Two New Cytotoxic Compounds from a Deep-Sea Penicillum citreonigrum XT20-134

Abstract

Penicillum citreonigrum XT20-134 (MCCC 3A00956) is a fungus with cytotoxic activity, derived from deep-sea sediment. Five new compounds, adeninylpyrenocine (1), 2-hydroxyl-3-pyrenocine-thio propanoic acid (2), ozazino-cyclo-(2,3-dihydroxyl-trp-tyr) (3), 5,5-dichloro-1-(3,5-dimethoxyphenyl)-1,4-dihydroxypentan-2-one (4), and 2,3,4-trihydroxybutyl cinnamate (5), together with 19 known compounds (6–24), were isolated from an ethyl acetate (EtOAc) extract of its fermentation. The structures of the new compounds were comprehensively characterized by high-resolution electrospray ionization-mass spectrometry (HR-ESI-MS), 1D and 2D nuclear magnetic resonance (NMR). All isolates were evaluated for their cytotoxic activities. The heteroatom-containing new compounds 2 and 4 showed potent cytotoxicity to the human hepatoma tumor cell Bel7402 with IC₅₀ values of 7.63 ± 1.46, 13.14 ± 1.41 μM and the human fibrosarcoma tumor cell HT1080 with IC₅₀ values of 10.22 ± 1.32, 16.53 ± 1.67 μM, respectively.

1. Introduction

Considering the extreme environment of high salinity, darkness, high pressure, and high/low temperature [1], the discovery of new lead compounds from deep-sea microorganisms has become a hot topic in natural products research. Deep-sea fungi are attracting great interest because of their relatively large genome size, which may produce more second metabolites compared with bacteria. Cancer is the leading lethal disease in the world and deep-sea microorganism-originated compounds are thought to be the new anti-tumor drugs repository [2,3]. Recently, the new antitumor compounds diketopiperazine, cytochalasan alkaloids, chromone-derived polyketides, etc., have been isolated from deep-sea fungi [4,5,6].

During the previous studies, several new cytotoxic compounds were characterized from deep-sea microbial resources [7,8,9,10,11]. In the present study, Penicillum citreonigrum XT20-134 (MCCC 3A00956), a fungal strain that originated from the deep-sea sediment in the southeast Indian Ocean, was found to possess cytotoxic activity. From its ethyl acetate (EtOAc) extract, five new compounds, adeninylpyrenocine (1), 2-hydroxyl-3-pyrenocine-thio propanoic acid (2), ozazino-cyclo-(2,3-dihydroxyl-trp-tyr) (3), 5,5-dichloro-1-(3,5-dimethoxyphenyl)-1,4-dihydroxypentan-2-one (4), and 2,3,4-trihydroxybutyl cinnamate (5) were isolated together with 19 known compounds (6–24) (Figure 1). The structures of the new compounds were comprehensively characterized by high-resolution electrospray ionization-mass spectrometry (HR-ESI-MS), 1D and 2D nuclear magnetic resonance (NMR). Their cytotoxic activities were studied. The new heteroatom-containing compounds 2 and 4 showed potent cytotoxicity to the human hepatoma tumor cell Bel7402 with IC₅₀ values of 7.63 ± 1.46 and 13.14 ± 1.41 μM, and the human fibrosarcoma tumor cell HT1080 with IC₅₀ values of 10.22 ± 1.32 and 16.53 ± 1.67 μM, respectively.

