinomycin analog featuring a benzoxazinone chromophore scaffold bearing an acrylic acid side chain.

Compound 9 afforded the molecular formula C₃₇H₅₀N₆O₁₁ as determined by the HR‐ESIMS data (Figure S88). The NMR data of 9 indicated the presence of a benzoxazinone chromophore and a pentapeptidolactone moiety (Tables 2 and 4; Figures S89–S95). HMBC cross‐peaks between H₃‐12/C‐8, H₃‐12/C‐14, H₃‐12/C‐15, and H₃‐16/C‐15 corroborated a methyl acrylate moiety attached at C‐8 of the morpholin‐3‐one motif. Thus, the chromophore of 9 was concluded to be a benzoxazinone scaffold bearing a methyl acrylate side chain. The extended sequence of amino acid residues in the pentapeptidolactone ring of 9 consists of Val, Pro, Sar, MeVal, and Thr. The residues were assigned as D‐Val, L‐MeVal, L‐Thr, L‐Pro, and non‐chiral Sar by Marfey's analyses (Figure S96). Collectively, the above findings established the structure of 9, named actinomycin H₉, as a new actinomycin analog featuring a benzoxazinone chromophore scaffold bearing a methyl acrylate side chain.

Compound 10 provided the molecular formula C₃₆H₄₉N₇O₁₀ as determined by analysis of the HR‐ESIMS data (Figure S98). The NMR data of 10 indicated the presence of a benzoxazinone chromophore and a pentapeptidolactone moiety (Tables 2 and 4; Figures S99–S105). HMBC cross‐peaks between H₃‐12/C‐8, H₃‐12/C‐14, H₃‐12/C‐15, and NH₂‐15/C‐15 corroborated an acrylamide moiety attached at C‐8 of the morpholin‐3‐one motif. Thus, the chromophore of 10 was concluded to be a benzoxazinone scaffold bearing an acrylamide side chain. The extended sequence of amino acid residues in the pentapeptidolactone ring of 10 consists of Val, Pro, Sar, MeVal, and Thr. The residues were assigned as D‐Val, L‐MeVal, L‐Thr, L‐Pro, and non‐chiral Sar by Marfey's analyses (Figure S106). Collectively, the above findings established the structure of 10, named actinomycin H₁₀, as a new actinomycin analog featuring a benzoxazinone chromophore scaffold bearing an acrylamide side chain.

Compound 11 had the molecular formula C₂₆H₄₁N₅O₉ as deduced by analysis of the HR‐ESIMS data (Figure S108). The NMR data of 11 indicated a pentapeptidolactone moiety (Tables 2 and 4; Figures S109–S115). The extended sequence of amino acid residues in the pentapeptidolactone ring of 11 consists of Val, Pro, Sar, MeVal, and Thr. HMBC correlation between the Thr NH and C‐1 of the 2‐methoxy‐2‐oxoacetyl moiety and that between the H₃‐3 and C‐2 revealed the modification of the Thr residue by a 2‐methoxy‐2‐oxoacetyl group (Figure 2). The residues were assigned as D‐Val, L‐MeVal, L‐Thr, L‐Pro, and non‐chiral Sar by Marfey's analyses (Figure S116). Collectively, the structure of 11, named actinomycin H₁₁, was concluded to be a truncated actinomycin analog featuring the attachment of an N‐terminal 2‐methoxy‐2‐oxoacetyl group at the Thr residue.

Compound 12 provided the molecular formula C₂₅H₃₉N₅O₉ as determined by the HR‐ESIMS data (Figure S118). The NMR data of 12 indicated the presence of a pentapeptidolactone moiety (Tables 2 and 4; Figures S119–S125). The extended sequence of amino acid residues in the pentapeptidolactone ring of 12 consists of Val, Pro, Sar, MeVal, and Thr. HMBC cross‐peak from the Thr NH to C‐1 of the oxalyl group revealed the modification of the Thr residue by an oxalyl group. The residues were assigned as D‐Val, L‐MeVal, L‐Thr, L‐Pro, and non‐chiral Sar by Marfey's analyses (Figure S126). Collectively, the structure of 12, named actinomycin H₁₂, was concluded to be a truncated actinomycin analog featuring the attachment of an N‐terminal oxalyl group at the Thr residue.

