Statins Diversity Revealed by the Deep-Sea-Derived Fungus Penicillium viridicatum

Abstract

Seven new (1–7) and six known (8–13) statin derivatives were obtained from the deep-sea-derived fungus Penicillium viridicatum MCCC 3A00265. The structures assigned to the new compounds were based on a comprehensive analysis of the spectroscopic data, with absolute configurations established by Mosher analysis and biogenetic consideration. Most of the new compounds (1–5 and 7) share an octohydronaphthalene backbone, except that viridecalin F (6) possesses an uncommon naphthalene core. Viridecalins C (3) and F (6) and the two known compounds 9 and 11 exhibit considerable ability in reactivating mutant p53 protein at 10 μM, while viridecalin C showcases the most potent reactivation activity, indicating the potential of application in cancer therapy.

1. Introduction

Statins are well-known agents for the treatment of hyperlipidemia, both as monotherapy and in combination therapy, representing a successful example of natural product-derived drug discovery, more specifically, from filamentous fungi [1]. Natural statins share a common hexahydro-naphthalene core and a 3,5-dihydroxy-hepta(e)noic acid side chain, which mimic the 3-hydroxy-3-methylglutaryl-coenzyme A(HMG-CoA), and act by occupying a portion of the binding site of the enzyme, thereby blocking access of this substrate (HMG-CoA) to the active site [2]. The modifications in the core structure, including substitution and dehydrogenation at the hydro-naphthalene ring and oxidation at the hydroxycarboxylate side chain, were thought to contribute to their bioactivity, such as half-life elimination and bioavailability [3]. Beyond their lipid-lowering effects, statins and their derivatives have demonstrated a wide range of additional pharmacological activities, including anti-inflammatory, antioxidant, and anticancer properties, which spurred interest in exploring structurally diverse statin-like compounds from novel sources [4,5]. Marine microorganisms, especially deep-sea-derived (>1000 m) fungi, are an enormous reservoir of structurally new and bioactive metabolites due to their extremely different environment compared to their terrestrial counterparts [6,7]. As an example, a previous chemical investigation of the deep-sea-derived Penicillium viridicatum resulted in the discovery of (i) viricyanoamides A and B, the sole representatives of quinazolinones that feature a nitrile group and exhibit significant mutant p53 reactivating activity; (ii) solitumidine F, an indole terpenoid with a rare pyrrolidinedione unit incorporated; (iii) and the herbicidal decline polyketides viridicatumones A–C [8,9] (Figure 1).

In a search of intriguing natural products from marine fungi, we identified an Atlantic abyssal sediment-derived Penicillium viridicatum as a statin derivative producer. A systematic investigation into this strain led to the discovery of seven new (1–7) and six known (8–13) decalin-type polyketides (Figure 2). Herein is an account of the isolation, structure elucidation, and bioactivities of the isolates.

2. Results and Discussion

The EtOAc extract of a scaled-up rice cultivation of Penicillium viridicatum MCCC 3A00265 was subjected to sequential solvent partitioning, followed by isolation and purification by column chromatography (CC) over silica gel, ODS, Sephadex LH-20, and by preparative thin-layer chromatography (PTLC) to yield 13 compounds (1–13). By comparison of the NMR, MS, and OR data with those reported in the references, six known compounds were identified, eujavanoic acid B (8) [10], solistatin (9) [11], dihydrocompactin (10) [10,12,13], desmethyl-dihydromonacolin L (11) [14], 3,5-dihydro-3-oxo-ML-236C (12) [10], and 6-demethylmonacolin J (13) [15], while seven new ones (1–7) were elucidated by detailed spectroscopic analysis and biogenetic considerations as summarized below.