2. Results

2.1. Structural Identification of New Compounds

Adeninylpyrenocine (1) was isolated as a white amorphous powder. The infrared (IR) spectrum of 1 indicated the presence of conjugated ketone and conjugated lactone carbonyl signals at 1695 and 1642 cm⁻¹, respectively. Its molecular formula of C₁₆H₁₇N₅O₄ was established by HR-ESI-MS at 344.1357 [M + H]⁺ (calcd. for 344.1353, C₁₆H₁₈N₅O₄). The unsaturation degree of 11 indicated the presence of heteroaromatic rings. With the aid of heteronuclear single quantum coherence (HSQC) spectra, three singlet signals in the low field of ¹H NMR at δ_H 8.21 (1H, s, H-8’), 8.11 (1H, s, H-2’), and 5.62 (1H, s, H-3) were attributed to olefinic protons. Their corresponding carbon signals were found in ¹³C NMR at δ_C 140.3, 152.6, and 87.8, respectively. The signal at δ_H 7.16 (2H, br.s, 6’-NH₂) should be assigned to the primary amino group due to the absence of carbon correlation in the HSQC spectrum. In the sp³ region of the ¹H NMR, two singlet signals, belonging to a methoxyl at δ_H 3.74 (3H, s, 4-OCH₃) and a methyl at δ_H 1.85 (3H, s, 6-CH₃), could be found. In addition, a CH₂–CH–CH₃ fragment could be established, based on the ABXC₃ spin system at δ_H [3.78 (1H dd, J = 17.6, 7.9 Hz) and 3.35 (1H dd, J = 17.6, 6.0 Hz), H-8], δ_H 5.06 (1H, m, H-9), and the doublet methyl signal and 1.53 (3H, d, J = 6.0 Hz, H-10). Considering the five nitrogen atoms in the molecular formula and the carbon signals at δ_C 156.4, 152.6, 149.5, 140.3, and 119.5, an adenine moiety is present in 1. With the help of distortionless enhancement by polarization transfer (DEPT-135) along ¹³C NMR, the four quaternary carbons at δ_C 156.4, 152.6, 149.5, 140.3, and the methine at δ_C 87.8 were attributed to an α-pyrone structure unit. In the ¹H–¹H homonuclear chemical shift correlation spectroscopy (COSY) spectrum, only the correlations in the CH₂CH–CH₃ were presented. In the ¹H detected heteronuclear multiple bond correlation (HMBC) spectrum, key correlations were found to reveal the total structure. The correlations between H-9 (δ_H 5.06) and C-4’ (δ_C 149.5) and C-8’ (δ_C 140.3) indicated that the adenine moiety was connected to C-9 at N-9’. The conjugated ketone signal at δ_C 198.8 (C-7) in ¹³C NMR, is considered to link to C-8, due to the HMBC correlation between H-8 and C-7. The other two methyl groups were also assigned, as shown in Figure 2. The theoretical electronic circular dichroism (ECD) spectra of 9R-1 and 9S-1 were further calculated and compared with the experimental ones to determine the absolute configurations. As shown in Figure 3, the experimental ECD spectrum was similar to the calculated ECD spectrum of 9S-1 and the absolute configuration of 1 was determined as 9S.

We isolated 2-hydroxyl-3-pyrenocine-thio propanoic acid (2, a pair of epimers with a ratio of 1:2) as a light yellow powder. In the IR spectrum of 2, unconjugated carbonyl and α-pyrone ketone signals emerged at 1714 and 1626 cm⁻¹, respectively. Its molecular formula of C₁₄H₁₈O₇S, which gave six degrees of unsaturation, was established by the positive HR-ESI-MS ion peaks at m/z 331.0845 [M + H]⁺, 353.0670 [M + Na]⁺ and negative ion peaks at m/z 329.0706 [M − H]⁻, respectively. The presence of a sulfur atom was supported by the isotope quasi-molecular ion peaks at m/z 333.0808 [M(³⁴S) + H]⁺, 355.0627 [M(³⁴S) + Na]⁺ and 331.0662 [M(³⁴S) – H]⁻, respectively. In the ¹H NMR spectrum of 2, an α-pyrone olefinic proton, a methyl and a methoxyl signal were found at δ_H 5.69 (1H, s, H-3), 1.23 (1H, d, J = 6.8 Hz, H-10) and 3.87 (3H, s, 4-OCH₃), respectively. In the ¹H–¹H COSY spectrum, the correlations between δ_H 5.69 (H-10), δ_H 3.30 (H-9) and δ_H 2.99 & 2.90 (H-8) revealed the presence of a CH₂CH–CH₃ structure unit. In the ¹³C NMR, signals belong to the pyrenocine moiety were similar to those in 1, except for the C-9 signal at δ_C 36.3, indicating that they differed in the substituent at C-9 (

Table 1. ¹H NMR and ¹³C NMR (DMSO-d₆) data of compounds 1 and 2.

Positions	1		2		2’
	δC	δH	δC	δH	δC	δH
2	162.0		162.2		162.2
3	87.8	5.62, s	87.9	5.69, s	87.9	5.69, s
4	168.3		168.5		168.5
5	115.1		115.4		115.4
6	162.8		163.2		163.2
7	198.8		199.5		199.5
8	49.3	3.78, dd (17.6, 7.9)	51.7	2.99, dd (17.1, 6.6)	51.6	2.99, dd (17.1, 6.6)
		3.35, dd (17.6, 6.0)		2.90, dd (17.1, 3.7)		2.89, dd (17.1, 3.7)
9	47.7	5.06, m	36.3	3.30, m	36.2	3.30, m
10	21.1	1.54, br. d (6.6)	21.8	1.23, d (6.8)	21.8	1.23, d (6.8)
6-CH3	18.0	1.85, s	18.5	2.18, d (2.6)	18.5	2.18, d (2.6)
4-OCH3	57.4	3.74, s	57.5	3.87, s	57.5	3.87, s
2′	152.6	8.11, br. s
4′	149.5
5′	119.5
6′	156.4
8′	140.3	8.21, br. s
6′-NH2		7.16, br. s
1′			174.5		174.5
2′			71.1	4.09, m	71.0	4.09, m
3′			34.7	2.83, dd (13.5, 5.1)	34.6	2.82, dd (13.5, 5.1)
				2.72, dd (13.6, 9.5)		2.71, dd (13.6, 9.5)

