ABSTRACT
Six new trichostatin analogs, designated karstmycins I−M (1−6), together with eight known congeners (7−14), were obtained from Streptomyces sp. DX6D14 isolated from the soil of the karst cave. The structures of the new compounds were elucidated through spectroscopic analysis, including high‐resolution electron spray ionization mass spectrometry (HRESIMS) and nuclear magnetic resonance (NMR), and electronic circular dichroism (ECD) calculations. All the isolates were evaluated for antibacterial and cytotoxic activities.
Six new trichostatin analogs, designated karstmycins I−M (1−6), together with eight known congeners (7−14), were obtained from Streptomyces sp. DX6D14 isolated from the soil of the karst cave.
1. Introduction
Caves represent extreme habitats characterized by low nutrients, stable temperatures, limited light, and high humidity. These unique conditions may promote the production of structurally novel and biologically active metabolites by resident microorganisms [1, 2]. Actinobacteria are reported to be a dominant microbial population in cave ecosystems [3]. Numerous bioactive metabolites, including polyketides [4, 5], pyranonaphthoquinones [6, 7], and alkaloids [8, 9], have been isolated from the cave‐associated actinobacteria, many of which exhibited antibacterial and cytotoxic activities. Among these, members of the genus Streptomyces are particularly prolific producers [1].
In our previous study, eight pairs of undescribed trichostatin analogues enantiomers, (±)‐karstmycins A−H, and three new enantiomers together with five known analogues were isolated from a karst cave‐derived Streptomyces sp. DX6D14 [10]. To further explore the metabolic potential of Streptomyces sp. DX6D14, we supplemented the culture medium with varying concentrations of CaCO3 to simulate the original habitat environment of this strain, thereby activating the cryptic biosynthetic gene clusters. HPLC analysis of the ethyl acetate extracts revealed that the medium containing 6 g/L CaCO3 yielded the most complex metabolite profile (Figure S43). From this culture, six new trichostatin analogs, karstmycin I−M (1−6), together with eight known congeners (7−14) (Figure 1), were obtained. Herein, the isolation, structure elucidation, and activity evaluation of these isolates were reported.
FIGURE 1.
The chemical structures of 1−14.
2. Results and Discussion
Karstmycin I (1) was isolated as a yellow oil with its molecular formula determined as C18H23NO3 from the HR‐ESI‐MS ion peak at m/z 324.1574 [M + Na]+ (calcd for C18H23NO3Na+, 324.1570), corresponding to eight degrees of unsaturation. The 1H and 13C NMR spectra, alongside the HSQC spectrum indicated the presence of a para‐substituted aromatic ring [δ H/C 6.73/112.0 (CH‐10/12), δ H/C 7.86/131.9 (CH‐9/13), δ C 125.5 (C‐8), and δ C 155.5 (C‐11)], an N,N‐dimethyl group (δ H/C 3.04/40.2), one trisubstituted olefin group [δ H/C 5.89/141.8 (CH‐5) and δ C 135.4 (C‐4)], one double bond [δ H/C 5.81/118.0 (CH‐2) and 7.26/150.7 (CH‐3)], one ketone group [δ C 201.1 (C‐7)], one methine group [δ H/C 4.40/49.1 (CH‐6)], one methylene [δ H/C 1.87/1.60/27.6 (CH2‐14)], two methyl groups [δ H/C 1.91/13.0 (CH3‐15) and 0.89/12.3 (CH3‐16)], and a carbonyl group [δ C 170.7 (C‐1)] (Figure 2). Four spin coupling systems of H‐2/H‐3, H‐6/H2‐14/H3‐16, and H‐9/13−H‐10/12 were constructed by the 1H‐1H COSY correlations (Figure 2). The HMBC correlations from H‐9/13 to C‐7, from CH 2‐14 to C‐7 C‐6, and C‐5, from 11‐N(CH 3)2 to C‐11, from CH 3‐15 to C‐3 C‐4, and C‐5, and from H‐2 and H‐3 to C‐1 established a structure related to trichostatin acid [11] with difference attributed to the that an ethyl in 1, instead of a methyl in trichostatin acid [11], attached at C‐6. The presence of an ethyl group at C‐6 in 1 is unusual among trichostatin analogues, which typically bear a methyl at this position. This ethyl group is likely derived from a non‐canonical extension of the polyketide chain, possibly through incorporation of ethylmalonyl‐CoA instead of methylmalonyl ‑CoA during biosynthesis [12]. To determine the absolute configuration of 1, we performed quantum chemical ECD calculation for 1 by the time‐dependent DFT method at the CAM‐B3LYP/TZVP//B3LYP/6‐311G(d,p) theoretical level. The computed ECD curve for (6R)‐1 closely matched the experimental spectrum, assigning the 6R configuration to 1 (Figure 3).