The positive HRESIMS analysis of 1 revealed the molecular formula C₁₈H₂₈O₄, requiring five degrees of unsaturation. The ¹H and ¹³C NMR spectroscopic data of 1 (Table 1, Figures S1 and S2) showcased resonances for one methyl doublet, seven sp³ methylene, seven sp³ methine, two sp² methine, and one carbonyl carbon, accounting for five degrees of unsaturation. Therefore, 1 incorporated three rings. A suite of ¹H-¹H COSY correlations and diagnostic HMBC cross-peaks observed from H₃-16 to C-1/2/3, H-1 to C-8, H₂-8 to C-6/4a, 6-OH to C-5/6/7, as well as H-4 to C-2/5/8a allowed for the connection of a 2-methyl-6-hydroxy-Δ³-octohydronaphthalene segment and a side chain terminated with an ester unit supported by HMBC signals of H-13/H₂-14 to the carbonyl carbon C-15 (Figure 3). Considering the deshielding of the oxygenated methine C-11 and the remaining one unsaturation, the lactone ring was inferred through a (C-11)-O-(C-15) linkage. In regards to the stereochemistry of 1, the trans configuration of the dihydrodecalin was indicated by the NOESY correlations observed from H-1 to H-5 and H-8a to H₂-10 (Figure 4). Additionally, the correlations of H-4a/H-6, H-4a/H-1 indicated those protons were on the same side, while H₃-16/H₂-9 suggested they were toward the opposite side of the former (Figure 4). The relative configuration of the lactone ring was implied by the NOESY signal between H₂-10 and H-13. Due to a convergent biosynthetic pathway of the trans-octohydronaphthalene and the hydroxycarboxylate side chain shared in this type of known statin derivative [3], the absolute configuration of C-4a/8a, C-11/13 was deduced to be the same as that of the cometabolites dihydrocompactin (10) and demethyl-dihydromonacolin L (11), for which the configurations of rest chiral centers could be indicated accordingly. To confirm this assignment, a modified Mosher’s analysis was performed. The deshielding of H₂-7 and H-11 (Δ_S-R > 0) and the shielding of H-2, H-3, H-4, H₂-5, and H₂-14 (Δ_S-R < 0) in the S-MTPA ester compared to the R-MTPA derivative unambiguously established the absolute configurations of C-6 and C-13 as 7S, 13R (Figure 5), thereby inferring the configurations of C-1/2/4a/8a/11. Therefore, the complete structure of 1 was established and named viridecalin A.

The positive HRESIMS spectrum of 2 returned an identical molecular formula, C₁₈H₂₈O₄, as that of 1. A close comparison of their NMR data revealed a high degree of similarities, with principal differences on the carbon chemical shifts of the octohydronaphthalene part. A scrutiny of the ¹H-¹H COSY and HMBC spectra of 2 indicated that the hydroxy group was substituted at C-8, which was evidenced by the COSY correlations of 8-OH/H-8/H-1/H-16 as well as HMBC cross-peaks from 8-OH to C-7/8/8a (Figure 3). The relative configuration of 8-OH was determined by the NOESY spectrum. The signals among H-8/H₂-9, H-8a/H₂-9/H₃-16 suggested these protons were on the same side of the plane, while 8-OH/H-4a/H-1 were on the opposite side, evident by correlations of 8-OH/H-4a, H-4a/H-4/H-1 (Figure 4). The absolute configuration of 2 was deduced to be the same as 1 and the known cometabolites based on a biogenetic consideration. Therefore, the structure of 2 was assigned and named viridecalin B.

Figure 3. ¹H-¹H COSY (bold line) and HMBC (blue arrow) correlations of 1–7.

Figure 3. ¹H-¹H COSY (bold line) and HMBC (blue arrow) correlations of 1–7.

Table 1. ¹H (400 Hz) and ¹³C (100 Hz) NMR spectroscopic data of 1–3 in DMSO-d₆.

Pos.	1		2		3
	1H a	13C	1H a	13C	1H a	13C
1	1.38, m	40.5 d	1.60, m	37.5 d	2.06, tt (11.8, 3.5)	32.2 d
2	2.21, m	31.5 d	2.20, m	31.5 d	2.23, qd (7.0, 5.0)	31.8 d
3	5.57, ddd (9.8, 4.6, 2.6)	132.6 d	5.52, m	132.4 d	5.68, dd (9.7, 5.0)	136.7 d
4	5.31, d (9.8)	130.5 d	5.32, d (9.8)	132.0 d	5.30, d (9.7)	131.6 d
4a	1.65, m	41.3 d	2.15, m	35.5 d		73.7 s
5	0.88, m; 1.82, m	42.0 t	0.90, dd (12.7, 2.8); 1.64, m	33.4 t	3.48, br s	72.2 d
6	3.41, m	68.8 d	1.39, m; 1.65, m	20.6 t	1.34, d (13.9) 2.03, tt (13.9, 3.3)	23.2 t
7	1.09, m; 1.90, m	35.9 t	1.35, m; 1.72, m	34.4 t	1.54, d (13.9); 1.71, m	28.5 t
8	0.91, m; 1.76, m	27.1 t	3.93, br s	64.1 d	3.89 b	66.9 d
8a	0.92, m	38.3 d	0.98, dd (10.8, 10.8)	42.9 d	1.58, dd (12.0, 2.1)	36.1 d
9	1.18, m; 1.54, tt (12.2, 3.8)	23.6 t	1.16, m; 1.71, m	23.1 t	1.19, m; 1.68, m	23.4 t
10	1.40, m; 1.63, m	32.5 t	1.40, m; 1.68, m	33.0 t	1.44, m; 1.66, m	33.1 t
11	4.54, tt (11.0, 3.8)	75.6 d	4.54, m	76.6 d	4.55, m	77.1 d
12	1.66, m; 1.80, m	35.2 t	1.68, m; 1.81, m	35.8 t	1.68, m; 1.82, d (13.9)	35.5 t
13	4.09, m	61.3 d	4.10, m	61.8 d	4.11 b	62.0 d
14	2.36, dd (3.3, 1.6) 2.62, dd (3.3, 1.6)	38.6 t	2.38, d (17.2) 2.62, dd (17.2, 4.4)	39.0 t	2.36 d (17.5) 2.61 dd (17.5, 4.6)	38.9 t
15		170.4 s		170.9 s		172.0 s
16	0.79, d (7.0)	14.6 q	0.78, d (7.0)	15.1 q	0.73, d (7.0)	13.1 q
4a-OH					5.53, s
5-OH					4.77, d (4.2)
6-OH	4.52, d (4.5)
8-OH			4.13 b		5.05, d (7.1)
13-OH	5.15, d (3.0)		5.17, d (2.9)		5.47, d (2.8)