Compound 13 afforded the molecular formula C₂₅H₄₀N₆O₈ as determined by the HR‐ESIMS data (Figure S128). The NMR data of 13 indicated the presence of a pentapeptidolactone moiety (Tables 2 and 4; Figures S129–S135). The extended sequence of amino acid residues in the pentapeptidolactone ring of 13 consists of Val, Pro, Sar, MeVal, and Thr. HMBC cross‐peaks between the Thr NH and C‐1 of the 2‐amino‐2‐oxoacetyl moiety and those between NH₂‐2/C‐1 and NH₂‐2/C‐2 indicated an additional 2‐amino‐2‐oxoacetyl group connected to the N‐terminus of the Thr residue. The residues were assigned as D‐Val, L‐MeVal, L‐Thr, L‐Pro, and non‐chiral Sar by Marfey's analyses (Figure S136). Collectively, the structure of 13, named actinomycin H₁₃, was concluded to be a truncated actinomycin analog featuring the presence of an N‐terminal 2‐amino‐2‐oxoacetyl group connected at the Thr residue. The same truncated scaffold of 11–13, along with the different N‐terminal modification patterns, indicated the possible degradation or transformation during the process of fermentation or isolation.

Compound 14 had the molecular formula C₃₁H₄₄N₆O₉ as deduced by analysis of the HR‐ESIMS data (Figure S138). The NMR data of 14 indicated the presence of a monocyclic chromophore and a pentapeptidolactone moiety (Tables 2 and 4; Figures S139–S144). HMBC cross‐peak from a methine proton (δ _H 6.61 s) to C‐1, C‐5, and C‐7 revealed a methine at C‐3. HMBC correlations from two exchangeable amino protons (δ _H 8.56 s, 10.30 s) to C‐1 and C‐5 indicated the 6‐NH₂ group. In addition, HMBC correlations from the methyl protons (δ _H 1.96 s) to C‐3, C‐4, and C‐5 corroborated the presence of the 7‐Me group (Figure 2). The C‐2 (δ _C 183.8 ppm) and C‐5 (δ _C 182.4 ppm) were consistent with two quinone carbonyl carbons. Thus, the chromophore of 14 was concluded to be a monocyclic 2‐amino‐6‐methylcyclohexa‐2,5‐diene‐1,4‐dione. The extended sequence of amino acid residues in the pentapeptidolactone ring of 14 consists of Val, Pro, Sar, MeVal, and Thr. The residues were assigned as D‐Val, L‐MeVal, L‐Thr, L‐Pro, and non‐chiral Sar by Marfey's analyses (Figure S145). Collectively, the above findings established the structure of 14, named actinomycin H₁₄.

Compound 15 afforded the molecular formula C₃₂H₄₄N₆O₉ as deduced by analysis of the HR‐ESIMS data (Figure S147). The NMR data of 15 indicated the presence of a bicyclic chromophore and a pentapeptidolactone moiety (Tables 2 and 4; Figures S148–S154). ¹H–¹H COSY correlations between H‐2/H‐3, along with HMBC correlations between H‐2/C‐4, H‐2/C‐6, H‐2/C‐11, H‐3/C‐1, H‐3/C‐5, H₃‐10/C‐3, H₃‐10/C‐4, and H₃‐10/C‐5, indicated the presence of a monomethyl‐substituted benzene ring. HMBC cross‐peaks from an exchangeable proton (δ _H 11.70 s) to C‐5, C‐6, and C‐8 revealed the presence of the 9‐NH group (Figure 2). The downfield‐shifted C‐8 (δ _C 154.1 ppm) was identified as a carbonyl carbon. Collectively, the above findings corroborated the presence of an oxazolidin‐2‐one motif. Thus, the bicyclic chromophore of 15 was concluded to be 7‐methylbenzo[d]oxazol‐2(3H)‐one. The extended sequence of amino acid residues in the pentapeptidolactone ring of 15 consists of Val, Pro, Sar, MeVal, and Thr. The residues were assigned as D‐Val, L‐MeVal, L‐Thr, L‐Pro, and non‐chiral Sar by Marfey's analyses (Figure S155). Collectively, the above findings established the structure of 15, named actinomycin H₁₅.