FIGURE 2.
Key HMBC (red arrow), 1H‐1H COSY (bold line), and ROESY (dash arrow) correlations of 1–6.
FIGURE 3.
The experimental and calculated ECD spectra of 1−6.
Karstmycin J (2) was obtained as a yellow oil. Its molecular formula was established as C17H23NO3 based on the HR‐ESI‐MS sodium adduct ion peak at m/z 312.1567 [M + Na]+ (calcd for C17H23NO3Na+, 312.1570). The 1D NMR data of 2 presented characteristic signals for an N,N‐dimethylbenzophenone group [δ H/C 6.74/112.0 (CH‐10/12), 7.87/131.7 (CH‐9/13), 3.06/40.1 (11‐N(CH3)2), δ C 125.1 (C‐8), 155.4 (C‐11), 205.0 (C‐7)], one trisubstituted olefin group [δ H/C 5.33/119.8 (CH‐3) and δ C 137.7 (C‐4)], two methylene groups [δ H/C 2.98/34.3 (CH2‐2) and 2.46/2.09/44.9 (CH2‐5)], one aliphatic methine group [δ H/C 3.72/39.0 (CH‐6)], and two methyl groups [δ H/C 1.10/16.5 (CH3‐14) and 1.66/17.8 (CH3‐15)]. The 1H‐1H COSY correlations of H3‐14/H‐6/H2‐5 and H2‐2/H‐3 constructed two spin coupling systems, and the key HMBC correlations from H3‐14 to C‐5, C‐6, and C‐7, and from H3‐15 to C‐3, C‐4, and C‐5 (Figure 2) connected the two spin coupling systems and the N,N‐dimethylbenzophenone group via C‐6‐C‐7 and C‐3−C‐4−C‐5 (Figure 2). The HMBC correlation between H‐2 and C‐1 attached the carbonyl (C‐1) at C‐2. The ECD spectrum of 2 matched well with the calculated curve for the 6R configuration (Figure 3).
Karstmycin K (3) was obtained as a yellow oil. Its HR‐ESI‐MS ion peak at m/z 326.1725 [M + Na] + (calcd for C18H25NO3Na+, 326.1727) established the molecular formula of C18H25NO3, indicating a mass increase of 14 Da (CH2) compared to that of 2. The 1D NMR data of 3 closely resemble that of 2, with the only difference being the presence of an extra methyl group [δ H/C 3.60/52.2 (1‐OCH3)] in 3. The HMBC correlation from 1‐OCH 3 to C‐1 (δ C 174.3) suggested the attachment of the methoxy group at C‐1. The similar ECD spectra for 2 and 3 indicated the same 6R absolute configuration of the two compounds (Figure 3).