^a δ in ppm, J in Hz within parentheses. ^b Resonance obscured by solvent peak.

Table 1. ¹H (400 Hz) and ¹³C (100 Hz) NMR spectroscopic data of 1–3 in DMSO-d₆.

Pos.	1		2		3
	1H a	13C	1H a	13C	1H a	13C
1	1.38, m	40.5 d	1.60, m	37.5 d	2.06, tt (11.8, 3.5)	32.2 d
2	2.21, m	31.5 d	2.20, m	31.5 d	2.23, qd (7.0, 5.0)	31.8 d
3	5.57, ddd (9.8, 4.6, 2.6)	132.6 d	5.52, m	132.4 d	5.68, dd (9.7, 5.0)	136.7 d
4	5.31, d (9.8)	130.5 d	5.32, d (9.8)	132.0 d	5.30, d (9.7)	131.6 d
4a	1.65, m	41.3 d	2.15, m	35.5 d		73.7 s
5	0.88, m; 1.82, m	42.0 t	0.90, dd (12.7, 2.8); 1.64, m	33.4 t	3.48, br s	72.2 d
6	3.41, m	68.8 d	1.39, m; 1.65, m	20.6 t	1.34, d (13.9) 2.03, tt (13.9, 3.3)	23.2 t
7	1.09, m; 1.90, m	35.9 t	1.35, m; 1.72, m	34.4 t	1.54, d (13.9); 1.71, m	28.5 t
8	0.91, m; 1.76, m	27.1 t	3.93, br s	64.1 d	3.89 b	66.9 d
8a	0.92, m	38.3 d	0.98, dd (10.8, 10.8)	42.9 d	1.58, dd (12.0, 2.1)	36.1 d
9	1.18, m; 1.54, tt (12.2, 3.8)	23.6 t	1.16, m; 1.71, m	23.1 t	1.19, m; 1.68, m	23.4 t
10	1.40, m; 1.63, m	32.5 t	1.40, m; 1.68, m	33.0 t	1.44, m; 1.66, m	33.1 t
11	4.54, tt (11.0, 3.8)	75.6 d	4.54, m	76.6 d	4.55, m	77.1 d
12	1.66, m; 1.80, m	35.2 t	1.68, m; 1.81, m	35.8 t	1.68, m; 1.82, d (13.9)	35.5 t
13	4.09, m	61.3 d	4.10, m	61.8 d	4.11 b	62.0 d
14	2.36, dd (3.3, 1.6) 2.62, dd (3.3, 1.6)	38.6 t	2.38, d (17.2) 2.62, dd (17.2, 4.4)	39.0 t	2.36 d (17.5) 2.61 dd (17.5, 4.6)	38.9 t
15		170.4 s		170.9 s		172.0 s
16	0.79, d (7.0)	14.6 q	0.78, d (7.0)	15.1 q	0.73, d (7.0)	13.1 q
4a-OH					5.53, s
5-OH					4.77, d (4.2)
6-OH	4.52, d (4.5)
8-OH			4.13 b		5.05, d (7.1)
13-OH	5.15, d (3.0)		5.17, d (2.9)		5.47, d (2.8)

^a δ in ppm, J in Hz within parentheses. ^b Resonance obscured by solvent peak.