Human MDA‐MB‐231 and murine 4T1 TNBC cancer cell lines were employed to assess the antiproliferative‌ effects of all the compounds. Compounds 1–6 and 9 exhibited antiproliferative activity against both cell lines in the CCK‐8 assay (Figure S156). In MDA‐MB‐231 cells, 1–6 showed more potent antiproliferative activity than that of the truncated analog 9. Compound 4 was the most active (IC₅₀ = 11.89 nM) (Figure 3A). Preliminary structure–activity relationship (SAR) investigations indicated that 1–6, containing two pentapeptidolactone moieties, exhibited marked antiproliferative activities, whereas among the truncated analogs 7–15, only 9 retained notable activity. Based on the above SAR analyses, 1 and 9 were selected for further mechanistic investigation, representing an actinomycin analog containing two pentapeptidolactone moieties and a truncated actinomycin analog, respectively. Although the inhibitory activity of 1 (IC₅₀ = 28.00 vs. 11.89 nM) was weaker than that of 4, it was also selected for further mechanistic investigation because 1 and 2 are positional isomers, only differing in the position of the L‐2‐MeAba residue. Cell cycle analyses revealed that the treatment of 1 and 9 induced marked S‐phase accumulation and decreased the G0/G1‐phase population in MDA‐MB‐231 cells (Figure 3B). This reduction in cell viability was accompanied by S‐phase arrest, suggesting that cell cycle disruption may contribute to the antiproliferative effects of 1 and 9.

FIGURE 3.

Antiproliferative effects in MDA‐MB‐231 cells. (A) Cell viability determined by the CCK‐8 assay. (B) Flow cytometric analysis of cell cycle arrest. Data are presented as mean ± SD (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001 compared to the control group.

Apoptosis is a major mechanism underlying the antitumor activity of many anticancer agents [24]. To investigate the effects of 1 and 9 on apoptosis in MDA‐MB‐231 cells, flow cytometry was employed. The proportion of apoptotic MDA‐MB‐231 cells increased upon treatment with both compounds (Figure 4A). Mitochondrial depolarization is an early hallmark of apoptosis [25]. Accordingly, MDA‐MB‐231 cells were treated with 1 and 9 and subsequently stained with the JC‐1 probe. Enhanced green fluorescence indicated mitochondrial membrane potential dissipation and early apoptotic changes (Figure 4B). The above findings suggested that mitochondrial dysfunction might contribute to the apoptosis induced by 1 and 9.

FIGURE 4.

Apoptosis and mitochondrial membrane potential alterations in MDA‐MB‐231 cells. (A) Apoptosis was quantified by Annexin V‐FITC/PI staining. (B) Mitochondrial membrane potential dissipation was assessed using JC‐1 staining. Data are presented as mean ± SD (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001 compared to the control group.

Ferroptosis is driven by iron‐dependent lipid peroxidation of membrane phospholipids [26, 27]. To determine whether 1 and 9 induce ferroptosis, intracellular lipid peroxidation, reactive oxygen species (ROS), and malondialdehyde were evaluated. BODIPY 581/591 C11 staining demonstrated that both compounds markedly increased lipid peroxidation (Figure 5A). In agreement with the above findings, DCFH‐DA staining revealed a concentration‐dependent elevation of ROS (Figure 5B). Furthermore, treatment with 1 and 9 significantly increased intracellular MDA levels (Figure 5C). Ferrostatin‐1 (Fer‐1), a ferroptosis inhibitor, significantly restored cell viability following the treatment with 1 and 9 (Figure 5D). The coordinated increases in ROS, lipid peroxidation, and MDA, together with the restoration of cell viability by Fer‐1, supported the involvement of ferroptosis in the cell death induced by 1 and 9. Taken together, these findings suggested that 1 and 9 suppressed TNBC cell growth through S‐phase arrest and induced cell death through apoptosis and ferroptosis.

FIGURE 5.

Ferroptosis‐related effects in MDA‐MB‐231 cells. (A) Lipid peroxidation assessed by BODIPY 581/591 C11 staining. (B) Intracellular ROS assessed by DCFH‐DA staining. (C) Malondialdehyde levels assessed by the TBARS assay. (D) Fer‐1‐mediated rescue assessed by the CCK‐8 assay. Data are presented as mean ± SD (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001 compared with the control or corresponding compound‐treated group.