Karstmycin L (4) was isolated as a yellow oil with molecular formula C16H19NO3 determined by the HR‐ESI‐MS ion peak at m/z 296.1262 [M + Na]+ (calcd for C16H19NO3Na+, 296.1257). The 1D NMR data of 2 revealed the presence of an N‐methylbenzophenone group [δ H/C 6.57/111.9 (CH‐10/12), 7.81/132.3 (CH‐9/13), 2.83/29.8 (11‐NCH3), δ C 124.8 (C‐8), 156.1 (C‐11), 201.1 (C‐7)], two olefinic groups [δ H/C 5.98/120.8 (CH‐2) and 7.92/141.9 (CH‐3); δ H/C 132.4 (C‐4) and δ C 5.84/140.3 (CH‐5)], one aliphatic methine group [δ H/C 4.73/40.6 (CH‐6)], and two methyl groups [δ H/C 1.26/19.2 (CH3‐14) and 1.89/20.2 (CH3‐15)]. These NMR data closely resembled that of trichostatin acid [11], except for the absence of an 11‐NCH3 group and the downfield shifted C‐15 (δ C 20.2) in 4, indicating a Z‐geometry for olefinic bond Δ4 [11], which was evidenced by the ROE correlation of H3‐15/H‐5. The planar structure of 4 was further confirmed via detailed analysis of the1H−1H COSY and HMBC analysis. The absolute configuration of 4 was determined by the ECD calculation (Figure 3), assigning its configuration to be 6R.
Karstmycin M (5) was purified as a yellow oil. The HR‐ESI‐MS ion peak at m/z 324.1562 [M + Na] + (calcd for C18H23NO3Na+, 324.1570) established its molecular formula as C18H23NO3. The NMR data of 5 showed similar structural features to those of 4, with the only difference being the presence of an extra methyl group [δ H/C 3.78/52.2 (1‐OCH3)] in 5. The HMBC correlation from 1‐OCH 3 to C‐1 (δ C 169.5) revealed the location of the OCH3 at C‐1. The similar ECD spectra of 5 and 4 indicated that they shared the same absolute configuration of 6R (Figure 3). Since methanol was used in the process of purification of compounds 3 and 5, the possibility that compounds 3 and 5 are artifacts cannot be excluded.
Karstmycin N (6) was isolated as a yellow oil. The molecular formula was determined to be C21H28N2O5 by the HR‐ESI‐MS m/z 411.1885 [M + Na]+ (calcd for C21H28N2O5Na+, 411.1890). The 1D NMR data of 6 exhibited the signals for a dehydroxytrichostatin A moiety, a methyl group [δ H/C 1.53/20.7 (2′‐CH3)], a carboxy carbonyl group [δ C 176.2 (C‐1′)], a monoprotonated carbon [δ C 61.8 (C‐2′)], and a hydroxymethyl group [δ H/C 3.97/3.86/65.8 (CH2‐3′)]. The HMBC correlations from the protons of hydroxymethyl (CH2‐3′) to the C‐1′, C‐2′, and 2′‐CH3 constructed an α‐methylserine motif. These NMR data closely resembled those of JBIR‐111 [13], except for the downfield‐shifted C‐15 (δ C 20.4) in 6, indicating a Z‐geometry for olefinic bond Δ4 [11], which was confirmed by the ROE correlation of H3‐15/H‐5. The configuration of C‐2′ in the α‐methylserine residue was tentatively defined as R based on the biosynthetic consideration and the similar chemical shifts of 6 and JBIR‐111 [13]. This assignment required further experimental validation (e.g., Marfey's analysis or total synthesis). The ECD curve of 6 matched those of 4 and 5, confirming the 6R configuration (Figure 3).
The known compounds, including JBIR‐111 (7) [13], N‐demethyl trichostatic acid (8) [14], dinor‐tetrahydro trichostatic acid (9) [13], 4‐ethyl trichostatic acid (10) [15], (±)‐(2E,4E)‐methyl 7‐(4‐(dimethylamino)phenyl)‐4,6‐dimethyl‐7 oxohepta‐2,4‐dienoate (11) [16], trichostatic acid B (12) [17], trichostatic acid (13) [12], and dehydroxytrichostatin A (14) [18], were identified by comparison of their NMR data with those reported in literature.
