Polyketides with a 6/6/6/6 Oxaphenalene Pyranone Skeleton from Marine-Derived Streptomyces sp. HDN150000

Abstract

Three new structures named naphpyrone I–K (1–3) that contain a 6/6/6/6 oxaphenalene pyranone skeleton were isolated and purified from a marine-derived Streptomyces sp. HDN155000. Their chemical structures, including configurations, were elucidated by extensive NMR, MS, single-crystal X-ray diffraction, theoretical NMR calculations, DP4+ probability analysis, and ECD analyses. Naphpyrone K (3) showed cytotoxic activities against L-02, K562, NCI-H446/EP, MDA-MB-231, and NCI-H446 cancer cells with IC₅₀ values of 5.13, 3.34, 2.50, 2.61, and 2.20 μM, respectively. These findings highlight the potential for screening and developing therapeutic drugs from aromatic polyketides derived from marine actinobacteria.

1. Introduction

Marine actinobacteria, a branch of actinobacteria, is widely distributed in various ecosystems in the marine environment, including seafloor sediments, rock surfaces, and seawater [1]. Compared to terrestrial actinobacteria, marine actinobacteria are more tolerant of hypoxia, oligonutrition, high salt, high pressure, low temperature, and other unusual living circumstances, which is closely related to their long-term survival in special environments [2]. This group of microorganisms exhibits a complex and elaborate cycle of morphological differentiation. Under suitable environmental and nutrient conditions, the mycelium of marine actinobacteria exhibits multinucleate, branched, slender, and non-septate characteristics [3]. Morphological differentiation in marine actinobacteria is frequently accompanied by complex physiological changes and the generation of a large number of metabolites [4,5]. In recent years, a variety of structurally diverse secondary metabolites with unique bioactivities originating from marine actinomycetes have been exploited [6,7]. Subramani summarized 167 new bioactive compounds produced by marine actinobacteria with antimicrobial, antitumor, anthelmintic, and antimalarial activities, of which the genus Microcystis is the richest source of chemical diversity and unique bioactivities [8]. Chen summarized 536 compounds isolated from marine actinomycetes, of which alkaloids (37%), polyketides (33%), and peptides (15%) accounted for the largest proportion, and Streptomyces (68%), Aeromonas (6%), and Nocardia (3%) were the major producers of secondary metabolites [9]. Some researchers have even begun to focus on the effect of marine Streptomyces secondary metabolites on drug-resistant bacteria. Cho extracted a new chromomycin from the marine-derived Streptomyces sp. MBTI36 with potent antimicrobial activity against Methicillin-resistant Staphylococcus aureus (MRSA) [10]. In addition, a study has isolated a novel tirandamycin with vancomycin-resistant Enterococcus faecalis inhibitory activity from the marine Streptomyces sp. 307-9 [11]. These findings have the potential to alleviate the antibiotic resistance crisis and demonstrate marine actinomycetes as a promising resource for lead compound discovery and development.

In the ongoing process of our group’s research for natural products with excellent bioactivity from marine-derived microorganisms, a strain of Streptomyces sp. HDN150000 isolated from a marine sediment sample caught our attention. The OSMAC strategy (one strain–many compounds) was employed to cultivate Streptomyces sp. HDN150000, resulting in compounds with significantly different UV absorption profiles in M1 liquid media compared to other conditions. Under the guidance of HPLC-UV and LC-MS, six compounds with a 6/6/6/6 oxaphenalene pyranone skeleton, including three novels named naphpyrone I–K (1–3), were isolated and purified (Figure 1). Among them, compound 3 showed cytotoxic activities against cells L-02, K562, NCI-H446/EP, MDA-MB-231, and NCI-H446 with IC₅₀ values of 5.13, 3.34, 2.50, 2.61, and 2.20 μM, respectively. Details of the isolation, structure elucidation, and bioactivities of these compounds are reported herein.

2. Results

Streptomyces sp. HDN15000 was isolated from a sediment sample collected from the South China Sea (125°28.550′ E, 29°1.618′ N). A comprehensive study of the metabolites produced by this Streptomyces species was carried out on various culture conditions. When culturing Streptomyces HDN150000 in M1 liquid medium, more differential peaks appeared (Figure 2).

To specifically identify the structures, we processed a larger-scale fermentation of this strain, and ethyl acetate extraction of the fermentation broth yielded 18.3 g crude extract. Subsequently, following HPLC results, the crude extracts were then separated stepwise using repeated silica gel column chromatography, Sephadex LH-20, and semipreparative HPLC to yield compounds 1–6 (Figure 1).