3. Conclusions

In summary, fifteen new actinomycin analogs, named actinomycins H₁–H₁₅, were identified from S. polyantibioticus S4D258. The structures of these compounds differ in the pentapeptidolactone moiety. Actinomycins H₇–H₁₅ are truncated actinomycin analogs. Actinomycins H₁–H₆ (1–6) and H₉ (9) exhibited significant antiproliferative activities against both MDA‐MB‐231 and 4T1 TNBC cells. Mechanistic investigations revealed that actinomycins H₁ (1) and H₉ (9) induced S‐phase arrest, apoptosis, and mitochondrial membrane potential disruption. In addition, treatment with both compounds increased intracellular ROS, promoted lipid peroxidation, and elevated malondialdehyde levels, whereas ferrostatin‐1 reversed the cytotoxic effects, suggesting that ferroptosis contributes to anticancer effects of these compounds. Collectively, these compounds broaden the chemical diversity of natural actinomycins and support their further exploration as potential anti‐TNBC leads.

4. Experimental Section

4.1. General Experimental Procedures

UV spectra were recorded on a Shimadzu UV‐2600i double‐beam spectrophotometer (Kyoto, Japan). LC–MS measurements were carried out using a Vanquish Auto‐liquid chromatography system coupled with a TSQ Quantis Plus mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). HR‐ESIMS spectra were obtained with a Bruker maXis Q‐TOF instrument (Karlsruhe, Germany). NMR spectra were recorded on a Bruker AVANCE 600 MHz spectrometer (Bruker, Karlsruhe, Germany). A Vanquish LC system (Thermo Scientific) was used for semi‐preparative and analytical HPLC with YMC‐Pack ODS‐A columns (5 µm; Kyoto, Japan; 250 × 10 mm i.d. and 250 × 4.6 mm i.d., respectively). Optical rotation values were recorded using a Model 341 polarimeter (PerkinElmer, Waltham, MA, USA).

4.2. Actinomycete Material

The marine actinomycete strain S4D258 was isolated from the South China Sea sediments by Prof. Gaiyun Zhang at the Third Institute of Oceanography, Ministry of Natural Resources, China. 16S rRNA gene sequence analysis and comparison with the NCBI database assigned strain S4D258 to S. polyantibioticus.

4.3. Extraction and Isolation

S. polyantibioticus S4D258 was cultivated in 100 mL of ISP2 seed medium for 5 days with shaking (28°C, 180 rpm). The seed cultures were dispensed into 100 Erlenmeyer flasks containing 250 mL of F2 production medium each (g/L: beef extract 1; soluble starch 10; yeast extract 10; corn steep liquor 3; glucose 20; CaCO₃ 2; MgSO₄·7H2O 0.5; and KH₂PO4 0.5; sea salt 2.5% w/v) and cultured for 14 days. Upon completion of fermentation, cultures were pooled and filtered under vacuum. The supernatant was concentrated and extracted with EtOAc, and the extract was evaporated to dryness, yielding 13.5 g of crude extract. Compound yields (%) were calculated on a w/w basis relative to the crude extract.

The resulting extract was fractionated on a YMC‐GEL ODS‐A‐HG column with a MeOH/H₂O gradient (30%–100% MeOH), yielding 58 fractions. Fraction 31, purified with MeCN/H₂O (47:53), yielded compound 5 (4.0 mg, 0.0296% yield, t _R = 74.3 min). Pooled fractions 32–33 (MeCN/H₂O, 50:50) afforded compounds 1 (5.2 mg, 0.0385% yield, t _R = 50.5 min) and 2 (5.0 mg, 0.0370% yield, t _R = 52.6 min). Pooled fractions 34–35 (MeCN/H₂O, 55:45) gave compounds 4 (4.8 mg, 0.0356% yield, t _R = 35.6 min) and 3 (6.3 mg, 0.0467% yield, t _R = 40.1 min). Fraction 50, purified with MeCN/H₂O containing 0.1% formic acid (52:48), provided compound 6 (12.0 mg, 0.0889% yield, t _R = 32.4 min).

