New Naphthalene Derivatives from the Mangrove Endophytic Fungus Daldinia eschscholzii MCZ-18

Abstract

Five new naphthalene derivatives dalesconosides A–D, F (1–4, 6), a known synthetic analogue named dalesconoside E (5), and eighteen known compounds (7–24) were isolated from Daldinia eschscholzii MCZ-18, which is an endophytic fungus obtained from the Chinese mangrove plant Ceriops tagal. Differing from previously reported naphthalenes, compounds 1 and 2 were bearing a rare ribofuranoside substituted at C-1 and the 5-methyltetrahydrofuran-2,3-diol moiety, respectively. Their structures were determined by detailed nuclear magnetic resonance (NMR) and mass spectroscopic (MS) analyses, while the absolute configurations were established by theoretical electronic circular dichroism (ECD) calculation. Compounds 1, 3, 13–17 and 19 showed broad ranges of antimicrobial spectrum against five indicator test microorganisms (Enterococcus faecalis, Methicillin-resistant Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans); especially, 1, 16 and 17 were most potent. The variations in structure and attendant biological activities provided fresh insights concerning structure−activity relationships for the naphthalene derivatives.

1. Introduction

Microorganisms from special ecological niches such as the mangrove endosymbionts have been demonstrated to be a reliable source of structurally novel and pharmacologically potent natural products (NPs) and have likewise drawn the attention of NP researchers [1,2]. Mangrove-associated fungi produce a larger number of bioactive secondary metabolites compared to any other mangrove-derived microbes; more than 70% were isolated from endophyte fungi [3,4]. Filamentous fungi of the species Daldinia eschscholtzii is known as an endophyte commonly found in multiple hosts such as dead trees [5], insects [6], alga [7], broad-leaved forest [8] and mangroves [9,10]. It has been demonstrated to be a prolific source of bioactive polyketides including immunosuppressive dalesconols A and B, acetodalmanols A and B, (+)-daeschol A, dalmanol A, 2,16-dihydroxyl-benzo[j]fluoranthene [6,11], free radical scavengers (±)-daeschol A [11], cytotoxic (±)-acetodalmanol B, 2,16-dihydroxyl-benzo[j]fluoranthene [11], fungistatic helicascolide C [7], stem cell differentiation inducer selesconol [12], NLRP3 inflammasome activation inhibitors spirodalesol [13] and α-glucosidase inhibitors chromones, naphthoquinones, and naphthofurans [14,15]. As part of our ongoing investigations on mangrove endophytic fungi [16,17,18], Daldinia eschscholtzii-Mcz18 was isolated from a fresh branch of the mangrove plant Ceriops tagal. Five new aromatic polyketide dalesconosides A–D, F (1–4, 6), a known synthetic analogue named dalesconoside E (5) (this is the first isolation from a natural source), as well as 18 known compounds (7–24) (Figure 1) were isolated and identified from the mangrove endophytic fungus Daldinia eschscholzii MCZ-18. These compounds were examined for antimicrobial and cytotoxic activities. Details of the isolation, structure elucidation, and antimicrobial and cytotoxic activities of these compounds are reported herein.

2. Results and Discussion

2.1. Structure Elucidation

Dalesconoside A (1) was isolated as yellow needles with the molecular formula C₁₇H₂₀O₇ established by HR-ESIMS (m/z 337.1289, calcd for [M+H]⁺ 337.1282). Consequently, 1 had eight degrees of unsaturation. The ¹H and ¹³C NMR data (

Table 1. ¹HNMR data of 1–6.