Compound 1 was isolated as a white powder. The chemical formula was determined to be C₁₇H₁₄O₄, supported by the cationic molecular ion peak at m/z 283.0962 [M + H]⁺ in the high-resolution electrospray ionization mass spectrometry (HRESIMS) (Figure S9), which signifies eleven degrees of unsaturation. The ¹H NMR spectrum of 1 displayed two methyls (δ_H 2.13 and δ_H 1.62), one methylene (δ_H 3.05 and 2.68), five aromatic methines (δ_H 8.96, d, J = 8.8 Hz; δ_H 7.48, m; δ_H 6.88, d, J = 7.2 Hz; δ_H 6.22, s; δ_H 6.41, s) (

Table 1. ¹H (500 MH_Z) and ¹³C (125 MH_Z) spectroscopic data for 1–3 in DMSO-d₆.

No	1		2		3
	δC Type	δH (J in Hz)	δC Type	δH (J in Hz)	δC type	δH (J in Hz)
2	101.5 qC		165.3 qC		163.4 qC
3	49.3 CH2	3.05 day (15.9)	111.3 CH	6.17 s	112.2 CH	6.22 day (1.0)
		2.68 day (15.9)
4	190.6 qC		178.6 qC		177.7 qC
4a	106.4 qC		117.7 qC		110.5 qC
4b	132.6 qC		140.2 qC		131.6 qC
5	121.2 CH	8.96 day (8.8)	36.7 CH2	3.69 m	122.7 CH	9.48 day (8.8, 1.0)
				3.64 m
6	131.8 CH	7.48 m	63.2 CH	4.15 m	131.3 CH	7.60 m
7	115.5 CH	6.88 day (7.2)	36.7 CH	2.97 m	118.0 CH	7.16 day (7.3, 1.0)
				2.80 m
7a	129.4 qC		153.7 qC		129.2 qC
7b	117.2 qC		114.2 qC		119.7 qC
8	104.4 CH	6.22 s	111.9 CH	6.32 s	104.2 CH	6.43 brs
9	152.3 qC		159.1 qC		156.3 qC
10a	158.6 qC		155.2 qC		156.9 qC
11	98.8 CH	6.41 s	103.1 CH	7.41 s	97.4 CH	6.88 s
11a	162.9 qC		158.8 qC		159.4 qC
12	18.7 CH3	2.13 s	19.5 CH3	2.34 s	74.1 CH	4.00 day (4.7)
13	27.3 CH3	1.62 s			67.4 CH	3.91 m
14					19.3 CH3	1.13 day (6.4)
15					19.2 CH3	2.35 s
2-OH		7.12 s

). The ¹³C NMR spectrum combined with the HSQC spectrum reveals the existence of two methyls, one methylene, five protonated sp² carbons, and nine non-protonated carbons (Table 1). The planar structure of 1 was established through a detailed analysis of its 2D NMR data. Firstly, the ¹H-¹H COSY correlations from H-5 (δ_H 8.98, d, J = 8.8 Hz) through H-6 (δ_H 7.48 m) to H-7 (δ_H 6.88, d, J = 7.2 Hz) suggest the existence of the 1,2,3-trisubstituted benzene ring (ring D). Further analysis of the key HMBC correlations from H-5 to C-7b/C-4a, from H-6 to C-7a/C-4b, and from H-7 to C-8/C-7b/C-5 identified the ring D and extended to the rings C and B. The HMBC correlations from H-12 to C-8/C-9, from H-8 to C-9/C-7a/C-7b, and from H-11 to C-7b/C-10a/C-4a/C-11a delineated rings C and B. Finally, the HMBC correlations from H-13 to C-2/C-3, from H-3 to C-4a/C-4/C-2, and from 2-OH (δ_H 7.12) to C-13/C-2 identified the ring A and its propagation to the ring B. Thus, the planar structure of compound 1 possessing 6/6/6/6 oxaphenalene pyranone was determined (Figure 3).

The absolute configuration of compound 1 was confirmed as 2S supported by the X-ray diffraction (Figure 4).