All the isolates were evaluated for the antibacterial activity against a panel of pathogenic bacteria comprising Staphylococcus aureus, methicillin‐resistant Staphylococcus aureus (MRSA), Escherichia coli, Pseudomonas aeruginosa, and Bacillus subtilis, and cytotoxicity toward human leukaemia cell line K562, hepatoma cell line BEL‐7402, gastric cancer cell line SGC‐7901, human non‐small cell lung cancer cell line A549, and cervical cancer cell line HeLa, but none of them showed significant antibacterial (MIC > 100 µg/mL) and cytotoxic (IC50> 50 µM) activities. Trichostatins are well‑known histone deacetylase (HDAC) inhibitors and display multiple biological effects, including antioxidant, antidiabetic, anti‐inflammatory, and anticancer activities [19]. The tested cell lines and bacterial strains may not be the optimal targets for these particular analogs. Future studies should explore other potential activities, including HDAC inhibition, antioxidant, or anti‑inflammatory effects.
3. Conclusions
Fourteen trichostatin anlogs, including six unreported ones, karstmycins I−M (1−6), and eight known congeners (7−14) were obtained from Streptomyces sp. DX6D14 isolated from the soil of the karst cave. The structures of the new compounds were elucidated by spectroscopic methods, including HRESIMS and NMR, and ECD calculations. All the isolated compounds were tested for antibacterial and cytotoxic activities, but none of them showed significant activity. These findings expanded the chemical diversity of the trichostatins.
4. Experimental Section
4.1. General Experimental Procedures
HR‐ESI‐MS data were acquired on a mass spectrometer API QSTAR Pulsar (Bruker, Bremen, Germany). ECD and UV spectra were recorded using a JASCO J‐715 spectropolarimeter. Optical rotations were measured on a JASCO P‐1020 digital polarimeter. NMR spectra were obtained on a Bruker AV‐500 spectrometer (Bruker, Bremen, Germany) with TMS as an internal standard. IR spectra were collected on a Shimadzu UV2550 spectrophotometer. Semi‐preparative HPLC was performed on an octadecyl silane (ODS) column (YMC‐pack ODS‐A, 10×250 nm, 5 µm, 4 mL/min). Silica gel (200–300 mesh, Qingdao Marine Chemical Inc., Qingdao, China), RP‐18 gel (40‐70 µm, Fujj Sihysia chemical Ltd, Japan), and Sephadex LH‐20 (40–70 µm, Merck, Darmstadt, Germany) were used for column chromatography. TLC was carried out using precoated silica gel GF254 plates (Qingdao Marine Chemical Co., Ltd, China), and the spots were visualized by heating after spraying with 10% (v/v) H2SO4.
4.2. Strain Material
The strain Streptomyces sp. DX6D14 was isolated from the soil of karst caves in Shaoguan, Guangdong province, China, and identified by the 16S rDNA sequence analysis (GenBank No. PX275816). A reference culture (No. DX6D14) is deposited at −80°C in the Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, and was maintained.
4.3. Fermentation, Extraction, and Isolation
The metabolic potential of Streptomyces sp. DX6D14 was evaluated using the AM3 medium (Soluble starch 10 g, soybean powder 5 g, peptone 15 g, glycerol 10 g) supplemented with varying concentrations of CaCO3 (0.2, 1.0, 2.0, 4.0, and 6.0 g). The fermentations were cultivated at 28°C and 200 rpm for 7 days. Then the fermentation broths were extracted thrice with ethyl acetate. The extracts were analyzed by the HPLC (C18 column (150 × 4.6 mm, 5 µm) with a DAD detector) with 0.1% (v/v) acetic acid (Solvent A) and MeOH (Solvent B) as the solvents with a linear gradient from 10% to 100% B over 25 min at a flow rate of 1.0 mL min−1. As shown in Figure S43, the metabolite profiles changed markedly with increasing CaCO3 concentration. At 0.2–1.0 g/L, only a few major peaks were observed. At 2.0 g/L, several new peaks appeared, and at 6.0 g/L, the chromatogram exhibited the highest number and intensity of detectable peaks. Higher concentrations led to precipitation. Therefore, 6 g/L CaCO3 was selected for large‑scale fermentation.