Position	1 a,d	2 a,c	3 b,c	4 b,c	5 b,c	6 a,c
1				7.33, d (7.6)
2	6.84, d (6.8)	2.46, m	6.59, d (8.0)	7.35, d (7.6)	6.72, s	Ha 3.18, dd (16.9, 2.8)
						Hb 2.64, dd (16.9, 3.2)
3	7.18, d (6.8)	Ha 2.76, m	6.37, d (8.0)			4.37, dd (3.2, 2.8)
		Hb 2.63, m
4						5.13, d (2.8)
5			7.91, d (8.7)
6	6.96, d (6.1)	7.32, dd (8.1, 1.0)	7.45, t (8.4, 7.7)	6.82, d (7.6)	6.87, d (7.7)	7.29, d (8.1)
7	7.39, t (6.5, 6.4)	7.42, t (8.1, 7.8)	6.86, d (7.7)	7.37, t (7.8)	7.39, t (8.3, 8.0)	7.42, t (8.1, 7.8)
8	8.00, d (6.4)	7.60, dd (7.8, 1.0)		7.43, d (7.6)	7.87, d (7.9)	7.56, d (7.8)
9		Ha 2.54, m
		Hb 2.33, m
10		Ha 2.56, m
		Hb 2.23, m
1′	5.63, d (3.6)
2′	4.24, m (overlap)	4.15, t (7.5)	Ha 2.38, m; Hb 1.86, m
3′	4.15, dd (6.6, 3.1)	Ha 2.00,m	Ha 1.83, m; Hb 1.78, m	7.06, s	7.05, s
		Hb 1.83, m
4′	4.23, m (overlap)	4.29, m	4.87, t (2.7)
5′	Ha 3.71, dd (9.7, 3.8) Hb 3.65, dd (9.7, 3.2)	1.21, d (6.3)	7.11, d (7.6)
6′			7.32, t (7.9)	7.78, dd (7.6, 2.9)	7.77, dd (7.6, 1.0)
7′			6.79, d (8.0)	7.68, t (8.2, 7.8)	7.68, t (8.3, 7.7)
8′				7.31, d (8.2)	7.32, d (8.1)
1-OCH3					3.96, s
4-OCH3	3.87, s
5-OCH3	3.92, s	3.89, s		4.05, s	4.03, s	3.93, s
8-OCH3			4.08, s
8′-OCH3			3.49, s	4.01, s	4.01, s
4-OH				9.82, s	9.42, s

^a In CD₃OD. ^b In CDCl₃.^{c 1}H (400 MHz). ^{d 1}H (500 MHz).

and

Table 2. ¹³C NMR data of 1–6.

Position	1 a,d	2 a,c	3 b,c	4 b,c	5 b,c	6 a,c
1	148.6, C	199.8, C	152.8, C	118.7, CH	147.9, C	198.9, C
2	108.2, CH	38.6, CH2	109.4, CH	128.8, CH	107.2, CH	41.8, CH2
3	113.1, CH	36.6, CH2	126.6, CH	116.2, C	114.9, C	71.4, CH
4	153.6, C	84.8, C	131.5, C	152.8, C	146.4, C	65.1, CH
4a	131.7, C	134.6, C	134.1, C	114.8, C	115.4, C	131.2, C
5	158.0, C	159.3, C	117.9, CH	156.7, C	156.5, C	160.1, C
6	108.4, CH	118.6, CH	125.5, CH	104.6, CH	105.6, CH	117.3, CH
7	127.2, CH	130.3, CH	103.8, CH	126.8, CH	126.4, CH	130.4, CH
8	116.5, CH	120.1, CH	156.9, C	121.9, CH	116.0, CH	118.7, CH
8a	119.7, C	134.7, C	115.6, C	137.4, C	129.0, C	133.8, C
9		36.1, CH2
10		35.1, CH2
1′	103.9, CH	115.8, C	33.7, CH	183.3, C	183.3, C
2′	73.7, CH	75.0, CH	23.5, CH2	150.4, C	151.0, C
3′	71.2, CH	40.4, CH2	26.7, CH2	134.5, CH	134.5, CH
4′	87.6, CH	73.3, CH	67.3, CH	185.4, C	185.4, C
4a′			140.4, C	134.5, C	134.4, C
5′	63.3, CH2	22.4, CH3	121.6, CH	118.7, CH	118.7, CH
6′			127.2, CH	134.6, CH	134.5, CH
7′			110.2, CH	117.8, CH	117.8, CH
8′			157.2, C	159.7, C	159.7, C
8a′			128.2, C	121.0, C	121.2, C
1-OCH3					56.0, CH3
4-OCH3	57.7, CH3
5-OCH3	56.9, CH3	56.1, CH3		56.2, CH3	56.2, CH3	56.5, CH3
8-OCH3			56.2, CH3
8′-OCH3			55.6, CH3	56.5, CH3	56.5, CH3

^a In CD₃OD. ^b In CDCl₃.^{c 13}C (100 MHz). ^{d 13}C (125 MHz).