Compound 2 was obtained as a white solid powder. The molecular formula C₁₆H₁₂O₅ deduced by HR-ESIMS, suggests 11 degrees of unsaturation. ¹H and ¹³C spectra reveal that compound 2 contains one methyl, two methylenes, one sp³ methine, three sp² methine, and nine non-protonated carbons (including two carbonyl and seven aromatic quaternary carbons). Detailed 2D NMR analysis showed that compound 2 has a similar 6/6/6/6 system with compound 1 except that the dehydration of C-2 and C-3 of the ring A to form a double bond, which can be determined by the HMBC correlations from H-12 (δ_H 2.34) to C-2 (δ_C 165.3) and C-3 (δ_C 111.3). The C-5 (δ_C 36.7), C-6 (δ_C 63.2), and C-7 (δ_C 36.7) of the ring D changed from sp² to sp³ carbon signals determined from the ¹H-¹H COSY correlations of H-5/H-6/H-7. Further consideration of the C-6 as the methenyl carbon signal suggests the presence of hydroxyl substitution [12]. Another difference lies in the low-field chemical shift of C-9 (δ_C 159.1) in combination with the HR-ESIMS data at m/z 285.0755 [M + H]⁺, identifying C-9 as a carbonyl carbon [13,14]. Thus, the planar structure of 2 was determined (Figure 3). To determine the absolute configuration of 2, ECD calculations of the possible configurations (6S*)-2 and (6R*)-2 were performed. Finally, the good agreement between the experimental and calculated ECD curves evidenced that the absolute configuration of 2 is 6R (Figure 5A).

Compound 3 was separated as a white amorphous solid powder. The molecular formula of C₁₉H₁₆O₅ is established by the HRESIMS data (m/z 325.1073 [M + H]⁺, calcd. 325.1071). The ¹H NMR spectrum of 3 showed two methyls (δ_H 1.13 and δ_H 2.53), two sp³ methines (δ_H 4.00 and δ_H 3.91), six sp² aromatic methines (δ_H 6.22; δ_H 9.48; δ_H 7.60; δ_H 7.16; δ_H 6.43; δ_H 6.88) (Table 1). The ¹³C NMR spectrum reveals the existence of two methyls, eight methines, and nine non-protonated carbons (including a carbonyl and eight non-protonated sp² carbons) (Table 1). 2D NMR showed that compound 3 has a 6/6/6/6/6 ring system structure similar to compound 1, except that C-2 (δ_C 163.4) and C-3 (δ_C 112.2) are dehydrated to form a double bond, in addition the substitution at C-9 (δ_C 156.3) with a propylene glycol side chain can be further determined by the HMBC correlations from H-14 (δ_H 1.13) to C-13 (δ_C 67.4) and C 12 (δ_C 74.1), from H-13 (δ_H 3.91) to C-9, and from H-12 (δ_H 4.00) to C-8 (δC 104.2), C-9, and C-13. At this point, the planar structure of compound 3 was determined.

Compound 3 has four possible configurations (12S*,13R*)-3, (12S*,13S*)-3, (12R*,13R*)-3, and (12R*,13S*)-3. To determine the relative structure of 3, the theoretical NMR calculations at PCM(DMSO)-mPW1PW91/6-311+G(d,p)//B3LYP/6-31G(d)-GD3BJ level and DP4+ probability analyses were employed on two possible configurations (12S*,13R*)-3 and (12S*,13S*)-3, and the calculations messages suggested that (12S*,13R*)-3 was the correct relative structure (Table S1). Subsequent ECD calculations were performed and compared to the experimental ECD curves to determine the absolute conformation of 3 as 12S,13R (Figure 5B).

Based on the comparison of NMR and MS data with those reported in the literature, the known compounds isolated in this study were identified as naphpyrone D (4), naphpyrone B (5), and naphpyrone C (6) [15].

All new compounds were evaluated for cytotoxicity against L-02, MDA-MB-231, K562, ASPC-1, NCI-H446, and NCI-H446/EP cell lines in vitro. Adriamycin was used as the positive control. As a result, compound 3 showed cytotoxic activity against cells L-02, K562, NCI-H446/EP, MDA-MB-231, and NCI-H446 with IC₅₀ values of 5.13, 3.34, 2.50, 2.61, and 2.20 μM, respectively (Table S2–S6).

3. Materials and Methods

3.1. General Experimental Procedures

Streptomyces sp. HDN150000 genomes were acquired using methods previously described in the literature [16].

An Agilent DD2-500 spectrometer was used to obtain NMR spectra, with tetramethylsilane (TMS) as the internal standard. UV-vis spectra were recorded on the UFLC system (Shimadzu, Tokyo, Japan). HRESIMS spectra were measured on a Thermo Scientific LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Specific rotations were obtained on a JASCO P-1020 digital polarimeter. The ECD spectra were obtained using a JASCO J-715 spectropolarimeter (JASCO, Tokyo, Japan). Column chromatography (CC) was performed using silica gel (300–400 mesh, Qingdao Marine Chemical Ltd., Qingdao, China), SiliaSphere C18 (Octadecylsilyl, ODS) monomeric (SiliCycle Inc., Québec City, Canada, 50 μm, 120 A), and Sephadex LH-20 (Amersham Biosciences, Buckinghamshire, UK). MPLC was performed on a Waters 1526. HPLC spectra were collected with an ODS column (YMC-Pack ODS-A, 10 × 250 mm, 5 μm, 3 mL min⁻¹, YMC Co., Ltd., Kyoto, Japan).