The large‐scale fermentation was performed in two hundred 2 L Erlenmeyer flasks with 1 L of fermentation medium (AM3 +6 g CaCO3). The fermented broth was extracted three times with ethyl acetate. The combined extracts were concentrated under reduced pressure to yield 73.0 g of crude extract. The extract was subjected to vacuum liquid chromatography (VLC) with a stepwise gradient elution of petroleum ether/ethyl acetate (10:1, 8:1, 6:1, 4:1, 2:1, 1:2, 0:1, v/v) followed by dichloromethane/methanol (10:1, 8:1, 6:1, 4:1, 2:1, 1:2, 0:1, v/v) (5 L of solvent mixture for each gradient) to afford six fractions (Fr.1–Fr.6).
Fr.3 was separated by reversed‐phase C18 column chromatography with a gradient of methanol/water (30% to 100% methanol) to give eight subfractions (Fr.3.1–Fr.3.8). Fr.3.7 was purified by semi‐preparative HPLC (30% acetonitrile/H2O) to yield compound 6 (10.3 mg, t R = 23.0 min).
Fr.4 was chromatographed over a reversed‐phase C18 column with a gradient of methanol/water (20% to 100% methanol) to afford twelve subfractions (Fr.4.1–Fr.4.12). Fr.4.2 was purified by semi‐preparative HPLC (35% acetonitrile/H2O) to give compound 7 (3.7 mg, t R = 11.5 min). Fr.4.11 was purified by semi‐preparative HPLC (35% acetonitrile/H2O) to give compound 12 (43.0 mg, t R = 11.8 min).
Fr.5 was subjected to reversed‐phase C18 column chromatography with a gradient of methanol/water (20% to 100% methanol) to yield twelve subfractions (Fr.5.1–Fr.5.12). Fr.5.5 was separated by gel column chromatography to give six subfractions (Fr.5.5.1–Fr.5.5.6). Fr.5.5.4 was purified by semi‐preparative HPLC (30% acetonitrile/H2O) to afford compound 9 (2.3 mg, t R = 45.0 min). Fr.5.5.5 was purified by semi‐preparative HPLC (30% acetonitrile/H2O) to afford compound 8 (34.2 mg, t R = 42.0 min) and compound 13 (17.9 mg, t R = 32.0 min). Fr.5.7 was separated by gel column chromatography to give six subfractions (Fr.5.7.1–Fr.5.7.6). Fr.5.7.4 was purified by semi‐preparative HPLC (75% methanol/H2O) to afford compound 5 (2.0 mg, t R = 14.0 min). Fr.5.8 was separated by gel column chromatography to give six subfractions (Fr.5.8.1–Fr.5.8.6). Fr.5.8.4 was purified by semi‐preparative HPLC (70% methanol/H2O) to afford compound 4 (2.0 mg, t R = 6.0 min). Fr.5.9 was purified by semi‐preparative HPLC (65% methanol/H2O) to afford compound 14 (98.7 mg, t R = 14.0 min). Fr.5.10 was purified by semi‐preparative HPLC (70% methanol/H2O) to afford compound 1 (5.5 mg, t R = 21.0 min), compound 2 (3.0 mg, t R = 26.0 min), compound 3 (2.1 mg, t R = 15.0 min), and compound 10 (2.0 mg, t R = 20.0 min). Fr.5.11 was purified by semi‐preparative HPLC (75% methanol/H2O) to afford compound 11 (8.7 mg, t R = 10.0 min).