Figure 4. NOESY correlations of 1–2, 4–5, and 7 (red: correlations among β-oriented protons; green: correlations among α-oriented protons).

Figure 4. NOESY correlations of 1–2, 4–5, and 7 (red: correlations among β-oriented protons; green: correlations among α-oriented protons).

Figure 5. Mosher’s analysis of 1, with the Δ_S−R value highlighted in blue (positive) and red (negative).

Figure 5. Mosher’s analysis of 1, with the Δ_S−R value highlighted in blue (positive) and red (negative).

Compound 3 was assigned the molecular formula C₁₈H₂₈O₆, accounting for five degrees of unsaturation. Comparison of the ¹H and ¹³C NMR spectroscopic data of 3 with 2 revealed great similarity, except for the major difference on the octohydronaphthalene unit: two additional hydroxy groups showing up at the C-4a and C-5 positions in 3. The assumption was supported by the COSY correlation of 6-OH/H-6 as well as HMBC correlations from 4a-OH to C-4/4a/5/8a, from H-3 to C-4a/5, and from 5-OH to C-4a/5/6 (Figure 3). On the basis of the small coupling constant of ³J_H-8/H-8a (2.1 Hz) and the broad singlet status of H-5, 5-OH and 8-OH were assigned as axial oriented. Due to a biogenetic ground, the absolute configurations of C-4a/8a and C-11/13 were deduced to be the same as those of 1 and 2. Thus, the structure of 3 was assigned and named viridecalin C.

The molecular formula of 4 was identified as C₁₉H₃₂O₅ based on an analysis of its positive HRESIMS spectrum, requiring four degrees of unsaturation. The ¹H and ¹³C NMR spectroscopic data of 4 (Table 2) revealed very similar resonances as those of 2, except for the presence of an additional methoxy group as well as the shielding effect of H-11 (Δδ_H − 1.0) and C-11 (Δδ_H − 8.1) in 4. This, along with the observation of a free hydroxy unit at C-11 that was evident from the ¹H-¹H COSY correlation of 11-OH/H-11, confirmed the opening of the lactone ring in 4. A scrutiny of the 2D NMR spectra of 4 established its planar structure as shown in Figure 3. The 8-OH was determined as axial based on the broad singlet peak of H-8 (δ_H 3.93, br s) and the NOESY correlation among 8-OH, H-4a, and H-1. All the chiral centers were assumed to retain the same absolute configurations as those of 2 based on the convergent biosynthetic consideration. Accordingly, the structure of 4 was determined and was given a trivial name, viridecalin D.

Analysis of the positive HRESIMS spectrum of 5 returned the same isomeric molecular formula as that of 4. Moreover, the ¹H and ¹³C NMR spectroscopic data of 5 showed almost the same as those of 4, except that the hydroxy group was located at the C-6 position in 4 instead of the C-8 position in 5. This was supported by the COSY correlation of 6-OH/H-6 and HMBC correlations from 6-OH to C-5/6/7 (Figure 3). The NOESY correlation of H-6/H-4a/H-1 suggested they were on the same side (Figure 4). On the basis of the above evidence, the structure of 5 was then assigned and name viridecalin E.

Table 2. ¹H (400 Hz) and ¹³C (100 Hz) NMR spectroscopic data of 4–7 in DMSO-d₆.