) of 1 indicated that seven out of eight degrees of unsaturation are derived from a naphthalene moiety. Therefore, the last unsaturation degree was attributed to an additional ring system. The ¹H and ¹³C NMR in combination with the ¹H-¹H COSY and HSQC spectra showed that the compound had five signals of aromatic protons at [δ_H 8.00 (d, J = 6.4 Hz), δ_C 116.5, d, CH-8; δ_H 7.39 (t, J = 6.5, 6.4 Hz), δ_C 127.2, d, CH-7; 7.18 (d, J = 6.8 Hz), δ_C 113.1, d, CH-3; δ_H 6.96 (d, J = 6.1 Hz), δ_C 108.4, d, CH-6; 6.84 (d, J = 6.8 Hz), δ_C 108.2, d, CH-2], an anomeric proton [δ_H 5.63 (d, J = 3.6 Hz), δ_C 103.9, d, CH-1′], two methoxy groups [δ_H 3.92, s, δ_C 56.9, q, 5-OCH₃; δ_H 3.87, s, δ_C 57.7, q, 4-OCH₃], and signals of a furanoside moiety at δ_H 3.65–4.24. Comparison of the ¹H and ¹³C NMR data with those of 1,8-dimethoxynaphthalene (7) co-isolated in the culture medium and isotorachrysone-6-O-α-D-ribofuranoside [19] revealed that 1 was a 1,8-dimethoxynaphthalene ribofuranoside. The HMBC correlations (Figure 2) observed from H-1′ (δ_H 5.63) to C-1 (δ_C 156.6) established that the connection of the furanoside and naphthalene moieties were through the ether bridge. The relative configuration of 1 was interpreted by the diagnostic NOE interactionsH-1′/H-2′, H-2′/H-3′, H-1′/H₂-5′, and H-3′/ H₂-5, which revealed their co-facial relationship (Figure 3). The α configuration of ribose was confirmed by the chemical shifts (δ_H 5.63, δ_C 103.9, CH-1′) and coupling constant of the anomeric proton J_1′,2′ value 3.6 Hz. The sample limitations precluded further acid hydrolysis to study the absolute configuration of the α-ribofuranoside in 1, whereas the calculated ECD spectrum method can be used to predict the absolute configuration of C-1′ (Figure 4, Tables S1 and S2). Consequently, the absolute configuration of C-1′ was assigned to be R, and the sugar moiety was assigned as α-D-ribofuranoside. Thus, compound 1 was elucidated as 4,5-dimethoxynaphthalen-1-O-α-D-ribofuranoside and named dalesconoside A (Figures S1–S8).

Dalesconoside B (2) was obtained as a colorless oil, and it possessed the same molecular formula of C₁₈H₂₄O₆ as 1 with seven degrees of unsaturation as determined by HRESIMS (m/z 359.1464, calcd for [M+Na] ⁺359.1465). The ¹H NMR spectrum of 1 (Table 1) exhibited resonances for three aromatic protons [δ_H 7.60 (dd, J = 7.8, 1.0 Hz), H-8; δ_H 7.42 (dd, J = 8.1, 7.8 Hz), H-7; δ_H 7.32 (dd, J = 8.1, 1.0 Hz), H-6], which indicated the presence of a trisubstituted benzene ring. The ¹H NMR spectrum further showed signals for 12 aliphatic protons, six oxygenated protons, including two oxygenated methines (δ_H 4.15 (t, J = 7.5 Hz), H-2′; δ_H 4.29, m, H-4′), one methoxy group (δ_H 3.89, s, 5-OCH₃) and one secondary methyl group [δ_H 1.21 (d, J = 6.3 Hz), H₃-5′]. The ¹³C NMR spectrum disclosed 18 carbon resonances, including two sp³ methyls, five sp³ methylenes, two sp³ methines, two sp³ quaternary carbons (including a dioxysubstituted sp³ carbon at δ_C 115.8 (C-1′)), three sp² olefinic methines, and four sp² nonprotonated carbons (including one ketone carbonyl), as supported by the DEPT and HSQC spectra. ¹H–¹H COSY spectrum (Figure 2) suggested the presence of fragments of –CH₂(2)–CH₂(3)–, –CH(5)–CH(6)–CH(7)–, –CH₂(9)–CH₂(10)–, and –CH(2′)–CH₂(3′)–CH(4′)–CH₃(5′), suggesting that 2 contains a tetralone skeleton and a 5-methyltetrahydrofuran-2,3-diol moiety. This was confirmed by the HMBC correlations from H₂-2 (δ_H 2.46, m) and H-7 (δ_H 7.42) to C-8a (δ_C 134.7); H₂-3 (δ_H 2.76, m; δ_H 2.63, m) and H-6 (δ_H 7.32) to C-4a (δ_C 134.6); and H₂-3 and H₂-9 (δ_H 2.54, m; δ_H 2.33, m) to C-4 (δ_C 84.8). Other correlations in the HMBC spectrum from H₂-9, H₂-10 (δ_H 2.56, m; δ_H 2.23, m) and H₂-3′ (δ_H 2.00, m; δ_H 1.83, m) to C-1′ (δ_C 115.8); 5-OCH₃ (δ_H 3.89) to C-5 (δ_C 159.3) further supported the atom connectivity in compound 2. The relative configuration of 2 was assigned by analysis of the NOESY spectrum (Figure 3). There were observed correlations between H-2′/ H-4′, H-2′/ H_b-3′, H_a-3′/ H₃-5′, H-2′/ H_a-10, H_a-10/ H_b-9, and H_a-9/ H_b-3, indicating that H-2′, H_b-3′, H-4′, H_b-9 and H_a-10 are on the same face and H_a-3′, H₃-5′, H_a-9 and H_b-3 are on the other face, which is consistent with a 4S^*,1′R^*,2′S^*,4′R^* relative configuration. The absolute configuration of 2 was then determined by comparing experimental and calculated ECD spectra using time-dependent density-functional theory (TDDFT) as shown in Figure 4, Tables S3 and S4. However, theoretical computations with the expected structure provided an ECD spectrum that was an excellent fit with the mirror image of the measured spectrum of 2. Finally, the structure of 2 was assigned to be (R)-4-(2-((1′S,2′R,4′S)-1′,2′-dihydroxy-4′-methyltetrahydrofuran-1′-yl)ethyl)-4-hydroxy-5-methoxy-3,4-dihydronaphthalen-1(2H)-one (Figures S9–S16).