). The adeninyl signals were absent in the NMR data of 2. Except for the adeninyl signals, the ¹H-NMR of 2 presented other ABX system signals at δ_H 4.09 (1H, m, H-2’) and δ_H [2.83 (1H, dd, J = 13.5, 5.1 Hz) and 2.72 (1H, dd, J = 13.5, 9.5 Hz), H-3’]; and three additional carbon signals were at δ_C 174.5 (C-1’), 71.1 (C-2’) and 34.7 (C-3’). With the help of ¹H–¹H COSY and HMBC spectra, they were attributed to a –CH₂–CH(OH)–COOH structure fragment. The HMBC correlations between H-3’ (δ_H 2.83, 2.71) and C-9 (δ_C 36.3), and between H-9 (δ_H 3.30) and C-3’ (δ_C 34.7) revealed that the two structure units connected at C-9 and C-3’. The ¹H-NMR and ¹³C-NMR signals at positions C-1’, 2’, 3’ and C-8, 9, whose peak intensities were halved and appeared in pairs, indicated that compound 2 is a pair of epimers (Figure 2). Considering the similar structure unit in 1, the absolute configuration of C-9 is S, while C-2’ contains the pair of R and S configurations.

Ozazino-cyclo-(2,3-dihydroxyl-trp-tyr) (3) was isolated as a white powder. The HR-ESI-MS cationized ion peaks revealed the presence of three nitrogen atoms. In the IR spectrum of 3, amide carbonyl signal was found at 1669 cm⁻¹. The ultravoilet (UV) maximum absorption wavelengths at λ_max (log ε) 240 (1.92) nm and 299 (1.55) nm, belong to the amide carbonyl and aromatic rings, respectively. In the ¹H NMR spectrum of 3, a set of mono-substituted benzene ring signals could be found at δ_H 6.98 (2H, br. d, J = 7.2 Hz, H-2’, 6’), 6.86 (2H, br. t, J = 7.4 Hz, H-3’, 5’) and 6.75 (1H, m, H-4’), respectively. Another ortho-substituted benzene ring was elucidated by the signals at δ_H 6.90 (1H, br. d, J = 7.2 Hz, H-4), 6.56 (1H, br. t, J = 7.0 Hz, H-5), 7.00 (1H, br. t, J = 7.5 Hz, H-6), and 6.56 (1H, br. d, J = 7.7 Hz, H-7). In addition, two ABX spin system signals were present at δ_H 4.19 (1H, dd, J = 5.0, 2.8 Hz, H-α′), [2.87 (1H, dd, J = 13.9, 2.8 Hz) and 2.74 (1H, dd, J = 13.8, 5.0 Hz), H-β′], and at δ_H 4.17 (1H, dd, J = 9.1, 4.8 Hz, H-α), [1.91 (1H, dd, J = 13.6, 4.8 Hz) and 1.03 (1H, dd, J = 13.5, 9.1 Hz) H-β], and were attributed to two –CH–CH₂– structure units. All these fragments were confirmed by the ¹³C NMR and DEPT spectra. In addition, two amide carbonyls at δ_C 166.1 and 161.5, together with a quaternary carbon at δ_C 74.7 and a methylidyne δ_C 99.3, indicated that 3 is a compound comprising two amino acid units, one of which is phenylalanine and the other one is a tryptophane-like structure unit. Similar structures have been isolated from P. citroviride [12] and from Penicillium sp. [13]. In the HSQC spectrum of 3, the proton signals at δ_H 7.96 (α′-NH) and δ_H 6.57 (1-NH) were attributed to two exchangeable protons, for the absence of correlation with carbon. With the help of the ¹H–¹H COSY spectrum, the signals belonging to the phenylalanine unit were attributed (Figure 2). The COSY correlation between δ_H 6.57 (1-NH) and δ_H 5.10 (1H, d, J = 2.8 Hz, H-2), together with the HMBC correlation between H-2 and C-3 (δ_C 74.6), revealed that C-2 and C-3 of the tryptophane unit were oxygen-connected. The HMBC correlations between δ_H 7.96 (α′-NH), 4.17 (H-α), 4.19 (H-α’), and δ_C 166.1 (C=O), 161.5 (C=O’) indicated the cyclo-dipeptide structure. Comparing the NMR data of 3 with those of cyclo-(L-tryptophyl-L-phenylalanyl) (19), a known compound reported previously [14], the chemical shift of 1-NH was significantly deduced from δ_H 10.83 to δ_H 6.57, indicating that the double bond between C-2 and C-3 in the indole ring disappeared. Moreover, absence of the α-NH signal (δ_H 7.96 in 19), and the high-field shifting of C=O’ (δ_C 167.3 in 19 and δ_C 161.5 in 3) were observed. Considering that the degree of unsaturation in 3 is 13, a six-membered ring between α-N and 2-O, connected by an ozazino bond, is prefered. Thus, the structrue of 3 is elucidated as ozazino-cyclo-(2,3-dihydroxyl-trp-tyr).