Fractions 5–27 (4.5 g) were pooled for further separation on an ODS‐A column with a MeOH/H₂O gradient (25%–70% MeOH), yielding 36 subfractions. Subfraction 1 was purified with MeCN/H₂O (20:80), yielding compound 12 (16.3 mg, 0.1207% yield, t _R = 24.8 min). Subfraction 2, purified with MeCN/H₂O (20:80), afforded compounds 13 (8.4 mg, 0.0622% yield, t _R = 46.2 min) and 11 (10.2 mg, 0.0756% yield, t _R = 60.0 min). Subfraction 4, purified with MeCN/H₂O (30:70), gave compounds 7 (7.8 mg, 0.0578% yield, t _R = 36.2 min), 10 (11.6 mg, 0.0859% yield, t _R = 42.3 min), and 14 (2.1 mg, 0.0156% yield, t _R = 56.6 min). Subfraction 9, purified with MeCN/H₂O (35:65), yielded compound 15 (2.0 mg, 0.0148% yield, t _R = 34.5 min). Pooled subfractions 10–11, purified with MeCN/H₂O (35:65), afforded compound 8 (5.6 mg, 0.0415% yield, t _R = 24.1 min). Subfraction 18, purified with MeCN/H₂O (65:35), provided compound 9 (13.6 mg, 0.1007% yield, t _R = 31.5 min).

1: Red amorphous powder; ${[a]}_{\mathrm{D}}^{25}$ = −317.5 (c 0.1, MeOH); UV (MeOH) λ _max (log ε) 207 (4.78), 239 (4.52), 442 (4.36) nm (Figure S1); HR‐ESIMS m/z 1241.6220 [M + H]⁺ (calcd for C₆₁H₈₅N₁₂O₁₆, 1241.6201).

2: Red amorphous powder; ${[a]}_{\mathrm{D}}^{25}$ = −310.0 (c 0.15, MeOH); UV (MeOH) λ _max (log ε) 208 (4.85), 239 (4.61), 442 (4.45) nm (Figure S14); HR‐ESIMS m/z 1241.6205 [M + H]⁺ (calcd for C₆₁H₈₅N₁₂O₁₆, 1241.6201).

3: Red amorphous powder; ${[a]}_{\mathrm{D}}^{25}$ = −375.0 (c 0.1, MeOH); UV (MeOH) λ _max (log ε) 205 (4.47), 240 (4.53), 448 (4.33) nm (Figure S24); HR‐ESIMS m/z 1241.6203 [M + H]⁺ (calcd for C₆₁H₈₅N₁₂O₁₆, 1241.6201).

4: Red amorphous powder; ${[a]}_{\mathrm{D}}^{25}$ = −388.8 (c 0.1, MeOH); UV (MeOH) λ _max (log ε) 207 (4.83), 238 (4.58), 442 (4.40) nm (Figure S35); HR‐ESIMS m/z 1241.6202 [M + H]⁺ (calcd for C₆₁H₈₅N₁₂O₁₆, 1241.6201).

5: Red amorphous powder; ${[a]}_{\mathrm{D}}^{25}$ = −313.3 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 206 (4.77), 239 (4.56), 444 (4.37) nm (Figure S46); HR‐ESIMS m/z 1257.6141 [M + H]⁺ (calcd for C₆₁H₈₅N₁₂O₁₇, 1257.6150).

6: Red amorphous powder; ${[a]}_{\mathrm{D}}^{25}$ = −191.3 (c 0.2, MeOH); UV (MeOH) λ _max (log ε) 211 (4.72), 255 (4.56), 450 (4.25) nm (Figure S57); HR‐ESIMS m/z 1384.6774 [M + NH₄]⁺ (calcd for C₆₇H₉₄N₁₃O₁₉, 1384.6783).

7: Yellow amorphous powder; ${[a]}_{\mathrm{D}}^{25}$ = −110.0 (c 0.2, MeOH); UV (MeOH) λ _max (log ε) 212 (4.40), 316 (3.67) nm (Figure S67); HR‐ESIMS m/z 768.3566 [M + H]⁺ (calcd for C₃₇H₅₀N₇O₁₁, 768.3563).

8: Yellow amorphous powder; ${[a]}_{\mathrm{D}}^{25}$ = −98.5 (c 0.5, MeOH); UV (MeOH) λ _max (log ε) 214 (4.41), 316 (3.76) nm (Figure S77); HR‐ESIMS m/z 741.3447 [M + H]⁺ (calcd for C₃₆H₄₉N₆O₁₁, 741.3454).