3.2. Materials and Culture Conditions

Streptomyces sp. HDN155000 was isolated from a marine sediment sample collected from the South China Sea at coordinates 125°28.550′ E and 29°1.618′ N. The strain was identified through genome sequencing and submitted to GenBank (No. OP001709) [17]. Wild type and their mutant strains were cultivated for 8 days at 28 °C on MS plates containing 2% soya bean flour, 2% mannitol, and 1.8% agar. For genome extraction, the strain was inoculated into 250 mL Erlenmeyer flasks with 50 mL of BF1 liquid medium (1% glucose, 1% tryptone, 2% starch, 0.5% yeast extract, and 0.5% CaCO₃) for 2 days.

3.3. Fermentation and LC/LC-MS Analysis

For small-scale analysis, all actinobacteria were cultured for 8 days at 28 °C in M1 medium (4 g/L yeast extract, 2 g/L peptone, 10 g/L starch), TSB medium (1.7% tryptone, 0.3% peptone, 0.25% glucose, 0.25% KH2PO4, and 0.3% NaCl) and BF1 medium after which the culture products were collected and extracted by ethyl acetate at least three times. The organic phase from the three extractions was evaporated, and the residue was redissolved in 200 μL MeOH. Then, 50 μL of dissolved extract was injected for high-performance liquid chromatography–photodiode array detection–mass spectrometry (HPLC-DAD-MS) analysis (C18 column, Shimadzu, 4.6 mm × 150 mm, 5 μm, 1 mL/min); the samples were separated on a linear gradient of 5–100% acetonitrile (MeOH) in water (0.1% trifluoroacetic acid) for 50 min at a flow rate of 1 mL/min, and HPLC analysis revealed a significant change in metabolite production in the mutant strain.

For compound separation, the selected strain was incubated in M1 medium at 28 °C for 2 days as a seed solution. Then, cultured seed solutions were transferred to 500 mL Erlenmeyer flasks containing 100 mL of M1 medium at 2% inoculum (total 18 L) for further fermentation. The broth was extracted three times with ethyl acetate to provide a total of 55 L of extract solution. The organic phase was evaporated under reduced pressure to afford a crude residue (18.3 g).

3.4. Extraction, Isolation, and Purification

To identify secondary metabolites, under the guidance of HPLC analysis, the crude extracts were separated with a step gradient elution of MeOH-H₂O, providing eight subfractions (Fr.1–Fr.8, 30% to 100%). The compounds are mainly concentrated in Fr.3–Fr.5. Fr.3 was purified by semi-preparative HPLC (41:59 MeOH-H₂O, 3 mL/min) to generate compound 1 (5.4 mg, t_R = 21.3 min). Fr.3 was purified by a semi-preparative C18 HPLC column (55:45 MeOH-H₂O), producing compound 3 (3.8 mg, t_R = 14.7 min) and Fr.4.1. This subfraction was separated with preparative C18 HPLC column (50:50 MeOH-H₂O) to yield compounds 2 (4.3 mg, t_R = 14.7 min) and 5 (3.4 mg, t_R = 18.5 min). Fr.5 was applied to a Sephadex LH-20 column and eluted with methanol, obtaining compound 4 (8.2 mg). Fr.5 was purified through semipreparative HPLC (66:34 MeOH-H₂O, 3 mL/min) to produce compound 6 (9.0 mg, t_R = 13.3 min).

Naphpyrone I (1): white powder, ${\left[\alpha \right]}_D^{25}$ –32.6 (c 0.03, CH₃OH); UV (DAD) λ_max 210 nm, 226 nm, 242 nm, 254 nm, 277 nm, 242 nm, and 399 nm; CD (MeOH); λ_max (∆ε) 210 (–12.46), 218 (+3.21), 226 (–1.78), 232 (–0.36), 240 (–2.09), and 263 (+2.35); ¹H and ¹³C NMR data, see Table 1; positive ion HRESIMS m/z 283.0962 [M + H]⁺ (calcd. for C₁₇H₁₅O₄⁺, 283.0965).