4.4. Spectroscopic Data
Karstmycin I (1): yellow oil; [α]25 D +6.0 (c 0.1, MeOH); UV (MeOH) λ max (log ε): 206 (2.91), 266 (3.27), 294 (2.78), 243(3.33) nm; ECD (MeOH) λ max (Δε) 217 (0.11), 242 (−5.95), 260 (0.65), 310 (−3.72) nm; IR (KBr) ν max cm−1 3437, 1690, 1195; 1H and 13C NMR data, see Table 1; HR‐ESI‐MS m/z 324.1574 [M + Na]+ (calcd for C18H23NO3Na+, 324.1570).
TABLE 1.
13C NMR (125 MHz) and 1H NMR (500 MHz) Spectroscopic Data for 1–3 (δ in ppm, J in Hz, CD3OD).
| No. | 1 | 2 | 3 | |||
|---|---|---|---|---|---|---|
| δ C, type | δ H (J in Hz) | δ C, type | δ H (J in Hz) | δ C, type | δ H (J in Hz) | |
| 1 | 170.7, C | 176.0, C | 174.3, C | |||
| 2a | 118.0, CH | 5.81, d (15.7) | 34.3, CH2 | 2.98, m | 34.2, CH2 | 3.00, dd (7.3, 2.7) |
| 2b | ||||||
| 3 | 150.7, CH | 7.26, d (15.7) | 119.8, CH | 5.33, t (7.4) | 119.3, CH | 5.31, t (7.3) |
| 4 | 135.4, C | 137.7 | 138.0 | |||
| 5a | 141.8, CH | 5.89, d (9.8) | 44.9, CH2 | 2.46, dd (13.7, 6.7) | 44.9, CH2 | 2.46, dd (13.7, 6.7) |
| 5b | 2.09, dd (13.7, 7.7) | 2.09, dd (13.7, 7.7) | ||||
| 6 | 49.1, CH | 4.40, ddd (9.8, 7.5, 6.5) | 39.0, CH | 3.72, m | 38.9, CH | 3.72, m |
| 7 | 201.1, C | 205.0, C | 204.9, C | |||
| 8 | 125.5, C | 125.1, C | 125.2, C | |||
| 9, 13 | 131.9, CH | 7.86, d (9.1) | 131.7, CH | 7.87, d (8.7) | 131.7, CH | 7.87, d (8.7) |
| 10, 12 | 112.0, CH | 6.73, d (9.1) | 112.0, CH | 6.74, d (8.7) | 112.0, CH | 6.75, d (8.7) |
| 11 | 155.5, C | 155.4, C | 155.4, C | |||
| 14a | 27.6, CH2 | 1.87, m | 16.5, CH3 | 1.10, d (6.8) | 16.5, CH3 | 1.09, d (6.8) |
| 14b | 1.60, m | |||||
| 15 | 13.0, CH3 | 1.91, s | 17.8, CH3 | 1.66, s | 17.9, CH3 | 1.66, s |
| 16 | 12.3, CH3 | 0.89, t (7.5) | ||||
| 11‐N(CH3)2 | 40.2, CH3 | 3.04, s | 40.1, CH3 | 3.06, s | 40.1, CH3 | 3.07, s |
| 1‐OCH3 | 52.2, OCH3 | 3.60, s | ||||
Karstmycin J (2): yellow oil; [α]25 D +232.0 (c 0.1, MeOH); UV (MeOH) λ max (log ε): 204 (2.58), 242 (2.52), 267 (2.25), 336 (3.02) nm; ECD (MeOH) λ max (Δε) 205 (12.94), 310 (−4.75), 340 (5.01) nm; IR (KBr) ν max cm−1 3420, 3291, 1656, 1597; 1H and 13C NMR data, see Table 1; HR‐ESI‐MS m/z 312.1567 [M + Na]+ (calcd for C17H23NO3Na+, 312.1570).