Pos.	4		5		6		7
	1H a	13C	1H a	13C	1H a	13C	1H a	13C
1	1.56, m	37.1 d	1.35, m	40.8 d		135.7 s	1.40, m	40.3 d
2	2.16, m	31.0 d	2.20, m	31.5 d		132.5 s	2.16, dd (9.0, 6.3)	30.9 d
3	5.51, m	132.1 d	5.57, m	132.9 d	7.32, d (8.4)	129.1 d	5.60, m	132.4 d
4	5.31, d (9.8)	131.4 d	5.31, d (9.8)	130.4 d	7.65, d (8.4)	125.7 d	5.90, d (10.0)	127.0 d
4a	2.14, m	35.0 d	1.65, dd (9.8, 9.0)	41.4 d		132.1 s	1.48, ddd (10.4, 10.4, 1.6)	50.8 d
5	0.88, m; 1.61, m	32.9 t	0.89, m; 1.83, d (12.9)	42.1 t	7.83, d (8.1)	128.4 d	2.96, ddd (14.5, 10.4, 4.8)	74.5 d
6	1.37, m; 1.64, m	20.1 t	3.42 b	68.9 d	7.42, m	124.5 d	1.14, dd (10.4, 10.4); 1.81, m	35.7 t
7	1.35, m; 1.72, d (13.0)	33.9 t	1.10, m; 1.90, d (12.0)	36.0 t	7.50, m	125.9 d	1.26, m	25.7 t
8	3.93, br s	63.7 d	0.90, m; 1.77, m	27.1 d	8.07, d (8.4)	123.5 d	0.83, dd (12.3, 3.1); 1.69, m	28.0 t
8a	0.94, m	42.6 d	0.90, m	38.4 d		131.6 s	1.03, m	36.6 d
9	1.09, m; 1.64, m	22.8 t	1.13, m; 1.50, m	23.9 t	2.98, td (13.3, 5.3) 3.17, td (9.9, 5.3)	24.0 t	1.26, m; 1.78, m	24.0 t
10	1.19, m; 1.34, m	34.4 t	1.22, m; 1.35, m	34.4 t	1.53, m; 1.65, m	37.3 t	2.20, dd (9.6, 6.5) 2.35, ddd (15.4, 10.0, 5.2)	31.2 t
11	3.54 b	68.5 d	3.55 b	68.0 d	3.78, m	67.9 d		173.6 s
12	1.47, m	44.3 t	1.48, m	44.2 t	1.58, m	44.0 t	3.59, s	51.3 q
13	4.00, m	65.8 d	4.00, m	65.8 d	4.05, m	65.6 d	0.77, d (7.1)	14.5 q
14	2.29, dd (15.0, 8.6) 2.45, dd (15.0, 4.4)	42.2 t	2.29, dd (14.5, 8.2) 2.47, dd (14.5, 4.3)	42.3 t	2.34, dd (14.6, 8.6)	42.3 t	1.41, m
15		171.8 s		170.9 s	2.50 b	171.8 s
16	3.57, s	51.1 q	3.57, s	51.1 q	3.57, s	51.1 q
17	0.76, d (7.0)	14.7 q	0.79, d (7.0)	15.1 q	2.46, s	19.6 q
5-OH							4.59, d (5.6)
6-OH			4.52, d (3.8)
8-OH	4.05, m
11-OH	4.45, d (4.7)		4.45, d (4.7)		4.79, d (5.0)
13-OH	4.77, d (4.8)		4.76, d (4.8)		4.85, d (5.0)

^a δ in ppm, J in Hz within parentheses. ^b Resonance obscured by solvent peak.

Table 2. ¹H (400 Hz) and ¹³C (100 Hz) NMR spectroscopic data of 4–7 in DMSO-d₆.

Pos.	4		5		6		7
	1H a	13C	1H a	13C	1H a	13C	1H a	13C
1	1.56, m	37.1 d	1.35, m	40.8 d		135.7 s	1.40, m	40.3 d
2	2.16, m	31.0 d	2.20, m	31.5 d		132.5 s	2.16, dd (9.0, 6.3)	30.9 d
3	5.51, m	132.1 d	5.57, m	132.9 d	7.32, d (8.4)	129.1 d	5.60, m	132.4 d
4	5.31, d (9.8)	131.4 d	5.31, d (9.8)	130.4 d	7.65, d (8.4)	125.7 d	5.90, d (10.0)	127.0 d
4a	2.14, m	35.0 d	1.65, dd (9.8, 9.0)	41.4 d		132.1 s	1.48, ddd (10.4, 10.4, 1.6)	50.8 d
5	0.88, m; 1.61, m	32.9 t	0.89, m; 1.83, d (12.9)	42.1 t	7.83, d (8.1)	128.4 d	2.96, ddd (14.5, 10.4, 4.8)	74.5 d
6	1.37, m; 1.64, m	20.1 t	3.42 b	68.9 d	7.42, m	124.5 d	1.14, dd (10.4, 10.4); 1.81, m	35.7 t
7	1.35, m; 1.72, d (13.0)	33.9 t	1.10, m; 1.90, d (12.0)	36.0 t	7.50, m	125.9 d	1.26, m	25.7 t
8	3.93, br s	63.7 d	0.90, m; 1.77, m	27.1 d	8.07, d (8.4)	123.5 d	0.83, dd (12.3, 3.1); 1.69, m	28.0 t
8a	0.94, m	42.6 d	0.90, m	38.4 d		131.6 s	1.03, m	36.6 d
9	1.09, m; 1.64, m	22.8 t	1.13, m; 1.50, m	23.9 t	2.98, td (13.3, 5.3) 3.17, td (9.9, 5.3)	24.0 t	1.26, m; 1.78, m	24.0 t
10	1.19, m; 1.34, m	34.4 t	1.22, m; 1.35, m	34.4 t	1.53, m; 1.65, m	37.3 t	2.20, dd (9.6, 6.5) 2.35, ddd (15.4, 10.0, 5.2)	31.2 t
11	3.54 b	68.5 d	3.55 b	68.0 d	3.78, m	67.9 d		173.6 s
12	1.47, m	44.3 t	1.48, m	44.2 t	1.58, m	44.0 t	3.59, s	51.3 q
13	4.00, m	65.8 d	4.00, m	65.8 d	4.05, m	65.6 d	0.77, d (7.1)	14.5 q
14	2.29, dd (15.0, 8.6) 2.45, dd (15.0, 4.4)	42.2 t	2.29, dd (14.5, 8.2) 2.47, dd (14.5, 4.3)	42.3 t	2.34, dd (14.6, 8.6)	42.3 t	1.41, m
15		171.8 s		170.9 s	2.50 b	171.8 s
16	3.57, s	51.1 q	3.57, s	51.1 q	3.57, s	51.1 q
17	0.76, d (7.0)	14.7 q	0.79, d (7.0)	15.1 q	2.46, s	19.6 q
5-OH							4.59, d (5.6)
6-OH			4.52, d (3.8)
8-OH	4.05, m
11-OH	4.45, d (4.7)		4.45, d (4.7)		4.79, d (5.0)
13-OH	4.77, d (4.8)		4.76, d (4.8)		4.85, d (5.0)