Naphpyrone J (2): white solid powder, ${\left[\alpha \right]}_D^{25}$ 19.5 (c 0.03, CH₃OH); UV (DAD) λ_max 210 nm, 225 nm, 258 nm, 263 nm, 269 nm, 274 nm, 279 nm, and 329 nm; CD (MeOH); λ_max (∆ε) 225 (–5.08), 266 (+1.81), 315 (–0.17), and 353 (+1.66); ¹H and ¹³C NMR data, see Table 1; positive ion HRESIMS m/z 285.0755 [M + H]⁺ (calcd. for C₁₆H₁₃O₅⁺, 285.0757).

Naphpyrone K (3): white amorphous solid powder, ${\left[\alpha \right]}_D^{25}$ –20.3 (c 0.03, CH₃OH); UV (DAD) λ_max 238 nm, 242 nm, 258 nm, 270 nm, 274 nm, and 274 nm; CD (MeOH); λ_max (∆ε) 224 (+2.16), 244 (+8.21), 251 (+1.66), 356 (+3.36), and 369 (–2.82); ¹H and ¹³C NMR data, see Table 1; positive ion HRESIMS m/z 325.1073 [M + H]⁺ (calcd. for C₁₉H₁₇O₇⁺, 325.1071).

3.5. X-Ray Crystallographic Analysis of Naphpyrone I

Naphpyrone I (1) was analyzed by X-ray diffraction on Cu Kα radiation. Compound 1 was concentrated in vacuo and redissolved in 1.5 mL CH₃OH that was transferred in a 2 mL clear screw autosampler vial. The lid with holes was then closely screwed on the vial, and white needle-like crystals were obtained after seven nights at room temperature.

Crystal Data: C₁₇H₁₄O₄ (M =282.28 g/mol): trigonal, space group R-3 (no. 148), a = 36.3042(8) Å, c = 5.5043(2) Å, V = 6282.7(4) ų, Z = 18, T = 120.00(10) K, μ (Cu Kα) = 0.789 mm⁻¹, Dcalc = 1.343 g/cm³, 7569 reflections measured (4.868° ≤ 2Θ ≤ 145.992°), 2693 unique (R_int = 0.0599, R_sigma = 0.0589) which were used in all calculations. The final R₁ was 0.0530 (I > 2σ(I)) and wR₂ was 0.1540 (all data).

The crystallographic data have been deposited in the Cambridge Crystallographic Data Centre as CCDC 2434219.

3.6. NMR and ECD Calculations

Conformational searches were conducted using Spartan′14, based on the MMFF force fields [18]. Compounds 1, 2, and 3 were further optimized with DFT calculations at the B3LYP/6-31G(d)-GD3BJ level, utilizing the Gaussian 16 program package [19]. Gauge-independent atomic orbital (GIAO) calculations of ¹³C NMR of conformers were accomplished by DFT at the mPW1PW91/6-311+G(d,p) level with the PCM model in DMSO. The calculated NMR data of these conformers were averaged according to the Boltzmann distribution theory and their relative Gibbs free energy. The ¹³C NMR chemical shifts for TMS were also calculated by the same procedures and used as the reference. After calculation, the experimental and calculated data were evaluated by the improved probability DP4+ method [20]. The ECD was calculated using time-dependent density functional theory (TDDFT) at a B3LYP/6-31+G(d) level in methanol with the IEFPCM model [21]. The calculated ECD curves were all generated using the SpecDis 1.71 program package, and the calculated ECD data of all conformers were Boltzmann averaged using Gibbs free energy [22].

3.7. Cytotoxicity Assay

The cytotoxic assay involved six human cancer cell lines: K562 (using the MTT method), L-02, MDA-MB-231, ASPC-1, NCI-H446, and NCI-H446/EP (using the SRB method). Adriamycin (ADM) was used as a positive control. The detailed procedures for biological testing were conducted as previously stated [23]. The cancer cell lines were purchased from the National Collection of Authenticated Cell Cultures of China (Shanghai).

4. Conclusions

Marine actinobacteria can produce structurally diverse secondary metabolites with unique biological activities, making them a new resource for the discovery of antibiotics. Six compounds containing a 6/6/6/6 oxaphenalene pyranone skeleton were isolated and determined from Streptomyces sp. HDN150000, which was isolated from a marine sediment sample collected from the South China Sea. Among them, compound 3 showed cytotoxic activity against cells L-02, K562, NCI-H446/EP, MDA-MB-231, and NCI-H446, providing an alternative lead compound for medical study. Our study highlights the potential of mining secondary metabolites from marine actinobacteria for screening and utilization of therapeutic molecules.