Karstmycin K (3): yellow oil; [α]25 D +3.0 (c 0.1, MeOH); UV (MeOH) λ max (log ε): 204 (2.67), 238 (2.56), 269 (2.18), 335 (2.98) nm; ECD (MeOH) λ max (Δε) 210 (+0.08), 240 (−0.80), 304 (−0.59), 346 (2.52) nm; IR (KBr) ν max cm−1 3464, 3376, 1681, 1597; 1H and 13C NMR data, see Table 1; HR‐ESI‐MS m/z 326.1725 [M + Na]+ (calcd for C18H25NO3Na+, 326.1727).
Karstmycin L (4): yellow oil; [α]25 D +144.0 (c 0.1, MeOH); UV (MeOH) λ max (log ε): 206 (2.09), 269 (2.39), 297 (2.07), 338 (2.48) nm; ECD (MeOH) λ max (Δε) 196 (6.76), 212 (−0.06), 219 (0.50), 239 (−0.46), 268 (1.78), 321 (−1.70) nm; IR (KBr) ν max cm−1 3452, 1654, 511; 1H and 13C NMR data, see Table 2; HR‐ESI‐MS m/z 296.1262 [M + Na]+ (calcd for C16H19NO3Na+, 296.1257).
TABLE 2.
13C NMR (125 MHz) and 1H NMR (500 MHz) Spectroscopic Data for 4–6 (δ in ppm, J in Hz, CD3OD).
| No. | 4 | 5 | 6 | |||
|---|---|---|---|---|---|---|
| δ C, type | δ H (J in Hz) | δ C, type | δ H (J in Hz) | δ C, type | δ H (J in Hz) | |
| 1 | 170.7, C | 169.5, C | 168.5, C | |||
| 2a | 120.8, CH | 5.98, d (15.5) | 120.1, CH | 6.01, d (15.6) | 123.4, CH | 6.24, d (15.6) |
| 2b | ||||||
| 3 | 141.9, CH | 7.92, d (15.5) | 141.8, CH | 7.94, d (15.6) | 137.7, CH | 7.81, d (15.6) |
| 4 | 132.4, C | 132.4, C | 132.5, C | |||
| 5 | 140.3, CH | 5.84, d (9.9) | 140.6, CH | 5.86, d (10.0) | 138.8, CH | 5.76, d (10.0) |
| 6 | 40.6, CH | 4.73, m | 40.7, CH | 4.73, m | 40.6, CH | 4.78, m |
| 7 | 201.1, C | 201.2, C | 201.6, C | |||
| 8 | 124.8, C | 124.5, C | 124.8, C | |||
| 9, 13 | 132.3, CH | 7.81, d (8.6) | 132.0, CH | 7.87, d (8.6) | 132.0, CH | 7.88, d (8.6) |
| 10, 12 | 111.9, CH | 6.57, d (8.6) | 111.9, CH | 6.73, d (8.6) | 112.0, CH | 6.72, d (8.6) |
| 11 | 156.1, C | 155.5, C | 155.3, C | |||
| 14 | 19.2, CH3 | 1.26, d (6.7) | 19.2, CH3 | 1.26, d (6.7) | 19.2, CH3 | 1.25, d (6.7) |
| 15 | 20.2, CH3 | 1.89, s | 20.2, CH3 | 1.89, s | 20.4, CH3 | 1.89, s |
| 11‐N(CH3)2 | 40.1, CH3 | 3.07, s | 40.1, CH3 | 3.05, s | ||
| 11‐NCH3 | 29.8, CH3 | 2.83, s | ||||
| 1‐OCH3 | 52.2, OCH3 | 3.78, s | ||||
| 1′ | 176.2, C | |||||
| 2′ | 61.8, C | |||||
| 3′a | 65.8, CH2 | 3.97, d (11.2) | ||||
| 3′b | 3.86, d (11.2) | |||||
| 2′‐CH3 | 20.7, CH3 | 1.53, s | ||||
Karstmycin M (5): yellow oil; [α]25 D +5.0 (c 0.1, MeOH); UV (MeOH) λ max (log ε): 204 (2.44), 220 (2.30), 269 (2.65), 296 (2.31), 344 (2.76) nm; ECD (MeOH) λ max (Δε) 213 (−0.36), 228 (0.64), 245 (−0.64), 275 (1.60), 322 (−1.84) nm; IR (KBr) ν max cm−1 3436, 1639, 1197; 1H and 13C NMR data, see Table 2; HR‐ESI‐MS m/z 324.1562 [M + Na]+ (calcd for C18H23NO3Na+, 324.1570).