Dalesconoside C (3) was obtained as yellow needles, which has the molecular formula C₂₂H₂₂O₄ established by HR-ESIMS (m/z 351.1597, calcd for [M+H]⁺ 351.1591). Consequently, 3 had 12 degrees of unsaturation. The ¹H and ¹³C NMR data of 3 (Table 1 and Table 2) indicated that 11 of the 12 units of unsaturation come from a naphthalene moiety and an aromatic ring. Thus, the last remaining degree of unsaturation must be attributed to an additional ring system. The 1D NMR spectroscopic data indicated the presence of a structure with two units, one of which is identical with those of 8-methoxy-1-naphthol (8), which was isolated from the same source. The second part showed signals corresponding to 1,2,3,4-tetrahydro-5-methoxynaphthalene with the substitution at C-4. The ¹H-¹H COSY and HSQC spectra of 3 allowed the assignments of the fragments –CH(2)–CH(3)–, –CH(5)–CH(6)–CH(7)–, –CH(1′)–CH₂(2′)–CH₂(3′)–CH(4′)– and –CH(5′)–CH(6′)–CH(7′)–. The HMBC correlations from H-1′ (δ_H 5.04) to C-3 (δ_C 126.6), C-4 (δ_C 131.5), C-2′ (δ_C 23.5), C-3′ (δ_C 26.7), C-4a′ (δ_C 140.4) and C-8a′ (δ_C 128.2); from 1-OH (δ_H 9.37) to C-1 (δ_C 152.8), C-2 (δ_C 109.4) and C-8a (δ_C 115.6); from 8-OCH₃ (δ_H 4.08) to C-8 (δ_C 156.9); and from 8′-OCH₃ (δ_H 3.49) to C-8′ (δ_C 157.2) enable the establishment of the planer structure of 3. The relative configuration of 3 was based on the NOESY correlations as indicated in Figure 3. The NOESY correlations observed of H-1′/H_b-2′(δ_H 1.86), H-1′/H-5 (δ_H 7.91), H_b-2′/H-5 and H_b-2′/H-4′ (δ_H 4.87) indicated that H-1′ and H-4′ were on the same side of the 1,2,3,4-tetrahydronaphthalene moiety. The absolute configuration of 1 were also determined by comparing experimental and calculated electronic circular dichroism (ECD) spectra for the truncated model (1′S, 4′S) -3a and the truncated model (1′R, 4′R) -3b using time-dependent density-functional theory (TDDFT) (Tables S5 and S6). The calculated electronic circular dichroism (ECD) curve for 1a was in good agreement with the experimental ECD spectrum of 3 (Figure 4). Therefore, the absolute configuration of 3 was firmly assigned as 1′S, 4′S. Thus, the complete structure of 3 was established (Figures S17–S24).