^a δ in ppm, J in Hz within parentheses. ^b Resonance obscured by solvent peak.

The positive HRESIMS spectrum of 6 assigned its molecular formula as C₁₉H₂₄O₄, requiring eight degrees of unsaturation. Analysis of the ¹H and ¹³C NMR spectroscopic data of 6 revealed resonances for an ortho-di-substituted naphthalene ring and a similar methoxylated hydroxycarboxylate side chain as 5, accounting for all the eight degrees of unsaturation. The key HMBC correlations observed from H₃-16 to C-1/2/3 and H₂-9 to C-1 determined the planar structure of 6. The absolute configurations of C-11 and C-13 were deduced to be the same as those of 5 due to the biogenetic ground. Accordingly, the structure of 6 was determined and named viridecalin F.

The positive HRESIMS spectrum of 7 revealed the molecular formula C₁₅H₂₄O₃, requiring four degrees of unsaturation. A suite of ¹H-¹H COSY correlations together with HMBC cross-peaks from H₃-16 to C-1/2/3, from 5-OH to C-4a/5/6, and from H₂-10 to the carbonyl carbon C-11 conformed a similar octohydronaphthalene segment as that of 6 and a C₃ hydroxycarboxylate chain. The connection of the two parts was implied by the COSY signals of H-1/H₂-9. Accordingly, the structure of 7 was established and was named viridecalin G.

Referred to as the “guardian of the genome,” p53 is a tumor suppressor protein encoded by the TP53 gene in humans and reported mutated in over 50% of tumor cells [16]. These mutations result in the loss of p53’s tumor-suppressing capabilities and contribute to tumor progression. Consequently, activating the p53 signaling pathway in tumor cells represents a promising strategy for cancer therapy [17]. Statins have been identified as degradation inducers for misfolded or conformationally incorrect p53 mutants and can also activate the p53 signaling pathway [18,19]. The antitumor potential of 11 statins (6 and 12 were not included due to a low quantity) from MCCC 3A00265 was evaluated by analyzing their ability to reactivate mutant p53 using relative fluorescence intensity as we reported previously [20,21]. Of note, compound 1 significantly enhanced the activity of the p53 response element p21 (>2-fold) in p53-deficient H1299 cells at 10 μM, while compounds 3, 7, 9, and 11 exhibited responsivity comparable to the positive control PRIMA-1, a known mutant p53 reactivator (Figure 6). The cytotoxicity of the new compounds 1–7 was also evaluated on the adenocarcinomic human alveolar basal epithelial cells A549 using the CCK-8 assay, returning no visible inhibition at the concentration of 20 μM.

3. Materials and Methods

3.1. General Experimental Procedures

The NMR spectra were recorded in DMSO-d₆ on Bruker 400 MHz or 600 MHz spectrometers (Billerica, Massachusetts, USA). The HRESIMS spectra were measured on a Waters Xevo G2 Q-TOF mass spectrometer (Milford, Massachusetts, USA). Optical rotations were measured with an Anton Paar MCP100 polarimeter (Graz, Austria). The semi-preparative HPLC was conducted on an Agilent technologies 1260 infinity instrument (California, USA) equipped with a DAD detector. The medium-pressure liquid chromatography (MPLC) was conducted on a Buchi Sepacore. Column chromatography (CC) was performed on silica gel (100–200 m, 200–300 m, 300–400 m; Qiaodao Marine Chemistry Co., Ltd. Qingdao, China), Sephadex LH-20 (Amersham Pharmacia Biotech AB, Uppsala, Sweden), and ODS (octadecylsilyl; 50 μm, Daiso), respectively.