Karstmycin M (6): yellow oil; [α]25 D −42.0 (c 0.1, MeOH); UV (MeOH) λ max (log ε): 269 (3.10), 298 (2.66), 342 (3.10) nm; ECD (MeOH) λ max (Δε) 196 (1.91), 206 (0.26), 212 (0.48), 241 (−0.29), 274 (2.14), 325 (−1.80) nm; IR (KBr) ν max cm−1 3436, 2937, 1595, 1374, 1259; 1H and 13C NMR data, see Table 2; HR‐ESI‐MS m/z 411.1885 [M + Na] + (calcd for C21H28N2O5Na+, 411.1890).
4.5. Antibacterial Assay
Antibacterial activities of compounds 1−14 were evaluated against a panel of pathogenic bacteria comprising S. aureus, methicillin‐resistant S. aureus (MRSA), E. coli, P. aeruginosa, and B. subtilis using the broth microdilution method [20] with vancomycin and gentamicin as positive controls.
4.6. Cytotoxicity Assay
MTT method [21] was used to assess the cytotoxicity of compounds 1−14 against the following human cancer cell lines: K562 (leukemia, RRID: CVCL_0004), BEL‐7402 (hepatoma, RRID: CVCL_3612), SGC‐7901 (gastric cancer, RRID: CVCL_0520), A549 (non‐small cell lung cancer, RRID: CVCL_0023), and HeLa (cervical cancer, RRID: CVCL_0030), with cisplatin as reference drug. All the cell lines were purchased from the Cell Bank of the Chinese Academy of Sciences, Shanghai, China
4.7. Statistical Analysis
All experiments were performed in triplicate. Data are presented as mean ± standard deviation (SD). For the antibacterial assay, MIC values were determined as the lowest concentration showing no visible bacterial growth. For the cytotoxicity assay, IC50 values were calculated by nonlinear regression. Since all tested compounds showed MIC > 100 µg/mL and IC50> 50 µM, no further statistical comparisons were performed.
Author Contributions
Y.‐X. Zhao, H.‐F. Dai, and Y.‐E. Hao conceived the study, designed the experiments, and finalized the manuscript. L. Yang analyzed the data, performed the ECD calculation, and wrote the first draft. H. Xie performed the isolation and structural identification of the chemical compounds. Q.‐Y. Xie performed strain identification. Q.‐Y. Ma and J.‐C. Guo performed the bioactivity assay.
Conflicts of Interest
The authors declare no conflicts of interest.
Supporting information
Supporting File: cbdv71334‐sup‐0001‐SuppMat.pdf
Acknowledgments
This work was supported by the National Natural Science Fund of China (82304349), Hainan Provincial Natural Science Foundation of China (326ZD1015), Special Financial Project of the Ministry of Agriculture and Rural Areas (No. NFZX2024), Chinese Academy of Tropical Agricultural Sciences for Science and Technology, the earmarked fund for CARS—Chinese Materia Medica (CARS‐21), and joint support from Hubei Provincial Natural Science Foundation and Xiangyang of China (2025AFD068).
Contributor Information
Yu‐E. Hao, Email: haohao200701@126.com.
You‐Xing Zhao, Email: zhaoyouxing@catasitbb.cn.
Data Availability Statement
The data that support the findings of this study are available in the Supporting Information of this article.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Supporting File: cbdv71334‐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.