We isolated 5,5-dichloro-1-(3,5-dimethoxyphenyl)-1,4-dihydroxypentan-2-one (4) as a white powder. The HR-ESI-MS cationized ion peaks indicated a molecular formula of C₁₃H₁₆Cl₂O₅. The presence of the two chlorine atoms was confirmed by the isotope ion peak relative high ratio of 9:6:1. In the IR spectrum of 4, an unconjuated ketone carbonyl signal was at 1714 cm⁻¹. A symmetrical 1,3,5-trisubstituted benzene ring was deduced by the ¹H NMR signal at δ_H 6.37 (2H, d, J = 2.2 Hz, H-2’, 6’) and δ_H 6.27 (1H, d, J = 2.2 Hz, H-4’). The methoxyl signal, identical at δ_H 3.57 (6H, s), was attributed to 3’, 5’-OCH₃. A –CH₂–CH–CH– structural unit could be revealed by the peak splitting and coupling constants of the proton signals at δ_H [2.66 (1H, dd, J = 17.4, 8.8 Hz) and 2.52 (1H, dd, J = 17.4, 2.8 Hz), H-3], 4.07 (1H, ddd, J = 8.8, 3.3, 2.8 Hz, H-4), and 5.96 (1H, d, J = 3.3 Hz, H-5). The fragments were confirmed by the ¹H–¹H COSY correlations of 4. The ¹³C NMR of 4 showed an unconjuated ketone carbonyl at δ_C 207.8. The correlation between δ_H 4.85 (1H, s, H-1) and δ_C 79.7 (C-1), found in the HSQC spectrum, revealed an oxygen-linked methylidyne. The HMBC correlations between δ_H 4.85 (H-1) and δ_C 141.7 (C-1’), 207.8 (C-2), and between δ_H 2.66 and 2.52 (H-3) and δ_C 207.8 (C-2), allowed the elucidation of the structure of 4 (Figure 2).

We isolated 2,3,4-trihydroxybutyl cinnamate (5) as a white powder. The molecular formula of C₁₃H₁₆O₅ was revealed by the HR-ESI-MS cationized ion peaks at m/z 275.0882 [M + Na]⁺ (calcd. for 275.0890 C₁₃H₁₆O₅Na) in positive mode, and m/z 251.0922 [M − H]⁻ (calcd. for 251.0925, C₁₃H₁₄O₅) in negative mode. A conjugated ester carbonyl IR signal was present at 1700 cm⁻¹. A mono-substituted benzene ring was deduced by the ¹H NMR signal at δ_H 7.72 (2H, dd, J = 6.3, 3.0 Hz, H-2, 6) and δ_H 7.74 (3H, overlapped, H-3,5 and H-4). A pair of trans-alkene proton signals were exhibited at δ_H 7.69 (1H, d, J = 16.0 Hz, H-7) and δ_H 6.64 (1H, d, J = 16.0 Hz, H-8). With the help of ¹H–¹H COSY spectrum, two ABX spin systems at δ_H [4.33 (1H, dd, J = 11.3, 2.7 Hz) and 4.11 (1H, dd, J = 11.3, 7.1 Hz), H-1′], 3.66 (1H, m, H-2′), and at δ_H [3.58 (1H, br. d, J = 9.2 Hz) and 3.42 (1H, br. d, J = 9.2 Hz), H-4′], 3.44 (1H, m, H-3′), were connected to a structure fragment as O–CH₂–CH(O)–CH(O)–CH₂–O (Figure 2). In the HMBC spectrum, correlations from H-1’ (δ_H 4.33 & 4.11), H-8 (δ_H 6.64) to C-9 (δ_C 166.9), and from H-3, 5 (δ_H 7.44), H-8 (δ_H 6.64) to C-1 (δ_C 134.5), allowed the elucidation of 2,3,4-trihydroxybutyl cinnamate. The chlorogenic acid contained in the potato medium may be the original precursor of this compound.