Dalesconoside D (4) was obtained as a red amorphous powder. Its molecular formula, C₂₂H₁₆O₅ (15 degrees of unsaturation), was established by HR-ESI-MS (m/z 361.1071, calcd for [M+H]⁺ 361.1071). Using the ¹H and ¹³C NMR (Table 1 and Table 2) combined with the HSQC spectra of 4, a phenolic hydroxyl signal at δ_H 9.82 (s, 4-OH), the signals for nine aromatic signals at [δ_H 7.78 (dd, J = 7.6, 2.9 Hz), δ_C 118.8, d, CH-5′; δ_H 7.68 (t, J = 8.2, 7.8 Hz), δ_C 134.6, d, CH-6′; 7.43 (d, J = 7.6 Hz), δ_C 121.9, d, CH-8; δ_H 7.37 (t, J = 7.8 Hz), δ_C 126.8, d, CH-7; δ_H 7.35 (d, J = 7.6 Hz), δ_C 118.7, d, CH-1; δ_H 7.33, s, δ_C 128.8, d, CH-2; δ_H 7.31 (d, J = 8.2 Hz), δ_C 117.8, d, CH-7′; δ_H 7.06, s, δ_C 134.5, d, CH-3′; 6.82 (d, J = 7.6 Hz), δ_C 104.6, d, CH-6], and signals of two methoxy groups [δ_H 4.05, s, δ_C 56.2, q, 5-OCH₃; δ_H 4.01, s, δ_C 56.5, q, 8′-OCH₃] were observed. The coupling constants and ¹H-¹H COSY spectrum showed the presence of three ¹H-¹H spin systems from –CH(1)–CH(2)–, –CH(6)–CH(7)–CH(8)– and –CH(5′)–CH(6′)–CH(7′)–. The NMR spectroscopic data indicated that 4 comprised one 7-hydroxy-1-methoxyanthracene-9,10-dione subunit and one 8-methoxy-1-naphthol (8) subunit [20]. The HMBC spectrum (Figure 2) and NOESY correlations (Figure 3) supported the assignments of the methoxy groups 5-OCH₃ at C-5 (δ_C 156.7) and 8′-OCH₃ at C-8′ (δ_C 159.7). Moreover, HMBC correlations of 4-OH with C-4 (δ_C 152.8) and C-3(δ_C 116.2), H-2 with C-3(δ_C 116.2) and C-2 (δ_C 150.4), H-2 with C-2′(δ_C 150.4), and H-3′ with C-3 (δ_C 116.2) provided evidence for the C-3-C-2′ linkage of 4. On the basis of the above results, the structure of dalesconoside D (4) was identified as 1′-hydroxy-8,8′-dimethoxy-[2,2′-binaphthalene]-1,4-dione (Figures S25–S32).

Dalesconoside E (5) was obtained as a red amorphous powder. Its molecular formula C₂₃H₁₈O₆ (i.e., differing from that of 4 by an additional OCH₃ group) was established from HRESIMS at m/z 391.1177 [M+H]⁺. The ¹H and ¹³C NMR data (Table 1 and Table 2) of 5 were similar to those of 4 except for the significant downfield shift of C-1(δ_C 147.9) and the presence of a methoxy signal (δ_H 3.96, s, δ_C 56.0, q, 1-OCH₃) indicative of 5 was a methoxylated analogue of 4. The HMBC correlations (Figure 2) of 1-OCH₃ with C-1 revealed that the extra methoxy group is bound to C-1. Hence, 5 was 1-methoxydalesconoside D (Figures S33–S40). Compound 5 is here reported for the first time as a natural product. It was previously prepared via a two-step sequence involving the oxidative dimerization of hydroquinone monomethyl ethers [21].

Dalesconoside F (6) was isolated as a colorless amorphous powder. Its molecular formula C₁₁H₁₂O₄ was deduced from HRESIMS at m/z 209.0806 [M+H]⁺, indicating six degrees of unsaturation. The planer structure of 6 was determined as 3,4-dihydroxy-5-methoxy-3,4-dihydronaphthalen-1(2H)-one [22], which was chemically synthesized from 3,4,5-trihydroxy-1-tetralone with diazomethane, on the basis of ¹H and ¹³C NMR observations, including 2D ¹H-¹H COSY and HSQC and HMBC spectral data (Figure 2). However, the observed NOESY correlations (Figure 3) of H-3 with H-4 suggested that these protons were on the same spatial orientation. Furthermore, the theoretical ECD spectrum (Figure 4, Tables S7 and S8) was also calculated by a quantum chemical method at the [B3LYP/ 6-311+G(2d,p)] level, and the predicted ECD curve of (3R,4S)-6 was in good agreement with that of the experimental one. Finally, the absolute configuration of 6, named dalesconoside F, was unambiguously determined to be 3R,4S (Figures S41–S48).