9: Yellow amorphous powder; ${[a]}_{\mathrm{D}}^{25}$ = −76.3 (c 0.2, MeOH); UV (MeOH) λ _max (log ε) 214 (4.43), 316 (3.79) nm (Figure S87); HR‐ESIMS m/z 755.3610 [M + H]⁺ (calcd for C₃₇H₅₁N₆O₁₁, 755.3610).

10: Yellow amorphous powder; ${[a]}_{\mathrm{D}}^{25}$ = −92.5 (c 0.1, MeOH); UV (MeOH) λ _max (log ε) 212 (4.39), 316 (3.65) nm (Figure S97); HR‐ESIMS m/z 740.3623 [M + H]⁺ (calcd for C₃₆H₅₀N₇O₁₀, 740.3614).

11: White amorphous powder; ${[a]}_{\mathrm{D}}^{25}$ = −57.5 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 218 (3.94) nm (Figure S107); HR‐ESIMS m/z 568.2979 [M + H]⁺ (calcd for C₂₆H₄₂N₅O₉, 568.2977).

12: White amorphous powder; ${[a]}_{\mathrm{D}}^{25}$ = −34.5 (c 1.0, MeOH); UV (MeOH) λ _max (log ε) 216 (3.67) nm (Figure S117); HR‐ESIMS m/z 554.2829 [M + H]⁺ (calcd for C₂₅H₄₀N₅O₉, 554.2821).

13: White amorphous powder; ${[a]}_{\mathrm{D}}^{25}$ = −68.8 (c 0.2, MeOH); UV (MeOH) λ _max (log ε) 219 (3.94) nm (Figure S127); HR‐ESIMS m/z 553.2979 [M + H]⁺ (calcd for C₂₅H₄₁N₆O₈, 553.2980).

14: Red amorphous powder; ${[a]}_{\mathrm{D}}^{25}$ = −20.0 (c 0.2, MeOH); UV (MeOH) λ _max (log ε) 211 (4.64), 260 (4.33), 456 (3.40) nm (Figure S137); HR‐ESIMS m/z 645.3249 [M + H]⁺ (calcd for C₃₁H₄₅N₆O₉, 645.3243).

15: Yellow amorphous powder; ${[a]}_{\mathrm{D}}^{25}$ = −102.5 (c 0.1, MeOH); UV (MeOH) λ _max (log ε) 218 (4.67), 249 (4.33), 298 (3.95) nm (Figure S146); HR‐ESIMS m/z 657.3255 [M + H]⁺ (calcd for C₃₂H₄₅N₆O₉, 657.3243).

4.4. Marfey's Analysis

The target compounds (0.5 mg each) were hydrolyzed in sealed vials under acidic conditions (6 M HCl, 110°C, 24 h). After hydrolysis, the samples were evaporated to dryness under a stream of N₂, mixed with 1 M NaHCO₃ solution (20 µL), and derivatized in the dark with 1% (w/v) Nα‐(2,4‐Dinitro‐5‐fluorophenyl)‐L‐alaninamide (FDAA, Marfey's reagent, Shanghai Macklin Biochemical Co., Ltd.) in acetone (50 µL) at 45°C for 60 min. The resulting derivatives were tested by LC–MS using a 20%–60% MeCN gradient in 0.1% formic acid (60 min; 1.0 mL/min). The absolute configurations were assigned by comparing the FDAA derivatives with authentic standards under the same conditions. The standards included the L‐ and D‐forms of Val, Thr, Pro, Hyp, MeVal, Ser, 2‐Aba, 2‐MeAba, allo‐Thr, and cis‐Hyp, together with Sar (Shanghai Macklin Biochemical Co., Ltd.), all derivatized under the same conditions.

4.5. Cell Culture

MDA‐MB‐231 (RRID: CVCL_0062) and 4T1 cells (RRID: CVCL_0125) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Both cell lines were cultured in DMEM supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) and 1% penicillin‐streptomycin (Solarbio Science & Technology Co., Ltd., Beijing, China), and maintained at 37°C in a humidified incubator with 5% CO₂.