The structures of compounds 6–24 were elucidated by the comparison of their MS and NMR data with those reported in the literature, and they were identified as: 4-methyl-5,6-dihydropyren-2-one (6) [15], citreo-g-pyrone (7) [16], pyrenocine B (8) [17], pyrenocine D (9) [18], pyrenocine A (10) [19], pyrenocine I (11) [20], pyrenocine E (12) [18], pyrenocine J (13) [21], citreovirenone (14) [12], citroethiolactone (15) [16], citreoviridin A (16) [22], isocitreoviridin A (17) [22], aurovertin U (18) [14], cyclo (phe-trp) (19) [14], N-(N-acetyl-valyl)-phenylalanine (20) [23], 2′,3′-dihydrosorbicillin (22) [24], citreoviranol (23) [25], and haenamindole (24) [26]. Most of the compounds have been isolated from the genus of Penicillum. Compound 21, a known compound without reported NMR data, was identified by MS and 1D, 2D NMR spectra data as 3,5-dihydroxy-2,4-dimethyl-6-(3-oxobutan-2-yl)benzaldehyde.

2.2. Cytotoxicity Evaluation

All of the isolated compounds were evaluated for their cytotoxic effects on four types of tumor cells; Bel7402, HT1080, CNE2 and A549. Compounds 2 and 4 showed potent cytotoxicity to the cell lines Bel7402 and HT1080, while they showed no obvious effects on Cne2 and A549 (

Table 2. Cytotoxic activities of compounds 2 and 4 (IC₅₀, μM).

Compd.	Bel7402	HT1080	Cne2	A549
2	7.63 ± 1.46	10.22 ± 1.32	73.14 ± 5.32	87.08 ± 7.32
4	13.14 ± 1.41	16.53 ± 1.67	83.56 ± 6.49	92.47 ± 6/33
paclitaxel	<1	<1	<1	<1
DMSO	None	None	None	None

). All other compounds exhibited much lower cytotoxicity to the four tumor cell lines, with IC₅₀ values larger than 100 μM.

3. Discussion

In this study, five new compounds, including two new heteroatom-containing compounds were isolated from the ethyl acetate extract of a deep-sea fungus P. citreonigrum XT20-134 (MCCC 3A00956). Chemically, the relative configuration of these compounds was confirmed by their NOESY spectra; the absolute configuration of compound 1 was revealed by comparison of its CD spectra with the calculated ECD. All of the compounds were evaluated for cytotoxic activity. The new heteroatom-containing compounds 2 and 4 showed potent cytotoxicity to the tumor cell lines Bel7402 and HT1080.

Marine microorganisms can utilize chloride ions or sulfate ions, the two most abundant anions in seawater, and produce heteroatom-containing compounds, with diverse chemical structures and various bioactivities [27]. The bioactive halogenated, mainly referring to chlorinated, natural products from microorganisms mainly manifest with cytotoxic and antibacterial activity, indicating that halogenated compounds produced from microorganisms, due to adaptability or defense from the extreme environment, have cytotoxic and antibacterial activity [28]. In this study, compound 4 that contained two choline atoms was also found to have potent cytotoxicity. The most abundant source of sulfur-containing natural products is also marine organisms. Sulfur can appear in a multitude of combinations and oxidation states: thiol, sulfide, disulfide, sulfoxide, sulfonate, etc. The diversity of sulfur-containing chemical structures leads to their various bioactivities [29]. Among them, psammaplin A, has been found to have a broad bioactive spectrum, especially in terms of antimicrobial and antiproliferative activities [30]. With a sulfur atom, compound 2 exhibited potent cytotoxic activity. Similar compounds without sulfur (1, 8, and 10) did not shown cytotoxic activity. However, compound 16 show no activity, indicating that the type of sulfur bond is important to the activity.

4. Materials and Methods

4.1. General Experimental Procedures

An electrospray ionization source (ESI)-equipped Q-Exactive mass spectrometer (Thermo Fisher Scientific Corporation, Waltham, MA, USA) was used to analyze the HR-ESI-MS data. A Shimadzu UV-260 spectrometer (Shimadzu Corporation, Tokyo, Japan) and a Perkin-Elmer 683 infrared spectrometer (PerkinElmer, Inc., Waltham, MA, USA) were used to obtain the UV and IR spectra, respectively. A JASCO P-200 polarimeter (JASCO Corporation, Tokyo, Japan) with a 5 cm cell was applied to measure the optical rotation value. The NMR spectra with TMS as the internal standard were taken on a Brucker Avance III 600 FT NMR spectrometer (Bruker Corporation, Billerica, MA, USA).