By comparing physical and spectroscopic data with those reported in the literature, the known compounds were elucidated as 1,8-dimethoxynaphthalene (7), 8-methoxy-1-naphthol (8) [23], regiolone (9) [24], nodulisporone (10) [25], nodulisporol (11) [25], xylariol A (12) [26], (4R)-4,8-dihydroxy- 3-hydro-5-methoxy-1-naphthalenone (13) [27], (4R)-O-methylsclerone (14) [28], (4R)-3,4-dihydro-4,5-dihydroxynaphthalen-1(2H)-one (15) [29], (3S)-3,8-dihydroxy-6,7-dimethyl-a-tetralone (16) [9], fusaraisochromenone (17) [30], 3R-3,4-dihydro-6,8-dihydroxy-3-methylisocoumarin (18) [31], 2-acetyl-7-methoxybenzofuran (19) [32], 4,8-dimethoxy-1H-isochromen-1-one (20) [22], (+)-citreoisocoumarin (21) [33], 5-hydroxy-8-methoxy-2-methyl-4H-1-benzopyran-4-one(22) [34], cytochalasin O (23) [35], and dankasterone A (24) [36].

2.2. Biological Activity

Compounds 1–24 were tested for antimicrobial activity against Pseudomonas aeruginosa, Enterococcus faecalis, methicillin-resistant Staphylococcus aureus and Escherichia coli as well as antifungal activity against Candida albicans. As shown in

Table 3. Antimicrobial activities of isolated compounds 1–24.

	MIC(μg/mL)
Compound	P. aeruginosa	E. faecalis	MRSA	E.coli	C. albicans
1	6.25	25	12.5	6.25	25
2	50	>50	50	>50	>50
3	25	25	25	12.5	>50
4	25	50	50	>50	>50
5	12.5	50	25	>50	50
6	>50	>50	50	50	>50
7	12.5	50	25	25	50
8	25	>50	25	50	12.5
9	>50	25	>50	>50	25
10	50	>50	50	50	>50
11	50	25	50	50	>50
12	25	25	25	>50	25
13	25	12.5	12.5	25	25
14	25	25	25	25	25
15	50	25	50	25	25
16	12.5	25	12.5	6.25	50
17	12.5	12.5	6.25	12.5	25
18	>50	>50	12.5	25	>50
19	12.5	25	25	12.5	>50
20	>50	>50	50	50	>50
21	50	>50	50	50	>50
22	>50	>50	25	50	>50
23	>50	>50	25	25	>50
24	50	>50	>50	>50	>50
Ciprofloxacin	0.78	0.625	0.3125	0.625
Amphotericin B					0.78

, all metabolites showed antimicrobial activity against at least one of the five tested indicator pathogens at the selected concentration of 50 μg/mL, but all of the antimicrobial activities were weaker than that of the positive control. Compounds 1, 3, 13–17 and 19 exhibited a wide range of antimicrobial activities; especially, 1 (against P. aeruginosa and E. coli), 16 (against E. coli) and 17 (against MRSA) were most potent, and their resistant strain with MIC values reached 6.25 µg/mL. The comparison between compounds 1 and 7 approved that the substitution of additional furanose was the critical structural component for its antimicrobial activity. The antibacterial results of the comparison of 2–16 suggested that the dihydronaphthalen-1-one nucleus may not be sensitive to the tested microbes in some cases, but the extra auxochromes, e.g., -OCH₃, -OH or CH₃ enhanced the antibacterial effect of the compound against the tested bacteria and fungi. Meanwhile, when the hydroxyl group is oxidized to a carbonyl group to a certain extent, the antibacterial activity of the metabolite against some indicator bacteria is weakened. Comparing the antimicrobial activities of compounds 17–22 indicated that the O-containing heterocyclic substructure may increase the compound’s effect on P. aeruginosa, E. faecalis, MRSA and E. coli. When the -OH and -OCH₃ groups are differently substituted on the benzene ring or the lactone/quionone of metabolites’ isocoumarin backbone, different indicator bacteria have different promotion and inhibition. In general, the broad antimicrobial activities of these naphthalene derivatives support their continued investigation so as to develop a deeper understanding of structure−activity relationships and mechanism of antimicrobial action.

3. Materials and Methods

3.1. General Experimental Procedure

Optical rotation was recorded at 20 °C using a WYA-2S digital Abbe polarimeter (Shanghai Physico-optical Instrument Factory, Shanghai, China). UV spectral data were obtained from online UV spectra acquired on a Shimadzu UV-2401 PC spectrophotometer. NMR spectra were recorded on a Bruker AV-400 and AV-500 (Bruker Corporation, Fällanden, Switzerland) instrument with TMS as an internal standard. ESI-MS spectra were recorded on a VG Auto Spec-3000 mass spectrometer (VG, Manchester, UK). High-resolution ESI-MS were recorded on an Agilent 6210 mass spectrometer (Agilent Technologies, Waldbronn, Germany) employing peak matching. The ECD spectra were measured on a JASCO J-715 spectra polarimeter (Japan Spectroscopic, Tokyo, Japan). SephadexLH-20 (Beijing Biotopped Science & Technology Co., Ltd., Beijing, China), SiliaSphere C18 (SiliCycle, Quebec, Canada) and Silica gel (200–300 mesh, Qingdao Marine Chemical Factory, Qingdao, China) were used for column chromatography (CC). Thin-layer chromatography (TLC) was performed on precoated silica gel GF254 plates (Qingdao Marine Chemical Factory, Qingdao, China).