3.2. Fungal Identification, Fermentation, and Extraction

The fungus strain Penicillium viridicatum was isolated from the deep-sea sediment collected in 2005 at a depth of 3039 m in the Atlantic. It was cultivated on PDA plates at 25 °C for 3 days. Then, the fresh mycelia were sliced into squares (0.5 × 0.5 × 0.5 cm³) and incubated in seed medium (PDB) on a rotating shaker (160 rpm). Large fermentation was carried out in 100 Erlenmeyer flasks (1 L), with each containing rice (200 g) and tape water (120 mL, 3% marine salt). After incubation for 30 days at 25 °C, the fermented broth was extracted three times with EtOAc. The organic solvent was evaporated to dryness and dissolved in MeOH. The filtrate was concentrated under reduced pressure to give a crude extract (53.4 g).

3.3. Isolation and Purification

The crude extract was subjected to MPLC (460 mm × 46 mm) over silica gel (200–300 mesh) using gradient PE/EtOAc (100% → 0%, 8 h, 10 mL/min) to provide four fractions (Fr.1−Fr.4). Fraction Fr.2 (18.1 g) was further separated by CC over ODS (460 mm × 26 mm, MeOH-H₂O, 5% → 50%, 1 h, 50% → 100%, 8 h, 10 mL/min) to give 15 subfractions (Fr.2.1–Fr.2.15). Subfraction Fr.2.4 (2.0 g), Fr.2.6 (130.3 mg), and Fr.2.14 (340.5 mg) were subsequently separated by CC over Sephadex LH-20 (150 cm × 2 cm, MeOH), silica gel (46 mm × 20 mm, PE-EtOAc), and PTLC (CH₂Cl₂-MeOH) to yield 7 (5.2 mg), 8 (12.5 mg), and 11 (37.6 mg). Compounds 6 (3.4 mg), 9 (6.1 mg), and 10 (11.3 mg) were separated by CC over Sephadex LH-20 (150 cm × 2 cm, MeOH), semi-prep HPLC (C-18, chiral column C4-5, 10 mm × 250 mm, MeOH-H₂O, 60% → 100%, 3 mL/min), and Prep. TLC (CH₂Cl₂-MeOH) from subfraction Fr.2.9 (439.0 mg), Fr.2.10 (234.5 mg), and Fr.2.15 (80.0 mg), respectively. Fraction Fr.3 (14.1 g) was isolated by reversed-phase MPLC (460 mm × 26 mm, MeOH-H₂O, 60% → 100%, 8 h, 10 mL/min) and Sephadex LH-20 (150 cm × 2 cm, MeOH), followed by semi-prep HPLC (5-PFP, 10 mm × 250 mm, MeOH-H₂O, 50% → 80%, 3 mL/min) to yield 12 (37.3 mg). Fraction Fr.4 (8.0 g) was further separated by CC over ODS (460 mm × 15 mm, MeOH-H₂O, 5% → 20%, 1 h, 20% → 90%, 8 h, 10 mL/min) to give seven subfractions (Fr.4.1–Fr.4.7). Compounds 3 (8.9 mg), 1 (9.1 mg), 2 (33.4 mg), 4 (5.1 mg), and 5 (6.2 mg) were separated by CC over Sephadex LH-20 (150 cm × 2 cm, MeOH) and Prep. TLC (CH₂Cl₂-MeOH, 10:1, 10:1, 15:1, 15:1, 20:1, v/v) from fraction Fr.4, respectively. Subfraction Fr.4.5 was separated by CC over Sephadex LH-20 (2 cm × 150 cm, MeOH), semi-prep HPLC (chiral column C4-5, 10 mm × 250 mm, MeOH-H₂O, 50% → 80%, 3 mL/min), and Prep. TLC to yield 13 (2.8 mg).

Viridecalin A (1): White solid; [α${]}_{\mathrm{D}}^{25}$ +78.0 (c 0.1, MeOH); ¹H and ¹³C NMR data, see Table 1. HRESIMS m/z 331.1879 [M + Na]⁺ (calcd for C₁₈H₂₈O₄Na, 331.1885).