4.6. Cell Viability Assay

MDA‐MB‐231 and 4T1 cells were seeded at 4.0 × 10³ cells/well and cultured for 24 h before treatment with compounds 1–15 (0–20 µM) for 48 h under standard culture conditions. For the Fer‐1 rescue experiment, MDA‐MB‐231 cells with 1 h pretreatment of Fer‐1 (1.0 µM) were subsequently treated with compound 1 (20 nM) or compound 9 (2.50 µM) for 48 h. After adding 10 µL of CCK‐8 solution (Beyotime Institute of Biotechnology, Shanghai, China) and incubating for 2 h, the OD450 was measured with a Thermo Fisher Scientific Multiskan SkyHigh microplate reader.

4.7. Cell Cycle Analysis

Following treatment, cells were subjected to overnight fixation in 70% ethanol at 4°C. After removing ethanol, the samples were resuspended in 500 µL of PI/RNase staining solution (Beyotime) and maintained under light‐protected conditions for 30 min. Flow cytometric data were acquired on a FACSCalibur instrument (BD Biosciences, CA, USA).

4.8. Apoptosis Analysis

Following treatment, the adherent and nonadherent cell fractions were collected and stained with the Annexin V‐FITC/PI kit (Beyotime) for 30 min at room temperature in the dark. Flow‐cytometric acquisition was performed immediately on a FACSCalibur system, followed by quantification of apoptotic fractions in FlowJo software.

4.9. Measurement of Mitochondrial Membrane Potential

Following treatment, the adherent and nonadherent cells were combined and pelleted. The cells were then stained with a JC‐1 kit (Beyotime) for 20 min at 37°C, washed twice with 1× staining buffer, and resuspended in fresh buffer. Flow‐cytometric data were acquired immediately on a FACSCalibur system.

4.10. Measurement of Lipid Peroxidation

Following two washes with PBS, BODIPY 581/591 C11 (2 µM, Beyotime) was added to the cells, and the mixture was incubated for 30 min in the dark. After incubation, the cells were resuspended in fresh PBS and immediately analyzed by flow cytometry on a FACSCalibur instrument. Lipid peroxidation was evaluated according to the increase in green fluorescence intensity.

4.11. Measurement of Intracellular ROS Content

Following treatment, the adherent and nonadherent cell fractions were pooled and exposed to 10 µM DCFH‐DA (Beyotime) in serum‐free DMEM for 20 min under light‐protected conditions. Following three rinses in serum‐free DMEM, the samples were brought into suspension with 500 µL of fresh medium and immediately subjected to flow cytometric analysis on a FACSCalibur instrument. Intracellular ROS levels were evaluated according to the fluorescence intensity of DCF.

4.12. Measurement of Intracellular Malondialdehyde Levels

Malondialdehyde accumulation was assessed following exposure to compounds 1 (20 nM) or 9 (2.50 µM). The harvested cells were adjusted to 2 × 10⁶ cells per sample and resuspended in extraction buffer. After sonication on ice, the lysates were centrifuged (8,000 × g, 10 min). The supernatants were used for TBARS analysis following the manufacturer's protocol (Solarbio). The reaction mixtures were heated (100°C, 1 h) and then centrifuged again (10,000 × g, 10 min) under ambient conditions. The resulting supernatants were analyzed at 532 nm using a Multiskan SkyHigh microplate reader, with readings at 600 nm for background correction.

4.13. Statistical Analysis

Data are presented as the mean ± standard deviation (SD) from at least three independent experiments. Two‑group comparisons were assessed by unpaired t‑tests, and multiple‑group differences by one‑way ANOVA with Tukey's test, using SPSS 25.0 and GraphPad Prism 8.0. Differences were regarded as significant when p < 0.05.

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. He Pang: investigation, visualization, writing – original draft, writing – review and editing. Xiao‐Min Huang: investigation, visualization. Usama Ramadan Abdelmohsen: methodology, visualization, writing – review and editing. Li Shen: conceptualization, methodology, investigation, visualization, supervision, writing – original draft, writing – review and editing, funding acquisition, resources. Jun Wu: conceptualization, methodology, investigation, visualization, supervision, writing – original draft, writing – review and editing, funding acquisition, resources.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

UV, HR‐ESIMS, 1D and 2D NMR, and Marfey's analysis spectra for compounds 1–15. Supporting File: cbdv71791‐sup‐0001‐SuppMat.pdf.

Data Availability Statement

The data that support the findings of this study are available in the Supporting Information of this article.