3.2. Fungal Material

The strain MCZ-18 was isolated from a fresh, healthy branch of Ceriops tagal collected from the Dong Zhai Gang-Mangrove Garden on Hainan Island, China. The fungus was isolated under sterile conditions from the inner tissue of the flower following an isolation protocol described previously [37] and identified as Daldinia eschscholzii (GenBank accession no. MH712260) by morphologic traits and molecular identification. A voucher strain was deposited in the School of Chemical Engineering and Technology, Hainan University, Haikou, China.

3.3. Fermentation, Extraction and Isolation

The Daldinia eschscholzii MCZ-18 strain was cultivated on an autoclaved rice solid-substrate medium, consisting of 80 1 L Erlenmeyer flasks. Each flask included 80 g of rice, 0.24 g of sea salt and 80 mL of water. The culture was maintained at a temperature of 25 °C. Following a fermentation period of 29 days, the mycelia and rice were subjected to three extractions using EtOAc. The extracts underwent filtration and evaporation under decreased pressure to yield a crude extract of 75 g. The crude extracts were then submitted to vacuum liquid chromatography (VLC) on silica gel employing a step gradient of petroleum ether/ethyl acetate (1:0–0:1, v/v) to obtain five fractions (Fr.1–Fr.5). Fr.1 was eluted equivalently with petroleum ether to give 8 (200 mg). Fr.2 was eluted equivalently with petroleum ether / ethyl acetate (50:1, v/v) to obtain 7 (150 mg). Fr.3 was eluted with a step gradient of petroleum ether/ethyl acetate (20:1, 10:1, 5:1, 3:1, 1:1, v/v) to give 3 fractions (Fr.3.1–Fr.3.3). Subsequently, Fr.3.1 was further separated by Sephadex LH-20 chromatography (CH₂Cl₂/MeOH, 1:1, v/v) and finally by an ODS column eluting with MeOH-H₂O (70:30, 80:20, 90:10, 100:0, v/v) giving 24 (4.5 mg) and 22 (20 mg). Fr.3.2 was eluted equivalently with petroleum ether/ethyl acetate (5:1, v/v) to give 3 subfractions (Fr.3.2.1–Fr.3.2.3). Fr.3.2.2 was further purified by preparative TLC (developing solvents: CH₂Cl₂/MeOH, 100: 3, v/v) to obtain 14 (12 mg) and 16 (24 mg). Fr.3.2.3 was purified by Sephadex LH-20 CC (CH₂Cl₂/MeOH, 1:1, v/v) and finally by an ODS column eluting with MeOH-H₂O (60:40, 70:30, v/v) to obtain 3 (2 mg), 19 (6 mg), 11 (1.0 mg), 5 (2.6 mg), 4 (1.2 mg) and 9 (2 mg). 2 (1.8 mg) and 6 (2.2 mg) was acquired by Sephadex LH-20 chromatography (CH₂Cl₂/MeOH, 1:1, v/v) and C-18 ODS column (MeOH/H₂O, 60:40, 70:30, v/v). Fr.3.3 was eluted with a step gradient of petroleum ether/ethyl acetate (3:1, 2:1, 1:1, v/v) to give 3 fractions (Fr.3.3.1–Fr.3.3.3). Fr.3.3.2 was purified by Sephadex LH-20 CC (CH₂Cl₂/MeOH, 1:1, v/v) and finally by an ODS column eluting with MeOH-H₂O (60:40, v/v) to obtain 13 (3 mg), 18 (3.2 mg) and 12 (2 mg). Fr.3.3.3 was purified by Sephadex LH-20 CC (MeOH) and finally by an ODS column eluting with MeOH-H₂O (45:55, v/v) to obtain 21 (3.3 mg), 10 (3.6 mg) and 17 (4.4 mg). Fr.4 was purified by an ODS column eluting with MeOH-H₂O (40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 100:0, v/v) to obtain 3 subfractions (Fr 4.1–Fr 4.3) and 23 (3.8 mg). Fr.4.2 was purified by Sephadex LH-20 CC (MeOH) and finally by an ODS column eluting with MeOH-H₂O (45:55, v/v) to obtain 15 (3 mg), 20 (2.6 mg) and 1 (5.5 mg).