4.2. Eletronic Circular Dichroism (ECD) Calculations

The theoretical electronic circular dichroism (ECD) spectra of the isolated compounds were calculated on the basis of the relative configurations determined by their NOESY spectra and J values in ¹H NMR. Conformational analyses and density functional theory (DFT) calculations were used to generate and optimize the conformers with energy. The ECD calculations were performed as previously described [31,32].

4.3. Fungal Strain and Fermentation

The strain Penicillium sp. XT20-134 was isolated from southeast Indian Ocean sediments at 2910 m by the tablet pour method. The internal transcribed spaces (ITS) region was amplified and sequenced using the general primers ITS1 and ITS4. The ITS region of the fungi is a 573 bp DNA sequence (GenBank Accession Number: KY 978587) that showed 99% identity to P. citreonigrum. The strain was deposited at the China Center for Type Culture Collection (CCTCC) as accession number M2017125 and Marine Culture Collection of China (MCCC) as accession number MCCC 3A00956. The fungus grew well on the rice medium in artificial seawater. Carbohydrate fermentation was conducted by subculturing the fungus in rice medium in artificial seawater and incubating at 28 °C for 30 days in a standing position.

4.4. Extraction and Isolation

The rice medium (10 kg) of P. citreonigrum XT20-134 was extracted with ethyl acetate (20 L) trice and concentrated under reduced pressure at 40 °C to yield 19.2 g of the residue.

The EtOAc extract (18 g) was fractionated over a column packed with silica gel (360 g, Yantai Chemical Industry Research Institute, Yantai, China), eluted with petroleum ether-ethyl acetate (v/v) (5:1, 1.0 L) and CHCl₃–CH₃OH (v/v) (5;1, 1.0 L) and CH₃OH (1.0 L), to afford PE eluent (0.5 g), CM eluent (12.3 g), and methanol eluent (3.4 g). The PE eluent was purified by a silica gel (30 g) column and eluted with petroleum ether-ethyl acetate (v/v) (10:1, 5:1, and 3:1, each 200 L) to give compound 22 (13 mg). The CM eluent was separated over a Cosmosil reversed-phase C18 (300 g, 75 μm, Nakalai Tesque Co. Ltd., Kyoto, Japan) column and eluted with CH₃OH/H₂O (10%–100%, each 1.5 L) to provide 12 fractions (Fr. 1–Fr. 12). Compound 24 (83 mg) was obtained from Fr. 11 after recrystallization. Other fractions were purified over a preparative Cosmosil ODS column (250 mm × 20.0 mm i.d., 5 μm, Cosmosil, Nakalai Tesque Co. Ltd., Kyoto, Japan), and isocratically eluted with a mobile phase system of acetonitrile–H₂O in different ratios. Eluting with 15% acetonitrile, preparative HPLC separation on Fr. 1, Fr. 2, and Fr. 3 resulted in the isolation of: compound 6 (6 mg) from Fr. 1, compounds 7 (8 mg), 8 (57 mg), 9 (5 mg) from Fr. 2, and compounds 1 (50 mg), 10 (90 mg), 11 (15 mg), 12 (8 mg) from Fr. 3, respectively. Preparative HPLC purification of Fr. 5, eluted with acetonitrile–H₂O (30:70, v/v), led to the isolation of compound 2 (15 mg) and compound 15 (150 mg). Fr. 4, Fr. 6, and Fr. 7 were separated with 30% acetonitrile. As a result, compounds 5 (5 mg), 13 (4 mg), and 14 (5 mg) were isolated from Fr. 4; compound 19 (6 mg) from Fr. 6, and compounds 20 (13 mg) and 21 (20 mg) from Fr. 7, respectively. Fr. 8 was isolated with 35% acetonitrile to obtain compound 4 (4 mg). Separation of Fr. 9 and Fr. 10, eluted with 40% acetonitrile, obtained compound 3 (8 mg) and compound 23 (13 mg), respectively. The other three compounds, 16 (15 mg), 17 (18 mg) and 18 (13 mg), were obtained from Fr. 12, by eluting with 45% acetonitrile.