Viridecalin B (2): Colorless oil; [α${]}_{\mathrm{D}}^{25}$ +71.0 (c 0.1, MeOH); ¹H and ¹³C NMR data, see Table 1. HRESIMS m/z 331.1879 [M + Na]⁺ (calcd for C₁₈H₂₈O₄Na, 331.1885).

Viridecalin C (3): White solid; [α${]}_{\mathrm{D}}^{25}$ +100 (c 0.1, MeOH); ¹H and ¹³C NMR data, see Table 1. HRESIMS m/z 363.1768 [M + Na]⁺ (calcd for C₁₈H₂₈O₆Na, 363.1778).

Viridecalin D (4): Colorless oil; [α${]}_{\mathrm{D}}^{25}$ +87.0 (c 0.1, MeOH); ¹H and ¹³C NMR data, see Table 2. HRESIMS m/z 363.2139 [M + Na]⁺ (calcd for C₁₉H₃₂O₅Na, 363.2147).

Viridecalin E (5): Colorless oil; [α${]}_{\mathrm{D}}^{25}$ +44.0 (c 0.1, MeOH); ¹H and ¹³C NMR data, see Table 2. HRESIMS m/z 363.2149 [M + Na]⁺ (calcd for C₁₉H₃₂O₅Na, 363.2147).

Viridecalin F (6): Colorless oil; [α${]}_{\mathrm{D}}^{25}$ +18.0 (c 0.1, MeOH); ¹H and ¹³C NMR data, see Table 2. HRESIMS m/z 339.1572 [M + Na]⁺ (calcd for C₁₉H₂₄O₄Na, 339.1572).

Viridecalin G (7): Colorless oil; [α${]}_{\mathrm{D}}^{25}$ +62.0 (c 0.2, MeOH); ¹H and ¹³C NMR data, see Table 2 HRESIMS m/z 275.1676 [M + Na]⁺ (calcd for C₁₅H₂₄O₃Na, 275.1623).

3.4. Luciferase Reporter Assay

H1299 cells (1 × 10³) were inoculated into 96-well plates and cultured for 24 h. Transfection was performed using EZ Trans (Life iLab Biotech, Shanghai, China) according to the manufacturer’s instructions. For transfection of 100 wells, 7500 ng of p53 R175H Expression Plasmid, 2500 ng of Luciferase Reporter Plasmid, and 120 ng of Renilla Plasmid were mixed thoroughly with 250 μL of Opti-MEM, while 30 μL of EZ Trans Transfection Reagent was mixed with 250 μL of Opti-MEM. Subsequently, the two parts of the mixture were mixed and incubated at room temperature for 10 min, and then, the mixture was added to 9 mL of room temperature DMEM medium and mixed thoroughly. All media in the 96-well plate was replaced with the mixture (100 μL per well). After transfection for 24 h, the cells were treated with the compounds for 24 h. The luciferase signal was detected using a luciferase assay kit (Vazyme, DL101-01, Nanjing, China). The luminescence signal of firefly luciferase for each sample was normalized to the luminescence signal of Renilla luciferase. Statistical analysis was performed using GraphPad Prism (version 10.4.1). Data are presented as the mean ± SD (n = 3), and significance was determined using one-way ANOVA. A p-value < 0.05 was considered statistically significant.

3.5. CCK8 Assay

A549 cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% antibiotics at 37 °C in a humidified incubator with 5% CO₂ until they reached the logarithmic growth phase. A549 cells were seeded into a 96-well plate at a density of 5 × 10³ cells per well and allowed to adhere overnight. The following day, cells were pre-treated with the compounds of interest for 24 h. Then, cell viability was assessed using the CCK8 assay, and absorbance at 480 nm was recorded with a Multimode Mithras LB 943 microplate reader (Berthold, Bad Wildbad, Germany), following the manufacturer’s protocol.

4. Conclusions

In summary, our investigation on the chemistry of a deep-sea-derived Penicillium viridicatum MCCC 3A00265 led to the discovery, characterization, and structure elucidation (including absolute configuration) of seven new and six known decalin-type polyketides. All isolates share an octohydronaphthalene backbone, except that viridecalin F (6) possesses a rare naphthalene core. Of particular note, viridecalin A (1) exhibits a potent reactivation activity of mutant p53 protein at 10 μM and no cytotoxicity activity, superior to the known mutant p53 reactivator PRIMA-1 that served as the positive control in the same study. This finding underscores the value of marine-derived fungi as a source of novel bioactive compounds and provides a promising starting point for the development of targeted cancer therapies. The discovery of these compounds not only enriches the structural diversity of decalin-type polyketides but also highlights the untapped potential of deep-sea-derived fungi in drug discovery.