Dalesconoside A (1): yellow needles; [α]²⁰_D +100 (c 0.0001, MeOH); UV (MeOH) λ_max 226 nm; ¹H and ¹³C NMR data, see Table 1 and Table 2; (+)-HRESIMS at m/z 337.1289 [M+H]⁺ (calcd for C₁₇H₂₁O₇ 337.1282).

Dalesconoside B (2): colorless oil; [α]²⁰_D +10 (c 0.0001, MeOH); UV (MeOH) λ_max 205 nm; ¹H and ¹³C NMR data, see Table 1 and Table 2; (+)-HRESIMS at m/z 359.1464 [M+Na] ⁺ (calcd for C₁₈H₂₄O₆Na 359.1465).

Alesconoside C (3): yellow needles; [α]²⁰_D +80 (c 0.0001, MeOH); UV (MeOH) λ_max 202 nm; ¹H and ¹³C NMR data, see Table 1 and Table 2; (+)-HRESIMS at m/z 351.1597 [M+H]⁺ (calcd for C₂₂H₂₃O₄ 351.1591).

Dalesconoside D (4): red amorphous solid; UV (MeOH) λ_max 201 nm; ¹H and ¹³C NMR data, see Table 1 and Table 2; (+)-HRESIMS at m/z 361.1071 [M+H]⁺ (calcd for C₂₂H₁₇O₅ 361.1071).

Dalesconoside E (5): red amorphous solid; UV (MeOH) λ_max 202 nm; [α]²⁰_D +70 (c 0.0001, MeOH); UV (MeOH) λ_max 201 nm; ¹H and ¹³C NMR data, see Table 1 and Table 2; (+)-HRESIMS at m/z 391.1177 [M+H]⁺ (calcd for C₂₃H₁₉O₆ 391.1176).

Dalesconoside F (6): colorless solid; [α]²⁰_D +80 (c 0.0001, MeOH); UV (MeOH) λ_max 221 nm; ¹H and ¹³C NMR data, see Table 1 and Table 2; (+)-HRESIMS at m/z 209.0806 [M+H]⁺ (calcd for C₁₁H₁₃O₄ 209.0808).

3.4. ECD Calculations

The Monte Carlo conformational searches were carried out by means of the Spartan’s 14 software (v1.1.4) using a Merck Molecular Force Field (MMFF). The conformers of 1–3 and 6 with a Boltzmann population of over 5% were chosen for ECD calculations, and then the conformers were initially optimized at B3LYP/6-31g (d, p) in gas. The theoretical calculation of ECD was conducted in MeOH using time-dependent density functional theory (TD-DFT) at the B3LYP/6-31+g (d, p) level for all conformers of compounds. Rotatory strengths for a total of 30 excited states were calculated. ECD spectra were generated using the program SpecDis 1.6 (University of Würzburg, Würzburg, Germany) and GraphPad Prism 5 (University of California San Diego, CA, USA) from dipole-length rotational strengths by applying Gaussian band shapes with sigma = 0.3 eV.

3.5. Antimicrobial Activity Assay

The microplate assay method was used to assess the antimicrobial activities of compounds 1–24 against four terrestrial pathogenic bacteria (Pseudomonas aeruginosa, Methicillin-resistant Staphylococcus aureus, Bacillus subtilis and Escherichia coli) and one pathogenic fungus (Candida albicans) (Guangdong Microbial Culture Collection Center, China, CDMCC) according to previously reported methods [38]. Ciprofloxacin and amphotericin B were used as a positive control.

4. Conclusions

In conclusion, five new naphthalene derivatives (1–4, 6), a new natural product (5) and eighteen known compounds (7–24) were isolated from mangrove endophytic fungus Daldinia eschscholzii MCZ-18. Compounds 1 and 2 are naphthalenes bearing a rare ribofuranoside substituted at C-1 and the 5-methyltetrahydrofuran-2,3-diol moiety, respectively. This is the first report of these naphthalene subtypes being isolated from mangrove-derived fungal sources. All the isolated metabolites showed more or less antimicrobial activities. Several naphthalene derivatives (1, 3, 13–17 and 19) showed broad ranges of antimicrobial spectrum. Based on the structure–activity relationship analysis, oxygen heterocyclic rings such as ribofuranoside and tetrahydropyran moieties, auxochromes, e.g., -OCH₃, -OH or CH₃ were considered as the pharmacophores. The results presented herein reinforce the importance of the mangrove microbial environment as a source of antimicrobial compounds with remarkably varied applications.