4.5. Structrural Elucidation of the New Compounds 1–5

Adeninylpyrenocine (1): white amorphous powder; [α${]}_{\mathrm{D}}^{25}$ − 14° (c = 0.1, CH₃OH); IR (KBr) (ν_max): 3441, 1695, 1642, 1402 and 1256 cm⁻¹; UV (MeOH) λ_max (log ε): 204 (2.49) and 261 (2.22) nm. HR-ESI-MS: m/z 344.1357 [M + H]⁺ (calcd. for 344.1353 C₁₆H₁₈N₅O₄) and 366.1172 [M + Na]⁺ (calcd. for 366.1173 C₁₆H₁₇N₅O₄Na) in positive mode, and m/z 342.1220 [M − H]⁻ (calcd. for 342.1208, C₁₆H₁₆N₅O₄) in negative mode. ¹H NMR (600 MHz, DMSO-d₆) and ¹³C NMR (150 MHz, DMSO-d₆) spectra data are listed in Table 1, and Figures S1–S9 in supporting materials.

2-Hydroxyl-3-pyrenocine-thio propanoic acid (2): light yellow powder; [α${]}_{\mathrm{D}}^{25}$ + 26° (c = 0.1, CH₃OH); IR (KBr) (ν_max): 3435, 2927, 1714, 1626, 1453, 1400, 1260, and 1096 cm⁻¹; UV (MeOH) λ_max (log ε): 203 (2.10) nm and 260 (2.52) nm. HR-ESI-MS: m/z 331.0845 [M + H]⁺ (calcd. for 331.0859 C₁₄H₁₉O₇S) and 353.0670 [M + Na]⁺ (calcd. for 353.0665 C₁₄H₁₈O₇SNa) in positive mode, and m/z 329.0706 [M − H]⁻ (calcd. for 329.0700, C₁₄H₁₇O₇S) in negative mode. ¹H NMR (600 MHz, DMSO-d₆) and ¹³C NMR (150 MHz, DMSO-d₆) spectra data are listed in Table 1, and Figures S10–S17 in supporting materials.

Ozazino-cyclo-(2,3-dihydroxyl-trp-tyr) (3): white powder; [α${]}_{\mathrm{D}}^{25}$ + 43° (c = 0.1, CH₃OH); IR (KBr) (ν_max): 3437, 1669, 1623, 1460, 1396, and 1081 cm⁻¹; UV (MeOH) λ_max (log ε): 204 (2.55) nm, 240 (1.92) nm, and 299 (1.55) nm. HR-ESI-MS: m/z 366.1449 [M + H]⁺ (calcd. for 366.1448 C₂₀H₂₀N₃O₄) and 388.1265 [M + Na]⁺ (calcd. for 388.1268 C₂₀H₁₉N₃ONa) in positive mode, and m/z 364.1303 [M − H]⁻ (calcd. for 364.1303, C₂₀H₁₈N₃O) in negative mode. ¹H NMR (600 MHz, DMSO-d₆) and ¹³C NMR (150 MHz, DMSO-d₆) spectra data are listed in

Table 3. ¹H NMR and ¹³C NMR (DMSO-d₆) data of compounds 3 and 19.

Positions	3		19
	δC	δH	δC	δH
2	99.3	5.10, d (2.8)	121.4	6.88, br. s
3	74.6		109.2
4	123.1	6.90, br. d (7.2)	118.9	7.43, br. d (7.2)
5	118.3	6.56, br. t (7.0)	119.2	6.93, br. t (7.0)
6	129.1	7.00, br. t (7.5)	124.9	7.01, br. t (7.4)
7	109.6	6.56, br. d (7.7)	111.8	7.26, br. d (7.7)
3a	131.9		128.0
7a	148.1		136.5
1′	136.0		137.0
2′, 6′	130.4	6.98, br. d (7.2)	130.2	7.10, overlapped
3′, 5′	128.2	6.86, br. t (7.4)	128.5	6.64, br. t (7.4)
4′	126.5	6.75, m	126.9	7.12, overlapped
C=O	166.1		166.7
C=O′	161.5		167.3
α	54.6	4.17, dd (9.1, 4.8)	55.8	3.92, m
α′	54.8	4.19, dd (5.0, 2.8)	56.1	3.79, m
β′	37.7	2.87, dd (13.9, 2.8)	39.5	2.40, overlapped
		2.74, dd (13.8, 5.0)		1.78, dd (13.5, 7.0)
β	36.0	1.91, dd (13.6, 4.8)	30.2	2.74, dd (14.0, 4.4)
		1.03, dd (13.5, 9.1)		2.44, overlapped
1-NH		6.57, br. s		10.83, br. s
α-NH				7.85, br. s
α′-NH		7.96, br. s		7.64, br. s

, and Figures S18–S25 in supporting materials.

5,5-Dichloro-1-(3,5-dimethoxyphenyl)-1,4-dihydroxypentan-2-one (4): white powder; [α${]}_{\mathrm{D}}^{25}$ − 23° (c = 0.1, CH₃OH); IR (KBr) (ν_max): 3447, 1608, 1396, and 1159 cm⁻¹; UV (MeOH) λ_max (log ε): 204 (2.36) nm and 275 (1.23) nm. HR-ESI-MS: m/z 323.0449 [M + H]⁺ (calcd. for 323.0448 C₁₃H₁₇O₅Cl₂) and 345.0261 [M + Na]⁺ (calcd. for 345.0267 C₁₃H₁₆O₅Cl₂Na) in positive mode, and m/z 321.0304 [M − H]⁻ (calcd. for 321.0302, C₁₃H₁₅O₅Cl₂) in negative mode. ¹H NMR (600 MHz, DMSO-d₆) δ_H: 6.37 (2H, d, J = 2.2 Hz, H-2′, 6′), 6.27 (1H, t, J = 2.2 Hz, H-4′), 5.96 (1H, d, J = 3.3 Hz, H-5), 4.85 (1H, s, H-1), 4.07 (1H, ddd, J = 8.8, 3.3, 2.8 Hz, H-4), 3.57 (6H, s, H-3′, 5′-OCH₃), [2.66 (1H, dd, J = 17.4, 8.8 Hz) & 2.52 (1H, dd, J = 17.4, 2.8 Hz), H-3]; ¹³C NMR (150 MHz, DMSO-d₆) δ_C: 207.8 (C-2), 160.9 (C-3′, 5′), 141.7 (C-1′), 105.3 (C-2′, 6′), 100.0 (C-4′), 79.7 (C-1), 77.7 (C-5), 71.1 (C-4), 55.6 (C-3′, 5′-OCH₃), 40.9 (C-3). The spectra are provided in Figures S26–S33 in supporting materials.

2,3,4-Trihydroxybutyl cinnamate (5): white powder; [α${]}_{\mathrm{D}}^{25}$ − 23° (c = 0.1, CH₃OH); IR (KBr) (ν_max): 3421, 1700, 1634, 1396, 1186 and 1085 cm⁻¹; UV (MeOH) λ_max (log ε): 216 (1.93) nm and 276 (1.99) nm. HR-ESI-MS: m/z 275.0882 [M + Na]⁺ (calcd. for 275.0890 C₁₃H₁₆O₅Na) in positive mode, and m/z 251.0922 [M − H]⁻ (calcd. for 251.0925, C₁₃H₁₄O₅) in negative mode. ¹H NMR (600 MHz, DMSO-d₆) δ_H: 7.69 (1H, d, J = 16.0 Hz, H-7), 7.44 (1H, overlapped, H-4), 7.44 (2H, overlapped, H-3, 5), 7.72 (2H, dd, J = 6.3, 3.0 Hz, H-2, 6), 6.64 (1H, d, J = 16.0 Hz, H-8), 3.44 (1H, m, H-3′), 3.66 (1H, m, H-2′), [4.33 (1H, dd, J = 11.3, 2.7 Hz) and 4.11 (1H, dd, J = 11.3, 7.1 Hz), H-1′], [3.58 (1H, br. d, J = 9.2 Hz) and 3.42 (1H, br. d, J = 9.2 Hz), H-4′]; ¹³C NMR (150 MHz, DMSO-d₆) δ_C: 166.9 (C-9), 144.8 (C-7), 134.5 (C-1), 130.9 (C-4), 129.4 (C-3, 5), 128.8 (C-2, 6), 118.8 (C-8), 72.8 (C-3′), 69.9 (C-2′), 67.0 (C-1′), 63.5 (C-4′). The spectra are provided in Figures S34–S42 in supporting materials.

4.6. Cytotoxicity Assay

Cytotoxicity assay was carried out according to the instructions of CCK-8 kit [7]. Briefly, compounds at different concentrations were added into the culture medium containing 10⁵ /mL HT1080, Cne2 and Bel7402 cells and incubated for 24, 48, 72 h. Then, 10 μL of CCK-8 solution was added into each well of the 96-well plate, incubated for 2 h and then the absorbance was measured at 450 nm using a microplate reader (BIO-RAD, Hercules, California, USA). The inhibition rate = (A _control − A _treated)/A _control × 100. The IC₅₀ was the concentration at which it caused 50% inhibition of cell proliferation (50% reduction in the absorbance value in the treated cells, in respect to the control).